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About the cover: DNA transcription by nRNA. Colored transmission electron micrograph of DNA and messenger RNA (mRNA) molecules forming a feather-like, transcriptionally active structure. This DNA is from the nucleus of an amphibian egg. The backbone of the feather, running down the image, is a long strand of DNA coated with protein. Numerous nRNA molecules extend in clusters from the DNA strand. Transcription of genetic information begins at one end of the gene, with the mRNA molecules growing longer as they approach completion. Tfanscription is the first step in protein synthesis. Magnification: approximately x30,000.
Libraryof CongressCatatogi ng-in- Publication Data Lewin, Benjamin. Genes IX / Benjamin Lewin. P'; cm' Includes bibliographical references and index. I S B N -I 3 : 9 78 - 0 - 76 3 7- 4 0 6 ) - 4 ( a l k . p a p e r ) I S B N -l 0 : 0 - 76 3 7 - 4 0 6 3 - 2 l.
Genetics.2. Genes. I. Title. II. Tirle: Genes9. III. Title: Genesnine. 4. Genome. 5. Proteins-genetics. [DNLM: l. Genes-physiology. 2. DNA-genetics. 3. Genetic Processes. 6. RNA-genetics. QU 470 L672g20061 QH430.L4 2006 576.5-dc22 20060t0787 6048 Printed in the United Statesof America rr I0 09 08 07 l0 9 8 7 6 5 43 2 |
BriefContents Contents vi Preface xvi
Is Connected Repl.ication @ aacterial to theCettCycle 408 428 Replication @ or,rn
Q
C.n., nre DNn L
forProteins23 @ C.n.,Code Gene 17 @ rn. rnrerrupreo of the Genome55 @ ff.r.Content
andGene Numbers 76 @ C.noreSequences andRepeats98 @ Ctusters @ u.rr.ngerRNA 1.27 Synthesis1,51. @ erotein Code 189 @ UtingtheGenetic Localization21.8 @ Protein @ Tr.nrrription 256 @ rh. 0peron 3oo RNA 331 @ negutatory @ ef,rg.Strategies34g @ rf.r.Repticon376 Replicons3gz @ *tractrromosomal
andSite-Specific @ Homotogous 457 Recombination Systems4gg @ nepair @rt.ntposons 521. 550 andRetroposons @ R.trouiruses @ trrrne Diversity570 andEnhancers609 @Promoters @ActivatingTranscription640 andProcessing667 @RNASpl.icing @CatatyticRNA 706 729 @Chr om osomes @Nucleosomes757 Structure 796 Chromatin @ControL[ing
(DEpigeneticEffectsAreInherited 818 Glossary845 Index 867
Contents Prefacexvi
1 Genes AreDNA 1, Introduction2 DNAIs the Genetic Material of Bacteria3 DNAIs the Genetic Material of Viruses4 IEl
DNAIs the Genetic Material of AnimalCe[[s5 Polynucteotide Chains HaveNitrogenous Bases Linked to a Sugar-Phosphate Backbone 6 DNAIs a Doubte Hetix 6 DNAReplication Is Semiconservative 8 DNAStrands Separate at the Replication Fork 9 Genetic Information CanBeProvided by DNAor RNA 10 Nucteic AcidsHybridize by BasePairing 1,2 Mutations Change the Sequence of DNA 1.4 Mutations MayAffectSingleBasePairsor Longer Sequences 75 TheEffects of Mutations CanBeReversed76 Mutations AreConcentrated at HotspotsIl ManyHotspots Result fromModified Bases18 SomeHereditary AgentsAreExtremety Smatl 79 Summary20
2 Genes Codefor Proteins23 Introduction24 A GeneCodes for a SinglePolypeptide24 Mutations in theSame Gene Cannot Complement 25 Mutations MayCause Loss-of-Function or Gain-of-Function 26 A LocusMayHaveManyDifferent MutantAlleles Zl A LocusMayHaveMorethanOneWitd-type Altete 28 Recombination 0ccursby Physicat Exchange of DNA 28 TheGenetic CodeIs Triptet 30 Every Sequence HasThreePossibte Reading Frames3l Prokaryotic Genes AreColinear withTheirProteins32
vi
Processes Several AreRequired to Express the Protein Product of a Gene 33 ProteinsAreTrans-acting, but Siteson DNA AreCrs-acting35 Summary36
3 TheInterrupted Gene 37 Introduction38 An InterruDted GeneConsists of Exons andIntrons 38 Restriction Endonucteases Area KeyToo[in Mapping DNA 39 0rganization of Interrupted Genes MayBeConserved 40 ExonSequences AreConserved but IntronsVary 42 Genes Showa WideDistribution of Sizes 43 SomeDNASequences Codefor MoreThanOne Protein45 HowDidinterrupted Genes Evolve?47 SomeExons CanBeEquated with ProteinFunctions49 TheMembers of a GeneFamity Havea Common 0rganization51. Is A[[Genetic Information Contained in DNA?53 Summary53
4 TheContent of the Genome55 Introduction56 Genomes CanBeMapped by Linkage, Restriction Cteavage, or DNASequence56 IndividuaI Genomes ShowExtensive Variation5l RFLPs andSNPs CanBeUsedfor Genetic Mapping58 WhyAreGenomes SoLarge?60 Eukaryotic Genomes Contain BothNonrepetitive andRepetitive DNASequences 61, Genes CanBeIso[ated bythe Conservation of Exons63 TheConservation of Genome 0rganization Hetps to IdentifyGenes65 0rganettes HaveDNA 67
HaveVeryShortIdenticaI Arthropod Satetlites Reoeats1.1.9 Consist of Hierarchical Satetlites Mammalian Repeats1.20 Mapping1'23 AreUsefulfor Genetic Minisate[lites
AreCircutar 0rganelte Genomes DNAs ThatCode for Organelte Proteins69 Mitochondrial DNA0rganization ]s Variabte70 TheChtoroptast Genome for ManyProteins Codes 71, andRNAs Mitochondria Evolved by Endosymbiosis 72
Summary725
Summarv73
RNA 1'27 7 Messenger 5 Genome Sequences andGene Numbers76
Introduction1,28 andIs Is Produced byTranscription mRNA 1,29 Trans[ated a Ctoverleaf130 Transfer RNAForms Areat Ends StemandAnticodon TheAcceptor of the TertiaryStructure 1,31, 1'32 by Ribosomes RNAIs Transtated Messenger
Introduction77 Bacterial GeneNumbers Range 0veran 0rder Magnitude 77 of TotalGeneNumber Is Knownfor Several Eukaryotes 79 HowManyDifferent Types of Genes AreThere?81
Bindto OnemRNA133 ManyRibosomes RNA 135 Messenger of Bacterial TheLifeCycte Duringor afterIts mRNA Is Modified Eukaryotic Transcriotion1'37 mRNA Is Capped138 The5'Endof Eukaryotic
TheHuman HasFewer Genome Genes ThanExpected83 HowAreGenes Distributed and0therSequences in theGenome? 85 TheY Chromosome HasSeveraI Male-Specific Genes86 MoreComplex Species Evotve by AddingNewGene Functions87 HowManyGenes AreEssential? 89
739 Is Po[yadenytated The3'Terminus InvolvesMultip[e Degradation mRNA BacteriaI Enzymes1.40 on Its Structure Depends mRNA StabiLity andSequence1,41, Activities743 InvolvesMultipte mRNA Degradation System1'44 Trigger a Surveiltance Mutations Nonsense
AreExpressed Levels92 Genes at WidetyDiffering HowManyGenes AreExpressed? 93 Expressed GeneNumber CanBeMeasured EnMosse93 Summarv94
AreTransported745 RNAs Eukaryotic Localized1'46 mRNA CanBeSpecifically
6 Clusters andRepeats98 Introduction99 Is a MajorForce in Evotution100 GeneDuptication AreFormed GlobinCtusters by Duplication and Divergence 101 Sequence Divergence Is the Basisfor the Evolutionary Clock 104 TheRateof Neutral Substitution CanBeMeasured fromDivergence 1.07 of Repeated Sequences Pseudogenes AreDeadEndsof Evolution108 Crossing-over Rearranges GeneCtusters109 UnequaI for rRNAFormTandem Repeats1.1.2 Genes TheReoeated Genes for rRNAMaintain Constant Sequence71,4 Fixation IdenticaIRepeats775 Crossover CoutdMaintain DNAs 1.17 Satettite 0ftenLiein Heterochromatin
Summary1.47
I
Synthesis15L Protein Introduction 1'52 0ccursby Initiation,Etongation, Synthesis Protein andTermination1'53 of Protein the Accuracy Control Mechanisms SpeciaI 156 Synthesis andAccessory Needs 30SSubunits Initiationin Bacteria Factors1.57 InitiatortRNAStartsthe Potypeptide A SpeciaI Chain 158 by IF-2andthe Is Controlted Useof fMet-tRNAt 160 Ribosome mRNA InitiationInvolvesBasePairingBetween andrRNA 1.61.
Contents vii
Sma[[Subunits Scanfor InitiationSiteson Eukarvotic mRNA1,62 Eukaryotes Usea Comptex of ManyInitiation Factors!64 Elongation Factor TuLoads Aminoacyt-tRNA into the A Site 167 ThePotypeptide ChainIs Transferred to Aminoacyl-tRNA 168 Transtocation Moves the Ribosome769 Etongation Factors BindAtternately to the Ribosome170 ThreeCodons Terminate Protein Synthesis772 Termination Codons AreRecognized by Protein Factors 173 RibosomaI RNAPervades BothRibosomaI Subunits775 G:t
I
Ribosomes HaveSeveral ActiveCenters177 165rRNAPtays an ActiveRolein Protein Synthesis179 23SrRNAHasPeptidyl Transferase Activity 792 RibosomaI Structures Chanqe WhentheSubunits Come Together183 Summary183
Usingthe Genetic Code 189 Introduction190 Related Codons Represent Related AminoAcids 190 Codon-Anticodon Recognition Invotves Wobbting1.92 tRNAs AreProcessed fromLongerPrecursors lg4 IRNAContains Modified Bases1,94 Modified Bases AffectAnticodon-Codon Pairing 796 ThereAreSporadic Alterations of the Universa[ Code 1.97 NovelAminoAcidsCanBeInsertedat Certain Stop Codons799 tRNAs AreCharged withAminoAcids by Synthetases 200 Aminoacyt-tRNA Synthetases Fat[into TwoGroupsZO1, Synthetases UseProofreading to ImproveAccuracy203 Suppressor tRNAsHaveMutated Anticodons ThatRead NewCodons206 ThereAreNonsense Suppressors for EachTermination Codon 207 Suppressors MayCompete with Witd-Type Reading of the Code 208 TheRibosome Inftuences the Accuracy of Translation209
viii
Contents
Recoding Changes CodonMeanings21.7 Frameshifting 0ccursat Stippery Sequences 21,3 Bypassing InvolvesRibosome Movement274 Summary 215
10 ProteinLocalization21,8 Introduction220 Passage Across a Membrane Requires a Special Apparatus220 Protein Transtocation MayBePosttranstational or Cotranslationa[ 227 Chaperones MayBeRequired for ProteinFolding 223 AreNeeded Chaperones by Newty Synthesized andby Denatured Proteins224 TheHsp70 Famity Is Ubiquitous226 SignaI Sequences InitiateTranslocation 227 TheSignalSequence Interacts withthe SRP 228 TheSRPInteracts withthe SRPReceptor229 TheTranslocon Forms a Pore 231, Transtocation Requires Insertioninto the Transtocon and(Sometimes) a Ratchet in the ER 233 Reverse Translocation SendsProteins to the CvtosoI for Degradation 234 Proteins Reside in Membranes by Means of Hydrophobic Regions235 AnchorSequences Determine Protein0rientation236 HowDoProteins Insertinto Membranes? 238 PosttranstationaI Membrane InsertionDepends on Leader Sequences 240 A Hierarchy of Sequences Determines Location within 0rgane[[es247 InnerandOuterMitochondriaI Membranes HaveDifferent Transtocons 243 Peroxisomes Employ Another Typeof Translocation System245 Bacteria UseBothCotranslationaI andPosttranstationa[ Trans[ocation 246 TheSecSystem Transports Proteins into andThrough theInnerMembrane247 Sec-Independent Transtocation Systems in E. coLi 249 Summary250
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Antisense RNACanBeUsedto Inactivate Gene Exoression 338 Smatl. RNAMotecules CanRegutate Trans[ation339 Bacteria ContainRegulator RNAs341, MicroRNAs AreRegulators in ManyEukaryotes 342 RNAInterference Is Related to GeneSilencinq343 Summary345
14 Phage Strategies349 Introduction350 LyticDevetopment Is Dividedinto TwoPeriods352 LyticDevetopment Is Controtled by a Cascade353 TwoTypesof Regutatory EventControlthe Lytic Cascade354 TheT7andT4Genomes ShowFunctiona[ Clustering355 Lambda Immediate EartyandDetayed EartyGenes Are Needed for BothLysogeny andthe LyticCycle 356 TheLyticCycteDepends on Antitermination357 Lysogeny Is Maintained by Repressor Protein 359 TheRepressor andIts 0perators Definethe Immunity Region360 TheDNA-Binding Formof Repressor Is a Dimer361 Repressor Usesa Helix-Turn-Helix Motifto Bind DNA362 TheRecognition HelixDetermines Specificity for DNA 363 Repressor Dimers BindCooperativety to the Operator364 Repressor at 0*2 Interacts with RNAPotymerase at P*, 365 Repressor Maintains an Autogenous Circuit 366 Cooperative Interactions Increase the Sensitivitv of Regulation367 Thecil andcIIi Genes AreNeeded to Establish Lysogeny368 A PoorPromoter Requires cII Protein 369 Lysogeny Requires SeveraI Events369 ThecroRepressor Is Needed for LyticInfection 311. WhatDetermines the Balance Between Lysogeny andthe LyticCycte?373 Summary374
Contents
15 TheReplicon376 Introduction377 Replicons CanBeLinearor Circutar378 0riginsCanBeMapped by Autoradiography andElectrophoresis 379 DoesMethytation at the OriginRegulate Initiation? 380 Origins MayBeSequestered afterReptication381 EachEukaryotic Chromosome Contains Many Repticons383 Reptication 0riginsCanBeIsolated in Yeast 384 Licensing Factor Controts Eukaryotic Rerep[ication 385 Licensing Factor Consists of MCMProteins386 D LoopsMaintain Mitochondrial 0rigins 388 Summary389
16 Extrachromosomal Repticons392 Introduction393 TheEndsof LinearDNAArea Prob[em for Reptication393 Termina[ Proteins Enabte Initiationat the Ends of ViraIDNAs394 Rol.ting Circles Produce Muttimers of a Replicon 396 Rotting Circtes AreUsedto Repticate Phage Genomes397 TheF Plasmid Is Transferred by Conjugation between Bacteria398 Conjugation Transfers Single-Stranded DNA 400 TheBacterial Ti Ptasmid Causes Crown Ga[[Disease in Ptants 401. T-DNA Carries Genes Required for Infection 402 Transfer of T-DNA Resembtes Bacteria[ Conjugation405 Summary407
17 Bacterial Replication Is Connected to the CeLL CycLe 408 Introduction 409 Reptication Is Connected to the Ce[[Cycte 410 TheSeptumDivides a Bacterium into Progeny ThatEach Contain a Chromosome 41,7 Mutations in Division or Segregation AffectCe[[ Shaoe 41,2 FtsZIs Necessary for SeptumFormation413 mrnGenes Regulate the Location of the Septum 475
MayRequire Site-Specific ChromosomaI Segregation 41.5 Recombination Partitioni ng Involves Separation 417 of theChromosomes Plasmids Havea Partitioning System479 Singte-Copy Incompatibitity Is Determined Plasmid by the Rep[icon421 by an RNA System Is Controtled TheCotErCompatibitity Regutator422 424 Replicate andSegregate? HowDoMitochondria Summary425
18 DNAReplication428
AreConnected Chromosomes Recombining ComP[ex465 bythe Synaptonema[ Forms afterDoubte-Strand Complex TheSynaptonemaI Breaks467 Are Formation Complex andSynaptonemal Pairing 469 Indeoendent Is Stimutated System RecBCD TheBacterial 470 by chiSequences Single-Strand Catalyze Proteins Strand-Transfer Assimilation471' Junctions473 Hol.l.iday Resolves TheRuvSystem for Intera[[e[ic Accounts GeneConversion 475 Recombination of DNA 476 the Structure Affects Supercoiting
Introduction 429 Arethe Enzymes ThatMakeDNA 430 DNAPolymerases Nuctease Activities 431' DNAPolymerases HaveVarious
Supercoits Relaxor Introduce Topoisomerases in DNA 478 Strands 480 BreakandReseal Topoisomerases
of Reptication432 DNAPotymerases Control the Fidel.ity Havea Common Structure433 DNAPotymerases
by Coi[Inversion481' Functions Gyrase Sites 482 Specific Invo[ves Recombination Specialized InvolvesBreakage Recombination Site-Specific andReunion484 Topoisomerase Resembles Recombination Site-Specific Activity 484 0ccursin an Intasome486 Recombination Lambda
Is Semidiscontinuous 434 DNASynthesis DNAIs HowSingle-Stranded The
andSite-Specific 19 Homologous 457 Recombination Introduction 459 Synapsed between Recombination Occurs Homotogous 460 Chromosomes DNA 462 InvolvesHeteroduptex Breakage andReunion 464 Breaks InitiateRecombination Doubte-Strand
YeastCanSwitchSitentandActiveLocifor Mating Type 488 Proteins490 for Regulator Ihe MATLocusCodes at HMLandHMRAreRepressed492 SitentCassettes Is Initiatedbythe Recipient Transposition UnidirectionaI MATLocus 493 Switching494 Controts of H0 Expression Regulation Summary496
Systems499 20 Repair Introduction500 to DNA 502 Damage Correct Systems Repair in E.coli 503 Systems Repair Excision Celts 504 in Mammalian Pathways Excision-Repair Is Usedby Methytases BaseFlipping 506 andGtycosytases 507 andMutatorPhenotypes Repair Error-Prone Repair 507 of Mismatch the Direction Controlling in E.coli 510 Systems Recombination-Repair to Recover Is an ImportantMechanism Recombination Errors 51'1' fromReplication the S0SSYstem513 RecATriggers Contents xi
Eukaryotic CetlsHaveConserved Repair Systems515 A Common System Repairs Doub[e-Strand Breaks51,6 Summary518
21 Transposons 521 introduction5Zz InsertionSequences AreSimpte Transposition Modutes524 Composite Transposons HaveIS Modutes525 Transposition 0ccursby BothRepticative andNonrepticative Mechanisms 527 Transposons Cause Rearrangement of DNA 5Zg Common Intermediates for Transposition 530 proceeds Repticative Transposition through a Cointegrate531 proceeds ]Zl.Fl Nonrepticative Transposition by Breakage andReunion533 TnATransposition Requires Transposase andResolvase 534 Transposition ofTn10HasMuttiple Controls536 Controlting Etements in MaizeCause Breakaqe andRearrangements 538 Controtling Etements FormFamilies of Transposons 540 SpmElements Inftuence GeneExpression 542 TheRo[eof Transposable Elements in Hybrid Dysgenesis 544 P E[ements AreActivated in the Germline545 Summary546
22 Retroviruses andRetroposons 550 Introduction551 TheRetrovirus LifeCycte Involves Transposition-Like Events 557 Retroviral Genes Codefor potyproteins552 ViratDNAIs Generated by Reverse Transcription 554 Viral. DNAIntegrates into the Chromosome 556 Retroviruses MayTransduce Cetlular Sequences 559 YeastIy Elements Resemble Retroviruses 559 ManyTransposable Etements Reside in Drosophilo melanogoster567 Retroposons Fat[into ThreeCtasses562 TheAtuFamilyHasManyWidetyDispersed Members564 Processed Pseudogenes 0riginated asSubstrates for Transposition 565
xii
Contents
LINES Usean Endonuclease to Generate a Priming End 566 Summary567
23 ImmuneDiversity570 Introduction572 C[onaI Se[ection Amplifies Lymphocytes ThatRespond to IndividuaI Antigens574 Immunogtobulin Genes AreAssembted fromTheirparts in Lymphocytes 575 LightChains AreAssembled by a Singte Recombination 577 Heavy Chains AreAssembled byTwo Recombinations 579 Recombination Generates Extensive Diversity580 ImmuneRecombination UsesTwoTypes of Consensus Sequence581 Recombination Generates Deletions or Inversions582 AtteticExclusion Is Triggered Productive by Rearrangement 582 TheRAGProteins Catalyze Breakage andReunion594 EartyHeavy ChainExpression CanBeChanged by RNA Processing 586 Class Switching Is Caused by DNARecombination 587 Switching 0ccursby a Nove[Recombination Reaction589 Somatic Mutation Generates Additionat Diversitv in Mouse andHuman Being 590 Somatic Mutation Is Inducedby Cytidine Deaminase andUraci[Glycosytase 597 AvianImmunogtobulins AreAssembled from Pseudogenes 593 B Ce[[Memory Altowsa RapidSecondary Response594 T Cet[Receptors AreRelated to Immunogtobulins 595 TheT Cet[Receptor Functions in Conjunction withthe MHC 597 TheMajorHistocompatibitity LocusCodes for ManyGenes of the ImmuneSystem599 InnateImmunityUtitizes Conserved Signating Pathways602 Summary604
24 Promoters andEnhancers 609 Introduction610 Eukaryotic RNAPotymerases Consist of Many Subunits61.2
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SomeGroupI IntronsCodefor Endonucteases That Sponsor Mobil.ity71.5 GroupII IntronsMayCodefor Multifunction Proteins 71.6 SomeAutospticing IntronsRequire Maturases717 TheCatatytic Activityof RNAase P Is Dueto RNA 71.8 ViroidsHaveCatatytic Activity 7tB RNAEditing0ccursat Individual Bases720 RNAEditingCanBeDirected by GuideRNAs721. Protein Spticing Is Autocatatytic724 Summary725
28 Chromosomes 729 Etrl ]I*,
Introduction 730 ViralGenomes ArePackaged into TheirCoats 731. TheBacterial Genome Is a Nucteoid734 TheBacterial Genome Is Supercoiled 135 Eukaryotic DNAHasLoopsandDomains Attached to a Scaffotd736 Specific Sequences AttachDNAto an Interphase Matrix 737 Chromatin Is Divided into Euchromatin andHeterochromatin 138 Chromosomes HaveBanding Patterns740 Lampbrush Chromosomes AreExtended747 Polytene Chromosomes FormBands 742 Polytene Chromosomes Expand at Sitesof Gene Expression 743 TheEukaryotic Chromosome Is a Segregation Device744 Centromeres MayContain Repetitive DNA 746 Centromeres HaveShortDNASequences in S. cerevisiae747 TheCentromere Bindsa ProteinComotex748 Tetomeres HaveSimpteRepeating Sequences 748 Tetomeres Sea[the Chromosome Ends 749 Te[omeres AreSynthesized by a Ribonucteoprotein Enzyme750 Tetomeres AreEssential for Survival752 Summary753
29 Nucleosomes 757 Introduction758 TheNucteosome Is the Subunitof At[Chromatin759 DNAIs Coited in Arraysof Nucleosomes 161. Nucteosomes Havea Common Structurel62 xtv
Contents
DNAStructure Varies on the Nucleosomal Surface763 ThePeriodicity of DNAChanges on the Nucteosome 766 Organization of the Histone Octamer767 ThePathof Nucleosomes in the Chromatin Fiber 769 Reproduction of Chromatin Requires Assemb[y of Nucleosomes 777 DoNucteosomes Lieat Specific Positions?774 AreTranscribed Genes Organized in Nucleosomes? 177 Histone 0ctamers AreDisptaced by Transcription 779 Nucteosome Disptacement andReassem btyRequi re SpeciaIFactors781. Insutators Blockthe Actionsof Enhancers andHeterochromatin 781. Insulators CanDefinea Domain783 Insulators MayActin OneDirection784 Insutators CanVaryin Strength785 DNAase Hypersensitive SitesReflect Changes in Chromatin Structure786 Domains DefineRegions ThatContain ActiveGenes788 An LCRMayContro[ a Domain789 WhatConstitutes a Regutatory Domain?790 Summary797
30 ControLLing Chromatin Structure796 Introduction797 Chromatin CanHaveAlternative States 797 Chromatin Remodeting is an ActiveProcess798 Nucteosome 0rganization MayBeChanged at the Promoter801 HistoneModification Is a KeyEvent 802 Histone Acetylation 0ccursin TwoCircumstances 805 Acetytases AreAssociated with Activators806 Deacetytases AreAssociated with Repressors 808 Methytation Histones of andDNAIs Connected808 Chromatin StatesAreInterconverted by Modification809 Promoter Activation Invotves an 0rdered Series of Events809 HistonePhosphorylation AffectsChromatin Structure810 SomeCommon MotifsAreFound in Proteins ThatModifv Chromatin 811 Summary81.2
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Preface Scienceis a wonderfully resilientventure. There are new and interesting developmentsto report in each revision of this book, and this revisionincludesmuch updatedmaterial to account for new findings in molecular research.The general organizationof material in this edition has been revised along the same lines as EssentialGenes,making it easierto use the two books in conjunction.With increasing sizebecoming a problem, the content has been more sharplyfocusedon genesand their expressionby eliminating the chaptersdealing with the consequencesof gene expressionfor cell biology. Striking changesoccur in the first part of the book, dealing with genomes,resulting from the successof many genome sequencingprojects.The importance of RNA as a regulator has become increasingly evident and now can be seento extend acrossall levelsof gene expressionin both prokaryotesand eukaryotes.Somewhat of a "missinglink," it castsfurther light on how the current apparatusfor gene expressionmust have evolved from the early RNA world. My policy in this book has been to cire researchand review articlesthat I believereaderswill reasonablybe abte to access.My preferenceis for articlesthat are free after six months; where that is not possible,the publication should be widely available. I thank the following individuals who servedasproofreadersand consultantsfor this revision: Elliott Goldstein Jocelyn Iftebs I(athleen Matthews
University of Arizona, Tempe University of Alaska, Anchorage Rice Universitv. Houston Benjamin Lewin January 2007
0rganization The new organization of GENESIX allows instructors and studentsto focus more sharply on genesand their expression with expandedcoverageof key topics.The number of chaptersand the order of topic coverageremains the same; however, severalchapterswere expandedinto two or more chapters.Thesechangesare as follows:
xvr
Chapter I in GENES7111,Genesare DNA, is expanded to two chaptersinGENESIX. Basicinformation on DNA structure, replication, and mutation remains in Chapter 1, whereasthe discussionof the gene'sfunction asthe unit of heredity appearsin the new Chapter 2, GenesCode for Proteins. Chapter 3 in GENESWII,The Content of the Genome, becomestwo chaptersin GENESIX. Chapter 4, The Content of the Genome,includes information on DNA sequences,genome mapping, and DNA in organelles. Chapter 5, Genome Sequencesand Gene Numbers, now contains genome size and expressioninformation for a number of organisms,as well as new material on genesin the Y chromosome. The new Chapter 12, The Operon, comprisesGENESWII Chapter I0, aswell information on regulation of transcription and translation from GENESWIIChapter I I, Regulatory Circuits. Material on regulatory RNA is now found in Chapter13. The material in GENESVIII Chapter 13, The Replicon, is expanded in three chaptersin GENESIX. Chapter 15, The Replicon,coversthe structure and function of the replicon, aswell asreplication origins. Chapter 16, Extrachromosomal Replicons,contains material on terminal proteins, rolling circle replication, plasmids, and T-DNA. Information on how bacterial replication is connected to the cell cycle is found in Chapter17. Recombination and Repair, Chapter l5 in GENES VIil, is now coveredin two chaptersinGENESIX. Chapter l9 covershomologous and site-specificrecombination, and Chapter 20 covers the repair systems,including new information on excision-repairpathways in mammalian cells. Chapter 23, Controlling Chromatin Structure, in GENES VIII is now Chapter 30, discussingthe relation between chromatin structure and gene expression.
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G nesIX
GenesAre DNA C H A P T EO RU T L I N E Introduction DNAIs the GeneticMaterialof Bacteria o Bacterial provided the first proofthat DNA transformation can is the geneticmateria[of bacteria.Geneticpropert'ies fromonebacterialstrainto anotherby betransferred DNAfromthe firststrainandaddingit to the extracting second strain. DNAIs the GeneticMaterialof Viruses o Phage of infectionproved that DNAis the geneticmaterial of bacterioviruses. Whenthe DNAandproteincomponents phages isotopes,on[y areLabeted with differentradioactive phages produced by to the progeny the DNAis transmitted infectingbacteria. DNAIs the GeneticMaterialof AnimalCe[[s o DNAcanbe usedto introduce into newgeneticfeatures anima[celtsor wholeanimats. r In someviruses, is RNA. the geneticmaterial
IE
Potynucleotide ChainsHaveNitrogenousBasesLinkedto a S u g a r - P h o s p h aBt ea c k b o n e . A nucleoside base[inked consists of a purineor pyrimjdine sugar. to position1 of a pentose . Positions witha prime(') on the riboseringaredescribed them. to distinguish r Thedifference DNAandRNAis in the groupat the between sugar 2' positionof the sugar.DNAhasa deoxyribose (2'-H);RNnhasa ribosesugar(2'-0H). o A nucleotide [inkedto a phosphate consists of a nucteoside groupon eitherthe 5' or 3' positionof the (deoxy)ribose. . Successive (deoxy)ribose chain residues of a potynucteotide groupbetween the 3'positionof arejoinedby a phosphate onesugarandthe 5' positionof the nextsugar. . Oneendof the chain(conventionatty the Left)hasa free5' e n da n dt h eo t h e re n dh a sa f r e e3 ' e n d . . DNAcontains guanine, cytosine, adenine, the fourbases andthymine;RNAhasuraciIinsteadof thymine. DNAis a DoubteHelix o TheB-formof DNAis a doublehetixconsisting of two polynucteotide chainsthat runantiparatlel. r Thenitrogenous bases of eachchainareftat purineor pyrimidine ringsthat faceinwardandpairwith oneanother bonding to formA-Tor G-Cpairson[y. by hydrogen
. Thediameter is 20 A, andthereis a hel.ix of the doubLe turn every34 A, with ten basepairsperturn. comptete . Thedoubtehelixformsa major(wide)grooveanda minor groove. (narrow) DNAReplicationIs Semiconservative o TheMeselson-StahI to useddensitylabeling experiment polynucteotide strandis the unit of provethat the singl.e duringreplication. DNAthat is conserved r Eachstrandof a DNAduptexactsasa templatet0 synthestrand. sizea daughter . Thesequences by aredetermined strands of the daughter parentaI basepairingwiththe separated comptementary strands. DNAStrandsSeparateat the RepticationFork . Replication of enzymes by a comptex of DNAis undertaken the the parentalstrandsandsynthesize that separate strands. daughter r Thereplication forkis the pointat whichthe parentaI areseparated. strands o Theenzymes DNApotyDNAarecal'ted that synthesize RNA RNAarecal'ted that synthesize the enzymes merases; po[ymerases. o Nucleases nucleicacids;they that degrade areenzymes andcanbedividedinto andRNAases includeDNAases andexonucleases' endonucteases GeneticInformationCanBe Providedby DNAor RNA . Cettular andviroidsmayhave genesareDNA,but viruses genomes of RNA. r DNAis converted andRNAmay into RNAby transcription. transcription. into DNAby reverse be converted . Thetranstation of RNAinto proteinis unidirectional'. NucteicAcidsHybridizeby BasePairing to r Heating of a DNAduptex the two strands causes separare. r TheI, is the midpointof thetemperature rangefor denaturation. . Complementary whenthe temcanrenature singtestrands is reduced. Derature . Denaturation canoccurwith andrenaturation/hybridization andcanbe combinations or RNA-RNA DNA-RNA, DNA-DNA, or intramolecutar. intermotecutar on nextpoge Continued
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. TheabiLity of two singte-stranded nucteic acidpreparations to hybridize is a measureof thejrcomptementarity. M u t a t i o n sC h a n g e t h e S e q u e n coef D N A r A [ [m u t a t i o ncso n s i sotf c h a n g ei ns t h e s e q u e n coef D N A . . Mutations mayoccurspontaneousty or m a yb ei n d u c ebdy m u t a g e n s . MutationsMayAffectSingteBasePairs r equences o r L o n g eS o A pointmutationchanges a singtebase pair. r Pointmutations canbe caused bythe chemicaI conversion of onebaseinto anotheror by mistakes that occurduring rep[ication. o A transition reptaces a G-Cbasepairwith an A-Tbasepairor viceversa. o A transversion reptaces a purinewith a pyrimidine, suchaschanging A-Tto T-A. o Insertions arethe mostcommon typeof mutation andresuttfromthe movement of transposabte e[ements. The Effectsof MutationsCanBe Reversed . Forward mutations inactivate a gene,and backmutations (or revertants) reverse their effects.
Introduction
The hereditary nature of every living organism is defined by its genome, which consists of a long sequence of nucleic acid that provides the informatior needed to construct the organism. We use the term "information" because the genome does not itself perform any active role in building the organism; rather it is the sequenceof the individual subunits (bases)of the nucleic acid that determines hereditary features. By a complex seriesof interactions.this sequenceis used to produce all the proteins of the organism in the appropriate time and place. The proteins either form part of the structure of the organism, or have the capacity to build the structures or to perform the metabolic reactions necessaryfor life. The genome contains the complete set of hereditary information for any organism. physically the genome may be divided into a numb e r o f d i f fe r e n t n u c l e i c a c i d m o l e c u l e s . Functionally it may be divided into genes. Each gene is a sequencewithin the nucleic acid that represents a single protein. Each of the discrete nucleic acid moleculescomprising the genome may contain a large number of genes.Genomes
CHAPTER 1 GenesAre DNA
. Insertionscanrevertby deletionof the inserted materia[, but detetions cannot revert. r Suppression occurs whena mutation in a genebypasses second the effectof mutation in the firstgene. MutationsAre Concentrated at Hotspots o Thefrequency of mutationat anyparticular basepairis determined by statistical ftuctuation.exceptfor hotspots,where the frequency is increased by at leastan orderof magnitude. ManyHotspotsResu[tfrom Modified Bases . A common cause of hotspots'is the modifiedbase5-methyLcytosine, whichis spontaneously deaminated to thymine. SomeHereditaryAgentsAre Extremety Smat[ . Someverysma[[hereditary agentsdo not codefor proteinbut consistof RNAor of proteinthat hashereditary properties. Summary
for living organismsmay contain as few as <500 genes (for a mycoplasma, a type of bacterium) to as many as >25,000 for a the human being. In this chapter, we analyze the properties of the gene in terms of its basic molecular construction. f,.i*l"jRf].1 summarizes the stagesin the transition from the historical concept of the gene to the modern definition of the genome. A genome consistsof the entire set of chromosomes for any particular organism. It, therefore, comprises a seriesof DNA molecules (one for each chromosome), each of which contains many genes. The ultimate definition ol a genome is to determine the sequence of the DNA of each chromosome . The first definition of the gene as a functional unit followed from the discovery that individual genesare responsiblefor the production of specific proteins. The difference in chemical nature between the DNA of the gene and its protein product led to the concept that a gene codesfor a protein. This in turn led to the discovery of the complex apparatus that allows the DNA sequence of a gene to generate the amino acid sequence of a protein. Understanding the processby which a gene is expressedallows us to make a more rigorous
1865Genesare particulatefactors 1871 Discoveryof nucleicacids 1903Chromosomesare hereditaryunits 1910Geneslie on chromosomes 1913Chromosomes are lineararravsof 1927Mutationsare physicalchangesin genes 1931Recombination occursby crossingover 1944DNA is the geneticmaterial 1945 A gene codesfor protein 1951Firstproteinsequence
that codes for an RNA; in protein-codinggenes,the RNA in turn codes for a protein. From the demonstration that a gene consists of DNA, and that a chromosome consistsof a long stretch of DNA representing many genes, we move to the overall organization of the genome in terms of its DNA sequence.In Chapter 3, The Interrupted Gene, we take up in more detail the organization of the gene and its representation in proteins. In Chapter 4, The Content of the Genome, we considerthe total number of genes, and in Chapter 6, Clustersand Repeats,we discuss other components of the genome and the maintenance of its organization.
1953DNA is a doublehelix 1958 DNA replicatessemiconservatively 1961Geneticcodeis triolet 1977 Eukaryoticgenes are interrupted 1977DNAcan be seouenced
DNAIs the Genetic of Bacteria MateriaL
1995Bacterialgenomessequenced
A briefhistoryof genetics.
Gene
Sequenceof nucleotides
DNA
RNA
Chemicalnature
I I
i:i+i:i".l: .l: Agene
codes foran RNA, whichmaycodefor
protein.
definition of its nature. ii::ii+ii:.li.;'ishows the basic theme of this book. A gene is a sequence of DNA that produces another nucleic acid, RNA. The DNA has two strands of nucleic acid, whereas the RNA has only one strand. The sequence of the RNA is determined by the sequence of the DNA. (In fact, it is identical to one of the DNA strands.)In many, but not all, cases,the RNA is in turn used to direct producof DNA tion of a protein. Thusa geneis a sequence
. Bacterial provided the first proof transformation of bacteria. that DNAis the geneticmaterial fromone properties canbetransferred Genetic DNAfrom bacterial strajnto anotherby extracting strain. the firststrainandaddinqit to the second The idea that genetic material is nucleic acid had its roots in the discovery of transformation in 1928. The bacterium Pneumococcuskllls mice by causing pneumonia. The virulence of the bacterium is determined by its capsularpolysaccharide.This is a component of the sudace that allows the bacterium to escapedestruction by the host. Severaltypes (I, II, and III) of,Pneuhave different capsular polysaccham0c0ccws rides. They have a smooth (S) appearance. types can Each of the smooth Pneumococcal give rise to variants that fail to produce the capsular polysaccharide.Thesebacteriahave arough (R) surface(consistingof the material that was beneath the capsular polysaccharide).They are avirulent. They do not kill the mice, because the absence of the polysaccharide allows the animal to destroy the bacteria. When smooth bacteriaare killed by heat treatment, they lose their ability to harm the animal. But inactive heat-killed Sbacteria and the ineffectual variant R bacteria together have a quite different effect from either bacterium by itself. lrii:r.ili i: il .-i shows that when they are j ointly inj ected into an animal, the mouse dies as the result of a infection. Virulent S bacteria can be Pneumococcal from the mouse postmortem. recovered
of Bacteria Material 1.2 DNAIs the Genetic
Pneumoccocustypes Injectionof cells Capsule ii smooth(S) :'i appearance
DNAIs the Genetic MateriaI of Viruses
LivingR
Nocalrsu rough(R) appearan i:*UFf 3.1 Neither heat-kitted S-typenorliveR-type bacteriacankit[mjce,but simuttaneous injectionof bothcan ki[ micejust aseffectively asthe liveS-type.
Mouseinjected with heafkilled S and livingR bacteria
LivingS bacteria recoveredfrom dead mouse
\BiTt \, R bacteria
Transform
s bacteria
f:*riftl 1..i TheDNAof S-typebacteriacantransform R-typebacteria into the sameS-type.
In this experiment, the dead S bacteriawere of type III. The live R bacteria had been derived from type II. The virulent bacteria recovered from the mixed infection had the smooth coat of type III. So some property of the dead type III S bacteria can transform tlne live R bacteria so that they make the type III capsular polysaccharide and as a result become virulent. g!*i#qt ':.ri shows the identification of the component of the dead bacteria responsible for transformation. This was called the transforrning principle. It was purified by developing a cell-free system, in which extracts of the dead S bacteria could be added ro the live R bacteria
CHAPTER L GenesAre DNA
before injection into the animal. Purification of the transforming principle in 1944 showed that it is deoxyribonucleic acid (DNA).
o Phage infectionproved that DNAjs the genetic material of viruses. Whenthe DNAandprotein components of bacteriophages are[abeted with differentradioactive isotopes, ontythe DNAis phages transmitted produced to the progeny by infectingbacteria. Having shown that DNA is the genetic material of bacteria, the next step was to demonstrate that DNA provides the genetic material in a quite different system. Phage T2 is a virus that infects the bacterium Escherichiacoli. When phage (virus) particlesare added to bacteria,they adsorbto the outside surface, some material enters the bacterium, and then -20 minutes later each bacterium bursts open (lyses) to release a large number of progeny phage. FgGiJft[ 1.5illustrates the results of an experiment in 1952 in which bacteria were infected with T2 phagesthat had been radioactively labeled either in their DNA component (with r2p) or in theirprotein component (with 35S).The infected bacteria were agitated in a blender, and two fractions were separatedby centrifugation. One fraction contained the empty phage coats that were releasedfrom the surfaceof the bacteria;the other consistedof the infected bacteria themselves. Most of the l2P label was present in the infected bacteria. The progeny phage particles produced by the infection contained -3O.h of the original l2P label. The progeny received very Iittle-less than 1"/,-of. the protein contained in the original phage population. The phage coats consist of protein and therefore carried the 35Sradioactive label. This experiment therefore showed directly that only the DNA of the parent phages enters the bacteria and then becomes part of the progeny phages, which is exactly the pattern of inheritance expected of genetic material. A phage reproduces by commandeering the machinery of an infected host cell to manufacture more copies of itself. The phage possesses genetic material whose behavior is analogous
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to that of cellular genomes: its traits are faithfully reproduced and are subject to the same rules that govern inheritance. The caseof T2 reinforces the general conclusion that the genetic material is DNA, whether part of the genome of a cell or virus.
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DNAIs the Genetic Material of AnimalCeLLs
o DNAcanbe usedto introduce newgenetic features into animalcettsor wholeanimats. . In someviruses, is RNA. the geneticmaterial When DNA is added to populations of single eukaryotic cells growing in culture, the nucleic acid enters the cells, and in some of them this resultsin the production of new proteins. When a purified DNA is used, its incorporation leads to the production of a particular protein. FiiiLlHl:.ir depictsone of the standard systems. Although for historical reasonsthese experiments are described as transfection when per-
str Colonyof IKr cells of Somec;ellstakeup IKgene; descendants transfected cellpileup intoa colony
a newphecettscanacquire i5*l.gFir i.r: Eukaryotic by addedDNA. notypeasthe resultof transfection
formed with eukaryotic cells, they are a direct counterpart to bacterial transformation. The DNA that is introduced into the recipient cell becomes part of its genetic material and is inherited in the same way as any other part. Its expression confers a new trait upon the cells (synthesis of thymidine kinase in the example of Figure 1.6). At first, these experiments were successfulonly with individual cells adapted to grow in a culture medium. Since then, however, DNA has been introduced into mouse eggs by microinjection, and it may become a stable part of the genetic material of the mouse. Such experiments show directly not only that DNA is the genetic material in eukaryotes, but also that it can be transferredbetweendifferent species and yet remainfunctional. The genetic material of all known organisms and many viruses is DNA. However, some viruses use an alternative type of nucleic acid, ribonucleicacid (RNA), as the genetic material. The general principle of the nature of the genetic material, then, is that it is always nucleic acid; in fact, it is DNA, except in the RNA viruses.
of AnimalCetls Material 1.4 DNAIs the Genetic
@
Polynucleotide Chains HaveNitrogenous Bases Linked to a Sugar-Phosphate Backbone
r A nucteoside consists of a purineor pyrimidine base[inkedto position1 of a pentose sugar. r Positions on the riboseringaredescribed witha prime(') to distinguish them. o Thedifference between DNAandRNAis in the groupat the 2' positionof the sugar.DNAhasa deoxyribose sugar(2'-H);RNAhasa ribosesugar (2',-0H). . A nucleotide consists of a nucteoside linkedto a phosphate groupon eitherthe 5' or 3' positionof the (deoxy)ribose. . Successive (deoxy)ribose residues of a polynucleotide chainarejoinedby a phosphate groupbetween the 3' positionof onesugarand the 5' positionof the nextsugar. r 0neendof the chain(conventionatly the left) has a free5'endandthe otherendhasa free3'end. o DNAcontains the fourbases guanine, adenine, clrtosine, andthymine;RNAhasuracjlinsteadof thymine.
The basic building block of nucleic acids is the nucleotide, which has three components: . a nitrogenous base, . a sugar, and . a phosphate. The nitrogenous base is a purine or pyrimidine ring. The base is linked to position I on a pentose sugar by a glycosidic bond from N1 of pyrimidines or Ne of purines. To avoid ambiguity between the numbering systemsof the heterocyclic rings and the sugar, positions on the pentose are given a prime ('). Nucleic acids are named for the type of sugar; DNA has 2'-deoxyribose, whereas RNA has ribose. The difference is that the sugar in RNA has an OH group at the 2'position of the pentose ring. The sugar can be linked by its 5' or 3'position to a phosphate group. A nucleic acid consistsof a long chain of nucleotides. F;*LiRgi-i shows that the backbone of the polynucleotide chain consistsof an alternating seriesof pentose (sugar) and phosphate residues. This is constructed by linking the 5' position of one pentose ring to the 3'position of the next pentose ring via a phosphate group.
CHAPTER 1 GenesAre DNA
So the sugar-phosphate backbone is said to consist of 5'-3' phosphodiester linkages. The nitrogenous bases "stick out" from the backbone. Each nucleic acid contains four types of base. The same two purines, adenine and guanine, are present in borh DNA and RNA. The two pyrimidines in DNA are cytosine and thymine; in RNA uracil is found instead of thymine. The only difference between uracil and thymine is the presence of a methyl substituent at position C:. The basesare usually referred to by their initial letters. DNA contains A, G, C, and T; RNA contains A, G, C, and U. The terminal nucleotide at one end of tne chain has a free 5'group; the terminal nucleotide at the other end has a free 3'group. It is conventional to wdte nucleic acid sequencesin the 5'to3' direction-that is, from the 5' terminus at the left to the 3'terminus at the right.
DNAIs a Doub[e HeLix o TheB-formof DNAis a doubtehelixconsistinq of two potynucteotide chainsthat runantiparattit. r Thenitrogenous bases of eachchainareflat purineor pyrimidine ringsthat faceinwardand pairwithoneanotherby hydrogen bonding to formA-Tor G-Cpairsonly. . Thediameter of the doubLe hel.ix is 20 A. and thereis a comptete turn every34 A, with ten base pairsperturn. r Thedoublehetixformsa major(wide)grooveand a minor(narrow) groove. The observation that the bases are present in different amounts in the DNAs of differenr species led to the concept that the sequenceof basesis theform in which geneticinformation is carried.By the 1950s,the conceprof geneticinformation was common: the twin problems it posed were working out the structure of the nucleic acid and explaining how a sequence of basesin DNA could represent the sequence of amino acids in a protein. Three notions converged in the construction of the double helix model for DNA by Watson and Crickin I953: r X-ray diffraction data showed that DNA has the form of a regular helix, making a complete turn every 34 A e.4 nm), with a diamerer of -2O A (2 nm). Since the distance between adiacent nucleo-
xrtaHalqnooe sI vNo 9'l
'l-l 'l--v'9-l 'v-I st ern6!J Joul(u P qlr1!\ xllaq elqnop P sIuJoJ Jaqloue are; aql ut sautptttlttfid aql uraluanbas aqf'sitPdasPql-g pueI-V AreluaualdLuor Juo punoJe spuPJls o&{l eqt Jo 8ul1slMl JqJ '.09€ urnl alalduroJ e e>leu sJledaseq xrteqelqnopaq1 l l lh;i-itij sAemle sauundosnplaqqlprMluelsuore surelulpur oS 0IJo 'Jred aseq xJu Jql 01 a^IleleJ xITJq eql Jo sIXe J q l p u n o r e 0 9 € - p J l P l o Js l J l e d J S P qq J P l 'sJleld;o agd e e>lrlJaqtoup euo rnoqe pe>lJets 's't aJe sJseq'xITJq aql 3uo1eSurpaeJord TEgl 'speeJl aql :*ij ul z(lerrleruaqJs palPJlsnlll se ruroy srted Jspq eql :eseJrlelslertds e Jo slrrJal 'xllaq Jq] uI xllaq Jlqnop eql JapISuo) Jo SIXP 'saJnpnJls Jql 01 relnrtpuadrad srrcd ur 3ur.d1 'aptsul eq seseqrqJ rq1 uo lel; are Laql 'llrr rql ul YNq Jo uorlezrue8ro aql Sututurralap uI eloJ luel -rodrur ue r{e1dsuralord asaql'arro; SuIZIIPJlneu eqt Jo aluos apllord sutalord pa8reqr ^lr^I1l -sod '1ar rqt uI '+eN [q,{pttd,{t 'suol Pleru Jo Surpurq aqr,{q pazllerlneu are sa8reqr er47'0111^ 'sdnor8 aleqdsoqd erruorlnlos q sI vN11 ueq6 Jql uo sa8reqr anrle8au saIrJPJpue JpISlno aql uo sI euoq>lJeq aleqdsoqd-:e3ns aq1 'uolt)Jrlp ' ?€ ,S ol ,t sunr rauued sll seeJeqM 'x{aq aql Suop 8ur ol ,E aqt ur sunJ pupJls auo ->loof '*"{ $itfit}3iuI palerlsnlll se'(1a1e'redltue) suorlJerlp altsoddo uI unJ suIPqJ eplloalJ VNO -nu,{1od omt eql leq1 pasodord laporu aq1 ',{leluauralduror eq 01 ples are (1 qtt,r') qllM O) saseqparrcd rql pue'Eu1.rpd V ro 'I qll.&l. esEq se paqlJJsep JJe suolDeeJ eseql ') qll,lr dpo z(lerrynads puoq ueJ V seereqm zlluo [1err;nads puoq uaSorpz(queJ J'sasoq 'epn.lloro snoua1outuaLfluaa/waqCurpuoqua6oq[q t(q aletr euoqlreqe uroJleql slurleleqdsoqd-rebns -ossexrlJq alqnop eql uI suleqf, aplroapnur{1od saseq aql qlrqMulor1 y ; t ,t*ili=:l:l ,E-,9lo sapasp JoslsrsuolureqrapqoalrnuAlod oml Jqt teql pasodord pu3 pue uoslPM 'sanads lueJJJJIp ro! "/oVLol o/o9Zruor; saSuer s l q - 1' ) + ) s l l e q l s a s e qs 1 t; o u o t t l o d -ord aqt l,q paqrnsap rq uel Ygg {ue 'J 1o uorllsodruoJ aI{1oS Jo leql se erues eql sz(eanpsl V Jo uoqrodord eql pue 'VNO ul 3 yo uorgodord aqt se erues Jql 'aseqqlee sI C Jo uoltJodord aql s,{.errzr1e Jo slunoue JlnlosqPJql Jo eAIDJdsa.r.rJ. ' (morreu oot) aulPnur.rzld -eurprunrdd ro (apr,rzr oo1) auund-auund ;o sdtqsrauged Sutptorre'autpturtr,{.d e atrsoddo slemle sr aurrnd e leql os paprJlser aJPpue prPMuI e)eJUIPqJqJPa u r s J s e qJ q l J I p J u t e l d x a a q u e ) x I I e q 'suleqJ aql Jo JalJruPIp luelsuoJ aqJ appoapnuz(1odou1 uletuoJ lsnu xIIJq eql lpqr slsaSEnsvN( Jo dltsuap 3vJ . 'urn1 rad saptl -oepnu 0I Jq lsnu eraql 'Y 7'€ sI sapll
groove (-12 A across) and a major groove (-22 A across),as can be seen from the scale model of fi$#€f, s.t*. The double helix is righthanded; the turns run clockwise looking along the helical axis. These features represent the accepted model for what is known as the Bform of DNA. It is important to realize that the B-form represents an average,not a precisely specified structure. DNA structure can change locally. If it has more base pairs per turn it is said to be overwound; if it has fewer base pairs per turn it is underwound. Local winding can be affected by the overall conformation of the DNA double helix in spaceor by the binding of proteins to specific sites. Phosphate
ii+ijF.i :.!i Ftatbasepairslieperpendicutar to thesugarphosphate backbone.
DNAReplication Is Semiconservative TheMese[son-StahI experiment useddensity tabetingto provethat the singtepol.ynucLeotide strandis the unit of DNAthat is conserved durinq replication. Eachstrandof a DNAduplexactsasa temptateto synthesize a daughter strand. Thesequences of the daughter strands are determined by complementary basepairingwith theseparated parentaI strands.
iilirsfiS1.:il Thetwostrands of DNAforma doubte hetix. Photo@Photodisc.
CHAPTER 1 GenesAre DNA
It is crucial that the genetic material is reproduced accurately. The two polynucleotide strands are joined only by hydrogen bonds; thus they are able to separatewithout requiring breakage of covalent bonds. The specificity of base pairing suggeststhat each of the separated parental strands could act as a template strand for the synthesis of a complementary daughter strand. FJ*iiRijt"i t shows the principle that a new daughter strand is assembledon each parental strand. The sequence of the daughter strand is dictated by the parental strand; an A in the parental strand causesa Tto be placed in the daughter strand, a parental G directs incorporation of a daughter C, and so on. The top part of Figure l.l I shows a parenral (unreplicated) duplex rhat consistsof the original two parental strands.The lower part shows the two daughter duplexes that are being produced by complementary basepairing. Each of the daughter duplexes is identical in sequence
Parental DNA
-
Parental
1 Generation
2 Generation
t
HYBRID
Density analysis - - - - Light - - - -'Hybrid --- Heavy
. I ,: ' B a s ep a i r i n gp r o v i d et sh e m e c h a n i sf o mr DNA. repl.icating
with the original parent and contains one parental strand and one newly synthesized strand. The structureof DNA carriesthe information neededt0 perpetuateitssequence. The consequencesof this mode of replication are illustrated in i i+iii'ri::. 1;:.The parental duplex is replicated to form two daughter duplexes, each of which consistsof one parental strand and one (newly synthesized)daughter strand. The unit conserved from onegenerationto the nextis oneof the two individual strandscomprising the parental duplex. This behavior is called semiconservative replication. Figure l.l2 illustrates a prediction of this model. If the parental DNA carries a "heavy" density label because the organism has been grown in medium containing a suitable isotope (such as r5N;, its strands can be distinguished from those that are synthesizedwhen the organism is transferred to a medium containing normal "light" isotopes. The parental DNA consists of a duplex of two heavy strands (red). After one generation of growth in light medium, the duplex DNA is "hybrid" in density-it consistsof one heavy parental strand (red) and one light daughter strand (blue). After a second generation, the two strands of each hybrid duplex have sepa-
- -' -' Light - -' Hybrid --'-'Heavy
- -- -"'-
Light Hybrid HeavY
rated. Each gains a light partner, so that now one half of the duplex DNA remains hybrid and the other half is entirely light (both strandsare blue). The individual strands of theseduplexesare entirely heavy or entirely light. This pattern was confirmed experimentally in the Meselson-Stahl experiment of 1958, which followed the semiconservative replication of DNA through three generations of growth of.E. coli.When DNA was extracted from bacteria and its density measured by centrifugation, the DNA formed bands corresponding to its density-healry for parental, hybrid for the first generation, and half hybrid and half light in the second generation.
Separate DNAStrands Fork at the Replication r Reptication of by a comptex of DNAis undertaken and strands the parental enzymes that separate strands. the daughter synthesize r Therep[ication forkis the pointat whichthe parentaI areseParated. strands . Theenzymes DNAarecattedDNA that synthesize RNAare that synthesize polymerases; the enzymes cattedRNApolymerases. . Nucleases acids; nucteic that degrade areenzymes andcanbe andRNAases theyinctudeDNAases andexonucleases. dividedinto endonucteases Replication requires the two strands of the parental duplex to separate.However, the disruption of structure is only transient and is reversedas the daughter duplex is formed. Only
Fork at the Reptication Separate 1.8 DNAStrands
-.+
Reolicationfork
f;*qJRti.1i Thereplication forkis the reqionof DNAin whichthereis a transitionfromthe unw6undDarentaI duptexto the newtyrepticated daughter duplexes.
i:*USil L.i4 An endonuctease cteaves a bondwithina nucleic acid.Thisexampte showsanenzyme that attacks onestrandof a DNAduotex.
sized: DNA polymerases synthesize DNA, and RNA polymerases synthesize RNA. Degradation of nucleic acids also requires specific enzymes: deoxyribonucleases (DNAases) degrade DNA, and ribonucleases (RNAases) degrade RNA. The nucleasesfall into the general classesof exonucleases and endonucleases: . Endonucleases cut individual bonds within RNA or DNA molecules, generating discretefragments. Some DNAases cleave both strands of a duplex DNA at the target site, whereas others cleave only one of the two strands. Endonucleasesare involved in cutting reactions, as shown in $l*Uftf;R.lt!. . Exonucleasesremove residues one at a time from the end of the molecule, generating mononucleotides. They always function on a single nucleic acid strand, and each exonucleaseproceedsin a specific direction, that is, starting either at a 5' or aI a 3' end and proceeding toward the other end. They are involved in trimming reactions, as shown in fIfii.3ftft"TS.
+-
f:*Lj*l i.i* Anexonuclease removes bases oneat a time by cteaving the lastbondin a polynucteotide chain.
a small stretch of the duplex DNA is separated into single strands at any moment. The helical structure of a molecule of DNA engaged in replication is illustrared in trI{:URft":3. The nonreplicatedregion consistsof the parental duplex, opening into the replicated region where the two daughter duplexes have formed. The double helical structure is disrupted at the junction between the two regions, which is called the replication fork. Replication involves movement of the replication fork along the parental DNA. so there is a continuous unwinding of the parental strands and rewinding into daughter duplexes. The synthesisof nucleic acidsis catalyzedby specificenzymes, which recognize the template and undertake the task of catalyzing the addition of subunits to the polynucleotide chain that is being synthesized.The enzymes are named according to the type of chain that is synthe-
10
CHAPTER 1 GenesAre DNA
Genetic Information Can BeProvided by DNA OTRNA . Cellutar genesareDNA,but viruses andviroids mayhavegenomes of RNA. . DNAis converted into RNAby transcription, and RNAmaybe converted into DNAby reverse transcriotion. r Thetranstation of RNAinto proteinis unidirectionat. The central dogma defines the paradigm of molecular biology. Genes are perpetuated as sequencesof nucleic acid, but function by being expressed in the form of proteins. Replication is responsible for the inheritance of genetic information. Transcription and translation are responsible for its conversion from one form to another. F:*uRil.:.€Sillustrates the roles of replication, transcription, and translation, viewed from the perspective of the central dogma: . Theperpetuationof nucleicacid may involve either DNA or RNA as theseneticmaterial.
ded
ii+i.ii-li: 1.3* Thecentral dogma states thatinformation or transferred, but in nucteicacidcanbe perpetuated into proteinis irreversibte. the transfer of information
Cells use only DNA. Some viruses use RNA, and replication of viral RNA occurs in the infected cell. . The expressionof cellular geneticinformation usually is unidirectional.Transcription of DNA generates RNA molecules that can be used further 7nly to generate protein sequences;generally they cannot be retrieved for use as genetic information. Translation of RNA into protein is always irreversible. These mechanisms are equally effective for the cellular genetic information of prokaryotes or eukaryotes and for the information carried by viruses. The genomes of all living organisms consist of duplex DNA. Viruses have genomes that consist of DNA or RNA, and there are examples of each type that are doublestranded (ds) or single-stranded(ss).Details of the mechanism used to replicate the nucleic acid vary among the viral systems,but the principle of replication via synthesisof complementary strands remains the same, as illustrated in l'Il.::--iF.:: :.. : .j.
Cellular genomes reproduce DNA by the mechanism of semiconservative replication. Double-stranded virus genomes, whether DNA or RNA, also replicate by using the individual strands of the duplex as templates to synthesize partner strands. Viruseswith single-strandedgenomes use the single strand as a template to synthesize a
Singleparentalstrand is used to synthesize complementarystrand
strand Complementary is usedto synthesize sirand copyof parental
nucleic andsingte-stranded iri{ijL'i i "11 Doubte-stranded strands of comptementary bysynthesis acidsbothreplicate governed by the ru[esof basepairing.
complementary strand; this complementary strand in turn is used to synthesizeits complement, which is, of course, identical with the original starting strand. Replication may involve the formation of stable double-stranded intermediates or use double-stranded nucleic acid only as a transient stage. The restriction to unidirectional transfer from DNA to RNA is not absolute. It is overcome by the retroviruses, whose genomes consist of single-stranded RNA molecules. During the infective cycle, the RNA is converted by the into a sinprocess of reverse transcription gle-stranded DNA, which in turn is converted into a double-stranded DNA. This duplex DNA becomes part of the genome of the cell and is inherited like any other gene. So reversetranscription allows a sequenceof RNA to be retrieved and usedasgeneticinformation. The existence of RNA replication and reverse transcription establishesthe general principle rhat inform ati on in th efo rm of eith er type can beconvertedinto the other ofnucleicacidsequence type.In the usual course of events, however, the cell relies on the processesof DNA replication, transcription, and translation. But on rare occasions(possiblymediated by an RNA virus), information from a cellular RNA is convefted into DNA and inserted into the genome.
by DNAor RNA CanBeProvided Information 1.9 Genetic
77
Genome
GeneNumber
Organisms Plants Mammals Worms Flies Fungi Bacteria Mycoplasma dsDNAViruses Vaccinia Papova(SV40) PhageT4
BasePairs <1011
-3 x 10e -108 1 . 6x 1 0 8 1 . 3x 1 0 7 <107 <106 187,000 5,226 165,000
ssDNAViruses Parvovirus PhagefX174
5,000 5,387
dsRNAViruses Reovirus
23,000
ssRNAViruses Coronavirus Influenza TMV PhageMS2 STNV
20,000 13,500 6,400 3,569 1,300
Viroids PSTV RNA
359
- ! i i i = : t i: . : i : T h ea m o u not f n u c t e iacc i di n ' t h eq e n o m e variesoveran enormous ranqe.
Although reverse transcription plays no role in the regular operations of the cell, it becomes a mechanism of potential importance when we consider the evolution of the genome. The same principles are followed to perpetuate genetic information from the massive genomes of plants or amphibians to the tiny genomes of mycoplasma and the yet smaller genetic information of DNA or RNA viruses. r:*:inin I.-'i.= summarizes some examples that illustrate the range of genome types and sizes. Throughout the range of organisms, with genomes varying in total content over a 100,000-fold range, a common principle prevalls: The DNA codesfor all the proteins that the cell(s)of the organismmust synthesize, and the proteinsin turn (directlyor indirectly) provide thefunctions neededfor survivaL A similar principle describesthe function of the genetic information of viruses, whether DNA or RNA: Thenucleic acid codesfor the protein(s)neededto packagethe genomeand ako for anyfunctions additional to those provided by the hostcell that are neededto reproduce the virus during its infectiverycle. (The smallest virus-the satellite tobacco necrosis virus ISTNV]-cannot replicate independently. It requires the simultaneouspresenceof a "helper"
72
CHAPTER 1 GenesAre DNA
virus-the tobacco necrosisvirus [TNV], which is itself a normally infectious virus.)
Nucleic AcidsHybridize by BasePairing r Heating causes the two strands of a DNAduplexto separate. . TheI, is the midpointof thetemperature range for denaturation. . Comptementary singtestrands canrenature when the temperature is reduced. r Denaturation andrenaturation/hybridization can occurwith DNA-DNA, DNA-RNA, or RNA-RNA combinations andcanbeintermolecutar or intramolecutar. r Theabitityof two single-stranded nucteic acid preparations to hybridize is a measure of their complementarity. A crucial property of the double helix is the ability to separatethe two strands without disrupting covalent bonds. This makes it possible for the strandsto separateand reform under physiological conditions at the (very rapid) rares needed to sustain genetic functions. The specificity of the process is determined by complementary base pairing. The conceptof basepairing is central to all prlcessesinvolving nucleic acids.Disruption of the basepairs is a crucial aspectofthe function of a double-strandedmolecule,whereasthe ability to form basepairs is essentialfor the activity of a singlestranded nucleic acid. Ft$# i{[ t. I $ shows that base pairing enablescomplementary single-stranded nucleic acids to form a duplex structure. . An intramolecular duplex region can form by basepairing between two complementary sequencesthat are part of a single-stranded molecule. . A single-strandedmolecule may base pair with an independent, complementary single-stranded molecule to form an intermolecular duplex. Formation of duplex regions from singlestranded nucleic acids is most important for RNA, but single-srranded DNA also exists (in the form of viral genomes). Base pairing between independent complementary single strands is not restricted to DNA-DNA or RNA-RNA, but can also occur between a DNA molecule and an RNA molecule. The lack of covalent links between complementary strands makes it possible to manipu-
DNA
DNA '
lntramolecular pairingwithin RNA
'
irt,',. ';"I .
Intermolecular pairingbetween short and long RNAs
, " ,.
... RNA
SinglestrandedDNA
,,., "Long RNA hortRNA
i i{:ijiji:l. i!} Basepairingoccurs in duplexDNAandalso in intra-andinter-molecutar interactions in sinole-stranded R N A( o r D N A ) . late DNA invitro. The noncovalent forces that stabilizethe double helix are disrupted by heating or by exposure to low salt concentration. The two strands of a double helix separate entirely when all the hydrogen bonds between them are broken. The processof strand separation is called denaturation or (more colloquially) melting. ("Denaturation" is also used to describeloss of authentic protein structure; it is a generalterm implying that the natural conformation of a macromolecule has been converted to some other form.) Denaturation of DNA occurs over a narrow temperature range and results in striking changes in many of its physical properties. The midpoint of the temperature range over which the strands of DNA separate is called thremelt(T^).It dependson the proporing temperature tion of G-C base pairs. Becauseeach G-C base pair has three hydrogen bonds, it is more stable than an A-T base pair, which has only two hydrogen bonds. The more G-C base pairs are contained in a DNA, the greater the energy that is needed to separate the two strands. In solution under physiological conditions, a DNA that is 40o/oG-C-a value typical of mammalian genomes-denatures with a T- of about 87'C. So duplex DNA is stableat the temperature prevailing in the cell. The denaturation of DNA is reversible under appropriate conditions. The ability of the two separated complementary strands to reform into a double helix is called renaturation. Renaturation depends on specificbasepairing between .i.'itjshows the complementary strands. :::i"!q-ii:ii place in two stages.First, that the reaction takes single strands of DNA in the solution encounter
Renatured DNA
li:i;i.i:tl.;.. i-: Denatured of DNAcanrenasingtestrands tureto givethe duptexform.
one another by chance; if their sequencesare complementary, the two strands base pair to generate a short double-helical region. Then the region of base pairing extends along the molecule by a zipper-like effect to form a lengthy duplex molecule. Renaturation of the double helix restores the original properties that were lost when the DNA was denatured. Renaturation describes the reaction between two complementary sequencesthat were separatedby denaturation. However, the technique can be extended to allow any two complementary nucleic acid sequencesto react with each other to form a duplex structure. This is sometimes called annealing, but the reaction is more generally described as hybridization whenever nucleic acidsof different sources are involved, as in the casewhen one preparation consists of DNA and the other consists of RNA. Tfteability of two nucleicacid preparations to hybridize clnstitutes a precisetestflr their comple' mentarity because only complementaryseq uences canform a duplex structure. The principle of the hybridization reaction is to expose two single-strandednucleic acid preparations to each other and then to measure the amount of double-strandedmaterial that forms. i iili'or'i:r."i:iillustrates a procedure in which a DNA preparation is denatured and the single strands are adsorbed to a filter. Then a second denatured DNA (or RNA) preparation is added. The filter is treated so that the second preparation can adsorb to it only if it is able to base pair with the DNA that was originally a d s o r b e d .U s u a l l y t h e s e c o n d p r e p a r a t i o n i s
by BasePairing AcidsHybridize 1.10 Nucleic
13
change in the phenotype of the organism may allow us to identify the function of the protein. The existenceof many mutations in a gene may DenatureDNA allow many variant forms of a protein to be comand adsorbto filter pared, and a detailed analysis can be used to identify regions of the protein responsible for individual enzymatic or other functions. All organisms suffer a certain number of mutations as the result of normal cellular operations or random interactions with the enviDipfilterin solution ronment. These are called spontaneous mutations; the rate at which they occur is characteristic for any particular organism and is sometimes called the background level. Mutations are rare events, and of course those that MeasureDNAbound damage a gene are selectedagainst during evolution. It is therefore difficult to obtain large numbers of spontaneousmutants to study from natural populations. The occurrence of mutations can be increased by treatment with certain compounds. These are called mutagens, and the changes they cause are referred to as induced mutations. Most i:ji.:rji:ri .1.::i Fitterhybridization estabtishes whether a sotutionof denatured DNA(or RNA)contains sequences mutagens act directly by virtue of an ability either comptementary to the strands immobitized on thefitter. to modify a particular baseof DNA or to become incorporated into the nucleic acid. The effectiveness of a mutagen is judged by how much it increasesthe rate of mutation above background. radioactively labeled, so that the reaction can be By using mutagens,it becomespossibleto induce measured as the amount of radioactive label many changesin any gene. retained by the filter. Spontaneous mutations that inactivate gene The extent of hybridization between two function occur in bacteriophages and bacteria single-strandednucleic acids is determined by at a relatively constant rate of 3-4 x 10-l per their complementarity. Two sequencesneed genome per generation. Given the large varianot be perfectlycomplementary ro hybridize. If tion in genome sizes between bacteriophages they are closely related but not identical, an and bacteria, this corresponds to wide differimperfect duplex is formed in which basepairences in the mutation rate per base pair. This ing is interrupted at positions where the two suggeststhat the overall rate of mutation has single strandsdo not correspond. been subject to selective forces that have balanced the deleterious effectsof most mutations against the advantageouseffectsof some mutations. This conclusion is strengthened by the observation that an archaeal microbe that lives under harsh conditions of high temperature and acidity (which are expected to damage o At[mutations consjstof changes DNA) does not show an elevated mutation rate, in the sequence of DNA. but in fact has an overall mutation rate just o Mutations mayoccurspontaneousty or maybe below the average range. induced by mutagens. Ft{;*f{il:i,tl shows that in bacteria,the mumtion rate corresponds to - I 0-6 events per locus Mutations provide decisive evidence that per generation or to an average rate of change DNA is the genetic material. When a change in per basepair of l0-e-10-10per generation. The the sequenceof DNA causesan alteration in the rate at individual base pairs varies very widely, sequence of a protein. we may conclude that over a 10,000-foldrange. We have no accurate the DNA codesfor that protein. Furthermore, a measurement of the rate of mutation in eukary-
I
I I
Mutations Change theSequence of DNA
74
CHAPTER 1 GenesAre DNA
Mutationrate Any base pair 1 ;n16s-1gto generations H_N
\
n
Any gene 1 in 105-106 generations
Thegenome 1 in 300 generations ril:1-;ttii.i:: A basepairismutated at a rateof 10-e-10r0 pergeneration, a geneof 1000bp is mutatedat -10-6 js mutatedat pergeneration, genome anda bacteriaI 3 x L0-3pergeneration.
otes,although usually it is thought to be somewhat similar to that of bacteria on a per-locus per-generation basis.
Mutations MayAffect SingleBasePairsor Longer Sequences . A pointmutationchanges a singlebasepair. o Pointmutations canbecaused bythe chemical of onebaseinto anotheror by conversjon mistakes that occurduringrep[ication. o A transition reptaces a G-Cbasepairwithan A-T basepairor viceversa. r A transversion replaces a purinewitha pyrimidine, A-Tto T-A. suchaschanging o Insertions arethe mostcommon typeof mutation of transposable andresultfromthe movement etements. Any basepair of DNA can be mutated.A point mutation changes only a single base pair and can be caused by either of two types of event: . Chemical modification oI DNA directly changes one base into a different base. . A malfunction during the replication of DNA causes the wrong base to be inserted into a polynucleotide chain during DNA synthesis.
wild type
modcanbeinducedbv chemica[ F:iri"iili::i..i:: Mutations ification of a base.
Point mutations can be divided into two types, depending on the nature of the change when one base is substituted for another: . The most common class is the transition, comprising the substitution of one pyrimidine by the other, or of one purine by the other. This replaces a GC pair with an A-T pair or vice versa. o The lesscommon classis the transversion, in which a purine is replaced by a pyrimidine or vice versa, so that an AT pair becomesa T-A or C-G Pair. The effects of nitrous acid provide a classic example of a transition causedby the chemical conversion of one baseinto another. F3{il"rfii.':,ti shows that nitrous acid performs an oxidative deamination that converts cytosine into uracil. In the replication cycle following the transition, the U pairs with an A, instead of with the G with which the original C would have paired. So the C-G pair is replacedby a T-A pair when the A pairs with the T in the next replication cycle. (Nitrous acid also deaminates adenine, causingthe reversetransition from A-T to G-C.) Transitions are also causedby base mispairing, when unusual partners pair in defiance of the usual restriction to WatsonCrick pairs. Base mispairing usually occurs as an aberration resulting from the incorporation into DNA of an abnormalbase that has ambigui"li4 shows the ous pairing properties. iriilr.iF:f
Sequences t 5 MayAffectSingteBasePairsor Longer 1.12 Mutations
BrdUpairswithA I at replication I
Keto-enolshift allowsBrdU to pair with G
vidual genes. However, we now know that insertions of stretches of additional material are quite frequent. The source of the inserted material lies with transposable elements, which are sequencesof DNA with the ability to move from one site to another (seeChapter 21, Transposons,and Chapter 22, Retroviruses and Retroposons.) An insertion usually abolishes the activity of a gene. Where such insertions have occurred, deletions of part or all of the inserted material, and sometimes of the adjacent regions, may subsequently occur. A significant difference between point mutations and the insertions/deletions is that the frequency of point mutation can be increasedby mutagens, whereas the occurrence of changes caused by transposable elements is not affected. However, insertions and deletions can also occurby othermechanisms-for example, those involving mistakes made during replication or recombination-although probably these are less common. In addition, a class of mutagens called the acridines introduce (very small) insertions and deletions.
TheEffectsof Mutations CanBeReversed
t':-{*itt f .i+ Mutations canbeinduced bvtheincornoration o f b a s ea n a t o qi n s t oD N A . example of bromouracil (BrdU), an analog of thymine that contains a bromine atom in place of the methyl group of thymine. BrdU is incorporated into DNA in place of thymine. However, it has ambiguous pairing properties,becausethe presence of the bromine atom allows a shift to occur in which the base changesstructure from a keto 1=O1form to an enol (-OH) form. The enol form can basepair with guanine, which leadsto substitution of the original A-T pair by a G-C pair. The mistaken pairing can occur either during the original incorporation of the base or in a subsequent replication cycle. The transition is induced with a certain probability in each replication cycle, so the incorporation of BrdU has continuing effectson the sequenceof DNA. Point mutations were thought for a long time to be the principal means of change in indi-
76
C H A P T E1RG e n e sA r e D N A
Forward mutations inactivate a gene.andback mutatjons(or revertants) reverse their effects. Insertionscanrevertby de[etionof the inserted materiat, but deletions cannotrevert. Suppression occurs whena mutation in a second genebypasses the effectof mutationin the first gene. f:GLl*l i.tS shows that the isolation of revertants is an important characteristic that distinguishes point mutations and insertions from deletions: . A point mutation can revert by restoring the original sequence or by gaining a compensatory mutation elsewhere in the gene. r An insertion of additional material can revert by deletion of the inserted material. . A deletion of part of a gene cannot revert. Mutations that inactivate a gene are called forward mutations. Their effects are reversed by back mutations, which are of two types: true reversion and second-site reversion. An exact reversal of the original mutation is called true reversion. So if an A-T nair has
ATCGGACTTACCGGTTA TAGCCTGAATGGCCAAT Point I mutation * ATCGGACOACCGGTTA TAGCCTGAGTGGCCAAT Reversion;
+
ATCGGAC CCGGTTA TAGCCTGAATGGCCAAT
ATCGGACTTACCGGTTA TAGCCTGAATGGCCAAT Insertion I V ATCGGACTTXXXXXACCGGTTA TAGCCTGAAYYYYYTG GCCAAT ReversionI bv deletionV ATCGGACTTACCGGTTA TAGCCTGAATGGCCAAT ATCGGACTTACCGGTTA TAGCCTGAATGGCCAAT
I
v ATCGGACGGTTA TAGCCTGCCAAT No reversionpossible
::i*i.ifrL'i .,.1:Pointmutations andinsertions canrevert. but deletions cannotrevert.
been replacedby a G-C pair, another mutation to restore the A-T pair will exactly regenerate the wild-type sequence. The secondtlpe of back mutation, secondsite reversion, may occur elsewhere in the gene, and its effects compensate for the first mutation. For example, one amino acid change in a protein may abolish gene function, but a second alteration may compensatefor the first and restore protein activity. A forward mutation results from any change that inactivates a gene, whereas a back mutation must restore function to a protein damaged by a particular forward mutation. So the demands forback mutation are much more specific than those for forward mutation. The rate of back mutation is correspondingly lower than that of forward mutation, typically by a factor of -10.
Mutations can also occur in other genes to circumvent the effects of mutation in the original gene. This effect is called suppression. A locus in which a mutation suppressesthe effect of a mutation in another locus is called a suppressor.
Mutations Are at Hotspots Concentrated o Thefrequency base of mutationat anyparticular pairis determined fluctuation, by statisticaI is exceptfor hotspots. wherethefrequency jncreased by at leastan orderof magnitude. So far we have dealt with mutations in terms of individual changesin the sequenceof DNA that influence the activity of the genetic unit in which they occur. When we consider mutations in terms of the inactivation of the gene, most genes within a speciesshow more or lesssimilar rates of mutation relative to their size. This suggests that the gene can be regarded as a target for mutation, and that damage to any part of it can abolish its function. As a result, susceptibility to mutation is roughly proportional to the size of t h e g e n e . B u t c o n s i d e rt h e s i t e so f m u t a t i o n within the sequence of DNA: are all base pairs in a gene equally susceptible,or are some more Iikely to be mutated than others? What happens when we isolate a large number of independent mutations in the same gene?Many mutants are obtained. Each is the result of an individual mutational event. Then the site of each mutation is determined. Most mutations will lie at different sites, but some will lie at the same position. Two independently isolated mutations at the same site may constitute exactly the same change in DNA (in which casethe same mutational event has happened on more than one occasion),or they may constitute different changes (three different point mutations are possibleat each basepair). .i.i:*shows the freThe histogram of l':iiiJ.qi' quency with which mutations are found at each base pair inthe lacl gene of E. coli.The statistical probability that more than one mutation occurs at a particular site is given by randomhit kinetics (asseenin the Poissondistribution). So some siteswill gain one, two, or three mutations, whereas others will not gain any. Some sites gain far more than the number of mutations expectedfrom a random distribution; they may have I0x or even l00x more mutations
at Hotspots 7 7 AreConcentrated 1.14 Mutations
a
540 Eso E
ozv
6 9t -i n l
z 250 300bp ! : ,; 1 : r r t::, - . , .$: p g n l 3 n g om u su t a t j o nosc c utrh r o u g h o u t the lacfgeneof E.colibutareconcentrated at a hotspot.
than predicted by random hits. These sitesare called hotspots. Spontaneousmutations may occur at hotspots,and different mutagens may have different hotsDots.
@
ManyHotspots Result fromModified Bases
. A common causeof hotspots is the modified base 5-methytcytosine, whichis spontaneousty deaminated to thymine. A major causeof spontaneousmutation results f r o m t h e p r e s e n c eo f a n u n u s u a l b a s e i n t h e D N A . I n a d d i t i o n t o t h e f o u r b a s e st h a t a r e inserted into DNA when it is synthesized,modified bases are sometimes found. The name reflectstheir origin; they are produced by chemically modifying one of the four basesalready present in DNA. The most common modified base is 5-methylcytosine, which is generated by a methylaseenzyme that addsa methyl group to certain cytosine residues at specificsites in the DNA. Sitescontaining 5-methylcytosineprovide hotspots for spontaneouspoint mutation in E. coli.In each case,the mutation takes the form of a G-C to A-T transition. The hotspotsare not found in strains of E. coli thatcannot methylate cytoslne. The reason for the existenceof the hotspots is that cytosinebasessuffer spontaneousdeamination at an appreciablefrequency. In this reaction, the amino group is replacedby a keto group. Recall that deamination of cytosine generatesuracil (seeFigure 1.23).f :iliiiii I ;lli compares this reaction with the deamination of
CHAPTER 1 GenesAre DNA
produces +itr:i1iilt:.:i.r Deamination of cytosine uraci[. w h e r e ads e a m i n a t i oonf 5 - m e t h y t c y t o s ipnreo d u c e s thymine.
5-methylcytosine where deamination generates thymine. The effect in DNA is to generate the basepairs G-U and G-T, respectively,where there is a mismatch between the partners. All organisms have repair systemsthat correct mismatched base pairs by removing and replacing one of the bases.The operation of these systems determines whether mismatched pairs such as G-U and G-T result in mutations. Ii{;iii:ii -i..jliishows that the consequencesof deamination are different for 5-methylcytosine and cytosine.Deaminating the (rare) 5-methylcytosine causesa mutation, whereas deamination of the more common cytosine does not have this effect.This happens becausethe repair systemsare much more effective in recognizing G-U than G-T. E coli conLainsan enzyme, uracil-DNA-glycosidase,that removes uracil residuesfrom DNA (see Section 20.5, Base Flipping Is Used by Methylasesand Glycosylases).This action leaves an unpaired G residue, and a "repair system" then inserts a C baseto partner it. the net result of these reactions is to restore the original sequenceof the DNA. This system protects DNA againstthe consequencesof spontaneousdeamination of cytosine. (This system is not, however, active enough to prevent the effectsof the increasedlevel of deamination causedby nitrous a c i d ; s e eF i g u r e 1 . 2 3 . ) Note that the deamination of 5-methylcytosine leaves thymine. This creates a mismatched basepair, G-T. If the mismatch is not corrected before the next replication cycle, a mutation results. At the next replication, the basesin the mispaired G-T partnership separate, and then they pair with new partners to
i:iii.itEi" 1,:j;r,i Thedeamination proof 5-methytcytosine (byC-Gto T-Atransitions), duces thymine whitethedeami n a t i o no f c y t o s i n ep r o d u c eusr a c i [( w h i c hu s u a t l iys removed andthenreplaced by cytosine).
produce one wild-type G-C pair and one mutant A-T pair. Deamination of 5-methylcytosine is the most common cause of production of G-T mismatched pairs in DNA. Repair systems that act on G-T mismatches have a bias toward replacing the T with a C (rather than the alternative of replacing the G with an A), which helps to reduce the rate of mutation (seeSection 20.7, Controlling the Direction of Mismatch Repair). However, these systems are not as effective as the removal of U from G-U mismatches. As a result, deamination of 5-methylcytosine leads to mutation much more often than does deamination of cytosine. 5-methylcytosine also creates hotspots in eukaryotic DNA. It is common at CpG dinucleotidesthat are concentrated in regions called CpG islands(seeSection24.I9, CpG IslandsAre Regulatory Targets).Although 5-methylcytosine accounts for -l% of the basesin human DNA, sites containing the modified base account f.or-30o/o of all point mutations. This makes the state of 5-methylcytosine a particularly important determinant of mutation in animal cells.
The importance of repair systemsin reducing the rate of mutation is emphasized by the effectsof eliminating the mouse enzyme MBD4, a glycosylasethat can remove T (or U) from mismatches with G. The result is to increasethe mutation rate at CpG sitesby a factor of 3x. (The reason the effect is not greater is that MBD4 is only one of severalsystemsthat act on G-T mismatches; we can imagine that elimination of all the systems would increase the mutation rate much more.) The operation of these systems casts an interesting light on the use of T in DNA compared with U in RNA. Perhaps it relates to the need of DNA for stability of sequence; the use of T means that any deaminations of C are immediately recognized becausethey generate a base (U) not usually present in the DNA. This greatly increasesthe efficienry with which repair systems can function (compared with the situation when they have to recognize G-T mismatches, which can be produced also by situations where removing the T would not be the appropriate response).Also, the phosphodiester bond of the backbone is more labile when the baseis U.
Agents SomeHereditary SmaL[ AreExtremely o Someverysmat[hereditary agentsdo not codefor proteinbut consistof RNAor of proteinthat has properties. hereditary Viroids are infectious agents that cause diseasesin higher plants. They are very small circular molecules of RNA. Unlike viruses-for which the infectious agent consistsof a virion, a genome encapsulated in a protein coat-the viroid RNA is itself the infectiousagent.Th'e viroid consistssolely of the RNA, which is extensively but imperfectly base paired, forming a characteristic rod like the example shown in I{{iijn{ t.itr. Mutations that interfere with the structure of the rod reduce infectivity. A viroid RNA consists of a single molecular speciesthat is replicated autonomously in infected cells. Its sequence is faithfully perpetuated in its descendants.Viroids fall into several groups. A given viroid is identified with a group by its similarity of sequence with other members of the group. For example, four viroids related to PSTV (potato spindle tuber viroid)
Sma[[ AgentsAreExtremely 1.16 SomeHereditary
t9
i::i-.:.:ii.ir :,,.:rl.PSTV structure, interrupted RNAis a circularmotecule that formsan extensive doub[e-stranded bv manyinteriorloops.Thesevere andmitdformsdifferat threesites.
haveT0oh-83% similarity of sequencewith it. Different isolates of a particular viroid strain vary from one another, and the change may affect the phenotlpe of infected cells.For example, the mild and severeslrains of PSTVdiffer bv three nucleotide substitutions. Viroids resemble viruses in having heritable nucleic acid genomes.They fulfill the criteria for genetic information. Yet viroids, which are sometimes called subviral pathogens, dif fer from viruses in both structure and function. Viroid RNA does not appear to be translated into protein, so it cannot itself code for the functions needed for its survival. This situation poses two questions:How doesviroid RNA replicate, and how does it affect the phenotype of the infected plant cell? Replication must be carried out by enzymes of the host cell, subverted from their normal function. The heritability of the viroid sequence indicatesthat viroid RNA provides the template. Viroids are presumably pathogenic because they interfere with normal cellular processes. They might do this in a relatively random way, for example, by sequesteringan essentialenz)'rne for their own replication or by interfering with the production of necessarycellular RNAs. Alternatively, they might behave as abnormal regulatory molecules, with particular effects upon the expressionof individual genes. An even more unusual agent is scrapie, the cause of a degenerative neurological diseaseof sheep and goats.The diseaseis related to the human diseasesof kuru and CreutzfeldtJakob syndrome, which affect brain function. The infectiousagent of scrapiedoesnot czntain nucleicacid.This extraordinary agent is called a
20
CHAPTER 1 GenesAre DNA
prion (proteinaceous infectious agent). It is a 28 kD hydrophobic glycoprotein, PrP. PrP is coded by a cellular gene (conserved among the mammals) that is expressedin normal brain. The protein exists in two forms: The product found in normal brain is called PrPc and is entirely degraded by proteases. The protein found in infected brains is called PrP" and is extremely resistantto degradation by proteases. PrPcis converted to PrPscby a modification or conformational change that confers proteaseresistance,and which has yet to be fully defined. As the infectious agent of scrapie,PrPSc must in some way modify the synthesis of its normal cellular counterpart so that it becomes infectious instead of harmless (see Section 3I.12, Prions CauseDiseasesin Mammals). Mice that lack a PrP gene cannot be infected to develop scrapie, which demonstrates that PrP is essential for development of the disease.
mary Sum Two classicexperiments proved that DNA is the genetic material. DNA isolated from one strain of.Pneumococcus bacteria can confer properties of that strain upon another strain. In addition, DNA is the only component that is inheritedby progeny phagesfrom the parental phages.DNA can be used to transfect new properties into eukaryotic cells. DNA is a double helix consisting of antiparallel strands in which the nucleotide units are linked by 5'ro-3' phosphodiester bonds. The backbone provides the exterior; purine and pyrimidine basesare stacked in the interior in
pairs in which A is complementary to T and G is complementary to C. The strands separate and use complementary basepairing to assemble daughter strands in semiconservative replication. Complementary basepairing is also used to transcribe an RNA representing one strand of a DNA duplex. A stretch of DNA may code for protein. The genetic code describes the relationship between the sequence of DNA and the sequence of the protein. Only one of the two strands of DNA codesfor protein. A codon consistsof three nucleotides that represent a single amino acid. A coding sequenceof DNA consistsof a seriesof codons, which are read from a fixed starting point. Usually only one o f t h e t h r e e p o s s i b l er e a d i n g f r a m e s c a n b e translated into protein. A mutation consists of a change in the sequenceof A-T and G-C basepairs in DNA. A mutation in a coding sequencemay change the sequence of amino acids in the corresponding protein. A frameshift mutation alters the subsequent reading frame by inserting or deleting a base; this causes an entirely new series of amino acids to be coded after the site of mutation. A point mutation changes only the amino acid represented by the codon in which the mutation occurs. Point mutations may be reverted by back mutation of the original mutation. Insertions may revert by loss of the inserted material, but deletions cannot revert. Mutations may also be suppressedindirectly when a mutation in a different gene counters the original defect. The natural incidence of mutations is increased by mutagens. Mutations may be concentrated at hotspots. A type of hotspot responsible for some point mutations is caused by deamination of the modified base 5methylcytosine. Forward mutations occur at a rate of - l0-6 per locus per generation; back mutations are rarer. Not all mutations have an effect on the phenotype. Although all genetic information in cells is carried by DNA, viruses have genomes of double-stranded or single-strandedDNA or RNA. Viroids are subviral pathogens that consist solely of small circular molecules of RNA, with no protective packaging. The RNA does not code for protein and its mode of perpetuation and of pathogenesis is unknown. Scrapie consistsof a proteinaceous infectious agent.
References Introduction Reviews Cairns,J., Stent, G., and Watson, J. D. (1966). Phage and the Origins of Molecular Biology. Cold Spring Harbor Symp Quant. Biol. Judson, H. ( I978) . TheEighth Day of Creation. I(nopf, New York. Olby, R. ll974l. ThePath to the DoubleHelix. M a c M i l l a n ,L o n d o n .
Materia[ of Bacteria DNAIs the Genetic Resea rcl-r Avery, O. T., Macleod, C. M., and McCarty, M. (1944). Studies on the chemical nature of the substance inducing transformation of pneumococcal types. J. Exp. Med 98, 451-460. Griffith, F. (1928). The significance of pneumococc a l t y p e s .J . H y g 2 7 , I I 3 - I 5 9 .
Material of Viruses DNAIs the Genetic Resea rch Hershey,A. D, and Chase,M. (1952).Independent functions of viral protein and nucleic acid in growth of bacteriophage J. Gen.Physiol.36, ]9-56.
Material of Animal DNAIs theGenetic Cet[s rcl-r Resea S. Pellicer, A.,Wigler,M., Axel,R.,andSilverstein, (197 8) . The transfer and stable integration of the HSV thymidine kinase gene into mouse cells. Cell 14, l)3-141.
DNAIs a DoubleHe[ix Resea rch Watson, J. D., and Crick, F. H. C. (1953). A structure for DNA. Nature 17|, 7)7-738. W a t s o n ,J . D . , a n d C r i c k , F . H . C . ( 1 9 5 3 ) .G e n e t i c implications of the structure of DNA. Nature t7t,964-967. Wilkins, M. F. H., Stokes, A. R., and Wilson, H. R. ( 195 3 ). Molecular structure of DNA. Nature t7r,738-740.
Is Semiconservative DNAReptication Review Holmes,F. (2001).Meselson, Stahl,andtheReplication of DNA:A Historyof theMostBeautifulExperimentin BiologyYaleUniversityPress,New Haven,CT.
References 2 l
Resea rch Meselson,M. and Stahl, F.w. (1958). The replication of DNA in E. coli. Proc. Natl. Acad. Sci.USA 44.67t-682.
Mutations Change the Sequence of DNA KCVICWS
Drake,J. W., Charlesworth,B., Charlesworth,D., and Crow,J.F. (1998).Ratesof spontaneous mutation. Genetics 148, | 667-l 686. Drake,J.W. and Balz,R. H. (1976\ . The biochemistry of mutagenesis. Annu.RevBiochem.45, tt-37. R e s erach Drake,J. W. ( I 99t ). A constantrate of spontaneousmutation in DNA-basedmicrobes.Proc. Natl.Acad.Sci.USA88,7160-7164. Grogan,D. W., Carver,G.T., and Drake,J. W. (2001).Geneticfidelity under harsh conditions: analysisof spontaneousmutation in the thermoacidophilicarchaeonSulfulobus acidocaldariusProc Natl Acad.Sci.USA98. 7928-79)3.
@
Mutations MayAffectSing[eBasePairs or Longer Sequences
Review Maki, H. (20021.Originsof spontaneousmutations: specificityand directionalityof base-substitution, frameshift,and sequencesubstitutionmutageneses. Annu.Rev.Genet. 36.279-30J.
22
CHAPTER 1 GenesAre DNA
ManyHotspots Result fromModified Bases Research Molecularbasis of base Coulondre, C.etal.(1978). hotspots in E coli.Nature274, substitution 775-780. Millar, C.B., Guy, J., Sansom,O. J., Selfridge,J., MacDougall,E., Hendrich,B., Keightley,P.D., Bishop,S.M., Clarke,A. R.,and Bird,A. (2002).EnhancedCpGmutability and tumorigenesisin MBD4 -deficient mice. Science 297, 403-405. SomeHereditaryAgentsAre Extremety Smat[ Reviews Diener,T. O. (1986).Viroidprocessing: a model involving the central conservedregion and hairpin. Proc.Natl. Acad.Sci.USA83, 58-62. Diener,T. O. ( 1999).Viroidsand the nature of s. Arch.Virol.Suppl | 5, 20)-220. viroid disease Prusiner,S.B. (1998).Prions.Proc.Natl.Acad.Sci. u s A 9 5 ,r ) ) 6 j - r 3 ) 8 ) . Resea rch Bueler,H. et al. (1993\. Mice devoidof PrPare resistantto scrapie.Cell73, I339-l)47. Mclfinley, M. P.,Bolton, D. C., and Prusiner,S.B. (1983).A protease-resistant proteinis a structural componentof the scrapieprion. Cell)5, 57-62.
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3 N I - I I N OU S I d V H J
@
Introduction
The gene is the functional unit of heredity. Each gene is a sequencewithin the genome that functions by giving rise to a discrete product (which may be a protein or an RNA). The basicbehavior of the gene was defined by Mendel more than a century ago. Summarized in his two laws, the gene was recognizedas a "particulate factor" that passesunchanged from parent to progeny. A gene may exist in alternative forms. These forms are called alleles. In diploid organisms, which have two sets of chromosomes, one copy of each chromosome is inherited from each parent. This is the same behavior that is displayedby genes.One of the two copies of each gene is the paternal allele (inherited from the father), the other is the maternal allele (inherited from the mother). The equivalence led to the discovery that chromosomes in fact carry the genes. Each chromosome consistsof a linear array of genes.Each gene residesat a particular location on the chromosome. The location is more formally called a genetic locus. The alleles of a
A chromosomeis a very long moleculeof DNA
c o n t a i nm s a n yg e n e s
Each gene is part of a continuous
F!.{'ij$li;i":t Eachchromosome hasa singte[ongmolecute of DNA genes. withinwhicharethe sequences of individuaI
24
CHAPTER 2 GenesCodefor Proteins
gene are the different forms that are found at its locus. The key to understanding the organization of genes into chromosomes was the discovery of genetic linkage-the tendency for genes on the same chromosome to remain together in the progeny instead of assorting independently as predicted by Mendel's laws. Once the unit of recombination (reassortment) was introduced as the measure of linkage, the construction of genetic maps became possible. The resolution of the recombination map of a higher eukaryote is restricted by the small number of progeny that can be obtained from each mating. Recombination occurs so infrequently between nearby points that it is rarely observed between different mutations in the same gene. As a result, classicallinkage maps of eukaryotes can place the genes in order, but cannot determine relationships within a gene. By moving to a microbial system in which a very large number of progeny can be obtained from each genetic cross, researchers could demonstrate that recombination occurs within genes.It follows the same rules that were previously deduced for recombination between genes. Mutations within a gene can be arranged into a linear order, showing that the gene itself has the same linear construction as the array of genes on a chromosome. So the genetic map is linear within as well as between loci: it consistsof an unbroken sequencewithin which the genes reside. This conclusion leads naturally into the modern view summarized in +'I{*#it[:J.1that the genetic material of a chromosome consists of an unintenupted length of DNA representingmany genes.
A GeneCodes for a Single PoLypeptide r Theonegene:oneenzyme hypothesis summarizes genetics: the basisof modern that a geneis a stretchof DNAcodingfor a singtepotypeptide chain. . Mostmutations genefunction. damage The first systematic attempt to associategenes with enzymes showed that each stagein a metabolic pathway is catalyzed by a single enzyme and can be blocked by mutation in a different gene. This led to the lne gene: oneenzymehypothesrs.Each metabolic step is catalyzed by a particular enzyme, whose production is the
responsibility of a single gene. A mutation in the gene alters the activity of the protein for which it is responsible. A modification in the hypothesis is needed to accommodate proteins that consist of more than one subunit. ff the subunits are all the same, the protein is a homomultimer, represented by a single gene. If the subunits are different, the protein is a heteromultimer. Stated as a more general rule applicable to any heteromultimeric protein, the one gene : one enzyme hypothesis becomes more precisely expressedas lne gene: onepolypeptidechain. Identifying which protein representsa particular gene can be a protracted task. The mutation responsible for creating Mendel's wrinkled-pea mutant was identified only in I990 as an alteration that inactivates the gene for a starch branching enzyme! It is important to remember that a gene does not directly generate a protein. As shown previously in Figure 1.2, agene codes for an RNA, which may in turn code for a protein. Most genescode for proteins, but some genescode for RNAs that do not give rise to proteins. These RNAs may be structural components of the apparatus responsible for synthesizing proteins or may have roles in regulating gene expression. The basic principle is that the gene is a sequence of DNA that specifiesthe sequence of an independent product. The process of gene
Mutant homozygote
Wild-type homozygote Both alleles produce activeprotein
t t
One (dominant) alleleproduces activeprotein
Neitherallele producesprotein
II
I
wib type
wildtype
mutant
wildtype
mutant
mutant
Wild phenotype
expression may terminate in a product that is either RNA or protein. A mutation is a random event with regard to the structure of the gene, so the greatest probability is that it will damage or even abolish gene function. Most mutations that affect gene function are recessive:they representan absence of function, becausethe mutant genehas beenpret"fi ventedfrom producingits usualprotein. StSLjftfi illustrates the relationship between recessive and wild-t1pe alleles. When a heterozygote contains one wild-t1pe allele and one mutant allele, the wild-type allele is able to direct production of the enzyme. The wild-type allele is therefore dominant. (This assumes that an adequate amlunt of protein is made by the single wildtype allele. When this is not true, the smalier amount made by one allele as compared to two alleles results in the intermediate phenotype of a partially dominant allele in a heterozygote.)
in the Same Mutations Complement GeneCannot A mutationin a geneaffectsonlythe protein codedby the mutantcopyof the geneanddoes not aftectthe proteincodedby anyothera[[ele. (produce Failureof two mutationsto comptement witdphenotype whentheyarepresentin trans meansthat they configuration in a heterozygote arepartof the samegene.
How do we determine whether two mutations that causea similar phenotype lie in the samegene?If they map closetogether,they may be alleles.However,they could alsorepresentmutations in two differentgeneswhose proteins are involved in the samefunction. The complementation test is usedto determine whether two mutationslie in the same gene or in different genes.The test consists of making aheterozygotefor the two mutations (by mating parentshomozygousfor each mutation). If the mutations lie in the samegene,the parentalgenotypescan be representedas: m,
ranomr
Wild phenotype
Mutant phenotype
F I S U R g? " 2 G e n e sc o d ef o r p r o t e i n sd; o m i n a n cies exptained by the properties of mutantproteins.A recessiveat[eledoesnot contribute to the phenotype because it produces no protein(or proteinthat is nonfunctionat).
,m, m2
allele Thefirst parentprovidesarrttT\rr-urarrt and the secondparentprovidesan m2 allele,so that the heterozygotehasthe constitution: [, m2
Complement 2 5 in the SameGeneCannot 2.3 Mutations
No wild-type gene is present, so the heterozygotehas mutant phenotype. If the mutations lie in different genes, the parental genotypescan be representedas: m,+
:
[, *
dflCl
, -+ m , *[,
Each chromosome has a wild-type copy of one gene (representedby the plus sign) and a mutant copy of the other. Then the heterozygote has the constitution: flr*
*[, in which the two parents between them have provided a wild-type copy of each gene. The heterozygote has wild phenotype, and thus the two genes are said to complement. The complementation test is sho\ rn in more detail in : :,,,-:!ii:. r. The basictest consistsof the comparison shown in the top part of the figure. If two mutations lie in the same gene, we see a difference in the phenotypes of lhe trans configuration and the crsconfiguration. The trans configuration is mutant, becauseeach allele has a (different) mutation. However, the as configuration is wild-type, becauseone allele has two mutations and the other allele has no mutations. The lower part of the figure shows that if the two mutations lie in different genes, we
MUTATIONS IN SAMEGENE
mutant1
No complementation r/ ,/ ,/ ,f Mutantphenotype ,/ ./ */ ,f
mutant2
doublemutant
,/ ,,{ */ r/ Onegeneis ,f ,J d -/ wild{ype ild phenotype wild type
MUTATIONS lN DIFFERENT GENES(transconfiguration) mutant1 Complementatio (wildphenotype) ./\f ./ , One copyof each ./ '/ '/. geneis wild-type wild type
jr;:iii,ritl :i ;i Thecistronis definedby the complementation test. Genes arerepresented byspira[s; redstarsidentifositesof mutation.
26
CHAPTER 2 GenesCodefor Proteins
always see a wild phenotype. There is always one wild-type and one mutant allele of each gene, and the configuration is irrelevant. Failure to complement means that two mutations are part of the samegenetic unit. Mutations that do not complement one another are said to comprise part of the same complementation group. Another term that is used to describe the unit defined by the complementation test is the cistron. This is the same as the gene. Basically these three terms all describe a stretch of DNA that functions as a unit to give rise to an RNA or protein product. The properties of the gene with regard to complementation are explained by the fact that this product is a single molecule that behaves as a functional unit.
MayCause Mutations Loss-of-Function or Gain-of-Function mutationsaredueto loss-of-function by Recessive the proteinproduct. Dominant mutations resultfroma gain-offunction. Testing whether requires a geneis essential a nutl its mutation(onethat comptetety eliminates function). Sitentmutationshaveno effect.eitherbecause the basechange doesnot change the sequence or amountof protein,or because the change in proteinsequence hasno effect. Leakymutationsdo affectthe functionof the geneproduct, in the but arenot revealed phenotype because sufficientactivityremains. The various possible effects of mutation in a gene are summarized in ;r'Ji.rt"iiii:.i,+. When a gene has been identified, insight into its function in principle can be gained by generating a mutant organism that entirely lacks the gene. A mutation that completely eliminates gene function-usually becausethe gene has been deleted-is called a null rnutation. If a gene is essential, a null mutation is lethal. To determine what effect a gene has upon the phenotype, it is essential to characterize a null mutant. When a mutation fails to affect the phenotype, it is always possible that this is becauseit is a leaky mutation-enough active product is made to fulfill its function, even though the activity is quantitatively reduced or qualitatively different from the wild type. However, if a null mutant fails to affect a phenotype, we may safely conclude that the gene function is not necessary.
If a recessivemutation is produced by every change in a gene that prevents the production Wild-typegene codes of an active protein, there should be a large number of such mutations in any one gene. Many amino acid replacements may change the structure of the protein sufficiently to impede its function. Different variants of the same gene are called Silentmutation multiple alleles, and their existence makes it does not affectprotein possibleto createa heterozygote between mutant alleles. The relationship between these multiV ple alleles takes various forms. In the simplest case, a wild-type gene codes for a protein product that is functional. Mutant allele(s) code for proteins that are Nullmutation nonfunctional. makes no protein But there are often casesin which a series of mutant alleles have different phenotypes. For example, wild-type function of. the white locus of Drosophilamelanogasteris required for development of the normal red color of the eye. ii,:r::iri i:',.r,Mutations that do notaffectproteinsequence The locus is named for the effect of extreme or functionaresitent.Mutations (null) mutations, which causethe fly to have a that abo[ish at[orotein a c t i v i t ya r en u [ t .P o i n tm u t a t i o ntsh a t c a u s e[ o s s - o f - white eye in mutant homozygotes. qain-of-function functjonarerecessive; thosethatcause To describe wild-type and mutant alleles, a r ed o m i n a n t . wild genotype is indicated by a plus superscript after the name of the locus (r,rz+ is the wild-type allele for [red] eye color in D. melanogaster). Sometimes + is used by itself to describe the wild-t1pe allele, and only the mutant allelesare Null mutations, or other mutations that indicated by the name of the locus. impede gene function (but do not necessarily An entirely defective form of the gene (or abolish it entirely), are called loss-of-function absence of phenotype) may be indicated by a mutations. A loss-of-function mutation is recesminus To distinguish among a varisuperscript. sive (as in the example of Figure 2.2). Someety of mutant alleleswith different effects,other times a mutation has the opposite effect and superscriptsmay be introduced, such as w' or wa. causesa protein to acquire a new function; such The rar'allele is dominant over any other a change is called a gain-of-function mutaallele in heterozygotes. There are many differtion. A gain-of -function mutation is dominant. ,:,:;shows a (small) ent mutant alleles. i:it'iii'i1r Not all mutations in DNA lead to a detectable A l t h o u g h a lleleshave no eye s a m p l e . s o m e change in the phenotype. Mutations without color, many allelesproduce some color. Each apparent effect are called silent mutations. They of these mutant alleles must therefore reprefall into two q,?es:One tlpe involves basechanges sent a different mutation of the gene, which in DNA that do not causeany changein the amino does not eliminate its function entirely, but acid present in the corresponding protein. The leaves a residual activity that produces a charsecond type changes the amino acid, but the acteristicphenotype. These alleles are named replacement in the protein does not affect its for the color of the eye in a homozygote. (Most activity; these are called neutral substitutions. w alleles affect the quantity of pigment in the eye. The examples in the figure are arranged in [roughly] declining amount of color, but others, such as wsP,affect the pattern in which it is deposited.) When multiple allelesexist, an animal may be a heterozygote that carries two different r Theexistence of muttipte a[[e[es allows mutant alleles. The phenotype of such a hetheterozygotes to occurrepresenting anypairwise erozygote depends on the nature of the residcombination of a[tetes. ual activity of each allele. The relationship
I I
I
A LocusMayHaveMany DifferentMutantALLeles
MutantAlleles 2.5 A LocusMayHaveManyDifferent
27
Allele w* wor wth wot wh w" we wl w' w"P w1
Phenotypeof homozygote red eye (wildtype) blood cherry buff honey apricot eosin ivory zeste (lemon-yellow) mottled,colorvaries white (no color)
l:iir-:.iiilr ,: r Ther,vlocushasan extensive seriesof at[e(red)cotor leswhosephenotypes extendfromwitd-type to comptete lackof pigment.
between two mutant alleles is in principle no different from that between wild-type and mutant alleles: one allele may be dominant, there may be partial dominance, or there may be codominance.
A LocusMayHaveMore i I{"litlRt: l.{i TheAB0btoodgroup[ocuscodes fora ga[acwhosespecificity determines the btood A[LeLe tosyltransferase thanOneWil.d-type gr0up.
@
. A [ocusmayhavea potymorphic distribution of atletes with no individual altetethat canbe considered to bethe solewitd-type. There is not necessarilya unique wild-type allele at any particular locus. Control of the human blood group systemprovides an example. Lack
This explains why,4 and B alleles are dominant in the AO and BO heterozygotes: the corresponding transferaseactivity createsthe A or B antigen. The A and B alleles are codominant in AB heterozygotes, because both transferase activities are expressed.The OO homozygote is a null that has neither activity and therefore Iacks both antigens. Neither A nor B can be regardedas uniquely wild type, because they represent alternative activities rather than loss or gain of function. A situation such as this, in which there are multiple functional alleles in a population, is describedas a polymorphism (seeSection4.3, Individual Genomes Show ExtensiveVariation).
ff,,:il1';:;H:i:,T:tJ::,i"'":iili:i;'"x';
B provide activities that are codominant with one another and dominant over O group. The basis for this relationship is illustrated in ! i:::i.il
r::.i:.
The O (or H) antigen is generatedin all indi-
:"x'H:*:',1Ti: ;*:lii:1,::1H:?:xifi
codes for a galactosyltransferaseenzyme that ldds a further sugar group to the O antigen. Ihe specificity of this enzyme determines the blood group. The A allele produces an enzyme that uses the cofactor UDP-N-acetylgalactose, creating the A antigen. The B allele produces an enzyme that uses the cofactor UDP-galactose, creating the B antigen. The A and B versions of the transferaseprotein differ in four amino acids that presumably affect its recognition of the type of cofactor. The O allele has a mutation (a small deletion) that eliminates activity, so no modification of the O antigen occurs. CHAPTER 2 GenesCodefor Proteins
Recombination 0ccurs bv Physical Exchange of DNA r Recombination is the resuttof crossing-over that occurs at chiasmata andinvolves two of the four chromatids. . Recombination occurs by a breakage andreunjon that proceeds viaanintermediate of hybridDNA.
ParentalDNA molecules chromatids,2 from each parent
a a
b D
Chiasma is causedby crossing-overbetween 2 ot the chromatids Two chromosomesremain parental(ABand ab). Recombinantchromosomes containmaterialfrom each parent,and have new genetic combinations(Ab and aB).
!::.Sijiti. li..' Chiasma formation is responsibte for generatingrecombinants. Genetic recombination describesthe generation of new combinations of alleles that occurs at each generation in diploid organisms. The two copies of each chromosome may have different alleles at some loci. By exchanging corresponding parts between the chromosomes,recombinant chromosomes can be generated that are different from the parental chromosomes. Recombination results from a physical exchange of chromosomal material. This is visible in the form of the crossing-over that occurs during meiosis (the specialized division that produceshaploid germ cells).Meiosis startswith a cell that has duplicated its chromosomes, so that it has four copies of each chromosome. Early in meiosis, all four copiesare closely associated (synapsed) in a structure called a bivalent. Each individual chromosomal unit is called a chromatid at this stage.Pairwise exchanges of material occur between the chromatids. The visible result of a crossing-overevent is called a chiasma and is illustrated diagrammatically in Fili*fi{ ;"1.1. A chiasma represents a site at which two of the chromatids in a bivalent have been broken at corresponding points. The broken ends have been rejoined crosswise, generating new chromatids. Each new chromatid consists of material derived from one chromatid on one side of the junction point, with material from the other chromatid on the opposite side.The two recombinant chromatids have reciprocal structures. The event is described as a breakage and reunion. Its nature explains why a single recombination event can produce only 50% recombinants:each individual recombination event involves only two of the four associatedchromatids.
I Recombinationintermediate
I Recombinants
l:i*ijSi: il"ji Recombinatjon invotvespairingbetween duplexDNAs. complementary strands of thetwo parentaI The complementarity of the two strands of DNA is essentialfor the recombination process. Each of the chromatids shown in Figure 2.7 consistsof a very long duplex of DNA. For them to be broken and reconnected without any loss of material requires a mechanism to recognize exactly corresponding positions. This is provided by complementary base pairing. Recombination involves a processin which the single strands in the region of the crossover t.li shows that exchange their partners. li:iriiF-h, this createsa stretch of hybrid DNA, in which the single strand of one duplex is paired with its complement from the other duplex. The mechanism, of course, involves other stages(strands must be broken and resealed),which we discuss in more detail in Chapter 20, Repair Systems, but the crucial feature that makes precise recombination possible is the complementarity of DNA strands.The figure shows only some stagesof the reaction, but we see that a stretch of hybrid DNA forms in the recombination intermediate when a single strand crossesover from one duplex to the other. Each recombinant consists of one parental duplex DNA at the left, which is connected by a stretch of hybrid DNA to the other parental duplex at the right. Each duplex DNA correspondsto one of the chromatids involved in recombination in Fig:e2.7. The formation of hybrid DNA requires the sequencesof the two recombining duplexes to
Exchange of DNA by Physical Occurs 2.7 Recombination
29
be close enough to allow pairing between the complementary strands. If there are no differencesbetween the two parental genomesin this region, formation of hybrid DNA will be perfect. However, the reaction can be tolerated even when there are small differences. In this case, the hybrid DNA has points of mismatch, at which a basein one strand facesa base in the other strand that is not complementary to it. The correction of such mismatches is another feature of genetic recombination (seeChapter 20 Repair Systems).
@
TheGenetic Code Is Triplet
. Thegeneticcodeis readin tripLetnucteotides catledcodons. . Thetripletsarenonoverlapping andarereadfrom a fixedstartingpoint. r Mutations that insertor deleteindividuaI bases causea shift in the triotetsetsafterthe siteof mutation. r Combinations of mutations that togetherinsertor (or multiples detetethreebases of three)insertor deleteaminoacids,but do not change the reading of the tripl.ets beyond the [astsiteof mutation. Each gene representsa particular protein chain. The conceptthat each protein consistsof a particular seriesof amino acids datesfrom Sanger's c h a r a c t e r i z a t i o no f i n s u l i n i n t h e 1 9 5 0 s .T h e discoverythat a gene consistsof DNA presents us with the issue of how a sequence of nucleotides in DNA representsa sequence of amino acidsin protein. A crucial feature of the general structure of DNA is that it is independentof the particular sequence of itsclmplnentnucleotides. The sequence of nucleotidesin DNA is important not because of its structure perse,but becauseit codesfor tne sequence of amino acids that constitutes the corresponding polypeptide. The relationship between a sequenceof DNA and the sequence of the corresponding protein is called the genetic code. The structure and/or enzymatic activity of each protein follows from its primary sequence of amino acids. By determining the sequence of amino acidsin each protein, the gene is able to carry all the information needed to specify an active polypeptide chain. In this way, a single tlpe of structure-the gene-is able to represent itself in innumerable polypeptide forms.
30
CHAPTER 2 GenesCodefor Proteins
Together the various protein products of a cell undertake the catalytic and structural activities that are responsiblefor establishingits phenotype. Of course,in addition to sequencesthat code for proteins, DNA also contains certain sequenceswhose function is to be recognized by regulator molecules, usually proteins. Here the function of the DNA is determined by its sequence directly, not via any intermediary code.Both types of region-genes expressedas proteins and sequencesrecognized as suchconstitute genetic information. The genetic code is decipheredby a complex apparatus that interprets the nucleic acid sequence.This apparatusis essentialif the information carried in DNA is to have meaning. In any given region, only one of the two strands of DNA codesfor protein, so we write the geneticcode as a sequenceof bases(rather than basepairs). The genetic code is read in groups of three nucleotides, each group representing one amino acid. Each trinucleotide sequenceis c a l l e d a c o d o n . A g e n e i n c l u d e s a s e r i e so f codons that is read sequentially from a starting point at one end to a termination point at the other end. Written in the conventional 5' to 3'direction, the nucleotide sequenceof the DNA strand that codesfor protein corresponds to the amino acid sequence of the protein written in the direction from N-terminus to C-terminus. The genetic code is read in nonoverlapping tripletsfrom a fixed starting point: . Nonoverlapping implies that each codon consistsof three nucleotides and that successivecodons are represented by successivetrinucleotides. . The use of a fixed starting point lrreans that assembly of a protein must start at one end and work to the other, so that different parts of the coding sequence cannot be read independently. The nature of the code predicts that two types of mutations will have different effects. If a particular sequence is read sequentially, such as: UUU AAA GGG CCC (codons) aal aa2 aa3 aa4 (amino acids) then a point mutation will affect only one amino acid. For example, the substitution of an A by some other base (X) causes aa2 to be replaced by aa5: UUU AAX GGG CCC aal aa5 aa7 aa4
because only the second codon has been changed. But a mutation that insertsor deletesa single basewill changethe triplet sets for the entiresubsequent sequence. A change of this sort is called a frameshift. An insertion might take the form: UUU AAX AGG GCC C a a l a a 5a a 6a a 7 Becausethe new sequenceof triplets is completely different from the old one, the entire amino acid sequence of the protein is altered beyond the site of mutation. So the function of the protein is likely to be lost completely. Frameshift mutations are induced by the acridines, compounds that bind to DNA and distort the structure of the double helix, causing additional basesto be incorporated or omitted during replication. Each mutagenic event sponsoredby an acridine results in the addition or removal of a single basepair. If an acridine mutant is produced by, say, addition of a nucleotide, it should revert to wild type by deletion of the nucleotide. However, reversion also can be causedby deletion of a different baseat a site closeto the first. Combinations of such mutations provided revealing evidence about the nature of the genetic code. i:iilii!-li:iri.::illustrates the properties of frameshift mutations. An insertion or deletion
Wildtype Ala Ala Ala, 'Ala ,Ala , ,Ala Ala ,i Ala.","',,
'_._] GCUGCUAGCUGCUGCUGCUGCUGCIU\,,{16;,:r:"1413:,, Ser Cys Cys Cys Cys Cys Deletion -.GCU
G G C UG C UG C U G C U C U GC U GC U G
Ala Ala Ala Ala Ala Leu Leu Leu
-
changes the entire protein sequence following the site of mutation. However, the combination of an insertion and a deletion causes the code to be read incorrectly only between the two sites of mutation; correct reading resumes after the second site. In 1961, geneticanalysisof acridine mutations in the rII region of the phage T6 showed that all the mutations could be classified into one of two sets,describedas (+) and (-). Either type of mutation by itself causes a frameshift: the (+) type by virtue of a base addition, and the (-) type by virtue of a base deletion. Double mutant combinations of the types (+ +) and (- -) continue to show mutant behavior. However, combinations of the types (+ -) or (- +) suppress one another, giving rise to a description in which one mutation is described as a frameshift supressor of the other. (In the context of this work, "suppressor" is used in an unusual sensebecausethe secondmutation is in the same gene as the first.) These results show that the genetic code must be read as a sequencethat is fixed by the starting point. Thus additions or deletions compensate for each other, whereas double additions or double deletions remain mutant. However, this does not reveal how many nucleotidesmake up each codon. When triple mutants are constructed, only (+ + + ) and (- - -) combinations show the wild phenotlpe, whereas other combinations remain mutant. If we take three additions or three deletions to correspond respectively to the addition or omission overall of a single amino acid, this implies that the code is read in triplets. An incorrect amino acid sequence is found between the two outside sitesof mutation and the sequence on either side remains wild type, as indicated in Fig:ure2.9.
GCUGCUAGCUGCUGCUCUGCUGCUFr Ala A,la Ser Cys Cys Ser Ala Ala
AIa Ata Cys Cys [4et His Ala Ala Wild-typesequence
Mutantsequence
::;iiii:ir .:.:1,Frameshift mutations showthatthegenetic codeis readin triptetsfroma fixedstartingpoint.
HasThree Every Sequence Frames Reading Possible . Usualty and translated frame'is ontyonereading termjnation by frequent the othertwo areblocked signa[s. If the genetic code is read in nonoverlapping triplets, there are three possible ways of translating any nucleotide sequence into protein, depending on the startingpoint. Theseare called reading frames. For the sequence ACGACGACGACGACGACG
Frames 3 1 Reading HasThreePossibte Sequence 2.9 Every
the three possible reading frames are ACG ACG ACG ACG ACG ACG ACG CGA CGA CGA CGA CGA CGA CGA GAC GAC GAC GAC GAC GAC GAC A reading frame that consistsexclusively of triplets representing amino acids is called an open reading frame or ORF. A sequence that is translated into protein has a reading frame that starts with a special initiation codon (AUG) and then extends through a seriesof triplets representing amino acids until it ends at one of three types of termination c o d o n ( s e eC h a p t e r 7 , M e s s e n g e rR N A ) . A reading frame that cannot be read into protein because termination codons occur frequently is said to be blocked.If a sequence is blocked in all three reading frames, it cannot have the function of coding for protein. When the sequence of a DNA region of unknown function is obtained, each possible reading frame is analyzed to determine whether it is open or blocked. Usually no more than one of the three possible frames of reading is open in any single stretch of DNA. ri$U R ft . t * s h o w s a n e x a m p l e o f a s e q u e n c et h a t can be read in only one reading frame because the alternative reading frames are blocked by frequent termination codons. A long open reading frame is unlikely to exist by chance; if it were not translated into protein, there would have been no selectivepressure to prevent the accumulation of termination codons. So the identification of a lengthy open reading frame is taken Io be prima facie evidence that the sequenceis translated into protein in that frame. An ORF for which no protein product has been identified is sometimes called an unidentified reading frame (URF).
Only one open readingframe
Termination
Secondreadingframe is blocked Third readingframe is blocked
FiGgJftil 2.1* An openreading framestartswithAUGandcontinuesin triptetsto a termination codon.Blocked reading frames may beinterrupted frequently by termination codons.
32
CHAPTER 2 Genes Codefor Proteins
Prokaryotic Genes AreCo[inear with TheirProteins r A prokaryotic geneconsists of a continuous [ength of 3fi nucteotides that codes for fi aminoacids. . Thegene,mRNA, andproteinarea[[cotinear. By comparing the nucleotide sequence of a gene with the amino acid sequence of a protein, we can determine directly whether the gene and the protein are colinear; that is, whether the sequenceof nucleotidesin the gene corresponds exactly with the sequenceof amino acidsin the protein. In bacteria and their viruses, there is an exact equivalence.Each gene contains a continuous stretch of DNA whose length is directly related to the number of amino acidsin the protein that it represents. A gene of 3N bp is required to code for a protein of N amino acids, according to the genetic code. The equivalence of the bacterial gene and its product means that a physical map of DNA will exactly match an amino acid map of the protein. How well do these maps fit with the recombination map? The colinearity of gene and protein was originally investigated in the tryptophan synthetase gene of E. coli. Genetic distance was measured by the percent recombination between mutations; protein distance was measured by the number of amino acids separating sites of replacement. FI€UftH?.3.1compares the two maps. The order of seven sites of mutation is the same as the order of the corresponding sitesof amino acid replacement, and the recombination distances are relatively similar to the actual distancesin the protein. The recombination map expands the distancesbetween some mutations, but otherwise there is little distortion of the recombination map relative to the physical map. The recombination map makes two further general points about the organization of the gene. Different mutations may cause a wildtype amino acid to be replaced with different substituents. If two such mutations cannot recombine, they must involve different point mutations at the same position in DNA. If the mutations can be separatedon the genetic map, but affect the same amino acid on the upper map (the connecting lines converge in the figure), they must involve point mutations at dif-
strands DNAconsistsof two base-paired top strand 5' ATGCCGTTAGACCGTTAGCGGACCTGAC 3' TACGGCAATCTGGCAATCGCCTGGACTG bottomstrand r II R N A sYnthesis * 3 5' AUGCCGUUAGACCGUUAGCGGACCUGAC top strand; as DNA sequence RNAhas same to Dl{A bottomstrafld is complementary by usingone strandof F;t-{;l,iii:i;;:,'i; RNAis synthesized basepairing. DNAas a temptatefor complementary
i6nt
"r I
Differentmutations at same position
Mutationscan be separatedbut aflectsame position
i:ii:;:,iF!-: ;:.i 1 Therecombination mapof thetryptophan genecorresponds synthetase withtheaminoacidsequence of the protein.
ferent positions that affect the same amino acid. This happens becausethe unit of geneticrecombination (actually I bp) is smaller than the unit coding for the amino acid (actually I bp).
Processes SeveraI Are
Required to Express the Protein Product of a Gene . A prokaryotic geneis expressed by transcription into mRNA andthenbytranstation of the mRNA into orotein. . In eukaryotes, a genemaycontaininternal regions that arenot represented in protein. . InternaIregions areremoved fromthe RNA transcript by RNAspticing to givean mRNA that is colinear withthe proteinproduct. e EachmRNA consists of a nontranstated 5' leader, a codingregion,anda nontranstated 3'traiter. In comparing gene and protein, we are restricted to dealing with the sequenceof DNA stretching between the points correspondingto the ends of the protein. However, a gene is not directly translated into protein, but is expressedvia the production of a rnessenger RNA (abbreviated to mRNA), a nucleic acid intermediate actu-
ally used to synthesizea protein (as we see in detail in Chapter 7, MessengerRNA). Messenger RNA is synthesizedby the same processof complementary base pairing used to replicate DNA. with the important difference that it corresponds to only one strand of the DNA double helix. flt{"illfii:l:":.;ishows that the sequence of mRNA is complementary with the sequence of one strand of DNA and is identical (apart from the replacement of T with U) with the other strand of DNA. The convention for writing DNA sequences is that the top strand runs 5'-+3', with the sequence that is the same as RNA. The processby which a gene gives rise to a protein is calledgene expression.In bacteria,it consistsof two stages.The first stage is transcription, when an nRNA copy of one strand of the DNA is produced. The second stage is translation of the nRNA into protein. This is the processby which the sequenceof an mRNA is read in triplets to give the series of amino acids that make the corresponding protein. A mRNA includes a sequenceof nucleotides that corresponds with the sequence of amino acidsin the protein. This part of the nucleic acid is called the coding region. However, the mRNA includes additional sequenceson either end; these sequencesdo not directly represent protein. The 5' nontranslated region is called the leader, and the 3' nontranslated region is called the trailer. The geneincludes the entire sequence representedin messengerRNA. Sometimesmutations impeding gene function are found in the additional, noncoding regions, confirming the view that these comprise a legitimate part of the genetic unit.
of a Gene the ProteinProduct to Express AreRequired 2.11 SeveraI Processes
33
Leader
I
rraiter RNA
Lengthof RNAdefinesregionof gene
I,'
rufficprotein
Proteindefinescodingregion
F I G U R?f. 1 3 T h eg e n em a yb el o n g e r t h at h nesequence codingfor protein.
Transcription
TRNAY 5',
CYTOPLASM Translation
FIGUftE 3.15 Geneexpression process. is a multistage
FIGURf i.14 Transcription andtranstation takeptace in thesame comoartment in bacteria.
F1GURE 2.13illustratesthis situation, in which the gene is considered to comprise a continuous stretch of DNA, needed to produce a particular protein. It includes the sequence coding for that protein, but also includes sequenceson either side of the coding region. A bacterium consists of only a single compartment, so transcription and translation occur in the same place, as illustrated in FIfiURIf .14. In eukaryotes transcription occurs in the nucleus, but the RNA product must be transportedto the cytoplasm in order to be translated. For the simplest eukaryotic genes (just like in bacteria) the transcript RNA is in fact the nRNA. However, for more complex genes, the immediate transcript of the gene is a pre-rnRNA that requires processing to generate the mature mRNA. The basic stagesof gene expression in a eukaryote are outlined in frl#{Jf{f, *.iS. This results in a spatial separation between transcrip-
34
CHAPTER 2 GenesCodefor Proteins
tion (in the nucleus) and translation (in the cytoplasm). The most important stage in processing is RNA splicing. Many genesin eukaryotes (and a majority in higher eukaryotes) contain internal regions that do not code for protein. The process of splicing removes these regions from the pre-mRNA to generate an RNA that has a continuous open reading frame (seeFigure 3. I ). Other processingevents that occur at this stage involve the modification of the 5'and 3'ends of the pre-mRNA (seeFigure 7.16). Translation is accomplished by a complex apparatus that includes both protein and RNA components. The actual "machine" that undertakes the process islhe ribosome,alarge complex that includes some large RNAs (ribosomalRNAs, abbreviated to rRN/s) and many small proteins. The process of recognizing which amino acid corresponds to a particular nucleotide triplet requires an intermediate transfer RNA (abbreviated to IRNA); there is at least one IRNA species for every amino acid. Many ancillary proteins are involved. We describe translation in Chap-
ter 7, Messenger RNA, but note for now that the ribosomes are the large structures in Figure 2.I4that move along the mRNA. The important point to note at this stage is that the process of gene expression involves RNA not only as the essentialsubstrate,but also in providing components of the apparatus. The rRNA and IRNA components are codedby genes and are generatedby the processof transcription (just like mRNA, except that there is no subsequentstageof translation).
ProteinsAreTran s-acting, but Siteson DNA AreCrs-acting . At[geneproducts (RNAor proteins) aretransacting.Theycanacton anycopyof a genein the ceIt. r crs-acting mutations identifysequences of DNA that aretargetsfor recognition by trons-acting products. Theyarenot expressed asRNAor protein andaffectonlythe contiguous stretchof DNA. A crucial step in the definition of the gene was the realization that all its parts must be present on one contiguous stretch of DNA. In genetic terminology, sites that are located on the same DNA are said to be in c/s. Sites that are located on two different molecules of DNA are described as being in trans. So two mutations may be in crs(on the same DNA) or in trans (on different DNAs). The complementation test usesthis concept to determine whether two mutations are in the same gene (seeFigure 2.3 in Section2.1, Mutations in the Same Gene Cannot Complement). We may now extend the concept of the difference between cis and trans eff-ectsfrom defining the coding region of a gene to describing the interaction between regulatory elements and a gene. Suppose that the ability of a gene to be expressed is controlled by a protein that binds to the DNA close to the coding region. In the example depictedin Fi*LtF.{ t.tS, nRNA can be synthesized only when the protein is bound to the DNA. Now suppose that a mutation occurs in the DNA sequence to which this protein binds, so that the protein can no longer recognize the DNA. As a result, the DNA can no longer be expressed. Soa genecan be inactivatedeither by a mutation in a control site or by a mutation in a codingregion. The mutations cannot be distinguished genetically, because both have the property of act-
Two types of DNA
RNA
ir{*j-i,-iI i]".i*; Controlsitesin DNAprovidebindingsites viathe syntheareexpressed for proteins; codingregions sisof RNA.
Both allelessynthesizeRNA in wild type
,, ,:'''";, ,,,'i
'..,,_.,'it-' ;., ,,it
"r.
NO RNASYNTHESISFROMALLELE1
RNA synthesis continuesfrom allele2 ,
r.1..,t'
' ,.,
,,1
o,
i!
DNA, the adjacent sitecontrots li I i A crs-acting l'i{it.iRir the othera[te[e. but doesnotinfluence ing only on the DNA sequence of the single allele in which they occur. They have identical properties in the complementation test, and a mutation in a control region is therefore defined as comprising part of the gene in the same way as a mutation in the coding region. f li:;tittf,ii.: r shows that a deficiency in the control site affectsonly the coding region to which it is connected;it doesnot affectthe ability of the other A mutation that acts solely alleleto be expressed. by affecting the properties of the contiguous sequence of DNA is called cis-acting.
butSiteson DNAAreCis-acting Arelrans-acting, 2.12 Proteins
The activeproteinacls on both alleles
tl Mutanlproteincannotbind to eitherallele
NO RNASYNTHESIZED FROMALLELE1
S--Mutant
protein
NO BNA SYNTHESIZED FROMALLELE2
i-iLi;-:$:i. .t.:i: A trons-acting mutationin a proteinaffects bothatteles of a genethat it controts.
codesfor protein. An RNA or protein product of a gene is said Io be trans-acting.A gene is defined as a unit of a single stretch of DNA by the complementation test. A site on DNA that regulates the activity of an adjacent gene is said to be czs-acting. When a gene codes for protein. the reiationship between the sequence of DNA and sequence of the protein is given by the genetic code. Only one of the two strands of DNA codes for protein. A codon consistsof three nucleotides that represent a single amino acid. A coding sequenceof DNA consistsof a seriesof codons, read from a fixed starting point. Usually one of the three possible reading frames can be transIated into protein. A gene may have multiple alleles.Recessive allelesare causedby loss-of-function mutations that interfere with the function of the protein. A null allele has total loss-of-function. Dominant alleles are causedby gain-of-function mutations that create a new property in the protein.
References TheGenetic CodeIs Triolet
We may contrast the behavior of the czsacting mutation shown in Figure 2.17 with the result of a mutation in the gene coding for the regulator protein. lii:i"iFI i.5* shows that the absenceof regulator protein would prevenl both allelesfrom being expressed.A mutation of this sorl is saidtobe trans-acting. Reversing the argument, if a mutation is trans-acting, we know that its effects must be exerted through some diffusible product (typically a protein) that acts on multiple targets within a cell. However, if a mutation is cli-acting, it must function via affecting directly the propefties of the contiguous DNA, which means that it is not expressed in theform of RNA or protein.
Summary A chromosome consists of an uninterrupted length of duplex DNA that contains many g e n e s .E a c h g e n e ( o r c i s t r o n ) i s t r a n s c r i b e d into an RNA product, which in turn is translated into a polypeptide sequence if the gene
36
CHAPTER 2 Genes Code for Proteins
Review Roth, J. R. ( 1974). Frameshift mutations. Annu. Rev.GeneL8, Jl9-346. Resea r ch B e n z e r ,S . a n d C h a m p e ,S . P . ( 1 9 6 1 ) .A m b i v a l e n t rII mutants of phage T4. Proc Natl. Acad Sci usA 47, 40)-416. Crick, F. H. C., Barnett, L., Brenner, S., and WattsTobin, R. J. ( I 96 I ). General nature of the genetic code for proteins. Nature 192, t227-1,2)2.
Prokaryotic Genes AreCotinear withTheirProteins R e s e a hr c Yanofsky, C., Drapeau, G.R., Guest,J. R., and Carlton, B.C. ,1967). The complete amino acid sequence of the tryptophan synthetase A protein (p subunit) and its colinear relationship with the genetic map of the A gene. Proc. Natl. Acad. Sci.USA 57, 2966-2968. Yanofsky, C. etal. (19641.On the colineariry of gene structure and protein structure. Proc Natl. Acad Sci USA, 51.266-272.
InterruptedGene C H A P T EO RU T L I N E Introduction An InterruptedGeneConsistsof Exonsand Introns o Intronsareremoved bythe process of RNAspLicing, which occurs onlyin crson anjndividual RNAmotecu[e. . Ontymutations in exonscanaffectproteinsequence; however.mutations in intronscanaffectprocessing of the RNA prevent production andtherefore of protein. RestrictionEndonucleases Are a KeyToo[in Mapping DNA . Restriction endonucleases DNAinto canbeusedto cteave definedfragments. . A mapcanbegenerated by usingthe overlaps between the generated fragments by differentrestrictionenzymes. 0rganizationof InterruptedGenesMayBe Conserved r Intronscanbe detected bythe presence of additional regions whengenesarecompared withtheirRNAproducts by restriction mapping or electron microscopy. Theuttimate definition, though,is basedon comparison of sequences. . Thepositions of intronsareusually whenhomolconserved ogousgenesarecompared between differentorganisms. Thelengthsof the corresponding intronsmayvarygreatty. o Intronsusualty do not codefor proteins. ExonSequences Are Conserved but IntronsVary r Comparisons genesin differentspecies of retated showthat the sequences of the corresponding exonsareusualty conserved butthe seouences of theintronsaremuchlesswe[[ retated. o Intronsevolvemuchmorerapidlythanexonsbecause of pressure the lackof selective to producea proteinwith a usefuIsequence. GenesShowa WideDistributionof Sizes r Mostgenesareuninterrupted in yeasts, but areinterrupted in highereukaryotes. r Exons areusuatty short,typicatty codingfor
o Nonhomologous from proteinsequences canbe produced of DNAwhenitis readin differentreadthe samesequence genes. ing framesby two (overtapping) . Homotogous proteins or absence that differbythe presence by differential(atternaof certainregionscanbe generated or excluded. whencertainexonsareincluded tive)spticing individuaL or excluding Thismaytakethe formof including exons. atternative between exonsor of choosing How Did InterruptedGenesEvolve? . Themajorevolutionary question is whethergenesorigithey interrupted byintronsor whether natedassequences uninterrupted. wereoriginatly o Mostprotein-coding genesprobab[y in an interoriginated genesthat codefor RNAmay ruptedform,butinterrupted haveorigina[ybeenuninterrupted. . A speciaI classof intronsis mobileandcaninsertitselfinto genes. SomeExonsCanBe Equatedwith ProteinFunctions . Factssuggesting that exonswerethe buitdingblocksof are: andthe firstgeneswereinterrupted evotution o Genestructure genesin very between is conserved distantspecies. . Manyexonscanbeequated with codingfor protein functions. that haveparticutar sequences r Retated exonsarefoundin differentgenes. T h eM e m b e ros f a G e n eF a m i t vH a v ea C o m m o n 0rganization o A common to identifya featurein a setof genesis assumed jn evolution. property theirseparation that preceded . Attgtobingeneshavea common with formof organization that theyare threeexonsandtwo introns,suggesting gene. froma singteancestral descended Is A[[ GeneticInformationContainedin DNA? o Thedefinitionof the genehasreversed from"onegene: oneprotein"to "oneprotein: onegene." r PositionaI in development. is alsoimportant information Summary
SomeDNASequences Codefor MoreThanOneProtein . Theuseof atternative initiationor termination codons atlows two proteins to be generated whereoneis equivatent to a fragment of the other.
37
saJuetsrp Jqt ot drqsuorlelar ou a.teq 'deru UOIIEUIqUO)E.I E UO USJS SP 'SJf,UP]SIPJI1JUJ9
'uratord Jqt ur sJruptsrpaqt qtlM IIp te puodsJJ -roJ lou op auaS )Lllursnualstp eqt tng 'uratord Jql ur sluauareldJJ prJe oururp Jo JepJo eql se Jures eql surpueJ aua8 aqt ur suortptnru Jo 'urpq) uratord eql )apJl ertrlwql sMoes : .
a u a 9p a t o n r a l u l e q ] € u l l d v H l 'suoxaaqlyoseruanbas aq1[1uoseq parqdsaresuoxoaq] uaqMpa^ourar a.lpsuorlul VNUUoq1'raq1a6o1 'y11X rosrnrarde eLnpassaldxe aresauaEpaldnralul t | ;i$!'tlti;j auebur peleredesa;e suor6erburpoc;enprnrpu; aue6 1ouorberseur;ep(yNgul 1ou)yyg rosrncerd]o tltOuel
yo sged Surpuodsarror aqt pup suoxa Ienprlrpur aql ueJMlJq pJurplureu sr uralord pue aua8 yo .dlrreaurlor Jqt oS 'VNC ul ar1Laql q)lqM uI JJpJo aues eql ur raqla3o1 pauroI s.{e,r,r1e are suoxJ Jqt '3uo11ds3ur,vro1o4laua8 aql Jo MarA rno a8ueqr suoJlur Jo eJualsrxaJql seop MoH 'uralord 1ouoLl:npord pupVNUaq11o6uLssarord lueneldalo1alaql lrage jeruanbes uet suo.llur ur suorlplnu'lanaMoq uraloidllo#e upl suoxeuLsuorlelnuAlug . 'alnrol0ur vNU '6uL:qds up uo so ur [1uosrnllo qrrqrvt lenpLnrpur y11g1ossaroldeq1fiq pa,rorueJ oJpsuo.llulr
VNO Z NOXf
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auagpaldnilaluluv E 'uorssardxaauaB3urleu -ItuJal (saruttauos) pup Surlprtrurro; parrnbar JJe teqt aua8 aqt Jo seprsqtoq uo suor8arLrol -eln8ar Jqt epnlJur ot peraprsuor sr aua8 aq1 ' (uotrel,{uapez(1o4 pue a3e.rea13,{qpalerauag eJV sYNUru Io spuf ,€ eqJ '6l'ge uorlres aJS) 'pua VNU rr{t;o a8eneap ,{q palerauaS srqrrqM ,€ aql puo^Jq spuJlxa ,,i.1ensnpue vNuur Jql Jo pua ,s Jql le suels uolldlrJsuert teql Mou>l aM 'YNUru Jrntelu Jo sJseq Ipururet ,€ pup ,E aql ol Surpuodsarror slurod uJaMlJq auouaS aql ur uor8ar aql sasrrdruor aua8 aq1 '(Surssaror4pue Sunrldg VNU '92 raldeq3 aas)alntaloru tJptuJ dllualenor e uJoJ ol eprs JJqlre uo vNU Jql Jo spua aqt SururoIueqt pup tdr.rrsuer]L.reur;d aqt urorJ uoJtur uB Surlalap z{lasrtardsJAIoAur pue (aueD p Jo DnpoJd urJtoJd aqt ssardxEo1 p a r r n b a g J J V s e s s J J o J Ide J a ^ e S' I I ' Z u o r l l J s aas) Suorlds VNU pJIIpJ sr ssarord srqJ 'suoxe Jo serJssaqt ;o zi.1uoslsrsuol leql vNu JeSues -seur e anr8 ol VNU aqt uoJJ pJ^otuJr eq tsntu suoJtul aqt tsJr{ 'uratord Sunnpord ro} pasn Jq louueJ tr jq8noqt 'rosrnra:d e ^d1uosl vNU slqJ 'aruanbas aruouaS aql sluasa;dar.,{1pexa leql (ldrnsuerl e) .{dor ue oJ JSrrsa.rr8aua8 17NU 'saua8 paldnual paldnr:a1ur up aqg VN( Jo -urun JoJpapJau lou sr lpq] dats leuorlrppe ue sarrnbalsaua8paldnuJlur Jo uorssardxaaq1
'suoJturJqt Jo eJuJsardaqt 1o esneJaqDnpord y1qg IpurJstr ueql ra8uol sr aua8 paldnualur ue tnq'VNU Jqt ur pup aua8 aql ur l J p r o J t u e sJ q l u r J l p s J ) u J n b J su o x J J q J .VNU JJnleru aql anr8ot passaro.rdsrldrrtsuerl Lrerurrd Jqt uJqM pelorueJ JJe teql saluanbas SuruarrralurJql eJp srroJlul r 'vNu aql Io spua,€ pue,E Jql o1 puods -eJJo) leql suoxa qlrM spuJ pup suels aua8 e'uorlrurJeprig'VNU arnlpu aql ur pJluaserdJJ saJuenbesJr{l JJe suoX![ o
_
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sarro8ateroMt eql otur paprlrp arB auaSpaldnr -Jalur ue Sursr.rdruor satuanbas aq1 VNO Io 'epoJ Jrlauetsaql Jo sJInJ Jqt o1Surprorre nnpord uratord eqt qlru Surpuodsarror Lltrexa JJuenbJSJplloJIJnu e sJpnlJul leql VNUIU uB SuIleJeue8 'uotssardxa auaS Surrnp lrnpord 1trNUJqt ruolJ paloruaJ JJe saJuenbas asaql 'uralord Jql sluesaJ -dar teqt aruanbas aqt stdnrrJlul qJIr{M 'uor8ar S u t p o r a q t u r q t r M a r l t e q l s a r u a n b a sI e u o r l -tppp apnpur,{eru aua8 p 'JJAeMoq 'sa1o.{re1na q 'spDp ourure 1g;o uralo.rd e sluJseJdar sJred aseqN€;o aruanbasSurpot snonurluoJe qlrqM ut 'ad1{l srql Jo sz{e,u.1e lsolule are saua8 lerr -JtJpg 'Dnpord ulaloJd sll qllM Jeaurlof,sr teq] VN11Jo er8ual e sr aua8 e Jo uroJ lsaldurs aq1
uoqlnpojlul
G e n o m i cD N A Exon1 AB
Intron1
A-B mRNA Exonl
Exon3
lntron2
Exon2
B-C Exon2
Exon3
|
L----r---------r+ A-B
B-C
rtri.i;liitirE . : xi "oi in s r e m a i n i n t h e s a m e o r d e r i n m R N A a s i n D N A , b u t d i s t a n c e s or proteinprodalongthegenedo notconespond to distances alongthemRNA fromB-C; ucts.Thedistance fromA-Bin thegeneis smatter thanthe distance (andprotein)is greater thanthe djsbutthe distance fromA-Bin the mRNA tancefromB-C.
between the correspondingpoints in the protein. The length of the gene is defined by the length of the initial (precursor) RNA instead of by the length of the messengerRNA(nRNA). All of the exons are representedon the same molecule of RNA, and their splicing together occurs only as anintramolecular reaction. There is usually no joining of exons carried by dffirer7lRNAmolecules, so the mechanism excludes any splicing together of sequencesrepresenting different alleles.Mutations located in different exons of a gene cannot complement one another; thus they continue to be defined as members of the same complementation group. Mutations that directly affect the sequence of a protein must lie in exons. What are the effects of mutations in the introns? The introns are not part of the messengerRNA, thus mutations in them cannot directly affect protein structure. However, they can prevent the production of the messengerRNA-for example, by inhibiting the splicing together of exons. A mutation of this sort acts only on the allele that carries it. As a result, it fails to complement any other mutation in that allele and constitutes part of the same complementation group as the exons. Mutations that affect splicing are usually deleterious.The majority are single-basesubstitutions at the junctions between introns and exons. They may causean exon to be left out of the product, cause an intron to be included, or make splicing occur at an aberrant site. The most common result is to introduce a termination codon that results in truncation of the protein sequence. About l5'h of the point mutations that causehuman diseasesare caused by disruption of splicing. Eukaryotic genesare not necessarilyinterrupted. Some correspond directly with the pro-
tein product in the same manner as prokaryotic genes. In yeast, most genes are uninterrupted. In higher eukaryotes most genes are interrupted, and the introns are usually much longer than exons. This creates genes that are noticeably larger than their coding regions.
Endonucleases Restriction
Area KeyToot in MappingDNA r Restriction canbe usedto cleave endonucleases fragments. DNAinto defined o A mapcanbe generated by usingthe overlaps generated by different thefragments between restriction enzymes. The characterization of eukaryotic genes was made possibleby the development of techniques for physically mapping DNA. The techniques can be extended to (single-stranded)RNA by making a (double-stranded)DNA copy of the RNA. A physical map of any DNA molecule can be obtained by breaking it at defined points whose distance apart can be accurately determined. Specificbreaks are made possibleby the ability of restriction endonucleases to recognize rather short sequences of doublestranded DNA as targetsfor cleavage. Each restriction enzyme has a particular target in duplex DNA, usually a specificsequence of four to six base pairs. The enzyme cuts the DNA at every point at which its target sequence occurs. Different restriction enzymes have different target sequences,and a large range of these activities (obtained from a wide variety of bacteria) now is available.
DNA Area KeyToo[in Mapping Endonucteases 3.3 Restriction
39
A
lri
1000 200
,1. 1900
600
ll
A 800
500
f,:*i:S:L-!.* A restriction mapis a [inearsequence of sites separated by defineddistances on DNA.Themapidentifiesthe sitescteaved by enzymes A andB, asdefinedby produced theindividuaI fragments bythesingleanddoub[edigests.
molecule in the form shown in {l$ijftUJ.*. The map shows the positions at which particular restriction enzymes cut DNA; the distances between the sites of cutting are measured in base pairs. So the DNA is divided into a seriesof regionsof defined lengths that lie betweensitesrecognizedby the restrictionenzymes.An important feature is that a restriction map can be obtained for any sequence of DNA, irrespective of whether mutations have been identified in i/, or, indeed, whether we have any knowledge of its function.
;iji: Jii: .-1..1 generatedby cleaving Fragments DNAwith a restrictionendonuctease can be separated according to their sizes.
A restriction map represents a linear sequenceof the sitesat which particular restriction enzymes find their targets.For short distances, the distance along such maps is measured directly in base pairs (bp). Longer distances are given in kilobases (kb), which correspondto kilobase ( 103)pairs in DNA or to kilobasesin RNA. At the level of the chromosome, a map is described in megabase pairs ( l M b = 1 0 6b p ) . When a DNA molecule is cut with a suitable restriction enzyme, it is cleaved into distinct fragments. These fragments can be separatedon the basisof their sizeby gel electrophoresis,as shown in ti**=iEt3."1. The cleaved DNA is placed on top of a gel made of agarose or polyacrylamide. When an electric current is passedthrough the gel, each fragment moves down at a rate that is inversely related to the log of its molecular weight. This movement producesa seriesof bands.Each band corresponds to a fragment of particular size,decreasingdown the gel. By analyzing the restriction fragments of DNA, we can generate a map of the original
40
CHAPTER 3 TheInterruptedGene
0rganization of Interrupted Genes MayBeConserved r Intronscanbedetected by the presence of additionaI regions whengenesarecompared with theirRNAproducts by restrict'ion mapping or electron microscopy. Theuttimatedefinition, though.is basedon comparison of sequences. . Theposjtions of intronsareusuatty conserved whenhomotogous genesarecompared between differentorganisms. Thelengthsof the corresponding intronsmayvarygreatty. o Intronsusuatly do not codefor proteins. When a gene is uninterrupted, the restriction map of its DNA corresponds exactly with the map of its mRNA. When a gene possessesan intron, the map at each end of the gene corresponds with the m a p a t e a c h e n d o f t h e m e s s a g es e q u e n c e . Within the gene, though, the maps diverge becauseadditional regions that are found in the gene are not representedin the message.Each such region corresponds to an intron. The example of F3*{}F*3,} comparesthe restriction maps of a B-globin gene and nRNA. There are two introns, each of which contains a seriesof restriction sites that are absent from the cDNA. The pattern of restriction sites in the exons is tne same in both the cDNA and the gene.
DNAfor f}*tJ*il 3.$ Comparison of therestriction mapsof cDNA andgenomic in mouseB globinshowsthat the genehastwo intronsthat arenot present the cDNA. Theexonscanbe aliqnedexactlvbetween cDNA andqene.
Exon Blocked Blocked
Blocked
Gene sequence
mRNAseouence Ser Leu Leu Ser Arg Asn Ser Trp Cys Ph€
(here present .Fg*LiRg 3"$ An intronis a sequence in the genebutabsentfromthe mRNA by the atternating shownin termsofthe cDNAsequence). Thereading frameis indicated by termiarebtocked openandshaded blocks; notethat altthreepossible reading frames n a t i o nc o d o nisn t h ei n t r o n .
Ultimately, a comparison of the nucleotide sequencesof the genomic and mRNA sequences precisely defines the introns. As indicated in ft$#ftEs"$,an intron usually has no open reading frame. An intact reading frame is created in the mRNA sequence by the removal of the introns. The structures of eukaryotic genes show extensive variation. Some genes are uninterrupted, so that the genomic sequence is colinear with that of the mRNA. Most higher eukaryotic genes are interrupted, but the introns vary enormously in both number and size. A l l c l a s s e so f g e n e s m a y b e i n t e r r u p t e d : nuclear genes coding for proteins, nucleolar genes coding for rRNA, and genes coding Ior IRNA. Interruptions also are found in mitochondrial genes in lower eukaryotes and in c h l o r o p l a s t g e n e s .I n t e r r u p t e d g e n e s d o n o t appear to be excluded from any classof eukaryotes and have been found in bacteria and bactedophages.They are. however, extremely rare in prokaryotic genomes.
Some interrupted genes possessonly one or a few introns. The globin genes provide an extensivelystudied example (seeSection 3.10, The Members of a Gene Family Have a Common Organization). The two general types of globin gene, o and B, share a common type of structure. The consistency of the organization of mammalian globin genes is evident from the structure of the "generic" globin gene summa:. i. rized in l;i;i.iltF, Interruptions occur at homologous positions (relative to the coding sequence) in all known active globin genes, including those of mammals, birds, and frogs. The first intron is always fairly short, and the second usually is longer, but the actual lengths can vary. Most of the variation in overall lengths between different globin genes results from the variation in the second intron. In the mouse, the second intron in the cr-globin gene is only 150 bp long, so the overall length of the gene is 850 bp, compared with the major p-globin gene for which the intron length of 585 bp gives the gene a
MayBeConserved 4 l Genes of Interrupted 3.4 0rganization
Intronlength
116-130
.+ Exon length 142-145
II
Y Contains
5'UTR + coding1-30
222
216-255
V
v
Amino acids 31-1 04
II
Coding105-end + 3' UTR
iI{iJR[ 3.? At[functionaL gtobingeneshaveaninterrupted structure withthreeexons. Thelengths pn - g t o b igne n e s . i n d i c a t ei d n t h ef i g u r ea p p t yt o t h em a m m a l i a
ExonSequences AreConserved but IntronsVary
12
3
4 5
6Exons
051015202530
o Comparisons genesin differentspecies of retated showthatthe sequences of the corresponding exonsareusua[[y conserved butthe sequences of the intronsaremuchlesswe[[related. r Intronsevotvemuchmorerapidlythanexons because of the lackof selective Dressure to produce a proteinwitha usefulsequence.
KO
iiGL;FA - : . S M a m m a t i ga en n e fso r D H F R h a v et h e s a m e retative organization of rathershortexonsandvery[ong jntrons,butvaryextensively in the lengthsof introns.
total length of 1382 bp. The vadarion in length of the genesis much greater than the range of lengths of the mRNAs (u-globin nRNA = 585 bases;B-globin nRNA = 620 bases). The example of DHFR, a somewhat larger gene, is shown in FIilURfl.*. The mammalian DHFR (dihydrofolate reductase)gene is organized into six exons that correspondto the 2000basemRNA. They extend over a much greater Iength of DNA becausethe introns are very long. In three mammals the exons remain essentially the same, and the relative positions of the introns are unaltered. The lengths of individual introns vary extensively, though, resulting in a variation in the length of the gene from25 ro 3l kb. The globin and DHFR genespresent examples of a general phenomenon: Genesthat are related by evolution have related organizations with conservationof the positions0f (at leastslme) of the introns. Variationsin the lengthsof thegenes are primarily determinedby the lengths of the introns.
42
CHAPTER 3 TheInterrupted Gene
Is a structural gene unique in its genome? The answer can be ambiguous. The entire length of the gene is unique as such, but its exons often are related to those of other genes.As a general rule, when two genes are related. the relationship between their exons is closer than the relationship between their introns. In an extreme case,the exons of two genesmay code for the same protein sequence, whereas the introns may be different. This implies that the two genes originated by a duplication of some common ancestralgene. Then differencesaccumulated between the copies, but they were restricted in the exons by the need to code for protein functions. As we will see later when we consider the evolution of the gene, exons can be considered basic building blocks that are assembledin various combinations. A gene may have some exons that are related to exons of another gene, but the other exons may be unrelated. Usually the introns are not related at all in such cases. Such genesmay arise by duplication and translocation of individual exons. The relationship between two genescan be plotted in the form of the dot matrix comparison of rifiLJft.fl "]"s.A dot is placed to indicate each position at which the same sequence is found
In corresponding introns, the pattern of divergence involves both changes in size (due to deletions and insertions) and base substitutions. Introns evolve much more rapidly than exons. When a gene is compared in different s p e c i e s ,t h e r e a r e t i m e s w h e n i t s e x o n s a r e homologous but its introns have diverged so much that correspondingsequencescannot be recognized. Mutations occur at the same rate in both exons and introns, but are removed more effectively from the exons by adverseselection.However, in the absenceof the constraints imposed by a coding function, an intron is able quite freely to accumulate point substitutions and other changes.These changes imply that the funcintron doesnot have a sequence-specific tion. Whether its presenceis at all necessaryfor sene function is not clear. ni.{'{.Jftil },.;;Thesequences of the mouse omarandam,n-gtobin genesarectoselyrelatedin codingregionsbut differin the fLanking regions andlongjntron.Dataprovided byPhitipLeder, Harvard MedicaI Schoo[.
in each gene. The dots form a line at an angle of,45" if two sequencesare identical. The line is broken by regions that lack similarity and is displaced laterally or vertically by deletions or insertions in one sequencerelative to the other. When the two p-globin genesof the mouse are compared, such a line extends through the three exons and through the small intron. The line peters out in the flanking regions and in the large intron. This is a typical pattern, in which coding sequencesare well related and the relationship can extend beyond the boundaries of the exons. The pattern is lost, though, in longer introns and the regions on either side of the gene. The overall degree of divergencebetween two exons is related to the differences between the proteins. It is causedmostly by basesubstitutions. In the translated regions, the exons are under the constraint of needing to code for amino acid sequences,so they are limited in their potential to change sequence. Many of the changes do not affect codon meanings, becausethey change one codon into another that represents the same amino acid. Changes occur more freely in nontranslated regions (corresponding to the 5'leader and 3'trailer of the mRNA).
Showa Wide Genes of Sizes Distribution o Mostgenesareuninterrupted but are in yeasts, interrupted in highereukaryotes. . Exons codingfor <100 short,typicaLty areusua[[y a m i n oa c i d s . r Intronsareshortin lowereukaryotes, but range in higher 10sof kbin Length upto several eukaryotes. o Theovera[[ [argety lengthof a geneis determined by its introns.
I'i{;r-ifiil;1,1i: shows the overall organization of genes in yeasts, insects, and mammals. In cerevisiae,the great majority of Saccharomyces genes (>96%) are not interrupted, and those that have exons usually remain reasonably compact. There are virtually no S. cerevisiaegenes with more than four exons. In insects and mammals the situation is reversed. Only a few geneshave uninterrupted c o d i n g s e q u e n c e s( 6 % i n m a m m a l s ) . I n s e c t genes tend to have a fairly small number of exons-typically fewer than 10. Mammalian genesare split into more pieces,and some have several l0s of exons. Approximately 50% of mammalian geneshave >10 introns. Examining the consequences of this type of.organization for the overall size of the gene, .l.':i that there is a striking difwe seein iir"-lJFli. ference between yeast and the higher eukaryotes.The averageyeastgene is 1.4 kb long, and
of Sizes Showa WideDistribution 3.6 Genes
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very few are longer than 5 kb. The predominance of interrupted genesin high eukaryotes, however, means that the gene can be much larger than the unit that codesfor protein. Relatively few genesin flies or mammals are shorter than 2 kb, and many have lengths between 5 kb and 100 kb. The average human gene is 2 7 k b l o n g ( s e eF i g u r e5 . 1 1 ) . The switch from largely uninterrupted to largely interrupted genes occurs in the lower e u k a r y o t e s .I n f u n g i ( e x c e p t t h e y e a s t s ) ,t h e majority of genesare interrupted, but they have a relatively small number of exons (<6) and are fairly short (<5 kb). The switch to long genes occurs within the higher eukaryotes, and genes become significantly larger in the insects.With this increase in the length of the gene, the relationship between genome size and organism complexity is lost (seeFigure 4.5). As genome size increases,the tendency is for introns to become rather large, whereas exons remain quite small. i l.ij-iiii '" i ,rshows that the exons coding for stretches of protein tend to be fairly small. In higher eukaryotes,the averageexon codesfor -50 amino acids,and the general distribution fits well with the idea that geneshave evolved
< 0 . 5< 1 < 2 < 5 < 1 0 < 2 5 < 5 0 < 1 0 0 > 1 0 0 Sizeof gene(kb) Yeastgenesareshort,but genesin fliesand mammalshavea dispersed distributjonextendingto very [ o n gs i z e s .
Exons codingforproteins usuatly areshort.
CHAPTER 3 TheInterrupted Gene
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Sometimes two pathways operate simultaneously, with a certain proportion of the RNA being spliced in each way. Sometimes the pathways are alternatives that are expressedunder different conditions-one in one cell type and one in another cell type. So alternative (or differential) splicing can generate proteins with overlapping sequences from a single stretch of DNA. It is curious that the higher eukaryotic genome is extremely spacious and has large genes that are often quite dispersed,but at the same time it may make multiple products from an individual Iocus. Alternative splicing expands the number of proteins relative to the number of genes by -I5% in flies and worms, but has much bigger effects in man, for which -60'h of genes may have altemative modes of expression(seeSection 5.5, The Human Genome Has Fewer Genes Than Expected). About 80% of the alternative splicing events result in a change in the protein seouence.
HowDidInterrupted Evolve? Genes o Themajorevotutionary genes question is whether by intronsor interrupted originated assequences uninterrupted. whether theywereoriginatty r Mostprotein-coding in genesprobably originated genesthat form.butinterrupted an interrupted been codefor RNAmayhaveoriginatly uninterruoted. . A special classof intronsis mobileandcaninsert itsetfinto genes. The highly interrupted structure of eukaryotic genes suggests a picture of the eukaryotic genome as a seaof introns (mostly but not exclusively unique in sequence), in which islands of exons (sometimes very short) are strung out in individual archipelagoesthat represent genes. What was the original form of genes that todav are interrupted?
Evo[ve? 4 7 Genes 3.8 HowDidInterrupted
. The "introns early" model proposesthat introns have always been an integral part of the gene. Genes originated as interrupted structures, and those without introns have lost them in the course of evolution. . The "introns late" model proposesthat the ancestralprotein-coding units cons i s t e d o f u n i n t e r r u p t e d s e q u e n c e so f DNA. Introns were subsequently inserted into them. A test of the models is to ask whether the difference between eukaryotic and prokaryotic genes can be accounted for by the acquisition of introns in the eukaryotes or by the loss of introns from the prokaryotes. The "introns early" model suggeststhat the mosaic structure of genes is a remnant of an ancient approach to the reconstruction of genes to make novel proteins. Supposethat an early cell had a number of separateprotein-coding sequences:One aspectof its evolution is likely to have been the reorganization and juxtaposition of different polypeptide units to build up new protelns. If the protein-coding unit must be a continuous serieso{ codons, every such reconstruction would require a precise recombination of DNA to place the two protein-coding units in
register, end to end in the same reading frame. Furthermore, if this combination is not successful, the cell has been damaged because it has lost the original protein-coding units. If an approximate recombination of DNA could place the two protein-coding units within the same transcription unit, splicing patterns could be tried out at the level of RNA to combine the two proteins into a single polypeptide chain. If these combinations are not successful, the original protein-coding units remain available for further trials. Such an approach essentially allows the cell to try out controlled deletions in RNA without suffering the damaging instability that could occur from applying this procedure to DNA. This argument is supported by the fact that we can find related exons in different genes,as though the gene had been assembledby mixing and matching exons (see Section 3.9, Some Exons Can Be Equated with Protein Functions). fl5$iJF*-:.iS illustrates the outcome when a random sequence that includes an exon is translocated to a new position in the genome. Exons are very small relative to introns, so it is Iikely that the exon will find itself within an intron. Only the sequencesat the exon-intron junctions are required for splicing, and as a result the exon is likely to be flanked by functional 3'
Intronsaremuchlongerthanexons 5' splicejunction
+rntron
i:I*{J#il":t..1*An exonsurroundedby ftankingsequences that is translocated into an intron may be sp[icedinto the RNAproduct.
CHAPTER 3 TheInterrupted Gene
and 5' splice junctions, respectively. Splicing junctions are recognized in pairs; thus the 5' splicing junction of the original intron is likely to interact with the 3' splicing junction introduced by the new exon, instead of with its original partner. Similarly, the 5' splicing junction of the new exon will interact with the 3'splicing junction of the original intron. The result is to insert the new exon into the RNA product between the original two exons. As long as the new exon is in the same coding frame as the original exons, a new protein sequence will be produced. This type of event could have been responsible for generating new combinations of exons during evolution. Note that the principle of this type of event is mimicked by the technique of exon trapping that is used to screen for functional exons (seeFigure 4.1 I ). Alternative forms of genes for rRNA and IRNA are sometimes found, both with and without introns. In the caseof the tRNAs, for which all the molecules conform to the same general structure, it seems unlikely that evolution brought together the two regions of the gene. After all, the different regions are involved in the base pairing that gives significance to the structure. So here it must be that the introns were inserted into continuous genes. Organelle genomes provide some striking connections between the prokaryotic and eukaryotic worlds. There are many general similarities between mitochondria or chloroplasts and bacteria, and becauseof this it seemslikely that the organelles originated by an endosymbiosisin which an early bacterial prototype was inserted into eukaryotic cytoplasm. Yet in contrast with the resemblanceswith bacteria-for example, as seen in protein or RNA synthesissome organelle genespossessintrons and therefore resembleeukaryotic nuclear genes. Introns are found in several chloroplast genes, including some that have homologies with genes of E. coli. This suggeststhat the endosymbiotic event occurred before introns were lost from the prokaryotic line. If a suitable gene can be found, it may be possible to trace gene lineageback to the period when endosymbiosis occurred. The mitochondrial genome presents a particularly striking case. The genes of yeast and mammalian mitochondria code for virtually identical mitochondrial proteins in spite of a considerable difference in gene organization. Vertebrate mitochondrial genomes are very small, with an extremely compact organization
of continuous genes,whereas yeast mitochondrial genomes are larger and have some complex interrupted genes. Which is the ancestral form? The yeast mitochondrial introns (and certain other introns) can have the property of mobility-they are self-contained sequences that can splice out of the RNA and insert DNA copies elsewhere-which suggeststhat they may have arisen by insertions into the genome (see Section 27 .5, Some Group I Introns Code for Endonucleases That Sponsor Mobility and Section 27.6, Group II Introns May Code for Multifunction Proteins).
CanBe SomeExons
withProtein Equated Functions r Factssuggesting that exonswerethe buitding btocksof evotutionandthe first geneswere interrupted are: r Genestructure genesin between is conserved verydistantspecies. . Manyexonscanbeequated with codingfor proteinsequences that haveparticutar functions. . Retated exonsarefoundin differentgenes. If current proteins evolved by combining ancestral proteins that were originally separate, the accretion of units is likely to have occurred sequentially over some period of time, with one exon added at a time. Can the different functions from which these genes were pieced together be seen in their present structures? In other words, can we equate particular functions of current proteins with individual exons? In some cases,there is a clear relationship between the structures of the gene and the prois provided by tein. The example par excellence the immunoglobulin proteins, which are coded by genes in which every exon corresponds exactly with a known functional domain of the protein. it*LlRf 3.?* comparesthe structure of an immunoglobulin with its gene. An immunoglobulin is a tetramer of two light chains and two heavy chains, which aggregate to generate a protein with several distinct domains. Light chains and heavy chains differ in structure, and there are several qpes of heavy chain. Each type of chain is expressed from a gene that has a series of exons corresponding with the structural domains of the protein.
with ProteinFunctions 49 CanBeEquated 3.9 SomeExons
L exon
V-J exon
C exon
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C1 exon
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C2 exon
C3 exon
ffGiJ&t3.19 Immunoglobulin lightchainsandheavychainsarecodedby geneswhosestructures (in theirexpressed forms)correspond withthedistinctdomains in the protein.Eachproteindomain corresponds to an exon;intronsarenumbered 1 to 5.
In many instances,some of the exons of a gene can be identified with particular functions. In secretory proteins, the first exon, coding for the N-terminal region of the polypeptide, often specifiesthe signal sequence involved in membrane secretion. An example is insulin. The view that exons are the functional building blocks of genes is supported by cases in which two genes may have some exons that are related to one another, whereas other exons are found only in one of the genes. f t S L ! f t 13 . t r * s u m m a r i z e s t h e r e l a t i o n s h i p between the receptor for human LDL (plasma low density lipoprotein) and other proteins. In the center of the LDL receptor gene is a series of exons related to the exons of the gene for the precursor for EGF (epidermal growth factor). In the N-terminal part of the protein, a s e r i e so f e x o n s c o d e s f o r a s e q u e n c er e l a t e d to the blood protein complement factor C9. So the LDL receptor gene was created by assembling modulesfor its various functions. Thesemodules also are used in different combinations in other proteins. Exons tend to be fairly smali (see Figure 4.ll), around the size of the smallest polypeptide that can assume a stable folded structure (-20 to 40 residues).Perhapsproteins were originally assembled from rather small modules. Each module need not necessarily correspond to a current function; several modules could have combined to qenerate a func-
CHAPTER 3 TheInterruptedGene
LDL receptor Cg complement EGF precursor homology homology
EGFprecursor FIGUft{ geneconsjsts }.*S TheLDLreceptor of 18exons. someof whichareretatedto EGFprecursor exonsand someof whicharerelatedto the C9btoodcomDtemenr gene.Triangles markthe positions of introns.
tion. The number of exons in a gene tends to increase with the length of its protein, which is consistent with the view that proteins acquire multiple functions by successivelyadding appropriate modules. This idea might explain another feature of protein structure. It appears that the sites represented at exon-intron boundaries often are located at the surface of a protein. As modules are added to a protein, the connections-at least of the most recently added modules-could tend to lie at the surface.
TheMembers of a Gene Family Havea Common 0rganization o A common featurein a setof genesis assumed to identifya property that preceded theirseparation in evolution. . Attgtobingeneshavea common formof organization withthreeexonsandtwointrons, suggesting that theyaredescended froma single gene. ancestraI
Many genesin a higher eukaryotic genome are related to others in the same genome. A gene family can be defined as a group of genes that code for related or identical proteins. A family originates when a gene is duplicated. Initially the two copies are identical, but then they diverge as mutations accumulatein them. Further duplications and divergence extend the family further. The globin genesare an example of a family that can be divided into two subfamilies (u globin and B globin), but all its members have the same basic structure and function. The concept can be extended further when we find genes that are more distantly related, but still can be recognized as having common ancestry;in this case,a group of gene families can be considered to make up a superfamily. A fascinatingcaseof evolutionary conservation is presentedby the u and B globins and two other proteins related to them. Myoglobin is a monomeric oxygen-binding protein of anim a l s w h o s e a m i n o a c i d s e q u e n c es u g g e s t sa common (though ancient) origin with the globin subunits. Leghemoglobinsare oxygen-binding proteins presentin the legume classof plants; like myoglobin, they are monomeric. They, too, share a common origin with the other hemebinding proteins. Together,the globins,myoglobin. and leghemoglobin constitute the globin superfamily-a set of gene families all descended from some (distant) common ancestor. Both cr- and B-globin genes have three e x o n s ( s e eF i g u r e 3 . 7 ) . T h e t w o i n t r o n s a r e located at constant positions relative to the coding sequence.The central exon representsthe heme-binding domain of the globin chain. Myoglobin is representedby a singlegene in the human genome whose structure is essentially the same as that of the globin genes.The threeexon structure therefore predatesthe evolution of separatemyoglobin and globin functions.
of globingenescorrel rilr;!:i:.'.1;, Theexonstructure hasan sponds withproteinfunction,butleghemogtobin extraintronin the centra[domain.
Leghemoglobin genescontain three introns, the first and last of which occur at points in the coding sequence that are homologous to the locations of the two introns in the globin genes. This remarkable similarity suggestsan exceedingly ancient origin for the heme-binding proteins in the form of a split gene, as illustrated in .::i,i-l:ir :,,: : .RoshanKeab02I-66950639 The central intron of leghemoglobin separates two exons that together code for the sequence corresponding to the single central exon in globin. Could the central exon of the globin gene have been derived by a fusion of two central exons in the ancestral gene? Or is the single central exon the ancestral form-in this case,an intron that must have been inserted into it at the start of plant evolution? Casesin which homologous genesdiffer in structure may provide information about their evolution. An example is insulin. Mammals and birds have only one gene for insulin, except for rodents,which have two. llliil'll li.ll;l illustrates the structuresof these genes. The principle we use in comparing the organization of related genesin different species is that a commonfeature identifiesa structurethat predatedtheevolwtionaryseparationofthe two speaes. In chickens, the single insulin gene has two introns; one of the two rat geneshas the same structure. The common structure implies that the ancestralinsulin gene had two introns. However, the second rat gene has only one intron. It must have evolved by a gene duplication in rodents that was followed by the precise removal of one intron from one of the copies. The organization of some genes shows e x t e n s i v e d i s c r e p a n c i e sb e t w e e n s p e c i e s .I n these cases,there must have been extensive removal or insertion of introns during evolution.
Organization 5 7 Havea Common of a GeneFamity 3.10TheMembers
A well-characterized caseis representedby the actin genes. The typical actin gene has a nontranslated leader of <100 bases,a coding r e g i o n o f - 1 2 0 0 b a s e s ,a n d a t r a i l e r o f - 2 0 0 bases. Most actin genes are interrupted; the positions of the introns can be aligned with regard to the coding sequence (except for a single intron sometimesfound in the leader). Fi*URf 3.t3 shows that almost every actin gene is different in its pattern of interruptions. Taking all the genes together, introns occur at l9 different sites.However, no individual gene has more than six introns; some genes have only one intron, and one is uninterrupted altogether. How did this situation arise? If we suppose that the primordial actin gene was interrupted, and that all current actin genesare related to it by loss of introns, different introns
Commoninsulingene (chickenand rat) Leader Coding exon Intron exon
Intron
Coding exon
Codingexon
Secondinsulingene in rat ili*iiftF :i.ii Theratinsutingenewith one intron evolved by lossof an intron from an ancestorwith two introns.
S pombe S cerevtsrae Acanthamoeba Thermomyces C elegans D melanogaster46 A1 A4 A2 S e au r c h i n C J Chickmuscle R a tm u s c l e Rat cytoplasmic H u m a ns m o o t hm u s c l e Humancardiacmuscle Soybean
:..
have been lost in each evolutionary branch. Probably some introns have been lost entirely, so the primordial gene could well have had 20 introns or more. The alternative is to suppose that a process of intron insertion continued independently in the different lines of evolution. The relationships between the intron locations found in different speciesmay be used ultimately to construct a tree for the evolution of the gene. The relationship between exons and protein domains is somewhat erratic. In some cases there is a clear l: I relationship; in others no pattern can be discerned. One possibility is that the removal of introns has fused the adjacent exons. This means that the intron must have been precisely removed, without changing the integrity of the coding region. An alternative is that some introns arose by insertion into a coherent domain. Together with the variations that we see in exon placement in casessuch as the actin genes,this arguesthat intron positions can be adjusted in the course of evolution. The equation of at least some exons with protein domains, and the appearanceof related exons in different proteins, leaves no doubt that the duplication and juxtaposition of exons has played an important role in evolution. It is possible that the number of ancestral exons-from which all proteins have been derived by duplication, variation, and recombination-could oe relatively small (a few thousand or tens of thousands). By taking exons as the building blocks of evolution, this view implicitly accepts the introns early model for the origin of genes coding for proteins.
.
Ft*ij;e$.t.*.?.Actingenesvarywidetyin theirorganization. Thesitesof intronsareindicated in purpl.e.
CHAPTER 3 The InterruptedGene
Is A[tGenetic Information in DNA? Contained o Thedefinitionof the genehasreversed from"one gene: oneprotein"to "oneprotein: onegene." o Positional is atsoimportant in information devetopment. The concept of the gene has evolved significantly in the past several years. The question of what's in a name is especiallyappropriate for the gene. We can no longer say that a gene is a sequence of DNA that continuously and uniquely codes for a particular protein. In situations in which a stretch of DNA is responsible for production of one particular protein, current usage regards the entire sequence of DNA-from the first point represented in the messengerRNA to the last point corresponding to its end-as cornprising the "gene," exons, introns, and all. When the sequencesrepresenting proteins overlap or have alternative forms of expression, we may reverse the usual description of the gene. Instead of saying "one gene--onepolypeptide," we may describe the relationship as "one polypeptide-one gene." So we regard the sequenceactually responsiblefor production of the polypeptide (including introns as well as exons) as constituting the gene while recognizing that from the perspective of another protein, part of this same sequence also belongs to i/s gene. This allows the use of descriptionssuch as "overlapping" or "alternative" genes. We can now see how far we have come from the original one gene : one enzyme hypothesis. Up to that time, the driving question was the nature of the gene. Once it was discovered that genes represent proteins, the paradigm became fixed in the form of the concept that every genetic unit functions through the synthesis of a particular protein. This view remains the central paradigm of molecular biology: A sequence of DNA functions either by directly coding for a particular protein or by being necessaryfor the use of an adjacent segment that actually codes for the protein. How far does this paradigm take us beyond explaining the basic relationship between genesand proteins? The development of multicellular organisms rests on the use of different genes to generate the different cell phenotypes of each tissue. The expression of genesis determined by a regulatory network that takes the form of a cas-
cade. Expression of the first set of genes at the start of embryonic development leadsto expression of the genes involved in the next stage of development, which in turn leads to a further stage,and so on until all the tissuesof the adult are functioning. The molecular nature of this regulatory network is largely unknown, but we assume that it consists of genes that code for products (probably protein, perhaps sometimes RNA) that act on other genes. Although such a series of interactions is almost certainly the means by which the developmental program is executed, we can ask whether it is entirely sufficient. One specific question concerns the nature and role of posiWe know that all parts tional information. of a fertilized egg are not equal; one of the features responsible for development of different tissue parts from different regions of the egg is Iocation of information (presumably specific macromolecules) within the cell. We do not know how these Particular regions are formed. We can, however, speculate that the existenceof positional information in the egg Ieadsto the differential expression of genesin the cells subsequently formed in these regions. This leads to the development of the adult organism, which in turn leads to the development of an egg with the appropriate positional information. This possibility prompts us to ask whether some information needed for development of the organism is contained in a form that we cannot directly attribute to a sequence of DNA (although the expression of particular sequences may be needed to perpetuate the positional information). Put in a more general way, we might ask the following: When we read out the entire sequence of DNA comprising the genome of some organism and interpret it in terms of proteins and regulatory regions, could we in principle construct an organism (or even a single living cell) by controlled expression of the proper genes?
Summary All types of eukaryotic genomes contain interrupted genes. The proportion of interrupted genesis low in yeastsand increasesin the lower eukaryotes; few genes are uninterrupted in higher eukaryotes. Introns are found in all classesof eukaryotic genes. The structure of the interrupted gene is the same in all tissues: exons are joined
3.12 Summary 5 3
together in RNA in the same order as their organization in DNA, and the introns usually have no coding function. Introns are removed from RNAby splicing.Some genesare expressed by alternative splicing patterns, in which a particular sequenceis removed as an intron in some situations,but retained as an exon in others. Positions of introns often are conserved when the organization of homologous genes is compared between species.Intron sequences vary-and may even be unrelated-although exon sequencesremain well related. The conservation of exons can be used to isolate related g e n e si n d i f f e r e n rs p e c i e s . The size of a gene is determined primarily by the lengths of its introns. Introns become larger early in the higher eukaryotes, when gene sizestherefore increase significantly. The range of gene sizes in mammals is generally from I to 100 kb, but it is possiblero have even larger genes: the longest known case is dystrophin, at 2000 kb. Some genesshare only some of their exons with other genes. suggesting that they have been assembledby addition of exons representing individual modules of the protein. Such modules may have been incorporated into a variety of different proteins. The idea that genes h a v e b e e n a s s e m b l e db y a c c r e t i o n o f e x o n s implies that introns were present in genes of primitive organisms. Some of the relationships between homologous genescan be explained by Ioss of introns from the primordial genes, with different introns being lost in different lines of descent.
Restriction Endonucleases Area KeyToot i n M a p p i nDgN A Reviews Nathans, D. and Smith, H. O. (1975). Resrriction endonucleasesin the analysis and restructuring of DNA molecules. Annu Rev Biochem-44, 27)-293. Wu, R. (1978). DNA sequenceanalysis.Annu. Rev Biochem47,607-734. Resea rch Danna, I(.J., Sack,G.H., andNathans, D. (1973\. Studies of SV40 DNA VII A cleavage map of the SV40 genome. J. Mol. Biol.78,363476.
0rganization of Interrupted Genes May BeConserved R e s erac h Berget, S.M., Moore, C., and Sharp, P. (1977). Spliced segments at the 5'terminus of adenovirus 2 late mRNA. Proc Natl Acad. Sci USA 74,3t7t-3175. Chow, L. T., Gelinas,R. E., Broker, T. R., and Roberts,R.J. (1977). An amazing sequence arrangement at the 5'ends of adenovirus 2 nRNA. Cell 12, l-8. Glover, D. M. and Hogness,D. S. (1977). A novel arrangement of the 8S and 28S sequencesin a repeating unit of D- melanogastar rDNA. Cell lO, r67-t7 6. Jeffreys,A.J. and Flavell, R.A. (1977). The rabbit B-globin gene contains a large insert in the coding sequence.Cell 12,1097-1 108. Wenskink, P. et al. (1974) . A sysrem for mapping DNA sequencesin the chromosomes of D. melanogaster. Cell ), )15-325.
SomeExons CanBeEquated with Protein Functions
References !!l
AnInterrupted Gene Consists of Exons andIntrons
Reviews Breathnach, R. and Chambon, P. (198I). Organization and expression of eukaryotic split genes coding for proteins. Annu Rev.Biochem.50, 349-)83. Faustino,N. A. and Cooper, T A. (2003). premRNA splicing and human disease.GenesDev. 17, 4t9437.
CHAPTER 3 TheInterrupted Gene
Review Blake, C. C. (1985). Exons and the evolution of proteins. Int. Rev.Cytol.93, 149-18i.
TheContentof the Genome C H A P T EO RU T L I N E Introduction CanBe Mappedby Linkage,Restriction Genomes C l e a v a g eo,r D N AS e q u e n c e Variation ShowExtensive IndividuatGenomes . Potymorphism [eve[ maybe detected at the phenotypic whena sequence affectsgenefunction,at the restriction target fragment[eve[whenit affectsa restrictionenzyme of DNA. [eve[by directanatysis site,andat the sequence . Theatletes potymorphism at the of a geneshowextensive changes do not affect levet,but manysequence sequence function. R F L Pasn d S N P sC a nB e U s e df o r G e n e t i cM a p p i n g . RFLPs andSNPs canbethe basisfor linkagemapsandare ps. relationshi ng parent-progeny usefuIfor estabtishi @
w h y A r eG e n o m eSs o L a r g e ? e Thereis no goodcorrelation genome sizeand between geneticcomptexity. . Thereis an increase genome sizerequired in the minimum of increasing comptexity. to makeorganisms . Therearewidevariations sizesof organisms in the genome withinmanyphyta. and Eukaryotic Genomes ContainBoth Nonrepetitive RepetitiveDNASequences . Thekinetics hasbeen aftera genome of DNAreassociation of repesequences bytheirfrequency denatured distinguish t i t i o ni n t h eg e n o m e . . Genes in nonrepetitive aregeneratty codedby sequences DNA. . Larger genomes withina phylumdo not containmore genes,but havelargeamounts DNA. of repetitive o A largepartof repetitive DNAmaybe madeup of transDOSOnS.
of Exons Genes CanBeIsotatedbythe Conservation . Conservation foridentifyas the basis can be used of exons whose fragments regions byidentifying ingcoding organisms. in muttipte sequences arepresent
of Genome0rganizationHetpsto TheConservation Identify Genes . Algorithms genesarenot perfectandmany for identifying mustbe madeto theinitiatdataset. corrections . Pseudogenes fromactivegenes. mustbe distinguished . Syntenic mouse and between extensive are retationships andmostactivegenesarein a syntenic humangenomes, reglon. 0rganel[esHaveDNA o Mitochondria that show havegenomes andchtoroptasts theyarematernatly Typicatty i nheritance. non-Mendelian i nherited. in r 0rgane[te segregation somatic genomes mayundergo D[ants. are r Comparisons that humans DNAsuggest of mitochondriaL years froma singtefema[ewhotived200,000 descended agoin Africa. Are CircularDNAsThat Codefor 0rgane[[eGenomes 0rganelteProteins o Organelte circutar (butnot atways) genomes areusualty of DNA. motecutes . 0rganette genomes codefor some,but not a[[,of the proteinsfoundin the organetle. MitochondriaIDNA0rganizationIs Variab[e and o Animalcetlmitochondriat compact DNAis extremely and22tRNAs. 2 rRNAs, codesfor 13 proteins, typicatLy r Yeastmitochondrial DNAis 5x longerthananimalcetl of longintrons' of the presence mtDNA because s nd C o d e sf o r M a n yP r o t e i n a s te n o m e T h eC h t o r o p t a G RNAs to . Chtoroptast genomes varyin size,but arelargeenough andtRNAs. aswetlasthe rRNAs codefor 50to 100proteins MitochondriaEvotvedby Endosymbiosis
Summary
55
@
Introduction
The key question about the genome is how many genesit contains. We can think about the total number of genesat four levels, which correspond to successivestagesin gene expression: . The genome is the complete set of genes of an organism. Ultimately it is defined by the complete DNA sequence,although as a practical matter it may not be possible to identify every gene unequivocally solely on the basisof sequence. . The transcriptome is the complete set of genesexpressedunder particular conditions. It is defined in terms of the set of RNA molecules that is present and can refer to a single cell type, or to any more complex assembly of cells, up to the complete organism. Because some genes generate multiple mRNAs, the transcriptome is likely to be larger than the number of genes defined directly in the genome. The transcriptome includes noncoding RNAs as well as mRNAs. . The proteome is the complete set of proteins. It should correspond to the mRNAs in the transcriptome, although there can be differencesof detail reflecting changes in the relative abundance or stabilitiesof mRNAs and proteins. It can be used to refer to the set of proteins codedby the whole genome or produced in any particular cell or tissue. . Proteins may function independently or as part of multiprotein assemblies.If we could identify all protein-prorein inreractions, we could define the total number of independent assembliesof proteins. The number of genes in the genome can be identified directly by defining open reading frames. Large-scalemapping of this nature is complicated by the fact that interrupted genes may consist of many separated open reading frames. We do not necessarilyhave information about the functions of the protein products-or indeed proof that they are expressed at all-so this approach is restricted to defining Ihe potentialof the genome. However, a strong presumption exists that any conserved open reading frame is likely to be expressed. Another approach is to define the number of genes directly in terms of the transcriptome (by directly identifying all the mRNAs) or proteome (by directly identifying all the proteins). This gives an assurancethat we are dealing with bonafide genesthat are expressedunder known circumstances.It allows us to ask how many
56
C H A P T E4RT h eC o n t e not f t h e G e n o m e
genes are expressedin a particular tissue or cell type, what variation exists in the relative levels of expression, and how many of the genes expressed in one particular cell are unique to that cell or are also expressedelsewhere. Concerning the types of genes,we may ask whether a particular gene is essential:what happens to a null mutant? If a null mutation is lethal, or the organism has a visible defect, we may conclude that the gene is essential or at Ieast conveys a selective advantage. But some genes can be deleted without apparent effect on the phenotype. Are these genes really dispensable,or does a selectivedisadvantageresult from the absenceof the gene, perhaps in other circumstances, or over longer periods of time?
Genomes CanBeMapped by Linkage, Restriction Cleavage, or DNA Sequence Defining the contents of a genome essentially means making a map. We can think about mapping genes and genomes at several levels of resolution: . A genetic (or linkage) map identifies the distancebetween mutations in terms of recombination frequencies. It is limited by its reliance on the occurrence of mutations that affect the phenotype. Becauserecombination frequencies can be distorted relative ro the physical distance between sites, it does not accurately representphysical distancesalong the genetic material. . A linkage map also can be constructed by measuring recombination between sites in genomic DNA. These sites have sequence variations that generate differencesin the susceptibility to cleavage by certain (restriction) enzymes. Because such variations are common, such a map can be prepared for any organism irrespective of the occurrence of mutants. It has the same disadvantage as any linkage map in that the relative distancesare based on recombination. . A restriction map is constructed by cleaving DNA into fragments with restriction enzymes and measuring the distancesbetween the sites of cleavage. This representsdistancesin terms of the length of DNA, so it provides a physical map of the genetic material. A restric-
tion map does not intrinsically identify sites of genetic interest. For it to be related to the genetic map, mutations have to be characterized in terms of their effects upon the restriction sites. Large changes in the genome can be recognized because they affect the sizes or numbers of restriction fragments. Point mutations are more difficult to detect. . The ultimate map is to determine the sequence of the DNA. From the sequence,we can identify genes and the distancesbetween them. By analyzing the protein-coding potential of a sequence of the DNA, we can deduce whether it represents a protein. The basic assumption here is that natural selection prevents the accumulation of damaging mutations in sequencesthat code for proteins. Reversing the argument, we may assume that an intact coding sequenceis likely to be used to g e n e r a t ea p r o t e i n . By comparing the sequence of a wild-type DNA with that of a mutant allele, we can determine the nature of a mutation and its exact site of occurrence. This defines the relationship between the genetic map (based entirely on sites of mutation) and the physical map (based on, or even comprising, the sequence of DNA). Similar techniques are used to identify and sequence genes and to map the genome, although there is of course a difference of scale. In each case,the principle is to obtain a series of overlapping fragments of DNA that can be connected into a continuous map. The crucial feature is that each segment is related to the next segment on the map by characterizing the overlap between them. so that we can be sure no segments are missing. This principle is applied both at the level of ordering large fragments into a map and in connecting the sequences that make up the fragments.
@
Individual Genomes Show Extensive Variation
. Potymorphism maybe detected at the phenotypic affectsgenefunction,at levelwhena sequence the restrictjonfragment[eve[whenit affectsa restrictionenzyme targetsite,andat the of DNA. sequence levetby directanatysis . Theatletes of a geneshowextensive potymorphism at the sequence [eve[,but many do not affectfunctjon. sequence changes
The original Mendelian view of the genome classifiedallelesas either wild-type or mutant. Subsequently we recognized the existence of multiple alleles, each with a different effect on the phenotype. In some casesit may not even be appropriate to define any one allele as "wildtype." The coexistenceof multiple allelesat a locus Any site at is called genetic polymorphism. which multiple alleles exist as stable components of the population is by definition polymorphic. An allele is usually defined as polymorphic if it is present at a frequency of >l % in the population. What is the basis for the polymorphism among the mutant alleles?They possessdifferent mutations that alter the protein function, thus producing changes in phenotype. If we compare the restriction maps or the DNA sequencesof these allelesthey, too, will be polymorphic in the sensethat each map or sequence will be different from the others. Although not evident from the phenotype, the wild type may itself be polymorphic. Multiple versions of the wild-type allele may be distinguished by differences in sequencethat do not affect their function, and which therefore do not produce phenotypic variants. A population may have extensive polymorphism at the level of genotype. Many different sequence variants may exist at a given locus; some of them are evident becausethey affect the phenotype, but others are hidden becausethey have no visible effect. So there may be a continuum of changesat a locus, including those that change DNA sequence but do not change protein sequence, those that change protein sequence without changing function, those that create proteins with different activities, and those that create mutant proteins that are nonfunctional. A change in a single nucleotide when alleles are compared is called a single nucleotide (SNP). One occurs every polymorphism -I130 basesin the human genome. Definedby their SNPs,every human being is unique. SNPs can be detectedby various means, ranging from direct comparisons of sequenceto mass spectroscopy or biochemical methods that produce differences based on sequence variations in a defined region. One aim of genetic mapping is to obtain a catalog of common variants. The observed frequency of SNPsper genome predicts that, over the human population as a whole (taking the sum of all human genomes of all living individuals). there should be >10 million SNPsthat
Variation 5 7 ShowExtensive 4.3 IndividualGenomes
DNA has 3 targetsites In regron
Mutationeliminates
1 targetsite
t tt Cleavagegenerates I 2 internalfragments|
CleavagegeneratesI 1 internalfragment I
f ragmentA fragmentB
l_l
V
fragmentC
Electrophoresis
V
FragmentsA+ B combined= C
i-:**t{ +.1 A pointmutation that affectsa restriction siteis detected bv a difference in restriction fraqments.
c
A
B
trl
The restriction map is independent of gene function; as a result, a polymorphism at this level can be detected irrespectiveof whether the sequence changezffectsthephenztype.Probably very few of the restriction site polymorphisms in a genome actually affect the phenotype. Most involve sequence changes that have no effect on the production of proteins (for example, because they lie between genes). A difference in restriction maps between two individuals is called a restriction fragment length polymorphism (RFLP). Basically, an RFLP is an SNP that is located in the target site for a restriction enzyme. It can be used as a genetic marker in exactly the same way as any other marker. Instead of examining some feature of the phenotype, we directly assessthe genotype, as revealed by the restriction map. f g#i.jfigri.t shows a pedigree of a restriction polymorphism followed through three generations. It displays Mendelian segregation at the level of DNA marker fragments.
Parents 3 are heterozygous, 1 is homozygousfor C
RFLPs andSNPs CanBe Usedfor Genetic Mapping
'rFr::
F2 inherit
e RFLPs andSNPs canbethe basisfor [inkage maps parent-progeny andareusefulfor estabtishing relationships.
AlleleB AlleleC AlleleD
It*t-;ft9+.i Restriction sitepol.ymorphisms areinherited according to Mendetian rules.Fouratle[es for a restriction markerarefoundin al.l. possibl.e pairwise photocourcombinations andsegregate independentty at eachgeneration. tesyof RayWhite,Ernest GatLo C[inicandResearch Center, Universitv of CaLjfornia,SanFrancisco.
occur at a frequency of >Ioh. Already >l million have been identified. Some polymorphisms in the genome can be detected by comparing the restriction maps of different individuals. The criterion is a change in the pattern of fragments produced by cleavagewith a restriction enzyme. f ifi*&[ +.1shows that when a target site is present in the genome of one individual and absent from another, the extra cleavage in the first genome will gener-
n:"ril"'#tril'i: ;H.;:#*y s8
CHAPTER 4 TheContent of the Genome
Recombination frequency can be measured between a restriction marker and a visible phenotlpic marker, as illustrated in f,.:*LtRil 4.i. Thus a genetic map can include both genotypic and phenotypic markers. Restriction markers are not restricted to those genome changes that affect the phenotype; as a result, they provide the basis for an extremely powerful technique for identifying genetic loci at the molecular level. A typical problem concerns a mutation with known effects on the phenotype, where the relevant genetic locus can be placed on a genetic map, but for which we have no knowledge about the corresponding gene or protein. Many damaging or fatal human diseasesfall into this category. For example, cystic fibrosis shows Mendelian inheritance, but the molecular nature of the mutant function was unknown until it could be identified as a result of characterizing the gene. If restriction polymorphisms occur at random in the genome, some should occur near any particular target gene. We can identify such restriction markers by virtue of their tight link-
tothesingre
Geneticcross
II + Parentallypes
ot
ScreenDNA patientswith
te as c( control
I
I I
I
Band is same in patientand unafiected Unlinkedpolymorphismvaries in all samoles Band is commonto patients Band is commonto unaffectedpeople
characteriswitha phenotypic f J{ri.lfii.',.;-r marker is associated If a restriction for the phenearthe generesponsibte sitemustbe tocated tic, the restrictjon peopte into in heatthy the bandthatis common notype. Themutation changing gene. [inkedto the disease is veryclosely in patients the bandthat is common Restrictionmarkeris 30 map unitsfrom eve colormarker
polymorphism :ri.i:i,:!-ti: +,--lA restriction canbe usedasa geneticmarker to measure recombination froma distance phenotypic marker(suchaseyecotor).Thefiguresimp[ifiesthe situationby showingonlythe DNAbandscorres p o n d i ntgo t h ea t t e t o e f o n eg e n o mien a d i p t o i d .
age to the mutant phenotype. If we compare the restriction map of DNA from patients suffering from a diseasewith the DNA of healthy people, we may find that a particular restriction site is always present (or always absent) from the patients. A hypothetical exarrrpleis shown in ilili-iFir4..;. This situation correspondsto finding I00% Iinkage between the restriction marker and the phenot1pe. It would imply that the restriction marker lies so close to the mutant gene that it is never separated from it by recombination. The identification of such a marker has two important consequences: . It may offer a diagnostic procedure tor detecting the disease.Some of the human diseasesthat are geneticallywell characterized but ill defined in molecular terms cannot be easily diagnosed. If a restriction marker is reliably linked to the phenotype, then its presencecan be used to diagnosethe disease. . It may lead to isolation of the gene.The restriction marker must lie relativelv
near the gene on the genetic map if the two loci rarely or never recombine. "Relatively near" in genetic terms can be a substantial distance in terms of basepairs of DNA; nonetheless,it provides a starting point from which we can Proceed along the DNA to the gene itself. The frequent occurrence of SNPs in the human genome makes them useful for genetic mapping. From the I.5 x 106 SNPsthat have already been identified, there is on average an SNP every I to 2 kb. This should allow rapid localization of new diseasegenes by locating them between the nearest SNPs. On the same principle, RFLP mapping has been in use for some time. Once an RFLP has been assignedto a linkage group, it can be placed on the genetic map. RFLP mapping in man and mouse has led to the construction of linkage maps forboth genomes. Any unknown site can be tested for linkage to these sites, and by this means can be rapidly placed on the map. There are fewer RFLPsthan SNPs;thus the resolution of the RFLP map is in principle more limited. The frequency of polymorphism means that every individual has a unique constellation of SNPs or RFLPs.The particular combination of sites found in a specific region is called a haplotype, a genotype in miniature. Haplotype was originally introduced as a concept to describe the genetic constitution of the major
Mapping 5 9 CanBeUsedfor Genetic andSNPs 4.4 RFLPs
histocompatibility locus, a region specifyingproteins of importance in the immune system (see Chapter 23, Immune Diversity). The concept now has been extended to describethe particular combination of alleles or restriction sites (or any other genetic marker) present in some d e f i n e d a r e a o f t h e g e n o m e . U s i n g S N P s ,a detailed haplotype map of the human genome h a s b e e n m a d e ; t h i s e n a b l e sd i s e a s e - c a u s i n g genesto be mapped more easily. The existence of RFLPsprovides the basis for a technique to establishunequivocal parent-progeny relationships.In casesfor which parentage is in doubt, a comparison of the RFLP map in a suitablechromosome region between potential parents and child allows absolute assignmentof the relationship.The use of DNA restriction analysis to identify individuals has been called DNA fingerprinting. Analysis of especiallyvariable "minisatellite" sequencesis used in mapping the human genome (seeSection 6.14, MinisatellitesAre Useful for Genetic Mapping).
@
Floweringplants Birds Mammals Reptiles Amphibians Bony fish Cartilaginousfish Echinoderms Crustaceans Insects Mollusks Worms Molds Algae Fungi Gram(+)bacteria Gram(-)bacteria Mycoplasma
ill
I
tl
I T
l
IT I
I
106
107 108
10s
1010 1011
r+.* DNAcontentofthe hapLoid FI{.jilJR* genome increases with morphological complexity of lowereukaryotes, but varies extensivety withinsomegroups of highereukaryotes. Therangeof DNAvalues withineachgroupis indicated by the shaded area.
WhyAreGenomes SoLarge? 1010
o Thereis no goodcorrelation genome between size andgeneticcomplexity. . T h e r ies a ni n c r e a si ne t h e m i n i m u m g e n o msei z e required to makeorganisms of increasing complexity. . Therearewidevariations in the genome sizesof organism wsi t h i nm a n yp h y t a . The total amount of DNA in rhe (haploid) genome is a characteristic of each living species known as its C-value. There is enormous variation in the range of C-values,from
l0ll bp for some plants and amphibians. !'-i{-ii-JE=iI +.1 summarizes the range of C-values found in different evolutionary phyla. There is an increasein the minimum genome sizefound in each group as the complexity increases.As absolute amounts of DNA increasein the higher eukaryotes, though, we see some wide variations in the genome sizeswithin some phyla. Plotting tl;re minimum amount of DNA required for a member of each group suggests in F3*,iitt:;.* that an increasein genome sizeis required to make more complex prokaryotes and lower eukaryotes. Mycoplasma are the smallest prokaryotes and have genomes only -3x the size of a large
C H A P T E4RT h eC o n t e not f t h e G e n o m e
1oe c)
H 108
o) E o
3
107
106
*o"***"o*"n*si"t"."d":."SC Fg*i"f*f .t.{i The minimumgenomesjze found in each phy[um increases from prokaryotes to mammats.
bacteriophage.Bacteria start at -2 x I06 bp. Unicellular eukaryotes (whose lifestylesmay resemble the prokaryotic) get by with genomes rhat are small, too, although they are larger than those of the bacteria. Being eukaryolic per se
Phylum
Species
Algae Mycoplasma Bacterium Yeast Slime mold Nematode lnsect Bird Amphibian Mammal
Pyrenomassalina M. pneumoniae E. coli S. cerevisiae D. discoideum C. elegans D. melanogaster G. domesticus X. laevis H. sapiens
Genome(bp) 6.6 x 1.0 x 4.2 x 1.3 x 5.4 x 8.0 x 1.4 x 1.2 x 3.1 x 3.3 x
10s 100 106 107 102 107 108 10e 10e 10s
FlSiJgfr+"7Thegenome sizesof somecommon experim e n t aoI r q a n i s m s .
does not imply a vast increase in genome size; a yeast may have a genome size of -1.3 x 107 bp, which is only about twice the size of an average bacterial genome. A further twofold increase in genome size is adequate to support the slime mold Dictyosteliumdiscnideum, which is able to Iive in either unicellular or multicellular modes. Another increase in complexity is necessaryto produce the first fully multicellular organisms;the nematode worm Caenorhabditiseleganshas a DNA content of 8 x 107bp. We also can see the steady increase in genome size with complexity in the listing in rIG*Rg+.f of some of the most commonly anaIyzed organisms. It is necessaryto increase the genome size in order to make insects, birds or amphibians, and mammals. After this point, though, there is no good relationship between genome size and morphological complexity of the organism. We know that genes are much larger than the sequences needed to code for proteins, because exons (coding regions) may comprise only a small part of the total length of a gene. This explains why there is much more DNA than is needed to provide reading frames for all the proteins of the organism. Large parts of an interrupted gene may not be concerned with coding for protein. In addition, there also may be significant lengths of DNA between genes.So it is not possible to deduce from the overall size of the genome anything about the number of genes. The C-value paradox refers to the lack of correlation between genome size and genetic complexity. There are some extremely curious variations in relative genome size. The toad Xenopusand man have genomes of essentially
the same size.We assume, though, that man is more complex in terms of genetic developmentl In some phyla there are extremely large variations in DNA content between organisms that do not vary much in complexity (see Figure 4.5). (This is especiallymarked in insects, amphibians, and plants, but does not occur in birds, reptiles, and mammals, which all show little variation within the group, with an -2x range of genome sizes.)A cricket has a genome I lx the size of a fruit fly. In amphibians, the smallest genomes are
Genomes Eukaryotic Both Contain and Nonrepetitive DNA Repetitive Sequences r Thekinetics aftera genome of DNAreassociation bytheir sequences distinguish hasbeendenatured in the genome. frequency of repetition r Genes in codedby sequences aregeneratty nonrepetitive DNA. r Larger genomes withina phylumdo not contain of repetitive moregenes.but have[argeamounts DNA. r A largepartof repetitive DNAmaybe madeup of transposons. The general nature of the eukaryotic genome can be assessedby the kinetics of reassociation of denatured DNA. This technique was used extensively before large scaleDNA sequencing became possible. Reassociation kinetics identifies two general types of genomic sequences: . Nonrepetitive DNA consists of sequences that are unique: there is only one copy in a haploid genome. . Repetitive DNA describessequences that are present in more than one copy in each genome. Repetitive DNA often is divided into two general types: . Moderately repetitive DNA consists of relatively short sequences that are repeated typically l0-1000x in the
DNASequences 6 7 andRepetitive ContainBothNonrepetitive Genomes 4.6 Eukaryotic
A significant part of the moderately repetitive DNA consists of transposons, short 1010 sequencesof DNA (-l kb) that have the ability to move to new locations in the genome and/or to make additional copies of themselves (see 10e Chapter 21, Transposons, and Chapter 22, Retroviruses and Retroposons). In some higher o .N in8 eukaryotic genomes they may even occupy s37 q) more than half of the genome (seeSection 5.5, E The Human Genome Has Fewer Genes Than .9 10', g Expected). Transposons are sometimes viewed as fit106 ting the concept of selfish DNA, which is defined as sequencesthat propagate themselves within a genome without contributing to the development of the organism. Transposonsmay sponsor genome rearrangements, and these could confer selectiveadvantages.It is fair to say, though, that we do not really understand why selective forces do not act against transposons becoming such a large proportion of the Theproportions of different sequence components genome. Another term that is used to describe s .h ea b s o t u tceo n t e not f n o n v a r yi n e u k a r y o tgi ce n o m eT the apparent excessof DNA isjunk DNA, meanr e p e t i t i vD e N Ai n c r e a s ewsi t h g e n o m e s i z eb u t r e a c h eas ptateau at -2 x 10ebp. ing genomic sequenceswithout any apparent function. Of course, it is likely that there is a balance in the genome between the generation of new sequencesand the elimination of unwanted sequences,and some proportion of DNA that apparently lacks function may be in genome. The sequencesare dispersed the processof being eliminated. throughout the genome and are responThe length of the nonrepetitive DNA comsible for the high degree of secondary ponent tends to increase with overall genome structure formation in pre-nRNA, when size as we proceed up to a total genome size -3 x 10e (characteristicof mammals). Further (inverted) repeatsin the introns pair to form duplex regions. increasesin genome size, however, generally . Highly repetitive DNA consists of very reflect an increasein the amount and proporshort sequences(typically<100 bp) rhar tion of the repetitive components, so that it is are present many thousandsof times in rare for an organism to have a nonrepetitive the genome, often organizedas long DNA component >2 x l0e. The nonrepetitive tandem repeats(seeSection6. I l, SatelDNA content of genomes therefore accordsbetlite DNAS Often Lie in Heterochroter with our senseof the relative complexity of matin). Neither classrepresentsprotein. the organism. E. colihas4.2 x I06 bp, C.elegans The proportion of the genome occupiedby increasesan order of magnitude to 6.6 x nonrepetitive DNA varieswidely. :,r,,i:.r ,,,:107 bp, D. melanogaslerincreases further to -108 bp, and mammals increaseanother order summarizesthe genome organization of some of magnitude Io -2 x lOe bp. What type of DNA corresponds to proteinmost of the DNA is nonrepetitive; <204 falls coding genes? Reassociation kinetics typically into one or more moderately repetitive comshows that mRNA is derived from nonrepetitive ponents.In animal cells,up to half of the DNA DNA. The amount of nonrepetitive DNA is often is occupiedby moderately and highly therefore a better indication than the total DNA repetitive components. In plants and amphibof the coding potential. (However, more detailed ians, the moderately and highly repetitive comanalysis based on genomic sequencesshows ponents may account for up to 80% of the that many exons have related sequencesin other genome, so that the nonrepetitive DNA is e x o n s [ s e e S e c t i o n ] . 5 , E x o n S e q u e n c e sA r e reduced to a minority component. Conservedbut Introns Varyl. Such exons evolve
::ilT:"-'::-:;:T,Tr?iii,i#I:Til.";.l.l
62
C H A P T E4RT h eC o n t e n o t f the Genome
by a duplication to give copies that initially are identical, but which then diverge in sequence during evolution.)
Genes CanBeIsolatedbv the Conservation of Exons e Conservation of exonscanbe usedasthe basisfor identifuing codingregions by identifuing fragments whosesequences arepresent in muttipte organisms. Some major approaches to identifying genes are basedon the contrast between the conservation of exons and the variation of introns. In a region containing a gene whose function has been conservedamong a range of species,the sequencerepresentingthe protein should have two distinctive properties : . It must have an open reading frame, . and it is likely to have a related sequence in other species, Thesefeatures can be used to isolate genes. Suppose we know by genetic data that a particular genetic trait is located in a given chromosomal region. If we lack knowledge about the nature of the gene product, how are we to identify the gene in a region that may be, for example, >l Mb? A heroic approachthat has proved successful with some genesof medical importance is to screen relatively short fragments from the region for the two properties expectedof a conserved gene. First we seek to identify fragments that c r o s s - h y b r i d i z ew i t h t h e g e n o m e s o f o t h e r species.then we examine these fragments for open reading frames. The first criterion is applied by performing a zoo blot. We use short fragments from the region as (radioactive) probes to test for related DNA from a variety of speciesby Southern blotting. If we find hybridizing fragments in several speciesrelated to that of the probe-the probe is usually human-the probebecomesa candidate for an exon of the gene. The candidatesare sequenced,and if they contain open reading frames they are used to isolate surrounding genomic regions. If these appear to be part of an exon, we may then use them to identify the entire gene, to isolate the corresponding cDNA or mRNA, and ultimately to identify the protein. This approach is especiallyimportant when the target gene is spreadout becauseit has many
l : " i i i l & i.:, f i T h eg e n ei n v o l v e idn D u c h e n nmeu s c u l a r and mapping wastracked downbychromosome dystrophy "watking" canbe identito a regionin whichdeletions ofthe disease. fiedwiththe occurrence
large introns. This proved to be the case with Duchenne muscular dystrophy (DMD), a degenerative disorder of muscle that is X-linked and affectst in 3500 human male births. The steps in identifying the gene are summarized in :l:::iil
Linkage analysislocalizedthe DMD locus to chromosomal band Xp2l. Patientswith the diseaseoften have chromosomal rearrangements involving this band. By comparing the ability of X-linked DNA probes to hybridize with DNA from patients with normal DNA, cloned fragments were obtained that correspond to the region that was rearranged or deleted in patients' DNA. Once some DNA in the general vicinity of the target gene has been obtained, it is possible to "walk" along the chromosome until the gene is reached. A chromosomal walk was used to construct a restriction map of the region on either side of the probe, which covered a region
of Exons CanBeIsotatedbythe Conservation 4.7 Genes
i : . i : , ' r T h eD u c h e n nmeu s c u t adry s t r o p hgye n e wascharacterized by zoobtotting,cDNAhybridization. genomic hybridization, andidentification oftheprotein.
of >100 kb. Analysis of the DNA from a series of patients identified large deletions in this region that extended in either direction. The most telling deletion is one that is contained entirely within the region, becausethis delineatesa segmentthat must be important in gene function and indicatesthat the gene-or at least p a r t o f i t - l i e s i n t h i sr e g i o n . Having now come into the region of the gene, we need to identify its exons and introns. A zoo blot identified fragments rhat crosshybridize with the mouse X chromosome and with other mammalian DNAs. As summarized ,i,.t,r,these were scrutinizedfor open 11 ;rir,3l,ii:i
64
CHAPTER 4 TheContent of the Genome
reading frames and the sequencestypical of exon-intron junctions. Fragments that met these criteria were used as probes to identify homologous sequencesin a cDNA library prepared from muscle mRNA. The cDNA corresponding to the gene identifies an unusually large nRNA of approximately l4 kb. Hybridization back to the genome shows that the mRNA is represented in >60 exons. which are spread over -2000 kb of DNA. This makes DMD the longest gene identified. The gene codes for a protein of -500 kD called dystrophin, which is a component of muscle and is present in rather low amounts. All patients with the diseasehave deletionsat this locus and lack (or have defective) dystrophin. Muscle also has the distinction of having the largest known protein, titin, with almost 2 7 , 0 0 0 a m i n o a c i d s .I t s g e n e h a s t h e l a r g e s t number of exons (178) and the longest single exon in the human genome (17,000 bp). Another technique that allows genomic fragments to be scanned rapidly for the presence of exons is called exon trapping. : i l i ; r i ! . : , , : ; s h o w s t h a t i t s t a r t sw i t h a v e c t o r that contains a strong promoter and has a single intron between two exons. When this vector is transfected into cells, its transcription generateslarge amounts of an RNA containing the sequencesof the two exons. A restrictioncloning site lies within the intron and is used to insert genomic fragments from a region of interest. If a fragment does not contain an exon, there is no change in the splicing pattern, and the RNA contains only the same sequencesas the parental vector. If the genomic fragment contains an exon flanked by two partial intron sequences,though, the splicing siteson either side of this exon are recognized and the sequenceof the exon is inserted into the RNA between the two exons of the vector. This can be detected readily by reverse transcribing the cytoplasmic RNA into cDNA and using PCR to amplify the sequencesbetween the two exons of the vector. So the appearancein the amplified population of sequencesfrom the genomic fragment indicates that an exon has been trapped. Becauseintrons are usually large and exons are small in animal cells, there is a high probability that a random piece of genomic DNA will contain the required structure of an exon surrounded by partial introns. In fact, exon trapping may mimic the events that have occurred naturally during evolution of genes (see Section 3.8, How Did Interrupted GenesEvolve?).
The vectorcontainstwo exonsthat are splicedtogetherin the transcript promoter
5' splicejunction
3' splicejunction
InIron Transcription and I splicingto removeintron Y
Genomicfragment intron exon Insertgenomic I fragmentinto I V intron
intron
-
I
V F
exonl
I
exon |
intron Transcription and I splicing to removeintron Y
|intron
rexon
il*{Jli[ *.31 A speciaI spticing vectoris usedfor exontrapping. If an exon is present in thegenomic fragment, its sequence in thecytowit[berecovered plasmic RNA. If thegenomic fragment consists fromwithin sotety of sequences an intron,though,spticing doesnot occur,andthe mRNA to is not exported the cytoplasm.
TheConservation of Genome 0rganization Helpsto IdentifyGenes . Atgorithms genesarenot perfect for identifying andmanycorrections mustbe madeto theinitiat dataset. . Pseudogenes mustbe distinguished fromactive genes. . Syntenic relationships areextensive between mouse andhumangenomes, andmostactive genesarein a syntenic region. O n c e w e h a v e a s s e m b l e dt h e s e q u e n c e o f a genome. we still have to identify the genes within it. Coding sequencesrepresent a very small fraction. Exons can be identified as uninterrupted open reading frames flankedby appropriate sequences. What criteria need to be satisfied to identify an active gene from a series of exons?
i":g';t.t5{!. +..:: shows that an active gene should consist of a series of exons for which the first exon immediately follows a promoter, the internal exons are flanked by appropriate splicing junctions, the last exon is followed by 3'processing signals, and a single open reading frame starting with an initiation codon and ending with a termination codon can be deduced by joining the exons together. Internal exons can be identified as open reading frames flanked by splicingjunctions. In the simplest cases,the first and Iast exons contain the start and end of the coding region, respectively (aswell as the 5'and 3' untranslated regions).In more complex cases, the first or last exons may have only untranslated regions and may therefore be more difficult to identify. The algorithms that are used to connect exons are not completely effective when the genome is very large and the exons maybe separated by very large distances.For example, the initial analysis of the human genome mapped
Hetpsto IdentifyGenes 6 5 0rganization of Genome 4.8 TheConservation
r;.i.l Exons genes ftanked i'ii.l-li.:i ascodingsequences byapproof protein-coding areidentified priatesignals(with untranstated an regionsat bothends).Theseriesof exonsmustgenerate framewithappropriate initjatjonandtermination codons. openreading
170,000 exons into 12,000 genes.This is unlikely to be correct becauseit gives an average of 5.1 exons per gene,whereas the average of individual genes that have been fully characterizedis 10.2. Either we have missed many exons, or they should be connecteddifferently into a smaller number of qenes in the whole genome sequence. Even when the organization of a gene is correctly identified, there is the problem of distinguishing active genesfrom pseudogenes. Many pseudogenescan be recognizedby obvious defectsin the form of multiple mutations that createan inactive coding sequence.Pseudogenesthat have arisen more recently have not accumulatedso many mutations and thus may be more difficult to recognize. In an extreme example, the mouse has only one active Gapdh g e n e ( c o d i n g f o r g l y c e r a l d e h y d ep h o s p h a t e d e h y d r o g e n a s e ) ,b u t h a s - 4 0 0 p s e u d o g e n e s . Approximately 100 of these pseudogenesinitially appearedto be active in the mouse genome sequence,and individual examination was necessaryto exclude them from the list of active genes. Confidence that a gene is active can be increasedby comparing regions of the genomes of different species.There has been extensive overall reorganization of sequencesbetween the mouse and human genomes,as seenin the simple fact that there are23 chromosomesin the human haploid genome and 20 chromosomes in the mouse haploid genome. However, at the local level the order of genes is generally the
C H A P T E4RT h eC o n t e not f t h e G e n o m e
same: When pairs of human and mouse homologuesare compared,the geneslocated on either side also tend to be homologues. This relationship is called synteny. ji.+.;i3shows the relationship between i].i:i-i3": mouse chromosome I and the human chromosomal set.We can recognize 2l segmentsin this mouse chromosome that have syntenic counterparts in human chromosomes. The extent of reshuffling that has occurred between the genomes is shown by the fact that the segments are spread among six different human chromosomes.The sametlpes of relationshipsare found in all mouse chromosomes except for the X chromosome, which is syntenic only with the human X chromosome. This is explained by the fact that the X is a specialcase,subject to dosage compensation to adjust for the difference between males (one copy) and females (two copies) (see Section 3L5, X Chromosomes Undergo Global Changes).This may apply selective pressure against the translocation of genes to and from the X chromosome. Comparison of the mouse and human genome sequencesshows rhal >90oh of each genome lies in syntenic blocks that range widely in size (from 300 kb to Ol Mb). There is a total of 342 syntenic segments, with an average Iength of 7 Mb (O.3% of the genome). Ninetynine percent of mouse geneshave a homologue in the human genome; for 96o/" that homologue is in a syntenic region. Comparing the genomes provides interesting information about the evolution of species.
HaveDNA 0rganelles 10 20 30 40 50 60 70 80 90 t00 Mb Mousechromosome1
' n llffi ...'. . 1 2145 2 Corresponding humanchromosome
:tEH 6
8
- I G * 8 f i . 1 3 M o u sceh r o m o s o m t hea s2 1s e g m e not sf 1 to 25 Mbthataresyntenic withregions corresponding to partsof six humanchromosomes.
The number of gene families in the mouse and human genomes is the same, and a major difference between the speciesis the differential expansion of particular families in one of the genomes. This is especially noticeable in genes that affect phenotypic features that are unique to the species.Of 25 families for which the size has been expanded in mouse, l4 contain genes specifically involved in rodent reproduction, and 5 contain genes specific to the immune system. A validation of the importance of syntenic blocks comes from pairwise comparisons of the genes within them. Looking for likely pseudogeneson the basisof sequencecomparisons,a gene that is not in a syntenic location (that is, its context is different in the two species)is twice as likely to be a pseudogene.Put another way, translocation away from the original locus tends to be associatedwith the creation of pseudogenes. The lack of a related gene in a syntenic position is therefore grounds for suspectingthat an apparent gene may really be a pseudogene. Overall, >l0o/o of the genes that are initially identified by analysis ol the genome are likely to turn out to be pseudogenes. As a general rule, comparisons between genomes add significantly to the effectiveness of gene prediction. When sequence features indicating active genes are conserved-for example, between Man and mouse-there is an increased probability that they identify active homologues. Identifying genes coding for RNA is more difficult becausewe cannot use the criterion of the open reading frame. It also is true that comparative genome analysis increased the rigor of the analysis.For example, analysis of either the human or the mouse genome alone identifies -500 genes coding for IRNA, but comparison of features suggeststhat
r Mitochondria havegenomes that andchtoroptasts Typicatly shownon-Mendetian inheritance. they arematernatty inherited. o Organelle genomes mayundergo somatic segregation in plants. o Comparisons DNAsuggest that of mitochondrial humans froma singtefemalewho aredescended yearsagoin Africa. tived200,000 The first evidence for the presenceof genesoutside the nucleus was provided by nonMendelian inheritance in plants (observed in the early years of the twentieth century, just after the rediscovery of Mendelian inheritance). Non-Mendelian inheritance sometimes is associated with the phenomenon of somatic segregation. Both have a similar cause: . Non-Mendelian inheritance is defined by the failure of the progeny of a mating to display Mendelian segregationfor parental characters. It reflects lack of association between the segregating character and the meiotic spindle. . Somatic segregation describesa phenomenon in which parental characters segregatein somatic cells and therefore display heterogeneity in the organism. This is a notable feature of plant development, as it reflects lack of association between the segregating character and the mitotic spindle. Non-Mendelianinheritanceand somaticsegregation are thereforetaken to indicate the presenceof genesthat resideoutsidethe nucleusand do not utilizesegregationon the meioticand mitotic spindlesto distribute replicasto gametesor to daughter cells, .,!.3:+ respectively. f,3.{;#Ri shows that this happens when the mitochondria inherited from the male and female parents have different alleles, and by chance a daughter cell receives an unbalanced distribution of mitochondria that representsonly one parent (seeSection 17.12, How Do Mitochondria Replicate and Segregate?). The extreme form of non-Mendelian inheritance is uniparental inheritance, which occurs when the genotype of only one parent is inherited and that of the other parent is permanently lost. In Iess extreme examples, the progeny of one parental genotlpe exceedthose of the other genotype. Usually it is the mother whose genotype is preferentially (or solely) inherited. This effect is sometimes described as maternal inheritance. The important point is that the genotype contributed by the parent of one
HaveDNA 4.9 0rqanettes
67
DNA genomes that are inherited independently of nuclear genes.In effect,the organellegenome Cell has mitochondriafrom both parents comprisesa length of DNA that has been physically sequesteredin a defined part of the cell and is subject to its own form of expressionand regulation. An organelle genome can code for some or all of the RNAs, but codesfor only some of the proteins needed to perpetuatethe organelle.The other proteins are coded in the nucleus, expressed via the cytoplasmic protein synthetic apparatus, and imported into the organelle. Genes not residing within the nucleus are generally described as extranuclear genes; they are transcribed and translatedintLre same organelle compartment (mitochondrion or chloroplast) in which they reside. By contrast, nucleargenes are expressedby means of cytoplasmicprotein synthesis. (The term cytoplasmic inheritance sometimes is used to describre the behavior of genes in organelies.We shall not use this description, though, because it is important to be able to distinguish between events in the general cytosol and those in specific organelles.) Higher animals show maternal inheritance, which can be explained if the mitochondria are : :,:! .. r.- When D a t e r naanLdm a t e r n m a ti t o c h o n d r i a l contributed entirely by the ovum and not at all a[[e[es differ,a cetlhastwo setsof mitochondriat DNAs. ,1r. i i.-. shows that the sperm by the sperm. tiii:r,Jiii: Mitosisusua[ygenerates daughter celtswith bothsets. a copy of the nuclear DNA. contributes only genSomatic variatjon mayresuttif unequaI segregation Thus the mitochondrial genesare derived exclueratesdaughter cettswith ontyoneset. sively from the mother, and in males they are discarded each generation. Conditions in the organelle are different from those in the nucleus, and organelle DNA particular sex predominates,as seen in abnortherefore evolves at its own distinct rate. If inherm a l s e g r e g a t i o nr a t i o s w h e n a c r o s si s m a d e itance is uniparental, there can be no recombination between parental genomes. In fact, recombination usually does not occur in those occurs when reciprocal crossesshow the casesfor which organelle genomes are inhercontributions of both parents to be equally ited from both parents. Organelle DNA has a inherited. different replication system from that of the The bias in parental genotypesis established nucleus; as a result, the error rate during repliat or soon after the formation of a zygote. There cation may be different. Mitochondrial DNA are various possiblecauses.The contribution of accumulates mutations more rapidly than maternal or paternal information to the nuclear DNA in mammals, but in plants the accuorganelles of the zygote may be unequal; in the mulation in the mitochondrion is slower than most extreme case,only one parent contributes. in the nucleus (the chloroplast is intermediate). ln other casesthe contributions are equal, but One consequence of maternal inheritance the information provided by one parent does is that the sequence of mitochondrial DNA is not survive. Combinations of both effects are more sensitive than nuclear DNA to reductions possible.Whatever the cause,the unequal repin the size of the breeding population. Comparresentation of the information from the two i s o n s o f m i t o c h o n d r i a l D N A s e q u e n c e si n a parents contrastswith nuclear genetic informarange of human populations allow an evolution, which derives equally from each parent. tionary tree to be constructed. The divergence Non-Mendelian inheritance results from the among human mitochondrial DNAs spans presencein mitochondria and chloroplastsof 0.57%. A tree can be constructedin which the
*::ilff :ffii[lx?i^:i["IJ"",]::ff Tn:';
C H A P T E4RT h eC o n t e n o t f the Genome
irii:l-:*i:.,.::i DNAfromthe spermentersthe oocyteto jn the fertil.ized formthe matepronucteus egg,but al.l. the mitochondria areprovided bythe oocyte.
mitochondrial variants diverged from a common (African) ancestor.The rate at which mammalian mitochondrial DNA accumulates mutations is2"h Io 4o/oper million years,which is >l0x fasterthan the rate for globin. Such a rate would generate the observeddivergence over an evolutionary period of 140,000 to 280,000 years. This implies that the human race is descendedfrom a single female who lived in Africa -200,000 years ago.
0rgane[Le Genomes Are Circular DNAs ThatCode for Organe[[e Proteins . Organelle genomes (butnot atways) areusualty circutar motecutes of DNA. . Organe[[e genomes codefor some,but not at[.of foundin the organetle. the proteins Most organellegenomestake the form of a single circular molecule of DNA of unique sequence (denoted mtDNA in the mitochondrion and
ctDNA in the chloroplast). There are a few exceptions for which mitochondrial DNA is a Iinear molecule; these generally occur in lower eukaryotes. Usually there are several copies of the genome in the individual organelle. There are multiple organellesper cell, therefore there are many organelle genomes per cell. Although the organelle genome itself is unique, it constitutes a repetitive sequence relative to any nonrepetitive nuclear sequence. Chloroplast genomes are relatively large, usually -140 kb in higher plants and <200 kb in lower eukaryotes. This is comparable to the size of a large bacteriophage, for example, T4 at -165 kb. There are multiple copies of the genome per organelle, typically 20 to 40 in a higher plant, and multiple copies of the organelle per cell, typically 20 to 40. Mitochondrial genomes vary in total size by more than an order of magnitude. Animal cells have small mitochondrial genomesapproximately 16.5 kb in mammals. There are several hundred mitochondria per cell. Each mitochondrion has multiple copies of the DNA. The total amount of mitochondrial DNA relative to nuclear DNA is small; it is estimated to be
er o t e i n s r N A sT h a tC o d ef o r O r g a n e l tP 4.10 0rganetle G e n o m eAs r eC i r c u l aD
Species
Size (kb)
Fungi Protists Plants Animals
19-100 6-100 186-366 16-17
Proteincoding genes
BNAcoding genes
8-14 3-62 27-34 13
10-28 2-29 21-30 4-24
.:.:.:-:i:i.- :, M'itochondriaL genomeshavegenescoding for (mostlycomplexI-IV) proteins.rRNAs. andtRNAs. ND3
c03 mitochondria use their l6 kb genomes to code for l3 proteins, whereas yeast mitochondria use their 60 to 80 kb genomes to code for as few as eight proteins. Plants, which have much larger mitochondrial genomes,code for more proteins. Introns are found in most mitochondrial genomes, although not in the very small mammalian genomes. The two major rRNAs are always coded by the mitochondrial genome. The number of tRNAs coded by the mitochondrial genome variesfrom none to the full complement (25 ro 26 in mitochondria). This accounts for the varia t i o n i n F i g u r e4 . 1 6 . The major part of the protein-coding activity is devoted to the components of the multisubunit assembliesof respiration complexes I-IV. Many ribosomal proteins are coded in protist and plant mitochondrial genomes,but there are few or none in {ungi and animal genomes. There are genes coding for proteins involved in import in many protist mitochondrial genomes.
MitochondriaL DNA 0rganization Is VariabLe . Animalcet[mitochondrial DNAis extremety compact andtypical.ty codesfor 13 proteins. 2 r R N A sa,n d2 2t R N A s . . Yeastmitochondrial DNAis 5x longerthananimal ceLtmtDNA because of the presence of long introns. Animal mitochondrial DNA is extremely compact. There are extensive differences in the detailed gene organization found in different animal phyla, but the general principle is maintained of a small genome coding for a restricted number of functions. In mammalian mitochondrial genomes, the organization is extremely compact. Ihere are no introns, some genesactually overlap, and almost every single base pair can be
70
C H A P T E4RT h eC o n t e not f t h e G e n o m e
ATPase 6 co2
uvl
# IRNAgenes ffi Cooingregions + Indicatesdirectionof gene,5' to 3' CO: cytochromeoxidase ND: NADHdehydrogenase
aN I Ah a s2 2 t R N A I l t S F i { ; ; . - :1r H u m a nm i t o c h o n d r iD genes, genes, regions. 2 rRNA and13protein-coding Fouror rRNA-coding regions are teenofthe 15protein-coding Fourteen in the samedirection. of the IRNA transcribed genesareexpressed in the clockwise direction and8 are readcounter-ctockwise.
assignedto a gene. With the exception of the D loop, a region concerned with the initiation of DNA replication, no more than 87 of the 16,569 bp of the human mitochondrial genome can be regarded as lying in intercistronic regions. The complete nucleotide sequencesof mitochondrial genomes in animal cells show extensive homology in organization. The map of the human mitochondrial genome is summarized +.i.r. There are l3 protein-coding in fjii;l"rFii, regions. All of the proteins are components of the apparatusconcemed with respiration. These include cytochrome b, three subunits of cytochrome oxidase, one of the subunits of ATPase,and sevensubunits (or associatedproteins) of NADH dehydrogenase. The fivefold discrepancy in size between (84kb) and mammalian (16 kb) the S.cerevisiae mitochondrial genomes alone alerts us to the fact that there must be a great difference in their genetic organization in spite of their common function. The number of endogenously synthesized products concerned with mitochondrial enzymatic functions appearsto be similar. Does the additional genetic material in yeast mitochondria represent other proteins, perhaps concerned with regulation. or is it unexpressed?
Genes RNA-coding 165 rRNA 23S rRNA 4.5S rRNA 55 rRNA tRNA oxi2 8,4kb pal
Gene Expression r-proterns RNA polymerase Others
Types 1 1 1 1
30-32 20-21 z
functions Chloroplast Rubiscoand thylakoids 31-32 NADHdehydrogenase 11 Total
105-113
Fil;"ift{:: i. .i!:;Thechloroptast genome in [andptantscodes for 4 rRNAs, 30 tRNAs, and-60 proteins. fl
Exons fft Intr,rns
o/l I = subunitsol oliqomvcrn-sensitive aapJATPase oxi = subunitsof cytochrome c Dox = cytochromeb par unknownlunctions var = smallribosomesubunitorotein
i iirL'ii lr .1.! ij ThemitochondriaI genomeof S.cerevisi oe c o n t a i nbs o t hi n t e r r u p t eadn du n i n t e r r u p t epdr o t e i n c o d i n gg e n e sr,R N Ag e n e sa, n dI R N Ag e n e s( p o s i t i o n s notindicated). Anowsindicate direction of transcription.
The map shown in : I{ri-{{iti +.; ii accounts for the major RNA and protein products of the yeast mitochondrion. The most notable feature is the dispersion of loci on the map. The two most prominent loci are the interrupted genes box (coding for cytochrome bl and oxi3 (coding for subunit I o f c y t o c h r o m e o x i d a s e ) .T o g e t h e r t h e s e t w o genesare almost as long as the entire mitochondrial genome in mammals! Many of the I o n g i n t r o n s i n t h e s e g e n e sh a v e o p e n r e a d ing frames in register with the preceding exon ( s e eS e c t i o n2 7 . 5 . S o m e G r o u p I I n t r o n s C o d e f o r E n d o n u c l e a s e sT h a t S p o n s o r M o b i l i t y ) . T h i s a d d s s e v e r a lp r o t e i n s , a l l s y n t h e s i z e di n Iow amounts. to the complement of the yeast mitochondrion. The remaining genes are uninterrupted. They correspond to the other two subunits of cytochrome oxidase coded by the mitochondrion, to the subunit(s) of the ATPase,and (in the caseof varl) to a mitochondrial ribosomal protein. The total numtrer of yeast mitochondrial genesis unlikely to exceed-25.
TheChloroplast Genome for ManyProteins Codes andRNAs . Chloroptast genomes varyin size,but are[arge enough aswetlas to codefor 50 to 100proteins the rRNAs andtRNAs. What genesare carried by chloroplasts?Chloroplast DNAs vary in length from 120 to 190 kb. The sequenced chloroplast genomes (>10 in total) have 87 to 183 genes.Flil*qi.'r,-l*summarizes the functions coded by the chloroplast genome in land plants. There is more variation in the chloroplast genomes of algae. The situation is generally similar to that of mitochondria, except that more genes are involved. The chloroplast genome codes for all the rRNA and IRNA speciesneeded for protein slmthesis.The ribosome includes two small rRNAs in addition to the major species.The IRNA set may indude all of the necessarygenes.The chloroplast genome codes for -50 proteins, including RNA polymerase and ribosomal proteins. Again, the rule is that organelle genes are transcribed and translatedby the apparatusof the organelie. About half of the chloroplast genescode for proteins involved in protein synthesis. The endosymbiotic origin of the chloroplast is emphasized by the relationships between these genes and their counterparts in bacteria. The organization of the rRNA genesin particular is closely related to that of a cyanobacterium, which pins down more precisely the last common ancestor between chloroplasts and bacteria.
GenomeCodesfor ManyProteinsand RNAs 4.1.2TheChtoroplast
7t
Introns in chloroplasts fall into two general c l a s s e s .T h o s e i n I R N A g e n e s a r e u s u a l l y (although not inevitably) located in the anticodon loop, like the introns found in yeast nuclear IRNA genes (see Section 26.I4, yeast IRNA Splicing Involves Cutting and Rejoining). T h o s e i n p r o t e i n - c o d i n g g e n e s r e s e m b l et h e introns of mitochondrial genes (seeChapter 27, Catalytic RNA). This placesthe endosymbiotic event at a time in evolution before the separation of prokaryotes with uninterrupted genes. The role of the chloroplast is to undertake photosynthesis.Many of its genescode for proteins of complexes located in the thylakoid membranes.The constitution of these complexes shows a different balance from that of mitochondrial complexes.Although some complexes are like mitochondrial complexesin having some subunits coded by the organelle genome and some by the nuclear genome, other chloroplast complexesare coded entirely by one genome.
@
Mitochondria Evolved by Endosymbiosis
How did a situation evolve in which an organelle contains genetic information for some of its functions, whereas others are coded in the .:;.=+ nucleus? f l{-ii+:i. shows the endosymbiosis model for mitochondrial evolution, in which primitive cells captured bacteria that provided the functions that evolved into mitochondria and chloroplasts.At this point, the protoorganelle must have contained all of the genes needed to specify its functions. Sequence homologies suggestthat mitochondria and chloroplastsevolved separately, from lineagesthat are common with eubacteria, with mitochondria sharing an origin with cr-purple bacteria and chloroplasts sharing an origin with cyanobacteria.The closestknown relative of mitochondria among the bacteria is Rickettsia(the causativeagent of typhus), which is an obligate intracellular parasite that is probably descended from free-living bacteria. This reinforcesthe idea that mitochondria originated in an endosymbiotic event involving an ancestor that is also common to Rickettsia. Two changes must have occurred as the bacterium became integrated into the recipient cell and evolved into the mitochondrion (or chloroplast).The organelleshave far fewer genes than an indenendent bacterium and have lost
72
Bacteriumevolvesinto losinggenesthat mitochondrion, are necessaryfor independentlite
CHAPTER 4 TheContent of the Genome
originated IItiJ*il +.i* Mitochondria by a endosymbiwascaptured otjc eventwhena bacterium by a eukaryoticce[t.
many of the gene functions that are necessary for independent life (such as metabolic pathways). The majority of genes coding for organelle functions are in fact now located in the nucleus, thus these genesmust have been transferred there from the organelle. Transfer of DNA between organelle and nucleus has occurred over evolutionary time periods and still continues. The rate of transfer can be measured directly by introducing into an organelle a gene that can function only in the nucleus, for example, because it contains a nuclear intron, or because the protein must function in the cytosol. In terms of providing the material for evolution, the transfer rates from organelle to nucleus are roughly equivalent to the rate of single gene mutation. DNA introduced into mitochondria is transferred to the nucleus at a rate of 2 x l0-5 per generation. Experiments to measure transfer in the reverse direction, from nucleus to mitochondrion, suggest that the rate is a much lower
a nuclear-specific antibiotic resistance gene is introduced into chloroplasts, its transfer to the nucleus and successful expression can be followed by screeningseedlingsfor resistanceto the antibiotic. This shows that transfer occurs atarate of I in 16,000 seedlings,or 6 x l0-5. Transfer of a gene from an organelle to the nucleus requires physical movement of the DNA, of course, but successfulexpression also requires changes in the coding sequence. Organelle proteins that are coded by nuclear genes have special sequencesthat allow them to be imported into the organelle after they have been synthesized in the cytoplasm (seeSection 10.16, PosttranslationalMembrane Insertion Depends on Leader Sequences). These sequencesare not required by proteins that are synthesized within the organelle. perhaps the process of effective gene transfer occurred at a period when compartnlents were less rigidly defined, so that it was easier both for the DNA to be relocated and for the proteins to be incorporated into the organelle irrespective of the site of synthesis. Phylogeneticmaps show that gene transfers have occurred independently in many different lineages. It appears that transfers of mitochondrial genes to the nucleus occurred only early in animal cell evolution, but it is possible that the processis still continuing in plant cells. The number of transfers can be large; there are >800 nuclear genes in Arabidopsis whose sequencesare related to genes in the chloroplasts of other plants. These genes are candidates for evolution frorn genes that originated in the chloroplast.
nonrepetitive DNA. The amount of nonrepetitive DNA is a better reflection of the complexity of the organism than the total genome size; the greatest amount of nonrepetitive DNA is genomes is -2 X l0e bp. Non-Mendelian inheritance is explained by the presence of DNA in organelles in the cytoplasm. Mitochondria and chloroplasts are membrane-bounded systems in which some proteins are synthesized within the organelle, while others are imported. The organelle genome is usually a circular DNA that codesfor all the RNAs and some of the proteins required by the organelle. Mitochondrial genomes vary greatly in size from the l6 kb minimalist mammalian genome to the 570 kb genome of higher plants. The Iarger genomes may code for additional functions. Chloroplast genomesrange from 120-200 kb. Those that have been sequenced have similar organization and coding functions. In both mitochondria and chloroplasts, many of the major proteins contain some subunits synthesized in the organelle and some subunits imported from the cytosol. Rearrangements occur in mitochondrial DNA rather frequently in yeast, and recombination between mitochondrial or between chloroplast genomes has been found. Transfers of DNA have occurred between chloroplasts or mitochondria and nuclear senomes.
References IndividuaI ShowExtensive Genomes Variation
Sum mary The DNA sequencescomposing a eukaryotic genome can be classifiedin three groups: . n o n r e p e t i t i v es e q u e n c e sa r e u n i q u e ; . moderately repetitive sequencesare dispersedand repeateda small number of times as related by not identical copies; . and highly repetitive sequencesare short and usually repeated as tandem arrays. The proportions of the types of sequences are characteristicfor each genome, although larger genomes tend to have a smaller proportion of nonrepetitive DNA. Almost 50% of the human genome consistsof repetitive sequences, the vast majority corresponding to transposons sequences.Most structural genes are located in
rch Resea Altshuler,D.,Brooks,L.D.,Chakravarti, A., Collins, F. S., Daly, M. J., and Donnelly, P. (2005). A haplotype map of the human genome. Nature 437, 1299-1320. Altshuler, D., Pollara, V. J., Cowles, C. R., Van Etten, W. J., Baldwin, J., Linton, L., and Lander, E. S. (2000). An SNP map of the human genome generated by reduced representation shotgun sequencing. Nature 407, 513-516. Mullikin, J. C., Hunt, S.E., Cole, C. G., Mortimore, B.J., Rice, C.M., Burton, J., Matthews, L.H., Pavitt, R., Plumb, R. W., Sims, S.K., Ainscough, R. M., Attwood, J., Bailey, J. M., BarIow, I(., Bruskiewich, R. M., Butcher, P. N., Carter, N. P., Chen, Y., and CIee, C. M. (2000). An SNP map of human chromosome 22. Nature 407, 516-520.
4.14Summary 7 3
@
RFLPs andSNPs CanBeUsedfor Genetic Mappi ng
Reviews
rch Resea
Gusella,J F. (I986). DNA polymorphism and human disease.Annu Rev Biochem.55, 8 3l - 8 5 4 . W h i t e , R , L e p p e r t ,M . , B i s h o p ,D . T . , e t a l . ( 1 9 8 5 ) . Construction of linkage maps with DNA markers for human chromosomes. Nature lr3. r0l-r05.
Buckler, A. J., Chang, D D., Graw, S.L., Brook, J. D., Haber, D. A., Sharp, P. A., and Housman, D. E. (1991). Exon amplification: a strategyto isolate mammalian genes based on RNA splicing. Proc Natl Acad. Sci.USA 88,4005-4009. I(unkel, L.M., Monaco, A.P., Middlesworth, W., Ochs,H. D., and Latt, S.A. (1985). Specific cloning of DNA fragments absent from the DNA of a male patient with an X chromosome deletion. Proc Natl Acad. Sci.USA 82, 4778-4782. Monaco, A. P., Bertelson, C. J., Middlesworth, W., Colletti, C. A., Aldridge, J., Fischbeck, K. H., Bartlett, R., Pericak-Vance,M. A., Roses,A. D., and I(unkel, L. M. ( 1985) . Detection of deletions spanning the Duchenne muscular dystrophy locus using a tightly linked DNA segment. Nature )16, 842-845.
Resea r ch Altshuler, D., Brooks, L. D., Chakravarti, A., Collins, F. S., Daly, M. J., and Donnelly, P. (2005). A haplotype map of the human genome. Nature437, 1299-1320. Dib, C., Faure, S., Fizames,C., et al. (1996). A comprehensive genetic map of the human genome based on 5,264 microsatellites. Nature )80. 152-r54 Dietrich, W. F., Miller, J., Steen,R., et al. (1996). A comprehensive genetic map of the mouse genome. Nature 380, 149-152. Donis-I(eller, J., Green, P., Helms, C , et al. (1987). A genetic linkage map of the human genome. Cell 51, )19-)37 . Hinds, D.A., Stuve, L.L., Nilsen, G B., Halperin, E., Eskin, E., Ballinger, D. G., Frazer,I(. A., a n d C o x , D . R . ( 2 0 0 5 ) .W h o l e - g e n o m ep a t terns of common DNA variation in three human populations. Science 307, 1072-1079. Sachidanandam,R., Weissman, D., Schmidt, S., et al. (2001). A map of human genome sequencevariation containing 1.42 million single nucleotide polymorphisms. The International SNP Map Working Group. Nature 409. 928-9)) .
@l
WhyAreGenomes SoLarge?
Revlews Gall, J G. ( I 981 ) Chromosome structure and the C-value paradox. J CellBiol. 9I, 3s-14s. Gregory, T. R. (2001) . Coincidence,coevolution, or causation? DNA content, cell size, and the C-value enigma. Biol. Rev.Camb.Philos.Soc.76, 65-10I.
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Eukaryotic BothNonGenomes Contain repetitive andRepetitive DNASequences
Reviews B r i t t e n ,R . J . a n d D a v i d s o n ,E . H . ( 1 9 7 1 ) .R e p e t i tive and nonrepetitive DNA sequencesand a speculation on the origins of evolutionary novelty. Q Rev.Biol.46, lll-l)3. Davidson,E.H. and Britten, R.J. (I971). Organization, transcription, and regulation in the animal genome. Q Rev Biol 48,565-613.
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Genes CanBeIsolatedbvthe Conservation of Exons
CHAPTER 4 TheContent of the Genome
HaveDNA 0rganetles Resea rch Cann, R. L., Stoneking,M., and Wilson, A. C. (1987i. Mitochondrial DNA and human evolution. Nature )25, )l-36.
DNAs AreCircutar 0rganette Genomes That Codefor 0rqane[[eProteins Review , .(1999). L a n g ,B . F . ,G r a y ,M . W . , a n d B u r g e r G Mitochondrialgenomeevolution and the origin of eukaryoles.Annu.Rev Genet33, )5r-397. M i t o c h o n d r i aDIN A0 r g a n i z a t i o n Is Variab[e Reviews Attardi,G. (1985).Animal mitochondrialDNA: an extremeexampleof economy.Int Rev Cytol. 9),9)-146. Boore,J.L. (1999).Animal mitochondrial genomes.Nucleic AcidsRes27, 1767-).780. of the mamClayton,D. A. (1984).Transcription malian mitochondrialgenome.Annu.Rev. Biochem. 53, 573-594. Gray,M. W. (1989).Originand evolutionof mitochondrialDNA.Annu.Rev.CellBiol.5,25-50. R e s e a hr c Anderson,S.,Bankier,A. T., Barrell,B. G.,et al. (I981). Sequence and organization of the human mitochondrialgenome.Nature290, 457-465.
TheChtoroptast Genome Codes for Many Proteins andRNAs Reviews Palmer, J. D. (1985). Comparative organization of chloroplast genomes. Annu. Rev.Genet 19, )25-354. Shimada, H. and Sugiura, M. (I991). Fine structural features of the chloroplast genome: comparison of the sequenced chloroplast genomes. NucleicAcids Res ll , 98J-995. Sugiura, M., Hirose, T., and Sugita,M. (I998). Evolution and mechanism of translation in chloroplasts.Annu. Rev.Genet.32,43'7459.
Mitochondria Evolved by Endosymbiosis Review L a n g ,B . F . ,G r a y ,M . W . , a n d B u r g e rG , .(1999). Mitochondrialgenomeevolution and the ori-
gin of eukaryotes. Annu. Rev.Genet.33, 35t-397. Research Adams, I(. L., Daley, D. O., Qiu, Y. L., Whelan. J., and Palmer, J. D. (2000). Repeated,recent and diverse transfers of a mitochondrial gene to the nucleus in flowering plants. Nature 408, j54-357. Arabidopsis Initiative (2000). Analysis of the genome sequence of the floweringplant Arabidopsisthaliana. N ature 408, 7 9 6-81 5. Huang, C. Y., Ayliffe, M. A., and Timmis, J. N. (2003). Direct measurement of the transfer rate of chloroplast DNA into the nucleus. Nature422,72-76. Thorsness,P. E. and Fox, T. D. (1990). Escapeof DNA from mitochondria to the nucleus in S. cerevisiae. Nature )46. 37 6-37 9.
References 7 5
Sequences Genome andGeneNumbers C H A P T EO RU T L I N E Introduction Bacteria[ GeneNumbers Ranqe 0veran Order of Magnitude TotalGeneNumber Is Knownfor Several Eukaryotes . There in a worm; 13,600 in are6000genes in yeasU 18.500 in thesma[[plantArabidopsrs,' a fty;25,000 andprobabty 20,000 to 25,000 in mouse andman. HowManyDifferent Types AreThere? of Genes TheHuman Genome HasFewer Genes ThanExpected r 0nty1%of thehuman genome regions. of coding consists -5olo r Theexons ptus (exons comprise of eachgene, sogenes
-25olo introns)comprise of the genome. . Thehumangenome has20,000to 25.000genes. o -60olo of humangenes areatternatively spticed. . Upto 800/o of the atternative spticeschangeprotein sequence, sothe proteome has-50,000to 60,000members. HowAre Genesand 0ther Seouences Distributedin the Genome? . Repeated (present sequences in morethanonecopy) for >50%of the humangenome. account . Thegreatbulkof repeated sequences consistof copies of nonfunctjonaI transposons. . Therearemanyduplications regions. of largechromosome
TheY Chromosome HasSeveralMale-Soecific Genes o TheY chromosome has-60 genes that areexpressed specificattyin thetestis. . Themate-specific genesarepresent in muttiplecopies in repeated chromosomaI segments. r Geneconversion muttipte between copies attows the active genes to be majntained duringevotution. MoreComptex SpeciesEvotveby AddingNewGene Functions . Comparisons genomes of djfferent increase in showa steady genenumber genesareaddedto makeeukaryasadditional otes,mutticellutar organisms, anima[s. andvertebrates.
76
o Mostof the genesthat areuniqueto vertebrates areconor nervous systems. withtheimmune cerned HowManyGenesAre Essential? . Nota[[genesareessential. of In yeastandfty.detetions effects. <50%of the geneshavedetectable . Whentwo or moregenesareredundant, a mutation'in any effects. oneof themmaynot havedetectable o Wedo notfuttyunderstand in the genome the survival of genes dispensable. that areapparently at Widel.yDiffering Levets GenesAre Expressed . In anygivence[t,mostgenesareexpressed at a low[eve[. . Ontya smatlnumber whoseproducts arespeciatof genes, izedfor the ce[[type.arehigh[yexpressed. HowManyGenesAre Expressed? r mRNAs whendifat lowlevetsoverlapextensively expressed ferentcetltypesarecompared. r Theabundantty mRNAs specific for the expressed areusuatly cetttype. . -L0,000expressed genesmaybe common to mostce[[types of a highereukaryote. En Masse Expressed GeneNumberCanBe Measured . "Chip"technology to betakenof the altowsa snapshot in a yeastce[[. of the entiregenome expression t -75olo (-4500genes) is expressed of theyeastgenome growthconditions. undernormaI . Chiptechnology comparisons of retated ania[tows detailed (forexampte) in malceltsto determine the differences a normalcetlanda cancer ce[t. exoression between
Summary
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homology with known genesin other species. These genes fall approximately equally into classeswhose produr:ts are concerned with metabolism, cell struclure or transport of components, and gene expressionand its regulation. In virtually every genome, >25o/oof the genes cannot be ascribedany function. Many of these genescan be found in related organisms,which implies that they have a conserved function. There has been sorne emphasison sequencing the genomes of pathogenic bacteria, given their medical importarLce.An important insight into the nature of pathogenicity has been provided by the demonst.ration that "pathogenicity islands" are a characteristic feature of their genomes.Theseare large regions,-10 to 200 kb, which are present in the genome of a pathogenic speciesbut absent from the genomes of nonpathogenic variants of the same or related species.Their G-C content often differs from that of the rest of the genome, and it is likely that they migrate between bacteria by a process of horizontal transfer. For example, the bacterium that causesanthrax (Bacillusanthracis)has two large plasmids (extrachromosomalDNA), one of which has a pathoge:nicityisland thar includes the gene coding for the anthrax toxin.
TotalGeneNumber Is Knownfor Several Eukaryotes . Thereare6000genes in yeas| 18,500in a worm; 13,600in a fty; 25,000in the smat[ptant -25,000in mouse Arobidopsis; andprobably a n om a n . As soon as we look at eukaryotic genomes,the relationship between genome size and gene number is lost. The genomes of unicellular eukaryotes fall in the same size range as the largest bacterial genornes.Higher eukaryotes have more genes,but the number does not correlate with genome size, as can be seen from tlti.:-stl:
,
The most extensive data for lower eukaryotes are available frorn the sequencesof the genomes of the yeasts Saccharomyces cerevisiae pombe.i'!r;rr*r r, :r sumand Schizosaccharomyce,s marizes the most important features. The yeast genomes ol12.5 Mb and 13.5 Mb have -6000 and -5000 genes,respectively.The averageopen reading frame (ORF) is -1.4 kb, so that -70o/o of the genome is occupied by coding regions.
40,000 o 30,000 o o
o zo,ooo 10,000
300 400 500 Genome size(Mb) varjesfrom6000 i ir,iiirli:, , Thenumber of genesin a eukaryote withthe genome sizeor the comto 40,000but doesnot corretate ptexityof the organism.
5% oi S. cerevisiaegenes have 1 intronon average
\ 43"koI S. pombegenes have introns Averageinterruptedgene has 2 introns
r i r l L . : i ' r : ' ;T r .h' re S . c e r e v i s i a e g e n o m e o f l 3 . 5 M b h a s 6 0 0 0 g e n e s . a l m o s t a [ [ u n i n almosthalfhaving of 12.5Mbhas5000genes, terrupted. Ihe 5. ponbegenome introns. arefairlysimjlar. Genesizesandspacing The major difference between them is that only 5t/. of.S.cerevisiaegeneshave introns, compared to 43o/oin S.pombe.The density of genesis high; organization is generally similar, although the spacesbetween genesare a bit shorter in S.cerevisiae. About half of the genes identified by sequence were either known previously or related to known genes.The remainder are new which gives some indication of the number of new types of genes that may be discovered. The identification of long reading frames on the basis of sequenceis quite accurate.However, ORFs coding for <100 amino acids cannot be identified solely by sequencebecause of the high occurrence of false positives. Analysis of gene expression suggeststhat -300 of 600 such ORFsin S.cerevisiae arelikely to be genuine genes. A powerful way to validate gene structure is to compare sequences in closely related species-if a gene is active, it is likely to be conserved. Comparisons between the sequencesof four closely related yeast species suggest that 503 of the genes originally identified in
Eukaryotes 7 9 Is Knownfor Several 5.3 TotalGeneNumber
ffi DNA manipulation I Transcription n Translation ! Proteinstructure I Cell cycle/death E Cytoskeleton I Enzymes tr Signaltransduction I
Cell adhesion I Transporters /channels tr Unknown
' -20oloof DrosophiLa genescodefor proteins genes,-20% concerned with majntaining or expressing codefor enzymes, and<100/o codefor proteins concerned withthecet[cycleorsignal Ha[fofthegenes transductjon. of DrosophiLa function. codefor products of unknown S.cerevisiae do not have counterparts in the other speciesand therefore should be deleted from the catalog.This reducesthe total gene number f,or S. cerevisiae to 5726. The genome oI CaenorhabditiselegansDNA variesbetween regionsrich in genesand regions in which genes are more sparsely organized. T h e t o t a l s e q u e n c ec o n t a i n s - 1 8 , 5 0 0 g e n e s . Only -42oh of the geneshave putative counterparts outside the Nematoda. Although the fly genome is larger than the w o r m g e n o m e ,t h e r e a r e f e w e r g e n e s( 1 3 , 6 0 0 ) in D. melanogaster.The number of different transcriptsis slightly larger ( 14,100) as the result of alternative splicing. We do not understand why the fly-a much more complex organism-has only 70o/oof the number of genes in the worm. This emphasizesforcefully the lack of an exact relationship between gene number and complexity of the organism. The plant Arabidopsisthaliana has a genome size intermediate between the worm and the fly, but has a larger gene number (25,000) than either. This again shows the lack of a clear relationship and also emphasizesthe specialquality of plants, which may have more genes (due to ancestralduplications) than animal cells.A majority of. the Arabidopsis genorr'e is found in duplicated segments,suggestingthat there was an ancient doubling of the genome (to give a tetraploid). Only 35ok oI Arabidopslsgenesare present as single copies. The genome of rice (Oryzasativa)is -4larger than Arabidopsis,but the number of genes is only -50% larger, probably -40,000. Repetitive DNA occupies 42o/o-45oh of the genome. More than 80% of the geneslotndinArabidop-
80
C H A P T E5RG e n o m S e e q u e n c easn d G e n eN u m b e r s
sls are represented in rice. Of these common genes, -8000 are found in Arabidopsrsand rice but not in any of the bacterial or animal genomes that have been sequenced.This is probably the set of genes that codes for plantspecific functions, such as photosynthesis. From the fly genome, we can form an impression of how many genes are devoted to each type of function. i l,ili.iii::..r breaks down the functions into different categories.Among the genes that are identified, we find 2500 enzymes,-750 transcription factors,-700 transporters and ion channels, and -700 proteins involved with signal transduction. Just over half of the genescode for products of unknown function. Approximately 2oo/oof the proteins reside in membranes. Protein sizeincreasesfrom prokaryotes and archaeato eukaryotes. The archaea M. jannaschi and bacterium E. coli have average protein lengths of 287 and 317 amino acids, respectively, whereas S. cerevisiaeand C eleganshave average lengths oI 484 and 442 amino acids, respectively. Large proteins (500 amino acids) are rare in bacteria, but comprise a significant component (-l /3) in eukaryotes.The increase in length is due to the addition of extra domains, with each domain typically constituting 100-300 amino acids.The increase in protein size,however, is responsible for only a very small part of t h e i n c r e a s ei n g e n o m e s i z e . Another insight into gene number is obtained by counting the number of expressed genes.If we rely upon the estimatesof the number of different mRNA species that can be counted in a cell, we would conclude that the average vertebrate cell expresses-I0,000 to 20,000 genes.The existence of significant overlaps between the messengerpopulations in dif ferent cell types would suggestthat the total expressedgene number for the organism should be within a few fold of this amount. The estimate for the total human genome number of 20 to 25,000 (seeSection5.5,The Human Genome Has Fewer GenesThan Expected) would imply that a significant proportion of the total gene number is actually expressedin any given cell. Eukaryotic genes are transcribed individually, with each gene producing a monocistronic messenger.There is only one general exception to this rule: in the genome of C. elegans,-l5oh of the genes are organized into polycistronic units (which are associatedwith the use oltranssplicing to allow expression of the downstream genes in these units; see Section 26.I3, transsplicing Reactions Use Small RNAs).
HowManyDifferent Types of Genes AreThere? Some genesare unique; others belong to families in which the other members are related (but not usually identical). The proportion of unique genes declines with genome size and the proportion of genes in families increases. The minimum number of gene families required to code a bacterium is >1000, a yeast is >4000, and a higher eukaryole I 1,000 to 14,000. Some genesare present in more than one copy or are related to one anothe4 thus the number of different t1,pesof genes is less than the total number of genes. We can divide the total number of genesinto setsthat have related members, as defined by comparing their exons. (A gene family arisesby duplication of an ancestral gene followed by accumulation of changes in sequencebetween the copies.Most often the members of a family zrrerelated but not identical.) The number of types of genes is calculated by adding the number of unique genes (for which there is no other related gene at all) to the numbers of farnilies that have two or more members. a;iii,il.li: rr.I compares the total number of genes with the number of distinct families in each of six genomes. In bacteria most genesare unique, so the number of distinct families is close to the total gene number. The situation is different even in the lower eukaryote S. cerevisiae,f.or which there is a significant proportion of repeated genes.The most striking effect is that the number of genes increases quite sharply in the higher eukaryotes, but the number of gene families does not change much. :';,i:ij+![:,.ii shows that the proportion of unique genes drops sharply with genome size. When genes are present in families, the number of members in a family is small in bacteria and lower eukaryotes, but is large in higher eukaryotes. Much of the extra genome size of Arabidopsisis accounted for by families with >4 members. If every gene is expressed,the total number of genes will account for the total number of proteins required to make the organism (the proteome). Thzoeffects mean. however, that the proteome is different from the total gene number. Genes are dtLplicated,and as a result some of them code for the same protein (although it may be expressed in a different time or place) and others may code for related proteins that again play the same role in differ-
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Unique genes
Families Families with >4 with 2-4 members memoers
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i n mu[ilgi;liii l-:ii.ii:lTheproportion thatarepresent of genes sizein higher eukaryotes. tiptecopies increases withgenome
ent times or places.The proteome can be larger than the number of genesbecausesome genes can produce more than one protein by means of alternative splicing. What is the core proteome-the basicnumber of the different types of proteins in the organism? A minimum estimate is given by the number of gene families, ranging from 1400 in the bacterium, >4000 in the yeast,and I1,000 to i4,000 for the fly and the worm. What is the distribution of the proteome among types of proteins? The 6000 proteins of the yeast proteome include 5000 soluble proteins and 1000 transmembrane proteins. About half of the proteins are cytoplasmic, a quarter
AreThere? 8 1 Types of Genes 5.4 HowManyDifferent
tags can be obtained by expression of cDNAs made by linking the sequencesof ORFsto appropriate expression vectors that incorporate the sequencesfor affinity tags. This allows array analysis to be used Io analyze the products. These methods also can be effective in comparing the proteins of two tissues-for example, a tissue from a healthy individual and one from a patient with disease-to pinpoint the differences. Once we know the total number of proteins, we can ask how they interact. By definiAdditionalin Common Soecific tion, proteins in structural multiprotein to all multicellular to oenus assembliesmust form stable interactions with eukaryotes eukaryotes one another. Proteins in signaling pathways interact with one another transiently. In both i:ii.i=ili::i.'-jThefly genome genes canbe dividedinto present that are(probabty) in a[[eukaryotes, additional cases,such interactions can be detected in test g e n e st h a t a r e( p r o b a b t yp)r e s e nj tn a t [ m u l t i c e t l u t a r systemswhere essentially a readout system mageukaryotes, andgenes thataremorespecific to subgroups nifies the effect of the interaction. One popuof species fties. that inctude lar such systemis the two hybrid assaydiscussed are in the nucleolus, and the remainder are split in Section25.3, Independent Domains Bind between the mitochondrion and the endoplasDNA and Activate Ttanscription. Such assays mic reticulum (ER)/Golgi system. cannot detect all interactions: for example, if How many genesare common to all organone enzyme in a metabolic pathway releasesa isms (or to groups such as bacteria or higher soluble metabolite that then interacts with the eukaryotes),and how many are specificfor the next enzyme, the proteins may not interact individual type of organism? ili*il9i:It.+ sumdirectly. marizesthe comparisonbetween yeast,worm, As a practical matter, assaysof pairwise and fly. Genesthat code for corresponding prointeractions can give us an indication of the teins in different organisms are called minimum number of independent structures orthologs. Operationally, we usually reckon or pathways. An analysis of the ability of all that two genes in different organisms can be 6000 (predicted) yeast proteins to interact in pairwise combinations shows that -I000 proconsideredto provide correspondingfunctions if their sequencesare similar over >807o of the teins can bind to at least one other protein. length. By this criterion, -20o/" of the fly genes Direct analysesof complex formation have idenhave orthologs in both yeast and the worm. tified I440 different proteins in 2)2 multiproThesegenesare probably required by all eukarytein complexes. This is the beginning of an otes. The proportion increasesIo 3oo/owhen fly analysis that will lead to definition of the numand worm are compared, probably representber of functional assembliesor pathways. A ing the addition of gene functions that are comcomparable analysis of 8I00 human proteins mon to multicellular eukaryotes.This still leaves identified 2800 interactions, but is more diffia major proportion of genesas coding for procult to interpret in the context of the larger proteome. teins that are required specificallyby either flies or worms, respectively. In addition to functional genes, there are The proteome can be deduced from the also copiesof genesthat have become nonfuncnumber and structures of genes, and can also tional (identified as such by interruptions in be directly measured by analyzing the total protheir protein-coding sequences).These are called tein content of a cell or organism. By such pseudogenes(seeSection6.6, Pseudogenes Are approaches,some proteins have been identiDead Ends of Evolution). The number of f i e d t h a t w e r e n o t s u s p e c t e do n t h e b a s i s o f pseudogenescan be large. In the mouse and genome analysis; this has led to the identificahuman genomes, the number of pseudogenes tion of new genes. Several methods are used is -10% of the number of (potentially) active for large scaleanalysisof proteins. Mass specg e n e s ( s e eS e c t i o n4 . 8 , T h e C o n s e r v a t i o no f trometry can be used for separating and idenGenome Organization Helps to Identify Genes). tifying proteins in a mixture obtained directly Besides needing to know the density of from cells or tissues.Hybrid proteins bearing genes to estimate the total gene number, we
C H A P T E5RG e n o m S e e q u e n c easn d G e n eN u m b e r s
must also ask: is it important in itself? Are there structural constraints that make it necessary{or genes to have a certain spacing, and does this contribute to the Iarge size of eukaryotic genomes?
TheHuman Genome HasFewer Genes ThanExpected r 0nty1%of the humanqenome consists of coding regrons. . Theexonscomprise -5% of eachgene,sogenes -25olo plusintrons)comprise (exons of the genome. r Thehumangenome has20,000to 25,000genes. c -60olo of humangenesarealternativety spticed. . Upto 80oio of the atternative splices change proteinsequence, sothe proteome has-50,000 to 60,000members. The human genome was the first vertebrate genome to be sequenced.This massive task has revealed a wealth of information about the genetic makeup of our speciesand about the evolution of the genome in general. Our understanding is deepened further by the ability to compare the human genome sequencewith the more recently sequencedmouse genome. Mammal and rodent genomes generally fall into a narrow size range, -3 x l0e bp (seeSection 4.5, Why Are Genomes So Large?). The mouse genome is -l4o/c,smaller than the human genome, probably becauseit has had a higher rate of deletion. The genomes contain similar gene families and genes,with most geneshaving an ortholog in the other genome, but with differencesin the number of members of a farrrily, especiallyin those casesfor which the functions are specificto the species(seeSection4.8, The Conservation of Genome Organization Helps to Identify Genes).Originally estimated to have -30,000 genes,the mouse genome is now thought to have ilbout the same number as the human genome, 20 to 25,000. i::.'.;i-:Frr ii.:.i; plots the distribution of the mouse genes. The 10,000 protein-coding genesare accompanied by -4000 pseudogenes.There are -800 genes representing RNAs that do not code for proteins; these are generally small (asidefrom the ribormal RNAs). Almost half of these genescode for transfer RNAs, for which a large number of pseudogenesalso have been identified.
All genes
30,000 25,000
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genes, genome has-30,000protein-coding ;:l:*!jiili:r.iil Themouse genes. There are-1600RNA-coding whichhave-4000pseudogenes. genesarereptottedon the right at an Thedatafor RNA-coding -350IRNA genes, expanded scate to showthatthereare-800 rRNA RNAgenes, genes and-450othernoncoding and150pseudogenes, i n c l u d i nsgn R N Aasn dm i R N A s .
The human (haploid) genome contains 22 autosomes plus the X or Y. The chromosomes range in size from 45 ro 279 Mb of DNA, making a total genome content of.3,286 Mb ( - 3 . 3 x l 0 e b p ) . O n t h e b a s i so f c h r o m o s o m e structure, the overall genome can be divided into regions of euchromatin (potentially containing active genes) and heterochromatin (see Section 28.7, Chromatin Is Divided into Euchromatin and Heterochromatin). The euchromatin comprises the majority of the genome, -2.9 x l0e bp. The identified genome sequencerepresents-90% of the euchromatin. In addition to providing information on the genetic content of the genome, the sequence also identifies features that may be of structural importance (see Section 28.8, Chromosomes Have Banding Patterns). i:ii\jili i:. r'i shows that a tiny proportion (-f/") of the human genome is accounted for by the exons that actually code for proteins. The introns that constitute the remaining sequences in the genes bring the total of DNA concerned with producing proteins to -25o/". As shown in fii;i.ifti:ii".i*1, the averagehuman gene is 27 kb long, with nine exons that include a total coding sequenceof 1,340 bp. The averagecoding sequence is therefore only 5o/oof the length of the gene.
ThanExpected 83 HasFewer Genes Genome 5.5 TheHuman
decline, and is now generally thought to be -20.000 to 25.000.
TWoindependent sequencing efforts for the human genome produced estimatesof -30,000 and -40,000 genes,respectively.One measure
By any measure, the total human gene number is much less than we had expectedmost estimates bef ore the genome was sequencedwere -100,000. It shows a relatively
of the accuracyof the analysesis whether they identify the same genes.The surprisinganswer is that the overlap between the two setsof genes is only -5O%, as summarizedin Ftr'li.Ril:.i.:i*. An earlier analysisof the human gene setbasedon RNA transcriptshad identified -l1,000 genes, almost all of which are present in both the large human gene sets,and which account for the major part of the overlap between them. So there is no question about the authenticity of half of each human gene set, but we have yet to establish the relationship between the other half of each set. The discrepanciesiilustrate the pitfalls of large scalesequenceanalysis!As the sequenceis analyzed further (and as other genomes are sequencedwith which it can be compared),the number of valid genesseemsto
iri.:-.i.:*{ .--n, i i Genes occupy 25%of the humangenome, but protein-coding sequences areon[ya tiny partof this fraction.
small increaseover flies and worms (I3,600 and I8,500, respectively),not to mention the plant (25,000) (seeFigure 5.2). However, Arabidopsis we should not be particularly surprised by the notion that it does not take a great number of additional genesto make a more complex organism. The difference in DNA sequencesbetween man and chimpanzee is extremely small (there is >99oh similarity), so it is clear that the functions and interactions between a similar set of genes can produce very different results. The functions of specific groups of genes may be especiallyimportant, becausedetailed comparisons of orthologous genes in man and chimpanzee suggestthat there has been accelerated evolution of certain classesof genes, including some involved in early development, olfaction, hearing-all functions that are relatively specific for the species. The number of genes is lessthan the number of potential proteins because of alternative splicing. The extent of alternative splicing is greater in man than in fly or worms; it may affect as many as 60oh of the genes,so the increase in size of the human proteome relative to the other eukaryotes may be larger than the increasein the number of genes.A sample of genesfrom two chromosomes suggeststhat the proportion of the alternative splicesthat actually result in changes in the protein sequence may be as high as 80%. This could increasethe size of the proteome to 50,000 to 6 0 , 0 0 0m e m b e r s . In terms of the diversity of the number of gene families, however, the discrepancy
7 internalexons of averagelength145 bp
lt
I
I
I
l::*U11[ i.i.-i!]Theaverage humangeneis 27 kb [ongandhasnineexons,usualty comprising two longerexons at eachendandseven internalexons. TheUTRs in theterminal exons arethe (noncoding) untranstated regions ateachendofthegene.(Thisis based ontheaverage. Some genesareextremely [ong,whichmakes the median length14 kb withsevenexons.)
84
C H A P T E5RG e n o m S e e q u e n c easn d G e n eN u m b e r s
:i,.ri Thetwo setsof genesidentifiedin the :;ii"li.ii'li: genome human overlap on[ypartiaLLy, asshown in thetwo largeuppercirctes. Theyinclude, however. almosta[[previouslyknowngenes.as shownby the overtap with the smalter, lowercircle.
between man and the other eukaryotes may not be so great. Many of the human genes belong to families. An analysisof -25,000 genes identified 3500 unique genesand 10,300 gene pairs.As can be seenfrom Figure 5.7, this extrapolates to a number of gene families only slightly larger than worm or f.[y.
HowAreGenes and0ther Sequences Distributed in the Genome? r Repeated (present sequences in morethanone copy)account for >50%of the humangenome. o Thegreatbulkof repeated sequences consistof copiesof nonfunctionaI transposons. . Therearemanyduplications of largechromosome regions.
Are genesuniformly distributed in the genome? Some chromosomes are relatively poor in genes and have >25"h oftheir sequencesas "deserts"regions longer than 500 kb where there are no genes.Even the most gene-rich chromosomes have >l0o/o of their sequencesas deserts. So overall. -20'h ol the human genome consistsof desertsthat have no genes. Repetitive sequencesaccount for >50% of the human genome, as seenin i:.ir.'iilil :.iir!. The repetitive sequencesfall into five classes: . Transposons (either active or inactive) account for the vast majority (45"h of the genome). r\ll transposonsare found in multiple copies.
genome ii{ii,i}'lir l:,l.'i Thetargest component ofthehuman inctude 0therrepetitive sequences consists of transposons. [argeduptications andsimpterepeats. . Processedpseudogenes (-3000 in all, account for -0. I % of total DNA). (These are sequencesthat arise by insertion of a copy of an nRNA sequence into the genome; see Section 6.6, Pseudogenes Are Dead Ends of Evolution.) . Simple sequence repeats (highly repetitive DNA such as (CA)n account for -3"/o\. . Segmental duplications (blocks of I0 to 300 kb that have been duplicated into a new region) account for -5oh. Only a minority of these duplications are found on the same chromosome; in the other cases,the duplicates are on different chromosomes. . Tandem repeatsform blocks of one type of sequence (especiallyfound at centromeres and telomeres). The sequence of the human genome emphasizes the importance of transposons. (Transposons have the capacity to replicate themselves and insert into new Iocations. They may function exclusively as DNA elements [see Chapter 2 l, Ttansposonslor may have an active form that is RNA [see Chapter 22, Retroviruses and Retroposonsl . Their distribution in the human genome is summarized in Figure 22.18.) Most of the transposons in the human genome are nonfunctional; very few are currently active. However, the high proportion of the genome occupied by these elements indicates that they have played an active role in shaping the genome. One interesting feature is that some present genes originated as transposons and evolved into their present condition after
Distributedin the Genome? 5.6 HowAre Genesand OtherSequences
85
losing the ability to transpose.Almost 50 genes appear to have originated in this manner. Segmental duplication at its simplest involves the tandem duplication of some region within a chromosome (typically because of an aberrant recombination event at meiosis; see Section 6.7, Unequal Crossing-overRearranges Gene Clusters). In many cases,however, the duplicated regions are on different chromosomes,implying that either there was originally a tandem duplication followed by a translocation of one copy to a new site, or that the duplication arose by some different mechanism altogether. The extreme case of a segmental duplication is when a whole genome is duplicated, in which casethe diploid genome initially becomes tetraploid. As the duplicated copies develop differences from one another, the genome may gradually become effectively a diploid again, although homologies between the diverged copies leave evidence of the event. This is especially common in plant genomes. The present state of analysis of the human genome identifies many individual duplicated regions, but does not indicate whether there was a whole genome duplication in the vertebrate lineage. One curious feature of the human genome is the presence of sequencesthat do not appear to have coding functions, but that nonetheless show an evolutionary conservation higher than the background level. As detected by comparison with other genomes (initially the mouse genome), these represent about 5 % of the total genome. Are these sequencesconnected with protein-coding sequences in some functional way? Their density on chromosome l8 is the same as elsewhere in the genome, although chromosome l8 has a significantly lower concentration of protein-coding genes. This suggests indirectly that their function is not connected with structure or expression of protein-coding genes.
TheY Chromosome Has Ma[e-Specific SeveraI Genes . TheY chromosome has-60 genes that are expressed specificatty in the testis. o Thema[e-specific genesarepresentin multiple copies in repeated chromosomaI segments. . Geneconversion betweenmuLtipte copiesa[tows the activegenesto be maintained during evotution.
86
CHAPTER 5 Genome Sequences andGeneNumbers
The sequence of the human genome has significantly extended our understanding of the role of the sex chromosomes. It is generally thought that the X and Y chromosomes have descended from a common (very ancient) autosome. Their development has involved a process in which the X chromosome has retained most of the original genes, whereas the Y chromosome has lost most of them. The X chromosome behaves like the autosomes insofar as females have two copies and recombination can take place between them. The density of genes on the X chromosome is comparable to the density of genes on other chromosomes. The Y chromosome is much smaller than the X chromosome and has many fewer genes. Its unique role results from the fact that only males have the Y chromosome, of which there is only one copy, so Y-linked loci are effectively haploid instead of diploid like all other human genes. For many years, the Y chromosome was thought to carry almost no genesexcept for one (or more) sex-determining genes that determine maleness.The vast majority of the Y chromosome (>95% of its sequence) does not undergo crossing-overwith the X chromosome, which led to the view that it could not contain active genes becausethere would be no means to prevent the accumulation of deleterious mutations. This region is flanked by short pseudoautosomal regions that exchange frequently with the X chromosome during male meiosis. It was originally called the nonrecombining region, but now has been renamed as the male-specific region. Detailed sequencing of the Y chromosome shows that the male-specific region contains three types of regions, as illustrated in F I G U R5E. 1 5 : . The X-transposedsequences consist of a total of 3.4li1'b comprising some large blocks resulting from a transposition from band q2 I in the X chromosome about 3 or 4 million years ago. This is specific to the human lineage. These sequences do not recombine with the X chromosome and have become largely inactive. They now contain only two active genes. . The X-degeneratesegmentsof the Y are sequences that have a common origin with the X chromosome (going back to the common autosome from which both X and Y have descended) and contain
L8
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yeast, the vast majority of architectures are concerned with intracellular proteins. About twice as many intracellular architectures are found in fly (or worm), but there is a very striking increase in transmembrane and extracellular proteins, as might be expected from the addition of functions required for the interactions between the cells of a multicellular organism. The increase in intracellular architectures required to make a vertebrate (man) is relatively small, but there is again a large increase in transmembrane and extracellular architectures. It has long been known that the genetic difference between man and chimpanzee (our nearest relative) is very small, with -99o/o identity between genomes. The sequence of the chimpanzee genome now allows us to investigate the I % of differences in more detail to see whether features responsible for "humanness" can be identified. The comparison shows 35 x 106 nucleotide substitutions (I.2oh sequencedifference overall), 5 x I0t' deletions or insertions (making -I.roh of the euchromatic sequence specific to each species),and many chromosomal rearrangements. Corresponding proteins are usually very similar;29oh are identical, and in most casesthere are only one or two amino acid changesin the protein between the species. In fact, nucleotide substitutions occur lessoften in genes coding for proteins than are likely to be involved in specificallyhuman traits, suggesting that protein evolution is not a major effect in human-chimpanzee differences. This leaves larger-scalechanges in gene structure and/or changesin gene regulation as the major candidates. Some 25ozl,of nucleotide substilutions occur in CpG dinucleotides (among which are many potential regulator sites).
@
HowManyGenes AreEssentia[?
o Notatl genesareessentiat. In yeastandfty, deletions of <50oio of the geneshavedetectabte effects. . Whentwo or moregenes areredundant. a mutation in anyoneof themmaynot havedetectable effects. . Wedo not fulty understand the survivalin the genome of genesthat areapparentty dispensabte. Natural selection is the force that ensures that useful genesare retained in the genome. Mutations occur at random, and their most common
effect in an ORF will be to damage the protein product. An organism with a damaging mutation will be at a disadvantage in evolution, and ultimately the mutation will be eliminated by the competitive failure of organisms carrying it. The frequency of a disadvantageousallele in the population is balanced between the generation of new mutations and the elimination of old mutations. Reversing this argument, whenever we see an intact ORF in the genome, we assume that its product plays a useful role in the organism. Natural selection must have prevented mutations from accumulating in the gene. The ultimate fate of a gene that ceasesto be useful is to accumulate mutations until it is no longer recognizable. The maintenance of a gene implies that it confers a selective advantage on the organism. In the course of evolution, though, even a small relative advantage may be the subject of natural selection, and a phenotypic defect may not necessarilybe immediately detectable as the result of a mutation. However, we should like to know how many genes are actually essential. This means that their absence is lethal to the organism. In the case of diploid organisms, it means of course that the homozygous null mutation is lethal. We might assume that the proportion of essential genes will decline with increase in genome size, given that larger genomes may have multiple related copies of particular gene functions. So far this expectation has not been borne out by the data (seeFigure 5.2). One approach to the issue of gene number is to determine the number of essential genes by mutational analysis.If we saturate some specified region of the chromosome with mutations that are lethal, the mutations should map into a number of complementation groups that correspond to the number of lethal loci in that region. By extrapolating to the genome as a whole, we may calculatethe total essentialgene number. In the organism with the smallest known genome (M.genitalium),random insertions have detectable effects only in about two thirds of the genes. Similarly, fewer than half of the genes of.E. coli appear to be essential.The proportion is even lower in the yeast S. cerevisiae.When insertions were introduced at random into the genome in one early analysis only l2o/o were lethal, and another l4% impeded growth. The majority (7o%) of the insertions had no effect. A more systematic survey based on completely deleting each of 5,9I6 genes (>96% of the
AreEssentiat? 89 5.9 HowManyGenes
identifiedgenes)showsthat only 18.7% are essential for growth on a rich medium (that is, when ,t shows nutrients are fully provided). r,..,, , that these include genesin all categories.The only
s Totalgenome r Slow growth
G e n ee x o r e s s t c i r
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, Essential , ' :.. yeastgenes arefoundin a[[classes. Btuebars showtotaIproportion of eachclass of genes, redbarsshows thosethat areessentiat.
n 90% no detectable effects W 7.Oo/o nonviable n 1.6"/" postembryonic phenotypes B t.6% growthdefects
rriir,i.li;,rl'l l;,l:i, A systematicanatysisof loss of function for 86% of worm genesshowsthat onl.y10% have detectab[e effects on the phenotype.
CHAPTER 5 Genome Sequences andGeneNumbers
notable concentration of defectsis in genescoding for products involved in protein synthesis, where -5O"/o are essential. Of course, this approach underestimates the number of genes that are essentialfor the yeast to live in the wild, when it is not so well provided with nutrients. i :,i,i:iii.:-....r,-: summarizesthe resultsof a systematic analysis of the effects of loss of gene function in the worm C.elegans.The sequences of individual genes were predicted from the genome sequence, and by targeting an inhibitory RNA against these sequences (see Section ll.l0, RNA Interference Is Related to Gene Silencing) a large set of worms were made in which one (predicted) gene was prevented from functioning in each worm. Detectable effects on the phenotype were only observed for l0% of these knockouts, suggesting that most genesdo not play essentialroles. There is a greater proportion of essential genes (2I%) among those worm genesthat have counterparts in other eukaryotes, suggesting that widely conserved genes tend to play more basic functions. There is also an increased proportion of essentialgenesamong those that are present in only one copy per haploid genome, compared with those where there are multiple copies of related or identical genes. This suggeststhat many of the multiple genes might be relatively recent duplications that can substitute for one another's functions. Extensive analysesof essentialgene number in a higher eukaryote have been made in Drosophilathrough attempts to correlate visible aspectsof chromosome structure with the number of functional genetic units. The notion that this might be possible arose originally from the presenceof bands in the polytene chromosomes of. D. melanogaster.(These chromosomes are found at certain developmental stagesand represent an unusually extended physical form, in which a series of bands [more formally called chromomeresl are evident; see Section 28.I0, Polytene Chromosomes Form Bands.) From the early concept that the bands might represent a I i n e a r o r d e r o f g e n e s ,w e h a v e c o m e t o t h e attempt to correlate the organization of genes with the organization of bands. There are -5000 bands in the D melanogaslerhaploid set; they vary in size over an order of magnitude, but on averagethere is -20 kb of DNA per band. The basic approach is to saturate a chromosomal region with mutations. Usually the mutations are simply collected as lethals, without analyzing the cause of the lethality. Any mutation that is lethal is taken to identify a locus that
is essentialfor the organism. Sometimes mutations cause visible deleterious effects short of lethality, in which casewe also count them as identifying an essentiallocus. When the mutations are placed into complementation groups, the number can be cornpared with the number of bands inthe region, orindividual complementation groups may even be assignedto individual bands.The purpose of these experiments has been to determine whether there is a consistent relationship between bands and genes.For example, does every band crlntain a single gene? Totaling the analyses that have been carried out over the past l0 years,the number of lethal complementation groups is -70% of the number of bands.It is an open question whether there is any functional significance to this relationship. Irrespective of the cause, the equivaIence gives us a reasonable estimate for the lethal gene number of -3600. By any measure, the number of lethal loci in Drosophilais significantly less than the total number of genes. If the proportion of essential human genes is similar to other eukaryotes, we would predict a range of 4000 to 8000 genes, in which mutations would be lethal or produce evidently damaging effects.At present, I300 geneshave been identified in which mutations cause evident defects.This is a substantial proportion of the expected total, especiallyin view of the fact that many lethal genesmay act so early that we never seetheir effects.This sort of bias may also explain the results in iii";i-:lii* :r:.,which show that the majority of known genetic defects are due to point mutations (where there is more Iikely to be at least some residual function of the gene). How do we explain the survival of genes whose deletion appears to have no effect? The most likely explanation is that the organism has alternative ways of fulfilling the same function. The simplest possibility is that there is redundancy, and that some genesare present in multiple copies.This is certainly true in some cases, in which multiple (r:elated) genes must be knocked out in order to produce an effect. In a slightly more complex scenario, an organism might have two separate pathways capable of providing some activity. Inactivation of either pathway by itself would not be damaging, but the simultaneous occurrence of mutations in genes from both pathways would be deleterious. Such situations can be tested by combining mutations. In principle, deletions in two genes, neither of which is lethal by itself, are introduced into the same strain. If the double
mutant dies, the strain is called a synthetic lethal. This technique has been used to great effect with yeast, where the isolation of double mutants can be automated. The procedure is called synthetic genetic array analysis (SGA). i.ii;;ri!ilir.,i,isummarizes the results of an analysis in which an SGA screen was made for each oI 132 viable deletions by testing whether it could survive in combination with any one of 4,700 viable deletions.Every one of the test genes had at least one partner with which the combination was lethal, and most of the test genes had many such partners; the median is -25 partners, and the greatest number is shown by one test gene that had 146 lethal partners. A small proportion (-I0%) of the interacting mutant pairs code for proteins that interact physically. This result goes some way toward explaining the apparent lack of effect of so many deletions. Natural selection will act against these deletions when they find themselves in lethal pairwise combinations. To some degree, the
Missense/nonsense Splicing Regulatory
587" 10% <1o/o
Smalldeletions Smallinsertions
16% 6%
Largedeletions Largerearrangements
5% 2o/o
genes genetic in human defects f;i{l!ii,'-11.;iiMostknown Themajoritydirecttyaffect aredueto point mutations. aredueto insertions, Theremainder theprotein sequence. sizes. of varying detetions, or rearrangements
7 q)
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Ez z1 10 20 30 40 50 60 70 80 90 100 110 120 130 140 genes(outof 4700) Numberof lethalinteracting , ' i i * t . j F i : i : . ,Ai i[rt 1 3 2 m u t a n t t e s t g e n e s h a v e s o m e c o m b i n a t i o n s t h a t a r e l e t h a [ Thechart mutations. with eachof 47Q0nontethaI whenthey arecombined genes therearefor eachtestgene. showshowmanylethalinteracting
AreEssentiat? 9 1 5.9 HowManvGenes
organism has protected itself againstthe damaging effectsof mutations by building in redundancy. However, it pays a price in the form of accumulating the "genetic load" of mutations that are not deleteriousin themselves,but that may cause serious problems when combined with other such mutations in future generations. The theory of natural selection would suggestthat the loss of the individual genesin such circumstancesproduces a sufficient disadvantage to maintain the active gene during the course of evolution.
@
Genes AreExpressed at WidetyDifferingLevels
. In anygivencet[.mostgenesareexpressed at a lowlevet. . 0nlya smatlnumber of genes, whoseproducts are speciatized for the ce[[type,arehigh[yexpressed.
The proportion of DNA representedin an nRNA population can be determined by the amount of the DNA that can hybridize with the RNA. Such a saturation analysis typically identifies -loh of the DNA as providing a template for nRNA. From this we can calculate the number of genes,so long as we know the averagelength of an nRNA. For a lower eukaryote such as yeast, the total number of expressedgenes is -4000. For somatic tissues of higher eukaryo t e s ,t h e n u m b e r u s u a l l y i s 1 0 , 0 0 0t o I 5 , 0 0 0 . The value is similar for plants and for vertebrates. (The only consistent exception to this type of value is presented by mammalian brain, for which much larger numbers of genesappear to be expressed,although the exact quantitation is not certain.) I(inetic analysisof the reassociationof an RNA population can be used to determine its sequence complexity. This type of analysis typically identifiesthree components in a eukaryotic cell. Just as with a DNA reassociationcurve, a single component hybridizes over about two decadesof Rot (RNA concentrationx time) values, and a reaction extending over a greater range must be resolvedby computer curvefitting into individual componenrs.Again, this representswhat is really a continuous spectrum of sequences. An example of an excessmRNA x cDNA reaction that generates three components is given in i il.ii.Jiitir i',,i:i:
C H A P T E5RG e n o m S e e q u e n c easn dG e n eN u m b e r s
. The first component has the same characteristicsas a control reaction of ovalbumin mRNA with its DNA copy. This suggeststhat the first component is in fact just ovalbumin nRNA (which indeed occupies about half of the messengermass in oviduct tissue). . The next component provides 15% of the reaction, with a total complexity of l5 kb. This correspondsto7 ro 8 mRNA speciesof averagelength 2000 bases. . The last component provides 35o/oof the reaction, which corresponds to a complexity of 26 Mb. This corresponds t o - 1 3 , 0 0 0 m R N A s p e c i e so f a v e r a g e length 2000 bases. From this analysis, we can see that about half of the mass of mRNA in the cell represents a single mRNA, -I5o/o of the mass is provided by a mere 7 to 8 mRNAs, and -35oh of the mass is divided into the large number of 13,000 nRNA species.It is therefore obvious that the mRNAs comprising each component must be present in very different amounts. The average number of molecules of each mRNA per cell is called its abundance. It can be calculated quite simply if the total mass of RNA in the cell is known. In the examole shown
First Second component component 50% at 15Yoat Roto 0.0015 Roto 0.04
Final component 35% at Rots 30
4zc
oo c N
Eso o
f25
j'iii:ij*i:1r.il; Hybridization between excess mRNA andcDNA identifies severaI components in chickoviductcetts,each characterized bythe Rot172 of reaction.
in Figure 5.2),Ihe total mRNA can be accounted for as I00,000 copies of the first component (ovalbumin nRNA), 4000 copies of each of the 7 to 8 mRNAs in the second component, but only -5 copies of each of the 13,000 mRNAs that constitute the last component. We can divide the nRNA population into two general classes, according to their abundance: . The oviduct is an extreme case,with so much of the mRNA represented in only one species,but most cells do contain a small number of RNAs present in many copies each. This abundant mRNA component typically consists of
HowManyGenes AreExpressed? mRNAs expressed at low [evetsovertapextensivety whendifferentcetltypesarecompared. Theabundantty expressed mRNAs areusualty specificfor the ce[[type. -10.000expressed genesmaybe common to most cetltypesof a highereukaryote. Many somatic tissuesof higher eukaryotes have an expressed gene number in the range of 10,000 to 20,000. How much overlap is there between the genes expressedin different tissues?For example, the expressedgene number of chick liver is -l1,000 to I7,000, compared with the value for oviduct of -l 1.000 to I 5.000. How many of these two sets of genes are identical? How many are specific for each tissue? These questionsare usually addressedby anaIyzingthe transcriptome-the set of sequences represented in RNA. We seeimmediately that there are likely to be substantial differences among the genes expressedin the aburrdant class.Ovalbumin, for example, is synthesized only in the oviduct, and not at all in the liver. This means lhat 50o/o
of the mass of mRNA in the oviduct is specific to that tissue. The abundant mRNAs represent only a small proportion of the number of expressed genes, though. In terms of the total number of genes of the organism, and of the number of changes in transcription that must be made between different cell types, we need to know the extent of overlap between the genes represented in the scarcemRNA classesof different cell phenotypes. Comparisons between different tissuesshow that, for example, -75'/o of the sequences expressedin liver and oviduct are the same. In other words, -I2,000 genes are expressedin both liver and oviduct, -5000 additional genes are expressed only in liver, and -3000 additional genes are expressedonly in oviduct. The scarce mRNAs overlap extensively. Between mouse liver and kidney, -90% of the scarcemRNAs are identical, Ieaving a difference between the tissues of only I000 to 2000 in terms of the number of expressed genes. The general result obtained in several comparisons of this sort is that only -l}oh of the nRNA sequencesof a cell are unique to it. The majority of sequences are common to manyperhaps even all-cell types. This suggests that the common set of expressedgene functions, numbering perhaps -10,000 in mammals, comprise functions that are needed in all cell types. Sometimes this type of function is referred to as a housekeeping gene or constitutive gene. It contrasts with the activities represented by specialized functions (such as ovalbumin or globin) needed only for particular cell phenotypes. These are sometimes called luxury genes.
Expressed GeneNumber CanBeMeasured EnMasse "Chip"technotogy to betaken atlowsa snapshot in a yeast of the expression of the entiregenome cetl. -75olo is (-4500genes)of the yeastgenome growthconditions. expressed undernormaI comparisons of detaited Chiptechnotogy attows (for exampte) the retatedanjmalceltsto determine betweena normalcetl in expression differences anda cancer cett.
EnMasse 9 3 CanBeMeasured GeneNumber 5.12 Exnressed
The most powerful new technology uses chips that contain high-density oligonucleotide arrays (HDAs). Their construction is made pos2000 sibleby knowledge of the sequenceof the entire 4OTo genome. In the case of.S. cerevisiae,each of 6 I 8 I o ORFs is represented on the HDA by 20 25-mer 357o z(E 1500 oligonucleotides that perfectly match the E s e q u e n c e o f t h e m e s s a g ea n d 2 0 m i s m a t c h oligonucleotides that differ at one baseposition. q) 1000 18% The expression level of any gene is calculated E l z by subtracting the average signal of a mismatch 500 from its perfect match partner. The entire yeast 8% genome can be represented on four chips. This technology is sensitive enough to detect tran<1 >100 scripts of 5460 genes (-90% of the genome), 1-10 10-100 and shows that many genes are expressedat Copiesof mRNAper yeastcell low levels,with abundancesof 0.I to 0.2 tran=:5;*fti.1.t& Theabundances ofyeastmRNAs varyfrom scripts/cell. An abundance of 100percett(codingfor the moreabundant s c r i p ta t a n y g i v e n m o m e n t . protei ns). The technology allows not only measurement of levels of gene expression,but also detection of differencesin expression in mutant cells compared with wild-type cells growing under different growth conditions, and so on. The results of comparing two states are expressed in the form of a grid, in which each square represents a particular gene and the relative change in expression is indicated by color. The left part of fitil!flfl 5.?5shows the effect of a mutation in RNA polymerase II, the enzyme that produces nRNA, which as might be expected causesthe expression of most genesto be heavily reduced. By contrast, the right part shows that a mutai:**fti l:.=5 HDAanalysis attows change in expression tion in an ancillary component of the transcripof eachgeneto bemeasured. Eachsquare represents one tion apparatus (SRB10)has much more restdcted gene(topleftis firstgeneon chromosome I, bottomright effects,causing increasesin expression of some is lastgeneon chromosome XVI).Change in expression genes. retative to wil.dtypeis indicated by red(reduction), white The extension of this technology to animal (nochange), or btue(increase). Photos courtesy of RickA. Young, Whitehead Institute,Massachusetts Instituteof cells will allow the general descriptions based on Iechno[ogy. RNA hybridization analysis to be replaced by exact descriptions of the genes that are Recent technology allows more systematic and expressed,and the abundances of their products, in any given cell type. A gene expression map of.D. melanogasterdetecls transcriptional activity in some stage of the life cycle in almost all tag to be used to identify each mRNA. The tech(93"/") of predicted genes and shows that 40'h nology then allows the abundance of each tag have alternatively spliced forms. to be measured.This approach identifies4,665 expressedgenes in S. cerevisiae growing under normal conditions, with abundances varying from 0.3 to >200 transcripts/cell.This means that -75o/o of the total gene number (-6000) is Genomes that have been sequenced include expressedunder these conditions. i:irl{iS[:$.irtri manybacteria and archaea,yeasts,and a worm, summarizes the number of different mRNAs atly, a mouse, and man. The minimum numthat is found at each different abundance level. ber of genes required to make a living cell (an
:il**riT,'#x',#i':idnrJ'"'Jffi
Summary
94
C H A P T E5RG e n o m S e e q u e n c easn d G e n eN u m b e r s
obligatory intracellular parasite) is -470. The minimum number required to make a free-living cell is -1700. A typical gram-negativebacterium has -1500 genes. Strains of E. coli vary from 4300 to 5400 genes.The averagebacterial gene is -1000 bp long and is separatedfrom the next gene by a spaceof - I 00 bp. The yeasts S. pombe and S. cerevisiae have 5000 and 6000 genes, respectively. Although the fly D. melanogasleris a more complex organism and has a larger genome than the worm C. elegans,the fly has fewer genes ( 1 3 , 6 0 0 ) t h a n t h e w o r m ( 1 8 , 5 0 0 ) .T h e p l a n t Arabidopsishas 25,000 genes, and the lack of a clear relationship between genome sizeand gene number is shovm by the fact that the rice genome is 4x larger but contains only a 507o increase in gene number, to -40,000. Mouse and man each have 20,000 to 30,000 genes,which is much less than had been expected. The complexity of development of an organism may depend on the nature of the interactions between genesas well as their total number. About 8000 genesare common to prokaryotesand eukaryotesand are likely to be involved in basic functions. A further 12,000 genes are found in multicellular organisms.Another 8000 genesare added to make an animal, and an additional8000 (largely involved with the immune and nervous systems)are found in vertebrates. In each organism that has been sequenced,only -50% of the genes have defined functions. Analysis of lethal genes suggeststhat only a minority of genesis essentialin each organism. The sequences comprising a eukaryotic genome can be classifiedin three groups: nonrepetitive sequencesare unique; moderately repetitive sequencesare dispersedand repeated a small number of times in the form of related, but not identical, copies; and highly repetitive sequencesare short and usually repeatedas tandem arrays. The proportions of the types of sequence are characteristicfor each genome, although larger genomes tend to have a smaller proportion of nonrepetitive DNA. Almost 50% of the human genome consists of repetitive sequences,the vast majority corresponding to transposon sequences.Most structural genes are located in nonrepetitive DNA. The complexity of nonrepetitive DNA is a better reflection of the complexity of the organism than the total genome complexity; nonrepetitive DNA reaches a maximum complexity of -2 x l0e bp. Genes are expressedat widely varying levels. There may be I05 copies of nRNA for an abundant gene whose protein is the principal
product of the cell, I03 copies of each mRNA for <10 moderately abundant messages,and <10 copiesof each nRNA for >10,000 scarcely expressedgenes. Overlaps between the mRNA populations of cells of different phenotypes are extensive; the majority of mRNAs are present in most cells. Non-Mendelian inheritance is explained by the presence of DNA in organelles in the cytoplasm. Mitochondria and chloroplastsboth represent membrane-bounded systemsin which some proteins are synthesized within the organelle, whereas others are imported. The organelle genome is usually a circular DNA that codesfor all of the RNAs and for some of the proteins that are required. Mitochondrial genomesvary greatly in size, from the l6 kb minimalist mammalian genome to the 570 kb genome of higher plants. It is assumedthat the larger genomes code for additional functions. Chloroplast genomes range from 120 to 200 kb. Those that have been sequencedhave a similar organization and coding functions. In both mitochondria and chloroplasts,many of the major proteins contain some subunits synthesizedin the organelle and some subunits imported from the cytosol. Mammalian mtDNAs are transcribed into a single transcript from the major coding strand, and individual products are generated by RNA processing.Rearrangementsoccur in mitochondrial DNA rather frequently in yeast, and recombination between mitochondrial or between chloroplast genomes has been found. Ttansfers of DNA have occurred from chloroplasts or mitochondria to nuclear genomes.
References 0ver Range GeneNumbers BacteriaI an 0rderof Magnitude Reviews Bentley,S. D. and Parkhill,J. (2004).Comparative genomicstructureof prokaryotes.Annu.Rev. Genet. )8, 77l-792. Hacker,J. and l(aper,J. B. (2000).Pathogenicity islandsand the evolution of microbes.Annu Rev.Microbiol.54, 641-679. Research Blattner,F.R. et al. (1997).The completegenome 277, coliI(12. Science sequenceof.Escherichia t453-1474. Deckert,G. et al. (I99S).The completegenomeof the hyperthermophilicbacteriumAquifexaeoli' cus.Nature392, 353-358.
References 9 5
Galibert, F. et al. (2001). The composite genome oi the legume symbiont Sinorhizobiummeliloti Science 29), 668-672.
![
TotalGeneNumber Is Knownfor Several Eukaryotes
Resea rch Adams, M. D. et al. (2000). The genome sequence of D- melanogaster. Science 287 , 2185-2195. ArabidopsisInitiative (2000). Analysis of the genome sequence of the floweringplant Arabidopsisthaliana Nature 408, 7 9 6-81 5. C elegansSequencingConsortium (1998). Genome sequence of the nematod,e C elegans:aplatform for investigating biology. Science282, 20t2-2022. Duffy, A., and Grof, P. (2001). Psychiatricdiagnosesin the context of genetic studiesof bipolar disorder.BipolarDisord.),270-275. Dujon, B. et al. (19941.Complete DNA sequence of yeast chromosome Xl. Nature j69, )7t-378. Goff, S. A. et al. (2002). A draft sequence of the rice genome (Oryza sativa L ssp.japonica) Scie n c e2 9 6 , 9 2 - l 1 4 Johnston, M. et al. (19941.Complete nucleotide sequence oI S. cerevisiae chromosome VIII. Scence265, 2077-2082. I(ellis, M., Patterson,N., Endrizzi, M., Birren, B., and Lander, E. S. (2003). Sequencingand comparison of yeast speciesto identify genes and regulatory elements. Nature 423, 241-254. Oliver, S. G. et al. (19921.The complete DNA sequence of yeast chromosome IlI. Nature 357,)8-46. Wilson, R. et al. (1994\.22 Mb oI conriguous nucleotide sequence from chromosome III of C elegans.Nature )68, )248. Wood, V. et al. (2002). The genome sequenceof S pombe Nature 415,871-880.
HowManyDifferent Types of Genes AreThere? Reference Rual, J. F., Venkatesan, I(., Hao, T., HirozaneICshikawa, T., Dricot, A., Li, N., Berriz, G. F., Gibbons,F. D., Dreze,M., Ayivi-Guedehoussou, N., et al. (2005). Towards a proteomescale map of the human protein-protein interaction network. Nature437. l17)-1178. Reviews Aebersold,R. and Mann, M. (2003). Mass spectrometry-based proteomics. N ature 422, 198-207. Hanash. S. (2003). Diseaseproreomics.Nature422, 226-2)2 Phizicky, E., Bastiaens, P. L, Zhtt, H., Snyder, M., and Fields,S. (2003). Prorein analysison a proteomic scale.Nature 422, 208-21 5.
96
CHAPTER 5 Genome Sequences andGeneNumbers
Sali, A., Glaeser,R., Earnest, T., and Baumeister, W (2003). From words to literature in structural proteomics. Nature 422, 216-225. Resea rch Agarwal, S., Heyman, J. A., Matson, S., Heidtman, M., Piccirillo, S., Umansky, L., Drawid, A., Jansen, R., Liu, Y., Miller, P., Gerstein, M., Roeder,G. S., and Snyder,M. (2002). Subcellular localization of the yeast proteome. Genes Dev.16,707-719. Arabidopsis Initiative (2000). Analysis of the genome sequence of the floweringplant Arabidopsisthaliana. N ature 408, 7 9 6-815. Gavin, A. C. et al. (2002). Functional organization of the yeast proteome by systematic analysis of protein complexes.Nature 4I5, l4l-147. Ho, Y. et al. (2002). Systematic identification of protein complexes in S. cerevisiae by mass spectrometry. Nature 415, 180-183. Rubin, G. M. et al. (2000). Comparative genomics of the eukaryotes. Science 287, 2204-2215. Uetz,P. et al. (2000). A comprehensiveanalysisof protein-protein interactions in S. cerevisiae Nature4Oj, 623-6)0. Venter, J. C. et al. (200I). The sequenceof the human genome. Science291, I 304-1 150.
TheHuman HasFewer Genome Genes ThanExoected Resea rch Clark,A. G.et al.(2003). Inferringnonneutral evolution f rom human-chimp-mouse orthologous gene trios. Science302, 1960-198. Hogenesch,J. B., Ching, I(. A., Batalov S., Su, A. I., Walker, J. R., Zhou, Y., I(ay, S. A., Schultz, P. G., and Cooke, M. P. (2001). A comparison of the Celera and Ensembl predicted gene sets reveals little overlap in novel genes.Cell 106, 4l)4I5. International Human Genome Sequencing Consortium (2001). Initial sequencingand analysis of the human genome. Nature 409, 860-921. International Human Genome Sequencing Consortium (2004). Finishing the euchromatic sequence of the human genome. Nature 4jl , 9)t-945. Venter, J. C. et al. (2001). The sequenceof the human genome. Science 291, 1304-1350. Waterston et al. (2002). Initial sequencingand comparative analysis of the mouse genome. Nature 420. 520-562.
HowAreGenes and0therSequences Distributedin the Genome? Reference Nusbaum,C.,Cody,M. C.,Borowsky,M. L., I(amal,M., I(odira, C. D., Taylor,T. D., Whittaker,C. A., Chang,J. L., Cuomo,C. A., Dewar,I(., et al. (2005).DNA sequence and
analysis of human chromosome 18. Nature 4)7, 55t-555.
TheY Chromosome HasSeveral Mate-Specific Genes Resea rch Skaletsky, H. et al. (2003).Themale-specific region of the human Y chromosome is a mosaic of discrete sequence classes.Nature 42), 825-837.
MoreComptex Species Evotveby Adding NewGeneFunctions Reference The Chimpanzee Sequencing and Analysis Consortium (2005). Initial sequence of the chimpanzee genome and comparison with the human genome. Nature 4)7, 69-87.
HowManyGenes AreEssentia[? Research Giaever et al. (2002). Functional profiling of the genome. Nature 418, )87-391. S cerevisiae Goebl, M. G. and Petes,T. D. (1986). Most of the yeast genomic sequencesare not essential for cell growth and division. Cell46,98)-992. Hutchison, C. A. et al. (1999). Global transposon mutagenesis and a rninimal mycoplasma genome. Science286, 2165-2169. Kamath, R. S., Fraser, A. G., Dong, Y., Poulin, G., Durbin, R., Gotta, M., Kanapin, A., Le Bot, N., Moreno, S., Sohrmann, M., Welchman, D. P., Zipperlen, P., and Ahringer, J. (2001). Systematic functional analysis of the C. elegans genome using RNAi. Nature 421,231-2]7. Tong, A. H. et al. (2OO4l. Global mapping of the yeast genetic interaction network. Science)0),
Diftering at Widel.y AreExpressed Genes Leve[s R e s erach Hastie,N. B. and Bishop,J. O. (1976\.The expresof mRNA in sion of three abundanceclasses Cell9,761-774. mousetissues.
CanBeMeasured Expressed GeneNumber En Masse Reviews Mikos,c. L. c. and Rubin,G. M. (I996). The role of the genomeprojectin determininggene function: insightsfrom model organisms.Cel/ 86,52t-529. Young,R. A. (2000).Biomedicaldiscoverywith DNA arrays.Cell102,9-L5. R e s erac h the reguHolstege, F. C. P.et al. (1998).Dissecting latory circuitry of a eukaryoticgenome. Cell95, 7 17-7 28. Hughes,T. R.,Marton,M. J., Jones,A. R.,Roberts, C. J., Stoughton,R., Armour, C. D., Bennett, H. A., Coffey,E., Dai,H., He, Y.D., Kidd,M. J., Ifing, A, M., Meyer,M. R., Slade,D., Lum, P.Y., Stepaniants,S. B., Shoemaker,D. D. et al. (2000).Functionaldiscoveryvia a compendium of expressionprofiles.Cell102, t09-t26. Stolc,V. et al. (2004).A geneexpressionmap for the euchromatic genome of.Drosophila 306,65 5-660. melanogaster. Science of Velculescu,V. E. et al. (1997).Characterization Cell88, 243-251. the yeasttranscriptosome.
808-8r 3.
References 9 7
Clusters andRepeats C H A P T EO RU T L I N E Introduction
GE
98
GeneDupticationIs a MajorForcein Evolution e Duplicated genesmaydiverge genes to generate different or onecopymaybecome inactive. GlobinCtusters Are Formedby Duplicationand Divergence . Attgtobingenes aredescended by duptication andmutation froman ancestral genethat hadthreeexons. . Theancestral genegaveriseto myogtobin, leghemogtobin, andcrandB gtobins. o Thec- andB-gtobin genes separated in the periodof early vertebrate evotution,afterwhichduptications generated the individuaI ctusters of separate cx-andB-tikegenes. o 0ncea genehasbeeninactivated by mutation.it mayaccumutatefurthermutations andbecome a pseudogene, whichis homotogous to the activegene(s)but hasnofunctionalrote. Sequence Divergence Is the Basisfor the Evo[utionary Clock r Thesequences of homologous genes in different species varyat reptacement sites(wheremutation causes amino acidsubstitutions) andsilentsites(wheremutation does notaffectthe proteinsequence). r Mutations accumutate at silentsites-10x fasterthanat reptacement sites. o Theevolutionary divergence between two proteins.is measuredby the percent of positions at whichthe correspondingaminoacidsdiffer. o Mutations accumutate at a moreor lessevenspeedafter genes separate, sothat the divergence between anypairof gtobinsequences is proportionaI to thetimesincetheir genesseparated. The Rateof NeutralSubstitutionCanBe Measured from Divergence of Repeated Sequences o Therateof substjtutionperyearat neutralsitesis greater i n t h em o u steh a ni n t h e h u m a g nenome. Pseudogenes Are DeadEndsof Evolution r Pseudogenes haveno codingfunction,buttheycanbe recognized by sequence similarities withexisting functional genes. Theyarisebythe accumutation of mutations in (formerly)functionaI genes. UnequaICrossing-over Rearranges GeneCtusters o Whena genome contains a clusterof genes with retated sequences, mispairing genes between nonatlelic cancause unequal crossing-over. Thisproduces a detetion in one recombinant chromosome anda conesponding duptication in the other.
r Differentthatassemias arecaused by variousdetetions that genes. eliminate o- or B-globin Theseverity of the disease depends on the individual deletion. Genesfor rRNAFormTandemRepeats o Ribosomal RNAis codedby a largenumberof identicalgenes repeated that aretandemly to formoneor moreclusters. r EachrDNActuster is organized sothattranscription units givinga joint precursor to the majorrRNAs atternatewith nontranscribed spacers. The RepeatedGenesfor rRNAMaintainConstant Seouence r Thegenes in an rDNActuster a[[havean identical sequence. r Thenontranscribed spacers consistof shorterrepeat'ing un'itswhosenumber variessothat the lengths of individuaI spacers aredifferent. Crossover FixationCoutdMaintainIdenticaIRepeats . Unequal crossing-over changes the sizeof a clusterof tandemreDeats. . IndividuaI repeating unitscanbeetiminated or canspread throughthe cluster. SatelliteDNAsOftenLiein Heterochromatin . Hightyrepetitive DNAhasa veryshortrepeating sequence andno codingfunction. . It occurs in largeblocks that canhavedistinctphysical properties. I It is oftenthe majorconstituent of centromeric heteroch romatin. ArthropodSatettitesHaveVeryShortIdenticaIRepeats r Therepeating un'itsof arthropod satetliteDNAs areon[ya fewnucleotides long.Mostof the copiesof thesequence are identical. Mammatian SatetlitesConsistof HierarchicaI Repeats r Mouse satettite DNAhasevotved by duplication andmutation of a shortrepeating unitto givea basicrepeating unit of 234bp in whichthe originaIha[f.quarter, andeighth repeats canbe recognized. Minisatetlites Are UsefuIfor GeneticMapping o Thevariationbetweenmicrosatettites or minisateltites in genomes individuaI canbe usedto identifyheredity unequivocatly by showing that 50%of the bandsin an individualarederived parent. froma particular Summarv
Introduction A set of genes descendedby duplication and variation from some ancestralgene is called a gene family. Its mernbers may be clustered together or dispersedon different chromosomes (or a combination of both). Genome analysis shows that many genesbelong to families; the 25,000 genesidentified in the human genome fall into -15,000 families, so the averagegene h a s a c o u p l e o f r e l a t i v e si n t h e g e n o m e ( s e e Figure 5.7). Gene fam;iliesvary enormously in the degree of relatednessbetween members, from those consistingof multiple identical members to those for which the relationship is quite distant. Genesare usu;llly related only by their exons, with introns having diverged (see Sect i o n 3 . 5 , E x o n S e q u e n c e sA r e C o n s e r v e db u t Introns Vary). Genesmay alsobe related by only some of their exons, w.hereasothers are unique (seeSection 3.9, Some Exons Can Be Equated with Protein Functions). The initial event tl:ratallows related exons or genes to develop is a duplication, when a copy is generatedof some sequencewithin the genome. Tandem duplication (when the duplicatesremain together) may arise through errors in replication or recombination. Separationof the duplicates can occur by a translocation that transfers material from one chromosonre to another. A duplicate at a new location may also be produced directly by a transposition event that is associatedwith copying a region of DNA from the vicinity of the transposon. Duplications may appl.geither to intact genesor to collectionsof exons or even individual exons. When an intact gene is involved, the act of duplication generates two copies of a gene whose activities are indistinguishable,but then usually the copies diverge as each accumulates different mutations. The members of a well-related structural gene family usually h;rve related or even identical functions, although they may be expressed at different times or in different cell types.As a result, different globin proteins are expressed in embryonic and adult red blood cells,whereas different actinsare utilized in muscle and nonmuscle cells.When geneshave divergedsignificantly, or when only some exons are related, the proteins may have different functions. Some gene families consist of identical members. Clusteringis a prerequisitefor maintaining identity between genes,although clustered genesare not necessarilyidentical. Gene clusters range from extremes in which a dupli-
cation has generatedtwo adjacent related genes to caseswhere hundreds of identical geneslie in a tandem array. Extensive tandem repetition of a gene may occur when the product is needed in unusually large amounts. Examples are the genes for rRNA or histone proteins. This creates a specialsituation with regard to the maintenance of identity and the effects of selective pressure. Gene clusters offer us an opportunity to examine the forces involved in evolution of the genome over larger regions than single genes. D u p l i c a t e d s e q u e n c e s ,e s p e c i a l l yt h o s e t h a t remain in the same vicinity. provide the substrate for further evolution by recombination. A population evolves by the classicalrecombination illustrated in ; l'iiil'"iI::ir.i and ii.;1,in which an exact crossing-overoccurs.The recombinant chromosomes have the same organization as the parental chromosome. They contain preciselythe same loci in the same order, but contain different combinations of alleles,providing the raw material for natural selection. However, the existenceof duplicated sequences allows aberrant events to occur occasionally, which changes the content of genes and not just the combination of alleles. Unequal crossing-over (also known as describes a nonreciprocal recombination) recombination event occurring between two sitesthat are not homologous. The feature that makes such events possible is the existence of repeatedsequences.r r,rilillir: "i shows that this allows one copy of a repeat in one chromosome to misalign for recombination with a different
chromatids,2 from each parent
a
D
d^ h
u
Chiasma is causedby crossing-overbetween 2 of the chromatids Two chromosomesremain parental(ABand ab). chromosomes Recombinant containmaterialfrom each parent,and have new genetic combinations(Ab and aB).
of recombinants.
6.1 Introduction
ParentalDNA molecules
I Becombinationintermediate
I Recombinants
FI6URE 6.t Recombinatio nn v o t v epsa i r i n gb e t w e e n comptementary strands of thetwo parentaI duptex DNAs.
The highly repetitive fraction of the genome consistsof multiple tandem copies of very short repeating units. Theseoften have unusual properties. One is that they may be identified as a separate peak on a density gradient analysis of DNA, which gave rise to the name satellite DNA. They often are associatedwith inert regions of the chromosomes and in particular with centromeres (which contain the points of attachment for segregation on a mitotic or meiotic spindle). As a result of their repetitive organization, they show some of the same behavior with regard to evolution as the tandem gene clusters. In addition to the satellite sequences, there are shorter stretchesof DNA called minisatellites that show similar behavior. They are useful in showing a high degree of divergence between individual genomes that can be used for mapping purposes. AII of these events that change the constitution of the genome are rare, but they are significant over the course of evolution.
GeneDuplication Is a MajorForce in Evolution r Dupticated genesmaydivergeto generate different genesor onecopymaybecome inactive.
I
ABCABCABCABCAB CABCABCABCABCABC ABCABCABCABCABCABC
fIfiUFf *"3 UnequaI crossing-over resultsfrompairing betweennonequivatent repeats in regions of DNAconsistingof repeating units.Herethe repeating unjtis the sequence ABC.andthe thirdrepeatof the bluechromosomehasatigned withthefirstrepeatof the blackchromosome. Throughout the regionof pairing,ABCunitsof onechromosome areatigned withABCunitsofthe other generates chromosome. Crossing-over chromosomes with ten andsix repeats each,insteadof the eightrepeats of eacnDarent.
copy of the repeat in the homologous chromosome, instead of with the corresponding copy. When recombination occurs, this increasesthe number of repeats in one chromosome and decreasesit in the other. In effect, one recombinant chromosome has a deletion and the other has an insertion. This mechanism is responsible for the evolution of clustersof related sequences. We can trace its operation in expanding or contracting the size of an array in both gene clusters and regions of highly repeated DNA.
100
CHAPTER 6 Clusters andReoeats
Exons behave like modules for building genes that are tried out in the course of evolution in various combinations. At one extreme, an individual exon from one gene may be copied and used in another gene. At the other extreme, an entire gene, including both exons and introns, may be duplicated. In such a case, mutations can accumulate in one copy without attracting the adverse attention of natural selection. This copy may then evolve to a new function; become expressedin a different time or place from the first copy, or acquire different activities. FISUSE S"4summarizesour present view of the rates at which these processesoccur. There is -l% probability that a given gene will be included in a duplication in a period of one milIion years. After the gene has duplicated, differencesdevelop as the result of the occurrence of different mutations in each copy. Theseaccumulate at a rate of -0.1"/o per million years (see Section 6.4, Sequence Divergence Is the Basis for the Evolutionary Clock). The organism is not likely to nee d to retain two identical copies of the gene. As differences
"?\r\ff\}\F\nl.}\t\if\?\?\}\ years Duplicationoccursat 1%/gene/million
years Divergenceaccumulatesat 0 1"/o/million
Silencingof one copy takes -4 millionyears Active
Silent
II*i-3RE {i"4 Aftera genehasbeendupticated, differences Thegenes mayacquire mayaccumulate between thecopies. d i f f e r e nftu n c t i o n os r o n eo f t h e c o p i e sm a yb e c o m e i nactive.
develop between the duplicated genes,one of two types of event is likely to occur: . Both of the genes become necessary. This can happen either becausethe dif ferences between them generate proteins with different functions, or because they are expressedspecificallyin different times or places. . If this does not Jrappen,one of the genes is likely to be eliminated becauseit will by chance gain a deleterious mutation, and there will be no adverseselectionto eliminate this copy. \pically this takes - 4 million years. In such a situation, it is purely a matter of chance in terms of which of the two copies becomes inactive. (This can contribute to incompatibility between different individuals, and ultimately to speciation, if different copies become inactive in different populations.) Analysis of the human genome sequence shows IhaI -5"/o comprisesduplications of identifiable segments ranging in length from l0 to 100 kb. These duplications have arisen relatively recently, that is, there has not been sufficient time for divergence between them to eliminate their relationship. They include a proportional share (-6%) of the expressedexons, which shows that the duplications are occurring more or less irrespective of genetic con-
tent. The genes in these duplications may be especially interesting because of the implication that they have evolved recently and therefore could be important for recent evolutionary developments (such as the separation of man from monkev).
Are Clusters GLobin by Duplication Formed andDivergence . Attgtobingenesaredescended and by duptication genethat hadthree froman ancestral mutation exons. r Theancestral genegaveriseto myoglobin, andcrandB gtobins. leghemogtobin, o Theq,-andp-gtobingenes in the period separated evotution,afterwhich of eartyvertebrate of clusters generated theindividual dupl.ications separate o- andB-[ikegenes. . 0ncea genehasbeeninactivated it by mutation, a andbecome furthermutations mayaccumutate to the active pseudogene, which'ishomotogous rote. gene(s) but hasnofunctional The most common tlpe of duplication generates a second copy of the gene closeto the first copy' In some cases,the copiesremain associatedand further duplication may generate a cluster of related genes.The best characterizedexample of a gene cluster is presented by the globin genes, which constitute an ancient gene family concerned with a function that is central to the animal kingdom: the transport of oxygen through the bloodstream. RoshanKetabO2L-66950639 The major constituent of the red blood cell is the globin tetramer, which is associatedwith its heme (iron-binding) group in the form of hemoglobin. Functional globin genes in all specieshave the same general structure: they are divided into three exons, as shown previously in Figure 3.7.We conclude that all globin genes are derived from a single ancestral gene, and by tracing the development of individual globin geneswithin and between specieswe may learn about the mechanisms involved in the evolution of gene families. In adult cells, the globin tetramer consists of two identical cr chains and two identical p chains. Embryonic blood cells contain hemoglobin tetramers that are different from the adult form. Each tetramer contains two identical crlike chains and two identical p-like chains, each of which is related to the adult polypeptide and is later replaced by it. This is an example of
andDivergence 101 by Duplication AreFormed 6.3 GtobinCtusters
functional ( gene, two d genes,two cr,nonfunctional genes, and the 0 gene of unknown func(1 rfct$tr ct2 o1 0 b genescode for the same protein. tion. The two clt, + trcluster 1i,,,.+++ TWo (or more) identical genes present on the e GrA't_F 6 B same chromosome are described as nonallelic + ++ ++ Bcluster I copres. The details of the relationship between embryonic and adult hemoglobins vary with 10 20 30 40 50 kb the organism. The human pathway has three stages:embryonic, fetal, and adult. The distinc+Functionalgene ; Pseudogene tion between embryonic and adult is common to mammals, but the number of pre-adult stages ; : : ' : . , :, , E a c ho f t h e s - t i k ea n dB - t i k eg t o b i ng e n e varies.In man, zeta and alpha are the two s-like families is organized intoa singte cluster thatincludes funcchains. Epsilon, gamma, delta, and beta are the tionaIgenes andpseudogenes (y). il:.i,shows how the chains B-like chains. l'g{iLiFii: are expressedat different stagesof development. In the human pathway, ( is the first o-like chain to be expressed,but it is soon replaced by Embryonic(<8 weeks):proteins €2{2 L2"12 o2e2 cr.In the B-pathway, e and y are expressedfirst. (o0 *& -e*"-*,.> f' with 6 and B replacing them larer. In adults, the | ., ^y^y € cr2p2form provides 97'h of the hemoglobin, -4' *18$@. ++ a262provides -2o/", and -1"/o is provided by persistence of the fetal lorm a2y2. Fetal(3-9 months):proteins o2"y2 ct0 What is the significance of the differences r-*--**+ + | between embryonic and adult globins? The f1 *&"&" .> + embryonic and fetal forms have a higher affin+ ity for oxygen. This is necessary in order to Adult (frombirth):proteins a262 azgz obtain oxygen from the mother's blood. This u0 .> _+,*w+ I explains why there is no equivalent in (for 6B example) chicken, for which the embryonic .> ->+ @.@ stages occur outside the body (that is, within + Functional gene ; Pseudogene the egg). *&. Activegene Functional genes are defined by their expression in RNA and ultimately by the proi r r . r : . : : i:: , ; . D i f f e r e nht e m o g l o b igne n e sa r ee x p r e s s e d teins for which they code. Nonfunctional genes duringembryonic, feta[,andaduttperiods of human devetopare defined as such by their inability ro code for ment. proteins; the reasons for their inactivity vary, and the deficiencies may be in transcription or developmental control, in which different genes translation (or both). They are calledpseudoare successivelyswitched on and off to provide genes and are indicated by the symbol y. A alternative products that fulfill the same funcsimilar general organization is found in other t i o n a t d i f f e r e n tt i m e s . vertebrate globin gene clusters, but details of The division of globin chains into a-like and the types, numbers, and order of genesall vary, p-like reflects the organization of the genes. as illustrated in il.{i,:*l{iii..t.Each cluster contains Each type of globin is coded by genes organized both embryonic and adult genes. The total into a single cluster. The structures of the two lengths of the clusters vary widely. The longest clustersin the higher primate genome are illusis found in the goat, where a basic cluster of trated in i:ii;:-:r'i.i ii,:1. four genes has been duplicated twice. The disStretching over 50 kb, the B cluster contribution of active genes and pseudogenes diftains five functional genes (e, two y, 5, and p) fers in each case,illustrating the random nature and one nonfunctional gene (yp). The two y of the conversion of one copy of a duplicated gene into the inactive state. The characterization of these gene clusters positionI16, whereasthe A varianthasalanine. makes an important general point. Theremay The more compactcr clusterextendsover bemoremembersof a genefamily, bothfunctional and 28 kb and includesone active( gene,one nonnonfunctional, thqn we would suspecton the basis
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702
CHAPTER 6 Ctusters andRepeats
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. At silent sites,mutation only substitutes one synonym codon for another, so there is no change in the protein. Usually the replacement sites account for 75'/. oIa coding sequenceand the silent sitesprovide 257o. In addition to the cr:ding sequence, a gene contains nontranslated regions. Here again. mutations are potentially neutral, apart from their effects on either secondary structure or (usually rather short) regulatory signals. Although silent mutations are neutral with regard to the protein, they could affect gene expression via the sequence change in RNA. For example, a change in secondarystructure might influence transcription, processing, or translation. Another possibility is that a change in synonym codons calls for a different IRNA to respond, influencing the efficiency of translation. The mutations in replacementsitesshould correspond with the amino acid divergence (determined by the percent of changesin the protein sequence).A nucleic acid divergenceof O.45ohat replacement sites correspondsto an amino acid divergence of I % (assuming that the average number oli replacement sites per codon is 2.25). Actually, the measured divergence underestimates the differencesthat have occurred during evolution, becauseof the occurrence of multiple events at one codon. Usually a correction is made for:this. To take the example of the human p- and 6-globin chains,there are l0 differencesin 146 residues, a divergence of 6.9oh. The DNA sequencehas 3l changesin44l residues.However, these changesare distributed very differently in the replacementand silent sites.There are I I changes in the 330 replacement sites, but 20 changesin only I I I silent sites.This gives (corrected)rates of divergence of 3.7'/. in the replacement sites and )2o/o in the silent sites. almost an order of magnitude in difference. The striking differe:ncein the divergence of replacement and silent sites demonstratesthe existence of much greater constraints on nucleotide positions that influence protein constitution relative to those that do not. So probably very few of the amino acid changes are neutral. Suppose we take the rate of mutation at silent sites to indicate the underlying rate of mutational fixation (this assumesthat there is no selectionat all at the silent sites).Then over the period since the p and 6 genes diverged, there should have been chansesat 32o/oof the
330 replacementsites,for a total of 105.All but l l of them have been eliminated, which means IhaI"-90o/o of the mutations did not survive. The divergence between any pair of globin sequencesis (more or less)proportional to the time sincethey separated.This provides an evolutionary clock that measures the accumulation of mutations at an apparently even rate during the evolution of a given protein. The rate of divergencecan be measured as the percent difference per million years, or as its reciprocal, the unit evolutionary period (UEP), the time in millions of years that it takes for lo/o divergence to develop. Once the clock has been established by pairwise comparisons between species(remembering the practical difficulties in establishing the actual time of speciation), it can be applied to related geneswithin a species.From their divergence,we can calculate how much time has passedsince the duplication that generatedthem. By comparing the sequencesof homologous genesin different species,the rate of divergence at both replacement and silent sites can i:"1. be determined, as plotted in i'riir,iiiir In pairwise comparisons, there is an average divergence of I 0 % in the replacement sites of either the a- or B-globin genesof mammals that have been separatedsince the mammalian radiation occurred -85 million years ago. This correspondsto a replacement divergencerate of 0.12% per million years. The rate is steady when the comparison is extended to genes that diverged in the more
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Ctock Is the Basisfor the Evolutionary Divergence 6.4 Sequence
105
zerl years of separation,we see that the rate of silent site divergence is much greater for the first -100 million years of separation. One interpretation is that a fraction of roughly half of the silent sites is rapidly (within I00 million years) saturated by mutations; this fraction behaves as neutral sites.The other fraction accumulates mutations more slowly, at a rate approximately the same as that of the replacement sites;this fraction identifies sites that are silent with regard to the protein, but that come under selective pressure for some other reason. Now we can reverse the calculation of divergence rates to estimate the times since genes within a specieshave been apart. The ., Replacement . :: : pairs sitedivergences between genes of B-gtobin attowthe historyof the humancluster difference between the human B and 6 genes o/" to bereconstructed. Thistreeaccounts fortheseparation is 3.7 for replacementsites.At a UEP of I0.4, of ctasses of globingenes. these genes must have diverged I0.4 x 3.7 = 40 million years ago-about the time of the separation of the lines leading to New World distant past. For example, the averagereplacemonkeys, Old World monkeys, great apes, and ment divergence between corresponding mamman. All of these higher primates have both malian and chicken globin genes is 237o. B and 6 genes, which suggeststhat the gene Relativeto a separation-270 million yearsago, divergence commenced just before this point this gives a rate of 0.O9o/o per million years. in evolution. Going further back, we can compare the o,Proceeding further back, the divergence with the B-globin geneswithin a species.They between the replacement sites of y and e genes have been diverging since the individual gene is l0%, which correspondsto a time of separatypes separated500 million years ago (seeFigtion -100 million years ago. The separation u r e 6 . 8 ) . T h e y h a v e a n a v e r a g er e p l a c e m e n t between embryonic and fetal globin genestheredivergenceof -50"/o,which givesa rate of 0. I % fore may have just preceded or accompanied per million years. the mammalian radiation. The summary of these data in Figure 6.9 An evolutionary tree for the human globin shows that replacement divergencein the glogenesis constructedin ,:;;i,:ii.i: ;a.':i.t. Featuresthat bin genes has an averagerate of -0.096oh per evolved before the mammalian radiation-such million years (or a UEP of 10.4). Considering as the separation of B/6 from y-should be found the uncertainties in estimating the times at in all mammals. Features that evolved afterwhich the speciesdiverged, the results lend ward-such as the separationof B- and 6-9logood support to the idea that there is a linear bin genes-should be found in individual lines clock. of mammals. The data on silent site divergence are much In each species,there have been comparaless clear. In every case,it is evident that the tively recent changes in the structures of the silent site divergenceis much greater than the clusters. We know this because we see differreplacement site divergence, by a factor that encesin gene number (one adult B-globin gene v a r i e s f r o m 2 t o 1 0 . T h e s p r e a do f s i l e n t s i t e in man, two in mouse) or in type (most often divergences in pairwise comparisons, though, concerning whether there are separateembryis too great to show whether a clock is applicaonic and fetal genes). ble (so we must basetemporal comparisonson When sufficient data have been collected t h e r e p l a c e m e n ts i r e s ) . on the sequencesof a particular gene, the arguFrom Figure 6.9, it is clear that the rate at ments can be reversed, and comparisons silent sitesis not linear with regard to time. f between genes in different speciescan be used we assumethat there must be zerl diverqenceat to assesstaxonomic relationshios.
106
CHAPTER 6 Clusters andRepeats
TheRateof Neutral Substitution Can BeMeasured from Divergence of Repeated Sequences
=0.18 cCCAGCGTAGCTTICATTACCCGTACGTTCATAT7 T 1C3G8G 0.16 c C T G G C G T A G C . T A C G T T A G C G G T A C G T G C A T A T6T/ 3G8G=G 3G 8 =G0 . 1 6 G G T A G C C T A a C T T A a G a T A C C G G T ! C G T G C T T G T6T/C G G T A G C C T A G C T T A G G T T A T T G G T A G G T G C A T G6T/C 38 C=G0G .16 cCTACCCTAGGTTACGTTATCGGTACGTGTCCGT 6T / 3C8G =G 0.16 G C C A C C C ' A G C T C A C G T T A C C G G C A C G T G C A T G7A/T3C G0C. 1 8 8= C C T A G C C T C G C T T T C G T T A G C G G T A C C T G C A T C7T/T3C C0G, 1 8 8= G C T T G C C T A G T T T A C G T T A C T G G T A C G C G C A T G 5TiT3G G0G, 1 3 8= GCCAGGCTAGCTTACGCCACCGGTACGTGGATG 6 /T3C8C - -G 0 .G1 6
. Therateof substitution peryearat neutralsitesis g r e a t ei nr t h em o u steh a ni n t h eh u m a n genome.
A T Calculateconsensussequence II
We can make the best estimate of the rate of substitution at neutral sites by examining sequencesthat do not codefor protein. (We use the term neutral here rather than silent, because there is no coding potr:ntial). An informative comparison can be made by comparing the members of a common repetitive family in the human and mouse genomes. The principle of the analysis is summarized in 'iir--i::ii:ir , :. \A'/sStart with a family of related sequencesthat have evolved by duplication and substitution from an original family member. We assumethat the cornmon ancestralsequence can be deduced by taking the basethat is most common at each position. Then we can calculate the divergence of each individual family member as the proportion of basesthat differ from the deduced ancestral sequence.In this example, individual members vary from 0.13 t o 0 . 1 8 d i v e r g e n c ea n d t h e a v e r a g ei s 0 . 1 6 . O n e f a m i l y u s e d f o r t h i s a n a l y s i si n t h e human and mouse genomes derives from a sequencethat is thought to have ceasedto be active at about the time of the divergence between man and rodents (the LINES family; see Section 22.9, RetroposonsFall into Three Classes).This means thLatit has been diverging without any selective pressure for the same length of time in both species.Its averagedivergencein man is -0.17 substitutionsper site,corresponding to a rate oI 2.2 x I0-e substitutions per baseper year over tl:re75 million years since the separation.In the rrrousegenome, however, neutral substitutions have occurred at twice this rate, corresponding to, 0.34 substitutions per site in the family, or a rate of 4.5 x l0-e. Note, however, that if we calculated the rate per generation instead of per 1,ear,it would be greater in man than in mouse (-2.2 x l0-8 as opposed to -10 e).
V
Calculate divergence trot
consensus
SEqUENCE GCTAGCCTAGCTTACGTTACcGGTACGTGCATGTTCGG
r'r.i r AnancestraI fora familyiscatsequence i I r,ilrr'ir consensus baseat eachposition.The cutatedby takingthe mostcommon of the fami[yis ca[divergence of eachexistingcurrentmember of basesat whichit differsfromthe cutatedas the proportion ancestraI sequence.
These figures probably underestimate the rate of substitution in the mouse; at the time of divergence the rates in both specieswould have been the same, and the difference must have evolved sincethen. The current rate of neutral substitution per year in the mouse is probably 2-)x grearerthan the historical average.These ratesreflect the balancebetween the occurrence of mutations and the ability of the genetic system of the organism to correct them. The differencebetween the speciesdemonstratesthat each specieshas systems that operate with a characteristic eff iciency. Comparing the mouse and human genomes allclwsus to assesswhether syntenic (corresponding)sequencesshow signsof conservation or have differed at the rate expected from accumulation of neutral substitutions. The proportion of sites that show signs of selectionis -5o/". This is much higher than the proportion that codes for protein or RNA (-l'/.).It implies that the genome includes many more stretcheswhose sequence is important for noncoding functions than for coding functions. I(nown regulatory elements are likely to comprise only a small part of this p r o p o r t i o n . T h i s n u m b e r a l s o s u g g e s t st h a t m o s t ( i . e . ,t h e r e s t ) o f t h e g e n o m e s e q u e n c e s do not have any function that depends on the exact seouence.
Sequences 707 of Repeated fromDivergence CanBeMeasured Substitution 6.5 TheRateof Neutral
@
Pseudogenes AreDead Endsof EvoLution
o Pseudogenes haveno codingfunction,butthey canbe recognized by sequence similarities with genes. ex'isting functional Theyarisebythe accumutation in (formerty) of mutations functional genes.
Pseudogenes(Y) are defined by their possession of sequencesthat are related to those of the functional genes.but that cannot be translated into a functional protein. Some pseudogeneshave the same general structure as functional genes,with sequences corresponding to exons and introns in the usual locations.They may have been rendered inactive by mutations that prevent any or all of the stagesof gene expression.The changescan take the form of abolishing the signals for initiating transcription. preventing splicing at the exon-intron junctions, or prematurely terminating translation. Usually a pseudogenehas severaldeleterious mutations. Presumablyonce it ceasedto be active, there was no impediment to the accumulation of further mutations. Pseudogenes that represent inactive versions of currently active geneshave been found in many systems, including globin, immunoglobulins, and histocompatibility antigens, where they are located in the vicinity of the gene cluster, often interspersedwith the active genes.
i i i ; t r i : r , i ri," i . M r a n yc h a n g ehsa v eo c c u r r eidn a B g t o b i ng e n es i n c ei t became a pseudogene.
108
CHAPTER 6 Ctusters and Repeats
A typical example is the rabbit pseudogene, YB2, which has the usual organization of exons and introns and is related most closely to the functional globin gene Bl. The rabbit pseudogene is not functional, though. ljl*i.iiil:ri,i:i summarizes the many changes that have occurred in the pseudogene.The deletion of a basepair at codon 20 of Yp2 has causeda frameshift that would lead to termination shortly after. Several point mutations have changed later codons representing amino acids that are highly conserved in the p globins. Neither of the two i n t r o n s a n y l o n g e r p o s s e s s e sr e c o g n i z a b l e boundaries with the exons, so probably the introns could not be splicedout even if the gene were transcribed. However, there are no transcripts corresponding to the gene, possibly becausethere have been changesin the 5'flankrng reglon. This list of defectsincludes mutations potentially preventing each stageof gene expression, thus we have no means of telling which event originally inactivated this gene. If we measure the divergence between the pseudogene and the functional gene, though, we can estimate when the pseudogene originated and when its mutations stafied to accumulate. If the pseudogene had become inactive as soon as it was generated by duplication from pI, we should expect both replacement site and silent site divergenceratesto be the same. (They will be different only if the gene is translated to create selective pressure on the replacement sites.) In fact, there are fewer replacement site substitutions than silent site substitutions. This suggests that at first (while the gene was expressed)there was selection against replacement site substitution. From the relative extents of substitution in the two types of site, we can calculatethat Yp2 diverged from Fl -55 million years ago, remained a functional gene for 22 million years, but has been a pseudogene for the last 33 million years. Similar calculations can be made for other pseudogenes.Some appear to have been active for some time before becoming pseudogenes; others appear to have been inactive from the very time of their original generation. The general point made by the structures of these pseudogenesis that each has evolved independently during the development of the globin gene cluster in each species.This reinforces the conclusion that the creation of new genesfollowed by their acceptanceas functional duplicates,variation to become new functional genes, or inactivation as pseudogenes-is a continuing
60I
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from the removal of exeLctly3.7 kb of DNA, the precise distance between the cx,land u,2 genes. It appearsto have been generatedby unequal crossing-over between the o,1 and c2 genes themselves. This is precisely the situation depicted in Figure 6.13,, Depending on the diploid combination of thalassemic chromosomes, an affected individual may have any number of o, chains from zero to three. There are few differences from the wild type (four o, genes) in individuals with three or two o genes. If an individual has only one o, gene, though, the excessp chains form the unusual tetramer F+, which causes hemoglobin H (HbH) disease.The complete absenceof crgenesresults in hydrops fetalis, which is fatal at or before birth. The same unequal crossing-overthat generated the thalassemicchromosome should also have generated a chromosome with three a genes.Individuals with such chromosomeshave been identified in severalpopulations. In some populations, the frequency of the triple o,Iocus is about the same as that of the single s locus; in others, the triple o(genes are much /esscommon than single crg , e n e s . T h i s s u g g e s t st h a t (unknown) selectivefactors operate in different populations to adjust the gene levels. Variations in the number of u genes are found relatively frequently, which argues that unequal crossing-overin the cluster must be fairly common. It occurs more often in the a cluster than in the B cluster. possibly because the introns in c, genes are much shorter and therefore present Iessirnpediment to mispairing between nonhomologous genes. The deletionsthat causeB-thalassemias are summarized in :;.!.iiiJ*,i -i:.tF.In some (rare) cases, only the p gene is affected.These have a deletion of 600 bp, extending from the secondintron through the l'flanking regions. In the other c a s e s ,m o r e t h a n o n e g e n e o f t h e c l u s t e r i s affected.Many of the deletions are very long. extending from the 5'end indicated on the map for >50 kb toward the right. The Hb Lepore type provided the classic evidence that deletion c'anresult from unequal crossing-overbetween linked genes.The p and 6 genes differ only -7"1>in sequence.Unequal recombination deletesthe material between the genes. thus fusing them together (see Figure 6.13). The fusedgene producesa singlep-like chain that consistsof the N-terminal sequence of 6 joined to the C-terminal sequenceof p. Severaltypes of Hb Leporenow are known, the difference between them lying in the point
detetions ii,.i,i o tha[assemias resuttfromvarious f :.irii=:1,: genec[uster. in the a-gl.obin
genecluster cause tJ{r;4"iil{i in theB-gtobin ii":.:, Deletions severaI typesof tha[assemia.
of transition from 6 to B sequences.Thus when the 6 and B genes pair for unequal crossingover, the exact point of recombination determines the position at which the switch from 6 to p sequence occurs in the amino acid chain. The reciprocal of this event has been found in the form of Hb anti-Lepore, which is produced by a gene that has the N-terminal part of B and the C-terminal part of 6. The fusion gene lies between normal 5 and B genes. Evidence that unequal crossing-over can occur between more distantly related genes is provided by the identification of Hb Kenya, another fused hemoglobin. This contains the
GeneClusters t7l Rearranges Crossing-over 6.7 UnequaI
N-terminal sequenceof the \gene and the Cterminal sequence of the p gene. The fusion must have resulted from unequal crossing-over between Ay and B, which differ -20% in sequence. From the differences between the globin gene clusters of various mammals, we see that duplication followed (sometimes) by variation has been an important feature in the evolution of each cluster. The human thalassemic deletions demonstrate that unequal crossing-over continues to occur in both globin gene clusters. Each such event generatesa duplication as well as the deletion, and we must account for the fate of both recombinant loci in the population. Deletions can also occur (in principle) by recombination between homologous sequenceslying on the samechromosome. This does not generate a corresponding duplication. It is difficult to estimate the natural frequency of these events,becauseselectiveforces rapidly adjust the levels of the variant clusters in the population. Generally a contraction in gene number is likely to be deleterious and selectedagainst.However, in some populations, there may be a balancing advantage that maintains the deleted form at a low frequency. The structures of the present human clusters show several duplications that attest to the importance of such mechanisms.Th'efunctional sequencesinclude two o,genescoding the same protein, fairly well related p and 6 genes, and two almost identical y genes. These comparatively recent independent duplications have survived in the population, not to mention the more distant duplications that originally generated the various types of globin genes. Other duplications may have given rise to pseudogenes or have been lost. We expect continual duplication and deletion to be a feature of all gene clusters.
@
Genes for rRNAForm Tandem Repeats
. Ribosomal RNAis codedby a largenumber of genesthat aretandemty identicaL repeated to form oneor moreclusters. o EachrDNActuster is organized sothat transcription unitsgivinga joint precursor to the majorrRNAs alternatewith nontranscribed spacers. In the caseswe have discussedthus far, there are differences between the individual mem-
tt2
CHAPTER 6 Ctusters and Repeats
bers of a gene cluster that allow selective pressure to act independently upon each gene. A contrast is provided by two casesof large gene clusters that contain many identical copies of the same gene or genes. Most organisms contain multiple copies of the genesfor the histone proteins that are a major component of the chromosomes, and there are almost always multiple copies of the genes that code for the ribosomal RNAs. These situations pose some interesting evolutionary questions. Ribosomal RNA is the predominant product of transcription, constituting some 80%-90% of the total mass of cellular RNA in both eukaryotes and prokaryotes. The number of major rRNA genes varies from seven in E. coli, 100 to 200 in lower eukaryotes, to several hundred in higher eukaryotes. The genes for the large and small rRNA (found in the large and small subunits of the ribosome, respectively) usually form a tandem pair. (The sole exception is the yeast mitochondrion.) The lack of any detectable variation in the sequencesof the rRNA molecules implies that all the copies of each gene must be identical, or at least must have differences below the level of detection in rRNA (-I%\.A point of major interest is what mechanism(s) are used to prevent variations from accruins in the individual sequences. In bacteria, the multiple rRNA gene pairs are dispersed.In most eukaryotic nuclei, the rRNA genesare contained in a tandem cluster or clusters. Sometimes these regions are called rDNA. (In some cases,the proportion of IDNA in the total DNA, together with its atypical base composition, is great enough to allow its isolation as a separate fraction directly from sheared genomic DNA.) An important diagnostic feature of a tandem cluster is that it generates a circular restriction map, as shown in FiSlJftf, S.3S. Suppose that each repeat unit has three restriction sites.When we map these fragments by conventional means, we find that A is next to B, which is next to C, which is next to A, generating the circular map. If the cluster is Iarge, the internal fragments (A, B, and C) will be present in much greater quantities than the terminal fragments (X and Y), which connect the cluster to adjacent DNA. In a cluster of 100 repeats,X and Y would be present at I % of the Ievel of A. B. and C. This can make it difficult to obtain the ends of a gene cluster for mappmg purposes. The region of the nucleus where rRNA synthesis occurs has a characteristic appearance,
l:.l.rii:: t-:.:.r'A tandemgenectusterhas an alternationof transcription unit and nontranscribed spacerand generates a cjrcularrestrictionmap.
with a core of fibrillar nature surrounded by a granular cortex. The fibrillar core is where the rRNA is transcribed from the DNA template, and the granular cortex is formed by the ribonucleoprotein particles irLto which the rRNA is assembled.The whole area is called the nucleolus. Its characteristic .morphology is evident lfl
!ir:i::r1: r:
The particular chromosomal regions associated with a nucleolus are called nucleolar organizers. Each nucleolar organizer corresponds to a cluster of tandemly repeated rRNA genes on one chromosome. The concentration of the tandemly repeated rRNA genes,together with their very intensive transcription, is responsible for creating the characteristicmorphology of the nucleoli. The pair of major rRNAs is transcribed as a single precursor in both bacteria and eukaryotic nuclei. Following transcription, the precursor is cleaved to release the individual rRNA molecules. The transcription unit is shortest in bacteria and is longest in mammals (where it is known as 45S RNA, accrrrdingto its rate of sedimentation). An rDNA cluster contains many transcription units, each separated from the next by a nontranscribed spacer. The alternation of transcription unit and nontranscribed spacer can be seen directly in electron micrographs. The example shown in i-ii,i:itt ii, iiii is taken from the newt Not|opthalmus viridescens,in which each transcription unit is intensively
,:1, rDNAunder i'ii i.t;r;1 l j Thenucleo[ar coreidentifies granutar codexconsists transcription, andthesurrounding Thisthinsectjon shows of assembling ribosomaI subunits. Photo viidescens. the nucteotus ofthe newtNotopthoLmus courtesy of 0scarMitter.
expressed,so that many RNA polymerases are simultaneously engagedin transcdption on one repeating unit. The polymerasesare so closely packed that the RNA transcripts form a characteristic matrix displaying increasing length along the transcription unit.
Repeats 773 for rRNAFormTandem 6.8 Genes
happens to be the sex chroD. melanogaster,Ihis mosome. The cluster on the X chromosome is Iarger than the one on the Y chromosome, so female flies have more copiesof the rRNA genes than male flies.) In mammals the repeating unit is very much larger, comprising the transcription unit of -13 kb and a nontranscribed spacerof -30 kb. Usually. the genes lie in several dispersed clusters-in the case of man and mouse the clusters reside on five and six chromosomes, respectively.One interesting (but unanswered) :j:..i=:*i: generates r;.i:-i Transcription of rDNActusters a eachcorresponding to onetranscrip- question is how the corrective mechanisms that seriesof matrices, fromthe nextby the nontran- presumably function within a single cluster to tion unit andseparated Photocourtesy scribed spacer. of 0scarMi[ter. ensure constancy of rRNA sequence are able to work when there are several clusters. The variation in length of the nontranscribed spacerin a single gene cluster contrasts with the conservation of sequence of the transcription unit. In spite of this variation, the Repetitious Bepetitious Repetitious s e q u e n c e so f l o n g e r n o n t r a n s c r i b e d s p a c e r s region1 ,"gion, region3 remain homologous with those of the shorter 8"n., B", island island nontranscribed spacers.This implies that each nontranscribed spaceris internally repetitious,so that the variation in length results from changes -500bp -300bp -300bp in the number of repeats of some subunit. 97 bp repeats 60/81bp repeats 60/81bp repeats Variable length Variable length length Variable The general nature of the nontranscribed spacer is illustrated by the example oI X. laevis. *" 1':*illustrates the situation. Regionsthat : i . . . . , - - i j : : = .Ti :hj e n o n t r a n s c r i b e d s p a c leareovf X r s. r D N A h a s a n i n t e r n a t ft+UFt[ ly are fixed in length alternate with regions that repetitious forits variation structure thatis responsibte in [ength.TheBam is[ands regions. vary. Each of the three repetitious regions comareshortconstant sequences that separate the repetitious prises a variable number of repeats of a rather short sequence. One type of repetitious region has repeats of.a 97 bp sequence;the other, which occurs in two locations, has a repeating unit found in two forms, 60 bp and 8l bp long. The variation in the number of repeating units in the repetitious regions accounts for the overall variation in spacer length. The repetitious . Thegenes in an rDNAclustera[[havean identical regions are separated by shorter constant sequence. sequences called Bam islands. (This descrip. Thenontranscribed spacers consist of shorter tion takes its name from their isolation via the repeating unitswhosenumber variessothat the use of the BamHI restriction enzyme.) From lengths of individuaI spacers aredifferent. this type of organization, we see that the cluster has evolved by duplications involving the The nontranscribed spacervaries widely in promoter region. Iength between and (sometimes) within We need to explain the lack of variation in species.In yeast there is a short nontranscribed the expressedcopiesof the repeated genes.One spacerthat is relatively constant in length. In model would suppose that there is a quantitaD. melanogasterIhere is almost a twofold varitive demand for a certain number of. " good" sequences. This would, however, enable mutated sequencesto accumulate up to a point A similar situation is seen in X. laevis.In each at which their proportion of the cluster is great of these cases,all of the repeating units are enough for selectivepressureto be exerted. We present as a single tandem cluster on one can exclude such models because of the lack of particular chromosome. (In the example of such variation in the cluster.
ffiffiffi
@
TheRepeated Genes for rRNAMaintain Constant Sequence
;:,Ti:ii:iT:*:j;[::?ilTl:;:Hil'ffi:
774
CHAPTER 6 Ctusters and Repeats
The lack of variation implies the existence of selectivepressure in some form that is sensitive to individual variations. One model would suppose that the entire cluster is regenerated periodically from one or from a very few members. As a practical matter, any mechanism would need to involve regeneration every generation. We can exclude such models because a regenerated cluster would not show variation in the nontranscribed regions of the individual repeats. We are left with a dilemma. Variation in the nontranscribed regions suggeststhat there is frequent unequal crossing-over.This will change the size of the cluster, but will not otherwise change the properties of the individual repeats. So how are mutations prevented from accumulating? We'll see in the next section that continuous contraction and expansion of a cluster may provide a mechanism for homogenizing its copies.
@
Crossover FixationCould Maintain ldenticaL Repeats
r UnequaI crossing-over changes the sizeof a ctuster of tandemrepeats. . Individual repeating unitscanbe eliminated or canspreadthroughthe cluster. The same problem is encountered whenever a gene has been duplicated. How can selection be imposed to prevent the accumulation of deleterious mutations? The duplication of a gene is likely ro result in an immediate relaxation of the evolutionary pressure on its sequence.Now that there are two identical copies, a change in the sequence of either one will not deprive the organism of a functional protein, because the original amino acid sequence continues to be coded by the other copy. Then the selective pressure on the two genesis diffused, until one of them mutates sufficiently away from its original function to refocus all the selective pressure on the other. Immediately following a gene duplication, changesmight accumulate more rapidly in one of the copies,leading eventually to a new function (or to its disuse in the form of a pseudogene). If a new function develops,the gene then evolves at the same, slower rate characteristic of the original function. Probably this is the sorr of mechanism responsible for the separation of
functions between embryonic and adult globin genes. Yet there are instancesin which duplicated genes retain the same function, coding for the identical or nearly identical proteins. Identical proteins are coded by the two human cr-globin genes,and there is only a single amino acid differencebetween the two y-globinproteins. How is selective pressure exerted to maintain their sequence identity? The most obvious possibility is that the two genes do not actually have identical functions, but instead differ in some (undetected) property, such as time or place of expression. Another possibility is that the need for two copies is quantitative, becauseneither by itself produces a sufficient amount of protem. In more extreme casesof repetition, however, it is impossible to avoid the conclusion that no single copy of the gene is essential.When there are many copies of a gene, the immediate effectsof mutation in any one copy must be very slight. The consequencesof an individual mutation are diluted by the large number of copies of the gene that retain the wild-type sequence.Many mutant copies could accumulate before a lethal effect is generated. Lethality becomes quantitative, a conclusion reinforced by the observation that half of the units of the rDNA cluster of.X. laevis or D. melanogaster canbe deleted without ill effect. So how are these units prevented from gradually accumulating deleterious mutations? What chance is there for the rare favorable mutation to display its advantages in the cluster? The basicprinciple of models to explain the maintenance of identity among repeated copies is to supposethat nonallelic genesare not independently inherited, but rather must be continually regenerated from oneof the copies of a preceding generation. In the simplest case of two identical genes, when a mutation occurs in one copy, either it is by chance eliminated (becausethe sequence of the other copy takes over), or it is spreadto both duplicates (because the mutant copy becomes the dominant version). Spreading exposesa mutation to selection. The result is that the two genesevolve together as though only a single locus existed. This is called coincidental evolution or concerted evolution (occasionally coevolution). It can be applied to a pair of identical genes or (with further assumptions) to a cluster containing many genes. One mechanism supposes that the sequences of the nonallelic genes are directly
ldenticalRepeats 115 Fixation CoutdMaintain 6.10 Crossover
compared with one another and homogenized by enzymes that recognize any differences.This can be done by exchanging single strands b e t w e e n t h e m t o f o r m g e n e s ,o n e o f w h o s e strands derives from one copy, and one from the other copy. Any differences are revealed as improperly paired bases,which attract attention from enzymes able to excise and replace a base, so that only A-T and G-C pairs survive. Ihis type of event is called gene conversion
events take place in spacersthat are internally mispaired. This can explain the homogeneity of the genes compared with the variability of the spacers.The genesare exposedto selection when individual repeating units are amplified within the cluster; however, the spacersare irrelevant and can accumulate changes. In a region of nonrepetitive DNA, recombination occurs between precisely matching points on the two homologous chromosomes, thus generating reciprocal recombinants. The basisfor this precision is the ability of two duplex DNA sequencesto align exactly. We know that unequal recombination can occur when there are multiple copies of genes whose exons are related, even though their flanking and intervening sequences may differ. This happens because of the mispairing between corresponding exons in nonallelicgenes. Imagine how much more frequently misalignment must occur in a tandem cluster of identical or nearly identical repeats. Except at the very ends of the cluster, the close relationship between successiverepeatsmakes it impossible even to define the exactly corresponding repeats! This has two consequences:there is continual adjustment of the size of the cluster; and there is homogenization of the repeating unit. Consider a sequenceconsisting of a repeating unit "ab" with ends "x" and "y." If we represent one chromosome in black and the other in color, the exact alignment between "allelic" sequenceswould be:
i^}.t,il#,ffi :"5i.Tj'1.?".i"',l..;:::S?:i:f;; events by comparing the sequencesof duplicate genes.If they are subjectto concertedevolution. we should not see the accumulation of silent site substitutions between them (because the homogenization processappliesto these as well as to the replacement sites).We know that the extent of the maintenance mechanism need
'ffi?,:",i"Ti,T; ::,'.','Ti'.i;',?#JT"Tff sequences are entirely different. Indeed, we may see abrupt boundaries that mark the ends of the sequencesthat were homogenized. We must remember that the existence of such mechanisms can invalidate the determination of the history of such genesvia their diver-
?ffii;lf iLTi,|,i;!:,:::,,:;,i;;!;:::;1,:l;:'::, the original duplication. The crossover fixation model supposes that an entire cluster is subject to continual rearrangement by the mechanism of unequal crossing-over.Such events can explain the concerted evolution of multiple genes if unequal crossing-overcausesall the copiesto be regenerated physically from one copy. Following the sort of event depicted in Figure 6.13, for example,the chromosome carrying a triple locus could suffer deletion of one of the genes. Of the two remaining genes, l% represent the sequence of one of the original copies; only k of the sequenceof the other original copy has survived. Any mutation in the first region now existsin both genesand is subject to selectivepressure. Thndem clusteringprovides frequent opportunities for "mispairing" of genes whose sequencesare the same. but that lie in different positions in their clusters. By continually expanding and contracting the number of units via unequal crossing-over,it is possiblefor all the units in one cluster to be derived from rather a small proportion of those in an ancestral cluster. The variable lengths of the spacersare consistent with the idea that unequal crossing-over
776
CHAPTER 6 Ctusters and Repeats
xababababababababababababababababy x a b a b a bbaab ab ab a b ab a b a b a bbaa b a b a b y It is likely, however, IhaI any sequence ab in one chromosome could pair with any sequenceab intL'e other chromosome. In a misalignment such as: xababababababababababababababababy x a b a b a b a b ab ab ab ab ab ab ab ab ab ab ab y the region of pairing is no lessstablethan in the perfectly aligned pair, although it is shorter. We do not know very much about how pairing is initiated prior to recombination, but very likely it starts between short corresponding regions and then spreads.If it starts within satellite DNA, it is more likely than not to involve repeating units that do not have exactly corresponding Iocationsin their clusters. Now suppose that a recombination event occurs within the unevenly paired region. The recombinants will have different numbers of
repeating units. In one case, the cluster has become longer; in the other, it has become shorter,
bababa ffi&&HbdhH$nbghabababa baby Xr,
xabababababa bab'c&iiFghti'&d dsa,bb,bfr: i xabababababalaUan*a lanabababababababa bv i
xababababababababababababababy where "x" indicates the site of the crossover. If this type of event is common, clusters of tandem repeats will undergo continual expansion and contraction. This can cause a particular repeating unit to spread through the cluster, as illustrated in iri::ljfitrr.r.;ii.Supposethat the cluster consists initially of a sequence abcde,
where each letter represents a repeating unit. The different repeating units are closely enough related to one another to mispair for recombination. Then by a seriesof unequal recombination events, the size of the repetitive region increasesor decreases,and one unit spreadsto replace all the others. The crossover fixation model predicts that presany sequenceof DNA that is not under selective will sure be taken over by a seriesof identical tandem repeatsgenerated in this way. T):recritical assumption is that the processof crossoverfixation is fairly rapid relative to mutation, so that new mutations either are eliminated (their repeats are lost) or come to take over the entire cluster.In the caseof the rDNA cluster,of course, a further factor is imposed by selection for an effective transcribedseouence.
SatelliteDNAs 0ftenLie in Heterochromatin . HightV repetitive DNAhasa veryshortrepeating sequence andno codingfunction. . It occurs in largeblocksthat canhavedistinct physicaI properties. . It is oftenthe majorconstituent of centromeric heteroch romati n.
I .ifficdcde affide
i1 abbbbbcdcde-
rl + dmmluitude 10 ffiSlffftdcde ,f
bbbbbr:&de
i.i{:iiiirr:.li: Unequal recombination altows oneparticutarrepeating unitto occupy theentirectuster. Thenumbersindicatethe Length of the repeating unit at each stage.
Repetitive DNA is defined by its (relatively) rapid rate of renaturation. The component that renatures most rapidly in a eukaryotic genome is called highly repetitiveDNA and consists of very short sequencesrepeated many times in tandem in large clusters. As a result of its short repeating unit, it is sometimes describedas simple sequence DNA. This type of component is present in almost all higher eukaryotic genomes, but its overall amount is extremely variable. In mammalian genomes it is typically
DNAs 0ftenLiein Heterochromatin 777 6.11 Sate[tite
M o u s eD N Ai s s e o a r a t eidn t o a m a i nb a n d and a sateltiteby centrifugationthrougha densitygradient of CsCL.
rs called satellite DNA. The term satellite DNA is essentiallysynonymous with simple sequence DNA. Consistentwith its simple sequence,this DNA is not transcribedor translated. Thndemly repeatedsequencesare especially liable to undergo misalignmentsduring chromosome pairing, and thus the sizesof tandem clus-
i;T:11"'*: *T#i ::ililffiil "-,: :,:,'f: " smaller clustersof such sequencescan be used to characterizeindividual genomesin the techrique of "DNA fingerprinting" (seeSection6.14, MinisatellitesAre Useful for GeneticMapping). The buoyant density of a duplex DNA d e p e n d s o n i t s G - C c o n t e n t a c c o r d i n gt o t h e emoirical formula p = 1 . 6 6 0+ 0 . 0 0 0 9 8( % c - c r g - c m - r Buoyant density usually is determined by centrifuging DNA through a density gradient of CsCl.The DNA forms a band at the position correspondingto its own density. Fractions of
usu. :,il13'i'"",:ii,:i :" #:::ri',f,iJran When eukaryotic DNA is centrifuged on a density gradient, two types of material may be distinsuished: . Nlurt of the genome forms a continuum of fragments that appear as a rather broad peak centered on the buoyant density corresponding to the averageGC content of the genome. This is called themainband . Sometimesan additional, smaller peak (or peaks) is seen at a different value. This material is the satelliteDNA.
118
CHAPTER 6 Ctusters and Reoeats
Satellitesare present in many eukaryotic genomes. They may be either heavier or lighter than the main band, but it is uncommon for them to represent>5% of the total DNA. A clear example is provided by mouse DNA. shown in i ii,riirr:: r:;,.i,. The graph is a quantitative scan of the bands formed when mouse DNA is centrifuged through a CsCl density gradient. The main band contains 92'k of the genome and is centeredon a buoyant density of 1.70I g-cm-; (correspondingto its averageG-C of 42%, typical for a mammal). The smaller peak represents 8'/. of.the genome and has a distinct buoyant density of 1.690 g-cm-;. It contains the mouse satelliteDNA, whose G-C content (30%) is much lower than any other part of the genome. The behavior of satellite DNA on density gradientsis often anomalous. When the actual basecomposition of a satelliteis determined, it is different from the prediction basedon its buoyant density.The reason is that p is a function not just of base composition, but of the constitution in terms of nearest neighbor pairs. For simple sequences,these are likely to deviate from the random pairwise relationships needed to obey the equation for buoyant density. In addition, satellite DNA may be methylated, which changesits density. Often, most of the highly repetitive DNA of a genome can be isolated in the form of satellites. When a highly repetitive DNA component does not separateas a satellite,on isolation its properties often prove to be similar to those of satellite DNA. That is to say, highly repetitive DNA consistsof multiple tandem repeatswith anomalous centrifugation. Material isolated in this manner is sometimes referred to as a cryptic satellite. Together the cryptic and apparent satellitesusually account for all the large tandemly repeated blocks of highly repetitive DNA. When a genome has more than one type of highly repetitive DNA, each exists in its own satellite block (although sometimes different blocks are adjacent). Where in the genome are the blocks of highly repetitive DNA located? An extension of nucleic acid hybridization techniques allows the location of satellitesequencesto be determined directly in the chromosome complement. In the technique of in situ hybridization, the chromosomal DNA is denatured by treating cells that have been squashedon a cover slip. Next, a solution containing a radioactively labeled DNA or RNA probe is added. The probe hybridizes with its complements in the denatured genome. The location of the sites of
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qPadau lPlrqlreralH JolsLsuol salrllalesuPrleuluehl 'et111alps aqt Sunuanbas ur InJesn aq ueJ srqJ '(T) puerrs tq8q,{retuarualdruor aql ruoJJ palpJedasaq uer (g) pueJls,{.,reaq srql uorlpJnleuep uodn leqt os z(ltsuapluer(onq s1t sasPJJf,ursrqJ 'seseqD puP J uI rJqJIr qJnlu sI spueJlseql Jo auo satlllJres.ro[eruJqt Jo tlJpJ uI '77'9 an8rg ur uMor{s satrllelpsstltlt^'e erqt lo aldruexa aq1 uI 'spueJls o,rl1eqi uo srted aseq Jo uorleluJrJo eqt ur drlaruru,{se parunouord e sr sJtrllJlesJsJql Jo .{.ueru1o JrnleJJ JUO pamquruw aq uat atuanbas '[1txa1du,tot atuanbas to huaqsuoc tptll/w utt1it/vt
10
20
30
40
50
60
70
^ 80
90
100
.t1o
GGACCTGGAATATGGCGAGAAAACTGAAAATCACC'GAAAATGAGAAATACACACTTT AGGACGTGAAATATGGCGAGIAAACTGAAAAAGGTGGAAAATTTGAAATGTccAcTGTA.
GGACGTGGAATATGGCAAGAAAAOTGAAAATCATGGAAAATGAGAAACATCCACTTG ACGACTTGAAAAATGACGAAATCACTAAAAAA@TGAAAAATGAGAAATGCACACTGAA
200
210
220
230
lI+:.j$ilti":?+Therepeating unitof mouse satelliteDNAcontains two half-repeats, (in btue). whichareatigned to showthejdentities
l:{-irin*i: ii.;::..Theatignmentof quarter-repeats identifieshomologies betweenthe first and second halfof eachhalf-repeat. Positions that arethesamein atlfourquarter-repeats areshown in gray;identities that extendontythroughthreequarter-repeats areindicated by btacktettersin the greenarea. quarter-repeatsare aligrredin $i.*#*l ri=itl,.The upper two lines represent the first half-repeat of Figure 6.24; the lower two lines represent the second half-repeat. We see that the divergence between the four quarter-repeats has increased to 23 out of 58 positions, or 407o. The first three quarter-repeats are somewhat better related, and a large proportion of the divergence is due to changes in the fourth quarter-repeat. Looking within the quarter-repeats, we find that each consistsof two related subunits (oneeighth-repeats),shown as the crand B sequences in Fli-=ijli:{r.;-:r-:. The O(sequencesall have an insertion of a C, and the p sequencesall have an insertion of a trinucleotide, relative to a common consensussequenc(:.This suggeststhat the quarter-repeat originated by the duplication of a sequence like the consensus sequence, after which changes occurred to generate the components we now seeas cx,and B. Further changes then took place between tandemly repeated ap sequences to generate t.he individual quarterand half-repeats that exist today. Among the one-eighth-repeats, the present divergence is 1 9l 3 I = 6 1 0 / o . The consensussequence is analyzed directly in l3:ii.rtiii*-"1,i,which denlonstratesthat the current satellite sequence can be treated as deriv-
atives of a 9 bp sequence. We can recognize three variants of this sequence in the satellite, as indicated at the bottom of the figure. If in one of the repeats we take the next most frequent base at two positions instead of the most frequent, we obtain three well-related 9 bp sequences: GAAAAAC GT GAAAAAT GA GAAAAAAC T The origin of the satellite could well lie in an amplification of one of these three nonamers. The overall consensussequence of the present satellite is GAAAAAf."t, *hi.h is effectively an amalgam of the three 9 bp repeats. The average sequence of the monomeric fragment of the mouse satellite DNA explains its properties. The longest repeating unit of 234bp is identified by the restriction cleavage. The unit of reassociationbetween single strands of denatured satellite DNA is probably the I I 7 bp half-repeat, because tl:'e 234 bp fragments can anneal both in register and in half-register (in the latter case,the first half-repeat of one strand renatures with the second half-repeat of the other). So far, we have treated the present satellite as though it consisted of identical copies of
Repeats t2t Satettites Consist of Hierarchical 6.13 Mammatian
g1
GGACCTGGAATATGGCGAGAA
pl
AATCACGGAAAATGA
CI2
GGACGTGAAATATGGCGAGAGA
92
AAAGGTGGAAAATTTA
O3
GGACGTGGAATATGGCAAGAA
O3
AATCATGGAAAATGA
A4
CGACTTGAAAAATGACGAAAT
F4
AAACGTGAAAAATGA
cont"n.ty.tt,#ffi$ffiffi
AACTGAA
GAAATACACACTTTA AACTGAA
GAAATGTCCACTGTA AACTGAA
GAAACATCCACTTGA CACTAAA
GAAATGCACACTGAA
ffiw.'..
:
ta. . . . .
Ancestral? AAACcTGAAAAATGA
GAAATGCACACTGAA
consists of an that eachquarter-repeat a:i:iiii+ii.iil Theatignment shows of eighth-repeats givesthe mostcommon baseat eachposition. sequence s ha[fanda B hatf.Theconsensus retated sequence, veryclosety to the consensus The"ancestra[" sequence showsa sequence is consequence to the cxandB units.(Thesatettite whichcoutdhavebeenthe predecessor wecantreatit asa cjrsequence theconsensus of deducing tinuous, sothatforthe purposes byjoiningthe lastGAAtriptetto the first6 bp.) cutarpermutation, asindicated
the 234 bp repeating unit. Although this unit accounts for the majority of the satellite, variants of it also are present. Some of them are scatteredat random throughout the satellite; others are clustered. The existence of variants is implied by our description of the starting material for the sequence analysis as the "monomeric" fragment. When the satellite is digested by an enzyme that has one cleavage site in the 234 bp sequence,it also generatesdimers, trimers, and tetramersrelative to the 2)4bp length. fhey arise when a repeating unit has lost the enzyme cleavagesite as the result of mutation. The monomeric 234 bp unit is generated when two adjacent repeatseach have the recognition site. A dimer occurs when one unit has Iost the site, a trimer is generated when two adiacent units have lost the site, and so on. With some restriction enzymes, most of the satellite is cleavedinto a member of this repeating series, as shown in the example of *ii,t::+*,*.::ij;.The declining number of dimers, trimers, and so forth shows that there is a random distribution of the repeats in which the enzyme's recognition site has been eliminated by mutation. Other restriction enzymes show a different type of behavior with the satellite DNA. They continue to generate the same series of bands. Ihey cleave, however, only a small proportion of the DNA, say 5oh-looh. This implies that a certain reqion of the satellite contains a con-
722
CHAPTER and Repeats 6 Ctusters
centration of the repeating units with this particular restriction site. Presumably the seriesof repeats in this domain all are derived from an ancestral variant that possessedthis recognition site (although in the usual way, some members since have lost it by mutation). A satellite DNA suffers unequal recombination. This has additional consequenceswhen there is internal repetition in the repeating unit. Let us return to our cluster consisting of "ab" repeats. Suppose that the "a" and "b" components of the repeating unit are themselves sufficiently well related to pair. Then the two clusters can align in half-register,with the " a" sequence of one aligned with the "b" sequence of the other. How frequently this occurs will depend on the closenessof the relationship between the two halves of the repeating unit. In mouse satellite DNA, reassociationbetween the denatured satellite DNA strands invitro commonly occurs in the half-register. When a recombination event occurs out of register, it changes the length of the repeating units that are involved in the reaction:
bababababababy ffisl*.ffiffi,$i*ffiffii*hg.i$aba '{' xabababababababiili*,F.i{,Hffitrtftii$iilHi*tifi$i#i J xababababababababaababababababababv t'
xababababa babababbabababababababv
GGACCT GGAATATGGC GAGAAAACT GAAAATCAC GGAAAATGA GAAATCACT TTAGGACGT GAAATATGGC G A G AGA A A C GAAAAAGGT G G A A A A TTT G A A A T-C A C GTAGGACGT GGAATATGGC AAGAAAACT GAAAATCAT GGAAAATGA G A A A C-C A C TGACGACTT GAAAAATGAC GAAATCACT AAAAAACGT GAAAAATGA G A A A T-C A C GAA
T A T
T
ir,,1 :l Djgestion iriirr.iii;l of mousesatetliteDNA with the restriction enzymeEcoRIIidentifiesa series of repeating units(1, 2, 3) that aremuttimersof 234 bp andatsoa minorseries('/,, 1'/,, 2%)thatinctudes (seetextthispage). half-repeats Thebandat the far left is a fractionresistantto digestion.
nant cluster, the "b" unit generates a fragment of half of the usual length. (The multiple fragT ments in the half-repeat seriesare generated in the same way as longer fragments in the inteGzoAro&r A2oA12A17T8 G11 As gral series,when some repeating units have lost Tz%&QTrs the restriction site by mutation.) Turning the argument the other way c? around, the identification of the half-repeat . indicatesinserted tripletin B sequence serieson the gel shows that the 234bp repeatC in position10 is extrabase in crsequence ing unit consistsof two half-repeatswell enough li...iiSi::r:.":.:,Theexistence of an overatIconsensus related to pair sometimes for recombination. sequence is shownby writingthe sateltitesequence in Also visible in Figure 6.28 are some rather faint termsof a 9 bp repeat. bands correspondingto',4-and Z-spacings.These will be generated in the same way as the zspacings,when recombination occurs between clusters aligned in a quarter-register. The decreasedrelationship between quarter-repeats In the upper recombinant cluster, an " ab" compared with half-repeats explains the reducunit has been replacedtry an "aab" unit. In the tion in frequency of r}":'e/^- and %-bandscomlower cluster, lhe "ab" unit has been replaced pared with the Z-bands. by a "b" unit. This type of event explains a feature of the restriction digest of rnouse satellite DNA. Figure 6.28 shows a fainter seriesof bands at lengths of Y', l'/r, 2Yr,an
AreUsefuL Minisatellites for Genetic Mapping
AreUsefulfor Genetic Mapping 723 6.14 Minisatettites
lites is that individual alleles have different numbers of the repeating unit. For example, one minisatellite has a repeat length of 64 bp and is found in the population with the following distribution: 7% 18 repeats II% 16 repeats 43% 14 repeats 360/o13 repeats 4% I0 repeats The rate of genetic exchange at minisatellite sequencesis high, -I0aperkb of DNA. (The frequency of exchanges per actual locus is assumed to be proportional to the length of the minisatellite.) This rate is -l0x greater than the rate of homologous recombination at meiosis, that is, in any random DNA sequence. The high variability of minisatellites makes them especially useful for genomic mapping, becausethere is a high probability that individuals will vary in their alleles at such a locus. An example of mapping by minisatellites is illustrated in FISUR{$"t9. This shows an extreme casein which two individuals both are heterozygous at a minisatellite locus, and in fact all four alleles are different. AII progeny gain one allele from each parent in the usual way, and it is possible unambiguously to determine the source of every allele in the progeny. In the terminolgenetics, the meioses described i i . i - - l , r i i i : : : .A, ;t.t:er t e s m a y d i f f e r i n t h e n u m b e r o f r e p e a t s a t a m i n i s a t e l l i t e t o c u sogy , s o of t h ahuman t cteavage on eithersidegenerates restriction fragments that differin length.Byusinga in this figure are highly informative because of parents, minisatettite with altetes the patternof inheritance canbe that differbetween the variation between alleles. fottowed. One family of minisatellites in the human genome share a common "core" sequence.The core is a G-C-rich sequenceof l0 to l5 bp, showoverall are much shorter-consisting of (for ing an asymmetry of purine/pyrimidine distriexample) 5 to 50 repeats-are common in mambution on the two strands. Each individual malian genomes. They were discoveredby minisatellite has a variant of the core sequence, chance as fragments whose size is extremely but -1000 minisatellitescan be detectedon variable in genomic libraries of human DNA. Southern blot by a probe consisting of the core The variability is seen when a population contains fragments of many different sizesthat repsequence. resent the same genomic region; when Consider the situation shoq'n in Figure 6.29, individuals are examined, it turns out that there but multiplied many times by the existence of is extensive polymorphism, and that many difmany such sequences.The effect of the variaferent alleles can be found. tion at individual loci is to create a unique patThe name microsatellite is usually used tern for every individual. This makes it possible to assignheredity unambiguously between parwhen the length of the repeating unit is <10 bp, ents and progeny by showing that 50% of the and the name minisatellite is used when the Iength of the repeating unit is -10 to 100 bp. bands in any individual are derived from a parThe terminology is not, however, precisely ticular parent. This is the basisof the technique known as DNA fingerprinting. defined. Thesetlpes of sequencesare also called variable number tandem repeat (VNTR) Both microsatellites and minisatellites are reglons. unstable, although for different reasons. Microsatellites undergo intrastrand mispairing, The cause of the variation between indiwhen slippage during replication leads to expanvidual genomes at microsatellites or minisatel-
124
CHAPTER 6 Ctusters and Repeats
sion of the repeat, as shown in i:jr,rljFri: ri .ii,r.Systems that repair dameLgeto DNA-in particular those that recognize mismatched base pairs-are important in reversing such changes, as shown by a large increasein frequency when repair genesare inactivated. Mutations in repair systems are an important contributory factor in the development of cancer, thus tumor cells often display variations in microsatellite s e q u e n c e s .M i n i s a t e l l i t e s u n d e r g o t h e s a m e sort of unequal crossing-overbetween repeats that we have discussed for satellites (seeFigure 6. 3 ) . One telling carseis that increased variation is associatedwit.h a meiotic hotspot. The recombination event is not usually associated with recombination b,etween flanking markers, but has a complex form in which the new mutant allele gains inlormation from both the sister chromatid and the other (homologous) chromosome. It is not clear at w.hat repeating length the cause of the variationLshifts from replication slippage to recombinartion.
@
Summary
Almost all genes belorrg to families, which are defined by the possessionof related sequences in the exons of indivirlual members. Families evolve by the duplicat.ionof a gene (or genes), followed by divergence between the copies. Some copies suffer inactivating mutations and become pseudogenesthat no longer have any function. Pseudogenesalso may be generated as DNA copiesof the mRNA sequences. An evolving set of genes may remain together in a cluster or may be dispersedto new locations by chromosomal rearrangement. The organization of existing clusterscan sometimes be used to infer the series of events that has occurred. These events act with regard to sequence rather than function, and therefore include pseudogenesas well as active genes. Mutations accumu.latemore rapidly in silent sitesthan in replacement sites (which affect the amino acid sequence).The rate of divergence at r e p l a c e m e n t s i t e s c a r Lb e u s e d t o e s t a b l i s ha clock, which can be calibrated in percent divergence per million years. The clock can then be used to calculate the time of divergence between any two members of t)re family. A tandem cluster consistsof many copies of a repeating unit that includes the transcribed s e q u e n c e ( s )a n d a n o n t r a n s c r i b e d s p a c e r ( s ) . rRNA gene clusters cocleonly for a single rRNA
L; . ., Rep[ication irri.il+lr: occurs whenthe daughter strandstipsback stippage onerepeating strand.Each slippage eventadds unitin pairingwjththetemplate onerepeating strand.Theextrarepeats areextruded asa unit to the daughter singte-strand of thjs daughter strandin the nextcyctegener[oop.Reptication atesa duplexDNAwithan increased number of repeats. precursor. Maintenance of active genesin clusters depends on mechanisms such as gene conversion or unequal crossing-over, which cause mutations to spread through the cluster so that they become exposed to evolutionary pressure. Satellite DNA consists of very short sequencesrepeated many times in tandem. Its distinct centrifugation properties reflect its biased basecomposition. SatelliteDNA is concentrated in centromeric heterochromatin, but its function (if any) is unknown. The individual repeating units of arthropod satellites are identical. Those of mammalian satellites are related and can be organized into a hierarchy reflecting the evolution of the satellite by the amplification and divergence of randomly chosen sequences. Unequal crossing-overappearsto have been a major determinant of satellite DNA organization. Crossover fixation explains the ability of variants to spread through a cluster. Minisatellites and microsatellites consist of even shorter repeating sequencesthan satellites:
6 . 1 5S u m m a r y 725
<10 bp for microsatellitesand I0 to 50 bp for minisatellites. The number of repeating units is usually 5 to 50. There is high variation in the repeat number between individual genomes. A microsatellite repeat number varies as the result of slippage during replication; the frequency is affected by systemsthat recognize and repair damage in DNA. Minisatellite repeat number variesas the result of recombination-like events. Variations in repeat number can be used to determine hereditary relationships by the technique known as DNA fingerprinting.
References Is a MajorForce GeneDuptication in Evolution Resea rch Bailey, J. A., Gu, 2., Clark, R. A., Reinert, I(., Samonte, R. V., Schwartz, S., Adams, M. D., Myers, E. W., Li, P. W, and Eichler, E. E. (20021. Recent segmental duplications in the human genome. Science 297, 1003-1007.
][
AreFormed GtobjnCtusters by Dup[icationand Divergence
Review from bacteriato Hardison,R. (1998) Hemoglobins man: evolution of differentpatternsof gene expression. J. Exp.Biol.2Ol,l}99-ll17.
@
TheRateof Neutral Substitution CanBe Measured fromDivergence of Repeated Seouences
Resea rch Waterston,R. H. et al. (2002\.Initial sequencing and comparative analysis of the mouse genome. Nature 420, 520-562.
726
CHAPTER 6 Clusters andReoeats
AreDeadEndsof Evotution Pseudogenes Research Hirotsune, S., Yoshida, N., Chen, A., Garrett, L., Sugiyama, F., Takahashi, S., Yagami, I(., Wynshaw-Boris, A., and Yoshiki, A. (2003). An expressedpseudogene regulates the messenger-RNA stability of its homologous coding gene. Nature 42), 9 l-96.
Fixation CoutdMaintain Crossover IdenticaIRepeats Review Charlesworth,B., Sniegowski,P.,and Stephan,W. (19941. The evolutionarydynamicsof repetiNature771,215-220. tive DNA in eukaryotes.
AreUsefu[ for Genetic Minisatettites Mapping Research Jeffreys, A. J., Murray, J., and Neumann, R. ( 1998). High-resolution mapping of crossoversin human sperm defines a minisatellite-associated recombination hotspot. Mol. Cell2, 267-273. Jeffreys,A. J., Royle, N. J., Wilson, V., and Wong, Z. (1988). Spontaneousmutation ratesto new length alleles at tandem-repetitive hypervariable loci in human DNA. Nature 332,278-281. Jeffreys, A. J., Tamaki, I(., Macleod, A., Monckton, D. G., Neil, D. L., and Armour, J. A. (L994). Complex gene conversion events in germline mutation at human minisatellites. N a t . G e n e L 6 ,l ) 6 - 1 4 5 . Jeffreys,A. J., Wilson, V., and Thein, S. L. (1985). Hypervariable minisatellite regions in human DNA. Nature il4, 67-73. Strand, M., Prolla, T. A., Liskay, R. M., and Petes, T. D. (1991). Destabilizationof tracts of simple repetitive DNA in yeast by mutations affecting DNA mismatch repair. Nature )65 , 27 4-27 6.
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Lueur ur VNU Jo tuJruJ^lolur aq1 'uorssardxa auaS;o sa8elsraqlo le seloJleuoll)unJ Jo IpJnl -rnr1s .,{.e1d teql perJ^oJsrp ueJq J^eq svNU :aqlo .{ueru uaql eJurs lnq 'srsaqluLs uralord ur elerpeurJlur up sp pezrJelf,eJeqJlsJrJseM 'uorssardxaaua8 ur radeld IeJtuJJ e sI vNU U
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(.vNuruot LreluarualdruoJ sr leqt VNU ro VNC Jo aruanbas p eqrJJSJpol turJl leraua8 e se pJSn s asuas!ruV)'pueJls asuasllup Jo puPJls aleldruat Jql pellet sr SurJred aseqfueluaualdruor erl Jqt Jo srs VNUtu -aqlu,{s stf,JJrpteql VN( Jo pueJts Jr{I . :r, j rrilitiihi ur palrrdap sp vNC Jo spuells o^{t Jql qstn8utlsrp e^A 'vNu ra8uassarue otur pJqIrJS -ueJl sr xaldnp VN( e Jo puelts auo z(1u6 'prf,e ourrue auo sluasardar uo6ar Surpot rrll Jo (uopor) 1a1drr1 eprloJlrnu qree :epoJ JrleueSaqt .dqe)uenbJs uralord p ol pateleJ sr teql uor8ar Surpor auo l s e J ll P s u r e l u o f v N u u r q ) p J l n q ' p J l p l
aua8 ur suorpunJ;o .d1aue.tp qtrm sygg 8ur -po)uou Jo sselJJepeorq qJnlu E ;o saldruexa are VNur pup VNUI 1nq 'anbrun sr VNuru Jo uorpunJ Surpor eqJ 'selnJaloruVNU Jeqlo rprM Llerr;nads DprJlul ol pue aJnlrnrls Lrepuoras str urJoJ 01 qloq 'Suured aseq uodn dllerrlrrr puadap suortJunJslr leql sr 'eJaqMeslepup srs -aqluzls uralord qloq ut 'yNu Jo saIlIAItJp Jql 'snteredde ;o 11eq8norqt sunr ler{l eueqt aqJ leur8rro aqt JoJsrseqeqt uJJq JAeq [eru sygg eql leql rJqruauJJ plnoqs rl!\ puP 'svNu rrqlo dq ro surelord raqlra .dquorleln8ar ro; 1a8re1e Jq upJ leqt pue JIoJ a^rlJe ue sz(e1d leql lueu -odruoJ p se lr ,lrerl ol JAeq aa,r'srsaqluLs ural -oJd ur VNU Jo JIor eql JeprsuoJ a,lr sp 'leql sl punorS>peq slql lnoqe Surqt tueuodrur aq1 'svNuJ Jql Jo Juo ur seprsar,{lrnrlresrql 'uralord olut palerodroJul sI prJe ourue ue qJrqm.{q puoq aprldad p Jo uorl -pruJoJ aql az[1e1er 01 ^UIqe aqt sr JruosoqrJ Jql Jo serlr^rpe IerJnrJ Jql Jo Juo 'auosoqrr Jql Jo sJrlr^rlre eql uI ,(lDarrp seledrJrued osIP 1I 'qJene suralord pruosoqu qJrqM ot >lro,lrerueJJe Suprnord ur 'lernlrnrts sl vNU Jo oloJ aug '.d1r -^rtJe Jo ad,{.1raqloue eJS JM 'yNUJ r{llM 'pr)P oulluP su dupues -a.rdar1a1drr1 epltoelrnu Jql qllM srted (uopor -uup Jql) JJuenbas1a1dr4goqs e uJqM'3utrrcd aseq .{q pJIIoJtuoJ sr pesn st yggl-lLreoulrue up q)rqm qtlm ^trJrJnads aq1 'stsaqluLs utal -ord ro; pesn sr tpql JrntJnJls eql se paztuSora.r sr qJrqM 'VNdl-1,{reoururp up seleeJJ e8e -Iull JqJ 'prJeourue rgnads e o1a8elurl ro; a1e -rrdordde sr teqt ta8rcl e Sutpurord se atu.{zua ue ,{.qlsrq pazruSorar sI eJnlJnJls leuolsuJrulp -aarqt rqJ'(VNUru) VNU taqtoue qltu rrcd aseq
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The TtyCarm is named for the presence of this triplet rsequence.(y stands for pseudouridine, a modified base.) The anticodon arm always contains the anticodon tdplet in the center of the loop. The D arm is.named for its content of the basedihydr:ouridine (another of the modified basesin IRNA). . The extra arm lies between the TyC and anticodon arms and varies from 3 to 2 I bases. The numbering system for IRNA illustrates the constancy of the structure. Positions are numbered from 5'to .l'according to the most common IRNA structule, which has 76 residues. The overall range of IRNA lengths is 74 to 95 bases.The variation in length is causedby dif ferences in the D arm and extra arm. The basepairing that maintains the secondary structure is showrL in Figure 7.4. Within a given IRNA, most of the base pairings are conv e n t i o n a l p a r t n e r s h i p so f A - U a n d G - C . b u t occasionalG-U, G-t+/,or A-y pairs are found. The additional types of base pairs are less stable than the regular pairs, but still allow a double-helical structure to form in RNA. When the sequences of tRNAs are compared, the basesfound at some positions are invariant (or conserved); almost always a particular base is founLdat the position. Some positions are describeclas semi-invariant (or semiconserved) becausethey are restricted to one type of base (purine versus pyrimidine), but either base of that type may be present. When a IRNA is chargedwith the amino acid corresponding to its anticodon, it is called aminoacyl-tRNA. The amino acid is linked by an ester bond from its carboxyl group to the 2' or 3'hydroxyl group of the ribose of the l' terminal baseof the IRNA (which is always adenine). The processof charging a IRNA is catalyzed by a specific enzyme, aminoacyl-tRNA synthetase. There are (at least) 20 aminoacylI R N A s y n t h e t a s e s .E a c h r e c o g n i z e sa s i n g l e amino acid and all the tRNAs on to which it can legitimately be placed. T h e r e i s a t l e a s t r c n eI R N A ( b u t u s u a l l y more) for each amino acid. A IRNA is named by using the three-letter abbreviation for the amino acid as a superscript.If there is more than one IRNA for the same amino acid. subscriptnumerals are used to distinguish them. So two tRNAs for tyrosine would be clescribedas IRNAlrrr 3ni tRNA2rvr.A IRNA carrying an amino acid-that is, an aminoacyl-tRNlr-is indicated by a pre-
i :,,r,irl isdetermine bd yi t s a n t i T h em e a n i nogf t R N A c o d o na n dn o tb yi t s a m i n oa c i d .
fix that identifies the amino acid. Ala-IRNA describestRy4ela carrying its amino acid. Does the anticodon sequencealone allow aminoacyl-tRNA to recognize the correct codon? A classicexperiment to test this question is illustrated in . Reductive desulfuration converts the amino acid of cysteinyl-tRNA into alanine, generating alanyl-IRNAcv'. The IRNA has an anticodon that responds to the codon UGU. Modification of the amino acid does not influence the specificityof the anticodon-codon interaction, so the alanine residue is incorporated into protein in place of cysteine. Oncea IRNA has beencharged,the amino acid plays no further role in itsspecificity,which is determinedgaslusivpht by the anticodon.
Stemand TheAcceptor Areat Ends Anticodon Structure of the Tertiary o Thectoverleaf tertiarystructure formsan L-shaped withthe acceptor armat oneendandthe anticodon armat the otherend.
The secondarystructure of each IRNA folds into a compact L-shaped tertiary structure in which the 3' end that binds the amino acid is distant from the anticodon that binds the mRNA. AII tRNAs have the same general tertiary structure, although they are distinguished by individual variations. The basepaired double-helical stems of the secondarystructure are maintained in the tertiary structure, but their arrangement in three dimensions essentially createstwo double helices at right angles to each other, as
7 .4 lhe AcceptorStemand AnticodonAre at Endsof the TertiaryStructure
131
Cloverlealhas four arms
2D projectionhas two perpendicular duplexes
.'ii:1;:.:: .r.:--Transfer RNAfotdsinto a compact L-shaped tertiarystructure withtheaminoacidat oneendandthe anticodon at the otherend.
illustrated in iii;ijil,r..:,i!.The acceptorstem and the TvC stem form one continuous double helix with a single gap; the D stem and anticodon stem form another continuous double helix, also with a gap. The region between the double helices, where the turn in the L-shape is made, contains the TyC loop and the D loop. So the amino acid residesat the extremity of one arm of the L-shape and the anticodon loop forms the other end. The tertiary structure is created by hydrogen bonding, mostly involving bases that are unpaired in the secondary structure. Many of the invariant and semi-invariant bases are involved in these H-bonds, which explains their conservation. Not every one of these interactions is universal, but probably they identify the general pattern for establishing IRNA structure. A molecular model of the structure of yeast lpy4rhe is shown in i:ii:i.-:iii:::.:. The left view correspondswith the bottom panel in Figure 7 .6. Differences in the structure are found in other tRNAs, thus accommodating the dilemma that all tRNAs must have a similar shape.yet it must be possible to recognize differences between them. For example, in tRNAe'p, the angle between the two axes is slightly grearcr,so the molecule has a slightly more open conformation. The structure suggestsa general conclusion about the function of IRNA: Its sitesfor exercising particular functions are maximally separated.Th'e amino acid is as far distant from the anticodon as possible,which is consistent with their roles in protein synthesis.
Messenger RNAIs Translated by Ribosomes
i:,..:::r': .' A space-fitting modeI shows thatthetRNAPhe tertiarystructure is compact. Thetwo viewsof IRNAare rotatedby 90". Photocourtesy of Sung-Hou Kim,University of Ca[ifornia, Berke[ey.
732
CHAPTER 7 Messenger RNA
. Ribosomes arecharacterized by their rateof (70Sfor bacteriaI sedimentation ribosomes and 80Sfor eukaryotic ribosomes). . A ribosome consists of a largesubunit(50Sor 605 for bacteria andeukaryotes) anda smatlsubunit (30Sor a0S). . Theribosome provides theenvironment in which aminoacyltRNAs addaminoacidsto the growing potypeptide chainin response to the corresponding triptetcodons. r A ribosome moves alonqan mRNA from5'to 3'.
Tlanslation of an mRNA into a polypeptide chain is catalyzed by the ribosome. Ribosomes are traditionally described in terms of their (approximate) rate of sedimentation (measuredin Sved-
ggI
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'VNUUaql 6uo1e uotsset6ord 'saurosoqu ;o e6elsluala#rpe lp srauosoquqlpl sr!lrl!ul u! r lerarasr\qpolplsuellAlsnoeuellnurs
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0E8I rre rraql JI'surrtord €- roJ sepoJ.{lqeqord q;ea 'a8eraneuO 'unrralreq rad ytr1gru qtea ]o sardor aeJqt ot ozltl ^luo Lllensn JJp atJql leql stsaSSnssIqJ 'runrJJlJpq e ul vNuru yo sadr{1 luareJJrp009- are JJaqJ 'uortrsodruorap pue srsaqlu.dsJo sJtels 3ur.{rBnul '00E I- aq plnoM ssanEalqeuospal p tnq 'sJlnJelour VNUtu Jo rJqrunu eql elelnJlp) ol tlnJr;lrp s1tr "hl1qelsur Jreql Jo llnseJ e sy 'srsaqluz{suralord ur aluo ]e pesn aq ot ,(pear 'sr leql 'syg51-1,{reourrue se luasard JJe ruaql Jo lsoru lploJuat tsotulp z(qsaruosoqrJeql Jeqrunutno saln)aloru VNU1 aql raqlaSolle pue 'vNUt q)eJ Jo sardor 000€< JJpJJeqJ'sseruIIaJ aqt Jo JJuenb e roy lunoJJe s a u l o s o q l Jo s J o 0 0 0 ' 0 2 e q J ' . : 1 ' , , i i ' r ; ' , . :u; f1 uanr8 sr unrJapeq Dplur aql ur srsaqluu{.s uralord 01palo^ap uorluJlte aql Jo 1vrar^IIpraAouv 'zbuanqya pue azrs VNUIU Jo sJlqerJe^eql Lq paunuralJp 'uortentJnlJ Ielusrlpls Jo JJltpu P Srlnq'salorfuelna Jo erral)pq Jeqlre ur 'pauru -ra1ap ,{.1asoardlou sr uralord relnlrued e 3ur -zrsaqluds aln)elour qf,ea uo sJurosoqrJ VNUru 'salrzb JJqlrnJ ro; Jo rJqrunu aq1 lood Jql ol pauJnlJJ uaql pue 'vNutu ue elelsuerl 01 pJSn '(slrunqnspruosoqu stsrsuoJ Jo lood aq1.{1pnpe) 'JruosoqrJ lood e uorJ uMpJp JJe sJruosoqrU aql alrzb a;u aqt salpJlsnlll , = ,. .:':jit,;t:i Jo 'Jrurt Juo .due peq)etlp sauosoqrJ g- spq te dlqeqord VNUrU rr1o,{re1naaSeraneuV 'qlpne setuosoqrJ qJIq,lt. qtrm druanbarJ ltsrralre -JEq) eql Jo pue (sator{re>1na ur uratord a13urse ,(po Suquasatdar,{11ensn)VNUUI aqr yo qfual 'ure8e 1erral qloq uorlJunJ e sr aql azrs rraql Jo -req ur asoql ueqt rJllprus eq or ,{1a41Jre IIef, ruo;hB>lna e yo ruseldotLr aqt u1 saruos,{1o4
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different soluble proteins, there must be on a v e r a g e > 1 0 0 0 c o p i e s o f each protein in a bacterium.
Component
Dry cell mass (%)
Molecules /cell
Different Copiesof types each type
TheLifeCycleof Bacterial Messenger RNA r Transcription andtransl,ation occursimultaneousty in bacteria, asribosomes begintranslating an mRNA beforeits synthesis hasbeencompleted. r Bacterial mRNA is unstabte andhasa hatf-Life of onlya fewminutes. r A bacterial mRNA maybe potycistronic in having severaI codingregions that represent different genes.
1 mRNA 3 IRNA rRNA 16 proteins 9 Ribosomal proteins sotubte Smallmolecules
46 3
1,500 200,000 38,000 106 ? 9 " 19: 7.5x 10'
600 60 2 52
2-3 >3,000 19,000 19,000
1,850 8OO
>1,ooo
E. coLiin termsof its macromolecular Considering c 0 mp 0 n e n r s . Messenger RNA has the same function in all cells, but there are important differences in the detailsof the synthesisand structure of prokaryotic and eukaryotic n.RNA. A major differenr:e in the production of mRNA depends on the locations where transcription and translation occur: . In bacteria, mRNA is transcribed and translated in the single cellular compartment; the two processesare so closely linked that they occur simultaneously. Ribosomes attach to bacterial mRNA even before its transcription has been completed, thr.rsthe polysome is likely still to be attached to DNA. Bacterial nRNA usually is unstable, and is therefore translated into nroteins for onlv a few minutes. . In a eukaryotic cell, synthesis and maturation of nR.NA occur exclusively in the nucleus. Only after these events are completed is the nRNA exported to the cytoplasm,where it is translatedby ribosomes. Eukarl'otic nRNA is relatively stableand continues to be translated lor severalhours. : r,'.. ,,rl j i :: shows that transcription and translation are intimately related in bacteria. Transcription begins when the enzyme RNA polymerasebinds to DNA and then moves along making a copy of one strand. As soon as transcription begins,ribosomesattach to the 5'end of the nRNA and start translation, even before the rest of the messagehas been synthesized.A bunch of ribosomes rnoves along the mRNA while it is being synthesized.The 3' end of the mRNA is generated when transcription termin a t e s . R i b o s o m e s c o r n t i n u et o t r a n s l a t e t h e nRNA while it survives, but it is degraded in
transtated, mRNAis transcribed. rriiiii*i:.,',-j,: Overview: in bacteria. and degradedsimuttaneously
Messenger RNA of Bacterial 7.7 TheLifeCycte
Transcription unitscanbevisuatized in bacteria. Photocourtesv of Oscar
the overall 5'-+3' direction quite rapidly. The mRNA is synthesized,translated by the ribosomes, and degraded, all in rapid succession. An individual molecule of mRNA survives for only a matter of minutes at most. Bacterial transcription and translation take placeat similar rates.At 37o C, transcription of mRNA occurs at -40 nucleotides/second.This is very close to the rate of protein synthesis, w h i c h i s r o u g h l y l 5 a m i n o a c i d s / s e c o n d .I t therefore takes -2 minutes to transcribe and translate an mRNA of 5000 bp, corresponding to 180 kD of protein. When expressionof a new
: ffili:5i*,ffi;:;:T*ift:+i[ :;li:m: ding protein will appear within perhaps another 0.5 minute. Bacterial translation is very efficient, and most mRNAs are translated by a large number of tightly packed ribosomes. In one example (/rp mRNA), about t5 initiations of transcription occur every minute, and each of the I5 mRNAs probably is translated by -30 ribosomes in the interval between its transcription and degradation. The instability of most bacterial mRNAs is striking. Degradation of mRNA closely follows its translation and likely begins within one minute of the start of transcription. The 5'end of the mRNA starts to decay before the 3' end has been synthesized or translated. Degradation seems to follow the last ribosome of the
It is usually expressedin terms of the half-life. The mRNA representing any particular gene has a characteristic half-life, but the average is -2 minutes in bacteria. T h i s s e r i e so f e v e n t s i s o n l y p o s s i b l e ,o f course, because transcription, translation, and degradation all occur in the same direction. The dynamics of gene expression have been caught in flagrantedelictoin the electron micrograph of ., i.:. In these (unknown) transcription ir.i.:t,,ii,ti: units, several mRNAs are under synthesis simultaneously, and each carries many ribosomes engagedin translation. (This correspondsto the stageshown in the secondpanel in Figure 7.13.) An RNA whose synthesishas not yet been completed is often called a nascent RNA. Bacterial mRNAs vary greatly in the number of proteins for which they code. Some mRNAs represent only a single gene: they are monocistronic. Others (the majority) carry sequencescoding for several proteins: they are polycistronic. In these cases,a single mRNA is transcribed from a group of adjacent genes. (Such a cluster of genesconstitutesan operon that is controlled as a single genetic unit; see Chapter I2, The Operon.) All mRNAs contain two types of region. The coding region consistsof a seriesof codons representing the amino acid sequence of the protein, starting (usually) with AUG and ending with a termination codon. The nRNA is always Ionger than the coding region, though, as extra regions are present at both ends. An additional sequence at the 5' end, preceding the start of the coding region, is described as the leader or 5'UTR (untranslated region). An additional sequencefollowing the termination signal, forming the 3' end, is called the trailer or f' UTR. Although part of the transcription unit, these sequencesare not used to code for protein. A polycistronic mRNA also contains intercistronic regions, as illustrated in irii-;l"it';i: .r.:i:ri. They vary greatly in size: They may be as long as 30 nucleotides in bacterial mRNAs (and even longer in phage RNAs). or they may be very short, with as few as one or two nucleotioes separating the termination codon for one protein from the initiation codon for the next. In an extreme case,two genesactually overlap, so that the last base of one coding region is also the first base of the next coding region. The number of ribosomesengagedin transIating a particular cistron depends on the efficiency of its initiation site. The initiation site for the first cistron becomes available as soon as the 5'end of the nRNA is synthesized.How are
liffi',HffiY;.T'i;,i,":T:J"ffi",1; ffi.'."ffi the speed of transcription or translation. The stability of mRNA has a major influence on the amount of protein that is produced.
136
C H A P T E7RM e s s e n q eRrN A
subsequent cistrons translated? Are the several coding regions in a polycistronic mRNA translated independently or is their expression connected? Is the mechanism of initiation the same for all cistrons, or is it different for the first cistron and the internal cistrons? T?anslation of a bilcterial mRNA proceeds sequentially through its cistrons. At the time when ribosomes attach to the first coding region, the subsequent codirLgregions have not yet even been transcribed. By the time the second ribosome site is available, translation is well under way through ttre first cistron. Tlpically ribosomes terminate translation at the end of the first cistron (and dissociateinto subunits), and a new ribosome assemblesindependently at the start of the next coding region. (We discussthe processesof initiation and termination in Chanter 8, Protein Synthesis.)
@
i l r , ; . . 1 ' , . , 1B , acterialmRNAincludesuntranslatedaswetlastranstatedregions A typicatmRNA signats. Eachcodingregionhasits owninitiationandtermination mayhaveseveraI codingregions.
Eukaryotic mRNA Is Modified Duringor afterIts Transcription
r A eukaryotic mRNA transcript is modified in the nucteus duringor shorttyaftertranscription. o Themodifications inctude the additionof a methylated capat the Ii'endanda sequence of poty(A)at the 3'end. . ThemRNA is exported fromthe nucleus to the cytoplasm on[yafterail modifications havebeen compteted.
The production of eukaryotic mRNA involves additional stagesafter transcription. Tfanscription occurs in the usual way, initiating a transcript with a 5'triphosphate end. However, the 3' end is generated by cleaving the transcript, rather than by terminating transcription at a fixed site. Those RNA.sthat are derived from interrupted genes require splicing to remove the introns, generatirLg a smaller nRNA that contains an intact coding sequence. ;''r.:,..ri:,: :: Shows that both ends of the transcript are modifierCby additions of further nucleotides (involving additional enzyme systems). The 5' end of the RNA is modified by the addition of a " cap" virtually as soon as it appears.This replacesthe triphosphate of the initial transcript with a nucleotide in reverse (3'-+5'\ orientation, thus "sealing" the end. The 3' end is modifierl by addition of a series of adenylic acid nucleotides [polyadenylic acid or poly(A)l immediately after its cleavage.
1.: r Eukaryotic is modified bythe addii :i,1r1r;,, mRNA t i o no f a c a pt o t h e5 ' e n da n dp o t y ( At)o t h e3 ' e n d .
Only after the completion of all modification and processing events can the nRNA be exported from the nucleus to the cytoplasm. The average delay in leaving for the cytoplasm is -20 minutes. Once the nRNA has entered the cytoplasm, it is recognized by ribosomes and translated. j r"r;.,r l shows that the life cycle of eukaryotic nRNA is more protracted than that of bacterial nRNA. Tfanscription in animal cells occurs at about the same speed as in bacteria, -40 nucleotides per second. Many eukaryotic genes are large; a gene of 10,000 bp takes -5 minutes to transcribe. Ttanscription of nRNA is not terminated by the releaseof enzyme from the DNA; instead the enzyme continues past the end of the gene. A coordinated series of events generates the 3' end of the nRNA by cleavage, and adds a length of poly(A) to the newly generated 3'end. Eukaryotic mRNA constitutes only a small proportion of the total cellular RNA (-3% ol the mass).Half-lives are relatively short in yeast, ranging from l-60 minutes. There is a substantial increase in stability in higher eukaryotes; animal cell nRNA is relatively stable,with halflives ranging from 4-24hours.
Duringor afterIts Transcription 137 Is Modified mRNA 7.8 Eukaryotic
Eukaryoticpolysomesarereasonablystable. The modificationsat both ends of the mRNA contributeto their stabilitv.
The5' Endof Eukaryotic mRNA Is Capped . A 5'capis formedby addinga Gto theterminal baseof the transcriptvtaa 5'-5' link. Oneto three methylgroups areaddedto the baseor riboseof guanosine. the newterminaI
Tlanscription starts with a nucleoside triphosphate (usually a purine, A or G). The first nucleotide retains its 5'triphosphate group and makes the usual phosphodiester bond from its 3' position to the 5' position of the next nucleotide. The initial sequence of the transcript can be represented as: 5'pppA/6pNpNpNp . When the mature mRNA is treated in vitro with enzymes that should degrade it into individual nucleotides, however, the 5' end does not give rise to the expectednucleoside triphosphate. Instead it contains two nucleotides, connected by a 5'-5' triphosphate linkage and also bearing methyl groups. The terminal base is i:iill:iii: .'.i;i Overview: Expression of mRNA in animaI guanine that is added to the original always a ce[[srequires processing, transcription, modification. nucleo-cytoplasmic RNA molecule after transcription. transport, andtranslation. Addition of the 5'terminal G is catalyzed by a nuclear enzyme, guanylyl transferase.The reaction occurs so soon after transcription has started that it is not possibleto detect more than Presentin allcaps trace amounts of the original 5'triphosphate end in the nuclear RNA. The overall reaction can be represented as a condensation between GTP and the original 5'triphosphate terminus of the RNA. Thus -, -, )) Gppp + pppApNpNp . v
)-) GpppApNpNp. . .+pp+p
i-i+i-litl i. ! il Thecap blocksthe 5' end of mRNAand maybe methylatedat severaIpositions.
CHAPTER 7 Messenqer RNA
The new G residue added to the end of the RNA is in the reverse orientation from all the other nucleotides. This structure is called a cap. It is a substrate for severalmethylation events. F5*iitri:.;.tS shows the full structure of a cap after all possible methyl groups have been added. \pes of caps are distinguished by how many of these methylations have occurred:
. The first methylation occurs in all eukaryotes, and consistsof the addition of a methyl group to the 7 position of the terminal guanine. A cap that possessesthis single methyl group is known as a cap 0. This is as far as the reaction proceedsin unicellular eukaryotes. The enzyme responsible for this modification is called guanine-7-methyltransferase. . The next step is to add another methyl group, to the 2'-O position of the penultimate base (which was actually the original first base of the transcripr before any modifications were made). This reaction is catalyzed by another enzyme (2'-O-methyl-transferase).A cap with the two methl'l groups is called cap l. This is the predominant type of cap in all eukaryotes except unicellular organrsms. o In a small minority of casesin higher eukaryotes, another methyl group is added to the second base. This happens only when the position is occupied by adenine; the reaction involves addition of a methyl group at the N6 position. The enzyme responsible acts only on an adenosine substrate that already has the methyl group in the 2'-O position. . In some species,a methyl group is added to the third base of the capped mRNA. The substrate for this reaction is the cap I mRNA that already possessestwo methyl groups. The third-base modification is always a2'-O ribose methylation. This createsthe cap 2 type. This cap usually representsless than I0%-I 5o/oof. the total capped population. In a population of eukaryotic mRNAs, every molecule is capped. The proportions of the different types of cap are characteristic for a particular organism. We do not know whether the structure of a particular mRNA is invariant or can have more than one type of cap. In addition to the methylation involved in capping, a Iow frequency of internal methylation occurs in the mRNA only of higher eukaryotes. This is accomplished by the generation of N6 methyladenine residues at a frequency of about one modification per 1000 bases.There are one or two methyladenines in a typical higher eukaryotic mRNA, although their presence is not obligatory; some mRNAs do not have any.
The3'Terminus Is Polyadenylated . A lengthof poty(A)-200 nucleotides longis added to a nuctear transcriptaftertranscription. . Thepoty(A)is boundby a specificprotein(PABP). . the poty(A)stabilizes against the mRNA degradation.
The 3'terminal stretch of A residues is often described as the poly(A) tail; mRNA with this feature is denoted poly(A)+. The poly(A) sequence is not coded in the DNA, but rather is added to the RNA in the nucleus after transcription. The addition of poly(A) is catalyzed by the enzyme poly(A) polymerase, which adds -200 A residues to the free 3'-OH end of the mRNA. The poly(A) tract of both nuclear RNA and mRNA is associatedwith a protein, the poly(A)-binding protein (PABP). Related forms of this protein are found in many eukaryotes. One PABP monomer of. -70 kD is bound every l0 to 20 basesof the poly(A) tail. Thus a common feature in many or most eukaryotes is that the 3' end of the nRNA consistsof a stretch of poly(A) bound to a large mass of protein. Addition of poly(A) occurs as part of a reaction in which the 3'end of the mRNA is generated and modified by a complex of enzymes (seeSection26.I9, The 3' Ends of mRNAs Are Generated by Cleavage and Polyadenylation). Binding of the PABP to the initiation factor eIF4G generates a closed loop, in which the 5' and3'ends of the mRNA find themselves held in the same protein complex (seeFigure 8.20 in Section 8.9, Eukaryotes Use a Complex of Many Initiation Factors). The formation of this complex may be responsible for some of the effects of poly(A) on the properties of nRNA. Poly(A) usually stabilizesmRNA. The ability of the poly(A) to protect mRNA against degradation requires binding of the PABP. Removal of poly(A) inhibits the initiation of translation in vitro, and depletion of PABP has the same effect in yeast invivo. These effects could depend on the binding of PABP to the initiation complex at the 5' end of nRNA. There are many examples in early embryonic development where polyadenylation of a particular mRNA is correlated with its translation. In some cases,mRNAs are stored in a nonpolyadenyIated form, and poly(A) is added when their translation is required; in other cases,poly(A)+
Is Potyadenylated1 3 9 7.10 The3'Terminus
Most oJ RNA population mRNAwith poly(A)is is rRNAthat lackspoly(A) small proportionof RNA
oligo(dT) Sepharose AA
-
POIY(A)+RNA sticks10column
The availability of a cloned DNA makes it easy to isolate the corresponding mRNA by hybridization techniques. Even mRNAs that are present in only very few copies per cell can be isolated by this approach. Indeed, only mRNAs that are present in relatively large amounts can be isolated directly without using a cloning step. Almost all cellular mRNAs possesspoly(A). A significant exception is provided by the mRNAs that code for the histone proteins (a major structural component of chromosomal material). These mRNAs comprise most or all of the poly(A)- fraction. The significance of the absence of poly(A) from histone mRNAs is not clear, and there is no particular aspect of their function for which this appearsto be necessary.
:'rr' ::. : Poty(A)+ RNAcanbeseparated fromother RNAs by fractionation on Sepharose-otigo(dT). mRNAs are de-adenylated, and their translation is reduced. The presence of poly(A) has an important practical consequence.The poly(A) region of mRNA can basepair with oligo(U) or oligo(dT), and this reaction can be used to isolate poly(A)+ mRNA. The most convenient technique is to immobilize the oligo (U or dT) on solid support material. Then, when an RNA population is applied to the colurnn, as illustrated in \ ir .ijitlj .r:, : |i, only the poly(A)+ RNA is retained.It can be retrieved by treating the column with a solution that breaks the bonding to releasethe RNA. The only drawback to this procedure is that it isolatesall the RNA that contains poly(A). If RNA of the whole cell is used, for example, both nuclear and cytoplasmicpoly(A)+ RNA will be retained.If preparationsof polysomesare used (a common procedure), most of the isolated poly(A)+ RNA will be active nRNA. However, in addition to mRNA in polysomes, there are also ribonucleoprotein particles in the cytosol that contain poly(A)+ nRNA, but which are not translated.This RNA may be "stored" for use at some other time. Isolation of total poly(A)+ nRNA therefore does not correspond exactly with the active nRNA population. The "cloning" approachfor purifying nRNA usesa procedure in which the mRNA is copied to make a complementary DNA strand (known as cDNA). Then the cDNA can be used asa template to synthesizea DNA strand that is identical with the original nRNA sequence.The product of these reactions is a double-stranded DNA corresponding to the sequenceof the nRNA. This DNA can be reproduced in large amounts.
C H A P T E7RM e s s e n g eRrN A
BacteriaI mRNA Degradation Involves MuLtiple Enzymes r Theoveralldirectionof degradation of bacterial mRNA is 5'+3'. r Degradation resutts fromthe combination of endonucteolytic foltowed cteavages by exonucleolytic degradation of thefragment rromJ -+5 .
Bacterial mRNA is constantly degraded by a combination of endonucleases and exonucleases.Endonucleasescleavean RNA at an internal site. Exonucleasesare involved in trimming reactions in which the extra residues are whittled away, baseby base,from the end. Bacterial exonucleasesthat act on single-strandedRNA proceed along the nucleic acid chain from the 3'end. The way the two types of enzymes work together to degrade an mRNA is shown in irii.iil:i-.,,iri. Degradation of a bacterial nRNA is initiated by an endonucleolytic attack. Several 3' ends may be generated by endonucleolytic cleavageswithin the mRNA. The overall direction of degradation (asmeasured by loss of ability to synthesize proteins) is 5'-+3'. This probably results from a successionof endonucleolytic cleavagesfollowing the last ribosome. Degradation of the releasedfragments of nRNA into nucleotides then proceeds by exonucleolytic attack from the free 3'-OH end toward the 5'terminus (that is, in the opposite direction from transcription). Endonucleolytic attack
releasesfragments that.may have different susceptibilities to exonucleases.A region of secondary structure withirr the mRNA may provide an obstacleto the exonuclease,thus protecting the regions on its 5'side. The stability of each mRNA is therefore determined by the susceptibility of its particular sequence to both endoand exonucleolytic cleavages. There are -12 ribonucleases in E. coli. Mutants in the endoribonucleases (except ribonuclease I, which is without effect) accumulate unprocessed precursors to rRNA and IRNA, but are viable. Mutants in the exonucleasesoften have apparently unaltered phenotypes, which suggeststhat one enzyme can substitute for the absence of another. Mutants lacking multiple enzymes sometimes are inviable. RNAase E is the key enzyme in initiating cleavageof mRNA. It nay be the enzyme that m a k e s t h e f i r s t c l e a v a g ef o r m a n y m R N A s . Bacterial mutants that have a defective ribonuclease E have increased stability (twoto threefold) of mRNA. However, this is not its only function. RNAase E was originally discovered as the enzyme that is responsible for processing5'rRNA. from the primary transcript by a specific enLdonucleolyticprocesslng event. T h e p r o c e s so f d e g r a d a t i o n m a y b e c a t alyzed by a multienzpne complex (sometimes called the degradosome) that includes ribonuc l e a s eE , P N P a s e ,a n d a h e l i c a s e .R N A a s e E plays dual roles. Its N-terminal domain provides an endonuclease activity. The C-terminal domain provides a scaffold that holds together the other components. The helicase unwinds the substrate RNA to make it availa b l e t o P N P a s e .A c c o r d i n g t o t h i s m o d e l . RNAase E makes the initial cut and then passes the fragments to the other components of the complex for processing. Polyadenylation may play a role in initiating degradation of some mRNAs in bacteria. Poly(A) polymeraseis associatedwith ribosomes inE. coli,and short (10 to 40 nucleotide) stretches of poly(A) are added to at least some mRNAs. Triple mutations thal remove poly(A) polymerase,ribonucleaseE, and polyrucleotide phosphorylase (PNPaseis a 3'-5' exonuclease) have a strong effect on stability. (Mutations in individual genesor pairs of geneshave only a weak effect.)Poly(A) polymerasemay createa poly(A) tail that acts as a binding site for the nucleases. The role of poly(A) in lbacteriawould therefore be different from that in eukaryotic cells.
Translation +
5t -?-
,-\.r'
-'- . r-\'a
.-'' '. ^
3'
i ii.,,ir'i' r,. Degradation is a two-stage mRNA of bacteriaI proceed 5'-3' behind process. cleavages Endonucteolytic t h e r i b o s o m eTsh. er e l e a s efdr a g m e n tasr ed e g r a d ebdy exonucteases that move3'-5'.
mRNA StabitityDepends
on Its Structure andSequence . Themodifications protectit at bothendsof mRNA by exonucteases. againstdegradation . Specific mayhave withinan mRNA sequences lizing effects. stabitizing or destabi . Destabitization maybetriggeredby lossof poty(A).
The major features of nRNA that affect its stability are summarized in i I l'r.iii ir ' ,:,r . Both struc" ture and sequenceare important. The 5'and 3' terminal structures protect againstdegradation, and specific sequenceswithin the mRNA may either serve as targets to trigger degradation or may protect against degradation: . The modifications at the 5'and 3' ends of mRNA play an important role in
andSequence 147 on lts Structure StabitityDepends 7.12 mRNA
preventing exonuclease attack. The cap prevents 5'-3' exonucleasesfrom attackCap protects Nonsense Endonuclease Poly(A) protects ing the 5'end, and the poly(A) prevents against5'-3' codons attacks against3'-5' 3'-5' exonucleases from attacking the exonuclease triggers destabilizing exonuclease urveillance J'end. sequence . Specific sequence elements within the mRNA may stabilize or destabilize it. The most common location for destabilizing elements is within the 3'untranslated region. The presence of such an element shortens the lifetime of the 3'UTR 5' UTR Codingregion mRNA. . Within the coding region, mutations FI{UR[?.li TheterminaI protectit against modifications of mRNA degradathat create termination codons trigger tion.InternaIsequences mayactivate degradation systems. a surveillance system that degradesthe mRNA (seeSection 7.14, NonsenseMutations Trigger a Surveillance System). Destabilizing elements have been found in several yeast mRNAs, although as yet we do not see any common sequencesor know how they destabilizethe mRNA. They do not necessarily act directly (by providing targets for endonucleases), but may function indirectly, perhaps by encouraging deadenylation. The criterion for defining a destabilizing sequenceelement is that its introduction into a new mRNA may causeit to be degraded. The removal of an element from an mRNA does not necessarily stabilize it, suggesting that an individual mRNA can have more than one destabilizing element. A commonfeature in some unstable mRNAs is the presence of an AU-rich sequence of -50 bases (called the ARE) that is found in the 3' trailer region. The consensus sequence in the ARE is the pentanucleotide AUUUA, repeated F l { , U R?[ . 2 ? A nA R Ei n a 3 ' n o n t r a n s l a treedq i o n initiatesdeqradation of mRNA. several times. FlGUftf?.fr?shows that the ARE triggers destabilization by a two-stage process: first the mRNA is deadenylated, and then it decays. The deadenylation is probably needed because it causesloss of the poly(A)-binding protein, whose presence stabilizesthe 3'region (seeSection 7.13, nRNA Degradation Involves proteinbindsIRSin absence IRE-binding of iroh Multiple Activities). In some cases,an mRNA can be stabilized by specifically inhibiting the function of a desta{4}$" bilizing element. Tlansferrin mRNA contains a IRE-binding proteindissociates in presence of iron sequence called the iron-responsive element (IRE), which controls the response of the nRNA to changes in iron concentration. The IRE is (A)n located in the 3'nontranslated region, and contains stem-loop structures that bind a protein whose affinity for the mRNA is controlled by iron. FIGURI 7.#3shows that binding of the protein to the IRE stabilizes the nRNA by inhibitf{*liRil?,fi AnIREin a 3'nontranstated region controls ing the function of (unidentified) destabilizing rRNAstabitity. sequencesin the vicinity. This is a general model
I
742
C H A P T E7RM e s s e n g eRrN A
for the stabilization of nRNA, that is, stability is conferred by inhibiting the function of destabilizing sequences. Decapping and degradation occur in large protein complexes, which may be localized in the form of discrete c'ytoplasmicunits called P-bodies, to which other enzymes involved in mRNA production or noetabolismmay be localized. In fact, P-bodiesmay provide a means for sequestering any mR|[As that are not actively involved in translation.
mRNA Deqradation InvoLves Multipl"e Activities . Degradation of yeastmRNA requires removal of the 5 ' c a pa n dt h e3 ' p o t y ( A ) . r Oneyeastpathwayinvolvesexonucteotytic from5'-+3'. degradation r Another yeastpathway usesa comptex of several exonucteases that workin the 3'-+5'direction. o Thedeadenytase of anirnal cetlsmaybinddirectty t o t h e5 ' c a p .
We know the most about the degradation of nRNA in yeast. There are basically two pathways. Both start with removal of the poly(A) tail. This is catalyzed by a specific deadenylase, which probably functions as part of a large protein complex. (The catalytic subunit is the exonucleaseCcr4 in yeast;it is also the exonucleasePARN in vertebrates, which is related to RNAaseD.) The enzyme action is processiveonce it has startedto degradea particular mRNA substrate, it continue:; to whittle away at that mRNA, baseby base. The major degradation pathway is summarized in : ,.,,ii;:-: ,,;r,.Deadenylationat the 3'end triggersdecappingat the 5'end. The basisfor this relationship is that th:e presence of the PABP (poly(A)-binding protein) on the poly(A) prevents the decapping enzyme from binding to the 5'end. PABP is releasedwhen the length of poly(A) falls below l0 to I 5 residues.Decapping occurs by cleaving the methylated base off the 5' end to leave a monophosphate in the . --) mTGDP + pX . reaction: mTGpppX . The enzyme requires the 7-methyl group. Each end of the rnRNA influences events that occur at the other end. This is explained by the fact that the trn,oends of the mRNA are held together by the factors involved in protein
II AAAATAAAAAAA
XRNl
r:i;,ii i decapping to occur, attows Deadenytation fromthe 5'end. cleavage whichleadsto endonucleotytic
synthesis (see Section 8.9, Eukaryotes Use a Complex of Many Initiation Factors).The effect of PABP on decapping allows the 3'end to have an effect in stabilizing the 5' end. There is also a connection between the structure at the 5' end and degradationat the 3'end. The deadenylase directly binds to the 5' cap, and this interaction is in fact needed for its exonucleolytic attack on the poly(A). What is the rationale for the connection between events occurring at both ends of an mRNA? Perhapsit is necessaryto ensure that the mRNA is not left in a state (having the structure of one end but not the other) that might compete with active nRNA for the proteins that bind to the ends. Removal of the cap triggers rh'e 5'-3'degradation pathway in which the mRNA is degraded rapidly from the 5'end, by the 5'-3'exonuciease XRNI. The decapping enzyme is concentrated in discrete cytoplasmic foci, which may be "processingbodies" where the mRNA is deadenylated and then degraded after it has been decapped. In the secondpathway, deadenylated yeast mRNAs can be degradedby rhe 3'-5' exonuclease activity of the exosome , a complex of >9 exonucleases.The exosome is also involved in processing precursors for rRNAs. The aggregation of the individual exonucleasesinto the exosome complex may enable 3'-5'exonucleolytic activities to be coordinately controlled. The exosome may also degrade fragments of mRNA
InvolvesMultipleActivities 7.13 mRNADegradation
743
Nonsense Mutations Trigger a SurveiL[ance
Deadenylation
System Endonucleolytic degradation
r Nonsense mutations causemRNA to be degraded. r Genes codingfor the degradation system have beenfoundin yeastandworms. 3'-5' exonucleolytic degradation
:::i:aji .=.r=:Deadenylation mayleaddirectty to endonucteol.ytic cleavage andexonucleotytic fromthe cteavage 3'end(s).
Wild-typemRNAhas normal Translation -.>
!,]**-t"-:l
Nonsensemutationtriggersdegradation
Premature termination
t
I
Degradationinitiates
i'ii-ii$:,1-liii:Nonsense mutations mavcausemRNA to be degraded.
releasedby endonucleolytic cleavage.ri;"il.s*iil :.tlI shows that the 3'-5' degradation pathway may actually involve combinations of endonucieolytic and exonucleolytic action. The exosome is also found in the nucleus, where it degrades unspliced precursors to mRNA. Yeastmutants lacking either exonucleolytic pathway degrade their mRNAs more slowly, but the loss of both pathways is lethal.
744
C H A P T E7RM e s s e n g eRrN A
Another pathway for degradation is identified by nonsense-mediated mRNA decay. FiillJR{l.I* shows that the introduction of a nonsense mutation often leads to increased degradation of the mRNA. As may be expected from dependence on a termination codon, the degradation occurs in the cytoplasm. It may represent a quality control or surveillance system for removing nonfunctional mRNAs. The surveillance system has been studied best in yeast and C. elegans,but may also be important in animal cells. For example, during the formation of immunoglobulins and T cell receptors in cells of the immune system, genes are modified by somatic recombination and mutation (seeChapter 23, Immune Diversity). This generatesa significant number of nonfunctional genes whose RNA products are disposed of by a surveillance system. In yeast,the degradation requires sequence elements (called DSE) that are downstream of the nonsense mutation. The simplest possibility would be that these are destabilizing elements and that translation suppressestheir use. When translation is blocked, however, tne mRNA is stabilized. This suggeststhat the process of degradation is linked to translation of the mRNA or to the termination event in some direct way. Genes that are required for the processhave been identified in S,cerevisiae(upfIocl) and C.elegans (smgloci)by identifying suppressorsof nonsense-mediateddegradation.Mutations in these genes stabilize aberrant mRNAs, but do not affect the stability of most wild-t1pe rranscriprs. One of these genes is conserved in eukaryotes (upfl /smg2) It codes for an AlP-dependent helicase(an enzyme that unwinds double-stranded nucleic acids into single strands). This implies that recognition of the mRNA as an appropriate larget for degradation requires a change in its structure. Upf linteracts with the releasefactors (eRFI and eRF3) th'al catalyze termination, which is probably how it recognizes the termination event. It may then "scan" the mRNA by mov-
ing toward the 3' end to look for the downstream sequenceelements. In mammalian cells, the surveillance system appearsto work only on mutations located prior to the last exon-in other words, there must be an intron after the site of mutation. This suggeststhat the system requires some event to occur in the nucleus, before the introns are removed by splicirrg. One possibility is that proteins attach to the nRNA in the nucleus at the exon-exon boundary when a splicing event :li:..iili:.:."; ShTJWS OCCUIS. a general mOdel fOr the operation of such a system. This is similar to the way in which an nRNA may be marked for export from the nucleus (seeSection26.10, Splicing Is Connected to Export of mRNA). Attachment of a protein to the exon-exon junction createsa mark of the event that persists into the cytoplasm. Human homologues of the yeast Upf2,3 proteins may be involved in such a system. They bind specifically to mRNA that has been spliced.
Eukaryotic RNAs AreTransported RNAis transported througha membrane asa particte. ribonucleoprotein A[[eukaryotic RNAs that functionin the cytoptasm mustbe exported fromthe nucteus. tRNAs andthe RNAcomponent of a ribonuctease areimported into mitochondria. plant mRNAs cantravellongdistances between cet[s.
A bacterium consists of only a single compartment, so all the RNAs f unction in the same environment in which they are synthesized. This is most striking in the caseof mRNA, where translation occurs simultaneously with transcription (seeSection7 .7,The Life Cycle of BacterialMessengerRNA). RNA is transported through membranes in the variety of instances summarized in i I i - : . i j - :' i , . I t p o s e s , as i g n i f i c a n t t h e r m o d y namic problem to transpoil a highly negative RNA through a hydrophobic membrane, and the solution is to transport the RNA packaged with proteins. In eukaryotic cel.[s,RNAs are transcribed in the nucleus, but t.ranslation occurs in the cytoplasm. Each typerof RNA must be transported into the cytoplasm to assemblethe apparatus for translation. l'he rRNA assembleswith ribosomal proteins into immature ribosome
J:jr'ii. system couldhavetwotypes iii: I ,' ,' A surveittance to mustbindin the nucteus Protein(s) of components. could event.0therproteins markthe resultof a spticing They or cytoplasm. bindto themarkeitherin thenucteus whenribosomes themRNA aretriggered to actto degrade premature[y. terminate
All RNA
Nucleus-+cytoplasm
All cells
IRNA
Nucleus-+ mitochondrion
Many cells
mRNA
Nursecell-+oocyte
mRNA
Anterior+posterioroocyte
mRNA
Cell-+cell
--d
i r i i . i i i i ! . : i : r r R N A s a r e t r a n s p o r t e d t h r om eh m b r a n e sa ' i vn a r i e t y o sf y s t e m s ' ug
subunits that are the substrates for the transport system. IRNA is transported by a specific protein system. nRNA is transported as a ribonucleoprotein, which forms on the RNA transcript in the nucleus (seeChapter 26, RNA Splicing and Processing).These processesare common to all eukaryotic cells. Many mRNAs are translated in the cytosol, but some are localized within the cell by means of attachment to
AreTransported 145 RNAs 7.15 Eukarvotic
a cytoskeletalelement. One situation in which localization occurs is when it is important for a protein product to be produced near to the site of its incorporation into some macromolecular structure. Some RNAs are made in the nucleus, exported to the cytosol. and then imported into mitochondria. The mitochondria of some organisms do not code for all of the tRNAs that are required for protein synthesis (seeSection 4.10, Organelle Genomes Are Circular DNAs That Code for Organelle Proteins). In these cases,the additional tRNAs must be imported from the cytosol. The enzyme ribonuclease P, which contains both RNA and protein subunits, is coded by nuclear genes, but is found in mitochondria as well as the nucleus. This means that the RNA must be imported into the mitochondria. We know of some situations in which nRNA is even transported between cells.During development of the oocyte in Drosophila, certain mRNAs are transported into the egg from the nurse cells that surround it. The nurse cells have specializedjunctions with the oocyte that allow passageof material needed for early development. This material includes certain mRNAs. Once in the egg, these mRNAs take up specific locations. Some simply diffuse from the anterior end where they enter, but others are transported the full length of the egg ro the posterior end by a motor attached to microtubules. The most striking caseof transport of mRNA has been found in plants. Movement of individual nucleic acidsover long distanceswas first discovered in plants, where viral movement proteins help propagate the viral infection by transporting an RNA virus genome through the plasmodesmata (connections between cells). Plants also have a defensesystem,which causes cells to silence an infecting virus. This, too, may involve the spread of components including RNA over long distance between cells. Now it has turned out that similar systems may transpofi mRNAs between plant cells. Although the existence of the systemshas been known for some time, it is only recently that their functional importance has been demonstrated. This was shown by grafting wild-type tomato plants onto plants that had the dominant mutationMe (which causesa change in the shapeof the leaf). mRNA from the mutant stock was transported into the leaves of the wild-type graft, where it changed their shape.
746
CHAPTER 7 Messenger RNA
mRNA CanBe Specifica[[y Locatized o Yeast Ashl mRNA formsa ribonucteoorotein that bindsto a myosinmotor. . A motortransports it alongactinfilaments into the daughter bud. r It is anchored in the bud,sothat andtranslated the proteinis foundonlyin the bud. An mRNA is synthesized in the nucleus but translated in the cytoplasm of a eukaryotic cell. It passesinto the cytoplasm in the form of a ribonucleoprotein particle that is transported through the nuclear pore. Once in the cytosol, the mRNA may associatewith ribosomes and be translated. The cytosol is a crowded place occupied by a high concentration of proteins. It is not clear how freely a polysome can diffuse within the cytosol, and most mRNAs are probably translated in random locations, determined by their point of entry into the cytosol, and the distance that they may have moved away from it. However, some mRNAs are translated at specific sites.This may be accomplished by several mechanisms: r An mRNA may be specifically transported to a site where it is translated. . It may be universally distributed but degraded at all sites except the site of translation. . It may be freely diffusible but become trapped at the site of translation. One of the best characterizedcasesof localization within a cell is that of AshI in yeast. Ashl repressesexpression of the HO endonuclease in the budding daughter cell, with the result that HO is expressedonly in the mother cell. The consequence is that mating type is changed only in the mother cell (see Section 19.24, Regulation of HO Expression Controls Switching). The cause of the restriction to the daughter cell is that all the Ashl mRNA is transported from the mother cell, where it is made, into the budding daughter cell. Mutations in any one of five genes, called SHEL-5,prevent the specificlocalization and cause AshI mRNA to be symmetrically distributed in both mother and daughter compartments. The proteins Shet, -2, and -l bind Ashl mRNA into a ribonucleoprotein particle that transports the mRNA into the daughter cell. fl*tJftil ;.;S shows the functions of the proteins. Shelp is a myosin (previously identified as Myo4), and She3 and
' 1: r.r:.:r' -j.1:'] :
into fromthenucleus is exported AshlmRNA with intoa complex it js assembled thecytoplasm, where it alongactinfitThecomplex transports theSheproteins. aments to the bud. ''...' ,.'r . .:, AshLmRNA formsa ribo-nucteoprotein cont a i n i n ga m y o s i nm o t o rt h a t m o v e si t a l o n ga n a c t i n fi[ament. She2 are proteins that connect the myosin to the nRNA. The myosin is a motor that moves the mRNA along actin filarnents. iri::,,I i:: I . : i; Summarizesthe overall process. Ashl mRNA is exported from the nucleus in the form of a ribonucleoprotein. In the cytoplasm it is first bound by She2, which recognizes some stem-loop secondarystructures within the mRNA. Then She3 binds to She2, after which the myosin Shel binds. Next, the particle hooks on to an actin filament and moves to the bud. When Ashl nRNA reachesthe bud, it is anchored there, probably by proteins that bind specificallyto themRNA. Similar principles govern other caseswhere mRNAs are transported to specific sites. The nRNA is recognized by means of czs-acting sequences,which usually are regions of secondary structure in the 3'untranslated region. (AshI mRNA is unusual in that the crs-acting regions are in the coding frame.) The mRNA is packaged into a ribonucleoprotein particle. In some cases,the transpOrtednRNA can be visualized in very large particles called nRNA granules. The particles are large enough (several times the size of a ribosome) to contain many protein and RNA components. A transported mRNP must be connected to a motor that moves it along a system of tracks. The tracks can be either actin filaments or micrt-rtubules. Whereas Ash.t usesa myosin motor on actin tracks. oscarmRNA in the Drosophilaegg usesa kinesin motor to move along microtubules. Once the mRNA reaches its destination, it needs to be anc.horedin order to prevent it from diffusing away. Lessis known about this,
but the processappearsto be independent of transport. An nRNA that is transported along microtubules may anchor to actin filaments at its destination.
mary Sum Genetic information carried by DNA is expressed in two stages:transcription of DNA into mRNA and translation of the mRNA into protein. Messenger RNA is transcribed from one strand of DNA. It is complementary to this (noncoding) strand and identical with the other (coding) strand. The sequence of nRNA, in triplet codons 5'-3', is related to the amino acid sequenceof protein, N- to C-terminal. The adaptor that interprets the meaning of a codon is transfer RNA, which has a compact L-shaped tertiary structure; one end of the IRNA has an anticodon that is complementary to the codon, and the other end can be covalently linked to the specific amino acid that corresponds to the target codon. A IRNA carrying an amino acid is called an aminoacyl-tRNA. The ribosome provides the apparatus that allows aminoacyl-tRNAs to bind to their codons on nRNA. The small subunit of the ribosome is bound to mRNA; the large subunit carriesthe nascent polypeptide. A ribosome moves along mRNA from an initiation site in the 5'region to a termination site in the 3'region, and the appropriate aminoaryl-tRNAs respond to their codons, unloading their amino acids, so that the growing pollpeptide chain extends by one residue for each codon traversed. The translational apparatus is not specificfor tissue or organism; an nRNA from one source can be translated by the ribosomes and tRNAs from another source. The number of times any
7.17Summary 747
mRNA is translated is a function of the affinity of its initiation site(s) for ribosomes and its stability. There are some casesin which translation of groups of nRNA or individual mRNAs is specifically prevented: this is called translational control. A typical mRNA contains both a nontranslated 5'leader and a 3'trailer as well as coding region(s). Bacterial mRNA is usually polycistronic, with nontranslated regions between the cistrons. Each cistron is represented by a coding region that starts with a specific initiation site and ends with a termination site. Ribosome subunits associateat the initiation site and dissociateat the termination site of each codine reglon. A growing E. coli bacterium has -20,000 ribosomesand -200,000 tRNAs, mostly in the form of aminoacyl-IRNA. There are -1500 nRNA molecules, representing two to three copiesof each of 600 different messengers. A single mRNA can be translated by many ribosomes simultaneously, generating a polyribosome (or polysome). Bacterial polysomes are large, typically with tens of ribosomes bound to a single mRNA. Eukaryotic polysomes are smaller, typically with fewer than ten ribosomes; each mRNA carries only a single coding sequence. Bacterial mRNA has an extremely short half-life of only a few minutes. The 5'end starts translation even while the downstream sequences are being transcribed. Degradation is initiated by endonucleasesthat cut at discretesites, following the ribosomes in the 5'-l'direction, after which exonucleasesreduce the fragments to nucleotides by degrading them from the released 3' end toward the 5' end. Individual sequencesmay promote or retard degradation in bacterial mRNAs. Eukaryotic nRNA must be processed in the nucleus before it is transported to the cytoplasm for translation. A methylated cap is added to the 5'end. It consistsof a nucleotide added to the original end by a 5'-5'bond, after which methyl groups are added. Most eukaryotic nRNA has an -200 base sequence of poly(A) added to its 3'terminus in the nucleus after transcription, but poly(A)- mRNAs appear to be translated and degraded with the same kinetics as poly(A)+ mRNAs. Eukaryotic mRNA exists as a ribonucleoprotein particle; in some casesmRNPs are stored that fail to be translated. Eukaryotic mRNAs are usually stable for several hours. They may have multiple
sequencesthat initiate degradation; examples are known in which the processis regulated. Yeast mRNA is degraded by (at least) two pathways. Both start with the removal of poly(A) from the 3' end, causinglossof poly(A)binding protein, which in turn leads to removal of the methylated cap from the 5' end. One pathway degrades the mRNA from the 5'end by an exonuclease.Another pathway degrades from the 3' end by the exosome, a complex containing several exonucleases. Nonsense-mediated degradation leads to the destruction of mRNAs that have a termination (nonsense) codon prior to the last exon. T}re upflociin yeast and the smgloci in worms are required for the process. They include a helicase activity to unwind mRNA and a protein that interacts with the factors that terminate protein synthesis.The features of the process in mammalian cells suggestthat some of the proteins attach to the mRNA in the nucleus when RNA splicing occurs to remove introns. mRNAs can be transported to specificlocations within a cell (especially in embryonic development). In the AshI system in yeast, mRNA is transported from the mother cell into the daughter cell by a myosin motor that moves on actin filaments. In plants, mRNAs can be transported long distancesbetween cells.
References Transfer RNAForms a Clover[eaf Review Soll, D. and RajBhandary,U. t. (1995). IRNA Structure, Biosynthesis,and Function. Washington, DC: American Society for Microbiology. Resea rch Chapeville, F. et al. (1962). On the role of soluble RNA in coding for amino acids. Proc Natl. Acad. Sci.USA 48, 1086-1092. Hoagland, M. B. et al. (1958). A soluble RNA intermediate in protein synthesis. J. Blol. Chem.2)I, 241-257. Holley, R. W. et al. (I965). Structure of an RNA. ScienceI47, 1462-1465
Messenger RNAIs Translated by Ribosomes Resea rch Dintzis, H. M. (1961). Assembly of the peptide chain of hemoglobin. Proc Natl. Acad. Sci.
usA47, 247-26r.
748
CHAPTER 7 Messenger RNA
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Resea rch Cui, Y., Hagan, K. W., Zhang, S., and Peltz, S. W. ( 1995). Identification and characterizationof genes that are required for the accelerated degradation of mRNAs containing a premature translational termination codon. Genes D e v . 9 ,4 2 ) - 4 ) 6 . Czaplinski, I(., Ruiz-Echevarria, M J., Paushkin, S. V., Han, X., Weng, Y., Perlick, H. A., Dietz, H. C., Ter-Avanesyan,M. D., and Peltz, S. W. ( I 998 ) . The surveillance complex interacts with the translation release factors to enhance termination and degrade aberrant mRNAs. GenesDev.12, 1665-1677. Le Hir, H., Moore, M. J., and Maquat, L. E. (2000). Pre-nRNA splicing alters mRNP composition: evidence for stable association of proteins at exon-exon junctions. GenesDey. ).4, r 0 9 8 - 11 0 8 . Lykke-Andersen,J., Shu, M. D., and Steirz,J. A. (2000). Human Upf proteins target an nRNA for nonsense-mediateddecay when bound downstream of a termination codon. Cell lO). I l2l-1 t 3l. Peltz, S. W., Brown, A. H., and Jacobson,A. (1993). mRNA destabilizationtriggeredby premature translational termination depends on at least three cls-actingsequence elements and one trans-acting f.acI-or.GenesDev.7 , t7)7-t7 54. Pulak, R. and Anderson, P. (19%1. mRNA surveillance by the C. eleganssmg genes.GenesDev.7, I 885-l 897 Ruiz-Echevarria,M. J. et al. (1998).Identifying the right stop: determining how the surveillance complex recognizes and degrades an a b e r r a n tm R N A . E M B OJ . 1 5 , 2 8 1 0 - 2 8 I 9 . Weng, Y., Czaplinski,K., and Peltz, S. (1996). Genetic and biochemical characterization of mutants in the ATPaseand helicase regions of the Upf I protein. Mol CellBiol. 16, 5477-5490. Weng, Y., Czaplinski,K., and Pelrz,S. (1996). Identification and characterization of mutations in the upfl gene that affect the Upf protein complex, nonsense suppression, but not mRNA turnover. Mol. CellBiol. 16. 549t-5506.
Eukaryotic RNAs AreTransported KCVICWS Ghoshroy, S., Lartey, R., Sheng, J., and Citovsky, V. (1997). Transport of proteins and nucleic acids
150
CHAPTER 7 Messenger RNA
through plasmodesma:a. Ann. Rev.Plant. Physiol.Plant Mol Biol 48,27-50. Jansen,R. P. (2001). mRNA localization:message on the move. Nat. Rev.Mol. CellBiol.2. 247-256. Lucas, W. J. and Gilbertson, R. L. (1994). Plasmodesmata in relation to viral movement within leaf tissues.Ann. Rev.Phytopathol.32, 387-4ll. Vance, V. and Vaucheret, H. (2001). RNA silencing in plants-defense and counterdefense. Science 292,2277-2280. Resea rch ICm, M., Canio, W., I(essler, S., and Sinha, N. (2001). Developmental changesdue to longdistance movement of a homeobox fusion transcript in tomato. Science 29), 287-289. Puranam, R. S. and Attardi, c. (2001). The RNase P associatedwith HeLa cell mitochondria contains an essential RNA component identical in sequence to that of the nuclear RNase P.Mol. CellBiol. 2l , 548-561.
mRNA CanBeSpecificatty Localized Reviews Chartrand, P., Singer, R. H., and Long, R. M. (2001). RNP localizationand transport in yeasL.Annu Rev.CellDev.Biol. 17, 297-310. Jansen, R. P. (2001 ) . mRNA localization: message on the move. Nat. Rev.Mol. CellBiol 2. 247-256. ICoc, M., Zearfoss,N. R., and Etkin, L. D. (2002). Mechanisms of subcellular mRNA localization. Cell 108, 533-544. Palacios,I. M. and Johnston, D. S. (2001). Getting the messageacross:the intracellular localization of mRNAs in higher eukaryoles. Annu. Rev.CellDev.Biol. 17, 569-614. Resea rch Bertrand, E., Chartrand, P., Schaefer,M., Shenoy, S. M., Singer,R. H., and Long, R. M. (1998). Localization of ASHI mRNA particles in living yeast. Mol. Cell 2, 4)7445. Long, R. M., Singer, R. H., Meng, X., Gonzalez,L, Nasmyth, I(., and Jansen,R. P. (1997). Mating type switching in yeast controlled by asymmetric localization of ASHI mRNA. Science 277, j83-387 .
ProteinSynthsis C H A P T EO RU T L I N E
l
Introduction ProteinSynthesis0ccursby Initiation, Etongation, and Termination o Theribosome hasthreeIRNA-binding sites. r An aminoacy[-tRNA enterstheA site. . Peptidyt-tRNA is boundin the P site. r Deacylated IRNAexitsviathe Esite. o An aminoacidis addedto the potypeptide chainbytransferringthe potypeptide frompeptidyt-tRNA in the P siteto in the A site. aminoacyltRNA GE
SpecialMechanisms Controlthe Accuracyof Protein Synthesis r Theaccuracy is controlted of proteinsynthesis by specific mechanisms at eachstage. Initiation in BacteriaNeeds30SSubunitsand Accessorv Factors r Initiationof proteinsynthesis requires separate 30Sand 50Sribosome subunits. -2, and-3),whichbindto 30Ssubr Initiationfactors(IF-1., unjts,areatsorequired. r A 30Ssubunitcarrying initiationfactorsbindsto an initiation siteon mRNA to formaninitiationcomptex. o IF-3mustbe reteased to atlow50Ssubunits to iointhe complex. 30S-mRNA A SpecialInitiator IRNAStartsthe Potypeptide Chain o Protein synthesis startswitha methionine aminoacidusuaLtycodedby AUG. r Different methionine in initiationand tRNAs areinvolved etongation. r TheinitiatortRNAhasuniouestructuraI features that distinguishit froma[[othertRNAs. r TheNHzgroupof the methionine initiaboundto bacteriaI tor IRNAis formytated. Is Controtted Useof fMet-tRNAr by IF-2 and the Ribosome r IF-2bindstheinitiatorfMet-tRNAr andalowsit to enterthe oartialP siteon the 305subunit. Initiation InvolvesBasePairingBetweenmRNAand rRNA r Aninitiationsiteon bacterial. mRNA consists of theAUGinitiationcodonpreceded with a gapof -10 bases by the potypurine Shine-Datgarno hexamer.
r TherRNAof the 30Sbacterial subunithasa comribosomal ptementary sequence that basepairswiththeShinesequence duringinitiation. Datgarno Sma[[SubunitsScanfor Initiation Siteson Eukaryotic mRNA . Eukaryotic bindto the 5'endof subunits 40Sribosoma[ untjltheyreachan initiationsite. andscanthe mRNA mRNA . A eukaryotic initiationsiteconsistsof a ten-nucteotide an AUGcodon. that includes seouence r 605ribosomaI at theinitiation the comptex subunitsjoin site. Usea Comptexof ManyInitiation Factors Eukaryotes e Initiationfactorsarerequired for a[[ stagesof initiation, bindingtheinitiatorIRNA,40Ssubunitattachinctuding andjoiningof alongthe mRNA. movement mentto mRNA, the 605subunit. r Eukaryotic that is different initiatorIRNAis a Met-tRNA butthe methionine usedin elongation, fromthe Met-tRNA is notformutated. r eIF2bindsthe initiatorMet-tRNAi andthe complex andGTP, with mRNA. it associates before bindsto the40Ssubun'it ElongationFactorTu LoadsAminoacyt-tRNA into the A Site r EF-Tu G proteinwhoseactiveform(boundto is a monomeric bindsaminoacyt-tRNA. GTP) . TheEF-Tu-GTP-aminoacy[-tRNA to the ribocomptex b'inds someA site. to Aminoacyt-tRNA ChainIs Transferred The Potypeptide r The50Ssubunithaspeptidyttransferase activity. r Thenascentpol,ypeptide frompeptidyLchainis transferred in theA site. IRNAin the P siteto aminoacyt-tRNA . Peptide generates IRNAin the P deacylated bondsynthesis in theA site. siteandpeptidyt-tRNA Movesthe Ribosome Transtocation . Ribosomal throughthe ribomoves the mRNA transtocation somebythreebases. r Translocation IRNAinto the Esite and movesdeacytated peptidyt-tRNA into the P site,andemptiesthe A site. . Thehybridstatemodelproposes occurs that transtocation relative to the 305, in whichthe 50Smoves in two stages, the origina[ to restore a[ongmRNA andthenthe 30Smoves conformation' on nerc pqge continued
151
GTET ElongationFactorsBindAtternate[y to the Ribosome o Transtocation requires EF-G, whosestructureresembles the aminoacyltRNA-EF-TuGTPcomotex. e Bindingof EF-Tu andEF-G to the ribosome is mutuatly exctusive. o Translocation requires GTPhydrotysis. whichtriggers a change in EF-G. whichin turntriggers a change in ribosome structure.
GEA
ThreeCodonsTerminateProtein Synthesis o Thecodons UAA(ochre), UAG(amber). proteinsynthesis. andUGAterminate r In bacteria theyareusedmostoftenwith retative frequencies UAA>UGA>UAG.
Gnil
Termination CodonsAre Recoqnized by ProteinFactors r Termination codons arerecognized by proteinretease factors,not by aminoacy[-tRNAs. r Thestructures of the ctass1 release factorsresemb[e aminoacyt-tRNA-EF-Tu and EF-G. . Theclass]. retease factorsrespond to specifictermjnation codons andhydrotyze the potypeptide-tRNA [inkage. o Theclass1 release factorsareassisted by class2 release factorsthat dependon GTP. o Themechanism is simi[arin bacteria (whichhavetwo typesof class1 retease factors) (whichhaveon[y andeukaryotes oneclass1 release factor).
r Virtuatty proteins at[ribosomal arein contactwith rRNA. . Mostof the contacts between ribosomal subunits aremadebetween the 165and 235rRNAs. Ribosomes HaveSeveraIActiveCenters r Interactions involvingrRNAarea keypart of ribosome function. r Theenvironment of thetRNA-binding sites is Largety determined by rRNA. 165 rRNAPtaysan ActiveRotein Protein Synthesis r L65rRNAplaysan activerotein thefunctionsof the 30Ssubunit.It interacts directty with mRNA, withthe 50Ssubunit, andwiththe anticodons of tRNAs in the P andA sites. 23SrRNAHasPeptidytTransferase Activity o Peptidyt transferase activityresides exctusivetyin the 23SrRNA. RibosomaI StructuresChanqeWhenthe SubunitsComeTogether o Theheadof the 30Ssubunitswivels around the neckwhencomplete ribosomes areformed. o Thepeptidyttransferase activesite of the 50Ssubun'it is moreactivein comotete ribosomes thanin individua[ 50Ssubunits. o Theinterfacebetweenthe 305and50S subunits is veryrichin solventcontacts. Summarv
Ribosomal RNAPervades Both Ribosomal
Subunits . EachrRNAhasseveraI distinctdomains that fotdindependent[y.
E
Introduction
An mRNA contains a seriesof codons that interact with the anticodons of aminoacyl-tRNAs so that a corresponding series of amino acids is incorporated into a polypeptide chain. The ribosome provides the environment for controlling the interaction between mRNA and aminoacyltRNA. The ribosome behaves like a smallmigrating factory that travels along the template engagingin rapid cyclesof peptide bond synthesis. Aminoacyl-tRNAs shoot in and out of the particle at a fearsome rate while depositing amino acids, and elongation factors cyclically associatewith and dissociatefrom the ribosome. Together with its accessoryfactors, the ribosome provides the full range of activities required for all the steps of protein synthesis.
752
CHAPTER 8 ProteinSynthesis
tr3{iJftf*.l shows the relative dimensions of the components of the protein synthetic apparatus. The ribosome consistsof two subunits that have specificroles in protein synthesis.Messenger RNA is associatedwith the small subunit; -30 basesof the nRNA are bound at any time. The mRNA threads its way along the surface close to the junction of the subunits. TWoIRNA molecules are active in protein synthesisat any moment, so polypeptide elongation involves reactions taking place at just two of the (roughiy) ten codons covered by the ribosome. The two tRNAs are inserted into internal sitesthat stretch acrossthe subunits. A third IRNA may remain on the ribosome after it has been used in protein synthesis before being recycled. The basic form of the ribosome has been conserved in evolution, but there are apprecia-
ble variations in the overall size and proportions of RNA and protein in the ribosomes of bacteria, eukaryotic cytoplasm, and organelles. i i;.:ii:i.:r.i Comparesthe components of bacterial and mammalian ribosomes. Both are ribonucleoprotein particles that contain more RNA than protein. The ribosomal proteins are known as r-proteins. Each of the ribosome subunits contains a major rRNA and a number of small proteins. The large subunit may also contain smaller R N A ( s ) . I n E . c o l i ,t h e s m a l l ( 3 0 S )s u b u n i t c o n sists of the l6S rRNA and 2l r-proteins. The large (50S) subunit contains 23S rRNA, the small 55 RNA, and 3I proteins. With the exception of one protein present at four copies per ribosome. there is one copy of each protein. The major RNAs constitute the major part of the mass of the bacterial ribosome. Their presence is pervasive, and probably most or all of the ribosomal proteins actually contact rRNA. So the major rRNAs form what is sometimes thought of as the backbone of each subunit-a continuous thread whose presencedominates the structure and which determines the positions of the ribosomal proteins. The ribosomes of higher eukaryotic cytoplasm are larger than those of bacteria.The total content of both RNA and protein is greater; the major RNA molecules are longer (called I8S and 28S rRNAs), and there are more proteins. Probably most or all of the proteins are present in stoichiometric amounts. RNA is still the predominant component by mass. Organelle ribosomes are distinct from the ribosomes of the cytosol and take varied forms. In some cases,they are almost the size of bacterial ribosomes and have 70o/oRNA; in other cases,they are only 605 and have <30% RNA. The ribosome possesses severalactive centers, each of which is constructed from a group of proteins associatedwith a region of ribosomal RNA. The active centers require the direct participation of rRNA in a structural or even catalytic role. Some catalytic functions require individual proteins, but none of the activities can be reproduced by isolatedproteins or groups of proteins; they function only in the context of the ribosome. Two types of information are important in analyzing the ribosome. Mutations implicate particular ribosomal proteins or basesin rRNA in participating in particular reactions. Structural analysis, including direct modification of components of the ribosome and comparisons to identify conserved features in rRNA, identi-
' r' , :r'r , S i z ec o m p a r i s o nssh o wt h a t t h e r i b o s o m ies [ a r o ee n o u o ht o b i n dt R N A sa n d m R N A .
Ribosomes
rRNAs
r-proteins
23S = 2904 bases 55 = 120 bases
31
165 = 1542bases
21
28S = 4718bases ' 605 5.8S= 160 bases Mammalian(B0S) mass: 4.2 MDa 55 = 120 bases 60% RNA . ' . : 4 0 S 1 B S= 1 8 7 4b a s e s
49
Bacterial(70S) '',,ir' 50S mass: 2.5 MDa ' rr..-.ri 66o/oRNA 30S
33
parti: i,:,,ririi,'i ,' Ribosomes arelargeribonuc[eoprotein into moreRNA thanproteinanddissociate clesthatcontain larqeandsmat[subunits.
fies the physical locations of components involved in particular functions.
0ccurs Protein Synthesis by Initiation,Elongation, andTermination . r . . o
sites. Theribosome hasthreetRNA-binding entersthe A site. An aminoacyt-tRNA is boundin the P site. Peptidyt-tRNA Deacytated IRNAexitsviathe Esite. chain An aminoacidis addedto the potypeptide frompeptidyt-tRNA the poLypeptide by transferring in the A site. in the Psiteto aminoacvltRNA
An amino acid is brought to the ribosome by an aminoacyl-tRNA. Its addition to the growing protein chain occurs by an interaction with the IRNA that brought the previous amino acid.
8.2 ProteinSynthesisOccursby Initiation, Etongation,and Termination
153
Codon"n" Codon"n+1" P siteholds A siteis entered peptidyl-tRNA by aminoacyFtRNA riiJ>
Ribosome movemenl
1 Beforepeptidebond formationpeptidyFtRNA occupiesP site; aminoacyl-tRNA occupiesA site
Aminoacidforcodonn+1
Nascentchain
polypeptide is transferred ;2 Peptidebondformation ! frompeptidyl-tRNA in P siteto aminoacyl-tRNA inA site .
J-^-
-^-!:i.,l
3 Translocation movesribosome onecodon; placespeptidyl-tRNA in P site;deacylated IRNA leavesviaE site;A siteis emptyfor nextaatRNA
Codon"n+1" Codon "n+2"
fifiURES"3 Theribosome hastwositesforbinding charged tRNA.
Aminoacyl-ends of IRNA interactwithinlarge
ribosome subunit
Anticodons are bound to adjacent tripletson mRNA in smallribosome subunit ilIfriJftn 8"4 ThePandA sitesposition thetwointeractingtRNAs across bothribosome subunits.
154
CHAPTER 8 Protein Synthesis
Each of these IRNA lies in a distinct site on the ribosome. riGUR[S,3 shows that the two sites have different features: . An incoming aminoacyl-tRNA binds to the A site. Prior to the entry of aminoacyl-tRNA, the site exposes the codon representing the next amino acid due to be added to the chain. . The codon representing the most recent amino acid to have been added to the nascent polypeptide chain lies in the P site. This site is occupied by peptidyltRNA, a IRNA carrying the nascent polypeptide chain. Fict"tR{S.4shows that the aminoacyl end of the IRNA is located on the large subunit, whereas the anticodon at the other end interacts with the mRNA bound by the small subunit. So the P and A sites each extend across both ribosomal subunits. For a ribosome to slmthesize a peptide bond, it must be in the state shown in step I in Figure 8.3, when peptidyl-tRNA is in the P site and aminoacyl-tRNA is in the A site. Peptide bond formation occurs when the pollpeptide carried by the peptidyl-tRNA is transferred to the amino acid carried by the aminoacyl-tRNA. This reaction is catalyzed by the large subunit of the ribosome. Ttansfer of the polypeptide generates the ribosome shown in step 2, in which the deacylated tRNA,lacking any amino acid, lies in the P site and a new peptidyl-tRNA has been created in the A site. This peptidyl-tRNA is one amino acid residue longer than the peptidyltRNA that had been in the P site in srep l. The ribosome now moves one triplet along the messenger.This stage is called translocation. The movement transfers the deacylated IRNA out of the P site and moves the peptidylIRNA into the P site (see step 3 in the figure). The next codon to be translated now lies in the A site, ready for a new aminoacyl-tRNA to enter, when the cycle will be repeated.FIGURf, S.5summarizes the interaction between tRNAs and tne ribosome. The deacylated tRNA leaves the ribosome via another tRNA-binding site, the E site. This site is transiently occupied by the IRNA en route between leaving the P site and being released from the ribosome into the cytosol. Thus the flow of IRNA is into the A site, through the P site, and out through the E site (see also Figure 8.28 in Section 8.12). FIGUR[S.Scompares the movement of IRNA and mRNA, which may be thought of as a sort of ratchet in which the reaction is driven by the codon-anticodon interaction.
r;fl.,;:,.1 i. -, AminoacyltRNA enterstheA site,receives the potypeptide chainfrompeptidytIRNA.andis transferred intothe P sitefor the nextcvcteof etonqation.
Initiation30S subuniton mRNAbindingsite joined by 50S subunitand aminoacyltRNAbinds
ElongationRibosomemovesalong mRNA,extending
to aminoacyl-tRNA by transterfrompeptidyl-tBNA ::ir.rr.i-.:. r:,t, tRNAand mRNAmovethrouqhthe ribosome in the samedirection.
Protein synthesis falls into the three stages :::'i: shown in ili'i,;":i::i: . Initiation involves the reactions that precede formation of the peptide bond between the first two amino acidsof the protein. It requires the ribosome to bind to the mRNA, which forms an initiation complex that contains the first aminoacyl-IRNA. This is a relatively slow step in protein synthesis and usually determines the rate at which an mRNA is translated. . Elongation includes all the reactions from synthesis of the first peptide bond to addition of the Iast amino acid. Amino acids are added to the chain one at a time; the addition of an amino acid is the most rapid step in protein synthesis. . Termination encompassesthe steps t h a t a r e n e e d e d t o r e l e a s et h e c o m pleted polypeptide chain; at the same time, the ribosome dissociatesfrom the mRNA. Different sets of accessoryfactors assistthe ribosome at each stage. Energy is provided at various stagesby the hydrolysis of guanine triphosphare (GTP).
Polypeptidechain is releasedfrom tRNA, ribosomedissociatesfrom mRNA
,
fatlsintothreestages. Protein synthesis
During initiation, the small ribosomal subunit binds to nRNA and then is joined by the 50S subunit. During elongation, the nRNA moves through the ribosome and is translated in triplets. (Although we usually talk about the ribosome moving along mRNA, it is more realistic to think in terms of the mRNA being pulled through the ribosome.) At termination the protein is released,nRNA is released,and the individual ribosomal subunits dissociatein order to be used again. 8.2 ProteinSynthesisOccursby Initiation, Etongation,and Termination
155
@
Special Mechanisms ControI the Accuracy of Protein Synthesis
o Theaccuracy of proteinsynthesis is controlled by specific mechanisms at eachstage.
We know that protein synthesis is generally a c c u r a t e , b e c a u s e o f t h e c o n s i s t e n c yt h a t i s found when we determine the sequence of a protein. There are few detailed measurements of the error rate in vivo,but it is generally thought to lie in the range of one error for every 104to I05 amino acids incorporated. Considering that most proteins are produced in large quantities, this means that the error rate is too low to have anv effect on the ohenotype of the cell. It is not immediately obvious how such a low error rate is achieved. In fact, the nature of discriminatory events is a general issue raisedby severalstepsin gene expression.How d o s y n t h e t a s e sr e c o g n i z ej u s t t h e c o r r e s p o n -
Wrongaminoacid WrongIRNA
I 1g-016-s1g-+
,NHZ R:C-H
I lt;r-iiiirlr:.i: Errors occurat ratesfrom10-6to 5 x 10-aat djfferent stages of proteinsynthesis.
t56
CHAPTER 8 ProteinSynthesis
ding tRNAs and amino acids?How does a ribosome recognize only the IRNA corresponding to the codon in the A site? How do the enzymes that synthesize DNA or RNA recognize only the basecomplementary to the template? Each caseposesa similar problem: how to distinguish one particular member from the entire set, all of which share the same general features. Probably any member initially can contact the active center by a random-hit process, but then the wrong members are rejected and only the appropriate one is accepted. The appropriate member is always in a minority (one of twenty amino acids, one oI-40 tRNAs, one of four bases),so the criteria for discrimination must be strict. The point is that the enzyme must have some mechanism for increasing discrimination from the level that would be achieved merely by making contacts with the available surfacesof the substrates. iirr i;.i:.summafizesthe errof fatesat the ::1i,, steps that can affect the accuracy of protein synthesis. Errors in transcribing nRNA are rareprobably <10-6. This is an important stage to control. because a single mRNA molecule is translated into many protein copies.We do not know very much about the mechanisms. The ribosome can make two types of errors in protein synthesis. It may cause a frameshift by skipping a basewhen it reads the mRNA (or in the reverse direction by reading a basetwiceonce as the last base of one codon and then again as the first base of the next codon). These errors are rare, occurring at -10-5. Or it may allow an incorrect aminoacyl-tRNA to (mis)pair with a codon, so that the wrong amino acid is incorporated. This is probably the most common error in protein synthesis, occurring at - 5 x l0a. It is controlled by ribosome structure and velocity (seeSection 9.15, The Ribosome Influences the Accuracy of Ttanslation). A IRNA synthetase can make two types of error: It can place the wrong amino acid on its IRNA, or it can charge its amino acid with the wrong IRNA. The incorporation of the wrong amino acid is more common, probably because the IRNA offers a larger surface with which the enzyme can make many more contacts to ensure specificity.Aminoacyl-IRNA synthetases have specific mechanisms to correct errors before a mischargedIRNA is released(seeSect i o n 9 . 1 1 , S y n t h e t a s e sU s e P r o o f r e a d i n g t o Improve Accuracy).
Initiationin Bacteria Needs 30SSubunits andAccessory Factors Initiationof proteinsynthesis requires separate 30Sand50Sribosome subunits. Initjationfactors(IF-1,-2, and-3),whichbindto 30Ssubunits, areatsorequired. A 305subunitcarrying initiationfactorsbindsto an initiationsiteon mRNA to forman initiation comDlex. IF-3mustbereleased to altow50Ssubunits to ioin the 30S-mRNA comp[ex. Bacterial ribosomes engaged in elongating a pollpeptide chain exist as 70S particles. At termination, they are releasedfrom the nRNA as free ribosomes. In growing bacteria, the majority of ribosomes are slnthesizing proteins; the free pool is likely to contain -20oh of.the ribosomes. Ribosomesin the free pool can dissociate into separatesubunits; this means that 70S ribosomesare in dynamic equilibrium with 30S and 50S subunits. Initiation of protein synthesisis not a function of intact ribosomes, but is undertakenby the separatesubunits,which reassociate during the initiation reaction. , i,.,i:i,,i': :r summarizes the ribosomal subunit cycle during protein synthesisin bacteria. Initiation occurs at a special sequence on nRNA called the ribosome-binding site. This is a short sequenceof basesthat precedesthe coding region (see Figure 8.1). The small and large subunits associateat the ribosomebinding site to form an intact ribosome. The reaction occurs in two steps: . Recognition of mRNA occurs when a small subunit binds to form aninitiation complex at the ribosome-binding site. . A large subunit then joins the complex to generatea complete ribosome. Although the 30S subunit is involved in initiation, it is not by itself competent to undertake the reactions of binding mRNA and IRNA. It requires additional proteins called initiation factors (IF). These factors are found only on 30S subunits, and they are releasedwhen the 30S subunits associatewith 50S subunits to generate 70S ribosomes.This behavior distinguishes initiation factors from the structural proteins of the ribosome. The initiation factors are concemed solely with formation of the initiation complex, they are absent from 70S ribosomes, and they play no part in the stagesof elongation.' ,, summarizesthe stagesof initiation.
; : i i . r : : : i , , ,I n i t i a t i o nr e q u i r ef rse er i b o s o mseu b u n i t s . the30Ssubat termination, Whenribosomes arereleased to generate unitsbindinjtiationfactorsanddissocjate reassociate to givea funcfreesubunits. Whensubunits thefactors. tionaIribosome at initiation.thevrelease
1 30S subunitbindsto mRNA
3 lFs are releasedand 50S subunitioins
r Initiation factorsstabitizefree 30Ssubcomptex. unjtsandbindinitiatorIRNAto the 30S-mRNA
Factors 157 andAccessory Needs 30SSubunits 8.4 initjationin Bacteria
Dynamic equilibrium rF-3 .,....-
Al :l
:+
lF-3 must be released before50S subunitcan ioin
'. -..-, ,:
Initiationrequires 30Ssubunits that carry
Lr-5.
Bacteria use three initiation factors, numberedIF-1, IF-2, and IF-3. They are neededlor both mRNA and 1RNA to enter the initiation complex: . IF-3 is needed for 30S subunits to bind specifically to initiation sites in mRNA. . IF-2 binds a special initiator IRNA and controls its entry into the ribosome. . IF- I binds to 30S subunits only as a part of the complete initiation complex. It binds to the A site and prevents aminoacyl-tRNe from entering. Its location also may impede the 30S subunit from binding to the 50S subunit. IF-3 has multiple functions: it is needed first to stabilize (free) 30S subunits; then it enables them to bind to mRNA; and as part of the 30SmRNA complex, it checks the accuracy of recognition of the first aminoacyl-IRNA (see Section8.6, Use of fMet-tRNAl Is Controlled by IF-2 and the Ribosome). The first function of IF-3 controls the equilibrium between ribosomal states,as shown in : . . , : ' i i ,: . I : . I F - l b i n d s t o f r e e l 0 S s u b u n i t st h a t
IF-3 binds to the surface of the 30S subunit in the vicinity of the A site. There is significant overlap between the basesin 165 rRNA protected by IF-3 and those protected by binding of the 50S subunit, suggestingthat it physically prevents junction of the subunits. IF-3 therefore behaves as an anti-association factor that causes a fOS subunit to remain in the pool of free subunits. The second function of IF-3 controls the ability of 30S subunits to bind to mRNA. Small subunits must have IF-3 in order to form initiation complexes with mRNA. IF-3 must be releasedfrom the 30S-mRNA complex in order to enable the 50S subunit to join. On its release, IF-l immediately recycles by finding another 30S subunit. IF-2 has a ribosome-dependentGTPase activity: It sponsorsthe hydrolysis of GTPin the presence of ribosomes, releasing the energy stored in the high-energy bond. The GTP is hydrolyzed when the 50S subunit joins to generate a complete ribosome. The GTP cleavage could be involved in changing the conformation of the ribosome, so that the joined subunits are converted into an active 70S ribosome.
A SpeciaL Initiator IRNAStartsthe Polypeptide Chain . Protein synthesis startswith a methionine amino acidusuatly codedby AUG. o Differentmethion'ine tRNAsareinvolvedin initiationandetongation. o TheinitiatorIRNAhasuniquestructuraI features that distinguish it fromat[othertRNAs. . TheNHzgroupof the methionine boundto bacteriaI initiatortRNAis formvlated. Synthesis of all proteins starts with the same amino acid: methionine. The signal for initiaring a polypeptide chain is a special initiation codon that marks the start of the reading frame. Usually the initiation codon is the triplet AUG, but in bacteria GUG or UUG are also used. The AUG codon represents methionine, and two types of IRNA can carry this amino acid. One is used for initiation, the other for recognizing AUG codons during elongation. In bacteria and in eukaryotic organelles, the initiator IRNA carries a methionine residue that has been formylated on its amino group, forming a molecule of N-formyl-methionyl-
il:Tl:ffi :J:? +rh?T 3ii,iTii; "?i,1"""i from reassociatingwith a 50S subunit. The reaction between IF-3 and the 30S subunit is stoi-
ilffi l?i;:.";L".\"":$;," 1,1,1,i i; "'#i'"i tF-3,so its availabilitydeterminesthe number of free 30Ssubunits.
158
CHAPTER 8 Protein Svnthesis
tRNA. The IRNA is known as tRNAflet. The name of the aminoacyl-IRNA is usually abbreviated to fMet-rRNAr. The initiator IRNA gains its modified amino acid in a two-stage reaction. First, it is charged with the amino acid to generate Met-tRNAr; and then the formylation reaction shown in i ii,-iriit i:,.i,: blocks the free NH2 group. Although the blocked amino acid group would prevent the initiator from participating in chain elongation, it does not interfere with the ability to initiate a protein. This IRNA is used only for initiation. It recognizesthe codons AUG or GUG (occasionally UUG). The codons are not recognized equally well: the extent of initiation declinesby about half when AUG is replaced by GUG, and declines by about half again when UUG is employed. The speciesresponsiblefor recognizingAUG codons in internal locations is tRNAmMet. This IRNA responds only to internal AUG codons. Its methionine cannot be formylated. What features distinguish the fMet-IRNA1 initiator and the Met-IRNA- elongator? Some characteristicfeatures of the IRNA sequenceare important, as summarizedin r-itJlri,,: t:r,,l:i. Some of these featuresare needed to prevent the initiator from being used in elongation, whereas others are necessaryfor it to function in initiation: . Formylation is not strictly necessary, becausenonformylated Met-IRNA1 can function as an initiator. Formylation improves the efficiency with which the Met-tRNAr is used, though, becauseit is one of the features recognized by the factor IF-2 that binds the initiator IRNA. . The bases that face one another at the last position of the stem to which the amino acid is connected are paired in ail tnNAs except tRNAtt'ret.Mutations that create a basepair in this position of tRNAtrvtet allow it to function in elongation. The absenceof this pair is therefore important in preventing tRNArM.t from being used in elongation. It is also needed for the formylation reaction. . A seriesof I G-C pairs in the stem that precedes the loop containing the anticodon is unique to tRNA#et. Thesebase pairs are required to allow the fMettRNAl to be inserted directly into the P site. In bacteria and mitochondria, the formyl residue on the initiator methionine is removed
Blockedamino group
"&"".', "U"n, . .C=O '"ry...!-r.r{
ii,.ritrl!ilt. r i TheinitiatorN-formyl-methionyt-tRNA (fMetof methionyL-tRNA, tRNAl)is generated by formylation hydrofolate ascofactor. usingformyt-tetra
I i,iill.,i ;r I , fMet-tRNAr that dishasuniquefeatures tinguishit astheinitjatorIRNA.
by a specificdeformylase enzyme to generate a normal NH2 terminus. If methionine is to be the N-terminal amino acid of the protein, this is the only necessary step. In about half the proteins, the methionine at the terminus is removed by an aminopeptidase, which creates a new terminus from R2 (originally the second amino acid incorporated into the chain). When both steps are necessary,they occur sequentially. The removal reaction(s) occur rather rapidly, probably when the nascent polypeptide chain has reached a length of I5 amino acids.
Chain InitiatorIRNAStartsthe Potypeptide 8.5 A Specia[
159
Useof fMet-tRNAr Is Controlted by IF-2 andthe Ribosome
@
. IF-2bindsthe initiatorfMet-tRNA1 andaltows it to enterthe partiatP siteon the 30Ssubunit. The meaning of the AUG and GUG codons depends on their context. When the AUG codon is used for initiation, it is read as formylmethionine; when used within the coding region, it represents methionine. The meaning of the GUG codon is even more dependent on its location. When present as the first codon, it is read via the initiation reaction as formylmethionine. Yet when present within a gene, it is read by Val-tRNA, one of the regular members of the IRNA set, to provide valine as required by the genetic code. How is the context of AUG and GUG codons interpreted? a:i;liti *..!+ illustratesthe decisive role of the ribosome when acting in conjunction with accessoryfactors. In an initiation complex, the small subunit alone is bound to mRNA. The initiation codon
lF-z {i,^+ "u'"'
*fi-i Jl b
Only fMet-tRNAr enters partialP siteon 30S boundto mRNA \subunit .1^^L
ilii.,i.lltl li. :.,40nty fMet-tRNA1 can be usedfor injtiatjon
ust ""-tRNA)m i: i::.iliilil';,'.lH xT'ff8 :yL'H:,| 160
CHAPTER 8 Protein Synthesis
lies within the part of the P site carried by the small subunit. the only aminoacyl-tRNA that can become part of the initiation complex is the initiator, which has the unique property of being able to enter directly into the partial P site to recognize its codon. When the Iarge subunit joins the complex, the partial tRNA-binding sites are converted into the intact P and A sites.The initiator fMettRNAr occupies the P site, and the A site is available for entry of the aminoacyl-tRNA complementary to the second codon of the gene. The first peptide bond forms between the initiator and the next aminoacyl-tRNA. Initiation prevails when an AUG (or GUG) codon lies within a ribosome-binding site, because only the initiator IRNA can enter the partial P site generated when the 30S subunit binds de nlvo to the mRNA. Internal reading prevails subsequently, when the codons are encountered by a ribosome that is continuing to translate an nRNA, because only the reguIar aminoacyl-tRNAs can enter the (complete) A site. Accessory factors are critical in controlling the usage of aminoacyl-tRNAs. All aminoacyltRNAs associatewith the ribosome by binding to an accessoryfactor. The factor used in initiation is IF-2 (seeSection 8.4, Initiation in Bacteria Needs 30S Subunits and Accessory Factors),and the correspondingfactor used at elongation is EF-Tu (seeSection 8.10, Elongation Factor Tir Loads Aminoacvl-IRNA into the A Site). The initiation factor IF-2 places the initiator IRNA into the P site. By forming a complex specifically with fMet-tRNAr, IF-2 ensures that only the initiator IRNA, and none of the regular aminoacyl-tRNAs, participates in the initiation reaction. Conversely,EF-Tu, which places aminoacyl-tRNAs in the A site, cannot bind fMet-tRNAr, which is therefore excluded from use during elongation. An additional check on accuracy is made by IF-3, which stabilizes binding of the initiator 1RNA by recognizing correct base pairing with the second and third basesof the AUG initiation codon. :$.ii details the seriesof events by F3|;i"tR{ which IF-2 places the fMet-tRNAl initiator in the P site. IF-2, bound to GTP,associateswith the P site of the 30S subunit. At this point, the 30S subunit carries all the initiation factors. fMet-tRNAr binds to the IF-2 on the 30S subunit, and then IF-2 transfers the IRNA into the partial P site.
30S-mBNAcomolex
tF_1
tF_3
I
Y lF2-GTPjoins complex
Addnuclease to digestall unprotected mRNA
GTP
InitiatortRNAjoins
r\.r'
\r^\ L----l--------l
lsolate fragmentof protected mRNA Determine
50S subunitjoins and lF1-3are released
G tr.r .
Codingregion Shine-Dalgarno <10 bases uostreamof AUG
tF-1 tF-2 tF-3 cDP Pi
i ::=i.;;ii. .'i.::i: IF-2is needed to bjndfMet-tRNAlto the30SmRNA complex. After505binding. a[[IFfuctors arereteased andGTP is cleaved.
InitiationInvolves Base Pairing Between mRNA andrRNA r An initiationsiteon bacterial mRNA consists of the AUGinitiationcodonpreceded with a gapof -10 bases bythe Shine-Datgarno potypurine nexamer, r TherRNAof the 30Sbacterial ribosomal subunit hasa complementary sequence that basepairs withthe Shine-Datgarno sequence during initiation. An nRNA contains many AUG triplets: How is the initiation codon recognizedas providing the starting point for translation? The siteson mRNA where protein synthesisis initiated can be identified by binding the ribosome to mRNA under conditions that block elongation. Then the ribosome remains at the initiation site. When ribonuclease is added to the blocked initiation complex, all the regions of mRNA outside the ribosome are degraded.Thoseactually bound to it are protected, though, as illustrated in i:l.i.i*,tii: i:]..ii.. The protected fragments can be recovered and characterized.
: l . i ; i i r r i i . : i r R i b o s o m e - b i n dsj n i tge so n m R N A c a nb e r e c o v e r ef d r o mi n i t i a t i o nc o m p l e x eTs h. e yi n c l u d et h e codon. upstream Shine-Datgarno sequence andtheinitiation
The initiation sequencesprotected by bacterial ribosomes are -10 baseslong. The ribosome-binding sites of different bacterial mRNAs display two common features: . The AUG (or less often, GUG or UUG) initiation codon is always included within the protected sequence. . Within ten basesupstream of the AUG is a sequence that corresponds to part or all of the hexamer. .3, 5, ,AGGAGG This polypurine stretch is known as the Shine-Dalgarno sequence. It is complementary to a highly conserved sequencecloseto the 3'end of 165 rRNA. (The extent of complementarity differs with individual mRNAs, and may extend from a four-base core sequence GAGG to a nine-base sequenceextending beyond each end of the hexamer. ) Written in reverse direction, the rRNA sequence is the hexamer: .5, 3, .UCCUCC. Does the Shine-Dalgarno sequence pair with its complement in rRNA during mRNAribosome binding? Mutations of both partners in this reaction demonstrate its importance in initiation. Point mutations in the ShineDalgarno sequencecan prevent an nRNA from
mRNA andrRNA 8.7 InitiationInvolvesBasePairingBetween
16l
being translated. In addition, the introduction of mutations into the complementary sequence in rRNA is deleterious to the cell and changes the pattern of protein synthesis.The decisive confirmation of the base-pairingreaction is that a mutation in the Shine-Dalgarno sequenceof an nRNA can be suppressedby a mutation in the rRNA that restoresbasepairing. The sequenceat the 3'end of rRNA is conserved between prokaryotes and eukaryotes, except that in all eukaryotesthere is a deletion o f t h e f i v e - b a s es e q u e n c eC C U C C t h a t i s t h e principal complement to the Shine-Dalgarno sequence.There doesnot appear to be basepairing between eukaryotic mRNA and 18S rRNA. This is a significant difference in the mechanism of initiation. In bacteria,a l0S subunit binds directly to a ribosome-binding site. As a result, the initiation complex forms at a sequencesurrounding the AUG initiation codon. When the mRNA is polycistronic, each coding region startswith a r i b o s o m e - b i n d i n gs i t e . The nature of bacterial gene expression means that translation of a bacterial nRNA proceedssequentially through its cistrons.At the time when ribosomesattach to the first coding region, the subsequent coding regions have not yet even been transcribed.By the time the second ribosome site is available,translation is well under way through the first cistron. What happens between the coding regions dependson the individual mRNA. In most cases, the ribosomes probably bind independently at the beginning of each cistron. The most common seriesof eventsiSillustratedin i i;:,ij;iii:, i .'.
ble at the next coding region and set out to transIate the next cistron. In some bacterial mRNAs, translation between adjacent cistrons is directly linked, because ribosomes gain accessto the initiation codon of the second cistron as they complete translation of the first cistron. This effect requires the spacebetween the two coding regions to be small. It may depend on the high local density of ribosomes, or the juxtaposition of termination and initiation sitescould allow some of the usual intercistronic events to be bypassed.A ribosome physically spans-30 basesof nRNA. so that it could simultaneously contact a termination codon and the next initiation site if thev are separated by only a few bases.
SmaL[ Subunits Scan for InitiationSites mRNA on Eukaryotic r Eukaryotic subunits bindto the 5' 40Sribosomal untiItheyreach endof mRNA andscanthe mRNA an initiationsite. . A eukaryotic initiationsiteconsistsof a tennucteotide sequence that includes an AUGcodon. . 605ribosomal join the complex subunits at the initiationsite.
Initiation of protein synthesis in eukaryotic cytoplasm resembles the process in bacteria, but the order of events is different and the number of accessoryfactors is greater. Some of the differences in initiation are related to a differWhen synthesisof the first protein terminates, ence in the way that bacterial 30S and eukarythe ribosomesleave the mRNA and dissociate otic 40S subunits find their binding sites for initiating protein synthesis on nRNA. In into subunits.Then a new ribosomemust assemeukaryotes, small subunits first recognize the 5'end of the mRNA and then move to the initiation site, where they are joined by large subunits. (In prokaryotes, small subunits bind directly to the initiation site.) Virtually all eukaryotic mRNAs are monocistronic, but each nRNA usually is substantially longer than necessaryjust to code for its protein. The averagemRNA in eukaryotic cytoplasm is 1000 to 2000 baseslong, has a methylated cap at the 5'terminus, and carriesI00 to 200 basesof poly(A) at the 3'terminus. The nontranslated 5'leader is relatively short, usually
CHAPTER 8 ProteinSvnthesis
The first feature to be recognized during translation of a eukaryotic nRNA is the methylated cap that marks the 5' end. Messengers whose caps have been removed are not translated efficiently invitro. Binding of 40S subunits to mRNA requires several initiation factors, including proteins that recognize the structure of the cap. Modification at the 5'end occursto almost all cellular or viral mRNAs and is essential for their translation in eukaryotic cytoplasm (although it is not needed in organelles).The sole exception to this rule is provided by a few viral mRNAs (such as poliovirus) that are not capped; only these exceptional viral mRNAs can be translated in vitro without caps.They use an alternative pathway that bypassesthe need for the cap. Some viruses take advantage of this difference. Poliovirus infection inhibits the translation of hosr mRNAs. This is accomplished by interfering with the cap-binding proteins rhat are needed for initiation of cellular mRNAs, but that are superfluous for the noncapped poliovirus nRNA. We have dealt with the processof initiation as though the ribosome-binding site is always freely available. However, its availability may be impeded by secondarystructure. The recognition of nRNA requires several additional factors; an important part of their function is to remove any secondary structure in the nRNA ( s e eF i g u r e 8 . 2 0 ) . Sometimes the AUG initiation codon lies within 40 basesof the 5'terminus of the nRNA, so that both the cap and AUG lie within the span of ribosome binding. In many mRNAs, however, the cap and AUG are farther apartin extreme cases,they can be as much as 1000 bases away from each other. Yet the presence of the cap still is necessaryfor a stable complex to be formed at the initiation codon. How can the ribosome rely on two sites so far apart? which supposesthat the 40S subunit initially recognizesthe 5'cap and then "migrates"along the mRNA. Scanning from the 5'end is a linear process.When 40S subunits scanthe leader region, they can melt secondary structure hairpins with stabilities <-30 kcal, but hairpins of greater stability impede or prevent migration. Migration stops when the 40S subunit encounters the AUG initiation codon. Usually, although not always, the first AUG triplet sequenceto be encountered will be the initiation codon. However. the AUG triplet by itself is not sufficient to halt migration; it is recog-
nized efficiently as an initiation codon only when it is in the right context. The most important determinants of context are the bases in positions --4 and +1. An initiation codon may be recognized in the sequence NNNPuNN,AUGG. The purine (A or G) 3 basesbefore the AUG codon, and the G immediately following it. can influence the efficiency of translation by l0x. When the leader sequenceis long, further 40S subunits can recognize t};^e5' end before the first has left the initiation site, creating a queue of subunits proceeding along the leader to the initiation site. It is probably true that the initiation codon is the first AUG to be encountered in the most efficiently translated mRNAs. What happens, though, when there is an AUG triplet in the 5'nontranslated region? There are two possible escapemechanisms for a ribosome that starts scanning at the 5'end. The most common is that scanning is leaky, that is, a ribosome may continue past a noninitiation AUG because it is not in the right context. In the rare case that it does recognize Lhe AUG, it may initiate translation but terminate before the proper initiation codon, after which it resumes scanning. The vast majority of eukaryotic initiation events involve scanning from the 5' cap, but
migratefromthe 5' ribosomes Eukaryotic bindinqsite,whichinctudes endof mRNA to theribosome a nA U Gi n i t i a t i o cn o d o n .
mRNA scanfor InitiationSiteson Eukarvotic 8.8 smatlSubunits
163
there is an alternative means of initiation, used especiallyby certain viral RNAs, in which a 40S subunit associatesdirectly with an internal site called an IRES. (This entirely bypassesany AUG codons that may be in the 5' nontranslated region.) There are few sequence homologies between known IRES elements. We can distinguish three types on the basis of their interaction with the 40S subunit: . One type of IRES includes the AUG initiation codon at its upstream boundary. The 40S subunit binds directly to it, using a subset of the same factors that are required for initiation at 5'ends. . Another is located as much as I00 nucleotides upstream of the AUG, requiring a 40S subunit to migrate, again probably by a scanning mechanism. o An exceptional type of IRES in hepatitis C virus can bind a 40S subunit directly. without requiring any initiation factors. The order of events is different from all other eukaryotic initiation. Following 40S-mRNA binding, a complex containing initiator factors and the initiator IRNA binds. Use of the IRES is especially important in picornavirus infection, where it was first discovered, becausethe virus inhibits host protein synthesis by destroying cap structures and inhibiting the initiation factors that bind them (seeSection 8.9, Eukaryotes Use a Complex of Many Initiation Factors). Binding is stabilized at the initiation site. When the 40S subunit is joined by a 605 subunit, the intact ribosome is located at the site identified by the protection assay.A 40S subunit protectsa region of up to 60 bases;when the 605 subunits join the complex, the protected region contracts to about the same length of 30 to 40 basesseen in prokaryotes.
@
Eukaryotes Usea Complex of ManyInitiationFactors
o In'itiationfactorsarerequired for a[[stagesof initiation,inctuding bindingthe initiatorIRNA. 40Ssubunitattachment to mRNA, movement alongthe mRNA, andjoiningof the 605subunit. r Eukaryotic initiatorIRNAis a Met-tRNA that is differentfromthe Met-tRNA usedin elongation, butthe methionine is notformutated. . eIF2bindsthe initiatorMet-tRNA1 andGTP, and the comptex bindsto the40Ssubunitbefore it associates with mRNA.
CHAPTER 8 ProteinSynthesis
Initiation in eukaryotes has the same general features as in bacteria in using a specific initiation codon and initiator IRNA. Initiation in eukaryotic cytoplasm usesAUG as the initiator. The initiator 1RNA is a distinct species,but its methionine does not become formylated. It is called tllNAiM.t. Thus the difference between the initiating and elongating Met-tRNAs lies solely in the IRNA moiety, with Met-tRNAi used for initiation and Met-tRNA. used for elongation. At least two features are unique to the initiator IRNA Met in yeast: it has an unusual rerriary structure, and it is modified by phosphorylation of the 2'ribose position on base64 (if this modification is prevented, the initiator can be used in elongation). Thus the principle of a distinction between initiator and elongator Met-tRNAs is maintained in eukaryotes, but its structural basis is different from that in bacteria (for comparison seeFigure 8.13). Eukaryotic cells have more initiation factors than bacteria-the current list includes l2 factors that are directly or indirectly required for initiation. The factors are named similarly to those in bacteria, sometimes by analogy with the bacterial factors, and are given the prefix "e" to indicate their eukaryotic origin. They act at all stagesof the process,including: . forming an initiation complex with the 5'end of nRNA; . forming a complex with Met-tRNAi; . binding the mRNA-factor complex to the Met-IRNA1-factor complex; . enabling the ribosome to scan mRNA from the 5'end to the first AUG; . detecting binding of initiator IRNA to AUG at the start site; and . mediating joining of the 605 subunit. i:l{,.ili;i:i:..:rl summarizes the stagesof initiation and shows which initiation factors are involved at each stage:eIF2 and eIF3 bind to the 40S ribosome subunit; eIF4A, eIF4B, and eIF4F bind to the mRNA; and eIFl and eIFIA bind to the ribosome subunit-mRNA complex. i'.ii.:il*i::;"i:i:shows the group of factors that bind to the 5'end of mRNA. The factor eIF4F is a protein complex that contains three of the initiation factors. It is not clear whether it preassemblesas a complex before binding to mRNA or whether the individual subunits are added individually to form the complex on mRNA. It includes the cap-binding subunit eIF4E, the helicase eIF4A, and the "scaffolding" subunit eIF4G. Aiter eIF4E binds the cap, eIF4A unwinds any secondary structure that exists in the first l5 basesof the mRNA. Energy for the unwinding is provided by hydrolysis of ATP. Unwind-
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of eIF2. Phosphorylation prevents eIF2B from regenerating the active form. This limits the action of eIF2B to one cycle of initiation, and thereby inhibits protein synthesis.
Etongation Factor Tu Loads Aminoacyl-tRNA into the A Site EF-Tu is a monomeric Gproteinwhoseactiveform (boundto GTP) bindsaminoacy[-tRNA. TheEF-Tu-GTP-aminoacyt-tRNA complex bindsto the ribosome A site. Once the complete ribosome is formed at the initiation codon, the stageis set for a cycle in which aminoacyl-tRNA enters the A site of a ribosome whose P site is occupiedby peptidylIRNA. Any aminoacyl-rRNA excepr the initiator can enter the A site.Its entry is mediated by an elongation factor (EF-Tu in bacteria). The processis similar in eukaryotes.EF-Tu is a highly conservedprotein throughout bacteria and mitochondria and is homologous to its eukaryotic counterpart. Just like its counterpart in initiation (IF-2), EF-Tu is associatedwith the ribosome only during the processof aminoacyl-tRNA entry. Once the aminoacyl-tRNA is in place. EF-Tu leaves the ribosome, to work again with another aminoaryl-tRNA. Thus it displaysthe cyclicassociation with, and dissociationfrom, the ribosome that is the hallmark of the accessory factors. The pathway for aminoacyl-tnXA entry to the A site is iilustrated in I ii,,.,r;':, i: ..: . EF-Tu carries a guanine nucleotide. The factor is a monomeric G protein whose activity is controlled by the state of the guanine nucleotide: . When GTP is present, the factor is in its actrve state. . When the GTP is hydrolyzed to GDp, the factor becomesinactive. . Activity is restored when the GDP is replaced by GTP. The binary complex of EF-Tu-GTP binds aminoacyl-IRNA to form a ternary complex of aminoacyl-tRNA-EF-Tu- GTP.The ternary complex binds only to the A site of ribosomeswhose P site is already occupiedby peptidyl-rRNA. This is the critical reaction in ensuring that the aminoacyl-IRNA and peptidyl-rRNA are correctly positioned for peptide bond formation. Aminoacyl-tRNA is loaded into the A site in two stages.First, the anticodon end binds to
Tu-GDP
aa-tRNAenters A site on 30S
CCA end moves inlo A site on 50S
i r ' i r r! " , ' r : E F - T u - GpTt aPc easm i n o a c y l t RoNnAt h er i b o s o maen dt h e n is reteased is required to mediate the replacement of as EF-Tu-GDP. EF-Ts GDP byGTP. Thereaction GTP andreteases GDP. Theontyaminoaconsumes cyltRNAthatcannotberecognized is fMet-tRNAr, whose fajtby EF-Tu-GTP ureto bindprevents to internalAUGor GUG codons. it fromresponding
the A site of the l0S subunit. Then, codonanticodon recognition triggers a change in the conformation of the ribosome. This stabilizes tRNA binding and causes EF-Tu to hydrolyze its GTP.The CCA end of the IRNA now moves into the A site on the 50S subunit. The binary complex EF-Tu-GDP is released.This form of EF-Tu is inactive and doesnot bind aminoacylIRNA effectively. Another factor,EF-Ts,mediatesthe regeneration of the used form. EF-TIr-GDP.into the active form EF-Tu-GTP.First, EF-Ts displaces the GDP from EF-Tu, forming the combined factor EF-Tu-EF-Ts.Then the EF-Tsis in turn displaced by GTP,reforming EF-Tu-GTP.The active binary complex binds aminoacyl-tRNA, and the releasedEF-Tscan recycle. There are -70,000 molecules of EF-Tu per bacterium (-5% of.the total bacterial protein), which approaches the number of aminoacylIRNA molecules.This implies that most aminoacyltRNAs are likely to be present in ternary complexes.There are only -10,000 molecules of EF-Tsper cell (about the same as the number of ribosomes). The kinetics of the interaction between EF-TU and EF-Ts suggestthat the EFTu-EF-Tscomplex existsonly transiently, so that the EF-Tu is very rapidly converted to the GTPbound form, and then to a ternary complex.
into the A Site Aminoacyt-tRNA Factor TuLoads 8.10 Elongation
767
The role of GTP in the ternary complex has been studied by substituting an analog that cannot be hydrolyzed. The compound GMP-PCP has a methylene bridge in place of the oxygen that links the p and yphosphates in GTP.In the presenceo{ GMP-PCP,a ternary complex can be formed that binds aminoacyl-tRNA to the ribosome. The peptide bond cannot be formed, though. so the presence of GTP is needed for aminoacyl-IRNA to be bound at the A site. The hydrolysis is not required until later. is an antibiotic that inhibits Kirromycin the function of EF-Tu. When EF-Tir is bound by kirromycin, it remains able to bind aminoacylIRNA to the A site. But the EF-Tu-GDP complex cannot be released from the ribosome. Its continued presence prevents formation of the peptide bond between the peptidyl-tRNA and the aminoacyl-tRNA. As a result, the ribosome becomes "stalled" on mRNA, bringing protein synthesisto a halt. This effect of kirromycin demonstrates that inhibiting one step in protein synthesis blocks the next step.The reason is that the continued presence of EF-Tu prevents the aminoacyl end of aminoacyl-IRNA from entering the A site on t h e 5 0 S s u b u n i t ( s e eF i g u r e 8 . 3 I ) . T h u s t h e release of EF-Tu-GDP is needed for the ribosome to undertake peptide bond formation. The same principle is seen at other stagesof protein
synthesis: one reaction must be completed properly before the next can occur. The interaction with EF-Tu also plays a role in quality control. Aminoacyl-tRNAs are brought into the A site without knowing whether their anticodons will fit the codon. The hydrolysis of EF-Tu-GTP is relatively slow: it takes longer than the time required for an incorrect aminoacyl-IRNA to dissociatefrom the A site, therefore most incorrect speciesare removed at this stage. The release of EFTu-GDP after hydrolysis also is slow, so any surviving incorrect aminoacyl-tRNAs may dissociate at this stage. The basic principle is that the reactions involving EF-Tu occur slowly enough to allow incorrect aminoacyl-tRNAs to dissociate before they become trapped in protein synthesis. In eukaryotes, the factor eEFlcx is responsible for bringing aminoacyl-tRNA to the ribosome, again in a reaction that involves cleavage of a high-energy bond in GTP.Like its prokaryotic homolog (EF-Tu), it is an abundant protein. After hydrolysis of GTP,the active form is regenerated by the factor eEFlBy, a counterpart to EF-Ts.
ThePoLypeptide Chain Is Transferred to AminoacyL-tRNA r The50Ssubunithaspeptidyttransferase activity. e ThenascentpoLypeptide from chainis transferred peptidyltRNA in the Psiteto aminoacyt-tRNA in the A site. . Peptidebondsynthesis generates deacytated IRNA in the P siteandpeptidyt-tRNA in the A site.
;$.I* Peptide ${'*l"JSil bondformation takesptaceby reaction betweenthe potypeptide of peptidyt-tRNA in the P siteandtheaminoacidof aminoacvt-tRNA in theA site.
168
CHAPTER 8 ProteinSvnthesis
The ribosome remains in place while the polypeptide chain is elongated by transferring the polypeptide attached to the IRNA in the P site to the aminoacyl-tRNA in the A site. The reaction is shown in Fi*SftS*.f b. The activity responsible for synthesis of the peptide bond is called peptidyl transferase. The nature of the transfer reaction is revealed by the ability of the antibiotic puromycin to inhibit protein synthesis. Puromycin resemblesan amino acid attached to the terminal adenosine of IRNA. l-:GtlR$&.f F shows that puromycin has an N instead of the O that joins an amino acid to IRNA. The antibiotic is treated by the ribosome as though it were an incoming aminoacyl-tRNA, after which the
polypeptide attached to pepridyl-rRNA is rransferred to the NH2 group of the puromycin. The puromycin moiety is not anchored to the A site of the ribosome, and as a result the pollpeptidyl-puromycin adduct is releasedfrom the ribosome in the form of polypeptidylpuromycin. This premature termination of protein synthesisis responsiblefor the lethal action of the antibiotic. Peptidyl transferaseis a function of the large (50S or 605) ribosomal subunit. The reacrion is triggered when EF-Tu releasesthe aminoacyl end of its IRNA. The aminoacyl end then swings into a location close to the end of the peptidylIRNA. This site has a peptidyl transferaseactivity that essentially ensures a rapid transfer of the peptide chain to the aminoacyl-rRNA. Borh rRNA and 50S subunit proteins are necessaryfor this activity, but the actual act of catalysis is a property of the ribosomal RNA of the 50S subunit (seeSection 8.19,235 rRNA Has Peptidyl TlansferaseActivitv).
r:iili,ii:ii: i:;,,.:.-Puromycin mimicsaminoacy[-tRNA because it resembles an aromatic aminoacid[inkedto a suqarbasemoiety.
Trans[ocation Moves the Ribosome . Ribosomal transtocation moves the mRNA through the ribosome bythreebases. o Translocation movesdeacytated IRNAinto the Esiteandpeptidyt-tRNA intothe P site,and empties the A site. . Thehybridstatemodelproposes that transtocation occurs in twostages, in whichthe 50Smoves relativeto the 30S.andthenthe 30Smovesalong mRNA to restore the oriqinalconformation.
The cycle of addition of amino acids to the growing polypeptide chain is completed by translocation, when the ribosome advances three nucleotides along the nRNA. l:ir,rtii;h i:::.;lE shows that translocation expels the uncharged IRNA from the P site, so that the new peptidyl-tRNA can enter. The ribosome then has an empty A site ready for entry of the aminoacyl-tRNA corresponding to the next codon. As the figure shows, in bacteria the dischargedIRNA is transferred from the P site to the E site (from which it is then expelled into the cytoplasm). In eukaryotes it is expelled directly into the cytosol. The A and P sites straddle both the large and small subunits; the E site (in bacteria) is located largely on the 50S subunit, but has some contacts in the l0S subunit. Most thinking about translocation follows the hybrid state model, which proposes that ij.i:i.t translocation occurs in two stages.i::l.i:!-iiiL shows that first there is a shift of the 50S subunit relative to the 30S subunit, followed by a second shift that occurs when the 30S subunit moves along nRNA to restore the original conformation. The basis for this model was the observation that the pattern of contacts that IRNA makes with the ribosome (measured by chemical footprinting) changes in two stages.When puromycin is added to a ribosome that has an aminoacylated IRNA in the P site, the contacts of IRNA on the 50S subunit change from the P site to the E site, but the contacts on the l0S subunit do not change. This suggeststhat the 50S subunit has moved to a posttransferstate,but the 30S subunit has not changed. The interpretation of these results is that first the aminoacyl ends of the tRNAs (located in the 50S subunit) move into the new sites (while the anticodon ends remain bound to their anticodons in the 30S subunit). At this stage, the tRNAs are effectively bound in
Moves the Ribosome 169 8.12 Translocation
ir:i1tii.ri: i:,,,iirModels invotve fortranstocation twostages. theaminoacyl endofthe First.at peptide bondformation retocated in the P site.SecIRNAin the A sitebecomes retocated in endof theIRNAbecomes ond.theanticodon the P site.
' ' r . ,' : , A b a c t e r i rai lb o s o mhea st h r e et R N A - b i n d ingsites.AminoacyltRNA enters theA siteof a ribosome that haspeptidyt-tRNA in the P site.Peptide bondsynpeptidytthesisdeacylates the PsjtetRNAandgenerates IRNAin theA site.Translocation moves thedeacyl"ated IRNA peptidyt-tRNA intothe Esjteandmoves into the P site.
h y b r i d s i t e s ,c o n s i s t i n go f t h e 5 0 S E / 3 0 SP a n d rhe 50SP/l0S A sites.Then movement is
:;:",x1".i ::rff:,il*lilf,,"i. li::liil?i: right site. The most likely means of creating the hybrid state is by a movement of one ribosomal subunit relative to the other, so that translocation in effect involves two stages, with the normal structure of the ribosome being restored by the second stage. The ribosome facesan interesting dilemma
: :::lHlTi li:i.iiffit.'.".: l"J :,i:ilL:'": ment. At the same time. however. it must
IRNA only at the right moment). One possibility is that the ribosome switches between alternative, discrete conformations. The switch could consistof changesin rRNA basepairing. The accuracy of translation is influenced by certain mutations that influence alternative base pairing arrangements. The most likely interpretation is that the effect is mediated by the tightness of binding to IRNA of the alternative conlormalions.
Factors Elongation BindAlternate[y to the Ribosome Transtocation requires EF-G, whosestructure resem btesthe aminoacy[-tRNA-EF-Tu-GTP comptex. Binding andEF-G to the ribosome is of EF-Tu mutualtyexctusive. Transtocation requires which GTPhydrotysis, triggers a change in EF-G, whichin turntriggers in ribosome a chanqe structure.
;ffi1T.::il'ff ,i"ffii":Tt*T.'"T,:T; 170
CHAPTER 8 Protein Svnthesis
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sites exist for some proteins. As each protein binds, it induces conformational changesin the RNA-rich rRNA that make it possiblefor other proteins to bind. In E. coli, virtually all the 30S ribosomal proteins interact (albeit to varying degrees)with I65 rRNA. The binding sites on the proteins show a wide variety of structural features, suggesting that protein-RNA recognition mechaFI6URIS.36 The30Ssubunithasa headseparated by a nisms may be diverse. ptatform. neckfromthe body.with a protruding The 70S ribosome has an asymmetric construction. FISIJftil*,3& shows a schematic of the structure of the l0S subunit, which is divided into four regions: the head, neck, body, and platform. FIGUR{*.3? shows a similar representation of the 50S subunit, where two prominent features are the central protuberance (where 55 rRNA is located) and the stalk (made of multiple copies of protein L7). FI*Ufi{ s.3* shows that the platform of the small subunit fits into the notch of the large subunit. There is a cavity between the subunits that contains some of the important sites. protuberance FIfiUR[$.3? The50Ssubunithasa centraI The structure of the 30S subunit follows where55 rRNAis located, separated by a notchfroma the organization of l65 rRNA, with each strucstatkmadeof copiesof the protein17. tural feature corresponding to a domain of the rRNA. The body is based on the 5'domain, the platform on the central domain, and the head on the 3'region. F:fitJRC S.3$shows that the 30S subunit has an asymmetrical distribution of RNA and protein. One important feature is that the platform of the l0S subunit that provides the interface with the 50S subunit is composed almost entirely of RNA. Only two proteins (a small part of 57 and possibly part of SI2) Iie near the interface. This means that the associFiGriR[8.3$ Theptatform ofthe30Ssubunitfits intothe ation and dissociation of ribosomal subunits notchof the 505subunitto formthe 70Sribosome. must depend on interactions with the I65 rRNA. Subunit associationis affected by a mutation in a loop of l65 rRNA (at position 791) that is located at the subunit interface, and other nucleotides in l65 rRNA have been shown to be involved by modification/interference experiments. This behavior supports the idea that the evolutionary origin of the ribosome may have been as a particle consisting of RNA rather than protein. The 50S subunit has a more even distribution of components than the 30S, with Iong rods of double-stranded RNA crisscrossingthe structure. The RNA forms a mass of tightly packed helices.The exterior surfacelargely consists of protein, except for the peptidyl transferasecenter (seeSection 8.19, 23S rRNA Has Fitu#Rg *.3# The30Sribosomal subunitis a ribonucteo- Peptidyl Tlansferase Activity). Almost all segments of the 23S rRNAinteractwithprotein, but proteinpariicle.Proteins arein yetlow.Photocoudesyof V.Ramakrishnan. MedicaI Research (UK). Councit many of the proteins are relatively unstructured.
176
CHAPTER 8 ProteinSynthesis
LLI
sraluala^L]lvlpra^aso^pHseuosoqru /I'g qJIqM 'sJlrs Y pue d aql uaaMlaq eqt sA1.ollP YNUI-lrbeouruY spPol I\l JoDed uotle8uolg '0I'8 uortJJ5 pJSeJIJT aas) aruosoqu Jql uoJJ vNuru eql ur {ul>l .gt e sMoqs arnlJnrls Iel -sLn aq1 'suopo) tua)elpe Sutpea; uI Jeqloue rq pup 419 az.dlorpAq or nJ-{iI ro; Lressa -Jeu sr uopor Jql qlrM Suured sll pue 'nI-{g ^q euo 01 txeu tlJ ueJ sVNUt,{11nq oanl llroq puPts -rJpun o1 alzznd 8rq e uaaq sleule seq U atISV Jql otur peuasur sry1qgl-1trrpoq1v 'pJAJaSUOJ LlleSramUn AJeleql alnlf,nJls JqrurvNut-lLreoutue aqt yo sued 1pVNut aqt stf,eluoJ vNUr eql 'a1ls eqtot JusO,q, ";?ilJ -llpttdad aql uoJJ pJrreJsuert rq ot ureqr qreJ uI 'VNUJ z(qpapnord [llsour srVNUI qJpe epu -dad aqt s.tr.ollesrql '(uaans Jq1 aueld aql Surpunorrns tuauruoJrlua JqJ'trunqns S0EJql Jo ot relnJrpurdrad) y urqlrM o1 a8ranuor.{aql ol punoq sl vNur qrea Jo lsar aqJ 'llunqns s0€ E arJqM 'spuJ,€ Jql le sJnrJosvNul eql sJuoq Jqt uo anoor8 Jql uI VNUIU aq1 ot punoq sdool Jo ->lJpqrqt uJeMlJq qJeordde uopoJrtup Jraqr qryu pau8rtp Jrp sVNUI JJJqI IIV JqJ 'suopoJ tsasop 'raqloup Juo 01 -Itup Jreql lp rJqto qJee 01anrleleJ ui.peauJJp sJlISd pue rc pelSue 1a11ered "9e eJe SJUS pue eql uI sVNUf a{J 'l r. aql ur svNul eqJ 'selrs Surpurq aerql eqt uI lr V .li.'rii:-11:i d v sVNUt yo suorlsod eqt qtl,t.t JruosoqlJ S0Z eql ;o uorsuedxJ Jqt ur uMoqs se lrJ ot sVNUl sMol{s ;j+'g -+}lil:l]..'sYNUl qll1\{ uolperelul rql punoJe pJraluel aJe suorlJunJ 'znll 'eruloJrlPl 'ta11o1t1 Ieluosoqlu eluPS rfueg 1ortlrstanrul ;o Asalnoro1oq6 'eJnlJUlS SU SruJJlUI SUOIIJUnJ '(uorleluauo eql ur srql) asLr'r1ro1r IPTUOSOqIJ JO 1rsMoqs lensnaqlJoas.la^el o06palplols! S0Eaql pue'asrmllollrolunol palplolsr oql 'uaellsor.1l o1telnrrpuadtad ,{.ueruro; lunoJf,e Mou upJ a714'sells ltunqns 1re1 o06 S0g Ieuollf,unJ -uorJ0aueldp ruo.lJ loppualeslleluololotlM saleJoqlMoqsol.laqloue auouor;ferne rql puP svNur aql Jo suollef,ol eql srlJlluapl palplorolpslrunqns oMlaql'ano11a[ urumoqs elesuralold ur 6uurlonuL sl]pluol'(aldtnd z(1reap'uorlnloseJy E'Stp 'JJnpruts IUJJJJlsoru ylx fq apeu[11sou ur'roqs) areslrunqns aql uoaMloq sl]eluol 11'r l,r{:a}Lj lprxosoqp aqJ 'VNUJ aql Jo JIoJ eqt aaseqdruJ pue uoll -ezruv?to ilerJno eql Jo uorssa:drur poo8 e sn arrrSsaurosoqrJIPrJapeqpue sllunqns pnpIAIpuI aql Jo sernDnrls plsrlD aq1 'sutalord leluosoqlJ eql uo se vNur rql uo qJnru se 1sn[ puadap .,i.erusartrnrlrepup uollf,nJlsuoJ asoqm suor8ar a8rel are daql 'saru,{zue lo sreluJf, a^IDp eql a111suor8ar eleJf,srp'lleurs lou aJp sJtls eAIlJe JqJ 'srsJqlu.dsuralord Surrnp sells eAIlJp sU Euorue sdrqsuoqelal eql q sa8ueqr uo spuadap leql eJnlJnrls a,rtleradooJ P sI lI lPrll sI Jruos -oqlr aql tnoqp JJqIUJTUJJ 01 a8essaurrtseq aq1
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lue^asa^eHsauosoqtu 't1unqnsq)pa a)eJJq1uo stutod peluoJ eqt Jo Jo suorlef,ol eql Moqs o1 (arn34 eql ul uMoqs sIXe eql punoJP asrM>lJolJpelelor llunqns s0€ aql pue Jsr!\>lJolJlalunoJ pJlelor llunqns s0E eql aur8erur) arnlJnJls Jql lno suado 1t'E SEfttl'j 'seJnDnrls VNUJ eqr uo slurod lJpluoJ Jql SJIJI1 'slJpluo) utatord-utalord ma; e -uJpI {.iir"iq .+s*1i;-{ pue teqto Jql q sulJlord qtt,u lpnqns I{JeJ Jo vNuJ uJeMlaq SuOrlJ€rJluIaruos oslPale aJaqJ 'vNUr S€Z pue (uorSar ruropeld aql ur dueur) vNur s9I uaaMlaq slJeluof, seAIoAuI auros - o q l J S 0 L J q r u t s l t u n q n s; o u o r t r u n I a q 1
.::.r,,iThe70Sribosome :.:::;j-.::: consists of the 505subunit(white)andthe305subunit (purpte) withthreetRNAs yettowin the A site,btuein the locatedsuperficiatly: P site,andgreenin the E site.Photocourtesy of Harry No[ter,University of Catifornia, SantaCruz.
: f[1ggtRNAs i:r.iii.irr1..l' havedifferent orientations on the ribosome. mRNA turnsbetween the P andA sitesto a[[owaminoacyltRNAs to bindadjacent Photo codons. courtesy of HarryNo[[er, University of CaUfornia. SantaCruz. into the A Site). EF-Tu inirially places the aminoacyl-IRNA into the small subunit, where the anticodon pairs with the codon. Movement of the IRNA is required to bring it fully inro rhe A site, when its 3'end enters the peptidyl transferase center on the large subunit. There are different models for how this processmay occur. One calls for the entire 1RNA to swivel, so that the elbow in the L-shaped structure made by the D and TYC arms moves into the ribosome, enabling the TYC arm to pair with IRNA.
778
CHAPTER 8 ProteinSvnthesis
Another calls for the internal structure of the IRNA to change, using the anticodon loop as a hinge, with the rest of the IRNA rotating from a position in which it is stackedon the J'side of the anticodon loop to one in which it is stacked on the 5'side. Following the transition, EF-Tu hydrolyzes GTP, allowing peptide synthesisto proceed. Translocation involves large movements in the positions of the tRNAs within the ribosome. The anticodon end of IRNA moves -28 A from the A site to the P site, and then a further 20 A from the P site to the E site. As a result of the angle of each IRNA relative to the anticodon, the bulk of the IRNA moves much larger distances: 40 A from A site to P site and 55 A from P site to E site. This suggeststhat translocation requires a major reorganization of structure. For many years, it was thought that translocation could occur only in the presence of the factor EF-G. However, the antibiotic sparsomycin (which inhibits the peptidyl transferase activity) triggers translocation. This suggests that the energy to drive translocation actually is stored in the ribosome after peptide bond formation has occurred. Usually EF-G acts on the ribosome to releasethis energy and enable it to drive translocation, but sparsomycin can have the same role. Sparsomycin inhibits peptidyl transferaseby binding to the peptidyl-IRNA, blocking its interaction with aminoacyl-IRNA. It probably createsa conformation that resembles the usual posttranslocation conformation, which in turn promotes movement of the peptidyl-tRNA. The important point is that translocation is an intrinsic property of the ribosome. T h e h y b r i d s t a t e s m o d e l s u g g e s t st h a t translocation may take place in two stages, with one ribosomal subunit moving relative to the other to create an intermediate stagein which there are hybrid tRNA-binding sites (50S E / 3 0 S P a n d 5 0 S P / 3 0 SA ) ( s e eF i g u r e 8 . 2 9 ) . Comparisons of the ribosome structure between pre- and posttranslocation states,and comparisons in 165 rRNA conformation between free l0S subunits and 70S ribosomes, suggestthat mobility of structure is especially marked in the head and platform regions of the 30S subunit. An interesting insight on the hybrid statesmodel is castby the fact that many basesin rRNA involved in subunit association are close to basesinvolved in interacting with IRNA. This suggeststhat IRNA-binding sites are close to the interface between subunits, and carries the implication that changesin subunit interaction could be connected with movement of IRNA.
Much of the structure of the ribosome is occupied by its active centers. The schematic ' ,. i shows view of the ribosomal sitesin they comprise about two thirds of the ribosomal structure. A IRNA enters the A site, is transferred by translocation into the P site. and then Ieaves the (bacterial) ribosome by the E site. The A and P sitesextend acrossboth ribosome subunits; IRNA is paired with mRNA in the 30S subunit, but peptide transfer takes place in the 50S subunit. The A and P sites are adjacent, enabling translocation to move the IRNA from one site into the other. The E site is located near the P site (representinga position en route to the surface of the 50S subunit). The peptidyl transferasecenter is located on the 50S subunit, close to the aminoacyl ends of the tRNAs in the A and P sites(seeSection8.18, l65 rRNA Plays an Active Role in Protein Synthesis). All of the GTP-binding proteins that function in protein synthesis (EF-Tu, EF-G, IF-2, and RFl, -2, and -3) bind to the same factorbinding site (sometimescalledthe GTPasecenter), which probably triggers their hydrolysis of GTP.This site is located at the base of the stalk of the large subunit, which consistsof the proteins L7 and Ll2. (L7 is a modification oILl2 and has an acetyl group on the N-terminus.) In addition to this region, the complex of protein Ll l with a 58-base stretch of 23S rRNA provides the binding site for some antibiotics that affect GTPaseactivity. Neither of these ribosomal structuresactually possesses GTPaseactivity, but they are both necessaryfor it. The role of the ribosome is to trigger GTP hydrolysis by factors bound in the factor-binding site. Initial binding of 30S subunits to nRNA requiresprotein Sl, which has a strong affinity for single-strandednucleic acid. It is responsible for maintaining the single-stranded state in nRNA that is bound to the 30S subunit. This action is necessaryto prevent the mRNA from taking up a base-pairedconformation that would be unsuitable for translation. SI has an extremely elongated structure and associates with S18 and 52l. The three proteinsconstitute a domain that is involved in the initial binding of nRNA and in binding initiator IRNA. This locates the mRNA-binding site in the vicinity of the cleft of the small subunit (seeFigure 8.3). The 3'end of rRNA, which pairs with the nRNA initiation site, is located in this region. The initiation factors bind in the same region of the ribosome. IF-l can be crosslinkedto the 3' end of the rRNA, as well as to severalribosomal proteins, including those probably involved in binding nRNA. The role of IF-3
I ' , ,. It hassevera[ activecenters. Theribosome mRNA takesa turn maybe associated with a membrane. asit passes throughthe A andP sjtes,whichareangted the Psjte. with regard to eachother.TheEsiteliesbeyond across stretches Thepeptidyt site(notshown) transferase the topsof the A andP sites.Partof the siteboundby EF-Iu/G ljesat the baseof the A andPsites. could be to stabilizemRNA-30S subunit binding; then it would be displacedwhen the 50S subunit joins. The incorporation of 5S RNA into 50S subunits that are assembled in vitro depends on the ability of three proteins-L5, L8, and L25-to form a stoichiometric complex with it. The complex can bind to 2lS rRNA, although none of the isolated components can do so. It lies in the vicinity of the P and A sites. A nascent protein debouches through the ribosome, away from the active sites, into the region in which ribosomesmay be attached to membranes (seeChapter 10, Protein Localization). A polypeptide chain emerges from the ribosome through an exit channel, which leads from the peptidyl transferasesite to the surface of rhe 50S subunit. The tunnel is composed I to mostly of rRNA. It is quite narrow-only 2 nm wide-and is -10 nm long. The nascent polypeptide emergesfrom the ribosome -I5 A away from the peptidyl transferase site. The tunnel can hold -50 amino acids, and probably constrains the polypeptide chain so that it cannot fold until it leavesthe exit domain.
165rRNAPlaysan Active Synthesis Ro[ein Protein . 165rRNAptaysan activerotein thefunctions of with mRNA, directty the 30Ssubunit.It interacts of withthe 50Ssubunit,andwiththe anticodons tRNAs in the PandA sites. The ribosome was originally viewed as a collection of proteins with various catalytic
Synthesis 179 an ActiveRotein Protein 8.18 165rRNAPtays
.
TERM suppressron
r:i:-:.i: .: ,i:: Somesitesin 165rRNAareorotected fromchemical orobes join 30Ssubunits when50Ssubunits or whenaminoacyltRNA bindsto the A site.0thersarethesitesof mutations thataffectproteinsynthesis. TERM suppression sitesmayaffecttermination at someor several termination codons. The[argecolored indicate btocks thefourdomains ofthe rRNA.
activitiesheld together by protein-protein inreractions and by binding to rRNA. The discovery of RNA molecules with catalytic activities (see Chapter 26, RNA Splicing and Processing) immediately suggests, however, that rRNA might play a more active role in ribosome function. There is now evidence that rRNA interacts with nRNA or tRNA at each stage of translation, and that the proteins are necessary to maintain the rRNA in a structure in which it can perform the catalytic functions. Several interactions involve specific regions of rRNA: . The 3' terminus of the rRNA interacts directly with mRNA at initiation. . Specific regions of l6S rRNA interact directly with the anticodon regions of tRNAs in both the A site and the P site. Similarly, 23S IRNA interacts with the CCA terminus of peptidyl-tRNA in both the P site and A site.
180
CHAPTER 8 ProteinSynthesis
Subunit interaction involves interactions between I65 and 23S rRNAs (see Section8.16, RibosomalRNA Pervades Both Ribosomal Subunits). Much information about the individual steps of bacterial protein synthesis has been obtained by using antibiotics that inhibit the process at particular stages.The target for the antibiotic can be identified by the component in which resistant mutations occur. Some antibiotics act on individual ribosomal proteins, but several act on rRNA, which suggeststhat the rRNA is involved with many or even all of the functions of the ribosome. The functions of rRNA have been investigated by two types of approach. Structural studies show that particular regions of rRNA are located in important sites of the ribosome, and that chemical modifications of these bases impede particular ribosomal functions. In addition, mutations identify basesin rRNA that are required for particular ribosomal functions. Fg*tJftil $.4* summarizesthe sitesin l6S rRNA that have been identified by these means. An indication of the importance of the 3' end of I65 rRNA is given by its susceptibility to the lethal agent colicin E3. Produced by some bacteria, the colicin cleaves-50 nucleotides from the 3'end of the I65 rRNA of E. coli.The cleavage entirely abolishesinitiation of protein synthesis. Several important functions require the region that is cleaved: binding the factor IF-3, recognition of mRNA, andbinding of IRNA. The 3' end of the 165 rRNA is directly involved in the initiation reaction by pairing with the Shine-Dalgarno sequencein the ribosome-binding site of mRNA (seeFigure 8.16). Another direct role for the 3'end of 165 rRNA in protein synthesis is shown by the properties of kasugamycin-resistant mutants, which lack certain modifications in l65 rRNA. I(asugamycin blocks initiation of protein synthesis. Resistantmutants of the type ksgAlacka methylase enzyme that introduces four methyl groups into two adjacent adenines at a site near the 3'terminus of the 165 rRNA. The methylation generatesthe highly conserved sequence G-m26A-m26A, found in both prokaryotic and eukaryotic small rRNA. The methylated sequence is involved in the joining of the 30S and 50S subunits, which in turn is connected also with the retention of initiator IRNA in the complete ribosome. I(asugamycin causesfMettRNAl to be released from the sensitive (methylated) ribosomes, but the resistant ribosomes are able to retain the initiator.
I8I
srsaqlur{s ura}ordur aloua^lllv ues,tpldvNulsgI gI.g uI sPeJaqM'punoq All)erJof, sr 1r ]Bql pJprJap xaldnp eql Jo aAooJBrourur Jql ut s;ted saseq seq aluosoqrrrqt 'Jlrs d aq1peq)eer seq vNul omt lsJrJJql Jo ernlJnrls JrIl ol spuodsar y1qgr e r { l e J u o J S n P J e q' a s u J s s a > l e u s l q J ' v N u l sgI aql Jo alnlJnJls aqr leql sI uollf,eJelul eql 'saxaldruo) uopoJllue-uopoJ - l ^ J e o u r r u e q t l u p e J e d r u o Jy N U t - 1 , { p r r d a d ;o aldnuud aq1 parred ,{gadord Surpalap JoJ Iuslueq)alu Jo ,{tlllqers paseerrul rqt roJ alqtsuodsar aq .{eru slqJ 'rtls V eqt ur sl tl ueqM ueql elrs Jr{t ur sr uar{M qll^^ slJeluoJ 1l d vNul JJOrUJJP eJerll 'DeJ uI 'JrnlJnJlS ui.rerualaql Surpurqtpnqns S0€-vNul1pqt os 'doo1-ruats ur Jeqtoue Juo Jeeu erl sJseqaqt ,{1a1r1 uopoJrtup aql Jo aplloelJnuo8rlo palelost tsotu luls eql uI aqt.dq parnpo;d aq uer 17NUrS9I uo seq Eur Lq papalord ere yNur d vNur s9I -pulq vNgl teql spaJJaeq1Jo IIV '(utdrrcq 3uo1e ;o suorlrsodtuereJlrp ur sJSeqIo ^talle^ y 'ells .dqpaDauuoJ Jre 1eql saqJlrrls papuerls-aputs v eqr qllM o1\^larP €6vI pue z6vl ]e seuluJpP aq1 puP YNU1 Jo uorlerJosseJql sazrlrqplsuorlJeJJlul JqJ'rnlJo touueJ uorlJeJJlursql pue pJuolslp Z6VI ol 66tI saseqesnerrq pa11et-os)uot8ar sr xaldruor uopolrtue-uopoJ aql Jo arnlJnJls 00SI ol 00tI rqt sIreqlo eql'(suopol VDnpue 'gvn 'Wn eql 1euollpulluJel sluJ^Jrd teql uotl eql 'JIIS V Jql srelue YNU] lJJJJoJur uP uaqM 'auoq{req -elnru e Jo elrs eqt sr osle qrlqm) dool 6EE aqt vNuru rql ur Ho-,z rql qlr1!{lrP -JJlur 01 auruJpe qJeJ sr auo '(97'g a;n8rg aas) alts y eqt ut punoq Jo IN eql smollp Suuted uopoJllue-uopo) leql sMoqs ,.. ti 'ir.,, : '(uotpe VNUI ^q papalord are suot8al pullslp uleuJJ 'pardnrro Jre sJlrs esaql uJqM Jn))o alnpnJls -ralul yNuru qJIqM) Jr{l serolsar ur saseq Surp -uodsarror Jqt Jo uortrsod aW tp aurronlJJo s1rur sa8ueqr luerrpu8ts pue 'uollJunJ JIIS d ,Z pue elrsv qroq uI peAIoAuIsI vNuJ s9I aI{J uorlf,nporlur aql .,{qpassarddnsaq UPJ €6vI Jo 'aruanbas 'rala,lroH 'elIS xaldnp e uoqs Jo JJnleu aql a8ueqr Z6VI rc suorlelnru v aql u1 3ur -purq uorJ YNul slua.tardvNur ul t 6vI to z6vl 01 lueruJ^Olu Ie)OI e SJAIOAUT l! 1,r"'g:ihtl-lg* ur uMoqs r srsaqluds uratord Sutrnp sJnrJo leql eseq reqtra ;o uorlrsod IN eql Jo uoue)rJrpolN 'uorlJeJalur p a u r o l auo 'suoruunJ asaql r4 17Ngryo uouedpp aSueqr u o p o r r t u p u o p o ) J q t l q -red parp alerrput dpressJJautou op .{aq1 'yggr arnsodxa Jq1 peJJp teqt uollpruroluoJ eruos 'lleralur louuPrvNUlx-vNUl 1o -oqrJ palelJosseJrp sluela asJql qll,lr 'vNUl ur sa8ueqr porreclsrur qlrMu0r1 lnq sgl to E6tl-z6tl sauruapP 'vNul;o Sutputq ro 'YNUITI -rptelursf oddns6uuLed uoporrlup-uopol r-f"l-r:lijili-ri:i teql atp)Ipq ^eqJ yo Surpurq 'sllunqns S0S qtp,t Sututol Iq para8 -31t yNUJ are S9I ut sa8ueqr Jq1 Jo eluos 'eJnlJnJls ,{ren,ral aqt ur raq1a3o1 asop Llpnpe eJeuorlJunJ erupseqt uI paAIoAuI srueas tl 'VNur 59I suortrsod eseq teql ,{1a111 ;o a;uanbas JeauII Jql uI pasradsrpaJPsuoll€Jol aqt q8noqrlv 'sulpruop IeJlueJ pue JouIuI ,€ Jql ur pJlpJluaJuoJ aJEleqt sdnor8 ^tal e olul IIeJselrspnprlrpul eqJ'>lJeltp 1ellruel{Jlsure8e uollceJoluloN rcln)ured yo uotlralord,{q uaJS sP 'slsJql saseq -uz(suralord ur pa8eSua eJe saurosoqlJ uaqm JnJJoVN5J 59I Jo aJntf,nJlsaql ur sa8ueq3
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068
the A site, the assessmentof binding is being made. The 1400 region can be directly crosslinked to pepridyl-tRNA, which suggesrsthat this region is a structural component of the P site. The basicconclusion to be drawn from these results is that rRNA has many interactions with both IRNA and nRNA, and that these inreractions recur in each cycle of peptide bond formation.
@
23SrRNAHasPeptidyl Transferase Activity
. Peptidyt transferase activityresides exctusivety in the 235rRNA. The sitesinvolved in the functions of 23S rRNA are lesswell identified than those of l6S rRNA, but the same generalpattern is observed:bases at certain positions affect specific functions. Basesat some positionsin 23S rRNA are affected by the conformation of the A site or P site. In particular, oligonucleotides derived from the 3' CCA terminus of IRNA protect a set of basesin 23S rRNA that essentiallyare the same as those protected by peptidyl-IRNA. This suggeststhat the major interaction of 2lS rRNA with peptidyl-tRNA in the P site involves the 3'end of the IRNA. The IRNA makes contacts with the 23S rRNA in both the P and A sites. At the P site, G2552 of 2I S rRNA base pairs with C74 of the peptidyl IRNA. A muration in the G in the rRNA prevents interaction with IRNA, but interaction is restored by a compensating mutation in the C of the amino acceptor end of the IRNA. At the A site. G2553 of the 23S rRNA base pairs with C75 of the aminoacyl-tRNA. Thus there is a close role for rRNA in both the tRNA-binding sites.Indeed, when we have a clearer structural view of the region, we should be able to understand the movements of IRNA between the A and P sitesin terms of making and breaking contacts with rRNA. Another site that binds IRNA is the E site, which is localizedalmost exclusively on the 50S subunit. Basesaffected by its conformation can be identified in 23S rRNA. What is the nature of the site on the 50S subunit that provides peptidyl transferasefunction? The involvement of rRNA was first indicated because a region of the 23S rRNA is the site of mutations that confer resistance to antibiotics that inhibit peptidyl transferase.
182
CHAPTER 8 ProteinSvnthesis
A long search for ribosomal proteins that might possessthe catalytic activity has been unsuccessful.Recent results suggestthat the ribosomal RNA of the large subunit has the catalytic activity. Extraction of almost all the protein content of 50S subunits leavesthe 23S rRNA associatedlargely with fragments of proteins, amounting to <5% of the massof the ribosomal proteins. This preparation retains peptidyl transferase activity. Tfeatments that damage the RNA abolish the catalytic activity. Following from theseresults,235 rRNAprepared by transcription in vitro can catalyze the formation of a peptide bond between Ac-PheIRNA and Phe-tRNA. The yield of Ac-Phe-Phe is very low, suggesting that the 23S rRNA requires proteins in order to function at a high efficiency. Given that the rRNA has the basic catalytic activity, though, the role of the proteins must be indirect, serving to fold the rRNA properly or to present the substratesto it. The reaction also works, although less effectively, if the domains of 23S rRNA are synthesized separately and then combined. In fact, some activity is shown by domain V alone, which has the catalytic center. Activity is abolished by mutations in position 2252 of domain V that lies in the P site. The crystal structure of an archaeal 50S subunit shows that the peptidyl transferase site basically consistsof 23S rRNA. There is no protein within 18 A of the active site where the transfer reaction occursbetween peptidyl-tRNA and aminoacyl-IRNA! Peptide bond synthesis requires an attack by the amino group of one amino acid on the carboxyl group of another amino acid. Catalysis requires a basic residue to accept the hydrogen atom that is released from the amino group, as shown in lg*i.ifigFi.+"{.If rRNA is the catalyst it must provide this residue, but we do not know how this happens. The purine and pyrimidine basesare not basic at physiological pH. A highly conserved base (at position 245I in E. colil had been implicated, but appears now neither to have the right properties nor to be crucial for peptidyl transf erase activity. Proteins that are bound to the 23S rRNA outside of the peptidyl transfer region are almost certainly required to enable the rRNA to form the proper structure invivo. The idea that rRNA is the catalytic component is consistent with the results discussedin Chapter 26, RNA Splicing and Processing,which identify catalytic properties in RNA that are involved with several RNA processingreactions.It fits with the notion
thesis). More directly, comparisons of the high resolution crystal structures of the individual subunits with the lower resolution structure of the intact ribosome suggeststhe existence of significant differences. These ideas have been peptidechain I confirmed by a crystal structure of.the E. coli H -IN ribosome ar 3.5 A, which furthermore identiH-C-R H-C.R I fies two different conformations of the ribox"\^ VU some, possibly representing different stagesin I tRNA ; IRNA protein synthesis. I The crystalcontains two ribosomesper unit, V each with a different conformation. The differences are due to changes in the positioning of Base domains within each subunit, the most important being that in one conformation the head of the small subunit has swiveled 6' around the IRNA neck region toward the E site. Also, a 6o rota,T tion in the opposite direction is seen in the (low peptidechain I resolution) structures of. Thermusthermophilus H-N I ribosomes that are bound to nRNA and have H-C.R Au tRNAs in both A and P sites,suggestingthat the t\ head may swivel overall by l2' depending on I H-C-R the stage of protein synthesis. The rotation of t-l a the head follows the path of tRNAs through the ol l d"b ribosome, raising the possibility that its swivelIRNA IRNA ing controls movement of nRNA and IRNA. ii.+;::Peptide irt{,il,rFil bondformation requires acid-base The changes in conformation that occur catalysis in whichan H atomjs transferred to a basic when subunits join together are much more residue. marked in the 30S subunit than in the 50S subunit. The changesare probably concerned with controlling the position and movement of nRNA. The most significant change in the 50S that the ribosome evolved from functions origsubunit concerns the peptidyl transferase ceninally possessedby RNA. ter. 50S subunits are -1000x less effective in catalyzing peptide bond synthesis than complete ribosomes; the reason may be a change in structure that positions the substrate more effectively in the active site in the complete ribosome. One of the main features emerging from the structure of the complete ribosome is the very high density of solvent contacts at their interface; this may help the making and break. Theheadof the 305subunitswivels around the ing of contacts that is essentialfor subunit assoneckwhencomplete ribosomes areformed. ciation and dissociation, and may also be . Thepeptidyltransferase activesite of the 505 involved in structural changes that occur duris more in ribosomes subunit active complete than in individual. 50Ssubunits. ing translocation. . Theinterface is between the 30Sand50Ssubunits veryrichin solventcontacts. Aminoacyl{RNA PeptidyltRNA
I
I
o
I
RibosomaL Structures Change Whenthe Together Subunits Come
Summary
Much indirect evidence suggeststhat the structures of the individual subunits change significantly when they join together to form a complete ribosome. Differencesin the susceptibilities of the rRNAs to outside agents are one of the strongest indicators (see Section 8.18, I6S rRNA Plays an Active Role in Protein Syn-
Ribosomes are ribonucleoprotein particles in which a majority of the mass is provided by rRNA. The shapesof all ribosomes are generally similar, but only those of bacteria (70S) have been characterizedin detail. The small (30S) subunit has a squashed shape, with a "body" containing about two thirds of the mass divided
8.21Summary 1 8 3
from the "head" by a cleft.The large (50S) subunit is more spherical,with a prominent "stalk" on the right and a "central protuberance." Locations of all proteins are known approximately in the small subunit. Each subunit contains a single major rRNA, I 65 and 2 35 in prokaryotes, and I 8S and 28S in eukaryotic cytosol. There are also minor rRNAs, most notably 5S rRNA in the large subunit. Both major rRNAs have extensive basepairing, mostly in the form of short, imperfectly paired duplex stemswith single-strandedloops. Conservedfeatures in the rRNA can be identified by comparing sequencesand the secondarystructures that can be drawn for rRNA of a variety of organisms. The l65 rRNA has four distinct domains; the 2lS rRNA has six distinct domains. Eukaryotic rRNAs have additional domains. The crystal structure shows that the 30S subunit has an asymmetrical distribution of RNA and protein. RNA is concentrated at the interface with the 50S subunit. The 50S subunit has a surface of protein, with long rods of double-stranded RNA crisscrossingthe structure. loSto-50S joining involves contacts between l65 rRNA and 235 rRNA. The interface between the subunits is very rich in contacts for solvent. Structural changes occur in both subunits when they join to form a complete ribosome. Each subunit has several active centers, which are concentrated in the translational domain of the ribosome where proteins are synthesized. Proteins leave the ribosome through the exit domain, which can associatewith a membrane. The major active sitesare the P and A sites,the E site, the EF-Tu and EF-G binding sites,peptidyl transferase,and the mRNA-binding site. Ribosome conformation may change at stagesduring protein synthesis;differencesin the accessibilityof particular regions of the major rRNAs have been detected. The tRNAs in the A and P sites are parallel to one another. The anticodon loops are bound to mRNA in a groove on the 30S subunit. The rest of each IRNA is bound to the 50S subunit. A conformational shift of IRNA within the A site is required to bring its aminoacyl end into
illr:T:'i,".1 ffirJffi$ :li*,::::'i* :iil* links the P- and A-binding siresis made of 23S rRNA, which has the peptidyl transferase cat-
somal function, interactions with mRNA or IRNA that can be detected by chemical crosslinking, and the requirement to maintain individual base pairing interactions with the IRNA or mRNA. The 3'terminal region of the rRNA base pairs with mRNA at initiation. Internal regions make individual contacts with the tRNAs in both the P and A sites.Ribosomal RNA is the target for some antibiotics or other agents that inhibit protein synthesis A codon in nRNA is recognized by an aminoacyl-tRNA, which has an anticodon complementary to the codon and carries the amino acid corresponding to the codon. A special initiator IRNA (fMet-tRNAr in prokaryotes or MettRNAi in eukaryotes) recognizesthe AUG codon, which is used to start all coding sequences.In prokaryotes, GUG is also used. Only the termination (nonsense) codons, UAA, UAG, and UGA, are not recognized by aminoacyl-tRNAs. Ribosomesare releasedfrom protein synthesis to enter a pool of free ribosomes that are in equilibrium with separate small and large subunits. Small subunits bind to mRNA and then are joined by large subunits to generate an intact ribosome that undertakes protein synthesis. Recognition of a prokaryotic initiation site involves binding of a sequenceat the J'end of rRNA to the Shine-Dalgarno motif, which precedesthe AUG (or GUG) codon in the mRNA. Recognition of a eukaryotic mRNA involves binding to the 5' cap; the small subunit then migrates to the initiation site by scanning for AUG codons. When it recognizes an appropriate AUG codon (usually, but not always, the first it encounters), it is joined by a large subunit. A ribosome can carry two aminoacyl-tRNAs simultaneously: its P site is occupied by a pollpeptidyl-tRNA, which carries the polypep tide chain synthesizedso far, whereas the A site is used for entry by an aminoacyl-tRNA carrying the next amino acid to be added to the chain. Bacterial ribosomesalso have an E site, through which deacylated IRNA passes before it is released after being used in protein synthesis. The polypeptide chain in the P site is transferred to the aminoacyl-tRNA in the A site, creating a deacylated IRNA in the P site and a peptidylIRNA in the A site. Following peptide bond synthesis,the ribosome translocates one codon along the nRNA, moving deacylated IRNA into the E site and peptidyl tRNA from the A site into the P site. Tlanslocation is catalyzedby the elongation factor EF-G and, like several other stagesof ribo-
i'JJ;:3',-i:';#,iTlJr1"i,:,T:,Xllo'oouo'' An activerole for the rRNAsin protein synthesisis indicatedby mutationsthat affectribo-
CHAPTER 8 Protein Synthesis
some function. requires hydrolysis of GTP.During translocation, the ribosome passesthrough a hybrid stagein which the 50S subunit moves relative to the 30S subunit. Protein synthesis is an expensive process. ATP is used to provide energy at several stages, including the charging of IRNA with its amino acid and the unwinding of mRNA. It has been estimated that up to 90o/" of all the ATP molecules synthesized in a rapidly growing bacterium are consumed in assemblins amino acids into protein! Additional factors are required at each stage of protein synthesis. They are defined by their cyclic association with, and dissociation from, the ribosome. IF factorsare involved in prokaryotic initiation. IF-l is needed for 30S subunits to bind to mRNA, and also is responsible for maintaining the l0S subunit in a free form. IF2 is needed for fMet-tRNAl to bind to the 30S subunit and is responsible for excluding other aminoacyl-tRNAs from the initiation reaction. GTP is hydrolyzed after the initiator IRNA has been bound to the initiation complex. The initiation factors must be releasedin order to allow a large subunit to join the initiation complex. Eukaryotic initiation involves a greater number of factors. Some of them are involved in the initial binding of the 40S subunit to the capped 5'end of the mRNA, at which point the initiator IRNA is bound by another group of factors. After this initial binding, the small subunit scansthe nRNA until it recognizes the correct AUG codon. At this point, initiation factors are releasedand the 60S subunit joins the complex. Prokaryotic EF factors are involved in elongation. EF-Tirbinds aminoacyl-tRNA to the 70S ribosome. GTP is hydrolyzed when EF-Tu is released,and EF-Ts is required to regenerate the active form of EF-Tu. EF-G is required for translocation. Binding of the EF-Tu and EF-G factors to ribosomes is mutually exclusive, which ensuresthat each step must be completedbefore the next can be started. Termination occurs at any one of the three specialcodons,UAA, UAG, and UGA. ClassI RF factors that specifically recognize the termination codons activate the ribosome to hydrolyze the peptidyl-tRNA. A class2 RF factor is required to release the class I RF factor from the ribosome. The GTP-binding factors IF-2, EF-Tu, EF-G, and RFI all have similar structures, with the latter two mimicking the RNA-protein structure of the first two when they are bound to IRNA.They all bind to the same ribosomal site, the G-factor binding site.
References Needs 30SSubunits Initiationin Bacteria and AccessoryFactors Review Maitra, U., ( 1982). Initiation factors in protein biosynthesis. Annu Rev.Biochem.51, 869-900. Research Carter,A. P.,Clemons, W. M., Brodersen,D. E., Morgan-Warren, R. J., Hartsch, T., Wimberly, B. T., and Ramakrishnan, V. (2001). Crystal structure of an initiation factor bound to the 30S ribosomal subunit. Science297, 498-5Ol . Dallas,A. and Noller, H. F. (2001). Interaction of translation initiation factor 3 with the 30S ribosomal subunit. Mol. Cell 8, 855-864. Moazed, D., Samaha,R. R., Gualerzi, C., and Noller, H. F. (I995). Specificprotection of I65 rRNA by translational initiation factots. J Mol. Biol. 248, 207-210.
InitiatortRNAStarts A SpeciaI the PotypeptideChain Research Lee, C. P., Seong,B. L., and RajBhandary,U. L. (t 991). Structural and sequenceelements important for recognition of E. coli formylmethionine IRNA by methionyl-tRNA transformylase are clustered in the acceptor stem. J. Biol. Chem 266, l80l2-l8}l7. Marcker, I(. and Sanger, F. (1964). N-Formylmethionyl-S-RNA. J. Mol Biol.8, 835-840. Sundari, R, M., Stringer,E. A., Schulman, L. H., and Maitra, U . (1976) .Interaction of bacterial initiation factor 2 with initiator 1RNA. J. Biol C h e m . 2 5 1 ,) ) ) 8 - 3 ) 4 5 .
Scanfor InitiationSites Smat[Subunits on EukaryoticmRNA Reviews Hellen, C. U. and Sarnow, P. (2001).Internal ribosome entry sites in eukaryotic mRNA molecules. GenesDev. 15, 159)-1612. I(ozak, M. (1978). How do eukaryotic ribosomes select initiation regions in mRNA? Cell 15, ll09-1123 I(ozak, M. (1983). Comparison of initiation of protein synthesis in prokaryotes, eukaryotes, and organelles. Microbiol. Rev.47, l-45. rch Resea I(aminski, A.. Howell, M. T., and Jackson, R. J. ( 1990) . Initiation of encephalomyocarditis virus RNA translation: the authentic initiation site is not selectedby a scanning mechanism. E M B OJ . 9 , J 7 5 ) - j 7 5 9 . Pelletier,J. and Sonenberg,N. (1988). Internal initiation of translation of eukaryotic mRNA
References 185
directed by a sequence derived from poliovirus RNA. Nalare )34, 320-325. Pestova,T. V, Hellen, C. U., and Shatsky,L N. (1996\. Canonical eukaryotic initiation factors determine initiation of translation by internal ribosomal entry. Mol. CellBiol. 16, 6859-6869. Pestova,T. V., Shatsky, L N., Fletcher, S. P., Jacks o n , R . J . , a n d H e l l e n ,C . U . ( 1 9 9 8 ) .A prokaryotic-like mode of cytoplasmic eukaryotic ribosome binding to the initiation codon during internal translation initiation of hepatitis C and classicalswine fever virus RNAs. GenesDev.\2, 67-8).
]l|
Eukaryotes Usea Complex of Many Initiation Factors
Reviews Dever, T. E. (2002). Gene-specificregulation by general translation factors. Cell lO8, 545-556. Gingras,A. C., Raught, B., and Sonenberg,N. (1999\. eIF4 initiation factors: effectors of mRNA recruitment to ribosomes and regulators of translation. Annu Rev.Biochem.68, 9),3-96). Hershey, J. W. B. ( I 99 I ) . Ttanslational control in mammalian ce]rls.Annu Rev.Biochem 60. 7t7-755 Merrick, w. C \1992). Mechanism and regulation of eukaryotic protein synthesis. Microbiol.Rev. 56. 29t-3r5. Pestova, T. V., I(olupaeva, V. G., Lomakin, I. B., Pilipenko, E. V., Shatsky, I. N., Agol, V. L, and Hellen, C. U. (2001). Molecular mechanisms of translation initiation in eukaryotes. Proc Natl Acad- Sci USA 98, 7029-786. Sachs,A., Sarnow, P.,and Hentze, M. W. ( 1997). Starting at the beginning, middle, and end: translation initiation in eukarvotes. Cell 89. 8l l-838 Resea r ch Asano, K., Clayton, J., Shalev A., and Hinnebusch, A. G. (2000). A multifactor complex of eukaryotic initiation factors, eIFl, eIF2, eIF3, eIF5, and initiator IRNA(Met) is an important translation initiation intermediate in vitro. GenesDev.I 4, 2t34-2546. Huang, H. K., Yoon, H., Hannig, E. M., and Donahue, T. F. (1997). GTP hydrolysis controls stringent selection of the AUG start codon during translation initiation in S cerevisiae GenesDev. l l, 2J96-241). Pestova,T. V., Lomakin, I. B., Lee, J. H., Choi, S. K., Dever, T. E., and Hellen, C. U. (2000). The joining o{ ribosomal subunits in eukaryotes requires elFlB. Nature 403, j32-j)r. Tarun, S. Z. and Sachs,A. B. (I996). AssociationoI the yeast poly(A) tail binding protein with translation initiation factor eIF-4G. EMBO J. 15, 7 168-7 777.
186
CHAPTER 8 ProteinSynthesis
Transtocation Moves the Ribosome Reviews Ramakrishnan, V. (2002). Ribosome structure and the mechanism of translation. Cell 108, 557-572. Wilson, I(. S. and Noller, H. F. (1998). Molecular movement inside the translational ensine. Cell q)
117_14q
Resea r ch Moazed, D., and Noller, H. F. (I986). TransferRNA shields specific nucleotides in l6S ribosomal RNA from attack by chemical probes. Cell47, 985-994. Moazed, D. and Noller, H. F. (1989). Intermediate states in the movement of IRNA in the ribosome. Nature J42, 142-148.
Elongation Factors BindAlternatety to the Ribosome Resea rch Nissen, P.,I(jeldgaard, M., Thirup, S., Polekhina, G. Reshetnikova, L., Clark, B. F., and Nyborg, J. ( 1995). Crystal structure of the ternary complex of Phe-tRNAPhe, EF-Tu, and a GTP analog. Science 270, 1464-1472. Stark, H., Rodnina, M. V., Wieden, H. J., van Heel, M., and Wintermeyer, W. (2000). Large-scale movement of elongation factor G and extensive conformational change of the ribosome during translocation.Cell lOO, 301-309.
Termination Codons AreRecoqnized by Protein Factors Reviews Eggertsson,G. and Soll, D. (1988). TransferRNAmediated suppression of termination codons in E coli.Microbiol.Rev.52,354=374. Frolova, L. et al. (1994). A highly conserved eukaryotic protein family possessingproperties of polypeptide chain release f.actor.Nature 372,70r-70). Nissen,P.,I(jeldgaard,M., and Nyborg, J. (2000). Macromolecular mimicry. EMBO J. 19, 489-495. Research Freistroffe4, D. V, I(wiatkowski M., Buckingham, R. H., and Ehrenberg, M. (2000). The accuracy of codon recognition by polypeptide release factors. Proc.Natl. Acad Sci.USA 97. 2046-205t. Ito, I(., Ebihara, I(., Uno, M., and Nakamura. Y. (1996). Conserved motifs in prokaryotic and eukaryotic polypeptide release factors: IRNAprotein mimicry hypothesis. Proc.Natl. Acad. Sci. USA 9j, 5443-5448. ICaholz, B. P., Myasnikov A. G., and Van Heel, M. (2004\. Visualization of release factor 3 on the
ribosome during termination of protein synthesis. Nature 427 , 862-865. Mikuni, O., Ito, I(., Moffat, J., Matsumura, I(., McCaughan, I(., Nobukuni, T., Tate, W., and Nakamura, Y. ( I 994). Identification of.th,eprfC gene, which encodes peptide-chain-release factor f of E. coli.Proc.Natl. Acad. Sci.USA 9l. 5798-5802. Milman, G., Goldstein, J., Scolnick, E., and Caskey, T. (1969). Peptide chain termination. 3. Stimulation of invitro termination. Proc NatI Acad. Sci USA 6), r9)-r90. Scolnick,E. et al. (I968). Releasefactors differing in specificity for terminator codons. Proc Natl. Acad. Sci.USA 61, 7 68-77 4. Selmer, M., Al-I(aradaghi, S., Hirokawa, G., I(aji, A., and Liljas, A. 11999). Crystal structure of Thermotog a maritima ribosome recycling factor: 286,2)49-2)52. a IRNA mimic. Science Song, H., Mugnier, P., Das, A. I(., Webb, H. M., Evans, D. R., Tuite, M. F., Hemmings, B. A., and Barford, D. (2000). The crystal structure of human eukaryotic release factor eRFlmechanism of stop codon recognition and peptidyl-IRNA hydrolysis. Cell lO0, jrt-)21.
RibosomaI RNAPervades Both R i b o s o m aS I ubunits Reviews Hill, W. E. et al. (1990). TheRibosome. Washington, DC: American Society for Microbiology. Noller, H. F. ( 1984) . Structure of ribosomal RNA. Annu. Rev.Biochem.5), 119-162. Noller, H. F. (2005). RNA structure: reading the ribosome. Science )09, I 508-1 5 14. Noller, H. F. and Nomura, M. (1987). E coliand S. typhimurium. Washington, DC: American Society for Microbiology. Wittman, H. G. (1983). Architecture of prokaryotic ribosomes. Annu Rev.Biochem 52. )5-65. R e s e ar c h Ban, N., Nissen,P.,Hansen,J., Capel,M., Moore, P. B., and Steitz,T. A. (1999). Placemenrof protein and RNA structuresinto a 5 Aresolution map of the 50S ribosomal subunit. Nature 4OO,841-847. Ban, N., Nissen, P., Hansen, J., Moore, P. B., and Sreitz, T. A. (2000). The complere atomic structure of the large ribosomal subunit at 2.4 A resoluti on. Science289, 905-920. Clemons, W. M. et al. ( I 999). Structure of a bacterial 30S ribosomal subunit at 5.5 A resolution. Nature 400,833-840. Wimberly, B. T., Brodersen, D. E., Clemons W. M. Jr., Morgan-Warren, R. J., Carter, A. P.,Vonrhein, C., Hartsch, T., and Ramakrishnan, V. (2000). Structure of the 30S ribosomal subunir. Nature 407, 327-)J9.
Yusupov, M. M., Yusupova,G.2., Baucom,A., Lieberman,A., Earnest,T. N., Cate,J. H. D., and Noller,H. F. (2001).Crystalstructure of the ribosomeat 5.5A resolution.Scierce 292,88)-896.
ActiveCenters HaveSeveraI Ribosomes Reviews Lafontaine, D. L. and Tollervey,D. (200I). The function and synthesis of ribosomes. Nat. Rev. Mol. CellBiol.2, 514-520. Moore, P.B. and Steitz,T. A. (2003). The structural basis of large ribosomal subunit function. Annu. Rev.Biochem.72,8l)-850. Ramakrishnan, V. (2002). Ribosome structure and the mechanism of translation. Cell 108, 557-572. Research Cate, J. H., Yusupov,M. M., Yusupova, G.2., Earnest, T. N., and Noller, H. F. ( I 999). X-ray crystal structures of 70S ribosome functional complexes. Science285, 209 5-2104. Fredrick, I(., and Noller, H. F. (2003). Catalysis of ribosomal translocation by sparsomycin. Sclence)00, ll59-1162. Sengupta,J., Agrawal, R. I(., and Frank, J. (200I). Visualization of protein SI within the 30S ribosomal subunit and its interaction with messenger RNA. Proc Natl. Acad Sci.USA 98, I I99l-r 1996. Simonson, A. B. and Simonson, J. A. (2002). The transorientation hypothesis for codon recognition during protein synthesis. Nature 416, 281-285. Valle, M., Sengupta, J., Swami, N. K., Grassucci, R. A.. Burkhardt, N., Nierhaus, I(. H., Agrawal, R. I(., and Frank, J. (2002). CryoEM reveals an active role for aminoacyl-tRNA in the accommodation process.EMBO J.21, 3557-3567. Yusupov,M. M., Yusupova, G.2., Baucom, A., Lieberman, A., Earnest,T. N., Cate, J. H. D., and Noller, H. F. (2001). Crystal structure of 292, the ribosome at 5.5 A resolution. Science 883-896.
an ActiveRo[e 165rRNAPtays Synthesis in Protein Reviews Noller. H. F. ( I 99I ). Ribosomal RNA and translatio\. Annu Rev.Biochem.60,I9l-227. Yonath, A. (2005). Antibiotics targeting ribosomes: resistance,selectivity, synergism and cellular regulation. Annu. Rev.Biochem 74, 649-679. Resea r ch Lodmell, J. S. and Dahlberg,A.E. (1997)r.A conformational switch in E. coli 163 rRNA during 277, 1262-1267. decoding of mRNA. Science
References 187
Moazed, D., and Noller, H. F. (1986). TfansferRNA shields specific nucleotides in I6S ribosomal RNA from attack by chemical probes. Cell 47, 985-994. Yoshizawa, S., Fourmy, D., and Puglisi, J. D. (1999). Recognition of the codon-anticodon helix by rRNA. Science285, 1722-1725.
@
23SrRNAHasPeptidylTransferase Activity
Resea rch Ban, N., Nissen, P., Hansen, J., Moore, P. B., and Sreirz,T. A. (2000). The complete atomic slructure of the large ribosomal subunit at 2.4 A resolution. Science289, 905-920. Bayfield, M. A., Dahlberg, A.E., Schulmeister,U., Dorner, S., and Barta, A. (2001). A conformational change in the ribosomal peptidyl transferase center upon active/inactive transition. Proc Natl. Acad. Sci.USA98, 10096-10l0l. Noller, H. F., Hoffarth, V., and Zimniak, L. (1992\. Unusual resistance of peptidyl transferase to protein extraction procedures. Science 256, t4t6-14t9.
188
CHAPTER 8 Protein Synthesis
Samaha,R. R., Green,R.,and Noller,H. F. (1995). A basepair betweenIRNA and 235 rRNA in the peptidyl transferasecenter of the ribosome.Nature)77, )09-314. Thompson,J., Thompson,D. F.,O'Connor,M., Lieberman,I(. R., Bayfield,M. A., Gregory,S.T., Green,R., Noller,H. F.,and Dahlberg,A. E. (2001). Analysisof mutationsat residues A245I and G2447of 23SrRNA in the peptidyltransferase activesite of the 50Sribosomal subunit. Proc.Natl. Acad.Sci.USA98. 9002-9007.
RibosomaI Structures Change Whenthe Subunits Come Together Reference Schuwirth,B. S.,Borovinskaya,M. A., Hau, C. W., Zhang,W., Vila-Sanjurjo,A., Holton, J. M., and Cate,J. H. (2005).Structures of the bacterial ribosomeat 3.5 A resolution.Science 3lO, 827-834.
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The genetic code is actually read on the mRNA, and thus it is usually described in terms of the four basespresent in RNA: U, C. A, and G. Codons representing the same or related amino acidstend to be similar in sequence.Often the base in the third position of a codon is not significant, because the four codons differing only in the third baserepresent the same amino acid. Sometimes a distinction is made only between a purine versus a pyrimidine in this position. The reduced specificity at the last position is known as third-base degeneracy. The interpretation of a codon requires base pairing with the anticodon of the corresponding aminoacyl-IRNA. The reaction occurs within the ribosome: complementary trinucleotidesin isolation would usually be too short to pair in a stable manner, but the interaction is stabilized by the environment of the ribosomal A site. Also, base pairing between codon and anticodon is not solely a matter of A-U and G-C base pairing. The ribosome controls the environment in such away that conventional pairing occurs at the first two positions of the codon. but additional reactions are permitted at the third base. As a result. a single aminoaryl-tnNAmayrecognize more than one codon, corresponding with the pattern of degeneracy. Furthermore, pairing interactions may also be influenced by the introduction of special bases into IRNA, especiallyby modification in or close to the anticodon. The tendency for similar amino acids to be represented by related codons minimizes the effects of mutations. It increases the probability that a single random base change will result in no amino acid substitution or in one involving amino acids of similar character. For example, a mutation of CUC to CUG has no effect, becauseboth codons represent leucine.A mutation of CUU to AUU results in replacement of leucine with isoleucine. a closelv related amino acid. i ' , : . ; n : :, J . lp: l o t s t h e n u m b e r o f c o d o n sr e p resenting each amino acid againstthe frequency with which the amino acid is used in proteins (in E. coli). There is only a slight tendency for amino acids that are more common to be representedby more codons,and therefore it does not seem that the genetic code has been optimized with regard to the utilization of amino acids. The three codons (UAA, UAG, and UGA) that do not represent amino acidsare used specifically to terminate protein synthesis. One of these stop codons marks the end of every gene.
CUUI
cuc| , ^.. cuAT'"" CUGI AUU-l AUC flle AUA-I AUG Met
GUUI cuc | ,,_, cu; fu"' GUGI
GAUIGCUI Gcc | ^,^GACI ccA fAra cAAL GAG-.I GCGI
GGUI GGCL.,,, ccA T"'Y GGGI
ir:lii,jii':;I ; Atl.the triplet codonshavemeaning:Sixty-one represent aminoacjds.andthreecausetermjnation(ST0P)'
10 '68
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o (!) o
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o I- a ) 2 IL
f : r . , i i i i rl : : . :T h en u m b eorf c o d o nfso r e a c ha m i n oa c i d of usein with its frequency cl"osely doesnot correlate oroteins.
Is the genetic code the same in all living organisms? Comparisons of DNA sequenceswith the corresponding protein sequencesreveal that the identical set of codon assignmentsis used in bacteria and in eukaryotic cytoplasm. As a result, mRNA from one speciesusually can be translated correcLly in vitro or in vivo by the protein
AminoAcids Related Represent Codons 9.2 Related
797
synthetic apparatus of another species.Thus the codons used in the nRNA of one species have the same meaning for the ribosomesand tRNAsof other species. The universality of the code argues that it must have been establishedvery early in evolution. Perhapsthe code started in a primitive form in which a small number of codons were used to represent comparatively few amino acids,possibly even with one codon corresponding to any member of a group of amino acids. More precise codon meanings and additional amino acids could have been introduced later. One possibility is that at first only rwo of the three basesin each codon were used; discrimination at the third position could have evolved later. (Originaily there might have been a stereochemical relationship between amino acids and the codons representingthem. Then a more complex system evolved.) Evolution of the code could have become " f . r o z e n "a t a p o i n t a t w h i c h t h e s y s t e m h a d become so complex that any changesin codon meaning would disrupt existing proreins by substituting unacceptable amino acids. Its universality implies that this must have happened at such an early stagethat all living organisms are descendedfrom a single pool of primitive cells in which this occurred.
UUU UUC UUA UUG CUU UUI-
CUA CUG AUU AUC AUA AUG GUU GUC GUA GUG
UCU UCA UUU
ccc ccA ACU ACC ACA GCC GCA \l tr Lr
Thrrd-baserelationship
UAU UAC UAA UAG
UGU UGC UGA UGG
cAc
cGc
EAA CAG AAU AAC AAA AAG GAU GAA GAG Thirdbases w r t hs a m e meanrng
-'l_ U, c, A, c _._ tnirc oase - I irrelevant U, C,A differ A or G l_ Purines *+; I frompyrimidines U or C Unique G only
U\fU
vuu
AGU AGC AGA AGG uuu
GGC GGA uuu Codon NumL:er
32 3 14 10 2
: : , i r r f : :. ; . T h i r d b a s ehsa v e t h e t e a s t i n f l u e nccoedoonn meanings. Boxes groups indicate of codons withinwhich third-base degeneracy ensures that the meaning is the same.
792
CHAPTER 9 Usingthe GeneticCode
Exceptions to the universal genetic code are rare. Changesin meaning in the principal genome of a speciesusually concern the termination codons. For example, in a mycoplasma, UGA codes for tryptophan; in certain speciesof the ciliates TetrahymenaarrdParamecium,UAA and UAG code for glutamine. Systematic alterations of the code have occurred only in mitochondrial DNA (see Section 9.7, There Are SporadicAlterations of the UniversalCode).
Codon-Anticodon Recognition Involves Wobb Ling . Muttipte codons that represent the sameamino acidmostoftendifferat the third baseposition. o Thewobblein pairingbetween thefirstbaseof the anticodon andthethirdbaseof the codon resutts fromthe structure of the anticodon [oop.
The function of IRNA in protein synthesisis fulfilled when it recognizesthe codon in the ribosomal A site.The interaction between anticodon and codon takesplace by basepairing, but under rules that extend pairing beyond the usual G-C and A-U partnerships. We can deduce the rules governing the interaction from the sequencesof the anticodons that correspond to particular codons. The ability of any IRNA to respond to a given codon can be measured directly by the trinucleotide binding assayor by its use in an in vitro protein synthetic system. The genetic code itself yields some important clues about the processof codon recognition. The pattern of third-base degeneracy is drawn in Jli!.iiti i:..-:,which shows that in almost all caseseither the third base is irrelevant or a distinction is made only between purines and pyrimidines. There are eight codon families in which all four codons sharing the same first two bases have the same meaning, so that the third base has no role at all in specifying the amino acid. There are seven codon pairs in which the meaning is the same regardlessof which pyrimidine is present at the third position, and there are five codon pairs in which either purine may be present without changing the amino acid that is coded. There are only three casesin which a unique meaning is conferred by the presenceof a par-
ticular base at the third position: AUG (for methionine), UGG (for tryptophan), and UGA (termination). So C and U never have a unique meaning in the third position, and A never signifies a unique amino acid. The anticodon is complementary to the codon; thus it is the first base in the anticodon sequence written conventionally in the direction from 5' to )'that pairs with the third base in the codon sequencewritten by the same convention. So the combination Codon Anticodon
It is therefore possible to recognize unique codons only when the third basesare G or U. This option is not used often, as UGG and AUG are the only examples of the first tlpe and there is none of the secondtype. (G-U pairs are common in RNA duplex structures. When only three base pairs can be formed the formation of stable contacts between codon and anticodon is more constrained. though, and thus G-U pairs can contribute only in the last position of the codon.)
5'ACG3' 3'UGC5'
is usually written as codon ACG/anticodon CGU, where the anticodon sequencemust be read backward for comnlementaritv with the codon. To avoid confusion, we shall retain the usual convention in which all sequencesare written 5'-3' , buI indicate anticodon sequenceswith a backward arrow as a reminder of the relationship with the codon. Thus the codon/anticodon nair shown above will be written as ACG and CGU, respectively. Does each triptet codon demand its own IRNA with a complementary anticodon? Or can a single IRNA respond to both members of a codon pair and to all (or at least some) of the four members of a codon family? Often one IRNA can recognize more than one codon. This means that the base in the first position of the anticodon must be able to partner alternative basesin the corresponding third position of the codon. Basepairing at this position cannot be limited to the usual G-C and A-U partnerships. The rules governing the recognition patterns are summarized in the wobble hypothesis, which states that the pairing between codon and anticodon at the first two codon positions always follows the usual rules, but that exceptional "wobbles" occur at the third position. Wobbling occurs because the conformation of the IRNA anticodon loop permits flexibility at the first base of the anticodon. i;ri,:..iri: :. :. shows that G-U pairs can form in additionto the usualpairs. This single change createsa pattern of base pairing in which A can no longer have a unique meaning in the codon (becausethe U that recognizesit must also recognizeG). Similarly, C also no longer has a unique meaning ( b e c a u s et h e G t h a t r e c o g n i z e si t a l s o m u s t recognize U). i:{.1:rii ir,:-:summarizes the pattern of recognition.
Standardbase pairsoccurat all positions
'HtNrW
G-U wobblepairingoccursonly at third codon position H
in basepairingaltowsG-Upairsto i:ilii.l.rlii,.t-Wobbte formbetween thethirdbaseof thecodonandthefirstbase of the anticodon.
Base in first position oi anticodon U A
Base(s)recognizedin third positionof codon AorG G only U only CorU
pairinginvotves wobb[ing li{.i-iir;:ir.:r Codon-anticodon at thethirdposition.
WobbLing 1 9 3 Invotves Recognition 9.3 Codon-Anticodon
@
tRNAs AreProcessed fromLonger Precursors
o A mature IRNAis generated by processing a precursor. o The5'endis generated by cleavage by the endonuclease RNAase P. e The3'endis generated by cteavage followed by trimmingof the lastfewbases, fol.lowed by additionof the common terminaI trinucleotide sequence CCA.
tRNAs are commonly synthesizedas precursor chains with additional material at one or both .:11,_:!:i - i: Showsthat the extra sequences "1619. are removed by combinations of endonucleolytic and exonucleolytic activities. One feature that is common to all tRNAs is that the three nucleotides at the 3'terminus, always the triplet sequenceCCA, are not codedin the genome,but are added as part of IRNA processing.
IRNAprecusorhas extra5' and 3' sequences
The 5'end of IRNA is generatedby a cleavage action catalyzed by the enzyme ribonuclease P. The enzymes that process the 3'end are best characterized in E. coli,where an endonucleasetriggers the reaction by cleaving the precursor downstream, and several exonucleases then trim the end by degradation in tlne 3'-5' direction. The reaction also involves several enzymes in eukaryotes.It generatesa IRNA that needs the CCA trinucleotide sequence to be a d d e dt o t h e 3 ' e n d . The addition of CCA is the result solely of an enzymatic process, that is, the enzymatic activity carries the specificity for the sequence of the trinucleotide, which is not determined by a template. There are several models for the process, which may be different in different organisms. In some organisms, the processis catalyzed by a single enzyme. One model for its action proposes that a single enzyme binds to the l' end, and sequentially adds C, C, and A, the specificity at each stage being determined by the structure of the 3' end. Other models propose that the enzyme has different active sites for cytosine triphosphate (CTP)and adenosine rriphosphare (ATP). In other organisms, different enzymes are responsible for adding the C and A residues, and they function sequentially. When a IRNA is not properly processed.it attracts the attention of a quality control system that degradesit. This ensuresthat the protein synthesis apparatus does not become blocked by nonfunctional tRNAs.
IRNAContains Modified Bases
CCA is added 3'3'-end { addition u,! enzyme(s)
'".ti ThetRNA3'endis generated i'ir":l"ii:li bycuttingand trimmingfollowed by additionof CCA; the 5'endis genoreicd
794
hrr rrrl-finn
CHAPTER 9 UsingtheGenetic Code
r tRNAs contain>50modified bases. r Modification usua[[y invotvesdirectatterationof the primary bases in IRNA,buttherearesome exceptions in whicha baseis removed and reptaced by anotherbase.
Transfer RNA is unique among nucleic acids in its content of "unusual" bases.An unusual base is any purine or pyrimidine ring except the usual A, G, C, and U from which all RNAs are synrhesized.All other basesare produced by modification of one of the four basesafter it has been incorporated into the polyribonucleotide chain. All classesof RNA display some degree of modification, but in all casesexcept IRNA this
is confined to rather simple events,such as the addition of methyl groups. In IRNA, there is a vast range of modifications, ranging from simple methylation to wholesale restructuring of the purine ring. Modifications occur in all parts of the IRNA molecule. There are >50 different types of modified basesin IRNA. I r , shows some of the more com!r,,' ,irr mon modified bases.Modifications of pyrimidines (C and U) are lesscomplex than those of purines (A and G). In addition to the modifications of the basesthemselves,methylation at Ihe 2'-O position of the ribose ring also occurs. The most common modifications of uridine are straightforward. Methylation at position 5 createsribothymidine (T). The baseis the same commonly found in DNA, but here it is attached to ribose rather than deoxyribose.In RNA, thymine constitutes an unusual base that originates by modification of U. Dihydrouridine (D) is generated by the saturation of a double bond, which changes the ring structure. Pseudouridine (y) interchanges the positions of N and C atoms (see Figure 26.40). In 4-thiouridine, sulfur is substituted for oxygen. The nucleoside inosine is found normally in the cell as an intermediate in the purine biosynthetic pathway. It is not, however, incorporated directly into RNA. Instead, its existencedepends on modification of A to createI. Other modifications of A include the addition of comolex groups. TWo complex seriesof nucleotides depend on modification of G. The Q bases.such as queuosine, have an additional pentenyl ring added via an NH linkage to the methyl group of 7-methylguanosine.The pentenyl ring may carry various further groups.The Y bases,such as wyosine, have an additional ring fused with the purine ring itself. This extra ring carries a long carbon chain; again, to which further groups are added in different cases. The modification reaction usually involves the alteration of, or addition to, existing bases in the IRNA. An exception is the synthesisof Q bases,for which a special enzyme exchanges {ree queuosine with a guanosine residue in the IRNA. The reaction involves breaking and remaking bonds on either side of the nucleoside. The modified nucleosidesare synthesizedby specificIRNA-modifying enzymes. The original nucleosidepresent at eachposition can be determined either by comparing the sequence of IRNA with that of its gene or (less efficiently) by isolating precursor molecules that lack some
or all of the modifications. The sequencesof precursors show that different modifications are introduced at different stagesduring the maturation of IRNA. Some modifications are constant features of all IRNA molecules-for example, the D residues that give rise to the name of the D arm and the y found in the TyC sequence.On the 3' side of the anticodon there is always a modified purine, although the modification varies widely. Other modifications are specific for particular tRNAs or groups of tnNAs. For example, c f t R N A P h 'i n w y o s i n e b a s e sa r e c h a r a c t e r i s t i o There are also yeast, mammals. bacteria, and patterns. some species-specific The many tRNA-modifying enzymes (-60 in yeast) vary greatly in specificity. In some cases,a single enzyme acts to make a particular modification at a single position. In other cases,an enzyme can modify basesat several different target positions. Some enzymes undertake single reactions with individual tRNAs;
in IRNAcanbe modified. Attof the fourbases
Bases 1 9 5 Modified 9.5 IRNAContains
others have a range of substratemolecules. The features recognized by the IRNA-modifying enzymes are unknown, but probably involve recognition of structural features surrounding the site of modification. Some modifications require the successiveactions of more than one enzyme.
:j i.. Inosinecan pair with any of U, C. and A.
Onebondis notenough
2thiouracil pairsonly withA
9Hs
:rli-ri"iilt r'.:i Modification to 2-thiouridine restr.icts oairingto A atonebecause on[yoneH-bond canformwithG.
796
CHAPTER 9 U s i n gt h e G e n e t i cC o d e
ModifiedBases Affect Anticodon-Codon Pairing r Modifications in the anticodonaffectthe Dattern pairingandtherefore of wobbLe are'important in determining tRNAspecifi city.
The most direct effect of modification is seen in the anticodon, where change of sequenceinfluences the ability to pair with the codon, thus determining the meaning of the IRNA. Modifications elsewhere in the vicinity of the anticodon also influence its pairing. When basesin the anticodon are modified, further pairing patterns become possible in addition to those predicted by the regular and wobble pairing involving A, C, U, and G. it*:Ji{[ t.ii shows the use of inosine (I), which is often present at the first position of the anticodon. Inosine can pair with any one of three bases,U, C, and A. This ability is especially important in the isoleucine codons, where AUA codes for isoleucine, whereas AUG codesfor methionine. It is not possible with the usual basesto recognize A alone in the third position, and as a result any IRNA with U starting its anticodon would have to recognize AUG as well as AUA. Thus AUA must be read together with AUU and AUC, a problem that is solved by the existence of IRNA with I in the anticodon. Actually, some of the predicted regular combinations do not occur, becausesome basesare always modified. There seemsto be an absolute ban on the employment of A; usually it is converted to I. In most cases,U at the first position of the anticodon is converted to a modified form that has altered pairing properties. Some modifications create preferential readings of some codons with respect to others. Anticodons with uridine-5-oxyacetic acid and 5-methoxyuridine in the first position recognize A and G efficiently as third basesof the codon, but recognize U lessefficiently. Another casein which multiple pairings can occur-but with some pairings preferred to others-is provided by the series of queuosine and its derivatives. These modified G bases continue to recognize both C and U, but pair with U more readily. A restriction not allowed by the usual rules can be achieved by the employment of. 2thiouridine in the anticodon. F3{iiiHt:n.'}shows that its modification allows the baseto continue to pair with A, but prevents it from indulging in wobble pairing with G.
Theseand other pairing relationships make the general point that there are multiple ways to construct a set of tRNAs able to recognize all the sixty-one codons representing amino acids. No particular pattern predominates in any given organism, although the absence of a certain pathway for modification can prevent the use of some recognition patterns. Thus a particular codon family is read by tRNAs with different anticodons in different organisms. Often the tRNAs will have overlapping responses,so that a particular codon is read by more than one IRNA. In such casesthere may be differences in the efficiencies of the alternative recognition reactions. (As a general rule, codonsthat are commonly used tend to be more efficiently read.) In addition to the construction of a set of tRNAs able to recognize all the codons, there may be multiple tRNAs that respond to the same codons. The predictions of wobble pairing accord very well with the observedabilities of almost all tRNAs. There are, however, exceptions in which the codons recognized by a IRNA differ from those predicted by the wobble rules. Such effects probably result from the influence of neighboring basesand/or the conformation of the anticodon loop in the overall tertiary structure of the IRNA. Indeed, the importance of the structure of the anticodon loop is inherent in the idea of the wobble hypothesis itself. Further support for the influence of the surrounding structure is provided by the isolation of occasionalmutants in which a change in a base in some other region of the molecule alters the ability of the anticodon to r e c o g n i z ec o d o n s . Another unexpected pairing reaction is presented by the ability of the bacterial initiator, fMet-tRNAr, to recognizeboth AUG and GUG. This misbehavior involves the third base oI the anticodon.
E
The universality of the genetic code is striking, but some exceptions exist. They tend to affect the codons involved in initiation or termination and result from the production (or absence) of tRNAs representing certain codons. The changesfound in principal (bacterialor nuclear) g e n o m e sa r e s u m m a r i z e di n i I 1 r r " I' ' : ' Almost all of the changesthat allow a codon to represent an amino acid affect termination codons: . In the prokaryote Mycoplasmacapricolum, UGA is not used for termination, but instead codes for tryptophan (Trp). In fact, it is the predominant Ttp codon, and UGG is used only rarely. Two TrpIRNA speciesexist, which have the antic o d o n s U C A e ( r e a d sU G A a n d U G G ) a n d C C A e ( r e a d so n l y U G G ) . Some ciliates(unicellular protozoa) read UAA and UAG as glutamine instead of termination signals. Tetrahymenathermoahila,which is one of the ciliates,contains three 1P54clu species. One recognizesthe usual codons CAA and CAG for glutamine, a secondrecognizes both UAA and UAG (in accordancewith the wobble hypothesis), and the third recognizes only UAG. We assume that a further change is that the releasefactor eRF has a restricted specificity,compared with that of other eukaryotes. In another ciliate (Euplotesoctacarinatus), UGA codes for cysteine. OnlY UAA is used as a termination codon, and UAG
UGU^..^ ucc "v" Cys,Sel UGA STOP-+Trp, UGG Trp
ThereAreSporadic Alterations of the Code UniversaL geneticcodehave in the universal Changes in somesoecies. occurred in mitochondriaI These aremorecommon changes genomes, treecanbe wherea phytogenetic for the changes. constructed genomes, the changes aresporadic and In nuctear codons. usuatty affectontytermination
, nuclear oreukaryotic codein bacterial in thegenetic Changes a codonso or change genomes assignaminoacidsto stopcodons usual.[y fromone in meaning an aminoacid'A change ihat it no [ongerspecifies a m i n oa c i dt o a n o t h ei sr u n u s u a l .
Code of the Universal Alterations 9.7 ThereAreSporadic
797
gent uses of the termination codons could represent their "capture" for normal coding purposes.If some termination codons were used only rarely, they could be recruited to coding purposesby changesthat allowed tRNAs to recognize them. Exceptions to the universal genetic code also occur in the mitochondria from several Ser r-i constructsa phylogeny for species.ilr,iii,:ir,.,', = Met the changes.It suggeststhat there was a universal code that was changed at various points in mitochondrial evolution. The earliest change was the employment of UGA to code for tryp. : - i i:-::-:., ! Changes in the genetic codein mitochontophan, which is common to all (nonplant) driacanbetracedin phytogeny. Theminimum number of mitochondria. independent changes is generated by supposing thatthe AUA=Met andthe AAA=Asn changes eachoccurred indeSome of these changesmake the code simpendentty twice,andthattheearLy AUA=Met change was pler by replacing two codons that had different reversed in echinoderms. meanings with a pair that has a single meaning. Pairs treated like this include UGG and UGA (both ftp instead of one Trp and one terminais not found. The change in meaning of tion) and AUG and AUA (both Met insread of UGA might be accomplishedby a modone Met and the other Ile). ification in the anticodon of tRNAcvsto Why have changes been able to evolve in allow it to read UGA with the usual the mitochondrial code? The mitochondrion codons UGU and UGC. synthesizesonly a small number of proteins . The only substitution in coding for (-I0), and as a result the problem of disruption amino acids occurs in a yeast (Candida), by changes in meaning is much less severe. It where CUG means serine instead of is likely that the codons that are altered were not leucine (and UAG is used as a sense used extensively in locations where amino acid codon). substitutions would have been deleterious. The Acquisition of a coding function by a termivariety of changesfound in mitochondria of difnation codon requires two types of change: a ferent speciessuggeststhat they have evolved IRNA must be mutated so as to recognizethe separately rather than by common descent from codon, and the class I releasefactor must be an ancestral mitochondrial code. mutated so that it does not terminate at this According to the wobble hypothesis, a mincodon. imum of 3I tRNAs (excluding the initiator) are The other common type of change is loss required to recognize all sixty-one codons (at of the IRNA that responds to a codon, so that least two tRNAs are required for each codon the codon no longer specifiesany amino acid. family and one IRNA is needed per codon pair What happens at such a codon will depend on or singlecodon). An unusual situation existsin whether the termination factor evolvesto rec(at least) mammalian mitochondria, however, ognize it. in which there are only twenty-two different All of these changes are sporadic, which is tRNAs. How does this limited set of tRNAs to say that they appear to have occurred indeaccommodate all the codons? pendently in specificlines of evolution. They The critical feature Iies in a simplification may be concentrated on termination codons, of codon-anticodon pairing, in which one IRNA becausethese changesdo not involve substiturecognizesall four members of a codon family. tion of one amino acid for another. Once the This reduces to twenty-three the minimum genetic code was established,early in evolunumber of tRNAs required to respond to all tion, any general change in the meaning of a usual codons.The use of AGA6 for termination codon would causea substitution in all the proreduces the requirement by one further IRNA, teins that contain that amino acid.It seemslikely to twenty-two. that the change would be deleterious in at least In all eight codon families, the sequence of some of these proteins, with the result that it the IRNA contains an unmodified U at the first would be strongly selected against. The diverposition of the anticodon. The remaining codons
198
CHAPTER 9 UsingtheGenetic Code
are grouped into pairs in which all the codons ending in pyrimidines are read by G in the anticodon, and all the codons ending in purines are read by a modified U in the anticodon, as predicted by the wobble hypothesis. The complication of the single UGG codon is avoided by the change in the code to read UGA with UGG as tryptophan. In mammals, AUA ceasesto represent isoleucine and instead is read with AUG as methionine. This allows all the nonfamily codons to be read as fourteen pairs. The twenty-two identified IRNA genes therefore code for fourteen tRNAs representing pairs and eight tRNAs representing families. This leaves the two usual termination codons UAG and UAA unrecognized by IRNA, together with the codon pair AGA6,.Similar rules are followed in the mitochondria of fungi.
NovelAminoAcidsCan BeInserted at Certain StopCodons r Changes in the reading codons can of specific genes. occurin individuaI . Theinsertion of seteno-Cys-tRNA at certainUGA proteins codons requires severaI to modifothe andinsertit intothe ribosome. Cys-tRNA . Pyrrotysine canbeinserted at certainUAGcodons.
Specific changes in reading the code occur in individual genes.The specificityof such changes implies that the reading of the particular codon must be influenced by the surrounding bases. In two cases,amino acids other than the classical twenty are inserted by special aminoacyltRNAs. A striking example is the incorporation of the modified amino acid seleno-cysteineat certain UGA codons within the genesthat code lor selenoproteinsin both prokaryotes and eukaryotes. Usually these proteins catalyze oxidationreduction reactions. and contain a single seleno-cysteineresidue, which forms part of the active site. The most is known about the use of the UGA codons in three E. coli genes coding for formate dehydrogenase isozymes. The internal UGA codon is read by a selenoCys-IRNA. This unusual reaction is determined by the local secondary structure of mRNA. in particular by the presence of a hairpin loop downstream of the UGA.
Mutations in 4 sel genescreate a deficiency in selenoprotein synthesis. sslCcodes for tRNA (with the anticodon ACU<-) that is charged with serine. selA and selD are required to modi f y t h e s e r i n e t o s e l e n o - c y s t e i n e .S e l B i s a n alternative elongation factor. It is a guanine nucleotide-binding protein that acts as a specific translation factor for entry of seleno-CysIRNA into the A site; it thus provides (for this single IRNA) a replacement for factor EF-T!t. The sequenceof SelB is related to both EF-Tu and IF-2. Why is seleno-Cys-IRNA inserted only at certain UGA codons? These codons are folIowed by a stem-loop structure in the mRNA. i : : i i , , i r jt.: .r , . s h o w s t h a t t h e s t e m o f t h i s s t r u c t u r e i s r e c o g n i z e db y a n a d d i t i o n a l d o m a i n in SelB (one that is not present in EF-Ttr or IF-2). A similar mechanism interprets some UGA codons in mammalian cells, except that two proteins are required to identify the appropriate UGA codons. One protein (SBP2) binds a stem-loop structure far downstream from the UGA codon, whereas the counterp a r t o f S e l B ( c a l l e dS E C I S )b i n d s t o S B P 2a n d simultaneously binds the IRNA to the UGA codon. Another example of the insertion of a special amino acid is the placement of pyrrolysine at an UAG codon. This happens in both an archaea and a bacterium. The mechanism is probably similar to the insefiion of seleno-cysteine. An unusual IRNA is charged with lysine, which is presumably then modified. The IRNA has a CUA anticodon, which respondsto UAG. There must be other components of the system that restricts its response to the appropriate UAG codons.
iri'i.iiririi i lrjSe[Bis an elongation factorthat specifito a UGAcodonthat is foLbindsSeteno-Cys-tRNA caLl.y in mRNA' structure lowedby a stem-toop
StopCodons 799 9.8 NovelAminoAcidsCanBeInsertedat Certain
@
tRNAs AreCharged withAminoAcids by Synthetases
. Aminoacyt-tRNA synthetases areenzymes that charge IRNAwithan aminoacidto generate aminoacyt-tRNA in a two-stage reaction that uses energyfromATP. . TherearetwentyaminoacyltRNA synthetases in eachcett.Eachcharges all thetRNAs that represent a particutar aminoacid. r Recognition of a IRNAis basedon a smatlnumber of pointsof contactin theIRNAsequence.
It is necessary for tRNAs to have certain characteristics in common, yet be distinguished by others. The crucial feature that confers this capacity is the ability of rRNA to fold into a specific tertiary structure . Changes in the details
ATP site
Synthetasehas 3 bindingsites
A n a m i n o a c y [ - t R NsAy n t h e t a s ec h a r g e s IRNAwith an am'inoacid.
200
CHAPTER 9 UsingtheGenetic Code
of this structure, such as the angle of the two arms of rhe "L" or the protrusion of individual bases,may distinguish the individual tRNAs. All tRNAs can fit in the P and A sites of the ribosome. At one end they are associatedwith nRNA via codon-anticodon pairing, and at the other end the polypeptide is being transferred. Similarly, all tRNAs (except the initiator) share the ability to be recognized by the translation factors (EF-Tiror eEFI ) for binding to the ribosome. The initiator IRNA is recognized instead by IF-2 or eIF2. Thus the IRNA set must possesscommon features for interaction with elongation factors, but the initiator IRNA can be distinguished. Amino acids enter the protein synthesis pathway through the aminoacyl-IRNA synthetases, which provide the interface for connection with nucleic acid. All synthetases function by the two-step mechanism depicted in i:li,."iii:r:i i.:;: o First, the amino acid reacts with ATP to form aminoacyl-adenylate, releasing pyrophosphate. Energy for the reaction is provided by cleaving the high energy bond of the ATP. . Then the activated amino acid is transferred to the IRNA, releasing AMP. The synthetasessort the tRNAs and amino acids into corresponding sets.Each synthetase recognizes a single amino acid and all the tRNAs that should be charged with it. Usually, each amino acid is represented by more than one IRNA. SeveraltRNAs may be needed to respond to synonym codons, and sometimes there are multiple speciesof IRNA reacting with the same codon. Multiple tRNAs representing the same amino acid are called isoaccepting tRNAs; because they are all recognized by the same synthetase, they are also described as its cognate tRNAs. Many attempts to deduce similarities in sequencebetween cognate tRNAs, or to induce chemical alterations that affect their charging, have shown that the basisfor recognition is not the same for different tRNAs, and does not necessarily lie in some feature of primary or secondary structure alone. We know from the crystal structure that the acceptor stem and the anticodon stem make tight contacts with the synthetase, and mutations that alter recognition of a IRNA are found in these two regions. (The anticodon itself is not necessarilyrecognized as such; for example, the "suppressor" mutations discussedlater in this chapter change a base in the anticodon, and therefore the codons to
which a IRNA responds, without altering its charging with amino acids.) A g r o u p o f i s o a c c e p t i n gt R N A s m u s t b e charged only by the single aminoacyl-tRNA synthetase specificfor their amino acid. So isoaccepting tRNAs must share some common feature(s) enabling the enzyme to distinguish them from the other tRNAs. The entire complement of tRNAs is divided into twenty isoaccepting groups, and each group is able to identify itself to its particular synthetase. tRNAs are identified by their synthetases by contacts that recognize a small number of bases,typically from one to five. Three types of features commonly are used: . Usually (but not always), at least one base of the anticodon is recognized. Sometimesall the positions of the anticodon are important. . Often one of the last three basepairs in the acceptor stem is recognized.An extreme caseis represented by alanine IRNA, which is identified by a single unique basepair in the acceptorstem. . The so-called discriminator base,which Iiesbetween the acceptorstem and the CCA terminus, is always invariant among isoacceptortRNAs. No one of these features constitutes a unique means of distinguishing twenty sets of tRNAs, or provides sufficient specificity, so it appears that recognition of tRNAs is idiosyncratic, with each following its own rules. Several synthetasescan specifically charge a "minihelix," which consistsonly of the acceptor and Try C arms (equivalent to one arm of the L-shaped molecule) with the correct amino acid. For certain tRNAs, specificity depends exclusively upon the acceptor stem. However, it is clear that there are significant variations between tRNAs, and in some casesthe anticodon region is important. Mutations in the anticodon can affect recognition by the classII Phe-IRNA synthetase. Multiple features may be involved; minihelices from the tRNAvuland tRNAMet(where we know that the anticodon is important in vivol can react specifically with their synthetases. Thus recognition depends on an interaction between a few points of contact in the IRNA, concentrated at the extremities, and a few amino acids constituting the active site in the protein. The relative importance of the roles played by the acceptor stem and anticodon is different for each IRNA-synthetase interaction.
Aminoacy[-tRNA Fa[[ Synthetases into TwoGroups r AminoacyltRNA intothe aredivided synthetases and classI andclassII groupsby sequence structuraI similarities.
In spite of their common function, synthetases are a rather diverse group of proteins. The individual subunits vary from 40 to 110 kD, and the enzymes may be monomeric, dimeric, or tetrameric. Homologies between them are rare. Of course, the active site that recognizes IRNA comprises a rather small part of the molecule. It is interesting to compare the active sites of different synthetases. Synthetases have been divided into two general groups, each containing ten enzymes, on the basis of the structure of the domain that contains the active site.A generaltype of organization that applies to both groups is repre'l s e n t e d i n r : i , r ' ' : ' :L i i ; . T h e c a t a l y t i c d o m a i n includes the binding sites for ATP and amino acid. It can be recognized as a large region that is interrupted by an insertion of the domain that binds the acceptor helix of the IRNA. This placesthe terminus of the IRNA in proximity to the catalytic site. A separate domain binds the anticodon region of IRNA. Those synthetases that are multimeric also possessan oligomerization domain. ClassI synthetaseshave an N-terminal catalytic domain that is identified by the presence o f t w o s h o r t , p a r t l y c o n s e r v e d s e q u e n c e so f amino acids, sometimes called "signature
contains i.i,ir,.,iiiri: synthetase il .I,ir An aminoacyt-tRNA (0ntymutfunctjons. withdifferent threeor fourregions domain.) possess an otigomerization timericsynthetases
Fa[[into TwoGroups 207 Synthetases 9.10 Aminoacyt-tRNA
allel B-sheet surrounded by c-helices. Again, the acceptor helix-binding domain that interrupts the catalytic domain has a structure that depends on the individual enzyme. The anticodon-binding domain tends to be N-terminal. The location of any oligomerization domain is widely variable. The lack of any apparent relationship between the two groups of synthetasesis a puzzle. Perhapsthey evolved independently of one another. This makes it seem possible even that an early form of life could have existed with proteins that were made up of just the ten amino acids coded by one type or the other. ,r,::-:i:,r-- , ,- CrystaI A general model for synthetase-IRNAbindstructures showthat classI andctassII aminoacV[tRNAsynthetases bindtheopposite facesoftheirIRNA ing suggeststhat the protein binds the IRNA substrates. TheIRNA js shown in redandtheproteinin b[ue.Photocourtesy of DinoMoras, Instialong the "side" of the L-shapedmolecule. The tute of Genetics andMotecu[ar andCe[[utar Biotogy. same general principle applies for all synthetaseIRNA binding: The IRNA is bound principally at its two extremities, and most of the IRNA sequence is not involved in recognition by a synthetase.However, the detailed nature of the interaction is different between classI and class II enzymes, as can be seen from the models of Acceptorstem lies in rli;iii:{ *.:.:i, which are based on crystal strucpocket protein deep in tures. The two types of enzyme approach the IRNA from opposite sides,with the result that 1' ott bindsnear the tRNA-protein models look almost like miracceptorstem ror images of one another. A classI enzyme (Gln-tRNA synthetase) approachesthe D-loop side of the IRNA. Ir recognizes the minor groove of the acceptor stem Anticodonlooo is distorted at one end of the binding site, and interacts with at U35-U36 the anticodon loop at the other end. ***' iFiil-ir.: n is a diagrammatic representation of the crystal structure of the tRNAcl"-synthetase complex. A Gln-tRNAsynthetase revealing feature of the structure is that conili.,r:iir::,,l ::, A classI IRNAsynthetase contactsIRNA tacts with the enzyme change the structure of a t t h e m i n o rg r o o v eo f t h e a c c e p t osrt e ma n da t t h e the IRNA at two important points. These can anticodon. be seen by comparing the dotted and solid lines sequences."The catalytic domain takes the form in the anticodon loop and acceptor stem: . B a s e sU 3 5 a n d U 3 6 i n t h e a n t i c o d o n of a motif called a nucleotide-binding fold (which is also found in other classesof enzymes that loop are pulled farther out of the IRNA bind nucleotides).The nucleotide fold consists into the protein. . The end of the acceptor stem is seriously of alternating parallel p-strands and cr-helices; the signature sequence forms part of the ATPdistorted, with the result that basepairbinding site. The insertion that contacts the ing between Ul and A72 is disrupred. acceptor helix of IRNA differs widely between T h e s i n g l e - s t r a n d e de n d o f t h e s t e m different class I enzymes. The C-terminal pokes into a deep pocket in the syndomains of the classI synthetases,which include thetaseprotein, which also contains the the IRNA anticodon-binding domain and any binding site for ATP. oligomerization domain, also are quite different This structure explains why changes in U35, from one another. G73, or the Ul-A72 basepair affect the recogClassII enzymes share three rather general nition of the IRNA by its synthetase. Ar all of similarities of sequence in their catalytic these positions. hydrogen bonding occurs domains. The active site contains a large antiparbetween the protein and IRNA.
Pair
7i';LiSrbase
202
C H A P T E9RU s i n gt h e G e n e t i cC o d e
A classII enzyme (Asp-tRNA synthetase) approachesthe IRNA from the other side;it recognizes both the variable loop and the major groove of the acceptor stem, as drawn in , lr i : : l i i r ,; ,l : . : , .T h e a c c e p t o rs t e m r e m a i n s i n i t s regular helical conformation. ATP is probably bound near to the terminal adenine. At the other end of the binding site, there is a tight contact with the anticodon loop, which has a change in conformation that allows the anticodon to be in close contact with the nrotein.
Synthetases Use Proofreading to Improve Accuracy o Specificity of recognition of bothaminoacidand IRNAis controtled by aminoacy[-tRNA synthetases by proofreading reactions that reverse the reaction if the wrongcomponent catatytic has beenincorporated.
Aminoacyl-IRNA synthetaseshave a difficult job. Each synthetasemust distinguish one out of twenty amino acids, and must differentiate cognate tRNAs (typically one to three) from the total set (perhaps 100 in all). Many amino acidsare closely related to one another, and all amino acids are related to the metabolic intermediates in their particular synthetic pathway. It is especially difficult to distinguish between two amino acids that differ only in the length ol the carbon backbone (that is, by one CH2group). Intrinsic discrimination based on relative energies of binding two such amino acidswould be only - I / 5. The synthetase enzymes improve this ratio -1000-fold. Intrinsic discrimination between tRNAs is better, becausethe IRNA offers a larger surface with which to make more contacts. It is still true, however, that all tRNAs conform to the same generalstructure, and there may be a quite limited set of featuresthat distinguish the cognate tRNAs from the noncognate tRNAs. We can imagine two general ways in which the enzyme might selectits substrate: . The cycle of admittance, scrutiny, and rejection/acceptancecould representa single binding step that precedesall other stagesof whatever reaction is involved. This is tantamount to saying that the affinity of the binding site is sufficient to control the entry of substrate. In the case of synthetases,this would mean
synthetase conr :, ,, I , ' A ctass II aminoacyltRNA hetixand of theacceptor tactsIRNAat the majorgroove looo. at the anticodon
that only the correct amino acids and cognate tRNAs could form a stable attachment at the site. . Alternatively, the reaction proceeds through some of its stages,after which a decision is reached on whether the correct speciesis present.If it is not present, the reaction is reversed, or a blpass route is taken, and the wrong member is expelled. This sort of postbinding scrutiny is generally describedas proofreading. In the example of synthetases, it would require that the charging reaction proceeds through certain stages even if the wrong IRNA or amino acid is present. Synthetasesuse proofreadingmechanisms to control the recognition of both types of substrates.They improve significantly on the intrinsic differences among amino acids or among tRNAs, but, consistent with the intrinsic differences in each group, make more mistakes in selecting amino acids (error rates are l0-4 to l0-5) than in selecting tRNAs (for which error rates are -10-6) (seeFigure 8.8). TransferRNA binds to synthetase by the two-stage reaction ':. CognatetRNAs have a depicted in greater intrinsic affinity for the binding site, so they are bound more rapidly and dissociate more slowly. Following binding, the enzyme scrutinizes the IRNA that has been bound. If the correct IRNA is present, binding is stabilized by a conformational change in the enzyme. This allows aminoacylation to occur rapidly. If the
to ImproveAccuracy 203 UseProofreading 9.11 Synthetases
t'
Adenylation
$"
Noncognate tRNA associates slowly dissociates rapidly
CognatetRNA associates rapidly dissociatesslowly
Aminoacyl-adenylate is hydrolyzed Wrong acid
,
tRNA charging
CognateIRNA triggerschange in conformation Aminoacylation withoutchange in conformation occursslowly
I
.rNHz RCH cxa)co
rf
AMP
IIS$R[ *.tr€ Recognition of the correctIRNAby synthetaseiscontrotted at two steps.First,the enzyme has a greater affinityforits cognate IRNA.Second, theaminoacylationof theincorrect IRNAis verystow.
wrong IRNA is present, the conformational change does not occur. As a result, the reaction proceeds much more slowly; this increasesthe chance that the IRNA will dissociate from the enzyme before it is charged. This type of control is called kinetic proofreading. Specificity for amino acids varies among the synthetases.Some are highly specific for initially binding a single amino acid, whereas others can also activate amino acids closely related to the proper substrate.The analog amino acid can sometimes be converted to the adenylate form, but in none of these casesis an incorrectly activated amino acid actually used to form a stable aminoacyl-IRNA. The presence of the cognate IRNA usually is needed to trigger proofreading, even if the reaction occurs at the stagebefore formation of aminoacyl-adenylate. (An exception is provided by Met-tRNA synthetase, which can reject CHAPTER 9 Usingthe GeneticCode
Aminoacyl-tRNA is hydrolyzed Wrong
Aminoacylation occursrapidly
204
&4nup n 3H
minoacid ao >q* +Xo
a
Correct amrnoacro
, NHz RCH
)co
A M P ftf Aminoacvl-tRNA
\) '
f,:*L€itt*.tG Whena synthetase bindsthe incorrect aminoacid,proofreading requires bindingofthecognate tRNA.It maytakeptaceeitherbya conformation change that causes hydrotysis of theincorrect aminoacyladenylateor by transferof the aminoacidto IRNA.followed by hydrotysis.
noncognate aminoacyl-adenylate complexes even in the absence of IRNA.) There are two stages at which proofreading of an incorrect aminoacyl-adenylate may occur during formation of aminoacyl-IRNA. F]"S{JRfl S"i* shows that both use chemical proofreading, in which the catalytic reaction is reversed. The extent to which one pathway or the other predominates varies with the individual synthetase: . The noncognate aminoacyl-adenylate may be hydrolyzed when the cognate IRNA binds. This mechanism is used predominantly by several synthetases, including those for methionine, isoleucine, and valine. (Usually, the reaction cannot be seen invivo, but it can be followed for Met-tRNA synthetase when the incorrectly activated amino acid is homocysteine, which lacks the methyl group of methionine). Proofreading releasesthe amino acid in an altered form, as homocysteine thiolactone. In fact, homocysteine thiolactone is pro-
duced in E. coli as a by-product of the charging reaction of Met-IRNA synthetase.This shows that continuous proofreading is part of the process of charging a IRNA with its amino acid. . Some synthetasesuse chemical proofreading at a later stage.The wrong amino acid is actually transferred to IRNA, is then recognizedas incorrect by its structure in the IRNA binding site, and so is hydrolyzed and released.The process requires a continual cycle of linkage and hydrolysis until the correct amino acid is transferred to the IRNA. A classicexample in which discrimination between amino acids depends on the presence of IRNA is provided by the Ile-IRNA synthetase of.E. coli. The enzyme can charge valine with AMP, but hydrolyzes the valyl-adenylate when ,ql.{4rleis added. The overall error rate depends on the specificitiesof the individual steps, as summarized in i::i.i;c,i.$.lr t;i.:i!.i.The overall error rate of I .5 x I 0-5 is lessthan the measuredrate at which valine is substituted for isoleucine (in rabbit globin), which rangesfrom 2 to 5 x I04. So mischarging probably provides only a small fraction of the errors that actually occur in protein synthesis. Ile-IRNA synthetaseusessizeas a basisfor discrimination among amino acids. i:ti.ijl:i,ri-i:,:.i shows that it has two active sites:the synthetic (or activation) site and the editing (or hydrolytic) site. The crystal structure of the enzyme shows that the synthetic site is too small to allow leucine (a closeanalog of isoleucine) to enter. All amino acids larger than isoleucine are excluded from activation because they cannot enter the synthetic site. An amino acid that can enter the synthetic site is placed on IRNA. Then the enzyme tries to transfer it to the editing site. Isoleucine is safefrom editing becauseit is too large to enter the editing site. However, valine can enter this site, and as a result an incorrect Val-tRNAIle is hydrolyzed. Essentially the enzyme provides a double molecular sieve, in which size of the amino acid is used to discriminate between closely related species. One interesting feature of Ile-IRNA synthetase is that the synthetic and editing sitesare a considerable distance apafi, -34 A. A crystal structure of the enzyme complexed with an edited analog of isoleucineshows that the amino acid is transported from the synthetic site to the editing site. i';r,;i-:qi;;.i.: shows thatthis involves a change in the conformation of the IRNA. The amino acid acceptor stem of tRNAIl" can exist in alternative conformations. It adoots an unusual
Frequencyof Error
Step Activationof valine to Val-AMPrre Releaseof Val-tRNA Overallrate of error
11225 11270 11225x 11270-- 1/60,000
!:it;i.ri':i:.: r:ii Theaccuracyof charging tRNAIte by'its synthetasedependson errorcontrolat two stages.
hastwo activesites. irir:-i+i:ri,' r l Ite-tRNAsynthetase because Aminoacidslarger thanIle cannotbeactivated site.Aminoacidssmalter theydo notfit in thesynthetic thevareabteto enterthe because thanI[e areremoved editinqsite.
fromthe synthetic iii iiiii: r,liii: An aminoacidjs transported in the bya change synthetase siteto theeditingsiteof Ite-tRNA stemof IRNA. of the aminoacceptor conformation
Accuracy to Improve UseProofreading 9.11 Synthetases
hairpin in order to be aminoacylated by an amino acid in the synthetic site. Then it returns to the more common helical structure in order to move the amino acid to the editing site. The translocationbetween sitesis the rate-limiting step in proofreading. Ile-tRNA synthetaseis a classI synthetase,but the double sieve mechanism is used also by classII synthetases.
Wild type: UUG codon is read by Leu-tRNA
AUG
XX.O
UAA
D o
or\
I v Leu
@
Suppressor tRNAsHave Mutated Anticodons ThatReadNewCodons
o A suppressor IRNAtypicatty hasa mutation in the anticodon that changes the codons to whichit respon0s. . Whenthe newanticodon corresoonds to a termination codon,an aminoacidis inserted and the potypeptide chainis extended beyond the termination codon.Thisresutts in nonsense suppression at a siteof nonsense mutation, or in readthrough at a naturaI termination codon. . Missense suppression occurs whentheIRNA recognizes a differentcodonfromusuat. sothat oneaminoacidis substituted for another.
Isolation of mutant tRNAs has been one of the most potent tools for analyzing the ability of a IRNA to respond to its codon(s) in nRNA, and for determining the effects that different parts of the IRNA molecule have on codon-anticodon recognition. Mutant tRNAs are isolatedby virtue of their ability to overcome the effects of mutations in genes coding for proteins. In general genetic terminology, a mutation that is able to overcome the effects of another mutation is called a suppressor. In IRNA suppressor systems, the primary mutation changes a codon in an mRNA so that the protein product is no longer functional. The secondary suppressor mutation changes the anticodon of a IRNA, so that it recognizes the mutant codon instead of (or as well as) its original target codon. The amino acid that is now inserted restoresprotein function. The suppressors are described as nonsense suppressors or missense suppressors, depending on the nature of the original mutation. In a wild-type cell, a nonsensemutation is recognized only by a release factor, which terminates protein synthesis.The suppressormutation creates an aminoacyl-tRNA that can recognize the termination codon; by inserting an amino acid, it allows protein synthesis to
206
C H A P T E9RU s i n gt h e G e n e t i cC o d e
Nonsensemutant:UAG codonterminates
@ffii:l'"
I
Suppressor mutation: changes Tyr-tRNA anticodon
AUG
Xog
UAA
i i1:rii:ri-j , rr Nonsense mutations canbesuppressed by a IRNAwitha mutantanticodon, whichinsertsanamino acidat themutantcodon,producing a fu[[lengthprotein in whichtheoriginal Leuresidue hasbeenreptaced byTyr.
continue beyond the site of nonsensemutation. This new capacity of the translation system allows a full-length protein to be synthesized, as illustrated in i:iriiiii r:.i:i'r.If the amino acid inserted by suppression is different from the amino acid that was originally present at this site in the wild-type protein, the activity of the protein may be altered. Missensemutations change a codon representing one amino acid into a codon representing another amino acid-one that cannot function in the protein in place of the original residue. (Formally, any substitution of amino acids constitutes a missensemutation, but in practice it is detectedonly if it changesthe activity of the protein.) The mutation can be suppressed by the insertion either of the original amino acid or of some other amino acid that is a c c e p t a b l et o t h e p r o t e i n .
'j":,i'ri i,ii:,i,lilil demonstratesthat missensesuppression can be accomplished in the same way as nonsense suppression,by mutating the anticodon of a IRNA carrying an acceptableamino acid so that it responds to the mutant codon. So missense suppression involves a change in the meaning of the codon from one amino acid to another.
ThereAreNonsense
Suppressors for Each Termination Codon Eachtypeof nonsense codonis suppressed by tRNAs with mutantanticodons. Someraresuppressor tRNAs havemutations in otherpartsof the motecule.
Nonsensesuppressorsfall into three classes,one i.l-lr: Ior each type of termination codon. l']iii"il1{: describesthe properties of some of the best characterizedsuppressors. The easiestto characterize]i^ave been amber suppressors.In E. coli, at least six tRNAs have been mutated to recognizeUAG codons.AII of the amber suppressortRNAs have the anticodon CUAe, in each case derived from wild type by a single base change. The site of mutation can be any one of the three basesof the anticodon, as seen from supD, supE, and srzpf Each suppressor IRNA recognizes only the UAG codon, instead of its former codon(s).The amino acids inserted are serine, glutamine, or tyrosine, the same as those carried by the correspondingwildtype tRNAs. Ochre suppressorsalso arise by mutations in the anticodon. The best known aresupC and supG,which insert tyrosine or lysine in response to both ochre (UAA) and amber (UAG) codons. This conforms with the prediction of the wobble hypothesis that UAA cannot be recognized alone. A UGA suppressorhas an unexpected property. It is derived from tRNArrp, but its only mutation is the substitution of A in place of G at position 24. This change replacesa G-U pair in the D stem with an A-U pair, increasing the stability of the helix. The sequence of the anticodon remains the same as the wild type, CCAe. So the mutation in the D stem must in some way alter the conformation of the anticodon loop, allowing CCAe to pair with UGA in an unusual wobble pairing of C with A. The
whentheantioccurs 611-i:r,:1.: suppression il.:.l.iMissense to thewrong sothatit responds codonof tRNAis mutated boththe is onLypartialbecause codon.Thesuppression to IRNAcanrespond witd-type IRNAandthe suppressor AGA.
Locus
IRNA WildType
SuPPressor
Codon/Anti Anti/Codon supD(su1) Ser UCG CGA CUA UAG supE(su2) Gln CAG CUG CUA UAG supF(su3) Tyr UAg cUA
cUA UAG
s u p C ( s u 4 )T y r U A g G U A U U A U A A supG(sus) Lys AAA UUU UUA UAA supU(su7) Trp UGG CCA UCA UGA tRNAs aregenerated suppressor l:+ijiti. r,i,r.riNonsense in the anticodon. by mutations
Codon for EachTermination Suppressors 9.13 ThereAreNonsense
207
suppressorIRNA continues to recognizeits usual codon, UGG. A related responseis seenwith a eukaryotic IRNA. Bovine liver contains a tRNAserwith the anticodon -CCA'. The wobble rules predict that this IRNA should respond to the tryptophan codon UGG, but in fact it responds to the termination codon UGA. So it is possiblethat UGA is suppressednaturally in this situation. The general importance of these observations lies in the demonstration that codon-anticodon recognition of either wild-type or mutant IRNA cannot be predicted entirely from the relevant triplet sequences,but is influenced by other features of the molecule.
@
Suppressors MayCompete with Wil.d-Type Reading of the Code
Suppressor tRNAs compete withwitd-type tRNAs that havethe sameanticodon to readthe corresponding codon(s). Efficient suppression is deteterious because it pastnormaI resutts in readthrough termination co00ns. TheUGAcodonis leakyandis misread by Trp-tRNA at 1%to 3olo frequency.
Wild-typetranslation AUG
c
UAG
Releasefactorterminates synthesisat stop codon
I
AUG SuppressortRNA reads UAG codon and proteinis extended to next stop codon
UAG AUC
/'f
ry.. I Tyr
riiliiiri; i,i.iili Nonsensesuppressors atso readthrough
svnthesi zinsprotei nsthat :*ili!::'Jl'#li'l il|3.''' 208
CHAPTER 9 UsinqtheGenetic Code
There is an interesting difference between the usual recognition of a codon by its proper aminoacyl-IRNA and the situation in which mutation allows a suppressor IRNA to recognize a new codon. In the wild-type cell, only one meaning can be attributed to a given codon, which representseither a particular amino acid or a signal for termination. In a cell carrying a suppressor mutation, though, the mutant codon has the alternatives of being recognized by the suppressorIRNA or of being read with its usual meanrng. A nonsense suppressorIRNA must compete with the releasefactors that recognize the termination codon(s). A missensesuppressor IRNA must compete with the tRNAs that respond properly to its new codon. The extent of competition influences the efficiency of suppression; thus the effectiveness of a particular suppressor depends not only on the affinity between its anticodon and the target codon, but also on its concentration in the cell, and on the parameters governing the competing terminat i o n o r i n s e r t i o nr e a c t i o n s . The efficiency with which any particular codon is read is influenced by its location. Thus the extent of nonsense suppression by a given IRNA can vary quite widely, depending on the context of the codon. We do not understand the effect that neighboring basesin nRNA have on codon-anticodon recognition, but the context can change the frequency with which a codon is recognized by a particular IRNA by more than an order of magnitude. The baseon the 3'side of a codon appearsto have a particularly strong effect. A nonsensesuppressoris isolatedby its ability to respond to a mutant nonsense codon. The same triplet sequence,however, constitutesone of the normal termination signals of the cell! The mutant IRNA that suppressesthe nonsense mutation must in principle be able to suppress natural termination at the end of any gene that u s e s t h i s c o d o n . i r i i i J f r i :5 . : + s h o w s t h a t t h i s readthrough results in the synthesis of a longer protein, with additional C-terminal material. The extended protein will end ar the next termination triplet sequence found in the phase of the reading frame. Any extensive suppression of termination is likely to be deleterious to the cell by producing extended proteins whose functions are thereby altered. Amber suppressorstend to be relatively efficient, usually in the range of l0o/o to 50oh, depending on the system.This efficiency is possible because amber codons are used relatively
infrequently to terminate protein synthesis in E, coli. Ochre suppressors are difficult to isolate. They are always much less efficient, usually with activitiesbelow I0%. AII ochre suppressors grow rather poorly, which indicates that suppressionof both UAA and UAG is damaging Io E. clli, probably because the ochre codon is used most frequently as a natural termination signal. UGA is the least efficient of the rermination codons in its natural function; it is misread by ftp-tRNA as frequently as I 7o to 3 % in wildtype situations. In spite of this deficiency, however, it is used more commonly than the amber triplet to terminate bacterial genes. One gene's missensesuppressoris likely to be another gene's mutator. A suppressorcorrects a mutation by substituting one amino acid for another at the mutant site. In other locations, though, the same substitution will replace the wild-type amino acid with a new amino acid. The change may inhibit normal protein function. This poses a dilemma for the cell: it must suppress what is a mutant codon at one location while failing to change too extensively its normal meaning at other locations.The absence of any strong missensesuppressorsis therefore explained by the damaging effects that would be caused by a general and efficient substitution of amino acids. A mutation that createsa suppressorIRNA can have two consequences.First, it ailows the IRNA to recognize a new codon. Second, it sometimes prevents the IRNA from recognizing the codons to which it previously responded. It is significant that all the high-efficiency amber suppressorsare derived by mutation of one copy of a redundant IRNA set. In these cases,the cell has several tRNAs able to respond to the codon originally recognized by the wild-type IRNA. Thus the mutation does not abolish recognition of the old codons, which continue to be served adequately by the tRNAs of the set. In the unusual situation in which there is only a single IRNA that responds to a particular codon, any mutation that prevents the response is lethal. Suppressionis most often consideredin the context of a mutation that changes the reading of a codon. There are, however, some situations in which a stop codon is read as an amino acid at a low frequency in the wild-type situation. The first example to be discovered was the coat protein gene of the RNA phage Q0. The formation of infective QF particles requires that the
stop codon at the end of this gene is suppressed at a low frequency to generate a small proportion of coat proteins with a C-terminal extension. In effect, this stop codon is leaky. The reason is that Tfp-IRNA recognizesthe codon at a low frequency. Readthrough past stop codons also occurs in eukaryotes, where it is employed most often by RNA viruses. This may involve the suppression of UAG/UAA by Tyr-tRNA, Gln-tRNA, or Leu-tRNA, or the suppression of UGA by TrpIRNA or Arg-tRNA. The extent of partial suppression is dictated by the context surrounding the codon.
Influences TheRibosome of the Accuracy TransLation r Thestructureof the 165rRNAat the PandA sites of the ribosome of influences the accuracv transtation.
The lack of detectable variation when the sequence of a protein is analyzed demonstrates that protein synthesis must be extremely accurate. Very few mistakes are apparent in the form of substitutions of one amino acid for another. There are two general stagesin protein synthesis at which errors might be made (seeFigure 8.8 in Section 8.3, SpecialMechanisms Control the Accuracy of Protein Synthesis): . Charging a IRNA only with its correct amino acid clearly is critical. This is a function of the aminoacyl-tRNA synthetase. The error rate probably varies with the particular enzyme, but in general mistakes occur in <1/105 aminoacylations. . The specificity of codon-anticodon recognition is crucial, but puzzling. Although binding constants vary with the individual codon-anticodon reaction, the specificity is always much too Iow to provide an error rate of <10-5. When free in solution, tRNAs bind to their trinucleotide codon sequencesonly rather weakly. Related, but erroneous, triplets (with two correct bases out of three) are recognizedl0-I to l0-2 times as efficiently as the correct triplets. Codon-anticodon base pairing therefore seems to be a weak point in the accuracy of
of Transtation 209 Influences the Accuracv 9.15 TheRibosome
IRNA-EF-Tu-GTP complex and the ribosome. If any complex, irrespective of its IRNA, can enter the A site, the number of incorrect entries must far exceed the number of correct entries. There are two basic models for how the ribosome might discriminate between correctly and incorrectly paired aminoacyl-tRNAs. The In addition to the role of the ribosome itself, place actual situation incorporates elements of both factors initiatorand aminoacylthe that models. in ribosome also may influence the tRNAs the . The direct recognition model supposes pairing reaction. for that the structure of the ribosome is There must be some mechanism stabiits designedto recognizeaminoacyl-tRNAs Iizing the correct aminoacyl-tRNA, allowing that are correctly paired. This would amino acid to be accepted as a substrate for mean that the correct pairing results in receipt of the polypeptide chain; contacts with some small change in the conformation an incorrect aminoacyl-tRNA must be rapidly of the aminoacyl-tRNA that the ribobroken, so that the complex leaves without some can recognize. Discrimination reacting. Suppose that there is no specificity in occurs before any further reaction the initial collision between the aminoacyloccurs. . The kinetic proofreading model proposes that there are at least two stages in the process, so that the aminoacylIRNA has multiple opportunities to disengage. An incorrectly paired aminoacyl-tRNA may pass through some stagesof the reaction before it is rejected. Overall selectivity can in principle be the product of the selectivities at each stage. FIfrURE9.27illustrates diagrammatically what happens to correctly and incorrectly paired aminoacyl-tRNAs. A correctly paired aminoacyl-tRNA is able to make stabilizing contacts The correctIRNA interactswith rRNA with rRNA. An incorrectly paired aminoacylIRNA does not make these contacts, and therefore is able to diffuse out of the A site. The path to discovering these interactions started with investigations of the effects of the antibiotic streptomycin in the I960s. Streptomycin inhibits protein synthesis by binding to i65 rRNA and inhibiting the ability of EF-G to catalyzetranslocation. It also increasesthe level lnteraction of misreading of the pyrimidines U and C (usuAn incorrect IRNAdiffuses out ally one is mistaken for the other, occasionally for A) . The site at which streptomycin acts is influenced by the SI2 protein; the sequence of this protein is altered in resistantmutants. Ribosomes with an Sl2 protein derived from resistant bacteria show a reduction in the level of misreading compared with wild-type ribosomes. In effect, Sl2 controls the level of misreading. When it is mutated to decreasemisreading, it FI6URE 9.27 AnyaminoacyltRNA in theA canbeplaced suppressesthe effect of streptomycin. site(byEF-Tu), butontyonethat pairswiththeanticodon SI 2 stabilizesthe structure of I 6 S rRNA in canmakestabilizing contacts with rRNA. In the absence the region that is bound by streptomycin. The of thesecontacts, the aminoacv[-tRNA diffuses out of tne important point to notehereis that the P/A siteregion A site.
translation. The ribosome has an important role in controlling the specificity of this interaction: It functions directly or indirectly as a "proofreader" in order to distinguish correct and incorrect codon-anticodon pairs, thus amplifying the rather modest intrinsic difference by -I000x.
2to
C H A P T E9RU s i n gt h e G e n e t i cC o d e
influencesthe accuraqtof translation:translation can be made more or lessaccurateby changingthe structure of 165rRNA. The combination of the effects of the Sl2 protein and streptomycin on the rRNA structure explains the behavior of different mutants in S12, some of which even make the ribosome dependentonthe presence of streptomycin for correct translation. We now know from the crystal structure of the ribosome that l65 rRNA is in a position ro make contacts with aminoacyl-tRNA. TWobases of l65 rRNA can contact the minor groove of the helix formed by pairing between the anticodon in IRNA with the first two bases of the codon in mRNA. This directly stabilizesthe structure when the correct codon-anticodon contacts are made at the first two codon positions, but it does not monitor contacts at the third position. The stabilization of correctly paired aminoacyl-tRNA may have two effects. By holding the aminoacyl-tRNA in the A site, it prevents it from escaping before the next stage of protein synthesis. The conformational change in the rRNA may help to trigger the next stage of the reaction, which is the hydrolysis of GTP by EF-Tu. Part of the proofreading effect is determined by timing. An aminoacyl-tRNA in the A site may in effect be trapped if the next stageof protein synthesis occurs while it is there. Thus a delay between entry into the A site and peptidyl transfer may give more opportunity for a mismatched aminoacyl-tRNA to dissociate.Mismatched aminoacyl-tRNA dissociatesmore rapidly than correctly matched aminoacyl-tRNA, probably by a factor of -5x. Its chance of escaping is therefore increased when the peptide transfer step is slowed. The specificity of decoding has been assumed to reside with the ribosome itself, but some recent results suggestthat translation factors influence the processat both the P site and A site. An indication that EF-Tu is involved in maintaining the reading frame is provided by mutants of the factor that suppressframeshifting. This implies that EF-Tu does not merely bring aminoacyl-tRNA to the A site, but also is involved in positioning the incoming aminoacyl-IRNR relative to the pepridyl-tRNA in the P site. A striking case in which factors influence meaning is found at initiation. Mutation of the AUG initiation codon to UUG in the yeast gene Il1S4prevents initiation. Extragenic suppressor mutations can be found that allow protein synthesis to be initiated at the mutant UUG codon.
TWo of these suppressorsprove to be in genes coding for the u and p subunits of eIF2, the factor that binds Met-tRNA to the P site. The mutation in eIFp2 residesin a part of the protein that is almost certainly involved in binding nucleic acid. It seems likely that its target is either the initiation sequence of mRNA as such or the base-paired association between the mRNA codon and tRNAlMet anticodon. This suggests that eIF2 participates in the discrimination of initiation codons as well as bringing the initiator IRNA to the P site. The cost of protein synthesis in terms of high-energy bonds may be increased by proof reading processes.An important question in calculating the cost of protein synthesis is the stageat which the decision is taken on whether to accept a IRNA. If a decision occurs immediately to releasean aminoacyl-tRNA-EF-TUGTP complex, there is little extra cost for rejecting the large number of incorrect tRNAs that are likely (statistically) to enter the A site before the correct 1RNA is recognized. If, however, GTP is hydrolyzed before the mismatched aminoacylIRNA dissociates,the cost will be greater.A mismatched aminoacyl-tRNA can be rejected either before or after the cleavage of GTP, although we do not know yet where on average it is rejected. There is some evidence that the use of GTP in vivo is greater than the three highenergy bonds that are used in adding every (correct) amino acid to the chain.
Recoding Changes Meanings Codon . Changes by in codonmeaning canbe caused mutanttRNAsor by tRNAswith specialproperLies. . Thereading framecanbechanged by frameshifting or bypassing, bothof which depend of the mRNA. on properties
The reading frame of a messenger usually is invariant. Tlanslation starts at an AUG codon and continues in triplets to a temination codon. Reading takes no notice of sense: insertion or deletion of a base causesa frameshift mutation, in which the reading frame is changed beyond the site of mutation. Ribosomes and tRNAs continue ineluctably in triplets, synthesizing an entirely different seriesof amino acids. There are some exceptions to the usual pattern of translation that enable a reading frame with an interruption of some sort-such as a
Meanings ztt 9.16 Recoding Changes Codon
is causedby mutatedanticodon
-1 frameshift in HIVretrovirus
NNNNUUU
GNNNNNNNN
frame Lastcodonreadin initialreading Specialfactor+ IRNA recognizescodon
s+cvs\sg
Nil
8ilil
NNNNNUGANNNNNNNNNN a:{i-jei'-:.:l:lA mutation in an individuat tRNA(usuatty in theanticodon) cansuppress theusuaImeaning ofthat codon.In a spec'iaI case,a specific IRNAis boundby an u n u s u ael l o n g a t i ofna c t o rt o r e c o g n i zaet e r m i n a t i o n codonadjacent to a hairpin[oop.
nonsense codon or frameshift-to be translated into a full-length protein. Recoding events are responsible for making exceptions to the usual rules, and can involve severaltypes of events. Changing the meaning of a single codon allows one amino acid to be substituted in place of another, or for an amino acid to be inserted at a termination codon. f.i*tiFtE*"t:s shows that these changes rely on the properties of an individual IRNA that responds to the codon: . Suppressioninvolves recognition of a codon by a (mutant) IRNA that usually would respond to a different codon (see Section 9.I2, SuppressortRNAs Have Mutated Anticodons That Read New Codons). . Redefinition of the meaning of a codon occurs when an aminoacyl-tRNA is modified (seeSection 9.8, Novel Amino Acids Can Be Inserted at Certain Stop Codons). Changing the reading frame occurs in two types of situations: . Frameshifting typically involves changing the reading frame when aminoacylIRNA slipsby one base,either +l forward or -l backward (seethe following section, Frameshifting Occurs at Slippery Sequences). The result shown in a:*i,jt[ *.iF is that translation continues past a termination codon. . Bypassinginvolves a movement of the ribosome to change the codon that is paired with the peptidyl-tRNA in the
212
CHAPTER 9 Usinqthe GeneticCode
lrame odonreadin newreading Readingwithoutf rameshift
NNNNUUUUUUAGGNNNNNNNN Readingafterframeshift
NNNNUUU F5*tjftil*.h-* A IRNAthat stipsonebasein pairingwith a frameshift that cansuppress terminaa codoncauses -5%. is usua[[y tion.Theefficiency
60 nucleotide bypassin phageT4 gene60
Last codon in originalreadingframe odon in new readingframe Readingwithoutframeshift
GAUGGAUGAC............AUUGGAUU Readingafterframeshift
GAUGGAUGAC...,......,.AUUGG agGti**9"3* Bypassing occurs whentheribosome moves in the P siteis atongmRNAso that the peptidyt-tRNA reteased frompairingwithits codonandthenrepairs with anothercodonfartheralonq.
P site. The sequencebetween the two codons fails to be represented in protein. As shown in F3fitiRF + . 3 { 1 ,t h i s allows translation to continue past any termination codons in the intervenins region.
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The sequence of the mRNA triggers the blpass. The important features are the two GGA codons for take-off and landing, the spacing between them, a stem-loop structure that includes the take-off codon, and the stop codon adjacent to the take-off codon. The protein under synthesis is also involved. The take-off stagerequires the peptidyl-tRNA to unpair from its codon. This is followed by a movement of the nRNA that prevents it from re-pairing. Then the ribosome scansthe mRNA until the peptidyl-tRNA can repair with the codon in the landing reaction. This is followed by the resumption of protein synthesis when aminoacyl-tRNA enters the A site in the usual way. Like frameshifting, the bypass reaction depends on a pause by the ribosome. The probability that peptidyl-tRNA will dissociarefrom its codon in the P site is increased by delays in the entry of aminoacyl-tRNA into the A site. Starvation for an amino acid can trigger bypassing in bacterial genes because of the delay that occurs when there is no aminoacyl-tRNA available to enter the A site. In phage T4 gene 60, one role of mRNA structure may be to reduce the efficiency of termination, thus creating the delay that is needed for the take-off reaction.
@
Summary
The sequence of mRNA read in triplets 5' -+ 3' is related by the genetic code to the amino acid sequenceof protein read from N- to C-terminus. Of the sixty-four triplets, sixty-one code for amino acids and three provide termination signals. Synonym codons that represent the same amino acids are related, often by a change in the third base of the codon. This third-base degeneracy, coupled with a pattern in which related amino acids tend to be coded by related codons, minimizes the effectsof mutations. The genetic code is universal and must have been establishedvery early in evolution. Changesin nuclear genomes are rare, but some changes have occurred during mitochondrial evolution. Multiple tRNAs may respond to a particular codon. The set of tRNAs responding to the various codons for each amino acid is distinctive for each organism. Codon-anticodon recognition involves wobbling at the first position of the anticodon (third position of the codon), which allows some tRNAs to recognize multiple codons. AII tRNAs have modified bases, introduced by enzymes that recognize targel basesin the IRNA structure. Codon-anticodon
pairing is influenced by modifications of the anticodon itself and also by the context of adjacent bases,especially on the 3'side of the anticodon. Taking advantage of codon-anticodon wobble allows vertebrate mitochondria to use only twenty-two tRNAs to recognize all codons, compared with the usual minimum of thirty-one tRNAs; this is assistedby the changes in the mitochondrial code. Each amino acid is recognized by a particular aminoacyl-tnNl synthetase, which also recognizesall of the tRNAs coding for that amino acid. Aminoacyl-tRNA synthetaseshave a proofreading function that scrutinizes the aminoacyl-IRNA products and hydrolyzes incorrectly joined aminoacyl-tRNAs. Aminoacyl-tRNA synthetasesvary widely, but fall into two general groups according to the structure of the catalytic domain. Synthetasesof each group bind the IRNA from the side, making contacts principally with the extremities of the acceptor stem and the anticodon stem-loop; the two types of synthetases bind tRNe from opposite sides.The relative importance attached to the acceptor stem and the anticodon region for specific recognition varies with the individual IRNA. Mutations may allow a IRNA to read different codons; the most common form of such mutations occurs in the anticodon itself. Alteration of its specificity may allow a IRNA to suppress a mutation in a gene coding for protein. A 1RNA that recognizes a termination codon provides a nonsense suppressor; one that changes the amino acid responding to a codon is a missense suppressor. Suppressorsof UAG and UGA codons are more efficient than those of UAA codons, which is explained by the fact that UAA is the most commonly used natural termination codon. The efficiency of all suppressors,however, depends on the context of the individual target codon. Frameshifts of the +l type may be caused by aberrant tRNAs that read "codons" of four bases.Frameshifts of either +l or -l may be caused by slippery sequencesin nRNA that allow a peptidyl-tRNA to slip from its codon to an overlapping sequencethat can also pair with its anticodon. This frameshifting also requires another sequence that causesthe ribosome to delay. Frameshifts determined by the nRNA sequencemay be required for expressionof natural genes. Bypassing occurs when a ribosome stops translation and moves along mRNA with its peptidyl-tRNA in the P site until the peptidyl-tRNA pairs with an appropriate codon; then translation resumes.
9.19 Summary 275
References ![
Introduction
Resea rch Nirenberg, M W. and Leder, P. (19641. The effect of trinucleotides upon the binding of sRNA to ribosomes. Science145, 1399-1407. Nirenberg, M. W. and Matthaei, H. J. ( 196 I ). The dependence of cell-free protein synthesis in E coli tpon naturally occurring or synthetic polyribonucieotides. Proc.Natl Acad- Sci.USA 47, r588-t602.
f[
Invotves Recognition Codon-Anticodon Wobbting
R e s erach Crick,F. H C. (1966).Codon-anticodonpairing: the wobble hypothesis.J Mol. Biol. 19, 548-555.
Etr
fromronger rxl\AsAreProcesseo Precu rsors
Review Hopper,A. I(. and Phizicky,E. M. (2003).IRNA Dev.17, transfersto the limelight. Genes I 62-t80.
lfl
Modified Bases IRNAContains
Reviews Hopper, A. I(. and Phizicky,E. M. (2003). IRNA transfers to the limelighr. GenesDev. 17, l 62-l 80.
Research Fagegaltier,D., Hubert, N., Yamada, I(., Mizutani, T., Carbon, P., and Iftol, A. (2000). Characterization of mSelB, a novel mammalian elongation factor for selenoprotein translation. E M B OJ . 1 9 , 4 7 9 6 - 4 8 0 5 . Hao, B., Gong, W., Ferguson, T. I(., James, C. M., ICzycki, J. A., and Chan, M. K. (2002). A new UAG-encoded residue in the structure of a 296, methanogen methyltransf erase.Science 1462-t466. Srinivasan, G., James, C. M., and Iftzycki, J. A. (2002). Pyrrolysine encoded by UAG in Archaea: charging of a UAG-decoding special296, 1459-1462. ized IRNA. Science
with AminoAcidsby AreCharged tRNAs Synthetases Review Schimmel,P. (I989). Parametersfor the molecular 28, recognitionof tRNAs.Biochemistry 2 74 7 - 2 75 9 .
Fa[[ Aminoacyt-tRNA Synthetases into TwoGrouos Review Schimmel,P.(I987). Aminoacyl-tRNAsynthetases:generalschemeof structure-function relationshipson the polypeptidesand recognition of tRNAs.Annu.Rev.Biochem.56, r25-r58.
Research Rould,M. A. et al. (1989).Structureof E. coliglutaminyl-IRNAsynthetasecomplexedwith 1p54clnand ATPat 28A resolution.Scierce Affect Anticodon-Codon Modjfied Bases !!l 246, rr)5-1142. Pairing Ruff, M. et al. ( l99l ). ClassII aminoacylIRNA Review crystalstructureof yeastaspartylsynthetases: Bjork, G. R. ( I 987) . TlansferRNA modification. Sci tRNA synthetasecomplexeswith TRNAA'p. 26j-287 Annu.Rev.Biochem.56, ence252, 1682-1689.
![
ThereAreSporadic Alterations Code of the Universa[
Reviews Fox,T. D. (1987).Naturalvariationin the genetic code.Annu.Rev.Genet21, 67-91. Osawa,S et al. (19921.Recentevidencefor evolution of the geneticcode.MicrobiolRev.56, 229-264.
@
NovelAminoAcidsCanBeInserted at Certain StopCodons
Reviews Bock, A. (1991). Selenoproteinsynthesis:an expansion of the genetic code. TrendsBiochem Sci.16, 46)-467. Ibba, M. and Soll, D. (2004). Aminoacyl-tRNAs: setting the limits of the genetic code. Genes Dev.18,731-738.
276
CHAPTER 9 Usingthe Genetic Code
UseProofreading to Improve Synthetases Accuracy Review Jakubowski,H. and Goldman,E. (19921.Editing of errorsin selectionof amino acidsfor protein synthesis.MicrobiolRev.56, 412-429. Resea rch Dock-Bregeon,A., Sankaranarayanan, R., Romby, P.,Caillet,J., Springer,M., Rees,B., Francklyn, C. S.,Ehresmann,C.,and Moras,D. (2000).TransferRNA-mediatededitingin threonyl-tRNAsynthetase.The classII solution to the doublediscriminationoroblem.Cel/ 103,877-884. Hopfield,J. J. (1974lr.I(ineticproofreading: a new mechanismfor reducingerrorsin biosynthetic processes requiringhigh specificiry. Proc.Natl. Acad.Sci.USA7l, 4lj5-41)9.
Jakubowski, H. (1990). Proofreading in vivo: editing of homocysteine by methionyl-rRNA synthetase in E. coli. Proc Natl. Acad. Sci USA 87, 4504-4508. Nomanbhoy, T. I(., Hendrickson, T. L., and Schimmel, P. (1999). Tiansfer RNA-dependent translocation of misactivated amino acids to prevent errors in protein synthesis. Mol. Cell4, 519-528. Nureki, O. et al. (1998). Enzyme structure with two catalytic sites for double sieve selection of substrate. Science280, 578-58 l. Silvian, L. F., Wang, J., and Sreitz,T. A. (1999). Insights into editing from an Ile-tRNA synthetase structure with tRNAIle and mupirocin. Science285, lO7 4-1077.
Ramakrishnan,V. (2002).Ribosomestructureand the mechanismof translation.Cell108. 557-572. Resea rch Carter,A. P.,Clemons,W. M., Brodersen,D. E., Morgan-Warren,R. J., Wimberly,B. T., and Ramakrishnan,V. (2000).Functionalinsights from the structureof the 30Sribosomalsubunit and its interactionswith antibiotics. Nature407,340-348. Ogle,J. M., Brodersen, D. E., Clemons,W. M., Tarry M. J., Carter,A. P.,and Ramakrishnan,V (2001).Recognitionof cognatetransferRNA by the 30Sribosomalsubunit.Science 292, 897-902.
Suppressors MayCompete with WiLd-type
Frameshifting 0ccursat SLippery
Readingof the Code
Sequences
Reviews Atkins,J. F. (I991). Towardsa geneticdissection of the basisof triplet decoding,and its natural subversion:programmedreadingf rameshifts and hops.Annu.Rev.Genet. 25, 2OI-228. Beier,H. and Grimm,M. (2001).Misreadingof termination codonsin eukaryotesby natural nonsensesuppressortRNAs.Nucleic AcidsRes. 29,47674782. Eggertsson, G. and Soll,D. ( I 988) . TtansferRNAmediatedsuppressionof termination codons in E. coli.Microbiol.Rev.52,354-374. Murgola,E. J. (1985).IRNA,suppression, and the cod,e. Annu.Rev.Genet.19, 57-80. Normanly,J. and Abelson,J. ( I 989). TransferRNA identity.Annu Rev.Biochem. 58, 1029-1049. R e s erac h Hirsh,D. (197ll . TryptophanrransferRNA asthe UGA suppressor. J.Mol.Bi01.58,419-458. Weiner,A. M. and Weber,K. (1973).A singleUGA codonfunctionsas a natural termination signal in the coliphageq beta coatprotein cistron.J.Mol.Biol.80,837-855.
TheRibosome Inftuences the Accuracv of Translation Reviews I(urland, C. G. (1992).Translationalaccuracyand the fitnessof bacteria.Annu.Rev.Genet.26. 29-50. Ogle,J. M., and Ramakrishnan,V. (2005).Sftuctural insightsinto translationaltidelity.Annu. Rev.Biochem. 74, 129-177.
Reviews Farabaugh, P. J. (1995). Programmed translational frameshifting . Microbiol. Rev.60, lO3-134. Farabaugh,P. J. and Bjorkk, G. R. (1999). How translational accuracy influences reading frame maintenance. EMBO J L8, 1427-14]4. Gesteland, R. F. and Atkins, J. F. (1996). Recoding: dynamic reprogramming of translation. Annu Rev.Biochem.65, 7 41-7 68.
Research Jacks,T., Power, M. D., Masiarz,F.R., Luciw, P. A., Barr, P. J., and Varmus, H. E. (1988). Characterization of ribosomal frameshifting in HIV- I gag-pol expression. Nature )31, 280-28).
Bypassing Invotves Ribosome Movement Review Herr, A. J., Atkins, J. F., and Gesteland, R. F. (2000). Coupling of open reading frames by translational bypassing.Annu. Rev.Biochem. 69, )4)-)72. Research Gallant, J. A. and Lindsley,D. (1998). Ribosomes can slide over and beyond "hungry" codons, resuming protein chain elongation many nucleotides downstream. Proc.Natl. Acad. Sci. usA95, t377t-t)776. Huang, W. M., Ao, S. 2., Casjens,S., Orlandi, R., Zeikus, R., Weiss, R., Winge, D., and Fang, M. (1988). A persistent untranslated sequence within bacteriophage T4 DNA topoisomerase gene 60. Science 2]9, I005-1012.
References 2t7
ProteinLocalization C H A P T EO RU T L I N E Introduction Passage Acrossa Membrane Requires a SpeciaIApparatus . Proteins passacross protein membranes throughspecialized structures embedded in the membrane. . Substrate proteinsinteractdirecttywith the transportapparatusof the ER,mitochondria. but require or chtoroplasts, carrierproteins to interactwith peroxisomes. e A muchlargerandcomptex apparatus is required for transoortinto the nucleus. tfaEf
tEE
ProteinTranslocation MayBe Posttranslational or CotranslationaI . Proteins that areimportedinto cytoplasmic organettes are synthesized on freeribosomes in the cytosot. o Proteins that areimpodedinto the ER-Gotgi system are synthesized withthe ER. on ribosomes that areassociated . Proteins associate with membranes by means of specific aminoacidsequences caltedsignaIsequences. . Signalsequences aremostoftenleaders that arelocated at the N-terminus. o N-terminaI signaIsequences areusuatly cleaved off the proteinduringtheinsertion process. Chaperones MayBe Requiredfor ProteinFotding . Protejns that canacquire theirconformation spontaneously aresaidto self-assembte. o Proteins canoftenassemble into atternative structures. . A chaperone pathway directsa proteininto oneparticular pathways. by excluding atternative . Chaperones prevent the formation of inconectstructures by interactingwith unfoldedprote'ins to preventthemfrom fotdingincorrectty. Chaperones Are Neededby NewLy Synthesized and by DenaturedProteins . Chaperones proteins, proteins acton newlysynthesized that arepassing throughmembranes, or proteins that havebeen denatured. o Hsp70 proteins andsomeassociated forma majorclass of chaperones that acton manytargetproteins. r Group I andgroupII chaperonins arelargeoligomeric assemblies that acton targetproteins theysequester in internaI cavities. . Hsp90 is a speciatized chaperone that actson proteins pathways. of signaItransductjon
278
The Hsp70Famil.y Is Ubiquitous o Members fami[yarefoundin the cytosol, of the Hsp70 in the ER,andin mitochondria andchloroplasts. . Hsp70 that functions on targetproteins is a chaperone with DnaJandGrpE. in conjunction Initiate Transtocation SignaISequences r Proteins with the ERsystemon[ycotranstationatty. associate . Thesigna[sequence proteinis responsibte of the substrate for membrane association. Interactswith the SRP TheSignalSequence . ThesignaIsequence bindsto theSRP(signaIrecognition paticte). . Signa[-SRP proteinsynthesis to pause. bindingcauses r Protein resumes whenthe SRPbindsto the SRP synthesis receDtor in the membrane. . Thesignalsequence profromthe translocating is cteaved located on the "insjde"face tein bythe signalpeptidase of the membrane. TheSRPInteractswith the SRPReceptor r TheSRPis a comptex of 75 RNAwith sixproteins. . ThebacteriaI to the SRPis a comptex of 4.55RNA equivalent with two oroteins. o TheSRPreceptor is a dimer. r GTPhydrolysis releases the SRPfromthe SRPreceptorafter theirinteraction. Formsa Pore TheTranslocon e TheSec61 provides for the channel trimericcomplex proteins to passthrougha membrane. o A transtocating proteinpasses directlyfromthe ribosome withoutexposure to the cytosot. to thetranslocon Insertioninto the Translocon and Transtocation Requires (Sometimes) a Ratchetin the ER o Theribosome, aresufficient to insert SRP, andSRPreceptor proteininto a transtocon. a nascent . Proteins posttranstationatty require addithat areinserted in the cytosolandBipin the ER. tionaIcomponents . Bipis a ratchetthat prevents a proteinfromstipping backward.
Reverse Transtocation SendsProteins to the Cytosolfor Degradation . Sec61 transtocons canbe usedfor reverse translocation of proteins fromthe ERinto the cytoso[. ProteinsResidein Membranes bv Means of Hydrophobic Regions r Group I proteins havethe N-terminus on the farsjdeof the membrane; groupII proteins havethe opposite orientation. . Someproteins havemultiptemembranes p a n n i ndgo m a i n s . AnchorSequences DetermineProtein 0rientation o An anchorsequence haltsthe passage of a proteinthroughthetranstocon. Typicattythisis located at the C-terminaI end andresutts in a groupI orientation in whichthe N-terminus haspassed through the membrane. r A combined signat-anchor sequence can be usedto inserta proteininto the membraneandanchor the siteof insertion. Typicatty thisis internaIandresults in a groupII orientation in whichthe Nterminus is cytoso[ic. HowDo ProteinsInsert into Membranes? r Transfer of transmembrane domains from thetranstocon intothe tipidbitayer is triggeredby the interactjonof the transmembrane regionwiththetranslocon. PosttranslationaI Membrane Insertion D e p e n dosn L e a d eS r equences o N-termina[ provide leader sequences the information that altowsproteins to associatewith mitochondriaI or chtoroolast mem branes. A Hierarchyof Sequences Determines Locationwithin 0rganelles e TheN-terminal partof a leadersequence targetsa proteinto the mitochondrial matrixor chloroptast [umen. o An adjacent sequence cancontrolfurther targeting to a membrane or theintermem0ranesDaces. r Thesequences arecleaved successivety fromthe protein. I n n e ra n d 0 u t e r M i t o c h o n d r i a l Membranes HaveDifferentTranslocons o Transpoft throughthe outerandinner mitochondriaI membranes usesdifferent receptor comptexes.
e TheTOM(outermembrane) is a complex proteins largecomptex in whichsubstrate aredirected to theTom40 channel by one of two subcomptexes. r Different TIM(innermembrane) complexes areuseddepending on whether the substrateproteinis targetedto the jnner membrane or to the lumen. r Proteins passdirect[yfromthe TOMto the TIMcomp[ex. Peroxisomes EmployAnotherTypeof Translocation System o Proteins areimported into peroxisomes jn theirfutlyfotdedstate. o Theyhaveeithera PTS1 sequence at the C-terminus or a PTS2 seouence at the N-termi nus. e Thereceptor Pex5p bindsthe PTS1 sequence andthe receptor PexTp binds the PTSZ seouence. o Thereceptors arecytosolicproteinsthat shuttleinto the peroxisome carrying a proteinandthenreturnto the substrate cytosoL. BacteriaUseBoth CotranstationaI and PosttranslationaI Translocation . Bacterial proteins that areexpoded to or throughmembranes usebothposttranstamechanisms. tionaIandcotranslationaI TheSecSystemTransports Proteinsinto a n dT h r o u g ht h e I n n e rM e m b r a n e . Thebacteriat translocon in the SecYEG innermembrane is retated to the eukaryoticSec61 translocon. r Various in directchaoerones areinvotved proteins ing secreted to thetrans[ocon. TranstocationSystems Sec-Independent in E. coLi t E. coliandorganetles haveretatedsystems for proteintranstocation. . Onesystemattowscertainproteinsto insertinto membranes withouta trans[ocationapparatus. r YidCis homologous to a mitochondrial proteinsinto the systemfor transferring innermembrane. r Thetat systemtransfers proteinswith a motifinto the periplasmic twin arginine sDace. Summarv
CHAPTER 10 ProteinLocalization 219
@
Introduction
Proteinsare synthesizedin two tlpes of location: . The vast majority of proteins are synthesized by ribosomes in the cytosol. . A small minority are sl,nthesizedby ribosomeswithin organelles (mitochondria or chloroplasts). Proteins synthesized in the cytosol can be divided into two general classeswith regard to localization: those that are not associatedwith membranes, and those that are associatedwith membranes. i-i:;ir::i ::J:: maps the cell in terms of the possibleultimate destinations for a newly synthesizedprotein and the systemsthat transport it: . Cytosolic (or "soluble") proteins are not localized in any particular organelle.
CoatedvesicletransDort
They are synthesizedin the cytosol, and remain there, where they function as individual catalytic centers, acting on metabolites that are in solution in the cytosol. Macromolecular structures may be Iocated at particular sites in the cytoplasm; for example, centrioles are associated with the regions that become the poles of the mitotic spindle. Nuclear proteins must be transported from their site of synthesisin the cytosol through the nuclear envelope into the nucleus. Most of the proteins in cytoplasmic organellesare synthesizedin the cytosol and transported specifically to (and through) the organelle membrane, for example, to the mitochondrion or peroxisome or (in plant cells) to the chloroplast. (Those proteins that are synthesized within the organelle remain within it.) The cytoplasm contains a seriesof membranous bodies, including endoplasmic reticulum (ER), Golgi apparatus, endosomes,andlysosomes.This is sometimes referred to as the "reticuloendothelial system." Proteins that reside within these compartments are inserted into ER membranes and then are directed to their particular locations by the transport system of the Golgi apparatus. Proteins that are secretedfrom the cell are transported to the plasma membrane and then must passthrough it to the exterior. They start their s)'nthesisin the same way as proteins associatedwith the reticuloendothelial system,but passentirely through the system instead of halting at some particular point within it.
Passage Across a Membrane Requires Apparatus a SpeciaI Posttranslational transDod
J;.:: r.::::i i-t : Overview: posttranslationalty Proteins are that arelocatized released intothe cytosoI aftersynthesis on freeribosomes. Somehavesignalsfor targeting Proto organeltes suchasthe nucteus or mjtochondria. teinsthat arelocatized cotranslatjonalty associate with the ERmembrane duringsynthesis, so theirribosomes are"membrane bound."Theproteins passintotheendoplasmic reticutum, andthen a[ongto theGotgiapparatus, throughthe plasma membrane. untess theyhavesignats that causeretention at oneofthestepson the pathway. Theymayalsobedirected to other organe[[es, suchasendosomes or lysosomes.
220
CHAPTER 10 ProteinLocalization
r Proteins passacross membranes through proteinstructures specialized embedded in the membrane. . Substrate proteinsinteractdirecttywith the of the ER,mitochondria, transport apparatus or but requirecarrierproteinsto chtoroptasts, interactwith peroxisomes. r A muchlargerandcomptex is required apparatus for transportinto the nucteus.
The processof inserting into or passingthrough a membrane is called protein translocation. The same dilemma must be solved for every situation in which a protein passesthrough a membrane. The protein presents a hydrophilic surface.but the membrane is hydrophobic. Like oil and water, the two would prefer not to mix. The solution is to createa specialstructure in the membrane through which the protein can pass. There are three different types of arrangements for such structures. The endoplasmic reticulum, mitochondria, and chloroplasts contain proteinaceous structures embedded in their membranes that allow proteins to passthrough without contacting the surrounding hydrophobic lipids. f l+i"tt{f:ti:i.,i shows that a substrate protein binds directly to the structure, is transported by it to the other side, and then is released. Peroxisomesalso have such structures in their membranes, but the substrateproteins do not bind directly to them. lii.;1;9q1 f i."1,"r shows that instead they bind to carrier proteins in the cytosol, the carrier protein is transported through the channel into the peroxisome, and then the substrateprotein is released. For transport into the nucleus, a much larger and more complex structure is employed. This is the nuclear pore. F:tirr.l!lt. i1,."*shows that, although the pore provides the environment that allows a substrate to enter (or to leave) the nucleus, it does not actually provide the apparatus that binds to the substrate proteins and moves them through. Included in this apparatus are carrier proteins that bind to the substrates and transport them through the pore to the other side.
l:tilliiil::i,i,ii Proteins enterthe ERor a mitochondrion by bindingto a transtocon that transports themacross the membrane.
ProteinTranslocation May BePosttranslationaI or CotransLationaI Proteins that areimported into cytoplasmic organelles aresynthesized on freeribosomes in the cytosot. Protejns that areimported into the ER-Gotgi system aresynthesized on ribosomes that are associated withthe ER. Proteins associate with membranes by means of specific aminoacidsequences cattedsignaI sequences. SignaI sequences aremostoftenleaders that are located at the N-terminus. N-termjnal signaIsequences areusuatly cleaved off process. the proteinduringthe insertjon
li{riiiti i,:.lt."i into peroxisomes Proteins aretransported passes proteinthatbindsthemin thecytosot, bya carrier with themthroughthe membrane channel, andreteases themon the otherside.
There are two ways for a protein to make its initial contact with a membrane: . The nascent protein may associatewith the translocation apparatus while it is still being synthesizedon the ribosome. This is called cotranslational translocation. MavBe Posttranstational or Cotranslational 227 10.3 ProteinTranslocation
The location of a ribosome depends on whether the protein under synthesisis associating with a membrane cotranslationally: . Cotranslational translocation is used for proteins that enter the endoplasmic reticulum (ER). The consequenceof this associationis that the ribosome is localized to the surfaceof the ER. The ribosomes are associatedwith the ER membranes during synthesis of these proteins, and therefore are found in membrane fractions of the cell; thus they are sometimes describedas "membrane bound." . AII other ribosomes are located in the cytosol; becausethey are not associated with any organelle and fractionate separately from membranes, they are sometimes called "free ribosomes."The free ribosomessynthesizeall proteins except those that are translocated cotranslationally. The proteins are released into : i a : - : i . : : i.- , . ! P r o t e i nesn t e rt h e n u c t e ubs y p a s s a g e the cytosol when their synthesisis compores. throughvery[argenuctear Thetransport apparatus pleted. Some of these proteins remain jnctudes is distinctfromthe poreitselfand components free in the cytosol in quasi-solubleform; that carrythe proteinthroughthe pore. others associatewith macromolecular cytosolic structures, such as filaments. microtubules, centrioles,etc., or are transported to the nucleus, or associate with membrane-bound organelles by posttranslational translocation. To associatewith a membrane (or any other type of structure), a protein requires an appropriate signal, typically a sequence motif that causesit to be recognizedby a translocation system (or to be assembledinto a macromolecular structure). *Ifillitfl 1*.5 summarizessome signalsused by proteins releasedfrom cytosolicribosomes. Import into the nucleus results from the presence of a variety of rather short sequenceswithin proteins. These "nuclear localization signals" Mitochondrion N-terminal Amphipathichelix 12-30 >25 Chloroplast N-terminal Charged enable the proteins to pass through nuclear Nucleus Internal Basicor bipartite 4-g pores. One type of signal that determines transPeroxisome C-terminal Short peptide 3-4 port to the peroxisome is a very short C-terminal sequence. Mitochondrial and chloroplast proi:i-i:i::i .ij.i:, Proteins rn synthesized on freeribosomes the cytosolaredjrected aftertheirrelease to specific desteins are synthesized on "free" ribosomes; after tinationsbv shortsiqnaImotifs. t h e i r r e l e a s ei n t o t h e c y t o s o l t h e y a s s o c i a t e with the organelle membranes by means of . The protein may be releasedfrom a riboN-terminal sequencesof -25 amino acids in some after translation has been comlength that are recognized by receptors on the pleted. Then the completed protein organelle envelope. diffuses to the appropriate membrane Proteins that reside within the reticuloenand associateswith the translocation dothelial system enter the ER while they are apparatus. This is called posttranslabeing synthesized. The principle of cotranstional translocation. lational translocation is summarized in
222
CHAPTER 10 ProteinLocalization
lir,,i,;ii:r. iir.i;. An important feature of this system is that the nascent protein is responsible for recognizing the translocation apparatus. This requires the signal for cotranslational translocation to be part of the protein that is first synthesized, and, in fact, it is usually located at the N-terminus. A common feature is found in proteins that u s e N - t e r m i n a l s e q u e n c e st o b e t r a n s p o r t e d cotranslationallyto the ER or posttranslationally to mitochondria or chloroplasts. The N-terminal sequencecomprisesa leader that is not part of the mature protein. The protein carrying this leader is called a preprotein and is a transient precursor to the mature protein. The leader is cleaved from the protein during protein translocation.
pathway l;jiii.iir ir ;,-r.,"1 on[y Proteins canentertheER-Gotgi reticulum whitethey by associating with the endoptasmic arebeingsynthesized.
Chaperones MayBe Required for Protein Fotding r Proteins that canacquire theirconformation spontaneousty aresaidto setf-assemb[e. r Proteins canoftenassembte intoatternative structures. . A chaperone directsa proteininto oneparticu[ar pathway pathways. by excluding alternative . Chaperones prevent the formation of jncorrect structures proteinsto by interactingwith unfoLded preventthemfromfotdingincorrectty. Some proteins are able to acquire their mature conformation spontaneously. A test for this ability is to denature the protein and determine whether it can then renature into the active form. This capacity is called selfassembly. A protein that can self-assemble can fold or refold into the active state frorn other conformations, including the condition in which it is initially synthesized. This implies that the internal interactions are intrinsically directed toward the right conformation. The classiccaseis that of ribonuclease;it was shown in the 1970s that, when the enzyme is denatured, it can renature in vitrl irrto the correct conformation. More recently the process of intrinsic folding has been described in detail for some small proteins. When correct folding does not happen, and alternative setsof interactions can occur, a protein may become trapped in a stable conformation that is not the intended final form. Proteins in this category cannot self-assemble.Their
regions of proteins areintrinsiliir,ilillr:r, r 1116roOnobic prevented wit[aggregate with caltyinteractive. andunless (ordenatured). oneanother whena proteinis synthesized
acquisition of proper structure requiresthe assistance of a chaperone. Protein folding takes place by interactions between reactive surfaces.Typically these surfacesconsist of exposed hydrophobic side chains. Their interactions form a hydrophobic core. The intrinsic reactivity of these surfacesmeans that incorrect interactions may occur unless the processis controlled. ;;:!iii.lir.ii: lri.l' illustrateswhat would happen. As a newly synthesizedprotein emerges from the ribosome, any hydrophobic patch in the sequenceis likely to aggregatewith another hydrophobic patch. Such associations are likely to occur at random and therefore will probably not represent the desired conformation of the protein. Chaperones are proteins that mediate correct assembly by causing a target protein to acquire one possible conformation instead of others. This is accomplished by binding to reactive surfaces in the Iarget protein that are exposed during the assemblyprocessand preventing those surfacesfrom interacting with
for ProteinFoLding 223 MayBeRequired 10.4Chaperones
AreNeeded Chaperones by NewlySynthesized andby Denatured Proteins proteins, Chaperones act on newlysynthesized proteins that arepassing throughmembranes. or oroteins that havebeendenatured. Hsp(heatshockprotein)70 andsomeassociated proteins forma majorclassof chaperones that act on manytargetproteins. I andgroupII chaperonins are[arge Group that acton targetproteins oligomeric assembties in internalcavities. theysequester Hsp90 is a speciatized chaperone that actson pathways. proteins of signaItransduction f,IG{Jft[ 1*.S Chaperones bindto interactive regions of proteins astheyaresynthesized The ability of chaperonesto recognize incorrect to preventrandom aggregation. Regions of the proteinarereleased to interactin an protein conformations allows them to play two orderlymanner to givethe properconformation. related roles concerned with protein structure: . When a protein is initially synthesizedother regions of the protein to form an incorrect that is to say,when it exits the ribosome conformation. Chaperonesf unction by preventto enter the cytosol-it appears in an ing formation of incorrect structuresrather than unfolded form. Spontaneous folding by promoting formation of correct structures. then occurs as the emerging sequence ffiSUftE10.8shows an example in which a chapinteracts with regions of the protein that erone in effect sequestersa hydrophobic patch, were synthesizedpreviously. Chaperthus allowing interactions to occur that would not ones influence the folding process by have been possiblein its presence,as can be seen controlling the accessibilityof the reacby comparing the result with Figure I0.7. tive surfaces.This processis involved in An incorrect structure may be formed either initial acquisition of the correct conforby the misfolding of a single protein or by intermation. . When a protein is denatured, new actions with another protein. The density of proteins in the cytosol is high. and "macromolregions are exposed and become able to ecular crowding" can increase the efficiencies interact. These interactions are similar of many reactions compared to the rates to those that occur when a protein (tranobserved in vitro. Crowding can cause folding siently) misfolds as it is initially syntheproteins to aggregate,but chaperonescan counsized.They are recognized by chaperones teract this effect. Thus one role of chaperones as comprising incorrect folds. This processis involved in recognizing a promay be to protect a protein so that it can fold without being adversely affected by the crowded tein that has been denatured and either conditions in the cytosol. assisting renaturation or leading to its We do not know what proportion of proremoval by degradation. teins can self-assemble,as opposedto those that Chaperones may also be required to assist require assistancefrom a chaperone. (It is not the formation of oligomeric structures and for axiomatic that a protein capableof self-assemthe transport of proteins through membranes. bly invitro actually self-assembles invivo,because A persistent theme in membrane passageis that there may be rate differences in the two condicontrol (or delay) of protein folding is an importions, and chaperones still could be involved in tant feature. fiG$RE1fl.Sshows that it may be vivo.There is, however, a distinction to be drawn necessaryto maintain a protein in an unfolded between proteins that can in principle selfstate before it enters the membrane because of assembleand those that in principle must have the geometry of passage:the mature protein a chaperone to assistin acquisition of the corcould simply be too large to fit into the available rect structure.) channel. Chaperones may prevent a protein
224
CHAPTER 10 ProteinLocatization
Proteinis inserted into closedchamber
ti:":ijr:{ !r-t.ii A proteinis constrained to a narrowpassageas it crosses a membrane.
formsa [argeoligomeric *li;Li$?il !i.:.,i'rA chaperon'in proteinwithinits interior. complex andfotdsa substrate
i:ili:!{i il} ii; Proteins emerge fromtheribosome orfrom passage througha membrane in an unfolded statethat attractschaperones to bindandprotectthemfrommjsfotding.
from acquiring a conformation that would prevent passagethrough the membrane; in this capacity, their role is basically to maintain the protein in an unfolded, flexible state. Once the protein has passed through the membrane, it may require another chaperone to assistwith folding to its mature conformation in much the same way that a cytosolicprotein requires assistance from a chaperone as it emerges from the ribosome. The state of the protein as it emerges from a membrane is probably similar to that as it emerges from the ribosome-f35i6allrz extended in a more or less linear condition. TWo major types of chaperones have been well characterized.They affect {olding through two different types of mechanism: o +'iii,liiii'.ij,:":iL: shows that the Hsp70 systemconsists of individual proteinsthat bind to, and act on, the substrateswhosefolding is to becontrolled.Ilrecognizesproteins as
they are synthesized or emerge from membranes (and also when they are denatured by stress).Basically,it controls the interactions between exposed reactive regions of the protein, enabling it to fold into the correct conformation in situ. The components of the system are Hsp70, Hsp40, and GrpE. The name of the system reflects the original identification of Hsp70 as a protein induced by heat shock. The Hsp70 and Hsp40 proteins bind individually to the substrate proteins. They use hydrolysis of ATP to provide the energy for changing the structure of the substrate protein and work in conjunction with an exchange factor that regeneratesATP from ADP. . li{:fili: lir'..1 I shows thatachaperoninsystem clnsists of a large oligomeric assembly (represented as a cylinder).This assembly forms a structure into which unfolded proteins are inserted. The protected environment directs their folding. There are two types of chaperonin system: GroEL/GroES is found in all classesof organism and TRiC is found in eukaryotic cytosol. The components of the systems are sum.i{i. marized in iitiii.ii;tt tii. The Hsp70 systemand the two chaperonin systems all act on many different substrateproteins. Another system, the Hsp90 protein, functions in conjunction
and by DenaturedProteins Synthesized Are Neededby NewLy 10.5 Chaperones
225
System
tein" is that increasein temperature causesproduction of heat shock proteins whose function is to minimize the damage causedto proteins by heat denaturation. Many of the heat shock proteins are chaperones and were first discovered, and named, as part of the heat shock response.)
Structure/function
Individual chaoerones Hsp70system Hsp70 (DnaK) ATPase Hsp40 (DnaJ) StimulatesATPase GrpE (GrpE) Nucleotideexchangefactor Functionson proteins involvedin sionaltransductior
Hsp90
Is Ubiquitous
Oligomericstructures(chaperonins) GroupI Hsp60 (GroEL) Formstwo heptamericrings; Hsp10 (GroES) Formscap G r o u pl l TR|C
. Members of the Hsp70 fami[yarefoundin the cytosot, in the ER.andin mitochondria and chtorootasts. o Hsp70 is a chaperone that functions on target proteins in conjunction with DnaJandGrpE.
Formstwo octamericrings
FIGURE 10.12 Chaperone famities haveeukaryotic and (named bacteriaI counterparts in parentheses).
Hsp70
GrpE disolacesADP
Hsp70-ATPbinds Hsp40+ substrate
Hsp40 is released
Hsp40binds substrate
Hsp70 dissociates i
y,. (gr Y. ()r
o.
FIGURT 10.13 Hsp40 bindsthesubstrate andthenHsp70. ATPhydrolysis drives conformationaI change. GrpE displaces theADP;thiscauses thechaperones to be released. Muttipte cyclesof association anddissociation mayoccurduring thefotdingof a substrate protein.
with Hsp70. but is directed against specificclasses of proteins that are involved in signal transduction, especially the steroid hormone receptors and signaling kinases.The Hsp90 system'sbasic lunction is to maintain its targets in an appropriate conformation until they are stabilizedby interacting with other components of the pathway. (The reason many of these proteins are named "Hsp," which standsfor "heat shockpro-
226
CHAPTER 10 ProteinLocatization
TheHsp70Family
The Hsp70 family is found in bacteria, eukaryotic cytosol, in the ER, and in chloroplasts and mitochondria. A typical Hsp70 has two domains: the N-terminal domain is an ATPaseand the Cterminal domain binds the substrate polypeptide. When bound to ATP, Hsp70 binds and releasessubstratesrapidly; when bound to ADP, the reactions are slow. Recycling between these statesis regulated by two other proteins, Hsp40 (DnaJ) and GrpE. FIGURE 10.13shows that Hsp40 (DnaJ) binds first to a nascent protein as it emerges from the ribosome. Hsp40 contains a region called the J domain (named for DnaJ), which interacts with Hsp70. Hsp70 (DnaK) binds to both Hsp40 and to the unfolded protein. In effect, two interacting chaperonesbind to the protein. The J domain accounts for the specificity of the pairwise interaction and drives a particular Hsp40 to select the appropriate pafiner from the Hsp70 family. The interaction of Hsp70 (DnaI() withHsp40 (DnaJ) stimulates the ATPaseactivity of Hsp70. The ADP-bound form of the complex remains associatedwith the protein substrateuntil GrpE displacesthe ADP. This causesloss of Hsp40 followed by dissociationof Hsp70. The Hsp70 binds another ATP and the rycle can be repeated. GrpE (or its equivalent) is found only in bacteria, mitochondria, and chloroplasts; in other locations, the dissociation reaction is coupled to ATP hydrolysis in a more complex way. Protein folding is accomplished by multiple cycles of association and dissociation. As the protein chain lengthens, Hsp70 (DnaK) may dissociate from one binding site and then reassociatewith another, thus releasing parts of the substrate protein to fold correctly in an ordered manner. Finally, the intact protein is
releasedfrom the ribosome folded into its mature conformation. Different members of the Hsp70 classfunction on various types of target proteins. CytosoIic proteins (the eponymous Hsp70 and a related protein called Hsc70) act on nascent proteins on ribosomes.Variantsin the ER (calledBiP or Grp78 in higher eukaryotes and I(ar2 in Saccharlmycescerevisiae), or in mitochondria or chloroplasts,function in a rather similar manner on proteins as they emerge into the interior of the organelle on passingthrough the membrane. What feature does Hsp70 recognizein a target protein? It binds to a linear stretch of amino acids embedded in a hydrophobic context. This is precisely the sort of motif that is buried in the hydrophobic core of a properly folded, mature protein. Its exposure therefore indicates that the protein is nascent or denatured. Motifs of this nature occur about every forty amino acids. Binding to the motif prevents it from misaggregating with another one. This mode of action explains how the Hsp70 protein Bip can fulfill two functions: to assistin oligomerization and/or folding of newly translocated proteins in the ER, and to remove misfolded proteins.Supposethat BiP recognizescertainpeptide sequencesthat are inaccessiblewithin the conformation of a mature, properly folded protein. Thesesequencesare exposedand attract BiP when the protein entersthe ER lumen in an essentially one-dimensional form. If a protein is misfolded or denatured, it may become exposed on its surfaceinstead of being properly buried.
SignaL Sequences Initiate Trans[ocation . Proteins withthe ERsvstem associate ontv cotrans[ationat[y. . Thesignalsequence proteinis of the substrate responsibte for membrane association.
Proteins that associatewith membranes via N-terminal Ieadersuse a hierarchy of signalsto find their final destination. In the case of the reticuloendothelial system,the ultimate location of a protein depends on how it is directed as it transits the endoplasmic reticulum and Golgi apparatus.The leader sequenceitself introduces the protein to the membrane; the intrinsic consequenceof the interaction is for the protein to passthrough the membrane into the compartment on the other side.For a orotein to reside
'li,:. ':, !r.ii"lii1;;|' pathway : Proteins thatenterthe ER-Gotgi a e m b r a noer m a yb e m a yf l o wt h r o u g ht o t h e p t a s m m by specific signa[s. diverted to otherdestinations
within the membrane, a further signal is required to stop passagethrough the membrane. Other types of signalsare required for a protein to be sorted to a particular destination, that is, to remain within the membrane or lumen of some particular compartment. The general process of finding its ultimate destination by transport through successivemembrane systems is called protein sorting or targeting, and is discussedin protein trafficking. The overall nature of the pathway is summ a r i z e di n ! , ' , i ; i i i i i ' , . . 1 rTrh. e " d e f a u l t p a t h w a y " takes a protein through the ER, into the Golgi apparatus, and on to the plasma membrane. Proteins that reside in the ER possessa C-terminal tetrapeptide (KDEL, which actually provides a signal for them to return to the ER from the Golgi apparatus).The signalthat diverts a protein to the lysosome is a covalent modification: the addition of a particular sugar residue. Other signals are required for a protein to become a permanent constituent of the Golgi apparatusor the plasma membrane. There is a common starting point for proteins that associatewith, or passthrough. the reticuloendothelial system 0f membranes. Theseproteinscan associatewith the membrane only while they are being synthesized.The ribosomes synthesizing these proteins become associatedwith the ER, enabling the nascent protein to be cotranslationally transferred to the membrane. Regions in which ribosomes are associatedwith the ER are sometimescalledthe "rough ER," in contrast with the "smooth ER" regionsthat lack associated polysomesand which have a tubular rather
InitiateTranslocation227 10.7SignaI Sequences
than sheetlikeappearance.i-:ilijiii;li:i.:i: shows ribosomesin the act of transferring nascentproteins to ER membranes. The proteins synthesizedat the rough ER pass from the ribosome directly to the membrane. Next they are transferred to the Golgi apparatus, and finally they are directed to their ultimate destination, such as the lysosome or secretoryvesicleor plasma membrane. The processoccurs within a membranous environment as the proteins are carried between organellesin small membrane-coatedvesicles. Cotranslational insertion is directed by a signal sequence. Most often this is a cleavable leader sequenceof l5 to 30 N-terminal amino acids.At or closeto the N-terminus are several polar residues, and within the Ieader is a hydrophobic core consisting exclusively or very largely of hydrophobic amino acids.There is no other conservation of sequence. iii--i=iF:i .iL:.:r:gives an example. The signal sequenceis both necessaryand sufficient to sponsor transfer of any attached polypeptide into the target membrane. A signal sequenceadded to the N-terminus of a glo-
i : + * * * i i * . . 1r T h ee n d o p l a s mr iect i c u t u m c o n s i s tosf a hightyfotdedsheetof membranes that extends fromthe nucteus. Thesma[[objects attached to the outersurface of themembranes areribosomes. Photocourtesy of LelioOrci. University of Geneva, Switzertand.
bin protein, for example, causesit to be secreted through cellular membranes instead of remaining in the cytosol. The signal sequenceprovides the connection that enablesthe ribosomes to attach to the membrane. There is no intrinsic difference between free ribosomes (synthesizing proteins in the cytosol) and ribosomes that are attached to the ER. A ribosome starts synthesis of a protein without knowing whether the protein will be synthesizedin the qtosol or transferred to a membrane. It is the synthesis of a signal sequencethat causes the ribosome to associatewith a membrane.
TheSignaI Sequence Interacts withthe SRP . Thesignalsequence bindsto the SRP(signat particte). recognition r Signa[-SRP proteinsynthesis bindingcauses to pause. r Protein synthesis resumes whenthe SRPb'inds to the SRPreceptor in the membrane. o Thesignalsequence is cleaved fromthe proteinbythe signalpeptidase translocating located on the "inside"faceof the membrane.
Protein translocation can be divided into two general stages:first, ribosomes carrying nascent polypeptides associatewith the membranes, and then the nascent chain is transferred to the channel and translocates through it. The attachment of ribosomes to membranes requires the signal recognition particle (SRP). The SRPhas two important abilities: o It can bind to the signal sequence of a nascent secretoryprotein. o It can bind to a protein (the SRPreceptor) located in the membrane. The SRPand SRPreceptor function catalytically to transfer a ribosome carrying a nascent
Initiation
{ Polar}<(-
Hydrophobiccore+
f l+Uf trit.]"i!ti ThesignaI sequence of bovinegrowthhormone consists of theN-termina[ 29amino acjdsandhasa central hightyhydrophobic region,preceded polar orflanked byregions containing a m i n oa c i d s .
228
CHAPTER 1O ProteinLocatization
protein to the membrane. The first step is the recognition of the signal sequence by the SRP. The SRPthen binds to the SRPreceptor and the ribosome binds to the membrane. The stagesof translation of membrane proteins are summarized in i ii.l'l;i r:r ! i.
The role of the SRP receptor in protein translocation is transient. When the SRPbinds to the signal sequence, it arrests translation. This usually happens when -70 amino acids have been incorporated into the polypeptide chain (at this point the 25-residue leader has become exposed,with the next -40 amino acids still buried in the ribosome). When the SRPbinds to the SRPreceptor, the SRPreleasesthe signal sequence.The ribosome becomesbound by another component of the membrane. At this point, translation can resume. When the ribosome has been passedon to the membrane, the combined role of SRPand SRP receptor has been played. They now recycle and are free to sponsor the associationof another nascent polypeptide with the membrane. This processmay be needed to control the conformation of the protein. If the nascent protein were releasedinto the cytoplasm, it could take up a conformation in which it might be unable to traverse the membrane. The ability of the SRPto inhibit translation while the ribosome is being handed over to the membrane is therefore important in preventing the protein from being releasedinto the aqueous environment. The signal peptide is cleavedfrom a translocating protein by a complex of five proteins called the signal peptidase. The complex is several times more abundant than the SRPand SRPreceptor.Its amount is equivalent roughly to the amount of bound ribosomes,suggesting that it functions in a structural capacity.It is located on the lumenal face of the ER membrane, which implies that the entire signal sequencemust crossthe membrane before cleavage occurs. Homologous signal peptidasescan be recognized in eubacteria, archaea, and eukaryotes.
The interaction between the SRP and the SRP receptor is the key event in eukaryotic translation in transferring a ribosome carrying a nascent protein to the membrane. An analogous interacting system exists in bacteria, although its role is more restricted. The SRPis an I I S ribonucleoprotein complex, containing six proteins (total mass240 kD) and a small (305 base, I00 kD) 75 RNA. rl{iiil'i ;i: ii, showsthat the 75 RNA provides the structural backbone of the particle; the individual proteins do not assemblein its absence. The 75 RNA of the SRP pafiicle is divided into two parts. The 100 basesat the 5'end and the 45 basesat the 3'end are closelyrelated to
TheSRPInteracts withthe SRPReceptor a a
a a
TheSRPis a comptex of 75 RNAwithsixproteins. Thebacterial equivatent to theSRPis a complex of 4.55RNAwithtwo proteins. TheSRPreceotor is a dimer. releases GTPhydrotysis the SRPfromtheSRP receotor aftertheirinteraction.
'' prosecretory synthesizing i ii.lilii:ri 1, I Ribosomes viathesignalsequence teinsareattached to themembrane potypeptide. on the nascent
withthe SRPReceptor 10.9 TheSRPInteracts
+
Alu RNAdomain.++
S RNAdomain*
Signalsequence emergesfrom ribosome; SRP approachesin extendedform Signalsequence
tI6URt10.18 75RNAoftheSRP hastwodomains. Proteins bindas shownon the two-dimensionaI diagram aboveto formthe crystalstructure shownbetow.Eachfunctionof the SRPis associated with a discrete oartof the structure. the sequence of Alu RNA, a common mammalian small RNA. They therefore define the Alu domain. The remaining part of the RNA comprises the S domain. Different parts of the SRPstructure depicted in Figure 10.l8 have separatefunctions in protein targeting. SRP54is the most important subunit. It is located at one end of the RNA structure and is directly responsible for recognizing the substrate protein by binding to the signal sequence.It also binds to the SRPreceptor in conjunction with the SRP68-SRP72dimer that is located at the central region of the RNA. The SRP9-SRPl4dimer is located at the other end of the molecule; it is responsible for elongation arrest. The SRPis a flexible structure. In its unengaged form (not bound to signal sequence),it is quite extended, as can be seen from the crystal structureof Figure 10.I 8. FIGURT 10.19shows t h a t b i n d i n g t o a s i g n a l s e q u e n c et r i g g e r s a change of conformation. The protein bends at a hinge to allow the SRP54end to contact the ribosome at the protein exit site, while the SRPI9 swings around to contact the ribosome at the elongation factor binding site. This enables it to causethe elongation arrest that gives time
230
CHAPTER 10 ProteinLocalization
bindsto stgnalsequence and bendsat hinge io contactribosome
F I G U R1I * " 1 9 S R Pb i n d st o a s i g n asl e q u e n caes i t emerges fromthe ribosome. Thebindingcauses the SRP to change conformation by bending at a "hinge,"altowing SRP54 to contactthe ribosome at the proteinexitsite whiteSRP19 makes a second setof contacts.
for targeting to the translocation site on the membrane. The SRPreceptor is a dimer containing sub(72 kD) and SRp (30 kD). The B subunirs SRcx, unit is an integral membrane protein. The amino-terminal end of the large u subunit is anchored by the B subunit. The bulk of the o protein protrudes into the cytosol. A large part of the sequence of the cytoplasmic region of the protein resembles a nucleic acid-binding protein with many positive residues. This suggests the possibility that the SRPreceptor recognizes the 75 RNA in the SRP. There is a counterpart to SRP in bacteria, although it contains fewer comporLerrts.E. coli contains a 4.5S RNA that associateswith ribosomes and is homologous to the 75 RNA of the SRP. It associateswith two proteins: Ffh is homologous to SRP54and FtsY is homologous to the cr subunit of the SRP receptor. In fact, FtsY replaces the functions of both the orand B SRPsubunits; its N-terminal domain substitutes for SRPp in membrane targeting, and the
C-terminal domain interacts with the target protein. The role of this complex is more limited than that of SRP-SRPreceptor. It is probably required to keep some (but not all) secreted proteins in a conformation that enablesthem to interact with the secretoryapparatus.This could be the original connection between protein synthesisand secretion;in eukaryotesthe SRPhas acquired the additional roles of causing translational arrest and targeting to the membrane. Why should the SRPhave an RNA component? The answer must lie in the evolution of the SRP: It must have originated very early in evolution, in an RNA-dominated world, presumably in conjunction with a ribosome whose functions were mostly carried out by RNA. The crystal structure of the complex between the protein-binding domain of 4.5S RNA and the RNA-binding domain of Ffh suggeststhat RNA continues to play a role in the function of SRP. The 4.55 RNA has a region (domain IV) that is very similar to domain IV in 7S RNA (seeFigure 10.18).Ffh consistsof three domains (N, G, and M). The M domain (named for a high content of methionines) performs the key binding functions. It has a hydrophobic pocket that binds the signal sequence of a target protein. The hydrophobic side chains of the methionine residues create the pocket by projecting into a cleft in the protein structure. Next to the pocket is a helix-turn-helix motif that is tlpical of DNAbinding proteins (seeSection 14.11,Repressor Usesa Helix-Turn-Helix Motif to Bind DNA). The crystal structure shows that the helixloop-helix of the M domain binds to a duplex region of the 4.5S RNA in domain IV. The negatively charged backbone of the RNA is adjacent to the hydrophobic pocket. This raisesthe possibility that a signal sequenceactually binds to both the protein and RNA components of the SRP.The positively charged sequencesthat start t h e s i g n a l s e q u e n c e( s e eF i g u r e 1 0 . 1 6 ) c o u l d interact with the RNA, while the hydrophobic region of the signal sequence could sit in the pocket. GTP hydrolysis plays an important role in inserting the signal sequence into the membrane. Both the SRPand the SRPreceptorhave GTPasecapability. The signal-binding subunit of the SRP.SRP54,is a GTPase.Both subunits o f t h e S R P r e c e p t o r a r e G T P a s e sA . ll of the GTPaseactivities are necessaryfor a nascent protein to be transferred to the membrane. i:ji-r.:;ilii''..,ti, Showsthat the SRPStartsout with GDP when it binds to the signal sequence.The ribosome then stimulates renlacement of the
w h e ni t b i n d tsh es i g i i { - i : ; i i , i : ,ri :1 l T h eS R P c a r r i eGsD P theGDP to bereptaced causes naIsequence. Theribosome with GTP.
iii";ri TheSRPandSRPreceptor bothhydrotyze r1:,r.ii!1i to themembrane. istransferred whenthesignat sequence GTP GDP with GTP.The signal sequence inhibits hydrolysis of the GT?.This ensures that the complex has GTP bound when it encounters the SRPreceptor. For the nascent protein to be transferred from the SRPto the membrane, the SRPmust i {.t;1I be releasedfrom the SRPreceptor. i:i-{:iiliri* shows that this requires hydrolysis of the GTPs of both the SRPand the SRPreceptor. The reaction has been characterizedin the bacterial system, where it has the unusual feature that Ffh activates hydrolysis by FtsY and FtsY reciprocally activates hydrolysis by Ffh.
TheTransLocon Forms a Pore o TheSec61 provides the channel trimericcomptex for proteins to passthrougha membrane. . A transtocating fromthe proteinpasses directty to withoutexposure ribosome to thetranslocon the cytosol. There is a basic problem in passinga (largely; hydrophilic protein through a hydrophobic membrane. The energeticsof the interaction between the chargedprotein and the hydrophobic lipids are highly unfavorable. However, a protein in the processof translocation across the ER membrane can be extracted by de-
Forms a Pore 10.10TheTranslocon
237
'':i.r.;j 'isa trimerof Sec6lthat j: Thetranslocon i:-i:.-:;::r formsa channel through themembrane. It is sealed onthe [ u m e n a( E [ Rs) i d e .
naturants that are effective in an aqueous environment. The same denaturants do not extract proteins that are resident components of the membrane. This suggeststhe model for translocation illustrated in i:*l,ii-:i:,:i.i.r,in which proteins that are part of the ER membrane form an aqueous channel through the bilayer. A translocating protein moves through this channei, interacting with the resident proteins rather than with the lipid bilayer. The channel is sealed on the lumenal side to stop free transfer of ions between the ER and the cytosol. The channel through the membrane is called the translocon. Its components have been identified in two ways: ResidenrER membrane proteins that are crosslinkedto translocating proteins are potential subunits of the channel, and sec mutants in yeast (named becausethey fail to secreteproteins) include a classthat causeprecursorsof secretedor membrane proteins to accumulate in the cytosol. These approaches together identified tli,e Sec6l complex,which consistsof three transmembrane proteins: Sec6lo,, p, and y. Sec6I is the major component of the translocon. In detergent (which provides a hydrophobic milieu that mimics the effectof a surrounding membrane), Sec61 forms cylindrical oligomers with a diameter of -85 A and a central pore of -20 A. tach oligomer c o n s i s t so I f o u r h e t e r o l r i m e r s . A similar trimeric structure for the channel is found in all organisms. In bacteria and archaeait is calledthe SecYcomplex. The Sec6lo subunit (or the correspondingSecYsubunit in b a c t e r i a / a r c h a e ap) r o v i d e s t h e p o r e t h r o u g h which the protein passesand is the best conserved in sequence.The pore is created from dimers of the trimeric complex, organizedback-
232
CHAPTER 10 ProteinLocatization
to-back so that the ot subunits are fused into a single channel. A channel complex in the membrane may contain two dimers, probably organized side-by-side,although only one can be accessedby the ribosome at any time. Is the channel a preexisting structure (as implied in the figure), or might it be assembled in responseto the associationof a hydrophobic signal sequencewith the lipid bilayer? Channels can be detectedby their ability to allow the passageof ions (measuredas a localizedchange in electrical conductance). Ion-conducting channels can be detected in the ER membrane, and their state depends on protein translocation. This demonstrates that the channel is a permanent feature of the membrane. A channel opens when a nascent polypeptide is transferred from a ribosome to the ER membrane. The translocating protein fills the channel completely, so ions cannot pass through during translocation. If. however, the protein is released by treatment with puromycin, then the channel becomes freely permeable. If the ribosomes are removed from the membrane, the channel closes,suggesting that the open state requires the presence of the ribosome. This suggeststhat the channel is controlled in responseto the presence of a t r a n s l o c a t i n gp r o t e i n . Measurements of the abilities of fluorescence-quenching agents of different sizes to enter the channel suggestthat it is large, with an inrernal diameter of 40 A ro -60 A. rhis is much larger than the diameter of an extended cr-helicalstretch of protein. It is also larger than the pore seen in direct views of the channel; this discrepancy remains to be explained. The aqueous environment of an amino acid in a protein can be measured by incorporating variant amino acids that have photoreactive residues.The fluorescenceof theseresiduesindicates whether they are in an aqueous or hydrophobic environment. Experiments with such probesshow that when the signalsequence is first synthesized in the ribosome, it is in an aqueous state, but is not accessibleto ions in the cytosol. It remains in the aqueous state throughout its interaction with a membrane. This suggeststhat the translocatingprotein travels directly from an enclosedtunnel in the ribosome into an aqueous channel in the membrane. In fact, accessto the pore is controlled (or "gated") onboth sidesof the membrane. Before attachment of the ribosome, the pore is closed on the lumenal side. *:3*tjfti:lt{-},;i}shows that when the ribosome attaches,it sealsthe pore on
the cytosolic side. When the nascent protein reachesa length of -70 amino acids-most likely, when it extends fully acrossthe channel-the pore opens on the lumenal side.Thus at all times, the pore is closedon one side or the other, maintaining the ionic integrities of the separate compartments. The translocon is versatileand can be used by translocatingproteins in severalways: . It is the means by which nascent proteins are transferred from cytosolic ribosomes to the lumen of ER (seeSection I 0. I I , Translocation Requires Insertion into the Translocon and (Sometimes) a Ratchet in the ER). r It is also the route by which integral membrane proteins of the ER system are transferred to the membrane; this requires the channel to open or disaggregate in some unknown way so that the protein can move laterally into the lipid bilayer (seeSectionI0.1 5. How Do Proteins Insert into Membranes?). . Proteins can also be transferred from the ER back to the cytosol; this is known as reverse translocation (see Section 10.12,ReverseTlanslocationSends Proteinsto the Cytosol for Degradation).
@
Translocation Requires Insertion into the Translocon and (Sometimes) a Ratchet in the ER
r Theribosome, SRP. andSRPreceptor aresufticient proteininto a transtocon. to inserta nascent o Proteins that areinsertedposttranstationatly require additionaI components in the cytosoland B i Pi n t h eE R . . BiPis a ratchetthat prevents a proteinfrom stipping backward.
The translocon and the SRP receptor are the basic components required for cotranslational translocation.When the Sec6I complex is incorporated into artificial membranes together with the SRPreceptor.it can support translocation of some nascent proteins. Other nascent proteins require the presenceof an additional component, the translocating chain-associatingmembrane (TRAM), which is a major protein that becomes crosslinked to a translocatinq nascent
ENDOPLASMIC RETICULUM
proteinis transferred {:l{.;{.1!{I directLy 1'"i.i.r,A nascent Theribosome seals fromthe ribosome to thetranslocon. side. the channel on the cvtosotic
SRP, l11:ilfirr requires thetranstocon, :il.;i:,r, Transtocation TRAM. andsignaIpeptidase. 5RPreceptor. Sec61, chain. TRAM stimulates the translocation of all proteins. The components of the translocon and their The functions are summarized in tr]*lJfi{:ii.r.;14. simplicity of this system makes severalimportant points. We visualize Sec6l as forming the channel and also as interacting with the ribosome. The initial targeting is made when the SRPrecognizesthe signalsequenceas the newly synthesized protein begins to emerge from the ribosome. The SRPbinds to the SRPreceptor, and the signal sequence is transferred to the translocon. When the signal sequence enters the translocon,the ribosomeattachesto Sec6l; this forms a seal.which prevents the pore from being exposed to the cytosol. Cleavageof the signal peptide does not occur in this system and therefore cannot be necessaryfor translocation
in the ER a Ratchet and(Sometimes) Insertion intotheTranstocon 10.11Translocation Reouires
irlr,ijlli:i;..:":i'ij Reverse transtocation usesthe trans[oproteinfromthe ERto thecytosot, conto sendanunfolded where it is degraded. Themechanism of puttingthetransloconinto reverse is not known. : : i r ' i , i r 'r ' : i ' B i Pa c t sa sa r a t c h e t t D o reveb na t ckward protein. diffusion of a transtocating p e r s e . I n t h i s s y s t e m ,c o m p o n e n t s o n t h e l u menal side of the membrane are not needed for translocation. Of course, the efficiency of the in vitro system is relatively low. Additional components could be required in vivo to achieve efficient transfer or to prevent other cellular proteins from interfering with the process. A more complex apparatus is required in certain casesin which a protein is inserted into a membrane posttranslationally.The same Sec6l complex forms the channel, but four other Sec proteins are also required. In addition, the chaperone BiP (a member of the Hsp70 class)and a supply of ATP are required on the lumenal side of the membrane. ii:r,i*i, ir,1,::-shows that BiP b e h a v e s a s a r a t c h e t . I n t h e a b s e n c eo f B i P , Brownian motion allows the protein to slip back into the cytosol.When BiP is present, though, it grabs the protein as it exits the pore into the ER. This stops the protein from moving backward. BiP does not pull the protein through; it just stops it from sliding back. (The reason why BiP is required for posttranslationaltranslocation, but not for cotranslationaltranslocation,may be that a newly synthesizedprotein is continuously extruded from the ribosome and therefore cannot slip backward.)
Reverse Translocation SendsProteins to the CytosoI for Degradation r Sec6l. transtocons canbe usedfor reverse transtocation of proteins fromthe ERinto the cytoso[.
234
CHAPTER 10 ProteinLocalization
Several important activities occur within the ER. Proteinsmove through the ER en route to a variety of destinations. They are glycosylated and folded into their final conformations. The ER provides a "quality control" system in which misfolded proteins are identified and degraded. The degradation itself, however, does not occur in the ER, but may require the protein to be exported back to the cytosol. The first indication that ER proteins are degraded in the cytosol and not in the ER itself was provided by evidence for the involvement of the proteasome, a large protein aggregate with several proteolytic activities. Inhibitors of the proteasomeprevent the degradationof aberrant ER proteins. Proteins are marked for cleavage by the proteasome when they are modified by the addition of ubiquitin, a small polypeptide chain. The important point to note now is that ubiquitination and proteasomal degradation both occur in the cytosol (with a minor proportion in the nucleus). Tlansport from the ER back into the cytosol o c c u r s b y a r e v e r s a l o f t h e u s u a l p r o c e s so f import. This is called reverse translocation (alsosometimes called retrotranslocation or dislocation). The Sec6I translocon is used. The conditions are different; for example, the translocon is not associatedwith a ribosome. Sonre mutations in Sec6l prevent reversetranslocation, but do not prevent forward translocation. This could be either because there is some differencein the processor (more likely) because these regions interact with other components that are necessaryfor reverse translocation. points out that we do not know irjlj{,iil--'ttl"}.1:-r how the channel is opened to allow insertion
of the protein on the ER side. Specialcomponents are presumably involved. One model is that misfoided or misassembled proteins are recognizedby chaperones,which transfer them to the translocon. In one particular case,human cytomegalovirus (CMV) codesfor cytosolicproteins that destroy newly synthesizedMHC class I (cellular major histocompatibility complex) proteins. This requires a viral protein product (USI 1), which is a membrane protein that functions in the ER. It interactswith the MHC proteins and probably conveys them into the translocon for reversetranslocation. The system involved in the degradationof aberrant ER proteins can be identified by mutations (in yeast) that lead to accumulation of aberrant proteins. In most casesa protein that misfolds (produced by a mutated gene) is degradedinstead of being transportedthrough the ER. Yeastmutants that cannot degradethe substrate fall into two classes:some identify components of the proteolytic apparatus,such as the enzymes involved in ubiquitination; others identify components of the transport apparatus, including Sec6l, BiP,and Sec63. Proteins involved in the system have also been identified by their interactions with the CMV system.The CMVprotein USI I passesthe MHC substratesto a protein called Derlin that is localizedin the ER membrane. Derlin in turn passesthe substratesto a cytosolicMPase called p97, which is probably responsiblefor pulling them out of the channel into the cytosol. Derlin is a homolog of yeastDerIp, one of the proteins identified by mutations as part of the reversetranslocation system.
Proteins Reside in Membranes by Means of Hydrophobic Regions r Group I proteins havethe N-terminus on thefar groupII proteins sideof the membrane; havethe opposite orientation. o Someproteins havemuttipte membrane-spanning domains.
All biological membranes contain proteins, which are held in the lipid bilayer by noncovalent interactions. The operational definition of an integral membrane protein is that it requires disruption of the lipid bilayer in order to be releasedfrom the membrane. A common feature in such proteins is the presence of at
Group I proteinshave the N-terminuson the far side and the C- terminusin ihe cvtosol
N{erminus i i { : i . i 1 i.i; i i , :t. ' G r o uIpa n dg r o u p I I t r a n s m e m b rparnoet e i n sh a v eo p p o s i t eo r i e n t a t i o nwsi t h r e g a r dt o t h e memDra ne.
least one transmembrane domain, consisting of an cr-helicalstretch of 2l to 26 hydrophobic amino acids.A sequencethat fits the criteria for membrane insertion can be identified by a hydropathy plot, which measuresthe cumulative hydrophobicity of a stretch of amino acids. A protein that has domains exposed on both sides of the membrane is called a transrnembrane protein. The association of a protein with a membrane takes severalforms. The topography of a membrane protein depends on the number and arrangement of transmembrane regions. When a protein has a single transmembrane region, its position determines how much of the protein is exposedon either side of the membrane. A protein may have extensive domains exposed on both sidesof the membrane or may have a site of insertion closeto one end, so that little or no material is exposedon one side.The length of the N-terminal or C-terminal tail that protrudes from the membrane near the site of insertion varies from insignificant to quite bulky. i:ti"i.llll:i;:,.:r'rshows that proteins with a single transmembrane domain fall into two classes. Group I proteins, in which the N-terminus faces the extracellular space,are more common than group II proteins, in which the orientation has
Regions 235 1 0 . 1 3 P r o t e i n sR e s i d ien M e m b r a n ebsy M e a n so f Hydrophobic
Proteinswith an odd numberof membranespanning domainshave the N- and C-terminion oppositesides
Proteins withan evennumberof membrane spanning domainshavetheN- andC-termini on thesameside
Theorientations of theterminiof muttip L em e m b r a n e - s p a n n p irnogt e i nds e p e n dosn w h e t h e r thereis anoddorevennumber oftransmembrane seqments.
been reversedso that the N-terminus facesthe cytoplasm. Orientation is determined during the insertion of the protein into the ER. - : :: .: shows orientations for proteins t h a t h a v e m u l t i p l e m e m b r a n e - s p a n n i n gd o mains. An odd number means that both termini of the protein are on opposite sidesof the membrane, whereas an even number implies that the termini are on the same face.The extent of the domains exposedon one or both sidesis determined by the locations of the transmembrane domains. Domains at either terminus may be exposed, and internal sequencesbetween the domains "loop out" into the extracellular spaceor cytoplasm. one common type of structure is the seven-membrane passage or "serpentine" receptor; another is the twelvemembrane passage component of an ion channel. Does a transmembrane domain itself play any role in protein function besidesallowing the protein to insert into the lipid bilayer? In the simple group I or II proteins, it has little or no additional function; often it can be replaced by any other transmembrane domain. However, transmembrane domains play an important role in the function of proteins that make
236
CHAPTER 10 ProteinLocatization
multiple passesthrough the membrane or that have subunits that oligomerize within the membrane. The transmembrane domains in such casesoften contain polar residues,which are not found in the single membrane-spanning domains of group I and group II proteins. Polar regions in the membrane-spanning domains do not interact with the lipid bilayer, but instead interact with one another. This enables them to form a polar pore or channel within the lipid bilayer. Interaction between such transmembrane domains can create a hydrophilic passage through the hydrophobic interior of the membrane. This can allow highly charged ions or molecules to pass through the membrane and is important for the function of ion channels and transport of ligands. Another casein which conformation of the transmembrane domains is important is provided by certain receptors that bind lipophilic ligands. In such cases,the transmembrane domains (rather than the extracellular domains) bind the ligand within the olane of the membrane.
Anchor Sequences Determine Protein 0rientation e An anchor sequence haltsthe passage of a protein throughthe translocon. Typicatty thisis located at the C-terminaI endandresutts in a groupI orientation in whichthe N-terminus haspassed throughthe membrane. o A combined signal-anchor sequence canbe usedto inserta oroteinintothe membrane andanchor the siteof insertion. Typicatty thisis internaI and resutts in a groupII orjentation in whichthe N-terminus is cytosolic.
Proteins that are secreted from the cell pass through a membrane while remaining in the aqueous channel of the translocon. By contrast, proteins that reside in membranes start the processin the same way, but then transfer from the aqueous channel into the hydrophobic environment. The challenge in accounting for insertion of proteins into membranes is to explain what distinguishes transmembrane proteins from secretedproteins and causesthis transfer. The pathway by which proteins of either type I or type II are inserted into the membrane follows the same initial route as that of secretory proteins, relying on a signal sequencethat functions cotranslationally. Proteins that are to
remain within the membrane, however, possessa second, stop-transfer signal. This takes the form of a cluster of hydrophobic amino acids a d j a c e n t t o s o m e i o n i c r e s i d u e s .T h e c l u s t e r seryesas an anchor that latcheson to the mernbrane and stops the protein from passing right through. A surprisingproperty of anchor sequences is that they can function as signal sequences when engineered into a different location. When placed into a protein lacking other signals,such a sequencemay sponsormembrane translocation. One possibleexplanation for these results is that the signal sequenceand anchor sequence interact with some common component of the apparatus for translocation. Binding of the signal sequence initiates translocation, but the appearanceof the anchor sequence displaces the signal sequenceand halts transfer. Membrane insertion startsby the insertion a of signal sequence in the form of a hairpin Ioop, in which the N-terminus remains on the cytoplasmic side. TWo features determine the position and orientation of a protein in the membrane: whether the signal sequenceis cleaved and the location of the anchor sequence. The insertion of type I proteins is illustrated 't i n i l i ' i 1 r i . : i :: . : . : 1 i .T h e s i g n a l s e q u e n c e i s N-terminal. The location of the anchor signal determines when transfer of the protein is halted. When the anchor sequence takes root in the membrane, domains on the N-terminal side will be locatedin the lumen, whereasdomains on the C-terminal side are located facing the cytosol. A common location for a stop-transfer sequenceof this type is at the C-terminus. As shown in the figure, transfer is halted only as the last sequencesof the protein enter the membrane. This type of arrangement is responsible for the location in the membrane of many proteins, including cell surface proteins. Most of the protein sequenceis exposed on the lumenal side of the membrane, with a small or negligible tail facing the cytosol. \pe II proteins do not have a cleavable leader sequenceat the N-terminus. The signal sequence is instead combined with an anchor sequence.We imagine that the general pathway for the integration of type I proteins into the membrane involves the steps illustrated in irii:,itii!-.i:.r::'-':. The signal sequence enters the membrane, but the joint signal-anchorsequence does not passthrough. It stays,instead, in the membrane (perhaps interacting directly with the lipid bilayer), while the rest of the growing polypeptide continues to loop into the ER.
enter in membranes that reside lil.;rji.il:r ii.r.,rl Protejns proteins, but transferis by the samerouteas secreted passes into the memhattedwhenan anchorsequence the butkof the is at the C-terminus, brane. If the anchor on proteinpasses andis exposed throughthe membrane thefar surface. The signal-anchorsequenceis usually internal, and its location determines which parts of the protein remain in the cytosol and which are e x t r a c e l l u l a r . E s s e n t i a l l ya l l t h e N - t e r m i n a l sequencesthat precede the signal-anchor are exposedto the cytosol.Most often the cytosolic tail is short, -6 to l0 amino acids. In effect the N-terminus remains constrainedwhile the rest of the protein passesthrough the membrane. This reversesthe orientation of the protein with regard to the membrane. The combined signal-anchor sequencesof type II proteins resemble cleavable signal s e q u e n c e s i.. i . i , i l i , l i .r I g i v e s a n e x a m p l e .A s with cleavable leader sequences, the amino acid composition is more important than the actual sequence.The regions at the extremities of the signal-anchor carry positive charges; the central region is uncharged and resembles
Protein0rientation 237 Determine 10.14AnchorSequences
a hydrophobic core of a cleavableleader. Mutations to introduce charged amino acids in the core region prevent membrane insertion; mutations on either side prevent the anchor from working, so the protein is secretedor Iocated in an incorrect compartment. The distribution of charges around the anchor sequencehas an important effect on the orientation of the protein. More positive charges are usuaily found on the cytoplasmic side (N-terminal side in type II proteins). If the positive chargesare removed by mutation, the orientation of the protein can be reversed. The effect of chargeson orientation is summarized by the "positive inside" rule, which statesthat the side of the anchor with the most positive chargeswill be located in the cytoplasm. The positive chargesin effect provide a hook that latcheson to the cytoplasmicside of the membrane, which in turn controls the direction in which the hydrophobic region is inserted, thus determining the orientation of the protein.
HowDoProteins Insert into Membranes? . Transfer of transmembrane domains fromthe transtocon into the tipidbitayer is triggered by the interaction of thetransmembrane reqionwith thetrans[ocon. A combined signa[-anchor sequence causes a proteinto reverseits orientation.so that the N-terminusremains on the innerfaceandthe C-term'inus is exoosed on the outerface of the membrane.
We have a reasonable understanding of the p r o c e s s e sb y w h i c h s e c r e t e d p r o t e i n s p a s s through membranes and of how this relatesto the insertion of the single-membrane spanning
ir,,r"ii4 -'nitiation I ,Astl I iiiri-l Cytoplasmic I
{:t I S!; V
HYdroPhobiccore --.-.....g
Membrane-spanning region
O
ThesignaL-anchor of influenza neuraminjdase is locatedctoseto the N-terminus andhasa hydrophobic
238
CHAPIER 10 ProteinLocalization
proteins Fttrj.tit{ are membrane synthesized ii},}.i Newly into the Lipid fromthetranstocon ab[eto transfer[ateral.ty is not known. of transfer bil"ayer. Themechanism
f 3*iilt* :i.#.,1i: Howdoesa transmembrane make Drotein thetransition frommoving through a proteinaceous channetto interacting directlywiththe tipidbitayer?
group I and group II proteins. We cannot yet explain the details of insertion of proteins with multiple membrane-spanning domains. W e u n d e r s t a n d h o w a s e c r e t e dp r o t e i n passesthrough a membrane without any conflict, but it is more difficult to apply the same model to a protein that resides in the memii}"lf illustrates the difference brane. F5{";iiF.f between the organization of a translocatingprotein, which is protected from the lipid bilayer by the aqueous channel, and a transmembrane protein, which has a hydrophobic segment directly in contact with the membrane. The critical question is how a protein is transferred from the proteinaceous channel into the lipid bilayer. The possibility that there is a mechanism for transferring hydrophobic transmembrane domains directly from the channel into the membrane is suggestedin t'I{ii}til it't.}"1.This idea is supported by observations of an in vitro system that measured transfer into a lipid environment for proteins with different transmemb r a n e d o m a i n s . W h e n t h e d o m a i n p a s s e da threshold of hydrophobicity, the protein could pass from a channel consisting of Sec6I and TRAM into the lipid bilayer. In addition to overall hydrophobicity, the locations of polar residues
within the transmembrane segments have an important effect. The simplest explanation is that the structure of the channel allows the translocating protein to contact the lipid bilayer, so that a sufficiently hydrophobic segment can simply partition directly into the lipid. The structure of the translocon suggeststhat there could be a gate located between two helices that is used for the transfer. It has always been a common assumption that, whatever the exact mechanism for transferring the transmembrane segment into the membrane, it is triggered by the presenceof the transmembrane sequencein the pore. However, changesin the pore occur earlier in responseto the synthesis of the transmembrane sequence in the ribosome. When a secreted protein passes through the pore, the channel remains sealed on the cytosolic side but opens on the lumenal side after synthesisof the first seventy residues. As soon as a transmembrane sequencehas been fully synthesized, though-that is, while it is still entirely within the ribosome-the pore closes on the lumenal side. How this change relates to the transfer of the transmembrane sequence into the membrane is not clear. The process of insertion into a membrane has been characterizedfor both type I and type II proteins, in which there is a single transmembrane domain. How is a protein with multiple membrane-spanning regions inserted into a membrane? Much lessis known about this process, but we assume that it relies on sequencesthat provide signal and/or anchor capabilities.One model is to supposethat there is an alternating series of signal and anchor sequences.Translocation is initiated at the first signal sequenceand continues until stopped by the first anchor. It then is reinitiated by a subsequent signal sequence until stopped by the next anchor. It is possible that there are multiple pathways for integration into the
Insertinto Membranes?239 10.15HowDoProteins
membrane, becausein some casesa transmembrane domain seemsto move into the lipid bilayer as soon as it enters the translocon.In other cases, though, there can be a delay until other transmembrane regions have been synthesized.
@
Posttranslational Membrane Insertion Depends on Leader Sequences
r N-termina[ provide leadersequences the information that attowsoroteins to associate with mitochondriaI or ch[oroplast membranes.
!:l+U*l i L:"i:.;Leader sequences attowproteins to recognizemitochondrial or chtoroplast surfaces by a posttrans[ationaI Drocess.
Mitochondria and chloroplasts synthesize only some of their proteins. Mitochondria synthesize only -10 organelle proteins; chloroplasts synthesize -50 proteins. The majority of organelle proteins are synthesizedin the cytosol by the same pool of free ribosomes that synthesize cytosolic proteins. They must then be imported into the organelle. Many proteins that enter mitochondria or chloroplastsby a posttranslational processhave Ieader sequencesthat are responsiblefor primary recognition of the outer membrane of the organelle. As shown in the simplified diagram of iris#R$t*"i+, the leader sequenceinitiates the interaction between the precursor and the organelle membrane. The protein passesthrough the membrane, and the leader is cleaved by a protease on the organelle side. The leadersof proteins imported into mitochondria and chloroplasts usually have both hydrophobic and basic amino acids.They consist of stretchesof uncharged amino acidsintenupted by basicamino acids,and they lack acidic amino acids.There is little other homology. An example is given in ili*{Jfif ':i}.i5. Recognition of the leader does not depend on its exact sequence,but rather on its ability to form an amphipathic helix, in which one face has hydrophobic amino acidsand the other face presentsthe basic amino acids. The leader sequence contains all the information needed to localize an organelle protein. The ability of a Ieader sequencecan be testedby constructing an artificial protein in which a leader from an organelle protein is joined to a cytosolic protein. The experiment is performed by constructing a hybrid gene, which is then translated into the hybrid protein. Severalleader sequenceshave been shown by such experiments to function independently to target any attached sequence to the mitochondrion or chloroplast. For example, if the Ieadersequencegiven in Figure 10.35is attached to the cytosolic protein DHFR (dihydrofolate reductase),the DHFR becomeslocalizedin the mitochondrion.
+
Initiation
Matrixtargetingsignal
fir:tji{.i:ill"3* Theleader sequence of yeastcytochrome c oxjdase subunitIV consjsts of twenty-five neutraI andbasicamino acids.Thefirsttwetveaminoacidsaresufficient to transport pol.ypeptide anyattached into the mitochondriaI matrix.
240
CHAPTER 10 ProteinLocatization
The leader sequence and the transported protein represent domains that fold independently. Irrespective of the sequence to which it is attached, the leader must be able to fold into an appropriate structure to be recognized by receptors on the organelle envelope. The attached polypeptide sequenceplays no part in recognition of the envelope. What restrictions are there on transporting a hydrophilic protein through the hydrophobic membrane? An insight into this question is given by the observation that methotrexate, a ligand for the enzyme DHFR, blocks transport into mitochondria of DHFR fused to a mitochondrial leader. The tight binding of methotrexate prevents the enzyme from unfolding when it is translocated through the membrane. The sequence of the transported protein is irrelevant for targeting purposes; however, in order to follow its leader through the membrane, the protein requires the flexibility to assume an unfolded conf ormation. Hydrolysis of ATP is required both outside and inside for translocation acrossthe membrane. It may be involved with pushing the protein from outside and pulling from inside. In the casesof mitochondrial import and bacterial export, there is also a requirement for an electrochemical potential acrossthe inner membrane to transfer the amino terminal part of the leader.
@
imported into a mitochondrion is to move through both membranes into the matrix. This property is conferred by the N-terminal part of the leader sequence.A protein that is localized within the intermembrane spaceor in the inner membrane itself requires an additional signal, which specifies its destination within the organelle. A multipart leader contains signals that function in a hierarchical manner, as sum1fi"}$. The first part of the marized in iri{-;,,iFil leader targets the protein to the organelle, and the second part is required if its destination is elsewhere than the matrix. The two parts of the leader are removed by successivecleavages. Cytochrome cl is an example. It is bound to the inner membrane and faces the intermembrane space.Its leader sequenceconsistsof sixtyone amino acidsand can be divided into regions with different functions. The sequence of the first thirty-two amino acids alone, or even the N-terminal half of this region, can transport DIIFR all the way into the matrix. Thus the first part of the leader sequence(thirty-two N-terminal amino acids) comprises a matrix-targeting signal. The
A Hierarchy of Sequences Determines Location within0rganelles
partof a leadersequence TheN-terminal targetsa proteinto the mitochondriaI matrixor chloroptast [umen. An adjacent sequence cancontrolfurthertargeting to a membrane or the intermembrane soaces. Thesequences arecteaved successivety fromthe orotein. The mitochondrion is surrounded by an envelope consisting of two membranes. Proteins imported into mitochondria may be located in the outer membrane, the intermembrane space, the inner membrane, or the matrix. A protein that is a component of one of the membranes may be oriented so that it facesone side or the other. What is responsible for directing a mitochondrial protein to the appropriate compartment? The "default" pathway for a protein
lnnermembrane
j #"}*, Mitochondria for protein havereceptors F:td;tJi;1{ t r a n s p o irnt t h e o u t e ra n di n n e rm e m b r a n eRse. c o g n i through mayleadto transport tionat theoutermembrane b o t h r e c e p t o risn t o t h e m a t r i x .w h e r et h e l e a d e irs signa[,it may If jt hasa membrane-targeting cleaved. be reexported. Locationwithin 0rganeltes Determines of Sequences 10.17 A Hierarchy
247
lnitiation
I
|
charged leaderregiontor mitochondrialtargeting-f>
Ser
9@ev Cleavage1
Continuous uncharged leaderregiontargetsinnermembrane
itnt
Cleavage 2
'lla; '
FIfi:JRii*.31 Theleader ofyeastcytochrome c1contains an N-terminaI regionthattargets theproteinto the mitochondrion, followed proteinto theinnermembya regionthattargets the (cteaved) Theleader brane. is removed by two cteavage events. intact leader, however, transports an attached sequence-such as murine DIIFR-into the intermembrane space. What prevents the protein from proceeding past the intermembrane space when it has an intact leader? The region following the matrixtargeting signal (comprising nineteen amino acids of the leader) provides another signal that localizes the protein at the inner membrane or within the intermembrane space.For working purposes, we call this the membrane-targeting signal. The two parts of a leader that contains both tlpes of signal have different compositions. As indicated in Fi*L:Hf ':*. j l, the thirty-five N-terminal amino acidsresemble other organelle leader sequencesin the high content of uncharged amino acids, punctuated by basic amino acids. The next nineteen amino acids,however, comprise an unintemrpted stretch of r.nchargedamino acids that is long enough to span a lipid bilayer. This membrane-targeting signal resembles the sequencesthat are involved in protein translocation into membranes of the ER (seeSection 10.7, Signal SequencesInitiate Translocation). Cleavageof the matrix-targeting signal is the sole processingevent required for proteins that reside in the matrix. This signal must also be cleavedfrom proteins that residein the intermembrane space;however, following this cleav-
242
CHAPTER 10 ProteinLocatization
age, the membrane-targeting signal (which is now the N-terminal sequenceof the protein) directs the protein to its destination in the outer membrane, intermembrane space, or inner membrane. The signal then, in turn, is cleaved. The N-terminal matrix-targeting signal functions in the same manner for all mitochondrial proteins. Its recognition by a receptor on the outer membrane leads to transport through the two membranes. Note that the same protease is involved in cleaving the matrix-targeting signal, irrespective of the final destination of the protein. This protease is a water soluble, Mg2+dependent enzyme that is located in the matrix. Thus the N-terminal sequence must reach the matrix, even if the protein ultimately will reside in the intermembrane space. Residence in the matrix occurs in the absence of any other signal. If there is a membrane-targeting signal, however, it is activated by cleavageof the matrix-targeting signal. The remaining part of the leader (which is now N-terminal) then causesthe protein to take up its final destination. The nature of the membrane-targeting signal is controversial. One model holds that tne entire protein enters the matrix, after which the membrane-targeting signal causesit to be reexported into or through the inner mem-
brane. An alternative model proposes that the membrane-targeting sequencesimply prevents the rest of the protein from following the leader through the inner membrane into the matrix. Whichever model applies, another protease (locatedwithin the intermembrane space)completes the removal of leader sequences. Passagethrough chloroplast membranes is achieved in a similar manner. 5li:lgS#t*.*{; illustrates the variety of locations for chloroplast proteins. They passthe outer and inner membranes of the envelope into the stroma, a process involving the same types of passageas into the mitochondrial matrix. Some proteins are transported yet further, though, acrossthe stacks of the thylakoid membrane into the lumen. Proteins destined for the thylakoid membrane or Iumen must crossthe stroma en route. Chloroplast targeting signalsresemblemitochondrial targeting signals.The leader consists of -50 amino acids, and the N-terminal half is needed to recognizethe chloroplast envelope. A cleavagebetween positions20 and 25 occurs during or following passageacrossthe envelope, and proteins destined for the thylakoid membrane or lumen have a new N-terminal Ieader that guides recognition of the thylakoid membrane. There are several (at least four) different systems in the chloroplast that catalyze import of proteins into the thylakoid membrane. The general principle governing protein transport into mitochondria and chloroplasts, therefore, is that the N-terminal part of the Ieader targets a protein to the organelle matrix, and an additional sequence (within the leader) is needed to localize the protein at the outer m e m b r a n e , i n t e r m e m b r a n e s p a c e ,o r i n n e r membrane.
.
.
.
comp[ex(es) formreceptor lT{,lJSl1iJ":,!jT0Mproteins across the mitochondrfor transtocation that areneeded iaI outermembrane.
There are different receptors for transport through each membrane in the chloroplast and mitochondrion. In the chloroplast they are called TOC and TIC, and in the mitochondrion they are called TOM and TIM, referring to the outer and inner membranes, respectively. The TOM complex consists of -9 proteins, many of which are integral membrane proteins. Transport throughthe outerandinner A general model for the complex is shown in mitochondriaI membranes usesdifferent f].ilLlq{ ;i}.;,ti}.The TOM aggregate has a size of receptor complexes. >500 kD, with a diameter of -138 A, and forms TheTOM(outermembrane) comptex is a large proteinsaredirected in whichsubstrate comptex an ion-conducting channel. A complex conto theTom40 channel by oneof two subcomptexes. tains 2 to I individual rings of diameter 75 A, TIM(innermembrane) Different complexes are e a c h w i t h a p o r e o f d i a m e t e r2 0 A . protein on whether useddepending the substrate Tom40 is deeply imbedded in the memis targeted to theinnermembrane or to the lumen. brane and provides the channel for translocapassdirectlyfromthe TOMto the TIM Proteins tion. It contacts preproteins as they passthrough comDtex. the outer membrane. It binds to three smaller
@
.
fromthe the chloroptast Fi*#flil 11.;.:i;t: A proteinapproaches hatfof the leader TheN-terminaI leader. cytosoI witha -50 residue it intothestroma. passage orthrough intotheenvetope sponsors passage. Cleavage occurs duringenvelope
Innerand0uter MitochondriaL Mem branes HaveDifferent TransLocons
Transtocons 243 M[ e m b r a n eHsa v eDifferent 1 0 . 1 8 I n n e ra n dO u t e rM i t o c h o n d r i a
FIGURE 10.40 Timproteins formthecomptex fortranstocationacross the mitochondriaI innermembrane.
proteins. Tom5, -6, and -7, which may be components of the channel or assembly factors. There are two subcomplexes that provide surface receptors. Tom20 and Tom22 form a subcomplex with exposed domains in the cytosol. Most proteins that are imported into mitochondria are recognized by the Tom20,22 subcomplex, which is the primary receptor and r e c o g n i z e st h e N - t e r m i n a l s e q u e n c e o f t h e translocatingprotein. Tom37,70,71provides a receptor for a smaller number of proteins that have internal targeting sequences. When a protein is translocated through the TOM complex, it passesfrom a state in which it is exposed to the cytosol into a state in which it is exposed to the intermembrane space.It is n o t , h o w e v e r , u s u a l l y r e l e a s e d ,b u t i n s t e a d is transferred directly to the TIM complex. It is possibleto trap intermediates in which the leader is cleaved by the matrix protease, leaving a major part of the precursor exposed on the cytosolic surface of the envelope. This suggeststhat a protein spans the two membranes during pass a g e .T h e T O M a n d T I M c o m p l e x e s d o n o t appear to interact directly (or at least do not form a detectable stable complex), and they may therefore be linked simply by a protein in transit. When a translocating protein reaches the intermembrane space,the exposed residues may immediately bind to a TIM complex, whereas the rest of the protein continues to translocate through the TOM complex. There are two TIM complexes in the inner membrane. The TimlT-23 complex translocates
CHAPTER 10 ProteinLocatization
proteins to the lumen. Substratesare recognized by their possessionof a positively charged N-terminal signal. Transmembrane proteins Timl7 through Tim23 comprise the channel. FIGURE 10-40shows that they are associatedwith Ttm44 on the matrix side of the membrane. Tim 44 in turn binds the chaperone Hsp70. This is also associatedwith another chaperone, Mge, the counterpart to bacterial GrpE. This association ensures that when the imported protein reaches the matrix, it is bound by the Hsp70 chaperone. The high affinity of Hsp70 for the unfolded conformation of the protein as it emerges from the inner membrane helps to "pull" the protein through the channel. A major chaperone activity in the mitochondrial matrix is provided by Hsp60 (which forms the same sort of structure as its counterpart GroEL). Association with Hsp60 is necessary for joining of the subunits of imported proteins that form oligomeric complexes. An imported protein may be "passed on" from Hsp70 to Hsp60 in the process of acquiring its proper conformation. The Tim22-54 complex translocatesproteins that reside in the inner membrane. How does a translocating protein finds its way from the TOM complex to the appropriate TIM complex? TWo protein complexes in the intermembrane spaceescort a translocating protein from TIM to TOM. The Tim9- l0 and Tim8I3 complexes act as escortsfor different sets of substrate proteins. Tim9- l0 may direct its substratesto either Tim22-54 orTtm23-17 , whereas Tim8-ll directs substratesonly to Tim22-54. Some substratesdo not use either Timg-I0 or Tim8-13, so other pathways must also exist. The pathways are summarized in FIGURH 1Q"41. What is the role of the escorting complexes? They may be needed to help the protein exit from the TOM complex as well as for recognizing the TIM complex. FIGURT 10"42shows that a translocating protein may pass directly from the TOM channel to the Tim9,l0 complex, and then into the Tim22-54 channel. A mitochondrial protein folds under different conditions before and after its passage through the membrane. Ionic conditions and the chaperones that are present are different in the cytosol and in the mitochondrial matrix. It is possible that a mitochondrial protein can attain its mature conformation only in the mitochondrion.
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terized for eukaryotic cells,and we can recognize some related components. litriiiii: !t.-i.,1. shows that proteins that are exported from the cytoplasm have one of four fates: . to be inserted into the inner membrane, . to be translocated through the inner membrane to rest in the periplasm, . to be inserted into the outer membrane, or o t o b e t r a n s l o c a t e dt h r o u g h t h e o u t e r membrane into the medium. Different protein complexes in the inner membrane are responsible for transport of proteins depending on whether their fate is to pass through or stay within the inner membrane. This resembles the situation in mitochondria, where different complexes in each of the inner and outer membranes handle different subsets of protein substratesdepending on their destin a t i o n s ( s e eS e c t i o n 1 0 . 1 6 , P o s t t r a n s l a t i o n a l Membrane Insertion Depends on Leader S e q u e n c e s )A d i f f e r e n c e f r o m i m p o r t i n t o organellesis that transfer in E. colimay be either co- or posttranslational.Some proteins are secretedboth cotranslationally and posttranslationally, and the relative kinetics of translation versus secretionthrough the membrane could determine the balance. Exported bacterialproteins have N-terminal leader sequenceswith a hydrophilic N-terminus and an adjacent hydrophobic core. The Ieader is cleavedby a signalpeptidasethat recognizes precursor forms of several exported proteins. The signal peptidase is an integral membrane protein located in the inner membrane. Mutations in N-terminal leadersprevent secretion; t h e y a r e s u p p r e s s e db y m u t a t i o n s i n o t h e r genes,which are thus defined as components of the protein export apparatus.Severalgenes g i v e n t h e g e n e r a l d e s c r i p t i o n s e ca r e i m p l i cated in coding for components of the secretory apparatusby the occurrenceof mutations that block secretion of many or all exported proterns.
@
TheSecSystem Transports Proteins intoandThrough theInnerMembrane
. Thebacteriat transtocon in the inner SecYEG Sec61 membrane is related to theeukarvotic transtocon. o Various in directing areinvolved chaperones proteins to the translocon. secreted
i 1r,i.liir.1 rrr r,', BacteridI ejtherposttransproteins maybeexported andmaybe [ocatedwithineither Lational"Ly or cotranslationat[y. or maybesecreted. space. membrane or the periplasmic
trans[oi:li;iiiiii r,:1,i,':TheSecsystemhasthe SecYEG protheSecA-associated in themembrane. conembedded theSecB proteins throughthe channet, teinthat pushes proteins andthe to SecA, nascent thattransfers chaperone signaIfrom the N-terminaI peptidase that cleaves signal. protein. thetranstocated
There are several systemsfor transport through the inner membrane. The best characterizedis the Secsystem,whose components are shown i n i i i , i . i i ; ii : , , , ' , .T h e t r a n s l o c o nt h a t i s e m b e d ded in the membrane consistsof three subunits that are related to the components of mammalian/yeast Sec6I. Each of the subunits is an integral transmembraneprotein. (SecYhas ten transmembrane segments;SecEhas three') The functional translocon is a trimer with one copy of each subunit. The major pathway for directing proteins to the translocon consistsof SecB and SecA.SecBis a chaperonethat binds to the
the InnerMembrane 247 into andThrough Proteins 10.21ThesecsystemTransports
;li.:i..:TlI proteins transfer to thetransto""-:: SecB/SecA conthat passthroughthe membrane. 4.4SRNAtransfers proteins that enterthe membrane. nascent protein to control its folding. It transfers the protein to SecA,which in turn transfers it to the translocon. r:j..ii:lii:ri-.,irir, Showsthat there are two pre_ dominant ways of directing proteins to the Sec channel: . the SecB chaperone,and . the 4.5S RNA-based SRP. Several chaperones can increase the efficiency of bacterial protein export by preventing premature folding; they include "trigger factor" (characterizedas a chaperonethat assistsexport), G r o E L ( s e e S e c t i o n I 0 . 5 , C h a p e r o n e sA r e Needed by Newly Synthesized and by Denat u r e d P r o t e i n s ,a n d S e c t i o n 1 0 . I 8 , I n n e r a n d Outer Mitochondrial Membranes Have Different Translocons),and SecB (identified as the product of one of the secmutants). SecBis the least abundant of these proteins; however, it has the major role in promoting export. This role comprisestwo functions: first, SecBbehaves as a chaperone and binds to a nascent protein to retard folding. It cannot reverse the change in structure of a folded protein, so it does not function as an unfolding factor. Its role is therefore ro inhibit improper folding of the newly synthesizedprotein. Second, SecBhas an affinity for the protein SecA.This allows it to target a precursor protein to the membrane. The SecBSecYEGpathway is used for translocation of proteins that are secreted into the periplasm and is summarized in ii{li:i:i: .:r:.4?. SecA is a large peripheral membrane protein that has alternative ways to associatewith the membrane. As a peripheral membrane protein, it associateswith the membrane bv virtue
248
CHAPTER 10 ProteinLocalization
FIS{JRf proteinto SecA, 1*"47 SecB transfers a nascent whjchinserts the proteinintothechannet. Translocation requires hydrotysis ofATPanda protonmotive force.SecA undergoes cyctes of association anddissociation withthe channel andprovides the motiveforceto pushthe protein through.
of its affinity for acidic lipids and for the Secy component of the translocon, which are part of a multisubunit complex that provides the translocase function. In the presence of other proteins (SecDand SecF),however, SecA can be found as a membrane-spanning protein. It probably provides the motor that pushes the substrate protein through the SecYEG translocon. SecArecognizesboth SecBand the precursor protein that it chaperones; most likely, features of the mature protein sequence as well as its leader are required for recognition. SecA has an ATPaseactivity that depends upon binding to lipids, Secl and a precursor protein. The MPase functions in a cyclical manner during translocation. After SecAbinds a precursor protein it binds ATP, and -20 amino acids are translocated through the membrane. Hydrolysis of ATP is required to release the precursor
from SecA. The cycle may then be repeated. Precursor protein is bound again to provide the spur to bind more ATP,translocate another segment of protein, and releasethe precursor. SecA may alternate between the peripheral and integral membrane forms during translocation; with each cycle. a 30 kD domain of SecAmay insert into the membrane and then retract. Another processcan also undertake translocation. When a precursor is releasedby SecA, it can be driven through the membrane by a protonmotive force (that is, an electrical potential acrossthe membrane). This processcannot initiate transfer through the membrane, but it can continue the processinitiated by a cycle of SecA ATPaseaction. Thus after or between cycles of the SecA-AIP driven reaction, the protonmotive force can drive translocation of the precursor. The E. coli ribonucleoprotein complex of 4.5S RNA with Ffh and FtsYproteins is a counterpart to the eukaryotic SRP(seeSection 10.9, The SRP Interacts with the SRP Receptor). It probably plays the role of keeping the nascent protein in an appropriate conformation until it interacts with other components of the secretory apparatus.It is needed for the secretionof some, but not all, proteins. As we see in Figure I0.46, its substratesare integral membrane proteins. The basisfor differential selection of substratesis that the E. coliSRPrecognizes a n a n c h o r s e q u e n c ei n t h e p r o t e i n ( a n c h o r sequencesby definition are present only in integral membrane proteins). Chloroplasts have counterparts to the Ffh and FtsY proteins, but do not require an RNA component.
@
Sec-Independent Translocation Systems in E. coli
o E. coliandorganetles haverelatedsystems for orotei n trans[ocation. o Onesystem al[owscertainproteins to insertinto membranes withouta transtocation apparatus. . YidCis homologous for to a mitochondrial system proteins into theinnermembrane. transferring r Thetat systemtransfers proteinswith a twin motifinto the periplasmic space. arginine
The most striking alternative system for protein translocation in E. coli is revealed by the liil"+i;shows coat protein of phage Ml3. iil"':"ifiil that this does not appear to require any translo-
fj-lri.:lii:I i,riir Ml,3coatproteininsertsinto the jnner fotcontact. aninitiaIetectrostatic membrane bymaking Transtocasequences. of hydrophobic I'owed byjnsertion c t e r a c t i o nas n d a t i o n i s d r i v e nb y h y d r o p h o b i n entersthe sequence protonmotive forceuntiIthe anchor memDrane. cation apparatus!It can insert posttranslationally into protein-free liposomes. Targeting the protein to the membrane requires specific sequences(comprising basicresidues) in the Nand C-terminal regions of the protein. They may interact with negatively charged heads of phospholipids. The protein then enters the membrane by using hydrophobic groups in its N-terminal leader sequence and an internal anchor sequence. Hydrophobicity is the main driving force for translocation, but it can be assistedby a protonmotive force that is generated between the positively chargedperiplasmic side of the membrane and an acidic region in the protein. This drives the protein through the membrane, and leader peptidasecan then cleavethe N-terminal sequence.The generality of this mechanism in bacteria is unclear; it may apply only
in E.coli Systems Transtocation 10.22Sec-Independent
249
to the specialcaseof bacteriophagecoat proteins. Some chloroplast proteins may insert into the thylakoid membrane by a similar pathway. Mutations in the gene yidCblock insertion of proteins into the inner membrane. YidC is homologous to the protein Oxalp that is required when proteins are inserted into the innermitochondrial membrane from the matrix. It can function either independently of SecYEG or in conjunction with it. The insertion of some of the YidC-dependentproteins requiresSecYEG, which suggeststhat YidC acts in conjunction with the translocon to divert the substrate into membrane insertion as opposed to secretion. Other proteins whose insertion dependson YidC do not require SecYEG:It seemslikely that some other (unidentified) functions are required instead of the translocon. The tat system is named for its ability to transport proteins bearing a twin arginine targeting motif. It is responsiblefor translocation of proteins that have tightly bound cofactors. This may mean that they have limitations on their ability to unfold for passagethrough the membrane. This would be contrary to the principle of most translocationsystems.where the protein passesthrough the membrane in an unfolded stateand then must be folded into its mature conformation after passage.This system is related to a system in the chloroplast thylakoid lumen called Hcf 106. Both of these systemstransport proteins into the periplasm.
@
Summary
A protein that is inserted into, or passesthrough, a membrane has a signal sequencethat is recognized by a receptor that is part of the membrane or that can associatewith it. The protein passes through an aqueous channel that is created by transmembrane protein(s) that reside in the membrane. In almost all cases.the protein passes through the channel in an unfolded form, and associationwith chaperoneswhen it emergesis necessaryin order to acquire the correct conformation. The major exception is the peroxisome. where an imported protein in its mature conformation binds to a cytosolic protein that carriesit through the channel in the membrane. Synthesis of proteins in the cytosol stafis on "free" ribosomes.Proteinsthat are secreted from the cell or that are inserted into membranes of the reticuloendothelial system start with an N-terminal signal sequencethat causes the ribosome to become attached to the mem-
250
CHAPTER 10 ProteinLocalization
brane of the endoplasmic reticulum. The protein is translocated through the membrane by cotranslationaltransfer.The processstartswhen the signal sequence is recognized by the SRP (a ribonucleoprotein particle). which interrupts translation. The SRPbinds to the SRPreceptor in the ER membrane and transfers the signal sequenceto the Sec6l/TRAM receptor in the membrane. Synthesisresumes, and the protein is translocated through the membrane while it is being synthesized,although there is no energetic connection between the processes.The channel through the membrane provides a hydrophilic environment and is largely made of the protein Sec6l. A secreted protein passes completely through the membrane into the ER lumen. Proteins that are integrated into membranes can be divided into two general tlpes basedon their orientation. For type I integral membrane proteins, the N-terminal signal sequenceis cleaved, and transfer through the membrane is halted later by an anchor sequence. The protein becomes oriented in the membrane with its N-terminus on the far side and its C-terminus in the cytosol. \pe II proteins do not have a cleavable N-terminal signal, but instead have a combined signal-anchor sequence,which enters the membrane and becomes embedded in it. This causesthe C-terminus to be located on the far side,whereas the N-terminus remains in the cytosol. The orientation of the signal-anchor is determined by the "positive inside" rule, which statesthat the side of the anchor with more positive charges will be located in the cytoplasm. Proteins that have single transmembrane spanning regions move laterally from the channel into the lipid bilayer. Proteins may have multiple membrane-spanning regions, with loops between them protruding on either side of the membrane. The mechanism of insertion of multiple segments is unknown. In the absence of any particular signal, a protein is releasedinto the cytosol when its synthesis is completed. Proteins are imported posttranslationally into mitochondria or chloroplasts. They possessN-terminal leader sequencesthat target them to the outer membrane of tne organelle envelope; they then are transported through the outer and inner membranes into the matrix. Translocation requires ATP and a potential across the inner membrane. The N-terminal leader is cleaved by a protease within the organelle. Proteins that reside within the membranes or intermembrane spacepossessa signal (which becomes N-terminal when the
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of substrate binding by the molecular chaperone DnaK. Science 272, 1606-1614.
@
SignaI Sequences InitiateTranslocation
Reviews Lee, C. and Beckwith, J. (1986). Cotranslational and posttranslational protein translocation in prokaryotic systems.Annu. Rev.CellBiol. 2, )t5-3)6. Palade,G. (197 5). Intracellular aspectsof the process of protein synthesis. Science189, )47-358. Resea r ch Blobel, G. and Dobberstein,B. (1975\. Tfansfer of proteins acrossmembranes. L Presenceof proteolytically processedand unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma. J. CellBiol 67 , 835-851 . Lingappa,V. R., Chaidez,J., Yost, C. S., and Hedgpeth, J. (1984). Determinanrs for protein localization: beta-lactamase signal sequence directs globin acrossmicrosomal membranes. Proc.Natl Acad. Sci.USA 8l, 456-460. von Heijne, G. (1985). Signal sequences.The limits of variation. J. Mol. Biol 184.99-\05.
IIIEI
TheSignal Sequence Interacts withtheSRP
Review Walter, P. and Johnson, A. E. ( I 994). Signal sequence recognition and protein targeting to the endoplasmic reticulum membrane. Annu Rev.CellBiol 10, 87-I 19. Resea r ch Tjalsma,H., Bolhuis, A., van Roosmalen,M. L., Wiegert, T., Schumann, W., Broekhuizen, C. P.,Quax, W J., Venema, G., Bron, S., and van Dijl, M. (1998). Functional analysisof the secretory precursor processing machinery of Bacillussubtilis:identification of a eubacterial homolog of archaeal and eukaryotic signal peptidases.GenesDev. 12, 2318-2))1. Walter, P. and Blobel, c. ( l98l ). Tlanslocation of proteins acrossthe ER III SRP causessignal sequence and site specific arrest of chain elongation that is released by microsomal membranes.J CellBiol.9l, 557-561.
@
TheSRPInteracts withthe SRPReceptor
Reviews Doudna, J. A and Batey, R. T. (2004). Structural insights into the signal recognition particle. Annu. Rev.Biochem 7), 539-557. I(eenan, R. J., Freymann, D. M., Stroud, R. M., and Walter, P. (2001). The signal recognirion particle. Annu. Rev.Biochem.7 0, 7 55-77 5.
252
CHAPTER 10 ProteinLocatization
rch Resea Batey, R. T., Rambo, R. P., Lucast, L., Rha, B., and Doudna, J. A. (2000). Crystal structure of the ribonucleoprotein core of the signal recognition particle. Science 287, 1232-1239. Halic, M., Becker, T., Pool, M. R., Spahn, C. M., Grassucci,R. A., Frank, J., and Beckmann, R. (2004lr. Structure of the signal recognition particle interacting with the elongationarrestedribosome. Nature427, 808-814. Keenan, R. J., Freymann, D. M., Walter, P., and Stroud, R. M. (1998). Crystal structure of the signal sequence-binding subunit of the signal recognition particle. Cell94, 181-l 91. Powers, T. and Walter, P. (1995). Reciprocal stimulation of GTP hydrolysis by two directly interacting GTPases.Science 269, 1422-1424. Siegel, V. and Walter, P. (1988). Each of the activities of SRP is contained within a distinct domain: analysis of biochemical mutants of SRP.Cel/ 52,39-49. Tajima, S., Lauffer, L., Rath, V. L., and Walter, P. ( t 986). The signal recognition particle receptor is a complex that contains two distinct polypeptide chains. J. CellBiol. 103, tt67-rt78. Walter, P. and Blobel, c. ( 1981) . Tlanslocation of proteins acrossthe ER III SRP causessignal sequence and site specific arrest of chain elongation that is released by microsomal membranes.J. CellBiol.91, 557-561. Walter, P. and Blobel, c. (1982). Signal recognition particle contains a 75 RNA essential for protein translocation acrossthe ER. Nature 299, 69t-698. Zopf,D., Bernstein, H. D., Johnson, A. E., and Walter, P. (1990). The methionine-rich domain of the 54 kd protein subunit of the signal recognition particle contains an RNA binding site and can be crosslinked to a signal sequence. EMBO J. 9, 451 l-4517.
TheTranslocon Forms a Pore Resea rch Crowley, K. S. (I994). Secretoryproteins move through the ER membrane via an aqueous, gated pore. Cell78, 461471. Deshaies,R. J. and Schekman, R. ( I 987). A yeast mutant defective at an early stage in import of secretory protein precursors into the endoplasmic reticulum. J. CellBiol.IO5, 63i-64i. Esnault, Y., Blondel, M. O., Deshaies,R. J., Scheckman,R., and I(epes,F. (I993). The yeast SSSI gene is essential for secretory protein translocation and encodes a conserved protein of the endoplasmic reticulum. EMBO J. 12,408?-4093. Hanein, D., Matlack, I(. E., Jungnickel, B., Plath, K., I(alies, I(. U., Miller, I(. R., Rapoport, T. A.,
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potential role in peroxisomal membrane assembly.Proc Natl. Acad Sci.USA 96,
2Lr6-2r2r. South, S. T. and Gould, S. J. (I999). Peroxisome synthesis in the absence of preexisting peroxisomes.J. CellBiol. 144,255-266. Walton, P. A., Hill, P. E., and Hill, S. (1995). Import of stably folded proteins into peroxisomes. Mol. Biol. Cell 6, 675-683.
TheSecSystem Transports Proteins i n t o a n d T h r o u g ht h e I n n e r M e m b r a n e Reviews Lee, C. and Beckwith, J. (1986). Cotranslational and posttranslational protein translocation in prokaryotic systems.Annu. Rev.CellBiol. 2, )t5-)36. Oliver, D. (1985). Protein secretioninE. coli.Annu Rey.Immunol J9, 615-648. Research Beck, I(., Wu, L. F., Brunner, J., and Muller, M. (2000). Discrimination between SRP-and SecA/SecB-dependent substratesinvolves selective recognition of nascent chains by SRP and trigger factor.EMBO J.19, 84-143. Brundage, L. et al. (1990). The purified E coli integral membrane protein SecY/E is sufficient for reconstitution of SecA-dependent precursor protein translocation.Cell62, 649-617. Collier, D. N. et al. (1988). The antifolding activity of SecB promotes the export of the E coli maltose-bindingprotein. Cell 53, 27 )-28). Crooke, E. et al. (1988). ProOmpA is stabilizedfor membrane translocation by either purified E. coli trigger Iactor or canine signal recognition particle. Cell 54, 1003-10l I Valent, Q. A., Scotti, P. A., High, S., von Heijne, G., Lentzen, G., Wintermeyer, W., Oudega, B., and Luirink, J. (1998). The E. coli SRPand
SecB targeting pathways converge at the translocon.EMBO J. 17,2504-2512. Yahr, T. L. and Wickner, W. T. (2000). Evaluating the oligomeric state of SecYEGin preprotein translocase.EMBO J. 19, 439)-4401.
Systems Transtocation Sec-Independent in E. coli Reviews Evolutionarily Dalbey, R.E.andI(uhn,A. (2000). related insertion pathways of bacterial, mitochondrial, and thylakoid membrane proteins. Annu Rev.CellDev.Biol. 16, 5l-87. Dalbey,R. E and Robinson, C. (1999). Protein translocation into and acrossthe bacterial plasma membrane and the plant thylakoid membrane. TrendsBiochem.Sci.24, 17-22. Resea rch Beck, I(., Wu, L. F., Brunner, J., and Muller, M. (2000). Discrimination betwe en SRP-and SecA/SecB-dependentsubstratesinvolves selective recognition of nascent chains by SRP and trigger factor. EMBO J. 19, 134-143. Samuelson,J. C., Chen, M., Jiang, F., Moller, I., Wiedmann, M., I(uhn, A., Phillips, G. J., and Dalbey, R. E. (2000). YidC mediatesmembrane protein insertion in bacteria. Nature 406,6)7-641. Scotti, P A., Urbanus, M. L., Brunner, J., de Gier. J. W., von Heijne, G., van der Does, C., Driessen,A. J., Oudega, B., and Luirink, J. (2000). YidC, the E. colihomologue of mitochondrial Oxalp, is a component of the Sec translocase.EMBO J 19,542-549. Soekarjo, M., Eisenhawer, M., I(uhn, A., and Vogel, H. (19961. Thermodynamics of the membrane insertion processof the Ml3 procoat protein, a lipid bilayer traversing protein containing a leader sequence. Biochemistry 35, 12)2-1241.
References 255
Transcription C H A P T EO RU T L I N E r Bindingconstants of RNApolymerase for differentpromotersvaryoversixorders of magnitude. corresponding to the frequency withwhichtranscription is initiatedat each 0romoter.
Introduction
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Transcription Occursby BasePairingin a "Bubb[e" of Unoaired DNA r RNApotymerase separates thetwo strands of DNAin a transient"bubb[e" andusesonestrandasa template to direct synthesis of a complementary sequence of RNA. . Thelengthof the bubbte is -i.2 to 14 bp,andthe length of RNA-DNA hybridwithinit is -8 to 9 bp. TheTranscription ReactionHasThreeStages o RNApotymerase initjatestranscription afterbindingto a promoter siteon DNA. r Duringetongation thetranscription bubbtemoves atong DNAandthe RNAchainis extended in the 5'-3'direction. r Whentranscription stops,the DNAduptexreforms andRNA polymerase dissociates at a terminator site. PhageT7 RNAPotymerase Is a UsefulModelSystem o T3andT7phageRNApolymerases aresingtepotypeptides with minimalactivjties in recognizing a sma[[number of phagepromoters. o Crysta[ structures of T7 RNApotymerase with DNAidentifo the DNA-binding regionandthe activesite. A Modetfor EnzymeMovementIs Suggested by the CrystaIStructure e DNAmoves througha groove in yeastRNApotymerase that makes a sharpturnat the activesite. . A proteinbridgechanges conformation to controlthe entry of nucleotides to theactivesite. BacterialRNAPotymerase Consists of MuttipteSubunits r Bacterial RNAcorepotymerases are-500 kDmuttisubunit comptexes withthe generaI structure o2Bp'. . D N Ai s b o u n di n a c h a n n ealn di s c o n t a c t ebdv b o t ht h eB a n dp ' s u b u n i t s . RNAPotymerase Consists of the CoreEnzymeand Sigma Factor r BacteriaI RNApotymerase canbedivjdedintothe a2Bp' coreenzyme that catatyzes transcription andthe sigma subunjtthat is required ontyfor initiation. r Sigmafactorchanges the DNA-binding properties of RNA potymerase sothat its affinityfor general DNAis reduced andits affinityfor promoters is increased.
256
TheAssociation with SigmaFactorChanges at Initiation . WhenRNApotymerase promoter, bindsto a it separates the DNAstrands to forma transcription bubbleandincorporates upto ninenucleotides into RNA. . Theremaybe a cycteof abortiveinitiationsbeforethe moves enzyme to the nextphase. r Sigmafactormaybe released fromRNApotymerase when the nascent RNAchainreaches eiqhtto ninebases in tength.
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P r o m o t eRr e c o g n i t i oD n e p e n dosn C o n s e n s uSse q u e n c e s . A promoter is defined by the presence of shortconsensus sequences at specific locations. o Thepromoter consensus sequences consistof a purineat the startpoint,the hexamer TATAAT centered at -L0, and anotherhexamer centered at -35. . IndividuaI promoters usuatly differfromthe consensus at oneor morepositions.
SltEr
PromoterEfficiencies CanBe Increasedor Decreased by Mutation o Downmutations promoter to decrease efficiency usuatly decrease conformance to the consensus sequences, whereas up mutations havethe opposite effect. o Mutatjons in the-35 sequence usually affectinitialbinding of RNApolymerase.
o Mutations in the -10 sequence usualty affectthe mettingreactionthat convertsa ctosed to an opencomplex. RNAPolymerase Bindsto OneFace O fD N A o Theconsensus sequences at -35 and-10 provide mostof the contactpointsfor RNA potymerase in the promoter. r Thepointsof contactlie on onefaceof t h eD N A . Supercoiting Is an ImportantFeature of Transcription e Negative supercoiting increases theefficiencyof somepromoters by assisting the meltingreaction. . Transcription generates positivesupercoils aheadof the enzyme andnegative supercoitsbehindit, andthesemustbe removed by gyrase andtopoisomerase. Substitutionof SigmaFactorsMay ControIInitiation t E.colihasseveral sigmafactors.eachof whichcauses RNApotymerase to initiate at a setof promoters definedby specific -35 and-10 sequences. . 070js usedfor generaI transcription, and the othersigmafactorsareactivatedby speciaI conditions. SigmaFactorsDirecttyContactDNA . o70changes its structure to retease its DNA-binding regions whenit associates with coreenzyme. . o7obindsboththe -35 and-i.0 sequences.
ll:A
SigmaFactorsMayBe 0rganizedinto Cascades o A cascade of sigmafactorsis created when onesigmafactoris required to transcribe the genecodingfor the nextsigmafactor. o Theearlygenesof phageSP01aretranscribed by hostRNApotymerase. r Oneof the earlygenescodes for a sigma factorthat causes RNApolymerase to transcribe the midd[egenes. o Twoof the middtegenescodefor subunits of a sigmafactorthat causes RNApolymerase to transcribe the lategenes.
ETEI SporulationIs Controttedby Sigma Factors . Sporulation divides a bacterium into a motherce[[that is tysedanda sporethat is released.
r Eachcomoartment advances to the next stageof devetopment by synthesizing a newsigmafactorthat displaces the previoussigmafactor. o Communication thetwo compartbetween mentscoordinates thetimingof sigma factorsubstitutions. BacteriaIRNAPotymerase Terminates at DiscreteSites o Termination mayrequire bothrecognition in DNAandthe sequence of theterminator formation in the of a hairoinstructure RNAoroduct. in ThereAreTwoTypesof Terminators E. coli r Intrinsicterminators consistof a G-C-rich followed hairpinin the RNAproduct by a U-richregionjn whichtermination occurs. HowDoesRhoFactorWork? o Rhofactoris a terminator oroteinthat RNAand bindsto a /uf siteon nascent it fromthe tracksalongthe RNAto retease RNA-DNA hybridstructure at the RNA poLymerase. Event AntiterminationIs a Regutatory o Termination is prevented whenantiterminationproteins acton RNApolymerase to it to readthrougha specific termicause natoror terminators. r Phage [ambda hastwo antitermination proteins, pNandpQ.that acton different transcription units. SitesThatAre AntiterminationReouires Indeoendentof the Terminators o Thesitewherean antiterminator protein sitein actsis uostream of theterminator thetranscription unit. . Thelocation of the antiterminator site andcanbein the variesin different cases promoter unit. or withinthetranscription Factors Termination and AntiTermination Interactwith RNAPotymerase o Severa[ for bacterialproteinsarerequired lambda oNto interactwith RNA poLymerase. . These proteins in areatsoinvolved of the in the rn operons antitermination hostbacterium. pQhasa differo Thetambda antiterminator that invotves ent modeof interaction bindingto DNAat the promoter. Summarv
11 Transcription 257 CHAPTER
Codingstrand
Templatestrand
RNAsequence is TRANSCRIPTION comptementarytotemplatestrand identicalto codingstrand RNAtranscript
i ii:j*i' i :.: Thefunctionof RNApolymerase is to copyonestrandof duptexDNAjnto RNA.
-35-10-1+1 +10
i:i.iitti : i -: A transcription unjt is a sequence of DNA into a sing[eRNA,startingat the promoter transcribed andendinoat theterminator.
@
Introduction
Tfanscription involves synthesis of an RNA chain representingone strand of a DNA duplex. When we say "representing," we mean that the RNA is identicalin sequence with one strand of the DNA, which is called the coding strand.It is clmplementaryto tlrreother strand. which provides the template strand for its synthesis. i::+:-liii: i. i recapitulates the relationship between double-strandedDNA and its singlestranded RNA transcript. RNA synthesis is catalyzed by the enzyme RNA polymerase. Transcription starts when RNA polymerase binds to a special region, the promoter, at the start of the gene. The promoter surrounds the first basepair that is transcribed into RNA, the startpoint. From this point, RNA polymerase moves along the template, synthesizing RNA, until it reaches a terminator (/) sequence. This action defines a transcription unit that extends from the pro-
258
CHAPTER 11 Transcriotion
moter to the terminator. The critical feature of I i..ll, the transcription unit, depicted in Fi{ili*?H is that it constitutes a stretch of DNA expressed via thep rodu ction of a single RNA molecule.A tr anscription unit may include more than one gene. Sequencesprior to the startpoint are described as upstream of it; those after the startpoint (within the transcribed sequence)are downstream of it. Sequencesare conventionally written so that transcription proceedsfrom left (upstream) to right (downstream). This corresponds to writing the mRNA in the usual 5' -+ 3' direction. The DNA sequenceoften is written to show only the coding strand, which has the same sequenceas the RNA. Basepositions are numbered in both directions away from the startpoint, which is assignedthe value +l; numbers increaseas they go downstream. The basebefore the startpoint is numbered -1, and the negative numbers increase going upstream. (There is no baseassignedthe number 0.) The immediate product of transcription is called the prirnary transcript. It consists of an RNA extending from the promoter to the terminator and possessesthe original 5' and 3' ends. The primary transcript is, however, almost always unstable. In prokaryotes, it is rapidly degraded (nRNA) or cleaved to give mature products (rRNA and tnNR;. In eukaryotes, it is modified at the ends (mRNA) and/or cleaved to give mature products (all RNA). Tfanscription is the first stagein gene expression and the principal step at which it is controlled. Regulatory proteins determine whether a particular gene is available to be transcribed by RNA polymerase. The initial (and often the only) step in regulation is the decision on whether or not to transcribe a gene. Most regulatory events occur at the initiation of transcription, although subsequent stages in
transcription (or other stagesof gene expression) are sometimesregulated. Within this context, there are two basic q u e s t i o n si n g e n e e x p r e s s i o n : . How does RNA polymerase find promoters on DNA? This is a particular example of a more general question: How do proteins distinguish their specific binding sites in DNA from other sequences? . How do regulatory proteins interact with RNA polyrnerase (and with one another) to activate or to repressspecific stepsin the initiation, elongation, or termination of transcription? In this chapter, we analyze the interactions of bacterial RNA polymerase with DNA from its initial contact with a gene, through the act of transcription, and then finally its releasewhen the transcript has been completed.Chapter 12, The Operon, describesthe various means by which regulatory proteins can assistor prevent bacterial RNA polymerase from recognizing a particular gene for transcription. Chapter 13, Regulatory RNA, discussesother means of regulation. including the use of small RNAs, and considershow these interactions can be connected into larger regulatory networks. In Chapt e r 1 4 , P h a g e S t r a t e g i e s ,w e c o n s i d e r h o w individual regulatory interactions can be connected into more complex networks. In Chapter 24, Promoters and Enhancers, and Chapter 25, Activating Transcription, we consider the analogous reactions between eukaryotic RNA polymerasesand their templates.
Transcription 0ccurs by BasePairing in a "BubbLe" of Unpaired DNA o RNApolymerase separates thetwo strands of DNA "bubbte" in a transient andusesonestrandasa temptate to directsynthesis of a comptementary sequence of RNA. o Thelengthof the bubbleis -12 to 14 bp,andthe lengthof RNA-DNA hybridwithinit is -8 to 9 bp.
Ttanscription takes place by the usual process of complementary basepairing. iri*Li$*t1".iillustrates the generalprinciple of transcription. RNA synthesis takes place within a "transcription bubble," in which DNA is transiently separated
DNA is melted
RNAchain is extended
j i..i DNAstrands t;i"i;r,iil:ri: to forma transcription separate bubbte. basepairing RNAis synthesized by comp[ementary with oneof the DNAstrands.
into its single strands and the template strand is used to direct synthesis of the RNA strand. The RNA chain is synthesized from the 5' end toward the 3'end. The 3'-OH group of the last nucleotide added to the chain reacts with an incoming nucleoside 5'triphosphate. The incoming nucleotide losesits terminal two phosphate groups (y and B); its a group is used in the phosphodiester bond linking it to the chain. The overall reaction rate is -40 nucleotides/second at 37" C (for the bacterialRNA polymerase); this is about the same as the rate of translation (i 5 amino acids/sec),but much slower than the rate of DNA replication (800 bp/sec). RNA polymerase creates the transcription 1i "q bubble when it binds to a promoter. Ijt{.it-ll't{ shows that as RNA polymerase moves along the DNA, the bubble moves with it and the RNA chain grows longer. The processof basepairing and baseaddition within the bubble is catalyzed and scrutinized by the enzyme. The structure of the bubble within RNA polymerase is shown in the expanded view of F.{i;l-Jfii; l.j.l,. As RNA polymerasemoves along the DNA template, it unwinds the duplex at the
DNA 1 1 . 2 T r a n s c r i p t i oOnc c u r b s y B a s eP a i r i n gi n a " B u b b t e "o f U n p a i r e d
259
with RNA at any given moment. Certainly the RNA-DNA hybrid is short and transient. As the enzyme moves on, the DNA duplex reforms, and the RNA is displacedas a free polynucleotide chain. Roughly the last twenty-five ribonucleotides added to a growing chain are complexed with DNA and/or enzyme at any moment.
TheTranscription HasThree Reaction Stages o RNApotymerase initiatestranscription after siteon DNA. bindingto a promoter r Duringetongation thetranscription bubblemoves in the aLong DNAandthe RNAchainis extended 5'-3'direction. o Whentranscription stops,the DNAduplexreforms dissociates at a terminator andRNApolymerase site.
i!ai-iiii i i.i Transcription in which takesp[acein a bubbl.e, RNAis synthesized by basepairingwithonestrandof DNAin the transiently region.Asthe bubbteprogresses, unwound the DNAduptexreforms the RNAin the behindit, displacing The transcription reaction can be divided into formof a singtepotynucteotide chain. the stagesillustrated in ri$tlftl: 1:i"{-i,in which a bubble is created, RNA synthesis begins, the bubble moves along the DNA, and finally the bubble is terminated: . Templaterecognitionbegins with the binding of RNA polymerase to the doublestranded DNA at a promoter to form a "closed complex." The strands of DNA are then separatedto form the "open complex" thatmakes the template strand available for base pairing with ribonucleotides. The transcription bubble is created by a local unwinding that begins at the site bound by RNA polymerase. . Initiation describesthe synthesis of the first nucleotide bonds in RNA. The RNA bindingsite enzyme remains at the promoter while l-i::iirii i,:.: Duringtranscription, is mainthe bubbl.e it synthesizes the first -9 nucleotide tainedwithinbacterjal RNApotymerase, whichunwinds bonds. The initiation phase is protracted andrewinds DNAandsynthesizes RNA. by the occurrence of abortive events, in which the enzyme makes short transcripts, releasesthem, and then starts synthesis of RNA again. The initiation front of the bubble (the unwinding point), and phase ends when the enzyme succeeds rewinds the DNA at the back (the rewinding in extending the chain and clears the point). The length of the transcription bubble promoter. Thesequence of DNA needed is -12 to l4bp, but the length of the RNA-DNA for RNApolymeraseto bind to thetemplateand hybrid region within it is shorter. accomplishthe initiation reactiondefinesthe There is a major change in the topology of prlmlter. Abortive initiation probably DNA extending over -l turn, but it is not clear involves synthesizing an RNA chain that how much of this region is actually base paired
CHAPTER lL Transcription
fills the acrivesite.If the RNA is released, the initiation is aborted and must start again. Initiation is accomplished if and when the enzyme managesto move along the template to move the next region of the DNA into the active site. . During elongation the enzyme moves along the DNA and extends the growing RNA chain. As the enzyme moves, it unwinds the DNA helix to expose a new segment of the template in singlestranded condition. Nucleotides are covalently added to the J'end of the growing RNA chain, forming an RNADNA hybrid in the unwound region. Behind the unwound region, the DNA template strand pairs with its original partner to reform the double helix. The RNA emerges as a free single strand. Elongationinvolvesthemovementof thetranscriptionbubbleby a disruptionof DNA structure, in which the template strand 0f the transientlyunwound regionis paired with the nascentRNA at thegrowing point. . Termination involves recognition of the point at which no further bases should be added to the chain. To terminate transcription, the formation of phosphodiesterbonds must cease,and the transcription complex must come apart. When the last baseis added to the RNA chain, the transcription bubble collapsesas the RNA-DNA hybrid is disrupted, the DNA reforms in duplex state, and the enzyme and RNA are both released.Thesequence of DNArequiredfor thesereactionsdefinesthe terminator. The traditional view of elongation has been that it is a monotonic process, in which the enzyme moves forward I bp along DNA Ior every nucleotide added to the RNA chain. Changesin this pattern occur in certain circumstances,in particular when RNA polymerase pauses. One type of pattern is for the "front end" of the enzyme to remain stationary while the "back end" continues to move, thus compressingthe footprint on DNA. After movement of severalbasepairs, the "front end" is released, restoring a footprint of full length. This gave rise to the "inchworm" model of transcription, in which the enzyme proceedsdiscontinuously, alternatively compressingand releasingthe footprint on DNA. It may, however, be the casethat these events describean aberrant situation rather than normal transcription.
Templaterecognition: RNA polymerasebindsto duplexDNA
DNA is unwoundat oromoter
Initiation: Veryshortchains aresvnthesized andreleased
Elongation:polymerasesynthesizesRNA
rtr*J Termination: RNA polymeraseand RNA are released
,,'' ,,
it' ]ir, ii' ,.i1"r,, ,,
'!n
'ir. n
jf
Theenzyme binds irl{iliitr*} i.ii Transcription hasfourstages: during to the promoter andmeltsDNA,remains stationary and initiation,moves duringetongation, a[ongthetemptate dissociates at termination.
Phage T7RNAPolymerase Is a UsefulModelSystem r T3andT7phageRNApotymerases aresingte jn potypeptides with minimalact'ivities recognizing of phagepromoters. a smat[number . Crystalstructures with DNA of T7 RNApotymerase identifythe DNA-bjnding regionandthe active site.
The existence of very small RNA polymerases, comprising single polypeptide chains coded by certain phages, gives some idea of the "minimum" apparatus necessaryfor transcription. These RNA polymerases recognize just a few promoters on the phage DNA, and they have no ability to change the set of promoters to which
Is a Usefu[ Mode[System 26r T7 RNAPotymerase 11.4 Phage
i:ii,::-ii1li ;' - 17 RNApotymerase hasa specificity Loop -7 to -11,ofthepromoter posiwh'ite thatbindspositions tions-1 to -4 enterthe activesite.
a crystal structure of a phage T7 RNA polymerase engaged in transcription. The T7 RNA polymerase recognizesits target sequencein DNA by binding to basesin the major groove at a position upstream from the ::",r. The enzyme startpoint, as shown in Ftr{:tiftfl usesa specificityloopthat is formed by a B ribbon. This feature is unique to the RNA polymerase (it is not found in DNA polymerases).The common point with all RNA polymerasesis that the enzyme recognizesspecificbasesin DNA that are upstream of the sequence that is transcribed. When transcription initiates, the conformation of the enzyme remains essentially the same while several nucleotides are added, and the transcribed template strand is "scrunched" in the active site. The active site can hold a transcript of six to nine nucleotides. The transition from initiation to elongation is defined as the point when the enzyme begins to move along DNA. This occurs when the nascent transcript extends beyond the active site and interacts with the specificity loop. The RNA emerges to the surfaceof the enzyme when twelve to fourteen nucleotideshave been synthesized.These features are similar to those displayed by bacterial RNA polymerase.
A Modelfor Enzyme Movement Is Suggested
Structure bytheCrystal o DNAmoves in yeastRNA througha groove potymerase that makes a sharpturnat the active site. o A proteinbridgechanges conformation to control to the activesite. the entryof nucteotjdes
) n dB ' s u b u n i (t p i n k )o f R N A : : l r , i - , : i:i :I i -. : i T h eB ( c y a n a polymerase havea channel for the DNAtemp[ate. Synthesis ( ct o p p e h r )a sj u s tb e g u nt ;h e D N At e m o f a n R N At r a n s c r i p jn a ptate(red)andcoding(yettow) strands areseparated transcription bubbLe. Photocourtesy ofSethDarst,Rockefetter Universitv. We now have much information about the structure and function of RNA polymerase as the they respond. They provide simple model result of the crystal structures of the bacterial and yeast enzymes. Bacterial RNA polymerase has systems for characterizing the binding of RNA polymerase to DNA and the initiation reaction. overall dimensions of -90 x 95 x 160 A; eukaryThe RNA polymerases coded by the related otic RNA polymeraseis larger but lesselongated. phagesT3 and T7 aresinglepolypeptide chains Structural analysisshows that they share a comof <100 kD each. They synthesizeRNA at rates mon tlpe of structure, in which there is a "chano f - 2 0 0 n u c l e o t i d e s / s e c o nadt 3 7 " C , a r a t e t h a t nel" or groove on the surface -25 A wide that is more rapid than that of bacterial RNA polycould be the path for DNA. An example of this merase. channel in bacterial RNA polymerase is illusThe T7 RNA polymerase is homologous to The Iength of the groove trated in f3*tiftfi.3.!,S. DNA polymerasesand has a similar structure, could hold 16 bp in the bacterial enzyme. and -25 bp in the eukaryotic enzyme, but this repin which DNA lies in a "palm" surrounded by "fingers" and a "thumb" (seeFigure 18.7). We resents only part of the total length of DNA now have a direct view of the active site from bound during transcription. The enzyme sur-
CHAPTER LL Transcriotion
face is largely negatively charged,but the groove is lined with positive charges, enabling it to interact with the negatively charged phosphate groups of DNA. The yeast enzyme is a large structure with twelve subunits (see Section 24.2, Eukaryotic RNA PolymerasesConsist of Many Subunits). Ten subunits of the yeast RNA polymerase II have been located on the crystal structure, as shown in irii;,.Jiii: ::i.',:.The catalytic site is formed by a cleft between the two large subunits (# I and #2), which grasp DNA downstream in "jaws" as it enters the RNA polymerase. Subunits 4 and 7 are missing from this structure; they form a subcomplex that dissociatesfrom the complete enzyme. The structure is generally simiIar to that of bacterial RNA polymerase. This can be seen more clearly in the crystal structure of iriiri-:iit:: i ti.I i:li.RNA polymerase surrounds the DNA, as seen in the view of f li,l.l:-1i.. ti i . ti i. A catalytic Mg2+ion is found at the active site. The DNA is clamped in position at the active site by subunits 1, 2, and 6. lt{i;,iitl-. t:.i:i shows that DNA is forced to take a turn at the entrance to the site becauseof an adjacent wall of protein. The length of the RNA hybrid is limited by another protein obstruction, called the rudder. Nucleotides probably enter the active site from below, via pores through the structure. The expanded view of the active site in ii;+ri3:f, i .. ; 5hs\,v5that the transcription bubble includes 9 bp of DNA-RNA hybrid. Where the DNA takes its turn, the basesdownstream are flipped out of the DNA helix. As the enzyme moves along DNA, the base in the template strand at the start of the turn will be flipped to face the nucleotide entry site.The 5'end of the RNA is forced to leave the DNA when it hits the p r o t e i n r u d d e r ( s e eF i g u r e I l . i 2 ) . Once DNA has been melted, the individual strands have a flexible structure in the transcription bubble. This enablesDNA ro rake its turn in the active site.Before transcription starts, though, the DNA double helix is a relatively rigid straight structure. How does this structure enter the polymerase without being blocked by the wall? The answer is that a large conformational shift must occur in the enzyme. Adjacent to the wall is a clamp. In the free form of RNA polymerase, this clamp swings away from the wall to allow DNA to follow a straight path through the enzyrne. After DNA has been melted to create the transcription bubble, the clamp must swing back into position against the wall. One of the dilemmas of any nucleic acid polymerase is that the enzyme must make tight
I i{,1:ilrl i .'. i; Tensubunits of RNApo[ymerase areptaced in positionfromthe crystaIstructure. Thecolorsof the subunits arethe sameasin the crystalstructures of the foltowinq fiqures.
ir:.il.l!{lii.i..i l: Thetopviewof thecrystaI structure of RNA potymerase II fromyeastshowsthat DNAis helddownstream by a pairofjawsandis ctamped in position in the activesite.whichcontains an Mg+ion. Photocourtesy of RogerKornberg, Stanford University Schoolof Medicine.
iiirlii;ir.i:i. ; .i Theendviewof thecrystal structure of RNA potymerase II fromyeastshowsthat DNAis surrounded by -270" of protein.Photocourtesy of RogerKornberg. Stanford of Medicine. University School
1 1 . 5 A M o d efl o r E n z y m e M o v e m e nI ts S u g g e s t eby d the CrystaI Structure
FI6URf11.1? DNAis forcedto makea turnat the active mayenterthe active site by a watlof protein.Nucteotides sitethrougha porein the protein.
of a nucleicacidpotymerase FI6URE 11.14 Movement andremaking bondsto the nucteotides requires breaking The at fixed positionsrelativeto the enzymestructure. in thesepositions change eachtimetheenzyme nucleotides moves a baseatongthetemptate.
FIGURf11.13 An exoanded viewofthe activesiteshows the sharpturnin the pathof DNA.
contacts with the nucleic acid substrate and product, but must break these contacts and remake them with each cycle of nucleotide addition. Consider the situation illustrated in F I G U R1E1 " 1 4 A . p o l y m e r a s em a k e s a s e r i e so f specific contacts with the bases at particular positions.For example, contact "l" is made with the base at the end of the growing chain and contact "2" is made with the base in the template strand that is complementary to the next baseto be added. Note, however, that the bases that occupy these locations in the nucleic acid chains change every time a nucleotide is added!
264
CHAPTER 11 Transcription
The top and bottom panels of the figure show the same situation: a base is about to be added to the growing chain. The difference is that the growing chain has been extended by one base in the bottom panel. The geometry of both complexes is exactly the same, but contacts "l" and"2" in the bottom panel are made to basesin the nucleic acid chains that are located one position farther along the chain. The middle panel shows that this must mean that, after the baseis added, and before the enzyme moves relative to the nucleic acid, the contacts made to specificpositions must be broken so that they can be remade to basesthat occupy those positions after the movement. The RNA polymerase structure suggestsan insight into how the enzyme retains contact with its substratewhile breaking and remaking bonds. A structure in the protein called the bridge is adjacent to the active site (see Figure I I.I2). This feature is found in both the bacterial and yeast enzymes, but it has differ-
ent shapesin the different crystal structures. In one it is bent, and in the other it is straight. iJGrJ*il3J..t:*suggeststhat the change in conformation of the bridge structure is closely related to translocation of the enzyme along the nucleic acid. At the start of the cycle of translocation, the bridge has a straight conformation adjacent to the nucleotide entry site. This allows the next nucleotide to bind at the nucleotide entry site. The bridge is in contact with rhe newly added nucleotide. The protein then moves one base pair along the substrate. The bridge changes its conformation, bending to keep contact with the newly added nucleotide. In this conformation, the bridge obscuresthe nucleotide entry site. To end the cycle, the bridge returns to its straight conformation, allowing accessagain to the nucleotide entry site. The bridge actsas a ratchet that releases the DNA and RNA strands for translocation while holding on to the end of the growing chain.
@
Bacterial RNAPoLymerase Consists of MuLtiple Subunits
r BacteriaI RNAcorepotymerases are-500 kD muttjsubunit complexes withthe general structure
uzFF'.
o D N Ai s b o u n d i n a c h a n n ealn di s c o n t a c t ebdy b o t ht h eB a n dB ' s u b u n i t s . The best characterized RNA polymerases are those of eubacteria,for which Escherichiacoliisa tlpical case.A singletypeof RNApolymeraseappears to beresponsible for almostall synthesisof wRNA, and all rRNA and IRNA,in a eubacterium.About7000 RNA polymerase molecules are present in an E. coli ceIL Many of them are engaged in transcription; probably 2000 to 5000 enzymes are syrrthesizingRNA at any one time, with the number depending on the growth conditions. The complete enzyme or holoenzyme in E. coli has a molecular weight of -465 kD. Its subunit composition is summarized in i i * # i i [ 3] i . : i : . The B and p'subunits together make up the catalytic center. Their sequencesare related to those of the largest subunits of eukaryotic RNA polymerases (seeSection 24.2,Eukaryotic RNA PolymerasesConsist of Many Subunits), suggesting that there are common features to the
r'{+{ifiili-i. i i: TheRNApolymerase e[ongation cycte startswitha straight bridge adjacent to thenucteotide entry site.Afternucteotide addjtjon,the enzymemovesone basepairandthe bridgebendsasit retains contactwith the newtyaddednucleotide. Whenthe bridgeis released, the cyctecanstartagain.
actions of all RNA polymerases. The B subunit can be crosslinked to the template DNA, the product RNA, and the substrateribonucleotides; mutations in rpoB affect all stagesof transcription. Mutations in rpoC show that B' also is involved at all stages. The s subunit is required for assembly of the core enzyme. When phage T4irrf.ecrsE. clli, the cr subunit is modified by ADP-ribosylation of an arginine. The modification is associated with a reduced affinity for the promoters formerly recognized by the holoenzyme, suggesting that the a subunit plays a role in promoter recognition. The c subunit also plays a role in the interaction of RNA polymerase with some regulatory factors.
of Muttipl.e Subunits 265 RNAPotymerase Consists 11.6 Bacteriat
2 ctsubunits (40kD each)
enzymeassembly promoterrecognition bindssome activators
B subunit ( 1 5 5k D )
B' subunit ( 1 6 0k D )
i ! + i j Q i 1 i . : * E u b a c t e rR i aN I Ap o l y m e r a sheasv ef o u r sizes typesof subuni|u. B. andB' haveratherconstant in different bacterial species. buto variesmorewidely.
of ii{ii-ir-:*: l l ii.r Boththetemplate andcodingstrands in the DNAarecontacted bytheB andB'subunits [argety regionof the transcription The bubbteanddownstream. js contacted There RNA mostLy in thetranscription bubble. is no downstream RNA,exceptin the speciaI casewhen the enzyme backtracks.
The o subunit is concerned specifically with promoter recognition, and we have more information about its functions than on any other subunit (seeSection 11.7, RNA Polymerase Consistsof the Core Enzyme and Sigma Factor). The crystal structure of the bacterial enzyme (see Figure lI.8) shows that the channel for DNA lies at the interface of the B and B' subunits. (The c, subunits are not visible in this view.) The DNA is unwound at the active site,
266
CHAPTER 11 Transcription
where an RNA chain is being synthesized. Crosslinking experiments identify the points at which the RNA polymerase subunits contact DNA. Theseare summarized in FtG#fifi i ":.;. The p and p' subunits contact DNA at many points downstream of the active site. They make several contacts with the coding strand in the region of the transcription bubble, thus stabilizing the separated single strands. The RNA is contacted largely in the region of the transcription bubble. The drug rifampicin (a member of the rifamycin antibiotic family) blocks transcription by bacterial RNA polymerase. It is a major drug used againsttuberculosis.The crystal structure of RNA polymerase bound to rifampicin explains its action: it binds in a pocket of the B subunit, >12 A away from the active site, but in a position where it blocks the path of the elongating RNA. By preventing the RNA chain from extending beyond two to three nucleotides,this blocks transcription. Originally defined simply by its ability to incorporate nucleotides into RNA under the direction of a DNA template, the enzyme RNA polymerase now is seen as part of a more complex apparatus involved in transcription. The ability to catalyze RNA synthesisdefinesthe miniasRNApolymum clmplnent that can be described m e r a s e . I t s u p e r v i s e st h e b a s e p a i r i n g o f t h e substrate ribonucleotides with DNA and catalyzes the formation of phosphodiester bonds between them. AII of the subunits of the basic polymerase that participate in elongation are necessaryfor initiation and termination. Transcription units differ, however, in their dependence on additional polypeptides at the initiation and termination stages. Some of these additional polypeptides are needed at all genes,whereas others may be needed specifically for initiation or termination at particular genes. The analogy with the division of labors between the ribosome and the protein synthesis factors is obvious. E. coli RNA polymerase can transcribe any one of many (>1000) transcription units. The enzyme therefore requires the ability to interact with a variety of host and phage functions that modify its intrinsic transcriptional activities. The complexity of the enzyme therefore, at least in part, reflects its need to interact with reguIatory factors, rather than any demand inherent in its catalytic activity.
RNAPolymerase Consists of the CoreEnzyme and SigmaFactor BacteriaI RNApotymerase canbe divjded intothe o2Bp'coreenzyme that catalyzes transcription andthe sigmasubunitthat is required on[yfor initiation. Sigma factorchanges properties the DNA-binding of RNApotymerase sothat'itsaffinityfor generaI DNAis reduced andits affinityfor promoters is i ncreased, Bindingconstants of RNApoLymerase for different promoters varyoversixorders of magnitude, corresponding to the frequency with which transcription is initiatedat eachpromoter.
Core enzymebindsto any DNA
Sigmadestabilizesbinding
,#\*\I\#\?\tu\#\#\#-
Holoenzymebindsto promoter
The holoenzyme (o2Bp'o) can be separatedinto two components, the core enzyme (cr2BB')and the sigma factor (the o polypeptide l. Only the holoenzymecan initiate transcription.Sigmafactor ensuresthat bacterial RNA polymerasebinds in a stablemannerto DNA only atpromolers.The sigma "factor" is usually releasedwhen the RNA chain reaches eight to nine bases,leaving the core enzyme to undertake elongation. Coreenzyme has the ability to synthesizeRNA on a DNA template, but cannotinitiate transcriptionat the propersites. The core enzyme has a general affinity for DNA, in which electrostatic attraction between the basic protein and the acidic nucleic acid plays a major role. Any (random) sequenceof DNA that is bound by core polymerase in this general binding reaction is describedas a loose binding site. No change occurs in the DNA, which remains duplex. The complex at such a site is stable, wirh a half-life for dissociation of the enzyme from DNA -60 minutes . Coreenzyme doesnot distinguish betweenprlmlters and other sequences of DNA. l:-rill-i;r rl :- ir, shows that the sigma factor introduces a major changein the affinity of RNA polymerase for DNA. Theholoenzyme hasa drastically reducedability to recognize loosebinding sitesthat is, to bind to any general sequenceof DNA. T h e a s s o c i a t i o nc o n s t a n t f o r t h e r e a c t i o n i s reduced by a factor of -104, and the half-life of the complex is
ii*{ilti :i.1..:iil Core enzyme bindsindiscriminately to any DNA.Sigmafactorreduces the affinityfor sequenceindependent forpromoters. binding andconfers specificity associationconstant increasedfrom that of core enzyme by (on average) 1000 times and with a half-life of severalhours. The specificityof holoenzyme for promoters compared to other sequencesis -I07, but this is only an average because there is wide variation in the rate at which the holoenzyme binds to different promoter sequences.This is an important parameter in determining the efficiency of an individual promoter in initiating transcription. The binding constantsrange from -1012 to -106. Other factors also affect the frequency of initiation, which varies from -llsec (rRNA genes) Io -llj0 min (the laclpromoter).
TheAssociation with Changes SigmaFactor at Initiation WhenRNApolymerase it bindsto a promoter, separates the DNAstrands to forma transcription bubbteandincorporates upto ninenucleotides into RNA. Theremaybe a cycleof abortiveinitiatjonsbefore the enzyme moves to the nextphase. fromRNApotymerase Sigmafactormaybe reteased e'iqhtto nine whenthe nascent RNAchainreaches in lenqth. bases
Changes at Initiation with SigmaFactor 11.8 TheAssocjation
267
Holoenzyme Equilibrium constant
A: : ; .V
DNAbinding
Ks = 1go-1gsY-t Closed binarycomplex
DNAmelting
Rate constant k2 = 1O-3-10-1sec-1 Open binarycomplex
Abortive
Rate constant k; - 10-3sec-l Ternary comprex
Releaseof slgma
Promoter clearance >1-2 sec RNA synthesisbegins
i i , : i : : : i i :- ' 1 , : + R N Ap o t y m e r aps ae s s etsh r o u g hs e v e r a I is constepspriorto elongation. A closed binarycomptex vertedto an openformandtheninto a ternarycomplex.
We can now describethe stagesof transcription in terms of the interactions between different forms of RNA polymerase and the DNA template. The initiation reaction can be described by the parameters that are summarized in i : : " , ; : : t"ar :. . i i . j : . The holoenzyme-promoter reaction starts by forming a closed binary complex. "Closed" means that the DNA remains duplex. The formation of the closedbinary complex is reversible;thus it is usually describedby an equilibrium constant (1(s).There is a wide range in values of the equilibrium constant for forming the closedcomplex. . The closedcomplex is converted into an open complexby "melting" of a short region of DNA within the sequence b o u n d b y t h e e n z y m e . T h e s e r i e so f events leading to formation of an open complex is called tight binding. For strong promoters, conversion into an open binary complex is irreversible, so this reaction is described by a rate con-
lL Transcriotion CHAPTER
stant (k2). This reaction is fast. Sigma factor is involved in the melting reaction (seeSection I l. i 6, Substitution of Sigma Factors May Control Initiation). . The next step is to incorporate the first two nucleotides; a phosphodiesterbond then forms between them. This generates a ternary complex that contains RNA as well as DNA and enzyme. Formation of the ternary complex is described by the rate constant ki; this is even faster than the rate constant k2. Further nucleotides can be added without any enzyme movement to generate an RNA chain of up to nine bases. After each baseis added, there is a certain probability that the enzyme will r e l e a s et h e c h a i n . T h i s c o m p r i s e s a n after which the abortive initiation, enzyme begins again with the first base. A cycle oI abortive initiations usually occurs to generate a seriesof very short oligonucleotides. . When initiation succeeds, sigma is no longer necessary,and the enzyme makes the transition to the elongation ternary complex of core polymerase-DNA-nascent RNA. The critical parameter here is how long it takesfor the polymeraseto leave can inithepromotersoanotherpolymerase tiate. This parameter is the promoter clearance time; its minimum value of one to two secondsestablishesthe maximum frequenry of initiation as
leaving only -60 bp of DNA coveredby the enzyme. This corresponds with the concept that the more upstream part of the promoter is involved in initial recognition by RNA polymerase, but is not required for the later stagesof initiation (seeSection I 1.13,Promoter Efficiencies Can Be Increased or Decreased by Mutation). . When the RNA chain extends to 15 to 20 bases,the enzyme makes a further transition, to form the complex that undertakes elongation; now it covers l0 to 40 bp (depending on the stagein the elongation cycle). It has been a tenet of transcription since soon after the discovery of sigma factor that it is releasedafter initiation. This may not be strictly true. Direct measurements of elongating RNA polymerase complexesshow that -70% of them retain sigma factor. A third of elongating polymerases lack sigma; hence the original conclusion is certainly correct that it is not necessary for elongation. In those caseswhere it remains associatedwith core enzyme, the nature of the associationhas almost certainly changed (see Section ll.Il, Sigma Factor Controls Binding ro DNA).
Initiationcomplexcontainssigma and covers 75-80 bo
Initialelongationcomplexforms at 10 bases,may lose sigma,and losescontactsfrom -35 to -55
Generalelongationcomplex forms al 15-20 basesand covers3H0
bp
Fi'i;i:Flf,: initiattycontactsthe 3i.:li:1RNApo[ymerase
@
thecore regionfrom-55 to +20.Whensigmadissociates, A StalLed RNAPoLymeraseenzyme moves a few contracts to -30; whenthe enzyme intothe morecompactly organized basepairs,jt becomes CanRestart generaI elongation comptex.
r An arrestedRNApolymerase canrestart transcription by cleaving the RNAtranscript to generaa t en e w3 ' e n d .
RNA polymerase must be able to handle situations when transcription is blocked. This can happen, for example, when DNA is damaged. A model system for such situations is provided by arresting elongation in vitro by omitting one of the necessaryprecursor nucleotides.When the missing nucleotide is restored, the enzyme can overcome the block by cleaving the 3'end of the RNA, to createa new J'terminus for chain elongation. The cleavageinvolves accessory factors in addition to the enzyme itself. In the caseof E. coliRNA polymerase, the proteins GreA and GreB release the RNA polymerase from elongation arrest. In eukaryotic cells,RNA p o l y m e r a s e I I r e q u i r e s a n a c c e s s o r yf a c t o r (TFrrS),which enablesthe polymerase to cleave a few ribonucleotidesfrom the 3'terminus of the RNA product.
The catalytic site of RNA polymerase undertakes the actual cleavagein each case.The roles of GreB and TFIS are to convert the enzyme's catalytic site into a ribonucleolytic site. Even though there is no sequencehomology between the factors,crystal structures of their complexes with the respective RNA polymerases suggest that they function in a similar way. Each of the factors inserts a narrow protein domain (in one case a zinc ribbon, in the other a coiled coil) deep into RNA polymerase, where it terminates within the catalytic site. The inserted domain positions two acidic amino acids close to the primary catalytic magnesium ion of the active site; this allows the introduction of a second magnesium ion, which converts the catalytic site to a ribonucleolytic site. The reason for this reaction may be that stalling causesthe template to be mispositioned,
CanRestart RNAPotymerase 11.9 A StaLted
269
so that the f'terminus is no longer located in the active site. Cleavage and backtracking is necessaryto place the terminus in the right location for addition of further bases. We see, therefore, that RNA polymerase has the facility to unwind and rewind DNA, to hold the separatedstrands of DNA and the RNA product, to catalyze the addition of ribonucleotides to the growing RNA chain, and to adjust to difficulties in progressing by cleaving the RNA product and restarting RNA synthesis (with the assistanceof some accessoryfactors).
500-1 000 core enzymes at loose complexes
I
500-1 000 holoenzymes at loose complexes
I 500-1000 holoenzymes at promoter comprexes
I
-2500core enzymes engaged in transcription
li€ilRf 11.:1 Coreenzyme andholoenzyme aredistributedon DNA.andveryLittteRNApolymerase is free.
i i.,ot M-1sec-1
F:fi*Rt 1-t.fit Theforwardrateconstant for RNApotymerasb e j n d i n gt o p r o m o t e ris f a s t e rt h a n r a n d o m diffusion.
274
C H A P T E1R1 T r a n s c r i p t i o n
HowDoesRNA FindPromoter Polymerase Sequences? r Therateat whichRNApolymerase bindsto promoters is too fastto be accounted for by random diffusion. . RNApotymerase probably bindsto random siteson DNAandexchanges themwith othersequences is found. veryrapidtyuntila promoter
How is RNA polymerase distributed in the cell? A (somewhat speculative) picture of the enzyme's situation is depicted in Fi$l*fi*11,F1: . Excess core enzyme exists largely as closed loose complexes because the enzyme enters into them rapidly and Ieaves them slowly. There is very little, if any, free core enzyme. . There is enough sigma factor for about one third of the polymerases to exist as holoenzymes, and they are distributed between loose complexes at nonspecific sites and binary complexes (mostly closed)at promoters. . About half of the RNA polymerases consist of core enzymes engaged in transcription. . How much holoenzyme is free? We do not know, but we suspect that the amount is very small. RNA polymerase must find promoters within the context of the genome. Supposethat a promoter is a stretch of -60 bp. How is it distinguished from the 4 x 106bp that comprise the E. coli genome? The next three figures illustrate the principle of some possible models. FliliJR[1l.tF shows the simplestmodel for promoter binding, in which RNA polymerase moves by random diffusion. Holoenzyme very rapidly associateswith, and dissociates from, loose binding sites. Thus it could continue to make and break a series of closed complexes until (by chance) it encounters a promoter, and its recognition of the specific sequence would allow tight binding to occur by formation of an open complex. For RNA polymerase to move from one binding site on DNA to another, it must dissociate from the first site, find the second site, and then associatewith it. Movement from one site to another is limited by the speed of diffusion through the medium. Diffusion sets an upper limit for the rate constant for associating with
a 60 bp target of < I 08 M-I sec-r. The actual forward rate constant for some promoters invitro, however, appears to be -I08 M-l sec-I, at or above the diffusion limit. If this value applies in vivo, the time required for random cycles of successiveassociation and dissociation at Ioose binding sitesis too great to account for the way RNA polymerase finds its promoter. RNA polymerase must therefore use some other means to seekits binding sites.rril-iJ*{tili,i:.,r shows that the process could be speeded up if the initial target for RNA polymerase is the whole genome, not just a specific promoter sequence.By increasing the target size,the rate constant for diffusion to DNA is correspondingly increased and is no longer limiting. If this idea is correct, a free RNA polymerase binds DNA and then remains in contact with it. How does the enzyme move from a random (loose) binding site on DNA to a promoter? The most likely model is to suppose that the bound sequence is directly displaced by another sequence.Having taken hold of DNA, the enzyme exchangesthis sequencewith another sequence very rapidly and continues to exchange sequencesuntil a promoter is found. The enzyme then forms a stable.open complex, after which initiation occurs.The searchprocess becomes much faster becauseassociationand dissociationare virtually simultaneous and time is not spent commuting between sites.Direct displacement can give a "directed walk," in which the enzyme moves preferentially from a weak site to a stronger site. Another idea supposes that the enzyme slidesalong the DNA by a one-dimensional randomwalk, as shonmin i.i*i,iftFi:..;i+,beinghalted only when it encounters a promoter. There is, however, no evidence that RNA polymerase (or other DNA-binding proteins) can function in this manner.
SigmaFactor Contro[s Binding to DNA
sequencesthat the enzyme encounters during riiillustrates how the transcription. irii i-itil: : L,;r' dilemma is solved by the reversible association between sigma factor and core enzyme. As mentioned previously (seeSection I 1.8, The Association with Sigma Factor Changes at Initiation), sigma factor is either released following initiation or changes its association with core enzyme so that it no longer participatesin DNA binding. There are fewer molecules of sigma than of core enzyme; thus the utilization of core enzyme requires that sigma recycles.This occurs immediately after initiation (as shown in the figure) in about one third of cases;presumably sigma and core dissociate at some later Doint in the other cases.
ililli-tlt5; bindsveryrapidtyto ranli i ,l:-li RNApolymerase by direct andcou[dfinda promoter domDNAsequences displacement of the boundDNAsequence.
. A change in association between sigmafactorand holoenzyme changes bindingaffinityfor DNAso that coreenzyme canmoveatongDNA.
RNA polymerase encounters a dilemma in reconciling its needs for initiation with those for eI on gatio n. Initiation re quire s tighl binding on ly to particular sequences(promoters), whereas elongation requires close associationwith 4//
il].{;ii*t-ii i ..1i RNApo[ymerase doesnots[idealongDNA.
Binding to DNA Factor Controts 11.11Sigma
277
{.............
rastI f s'o' Core enzyme storedon DNA
Sigmafactor associates with core enzyme
VerytastI Holoenzyme movesIo promoters
Core enzyme synthesizes RNA
Core enzyme terminates and is released
::i:":i,::=-:i I : -.::r Sigmafactorandcoreenzyme recycte at different pointsin transcription. Irrespectiveof the exact timing of its release from core enzyme, sigma factor is involved only i n i n i t i a t i o n . I t b e c o m e s u n n e c e s s a r yw h e n abortive initiation is concluded and RNA synthesishas been successfullyinitiated. We do not know whether the state of polymerase changes as a consequence of overcoming abortive initiation, or whether instead it is the change in state that ends abortive initiation and allows elongation to commence. When sigma factor is released from core enzyme, it becomes immediately available for u s e b y a n o t h e r c o r e e n z y m e . R e g a r d l e s so I whether sigma factor is released or remains more loosely associatedwith core enzyme, the core enzyme in the ternary complex is bound very tightly ro DNA. Ir is essentially"locked in" until elongation has been completed. When transcription terminates, the core enzyme is released.It is then "stored" by binding to a loose
272
CHAPTER 11 Transcriotion
site on DNA. If it has lost its sigma factor, it must find another sigma factor in order to undertake a further cycle of transcription. Core enzyme has a high intrinsic affinity for DNA, which is increased by the presence of nascent RNA. Its affinity for loose binding sites is, however, too high to allow the enzyme to distinguish promoters efficiently from other sequences.By reducing the stability of the loose complexes, sigma factor allows the process to occur much more rapidly; and by stabilizing the association at tight binding sites,the factor drives the reaction irreversibly into the formation of open complexes. When the enzyme releases sigma factor (or changesits associationwith it), it reverts to a general affinity for all DNA, irrespective of sequence, that suits it to continue transcription. What is responsible for the ability of holoenzyme to bind specifically to promoters? Sigma factor has domains that recognize the promoter DNA. As an independent polypeptide, sigma factor does not bind to DNA, but when holoenzyme forms a tight binding complex, o contacts the DNA in the region upstream of the startpoint. This difference is due to a change in the conformation of sigma factor when it binds to core enzyme. The N-terminal region of free sigma factor suppressesthe activity of the DNAbinding region; when sigma binds to core, this inhibition is released,and it becomes able to bind specifically to promoter sequences (see a l s oF i g u r e I I . 3 5 i n S e c t i o nI l . t 7 ) . T h e i n a b i l ity of free sigma factor to recognize promoter sequencesmay be important: if o could freely bind to promoters, it might block holoenzyme from initiating transcription.
Promoter Recognition Depends on Consensus Sequences . A promoter is definedby the presence of short consensus sequences at specific locations. . Thepromoter consensus sequences consistof a purineat the startpoint,the hexamer TATAAT centered at -10, andanotherhexamer centered at -35. o Individual promoters usually differfromthe consensus at oneor moreoositions.
As a sequence of DNA whose function is to be recognizedby proteins,a promoter differs from sequenceswhose role is to be transcribed or
translated. The information for promoter function is provided directly by the DNA sequence: its structure is the signal. This is a classicexample of a crs-actingsite, as defined previously in Figure 2.16 and Figure 2.17. By contrast, expressedregions gain their meaning only after the information is transferred into the form of some other nucleic acid or protein. A key question in examining the interaction between an RNA polymerase and its promoter is how the protein recognizes a specific promoter sequence.Does the enzyme have an active site that distinguishes the chemical structure of a particular sequenceof basesin the DNA double helix? How specific are its requirements? One way to design a promoter would be for a particular sequence of DNA to be recognized by RNA polymerase. Every promoter would consistof, or at leastinclude, this sequence.In the bacterial genome, the minimum length that could provide an adequatesignalis l2 bp. (Any shorter sequence is likely to occur-just by chance-a sufficient number of additional times to provide false signals. The minimum length required for unique recognition increaseswith the size of genome.) The l2 bp sequenceneed not be contiguous. If a specific number of base pairs separatestwo constant shorter sequences, their combined length could be lessthan I2 bp, becausethe distanceof separation itself provides a part of the signal (even if the intermediate is itself irrelevant). sequence Attempts to identify the features in DNA that are necessaryfor RNA polymerase binding started by comparing the sequences of different promoters. Any essential nucleotide sequenceshould be present in all the promoters. Such a sequenceis said to be conserved. However, a conserved sequence need not necessarilybe conserved at every single position; some variation is permitted. How do we analyze a sequence of DNA to determine whether it is sufficiently conserved to constitute a recognizable signal? Putative DNA recognition sites can be defined in terms of an idealizedsequencethat represents the base most often present at each position. A consensus sequence is defined by aligning all known examples so as to maximize their homology. For a sequenceto be accepted as a consensus,each particular base must be reasonably predominant at its position, and most of the actual examplesmust be related to the consensusby only one or two substitutions. The striking feature in the sequenceof proconsermoters in E. coli is the lackof any extensive
vationof sequence over the 60 bp associatedwith polymerase. The sequenceof much of the RNA binding site is irrelevant. Some short stretches within the promoter are conserved, however, and they are critical for its function. Conservais a typisequences tion of only very short consensus calfeature of regulatory sites(such as promoters)in both prokaryoticand eukaryoticAenlmes. There are four (perhapsfive) conservedfeatures in a bacterial promoter: the startpoint, the -I0 sequence,the -35 sequence,the separation between the -10 and -35 sequences,and (sometimes)the UP element: . The startpoint is usually (>90o/oof the time) a purine. It is common for the startpoint to be the central base in the sequenceCAT,but the conservation of this triplet is not great enough to regard it as an obligatory signal. . Just upstream of the startpoint, a 6-bp region is recognizable in almost all promoters. The center of the hexamer generally is close to l0 bp upstream of the startpoint; the distancevaries in known promoters from Position -18 to -9. Named for its location, the hexamer is often called the -10 sequence. Its consensus is TATAATand can be summarized in the form TeoAqr T+r Aoo Aro Tso where the subscriptdenotes the percent occurrenceof the most frequently found base,which variesfrom 45 % to 96%. (A position at which there is no discernible preference for any base would be indicated by N.) If the frequency of occurrence indicates likely importance in binding RNA polymerase, we would expect the initial highly conserved TA and the final almost completely conservedT in the -10 sequenceto be the most important bases. . Another conservedhexamer is centered -35 bp upstream of the startpoint. This is called the -35 sequence. The consensusis TTGACA;inmore detailed form, the conservation is TezTs+GzsAot Csa,Aa,s. The distanceseparatingthe -35 and -10 sitesis between l6 and I8 bp in 90"h oI promoters; in the exceptions, it is as little as l5 bp or as great as 2obp. Although in the intenteningregion the actualsequence is unimportant, the distanceis critical in
Sequences 273 on Consensus Depends Recognition 11.12Promoter
in one promoter might never have been isoIated in another (which in its wild-type state Startpoint could be even less efficient than the mutant -35 -10 | form of the first promoter). Information gained TTGACA 16-19 bp TATAAT 5-9 bp from studies in vivo simply identifies the overall direction of the change causedby mutation. r:il*i:i i i.:'* A typicaIpromoter hasthreecomponents. Is the most effective promoter one that has -35. -10. consisting of consensus sequences at andthe the actual consensussequences?This expectastartpoint. tion is borne out by the simple rule that up mutations usually increasehomology with one holding the two sitesat the appropriate separationfor thegeometryof RNApolymerase. of the consensussequencesor bring the distance between them closer to l7 bp. Down . Some promoters have an A-T-rich mutations usually decreasethe resemblance of sequencelocated farther upstream. This either site with the consensusor make the disis calledthe UP element. It interactswith tance between them more distant from l7 bp. the cr subunit of the RNA polymerase. Down mutations tend to be concentrated in the It is typically found in promoters that most highly conserved positions, which conare highly expressed,such as the profirms their particular importance as the main moters for rRNA genes. determinant of promoter efficiency. There are, The optimal promoter is a sequence conhowever, occasional exceptions to these rules. -3 sisting of the 5 hexamer, separatedby l7 bp To determine the absolute effects of pro-10 from the hexamer, lying 7 bp upstream of moter mutations, we must measure the affinity the startpoint. The structure of a promoter, polymerase of RNA for wild-type and mutant showing the permitted range of variation from -100-fold variation promoters invitro. There is this optimum, is illustrated in ll{LJQfltt.fti. in the rate at which RNA polymerase binds to different promoters in vitro, which correlates well with the frequencies of transcription when their genes are expressed in vivo. Taking this analysis further, we can investigate the stageat which a mutation influences the capacity of the promoter. Does it change the affinity of the promoter for binding RNA polyrnerase?Does it leave r Downmutations promoter to decrease efficiency the enzyme able to bind but unable to initiate? usua[[y decrease conformance to the consensus sequences, whereas up mutations Is the influence of an ancillary factor altered? havethe oppositeeffect. By measuring the kinetic constants for foro Mutations in the -35 sequence usuatty affect mation of a closed complex and its conversion initiatbindingof RNApolymerase. to an open complex, as defined in Figure I 1.19, r Mutations in the-10 sequence usuatty affectthe we can dissect the two stagesof the initiation mettingreaction that converts a ctosed to an open reaction: compLex. . Down mutations in the -35 sequence reduce the rate of closed complex forMutations are a major source of information mation (they reduce 1(s), but they do about promoter function. Mutations in promotnot inhibit the conversion to an open ers affect the level of expressionof the gene(s) complex. . Down mutations in the -10 sequencedo they control without altering the gene products t h e m s e l v e s .M o s t a r e i d e n t i f i e d a s b a c t e r i a l not affect the initial formation of a closed mutants that have lost, or have very much complex, but they slow its conversion reduced, transcription of the adjacent genes. to the open form (they reduce k2). They are known as down mutations. Less These results suggest the model shown in often, mutants are found in which there is fI**R[ X1-f;=.The function of the -35 sequence increasedtranscription from the promoter. They is to provide the signal for recognition by RNA have up mutations. polymerase,whereas the -10 sequenceallows It is important to remember that "up" and the complex to convert from closed to open "down" mutations are defined relative to the form. We might view the -35 sequenceas comusual elliciency with which a particular proprising a "recognition domain," whereas the -I0 sequencecomprisesan "unwinding domain" moter functions. This varies widely. Thus a change that is recognized as a down mutation of the nromoter.
@
274
CHAPTER 11 Transcription
Promoter Efficiencies CanBeIncreased or Decreased by Mutation
complex lsolateDNA and RNApolymerase-promoter partially attacked by DNAaseI denatureto singlestrands jdq rrr+ jd!" y'"
li -/" Sf{ita
1q
/"a
.r!
y'\
a a
{
/r /1 ttra
/r
,/\
+
r'*
/l
/,. ELECTROPHORESIS
i:i'ii,i.i lti. :i l..a:i The-3 5 sequence is usedfor i nitialrecogjs usedforthemetting nitionandthe-10 sequence reactjonthat converts a closed comptex to an opencomp[ex. The consensussequenceof the -10 site consistsexclusively of A-T base pairs, a configuration that assiststhe initial melting of DNA into singlestrands.The lower energy needed to disrupt A-T pairs compared with G-C pairs means that a stretch of A-T pairs demands the minimum amount of energy for strand separation. The sequence immediately around the startpoint influences the initiation event. The initial transcribedregion (from +l to +30) influences the rate at which RNA polymerase clears the promoter and therefore has an effect upon promoter strength. Thus the overall strength of a promoter cannot be predicted entirely from its -15 and -10 consensussequences. A "typical" promoter relies upon its -35 and -10 sequencesto be recognizedby RNA polymerase,but one or the other of these sequences can be absent from some (exceptional) promoters. In at least some of these cases,the promoter cannot be recognizedby RNA polymerasealone; the reaction requires ancillary proteins, which overcome the deficiency in intrinsic interaction between RNA polymerase and the promoter.
Binds RNAPol.ymerase to OneFaceof DNA Theconsensus sequences at -35 and-L0 provide in mostof the contactpointsfor RNApotymerase the promoter. Theoointsof contactlie on onefaceof the DNA.
DNAlabeledat one end of one strand
-
Farthestfrom labeledend
g EXPERIMENTAL GEL
:
CONTBOL GEL DNA not bound to polymerase has bandsat positions corresponding to breakageof every bond
Missingbands identify bindingsite
ffi Nearestto labeledend
sitesfor proteins I.lr,;i.lft:ii DNA-binding identifies I i .l:r Footprinting againstnicking. bytheirprotection The ability of RNA polymerase (or indeed any protein) to recognize DNA can be characterized by footprinting. A sequenceof DNA bound to the protein is partially digestedwith an endonucleaseto attack individual phosphodiesterbonds within the nucleic acid. Under appropriate conditions, any particular phosphodiester bond is broken in some,but not in all, DNA molecules. The positions that are cleaved are recognized by using DNA labeled on one strand at one end only. The principle is the same as that involved in DNA sequencing: partial cleavageof an endIabeled molecule at a susceptible site creates a fragment of unique length. As i ii,i.ii:.i::1 .;ii:.shows, following the nuclease treatment, the broken DNA fragments are recovered and electrophoresed on a gel that separatesthem according to length. Each fragment that retains a labeled end produces a radioactive band. The position of the band corresponds to the number of bases in the
Bindsto OneFaceof DNA 11.14RNAPolvmerase
275
-1 0 sequence
r rlll
I
I
l . l " l J l l l l lI A A T X X X X A 1 T Codingstrandlu
T
ilririffi;;;l ATA
I
I
Template stranc
TTAXXXX
il
lr
Most pointsof contactlie on one face of DNA (on the nontemplatestrand)
Modificationsthat preventRNApolymerase from binding Siteswhere RNA polymeraseprotectsagainstmodification Mutationsthat abolishor reducepromoteractivtty
: : = r . . . : : r: , r i 0 n ef a c eo f t h ep r o m o t ec ro n t a i nt h s ec o n t a cpt o i n t fso rR N A . fragment. The shortestfragments move the fastest,so distance from the labeled end is counted up from the bottom of the gel. In a free DNA, everysusceptiblebond position is broken in one or another molecule. When the DNA is complexed with a protein, though, the region covered by the DNA-binding protein is protected in every molecule. Thus two reactions are run in parallel: a control of DNA alone, and an experimental mixture containing molecules of DNA bound to the protein. When a bound protein blocksaccess of the nucleaseto DNA, the bondsin the boundsequence fail to be brokenin the experimentalmixture. In the control, every bond is broken. This generatesa seriesof bands,with one band representing each base.There are thirty-one bands in the figure. In the protected fragment, bonds cannot be broken in the region bound by the protein, so bands representing fragments of the correspondingsizesare not generated.The absenceof bands 9-18 in the figure identifiesa protein-binding site covering the region located 9-18 basesfrom the labeled end of the DNA. By comparing the control and experimental lanes with a sequencingreaction that is run in parallel, it becomes possible to "read off" the corresponding sequencedirectly, thus identifying the nucleotide sequenceof the binding site. As describedpreviously (seeFigure Il.20l, RNA polymerase initially binds the region from -50 to +20. The points at which RNA polymerase
fi:'ffir'-:".Td;ili:fl :lilililT.niff ::: treat RNA polymerase-promotercomplexes
276
CHAPTER 11 Transcription
with reagents that modify particular bases.We can perform the experiment in two ways: . The DNA can be modified before it is bound to RNA polymerase.If the modification prevents RNA polymerase from binding, we have identified a baseposition where contact is essential. . The RNA polymerase-DNA complex can be modified. We then compare the pattern of protected bands with that of free DNA and the unmodified complex. Some bands disappear,thus identifying sitesat which the enzyme has protected the promoter againstmodification. Other bands increase in intensity, thus identifying sites at which the DNA must be held in a conformation in which it is more exposed. These changes in sensitivity reveal the geometry o{ the complex, as summarized in i:ltur.j*i: ri:.!l'i for a typical promoter. The regions at -35 and -I0 contain most of the contactpoints for the enzyme. Within these regions, the same setsof positions tend both to prevent binding if previously modified, and to show increased or decreasedsusceptibility to modification after binding. The points of contact do not coincide completely with sites of mutation; however, they occur in the same limited region. It is noteworthy that the same positionsin different promoters provide the contact points, even though a different base is present. This indicates that there is a common mechanism {or RNA polymerase binding, although the reaction does not depend on the presence of particular basesat some of the points of contact. This model explains why some of the points of contact are not sites of mutation. In addition, not every mutation lies in a point of contact; the mutations may influence the neighborhood without actually being touched by the enzyme. It is especially significant that the experiments with prior modification identify only sites in the same region that is protected by the enzyme againstsubsequentmodification. These two experiments measure different things. Prior modification identifies all those sites that the enzyme must recognizein order to bind to DNA. Protection experiments recognize all those sites that actually make contact in the binary complex. The protected sites include all the recognition sites and also some additional positions, which suggeststhat the enzyme first recognizes a set of basesnecessaryfor it to "touch down" and then extends its points of contact to additional bases.
The region of DNA that is unwound in the binary complex can be identified directly by chemical changes in its availability. When the strandsof DNA are separated,the unpaired bases become susceptibleto reagentsthat cannot reach them in the double helix. Such experiments implicate positions between -9 and +3 in the initial melting reaction. The region unwound during initiation therefore includes the right end of the -10 sequenceand extendsjust past the startpoint. Viewed in three dimensions, the points of contact upstream of the -10 sequenceall lie on one face of DNA. This can be seen in the lower drawing in Figure I1.29, in which the contact points are marked on a double helix viewed from one side. Most lie on the coding strand. These basesare probably recognized in the initial formation of a closed binary complex. This would make it possible for RNA polymerase to approach DNA from one side and recognizethat face of the DNA. As DNA unwinding commences, further sites that originally lay on the other face of DNA can be recosnizedand bound.
Is an Supercoiling ImportantFeature of Transcription . Negative increases supercoiting the efficiency of somepromoters by assisting the meltingreaction. r Transcription generates positivesupercoi[s ahead of the enzyme andnegative supercoits behindit, andthesemustbe removed by gyrase and toooisomerase,
The importance of strand separation in the initiation reaction is emphasized by the effects of supercoiling. Both prokaryotic and eukaryotic RNA polymerases can initiate transcription more efficiently in vitro when the template is superc o i l e d , p r e s u m a b l y b e c a u s et h e s u p e r c o i l e d structure requires Iess free energy for the initial melting of DNA in the initiation complex. The efficiency of some promoters is influenced by the degree of supercoiling. The most common relationship is for transcription to be aided by negative supercoiling. We understand in principle how this assiststhe initiation reaction. Why, though, should some promoters be influenced by the extent of supercoiling whereas others are not? One possibility is that the dependence of a promoter on supercoiling is determined by its sequence.This would predict
(Negative supercoils)
Transcribing Overwound (Positivesupercoils) DNA
-
Topoisomerase I relaxesnegative I V supercoils
Gyrase positive introduces supercoils
DuplexDNA(10.4bp/turn) generates moretightlywound f,f{ii"5ii!. ::i ..li:iTranscription (positivety DNAaheadof RNApolymerase, supercoi[ed) lesstighttywound(negwhitethe DNAbehindbecomes ativetysupercoil"ed).
that some promoters have sequences that are easierto meit (and are therefore lessdependent on supercoiling), whereas others have more difficult sequences(and have a greater need to be supercoiled).An alternative is that the location of the promoter might be important if different regionsof the bacterialchromosome have different degreesof supercoiling. Supercoiling also has a continuing involvement with transcription. As RNA polymerase transcribes DNA, unwinding and rewinding occurs,as illustrated in Figure I 1.4.This requires that either the entire transcription complex rotates about the DNA or the DNA itself must rotate about its helical axis. The consequences of the rotation of DNA are illustrated in f: i.L. i.rin t]netwin domain model for tranlj}{:i#fi scription. As RNA polymerase pushes forward along the double helix, it generates positive supercoils (more tightly wound DNA) ahead and leaves negative supercoils (partially unwound DNA) behind. For each helical turn traversed by RNA polymerase, +l turn is generated ahead and -l turn behind. Transcription therefore has a significant effect on the (local) structure of DNA. As a result, the enzymes gyrase (introduces negative supercoils) and topoisomeraseI (removes negative supercoils) are required to rectify the situation in front of and behind the polymerase, respectively. Blocking the activitiesof gyraseand topoisomerase causes major changes in the supercoiling of DNA. For example, in yeastlacking an enzyme that relaxes negative supercoils, the density of negative supercoiling doubles in a transcribed region. A possible implication of these results is that transcription is responsible
of Transcription 277 Is an ImportantFeature 11.15Supercoiting
for generating a significant proportion of the supercoilingthat occurs in the cell. A similar situation occurs in replication, when DNA must be unwound at a moving replication fork so that the individual single strands can be used as templates to synthesizedaughter strands. (Solutions for the topological cons t r a i n t s a s s o c i a t e dw i t h s u c h r e a c t i o n s a r e indicated later, in Figure 19.20.)
@
Substitution of Sigma Factors MayControl Initiation
. E. coLihasseveral sigmafactors,eachof which causes RNApotymerase to initiateat a setof -35 and-10 promoters definedby specific sequences. . o70is usedfor generaI transcription. andthe other sigmafactorsareactivated by speciaI conditions.
The division of labors between a core enzyme that undertakes chain elongation and a sigma factor involved in site selection immediately raises the question of whether there is more than one type of sigma factor, each specific for a different classof promot".r. ;;i-:,riii:: :, -;: shows the principle of a system in which a substitution of the sigma factor changes the choice of Dromoter.
Holoenzymewith o7orecognizes one set of oromoters
Substitutionof sigmafactor causesenzymeto recognize a differentset of promoters
::i1;1.;liir i"l.:l i Thesigmafactorassociated with core enzyme determines thesetof promoters at whichtranscriptionis initiated.
278
CHAPTER 11 Transcription
E. coli uses alternative sigma factors to respond to general environmental changes;they are listed in :ri*ljF.It:,1ii. (They are named either by molecular weight of the product or for the gene.) The general factor, which is responsible for transcription of most genes under normal conditions, is o70.The alternative sigma factors 6s, 6)2, oE, and o54are activated in response to environmental changes;o28is used for expression of flagellar genes during normal growth, but its level of expression responds to changes in the environment. AII the sigma factors except o5abelong to the same protein family and function in the same general manner. Temperature fluctuation is a common type of environmental challenge. Many organisms, both prokaryotic and eukaryotic, respond in a similar way. Upon an increase in temperature, synthesis of the proteins currently being made is turned off or down, and a new set of proteins is synthesized. The new proteins are the products of the heat shock genes, which play a role in protecting the cell against environmental stress.Heat shock genes are synthesized in response to conditions other than heat shock as well. Several of the heat shock proteins are chaperones. In E. coli,the expression of seventeen heat shock proteins is triggered by changes at transcription. The gene rpoH is a regulator needed to switch on the heat shock response. Its product is o32,which functions as an alternative sigma factor that causestranscription of the heat shock genes. The heat shock responseis accomplished increasing by the amount of o32when the temperature increasesand decreasingits activity when the temperature change is reversed. The basic signal that induces production of ol2 is the accumulation of unfolded (partially denatured) proteins that results from increase in temperature. The or2 protein is unstable,which is important in allowing its quantity to be increased or decreasedrapidly. The proteins o70and ol2 can compete for the available core enzyme, so that the set of genes transcribed during heat shock depends on the balance between them.Changing sigma factors is a serious matter that has widespread implications for gene expression in the bacterium. It is not surpdsing, therefore, that the production of new sigma factors can be the target of many regulatory circuits. The factor os is induced when bacteria make the transition from growth phase to stationary phase and also in other stressconditions. It is controlled at two levels. Translation of. the rpoS mRNA is increased by low temperature or high
osmolarity. Proteolysisof the protein product is inhibited by carbon starvation (the typical signal of stationary phase) and by high temperature. Another group of heat-regulated genes is controlled by the factor oE.It respondsto more extreme temperature shifts than ol2 and is induced by accumulation of unfolded proteins in the periplasmicspaceor outer membrane. It is controlled by the intricate circuit summar i z e d i n r 1 i . .i i : t ' : r . T h e f a c t o r o E b i n d s t o a protein (RseA) that is located in the inner membrane. As a result, it cannot activate transcription. The accumulation of unfolded proteins activatesa protease (DegS) in the periplasmic space,which cleavesoff the C-terminal end of the RseA protein. This cleavage activates another protein in the inner membrane (YaeL), which cleavesthe N-terminal region of RseA. When this happens, the oE factor is released and can then activate transcription. The net result is that the accumulation of unfolded proteins at the periphery of the bacterium is responsible for activating the set of genes controlled by the sigma factor. This circuit has two interesting parallels with other regulatory circuits.The responseto unfolded proteins in eukaryotic cells also uses a pathway in which an unfolded protein (within the endoplasmic reticulum) activatesa membrane protein. In this case.the membrane protein is an endonucleasethat cleavesan RNA, Ieading ultimately to a change in splicing that causesthe production of a transcription factor (see Section 26.17, The Unfolded Protein ResponseIs Relatedto IRNA Splicing).A more direct parallel is with the first caseto be discovered, in which cleavageof a membrane protein activates a transcription {actor. In this case,the transcription factor itself is synthesized as a membrane protein, and the level of sterols in the membrane controls the activation of proteases that releasethe transcription factor from the cytosolic domain of the protein. Another sigma factor is used under conditions of nitrogen starvation. E coli cellscontain a small amount of o5a,which is activated when ammonia is absent from the medium. In these conditions, genes are turned on to allow utilization of alternative nitrogen sources.Counterpartsto this sigmafactor have been found in a w i d e r a n g e o f b a c t e r i a , s o i t r e p r e s e n t sa responsemechanism that has been conserved in evolution. Another caseof evolutionary conservation of sigma factors is presented by the factor oF,
which is present in small amounts and causes RNA polymerase to transcribe genes involved in chemotaxis and flagellarstructure. Its counterpart in Bacillussubtilis is oD, which controls flagellar and motility genes; factors with the same promoter specificity are present in many speciesof bacteria. Each sigma factor causesRNA polymerase to initiate at a particular set of promoters. By analyzingthe sequencesof these promoters, we can show that each set is identified by unique sequence elements. Indeed, the sequence of each type of promoter ensuresthat it is recognized only by RNA polymerase directed by the appropriate sigma factor. We can deduce the general rules for promoter recognition from the identification of the genesresponding to the sigma factors found in E. coli andthose involved
rpoD
o7o
general
rpoS
os
stress
rpoH
o32
heat shock
rpoE
oE
rpoN
o54
heat shock nitrogenstarvation
fliA
o" (ot)
flagellarsYnthesis
sigma E.collhasseverat irril.iirr I ,. In addition to oT0, conenvironmentaI byparticu[ar factors thatareinduced its in the nameof a factorindicates djtions.(A number m a s s) .
as a proteinin the i'ii:,!.iirr, i t, rir RseAis synthesized bjndstheoEfacdomain Its cytoplasmic innermembrane. space in the periptasmic is cleaved sequentialty tor.RseA mh. ec y t o p [ a s mci ct e a v a g e a n dt h e ni n t h e c y t o p L a s T reteases o'.
Initiation MayControl ofsigmaFactors 11.16Substitution
279
Gene Factor -35 SequenceSeparation-10 Sequence rpoD
o7o
TTGAoA
16-18 bp
TATAAT
rpoH
o32
cccrrcAA
13-15 bp
ccccATNT
rpoN
o54
CTGGNA
fliA
o28(oF)
CTAAA
sigH
on
AGGANPUPu 11-12bp
6 bp
TTGoA
15 bp
ccccATAA
Core enzymebinding
+ I
_ l!J"'T!lys_*_?qq-____-"" . 100_ llgYgntt
i90*
2.12.22.4 4.14.2 Interactionswith promoter
DNA-bindins
:..... €Gene
I
I
...-10 J.......Y.. -35 1 TAATAT.ACAGTT5'
i:...i-;ii :: :i A mapof the E. colio70factoridentifies conserved regions. Regions 2.1and2.2contactcorepotymerase, 2.3is required for metting,and2.4and4.2 con-
XJT :.iJ.il'# illi;'ff J;l' il'Ji i ntj! #T*:',-;', in sporulation in B subtilis(see Section I1.19, Sporulation Is Controlled by Sigma Factors). A significant feature of the promoters for each enzyme is that they have the samesizeand Iocationrelativeto the startpoint, and theyshow conseruedsequences only around the usualcenters of-35 and-10. (The factor o5ais an exception for which the consensussequencesare closertogether and are positioned at -24 and -l 2; seeSection I L I 7, Sigma Factors Directly Conract DNA.) As summarized in ilii=i:r .::",-:+, the consensussequences for each set of promoters are different from one another at either or both of the -35 and -10 positions. This means that an enzyme containing a particular sigma factor can recognize only its own set of promoters, so that transcription of the different groups is mutually exclusive. Substitution of one sigma factor by another therefore turns off transcription of the old set of genes as well as turning on transcription of a new set of genes. (Some genes are expressedby RNA polymerases with different sigma factors because they have more than one promoter, each with a different set of consensussequences.)
280
CHAPTER 1.1 Transcription
o70changes its structure to retease its DNAbindingregions whenit associates withcore enzyme. o70bindsboththe -35 and-10 sequences.
GCTGAATCA
r-t:-.i-,il:: .::.:..=E.colisigma factorsrecognize promoters withdiffere n tc 0 n s e n ssuesq u e n c e s .
1.1 1.2
SigmaFactors Directly Contact DNA
The definition of a seriesof different consensus sequencesrecognizedat -35 and -10 by holoenzymes containing different sigma factors (see Figure I1.34) carries the immediate implication that the sigma factor subunit must itself contact DNA in these regions. This suggeststhe general principle that there is a common type of relationship between sigma factor and core enzyme, in which the sigma factor is positioned in such a way as to make critical contacts with the promoter sequencesin the vicinity of -35 and -10. Direct evidence that sigma factor contacts the promoter directly at both the -35 and -10 consensussequencesis provided by mutations in sigma factor that suppressmutations in the consensus sequences.When a mutation at a particular position in the promoter prevents recognition by RNA polymerase, and a compensating mutation in sigma factor allows the polymerase to use the mutant promoter, the most likely explanation is that the relevant base pair in DNA is contacted by the amino acid that has been substituted. Comparisons of the sequencesof several bacterial sigma factors identify regions that have been conserved.Their locations in E. coli o70are summarized in F{{itJFti t:i.:i;. The crystal structure of a sigma factor fragment from the bacterium Thermus aquaticus shows that these regions fold into three independent domains in the protein: domain 02 contains \.2-2.4,63 Contains 3.0-1.3, and oa contains 4.14.2. Figure I I .3 5 shows that two short parts of regions2 and4 (named 2.4 and4.2) are involved in contacting basesin the -10 and -l5 elements, respectively. Both of these regions form short stretchesof s-helix in the protein. Experiments with heteroduplexes show that o70makes contactswith basesprincipally on the coding strand, and it continues to hold these contacts after the DNA has been unwound in this region. This suggeststhat sigma factor could be important in the melting reaction. The use of a,-helicalmotifs in proteins to recognize duplex DNA sequencesis common
( s e e S e c t i o n 1 4 . I l , R e p r e s s o rU s e s a H e l i x T\rrn-Helix Motif to Bind DNA). Amino acids separatedby three to four positions lie on the same face of an s-helix and are therefore in a position to contact adjacent base pairs. lril',l,jiil5i.:ri::shows that amino acids lying along one face of the 2.4 region u-helix contact the b a s e sa t p o s i t i o n s- 1 2 t o - 1 0 o f t h e - 1 0 p r o moter sequence. Region 2.3 resemblesproteins that bind single-strandednucleic acidsand is involved in the melting reaction. Regions 2.1 and 2.2 (which comprise the most highly conserved part of sigma) are involved in the interaction with core enzyme. It is assumedthat all sigma factorsbind the same regions of the core polymerase, which ensures that the reactions are competitive. The N-terminal region of o70has important regulatory functions. If it is removed, the shortened protein becomes able to bind specifically to promoter sequences.This suggeststhat the N-terminal region behaves as an autoinhibition domain. It occludesthe DNA-binding domains when o70is free. Association with core enzyme changes the conformation of sigma so that the inhibition is released, and the DNA-binding domains can contact DNA. t::. ::j- schematizesthe conformational i:,i.-:-!iiil: change in sigma factor at open complex formation. When sigma factor binds to the core poly-merase, the N-terminal domain swings -2O A away from the DNA-binding domains, and the DNA-binding domains separate from one another by -15 A, presumably to acquire a more elongated conformation appropriate for contacting DNA. Mutations in either the - I 0 or -3 5 sequencesprevent an (N-terminal-deleted)o70 from binding to DNA, which suggeststhat o70 contacts both sequences simultaneously. This implies that the sigma factor must have a rather elongated structure, extending over the -68 A of two turns of DNA. In the free holoenzyme, the N-terminal domain is located in the active site of the core enzyme components, essentiallymimicking the location that DNA will occupy when a transcription complex is formed. When the holoenzyme forms an open complex on DNA, the N - t e r m i n a l s i g m a d o m a i n i s d i s p l a c e df r o m the active site. Its relationship with the rest of the protein is therefore very flexible; the relationship changes when sigma factor binds to core enzyme and again when the holoenzyme binds to DNA. Comparisons of the crystal structures of the core enzyme and holoenzyme show that
Protein
Position -1T12-11-1H-a-7
of o70conilii-iililir :l i,lr:r:Aminoacidsin the2.4cr-helix in the codingstrandof the -10 probases tact specjfic motersequence.
Nterminalregionbinds
1.l "-i,:TheN-terminus of sigmablocksthe DNAi:iir,i"iiiifrombindingto DNA.Whenan opencombindingregions swings20.A away,andthetwo ptexforms,the N-terminus by 15 A. regions separate DNA-binding
sigma factor lies largely on the surface of the i i.;itr shows that it has core enzyme. iri:i,iia;i:a an elongated structure that extends past the DNA-binding site. This places it in a position to contact DNA during the initial binding. The DNA helix has to move some l6 A from the initial position in order to enter the active site. iii.i.ri:.i::: r'i illustrates this movement, looking in cross-sectiondown the helical axis of the DNA. An interesting difference in behavior is found with the o5afactor.This causesRNA polymerase to recognize promoters that have a distinct consensus sequence, with a conserved element at -12 and another close by at -24 (given in the "-35" column of Figure 11.32). Thus the geometry of the polymerase-promoter complex is different under the direction of this sigma factor. Another diff erence in the
DNA Contact Directly 11.17SigmaFactors
287
SigmaFactors MayBe 0rganized into Cascades . A cascade of sigmafactorsis created whenone sigmafactoris required to transcribe the gene codingforthe nextsigmafactor. . Theeartygenesof phageSP01aretranscribed by hostRNApolymerase. . 0neof theeartygenescodes for a sigmafactor that causes RNApotymerase to transcribe the midd[egenes. r Twoof the midd[egenes codefor subunits of a sigmafactorthat causes RNApolymerase to transcribe the [ategenes.
Sigma factors are used extensively to control initiation of transcription in the bacterium B. subtilis,for which -10 different o factors are known. Some are present in vegetative cells; Sigmafactorhasan elongated structure others are produced only in the special circumthatextends atongthesurface of thecoresubunits when stances phage of infection or the change from the hoLoenzvme is formed. vegetative growth to sporulation. The major RNA polymerase found in B. subtilis cells engaged in normal vegetative growth has the same structure as that of.E. coli,szFF'o. Its sigma factor (describedas oa3or oA) recognizes promoters with the same consensus sequencesused by the E. coli enzyme under direction from o70.Severalvariants of the RNA polymerase that contain other sigma factors are found in much smaller amounts. The variant enzymes recognizedifferent promoters on the basisof consensussequencesat-35 and -I0. Transitions from expression of one set of , DNAinitiattycontacts sigma factor(pink) genes to expressionof another set are a comandcoreenzyme (gray).It movesdeeper into the core mon feature of bacteriophageinfection. In all enzyme to make contacts at the-10 sequence. When sigma but the very simplest cases,the development i s r e l e a s e tdh, e w i d t ho f t h e p a s s a gceo n t a i n i nD g NA increases. Reproduced fromVassylye4 D.G.,et al.Nature. of the phage involves shifts in the pattern of 2 0 0 2 .4 1 . 7 :7 1 , 2 - 7 1 . 9 P.h o t oc o u r t e s yo f S h i g e y u k i transcription during the infective cycle. These Yokoyama, TheUniversity of Tokyo. shifts may be accomplished by the synthesis of a phage-encoded RNA polymerase or by the efforts of phage-encodedancillary factors that mechanism of regulation is that high-level trancontrol the bacterial RNA polymerase.A wellscription directed by otn requires other activacharacterizedexample of control via the protors to bind to sitesthat are quite distant from duction of new sigma factors occurs during the promoter. This contrastswith the other types infection of.B. subtilisby phage SPOL of bacterial promoter, for which the regulator The infective cycle of SPOl passesthrough sitesare always in close proximity to the prothree stagesof gene expression.Immediately moter. The behavior of o5aitself is different from on infection, the early genes of the phage are other sigma factors,most notably in its ability transcribed.After four to five minutes, the early to bind to DNA independently of core polygenesceasetranscription and the middle genes merase. In this regard, o5ais more like the are transcribed.At eight to twelve minutes, mideukaryotic regulators we discussin Chapter 24, dle gene transcription is replaced by transcripPromoters and Enhancers, than the typical tion of late genes. prokaryotic regulators discussedin Chapter I 2, The early genes are transcribed by the Ihe Operon. holoenzyme of the host bacterium. They are CHAPTER L1 Transcriotion
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r: ,,': . . Sporutation invotves thedifferentiation of a vegetative bacterium into a mothercetlthat is tysed anda sDore that is reteased.
cell is eventually lysed, and the spore that is releasedhas an entirely different structure from the original bacterium. Sporulation involves a drastic change in the biosynthetic activitiesof the bacterium, in which many genesare involved. The basiclevel of control lies at transcription. Some of the genesthat functioned in the vegetativephase are turned off during sporulation,but most continue to be expressed.In addition, the genes specific for s p o r u l a t i o n a r e e x p r e s s e do n l y d u r i n g t h i s period. At the end of sporulation, -40% of the bacterialmRNA is sporulation specific. New forms of the RNA polymerase become
marized in ijrli,ljiii I j.r,.l'.The principle is that in each compartment the existing sigma factor is successivelydisplacedby a new factor that causes transcription of a different set of genes. Communication between the compartments occurs in order to coordinate the timing of the changes in the foresporeand mother cell. The sporulation cascadeis initiated when environmental conditions trigger a phosphorelay, in which a phosphate group is passed along a seriesof proteins until it reachesSpoOA. (Severalgene products are involved in this process,whose complexity may reflect the need to avoid mistakes in triggering sporulation unnecessarily.)SpoOA is a transcriptional regulator whose activity is affected by phosphorylation. In the phosphorylated form. it activates transcription of two operons, each of which is transcribed by a different form of the host RNA polymerase. Under the direction of phosphorylated SpoOA, host enzyme utilizing the general dl transcribesthe gene coding for the factor oF, and host enzyme under the direction of a minor factor, oH, transcribes the gene coding for the factor pro-oE. Both of these new sigma factors are produced before septum formation, but become active later. Factor oF is the first one to become active in the forespore compartment. It is inhibited by an antisigma factor that binds to it; in the forespore, an anti-antisigma factor removes the inhibitor. This reaction is controlled by a series of phosphorylation/dephosphorylation evenrs. The initial determinant is a phosphatase(SpotrE) that is an integral membrane protein that accumulates at the pole, with the result that its phosphatase domain becomesmore concentrated in the forespore. It dephosphorylates,and thereby activates, SpoIIAA, which in turn displacesthe antisigma factor SpoIIAB from the complex of SpoIIAB-oF.Releaseof oF activatesit. Activation of oF is the start of sporulation. Under the direction of oF, RNA polymerase transcribesthe first set of sporulation genes instead of the vegetative genes it was previously transcribing. The replacement reaction probably affects only part of the RNA polymerase popuIation, because oF is produced only in small amounts. Some vegetative enzyme remains present during sporulation. The displaced oar is not destroyed; it can be recovered from extractsof sporulating cells. TWoregulatory events follow from the activity of oF, as detailed in i i i;l,iii!.'i :t i!ti..In the forespore itself, another factor, oG. is the product of one of the early sporulation genes. Factor oc
::l'ff;trJ:ll&Hlf J:""',J?1',1*"Jft:: ent proteinsin placeof the vegetativeo4l. The changesin transcriptionalspecificityare sum-
284
CHAPTER 1.1Transcription
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BacteriaI RNAPolymerase Terminates at Discrete Sites r Termination mayrequirebothrecognition of the terminator sequence in DNAandtheformation of a hairpinstructure in the RNAproduct.
ii*-*i : :.'t-: oFtriggers synthesis of thenextsigmafactor in theforespore (oG)andturnsonSpoIIR, whichcauses SpoIIGAto cleaveoro-oE.
MOTHER
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FORESPORE
Proteolysis
a-ILiii:s::.ilr; Thecrisscross regutation of sporulation c o o r d i n a t et ism i n go f e v e n t si n t h e m o t h e cr e [ [a n d forespore.
compartment are successivelyactivated, each directing the synthesis of a particular set of genes. -i!Liq{ ::..++outlines how the two cascadesare connected by the transmission of signals from one compartment to the other. As new sigma factors become active, old sigma factors are displaced, so that transitions in sigma factors turn genes off as well as on. The incorporation of each factor into RNA polymerase dictateswhen its set of target genesis expressed, and the amount of factor available influences the level of gene expression. More than one sigma factor may be active at any time, and the specificities of some of the sigma factors overlap. We do not know what is responsible for the ability of each sigma factor to replace its predecessor.
286
CHAPTER 11 Transcription
Once RNA polymerase has started transcription, the enzyme moves along the template, synthesizing RNA, until it meets a terminator sequence.At this point, the enzyme stops adding nucleotides to the growing RNA chain, releases the completed product, and dissociatesfrom the DNA template. Termination requires that all hydrogen bonds holding the RNA-DNA hybrid together must be broken, after which the DNA duplex reforms. It is difficult to define the terminarion point of an RNA molecule that has been synthesized in the living cell. It is always possible rhat the 3' end of the molecule has been generatedby cleavageof the primary transcript, and therefore does not represent the actual site at which RNA polymerase terminated. The best identification of termination sites is provided by systemsin which RNA poll.rnerase terminates invitro. The ability of the enzyme to terminate is strongly influenced by parameters such as the ionic strength; as a result, its termination at a particular point in vitro does not prove that this same point is a natural terminator. We can, however, identify authentic J' ends when the same end is generated in vitro and in vivo. fi*LFR*1.1-:i$ summarizes the two types of features found in bacterial terminators. . Terminators in bacteria and their phages have been identified as sequencesthat are needed for the termination reaction (in vitro or in vivo). The sequences ar prokaryotic terminators show no similarities beyond the point at which the last base is added to the RNA. The responsibility for termination lies with the sequences alreadytranscribedbyRNA polymerase. Thus termination relies on scrutiny of the template or product that the polymerase is currently transcribing. . Many terminators require a hairpin to form in the secondary structure of the RNA being transcribed. This indicatesthat termination dependson the RNA product and is not determinedsimply by scrutiny of the DNA sequenceduring transcription.
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very end of the unit that is rich in U residues. Both featuresare needed for termination. The hairpin usually contains a G-C-rich region near the base of the stem. The typical distance between the hairpin and the U-rich region is sevento nine bases.There are -l 100 sequences in the E. coli genome that fit these criteria, which suggeststhat about half of the geneshave intrinsic terminators. Point mutations that prevent termination occur within the stem region of the hairpin. What is the effect of a hairpin on transcription? It is likely that all hairpins that form in the RNA product causethe polyme raseto slow (and perhaps to pause) in RNA synthesis. Pausingcreatesan opportunity for termination to occur. Pausing occurs at sitesthat resemble terminators but have an increased separation (typically ten to eleven bases)between the hairpin and the U-run. If the pause site does not correspond to a terminator, though, the enzyme usually moves on again to continue transcription. The length of the pause varies, but at a typical terminator lasts-60 seconds. A downstream U-rich region destabilizes the RNA-DNA hybrid when RNA polymerase pauses at the hairpin. The rU-dA RNA-DNA hybrid has an unusually weak base-pairedstructure; it requires the least energy of any RNA-DNA hybrid to break the association between the two strands.When the polymerase pauses, the RNA-DNA hybrid unravels from the weakly bonded rU-dA terminal region. Often, the actual termination event takes place at any one of several positions toward or at the end of the U-rich region, as though the enzyme "stutters" during termination. The U-rich region in RNA corresponds to an A-T-rich region in DNA, so we seethat A-T-rich regions are important in intrinsic termination as well as initiation. Both the sequence of the hairpin and the length of the U-run influence the efficiency of termination. Termination efficiency in vitro, however, varies from 2o/olo 90oh and does not correlate in any simple way with the constitution of the hairpin or the number of U residues in the U-rich region. The hairpin and U-region are therefore necessary,but not sufficient, and additional parameters influence the interaction with RNA polymerase. In particular, the sequencesboth upstream and downstream of the intrinsic terminator influence its efficiency. Less is known about the signals and ancillary factors involved in termination for eukaryotic polymerases.Each classof polymerase uses a different mechanism (see Chapter 26, RNA Splicing and Processing). ption CHAPTER 11 Transcri
HowDoesRhoFactor
Work? o Rhofactoris a terminator proteinthat bindsto a ruf siteon nascent RNAandtracksatongthe RNA to retease it fromthe RNA-DNA hvbridstructure ar the RNApotymerase.
Rho factor is an essential protein in E. coli that functions solely at the stage of termination. It acts at rho-dependent terminators, which account for about half of E. coli Lerminators. FIGURE x t -4?shows how rho functions. First it binds to a sequence within the transcript upstream of the site of termination. This sequence is called a rut site (an acronym for rho utilization). The rho then tracks along the RNA until it catches up ro RNA polymerase. When the RNA polymerase reaches the termination site, rho acts on the RNA-DNA hybrid in the enzyme to causereleaseof the RNA. Pausing by the polymerase at the site of termination allows time for rho factor to translocate to the hybrid stretch and is an important feature of termination. We seean important general principle here. When we know the site on DNA at which some protein exercisesits effect, we cannot assume that this coincides with the DNA sequencerhat it initially recognizes.They can be separate,and there need not be a fixed relationship between them. In f.act,rut sitesin different transcription units are found at varying distancespreceding the sites of termination. A similar distinction is made by antitermination factors (see Section 11.24,Antitermination Requires SitesThat Are Independent of the Terminators). The common feature of.rut sites is that the sequence is rich in C residues and poor in G residues and has no secondary structure. An example is given in FSGttRil it.4S. C is by far the most common base (4lo/o) and G is the least common base (Ia%|. rut sitesvary in length. As a general rule, the efficiency of a rutsile increases with the length of the C-rich/G-poor region. Rho is a member of the family of hexameric ATP-dependent helicases.The subunit has an RNA-binding domain and an ATP hydrolysis domain. The hexamer functions by passing nucleic acid through the hole in the middle of the assembly formed from the RNA-binding domains of the subunits. f,cfruRg 11.4s shows that the structure of rho gives some indications of how it functions. It winds RNA from the 3' end around the exterior of the N-terminal
domains, and pushes the 5' end of the bound region into the interior, where it is bound by a secondaryRNA-binding domain in the C-terminal domains. The initial form of rho is a gapped ring, but binding of the RNA converts it to a closedring. After binding to the rut site, rho uses its helicase activity, driven by ATP hydrolysis, to translocate along RNA until it reaches the RNA-DNA hybrid stretch in RNA polymerase. It then unwinds the duplex structure. We do not know whether this action is sufficient to releasethe transcript or whether rho also interactswith RNApolymerase to help releaseRNA. Rho functions as an ancillary factor for RNA polymerase; typically its maximum aclivity in vitro is displayed when it is present at -l0o/o of the concentration of the RNA polymerase. Rho needs to translocate along RNA from threrut site to the actual point of termination. This requires the factor to move faster than RNA polymerase. The enzl.rne pauseswhen it reaches a terminator, and termination occurs if rho catchesit there. Pausing is therefore important in rho-dependent termination, just as in intrinsic termination, because it gives time for the other necessaryevents to occur. The idea that rho moves along RNA leads to an important prediction about the relationship between transcription and translation. Rho must first have accessto a binding sequence on RNA and then must be able to move along the RNA. Either or both of these conditions may be prevented if ribosomes are translating an RNA. Thus the ability of rho factor to reach RNA polymerase at a terminator depends on what is happening in translation. This model explains apluzzlir'g phenomenon. In some cases,a nonsensemutation in one gene of a transcription unit prevents the expression of subsequent genesin the unit. This effect is called polarity. A common cause is the
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iI+i.il:l i i.ri.j Rhofactorbindsto RNAat a ruf siteand trans[ocata e tso n gR N Au n t j ti t r e a c h etsh e R N A - D N A the RNAfrom it reteases where hybridin RNApoLymerase, t h eD N A .
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Work? 17.22HowDoesRhoFactor
Rho monomerhas two domains domain
Hexamericringbinds RNA
iiiL:il : :.+"s RhohasanN-terminaL RNA-binding domain anda C-terminaI ATPase domain. A hexamer in theformof a gapped ringbindsRNAalongthe exterior of the N-term j n adl o m a i nT s .h e5 ' e n do f t h e R N Ai s b o u n db y a s e c ondarybindingsitein thejnteriorof the hexamer.
absenceof the nRNA corresponding to the subsequent (distal) pafis of the unit. Supposethat there are rho-dependent terminators within the transcription unit, that is, before the terminator that usually is used. The c o n s e q u e n c e sa r e i l l u s t r a t e d i n n : { * { t * 1 1 , 5 * . Normally these earlier terminators are not used, becausethe ribosomes prevent rho from reaching RNA polymerase. A nonsense mutation, however, releasesthe ribosomes, so that rho is free to attach to and/or move along the mRNA, enabling it to act on RNA polymerase at the terminator. As a result, the enzyme is released, and the distal regions of the transcription unit are never expressed.(Why should there be internal terminators? Perhaps they are simply sequencesthat by coincidence mimic the usual rho-dependent terminator.) Some stable RNAs that have extensive secondarystructure are preserved from polar effects, presumably because the structure impedes rho attachment or movement. rho mutations show wide variations in their influence on termination. The basic nature of the effect is a failure to terminate. The magnitude of the failure, however, as seen in the percent of readthrough in vivo, depends on the particular target locus. Similarly, the need for rho
WILDTYPE
NONSENSEMUTANT Ribosomespack mRNA behindRNA polymerase
Ribosomes impederho attachment and/or movemenl
Ribosomes dissociate at mutation
Rho attaches I Rho obtains
butribosomes I accessto impedeits I RNA movement I polymerase
TranscriptionlTranscription continues I terminates prematurely
f l ; # l J f t i : ' i ' 1 . lTi #h e a c t i o n o f r h o f a c t o r m a y c r e a t e a l i n k b e t w e e n t r a n s c r i p t i o n a n d t r a n s t a t i o n w h e n a rho-dependent terminator liessoonaftera nonsense mutation.
290
CHAPTER 11 Transcription
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that it enablesRNA polymerase to read through a terminator, thus extending the RNA transcript. In the absence of the antitermination protein, RNA polymerase terminates at the terminator (top panel). When the antitermination protein is present, it continues past the terminator (middle panel). The best-characterizedexample of antitermination is provided by phage lambda, with which the phenomenon was discovered.It is used at two stagesof phage expression. The antitermination protein produced at each stage is specific for the particular transcription units that are expressedat that stage,as summarized in the bottom panel of Figure ll.r2. The host RNA polymerase initially rranscribestwo genes, which are called the immediate early genes. The transition to the next stage of expression is controlled by preventing termination at the ends of the immediate early genes, with the result that the delayed early genes are expressed. (We discussthe overall regulation of lambda development in Chapter 14, Phage Strategies.) The regulator gene that controls the switch from immediate early to delayed early expression is identified by mutations in lambda gene N that can transcribe only the immediate early genes; they proceed no further into the infective cycle. There are two transcription units of immediate early genes (transcribed from the promoters P1 and Pp). Ttanscription by E. coli RNA polymerase itself stops at the terminators at the ends of these transcription units (41 and /p1,respectively.) Both terminators depend on rho; in fact, these were the terminators with which rho was originally identified. The situation is changed by expression of the N gene. The product pN is an antitermination protein that acts on both of the immediate early transcription units and allows RNA polymerase to read through the terminators into the delayed early genesbeyond them. As for other phages, still another control is needed to express the late genes that code for the components of the phage particle. This switch is regulated by gene Q, itself one of the delayed early genes.Its product, pQ, is another antitermination protein, one that specifically allows RNA polymerase initiating at another site, the late promoter Pp,.to read through a terminator that lies between it and the late genes. The different specificitiesof pN andpe establish an important general principle: RNA polymeraseinteracts with transcription units in such a
292
CHAPTER L1 Transcriotion
way that an ancillaryfactlr cansplnslr antiterminationspecifically Termination for sometranscripts. can be controlledwith the samesort of precision asinitiation.
Antitermi nationRequi res SitesThatAre Independent of the Terminators o Thesitewherean antiterminator proteinacts is upstream of the terminator sitein the transcription unit. o Thelocationof the antiterminator sitevariesin differentcasesandcanbe in the oromoter or withinthe transcription unit.
Which sitesare involved in controlling the specificity of antitermination? The antitermination activity of pN is highly specific, b:ut the antitermination eventis not determinedby the terminators 11 and 4p1;the recognitionsiteneeded for antitermination liesupstreamin the transcriptionunit, that is, at a different placefrom the terminator site at which the actioneventuallyis accomplished. FI€USIti"5$ shows the locations of the sites required for antitermination in phage lambda. The recognition sitesrequired for pN action are called nut (f.or N utilization). The sites responsiblefor determining leftward and rightward antitermination are describedas nutL and nutR, respectively. Mapping of nut mutations locates nutL between the startpoint of P1 and the beginning of the N coding region. By contrast, nutR lies between the end of the cro gene and [1. This means that the two nut sites lie in different positions relative to the organization of their transcription units. Whereas nutL is near the promoter, nutRis near to the terminator. (qut is different yet again, and lies within the promoter.) How does antitermination occur? When pN recognizes the nut site, it must act on RNA polymerase to ensure that the enzyme can no longer respond to the terminator. The variable Iocations of.t]:renut siles indicate that this event is linked neither to initiation nor to termination, but can occur to RNA polymerase as it elongates the RNA chain past the nut site. As illustrated in FI6URE 11"54,the polymerase then becomes a juggernaut that continues past the terminator, heedlessof its signal. (This reaction involves antitermination at rho-dependent ter-
minators, but pN also suppressestermination at intrinsic terminators. ) Is the ability of pN to recognizea short sequence within the transcription unit an example of a more widely used mechanism for antitermination? Phagesthat are related to lambda have different N genes and different antitermination specificities.The region of the phage genome in which rlae nut sites lie has a different sequence in each of these phages, and each phage must therefore have characteristic nut sitesrecognized specificallyby its own pN. Each of these pN products must have the same general ability to interact with the transcription apparatus in an antitermination capacity, but also have a different specificityfor the sequence of DNA that activatesthe mechanism.
@
Termination andAntitermination Factors Interactwith RNAPoLymerase
. Severa[ proteins bacterial arerequired for lambda pNto interactwith RNApotymerase. . These in antitermination oroteins areatsoinvotved in thern operons of the hostbacterium. r Thelambda pQhasa different mode antiterminator of interaction that involves bindinqto DNAat the oromoter.
Termination and antitermination are closely connected and involve bacterial and phage proteins that interact with RNA polymerase. Several proteins concerned with termination have been identified by isolating mutants of E. coliin which pN is ineffective. Severalof these mutations lie in the rpoB gele. This argues that pN (like rho factor) interacts with the B subunit of the core enzyme. Other E coli mutations that prevent pN function identify rhe nusloci: nusA, nusB, nusE, and nusG. (The term "nus" is an acronym tor N utllization substance.) A lambda nut site consistsof two sequence elements called boxA and boxB. Sequence elements related to boxA are also found in bacterial operons.boxAis required for binding bacterial proteins that are necessaryfor antitermination in both phage and bacterial operons. boxB is specific to the phage genome, and mutations in boxB abolish the ability of pN to cause antitermination.
genesandtermi[ambda transcribes lrll,i.iliirr.;,.,i HostRNApotymerase in theL andR1units; terminators it to readthrough natesat t sites.pNal.l.ows Thesitesat whichpNacts pQallowsit to readthroughthe R'terminator. (nut) andat whichpQacts(qut) arelocatedat differentrelativepositions units. in thetranscription
Promoter
nut site
Terminator
Factorsact on RNA polymerase
i r l i : * i : r l . ; i . : ; .A 1n , citl"aryfactorsbindtoRNApolymeraseasitpassesthenut reaches whenthepotymerase termination rhofromcausing site.Theyprevent terminator. the
11.25 Terminationand AntiterminationFactorsInteractwith RNAPotymerase 293
Terminator
CGCTCTTANNNNNNNNNAGCCCTGAAPuAAGGGCA GCGAGAATNNNNNNNNNTCGGGACTTPvT TCCCGT
NusB-S10 bind to boxA
NusG Facilitates assembly of comolex
DNbinds General at boxB termination factor
ii::a:-.i:; i.::1i,Ancitlary factorsbindto RNApotymerase asit passes certain sites.Thenuf siteconsists joinscoreenzyme of two sequences. NusB-S10 asit passes boxA.Ihen NusA andpNproteinbindaspotymerase passes boxB. Thepresence of pNallowsthe enzyme to readthroughthetermjnator, prod u c i n ga j o i n t m R N A t h a t c o n t a j nism m e d i a teea r t ys e q u e n cjeosi n e dt o deLayed earLy sequences. The nus loci code for proteins that form part of the transcription apparatus,but that are not isolated with the RNA polymerase enzyme. The nusA, nusB, and nusG functions are concerned solely with the termination of transcription. nusE codesfor ribosomal protein Sl0; the relationship between its location in the l0S subunit and its function in termination is not clear. The Nus proteins bind to RNA polymerase ar the nut site, as summarized in i:lf:i-:tiir : :.i,ij. NusA is a general transcription factor that increasesthe efficiency of termination, probably by enhancing RNA polymerase's tendency to pause at terminators (and indeed at other regionsof secondarystructure; seebelow). NusB and Sl0 form a dimer that binds specificallyto RNA containing a boxA seqtence. NusG may be concerned with the general assembly of all the Nus factors into a complex with RNA polymerase. Intrinsic and rho-dependent terminators have different requirements for the Nus factors. NusA is required for termination at intrinsic terminators, and the reaction can be prevented by pN. At rho-dependent terminators, all four Nus proteins are required, and again pN alone can inhibit the reaction. The common feature of pN at both types of terminator is to prevent the role of NusA in termination. Binding of pN to NusA inhibits the ability of NusA to bind RNA, which is necessaryfor termination. Antitermination occurs in the rrn (rRNA)
ognize these sequencesand bind to RNA polymerase as it elongates past boxA. Ttirischanges the properties of RNA polymerase in such a way that it can now read through rho-dependent terminators that are present within the transcription unit. T}:reboxA sequence of lambda RNA does not bind NusB - SI 0 and is probably enabled to do so by the presence of NusA and pN; Ihe boxB sequencecould be required to stabilizethe reaction. Thus variations in boxA sequencesmay determine which particular set of factors is required for antitermination. The consequences are the same:when RNApolymerase passesthe nut sile, it is modified by addition of appropriate factors and fails to terminate when it subsequently encounters the terminator sites. Antitermination in lambda requires pN to bind to RNA polymerase in a manner that depends on the sequence of the transcription unit. Does pN recognizetl:'e boxBsite in DNA or in the RNA transcript? It does not bind independently to either type of sequence,but does bind to a transcription complex when core enzyme passes t]:reboxB site. pN has separate domains that recognizethe boxBRNA sequence and the NusA protein. After joining the transcription complex, pN remains associatedwith the core enzyme, in effect becoming an additional subunit whose presence changes recognition of terminators. It is possible that pN in fact continues to bind to both rhe &oxBRNA sequenceand to RNA polymerase,which maintains a loop in the RNA; thus the role of.boxB RNA would partly be to tether pN in the vicinity, effectively increasingits Iocal concentration. pQ, which prevents termination later in phage infection by acting aI qut, has a different mode of action. The 4rl sequencelies at the start of the late transcription unit. The upstream part of qut lies within the promoter, whereas the downstream part lies at the beginning of the transcribed region. This implies that pQ action involves recognition of DNA; it also implies that its mechanism of action, at least concerning the initial binding to the complex, musr be different from that of pN. pQ interacts with the holoenzyme during the initiation phase. In fact, o70is required for the interaction with pe. This reinforces the view of RNA polymerase as an interactive structure in which conformational changes induced at one phase may affect its activity at a later phase. The basic acrion of pQ is to interfere with pausing. Once pQ has acted upon RNA polymerase,the enzyme shows much reducedpaus-
d*1"#,#:1ift",',;:';lJl;ffi"}i#i 294
CHAPTER 11 Transcriotion
ing at all sites, including rho-dependent and intrinsic terminators. This means that pQ does not act directly on terminatior'per se,but instead allows the enzyme to passthe terminator more quickly, thus depriving the core poiymerase and/or accessoryfactor of the opportunity to causetermination. The general principle is that RNA polymerase may exist in forms that are competent to undertake pailicular stagesof transcription, and its activities at these stagescan be changed only by modifying the appropriate form. Thus substitutions of sigma factors may change one initiation-competent form into another; and additions of Nus factors may change the properties of termination-competent forms. Termination seemsto be closely connected with the mode of elongation. In its basic transcription mode, core polymerase is subject to many pauses during elongation, and pausing at a terminator site is the prerequisite for termination to occur. Under the influence of factors such as NusA, pausing becomes extended, increasing the efficiency of termination; while under the influence of pN or pQ, pausing is abbreviated, decreasingthe efficiency of termination. Recognition sites for these factors are found only in certain transcription units, and as a result pausing and consequently termination are altered only in those units.
@
Summary
A transcription unit comprises the DNA between a promoter, where transcription initiates, and a terminator, where it ends. One strand of the DNA in this region servesas a template for synthesis of a complementary strand of RNA. The RNA-DNA hybrid region is short and transient, as the transcription "bubble" moves along DNA. The RNA polymerase holoenzyme that synthesizes bacterial RNA can be separated into two components. Core enzyme is a multimer of structure o2pB'that is responsiblefor elongating the RNA chain. Sigma factor (o) is a single subunit that is required at the stage of initiation for recognizing the promoter. Core enzyme has a general affinity for DNA. The addition of sigma factor reduces the affinity of the enzyme for nonspecific binding to DNA, but increasesits affinity for promoters. The rate at which RNA polymerase finds its promoters is too great to be accounted for by diffusion and random contacts with DNA; direct exchangeof DNA sequencesheldby the enzyme may be involved.
Bacterial promoters are identified by two short conserved sequencescentered at -35 and -10 relative to the startpoint. Most promoters have sequencesthat are well related to the consensus sequencesat these sites. The distance separating the consensus sequences is l6 to l8 bp. RNA polymerase initially "touches dor,rm" at the -35 sequenceand then extends its contacts over the -l 0 region. The enzlrne covers-77 bp of DNA. The initial "closed" binary complex is converted to an "open" binary complex by melting of a sequenceof -12 bp that extends from the -10 region to the staftpoint. The A-Trich base pair composition of the -I0 sequence may be important for the melting reaction. The binary complex is converted to a ternary complex by the incorporation of ribonucleotide precursors. There are multiple cycles of abortive initiation, during which RNA polymerase synthesizesand releasesvery short RNA chains without moving from the promoter. At the end of this stage,there is a change in structure, and the core enzyme contracts to cover -50 bp. Sigma factor is either releasedQ0% of' cases) or changes its form of association with the core enzyme. The core enzyme then moves along DNA, synthesizing RNA. A locally unwound region of DNA moves with the enzyme. The enzyme contracts further in size to cover only 30 to 40 bp when the nascent chain has reached I5 to 20 nucleotides;then it continues to the end of the transcription unit. The "strength" of a promoter describesthe frequency at which RNA polymerase initiates transcription; it is related to the closenesswith which its -35 and -i0 sequencesconform to the ideal consensussequences,but is influenced also by the sequencesimmediately downstream of the startpoint. Negative supercoiling increases the strength of certain promoters. Transcription generatespositive supercoils ahead of RNA polymerase and leaves negative supercoils behind the enzyme. The core enzyme can be directed to recognize promoters with different consensus s e q u e n c e sb y a l t e r n a t i v e s i g m a f a c t o r s . I n E. coli,these sigma factors are activated by adverse conditions, such as heat shock or nitrogen starvation. B. subtiliscontains a single major sigma factor with the same specificity as the E. coli sigma factor and also contains a variety of minor sigma factors. Another seriesof factors is activated when sporulation is initiated; sporulation is regulated by two cascadesin which sigma factor replacementsoccur in the forespore and mother cell. A cascadefor
11.26Summary 295
regulatingtranscriptionby substitutionof sigma factorsis alsousedby phageSPOI. The geometry of RNA polymerase-promoter recognition is similar for holoenzymes containing all sigma factors (except o5a).Each sigma factor causesRNA polymerase to initiate transcription at a promoter that conforms to a particular consensusat-35 and-10. Direct contacts between sigma and DNA at these sites have been demonstrated for E. coli o70. The o70 factor of E. coli has an N-terminal autoinhibitory domain that prevents the DNA-binding regions from recognizing DNA. The autoinhibirory region is displaced by DNA when the holoenzyme forms an open complex. Bacterial RNA polymerase terminates transcription at two types of sites.Intrinsic terminators contain a G-C-rich hairpin followed by a U-rich region. They are recognized in vitro by core enzyme alone. Rho-dependent terminators require rho factor both in vitro and in vivo; rho binds ro rut silesthat are rich in C and poor in G residues and that precede the actual site of termination. Rho is a hexameric ATpdependent helicase activity that translocates along the RNA until ir reaches the RNA-DNA hybrid region in the transcription bubble of RNA polymerase, where it dissociatesthe RNA from DNA. In both types of termination, pausing by RNA polymerase is important in order to allow time for the actual termination event to occur. The Nus factors are required for termination. NusA is required for intrinsic terminators, and in addition NusB-Sl0 is required for rhodependent terminators. The NusB-Sl0 dimer recognizesthe boxAsequenceof a n rzlsite in the elongating RNA; NusA joins subsequently. Antitermination is used by some phages to regulate progression from one stage of gene expressionto the next. The lambda gene Ncodes for an antitermination protein (pN) that is necessaryto allow RNA poll'rnerase to read through the terminators located at the ends of the immediate early genes.Another antitermination protein, pQ, is required later in phage infection. pN and pQ act on RNA polymerase as it passes specific sites (nut and qut, respecrively). These sites are located at different relative positions in their respective transcription units. pN recognizes RNA polymerase carrying NusA when the enzyme passesthe sequenceboxB.pN then binds to the complex and prevents termination by antagonizing the action of NusA when the polymerase reachesthe rho-dependent terminator.
296
CHAPTER 11 Transcription
Refe fe n CgS Transcription 0ccursby BasePairing i n a " B u b b l e "o f U n o a i r e d DNA Review Losick,R. and Chamberlin,M. (1976).RNA Polymerase.ColdSpringHarborSympQuant.Biol. Researc h Korzheva,N., Mustaev A., Kozlov M., Malhotra, A., Nikiforov V., Goldfarb,A., and Darst.S. A. (2000).A structuralmodel of transcription elongation.Science 289, 619-625.
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Phage T7 RNAPolymerase Is a UsefuI ModeISvstem Resea rch Cheetham, G. M., Jeruzalmi, D., and Steitz, T. A. (1999\. Structural basis for initiation of transcription from an RNA polymerase-promoter complex. Nature )99, 80-83. Cheetham, G. M. T. and Steitz,T. A. (I999). Structure of a transcribing T7 RNA polymerase initiation complex. Science 286, 2)05-2)09. Temiakov D., Mentesana, D., Temiakov D., Ma, K., Mustaev A., Borukhov S., and McAllister, W. T. (2000). The specificity loop of T7 RNA polymerase interacts first with the promoter and then with the elongating transcript, suggesting a mechanism for promoter clearance. Proc. Natl. Acad. Sci.USA 97 . t4to9-r4tI4.
A Modelfor Enzyme Movement Is Suggested by the CrystalStructure Review Shilatifard,A., Conaway,R. C., and Conaway,J. W. (2003).The RNA polymeraseII elongation complex.Annu.Rev.Biochem. 72, 693-7li. Research Cramer,P.,Bushnell,D. A., Fu, J., Gnatt,A. L.. Maier-Davis,B., Thompson,N. E.,Burgess, R. R.,Edwards,A. M., David,P.R., and I(ornberg,R. D. (2000).Architecture of RNA polymeraseII and implicationsfor
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TheAssociation with SiqmaFactor Changesat Initiation Resea r ch Bar-Nahum, G. and Nudler, E, (2001). Isolation and characterization of sigma(70)-retaining transcription elongation complexes lrom E. coli.Cell 106, 44j-451. I(rummel, B. and Chamberlin, M. J. (1989). RNA chain initiation by E. coli RNA polymerase. Structural transitions of the enzyme in early ternary complexes. Biochemistry28, 7829-7842. Mukhopadhyay, J., I(apanidis, A. N., Mekler, V., I(ortkhonjia, E., Ebright, Y. W., and Ebright, R. H. (2001).Tfanslocationof sigma(70)with RNA polymerase during transcription. Fluores-
cenceresonanceenergytransferassayfor movementrelativeto DNA. Cell106,45J463.
CanRestart RNAPolvmerase A Statted rch Resela I(ettenberger, H., Armache, I(. J., and Cramer, P. (2003). Architecture of the RNA polymerase II-TFIIS complex and implications for mRNA cleavage.Cell ll4, 347-357. Opalka, N., Chlenov M., Chacon, P., Rice, W. J., Wriggers,W., and Darst, S. A. (2003). Structure and function of the transcription elongation factor GreB bound to bacterial RNA polymerase.Cell ll4, 3)5-345.
Bindingto DNA Controls SigmaFactor rch Resr:a Bar-Nahum, G. and Nudler, E. (2001). Isolation and characterization of sigma(70)-retaining transcription elongation complexes from E coli.Cell 106, 443-451. Mukhopadhyay, J., I(apanidis, A. N., Mekler, V., I(ortkhonjia, E., Ebright, Y. W., and Ebright, R. H. (2001). Translocationof sigma(70) with RNA polymerase during transcription. Fluorescenceresonance energy transfer assayfor movement relative to DNA. Cell lO6, 45j-463.
DePends Recognition Promoter 0 n C o n s e n s uSse q u e n c e s Rev1ew McClure,W. R. (1985).Mechanismand controlof transcriptioninitiation in prokaryotesAnnu 54, 17l-204. Rev. Biochem. R e s erac h Ross,W., Gosink,I(. K., Salomon,J., Igarashi,I(., Z o u ,C . , I s h i h a m aA, . , S e v e r i n o vI (, . .a n d Gourse,R. L. (1991).A third recognitionelement in bacterialpromoters:DNA binding by the alpha subunit of RNA polymerase. 262. l4O7-l4l). Science
CanBeIncreased Efficiencies Promoter bY Mutation or Decreased Review McCIure,W. R. (1985).Mechanismand controlof transcriptioninitiation in prokaryotes.Annu 54, 17l-204. Rev.Biochem. Bindsto OneFace RNAPotymerase of DNA Review Siebenlist,U., Simpson,R. B., and Gilbert,W. ( I9S0). E. coliRNA polymeraseinteracts homologouslywith two differentpromoters. Cell20,269-281.
References 297
@
Supercoiting Is an ImportantFeature of Transcription
R e s erach Wu, H.-Y.et al. (1988).Transcription generates positivelyand negativelysupercoileddomains in the remplate.Cellfi,4)i-440.
@
Substitution ofSigmaFactors May ControI Initiation
Review Hengge-Aronis,R. (2002). Signal transduction and regulatory mechanisms involved in control of the sigma(S) (RpoS) subunit of RNA polymerase.Microbiol.Mol. Biol. Rev.66.37)-j%. Resea r ch Alba, B. M., Onufryk, C., Lu, C. 2., and Gross, C. A. (2002). DegS and YaeL participate sequentially in the cleavage of RseA to activate the sigma(E)-dependent extracytoplasmic stressresponse.GenesDev.16,2156-2168. Grossman,A. D., Erickson, J. W., and Gross,C. A. (1984). The htpR gene producr of E. coliisa sigma factor for heat-shock promoters. Cell 38, )83-390. I(anehara, K., Ito, K., and Akiyama, Y. (2002). YaeL (EcfE) activates the sigma(E) pathway of stressresponse through a site-2 cleavage of anti-sigma(E),RseA. GenesDev.\6, 2t47-2155. Sakai,J., Duncan, E. A., Rawson, R. B., Hua, X., Brown, M. S., and Goldstein,J.L. (19961. Sterol-regulatedreleaseof SREBP-2from cell membranes requires two sequential cleavages, one within a transmembrane segment. Cell 85, IO37-1046.
@
SigmaFactors Directty Contact DNA
Resea rch Campbell, E. A., Muzzin, O., Chlenov M., Sun, J. L., Olson, C. A., Weinman. O.. TresterZedlitz,M. L., and Darsr, S. A. (2002). Structure of the bacterial RNA polymerase promoter specificity sigma subunit. Mol. Cell 9 , 527 -539 . Dombrowski, A. J. et al. (19921. Polypeptides containing highly conserved regions of transcription initiation factor o70 exhibit specificity of binding to promorer DNA. Cel/70, i}t-il2. Mekler, V., I(ortkhonjia, E., Mukhopadhyay, J., I(night, J., Revyakin, A., Iftpanidis, A. N., Niu, W., Ebright, Y. W., Levy, R., and Ebright, R. H. (2002). Structural organization of bacterial RNA polymerase holoenzyme and the RNA polymerase-promoter open complex. Cel/ to8, 599-614. Vassylyev,D. G, Sekine, S., Laptenko, O., Lee, J., Vassylyeva,M. N., Borukhov, S., Yokoyama S. (2002). Crystal strucrure of a bacterial RNA
298
CHAPTER l.L Transcription
polymeraseholoenzymeat 2.6 Aresolution. Nature417,712-719.
Sporutation Is Controlted by SigmaFactors Reviews Errington,J. (I993). B subtilis sporulation:regulation of geneexpressionand control of morphogenesis. MicrobiolRev.57, l-j}. Haldenwang, W. G. (1995).The sigmafactorsof B. subtilis.Microbiol. Rev.59,140. Losick,R. and Stragier,P. (19921.Crisscross regulation of cell-typespecificgeneexpressionduring developmentin B. subtilis.NatureJ55, 60r-604. Losick,R. et al. (1986).Genetics of endospore formation in B. subtilis.Annu Rev.Genet.20. 625-669. Stragier,P and Losick,R. ( I 996). Moleculargenetics of sporulationin B. subtilis.Annu.Rev.Genet. )0,297-34t. Rese arch Haldenwang,W. G., Lang,N., and Losick,R. ( 1981). A sporulation-induced sigma-likeregulatory protein from B. subtilis.Cell2), 615-624. Haldenwang, W G. and Losick,R. (1980).A novel RNA polymerasesigmafactorfrom B. subtilis. Proc.Natl.Acad Sci.USA77,7OOO-7004.
BacteriaI RNAPotvmerase Terminates at Discrete Sites Reviews Adhya, S. and Gottesman,M. (1978). Control of transcription termination. Annu. Rev.Biochem. 47,967-996. Friedman, D. L, Imperiale, M. J., and Adhya, S. L. (19871.RNA 3'end formation in the control of gene expression.Annu Rev.Genet.2l, 45)488. Platt, T. (1986). Transcription termination and the regulation of gene expression. Annu. Rev. B i o c h e m . 5 5),) 9 3 7 2 .
ThereAreTwoTypesof Terminators in E. coli Review von Hippel, P. H. (1998). An integrated model of the transcription complex in elongation, termination, and editing. Science281, 660-66i . Resea rch Lee, D. N., Phung, L., Stewart, J., and Landick, R. ( I 990). Transcription pausing by E. coli RNA polymerase is modulated by downstream DNA sequences.J. Biol. Chem.265, 15145-15153. Lesnik, E. A., Sampath, R., Levene, H. B., Henderson, T. J., McNeil, J. A., and Ecker. D. J.
(2001 ). Prediction of rho-independent transcriptional terminators in E coli NucleicAcids Res.29,)58j-3594. Reynolds, R., Bermadez-Cruz, R. M., and Chamberlin, M. J. (1992). Parameters affecting transcription termination by E. coli RNA polymerase. I. Analysis of l3 rho-independent terminators. J. Mol. Biol.224. )l-51.
HowDoesRhoFactor Work? Reviews Das, A. (199)). Control of transcription termination by RNA-binding proteins. Annu. Rev. Biochem.62,893-9]0. Richardson, J. P. (1996). Structural organization of transcription termination factor Rho. J. Biol. Chem.27l, 125I-1254. von Hippel, P. H. (1998). An integrated model of the transcription complex in elongation, termination, and editing. Science281, 660-665. Research Brennan, C. A., Dombroski, A. J., and Platt, T. ( I 987 ) . Transcription termination factor rho is an RNA-DNA helicase.Cell 48,945-952. Geiselmann, J., Wang, Y., Seifried, S. E., and von Hippel, P. H. (1993). A physical model for the translocation and helicase activities of E. coli transcription termination protein Rho. Proc Natl Acad Sci.U9A90,7754-7758. Roberts, J. w. (1969). Termination factor for RNA synthesis.Nature224, I 168-1 174. Skordalakes,E. and Berger,J. M. (2003). Structure of the Rho transcription terminator: mecha-
nism of mRNA recognition and helicase load' ing. Cell ll4, I)5-146.
Factors Termination andAntitermination Interact with RNA Polvmerase Review Greenblatt, J., Nodwell, J. R., and Mason, S. W. ( 1993). Ttanscriptional antitermination. Nature 364, 401-406. Resea rch Legault, P.,Li, J., Mogridge, J.,Kay, L. E., and Greenblatt, J. ( 1998) . NMR structure of the bacteriophage lambda N peptide/boxB RNA complex: recognition of a GNRA fold by an arginine-rich motif . Cell 9), 289-299 . Mah, T. F., I(uznedelov I(., Mushegian, A., Severinov I(., and Greenblatt, J. (2000). The alpha subunit oI E coli RNA polymerase activates RNA binding by NusA. GenesDeY.14, 2 6 6 4 - 2 6 75 . Mogridge, J., Mah, J., and Greenblatt,J. (1995). A protein-RNA interaction network facilitates the template-independent cooperative assembly on RNA polymerase of a stable antitermination complex containing the lambda N protein. GenesDev.9, 2831-2845. Olson, E. R., Flamm, E. L., and Friedman, D. I. (19821. Analysis of nutR: a region of phage lambda required for antitermination of transcription. Cell 3I, 6l-70.
References 299
TheOperon I
C H A P T EO RU T L I N E ] Introduction
@
RegulationCanBe Negativeor Positive r In negative regulation, proteinbindsto an opera repressor atorto prevent a genefrombeingexpressed. o In posjtive regulation, a transcription factoris required to bindat the promoter in orderto enabte RNApolymerase to initiatetranscriotion. StructuraIGeneClustersAre Coordinatety Controtted r Genes codingfor proteins that functionin thesamepathwaymaybe located adjacent to oneanother andcontrotted asa sing[eunitthat is transcribed into a potycistronic mRNA. The/ac GenesAre Controltedby a Repressor . Transcription genectusteris controtled of theIacZYA by proteinthat bindsto an operator a repressor that overlaps the promoter at the startof the cluster. r Therepressor proteinis a tetramer of identical subunits codedby the genelacl. Thelac 0peronCanBe Induced o Sma[[ molecules that inducean oDeron areidentical with or relatedto the substrate for its enzymes. r B-gatactosides arethe substrates for theenzymes codedby LocZYA. . In the absence of B-galactosides, the locoperonis expressed on[yat a verylow (basat)tevel. e Additionof specific induces transcription of B-galactosides atlthreegenesof the operon. c ThelacmRNA is extreme[y unstable; asa result,induction canbe rapidlyreversed. . Thesametypesof systems that altowsubstrates to induce operons codingfor metabotic enzymes canbe usedto attow end-products to repress the operons that codefor biosyntheticenzymes. Repressor Is Controtted by a Smat[-Motecute Inducer o An inducer functjons protein by converting the repressor into a formwith loweroperatoraffinity. . Repressor hastwo bindingsites.onefor the operator and another for the inducer. js inactivated r Repressor by an allosteric interaction in which bindingof inducer at its sitechanges the properties of the DNA-bindinq site.
300
@
''.'iN
c6-ActingConstitutiveMutationsIdentifothe 0perator r Mutations in the operatorcauseconstitutive expression of genes. a[[threelacstructural r Thesemutations areos-actingandaffectonlythosegenes on the contiguous stretchof DNA. trans-ActingMutationsIdentifythe RegulatorGene . Mutations in the /ocl genearetrans-acting andaffect exDression of alllocZYA ctusters in the bacterium. . Mutations that eliminate /aclfunctioncauseconstitutive expression andarerecessive. r Mutations in the DNA-binding siteof the repressor areconstitutivebecause the repressor cannotbindthe operator. o Mutations in theinducer-binding presiteof the repressor ventit frombeinginactivated andcauseuninducibi[ity. r Mutations in the promoter areun'inducibte andcrs-acting. MultimericProteinsHaveSpecialGeneticProperties r Activerepressor is a tetramer of identicaI subunits. o Whenmutantandwitd-type subunits arepresent, a singte /ocf-dmutantsubunitcaninactivatea tetramerwhoseother subunits arewitd-type. t locl-dmutations occurin the DNA-binding site.Theireffect is exptained by the factthat repressor activityrequires at[ DNA-binding sitesin thetetramer to beactive. The Repressor MonomerHasSeveraIDomains o A singterepressor subunitcanbedividedintothe N-terminaI DNA-binding domajn. a hinge.andthe core of the orotein. o TheDNA-binding domaincontains two shortcx-hetical. regions that bindthe majorgrooveof DNA. . Theinducer-binding siteandthe regions responsib[e for multimerization arelocated in the core. Repressor Is a TetramerMadeof Two Dimers o Monomers forma dimerby making contacts between core domains 1.and2. r Dimers forma tetramer bv interactions between the o[igomerization hetices. DNA-Binding Is Regutated by an AltostericChange in Conformation r T h eD N A - b i n d idnogm a i on f e a c hm o n o m e wri t h i na d i m e r insertsintothe majorgrooveof DNA.
r0€
uorado 3ql zI uttdvHl
tueuuns 'vNUtlt qlqlqul 'urelo.ld urtnqnlJ0 uorlPlsuetl urlnqnlaalj 'salnqnlo.llrut ol tostnta.ld eq1 . sallquassv lplnlalouollpt4Jo srsaqlu^slotluol o1 pasn ualJ0sI uoqpln6ausnouabolnv 'uorlPlsueil uorlerl J0 -rurlua^oldol vNUruuMosll ol spurqztd r ]rnlll snouabolnyue r{q pellorluol s17gd 71 abeq6 'VNUur lruorlsrrflod aq1uo alrse ol spurq 1eq1uoredoaq1;opnpotde [q pallorluor aq uer uoradouralotd-r ueJouorlelsupll. u o q e l n 6 a gs n o u e b o l n^vq pallorluol s1srsaqluri5 uralold-l 'uorlelsupll Jollnsolaql splntlo eqlJoainllnilsaql ur se6ueqr tptll VNUrU Aqpallorluor aq uprVNUut rLuo.rlsoAlod e ur suopoluoqerlrut;o flqrqrssely o 'uopoluorlprlrut ue o16ut -purq uolJ auosoqu e Fuqualeld fq uoq -elsupllalelnbar uer urelordrossalda: y . palplnbauag upl uorlelsuell :ralouord aql puealrs 6urputq-493 aqlJosuorlplole^rlplaloql u0 pueoap uor]lplalur aqlJoslrelapeql 'asetaur\1od qrrytslrpralur 1nq vruu l!l o 'lelouo.loeql 01a^rlplalsuorlelol alqeup^ f1q61q o 1eortsolrs6urpuLq-693 'alrs Durpurq $! le vNo olurpuaq006p solnpollu! dul o suoledOlablpl lualaJJl0ul sApMtualaJJloul suotpunl dul 261yrrlrfr Joalnraloul al6urse fiq palenrlre sl dul Joraurp! o 'talouo.rde aruanbes 1e 1abte1 p 0l spurq1eq1uralord.lolp^qleupsrdul .
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'0ul11 aql lossaidol Jo o/og6 fq punoqsrrolerado oql setnsua leql llol lad srauellalrossaldar uol JoleAola{l r 'a1rsr{1rug1e Molp Jolpql x10I sr1eq1 tossardet rol Alruqleueseq roleladooql lernpurJoaluasqp eql uI . lossaldauputg ol salrs f1ru1g1y-mo1 q]rm satadurol rolprad0eql ' uorlnlos ruor;6urlelqrlrnba fq ueqllaqleralrsflruqe-ruo1e uol; 6uLnou Aq.roleredo eql ol spurqtossaldeg . 'vN00l punoqsr ureloldrossardal llp lpql saJnsua selLsr{1rug1e-r'rol Joloqurnuebrelaql r '.lossel0al
ro; elLs-6urpuLq Alruqe-mo1 e Joilpls aql sreuoueblpuallpqaq1urtredaseqA.ran1 r 'saluanbas VN0raqlotol AlLug;e Molp o^eqoslearuanbas y16 rqLrads e ro1Alruu-le qELqe eneq]e!l suralo.llo
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Introduction
@
Gene expressioncan be controlled at any of several stages,which we divide broadly into transcription. processing, and translation: . Tianscription often is controlled at the stage of initiation. Tlanscription is not usually controlled at elongation. but may be controlled at termination to determine whether RNA polymerase is allowed to proceedpast a terminator to the gene(s)beyond. . In eukaryotic cells, processing of the RNA product may be regulated at the stages of modification, splicing, transport, or stability. In bacteria, an mRNA is in principle available for translation as soon as (or even while) it is being synthesized, but during this time the stagesof control are not available. . Translation may be regulated, usually at the stagesof initiation and termination (like transcription). Regulation of initiation is formally analogous to the regulation of transcription: the circuitry can be drawn in similar terms for regulating initiation of transcription on DNA or initiation of translation on RNA. The basic concept for how transcription is controlled in bacteria was provided by the classic formulation of the model for control of gene expressionby Jacoband Monod in 1961. They distinguishedbetween two types of sequences in DNA: sequencesthat code for trans-acting products and crs-acting sequences that function exclusively within the DNA. Gene activity is regulated by the specific interactions of the trans-actingproducts (usually proteins) with the cls-actingsequences(usually sitesin DNA). In more formal terms:
Regulatorgene
II
v mRNA
II
v ..i:i
+
Regulatorprotein
Targetsite Structural gene
f ii:,1j i'tLri ir.i, A regutator genecodes for a proteinthatacts at a targetsiteon DNA.
302
C H A P T E1R2 T h e0 p e r o n
. A gene is a sequence of DNA that codes for a diffusible product. This product may be protein (as in the case of the majority of genes) or may be RNA (asin the caseof genesthat code for IRNA and rRNA). Thecrucialfeatureis that theproduct dffises away from its site of synthesisto act elsewhere.Any gene product that is free to diffuse to find its target is described as tr ans- acting. . The description cls-actingapplies to any sequence of DNA that is not converted into any other form, but that functions exclusively as a DNA sequence in situ, affecting only the DNA to which it is physically linked. (In some cases,a crsacting sequence functions in an RNA rather than in a DNA molecule.) To help distinguish between the components of regulatory circuits and the genesthat they regulate, we sometimes use the terms structural gene and regulator gene. A structural gene is simply any gene that codesfor a protein (or RNA) product. Structural genesrepresent an enormous variety of protein structures and functions, including structural proteins, enzymeswith catalytic activities, and regulatory proteins. A regulator gene simply describesa gene that codes for a protein (or an RNA) involved in regulating the expression of other genes. The simplest form of the regulatory model is illustrated in Ft{:*&$.'t'l.ii a regulatorgenecodes for a protein that controlstranscription by binding to particular site(s)on DNA. This interaction can regulate alargel gene in either a positive manner (the interaction turns the gene on) or in a negative manner (the interaction turns the gene off). The siteson DNA are usually (but not exclusively) located just upstream of the target gene. The sequencesthat mark the beginning and end of the transcription unit, the promoter and terminator, are examples of cls-acting sites. 4 prlmlter servesto initiate transcription only of the gene 0r genesphysically connectedto it on the same stretchof DNA. In the same way, a terminator can terminate transcription only by an RNA polymerase that has traversed the preceding gene(s).In their simplestforms, promoters and terminators are cls-actingelements that are recognized by the same trans-actingspecies,that is, by RNA polymerase (although other factors also participate at each site). Additional cls-actingregulatory sitesare often juxtaposed to, or interspersedwith, the promoter. A bacterial promoter may have one or more such sites located close by, that is, in the immediate
vicinity of the startpoint. A eukaryotic promoter is likely to have a greater number of sites that are spread out over a longer distance.
precedesstructuralgene(s) cr+actingoperator/promoter Structuralgene(s)
PromoterOperator
Regulation CanBe Negative or Positive proteinbinds In negative regulation, a repressor to an operator to prevent a genefrombeing expresseo. In positiveregulation, a transcription factoris required to bjndat the promoter in orderto enable RNApotymerase to initiatetranscription. A classicmode of control in bacteria is negative: a repressor protein prevents a gene from being expressed.i:i+ijiii;: ll ,;: shows that the "default state" for such a gene is to be expressedvia the recognition of its promoterby RNApolymerase. CIoseto the promoter is another cli-actingsite called the operator, which is the target for the repressorprotein. When the repressorbinds to the operator, RNA polymerase is prevented from initiating transcription and geneexpression is therefore turned off. An alternative mode of control is positive. This is used in bacteria (probably) with about equal frequency to negative control, and it is the most common mode of control in eukaryotes. A transcription factor is required to assist RNA polymerase in initiating at the promoter. i::i{:ijiirl1;:,.rishows that the typical default state of a eukaryotic gene is inactive: RNA polymerase cannot by itself initiate transcription at the promoter. Several trans-acling factors have target sites in the vicinity of the promoter, and binding of someor all of thesefactorsenablesRNA polymeraseto initiate transcription. The unifying theme is that regulatory proteins are trans-acting factors that recognize clsacting elements (usually) upstream of the gene. The consequencesof this recognition are to activate or to repress the gene, depending on the individual type of regulatory protein. A typical feature is that the protein functions by recognizing a very short sequence in DNA, usually <10 bp in length, although the protein actually binds over a somewhat greater distanceof DNA. The bacterialpromoter is an example: RNA polymerasecovers>70 bp of DNA at initiation, but the crucial sequencesthat it recognizesare the hexamers centered at -35 and -I0. A significant difference in gene organization between prokaryotes and eukaryotesis that
Gene on: RNA polymeraseinitiatesat promoter
RNA
i,
l'',, ,
ln,
'
j'r' :1,.,r"'r-..,
erotein 6ffiffi
I
...,"'
bindsto operalor Geneis turnedoffwhenrepressor Repressor
I repressor rir.;i,lFili. t::r.;: In negativecontrot,a frons-acting operator to turnoff transcription. bindsto thecrs-acting
GENETURNEDON BYACTI
ORS
Factorsinteractwith RNA polymerase
RNA
II
v factorsmust frlt i.iiiii l;l.ri In positivecontrot,trans-acting to inibindto crs-acting sitesin orderfor RNApotymerase tiatetranscription at the promoter.
structural genes in bacteria are organized in clusters.whereas those in eukaryotesoccur individually. Clustering of structural genes allows them to be coordinately controlled by means of interactions at a single promoter: as a result of these interactions, the entire set of genes is either transcribed or not transcribed. In this
or Positive 303 CanBeNegative 12.2 Regutation
chapter, we discussthis mode of control and its use by bacteria.The means employed to coordinate control of dispersedeukaryotic genesare discussedin Chapter 25, Activating Tfanscription.
@
Structural Gene Clusters AreCoordinateLy ControLLed
r Genes codingfor proteins that functionin the samepathway maybe [ocated adjacent to one another andcontrolted asa singteunitthat is transcribed into a polycistronic mRNA.
Bacterial structural genes are often organized into clustersthat include genescoding for proteins whose functions are related. It is common for the genescoding for the enzymes of a metabolic pathway to be organizedinto such a cluster. In addition to the enzymesactually involved in the pathway, other related activities may be included in the unit of coordinate control; for example, the protein responsiblefor transporting the small molecule substrateinto the cell. The cluster of the three lac structural genes, lacZYA,is typical. 'i,rlj:ri. i..lr'i.gsppurizes the organizationof the structural genes,their associated cli-acting regulatory elements, and the trans-acting regulatory gene. The keyfeature is that theclusteris transcribedinto a singlepolycistrontc wRNA from a promoterwhere initiation of transcription is regulated. The protein products enable cells to take up and metabolize B-galactosides, such as lactose.The roles of the three structuralgenesare: . lacZ codes for the enzyme B-galactosidase,whose activeform is a tetramer of -500 kD. The enzyme breaksa B-galactoside into its component sugars. For example, lactoseis cleavedinto glucose
and galactose (which are then further metabolized).This enzyme alsoproduces an important by-product, p-1, 6-allolactase,which has a role in regulation. . lacY codesfor the p-galactosidepermease,a l0-kD membrane-bound protein constituent of the transport system.This transports B-galactosidesinto the cell. . lacA codesfor B-galactosidetransacetylase, an enzyme that transfers an acetyl group from acetyl-CoAto B-galactosides. Mutations in either lacZ or lacY can create the lac genolype, in which cells cannot utilize lactose. (The genotlpic description "lac" withoul a qualifier indicates loss-of-funclion.\ Ti;re lacZ mutations abolish enzyme activity, directly preventing metabolism of lactose.The /acYmutants cannot take up lactose from the medium. (No defect is identifiable in lacA cells,which is puzding. It is possible that the acetylation reaction gives an advantagewhen the bacteria grow in the presence of certain analogs of B-galactosidesthat cannot be metabolized, because the modification results in detoxification and excretion.) The entire system, including structural genesand the elements that control their expression, forms a common unit of regulation called an operon. The activity of the operon is controlled by regulator gene(s)whose protein products interact with the as-acting control elements.
ThelqcGenes Are Contro[Led by a Repressor geneclusteris Transcription of the lacZYA proteinthat bindsto an controlled by a repressor operatorthat overlaps the promoter at the startof the ctuster. proteinis a tetramerof identical Therepressor subunits codedbythe genelacf.
-6000bpof DNA.Atthe Leftthelaclgenehasits ownpro;:.:{ii"!i:i,,r .1..r.,,, Thelacoperon occupies moterandterminator. Theendof the locl regionis adjacent to the promoter, P.Theoperator, 0 occupies the first26 bp of thetranscription unit.ThelongIacZgenestartsat base39,andis fotlowedbv the LacY andlac,4qenesanda terminator.
304
CHAPTER 12 The0peron
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Mutations in the Lac repressoridentified the existence of different domains even before the structure was known. We can now explain the nature of the mutations more fully by reference to the structure,as summarizedin , lr,l:':r : L .,. Recessive mutations of the lacl- Iype can occur anl.where in the bulk of the protein. Basically, any mutation that inactivates the protein will have this phenotype. The more detailed mapping of mutations on to the crystal structure in Figure 12.13 identifies specificimpairments for some of these mutations, for example, those that affect oligomerization. The specialclassof dominant-negalive lacla mutations lie in the DNA-binding site of the repressorsubunit (seeSection12.9,Multimeric ProteinsHave SpecialGeneticProperties).This explains their ability to prevent mixed tetramers from binding to the operator; a reduction in the number of binding sites reduces the specific affinity for the operator. The role of the N-terminal region in specifically binding DNA is shown also by its location as the site of occurrence of "tight binding" mutations. These increase the affinity of the repressor for the operator, sometimes so much that it cannot be releasedby inducer. They are rare. Uninducible /aclsmutations map largely in a region of the core domain I extending from the inducer-binding site to the hinge. One group Iies in amino acidsthat contact the inducer, and these mutations function by preventing binding of inducer. The remaining mutations lie at sites that must be involved in transmitting the allosteric change in conformation to the hinge when inducer binds.
Repressor ProteinBinds to the Operator o Repressor proteinbindsto the doubte-stranded DNAsequence of the operator. r Theoperator is a palindromic sequence of 26 bp. o Eachinvertedrepeatof the operator bindsto the DNA-binding siteof onerepressor subunit.
The repressor was isolated originally by purifying the component able to bind the gratuitous inducer IPTG. (The amount of repressorin the cell is so small that in order to obtain enough material, it was necessaryto use a promoterup mutation to increase lacl Iranscription and to place this /acllocuson a DNA molecule pres-
' ,l,l-li::,1,, The[ocations of threetypeof mutations in lactose repressor on the domain structure aremapped ofthe protein.Recessive lacf-mutants that cannotrepress canmapanywhere in theprotein.Dominant negative lacli m u t a n t tsh a t c a n n o rt e p r e sm s a pt o t h e D N A - b i n d i n g d o m a i nD . o m i n a nl o t c f ' m u t a n t st h a t c a n n o it n d u c e because theydo not bindinducer or cannotundergo the allosteric mapto coredomain1. change ent in many copies per cell. This results in an overall overproduction of 100 to 1000-fold.) The repressorbinds to double-strandedDNA containing the sequence of the wild-type lac operator.The repressordoesnot bind DNA from an OCmutant. The addition of IPTG releasesthe repressor from operator DNA in vitro. The in vitro reacion between repressor protein and operator DNA therefore displays the characteristics of control inferred in vivo;thus it can be used to establishthe basisfor repression. How doesthe repressorrecognizethe specific sequence of operator DNA? The operator has a feature common to many recognition sites for bacterial regulator proteins: it is a palindrome. The inverted repeats are highlighted in i ri ri r , . Each repeat can be regardedas a half-site of the operator. We can use the same approaches to define the points that the repressor contacts in the operator that we used for analyzing the polymerase-promoterinteraction (seeSection I l. 14, RNA PolymeraseBinds to One Face of DNA). Deletions of material on either side define the end points of the region; constitutive point mutations identify individual base pairs that must be crucial. Experiments in which DNA bound to repressor is compared with unbound
Protein Bindsto the0perator 313 12.14Reoressor
MRNA
T G T T G T G T GG A A T T ACAACACACCTTA -10
-5
+1
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CT GA
t+
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tttt
of the operator makes the same pattern of contacts with a repressor monomer. This is shown by symmetry in the contacts that repressor makes with the operator (the pattern between +l and +6 is identical with that between +21 and +16) and by matching constitutive mutations in each inverted repeat. (The operator is not perfectly symmetrical, though; the left side binds more strongly than the right side to the repressor.A stronger operator is created by a perfect inverted duplication of the left side and eliminated of the central base pair.)
Binding of Inducer Repressor Releases fromthe 0perator r Inducer in repressor a change bindingcauses its affinityfor DNAanc that reduces conformation releases it fromthe operator.
t tfttf t
Various inducers causecharacteristicreductions in the affinity of the repressor for the operator -10 -5 +1 +5 +15 +2O +25 invitro. These changes correlate with the effectiveness of the inducers in vivo. This suggests that induction results from a reduction in the attraction between operator and repressor.Thus i:liri.ii1li. -i,i"l:;riBases that contactthe repressor canbe identifiedbv chemicaI when inducer enters the cell, it binds to free prevents They cross[inking or byexperiments to seewhether modification binding. repressors and in effect prevents them from identifypositions from+1 to +23.Constitutive on bothstrands of DNAextending finding their operators. Consider, though, a +5 and+17. mutations in the operator occurat eightpositions between repressortetramer that is already bound tightly to the operator. How does inducer cause this repressorto be released? DNA for its susceptibility to methylation or UV TWo models for renressor action are illuscrosslinking identify bases that are either :.;:.:*s: protectedor more susceptiblewhen associated trated in FI"";iisi. . The equilibrium model (left) calls for with the protein. iii".iiiii:::j.:ii shows that the region of DNA repressor bound to DNA to be in rapid protected from nucleasesby bound repressor equilibrium with free repressor.Inducer would bind to the free form of represlies within the region of symmetry, comprising sor and thus unbalance the equilibrium the 26 bp region from -5 to +2 l. The area idenby preventing reassociationwith DNA. tified by constitutive mutations is even smaller. . The rate of dissociation of the repressor Within a central region extending over the l3 bp from the operator, however, is much too from +5 Io +I7 , there are eight sitesat which sinslow to be compatible with this model (the half-life in vitro in the absence of promoter mutations summarized earlier in Figinducer is >I5 min). This means that instead the inducer must bind directly to ure I I.29. A small number of essential specificcontactswithin a larger region can be responsiblefor represslrprotein complexedwith the opera/or As indicated in the model on the right, sequence-specific associationof DNA with protein The symmetry of the DNA sequencereflects inducer binding must produce a change the symmetry in the protein. Each of the idenin the repressorthat makes it releasethe tical subunits in a repressortetramer has a DNAoperator. Indeed, addition of IPTG causes binding site. TWo of these sites contact the an immediate destabilization of the repressor-operatorcomplex in vitro. operator in such a way that each inverted repeat Protectedby
-
repressor +
+[iffi ,ffni:Htx Jffi::1T:"':nfi
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e r s ( s e eF i g u r e 1 1 . 2 2 a n d F i g u r e I 1 . 2 3 ) . T h e same solution is likely: movement could be accomplished by reducing the dimensionality of the search by sliding along DNA or by direct displacement from site to site (asindicated in Figure 12.241. A displacement reaction might be aided by the presence of more binding sitesper tetramer (four) than are actually needed to contact DNA at any one time (two). The parameters involved in finding a highaffinity operator in the face of competition from many low-affinity sites pose a dilemma for repressor.Under conditions of repression,there must be high specificity for the operator. Under conditions of induction, however, this specificity must be relieved. Suppose,for example, thatthere were 1000 moleculesof repressorper cell: only 0.04oh of operators would be free under conditions of repression. Upon induction, though, only 40o/oof operators would become free. We therefore see an inverse correlation between the ability to achieve complete repression and the ability to relieve repressioneffectively.We assumethat the number of repressorssynthesizedin vivo has been subject to selective forces that balance these demands. The difference in expression of the lactose operon between its induced and repressedstates in wvo isactually I 03x.In other words, even when inducer is absent,there is a basallevel of expression of -O.l% of the induced level. This would be reduced if there were more repressorprotein present and increased if there were less.Thus it could be impossible to establishtight repression if there were fewer repressorsthan the ten found per cell, and it might become difficult to induce the operon if there were too many. It is possible to introduce t};relac operator-repressor system into the mouse. When Lhe lac operator is connected to a tyrosinasereporter gene,the enzyme is induced by the addition of IPTG. This means that the repressoris finding its target in a genome l0r times larger than that of E coli.Induction occurs at approximately the same concentration of IPTG as in bacteria. We do not. however. know the concentration of Lac repressorand how effectively the target is induced. In order to extrapolate invivo fromthe affinity of a DNA-protein interaction in vitro, we need to know the effective concentration of DNA ln vivo.T];re"effective concentration" differs frt-rm the mass/volume becauseof severalfactors.The effective concentration is increased, for example, by molecular crowding, which occurswhen polyvalent cations neutralize -90% of the
charges on DNA, and the nucleic acid collapses into condensed structures. The major force that decreasesthe effectiveconcentration is the inaccessibility of DNA that results from occlusion or sequestration by DNA-binding proteins. One way to determine the effective concentration is to compare the rate of a reaction in vitro and in vivo thar depends on DNA concentration. This has been done using intermolecular recombination between two DNA molecules. To provide a control, the same reaction is followed as an intramolecular recombination, that is, the two recombining sites are presented on the same DNA molecule. We assume that concentration is the same in vivo and in vitro for t]ne intramolecular reaction, and therefore any difference in the ratio of intermolecular/ intramolecular recombination rates can be attributed to a change in the effective concentrationinvivo. The results of such a comparison suggestthat the effective concentration of DNA is reduced >10-fold invivo. This could affect the rates of reactions that depend on DNA concentration, including DNA recombination and protein-DNA binding. It emphasizes the problem encountered by all DNA-binding proteins in finding their targets with sufficient speedand reinforces the conclusion that diffusion is not adequate (see Figure I L22).
Can0ccur Repression at MultipleLoc' o A repressor will acton a[[locithat havea copyof sequence, its targetoperator The lac repressor acts only on the operator of tine lacZYA cluster. Some repressors,however, control dispersedstructural genesby binding at more than one operator. An example is lhe trp repressor, which controls three unlinked sets of genes: . An operator at the cluster of structural genestrpEDBC, controls coordinate s1mthesis of the enzymes that synthesize tryptophan from chorismic acid. . An operator at another locus controls tine aroH gene, which codes for one of the three enzymes thatcatalyze the initial reaction in the common pathway of aromatic amino acid biosynthesis. o The trpRregulator gene is repressedby its own product, tlnetrp repressor' Thus
Loci at Multipte CanOccur 12.19Repression
319
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Operator region+
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ri*iJftf i?.t5 Thefrp repressor recognizes operators at threeloci.Conserved bases areshownin red.The[ocation of the startpoint andmRNA varies, asindicated bvthe whitearrows.
Startpoint
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Operat6ylq66lisns-r>
fiGiJtf 1i.I6 0perators positions maylieat various rel ativeto the promoter.
the repressor protein acts to reduce its own synthesis. This circuit is an example of autogenous control. Such circuits are quite common in regulatory genesand may be either negative or positive (seeSection 12.23,r-Protein Synthesis Is Controlled by Autogenous Regulation and Section l4.lr, Repressor Maintains an Autogenous Circuit). A related 2i bp operator sequenceis present at each of the three loci at which the trp repressoracts.The conservationof sequenceis indicated in FIiliJR[:.?..J5.Each operator contains appreciable(but not identical) dyad symmetry. The features conserved at all three operators include the important points of contact for /rp repressor. This explains how one
320
C H A P T E1R2 T h e0 o e r o n
repressor protein acts on several loci: eachlocus has a copyof a specificDNA-binding sequencereclgnizedby therepressor(just as each promoter shares consensussequenceswith other promoters). FtilLiRE X?"?Ssummarizesthe variety of relationships between operatorsand promoters. A notable feature of the dispersed operators recognized by TrpR is their presence at different Iocations within the promoter in each locus. In trpRlh.eoperator lies between positions -I2 and +9, whereas inthe trp operon it occupies positions -23 to -3.In the aroH locus it lies farther upstream, between -49 and-29.In other cases, the operator lies downstream from the promoter (asin lac), or apparently just upstream of the promoter (asingal, for which the nature of the repressiveeffect is not quite clear). The ability of the repressorsto act at operators whose positions are different in each target promoter suggeststhat there could be differences in the exact mode of repression, the common feature being that RNA polymerase is prevented from initiating transcription at the promoter.
CyclicAMPIs an Effector ThatActivates CRP to Act at ManyOperons o CRP proteinthat bindsto a target is an activator sequence at a promoter. r A dimerof CRP is activated by a singlemotecute of cyclicAMP.
Thus far we have dealt with the promoter as a DNA sequence that is competent to bind RNA polymerase, which then initiates transcription. There are, however, some promoters at which
RNA polymerase cannot initiate transcription without assistancefrom an ancillary protein. Such proteins are positive regulators,because their presenceis necessaryto switch on the transcription unit. T\.pically, the activator overcomes a deficiency in the promoter, for example, a poor consensussequenceat -35 or -I0. One of the most widely acting activators is a protein called CRP activator that controls the activity of a large set of operonsinE. coli.The protein is a positive control factor whose prese n c e i s n e c e s s a r yt o i n i t i a t e t r a n s c r i p t i o n a t dependent promoters. CRP is active only in the presence of qclic AMP, which behaves as a classic small-molecule inducer for positive control (see i : : i - , : + ,r : ' . . : ; U p p e f f i g h t ) . Cyclic AMP is synthesized by the enzyme adenylate cyclase. The reaction uses ATP as substrateand introduces a 3'-5'link via phosphodiester bonds, which generatesthe strucji:i: t,i ilri. Mutations in the ture drawn in i:irl..i gene coding for adenylate cyclase (cya-)do not respond to changesin glucoselevels. The level of cyclic AMP is inversely related to the level of glucose. The basis for this effect lies with the same component of the Pts system that is responsiblefor controlling lactoseuptake. The phosphorylated form of protein IIAGI'stimulates adenylate cyclase.When glucose is imported, the dephosphorylation of IIAGI' leads to a fall in adenylate cyclaseactivity. ir,',::;:i . .. shows that reducing the Ievel of cyclic AMP renders the (wild-type) protein unable to bind to the control region, which in turn prevents RNA polymerase from initiating transcription. Thus the effectof glucosein reducing cyclic AMP levels is to deprive the relevant operons of a control factor necessaryfor their expression.
REPRESSED ----l
TNDUCEDI INDUCED
nepnesseo REPRESSEIJ --
INDUL;EU. INDUCED
Corepressor INDUCED
REPRESSED
i r i l j i t i r t lj , . r . . r C o n t r o t c i r c u i t s a r e v e r s a t i t e a n d c a n b e d e s i g n e d t o a t l o w p o s i t i v e o r n e g ative controlof inductionor repression.
Adenine
Functions in CRP Different Waysin Different Target0perons . CRP introduces a 90obendinto DNAat its bindinq site. o CRP-binding siteslie at hightyvariable [ocations retativeto the promoter. o CRP interacts with RNApotymerase, butthe detaits depend on the relative locations of theinteraction siteandthe promoter. of the CRP-bjnding The CRP factor binds to DNA, and complexes of cyclic AMP.CRP DNA can be isolated at each
\l
ll
_!
/
6'
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promoter at which it functions. The factor is a dimer of two identical subunits of.22.5 kD, which can be activated by a single molecule of cyclic AMP. A CRP monomer contains a DNA-binding region and a transcription-activating region.
12.2! CRPFunctionsin DifferentWaysin DifferentTarget0perons
327
Glucose
t
cl
I
I
V Reduced cAMP
I
V
ts ActiveCRP
InactiveCRP
I *t,
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irIGUfiS 1?.?$ By reducing the [eve[of cyclicAMP,gtucoseinhibitsthetranscription of operons thatrequire CRP activitv.
Transcription
+
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fi6iJftt 1*.3* Theconsensus sequence for CRP contains pentamer thewet[conserved TGTGA and(sometimes) an inversion (TCANA). of thissequence
Centerot dyadsymmetry
f,gl'#l?il:t.]i symmetry.
322
CHAPTER 12 TheOperon
CRPbendsDNA>90oaroundthe centerof
A CRP dimer binds to a site of -22bp at a responsive promoter. The binding sitesinclude variations of the consensus sequence given in FIfiLiRf; t*.3il. Mutations preventing CRPaction usually are located within the well-conserved TGTGA pentamer which appears to be the ;;;;i, essentialelement in recognition. CRPbinds most strongly to sitesthat contain two (inverted) versions of the pentamer, becausethis enablesboth subunits of the dimer to bind to the DNA. Many binding sites lack the second pentamer, however, and in these the secondsubunit must bind a different sequence (if it binds to DNA). The hierarchy of binding affinities for CRP helps to explain why different genes are activated by different Ievels of cyclic AMP in vivo. CRP introduces a large bend when it binds DNA. In Ihe lac promoter, this point lies at the center of dyad symmetry. The bend is quite severe, >90o, as illustrated in the model of F I f i U f t [1 e . 5 3 .T h e r e i s , t h e r e f o r e , a d r a m a t i c change in the organization of the DNA double helix when CRPprotein binds. The mechanism of bending is to introduce a sharp kink within the TGTGA consensussequence.When there are inverted repeats of the consensus,the two kinks in each copy present in a palindrome cause the overall90" bend. It is possiblethat the bend has some direct effect upon transcription, but it could be the casethat it is needed simply to allow CRPto contact RNA polymerase at the promoter. The action of CRP has the curious feature that its binding sites lie at different locarions relative to the startpoint in the various operons that it regulates. The TGTGA pentamer may lie in either orientation. The three examples summarized in FIG{Jftil 1f,3f encompassthe range of locations: . The CRP-binding site is adjacent to the promoter, as in the lacoperon, in which the region of DNA protected by CRP is centered on -61. It is possiblethat two dimers of CRP are bound. The binding pattern is consistent with the presence of CRP largely on one face of DNA, which is the same face that is bound by RNA polymerase. This location would place the two proteins just about in reach of each other. o Sometimes the CRP-binding site lies within the promoter, as in tine gallocus, where the CRP-binding site is centered on -41 . It is likely rhat only a single CRp dimer is bound, probably in quite intimate contact with RNA polymerase,
because the CRP-binding site extends well into the region generally protected by the RNA polymerase. . In other operons, the CRP-binding site lies well upstream of the promoter. In tll'e ara region, the binding site for a single CRP is the farthest from the startpoint, centeredat-92. Dependence on CRPis related to the intrinsic efficiency of the promoter. No CRP-dependent promoter has a good -3 5 sequenceand some also lack good -10 sequences.In fact, we might argue that effective control by CRP would be difficult if the promoter had effective -35 and -10 regions that interacted independently with RNA polymerase. There are in principle two ways in which CRP might activate transcription: it could interact directly with RNA polymerase, or it could act upon DNA to change its structure in some way that assistsRNA polymerase to bind. In fact, CRPhas effectsupon both RNA polymerase and DNA. Binding sites for CRP at most promoters resemble either lac (centered at -6I ) or gal (centered at - I bp). The basic differencebetween them is that in the first type (called classI) the CRP-binding site is entirely upstream of the promoter, whereas in the second type (called class II) the CRP-binding site overlaps the binding site for RNA polymerase. (The interactions at the ara promoter may be different.) In both types of promoter, the CRP binding site is centered an integral number of turns of the double helix from the startpoint. This suggeststhat CRP is bound to the same face of DNA as RNA polymerase. The nature of the interaction between CRP and RNA polymerase is, however, different at the two types of promoter. When the cr subunit of RNA polymerase has a deletion in the C-terminal end, transcription appears normal except for the loss of ability to be activated by CRP.CRPhas an "activating region" that is required for activating both types of its promoters. This activating region, which consistsof an exposed loop of - I 0 amino acids, is a small patch that interacts directly with the cr subunit of RNA polymerase to stimulate the enzyme. At classI promoters, this interaction is sufficient. At classII promoters, a second interaction is required, which involves another region of CRP and the N-terminal region of the RNA polymerase cr subunit. Experiments using CRP dimers in which only one of the subunits has a functional tran-
Startpoint gal
Iac
ara
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scription-activating region shows that, when CRPis bound attine lacpromoter, only the activating region of the subunit nearer the startpoint is required, presumably becauseit touches RNA polymerase. This offers an explanation for the lack of dependence on the orientation of the binding site: the dimeric structure of CRP ensures that one of the subunits is available to contact RNA polymerase, no matter which subunit binds to DNA and in which orientation. The effect upon RNA polymerase binding depends on the relative locations of the two proteins. At classI promoters, where CRPbinds adjacent to the promoter, it increases the rate of initial binding to form a closed complex. At classII promoters, where CRPbinds within the promoter, it increasesthe rate of transition from the closedto open complex.
Can Translation BeRegulated o A repressor by proteincanregulatetranslation preventing frombindingto an a ribosome initiationcodon. o Accessibility of initiationcodonsin a polycistronic in the by changes mRNA canbecontrolled that occurasthe result structure of the mRNA of transtation. Ttanslational control is a notable feature of operons coding for components of the protein synthetic apparatus. The operon provides an regulation of a group arrangementf.or coordinale of structural genes. Further controls superimposed on the operon, though, such as those at tn the level of translation, may create differences
CanBeRegulated 323 1.2.22TransLation
the extent to which individual genes are expressed. A similar type of mechanism is used to achieve translational control in several systems. Repressor function is providedby a protein that binds to a targetregionon wRNA to preventribosomes from recognizingthe initiation region.Formally this is equivalent to a repressor protein binding to DNA to prevent RNA polymerase from utilizing a p r o m o t e r . a i S i l R i :l : . 3 : i l l u s t r a t e st h e m o s t common form of this interaction, in which the regulator protein binds directly to a sequence that includes the AUG initiation codon, thereby preventing the ribosome from binding. Some examples of translational repressors and their targetsare summarized in ijl**ft{ ti..:i4. A classicexample is the coat protein of the RNA phage Rl7; it binds to a hairpin that encompassesthe ribosome-binding site in the phage mRNA. Similarly, the T4 RegA protein binds to a consensussequence that includes the AUG initiation codon in severalT4 early mRNAs, and T4 DNA polymerase binds to a sequence in its own mRNA that includes the Shine-Dalgarno element needed for ribosome bindins.
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Another form of translational control occurs when translation of one cistron requires changes in secondary structure that depend on translation of a preceding cistron. This happens during translation of the RNA phages, whose cistrons always are expressed in a set order. F{*#R[ if-3* shows that the phage RNA takes up a secondary structure in which only one initiation sequence is accessible;the second cannot be recognized by ribosomes because it is
Only one initiationsite is availableinitially Secondinitiation site is blocked
Firstinitiation site is accessible
Regulato,r bindingsite+
55{;tji?l .:il"i} A regutator proteinmaybtocktranstation by binding to a siteon mRNA thatovertaps theribosomebindingsiteat theinitiationcodon.
i::fiilltf tI=3S Secondary structure cancontrolinitiation.0ntyoneinitiation sjtejs avaitable in theRNAphage. buttranslation ofthefirstcistronchanqes theconformation of the RNAso that otherinitjatio-n site(s)become avai[abte.
Repressor
TargetGene
Site of Action
R17coat protein
R17 replicase
hairpinthat includesribosomebindingsite
T4 RegA
earlyT4 mRNAs
varioussequencesincludinginitiation codon
T4 DNA polymerase T4 DNA polymerase Shine-Dalgarno sequence T4 p32
gene32
single-stranded 5' leader
il3{"i#Flil 1t";1.{Proteins that bindto sequences withinthe initiationregions of mRNAs may functionastranslationaI reoressors.
324
CHAPTER 12 The0oeron
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A feature of autogenous control is that each regulatory interaction is unique: a protein acts mRNA ssDNA Bindingto: only on the nRNA responsible for its own synP32 thesis. PhageT4 provides an example of a more general translational regulator, coded by the gele regA, which represses the expression of several genes that are transcribed during early infection. RegA protein prevents the transla10 6 10 5 10 8 10 7 1O-4 1O-3 tion of mRNAs for these genes by competing Proteinconcentration(Mola0 + with 30S subunits for the initiation siteson the mRNA. Its action is a direct counterpart to the i ; : . l i : l i i l,ii:. i . : i : ,6:. n . 3 2 o r o t e i nb i n d st o v a r i o u s u b wjthdifferent affinities, in theordersingte-stranded function of a repressor protein that binds mulstrates tiple operators. D N Ai ,t s o w nm R N Aa,n do t h e m r R N ABs i. n d i ntgo i t so w n prevents mRNA the [eve[of p32fromrising>10-6M.
lation of its own nRNA. The effect is mediated directly by p32 binding to mRNA to prevent initiation of translation. In all likelihood this occurs at an A-T-rich region that surrounds the ribosome binding site. TWo features of the binding of.p32 to the site on nRNA are required to make the control loop work effectively: . The affinity of p32 for the site on gene 32 mRNA must be significantly lower than its affinity for single-strandedDNA. The equilibrium constant for binding RNA is in fact almost two orders of magnitude below that for single-strandedDNA. . The affinity ofp32 for the mRNA, however, must be significantly greater than the affinity for other RNA sequences.It is influenced by base composition and by secondary structure; an important aspect of the binding to gene J2 mRNA is that the regulatory region has an extended sequence lacking secondary structure. Using the known equilibrium constants, we can plot the binding of p32 to its target sites as a function of protein concentration. iii.,i-jtir; "::.-.ti] shows that at concentrations below I0-6 M, p32 binds to single-strandedDNA. At concentrations >10-6 M, it binds to ge\e 32 nRNA. At yet greater concentrations, it binds t o o t h e r n R N A s e q u e n c e s ,w i t h a r a n g e o f affinities. These results imply that the level of.p32 should be autoregulatedto be <10-6M, which correspondsto -2000 molecules per bacterium. This fits well with the measured level of 1000 to 2000 molecules/cell.
Regulation Autogenous Is OftenUsedto Control of Synthesis Macromolecu[ar AssembLies . Theprecursor freetubulin to microtubutes, of tubutinmRNA. protein,inhibitstranslation Autogenous regulation is a common type of control among proteins that are incorporated into macromolecular assemblies.The assembled particle itself may be unsuitable as a regulator becauseit is too large, too numerous, or too restricted in its location. The need for synthesis of its components, though, may be reflected in the pool of free precursor subunits. If the assemblypathway is blocked for any reason, free subunits accumulate and shut off the unnecessary synthesis of further components. Eukaryotic cells have a common system in which autogenous regulation of this tlpe occurs. Tubulin is the monomer from which microtubules, a major filamentous system of all eukaryotic cells, are synthesized. The production of tubulin nRNA is controlled by the free tubulin pool. When this pool reachesa certain concentration, the production of further tubulin mRNA is prevented. Again, the principle is the same: tubulin sequesteredinto its macromolecular assemblyplays no part in regulation, but the level of the free precursor pool determines whether further monomers are added to it. The target site for regulation is a short sequence at the start of the coding region. We do not know yet what role this sequenceplays, .irl-+-l}. but two models are illustrated in Ih*{"1ftil Tirbulin may bind directly to the mRNA, or it
Assembties 327 of Macromotecutar Synthesis Is 0ftenUsedto Control Regutation 12.25Autogenous
tions by being recognizedin situ.It has no coding function and can regulate only those sequenceswith which it is physically contiguous. Bacterial genescoding for proteins whose functions are related, such as successiveenzymes Freetubulinbinds rs to ro either erlner ^ o --oo in a pathway, may be organized in a cluster that O6 o is transcribed into a polycistronic mRNA from f a single promoter. Control of this promoter regulates expression of the entire pathway. The )tein unit of regulation, which contains structural genes and crs-acting elements, is called the operon. Tubulinis assembled into microtubules Initiation of transcription is regulated by interactions that occur in the vicinity of the promoter. The ability of RNA polymerase to initiate at the promoter is prevented or activated by other proteins. Genesthat are active unless they are turned off are said to be under negative control. Genes that are active only when specifically turned on are said to be under positive i-i*i":,1ii.i.+t Tubutin is assembted into microtubuLes control. The type of control can be determined whenit is synthesized. Accumutation of excess freetububy the dominance relationships between wild jn [in induces instabiLity the tubu[inmRNA by actingat type and mutants that are constitutive/derea siteat thestartof the reading framejn mRNA or at the positionin the nascent corresponding protein. pressed(permanently on) or uninducible/superrepressed(permanently off). A repressor protein prevents RNA polymerase either from binding to the promoter or from activating transcription. The repressor binds to a target sequence, the operator, that usually is located around or upstream of the startpoint. Operator sequencesare short and may bind to the nascent polypeptide representoften are palindromic. The repressor is often a ing this region. Whichever model applies,excess homomultimer whose svmmetrv reflectsthat of tubulin causestubulin nRNA that is located on its target. polysomesto be degraded,so the consequence The ability of the repressor prorein to bind of the reaction is to make the tubulin mRNA to its operator is regulated by a small molecule. unstable. An inducer prevents a repressor from binding; Autogenous control is an intrinsically selfa corepressoractivatesit. Binding of the inducer Iimiting system, by contrast with the extrinsic or corepressor to its site produces a change in control that we discussedpreviously.A represrhe strucrure of the DNA-binding site of the sor protein's ability to bind an operator may be repressor.This allosteric reaction occurs in both controlled by the level of an extraneous small free repressor proteins and directly in represmolecule, which activates or inhibits its activsor proteins already bound to DNA. ity. In the caseof autogenousregulation, though, The lactosepathway operatesby induction, the critical parameter is the concentration of when an inducer B-galactosideprevents the the protein itself. repressor from binding its operator; transcription and translation of the lacZ gene then p r o d u c e B - g a l a c t o s i d a s e t, h e e n z y m e t h a t metabolizes p-galactosides.The tryptophan Transcription is regulated by the interaction pathway operates by repression; the corepresb etwe en tr ans- acling f actors and cli - acting sites. sor (tryptophan) activates the repressor proA trans-acing factor is the product of a regulatein, so that it binds to the operator and prevents tor gene. It is usually protein but also can be expression of the genes that code for the RNA. It diffuses in the cell, and as a result it can enzymes that biosynthesize tryptophan. A act on any appropriate target gene. A cis-acting repressorcan control multiple targets that have site in DNA (or RNA) is a sequence that funccopiesof an operator consensussequence.
nffif
Sum mary
328
C H A P T E1R2 T h e0 p e r o n
A protein with a high affinity lor a particular target sequencein DNA has a lower affinity for all DNA. The ratio defines the specificity of the protein. There are many more nonspecific sites (any DNA sequence) than specific target sites in a genome; as a result, a DNAbinding protein such as a repressor or RNA polymerase is "stored" on DNA. (It is likely that none, or very little, is free.) The specificity for the target sequencemust be great enough to counterbalance the excess of nonspecific sites over specificsites.The balance for bacterial proteins is adjusted so that the amount of protein and its specificity allow specificrecognition of the target in "on" conditions, but allow almost complete releaseof the target in "off" conditions.
Weber, I(. and Geisler,N. (1978). Lac repressor fragments produced in vivo and in vitro: an approach to the understanding of the interaction of repressor and DNA. In The Operon,eds. Miller, J. and Reznikoff, W New York: Cold Spring Harbor Laboratory, 155-17 6. Wilson, C. J., Zahn, H., Swint-I(ruse, L., and Matthews, K. S. (2006). The lactoserepressor system: paradigms for regulation, allosteric behavior and protein folding. Cell.Mol Life Sci November 13. Research Jacob, F. and Monod, J. ( l96l ) . Genetic regulatory mechanisms in the synthesis of proteins. J. Mol. Biol. ), jl8-)89.
HasSeveral Monomer TheReoressor Domains rch Resea
References Introduction Research Jacob, F. and Monod, J. ( 196 I ). Genetic regulatory mechanisms in the synthesis of proteins. J. Mol. Biol ),318-)89.
Regutation CanBeNegative or Positive Review Miller, J. and Reznikoff, W, eds (1980). The Operon, 2nd ed., Woodbury, NY: Cold Spring Harbor Laboratory Press.
ThelacGenes AreControtled by a Repressor Reviews Barkley, M. D. and Bourgeois, S. (1978). Repressor recognition of operator and effectors. In Tfte Operon,eds. Miller, J. and Reznikoff, W. New York: Cold Spring Harbor Laboratory, t77-220. Beckwith, J. (1978). lac: Ihe genetic system. In Tfte Operon,eds. Miller, J. and Reznikoff, W. New York: Cold Spring Harbor Laboratory, I I-30. Beyreuther, I(. (1978). Chemical structure and functional organization of the lac repressor from E. coli.ln The Operon,eds. Miller, J. and Reznikoff, W. New York: Cold Spring Harbor Laboratory, 123-154. Miller, J. H. (I978). The lacl gene: its role in lac operon control and its use as a genetic system. ln The Operon,eds. Miller, J. and Reznikoff, W. New York: Cold Spring Harbor Laboratory, 3l-88.
Friedman, A. M., Fischmann, T. O., and Steitz, T. A. ( 1995). Crystal structure of lac repressor core tetramer and its implications for DNA 268, 172l-1727. Iooping. Science Lewis, M. et al ( 1996). Crystal structure of the lactose operon repressor and its complexes with DNA and inducer. Science271, 1247-1254.
Corretate MutantPhenotYPes with the Domain Structure Reviews Pace,H. C., I(ercher, M. A., Lu, P., Markiewicz, P., Miller, J. H., Chang, G., and Lewis, M' (1997)r. Lac repressor genetic map in real space.Trends Biochem.Sci.22, 3)4-)J9. Markiewicz, P, ICeina, L. G., Cruz, C., Ehret, S., and Miller, J. H. (1994\ . Genetic studies of the lac repressor.XIV. Analysis of 4000 altered E. coli lac repressorsreveals essential and nonessential residues, as well as spacerswhich do not require a specific sequence. J Mol. Biol240,42t-4)3. Suckow, J., Markiewicz, P, ICeina, L. G., Miller, J., ICsters-Woike, B., and Miiller-Hill. B. (19961. Genetic studies of the Lac repressor. XV: 4000 single amino acid substitutions and analysis of the resulting phenotypes on the basis of the protein structure. J. Mol Biol' 26r,509-123.
ProteinBindsto the 0perator Repressor rch Resea Gilbert, W. and Miiller-Hill, B. (I966). Isolation of the lac repressor.Proc Natl Acad. Sci'USA 56' I 8 9l - l 8 9 8 .
References 329
Gilbert, W. and Mtiller-Hill, B. (1967). The lac operator is DNA. Proc.Natl Acad. Sci.USA 58, 24t5-2421.
@
Repressor Bindsto Three0perators and Interactswith RNAPotymerase
R e s erach Oehler,S.et al. (1990).The threeoperatorsof the lac operoncooperate in repression. EMBOJ.9 97)-979.
@
The0perator Competes with Low-Affinity Sites to Bind Repressor
Resea r ch Cronin, C. A., Gluba, W., and Scrable,H. (2001). The lac operator-repressorsystem is functional in the mouse. GenesDev. 15, 1506-1517. Hildebrandt, E. R. er al. (1995). Comparison of recombination in vitro and in E. coli cells: measure of the effective concentration of DNA in vivo.Cell 81, ,I-340. Lin, S.-Y.and Riggs,A. D. (1975). The general affinity of lac repressor for E. coli DNA: implications for gene regulation in prokaryotes and eukarvotes.Cell4. 107-111.
@
CRPFunctions in Different ways in DifferentTargetOperons
Reviews Botsford,J. L. and Harman,J. G. (1992).Cyclic AMP in prokaryotes.Mitobiol Rev.56, t00-122. I(olb, A. (1993).Transcriptionalregulationby cAMP and its receptorprotein.Annu Rev. Biochem 62,749-795.
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CHAPTER 12 The0peron
Researc h Niu, W., Ifim, Y., Tau, G., Heyduk, T., and Ebright, R. H. (t 996). Tlanscription activation at class II CAP-dependent promoters: two interactions between CAP and RNA polymerase. Cell 87,
rr23-t134. Zhou,Y., Busby, S.,andEbright, R.H. (1993). Identification of the functional subunit of a dimeric transcription activator protein by use of oriented heterodimers. CellT), )75479. Zhou, Y., Merkel, T. J., and Ebright, R. H. ( 1994) . Characterization of the activating region of E coli catabolite gene activator protein (CAP). II. Role at ClassI and classII CAP-dependent promoters. J. Mol Biol.243, 603-610.
r-Protein Synthesis Is Controlted by AutogenousRegutation Review Nomura,M. et al. (1984).Regulationof the synthesisof ribosomesand ribosomalcomponelf,ts.Annu.Rev.Biochem. 53.75-ll7 . Research Baughman,G. and Nomura,M. (1983).Localization of the targetsitefor translationalregulation of the L I I operonand direct evidencefor translationalcouplingin E. coli.Cell)4, 979-988.
Autogenous Regutation Is 0ftenUsed to ControI Synthesis of Macromolecular Assemb[ies Review Gold, L. (1988). Posttranscriptionalregulatory mechanisms in E. coli.Annu. Rev.Biochem 57. 199-223.
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small molecule. Binding of the small molecule to its site changes the conformation in such a way as to alter the affinity of the other site for r RNAfunctions asa regulator by forminga the nucleic acid. The way in which this hapregionof secondary (eitherinter-or structure pens is known in detail for the Lac repressor. intramolecular) that changes the properties of a Protein regulators are often multimeric, with a targetsequence. symmetrical organization that allows two subunits to contact a palindromic target on DNA. The basic principle of regulation in bacteria is This can generate cooperative binding effects that gene expressionis controlled by a regulathat create a more sensitive response to tor that interacts with a specific sequence or regulation. structure in DNA or nRNA at some stageprior Regulation via RNA uses changes in secto the synthesisof protein. The stageof expresondary structure as the guiding principle. The sion that is controlled can be transcription, when ability of an RNA to shift between different conthe target for regulation is DNA, or it can be at formations with regulatory consequencesis the translation, when the target for regulation is nucleic acid's alternative to the allosteric changes RNA. When control is during transcription, it of protein conformation. The changes in struccan be at initiation or at termination. The regture may result from either intramolecular or ulator can be a protein or an RNA. "Controlled" intermolecular interactions. can mean that the regulator turns off (represses) The most common role for intramolecular the target or that it turns on (activates)the tarchangesis for an RNA molecule to assumealterget. Expression of many genes can be coordinative secondary structures by utilizing differnately controlled by a single regulator gene on ent schemesfor base pairing. The properties of the principle that each target contains a copy the alternative conformations may be different. of the sequenceor structure that the regulator Changes in secondary structure of an mRNA recognizes.Regulatorsmay themselvesbe regcan result in a change in its ability to be transulated, most typically in responseto small mollated. Secondarystructure also is used to regueculeswhose supply respondsto environmental late the termination of transcription, when the conditions. Regulators may be controlled by alternative structures differ in whether they other regulators to make complex circuits. permit termination. Let's compare the ways that different types In intermolecular interactions, an RNA regof regulators work. ulator recognizesits target by the familiar prinProtein regulators work on the principle of ciple of complementary basepairing. ti*ijfil ]i"r".i allostery. The protein has two binding sitesshows that the regulator is usually a small RNA one for a nucleic acid target, the other for a molecule with extensive secondary structure, but with a single-strandedregion(s) that is complementary to a single-stranded region in its target. The formation of a double helical region between regulator and target can have two t).pes of consequence: o Formation of the double helical structure may itself be sufficient. In some cases,protein(s) can bind only to the single-stranded form of the target sequence and are therefore prevented from acting by duplex formation. In other cases,the duplex region becomes a target for binding; for example, by nucleases that degrade the RNA and therefore prevent its expression. . Duplex formation may be important becauseit sequestersa region of the target RNA that would otherwise partici:r:',ii1;r:i li:.': A regulator RNAis a smat[RNAwitha single-stranded pate in some alternative secondary regionthat canpairwith a singte-stranded regionin a targetRNA. structure.
Introduction
332
CHAPTER 13 RegutatoryRNA
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ALTERNATIVESTRUCTURES Regions2&3pair; Regions3&4 to 1 & 3 region Region2 is complementary pairto formthe terminator to 2 and4 is single-stranded terminator hairpin Region3 is complementary The conformations. base-paired ili*iJRil1.lt-iiThetrp leaderregioncanexistin atternative to region2, which 1 is complementary thatcanbasepair.Region centershows thefourregions to region4. 0n the leftis the conforis complementary to region3, whichis comptementary mationproduced whenregion1 pairswith region2 andregion3 pairswith region4.0n the 1 and4 unpaired. regions rightistheconformation whenregion2 pairswith region3, [eaving
4 generates the hairpin that precedes the Ug sequence: this is the essential signal for intrinsic termination. It is likely that the RNA would take up this structure in lieu of any outside intervention. A dilferent structure is formed if region I is prevented from pairing with region 2. In this case,region 2 is free to pair with region 3. Region 4 then has no available pairing partner, so it is compelled to remain single-stranded. Thus the terminator hairpin cannot be formed. fi{:ijRgri"* shows that the position of the ribosome can determine which structure is formed in such a way that termination is attenuated only in the absence of tryptophan. The crucial feature is the position of the Ttp codons in the leader peptide coding sequence. When try?tophan is present, ribosomes are able to synthesizethe leader peptide. They continue along the leader section of the nRNA to the UGA codon, which lies between regions I and 2. As shown in the lower part of the figure, by progressing to this point, the ribosomes extend over region 2 and prevent it from base pairing. The result is that region 3 is available to base pair with region 4, which generatesthe terminator hairpin. Under these conditions, therefore, RNA polymerase terminates at the attenuator. When there is no tryptophan ribosomes stall at the Trp codons, which are part of region l, as shown in the upper part of the figure. Thus
U U
Ribosomehaltsat Trp codons PRESENT TRYPTOPHAN
Ribosomemovement disrupts2:3 pairing 3:4 pairingforms terminatorhairPin
at the for RNApotymerase i.*.$ Theatternatives F;*iJfr.fl which oftheribosome, onthelocatjon depend attenuator regions 3 and4 canpairto formthe whether determines hairpin. terminator
by Transtation 337 CanBeControlted 13.5 Attentuation
i : i . i : i i L: : , t i . I n t h ep r e s e n o ce f t r y p t o p h aI R n N Ar,i b o somes translate the leaderpeptide andarereteased. This altowshairpinformatjon, sothat RNApotymerase terminates.In the absence of tryptophan IRNA,the ribosome is btocked, thetermination hairpincannotform,andRNA polymerase continues.
region I is sequesteredwithin the ribosome and cannot basepair with region 2. This means that regions 2 and 3 become base paired before region 4 has been transcribed. This compels region 4 to remain in a single-strandedform. In the absence of the terminator hairpin, RNA polymerase continues transcription past the attenuator. Control by attenuation requires a precise timing of events. For ribosome movement to determine formation of alternative secondary structures that control termination, translation of the leadermust occurat the sametime when RNA polymeraseapproachesthe terminator site. A crilical event in controlling the timing is the presence of a site that causesthe RNA polymerase to pause at base 90 along the leader.The RNA polymerase remains paused until a ribosome translatesthe leader peptide. The polymerase is then releasedand move s off toward the attenuation site. By the time it arrives there, secondary structure of the attenuation region has been determined. ;-; i:iis1111tutiresthe role of Trp_tRNA iri:,::.ii:ii: in controlling expression of the operon. By providing amechanismtosensethe inadequaqtof thesupply of Trp-tRNA, attenuation respondsdirectly to the needof the cellfor tryptophan in protein synthesis.
338
C H A P T E1R3 R e g u t a t o rRy N A
How widespread is the use of attenuation as a control mechanism for bacterial operons? It is used in at least six operons that code for enzymes concerned with the biosynthesis of amino acids. Thus a feedback from the level of the amino acid available for protein synthesis (asrepresentedby the availability of aminoacylIRNA) to the production of the enzymes may be common. The use of the ribosome to control RNA secondary structure in response to the availability of an aminoacyl-IRNA establishesan inverse relationship between the presence of aminoacyl-tnNA and the transcription of the operon, which is equivalent to a situation in which aminoacyl-IRNA functions as a corepressor of transcription. The regulatory mechanism is mediated by changesin the formation of duplex regions; thus attenuation provides a striking example of the importance of secondary structure in the termination event and of its use in regulation. E. coli and B. subtilis,therefore, use the same types of mechanisms, which involve control of nRNA structure in response to the presence or absenceof a IRNA, but they have combined the individual interactions in different ways. The end result is the same: to inhibit production of the enzymes when there is an excesssupply of the amino acid. and to activate production when a shortage is indicated by the accumulation of uncharged tRNAftp.
Antisense RNACan BeUsedto Inactivate GeneExpression r Antisense genesbtockexpression of theirtargets whenintroduced into eukarvotic cetts. Base pairing offers a powerful means for one RNA to control the activity of another. There are many casesin both prokaryotes and eukaryotes where a (usually rather short) singlestranded RNA basepairs with a complementary region of an mRNA, and as a result it prevents expression of the nRNA. One of the early illustrations of this effect was provided by an artificial situation in which antisense genes were introduced into eukaryotic cells. Antisense genes are constructed by reversing the orientation of a gene with regard to its promoter, so that the "antisense" strand is tran-
ii r:1i 1 i i . Synthesis scribed, as illustrated in f:Il' 1.; of antisenseRNA can inactivate a target RNA in either prokaryotic or eukaryotic cells.An antisenseRNA is in effect a synthetic RNA regulator. An antisense thymidine kinase gene inhibits synthesisof thymidine kinase from the endogenous gene. Quantitation of the effect is not entirely reliable, but it seems that an excess (perhaps a considerableexcess)of the antisense RNA may be necessary. At what level does the antisenseRNA inhibit expression?It could in principle prevent transcription of the authentic gene. processingof its RNA product, or translation o{ the messenger. Results with different systems show that the inhibition depends on formation of RNA-RNA duplex molecules,but this can occur either in the nucleus or in the cytoplasm. In the caseof an antisensegene stably carried by a cultured cell, sense-antisenseRNA duplexes form in the nucleus, preventing normal processing and/or transport of the senseRNA. In another case,injection of antisense RNA into the cytoplasm inhibits translation by forming duplex RNA in the 5'region of the nRNA. This technique offers a powerful approach for turning off genes at will; for example, the function of a regulatory gene can be investigated by introducing an antisenseversion. An extension of this technique is to place the antisensegene under the control of a promoter that is itself subject to regulation. The target gene can then be turned off and on by regulating the production of antisense RNA. This technique allows investigation of the importance of the timing of expressionof the target gene.
Can RNAMotecules SmaLL Translation ReguLate o A regulator RNAfunctions by forminga duplex regionwith a targetRNA. . Theduptexmayblockin'itiation of transtation, or createa termination of transcription, cause targetfor an endonuctease. Repressorsand activators are trans-acting proteins, yet the formal circuitry of a regulatory network could equally well be constructed by using an RNA as regulator. In fact, the original model for the operon left open the question of whether the regulator might be RNA or protein. Indeed, the construction of synthetic antisense RNAs turns out to mimic a classof RNA
Promoter
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regulators that is becoming increasingly important. Like a protein regulator, a small regulator RNA is an independently synthesizedmolecule that diffusesto a target site consistingof a specific nucleotide sequence. The target for a regulator RNA is a single-stranded nucleic acid sequence.The regulator RNA functions by complementarity with its target, at which it can form a d o u b l e - s t r a n d erde g i o n . We can imagine two general mechanisms for the action of a regulator RNA: . Formation of a duplex region with the target nucleic acid directly prevents its ability to function by forming or sequestering a specific site. !'i{;iililiri i'i";: illustrates the situation in which a protein that binds to single-strandedRNA is prevented from acting by formation of a li..r:irlrshows the oppoduplex. ',irri,ji-lisite type of relationship, in which the formation of a double-strandedregion createsa target site for an endonuclease that destroYsthe RNA target. ' Formation of a duplex region in one part of the target molecule changesthe conformation of another region, thus indi,1:i.'i rectly affecting its function. i ll'i-.:fiS" "1 shows an examPle. The mechanism is essentiallysimilar to the use of secondary structure in attenuation (see
Translation 339 CanRegutate RNAMolecutes 13.7Smatt
Section I 3.2, Alternative Secondary Structures Control Attenuation), except Proteinbinds that the interacting regions are on difsingle-stranded regionin target ferent RNA molecules instead of being part of the same RNA molecule. The feature clmmln to both types of RNAmediated regulation is that changesin secondary structure ofthe target control its activity. A small RNA regulator typically can be turned on by controlling transcription of its gene or turned off by an enzyme that degrades the RNA regulator product. Usually it is not possible otherwise to regulate the activity of an RNA regulator. In fact, it used to be thought that it cannotbind to target would not be possible for an RNA to have Fl*tlR[ 13.1? A proteinthat bindsto a singte-stranded allosteric propefiies; unlike repressor proteins regionin a targetRNAcoul.d be excluded by a regulator that control operons, an RNA usually cannot RNAthat formsa duptex in this reoion. respond to small molecules by changing its ability to recognize its target. The discovery of the riboswitch provides g3.Ii summaan exception to this rule. FISURE rizes the regulation of the system that produces the metabolite GlcN6P.The geneglms codesfor an enzyme that synthesizes GlcN6P (Glucosamine-6-phosphate) from fructose-6phosphate and glutamine. The mRNA contains a long, 5'untranslated region (UTR) before the coding frame. Within the UTR is a ribozymea sequence of RNA that has a catalytic activity (seeSection 27.4,Rlbozymes Have Various Catalytic Activities). In this case,the catalytic activity is an endonucleasethat cleavesits own RNA. =ffitr 04 It is activatedby binding of the metabolite product, GlcN6P,to the ribozyme. The consequence FIfiIJR[ 13.1] Bybinding to a targetRNA to forma duptex is that accumulation of GlcN6P activates the region, a regulator RNAmaycreate a sitethatis attacked ribozyme, which cleaves the mRNA, which in bv a nuclease. turn prevents further translation. This is an exact parallel to allosteric control of a repressor protein by the end product of a metabolic pathway. There are several examples of such riboswitches in bacteria. 'j'l 'i j Another regulatory mechanism that involves transcription of a noncoding RNA works indirectly. Initiation at a target promoter can be suppressedby transcription from another promoter upstream from it, as shown in Fi$ljftE13.36.The causeof the inhibition is that Secondary slructure the RNA polymerase initiating at the upstream formsin absence promoter reads through the downstream proof regulator moter, which prevents transcription factors and RNA polymerase from binding ro it. This type of effect has been demonstrated in eukaryotic r:6URt 13.14 Thesecondary structure formedby base systems and may depend on the disruption of pairingbetween two regions of the targetRNAmaybe prevented fromformingby basepairingwitha regutator chromosomal structure at the target promoter. RNA. In thisexample. The RNA that is transcribed from the upstream theabitityof the3' endof theRNA to pairwiththe 5' endis prevented by the regulator. (regulatory) promoter has no coding function.
340
CHAPTER 13 Regutatory RNA
This may explain the presence of some of the noncoding RNAs in eukaryotic nuclei; they are not regulator RNAs as such, but are the indirect products of a regulatory system.
Bacteria Contain Regulator RNAs a a
BacteriaI regutator RNAs arecatledsRNAs. Several of the sRNAs areboundby the proteinHfq, whichincreases theireffectiveness. TheOxySsRNAactivatesor represses expression of >10tociat the posttranscriptiona[ [eve[.
In bacteria, regulator RNAs are short molecules that are collectively known as sRNAs; E. coli contains at least seventeen different sRNAs. Some of the sRNAs are general regulators that affect many target genes.They function by basepairing with target RNAs (typically mRNAs) to control either their stability or function. An example of stability control is provided by the small antisenseregulator RyhB. which regulates six mRNAs coding for proteins concerned with iron storage in E. coli. RyhB base pairs with each of the target mRNAs to form double-strandedregionsthat are substratesfor RNAase E. An interesting feature of the circuit is that the ribonuclease destroys the regulator RNA as well as the mRNA. O x i d a t i v e s t r e s sp r o v i d e s a n i n t e r e s t i n g example of a general control system in which RNA is the regulator. When exposed to reactive oxygen species,bacteria respond by inducing antioxidant defense genes. Hydrogen peroxide activates the transcription activator OxyR, which controls the expression of several inducible genes. One of these genes is oxyS, which codes for a small RNA. i::Ir-:i'i.j :r.:..r'shows two salient features of the control of oxySexpression. In a wild-type bacterium under normal conditions, it is not expressed.The pair of gels on the left side of the figure show that it is expressedat high levels in a mutant bacterium with a constitutively active oxyR gene. This identifies oxyS as a target for activation by oxyR The pair of gels on the right side of the figure show that OxyS RNA is transcribed within one minute of exposure to hydrogen peroxide. T h e O x y S R N A i s a s h o r t s e q u e n c e( 1 0 9 nucleotides) that does not code for protein. It
for the regionof the mRNA l':i;iiiil: .irr.11 :- The5' untranstated thatis actja ribozyme contains GtcN6P enzyme thatsynthesizes the inactivates product.Theribozyme vatedby the metabotic it. mRNA by cteaving
Regulatory_I@-. Promoter
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promoter mayinhibitinitiafroman upstream ::'liii"liii: li;i.'ir Transcription reading throughthe downfromit, because downstream tion at a promoter frombindingto factors transcription prevents promoter the necessary stream promoter hasno codingfunction. fromthe upstream it. TheRNAtranscribed
RNAs 347 Regulator Contain 13.8 Bacteria
the 3'terminus is complementary to a sequence just preceding the initiation codon of FIhA Add H2o2 Wtld- oxyR mRNA. Base pairing between OxyS RNA and type mutant FlhA RNA prevents the ribosome from binding to the initiation codon and therefore represses oxyR translation. There is also a second pairing intermRNA * action that involves a sequencewithin the coding region of FlhA. Another target for oxyS is rpoS, the gene coding for an alternative sigma factor (which activates a general stressresponse). By inhibiting production of the sigma factor, oxy.S ensures that the specificresponseto oxidative stressdoes not trigger the response that is appropriate for other stressconditions. The rpoS gene is also regulated by two other sRNAs (DsrA and RprA), which activate it. These three sRNAs appear to global regulators that coordinate responses be oxyS t to various environmental conditions. RNA (109nts) The actions of all three sRNAs are assisted by an RNA-binding protein called Hfq. The Hfq protein was originally identified as a bacterial host factor needed for replication of the RNA bacteriophage QB. It is related ro rhe Sm proteins of eukaryotes that bind to many of the Fl*LlRg1i.:i Thegetson the left showthat oxySRNA snRNAs (small nuclear RNAs) that have reguis inducedin an oxyRconstitutive mutant.Theqetson latory roles in gene expression (see Section therightshowthatoxySRNA isinduced wjthinone-minute 26.5, snRNAs Are Required for Splicing).Mutaof addinghydrogen peroxide to a wild-typeculture.Reprotions in its gene have many effects,which idenducedfromCell,vol.90,Altuvia,S.,et at.,A smallstable RNA.. . , pp.44-53.Copyright 1997.with permission tifies it as a pleiotropic protein. Hfq binds to fromElsevier. Photocourtesy of GiselaStorz,National many of the sRNAs of E. coli, and it increases the Institutes of Health. effectiveness of OxyS RNA by enhancing its ability to bind to irs rarget mRNAs. The effect of Hfq is probably mediated by causing a small change in the secondary structure of OxyS RNA that improves the exposure of the singlestranded sequencesthat pair with the target mRNAs.
MicroRNAs AreRegulators flE*lJS*. 33-3* oxySRNAinhibitstranslation oflhl mRNA by basepairing just upstream witha sequence of theAUGinitiationcodon.
is a trans-acting regulator that affects gene expression at posttranscriptional levels. It has >10 target loci; at some of them, it activates expression; at others it repressesexpression. F?{:iJf;5 1;*1..i8 shows the mechanism of repression of one target, the FlhA mRNA. Three stemloop structures protrude in the secondary structure of OxyS mRNA, and the loop close to
342
CHAPTER 13 RegutatoryRNA
in ManyEukaryotes . Animalandptantgenomes codefor manyshort (-22 base)RNAmolecules cattedmicroRNAs. r MicroRNAs geneexpression regutate by base pairingwith complementary sequences in target mRNAs. Very small RNAs are gene regulators in many eukaryotes. The first example was discoveredin the nematode Caenorhabditiselegansas the result of the interaction between the regulator gene lin4 and its target gene, lin14. FIfrl"]ftgi .t].r*
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Martens, J. A., Laprade, L., and Winston, F. (2004). Intergenic transcription is required to repress Ilrle Saccharomyces cerevisiaeSERS gene. Nature429, 571-574. Winkler, W. C., Nahvi, A., Roth, A., Collins, J. A.. and Breaker, R. R. (2004). Control of gene expression by a natural metabolite-responsive ribozyme. Nature 428, 281-286.
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TheDNA-Binding Form of Repressor Is a Dimer r A repressor monomer hastwo distinctdomains. o TheN-terminaI domaincontains the DNA-bindinq site. r TheC-terminal domaindimerizes. o Binding to the operator requires the dimeric form sothat two DNA-binding domains cancontactthe operator simultaneousty. r Cleavage of the repressor between the two domains reduces the affinitvfor the operator and inducesa lytic cycte.
The repressorsubunit is a polypeptide of 27 kD with the two distinct domains summarized in !:?GLiR[ -!'.:.i.fi. . The N-terminal domain, residuesl-92, provides the operator-binding site. . The C-terminal domain, residues I)2-236, is responsiblefor dimerization. The two domains are joined by a connector of forty residues.When repressoris digested by a protease,each domain is releasedas a separate fragment. Each domain can exerciseits function independently of the other. The C-terminal fragment can form oligomers. The N-terminal fragment can bind the operators, although with a lower affinity than the intact repressor.Thus the information for specificallycontacting DNA is contained within the N-terminal domain, but the efficiency of the processis enhanced by the attachment of the C-terminal domain. The dimeric structure of the repressoris crucial inmaintaining lysogenyThe induction of a lysogenic prophage to enter the lytic cycle is caused by cleavageof the repressor subunit in the connector region, between residues I I I and t 13. (This is a counterpart to the allosteric change in conformation that results when a small-molecule inducer inactivates the repressorof a bacterial operon, a capacity that the lysogenic repressordoes not have.) Induction occurs under certain adverseconditions, such as exposure of lysogenic bacteria to UV irradiation, which leads to proteolytic inactivation of the repressor. In the intact state, dimerization of the Cterminal domains ensuresthat when the repressor binds to DNA, its two N-terminal domains each contact DNA simultaneously. Note, howe v e r , t h a t c l e a v a g e r e l e a s e st h e C - t e r m i n a l domains from the N-terminal domains. As illustrated in ri*',lRF i.4"::.',this means that the
ti;i;iJi{i:,:+.I r TheN-terminaI andC-terminat regionsof repressor formseparate TheC-terminaI domains. domains associate to formdimers; theN-terminal domains bindDNA.
Fli:t'!ll :r.,.1; Repressor The dimersbindto the operator. affinityof the N-terminaI for DNAis controlted by domains the dimerization domains. of the C-terminaI N-terminal domains can no longer dimerize, which upsetsthe equilibrium between monomers and dimers. As a result, repressordissociatesfrom DNA, which allows lytic infection to start. (Another relevant parameter is the loss of cooperative effectsbetween adjacent dimers.) The balance between lysogeny and the Iytic cycle depends on the concentration of
Is a Dimer Formof Repressor 14.10TheDNA-Binding
361
repressor.Intact repressoris present in a lysogenic cell at a concentration sufficient to ensure that the operators are occupied. If the repressor is cleaved, however, this concentration is inadequate, becauseof the lower affinity of the separateN-terminal domain for the operator. A concentration of repressor that is too high would make it impossible to induce the lytic cycle in this way; a level too low, of course, would make it impossible to maintain lysogeny.
Usesa HelixRepressor Turn-Helix Motifto BindDNA
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regionin the repressor contacts EachDNA-binding a half-site in the DNA. inctudes two TheDNA-binding siteof the repressor regions that fit into the successive shorta,-hetical turnsof the majorgrooveof DNA. pa[indromic A DNA-binding siteis a (partial.ty) s e q u e n coef 1 7b p .
: i : - - . . : i l : , .. . : . T h eo p e r a t o r i sLa7 - b p s e q u e nwci teha n axisof symmetry through thecentraI basepair.Eachha[fs'iteis marked in tightb[ue.Basepairsthat areidenticaI in eachoperator hatfarein darkbtue.
A repressordimer is the unit that binds to DNA. It recognizesa sequenceof I 7 bp displaying partial symmetry about an axis through the central base pair. tir**ft{ :'""}* shows an example of a binding site. The sequence on each side of the central basepair is sometimes called a "half site." Each individual N-terminal region contacts a half-site. Several DNA-binding proteins that regulate bacterial transcription share a similar mode of holding DNA, in which the active domain contains two short regions of o-helix that contact DNA. (Some transcription factors in eukaryotic cells use a similar motif; see Section25.I4, Homeodomains Bind RelatedThrgets in DNA.) The N-terminal domain of lambda repressor contains several stretchesof u-helix, which are arranged as illustrated diagrammatically in ri'"iit'il li:i.iii. Two of the helical regions are responsible for binding DNA. The helix-turnhelix model for contact is illustrated in ':+"fti. rr:r:t-iFl Looking at a single monomer, ohelix-3 consistsof nine amino acids, each of which lies at an angle to the preceding region of seven amino acids that forms u-helix-2. In the dimer, the two apposed helix-3 regions lie 34 A. apart, enabling them to fit into successive major grooves of DNA. The helix-2 regions lie at an angle that would place them across the groove. The symmetrical binding of dimer to the site means that each N-terminal domain of the dimer contacts a similar set of basesin its half -site.
N-terminal domain consistsof five s-helices
iii.i1-rrilii ;.i :ii Lambda repressor's N-terminaI domain 2 and3 bindDNA. contains fivestretches ofo-hetix;hetices
362
CHAPTER 14 PhageStrategies
i.i{ji"J fii ii.i"r., iii.i In thetwo-hetixmodelfor DNAbinding. hetix3 of eachmonomer liesin the widegrooveon the sameface of DNA,andhelix-Zliesacross the qroove.
TheRecognition Helix Determines Specificity for DNA . Theaminoacidsequence of the recognition helix makes contacts with particutar bases in the operator sequence that it recognizes. Related forms of the o,-helicalmotifs employed in the helix-turn-helix of the lambda repressor are found in several DNA-binding proteins, including cyclicAMP receptorprotein (CRp), rhe /acrepressor,and severalother phage repressors. By comparing the abilities of these proteins to bind DNA, we can define the roles of each helix: o Contactsbetween helix-l and DNA rely on hydrogen bonds between the amino acid side chains and the exposed positions of the base pairs. This helix is responsible for recognizing the specific target DNA sequence and is therefore also known as the recognition helix. . Contactsfrom helix-2 to the DNA take the form of hydrogen bonds connecting with the phosphate backbone.These interactions are necessaryfor binding, but do not control the specificityof target recognition. In addition to these contacts, a large part of the overall energy of interaction with DNA is provided by ionic interactions with the phosphate backbone. What happens if we manipulate the coding sequenceto construct a new protein by substituting the recognition helix in one repressor with the corresponding sequencefrom a closely related repressor?The specificity of the hybrid protein is that of its new recognition helix. The amino acid sequence of this short regiondetermines the sequence specificities of the individual proteins and is able to act in conjunctionwith the restof the nnlvnpntiip
. Amino acidsin helix-3 of the repressor make contacts with specificbasesin the operator. Three amino acids in repressor recognize three basesin DNA; the amino acidsat thesepositions,and also at additional positions in Cro, recognize five (or possibly six) basesin DNA. TWo of the amino acids involved in specific recognition are identical in repressor and Cro (Gln and Ser at the N-terminal end of the helix), whereas the other contacts are different (Ala in repressor versus Lys and the additional Asn in Cro). In addition, a Thr in helix-2 of Cro directly contactsDNA. The interactions shown in Figure 14.21 represent binding to the DNA sequencethat each protein recognizesmost tightly. The sequences shown at the bottom of the figure with the contact points in color differ at three of the nine basepairs. The use of overlapping, but not identical, contacts between amino acids and bases shows how related recognitioq helices confer r e c o g n i t i o n o f r e l a t e d D N A s e q u e n c e s .T h i s enablesrepressorand Cro to recognizethe same set of sequences,but with different relative affinities for particular members of the group. The basescontactedby helix-3 of repressor or Cro lie on one face of DNA, as can be seen from the positions indicated on the helical diag r a m i n F i g u r e 1 4 . 2 1. N o t e , h o w e v e r , t h a t repressor makes an additional contact with the other face of DNA. Removing the last six N-terminal amino acids (which protrude from
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i. , ,. r , shows the details of the binding to DNA of two proteins that bind similar DNA sequences.Both lambda repressorand Cro protein have a similar organization of the helixturn-helix motif, although their individual specificities for DNA are not identical: . Each protein uses similar interactions between hydrophobic amino acids to maintain the relationship between h e l i x - 2 a n d h e l i x - 3 : r e p r e s s o rh a s a n Ala-Val connection, whereas Cro has an Ala-Ile association.
: rr,itrrrr r r, , r Twoproteins thatusethetwo-hetix arrangementto contact operators withaffiniDNArecognize [ambda of heLix-3. tiesdetermjned bythe aminoacjdsequence
for DNA HelixDetermines Specificity L4.1.2Ihe Recognition
363
lIGuftt 14.2t A viewfromthe backshows that the bul.k o f t h e r e p r e s s ocro n t a c t so n e f a c e o f D N A ,b u t i t s to the otherface. N-terminal armsreacharound helix-l) eliminates some of the contacts.This observation provides the basis for the idea that the bulk of the N-terminal domain contacts one face of DNA, whereas the last six N-terminal amino acids form an " arm" extending around 14.22shows the view from the the back. FIGURE back. Lysine residues in the arm make contacts with G residues in the major groove and also with the phosphate backbone. The interaction between the arm and DNA contributes heavily to DNA binding; the affinity of the armless repressorfor DNA is reduced by -1000-fold. Bases that are not contacted directly by repressorprotein may have an important effect on binding. The related phage 434 repressor binds DNA via a helix-turn-helix motif, and the crystal structure shows that helix-3 is positioned at each half-site so that it contacts the five outermost basepairs, but not the inner two. Operators with A-T basepairs at the inner positions, however, bind434 repressor more strongly than operators with G-C base pairs. The reason is that 434 repressor binding slightly twists DNA at the center of the operator, which widens the angle between the two half -sitesof DNA by -3'. This is probably needed to allow each monomer of the repressordimer to make optimal contacts with DNA. A-T base pairs allow this twist more readily than G-C pairs, thus affecting the affinity of the operator for repressor.
Dimers Bind Repressor CooperativeLy to the 0perator Repressor bindingto oneoperator increases the affinityfor bindinga secondrepressor dimerto the adjacentoperator. Theaffinityis 10x greaterfor 0r1 and0*1than otheroperators, sotheyareboundfirst. Cooperativity to bindthe 07/02 attowsrepressor sitesat [owerconcentrations.
364
CHAPTER 14 Phage Strategies
Each operator contains three repressor-binding sites.As can be seen from FI6i",Rf14-33,no two of the six individual repressor-binding sites are identical, but they all conform with a consensus sequence.The binding siteswithin each operator are separatedby spacersof I to 7 bp that are rich in A-T basepairs. The sitesat each operator are numbered so that O* consistsof the seriesof binding sites O*1-O*2-OR3,whereas O, consists of the seriesOLI-OL2-OL3.In each case,site I lies closest to the startpoint for transcription in the promoter, and sites2 and3lie farther upstream. Faced with the triplication of binding sites at each operator, how does repressor decide where to start binding? At each operator, site I has a greater affinity (roughly tenfold) than the other sitesfor the repressor.Thus the repressor always binds first to Orl and o*I. siteswithin bindstosubsequent Lambda repressor manner. The preseachoperatorin a cooperative ence of a dimer at site I greatly increases the affinity with which a second dimer can bind to site 2. When both sites I and 2 are occupied, this interaction does not extend farther, to site 3. At the concentrations of repressor usually found in a lysogen, both sites I and2 are filled at each operator, but site 3 is not occupied. If site I is inactive (because of mutation), then repressorbinds cooperatively to sites2 and 3. That is, binding at site 2 assistsanother dimer to bind at site 3. This interaction occurs directly between repressor dimers and not via conformational change in DNA. The C-terminal domain is responsiblefor the cooperative interaction between dimers, as well as for the dimer 14-24shows formation between subunits. FISURS involves both subunits of each dimer, that it contacts its counterpart in that is, each subunit forming a tetrameric structure. the other dimer, A result of cooperative binding is to increase the effective affinity of repressor for the operator at physiological concentrations. This enables a lower concentration of repressor to achieve occupancy of the operator. This is an important consideration in a system in which release of repression has irreversible consequences.In an operon coding for metabolic enzymes, after all, failure of repression will merely allow unnecessary synthesis of enzymes. Failure to repress Iambda prophage, however, will lead to induction of phage and lysis of the cell. From the sequencesshown in Figure 14.23, we see that O.l and O^l lie more or less in the center of the RNA polymerase binding sites of P, and P*, respectively. Occupancy of.OrI-Or2 and O* I -O*2 thus physically blocks accessof RNA polymerase to the corresponding promoters.
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tor protein that is necessaryfor transcription of the clgene (seeSection 14.14,Repressorat Oo2 Interacts with RNA Polymerase at P**1 . The represslris the producl o/ cI; thus this interaction createsa positiveautogenouscircuit, in which the presenceof repressoris necessaryto support its own continuedsynthesis. The nature oI this control circuit explains the biological features of lysogenic existence. Lysogeny is stable becausethe control circuit ensuresthat as long as the level of repressoris adequate,there is continued expressionof the clgene. The result is that O. and O* remain occupied indefinitely. By repressing the entire lytic cascade,this action maintains the prophage in its inert form.
boundat 0r1 i:il..iii-ili i In the [ysogenic state,the repressors r,,.,:1 and0r2interactwiththoseboundat 0*1and0R2.RNApotymerase with the with 0*3) andinteracts is boundat P*, (whichovertaps repressor boundat 0*2.
Interactio ns Cooperative Increase the Sensitivity of ReguLation
or_1J+
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o Repressor dimersboundat 0r1and0r2interact with dimersboundat 0*1and0*2to form 0clamers. r Octamer formation brings0,3 ctoseto 0*3, interactions a[[owing between dimersboundthere. o These cooperative interactions increase the sensitivity of regutation.
C o o p e r a t i v ei n t e r a c t i o n s b e t w e e n r e p r e s s o r dimers occur at both the left and right operators, so that their normal condition when occupied by repressoris to have dimers at both the I and 2 binding sites.In effect, each operator has a tetramer of repressor.This is not the end of the story, though. The two dimers interact with one another to form an octamer. The interaction occurs between the C-terminal domains, which can form an octamer as a crystal structure. : ii:.i:!i. . r , , shows the distribution of repressorsat the operator sites that are occupied in a lysogen. Repressorsare occupying O.1, OL2,ORI,and O*2, and the repressorat the last of these sitesis interacting with RNA polymerase, which is initiating transcription at Pono. The interaction between the two operators has several consequences.It stabilizesrepressor binding, thereby making it possible for repressorto occupy operatorsat Iower concentrations. Binding at OR2stabilizesRNA polymerase binding at P*r, which enables low concentrations of repressor to autogenously stimulate their own production.
byformation into proximity lii;'iiii1 i;,.i.;i'r0r3and0*3arebrought concentration in repressor ind anincrease octamer, of the repressor to bindat thesesitesandto interact. attows dimers
The DNA between the O, and Oo sites (that is. the gene c1)forms alarge loop, which is held together by the repressoroctamer. The octamer brings the sites Or3 and O*3 into proximity. As a result, two repressordimers can bind to these sites and interact with one another, as shown in i:ii:l.iriii ; :,::.The occupationof O*3 prevents RNA polymerase from binding to P** and therefore turns off expressionof repressor. This shows us how the expression of the cI gene becomes exquisitely sensitive to repressor concentration. At the lowest concentrations, it forms the octamer and activates RNA polymerase in a positive autogenous regulation. An increasein concentration allows binding to Ot3 and O*3 and turns off transcription in a negative autogenousregulation. The threshold levels of repressor that are required for each of these events is reduced by the cooperativeinteractions,which makes the overall regulatory system much more sensitive. Any change in repressor level triggers the appropriate regulatory responseto restore the lysogeniclevel.
14.16 cooperativeInteractionsIncreasethe sensitivityof Regutation
367
The overall level of repressorhas been reduced (about threefold from the level that would be required if there were no cooperative effects),and as a result there is less repressor that has to be eliminated when it becomes necessary to induce the phage. This increases the efficiency of induction.
@
ThecII andcIII Genes AreNeeded to Establish Lysogeny
o Thedetayed earlygeneproducts cII andcIII are necessary for RNApotymerase to initiate transcription at the promoter P*r. o cII actsdirectat the promoter andcIII protects cII fromdegradation. o Transcription fromP*,[eadsto synthesis of repressor andatsoblocksthe transcription of cro.
The control circuit for maintaining lysogeny presents a paradox. Thepresence proof repressor tein is necessary for itsown synthesis.This explains how the lysogenic condition is perpetuated. How, though, is the synthesisof repressorestabIished in the first place? When a lambda DNA enters a new host cell, RNA polymerase cannot transcribe c1because there is no repressor present to aid its binding at P*r. This same absenceof repressor,however, means that P* and P, are available. Thus the first event after lambda DNA infects a bacterium is when genesNand crl are transcribed. After this, pN allows transcription to be extended
tT F\
Cll protein
FiGlJRt14.29 Repressor synthesis is estabHshed bytheaction of cII andRNApolymerase at P*,to jnitiatetransciiption that extends fromthe antisense strandof crothrouqhthec/ qene.
368
CHAPTER 14 Phage Strategies
farther. This allows cIII (and other genes) to be transcribed on the left, whereas cI1 (and other genes) are transcribed on the right (see Figure 14.14). The c1land cIII genesshare with clthe property that mutations in them causeclear plaques. There is, however, a difference. The clmutants can neither establish nor maintain lysogeny. The c11or cIII mutants have some difficulty in establishing lysogeny, but once it is established they are able to maintain it by the c1autogenous circuit. This implicates the cII andclllgenes as positive regulators whose products are needed for an alternative system for repressor synthesis. The system is needed only to initiatethe expression of clin order to circumvent the inability of the autogenous circuit to engagein denovo synthesis. They are not needed for continued expresslon. The cII protein acts directly on gene expression. Between the cro and c1Igenes is another promoter, called P*.. (The subscript "RE" stands for repressorestablishment.) This promoter can be recognized by RNA polymerase only in the presence of cII, whose action is illustrated in F I G U R1E4 - 2 9 . The cII protein is extremely unstable invivo, becauseit is degraded as the result of the activity of a host protein called HflA. The role of cIII is to protect cII against this degradation. Tlanscription from P*u promotes lysogeny in two ways. Its direct effect is that cI is transIated into repressorprotein. An indirect effect is that transcription proceeds through the to gene in the "wrong" direction. Thus the 5' part of the RNA corresponds to an antisense transcript of cro; in fact, it hybridizes to authentic cromRNA, which inhibits its translation. This is important because cro expression is needed to enter the lytic cycle (see S e c t i o n 1 4 . 2 0 , T h e c r o R e p r e s s o rI s N e e d e d for Lytic Infection). The cI coding region on the P*, transcript is very efficiently translated, in contrast with the weak translation of the P*, transcript. In fact, repressor is synthesized approximately seven to eight times more effectively via expression from P*. than from P*n,,.This reflects the fact that the P*u transcript has an efficient ribosome-binding site, whereas the P*, transcript has no ribosome-binding site and actually starts with the AUG initiation codon.
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We shall not at this point deal in detail with the other functions needed to establishlysogeny, but we can just briefly remark that the infecting lambda DNA must be inserted into the bacterial genome (see Section 19.I7, Specialized Recombination Involves Specific Sites). The insertion requires the product of gene int,which is expressedfrom its own promoter {, at which cII also is necessary.The sequence of P, shows homology with P*" in the cII binding site (although not in the -10 region). The functions necessaryfor establishing the lysogenic control circuit are therefore under the same control as the function needed to integrate the phage DNA into the bacterial genome. Thus the establishment of lysogeny is under a control that ensures all the necessaryevents occur with the same timing. Emphasizing the tricky quality of lambda's intricate cascade.we now know that cII promotes lysogeny in another, indirect manner. It sponsors transcription from a promoter called Pu,ti-q,which is located within the Q gene. This transcript is an antisenseversion of the Q region, and it hybridizes with Q mRNA to prevent translation of Q protein, whose synthesis is essential for lytic development. Thus the same mechanisms that directly promote lysogeny by causing transcription of the cI repressorgene also indirectly help lysogeny by inhibiting the expressionof cro (seeabove) and Q, the regulator genes needed for the antagonistic lytic nathwav.
ThecroRepressor for Lytic Is Needed Infection . Crobindsto the sameoperators but asrepressor. with differentaffinities. . WhenCrobindsto 003,it prevents RNApotymerase frombindingto P*.,, andblocksmaintenance of reDreSs0r. . WhenCrobindsto otheroperators at 0* or 0r, fromexpressing it prevents RNApotymerase bl.ocks immediate eartygenes, which(indirectty) reDressor estabtishment. Lambda has the altematives of entering lysogeny or starting a lytic infection. Lysogeny is initiated by establishing an autogenous maintenance circuit that inhibits the entire lytic cascade through applying pressure at two points. The program for establishing lysogeny proceeds
through some of the same events that are required for the lytic cascade (expression of delayed early genes via expression of N is needed). We now face a problem. How does the phage enter the lytic cycle? The key influence on the lytic cycle is the role of genecro,which codesfor another repressor. Cro is responsiblefor preventing the synthesisof protein;Ihis action shuts off the posthe repressor sibility of establishing lysogeny. cromutants usually establishlysogeny rather than entering the lytic pathway, because they lack the ability to switch events away from the expression of repressor. Cro forms a small dimer (the subunit is 9 kD) that acts within the immunity region.It has two effects: . It prevents the synthesisof repressorvia the maintenance circuit; that is, it prevents transcriPtion via P**. . It also inhibits the expression of early genes from both Pr and P*. This means that, when a phage enters the lytic pathway, Cro has responsibility both for preventing the synthesis of repressor and (subsequently) for turning down the expression of the early genes. Cro achieves its function by binding to the same operators as (cI) repressor protein' Cro includes a region with the same general structure as the repressor;a helix-2 is offset at an angle from recognition helix-1. (The remainder of the structure is different, which demonstrates that the helix-turn-helix motif can operate within various contexts.) As for repressor. Cro binds symmetrically at the operators. The sequencesof Cro and repressorin the helix-turn-helix region are related, which explains their ability to contact the same DNA sequences(seeFigure I4.2I\. Cro makes similar contacts to those made by repressor,but binds to only one face of DNA; it lacks the Nterminal arms by which repressor reaches around to the other side. How can two proteins have the same sites of action, yet have such opposite effects? The answer lies in the different affinities that each protein has for the individual binding sites within the operators. Let us just consider O*, for which more is known, and where Cro exerts both its effects.The seriesof events is illustrated x'i.3}. (Note that the first two stages in F3fr{Jsf, are identical to those of the lysogenic circuit shown in Figure 14.32.) The affinity of Cro for O*3 is greater than its affinity for O*2 or O*1. Thus it binds first to
for LyticInfection Is Needed 74.20Ihe croRepressor
377
N antiterminates; cll andclll aretranscribed
EXPRESSION cll Pe, Cto rePressesc/ " and all earlv
FIGU*E 14.33 Thelyticcascade requires Croprotein, prevents whichdirectty repressor maintenance viaP*r,aswetlasturningoff delayed earlygeneexpression, preventing indirectLy repressor estab[ishment. O*3. This inhibits RNA polymerase from binding to P"*. As a result, Cro's first action is to prevent the maintenance circuit for lysogeny from coming into play. Cro then binds to O*2 or O*1. Its affiniry for these sites is similar, and there is no coop-
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P*. This in turn stopsthe production of the early lunctions (including Cro itself). As a result of cII's instability, any use of p*, is brought to a
372
CHAPTER 14 PhageStrategies
halt. Thus the two actions of Cro together block a/i production of repressor. As far as the lytic cycle is concerned, Cro turns down (although it does not completely eliminate) the expression of the early genes.Its incomplete effect is explained by its affinity for O*l and O*2, which is about eight times lower than that of repressor. This effect of Cro does not occur until the early genes have become more or less superfluous, becausepe is present; by this time, the phage has started late gene
expression and is concentrating on the production of progeny phage particles.
WhatDetermines the Balance Between Lysogeny andthe LyticCycle? Thedelayed earlystagewhenbothCroand is common repressor arebeingexpressed to andthe lyticcycte. lysogeny ThecriticaIeventis whether cII causes sufficient of repressor to overcome the action synthesis of Cro.
The programs for the lysogenicand lytic pathways are so intimately related that it is impossible to predict the fate of an individual phage genome when it enters a new host bacterium. Will the antagonism between repressorand Cro b e r e s o l v e d b y e s t a b l i s h i n gt h e a u t o g e n o u s maintenance circuit shown in Figure 14.32,or by turning off repressor synthesis and entering the late stage of development shown in Figure 14.33? The same pathway is followed in both cases right up to the brink of decision. Both involve the expression of the immediate early genes and extension into the delayed early genes.The difference between them comes down to the question of whether repressoror Cro will obtain occupancy of the two operators. The early phase during which the decision is made is limited in duration in either case.No matter which pathway the phage follows, expressionof all early geneswill be prevented as P. and P* are repressedand, as a consequence of the disappearanceof cII and cIII, production of repressorvia P*u will cease. The critical question comes down to whether the cessationof transcription from P*. is followed by activation of P** and the establishment of lysogeny, or whether P** fails to become active and the pQ regulator commits :.'rr,{l the phage to lytic development. :rJ{rl"iiitrr" shows the critical stage, at which both repressor and Cro are being synthesized. The initial event in establishing lysogeny is the binding of repressorat OLI and OoI . Binding at the first sitesis rapidly succeededby cooperative binding of further repressordimers at O12 and O*2. This shuts off the synthesis of Cro and starts up the synthesis of repressorvia Po*.
andlysisis whendel'ayed lysogeny between i ir:i,ifiii:;.:r,rThecriticalstagein deciding l"ysogeny of repressor, synthesis sufficient If cII causes earlygenesarebeingexpressed. theoperators, Crooccupies Otherwise theoperators. occupies repressor wjttieiuttbecause in a lytic cycl.e. resutting The initial event in entering the lytic cycle is the binding of Cro at OR3.This stopsthe lysogenic-maintenance circuit from starting up at P*r. Cro must then bind to Ool or Oo2,and to O.l or Or2, to turn down early gene expression. By halting production of cII and CIII, this action leadsto the cessationof repressorsynthesis via P*u. The shutoff of repressor establishment occurs when the unstable cII and cIII proteins decay. The critical influence over the switch between lysogeny and lysis is cII. If cII is active, synthesisof repressorvia the establishmentpromoter is effective,and as a result, repressorgains occupancy of the operators.If cII is not active
and the LyticcycLe? the BalanceBetweenLysogeny 1.4.21.WhatDetermines
373
repressor establishment fails, and Cro binds to the operators. The level of cII protein under any particular set of circumstancesdetermines the outcome of an infection. Mutations that increasethe stability of cII increase the frequency of lysogenization. Such mutations occur in c1Iitself or in other genes.The causeof cIIs instability is its susceptibility to degradationby host proteases.Its level in the cell is influenced by cIII as well as by host functions. The effect of the lambda protein cIII is secondary: it helps to protect cII against degradation. The presence of cIII does not guarantee the survival of cII; however, in the absence of cIII, cII is virtually always inactivated. Host gene products act on this pathway. Mutations in the host genes hflA and hflB in crease ly so geny-hfl stand s f or high ft equ en cy /ysogenization. The mutations stabilize cII because they inactivate host protease(s) that degradeit. The influence of the host cell on the level of cII provides a route for the bacterium to interfere with the decision-takingprocess.For example, host proteasesthat degrade cII are activated by growth on rich medium. Thus lambda tends to lyse cells that are growing well, but is more Iikely to enter lysogeny on cells that are starving (and which lack components necessaryfor e f f i c i e n rl y t i c g r o w t h 1 .
Sum mary Phages have a lytic life cycle, in which infection of a host bacterium is followed by production of a large number of phage particles, Iysis of the cell, and release of the viruses. Some phages also can exist in lysogenic form, in which the phage genome is integrated into the bacterial chromosome and is inherited in this inert. latent form like any other bacterial gene. In general, lytic infection falls into three phases. In the first phase a small number of phage genes are transcribed by the host RNA polymerase. One or more of these genes is a regulator that controls expression of the group of genes expressedin the second phase. The pattern is repeated in the second phase, when one or more genes is a regulator needed for expressionof the genesof the third phase.Genes of the first two phasescode for enzymes needed to reproduce phage DNA; genes of the final phase code for structural components of the phage particle. It is common for the very early genes to be turned off during the later phases.
374
CHAPTER 14 PhageStrategies
In phage lambda, the genes are organized into groups whose expression is controlled by individual regulatory events. The immediate early gene N codes for an antiterminator that allows transcription of the leftward and rightward groups of delayed early genes from the early promoters PR and Pr. The delayed early gene Q has a similar antitermination function that allows transcription of all late genes from the promoter P*,. The lytic cycle is repressed, and the lysogenic state maintained, by expression of the cI gene, whose product is a repressor protein that acts at the operators O* and O, to prevent use o{ the promoters P* and P, respectively. A lysogenic phage genome expressesonly the cI gene from its promoter, P*no.Transcription from this promoter involves positive autogenous regulation, in which repressor bound at O* activates RNA polymerase at p . RM.
Each operator consistsof three binding sites for repressor.Each site is palindromic, consisting of symmetrical half-sites.Repressorfunctions as a dimer. Each half binding site is contacted by a repressor monomer. The N-terminal domain of repressor contains a helixturn-helix motif that contacts DNA. Helix-3 is the recognition helix and responsible for making specificcontacts with basepairs in the operator. Helix-2 is involved in positioning helix-3; it is also involved in contacting RNA polymerase at P*r. The C-terminal domain is required for dimerization. Induction is caused by cleavage between the N- and C-terminal domains, which prevents the DNA-binding regions from functioning in dimeric form, thereby reducing their affinity for DNA and making it impossible to maintain lysogeny. Repressor-operatorbinding is cooperative, so that once one dimer has bound to the first site, a seconddimer binds more readily to the adjacent site. The helix-turn-helix motif is used by other DNA-binding proteins, including lambda Cro. Lambda Cro binds to the same operators but has a different affinity for the individual operator sites,which are determined by the sequence of helix-3. Cro binds individually ro operator sites, starting with O*3, in a noncooperative manner. It is needed for progression through the lytic cycle. Its binding to O*3 first prevents synthesis of repressor from P*r, and then its binding Io O^2 and O*I prevents continued expression of early genes, an effect also seen in its binding to O.l and Or2. Establishment of repressor synthesis requires use of the promoter P*", which is acti-
vated by the product of the c11gene. The product of c111isrequired to stabilize the cll product against degradation. By turning off cI1and c111 expression, Cro acts to prevent lysogeny. By turning off all transcription except that of its own gene, repressoracts to prevent the lytic cycle. The choice between lysis and lysogeny depends on whether repressor or Cro gains occupancy of the operators in a particular infection. The stability of cII protein in the infected cell is a primarv determinant of the outcome.
References TwoTypes EventControl of Regutatory the LyticCascade Review Greenblatt,J., Nodwell,J. R., and Mason,S.W. (l 99J). Transcriptionalantitermination. Nature)64, 40),-406.
Immediate Earty andDetayed Lambda Needed for Both EartyGenes Are Lysogeny andthe LyticCycte Review Ptashne, M. (2004). The GeneticSwitch:Phage Lambda Revisited.Cold Spring Harbor, NY: Cold Spring Harbor Press
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Lysogeny Is Maintained by Repressor Protein
Motif Repressor Usesa Helix-Turn-Hetix to BindDNA rch Resea Sauer,R. T. et al. ( 1982) . Homology among DNAbinding proteins suggestsuse of a conserved super-secondarystructure. N ature29 8, 44745 1.
HetixDetermines TheRecognition Specificity for DNA Research Brennan, R. G et al. ( I 990). Protein-DNA conformational changes in the crystal structure of a lambda Cro-operator complex. Proc Natl' Acad. Sci USA 87. 8165-8169. Wharton. R. L , Brown, E. L., and Ptashne, M. (1984). Substitutingan cx-helixswitchesthe s e q u e n c es p e c i f i cD N A i n t e r a c t i o n so I a repressor.Cell)8, 361-)69.
BindCooperativety Dimers Repressor to the 0perator rch Resea Bell, C. E., Frescura,P.,Hochschild,A., and Lewis, M. (2000). Crystal structure of the lambda repressor C-terminal domain provides a model for cooperative operator binding. Cell I 0 l , 8 0 1 - 8 1I . Johnson, A. D., Meyer, B. J., and Ptashne,M. (1979). Interactionsbetween DNA-bound repressorsgovern regulation by the phage lambda repressor.Proc.Natl. Acad. Sci USA 76' 5061-5065.
with RNA at 0*2Interacts Repressor at P*t Potymerase
rch Resea Pirrotta, V., Chadwick, P.,and Ptashne,M. (1970). Active form of two coliphage repressors. Nature227, 4l-44. Ptashne, M. ( I 967 ) . Isolation of the lambda phage repressor.Proc Natl. Acad. Sci USA 57, 306-)l). Ptashne,M. (1967). Specificbinding of the lambda phage repressor to lambda DNA. Nature 214, 2j2-234.
Define TheRepressor andIts 0perators t h e I m m u n i t yR e g i o n Review M. (I982). Friedman,D. I. and Gottesman, Lambda11.Cambridge,MA: Cell Press. @
T h eD N A - B i n d i nFgo r mo f R e p r e s s o r Is a Dimer
R e s e ahr c Pabo,C. O. and Lewis,M (1982).The operatorbinding domain of lambdarepressor:structure and DNA recognition.Nature298, 44)-447.
rch Resea Hochschild,A., Irwin, N., and Ptashne,M. (1983). Repressor structure and the mechanism of positive control. cell J2, 319-325. Li, M., Moyle, H, and Susskind,M' M. (1994). Target of the transcriptional activation function of 26),75-77. phage lambda cI protein. Science
the Increase Interactions Cooperative of Regutation Sensitivity Review Ptashne, M. (2004). The GeneticSwitch:Phage Lambda Revisited.Cold Spring Harbor, NY: Cold Spring Harbor Press. Research Bell, C. E. and Lewis, M. (2001). Crystal structure of the lambda repressor C-terminal domain o c t a m e r .J M o l . B i o l . 3 l 4 , 1 1 2 7 - 1 1 3 6 . Dodd, I. B., Perkins,A. J., Tsemitsidis,D., and Egan, J. B. (200I). Octamerizationof lambda CI repressor is needed lor effective repression of P(RM) and efficient switching from lysogeny.GenesDev.| 5, )Ol3-3022'
References 375
TheRepticon 1
C H A P T EO RU T L I N E
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Repticons CanBe Linearor Circutar r A replicated regionappears asan eyewithinnonreplicated DNA. r A replication forkis initjatedat the originandthenmoves sequentiatly atongDNA. . Replication is unidjrectional whena singtereptication fork is created at an origin. . Reptication is bidirectjonal whenan origincreates two repticationforksthat movein opposite directjons. OriginsCanBe Mappedby Autoradiography and Etectrophoresis . Reptication forkmovement canbedetected by autoradiographyusingradioactive putses. . Replication forkscreate Y-shaped structures that change the electrophoretic migration of DNAfragments. DoesMethylationat the OriginRegulateInitiation? carr o onCcontains r'rteven repeats that aremethylated ;iA; on adenine on bothstrands. r Replication generates hemimethylated DNA,whichcannot initiatereotication. . There is a 13-minute detavbeforethe a'e fflf ,eneats remethvtated. 0riginsMayBe Sequestered after Reptication . SeqAbindsto hemimethylated DNAandis required for detaying rerep[ication. . SeqAmayinteractwith DnaA. r Asthe originsarehemimethytated theybindto the cetl membrane andmaybe unavaitable to methytases. o Thenatureof the connection between the originandthe membrane is sti[[unctear. EachEukaryotic Chromosome ContainsManyRepticons . Eukaryotic replicons are40 to 100kb in tength. e A chromosome js dividedinto manyrepticons. o IndividuaI repticons areactivated at characteristic times during5 phase. o RegionaI activation patterns suggest that repticons nearone another areactivated at the sametime.
376
Replication0riginsCanBe Isolatedin Yeast . Originsin S. cerevisiae areshortA-T-richsequences that havean essentia[ 11-bpsequence. e TheORC is a comptex of sixproteins that bindsto anARS. LicensingFactorControtsEukaryotic Rereptication r Licensing factoris necessary for initiationof reptication at eachorigin. r It is present priorto reptication. in the nucteus butis inactivatedor destroyed by replication. o Initiationof another reptication possibte cyclebecomes ontyafterlicensing factorreenters the nucteus after mitosis. LicensingFactorConsists of MCMProteins . TheORC is a proteincomplex that is associated withyeast originsthroughout the cetlcycle. e Cdc6proteinis an unstabte proteinthat is synthesized on[y inG1. e Cdc6bindsto ORC anda[tows MCMproteins to bind. o Whenreptication is initiated.Cdc6andMCMproteins are disptaced. Thedegradation of Cdc6prevents reinitiation. o SomeMCMproteins arein the nucteus throughout the cycte, but othersmayenterontyaftermitosis. D LoopsMaintainMitochondria[ Origins o Mitochondria usedifferent originsequences to initiate reptication of eachDNAstrand. o Reptication ofthe H strandis initiatedin a D [oop. r Replication of the L strandis initjatedwhenits originis exposed by the movement of the first reptication fork. Summary
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f l G # f t *t * . 3 T h e5 ' t e r m i n apIh o s p h aat et e a c he n do f in the55kD to serine tinked DNAis covalenttv adenovirus protein. Ad-binding
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DNAreplication is initiatedsep$ISUi{il1S,f Adenovirus by andproceeds aratelyat the two endsof the motecute stranddisp[acement. adenovirus DNA. This suggeststhe model illust r a t e d i n F t * L l f t *1 * . d ; .T h e c o m p l e x o f p o l y merase and terminal protein, bearing the priming C nucleotide, binds to the end of the adenovirus DNA. The free 3'-OH end of the C nucleotide is used to prime the elongation reaction by the DNA polymerase.This generatesa new strand whose 5' end is covalently linked to the initiating C nucleotide. (The reaction actually involves displacement of protein from DNA rather than binding denovo.The 5' end of adenovirus DNA is bound to the terminal protein that was used in the previous replication cycle. The old terminal protein is displaced by the new terminal protein for each new replication cycle.) Terminal protein binds to the region located between 9 and l8 bp from the end of the DNA. The adjacentregion, between positions l7 and 48, is essentialfor the binding of a host protein, nuclear factor I, which is also required for the
proteinbindsto the5' terminaI $;Stlftili*"si Adenovirus endto primesynthesis a C-OH endof DNAandprovides of a newDNAstrand.
initiation reaction. The initiation complex may therefore form between positions 9 and 48, a fixed distance from the actual end of the DNA.
Initiationat the Endsof ViratDNAs Enab[e Proteins 16.3TerminaI
RolLing Circles Produce Multimers of a Replicon
@
. A rotlingc'irctegenerates singte-stranded muttimers of the originaI sequence.
The structures generated by replication depend on the relationship between the template and the replication fork. The critical features are whether the template is circular or linear, and whether the replication fork is engagedin synthesizingboth strandsof DNA or only one. Replication of only one strand is used to generate copies of some circular molecules.A nick opens one strand, and then the free 3'-OH end generated by the nick is extended by the DNA polymerase.The newly synthesizedstrand
Templateis circularduplexDNA
Initiationoccurson one strand
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displacesthe original parental strand. The ensuing events are depicted in i*3fi["1ft{ t$.*. This type of structure is called a rolling circle, becausethe growing point can be envisaged as rolling around the circular template strand. It could in principle continue to do so indefinitely. As it moves, the replication fork extends the outer strand and displacesthe previous partner. An example is shown in the electron micrograph of Fl$;#Ril i$"f'. The newly synthesized material is covalently linked to the original material, and as a result the displaced strand has the original unit genome at its 5' end. The original unit is followed by any number of unit genomes, synthesized by continuing revolutions of the template. Each revolution displacesthe material synthesized in the previous cycle. The rolling circle is put to several uses in vivo. Some pathways that are used to replicate DNA are depicted in Filii.jH& tii.l. Cleavageof a unit length tail generatesa copy of the original circular replicon in linear form. The linear form may be maintained as a single strand, or may be converted into a duplex by synthesis of the complementary strand (which is identical in sequence to the template strand of the original rolling circle). The rolling circle provides a means for amplifying the original (unit) replicon. This mechanism is used to generate amplified ribosomal DNA (rDNA) inthe Xenopzsoocyte. The genesfor ribosomal RNA (rRNA) are organized as a large number of contiguous repeats in the genome. A single repeating unit from the genome is converted into a rolling circle. The displaced tail, which contains many units, is converted into duplex DNA; later it is cleaved from
Displacedstrand After one revolutiondisplacedstrand reachesunit length
Continuedelongationgenerates displacedstrandof multipleunit
i:ii:t"iHI"tj.."tTherottingcirclegenerates a multjmeric single-stranded tait.
396
C H A P T E1R6 E x t r a c h r o m o s o m RaeIp t i c o n s
l:.1{;iJFI :$"* A rottingcircleappears asa circutar mo[ecu[ewitha [ineartaiIbyelectron microscopy. Courtesy of Ross B.Inman,Instituteof Molecutar Virotogy, BockLaboratoryand Department of Biochemistry, University of Wisconsin. Madison. Wisconsin, USA.
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nation at relatively high frequencies (compared to strains that lack integrated F factors); such strains are describedas Hfr (for high frequency recombination). Each position of integration for the F factor gives rise to a different Hfr strain, with a characteristicpattern of transferring bacterial markers to a recipient chromosome. Contact between conjugating bacteria is usually broken before transfer of DNA is complete. As a result, the probability that a region of the bacterial chromosome will be transferred depends upon its distance from oriT. Bacterial genes located close to the site of F integration (in the direction of transfer) enter recipient bacteria first, and are therefore found at greater frequencies than those that are located farther away and enter later. This gives rise to a gradient of transfer frequencies around the chromosome, declining from the position of F integration. Marker positionson the donor chromosome can be assayedin terms of the time at which transfer occurs; this gave rise to the standard description of the E. coli chromosome as a map divided into 100 minutes. The map refers to transfer times from a particular Hfr strain; the starting point for the gradient of transfer is different for each Hfr strain becauseit is determined by the site where the F factor has integrated into the bacterial genome.
RECIPIENTBACTERIUM
DONORBACTERIUM F FACTOR fra region
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Singiestrandsare convertedto doublestrandsin both bacteria
whenaninteDNAoccurs Sl{i;*itil1*.i.i Transfer of chromosomal TheBacterial Ti Plasmid grated of DNAstartswitha short Ffactoris njcked at onJiTransfer by lossof conuntilprevented of F DNAandcontjnues Causes Crown Ga[[Disease sequence tact between the bacteria. in PLants o Infectionwith the bacterium A. tumefociens can ptantcetlsintotumors. transform o Theinfectious agentis a ptasmid carried by the bacterium. o Theplasmid genes atsocarries for synthesizing andmetabotizing opines(arginine derivatives) that areusedbythe tumorcett.
Most events in which DNA is rearranged or amplified occur within a genome, but the interaction between bacteria and certain plants involves the transfer of DNA from the bacterial genome to the plant genome. Crown gall disease, shown in trI*i.JR{ 1$.'i3,can be induced in most dicotyledonous plants by the soil bacIerium Agr obacteriumtumefaciens.The bacterium is a parasite that effects a genetic change in the eukaryotic host cell, with consequenceslor both parasite and host: It improves conditions for
survival of the parasite and causesthe plant cell to grow as a tumor. Agrobacteria are required to induce tumor formation, but the tumor cellsdo not require the continued presence of bacteria. As with animal tumors, the plant cells have been transformed into a state in which new mechanisms govern growth and differentiation. Tlansformation is caused by the expression within the plant cell of genetic information transferred from the bacterium. The tumor-inducing principle of.Agrobacterium resides in the fi plasmid, which is perpetuated as an independent replicon within the bacterium. The plasmid carries genes involved in various bacterial and plant cell activities, including those required to generate the transformed state, and a set of genesconcerned with synthesis or utilization of opines (novel derivatives of arginine).
in P[ants CrownGa[[Disease 16.8 The BacteriaITi PtasmidCauses
401
ir:;.;i;iiLi r.1 .j AnAgrobacteium carryinga Ti plasmidof jn whjchdjfferenthe nopatine typeinduces a teratoma, t'iated structures devetop. Photocourtesy of theestateof JeffSchett. Usedwithoermission Instiof theMaxP[anck tute for PtantBreeding Research, Cologne.
Locus
Function
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all all all
nos noc ocs occ
nopalinesynthesis nopalinecatabolism octopinesynthesis octopinecatabolism
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all all all
i-ii:l-:{i r i=-.:.i; Ti ptasmids carrygenes involved in both p[antandbacterial functions. Ti plasmids (and thus tlire Agrobacteriain which they reside) can be divided into four groups, according to the types of opine that are made: . Nopaline plasmids carry genesfor synthesizing nopaline in tumors and for utilizing it in bacteria. Nopaline tumors can differentiate into shootswith abnormal structures. They have been called teratomas by analogy with certain mammalian tumors that retain the ability to differentiate into early embryonic structures. . Octopine plasmids are similar to nopaline plasmids, but the relevant opine is different. Octopine tumors are usually
402
C H A P T E1R. 6 E x t r a c h r o m o s o m Raelo t i c o n s
undifferentiated, however, and do not form teratoma shoots. . Agropine plasmids carry genes for agropine metabolism; the tumors do not differentiate, and they develop poorly and die early. . Ri plasmids can induce hairy root diseaseon some plants and crown gall on others. They have agropine type genes, and may have segments derived from both nopaline and octopine plasmids. The types of genes carried by a Ti plasmid are summarized in fl'i*lifit t*.i'i. Genesutilized in the bacterium code for plasmid replication and incompatibility, transfer between bacteria, sensitivity to phages, and synthesis of other compounds, some of which are toxic to other soil bacteria. Genes used in the plant cell code for transfer of DNA into the plant, induction of the transformed state, and shoot and root induction. The specificity of the opine genes depends on the type of plasmid. Genesneeded for opine synthesis are linked to genes whose products catabolize the same opine; thus each strain of Agrobacteriumcausescrown gall tumor cells to synthesize opines that are useful for survival of the parasite.The opines can be used as the sole carbon and/or nitrogen source for the inducing Agrobacterium strain The principle is that the transformed plant cell synthesizes those o p i n e st h a t t h e b a c t e r i u m c a n u s e .
T-DNA Carries Genes for Infection Required . Partof the DNAof the Ti plasmid is transferred to the olantce[[nucleus. r Thevrrgenesof the Ti ptasmid arelocatedoutside the transferred regionandarerequired for the transferprocess. o Thevirgenesareinduced by phenotic compounds reteased by plantsin response to wounding. o Themembrane proteinVirAis autophosphorytated on histidine whenit bindsan inducer. r VirAactivates VirGby transferring the phosphate groupto it. r TheVirA-VirG is oneof several bacterialtwo component systems that usea phosphohistidine reray.
The interaction between Agrobacterium and a plant cell is illustrated in E{#liSf;i*"1h. The bacterium does not enter the plant cell. but rather transfers part of the Ti plasmid to the plant
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d a u g h t e r c h r o m o s o m e s t o s e p a r a t e ;e a c h o f them has already been partially replicated by the new replication forks (which now are the only replication forks). Theseforks continue to advance. At the point of division, the two partially replicated chromosomessegregate.This recreates the point at which we started. The single replication fork becomes"old," it terminates at l5 minutes, and 20 minutes later there is a division. We seethat the initiation event occurs l,Zs cell cyclesbefore the division event with which it is associated. The general principle of the link between initiation and the cell cycle is that, as cells grow more rapidly (the cycle is shorter), the initiarion event occurs an increasing number of cycles before the related division. There are correspondingly more chromosomes in the individual bacterium. This relationship can be viewed as the cell's response to its inability to reduce the periods of C and D to keep pace with the shorter cycle.
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TheSeptum Divides a Bacterium into Progeny ThatEachContain a Chromosome
formation Septum is initiatedat the annu[us, whichis a ringaroundthe cetlwherethe structure of the envelope is a[tered. Newannutiareinitiatedat 50%of the distance fromthe septum to eachendofthe bacterium. Whenthe bacterium divjdes, eachdaughter hasan position. annulus at the mid-center Septation startswhenthe ce[[reaches a fixed tength. Theseptumconsjsts of thesamepeptidogLycans that comprise the bacterjaI envelope.
Chromosome segregationin bacteria is especially interesting because the DNA itself is involved in the mechanism for partition. (This contrasts with eukaryotic cells, in which segregation is achievedby the complex apparatusof mitosis.) The bacterial apparatusis quite accurate; however, anucleate cellsform <0.03% of a b a c t e r i a lp o p u l a t i o n . The division of a bacterium into two daughter cells is accomplishedby the formation of a septum, a structure that forms in the center of the cell as an invagination from the surrounding envelope. The septum forms an impenetra-
ble barrier between the two parts of the cell and provides the site at which the two daughter cells eventually separateentirely. TWo related questions addressthe role of the septum in division: What determines the location at which it forms, and what ensures that the daughter chromosomeslie on opposite sidesof it? The formation of the septum is preceded by the organization of the periseptal annulus. This is observed as a zone in E. coli or Salmonellatyphimurium, for which the structure of the envelope is altered so that the inner membrane is connected more closely to the cell wall and outer membrane layer. As its name suggests,the annulus extendsaround the cell. i'ir.ilirii r i' summarizesits development. The annulus starts at a central position in a new cell. As the cell grows, two events occur: A septum forms at the mid-cell position defined by the annulus, and new annuli form on either side of the initial annulus. These new annuli are displacedfrom the center and move along the cell to positions at one quarter and three quarters of the cell length. These will become the mid-cell positions after the next division. The displacement of the periseptal annulus to
I I I I I I I I I I I I I I I I I t I
Cell startswith annulusat midcenter New annuliare generated New annulimove in polardirection New annulistop movemenl Centralannulus developsinto seplum
Side view showsthat annulusextendsaround circumlerence of cell Cross-section shows thatannulus connectsmembranes
perisepi::i::rtl dispLacementofthe l t i r Duplicationand giveriseto the formation of a septumthat tal annutus divides the cetl..
ThatEachContain a Chromosome4tt into Proqenv 17.3TheSentum Divides a Bacterium
Originsof replicating chromosomes to membrane attached Daughter chromosomes attachedto envelope
Septumgrows between chromosomes
Septumdividescell
Chromosomes distributedto daughtercells
': ..- Attachment i:i.:i:i:::l of bacteriaI DNAto the membranecouldprovide a mechanism for segregation.
the correct position may be the crucial event that ensures the division of the cell into daughters of equal size. (The mechanism of movement is unknown.) Septationbeginswhen the cell reachesa fixed length (2L), and the distance between the new annuli is always L. We do not know how the cell measures length, but the relevant parameter appearsto be linear distance as such (not area or volume). The septum consistsof the same components as the cell envelope:There is a rigid layer of peptidoglycan in the periplasm, between the inner and outer membranes. The peptidoglycan is made by polymerization of tri- or pentapeptide-disaccharideunits in a reaction involving connections between both types of subunit (transpeptidation and transglycosylation). The rodlike shape of the bacterium is maintained by a pair of activities, PBP2 and RodA. They are interacting proteins and are coded by the same operon. RodA is a member of the SEDSfamily (SEDSstandsfor shape,elongation, division, and sporulation) that is present in all bacteria that have a peptidoglycan cell wall. Each SEDSprotein functions together with a specifictranspeptidase,which catalyzesthe formation of the crosslinksin the peptidoglycan. PBP2 (penicillin-binding protein 2) is the transpeptidasethat interacts with RodA. Mutations in the gene for either protein cause the bacterium to Iose its extended shape and become round. This demonstrates the important principle that shape and rigidity can be determined
472
CHAPTER 17 BacterialReptication Is Connected to the Ce[[Cycte
by the simple extension of a polymeric structure. Another enzyme is responsible for generating the peptidoglycan in the septum (see Section 17.5,FtsZIsNecessaryfor Septum Formation). The septum initially forms as a double layer of peptidoglycan, and the protein EnvA is required to split the covalent links between the layers so that the daughter cells may separate. The behavior of the periseptal annulus suggeststhat the mechanism for measuring position is associatedwith the cell envelope. It is plausible to suppose that the envelope could also be used to ensure segregationof the chromosomes. A direct link between DNA and the membrane could account for segregation. If daughter chromosomes are attached to the membrane, they could be physically separated shows that when the septum forms. Fl+#F{1}"s+ the formation of a septum could segregatethe chromosomes into the different daughter cells if the origins are connected to sites that lie on either side of the periseptal annulus.
Mutations in Division AfFect or Segregation Ce[[Shape o jfs mutantsformlongfitamentsbecause the failsto formto dividethe daughter septum bacteria. o Minicelts formin mutants that produce too many septa;theyaresma[[andLackDNA. o Anucteate cettsof normalsizearegenerated by partitionmutants in whichthe dupticate faiIto seoarate. chromosomes
A difficulty in isolating mutants that affect cell division is that mutations in the critical functions may be lethal and/or pleiotropic. For example, if formation of the annulus occurs at a site that is essential for overall growth of the envelope, it would be difficult to distinguish mutations that specifically interfere with annulus formation from those that inhibit envelope growth generally. Most mutations in the division apparatus have been identified as conditional mutants (whose division is affected under nonpermissive conditions; typically they are temperature sensitive). Mutations that affect cell division or chromosome segregation cause striking phenotypic changes. Ftr{itlfito" l;.* and It"$ul{F. i,r"ii illustrate the opposite consequences of failure in the division Drocessand failure in segregation:
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Cellcycleof one plasmid
plasmids Cellcycleof incompatible
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I Cellgrowsand Y plasmidreplicates Loss of plasmid leaveskillerand antidote
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Celldivides
Cellgrowsbut r olasmidsdo not I replicatebecause two originsare alreadyPresent
T J Celldivides V
plasmidshavebeen Eachcellhas copyo{ sameplasmid lncompatible to ditferenlceils drslributed
(theybetongto the same areincompatib[e ;:iirijiii: r.' i.: Twoptasmjds at the stage group)if theiroriginscannotbe distinguished compatibility of initiation.ThesamemodeIcouldapptyto segregation.
li:..r.iiri: .;.::" Ptasmids mayensure that bacteria cannot livewithoutthemby synthesizing kitter a [ong-tived anda short-lived antidote.
P[asmid IncompatibiLity Is Determined bythe Replicon . Plasmids grouphave in a singtecompatibility originsthat areregulated by a common control system. The phenomenon of plasmid incompatibility is related to the regulation of plasmid copy n u m b e r a n d s e g r e g a t i o n .A c o m p a t i b i l i t y group is defined as a set of plasmids whose members are unable to coexist in the same b a c t e r i a lc e l l . T h e r e a s o nf o r t h e i r i n c o m p a t ibility is that they cannot be distinguished from one another at some stagethat is essential for plasmid maintenance. DNA replication and segregation are stagesat which this may apply. The negative control model for plasmid incompatibility follows the idea that copy number control is achieved by synthesizing a repres-
sor that measuresthe concentration of origins. (Formally, this is the same as the titration model for regulating replication of the bacterial chromosome.) The introduction of a new origin in the form of a secondplasmid of the same compatibility group mimics the result of replication of the resident plasmid; two origins now are present. Thus any further replication is prevented until after the two plasmids have been segregatedto different cells to create the correct prereplication copy number, as illustrated i n i : . 1 1 1 . 1 1I! i:i .- ,i : .
A similar effect would be produced if the system for segregatingthe products to daughter cellscould not distinguish between two plasmids. For example, if two plasmids have the same cis-acting partition sites, competition between them would ensure that they would be segregatedto different cells, and therefore could not survive in the same line. The presence of a member of one compatibility group does not directly affect the survival of a plasmid belonging to a different group. Only one replicon of a given compatibility group (of a single-copy plasmid) can be maintained in the bacterium, but it does not interact with replicons of other compatibility groups.
bythe Replicon Is Determined Incompatibitity 17.10Ptasmid
@
TheCoLEI Compatibility System Is ControL[ed by an RNARegulator
. Reptication of ColEl. requires transcription to pass throughthe origin,wherethetranscript is cteaved by RNAase H to generate a primerend. r Theregutator RNAI is a shortantisense RNAthat pairswiththetranscript andprevents the cteavage that generates the primingend. o TheRomproteinenhances pairingbetween RNAI andthetranscriot.
The best characterizedcopy number and incompatibility systemis that of the plasmid ColEl, a multicopy plasmid that is maintained at a steady Ievel of -20 copies per E. coli cell.The system for maintaining the copy number depends on the mechanism for initiating replication at the ColEl origin, as illustrated in ilil*;:l=li: I l;.ii*. Replication starts with the transcription of an RNA that initiates 555 bp upstream of the origin. Transcription continues through the origin. The enzyme RNAase H (whose name reflects its specificity for a substrate of RNA
f:gi#i.Jfii: :t'i'.1i}Repl"ication of Co[E1 DNAis initiatedby cteaving the primerRNAto generate a 3'-0H end.The primerformsa persistent hybridin the originregion.
422
CHAPTER 17 BacteriaIRepticationIs Connected to the Ce[[Cycte
hybridized with DNA) cleavesthe transcript at the origin. This generates a l'-OH end that is used as the "primer" at which DNA synthesis is initiated (the use of primers is discussedin more detail in Section 18.8, Priming Is Required to Start DNA Synthesis).The primer RNA forms a persistent hybrid with the DNA. Pairing between the RNA and DNA occurs just upstream of the origin (around position -20) and also farther upstream (around position -265). TWo regulatory systems exert their effects on the RNA primer. One involves synthesis of an RNA complementary to the primer; the other involves a protein coded by a nearby locus. The regulatory speciesRNA I is a molecule of -108 basesand is codedby the oppositestrand from that specifying primer RNA. The relationship between the primer RNA and RNA I is illustrated in l3Gtifril1.:.:$. The RNA I molecule is initiated within the primer region and terminates close to the site where the primer RNA initiates. Thus RNA I is complementary to the 5'-terminal region of the primer RNA. Base pairing between the two RNAs controls the availability of the primer RNA to initiate a cycle of replication. An RNA molecule such as RNA I that functions by virtue of its complementarity with another RNA coded in the same region is called a countertranscript. This type of mechanism, of course, is another example of the use of antisenseRNA (seeSection 13.7, Small RNA MoleculesCan RegulateTranslation). Mutations that reduce or eliminate incompatibility between plasmids can be obtained by selecting plasmids of the same group for their ability to coexist. Incompatibility mutations in ColEl map in the region of overlap between RNA I and primer RNA. This region is represented in two different RNAs, so either or both might be involved in the effect. When RNA I is added to a system for replicating ColEl DNA invitro, it inhibirs the formation of active primer RNA. The presence of RNA I, however, does not inhibit the initiati<,rn
F3#f-jgqfl 1?,J$ Thesequence of RNAI is comp[ementary to the 5' regionof primerRNA.
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synthesisof one Okazaki fragment is completed, synthesis of the next Okazaki fragment is required to start at a new location approximately in the vicinity of the growing point for the leading strand. This requires a translocation relative to the DNA of the enzyme unit that is synthesizing the lagging strand. The term "e\zyme unit" avoids the issue of whether the DNA polymerase that synthesizes the leading strand is the same type of enzyme as the DNA polymerase that synthesizesthe lagging strand. In the case that we know best, E. coli, there is only a single type of DNA polymerase catalytic subunit used in replication. the DnaE protein. The active replicase is a dimer, and each half of the dimer contains DnaE as the catalytic subunit. The DnaE is supported by other proteins (which differ between the leading and lagging strands). The use of a single type of catalytic subunit, however, may be atypical. In the bacterium Bacillussubtills,there are two different catalytic subunits. PolC is the homolog Io E. coli'sDnaE, and is responsible for synthesizing the leading strand. A related protein, DnaEss. is the catalltic subunit that synthesizesthe lagging strand. Eukaryotic DNA polymerases have the same general structure, with different enzyme units synthesizing the leading and lagging strands, but it is not clear whether the same or different FtSUftfi8.18 Eachcatalytic coreof Po[III synthesizes types of catalytic subunits are used (see Seca daughter strand.DnaB is responsibte forforward movetion 18.13, separate Eukaryotic DNA polymentat the reptication fork. merases Undertake Initiation and Elongation). A major problem of the semidiscontinuous mode of replication follows from the use of different enzyme units to synthesize each new DNA strand: How is synthesis of the lagging strand coordinated with synthesis of the leading strand? As the replisome moves along DNA, r Differentenzymeunitsarerequired to synthesize unwinding the parental strands, one enzyme the [eading andlaggingstrands. unit elongatesthe leading strand. Periodically t ln E.coLiboththeseunitscontainthe same primosome the activity initiates an Okazaki catatytic subunit(DnaE). fragment on the lagging strand, and the other o In otherorganisms, differentcatatytic subunits enzyme unit must then move in the reverse maybe required for eachstrand. direction to synthesizeDNA. FIG{JRf, ?S.*i.proposestwo types of model for what happens to Each new DNA strand is synthesizedby an indithis enzyme unit when it completes synthesis vidual catalytic unit. fIG#Rf ts.I# shows thar of an Okazaki fragment. The same complex the behavior of these two units is different may be reutilized for synthesis of successive becausethe new DNA strands are growing in Okazaki fragments. Another possibility is that opposite directions. One enzyme unit is movthe complex might dissociate from the teming with the unwinding point and synthesizing plate, so that a new complex must be assemthe leading strand continuously. The other unit bled to elongate the next Okazaki fragment. is moving "backward" relative to the DNA, along We see in Section 18.I0, The Clamp Controls the exposed single strand. Only short segments Association of Core Enzyme with DNA that the of template are exposed at any one time. When first model applies.
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The roles of RF-C and PCNA are analogous to the E. coliy clamp loader and B processivity u n i t ( s e eS e c t i o n 1 8 . 1 0 ,T h e C l a m p C o n t r o l s Associationof Core Enzyme with DNA). RF-C is a clamp loader that catalyzesthe loading of PCNA on to DNA. It binds ro the 3'end of the iDNA and usesATP-hydrolysis to open the ring of PCNA so that it can encircle the DNA. The processivity of DNA polymerase 6 is maintained by PCNA, which tethers DNA polymerase 6 to the template. (PCNA is called proliferating cell nuclear antigen for historical reasons.)The cryst a l s t r u c t u r e o f P C N A c l o s e l y r e s e m b l e st h e E. colip subunit: A trimer forms a ring that surrounds the DNA. The sequence and subunit organization are different from the dimeric p clamp; however, the function is likely to be similar. We are lesscertain about events on the lagging strand. One possibility is that DNA polymerase 6 also elongates the lagging strand. It has the capability to dimerize, which suggests a model analogous to the behavior of.E. coli replicase (see Section 18.9, DNA Polymerase Holoenzyme Has Three Subcomplexes).There are, however, some indications that DNA polymerase s may elongate the lagging strand, although it also has been identified with other roles. A general model suggeststhat a replication fork contains one complex of DNA polymerase c,/primase and two other DNA polymerase complexes. One is DNA polymerase 6 and the other is either a secondDNA polymerase 6 or may possiblybe a DNA polymerase e. The two complexesof DNA polymerase 6/e behave in the same way as the two complexes of DNA polymerase III in the E. coli replisome: one synthesizes the leading strand, and the other synthesizesOkazaki fragments on the lagging strand. The exonucleaseMFI removes the RNAprimers of Okazaki fragments.The enzyme DNA ligase I is specificallyrequired to seal the nicks between the completed Okazaki fragments.
Phage T4 Provides Its OwnReplication Apparatus r Phage T4provides its ownreptication apparatus, whichconsists of DNApoLymerase, the genej2 55B.a heUcase, proteins a primase, andaccessory that increase speedandprocessivity.
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prim'ing. removaI fg$#R[i*.tt Synthesis fragments requires extension, ofOkazakj of RNA,gapfitting,andnickligation. When phage T4 takes over an E. coli cell, it provides several functions of its own that either replace or augment the host functions. The phage placeslittle reliance on expressionof host functions. The degradation of host DNA is important in releasing nucleotides that are r e u s e d i n t h e s y n t h e s i so f p h a g e D N A . ( T h e phage DNA differs in base composition from cellular DNA in using hydroxymethylcytosine instead of the customary cytosine.) The phage-codedfunctions concerned with DNA synthesis in the infected cell can be identified by mutations that impede the production of mature phages.Essentialphage functions are identified by conditional lethal mutations, which fall into three phenotypic classes: . Those in which there is no DNA synthesisat all identify geneswhose products either are components of the replication apparatus or are involved in
Apparatus 445 18.14Phage T4 Provides lts OwnReptication
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poceloslpst lou.lud
Highfidelityreplicases Nuclearreplication 350 kD tetramer "
250 kD tetramer
"
350 kD tetramer
Mitochondrial replication
200 kD dimer
Highfidelityrepair Baseexcisionrepair 39 kD monomer Low fidelityrepair Thyminedimer bypassheteromer Base damagerepair
monomer
Requiredin meiosis
monomer
Deletionand base substitution
monomer
F t S i l * e1 * * i 5 E u k a r y o tci ce [ [ sh a v em a n yD N Ap o t y Therepticative merases. enzymes operate withhighfidetity. forthe p enzyme, Except the repairenzymes a[[havelow fidel.ity.Replicative enzymes have[argestructures, with subunits fordifferent activities. Repair enzymes separate havemuchsimoter structures. mase activity, which is analogous to DnaG of E. coli. The primase recognizes the template sequence 3'-TTG-5' and synthesizespentaribonucleotide primers that have the general sequence pppApCpNpNpNp. If the complete replication apparatus is present, these primers are extended into DNA chains. The gene 43 DNA polymerase has the usual 5'-3'synthetic activity, which is associatedwith a 3'-5' exonucleaseproofreading activity. It catalyzesDNA synthesisand removes the primers. When T4 DNA polymerase uses a singlestranded DNA as template, its rate of progress is uneven. The enzyme moves rapidly through s i n g l e - s t r a n d e dr e g i o n s , b u t p r o c e e d sm u c h more slowly through regions that have a basepaired intrastrand secondary structure. The accessoryproteins assistthe DNA polymerase in passing these roadblocks and maintaining its speed. The remaining three proteins are referred to "polymerase accessoryproteins." They increase as the affinity of the DNA polymerase for the DNA, aswell as increaseits processivityand speed.The gene 45 product is a trimer that acts as a sliding clamp. The structure of the trimer is similar to that of the E. coli $ dimer, in that it forms a circle around DNA that holds the DNA polymerase subunit more tightly on the template.
Function
E.coti
HeLa/SV40
PhageT4
Helicase Loadinghelicase/primase Singlestrandmaintenance Priming
DnaB DnaC SSB DnaG
T antigen T antigen RPA Polo/Primase
41 59 32 61
Slidingclamp Clamp loading(ATPase)
P 16 complex
PCNA RFC
45 44162
Pol6 ?
43 43
MF1 Ligase1
43 T4 ligase
Pol lll core Catalysis Holoenzymedimerization r RNA removal Ligation
Pol I Ligase
forks' at a[[replication arerequired functions f;IilLlRg 1$"fi* SjmiLar
The products ol genes44 and 62 form a tight complex that has ATPaseactivity. They are the equivalent of the y6 clamp loader complex, and their role is to load p45 onto DNA. Four molecules of ATP are hydrolyzed in loading the p45 clamp and the p43 DNA polymerase on to DNA. The overall structure of the replisome is similar to that of.E. coli.It consists of two coupled holoenzyme complexes, one synthesizing the leading strand and the other slmthesizing the lagging strand. In this case,the dimerization involves a direct interaction between the p43 DNA polymerase subunits, and p32 plays a role in coordinating the actions of the two DNA polymerase units. Thus far we have dealt with DNA replication solely in terms of the progression of the replication fork. The need for other functions is shown by the DNA-delay and DNA-arrest mutants. Three of the four genes of the DNAdelay mutants are 39, 52, and 60, which code for the three subunits of T4 topoisomeraseII, an activity needed for removing supercoils in the template (see Section 19.l), Topoisomerases Relax or Introduce Supercoils in DNA). The essential role of this enzyme suggeststhat T4 DNA does not remain in a linear form, but rather becomestopologically constrained during some stage of replication. The topoisomerase could be needed to allow rotation of DNA ahead of the replication fork. Comparison of the T4 apparatus with the E. coli apparatus suggeststhat DNA replication posesa set of problems that are solved in analogous ways in different systems.We may now compare the enzymatic and structural activities found at the replication fork in E. coli, T4, and "I*.td summarizes HeLa (human) cells. Fe&Lift[
Apparatus 447 Its OwnReplication T4 Provides 18.14Phage
the functions and assignsthem to individual proteins. We can interpret the known properties of replication complex proteins in terms of similar functions that involve the unwinding, priming. catalytic, and sealing reactions. The components of each system interact in restricted ways, as shown by the fact that phage T4 requires its own helicase,primase, clamp, and so on, and by the fact that bacterial proteins cannot substitute for their phage counterparts.
@
involves action at two types of sequences:9 bp and l3 bp repeats.Togetherthe 9 bp and l3 bp repeats define the limits of the 245 bp minimal origin, as indicated in F?GtJftf, 3S.:T.An origin is activated by the sequence of events summarized in fgGilft*1S.iltr,in which binding of DnaA is succeeded by association with the other proteins. The four 9 bp consensussequenceson the right side of oriCprovide the initial binding sites for DnaA. It binds cooperatively to form a central core around which oriC DNA is wrapped.
Creating the Reptication Forks at an Origin
r Initiatjonat onCrequires the sequentiaI assembly of a largeproteincomptex. o DnaAbindsto shortrepeated sequences andforms an otigomeric comptex that mel.ts DNA. o SixDnaC monomers bindeachhexamer of DnaB. andthis complex bindsto the origin. . A hexamer of DnaBformsthe reptication fork. Gyrase andSSBarealsorequired.
13-mers 245 bp
F€##ftf, i*.P? TheminimaI originis defined bythe distancebetween the outsidemembers of the 13-merand 9-merrepeats.
Starting a cycle of replication of duplex DNA requires severalsuccessiveactivities: . The two strandsof DNA must suffer their GATCTNTTNTTTT TTATNCANA Theoriginhas i n i t i a l s e p a r a t i o n .T h i s i s , i n e f f e c t , a three 13-bprepeats melting reaction over a short region. and four 9-bp repeats . An unwinding point begins to move along the DNA; this marks the generation of the replication fork, which conDnaAmonomers tinues to move during elongation. bind at g-bp . The first nucleotides of the new chain repears must be synthesized into the primer. This action is required once for the leadDnaAbinds ing strand,but is repeatedat the start of to 13-bprepeats each Okazaki fragment on the lagging strand. Some events that are required for initiation therefore occur uniquely at the origin; others recur with the initiation of each Okazaki fraeDNA strands ment during the elongation phase. separateat 13-bprepeats Plasmids carrying the E coli oriC sequence have been used to develop a cell-free system for replication. Initiation of replication at oriCin vitro slarts with formation of a complex that DnaB/DnaC requires six proteins: DnaA, DnaB, DnaC, HU, joins complex, Gyrase,and SSB. Of the six proteins involved forming in prepriming, DnaA draws our attention as the replicationforks only one uniquely involved in initiation vis-)vis elongation. DnaB/DnaC provides the "engine" of initiation at the origin. f lfi{if,{il3ii"ii* Prepriming involvesformationof a comptexby The first stagein complex formation is bind- sequentiaIassociation proteins, of which leadsto the separaing to oriC by DnaA protein. The reaction tion of DNAstrands.
CHAPTER 18 DNAReptication
The region of strand separation in the open complex is large enough for both DnaB hexamers to bind, which initiates the two replication forks. As DnaB binds, it displacesDnaA from the l3 bp repeats and extends the length of the open region. It then usesits helicaseactivity to extend the region of unwinding. Each DnaB activatesa DnaG primase-in one case to initiate the leading strand, and in the other to initiate the first Okazaki fragment of the lagging strand. TWofurther proteins are required to support the unwinding reaction. Gyraseprovides a swivel that allows one strand to rotate around the other (a reaction discussedin more detail in Section 19.15,GyraseFunctionsby Coil Inversion); without this reaction, unwinding would generate torsional strain in the DNA. The protein SSBstabilizes the single-strandedDNA asit is formed. The length of duplex DNA that usually is unwound to initiate replication is probably <60 bp. The protein HU is a general DNA-binding protein in E. coli Its presence is not absolutely :::.{!LJfi{ ii:1.1+Thecomplex at onCcanbe detected by required to initiate replication in vitro, but ir etectron microscopy. Bothcomptexes werevisuatized with antibodies againstDnaBprotein.Topphotoreproduced stimulates the reaction. HU has the capacity to f r o mF u n n e tB, . E . ,e t a l .J . B i o l .C h e m . 1 9 8 7 . 2 6 2 : bend DNA, and is involved in building the struc1.0327-70334. Copyright 1987by American for ture that leads to formation of the open complex. Society Biochemistry & Motecu[ar Biotogy. Photocoudesy of BarInput of energy in the form of ATP is required baraE.Funnett, University ofToronto. Bottomphotoreproat several stagesfor the prepriming reaction, and ducedfromBarker, T.A., et at.J. Biol.Chem. 7987.262: it is required for unwinding DNA. The helicase 6877-6885. Copyright 1987byAmerican for BioSociety chemistry Biotogy. & Motecutar Photocourtesy of Barbara action of DnaB depends on ATP hydrolysis, and E.Funne[t, University of Toronto. the swivel action of gyrase requires AIP hydrolysis.ATP alsois needed for the action of primase DnaA then actsat three A-T-rich l3 bp tandem and to activate DNA polymerase III. repeats located in the left side oI oriC.In the Following generation of a replication fork presence of ATP, DnaA melts the DNA strands as indicated in Figure 18.28, the priming reacat each of these sitesto form an open complex. tion occurs to generate a leading strand. We All three l3 bp repeatsmust be opened for the know that synthesisof RNA is used for the primreaction to proceed to the next stage. ing event, but the details of the reaction are not Altogether, two to four monomers of DnaA known. Some mutations in dnaA can be supbind at the origin, and they recruit two pressedby mutations in RNA polymerase,which "prepriming" complexesof DnaB-DnaC to bind, suggeststhat DnaA could be involved in an iniso that there is one for each of the two (bidirectiation step requiring RNA synthesis invivo. tional) replication forks. Each DnaB-DnaC comRNA polymerase could be required to read plex consistsof six DnaC monomers bound to into the origin from adjacent transcription units; a hexamer of DnaB. Each DnaB-DnaC comby terminating at sites in the origin, it could plex transfersa hexamer of DnaB to an oppoprovide the 3'-OH ends that prime DNA polysite strand of DNA. DnaC hydrolyzes ATP in merase III. (An example is provided by the use order to releaseDnaB. of D loops at mitochondrial origins, as discussed The prepriming complex generates a proin Section l5.l l, D LoopsMaintain Mitochontein aggregateof 480 kD, which correspondsto drial Origins.) Alternatively, the act of transcripa sphere of radius 6 nm. The formation of a tion could be associatedwith a structural change complex ar lric is detectable in the form of the that assistsinitiation. This latter idea is suplarge protein blob visualized in F:*Liq{ ?*.,?+. ported by observations that transcription does When replication begins, a replication bubble not have to proceed into the origin; it is effecbecomes visible next to the blob. tive up to 200 bp away from the origin, and can
Forks at an 0rigin the Reptication 18.15Creating
449
use either strand of DNA as template in vitro. The transcriptional event is inversely related to the requirement for supercoiling invitro, which suggeststhat it actsby changing the local DNA structure so as to aid meltins of DNA.
@
Common Events in PrimingReptication at the 0rigin
. ThegeneraI principte of bacteriaI initiationis that the originis initiaLty recognized by a proteinthat formsa largecomptex with DNA. o A shortregionof A-T-rich DNAis melted. . DnaBis boundto the complex andcreates the reotication fork.
Another system for investigating interactions at the origin is provided by phage lambda. A map of the region is shown in :rT*t-i*i: :9. ii:. Initiation of replication at the lambda origin requires "activation" by transcription startingfrom Pp.As with
For replication to initiatehere i:i:r-ii:i :iS.-ii"rTranscription initiating at Pqis required to activatethe origin of Lambda DNA.
the events at lric, this doesnot necessarilyimply that the RNA provides a primer for the leading strand. Analogies between the systems suggest that RNA synthesiscould be involved in promoting some structural change in the region. Initiation requires the products of phage genesO and.B as well as severalhost functions. The phage O protein binds to the lambda origin; the phage P protein interacts with the O protein and with the bacterial proteins. The origin lies within gene O, so the protein acts close to its site of synthesis. Variants of the phage called Xdv consist of shorter genomes that carry all the information needed to replicate,but lack infective functions. l,dv DNA survives in the bacterium as a plasmid, and can be replicated in vitro by a system consistingof the phage-codedproteins O and P together with bacterial replication functions. Lambda proteins O and P form a complex together with DnaB at the lambda origin, ori)". The origin consistsof two regions, as illustrated in F3*iiill i*.-i1: A region comprising a seriesof four binding sites for the O protein is adjacent to an A-T-rich region. The first stagein initiation is the binding of O to generate a roughly spherical structure of diameter -l I nm, a structure sometimes called the O-some. The O-some contains -100 bp or 60 kD of DNA. There are four I8 bp binding sites for O protein, which is -34 kD. Each site is palindromic, and probably binds a symmetrical O dimer. The DNA sequencesof the Obinding sites appear to be bent. and binding of O protein induces further bending.
O-bindingsites18 bp
rvilt
[|
liit-;i;1;iJ.*,-ii The[ambda originfor reptication comprises two regions. Early events arecatalyzed by0 protein, whichbindsto a series of foursites,andthen DNAis mettedin the adjacent A-T-rich region.TheDNAis drawnasa straight duptex; it is, however, actua[[y bentat the origin.
450
CHAPTER 18 DNAReolication
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ment, whereas the nearest lagging strand is initiated 50 tol00 bp before reachingter. The result of this inhibition is to halt movement of the replication fork and (presumably) to cause disassemblyof the replication apparatus. F:*:-,!Sii i*"ii* reminds us that Tus stops the movement of a replication fork in only one direction. The crystal structure of a Ttrs-ter complex shows that the Tus protein binds to INA asymmetrically; a-helices of the protein protrude around the double helix at the end that blocks the replication fork. Presumably a fork proceeding in the opposite direction can displace Tus and thus continue. A difficulty in understanding the function of.the system in vivo i s t h a t i t a p p e a r s t o b e d i s p e n s a b l e ,b e c a u s e mutations if.the ter sitesor intus are not lethal.
Summary DNA synthesis occurs by semidiscontinuous replication, in which the leading strand of DNA growing 5'-)'is extended continuously, but the lagging strand that grows overall in the opposite J'-5' direction is made as short Okazaki fragments, each synthesized 5'-3'. The leading strand and each Okazaki fragment of the lagging strand initiate with an RNA primer that is
DNA blocked DNA accessible Replicationproceeds Replicationterminates
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extended by DNA polymerase. Bacteria and eukaryotes each possessmore than one DNA polymerase activity. DNA polymerase III synthesizesboth lagging and leading strands inE. coli. Many proteins are required for DNA polymerase III action and severalconstitute part of the replisome within which it functions. The replisome contains an asymmetric dimer of DNA polymerase III; each new DNA strand is synthesized by a different core complex containing a catalytic (o) subunit. Processivity of the core complex is maintained by the B clamp, which forms a ring around DNA. The clamp is loaded onto DNA by the clamp Ioader complex. Clamp/clamp loader pairs with similar structural features are widely found in both prokaryotic and eukaryotic replication systems. The looping model for the replication fork proposesthat, as one half of the dimer advances
18.18Summary 453
to synthesizethe leading strand, the other half of the dimer pulls DNA through as a single loop that provides the template for the lagging strand. Ihe transition from completion of one Okazaki fragment to the start of the next requires the Iagging strand catalytic subunit to dissociate from DNA and then reattach to a p clamp at the priming site for the next Okazaki fragment. DnaB provides the helicase activity at a replication fork; this depends on ATP cleavage. DnaB may function by itself in oriC replicons to provide primosome activity by interacting periodically with DnaG, which provides the primase that synthesizesRNA. Phage t+ codes for a replication apparatus consistingof sevenproteins: DNA polymerase. helicase,single-strandbinding protein, priming activities, and accessoryproteins. Similar functions are required in other replication systems, including a HeLa cell system that replicates SV40 DNA. Different enzymes-DNA polymerase o and DNA polymerase 6-initiate and elonsate the new strandsof DNA. fhe
454
CHAPTER 18 DNAReotication
Following initiation of replication, DnaA hydrolyzes its AIP under the stimulus of the p sliding clamp, thereby generating an inactive form of the protein. In addition, oriCmust compete with Ihe dat site for binding DnaA. Several sites that are methylated by the Dam methylase are present in the E. coli origin, including those of the l3-mer binding sitesfor DnaA. The origin remains hemimethylated and is in a sequesteredstate for -10 minutes following initiation of a replication cycle. During this period it is associatedwith the membrane and reinitiation of replication is repressed.The protein SeqA is involved in sequestration and may interact with DnaA.
References Introduction Resea rch Hirota, Y., Ryter, A., and Jacob,F. (1968). Thermosensitive mutants of E coli affected in the processesof DNA synthesis and ceilular diviston. ColdSpringHarbor Symp Quant Biol. 3),677-69).
DNAPotymerases Havea Common Structure Reviews Hubscher,U., Maga, G., and Spadari,S. (2002). Eukaryotic DNA polymerases. Annu. Rev. B i o c h e m7 1 , l ) 3 - 1 6 ) . Johnson, K. A. (I991). Conformational coupling in DNA polymerase fidelity. Annu Rev. B i o c h e m . 6 26, 8 5 - 7 | j . Joyce, C. M. and Steitz,T. A. (1994) Function and structure relationships in DNA polymerases. Annu Rey.Biochem 6J, 777-822. rch Resea shamoo, Y. and Steitz,T. A. (1999). Building a replisome from interacting pieces: sliding clamp complexed to a peptide from DNA polymerase and a polymerase editing complex. Cal/ 99,155-r66.
The
Singleton, M. R., Sawaya, M. R., Ellenberger, T., and Wigley, D. B. (2000). Crystal structure of T7 gene 4 ring helicase indicates a mechanism for sequential hydrolysis of nucleotides. Cel/ 10r,589-600.
@
KEVIEW Liu, Y., I(ao, H. I., and Bambara, R. A. (2004). FIap endonuclease l: a central component of DNA metabolism.Annu Rev.Biochem.73.589-615.
DNAPotymerase Hotoenzyme HasThree Subcomptexes
Refere nce Johnson, A.,andO'Donnell, M. (2005).Cellular DNA replicases:components and dynamics at the replication fork. Annu RevBiochem.7 4, 283-]15. Resea rch Studwell-Vaughan, P. S. and O'Donnell, M. (199 t ) Constitution of the twin polymerase of DNA polymerase III holoenzyme. J. Biol. Chem.266, 1983?-19841. Stukenberg, P. T., Studwell-Vaughan, P. S., and O'Donnell, M. (1991). Mechanism of the sliding beta-clamp of DNA polymerase III holoenzyme. J Biol. Chem 266,ll?28-11l-)4.
TheClamp Controts Association of Core Enzymewith DNA Reviews Benkovic, S. J., Valentine, A. M., and Salinas. F. (2001). Replisome-mediatedDNA replication. Annu Rev.Biochem70, l8l-208. Davey, M. J., Jeruzalmi, D., I(uriyan, J., and O'Donnell, M. (2002). Motors and switches: AAA+ machines within the replisome. Nal Rev.Mol. CellBiol. J, 826-835. Resea r ch Bowman, G. D., O'Donnell, M., and I(uriyan, J. (20041. Structural analysis of a eukaryotic sliding DNA clamp-clamp loader complex. N a t u r e4 2 9 , 7 2 4 - 7 3 0 . Jeruzalmi, D., O'Donnell, M., and Kuriyan, J. (200 I ) . Crystal structure of the processivity clamp loader gamma (gamma) complex of E. coli DNA polymerase lIL Cell 106, 429441. Kong, X. P., Onrust, R., O'Donnell, M., and I(uriyan, J. (1992\. Three-dimensional structure of the beta subunit ol E. coli DNA polymerase III holoenzyme: a sliding DNA clamp Cell69. 425437.
@
Fragments AreLinkedby Ligase Okazaki
Coordinating Synthesis ofthe Lagging and LeadingStrands
Resea rch Dervyn,E., Suski,C.,Daniel,R.,Bruand,C., Chapuis,J., Errington,J., Janniere,L., and Ehrlich,S.D. (2001).TWoessentialDNApolymerasesat the bacterialreplicationfork. Sci e n c 2e 9 4 ,1 7 1 6 - 1 7 1 9 .
Eukaryotic DNAPotymerases Separate Undertakeinitiation and Etonqation Reviews Goodman, M. F. (2002). Error-prone repair DNA polymerases in prokaryotes and eukaryotes. Annu. Rev.Biochem71, 17-50. Hubscher,U., Maga, G., and Spadari,S. (2002). Eukaryotic DNA polymerases. Annu. Rev. Biochem.T l, 133-163 . I(aguni, L. S. (2004). DNA polymerase gamma, the mitochondrial replicase.Annu. Rev.Biochem. 7),293-)20. Resea r ch I(arthikeyan, R., Vonarx, E. J., Straffon, A. F., Simon, M., Faye, G., and I(unz, B. A. (2000). Evidence from mutational specificity studies that yeast DNA polymerases delta and epsilon replicate different DNA strands at an intracellular replication fork. J. Mol. Biol.299, 405-419. Shiomi, Y., Usukura, J., Masamura, Y., Takeyasu, I(., Nakayama, Y, Obuse, C., Yoshikawa,H., and Tsurimoto, T. (2000). ATP-dependent structural change of the eukaryotic clamploader protein, replication factor C. Proc.Natl. Acad. Sci.USA 97, 14127-141)2. Waga, S., Masuda, T., Takisawa, H., and Sugino, A. (2001 ) . DNA polymerase epsilon is required for coordinated and efficient chromosomal DNA replicationin Xenopus egg exfiacts. Proc. Natl. Acad. Sci.USA98,4978-498). Ztto,5., Bermudez, Y., Zhang, G., I(elman, 2., and Hurwitz, J. (2000). Structure and activity associatedwith multiple forms of S.pombe DNA polymerasedelta. J Biol Chem.275, 5l5J-5r62.
Its OwnReplication Phage T4 Provides Apparatus Resea rch Ishmael, F. T., Alley, S. C , and Benkovic, S. J. (2002) . Assembly of the bacteriophage T4 helicase: architecture and stoichiometry of the gp4l-gp59 complex. J. Biol. Chem.277, 20555-20562. Salinas,F., and Benkovic, S. J. (2000). Charactenzation of bacteriophage T4-coordinated leading- and lagging-strand synthesis on a minicircle substrate. Proc Natl. Acad. Sci.USA 97, 7 196-7201. S c h r o c k ,R . D . a n d A l b e r t s , B . ( 1 9 9 6 ) . P r o c e s sivity of the gene 4l DNA helicase at the
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bacteriophage T4 DNA replication fork. J. Biol. Chem 271,16678-16682.
@
Forks Creating the Reptication at an 0rigin
rch Resea Duplex Bramhill, D. andKornberg, A. (1988). opening by dnaA protein at novel sequences in initiation of replication at the origin of the E. colrchromosome. Cel/52,743-755. Fuller, R. S., Funnell, B. E., and Kornberg, A. ( I 984). The dnaA protein complex with the E coli chromosomal replication origin (oriC) and other DNA sites.Cell)8,889-900. Funnell, B. E. and Baker, T. A. (1987). In vitro assembly of a prepriming complex at the origin of the E coli chromosome. J. Biol. Chem. 262, t0)27-10334. Sekimizu, K, Bramhill, D., and I(ornberg, A. ( 1987). ATP activates dnaA protein in initiating replication of plasmids bearing the origin of the E coli chromosome. Cell 50, 259-265. Wahle, E., Lasken, R. S., and Kornberg, A. (1989). The dnaB-dnaC replication protein complex of Escherichiacoli II. Role of the complex in mobilizing dnaB functions. J. Biol. Chem. 264,2469-2475.
456
CHAPTER 18 DNAReotication
Is Needed ThePrimosome to Restart Reo[ication Reviews Cox, M. M. (200I). RecombinationalDNA repair of damagedreplication forks in E. coli: qtestions.Annu.Rev.Genet.)5, 5)-82. Cox, M. M., Goodman,M. F.,I(reuzer,I(. N., Sherratt, D. J., Sandler,S.J., and Marians,K. J. (2000).The importanceof repairingstalled replicationf.orks.Nature404, 37-4L I(uzminov A. (I995). Collapseand repair of replication forks in E. coli.Mol. Microbiol 16, 37)-i84. McGlynn, P.and Lloyd, R. G. (2002).Recombinational repair and restartof damagedreplication forks. Nat. Rev.Mol. CellBiol ), 859-870. Research Seigneur,M., Bidnenko,V.,Ehrlich, S. D., and Michel,B. (1998).RuvABactsat arrested replicationforks.Cell95, 419430.
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its existence means that the point of branching cannot be establishedby examining a molecule invitro (becausethe branch may have migrated since the molecule was isolated). Branch migration could allow the point of crossoverin the recombination intermediate to Genomesare not move in either direction. The rate of branch recombinant, but migration is uncertain, but as seen in vitro is coniainheteroduplex probably inadequate to support the formation regron of extensive regions of heteroduplex DNA in natural conditions. Any extensive branch migraReciprocalrecombinant Iion in vivo must therefore be catalyzed by a genomesare generated recombination enzyme. The joint molecule formed by strand f l * * R * 3 l l . S R e c o m b i n a t jboent w e etnw o p a i r e dd u p l e x DNAs coutdinvotve reciprocal singte-strand exchange. branch exchange must be resolvedinto two separate migratioa nn , dn i c k i n g . duplex molecules. Resolution requires a {urther pair of nicks. We can most easily visualize the outcome by viewing the joint molecule in of the parental DNA molecules. This region is one plane as a Holliday junction. This is illuscalled hybrid DNA or heteroduplex DNA. trated in Fi*{Jltfii}.i}, which representsthe strucAn important feature of a recombinant ture of Figure I9.6 with one duplex rotated joint is its ability to move along the duplex. relative to the other. The outcome of the reacSuch mobility is called branch migration. tion depends on which pair of strandsis nicked. Fi*13fi[1*.; illustratesthe migration of a single If the nicks are made in the pair of strands strand in a duplex. The branching point can that were not originally nicked (the pair rhat migrate in either direction as one strand is disdid not initiate the strand exchange), all four placed by the other. of the original strands have been nicked. This Branch migration is important forboth thereleasessplice recombinant DNA molecules. oretical and practical reasons.As a matter of The duplex of one DNA parent is covalently principle, it confers a dynamic property on linked to the duplex of the other DNA parent recombining structures. As a practical feature, via a stretch of heteroduplex DNA. There has
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stantially reduced if the homologous region is <75bp. This distance is appreciably longer than the -I0 bp required for association between complementary single-strandedregions, which suggeststhat recombination imposes demands beyond annealing of complements as such.
Double-Strand Breaks InitiateRecombi nation Recombination is initiatedby making a doubteDNAduptex. strandbreakin one(recipient) Exonuclease actiongenerates 3'-single-stranded endsthat invadethe other(donor)dup[ex. NewDNAsynthesis reptaces the material that has beendegraded. Thisgenerates a recombinantjoint motecute in whichthetwo DNAduptexes areconnected by heteroduolex DNA.
Nickingcontrolsoutcome Nicksin same strandsrelease patch recombinants
i:+ij*lF 1*.* Resotution ofa Hotlidayjunction cangenerateparentaI or recombinant duplexes, depending on which strands arenicked.Bothtypesof producthavea regionof heteroduotex DNA. been a conventional recombination event between markers located on either side of the heteroduplex region. If the same two strandsinvolved in the original nicking are nicked again, the other two
l'",:l*,;xniT,1il;lllff [T?.;T,ff :,'l; with the exception that each has a residuum of the event in the form of a length of heteroduplex DNA. These are called patch recombinants. These alternative resolutions of the joint molecule establish the principle that a strand
The general model of Figure 19.4 shows that a break must be made in one duplex in order to generate a point from which single strands can unwind to participate in genetic exchange.Both strands of a duplex must be broken to accomplish a genetic exchange.Figure I9.6 shows a model in which individual breaks in single strands occur successively.Genetic exchange, however, is actually initiated by a doublestrand break (DSB). The model is illustrated in il5$l.JR* t$.*. Recombination is initiated by an endonuclease that cleaves one of the partner DNA duplexes, the "recipient." The cut is enlarged to a gap by exonucleaseaction. The exonuclease(s)nibble away one strand on either side of t h e b r e a k , g e n e r a t i n g 3 ' s i n g l e - s t r a n d e dt e r mini. One of the free 3' ends then invades a homologous region in the other ("donor") duplex. This is called single-strand invasion. The formation of heteroduplex DNA generates a D loop, in which one strand of the donor duplex is displaced. The D loop is extended by repair DNA synthesis,using the free J'end as a primer to generatedouble-strandedDNA. Eventually the D loop becomes large enough to correspond to the entire length of the gap on the recipient chromatid. When the extruded single strand reaches the far side of the gap, the complementary single-stranded sequencesanneal. Now there is heteroduplex DNA on either side of the gap, and the gap itself is representedby the single-stranded D loon.
':'l :;#;:: ;,:!; !:;l:::tri';y#::::;';:;'#l
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What is the minimum length of the region required to establishthe connection between the recombining duplexes?Experiments in which short homologous sequencescarried by plasmids or phages are introduced into bacteria suggestthat the rate of recombination is sub-
C H A P T E1R9 H o m o l o g o uasn dS j t e - S p e c i fR i ce c o m b i n a t i o n
The duplex integrity of the gapped region can be restoredby repair synthesisusing the 3' end on the left side of the gap as a primer. Overall, the gap has been repaired by two individual rounds of single-strandDNA synthesis. Branch migration converts this structure into a molecule with two recombinant joints. The joints must be resolvedby cutting. If both joints are resolved in the same way, the original noncrossover molecules will be released, each with a region of altered genetic information that is a footprint of the exchange event. If the two joints are resolvedin opposite ways, a genetic crossoveris produced. The structure of the two-jointed molecule before it is resolved illustrates a critical difference between the double-strand break model and models that invoke only single-strand exchanges. . Following the double-strand break, heteroduplex DNA has been formed at each end of the region involved in the exchange.Between the two heteroduplex segmentsis the region corresponding to the gap, which now has the sequenceof the donor DNA in both molecules (Figure 19.9).Thus the arrangem e n t o f h e t e r o d u p l e x s e q u e n c e si s asymmetric, and part of one molecule has been converted to the sequenceof the other (which is why the initiating chromatid is called the recipient). . Following reciprocal single-strand exchange,each DNA duplex has heteroduplex material covering the region from the initial site of exchangeto the migrating branch (Figure 19.6). In variants of the single-strand exchange model in which some DNA is degradedand resynthesized, the initiating chromatid is the donor of genetic information. The double-strand break model does not reduce the importance of the formation of heteroduplex DNA, which remains the only plausiblemeans by which two duplex molecules can interact. By shifting the responsibility for initiating recombination from single-strand to double-strand breaks,though, it influences our perspectiveabout the ability of the cell to manipulate DNA. The involvement of double-strand breaks at first seems surprising. Once a break has been made right acrossa DNA molecule, there is no going back. Compare the events of Figure 19.6 and Figure 19.9.At no point in the single-strand exchange model has any information been lost.
lilirliill. .L',r,iiRecombination is initiatedbya double-strand 3' ends, break,fotlowedby formationof single-stranded duplex. to a homologous oneof whichm'igrates
In the double-strandbreak model, though, the initial cleavageis immediately followed by loss of information. Any error in retrieving the information could be fatal. On the other hand, the very ability to retrieve lost information by resynthesizing it from another duplex provides a maior safetvnet for the cell.
Recombining Are Chromosomes bythe Connected Complex SynaptonemaI . Duringtheearlypartof meiosis. homologous arepairedin the synaptonemaI chromosomes comotex. . Themassof chromatin is of eachhomotog fromthe otherby a proteinaceous separated complex. A basic paradox in recombination is that the parental chromosomesnever seem to be in close enough contact for recombination of DNA to
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465
ently proteinaceous. The axial elements of corresponding chromosomes then become aligned, and the synaptonemal complex forms as a tripartite structure, in which the axial elements, now called lateral elements, are separated from each other by a central element. F:*iififl:il"3i: shows an example. Each chromosome at this stage appears as a mass of chromatin bounded by a lateral element. The two lateral elements are separated from each other by a fine, but dense, central element. The triplet of parallel dense strands lies in a singleplane that curves and twists along its axis. The distance between the homologous chromosomes is considerable in molecular iia::-:rii::-:.-::: ThesynaptonemaL complex bringschromosomes into terms, at more than 200 nm (the diameter of juxtaposition. Reproduced fromD.vonWettstejn. 1.971.. Proc.Natl. DNA is 2 nm). Thus a major problem in underAcad.Sci.USA.68:851-855.Photocourtesy of D. von Wettstein, standing the role of the complex is that, although Washington StateUniversity. it alignshomologous chromosomes,it is far from bringing homologous DNA molecules into contact. The only visible link between the two sides of the synaptonemal complex is provided by spherical or cylindrical structures observed in fungi and insects. They lie across the complex and are called nodes or recombination nodules; they occur with the same frequency and distribution as the chiasmata. Their name reflects the hope that they may prove to be the sites of recombination. From mutations that affect synaptonemal complex formation, we can relate the types of proteins that are involved to its structure. IItuiJiqil i.*"1: presents a molecular view of the synaptonemal complex. Its distinctive structural features are due to two groups of proteins: . The cohesins form a single linear axis j,;.i ;:.ii:jiii pair ir .i*Each of sisterchromatids hasan axismadeof for each pair of sister chromatids from cohesins. Loopsof chromatin projectfromthe axis.Thesynaptonemalcomplex is formedby tinkingtogether which loops of chromatin extend. This the axesviazipproteins. is equivalent to the lateral element of occur. The chromosomes enter meiosis in the Figure I9. 10. (The cohesinsbelong to a form of replicated (sisterchromatid) pairs,which general group of proteins involved in are visible as a mass of chromatin. They pair to connecting sister chromatids so that they form the synaptonemal complex, and it has segregateproperly at mitosis of meiosis.) . The lateral elements are connected by been assumedfor many years that this representssome stageinvolved with recombinationtransversefilaments that are equivalent possibly a necessarypreliminary to exchange to the central element of Figure 19.10. of DNA. A more recent view is that the synapThese are formed from Zip proteins. tonemal complex is a consequencerather than Mutations in proteins that are needed lor a causeof recombination, but, we have yet to lateral elements to form are found in the genes define how the structure of the synaptonemal coding for cohesins.The cohesins that are used complex relatesto molecular contactsbetween in meiosis include Smc3p (which is also used DNA molecules. in mitosis) and Rec8p (which is specificto meioSynapsisbegins when each chromosome sisand is related to the mitotic cohesin Scclp). (sisterchromatid pair) condensesaround a strucThe cohesins appear to bind to specific sites ture called the axial element, which is apparalong the chromosomes in both mitosis and
466
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suggeststhat the enzyme can mediate reciprocal strand transfer. We know less about the geometry of four-strand intermediates bound by RecA, but presumably two duplex molecules can lie side by side in a way consistent with the requirements of the exchange reaction. The biochemical reactions characterized in vitro leave open many possibilities for the functions of strand-transfer proteins in vivo. Their involvement is triggered by the availability of a single-stranded3' end. In bacteria,this is most likely generatedwhen RecBCDprocesses a double-strand break to generate a singlestranded end. One of the main circumstancesin which this is invoked may be when a replication fork stallsat a site of DNA damage (seeSection 20.9, Recombination Is an Important Mechanism to Recoverfrom ReplicationErrors). The introduction of DNA during conjugation, when RecA is required for recombination with the host chromosome, is more closely related to conventional recombination. In yeast, doublestrand breaks may be generated by DNA damage or as part of the normal process of recombination. In either case,processingof the break to generate a 3'single-stranded end is followed by loading the single strand into a filament with Rad5l, followed by a search for matching duplex sequences.This can be used in both reuair and recombination reactions. '.1 $:i:]Li+t F.tti RecA-mediated between strandexchange partiatty andentire[y dupl.ex DNAgenerates ajoint duplex m o l e c u [w e i t h t h e s a m es t r u c t u raes a r e c o m b i n a t i o n i ntermediate. act with each other under the sponsorship of RecA, provided that one of them has a singlestranded region of at leastfifty bases.The singlestranded region can take the form of a tail on a linear molecule or of a gap in a circular molecule. The reaction between a paftially duplex molecule and an entirely duplex molecule leads to the exchange of strands. An example is illus.1+.it. Assimilation starts at one trated in Fi+{.FFtil end of the linear molecule, where the invading single strand displacesits homolog in the duplex in the customary way. When the reaction reaches the region that is duplex in both molecules, though, the invading strand unpairs from its partner, which then pairs with the other displacedstrand. At this stage, the molecule has a structure indistinguishable from the recombinant joint in Figure 19.8. The reaction sponsoredinvitro by RecA can generateHolliday junctions, which
TheRuvSystemResolves Junctions Hol.l.iday o TheRuvcomptex actson recombinantjunctions. . RuvArecognizes of thejunctionand the structure branchmigration. that catalyzes RuvBis a heticase r RuvC junctions generate recombination to c[eaves intermediates. One of the most critical stepsin recombination is the resolution of the Holliday junction, which determines whether there is a reciprocal recombination or a reversalof the structure that Ieaves only a short stretch of hybrid DNA (see Figure 19.6 and Figure 19.8). Branch migration from the exchangesite (seeFigure I9.7) determines the length of the region of hybrid DNA (with or without recombination).The proteins involved in stabilizing and resolving Holliday junctions have been identified as the products of the ruv genes \n E. coli. RuvA and RuvB increase the formation of heteroduplex structures. RuvA recognizes the structure of the
Junctions 473 Hol.tiday Resolves 19.10TheRuvSystem
RuvB hexamerbinds as ringaroundDNA iirlijili l:i.iI RuvABis an asymmetriccomplexthat promotesbranchmigrationof a Hotlidayjunction.
Gap generatedby replicationof damagedDNA
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RuvO CleaveHolliday junction i: {:ii* l i *-"i S BacteriaI enzymes cancatatyze a[[stages of recombination in the repairpathway fo[towing the production of suitabte substrate DNAmolecutes.
Holliday junction. RuvA binds to all four strands of DNA at the crossover point and forms two tetramers that sandwich the DNA. RuvB is a hexameric helicasewith an ATPaseactivity that provides the motor for branch migration. Hexameric rings of RuvB bind around each duplex of DNA upstream of the crossoverpoint. A diagram of the complex is shown in li{:i"rH*'ti}":tt.
474
CHAPTER 19 Homologous andSite-Specific Recombination
The RuvAB complex can cause the branch to migrate as fast as l0 to 20 bp/sec.A similar activity is provided by another helicase, RecG. RuvAB displaces RecA from DNA during its action. The RuvAB and RecG activities both can act on Holliday junctions, but if both are mutant, E. coliis completely defective in recombination activity. The third gene, ruvC, codesfor an endonucleasethat specificallyrecognizesHolliday junctions. It can cleave the junctions in vitro Io resolve recombination intermediates. A common tetranucleotide sequence provides a hotspot for RuvC to resolve the Holliday junction. The tetranucleotide (ATTG) is asymmetric, and thus may direct resolution with regard to which pair of strands is nicked. This determines whether the outcome is patch recombinant formation (no overall recombination) or splice recombinant formation (recombination between flanking markers). Crystal structures of RuvC and other junction-resolving enzymes show that there is little structural similarity among the group, in spite of their common function. All of this suggeststhat recombination uses a "resolvasome"complex that includes enzymes catalyzingbranch migration as well asjunctionresolving activity. It is possiblethat mammalian cells contain a similar complex. We may now account for the stages of recombination in E. coli in terms of individual proteins. f+i;{JftE i*.18 shows the events that are involved in using recombination to repair a gap in one duplex by retrieving material from the other duplex. The major caveat in applying these conclusions to recombination in eukaryotes is that bacterial recombination generally involves interaction between a fragment of DNA and a whole chromosome. It occurs as a repair reaction that is stimulated by damage to DNA, bur this is not entirely equivalent to recombination between genomes at meiosis.Nonetheless,similar molecular activities are involved in manipulating DNA. Another systemof resolvaseshas been characterized in yeast and mammals. Mutants in S. cerevisiae mus81are defective in recombination. Mus8l is a component of an endonuclease that resolves Holliday junctions into duplex structures. The resolvaseis important both in meiosis and for restarting stalled replication forks (seeSection20.9, Recombination Is an Important Mechanism to Recover from Renlication Errors).
GeneConversion Accounts for Intera[[el.ic Recombination
No recombination
Heterodup[ex DNAthat is created by recombination canhavemismatched seouences wherethe recombining al[etes arenotidenticat. Repair systems mayremove mismatches by changing oneof the strands soits sequence is comptementary to the other.
The involvement of heteroduplex DNA explains the characteristicsof recombination between alleles; indeed, allelic recombination provided the impetus for the development of the heteroduplex model. When recombination between alleles was discovered, the natural assumption was that it takes place by the same mechanism of reciprocal recombination that applies to more distant loci. That is to say, an individual breakageand reunion event occurs within the locus to generatea reciprocalpair of recombinant chromosomes.In the closequarters of a single gene. however, the formation of heteroduplex DNA itself is usually responsible for the recombination event. Individual recombination events can be studied in the ascomycetesfungi, becausethe products of a single meiosis are held together in a large cell called the ascus(or Iesscommonly. the tetrad). Even better is that in some fungi, the four haploid nuclei produced by meiosis are arranged in a linear order. (Actually, a mitosis occurs after the production of these four nuclei, giving a linear seriesof eight haploid nuclei.) i:iiiiifii .i-:.i,-ishows that each of these nuclei effectively represents the genetic character of one of the eight strands of the four chromos o m e sp r o d u c e db y t h e m e i o s i s . Meiosis in a heterozygoteshould generate four copies of each allele. This is seen in the m a j o r i t y o f s p o r e s .T h e r e a r e s o m e s p o r e s , though, with abnormal ratios. They are explained by the formation and correction of heteroduplex DNA in the region in which the alleles differ. The figure illustrates a recombination event in which a length of hybrid DNA occurs on one of the four meiotic chromosomes, a possibleoutcome of recombination initiated by a double-strandbreak. Suppose that two alleles differ by a single point mutation. When a strand exchange occurs to generate heteroduplex DNA, the two strands
4:4 parentalratio
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of atlowsdetermination fii;;l,i.it5 in the ascomycetes :ii;,:lr Sporeformation jnvolved in meiosis. of eachof the DNAstrands the genetic constitution
of the heteroduplex will be mispaired at the site of mutation. Thus each strand of DNA carries different genetic information. If no change is made in the sequence,the strands separateat the ensuing replication, each giving rise to a duplex that perpetuates its information. This segregation, event is called postmeiotic of becauseit reflectsthe separation DNA strands after meiosis. Its importance is that it demonstrates directly the existence of heteroduplex DNA in recombining alleles. Another effect is seen when examining recombination between alleles:the proportions of the alleles differ from the initial 4:4 ratio. This effect is called gene conversion. It describesa nonreciprocal transfer of information from one chromatid to another. Gene conversion results from exchange of strands between DNA molecules, and the change in sequence may have either of two causesat the molecular level: . As indicated by the double-strand break model in Figure I9.9, one DNA duPlex may act as a donor of genetic information that directly replacesthe corresponding sequencesin the recipient duplex by a processof gap generation, strand exchange, and gap filling. . As part of the exchange process,heteroduplex DNA is generatedwhen a single strand from one duplex pairs with its complement in the other duplex.
Recombination 475 for Interattetic Accounts 19.11GeneConversion
Repair systems recognize mispaired basesin heteroduplex DNA, and may then excise and replace one of the strands to restore complementarity. Such an event converts the strand of DNA representing one allele into the sequenceof the other allele. Gene conversion does not depend on crossingover, but is correlated with it. A large proportion of the aberrant asci show genetic recombination between two markers on either side of a site of interallelic gene conversion. This is exactly what would be predicted if the aberrant ratios result from initiation of the recombination processas shown in Figure 19.6, but with an approximately equal probability of resolvingthe structure with or without recombination (asindicated in Figure I9.8). The implication is that fungal chromosomes initiate crossingover about twice as often as would be expected {rom the measured frequency of recombination between distant genes. Variousbiasesare seenwhen recombination is examined at the molecular level. Either direction of gene conversion may be equally likely, or allele-specificeffectsmay create a preference for one direction. Gradients of recombination may fall away from hotspots. We now know that hotspots represent sitesat which doublestrand breaks are initiated, and that the gradient is correlated with the extent to which the gap at the hotspot is enlargedand converted to long single-strandedends (seeSection19.6,The Synaptonemal Complex Forms after DoubleS t r a n dB r e a k s ) . Some information about the extent of gene c o n v e r s i o n i s p r o v i d e d b y t h e s e q u e n c e so f members of gene clusters. Usually, the products of a recombination event will separateand become unavailable for analysisat the level of DNA sequence.When a chromosome carries two (nonallelic)genesthat are related,though, they may recombine by an "unequal crossingover" event (see Section 6.7, Unequal Crossi n g - O v e r R e a r r a n g e sG e n e C l u s t e r s ) .A l l w e need to note for now is that a heteroduplex may be formed between the two nonallelic genes. Gene conversion effectively converts one of the nonallelic genesto the sequenceof the other. The presenceof more than one gene copy r-1-. chromosome provides a footprint :"^rl: to trace theseevents.For example,if heteroduplex formation and gene conversion occurred
shows more divergence. Available sequences suggestthat gene conversion events may extend for considerable distances,up to a few thousand bases.
SupercoiLing Aftects the Structureof DNA . Supercoiting occurs on[yin a closedDNAwith no freeends. r A ctosed DNAcanbea circutar DNAmotecule or a [inearmolecute wherebothendsareanchored in a proteinstructure. o Anyctosed DNAmolecute hasa [inkingnumber, whichjs the sumof thetwistingnumber and writhingnumber. r Turnscanbe repartitioned betweenthe twisting number andwrithingnumber, sothat a change in the structure of the doublehelixcancomoensate for a change in its coitingin space. o Thelinkingnumber canbechanged onl.yby b r e a k i nagn dm a k i nbgo n d isn D N A .
The winding of the two strands of DNA around each other in the double helical structure makes it possible to change the structure by influenc-
M:i"9fttt S.Iii Linear DNAis extended (a),a circular DNA jfit is retaxed remains extended (nonsupercoiled) (b),but a supercoiled DNAhasa twistedandcondensed form(c;. Photos courtesy of Nirupam RoyChoudhury. InternatjonaL Centre forGenetic Engineering andBiotechnology (ICGEB).
:;:,x":#;s:r'ffi.:;":li:',:H*#liT 476
CHAPTER 19 Homotogous andSite-Specific Recombination
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Phageand bacterialDNAsalign
Staggeredcleavageslead to crosswisepairing
PhageDNA
GCTTTTTTATACTAA C G A A A A A A T A T GA T T BacterialDNA
Recombinantjunctionsare sealedto generateintegratedprophageDNA
q3;i,Jli= of attP andattBa[[owcrosscoresequence in the common i.ii,::SStaggered cteavages reciprocaI recom binantjunctions. wisereunion to generate
Integrases use a mechanism similar to that of type I topoisomerases,in which a break is made in one DNA strand at a time. The difference is that a recombinasereconnectsthe ends crosswise, whereas a topoisomerasemakes a break, manipulates the ends, and then rejoins the original ends. The basic principle of the system is that four molecules of the recombinase are required, one to cut each of the four strands of the two duplexes that are recombining. iltl:*ft| .1-'-.:.;r-il shows the nature of the reaction catalyzedby an integrase.The enzyme is a monomeric protein that has an active site capableof cutting and ligating DNA. The reaction involves an attack by a tyrosine on a phosphodiester bond. The 3'end of the DNA chain is linked through a phosphodiesterbond to a tyrosine in the enzyme. This releasesa free 5' hydroxyl end. TWo enzyme units are bound to each of the recombination sites. At each site, only one of the units attacks the DNA. The symmetry of the system ensuresthat complementary strandsare broken in each recombination site. The free 5'-OH end in each site attacks the 3'-phosphotyrosine link in the other site. This generates a Holliday junction. The structure is resolved when the other two enzyme units (which had not been involved in the first cycle of breakage and
reunion) act on the other pair of complementary strands. The successiveinteractions accomplish a conservative strand exchange, in which there are no deletions or additions of nucleotides at the exchange site, and there is no need for input of energy. The transient 3'-phosphotyrosine link between protein and DNA conservesthe energy of the cleaved phosphodiester bond. f]:i;i-iilfli.1;.]f.ishows the reaction intermediate, based on the crystal structure. (Trapping the intermediate was made possible by using a "suicide substrate,"which consistsof a synthetic DNA duplex with a missing phosphodiester bond, so that the attack by the enzyme does not generatea free 5'-OH end.) The structure of the Cre-/oxcomplex shows two Cre molecules, each of which is bound to a I5 bp length of DNA. The DNA is bent by - 100' at the center of symmetry. TWo of these complexes assemblein an antiparallel way to form a tetrameric protein structure bound to two synapsed DNA molecules. Strand exchange takes place in a central cavity of the protein structure that contains the central six basesof the crossover region. The tyrosine that is responsiblefor cleaving DNA in any particular half site is provided by the enzyme subunit that is bound to that half site. This is called czscleavage. This is true also for the Int integrase and XerD recombinase. The Activity Topoisomerase bination Resembles 19.18 site-specific Recom
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@
Lambda Recombination 0ccurs in an Intasome
r Lambda integration takesptacein a [argecomptex that atsoinctudes the hostproteinIHF. . Theexcision reaction requires Int andXisand recognizes the endsof the prophage DNAas substrates.
486
CHAPTER 19 H o m o t o g o uasn d S i t e - S p e c i fR i ce c o m b i n a t i o n
fl$il&$ tr.ff.;t*A synapsed loxArecombination comptex nas a tetramer of Crerecombinases, withoneenzvme monomer boundto eachhalfsite.Twoof the fouractivesitesarein use,actingon comptementary strands ofthetwo DNAsites.
Unlike the CreI lox recombination system, which requires only the enzyme and the two recombining sites,phage lambda recombination occurs in a large structure and has different components for each direction of the reaction (integration versus excision). A host protein called IHF is required for both integration and excision. IHF is a 20 kD protein of two different subunits, which are coded by the genes himA and himD.IHF is not an essentialprotein in E. coli,and is not required for homologous bacterial recombination. It is one of several proteins with the ability to wrap DNA on a surface. Mutations inthe him genes prevent lambda site-specificrecombination and can be suppressedby mutationsinl,int, which suggeststhat IHF and Int interact. Site-specific recombination can be performe d in vitro by Int and IHF. The in vitro reaclion requires supercoiling in attP,but not in attB. When the reaction is performed in vitro between two supercoiled DNA molecules, almost all of the supercoiling is retained by the products. Thus there cannot be any free intermediates in which strand rotation could occur. This was one of the early hints that the reaction proceeds through a Holliday junction. We now know that the reaction proceeds by the mechanism tlpical of this classof enzy'rnes, which is related to the topoisomeraseI mechanism (seeSectionI9.18, Site-SpecificRecombination ResemblesTopoisomeraseActivity). Int has two different modes of binding. The C-terminal domain behaves like the Cre recombinase. It binds to inverted sites at the core sequence,positioning itself to make the
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RepairSystems C H A P T EO RU T L I N E Introduction Repair Systems Correct Damage to DNA . Repair systems recognize DNA sequences thatdonotconformto standard basepairs. r Excision systems remove onestrand of DNAat thesiteof damage andthenreplace it. r Recombination-repair systems userecombination to reptace the double-stranded regionthat hasbeendamaged. o At[thesesystems areproneto introducing errorsduringthe repairprocess. r Photoreactivation is a nonmutagenic repairsystem that acts specificatty on pyrimidine dimers. ExcisionRepairSystemsin E. coli o TheUvrsystem -12 bases makes incisions aparton both sidesof damaged DNA,removes the DNAbetween them,and resynthesizes newDNA. Excision-Repair Pathways in Mammalian Cetls . Mammatian excision repairis triggered by directlyremoving a damaged basefromDNA. r Baseremoval triggers the removal andreptacement of a stretchof potynucleotides. r Thenatureof the baseremoval reaction determines whichof two pathways for excisionrepairis activated. . Thepot6/epathwayreptaces a longpotynucleotide stretch; the potppathway replaces a shortstretch.
r TheDNAsynthesized by the repairDNApotymerase often haserrorsin its sequence. o Proteins maybeidentithat affectthe fidetityof reptication in whichmutation causes an fiedby mutatorgenes, mutation. increased rateof spontaneous the Directionof MismatchRepair Controtting . Themut genescodefor a mismatch-repair systemthat dea[s with mismatched basepairs. . There at is a biasin the setection of whichstrandto reptace mismatches. r Thestrandtackingmethytation at a hemimethytat.a fflf is rep[aced. usuatty . Thisrepairsystemis usedto removeerrorsin a newlysyntheT is strandof DNA.At G-TandC-Tmismatches, thesized preferentiatty removed. Systemsin E. coli Recombination-Repair . Therecgenesof E.coli codefor the principalretrieva[ system. r Theprincipalretrieva[systemfunctionswhenreptication strandthat is opposite leavesa gapin a newtysynthesized sequence. a damaged r Thesingtestrandof another is usedto reptace duptex the gap. r Thedamaged is thenremoved andresynthesized. sequence
BaseFlippingIs Usedby Methylases and Glycosytases o UraciIandatkyl"ated bases arerecognized by glycosytases andremoved directty fromDNA. o Pyrimidine dimersarereversed by breaking the covatent bondsbetween them. r Methylases adda methylgroupto cytosine. e A[[thesetypesof enzyme actby ftippingthe baseout of the jt is either doubtehetixwhere, depending on the reaction, removed or is modified andreturned to the hetix.
to Recover Is an ImoortantMechanism Recombination from ReplicationErrors r A replication a damaged fork maystatlwhenit encounters siteor a nickin DNA. . A stalledfork mayreverse by pairingbetweenthe two newty strands. synthesized o A stalledforkmayrestartafterrepairingthe damage and to movethe forkforward. usea heticase o Thestructureof the stattedforkis the sameasa Holtiday junctionandmaybe converted to a duplexandDSBby reso[va5es.
Error-Prone Repairand MutatorPhenotypes . Damaged DNAthat hasnot beenrepaired DNApotycauses merase III to stallduringreplication. . DNApotymerase V (codedby umuCD) IV or DNApotymerase (codedbydinB)cansynthesize a comptement to the damaqedstrand.
RecATriggersthe S0SSystem . Damage to triggerthe S0Sresponse. RecA to DNAcauses of genescodingfor manyrepairenzymes. whichconsists r RecAactivates activityof LexA. the autocteavage o LexArepresses activates its the S05system; autocteavage thosegenes. on nextpoge Continued 499
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Eukaryotic CettsHaveConserved Repair Systems o TheyeastR/Dmutations, identified by phenotypes, radiation sensitjve arein genes that codefor repairsystems. . Xeroderma pigmentosum (XP)is a human disease caused by mutatjons in anyoneof repairgenes. severaI . A complex of proteins inctuding XPproductsandthe transcription factorTFnrprovidesa humanexcision-repair mechanism. r Transcriptionatly activegenesarepreferrepaired. entiatty
Introduction
Any event that introduces a deviation from the usual double-helical structure of DNA is a threat to the genetic constitution of the cell. Injury to
r!**lt[ f*.i Repairgenescan be ctassified into pathwaysthat usedifferentmechanisms to reverse or bypassdamageto DNA.
s00
CHAPTER 20 Repair Systems
A Common System Repairs Double-Strand Breaks o TheNHEJ pathway canligatebluntendsof DNA. dupLex o Mutations pathway in the NHEJ cause humandiseases. Summary
DNA is minimized by systems that recognize and correct the damage. The repair systemsare as complex as the replication apparatus itself, which indicates their importance for the survival of the cell. When a repair system reverses a change to DNA, there is no consequence.A mutation may result, though, when it fails to do so. The measured rate of mutation reflectsa balance between the number of damaging events occurring in DNA and the number that have been corrected (or miscorrected). Repair systemsoften can recognize a range of distortions in DNA as signals for action, and a cell is likely to have several systems able to deal with DNA damage. The importance of DNA repair in eukaryotes is indicated by the identification of >130 repair genesin the human genome. We may divide the repair systemsinto several general types, as summarized in g*.1 F:ili-8ftil . Some enzymes directly reverse specific sorts of damage to DNA. . There are pathways for base excision repair, nucleotide excision repair, and mismatch repair, all of which function by removing and replacing material. . There are systems that function by using recombination to retrieve an undamaged copy that is then used to replace a damaged duplex sequence. . The nonhomologous end-joining pathway rejoins broken double-stranded ends. . Several different DNA polymerases can resynthesize stretches of replacement DNA. Directrepair is rare and involves the reversal or simple removal of the damage. Photoreactivation of pyrimidine dimers, in which the offending covalent bonds are reversed by a light-dependent enzyme. is a good example.
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510
CHAPTER 20 RepairSystems
Gap in normalcopy is repaired
An E coliretrieval systemusesa normal strandof DNAto replace thegap[eftin a newtysynthesized strandopposite a siteof unrepaired damage.
prevents the damaged site from acting as a template. Replication is forced to skip past it. DNA polymerase probably proceeds up to or close to the pyrimidine dimer. The poll'rrerase then ceases synthesis of the corresponding daughter strand. Replication restarts some distance farther along. A substantial gap is left in the newly synthesized strand. The resulting daughter duplexes are different in nature. One has the parental strand containing the damaged adduct, which faces a newly synthesized strand with a lengthy gap. The other duplicate has the undamaged parental strand, which has been copied into a normal complementary strand. The retrieval system takes advantage of the normal daughter. The gap opposite the damaged site in the first duplex is filled by stealing the homologous single strand of DNA from the normal duplex. Following this single-strand exchange, the recipient duplex has a parental (damaged) strand facing a wild-type strand. The donor
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Some of the genesfor theseproteins are mutated in patients who have diseasesdue to deficiencies in DNA repair. The I(u heterodimer is the sensor that detects DNA damage by binding to the broken ends. The crystal structure in Ft{liltL }i.:.i5 shows why it binds only to ends. The bulk of the protein extends for about two turns along one face of DNA (lower). but a narrow bridge between the subunits, located in the center of the structure, completely encirclesDNA. This means that the heterodimer needs to slip onto a free end. I(u can bring broken ends together by binding two DNA molecules. The ability of I(u heterodimers to associatewith one another suggests that the reaction might take place as illustrated .".ii.i*. in iiiirlhli= This would predict that the ligasewould act by binding in the region between the bridgeson the individual heterodimers. Presumably I(u must change its structure in order to be releasedfrom DNA. Deficiency in DNA repair causes several human diseases.The common feature is that an inability to repair double-strand breaks in DNA leads to chromosomal instability. The instability is revealed by chromosomal aberrations, which are associatedwith an increased rate of mutation, which in turn leads to an increased susceptibility to cancer in patients with the disease.The basiccausecan be mutation in pathways that control DNA repair or in the genes that code for enzymesof the repair complexes. The phenotypes can be very similar, as in the c a s e o f A t a x i a t e l a n g i e c t a s i a( A T ) , w h i c h i s causedby failure of a cell cycle checkpoint pathway, and Nijmegan breakagesyndrome (NBS), which is caused by a mutation of a repair enzyme. One of the lessons that we learned from characterizing the repair pathways is that t h e y a r e c o n s e r v e di n m a m m a l s , y e a s t , a n d bacteria. The recessivehuman disorder of Bloom's syndrome is causedby mutations in a helicase gene (called BLM) that is homologous to recQ oI E coli. The mutation results in an increased frequency of chromosomal breaks and sister with other chromatid exchanges.BLM associates repair proteins as part of a large complex. One of the proteins with which it interactsis hMLHl, a mismatch-repair protein that is the human homolog of bacterialmutL.The yeasthomologs of these two proteins, Sgsl and MLHI, also associate, identifying these genes as parts of a wellconserved repair pathway. Nijmegan breakage syndrome results from mutations in a gene coding for a protein
recogendjoiningrequires Nonhomologous i:*li*:i: .r:i-:"ij'+ ends nitionof the brokenends,trimmingof overhanging by tigation. and/orfilting,fotlowed
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Breaks 517 Doubte-Strand Repairs System 20.1.2A Common
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(variously called Nibrin , p95, or NBS 1) that is a component of the Mre I I /Rad50 repair complex. Its involvement in repairing doublestrand breaks is shown by the formation of foci containing the group of proteins when human cells are irradiated with agents that induce double-strand breaks.After irradiation, the kinase ATMP (codedby the ,4Tgene) phosphorylates NBSl; this activates the complex, which localizesat sites of DNA damage. Subsequent stepsinvolve triggering a checkpoint (a mechanism that prevents the cell cycle from proceeding until the damage is repaired) and recruiting other proteins that are required to repair the damage
@
Summary
Bacteria contain systems that maintain the integrity of their DNA sequencesin the face of damage or errors of replication and that disting u i s h t h e D N A f r o m s e q u e n c e so f a f o r e i g n source. Repair systems can recognize mispaired, altered, or missing basesin DNA, as well as other structural distortionsof the double helix. Excision repair systemscleave DNA near a site of damage, remove one strand, and synthesize a new sequenceto replace the excisedmaterial. The Uvr system provides the main excisionrepair pathway in E. coli. The dam system is involved in correcting mismatches generated by incorporation of incorrect basesduring replication and functions by preferentially removing the base on the strand of DNA that is not methylated atthe dam target sequence.Eukaryotic homologs of the E. coli MrttSL system are involved in repairing mismatches that result from replication slippage;mutations in this pathway are common in certain types of cancer.
518
CHAPTER 20 RepairSystems
Recombination-repair systems retrieve information from a DNA duplex and use it to repair a sequence that has been damaged on both strands. The RecBC and RecF pathways both act prior to RecA, whose strand-transfer function is involved in all bacterial recombination. A major use of recombination-repair may be to recover from the situation created when a replication fork stalls. The other capacity of recA is the ability to induce the SOS response.RecA is activatedby damaged DNA in an unknown manner. It triggers cleavage of the LexA repressor protein, thus releasing repression of many loci, and inducing synthesisof the enzymes of both excision repair and recombination-repair pathways. Genesunder LexA control possessan operator SOSbox. RecA also directly activates some repair activities. Cleavageof repressorsof lysogenic phagesmay induce the phagesto enter the lytic cycle. Repair systemscan be connected with transcription in both prokaryotes and eukaryotes. Human diseasesare caused by mutations in genes coding for repair activities that are associated with the transcription factor TFIIH. They have homologs in the RAD genes of yeast, which suggeststhat this repair system is widespread. Nonhomologous end-joining (NHEJ) is a general reaction for repairing broken ends in (eukaryotic) DNA. The I(u heterodimer brings the broken ends together so they can be ligated. Several human diseasesare caused by mutations in enzymes of this pathway.
References Repair Systems Correct Damage to DNA Reviews Sancar, A., Lindsey-B oltz, L. A., Unsal-Kagmaz, K., and Linn, S. (2004). Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints. Annu. Rev.Biochem 73, )9-85. Wood, R. D., Mitchell, M., Sgouros, J., and Lindahl, T. (2001). Human DNA repair genes. Sclence29I, 1284-1289.
Excision-Repair Pathways i n M a m m a l i aC ne [ t s Reviews Barnes,D. E. and Lindahl,T. (2004t.Repairand geneticconsequences of endogenousDNA basedamagein mammaliancells.Annu Rev. G e n e )t 8 , 4 4 5 - 4 7 6 .
McCullough, A. I(., Dodson, M. L., and Lloyd, R. S. (1999\. Initiation of base excision repair: glycosylasemechanisms and structures. Annu Rev.Biochem 68, 255-285. Sancar, A., Lindsey-BoItz,L. A., Unsal-I(agmaz, I(., and Linn, S. (2004). Molecular mechanismsof mammalian DNA repair and the DNA damage checkpoints. Annu Rey.Biochem.T), )9-85. r ch Resea ICungland, A. and Lindahl, T. (1997lr. Second pathway for completion oI human DNA base excision-repair: reconstitution with puri{ied proteins and requirement for DNase IV ( F E N I ) . E M B OJ . 1 6 , j ) 4 r - 3 3 4 8 . Matsumoto, Y. and I(im, K. (1995). Excision of deoxyribose phosphate residues by DNA polymerase beta during DNA repair. Science 269, 699-702.
BaseFtipping Is Usedby Methytases andGlycosytases Resea rch Aas, P. A., Otterlei, M., Falnes,P A., Vagbe,C. B., Skorpen, F., Akbari, M., Sundheim, O., Bjoras, M., Slupphaug, G., Seeberg,E., and I(rokan, H. E.,(2003). Human and bacterial oxidative demethylases repair alkylation damage in both RNA and DNA. Nature 421,859-863. Falnes,P. A., Johansen,R. F., and Seeberg,E. (2002\. AlkB-mediated oxidative demethylation reverses DNA damaee in E. coli.Nature 4r9, t78-182. ICimasauskas,S., Kumar, S., Roberts,R. J. and Cheng, X. (1994\. HhaI methyltransferase flips its target base out of the DNA helix. Cel/ 76,357-J69. Lau, A. Y., Glassner,B. J., Samson,L. D., and Ellenberger,T. (2000). Molecular basisfor discriminating between normal and damaged basesby the human alkyladenine glycosyiase,AAG. Proc Natl Acad. Sci.USA 97 , tj57)-t)578. Lau, A. Y., Scherer,O. D., Samson,L., Verdine, G. L., and Ellenberger,T. (1998). Crystal structure of a human alkylbase-DNA repair enzyme complexed to DNA: mechanisms for nucleotide flipping and base excision. Cell 95, 249-258. Mol, D. D. et al. (1995). Crystal structure and mutational analysis of human uracil-DNA glycosylase:structural basis for specificity and catalysis.Cell 80, 869-878. Park, H. W., ICm, S. T., Sancar, A., and Deisenhofer, J. (1995). Crystal structure of DNA photolyase from E. coli.Science268, t866-1872. Sawa, R. et al. ( I 99 5 ) . The structural basis of specific base-excision repair by uracil-DNA glycosy\ase.Nature 373, 487-49).
Trewick, S. C., Henshaw, T. F., Hausinger, R. P., Lindahl, T., and Sedgwick, B. (2002). Oxidative demethylation by E coli AlkB directly reverts DNA base damage. Nature 419,
t74-r78. Vassylyev,D. G. et al. (1995). Atomic model of a p y r i m i d i n e d i m e r e x c i s i o nr e p a i re n z y m e complexed with a DNA substrate: structural basis for damaged DNA recognition. Cell 8), 773-782.
Repair Error-Prone and Mutator PhenotYPes Research Friedberg, E. C., Feaver, W. J., and Gerlach, V. L. (2000). The many facesof DNA polymerases: strategiesfor mutagenesis and for mutational avoidance. Proc Natl. Acad. Sci.USA 97, 5681-568). Goldsmith, M., Sarov-Blat,L., and Livneh, Z. (2000). Plasmid-encodedMucB protein is a DNA polymerase (pol RI) specializedfor lesion bypass in the presence oI MucA, RecA, and SSB. Proc.Natl Acad Sci.USA 97, 11227-tt23t. Maor-Shoshani, A., Reuven, N. B., Tomer, G., and Livneh, Z. (2000\ . Highly mutagenic replication by DNA polymerase V (UmuC) provides a mechanistic basisfor SOS untargeted mutagenesis.Proc.Natl. Acad Sci.USA97, 565-570' Wagner, J., Grtrz, P., Kim, S. R., Yamada, M., Matsui, K., Fuchs, R. P., and Nohmi, T. (1999). The dinB gene encodes a novel E. coli DNA polymerase, DNA pol IV involved in mutagenesis.Mol. Cell4,281-286.
the Direction Controlting RePair of Mismatch Review I(unkel,T. A., and Erie,D. A. (2005).DNA mismatchrepair.Annu Rev.Biochem.74,681-710' Resea rch Strand,M., Prolla,T. A., Liskay,R. M., and Petes, of tractsof simple T. D. (t993). Destabilization repetitiveDNA in yeastby mutationsaffecting DNA mismatchrepair.Nature)65,274-276. in E' coli Systems Recombination-Repair Review recombination West,S. C (1997i.Processingof intermediatesby the RuvABC proteins.Annu. Rev.Genet. ) l, 2lJ-244. rch Resea Bork,J. M. and Inman,R. B. (2001).The RecOR proteinsmodulateRecAprotein function at 5' DNA. EMBOJ.20, endsof single-stranded 7)t)-7)22.
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@
Recombination Is anImportant Mechanism to Recover fromReptication Errors
Reviews Cox, M. M., Goodman, M. F., Kreuzer. I(. N.. Sherratt, D. J., Sandler,S. J., and Marians, I(. J. (2000). The importance of repairing stalled replication forks. Nature 404, )7-41 . McGlynn, P and Lloyd, R. c. (2002). Recombinational repair and restart of damaged replication forks. Nat Rev.Mol. CellBiol. ), 859-870. Michel, B,, Viguera, E., Grompone, G., Seigneur,M., and Bidnenko, V (2001). Rescueof arrested replication forks by homologous recombination. Proc Natl Acad Sci.USA 98, 8l8l-8188. Resea r ch Courcelle,J. and Hanawalr, p C. (2003). RecAdependent recovery of arrested DNA replication forks. Annu. Rev.Genet 37, 6ll-646. I(uzminov, A. (2001 ). Single-strand interruptions in replicating chromosomes cause doublestrand breaks. Proc.Natl. Acad. Sci.USA 98. 8241-8246. Rangarajan, S., Woodgate, R., and Goodman, M. F. (19991.A phenotype for enigmaric DNA polymerase II: a pivotal role for pol II in replication restart in UV-irradiated Escherichiacoli Proc. Natl Acad Sci.USA 96, 9224-9229.
RecA Triggers theS0SSystem Research Tang,M. et al. ( I 999). UmuD'2C is an error-prone DNA polymerase, E. coli polY. Proc.Natl. Acad. Sci.USA 96, 8919-8924.
W
Eukaryotic CeltsHaveConserved Repair Systems
Reviews Iftogh, B. O. and Symington, L. S. (2004). Recombination proteins in yeast. Annu Rev.Genet. )8,2)j-27t. Prakash, S. and Prakash,L. (2002\. Tfanslesion DNA synthesis in eukaryotes: a one- or twopolymerase aff.air.GenesDev. 16, 1872-188). Sancar, A., Lindsey-B oltz, L. A., Unsal-I(agmaz, K., and Linn, S. (2004). Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints. Annu Rev.Biochem 7), j9-85. Resea r ch Friedberg, E. C., Feaver, W. J., and Gerlach, V. L. (2000). The many facesof DNA polymerases: strategiesfor mutagenesis and for mutational avoidance. Proc Natl. Acad. Sci.USA 97. 5 6 8l - t 6 8 3 . Johnson, R. E , Prakash, S., and Prakash, L. (1999). Efficient bypass of a thymine-thymine dimer by yeast DNA polymerase, Pol eta. Sclence28), 1001-1004,
520
CHAPTER 20 Repair Systems
Rattray,A. J. and Strathern, J. N. (2003). Errorprone DNA polymerases: when making a mistake is the only way to get ahead. Annu. Rev. Genet.37, )l-66. Reardon, J. T. and Sancar,A. (2003). Recognition and repair of the cyclobutane thymine dimer, a major cause oI skin cancers,by the human excision nuclease. GenesDev. 17, 2*9-2551. Wolner, 8., van I(omen, S., Sung, P., and Peterson, C. L. (2003). Recruitment of the recombinational repair machinery to a DNA doublestrand break in yeast.Mol Cell 12,221-232.
A Common System Repairs Doubte-StrandBreaks Review D'Amours, D. and Jackson,S. P. (2002). The Mrel I complex: at the crossroadsof DNA repair and checkpoint signalling. Nat. Rev.Mol. C e l lB i o l ) , 3 1 7 - 3 2 7 . Research Carney, J. P.,Maser, R. S., Olivares, H., Davis, E. M., Le Beau, M., Yates,J. R., Hays,L., Morgan, W. F., and Petrini, J. H. (1998). The hMrel I /hRad5O protein complex and Nijmegen breakage syndrome: linkage of double-strand break repair to the cellular DNA damage response.Cell93, 477 486. Cary, R. B., Peterson,S. R., Wang, J., Bear, D. G., Bradbury, E. M., and Chen, D. J. (1997). DNA looping by Ku and the DNA-dependenr protein kinase. Proc.Natl Acad. Sci.USA 94. 4267-4272. Ellis, N. A., Groden, J., Ye, T. 2., Straughen, J., Lennon, D. J., Ciocci, S., Proytcheva, M., and German, J. (1995). The Bloom's syndrome gene product is homologous to RecQ helicases.Cell 83, 655-666. Ma, Y., Pannicke, U., Schwarz, I(., and Lieber, M. R. (2002). Hairpin opening and overhang processingby an Artemis/DNA-Dependent protein kinase complex in nonhomologous end joining and V(D)J Recombinarion. Cell 108, 78t-794. Ramsden,D. A. and Gellert, M. (199S).I(u prorein stimulates DNA end joining by mammalian DNA ligases:a direct role for I(u in repair of DNA double-strand breaks. EMBO J. 17, 609-6t4. Varon, R. et al. (1998). Nibrin, a novel DNA double-strand break repair protein, is mutated in Nijmegen breakage syndrome. Cell 93, 467-476. Walker, J. R., Corpina, R. A., and Goldberg, J. (2001 ) . Structure of the I(u heterodimer bound to DNA and its implications for doublestrand break repair. Nature 412, 607-614.
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Unequal recombination results from mispairing by the cellular systemsfor homologous recombination. Nonreciprocal recombination results in duplication or rearrangement of loci (see Section 6.7, Unequal Crossing-Over RearrangesGene Clusters). Duplication of sequences within a genome provides a major s o u r c e o f n e w s e q u e n c e s .O n e c o p y o f t h e sequence can retain its original function, whereas the other may evolve into a new function. Furthermore, significant differences between individual genomes are found at the molecular level because of polymorphic variations causedby recombination. We saw in Section 6.14, MinisatellitesAre Useful for Genetic Mapping, that recombination between minisatellitesadjuststheir lengths so that every individual genome is distinct. Another major cause of variation is provided by transposable elements or transposons: these are discrete sequencesin the genome that are mobile-they are able to transport themselves to other locations within the genome. The mark of a transposon is that it does not utilize an independent form of the element (such as phage or plasmid DNA), but moves directly from one site in the genome to another. Unlike most otherprocessesinvolved in genome restructuring, transposition does not rely on any relationship between the sequencesat the donor and recipient sites. Transposons are restrictedto moving themselves,and sometimes additional sequences,to new sites elsewhere within the same genome; they are, therefore, an internal counterpart to the vectors that can transport sequences from one genome to another. They may provide the major source of mutations in the genome. Transposonsfall into two general classes. The groups of transposons reviewed in this chapter exist as sequencesof DNA coding for proteins that are able directly to manipulate DNA so as to propagate themselves within the genome. The transposons reviewed in Chapter 22, Retrovirusesand Retroposons, are related to retroviruses, and the source of their mobility is the ability to make DNA copies of their RNA transcripts; the DNA copies then become integrated at new sites in the genome. Transposons that mobilize via DNA are found in both prokaryotes and eukaryotes. Each bacterial transposon carries gene(s) that code for the enzyme activities required for its own transposition, although it may also require ancillary functions of the genome in which it
resides (such as DNA polymerase or DNA gyrase). Comparable systemsexist in eukaryotes, although their enzymatic functions are not so well characterized.A genome may contain both functional and nonfunctional (defective) elements. Often the majority of elements in a eukaryotic genome are defective, and have Iost the ability to transpose independently, although they may still be recognized as substrates for transposition by the enzymes produced by functional transposons. A eukaryotic genome contains a large number and variety of transposons.The fly genome has >50 types of transposons, with a total of several hundred individual elements. Transposable elements can promote rearrangements of the genome directly or indirectly: . The transposition event itself may cause deletions or inversions or lead to the movement of a host sequence to a new location. . Transposons serve as substrates for cellular recombination systemsby functioning as "portable regions of homology"; lwo coPies of a transPoson at different locations (even on different chromosomes) may provide sites for reciprocal recombination. Such exchanges result in deletions, insertions, inversions, or translocations. The intermittent activities'ofa transposon seem to provide a somewhat nebulous target f or natural selection. This concern has p r o m p t e d s u g g e s t i o n st h a t ( a t I e a s t s o m e ) transposableelements confer neither advantage nor disadvantageon the phenotype, but could constitute "selfish DNA"-DNA concerned only with their own propagation. Indeed, in considering transposition as an event that is distinct from other cellular recombination systems, we tacitly accept the view that the transposon is an independent entity that residesin the genome. Such a relationship of the transposon to the genome would resemble that of a parasite with its host. Presumably the propagation of an element by transposition is balanced by the harm done if a transposition event inactivates a necessary gene, or if the number of transposons becomes a burden on cellular systems' Yet we must remember that any transposition event conferring a selectiveadvantage-for example, a genetic rearrangement-will lead to preferential survival of the genome carrying the active transDoson.
21.1 Introduction
@
they were detected). Each type is given the preInsertion Sequences fix IS, followed by a number that identifies the AreSimpLeTransposition type. (The original classeswere numbered IS I to IS4; later classeshave numbers reflecting the Modules history
. An insertion sequence is a transposon that codes for theenzyme(s) needed for transposition ftanked by shortinverted terminaI repeats. r Thetargetsiteat whicha transposon is inserted is dupl.icated process duringthe insertion to form two repeats in directorientation at the endsof the transooson. o Thelengthof the directrepeatis 5 to 9 bp andis characteristic for any particu [ar transposo n.
Transposableelements were first identified at the molecular level in the form of spontaneous insertions in bacterial operons. Such an insertion prevents transcription and/or translation of the gene in which it is inserted. Many different types of transposableelementshave now been characterized. The simplest transposons are called insertion sequences (reflecting the way in which
| I
a i . '123456789
987654321 Transposase gene
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\ \
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987654321TACGT
Target repeat
Target repeat
Target Transooson ' repeat(bp)
Inverted repeat(bp)
Overall Target length(bp) setection
ls1 lS2
g
23
768
s
41
ls4 lS5
11-13 4
18 16
1927 1428
lS10R
9
22
1195 1329
AAAN2oTTT horspots NGCTNAGCN
lS50R 15903
I
I 18
1531 1057
hotspots random
I
random hotsoots
FJ{-;ltFil I 3.} Transposons haveinverted terminal repeats andgenerate direct repeats of flankingDNAat the targetsite.In this exampte. thetargetis a 5 bp sequence. Theendsof thetransposon consist of invertedrepeatiof 9 bp, wherethe numbers 1 through9 indjcatea sequence of basepairs.
524
C H A P T E2RL T r a n s p o s o n s
of their isolation. but not corresponding to the total number of elements so far isolated!) The IS elements are normal constituents of bacterialchromosomesand plasmids.A standard strain of E. coli is likely to contain several (<10) copies of any one of the more common IS elements. To describean insertion into a particular site, a double colon is used; so 7,::ISI describesan IS I element inserted into phage lambda. The IS elements are autonomous units, each of which codes only for the proteins needed to sponsor its own transposition. Each IS element is different in sequence,but there are some common features in organization. The structure of a generic transposon before and after insertion at a target site is illustrated in FSGiJftr ? 1-F,which also summarizes the details of some common IS elements. An IS element ends in short inverted terminal repeats; usually the two copies of the repeat are closely related rather than identical. As illustrated in the figure, the presence of the inverted terminal repeats means that the same sequenceis encountered proceeding toward the element from the flankine DNA on either side of it. When an IS element transposes,a sequence of host DNA at the site of insertion is duplicated. The nature of the duplication is revealed by comparing the sequenceof the target site before and after an insertion has occurred. Figure 2I.2 shows that at the site of insertion, the IS DNA is always flanked by very short direct repeats. (In this context, "direct" indicates that two copies of a sequence are repeated in the same orientation, not that the repeatsare adjacent.) In the original gene (prior to insertion), however, the target site has the sequenceof only one of these repeats. In the figure, the target site consistsof ATGCA , the sequenc. After rransposirion,one i;CGi. copy of this sequence is present on either side of the transposon. The sequence of the direct repeat varies among individual transposition events undertaken by a transposon, but the Iength is constant for any particular IS element (a reflection of the mechanism of transposition). The most common length for the direct repeats is 9 bp. An IS element therefore displays a characteristic structure in which its ends are identified by the inverted terminal repeats, whereas the adjacent ends of the flanking host DNA are
identified by the short direct repeats. When observed in a sequence of DNA, this type of organization is taken to be diagnostic of a transposon, and suggeststhat the sequence originated in a transposition event. Most IS elements insert at a variety of sites within host DNA. Some, though, show (varying degreesof ) preferencefor parlicular hotspots. The inverted repeats define the ends of a transposon. Recognition of the ends is common to transposition events sponsoredby all types of transposon.Cli-acting mutations that prevent transposition are located in the ends, which are recognizedby a protein(s) responsiblefor transposition. The protein is called a transposase. All the IS elements except IS I contain a single long coding region, which starts just inside the inverted repeat at one end and terminates just before or within the inverted repeat at the other end. This codesfor the transposase.IS I has a more complex organization. with two separate reading frames; the transposaseis produced by making a frameshift during translation to allow both reading frames to be used. The lrequencyof lranspositionvariesamong different elements. The overall rate of transposition is -I0-l to l0a per element per generation. Insertions in individual targets occur at a level comparable with the spontaneous mutation rate, usually - l0-5 to l0-7 per generation. R e v e r s i o n ( b y p r e c i s ee x c i s i o n o f t h e I S e l e ment) is usually infrequent, with a range of rates of l0-6 to l0-10 per generation, which is -103 times lessfrequent than insertion.
drug marker(s) is flanked on either side by " arms" that consist of IS elements. The arms may be in either the same or (more commonly) inverted orientation. Thus a composite transposon with arms that are direct repeatshas the structure
If the arms are inverted repeats, the structure is
The arrows indicate the orientation of the arms, which are identified as L and R according to an (arbitrary) orientation of the genetic map of the transposon from left to right. The structure of a composite transposon is illustrated in more detail in j'ii.l.i!ii :r':.:i,which also summarizes
Transposons Composite HaveIS Modules in additionto Transposons cancarryothergenes thosecodingfor transposition. havea centralregion transposons Composite flankedby an IS etement at eachend. of a Eitheroneor bothof the IS etements maybe abteto undertake transposon composite transposition. maytranspose asa unit. A composite transposon at eitherendmavalso but an activeIS element i ndependent[y. transpose s o m e t r a n s p o s o n s c a r r y d r u g r e s i s t a n c e( o r other) markers in addition to the functions concerned with transposition. These transposons are named Tn followed by a number. One class of larger transposonsare called cornposite elements, because a central region carrying the
hasa centraIregion transposon :l:i.;:IA composite +:j{:1-lIil ftanked byIS mod(suchasdrugresistance) markers carrying If haveshortjnvertedtermjnalrepeats. utes.Themodules theshort orientation, arein inverted themsetves themodules are at the endsof the transposon inverted termina[repeats identicat.
HaveIS Modules 525 Transposons 21.3 Composite
transposon movesagain
New transposoncreatedby mobilization of lS10 modulesin alternativeorientation
r:.r:-;iiri,. i: I ..: Two15L0modules createa composite transp o s o nt h a t c a n m o b i t i z a e n y r e g i o no f D N At h a t L i e s between them.WhenTn10is partof a smatlcircular motecule,the IS10repeats cantranspose ejthersideof the circ[e.
the properties of some common composite transposons. Arms consist of IS modules, and each module has the usual structure ending in inverted repeats; as a result the composite transposon also ends in the same short inverted repeats. In some cases,the modules of a composite transposon are identical, such as Tn9 (direct r e p e a t so f I S I ) o r T n 9 0 3 ( i n v e r t e d r e p e a t so f I S 9 0 l ) . I n o t h e r c a s e st,h e m o d u l e s a r e c l o s e l y related, but not identical. Thus we can distinguish the L and R modules in Tnl0 or in Tn5. A functional IS module can transposeeither itself or the entire transposon.When the modules of a composite transposon are identical, presumably either module can sponsormovement of the transposon, as in the caseof Tn9 or Tn903. When the modules are different. they may differ in functional ability, so transposition
526
C H A P T E2R L Transposons
can depend entirely or principally on one of the modules, as in the caseof Tnl0 or Tn5. we assume that composite transposons evolved when two originally independent modules associatedwith the central region. Such a situation could arise when an IS element transposes to a recipient site close to the donor site. The two identical modules may remain identical or diverge. The ability of a single module to transpose the entire composite element explains the lack of selective pressure for both modules to remain active. What is responsible for transposing a composite transposon instead of just the individual module? This question is especiallypressing in caseswhere both the modules are functional. In the example of Tn9, where the modules are IS I elements, presumably each is active in its own right as well as on behalf of the composite transposon. Why is the transposon preserved as a whole, instead of each insertion sequence looking out for itself? TWo IS elements in fact can transpose any sequence residing between them, as well as t h e m s e l v e s . i : * i . j i t l ; l : : . r is h o w s t h a t i f T n l 0 resides on a circular replicon, its two modules can be considered to flank either the /e/Rgene of the original Tnl0 or the sequencein the other part of the circle. Thus a transposition event can involve either the original Tnl0 transposon (marked by the movement of rerR)or the creation of the new "inside-out" transposonwith the alternative central region. Note thatboth the original and "inside-our" transposons have inverted modules, but these modules evidently can function in either orientation relative to the central region. The frequency of transposition for composite transposons declines with the distance between the modules. Thus length dependence is a factor in determining the sizesof the common composrte transposons. A major force supporting the transposition of composite transposons is selection for the marker(s) carried in the central region. An ISI0 module is free to move around on its own, and mobilizes an order of magnitude more frequently than TnI0. Tnl0 is held together by selection for tetR,though, so that under selective conditions, the relative frequency of intact Tnl0 transposition is much increased. The IS elements code for transposaseactivities that are responsibleboth for creating a target site and for recognizing the ends of the transposon. Only the ends are needed for a transposon to serve as a substrate for transposition.
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the transposon, separating its strands and terminating at its ends. Replication is probably accomplishedby host-codedfunctions. At this juncture, the structure has become a cointegrate, possessingdirect repeatsof the transposon at the junctions between the replicons (as can be seen by tracing the path around the cointegrate).
Nonrep[icative Proceeds Transposition andReunion by Breakage jf a crossover resutts transposition Nonrepticative pairof donor is nickedon the unbroken structure on eithersideof the andthetargetstrands strands areligated. transposon transposition for nonreplicative Twopathways thefirst pairof to whether differaccording strands arejoinedto thetargetbefore transposon pairarecut (Tn5).or whether a[[four the second joiningto thetarget(Tn10). strands arecut before
The crossoverstructure can also be used in nonreplicative transposition. The principle of nonreplicative transposition by this mechanism is that a breakageand reunion reaction allows the target to be reconstructed with the insertion of the transposon; the donor remains broken. No cointegrateis formed. :-ir,.ii-:i: ,,i- ::: shows the cleavageeventsthat generate nonreplicative transposition of phage Mu. Once the unbroken donor strands have been nicked, the target strands on either side of the transposon can be ligated. The singlestranded regions generated by the staggered cuts must be filled in by repair synthesis. The product of this reaction is a target replicon in which the transposonhas been insertedbetween repeats of the sequence created by the original single-strand nicks. The donor replicon has a double-strand break acrossthe site where the transposonwas originally located. Nonreplicative transposition can also occur by an alternative pathway in which nicks are made in target DNA, but a double-strand break is made on either side of the transposon, releasing it entirely from flanking donor sequences(as envisagedin Figure 21.7). This "cut and paste" pathway is used by Tnl0, as illustrated in ;;;l--;1'r::.iir.
A neat experiment to prove that Tnl0 transposesnonreplicatively made use of an artificially constructed heteroduplex of Tnl0 that contained singlebasemismatches.If transposition involves
when results transposition i:"ri.tiiilii !. l5 Nonrepticative by nicking.Thisinserts is reteased structure a crossover bythedirect intothetargetDNA,ftanked thetransposon repeats ofthetarget.andthedonoris teftwitha doubLestrandbreak.
Tn is joinedto target
sequenof Tn10arecteaved i:i-li-ilii:.ii,ii: Bothstrands tarisjoinedto thenjcked andthenthetransposon tiaLty, get site.
replication, the transposon at the new site will contain information from only one of the parent Tnl0 strands. If. however, transposition takes place by physical movement of the existing transposon, the mismatches will be conserved at the new site. This proved to be the case. and Reunion by Breakage Proceeds Transposition 21..8Nonrepticative
533
r : i.ij *i .'l't..:.: Cteavage of Tn5 fromflankingDNAinvolves nicking,interstrand reaction,and hairpjncleavaqe.
i:!r.: : ;+-: :: Lr'i. l::r Each subunit of theTn5 transDosase nas oneendof thetransposon located in its activesiteano alsomakescontactat a differentsite with the otner endof thetransposon.
The basicdifferencein Figure 2l.l6 from the m o d e l o f F i g u r e 2 1 . 1 5 i s t h a t b o t h s t r a n d so f Tn10 are cleavedbefore any connection is made to the target site. The first step in the reaction is recognition of the transposon ends by the transposase,forming a proteinaceous structure within which the reaction occurs. At each end of the transposon,the strandsare cleavedin a specificorder: the transferred strand (the one to be connected to the target site) is cleavedfirst, followed by the other strand. (This is the same order as in the Mu transposition of Figure 2 I . l4 a n d F i g u r e2 1 . 1 5 . ) Tn5 also transposesby nonreplicative transposition, and ;'i*l.it:tf,l i . ].trshows the interesting cleavagereaction that separatesthe transposon
534
C H A P T E2R 1 Transposons
from the flanking sequences.First one DNA strand is nicked. The 3'-OH end that is released then attacks the other strand of DNA. This releasesthe flanking sequence and joins the two strands of the transposon in a hairpin. An activated water molecule then attacks the hairpin to generate free ends for each strand of the transposon. In the next step, the cleaved donor DNA is released,and the transposon is joined to the nicked ends at the target site. The transposon and the target site remain constrained in the proteinaceous structure created by the transposase(and other proteins).The double-strand cleavage at each end of the transposon precludes any replicative-type transposition and forces the reaction to proceed by nonreplicative transposition, thus giving the same outcome as in Figure 21.14, but with the individual cleavageand joining steps occurring in a different order. The Tn 5 and Tn I 0 transposases both function as dimers. Each subunit in the dimer has an active site that successivelycatalyzesthe double-strand breakage of the two strands at one end of the transposon, and then catalyzesstaggered cleavageof the target site. {:i{llJfti;ii.'lil illustrates the structure of the Tn5 transposase bound to the cleavedtransposon.Each end of the transposon is located in the active site of one subunit. One end of the subunit also contactsthe other end of the transposon.This controls the geometry of the transposition reaction. Each of the active siteswill cleave one strand of the target DNA. It is the geometry of the complex that determines the distancebetween these siteson the two target strands (nine basepairs in the caseof Tn5).
TnATransposition
Requires Transposase andResolvase . Repticative transposition of TnArequires a transposase to formthe cointegrate structure and a resotvase to retease the two replicons. r Theactionof the resolvase resembtes [ambda Int proteinandbetongs to thegeneral famityof topoisomerase-tike site-specifi c recombination reactions. whichpassthroughan intermediate in whichthe proteinis covalently boundto the DNA. Replicative transposition is the only mode of mobility of the TnA family, which consists of
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by a sector of the new phenotlpe residing within the tissue of the original phenotype. The size of the sector depends on the number of divisions in the lineage giving rise to it, so the size of the area of the new phenotype is determined by the timing of the change in genotype. The earlier its occurrence in the cell lineage, the greater the number of descendantsand thus the sizeof patch in the mature tissue.This is seen most vividly in the variation in kemel color, when patchesof one color appear within another color. Insertion of a controlling element may affect the activity of adjacentgenes.Deletions,duplications, inversions, and translocations all occur at the siteswhere controlling elements are present. Chromosome breakageis a common consequenceof the presenceof some elements.A unique feature of the maize system is that the activities of the controlling elements are regulated during development. The elements transpose and promote genetic rearrangements at characteristic times and frequencies during plant development. The characteristicbehavior of controlling elements in maize is typified by the Ds element, which was originally identified by its ability to provide a site for chromosome breakage. The consequencesare illustrated in r:i,.::.:i . . :. Consider a heterozygote in which Ds lies on one homolog between the centromere and a seriesof dominant markers. The other homolog lacks Ds and has recessivemarkers (C bz, andwx). Breakage at Ds generatesan acentric fragment carrying the dominant markers. As a result of its lack of a centromere, this fragment is lost at mitosis. Thus the descendant cellshave only the recessivemarkers carried by the intact chromosome. This gives the type of situation whose results are depicted in F i g u r e2 I . 2 2 . :' :.!.:.i'ri, ,., shows that breakageat Ds leads to the formation of two unusual chromosomes. These are generated byjoining the broken ends of the products of replication. One is a U-shaped acentric fragment consisting of the joined sister chromatids for the region distal to Ds (on the left as drawn in the figure). The other is a U-shaped dicentric chromosome comprising the sister chromatids proximal to Ds (on its right in the figure). The latter structure leadsto the classic cycle illustrated in breakage-fusion-bridge the figure. Follow the fate of the dicentric chromosome when it attempts to segregate on the mitotic spindle. Each of its two centromeres pulls toward an opposite pole. The tension
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. Eachfamilyof transposons in maizehasboth autonomous andnonautonomous controtting e[ements. o Autonomous controlting elements codefor proteins that enabte themto transoose. r Nonautonomous controtting elements have mutations that eliminate theircapacity to catalyze transposition, buttheycantranspose whenan provides autonomous element the necessary proteins. r Autonomous controtling elements havechanges of phase, whentheirproperties alterasa resuttof changes in the stateof methytation.
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'r' . ' Dsprovides -, a siteto initiatethe chromatidbreakage-fusion-bridge cycle.Theproducts canbe fotlowed by clonaIanatysis. breaks the chromosome at a random site between the centromeres. In the example of the figure, breakageoccursbetween loci A and B, with the result that one daughter chromosome has a duplication of A, whereas the other has a deietion. If A is a dominant marker, the cells with the duplication will retain a phenotype, but cells with the deletion will display the recessive a phenotype. The breakage-fusion-bridgecycle continues through further cell generations, allowing genetic changesto continue in the descendants. For example, consider the deletion chromo-
C H A P T E2RL T r a n s p o s o n s
The maize genome contains several families of controlling elements. The numbers, types, and locations of the elements are characteristic for each individual maize strain. They may occupy a significant part of the genome. The members of each family are divided into two classes: . Autonomous controlling elements have the ability to exciseand transpose. As a result of the continuing activity of an autonomous element, its insertion at any locus createsan unstable or "mutable" allele. Loss of the autonomous element itself, or of its ability to transpose, converts a mutable allele to a stableallele. . Nonautonomous controlling elements are stable;they do not transpose or suffer other spontaneous changes in condition. They become unstable only when an autonomous member of the same family is present elsewhere in the genome. When complemented it trans by an autonomous element, a nonautonomous element displays the usual range of activities associated with autonomous elements, including the ability to transposeto new sites.Nonautonomous elements are derived frorn autonomous elements by loss of transacting functions needed for transposition.
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they are located and often causechromosomal breaks. The result of the transpositions is therefore to inactivate the genome.
P Elements AreActivated in the GermLine o P elements areactivated in the germtine of P malex M femate crosses because a tissuespecific spticing eventremoves oneintron, whichgenerates the codingsequence for thetransoosase. . TheP etement alsoproduces a repressor of transposition, whichis inherited maternatty in the cytoptasm. e Thepresence of the repressor exptains whyM male x Pfematecrosses remainferti[e.
lntron1 Intron2 Intron3
I
I Transcription V
I 66 kD repressor t.+-+:;5!igqFffi
Germline
mRNA
Activation of P elements is tissue-specific:it occursonly in the germline. P elementsare transcribed, though, in both germline and somatic t i s s u e s . T i s s u e - s p e c i f i c i t yi s c o n f e r r e d b y a change in the splicing pattern. riiii:riii: , : ; I depicts the organization of the element and its transcripts. The primary transcript extends for 2.5 kb or 3.0 kb, the difference probably reflecting merely the leakiness of the termination site. TWo protein products can be produced: . In somatic tissues, only the first two introns are excised, creating a coding region of ORF0-ORFI -ORF2. Translation of this RNA yields a protein of 6 6 k D . T h i s p r o t e i n i s a r e p r e s s o ro { transposon activity. . In germline tissues,an additional splicing event occurs to remove intron 3. This connects all four open reading frames into an mRNA that is translated to generate a protein of 87 kD. This protein is the transposase. TWo types of experiment have demonstrated that splicing of the third intron is needed for transposition. First, if the splicing junctions are mutated in vitro and the P element is reintroduced into flies, its transposition activity is abolished. Second, if the third intron is deleted, so that ORF3 is constitutively included in the mRNA in all tissues, transposition occurs in somatic tissues as well as the germline. Thus whenever ORFI is splicedto the precedingreading frame, the P element becomesactive. This is the crucial regulatory event, and usually it occurs only in the germline.
.e* irr
Short RNA Long RNA
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IT
Y 87 kDtransposase i,iiii.i{'11 .' 1,,:rrr hasfourexons.Thefirst TheP etement at[four in somatic expression; together threearespliced expression. in germtine aresp[iced together
What is responsiblefor the tissue-specific splicing? Somatic cells contain a protein that binds to sequencesin exon 3 to prevent splicing of the last intron (seeSection26.I2, AIter' native Splicing Involves Differential Use of Splice Junctions). The absenceof this protein in germline cells allows splicing to generate the mRNA that codesfor the transposase. Ttanspositionof a P element requires -I50 bp of terminal DNA. The transposasebinds to l0 bp sequencesthat are adjacent to the 3l bp inverted repeats.Transpositionoccursby a nonreplicative "cut and paste" mechanism resembling that of Tnl0. (It contributes to hybrid dysgenesisin two ways: Insertion of the transposed element at a new site may causemutations, and the break that is left at the donor site-see Figure 21 .7-h'as a deleterious effect.) It is interesting that, in a significant proportion of cases,the break in donor DNA is repaired by using the sequenceof the homologous chromosome. If the homolog has a P element, the presenceof a P element at the donor site may be restored (so the event resemblesthe result of a replicative transposition). If the homolog lacks a P element, repair may generate a sequence lacking the P element, thus apparently
in the Germline 545 AreActivated P Elements 21..1,5
providing a preciseexcision (an unusual event in other transposablesystems). The dependence of hybrid dysgenesison the sexual orientation of a crossshows that the cytoplasm is important, as well as the P factors themselves.The contribution of the cytoplasm is describedas the cytotype; a line of flies containing P elements has P cytotype, whereas a line of flies lacking P elementshas M cytotype. Hybrid dysgenesisoccurs only when chromosomes containing P factors find themselvesin M cytotype, that is, when the male parent has P elements and the female parent does not. Cytotype shows an inheritable cytoplasmic e{fect; when a cross occurs through P cytotype (the female parent has P elements),hybrid dysgenesisis suppressedfor severalgenerationsof crosseswith M female parents. Thus something in P cytotype, which can be diluted out over some generations,suppresseshybrid dysgenesis. The effect of cytotlpe is explained in molecu l a r t e r m s b y t h e m o d e l o f i i { , i . i i : i i.:: i " - : i l .I t depends on the ability o{ the 66 kD protein to represstransposition. The protein is provided
as a maternal factor in the egg.In a P line, there must be sufficient protein to prevent transposition from occurring, even though the P elements are present. In any cross involving a P female, its presenceprevents either synthesis or activity of the transposase.When the female parent is M type, though, there is no repressor in the egg, and the introduction of a P element from the male parent results in activity of transposasein the germline. The ability of P cytotlpe to exert an effect through more than one generation suggeststhat there must be enough repressorprotein in the egg, and it must be stable enough, to be passedon through the adult to be present in the eggsof the next generation. Strains of.D. melanogasterdescended from flies caught in the wild more than 30 years ago are always M. Strains descendedfrom flies caught in the past l0 years are almost always P.Does this mean that the P element family has invaded wild populations of D. melanogaster in recent years? P elements are indeed highly invasive when introduced into a new population; the source of the invading element would have to be another species. Hybrid dysgenesis reduces interbreeding, and thus it is a step on the path to speciation. Supposethat a dysgenicsystem is createdby a transposable element in some geographic Iocation. Another element may create a different system in some other location. Flies in the two areaswill be dysgenicfor two (or possiblymore) systems.If this renders them intersterile and the populations become genetically isolated, further separationmay occur. Multiple dysgenic systems therefore lead to inability to mateand to speciation.
Summary
::iliriltii;i i.:;i; Hybriddysgenesis is determined by theinteractions between P etements in the genome and66 kDrepressor in the cytotype.
C H A P T E2R L Transposons
Prokaryotic and eukaryotic cells contain a variety of transposonsthat mobilize by moving or copying DNA sequences.The transposon can be identified only as an entity within the genome; its mobility doesnot involve an independent form. The transposon could be selfish DNA, concerned only with perpetuating itself within the resident genome; if it conveys any selectiveadvantage upon the genome, this must be indirect. All transposons have systems to limit the extent of transposition, because unbridled transposition is presumably damaging, but the molecular mechanisms are different in each case. The archetypal transposon has inverted repeats at its termini and generates direct repeats
of a short sequence at the site of insertion. The simplest types are the bacterial insertion sequences(IS), which consistessentiallyof the inverted terminal repeats flanking a coding frame(s) whose product(s) provide transposition activity. Composite transposons have terminal modules that consist of IS elements; one or both of the IS modules provides transposase activity, and the sequencesbetween them (often carrying antibiotic resistance)are treated as passengers. The generation of target repeats flanking a transposon reflects a common feature of transposition. The target site is cleaved at points that are staggeredon each DNA strand by a fixed distance (often five or nine base pairs). The transposon is in effect inserted between protruding single-stranded ends generated by the staggeredcuts. Thrget repeats are generated by filling in the single-stranded regions. IS elements, composite transposons,and P elements mobilize by nonreplicative transposition, in which the element moves directly from a donor site to a recipient site. A single transposase enzyme undertakes the reaction. It occurs by a "cut and paste" mechanism in which the transposon is separated from flanking DNA. Cleavage of the transposon ends, nicking of the target site, and connection of the transposon ends to the staggered nicks all occur in a nucleoprotein complex containing the transposase.Loss of the transposon from the donor createsa double-strand break whose fate is not clear.In the caseof Tn I0, transposition becomespossibleimmediately after DNA replication, when sites recognized by t}i'e dam methylation system are transiently hemimethylated. This imposesa demand for the existence of two copiesof the donor site, which may enhance the cell's chances for survival. The TnA family of transposons mobilize by replicative transposition. After the transposon at the donor site becomes connected to the target site, replication generatesa cointegrate molecule that has two copies of the transposon. A resolution reaction that involves recombination between two particular sitesthen frees the two copies of the transposon, so that one remains at the donor site and one appears at the target site. TWoenzymes coded by the transposon are required: Ttansposaserecognizesthe ends of the transposon and connects them to the target site, and resolvaseprovides a sitespecific recombination function. Phage Mu undergoes replicative transposition by the same mechanism as TnA. It also can use its cointegrate intermediate to transposeby
a nonreplicative mechanism. The difference between this reaction and the nonreplicative transposition of IS elements is that the cleavage events occur in a different order. The best characterizedtransposonsin plants are the controlling elements of maize, which fall into several families. Each family contains a single type of autonomous element that is analogous to bacterial transposons in its ability to mobilize. A family also contains many different nonautonomous elements that are derived by mutations (usually deletions) of the autonomous element. The nonautonomous elements lack the ability to transpose, but display transposition activity and other abilities of the autonomous element when an autonomous element is present to provide the necessary tr ans- acting f unctions. In addition to the direct consequences of insertion and excision, the maize elements may also control the activities of genesat or near the sites where they are inserted; this control may be subject to developmental regulation. Maize elements inserted into genes may be excised from the transcripts, which explains why they do not simply impede gene activity. Control of target gene expression involves a variety of molecular effects, including activation by provision of an enhancer and suppressionby interference with posttranscriptional events. Tiansposition of maize elements (in particular Ac) is nonreplicative, and probably requires only a single transposaseenzyme coded by the element. Transposition occurs preferentially after replication of the element. It is likely that there are mechanisms to limit the frequency of transposition. Advantageous rearrangements of the maize genome may have been connected with the presence of the elements. P elements in D. melanogasterare responsible for hybrid dysgenesis,which could be a forerunner of speciation. A cross between a male carrying P elements and a female lacking them generates hybrids that are sterile. A P element has four open reading frames, which are separated by introns. Splicing of the first three ORFs generatesa 66 kD repressorand occurs in all cells. Splicing of all four ORFs to generate the 87 kD transposaseoccurs only in the germline by a tissue-specific splicing event. P elements mobilize when exposed to cytoplasm lacking the repressor.The burst of transposition events inactivates the genome by random insertions. Only a complete P element can generate transposase,but defective elements can be mobilized intrans bv the enzvme.
21.16Summary 547
References @
Introduction
Reviews Campbell, A. ( I98l ). Evolutionary significanceof accessoryDNA elements inbacteria. Annu. Rev.Immunol. 35, 55-83 Finnegan, D. J. (1985). Transposableelements in eukaryotes. Int Rev.Cytol 9), 281-)26.
@
Reviews B e r g ,D . E . a n d H o w e , M . , e d s . ( I 9 8 9 ) . M o b i l eD N A Washington, DC: American Societyfor Microbiology Press. Calos,M. and Miller, J. H. (1980). Transposable elemenrs.Cell20, 579-595 Craig, N. L. (1997). Targetsite selectionin transposilio}l. Annu Rey.Biochem.66, 437 47 4. Galas,D. J. and ChandleaM. (1989).Bacterialinsertion sequence.In Berg, D. E and Howe, M., eds.,Mobile DNA Washington, DC: American Society for Microbiology Press,pp. 109-I62. I(leckner, N.( 1977). Ttanslocatableelements in prokaryotes. Cell ll, ll-23. I(leckner, N. (1981). Transposableelements in prokaryotes.Annu Rev.Genet.15,341-404. r ch Resea G r i n d l e y ,N . D . ( 1 9 7 8 ) . I S l i n s e r t i o ng e n e r a t e s duplication of a 9 bp sequenceat its target site. Cell 13, 419-426. Johnsrud, L , Calos,M. P.,and Miller, J. H. (1978t. The transposon Tn9 generates a 9 bp repeated sequence during integration. Cell 15, t209-12r9.
Composite Transposons Have ISModutes
KCVIEW I(eckner, N. (1989).Tnl0 transposon.In Berg, D. E. and Howe, M. M., eds.,Mobile DN,4. Washington, DC: American Society for Microbiology Press,pp. 227-268.
@
Transposition 0ccursby BothRepticative andNonrepticative Mechanisms
Reviews Craig, N. L. (19971.Targetsite selectionin transposilion Annu Rev.Biochem 66,4)7-474. Grindley, N. D. and Reed, R. R. (1985). Transpositional recombination in prokaryotes Annu. Rev.Biochem 54, 86)-896. Haren, L., Ton-Hoang, B., and Chandler, M. (19991.Integrating DNA: transposasesand retroviral integrases.Annu Rev.Microbiol. 5), 245-28t. Scott,J. R. and Churchward, G. G. (1995). Conjugative transposition.Annw. Rev.Immunol 49, 367-J97.
548
CHAPTER 21 Transoosons
Reviews Mizuuchi, K. (1992). Transpositionalrecombination: mechanistic insights from studies of Mu and other elements. Annu. Rev.Biochem.61. l0I l-I051. Pato, M. L. ( 1989). Bacteriophagemu. In Berg, D. E. and Howe, M., eds.,Mobile DNA. Washington, DC: American Society for Microbiology Press, pp.23-52.
Insenron nresrmpre sequences TransoositionModules
t@
Intermediates for Transposition Common
Research Aldaz, H., Schuster,E., and Baker, T. A. (1996). The interwoven architecture of the Mu transposase couples DNA synthesis to catalysis. Cel/ 85,257-269. Savilahti,H. and Mizuuchi, K. (1996). Mu transpositional recombination: donor DNA cleavage and strand transfer in trans by the Mu transpose. Cell 85, 27 l-280.
Nonrepticative Transposition Proceeds by Breakage andReunion Resea rch Bender, J. and I{eckner, N. (I986). Genetic evidence that Tnl0 transposesby a nonreplicative mechanism Cell 45,801-815. Bolland, S. and Ifleckner, N. (1996). The three chemical steps of Tn I 0/IS I 0 transposition involve repeated utilization of a single active site. cell 84,223-2)3. Davies, D. R., Goryshin, L Y., Reznikoff, W. S., and Rayment, I. (2000). Three-dimensional structure of the Tn5 synaptic complex transposition intermediate. Science289, 77 -85. Haniford, D. B., Benjamin, H. W, and I(leckner, N. ( I99 I ) . I(netic and structural analysis of a cleaved donor intermediate and a strand transfer intermediate in Tnl0 transposition. Cell64, 17l-179. I(ennedy, A. I(., Guhathakurta, A., I(leckner, N., and Haniford, D. B. (1998). Tnl0 transposition via a DNA hairpin intermediate. Cell 95, 125-tj4.
TnATransposition Requires Transposase andResotvase Review Sherratt, D. (1989). Tn3 and related transposabie elements: site-specificrecombination and transposition.In Berg, D. E. and Howe, M. M., eds.,Mobile DN,4.Washington, DC: American Societyfor Microbiology Press,pp. 163-l 84. Resea r ch Droge, P. et al. (1990). The two functional domains of gamma delta resolvase act on the same recombination site: implications for the mechanism of strand exchange. Proc.Natl Acad. Sci. usA 87, 5336-5340.
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anism from retroviruses to prime the reverse transcription reaction. They are derived from RNA polymerase II transcripts. A minority of the elements in the genome are fully functional and can transpose autonomously; others have mutations, and thus can only transpose as the result of the action of.a transacting autonomous element. . Members of the nonviral superfamily are identified by external and internal featuresthat suggestthat they originated in RNA sequences.In these cases, though. we can only speculate on how a DNA copy was generated.We assume that they were targets for a transposition event by an enzyme system coded elsewhere, that is, they are always nonautonomous. They originated in cellular transcripts. They do not code for proteins that have transposition functions. The most prominent component of this family is called short interspersed repeatedsequences(SINES).Thesecomponents are derived from RNA polymerase III transcripts. i:i{,,rif.i.r r.:.i..r shows the organization and sequencerelationships of elements that code for reversetranscriptase.Like retroviruses,the LIRcontaining retroposons can be classifiedinto groups according to the number of independent reading frames for gag,pol, and int, and the order of the genes.In spite of these superficial differencesof organization,the common feature is the presence of reverse transcriptase and integrase activities. \pical mammalian LINES elements have two reading frames; one codesfor a nucleic acid-binding protein and the other codes for reversetranscriptaseand endonucleaseactivity. LTR-containing elements can vary from integrated retroviruses to retroposonsthat have lost the capacity to generate infectious particles. Yeastand fly genomes have the Ty and copia elements that cannot generate infectious particles. Mammalian genomes have endogenous retroviruses that, when active, can generate infectious particles.The mouse genome has several active endogenous retroviruses, which are able to generate particles that propagate horizontal infections. By contrast,almost all endogenous retroviruses lost their activity some 50 million years ago in the human lineage, and the genome now has mostly inactive remnants o f t h e e n d o g e n o u sr e t r o v i r u s e s . LINES and SINES comprise a major part of the animal genome. They were defined origi-
nally by the existence of a large number of relatively short sequencesthat are related to one another (comprisingthe moderately repetitive DNA described in Section 4.6, Eukaryotic Genomes Contain Both Nonrepetitive and Repetitive DNA Sequences).The LINES comprise long interspersed sequences,and the SINES comprise short interspersedsequences.(They are describedasinterspersedsequencesor interspersed repeats because of their common occurrence and widespread distribution.) Plants contain another type of small mobile element, called MITE (for miniature invertedrepeat transposableelement). Such elements terminate in inverted repeats, have a 2 or 3 bp target sequence,do not have coding sequences, and are 200 to 500 bp long. At least nine such families exist in (for example) the rice genome. They are often found in the regions flanking protein-coding genes.They have no relationship to SINESor LINES. LINES and SINES comprise a significant part of the repetitive DNA of animal genomes. In many higher eukaryotic genomes, they occupy -50% of the total DNA. i:iil"tfli:;i:.,l'* summarizes the distribution of the different types of transposons that constitute almost half of the human genome. Except for the SINES,which are always nonfunctional, the other types of elements all consist of both functional elements and elements that have suffered deletions that eliminated parts of the reading frames that code for the protein(s) needed for transposition. The relative proportions of these types of transposons are generally similar in the mouse genome. A common LINES in mammalian genomes is called Ll. The typical member is -6500 bp long and terminates in an A-rich tract. The two open reading frames of a full-length element
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are calledORFI and ORF2.The number of fulllength elementsis usually small (-50), and the remainder of the copies are truncated. TIanscripts can be found. As implied by its presence in repetitive DNA, the LINES family shows sequencevariation among individual members. The members of the family within a species, however, are relatively homogeneous compared to the variation shown between species.LI is the only member of the LINES family that has been active in either the mouse or human lineages.It seems to have remained highly active in the mouse, but has declined in the human lineage. Only one SINES has been active in the human lineage: the common Alu element. The mouse genome has a counterpart to this elem e n t ( B l ) , a n d a l s o o t h e r S I N E S( B 2 , I D , B 4 \ that have been active. Human Alu and mouse Bl SINES are probably derived from the 7SL RNA (see Section 22.10, The AIu Family Has Many Widely DispersedMembers). The other mouse SINES appear to have originated from reverse transcripts of tRNAs. The transposition of the SINESprobably results from their recognition as substratesby an active Ll element.
TheALuFamilyHas ManyWideLy Dispersed Members . A majorpartof repetitiveDNAin mammatian genomes consists of repeatsof a singlefamity organized [iketransposons andderived fromRNA potymerase pts. III transcri
Ihe most prominent SINES comprises members of a single family. Its short length and high
individual members of the family are dispersed around the genome instead of being confined to tandem clusters. Again, there is significant similarity between the members within a species compared with variation between species. In the human genome, a large part of the moderately repetitive DNA exists as sequences of -300 bp that are interspersedwith nonrepetitive DNA. At Ieasthalf of the renatured duplex material is cleaved by the restriction enzyme AluI at a single site located 170 bp along the sequence.The cleaved sequencesall are members of a single family known as the Alu family, after the means of its identification. There are -100,000 members in the haploid genome (equivalent to one member per 6 kb of DNA). The individual Alu sequences are widely dispersed. A related sequence family is present in the mouse (where the 50,000 members are called the Bl family), in the Chinese hamster (where it is called the Alu-equivalent family), and in other mammals. The individual members of the Alu family are related rather than identical. The human family seems to have originated by means of a 130 bp tandem duplication, with an unrelated sequence of 3I bp inserted in the right half of the dimer. The two repeats are sometimes called the "left half" and the "right half" of the Alu sequence. The individual members of the Alu family have an average identity with the consensussequence of.87'/". The mouse Bl repeating unit is 130 bp long and corresponds to a monomer of the human unit. IthasT0o/o-80o/o homology with the human sequence. The Alu sequence is related to 7SL RNA, a component of the signal recognition particle (see Section I0.9, The SRP Interacts with the SRP Receptor). The 7SL RNA corresponds ro the left half of an Alu sequence with an insertion in the middle. Thus the ninety 5'terminal bases of 7SL RNA are homologous to the left end of Alu, the central 160 bases of 7SL RNA
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CHAPTER 22 Retroviruses andRetroposons
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sue culture cells suggeststhat a transposition event can introduce several types of collateral damage as well as inserting into a new site; the damage includes chromosomal rearrangements and deletions. Such events may be viewed as agents of genetic change. Neither DNA transposonsnor retroviral-like retroposonsseem to have been active in the human genome for 40 to 50 million years, but several active examples of both are found in the mouse. Note that for transpositionsto survive, they must occur in the germline. Presumably simiIar events occur in somatic cells,but do not survive beyond one generation. r i , , , : r , . , ' A t r a n s p o s o n i s t r a n s c r i bReNdAi nt h t oaat n is transtated into proteins that moveindependently to the nucleus, where theyactonanypairofinverted repeats with thesamesequence astheoriginaI transposon.
insertion of an active copy, becausethe proteins are acting on a transcript of the original active element. By contrast, the proteins produced by the DNA transposons must be imported into the nucleus after being synthesized in the cytoplasm, but they have no means of distinguishing full-length transposonsfrom inactive deleted transposons.r-r;,r:ril,r:t .r i shows that insteadof distinguishing these two types of transposons, the proteins will indiscriminately recognize any element by virtue of the repeats that mark the ends. This greatly reducestheir chance of acting on a full-length element as opposedto one that has been deleted.The consequenceis that inactive elements accumulate, and eventually the family dies out becausea transposasehas such a small chance of finding a target that is a fully functional transposon. Are transposition events currently occurring in these genomes, or are we seeing only the footprints of ancient systems?This varies with the species.There are only a few currently active transposonsin the human genome, but by contrast several active transposons are known in the mouse genome. This explains the fact that spontaneousmutations causedby LINES insertions occur at a rate of -3o/" in mouse, but only 0.17o in man. There appear to be -10 to 50 activeLINES elementsin the human genome. Some human diseasescan be pinpointed as the result of transposition of LI into genes,and others result from unequal crossing-over events involving repeated copies of LI. A model system in which LINES transposition occurs in tis-
Summary Reverse transcription is the unifying mechanism for reproduction of retrovirusesand perpetuation of retroposons.The cycle of each type of element is in principle similar, although retroviruses are usually regardedfrom the perspective of the free viral (RNA) form, whereas retroposons are regarded from the stance of the genomic (duplex DNA) form. Retroviruses have genomes of singlestranded RNA that are replicated through a double-stranded DNA intermediate. An individual retrovirus contains two copies of its genome. The genome contains the gag,pol, and env ge\es that are translated into polyproteins, each of which is cleaved into smaller functional proteins. The Gagand Env components are concerned with packing RNA and generating the virion; the Pol components are concerned with n u c l e i ca c i d s y n t h e s i s . Reversetranscriptaseis the major component of Pol, and is responsible for synthesizing a DNA (minus strand) copy of the viral (plus strand) RNA. The DNA product is longer than the RNA template; by switching template strands, reverse transcriptase copies the 3' sequenceof the RNA to the 5'end of the DNA, and copiesthe 5'sequenceof the RNA to the l' end of the DNA. This generates the characteristic LTRs (long terminal repeats) of the DNA. A similar switch of templates occurs when the plus strand of DNA is synthesized using the minus strand as a template. Linear duplex DNA is inserted into a host genome by the integrase enzyme. Transcription of the integrated DNA from a promoter in the left LTR generates further copiesof the RNA sequence. Switches in template during nucleic acid synthesisallow recombination to occur by copy
2 2 . 1 3S u m m a r y 567
choice. During an infective cycle, a retrovirus may exchange part of its usual sequencefor a cellular sequence;the resulting virus is usually replication-defective,but can be perpetuated in the course of a joint infection with a helper virus. Many of the defectiveviruses have gained an RNA version (v-onc)of a cellular gene (c-onc) The oncsequence may be any one of a number of geneswhose expressioninv-oncform causes the cell to be transformed into a tumorisenic phenotype. The integration event generates direct target repeats (like transposons that mobilize via DNA). An inserted provirus therefore has direct terminal repeats of the LTRs, flanked by short repeats of target DNA. Mammalian and avian genomeshave endogenous (inactive) proviruses with such structures.Other elementswith this organization have been found in a variety of genomes, most notably in S. cerevisiaearrd p elements of yeast and copiaeleD. melanogaster. m e n t s o f f l i e s h a v e c o d i n g s e q u e n c e sw i t h homology to reverse transcriptaseand mobiIize via an RNA form. They may generate particles resembling viruses, but do not have infectious capability. The LINES sequencesof mammalian genomes are further removed from the retroviruses, but retain enough similarities to suggesta common origin. They use a different type of priming event to initiate reverse transcription, in which an endonuclease activity associatedwith the reverse transcriptase makes a nick that provides a 3'-OH end for priming synthesison an RNA template. The frequency of LINES transposition is increased becauseits protein products are cls-acting;they associatewith the mRNA from which they were translated to form a ribonucleoprotein complex that is transported into the nucleus. T h e m e m b e r s o f a n o t h e r c l a s so f r e t r o posons have the hallmarks of transposition via RNA, but have no coding sequences(or at least none resembling retroviral functions). They may have originated as passengersin a retroviral-like transposition event, in which an RNA was a target for a reverse transcript a s e . P r o c e s s e dp s e u d o g e n e s a r i s e b y s u c h events. A particularly prominent family that appears to have originated from a processing event is the mammalian SINES;it includes the human Alu family. Some snRNAs, including 7SL snRNA (a component of the SRP),are related to this family.
CHAPTER 22 Retroviruses andRetroposons
References TheRetrovirus LifeCvcleInvolves Transposition-Like Events Review Varmus,H. E. and Brown, P.O. (1989).Retroviruses.In Howe,M. M. and Berg,D. E., eds., MobileDNA Washington,DC: American Socipp. 53-108. ety for Microbiology, Resea rch Baltimore,D. (1970).RNA-dependent DNA polymerasein virions of RNA tumor viruses. Nature226, 1209-l2ll. Temin,H. M. and Mizutani,S. (1970).RNAdependentDNA polymerasein virions of Rous sarcomavirlus.Nature226, l2ll-1213.
ViratDNAis Generated bv Reverse Transcription Reviews I(atz, R. A. and Skalka,A. M. (1994). The retroviral enzymes. Annu. Rev.Biochem.63, B3-17J. Lai, M. M. C. ( 1992). RNA recombination in animal and plant viruses. Microbiol Rev.56, 6l-79. Negroni, M. and Buc, H. (2001). Mechanisms of retroviral recombination. Annu. Rev.Genet.3 5. 275-302. Resea r ch Hu, W. S. and Temin, H. M. (1990). Retroviral recombination and reverse transcription. Sci e n c e2 5 0 , 1 2 2 7 - 1 2 ) ) . Negroni, M. and Buc, H. (2000). Copy-choice recombination by reverse transcriptases: reshuffling of genetic markers mediated by RNA chaperones. Proc Natl. Acad Sci.USA 97 6385-6390.
Viral. DNAIntegrates into the Chromosome Review Goff, S. P. (1992).Geneticsof retroviralintegraIion. Annu.Rev.Genet.26,527-544. Resea rch Craigie,R.,Fujiwara,T.,and Bushman,F. (1990). The IN protein of Moloney murine leukemia virus processes the viral DNA endsand accomplishes their integrationin vitro Cell62, 829-837.
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plement also provides a means of attracting macrophages, which scavenge the target cells or their products. Alternatively, the antigenantibody complex may be taken up directly by macrophages(scavengercells) and destroyed. The cell-mediated response is executed by a classof T lymphocytes called cytotoxic T cells (also called killer T cells).The basicfunction of the T cell in recognizing a target antigen i s i n d i c a t e d i n r ; r . . , i r i 'lr:i;rr . l i .A c e l l - m e d i a t e d response typically is elicited by an intracellular parasite, such as a virus that infects the body's own cells.As a result of the viral infection, fragments of foreign (viral) antigens are displayed on the surface of the cell. These fragments are recognized by the T cell receptor (TCR), which is the T cells' equivalent of the antibody producedbyaBcell. A crucial feature of this recognition reaction is that theantigenmust bepresented by a cellular protein that is a memberof the IVIHC (maj or histocompatibility complex). The MHC protein has a groove on its surfacethat binds a peptide fragment derived from the foreign antigen. The combination of peptide fragment and MHC protein is recognized by the T cell receptor. Every individual has a characteristic set of MHC proteins. They are important in graft reactions; a graft of tissue from one individual to another is rejectedbecauseof the differencein MHC proteins between the donor and recipient, an issue of major medical importance. The demand that the T lymphocytes recognize both foreign antigen and MHC protein ensures that the cellmediated response acts only on host cells that have been infected with a foreign antigen. (We discuss the division of MHC proteins into the general types of classI and classII later in Section 2).20, The Maj or Histocompatibility Locus Codesfor Many Genesof the Immune System.) The purpose of each type of immune response is to attack a foreign target. Target recognition is the prerogative of B-cell immunoglobulins and T cell receptors.A crucial aspect of their function lies in the ability to distinguish "self" from "nonself." Proteins and cells of the body itself must neverbe attacked. Foreign targets must be destroyed entirely.TLreprop"self" is calledtolerance. erty of failing to attack Loss of this ability results in an autoimmune disease, in which the immune system attacks i t s o w n b o d y , o f t e n w i t h d i s a s t r o u sc o n s e quences. What prevents the lymphocyte pool from responding to "self" proteins? Toleranceprob-
ably arises early in lymphocyte cell development when B cells and T cells that recognize " s e l f " a n t i g e n s a r e d e s t r o y e d .T h i s i s c a l l e d clonal deletion. In addition to this negative selection, there is also positive selection for T cells carrying certain sets of T cell receptors. A corollary of tolerance is that it can be difficult to obtain antibodies against proteins that are closelyrelated to those of the organism itself. As a practical matter, therefore, it may be difficult to use (for example) mice or rabbits to obtain antibodies against human proteins that have been highly conservedin mammalian evolution. The tolerance of the mouse or rabbit for its own protein may extend to the human protein in such cases. Each of the three groups of proteins required for the immune response-immunoglobulins, T cell receptors, and MHC proteins-is diverse. Examining a Iarge number of individuals, we find many variants of each protein. Each protein is coded by a large family of genes; in the caseof antibodies and the T cell receptors, the diversity of the
immunity,k'itterT cel.l.s i:il.i"l[{fi]jl.i.rIn cet[-mediated of thefora fragment to recognize usetheT ce[[receptor of thetaron the surface that is presented eignantigen getcetlby the MHCprotein.
23.1Introduction 573
population is increasedby DNA rearrangements that occur in the relevant lymphocytes. Immunoglobulins and T cell receptorsare direct counterparts, each produced by its own type of lymphocyte. The proteins are related in structure, and their genes are related in organization. The sourcesof variability are similar. The MHC proteins also share some common features with the antibodies, as do other lymphocyte-specificproteins. In dealing with the genetic organization of the immune system, we are therefore concerned with a series of related gene families, indeed a superfamily that may have evolved from some common ancestor representing a primitive immune resDonse.
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:.:i.r-:i;ir .l:i-:iThepooL of immature [ymphocytes contains B ce[[sandT cetLs makingantjbodies andreceptors with a varietyof specificities. Reaction with an antigenleadsto clonaIexpansion of the lymphocyte with the antibooy (Bcett)or receptor (Tcetl)thatcanrecognize theantigen.
574
C H A P T E2R3 I m m u n eD i v e r s i t v
C[onaL Selection Amplifies Lymphocytes That Respond to IndividuaL Antigens r EachB lymphocyte expresses a single immunoglobutin andeachT lymphocyte expresses a singteT cetlreceptor. . There is a very[argevarietyof immunoglobutins andT ce[[receptors. e Antigenbindingto animmunogtobutin or T ce[[ receptor triggerscLonal. multiptication of the cet[. The name of the immune response describes one of its central features. After an organism has been exposedto an antigen, it becomes immune to the effectsof a new infection. Before exposure to a particular antigen, the organism lacks adequate capacity to deal with any toxic effects. This ability is acquired during the immune response.After the infection has been defeated, the organism retains the ability to respond rapidly in the event of a reinfection. These features are accommodated by the clonal selection theory illustrated in F:Iili-Jiis f i"5. The pool of lymphocytes contains B cells and T cells carrying a large variety of immunoglobulins or T cell receptors. Any individual B lymphocyteproducesoneimmunoglobulin, however,which is capableof recognizingonly a single antigen; similarly, any individual T lymphoqtte producesonly oneparticular T cell receptor. In the pool of immature lymphocytes, the unstimulated B cells and T cells are morphoIogically indistinguishable. On exposure to antigen, though, a B cell whose antibody is able to bind the antigen, or a T cell whose receptor can recognize it, is stimulated to divide, probably by some feedback from the surface of the cell, where the antibody/receptor-antigen reaction occurs. The stimulated cells then develop into mature B or T lymphocytes; this includes morphological changes involving (for example) an increasein cell size (especiallypronounced for B cells). The initial expansion of a specificB or T cell population upon first exposure to an antigen is called the primary immune response. Large numbers of B or T lymphocytes with specificity for the target antigen are produced. Each population represents a clone of the original responding cell. Antibody is secreted from the B cellsin large quantities, and it may even come to dominate the antibody population.
919
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event involves the genes of or'ly 7ne of the homologous chromosomes, and as a resulr the in are not expressed alleleson the otherchromzsome thesamecell.This phenomenon is called allelic exclusion. The occurrence of allelic exclusion complicates the analysis of somatic recombination. A probe reacting with a region that has rearranged on one homolog will also detect the allelic sequenceson the other homolog. We are therefore compelled to analyze the different fates of the two chromosomes together. The usual pattern displayedby a rearranged active gene can be interpreted in terms of a deletion of the material between the recombining V and C loci. Two types of gene organization are seen in active cells: . Probes to the active gene may reveal one rearranged copy and one germline copy. We assume,then, that joining has occurred on one chromosome, whereas the other chromosome has remained unaltered. . TWo different rearranged patterns may be found, indicating that the chromosomes have suffered independent rearrangements. In some of these instances,material between the recombining V and C gene segmentsis entirely absent from the cell line. This is most easily explained by the occurrence of independent deletions (resulting from recombination) on each chromosome. When two chromosomes both lack the germline pattern, usually only one of them has passedthrough a productive rearrangement to generate a functional gene. The other has rearrangement; suffered a nonproductive this may take several forms, but in each case the gene sequencecannot be expressedas an immunoglobulin chain. (It may be incomplete, for example becauseD-J joining has occurred but V-D joining has not followed; or it may be aberrant, with the process completed but failing to generate a gene that codes for a functional protein.) The coexistenceof productive and nonproductive rearrangements suggeststhe existence of a feedback loop to control the recombination ' p r o c e s s .A m o d e l i s o u t l i n e d i n , , l : r : : r Supposethat each cell starts with two loci in the unrearranged germline configuration Ig0. Either of these loci may be rearrangedto generate a productive gene Ig+ or a nonproductive gene Ig-.
If the rearrangement is productive, the synthesis of an active chain provides a trigger to prevent rearrangement of the other allele. The a c t i v ec e l l h a s t h e c o n f i g u r a t i o nI g 0 / l g + . If the rearrangement is nonproductive, it creates a cell with the configuration Ig0ttg-. There is no impediment to rearrangement of the remaining germline allele. If this rearrangement is productive, the expressing cell has the configurationlg+llg-. Again, the presenceof an active chain suppressesthe possibility of further rearrangements. TWo successivenonproductive rearrangements produce the cell Ig-lIg-.In some casesan Ig-lIg- cell can try yet again. Sometimes the observed patterns of DNA can only have been generatedby successiverearrangements. The crux of the model is that the cell keeps trying to recombine V gene segments and C gene segmentsuntil a productive rearrangement is achieved. Allelic exclusion is caused by the suppression of further rearrangement as soon as an active chain is produced. The use of this mechanism in vivo is demonstrated by the creation of transgenic mice whose germline has a rearranged immunoglobulin gene.
produce an activelightor heavy i';.:,.,,'r,, li A successful rearrangementto jn aLletic of thesametype,andresults furtherrearrangements chainsuppresses exctusion.
Rearrangement583 by Productive Is Triggered 23.9 AtteticExclusion
Expression of the transgene in B cells supp r e s s e st h e r e a r r a n g e m e n t o f e n d o g e n o u s genes. Allelic exclusion is independent for the heavy- and light-chain loci. Heavy-chain genes usually rearrange first. Allelic exclusion for light chains must apply equally to both families (cells may have either aclive r or i,light chains). Ir is Iikely that the cell rearranges its r genesfirst, and tries to rearrange l, only if both K attempts are unsuccessful. There is an interesting paradox in this series of events. The same consensus sequencesand the same V(D)J recombinase are involved in the recombination reactions at H, r, and l, loci, and yet the three loci rearrange in a set order. What ensures that heavy rearrangement precedeslight rearrangement, and that r precedes l,? The loci may become accessibleto the enz).rne at different times. possiblyas the result of transcription. Transcription occurs even before rearrangement, although of course the products have no coding function. The transcriptional event may change the structure of chromatin, making the consensussequences for recombination available to the enzyme.
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r TheRAGproteinsarenecessary andsufficientfor the cleavage reaction. . RAGlrecognizes the nonamer consensus sequences for recombination. RAG2 bindsto RAGl andcteaves at the heptamer. . Thereaction resembles thetopoisomerase-tike resotution reaction that occurs in transposition. r It proceeds througha hairpinintermediate at the codingend;openingof the hairpinis responsibte for insertion of extrabases (P nucleotides) in the recombined gene. o Deoxynucteoside transferase insertsadditional. N nucteotides at the codingend. o Thecodonat the siteof the V-(D)J joining reactionhasan extremely variabtesequence and codes for aminoacid96 in the antigen-binding site. . Thedoubte-strand breaks at the codingjointsare repaired by the samesysteminvotvedin nonhornologous end-joining of damaged DNA. o An enhancer in the Cgeneactivates the promoter of the V geneafterrecombination hasqenerated theintactimmunogtobutin gene. The proteins RAGI and RAG2 are necessary and sufficient to cleave DNA for V(D)J recom-
584
CHAPTER 23 ImmuneDiversitv
bination. They are coded by two genes. separated by <10 kb on the chromosome, whose transfection into fibroblasts causes a suitable substrate DNA to undergo the V(D)J joining reaction. Mice that lack either RAGL or RAG2 are unable to recombine their immunoglobulins or T cell receptors, and as a result have immature B lymphocytes and T lymphocytes. The RAG proteins together undertake the catalytic reactions of cleaving and rejoining DNA, and also provide a structural framework within which the reactions occur. RAGI recognizes the heptamer/nonamer signals with the appropriate l2l23 spacing and recruits RAG2 to the complex. The nonamer provides the site for initial recognition, and the heptamer directs the site of cleavage. The reactions involved in recombination are shown in Fl#{Jftilf^1.1.+. The complex nicks one strand at eachjunction. The nick has 3'-OH and 5'-P ends. The free 3'-OH end then attacks the phosphate bond at the corresponding posiIion in the other strand of the duplex. This creates a hairpin at the coding end, in which the 3'end of one strand is covalently linked to the 5' end of the other strand; it leaves a blunt doublestrand break at the signal end. This second cleavageis a transesterification reaction in which bond energiesare conserved. It resembles the topoisomerase-like reactions calalyzedby the resolvaseproteins of bacterial transposons(seeSection 2I.9, TnATfansposition Requires Tlansposaseand Resolvase).The parallel with these reactions is supported further by a homology between RAGI and bacterial invertase proteins (which inyert specific segments of DNA by similar recombination reactions). In fact, the RAG proteins can insert a donor DNA whose free ends consist of the appropriate signal sequences(heptamer- | 2 | 23 spacernonamer) into an unrelated target DNA in an in vitro transposition reaction. This suggests that somatic recombination of immune genes evolved from an ancestral transposon. It also suggeststhat the RAG proteins are responsible for chromosomal translocations in which Ig or TCR loci are connected to other loci. The hairpins ar the coding ends provide the substrate for the next stageof reaction. If a single-strand break is introduced into one strand close to the hairpin, an unpairing reaction at the end generatesa single-strandedprotrusion. Synthesis of a complement to the exposed single strand then converts the coding end to an extended duplex. This reaction explains the introduction of P nucleotides at coding ends;
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gene whose sequence is different must have been generatedby somatic changes. One difficulty is to ensure that every potential contributor in the germline V gene segments actually has been identified. This problem is overcome by the simplicity of the mouse l. chain system. A survey of several myelomas producing 1"1chains showed that many have the sequence of the single germline gene segment. Others,however,havenew sequences that musthave beengeneratedby mutation of the germlinegene segment. To determine the frequency of somatic mutation in other cases,we need to examine a large number of cells in which the same V gene segment is expressed.A practical procedure for identifying such a group is to characterize the immunoglobulins of a seriesof cells, all of which express an immune response to a particular antigen. Epitopes used for this purpose are small molecules-haptens-whose discrete structure is likely to provoke a consistentresponse,unlike a large protein, different parts of which provoke different antibodies. A hapten is conjugated with a nonreactive protein to form the antigen. The cells are obtained by immunizing mice with the antigen, obtaining the reactive Iymphocytes, and sometimes fusing these lymphocytes with a myeloma (immortal tumor) cell to generate a hybridoma that continues to express the desired antibody indefinitely. In one example, l0 out of l9 different cell lines producing antibodies directed against the hapten phosphorylcholine had the same Vs seeuence. This sequence was the germline V gene segmentTI5. one of four related Vs genes.The other nine expressedgene segments differed from each other and from all four germline members of the family. They were more closely related to the Ti5 germline sequence than to any of the others, and their flanking sequences were the same as those around TI5. This suggestedthat they arosefrom the T15 member by somatic mutation. I shows that sequencechanges Fi*tjtI r:1.:1 are localized around the V gene segment, extending in a region from -I50 bp downstream of the V gene promoter for -1.5 kb. They take the form of substitutions of individual nucleotide pairs. Usually there are -3 to - I 5 substitutions, corresponding to <10 amino acid changesin the protein. They are concentrated in the antigenbinding site (thus generating the maximum diversity for recognizing new antigens). Only some of the mutations affect the amino acid
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$3{rl,iF.tf, il;i.illi Somatjcmutationoccursin the region overthejoined andextends surrounding the V segment VDJsegments. sequence,becauseothers lie in third-base coding positions as well as in nontranslated regions. The large proportion of ineffectual mutations suggeststhat somatic mutation occurs more or lessat random in a region including the V gene segmentand extending beyond it. There is a tendency for some mutations to recur on multiple occasions.These may represent hotspots as a result of some intrinsic preferencein the system. Somatic mutation occurs during clonal proliferation, apparently at a rate - t g-r per bp per cell generation. Approximately half of the progeny cells gain a mutation; as a result, cells expressing mutated antibodies become a high fraction of the clone. In many cases,a single family of V gene segments is used consistently to respond to a particular antigen. Upon exposure to an antigen, presumably the V region with highest intrinsic affinity provides a starting point. Somatic mutation then increases the repeiloire. Random mutations have unpredictable effects on protein function; some inactivate the protein, whereas others confer high specificity for a particular antigen. The proportion and effectivenessof the lymphocytes that respond is increased by selection among the lymphocyte population for those cells bearing antibodies in which mutation has increased the affinity for the antigen.
Mutation Somatic by Is Induced Deaminase Cytidine andUraciIGlycosylase for somatjc is required A cytidinedeaminase mutation aswe[[asfor ctassswitching. the activityi nftuences Uracit-DNAglycosytase patternof somatic mutations. maybeinitiatedbythe sequentiaI Hypermutation actionof theseenzvmes.
andUracilGtycosylase 59r Deaminase by Cytidine Is Induced Mutation 23.15Somatic
Somatic mutation has many of the same requirements as class switching (see SecIion23.l3, Switching Occursby a Novel Recombination Reaction: o transcription must occur in the target r e g i o n ( a s s h o w n i n t h i s c a s eb y t h e demand for the enhancer that activates transcription at each Ig locus; . it requires the enzymes AID and UNG and r the MSH mismatch-repair system is involved. The way in which the removal of the deaminated base leads to somatic mutation is suggested by the experiment summarized in FiGuftE23.2?.When AID deaminates cytosine, it generatesuracil, which then is removed from the DNA by UNG. Normally all possiblesubstitutions occur at the abasicsite. If the action of uracil-DNA glycosylaseis blocked, though, we see a different result. If uracil is not removed from DNA, it should pair with adenine during replication. The ultimate result is to replace the original C-G pair with a T-A pair. Uracil-DNA glycosylasecan be blocked by introducing into cells the gene coding for a protein that inhibits
the enzyme. (The gene is a component of the bacteriophagePSB-2,whose genome is unusual in containing uracil, so that the enzyme needs to be blocked during a phage infection.) When the gene is introduced into a lymphocyte cell line, there is a dramatic change in the pattern of mutations, with almost all comprising the predicted transition from C-G to A-T. The key event in generating a random spectrum of mutations is therefore to create the abasic site. The MSH repair system is then recruited to excise and replace the stretch of DNA containing the damage. The simplest possibility is that if the replacement is performed by an errorprone DNA polymerase, mutations may be introduced. Another possibility is that so many abasic sites are created that the repair systems are ovenvhelmed. When replication occurs, this could lead to the random insertion of bases opposite the abasic sites. We don't know yet what restricts the action of this system to the target region for hypermutation. The difference in the systems is at the end of the process,when double-strand breaks are introduced in class switching. but individual point mutations are created during somatic
Cytidinedeaminase createsa U-G pair
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592
CHAPTER 23 Immune Diversitv
mutation. We do not yet know exactly where the systemsdiverge.One possibilityis that breaks are introduced at abasicsitesin classswitching, but the sitesare erratically repaired in somatic mutation. Another possibilityis that breaksare introduced in both cases,but are repaired in an e r r o r - D r o n em a n n e r i n s o m a l i cm u t a t i o n .
rearranged V11 seeuence has four to six converted segments spanning its entire length, which are derived from different donor pseudogenes.If all pseudogenesparticipate, this allows 2.5 x 108possiblecombinations! The enzymatic basis for copying pseudogene sequencesinto the expressedlocus depends on enzymes involved in recombination and is related to the mechanism for somatic hypermutation that introduces diversity in mouse and man. Some of the genesinvolved in recombination are required for the gene conversion process;for example, it is prevented by deletion of R.4D54.Deletion of other recombination genes(XRCC2,XRCC3,andP.1'D5LB)has another, very interesting effect: Somatic mutation occurs at the V gene in the expressedlocus. The frequency of the somatic mutation is - I 0x greater t h a n t h e u s u a lr a t eo f g e n ec o n v e r s i o n . T h e s e r e s u l t s s h o w t h a t t h e a b s e n c eo f somatic mutation in chick is not due to a deficiency in the enzymatic systemsthat are responsible in mouse and man. The most likely explanation for a connection between (lack of ) recombination and somatic mutation is that unrepaired breaks at the locus trigger the induction of mutations. The reason why somatic mutation occurs in mouse and man but not in chick may therefore lie with the details of the operation of the repair system that operates on
AvianImmunogLobu[ins AreAssembled from Pseudogenes r An immunogtobulin genein chicken is generated by copying a sequence fromoneof 25 pseudogenes intothe V geneat a singleactive r.ocus.
The chick immune system is the paradigm for rabbits, cows, and pigs, which rely upon using the diversity that is coded in the genome. A similar mechanism is used by both the single light chain locus (of the l" tlpe) and the H chain locus. The organization of the l, locus is drawn in :rii:riir::.,'::.i .. It has only one functional V gene segment, J segment, and C gene segment. Upstream of the functional V11gene segment lie 25 Vr pseudogenes,organizedin either orie n t a t i o n . T h e y a r e c l a s s i f i e da s p s e u d o g e n e s becauseeither the coding segmentis deletedat one or both ends, or proper signalsfor recombination are missing, or both. This assignment is confirmed by the fact that only the V1y gene segment recombines with the J-Cr gene segment. Sequencesof active rearrangedV1-J-C1gene segments show considerable diversity, though! A rearranged gene has one or more positions at which a cluster of changeshas occurred in the sequence.A sequenceidentical to the new sequencecan almost aiways be found in one of t h e p s e u d o g e n e s( w h i c h t h e m s e l v e sr e m a i n unchanged). The exceptional sequencesthat are not found in a pseudogenealways represent changesat the junction between the orioinal sequenceand the altered sequence. Thus a novel mechanism is employed to generate diversity. Sequencesfrom the pseudogenes, between l0 and 120 bp in length, are substituted into the active V11region by gene conversion. The unmodified V11sequenceis not e x p r e s s e d ,e v e n a t e a r l y t i m e s d u r i n g t h e i m m u n e r e s p o n s e .A s u c c e s s f u lc o n v e r s i o n event probably occurs every ten to twenty cell divisions to every rearrangedVtl sequence.At the end of the immune maturation period, a
vrlJ
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i 1 { - ; i . i i r i I i : i ,Ti .hi e c h i c k e n t a m b d a l i g h t l ' o c u s h a s 2 5 V p s e u d o g e n e s u p s t r e a m fromthe pseudoderived V-J-Cregion.Sequences of the sing[efunctionaI V-J-Cgenes. genes, however, arefoundjn activereananged
fromPseudogenes5 9 3 AreAssembted 23.16AvianImmunogtobutins
breaks at the locus. It is more efficient in chick, so that the gene is repaired by gene conversion before mutations can be induced.
Memory Altows l@ B Cel"L a RapidSecondary Response o Theprimary response to an antigenis mounted by B cetlsthat do not survive beyond the response oeriod. Memory B cetlsareproduced that havespecificity for the sameantigen,butthat areinactive. A reexposure to antigentriggers the secondary response in whichthe memory ce[[sarerapidty activated. We are now in a position to summarize the relationship between the generation of high-affinity antibodies and the differentiation of the B cell. :.r-*ii-:=l i3.l:* shows that B cellsare derived from a self-renewing population of stem cells in the bone marrow. Maturation to give B cells depends upon Ig gene rearrangement,which requires the functions of the SC/D arLdRAG1,2 (and other) genes.If gene rearrangement is blocked, mature B cells are not produced. The antibodies carried by the B cellshave specificitiesdetermined by the particular combinationsof V(D)J regionsand any additional nucleotides incorporated during the lolnng process. Exposure to antigen triggers two aspectsof the immune response.The primary immune response occursby clonal expansion of B cells responding to the antigen. This generatesa large number of plasma cells that are specific for the
i*{i{.lFlIt-t.:+ B ce[[differentiation is responsib[e for acquired immunity. Pre-Bcetlsareconverted to B ce[[s byIg generearrangement. Initialexposure to antigenprovokesboththe primary response andstorage of memory ce[[s.Subsequent exposure to antigenprovokes the secondaryresponse of the memory ce[[s.
l#'3il; ii,Xlli;',il51'l?"T;H,Ti# lt : population of cells concerned with the primary responseis a dead end; these cells do not live beyond the primary response itself . Provision for a secondary immune response is made through the phenomenon
further somatic mutation or isotype switching occurs during the secondary response. The pathways summarized in Figure 23.24 show the development of acquired immunity, that is, the response to an antigen. In addition to these cells, there is a separate set of B cells, named the Ly-1 cells. These cells have gone through the process of V gene rearrangement, and apparently are selectedfor expression of a particular repertoire of antibody specificities. They do not undergo somatic mutation or the memory response. They may be involved in natural immunity, that is, an intrinsic ability to r e s p o n dt o c e r t a i na n t i g e n s .
:iJ;::1,ffiTi:';.":1X::#:lililf"11": antigen. These cells do not trigger an immune responseat this time, although they may undergo isotype switching to selectother forms of Cs region. They are stored as memory cells, with appropriate specificity and effector response type, but are inactive. They are activated if there is a new exposure to the same antigen. They are preselectedfor the antigen, and as a result
ffI".:ilff,,#ilT;:"'"lT:':, 3 """T::lT 594
CHAPTER 23 Immune Diversitv
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is activated by binding antigen. Our present picture of the components of the receptor complex on a T cell is illustrated in FSftljtttj.:jj.li;:.The important point is that the interaction of the TCR variable regions with antigen causesthe ( subunits of the CDI complex to activate the T cell response.The activation of CD3 provides the means by which either crBor y6 TCR signalsthat it has recognized an antigen. This is comparable to the constitution of the B cell receptor, in which immunoglobulin associateswith the IgoB signaling chains (seeFigure 2j.26). A central dilemma about T cell function remains to be resolved.Cell-mediatedimmunity requires two recognition processes.Recognition of the foreign antigen requires the ability to respond to novel structures. Recognition of the MHC protein is of course restrictedto one of those coded by the genome, but even so there are many different MHC proteins. Thus considerable diversity is required in both recognition reactions.Helper and killer T cellsrely upon different classesof MHC proteins; however, they use the same pool of cr and B gene segmentsto assembletheir receptors. Even allowing for the introduction of additional variation during the TCR recombination process,it is not clear how enough different versionsof the T cell receptor are made available to accommodate all these demands.
V-regionof TCR recognizesantigen
( chain is efiector
i:ii:,iii: l: ;;..:;rThetwochains of theT ce[[receotor associatewiththepotypeptides oftheCD3compLex. Thevariabteregions of the TCRareexposed on the ce[[surface. Thecytoplasmic domains of the ( chainsof CD3provide function. the effector
TheMajor patibitityLocus Histocom for ManyGenes Codes of the ImmuneSystem r TheMHClocuscodes for the ctassI andclassII proteins of the aswe[[asfor otherproteins immune system. . Ctass antigens I proteins arethetransplantation "self'from that areresponsible for distinguishing "nonself'tissue. . An MHCclassI proteinis activeasa heterodimer wjth Fzmicrogtobu[in. . Class in interactions II oroteins areinvotved between T ce[ts. . An MHCclassII proteinis a heterodimer of g and c h a i n s . B
The major histocompatibility locus occupies a small segment of a single chromosome in the mouse (where it is called the H2 locus) and in man (called the HLA locus). Within this segment are many genescoding for functions concerned with the immune response.At individual gene Ioci whose products have been identified, many alleles have been found in the population; the locus is described as highly polymorphic,mearring that individual genomes are likely to be different from one another. Genes coding for certain other functions also are located in this region. Histocompatibility antigens are classified into three types by their immunological properties.In addition, other proteins found on lymphocytes and macrophages have a related structure and are important in the function of cells of the immune system: MHC class I proteins are the transplantation antigens. They are present on every cell of the mammal. As their name suggests, these proteins are responsiblefor the rejection of foreign tissue, which is recognized as such by virtue of its particular array of transplantation antigens.In the immune system,their presence on target cells is required for the cell-mediated response.The types of classI proteins are defined serologically (by their antigenic properties). The murine classI genescode for the H2-I( and H2-DlL proteins.Each mouse strain has one of severalpossibleallelesfor each of these functions. The human classI functions include the classicaltransplantation antigens, HLA-A. B. and C.
of the ImmuneSystem 5 9 9 for ManyGenes Locus Codes 23.20TheMajorHistocompatibitity
NIHC class II proteins are found on the surfacesof both B lymphocytes and T lymphocytes, as well as on macrophages.These proteins are involved in communications between cellsthat are necessaryto execute the immune response; in particular, they are required for helper T cell function. The murine classII functions are defined geneticallyas I-A and I-E. The human classII region (also called HLA-D) is arrangedinto four subregions,DR, DQ, DZIDO, and DP. The complement proteins are coded by a genetic locus that is also known as the S region; S standsfor serum, indicating that the proteins are components of the serum. Their role is to interact with antibody-antigen complexes to cause the lysis of cells in the classicalpathway of the humoral response. The Qa and Tla loci proteins are found on murine hematopoietic cells. They are known as differentiation antigens,becauseeach is found only on a particular subset of the blood cells, presumably related to their function. They are structurally related to the classI H2 proteins, and like them are polymorphic. We can now relate the types of proteins to the organization of the genesthat code for them. The MHC region was originally defined by geneticsin the mouse, where the classicalH2 region occupies 0.3 map units. Together with the adjacent region where mutations affecting immune function are also found, this corresponds to a region of -2000 kb of DNA. The MHC region has been completely sequencedin severalmammals, as well as in some birds and fish. By comparing these sequences,we find
that the organization has been generally conserved. The gene organization in mouse and man is summarized in il{iti't!: ii;-,:.1.The genomic regions where the classI and classII genes are Iocated mark the original boundaries of the locus (going in the direction from telomere to centromere; right to left as shown in the figure). The genes in the region that separatesthe class I and classII genes code for a variety of functions; this is called the classIII region. Defining the ends of the locus varies with the species, and the region beyond the classI genes on the t e l o m e r i c s i d e i s c a l l e d t h e e x t e n d e d c l a s sI region. Similarly, the region beyond the classII genes on the centromeric side is called the extended classII region. The major difference between mouse and human is that the extended classII region contains some classI (H2-I() genes in the mouse. There are several hundred genes in the MHC regions of mammals, but it is possible for MHC functions to be provided by far fewer genes, as in the caseof the chicken, where the MHC region is 92 I(b and has only nine genes. As in comparisons of other gene families, we find differences in the exact numbers of genes devoted to each function. The MHC locus shows extensive variation between individuals, and as a result the number of genes may be different in different individuals. As a general rule, however, a mouse genome has fewer active H2 genesthan a human genome. The classII genes are unique to mammals (except for one subgroup), and as a rule, birds and fish have different genesin their place. There are -8 functional
i':i.l:.i!ilil.il.i,:;The MHCregionextendsfor >2 Mb. MHCproteinsof classesI and II are codedbV two separateregions.TheclassIII regionis definedas the segmentbetweenthem. Theextended regionsdescribesegmentsthat are syntenicon either end of the ctuster.The major difference betweenmouseand humanis the presence of H2 classI genesin the extendedregionon the [eft. Themurine[ocusis [ocatedon chromosome 1.7,andthe humanlocusis locatedon chromosome 6.
CHAPTER 23 ImmuneDiversitv
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Toll, which is related to mammalian ILI receptor, triggers the pathway in Drosophilathat controls dorsal-ventraldevelopment. This leadsto activation of the transcription factor dorsal, a member of the Rel family, which is relaredto the mammalian factor NF-rB. The pathway of innate immunity is parallel to the Toll pathway, with similar components. In fact, one of the first indications of the nature of innate immunity in flies was the discovery of the transcription factor Dif (dorsal-relatedimmunity factor), which is activatedby one of the pathways. Flies have no system of adaptive immunity, but are resistantto microbial infections.This is becausetheir innate immune systemstrigger synthesisof potent antimicrobial peptides.Seven distinct peptides have been identified in Drosophila,where they are synthesized in the fat body (the equivalent organ ro rhe liver). Two of the peptides are antifungal, and five act largely on bacteria. The general mode of action is to kill the target organism by permeabilizing irs membrane. AII of these peptides are coded by genes whose promoters respond to transcription factorsof the Rel family. ; ri,,rrilr,1,;r ,'summarizes the components of the innate pathways in Drosophila. TWo innate responsepathways function in Drosophila;one responds principally to fungi, whereas the other responds principally to gram-negative bacteria. Gram-positive bacteria may be able to trigger both pathways. : i . . r i : : ' , , o u t l i n e s t h e s t e p si n e a c h p a t h way. Fungi and gram-positivebacteriaactivate a proteolytic cascadethat generates pepticles that activate a TLR. This is the NF-rB-like pathway. The dToll receptor activates the transcription factor Dif (a relative of NF-rB), leading ultimately to activation of the antifungal peptide drosomycin. Gram-negative bacteria trigger a pathway via a different receptor that activatesthe transcription factor Relish, Ieading to production of the bactericidal peptide attacin. This pathway is called the Imd pathway after one of its components, a protein that has a "death domain" related to those found in the pathways for apoptosis. The key agentsin responding to the bacteria are proteins called PGRPsbecauseof their high affinities for bacterialpeptidoglycans.There are two types of these proteins. PGRP-SAsare short extracellularproteins. They probably function by activating the proteasesthat trigger the Toll pathway. PGRP-LCsare transmembrane proteins with an extracellular PGRP domain. Their exact role has to be determined.
i : , i , ; , r i ''i' , I n n a t ei m m u n i tiys t r i g g e r ebdy P A M P s . In flies,theycause of peptides that actithe production vateTotll.ike receptors. Thereceptors leadto a pathway thatactivates factoroftheRe[famity. Tara transcription getgenes forthisfactorinctude bactericidal andantifungalp " e p t i d e sT.h ep e p t i d easc t b y p e r m e a b i L i ztihneg membran oe f t h ep a t h o g e noi cr g a n i s m .
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Gram-negative
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'r jnnate immunity rri'i,ili,ir: .r :,il One of the Drosophilo pathway pathwaysis closelyrelatedto the mammaUan for activatingNF-rB;the other has componentsretated to thoseof apoptosispathways.
Pathways Signating Conserved 23.21InnateImmunityUtitizes
The innate immune responseis highly conserved. Mice that are resistant to septic shock when they are treated with LPS have mutations in the Toll-like receptor TLR4. A human homolog of the Toll receptor can activate some immune-response genes, suggestingthat the pathway of innate immunity may also function in man. The pathway downstream of the TLRs is generally similar in all cases,typically leading to activation of the transcription factor NFrB. We do not yet know whether the upstream pathway is conserved,and in particular whether the PAMPs function by generating ligands that in turn activate the TLRs or whether they might interact directly with them. The pathway upstream of the TLRs is different in mammals and flies,becausethe pathogensdirectly activate mammalian TLRs. In the case of LPS, the pathogen binds to the surface protein CDl4; this enables CDI4 to activate TLR4, triggering the innate response pathway. There are -20 receptorsin the TLR classin the human genome, which gives some indication of how many pathogens can trigger the innate response. Plants have extensive defensemechanisms, among which are pathways analogous to the innate responsein animals. The same principle applies that PAMPs are the motifs that identify the infecting agent as a pathogen. The proteins that respond to the pathogens are coded by a classof genescalled the diseaseresistancegenes. Many of these genes code for receptors that share a property with the TLR class of animal receptors: The extracellular domain has a motif called the leucine-rich region (LLR). The response mechanism is different from animal cells, and is directed to activating a mitogenactivated protein kinoset (MAPK) cascade. Many different pathogensactivatethe same cascade,which suggeststhat a variety of pathogenreceptor interactions converge at or before the activation of the first MAPK.
mary Sum Immunoglobulins and T cell receptors are proteins that play analogous functions in the roles of B cells and T cells in the immune system. An Ig or TCR protein is generated by rearrangement of DNA in a single lymphocyte; exposure to an antigen recognized by the Ig or TCR leads to clonal expansion to generate many cells that have the same specificity as the original cell.
il:::,ffi :i:H; ,,1":li:iii11ffiffJ u;:H, CHAPTER 23 Immune Djversitv
creating a large repertoire of cells of different specificities. Each immunoglobulin protein is a tetramer containing two identical light chains and two identical heavy chains. A TCR is a dimer containing two different chains. Each polypeptide chain is expressedfrom a gene created by linking one of many V segments via D segments and J segments to one of a few C segments. Ig L chains (either x or l,) have the general structure V-J-C, Ig H chains have the structure V-DJ-C. TCR cr and y have components like Ig L chains, and TCR 6 and B are like Ig H chains. Each type of chain is coded by a large cluster of V genes separated from the cluster of D, J, and C segments. The numbers of each type of segment and their organization are different for each type of chain, but the principle and mechanism of recombination appear to be the same. The same nonamer and heptamer consensus sequencesare involved in each recombination; the reaction always involves joining of a consensuswith 23 bp spacing to a consensus with l2 bp spacing. The cleavage reaction is catalyzed by the RAGI and RAG2 proteins, and the joining reaction is catalyzed by the same NHEJ pathway that repairs double-strand breaks in cells. The mechanism of action of the RAG proteins is related to the action of site-specific recombination catalyzed by resolvases. Considerablediversity is generated by joining different V D, and J segments to a C segment; however, additional variations are introduced in the form of changes at the junctions between segmentsduring the recombination process. Changes are also induced in immunoglobulin genes by somatic mutation, which requires the actions of cytidine deaminase and uracil glycosylase.Mutations induced by cytidine deaminase probably lead to removal of uracil by uracil glycosylase,followed by the induction of mutations at the siteswhere bases are mlsslng. Allelic exclusion ensures that a given lymphocyte synthesizes only a single Ig or TCR. A productive rearrangement inhibits the occurrence of further rearrangements.The use of the V region is fixed by the first productive rearrangement, but B cells switch use of Cs genes from the initial p chain to one of the H chains coded farther downstream. This process involves a different type of recombination in which the sequencesbetween the VDJ region and the new Cn gene are deleted. More than one switch occurs in Cs gene usage. Class switching requires the same cytidine deami-
nase that is required for somatic mutation, but its role is not knonm. At an earlier stageof Ig production, switches occur from synthesis of a membrane-bound version of the protein to a secreted version. These switches are accomplished by alternative splicing of the transcript. Innate immunity is a response triggered by receptorswhose specificityis predefined for certain common motifs found in bacteria and other infective agents. The receptor that triggers the pathway is typically a member of the Toll-like class,and the pathway resembles the pathway triggered by Toll receptors during embryonic development. The pathway culminates in activation of transcription factors that causegenes to be expressedwhose products inactivate the infective agent, typically by permeabilizing its membrane.
constant region gene. Proc.Natl. Acad. Sci USA 76,3450-)454.
TwoTypes Immune Recombination Uses o f C o n s e n s uSse q u e n c e Research Lewis,S.,Gifford,A., and Baltimore,D. (1985). DNA elementsare asymmetricallyjoined durrecombinationof kappa ing the site-specific 228, immunoglobulin genes.Science 677-685.
Is Triggered AtteticExclusion by Productive Rearrangement Review Storb, U. (1987\. Transgenic mice with immunoglobulin genes.Annu Rev.Immunol. 5, t5t-t7 4. The RAGProteins CatatyzeBreakage and Reunion
References Immunogtobutin Genes AreAssembled from Their Parts in Lymphocytes Reviews AIt, F. W., Blackwell, T. I(., and Yancopoulos, G. D. (1987). Development of the primary antibody repertoire. Science 2)8, lO7 9-1087 . Blackwell, T. I(. and Alr, F. W. (1989). Mechanism and developmental program of immunoglobulin gene rearrangement in mammals. Annu. Rev.Genet.23, 605-636. Hood, L., I(ronenberg, M., and Hunkapiller, T. (1985). T cell antigen receptorsand the immunoglobulin supergene family. Cell 40, 225-229. Tonegawa,S. (1983). Somatic generation of antibody diversity. Nature 302, 575-581. Yancopoulos,G. D. andAlr, F. W (1986). Regulation of the assembly and expression of variable-region genes.Annu. Rev.Immunol. 4, )39-368. Resea rch Hozumi, N. andTonegawa, S. (1976). Evidence for somatic rearrangement of immunoglobulin genes coding for variable and constant regions. Proc.Natl Acad Sci.USA73, 1628-3632.
LightChains AreAssembled by a Singte Recombination Resea rch Max, E. E., Seidman, J. G., and Leder, P. (L979). SequencesoI five potential recombination sites encoded close to an immunoslobulin r
Reviews Gellert, M. (1992). Molecular analysis of VDJ recombination Annu. Rev.Genet.26,425-446. J eggo, P. A. ( I 998 ). DNA breakage and repair. Adv. G e n e t) 8 , 1 8 5 - 2 1 8 . Schatz,D. G., Oettinger,M. A., and Schlissel,M. S. 11992).VDJ recombination: molecular biology and regulation. Annu. Rev.Immunol. lO, )59-)83 Research Agrawal, A., Eastman, Q. M., and Schatz, D. G. ( 1998). Transpositionmediated by RAGI and RAG2 and its implications for the evolution of the immune system. Nature )94, 7 44-7 5I . Hiom, K., Melek, M., and Gellert, M. (I998). DNA transposition by the RAGI and RAG2 proteins: a possible source of oncogenic translocations. Cell 94, 463-470. Ma, Y., Pannicke, U., Schwarz, I(., and Lieber, M. R. \2002). Hairpin opening and overhang processingby an Artemis/DNA-dependent protein kinase complex in nonhomologous end joining and V(D)J recombination. Cell 108, 78t-794. Melek, M. and Gellert, M. (2000). RAGI/2-mediated resolution of transposition intermediates: two pathways and possible consequences. Cell l0l, 625-6)). Qiu, J. X., I(ale, S. B., Yarnell Schultz, H., and Roth, D. B. (200I). Separation-of-function mutants reveal critical roles for RAG2 in both the cleavage and joining steps of V(D)J recombination. Mol. Cell T, 77-87 . Roth, D. B., Menetski, J. P., Nakajima, P. B., Bosma, M. J., and Gellert, M. (1992). V D J recombination: broken DNA molecules with covalently sealed (hairpin) coding ends in SCID mouse thymocytes. Cell 70, 983-991.
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Schatz,D. G. and Baltimore, D. (1988). Stable expressionof immunoglobulin gene V(D)J recombinase activity by gene transfer into lT3 f i b r o b l a s t sC . e l l5 ) , I 0 7 - I 1 5 . Schatz,D. G., Oettinger,M. A., and Baltimore, D. (1989). The V(D)J recombination activating gene, RAG- I . Cell 59, I03 5-l 048. Tsai,C. L., Drejer, A. H., and Drejer, A. H. (2002). Evidence of a critical architectural function for the RAG proteins in end processing, protection, and joining in V(D)J recombination. GenesDev. 16, 19)4-1949. Yarnell Schultz,H., Landree, M. A., Qiu, J. X., I(ale, S. B., and Roth, D. B. (2001). Joiningdeficient RAGI mutants block V(D)J recombination in vitro and hairpin opening in vitro. M o l C e l l7 , 6 5 - 7 5 .
@
Switching 0ccursby a Novel Recombi nationReaction
Reviews Honjo, T, I(inoshita, I(., and Muramatsu, M. (2002) . Molecular mechanism of classswitch recombination: linkage with somatic hypermutation. Annu Rev.Immunol.20, 165-196. Li, 2., Woo, C. J., Iglesias-Ussel, M. D., Ronai, D., and Scharff,M. D. (2004). The generation of antibody diversity through somatic hypermutation and classswitch recombination. Genes Dev.),8, l-ll. Resea r ch Bransteitter, R , Pham, P., Scharff, M. D., and Goodman, M F. (2003). Activation-induced cytidine deaminase deaminates deoxycytidine on single-stranded DNA but requires the action of RNase. Proc Natl Acad. Sci.US,4100. 4to2-4107. G u , H . , Z o t , Y . R . , a n d R a j e w s k y ,K . ( I 9 9 3 ) . I n d e pendent control of immunoglobulin switch recombination at individual switch regions evidencedthrough Cre-loxP-mediatedgene targeting. Cell7), I 155-l 164. Iwasato, T., Shimizu, A., Honjo, T., and Yamagishi, H. (I990). Circular DNA is excisedby immunoglobulin classswitch recombination. Cell 62, 14)-149. I(inoshita, I(., Tashiro, J., Tomita, S., Lee, C. G., and Honjo, T. (1998). Targetspecificityof immunoglobulin classswitch recombination is not determined by nucleotide sequencesof S regions. Immunity 9, 849-858. Manis, J. P.,Gu, Y., Lansford, R., Sonoda,E., Ferrini, R., Davidson, L., Rajewsky, I(., and Alt, F. W. (1998). I(u70 is required for late B cell development and immunoglobulin heavy chain class switching. J. Exp. Med. 187, 2081-2089. Matsuoka, M., Yoshida, I(., Maeda, T., Usuda, S., and Sakano,H. (1990). Switch circular DNA formed in cytokine-treated mouse splenocytes: evidence for intramolecular DNA dele-
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CHAPTER 23 ImmuneDiversitv
tion in immunoglobuiin classswitching. Cel/ 62, t35-t42. Muramatsu, M., I(noshita, I(., Fagarasan,S., Yamada, S., Shinkai, Y., and Honjo, T. (2000). Classswitch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell lO2, 55j-56). Pham, P., Bransteitter, R., Petruska, J., and Goodman, M. F. (2003). ProcessiveAID-catalysed cytosine deamination on single-standed DNA simulates somatic hypermutation. Nature 424, I 03-l 07. Revy, P., Muto, T., Levy, Y., Geissmann, F., Plebani, A., Sanal, O., Catalan, N., Forveille, M., Dufourcq-Labelouse, R., Gennery, A., Tezcan, I., Ersoy, F., I(ayserili, H.,Ugazio, A. G., Brousse, N., Muramatsu, M., Notarangelo, L. D., I(noshita, K., and Honjo, T. (2000). Activation-induced cytidine deaminase (AID) deficiency causesthe autosomal recessive form of the Hyper-IgM syndrome (HIGM2). Cell lO2, 565-575. Rolink, A., Melchers, F., and Andersson, J. (19961. The SCID but not the RAG-2 gene product is required for S mu-S epsilon heavy chain class switching. Immunity 5, )19-)30. von Schwedler, U., Jack, H. M., and Wabl, M. (1990). Circular DNA is a product of the immunoglobulin classswitch rearrangement. Nature 345,452-456. Xu, L., Gorham, B., Li, S. C., Bottaro, A., Alt, F. W., and Rothman, P. (19931.Replacementof germ-line epsilon promoter by gene targeting alters control of immunoglobulin heavy chain classswitching. Proc.Natl. Acad Sci.USA 90, 3705-3709.
Somatic MutationGenerates Additional D i v e r s i tiyn M o u s ea n d H u m a nB e i n g Reviews French,D. L., Laskov R., and Scharff,M. D. ( I 989).The role of somatichypermutationin the generationo{ antibody diversity.Science 244, tt52-tt57. I(ocks,C. and Rajewsky, K. (1989).Stableexpression and somatichypermutationof antibody V regionsin B-cell developmentalpathways. Annu.Rev.Immunol.T,5)7-559. rch Resea I(m, S.,Davis,M., Sinn,E., Patten,P.,and Hood,L. ( l98l ) . Antibody diversity:somatichypermutation of rearrangedVH genes.Cell22, 573-581.
Somatic Mutation Is Inducedby Cytidine Deaminase and UraciIGlycosytase Reviews Honjo, T.,Ifinoshita,I(., and Muramatsu,M. (2002\. Molecularmechanismof classswitch
recombination: Iinkage with somatic hypermutation. Annu. Rev.Immunol.20, 165-196. I(inoshita, I(. and Honjo, T. (200I). Linking classswitch recombination with somatic hypermutation. Nal. Rev.Mol. CellBiol 2, 49)-50). Li,2., Woo, C J., Iglesias-Ussel, M. D., Ronai, D., and Scharff,M. D. (2004). The generation of antibody diversity through somatic hypermutation and classswitch recombination. Genes D e v . 1 8 ,l - l l .
I(ronenberg, M., Siu, G., Hood, L. E., and Shastri, N. (1986). The molecular geneticsof the T-cell antigen receptor and T-cell antigen recognition. Annu Rev.Immunol. 4, 529-591. Marrack, P and I(appler, J. (1987lt. The T-cell receptor.Science 238, lO7)-1079. Raulet, D. H. (1989). The structure, function, and molecular genetics of the gamma/delta T-cell receptor. Annu. Rev.Immunol. T, 175-207.
Functions TheT CettReceptor Research Di Noia, J. and Neuberger, J. (2002). Altering the pathway of immunoglobulin hypermutation by inhibiting uracil-DNA glycosylase.Nature 4t9,43-48. Muramatsu, M., I(noshita, I(., Fagarasan,S., Yamada,S., Shinkai, Y., and Honjo, T. (2000). Classswitch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell 102, 553-563. Peters, A. and Storb, U. (19961. Somaric hypermutation of immunoglobulin genes is linked to transcription initiation. Immunity 4, 57-65. Revy, P.,Muto, T., Levy, Y., Geissmann, F., Plebani, A., Sanal, O., Catalan,N., Forveille, M., Dufourcq-Labelouse, R., Gennery, A., Tezcan, I., Ersoy, F., I(ayserili, H., Ugazio, A. G., Brousse,N., Muramatsu, M., Notarangelo, L. D., ICnoshita, I(., and Honjo, T. (2000). Activation-induced cytidine deaminase (AID) deficiency causesthe autosomal recessive form of the Hyper-IgM syndrome (HIGM2). Cell 102, 565-575.
AvianImmunoglobulins AreAssembled fromPseudogenes R e s erach Reynaud, C. A., Anquez, V., Grimal, H., and Weill, J. C. (1987). A hyperconversion mechanism generates the chicken light chain preimmune repertoire. Cell 48, 37 9-388. Sale, J. E., Calandrini, D. M., Takata, M., Takeda, S., and Neuberger,M. S. (2001). Ablation of XRCC2/3 transforms immunoglobulin V gene conversion into somatic hypermutation. N6ture 412.921-926.
B Ce[[Memory A[lowsa RapidSecondary Response Review Rajewsky, K. ( 1996) . Clonal selection and learning in the antibody system.Nature 381,751-758.
i n C o n j u n c t i ow ni t h t h e M H C Review Goldrath,A. W and Bevan,M. J. (1999).Selecting and maintaininga diverseT cell repertoire. Nature402,255-262.
Locus TheMajorHistocompatibitity
oftheImmune Codes forMany Genes System Reviews Flavell, R. A., AIIen, H., Burkly, L. C., Sherman, D. H., Waneck, G. L., and Widera, G. (1986). Molecular biology of the H-2 histocompatibility complex. Science23j, 437-443. I(umnovics, A., Takada, T., and Lindahl, I(. F. (2003). Genomic organization of the mammalian MHC. Annu. Rev.Immunol.2l, 629-657. Steinmetz,M. and Hood, L. (1983). Genesof the 222, MHC complex in mouse and man. Science 727-7)2. rch Resea I(aufman, J. et al. (19991. The chicken B locus is a minimal essential maj or histocompatibility complex. Nature 401, 923-925.
Conserved InnateImmunitvUtilizes Signating Pathways Reviews Aderem, A. and Ulevitch, R. J. (2000). Toll-like receptors in the induction of the innate immune response. Nature 406, 7 82-7 87. Dangl, J. L. and Jones,J. D. (2001). Plant pathogens and integrated defence responses to infection. Nature 4ll,826-83). Hoffmann, J. A., Kafatos, F. C., Janeway, C. 4., and Ezekowitz, R. A. (19991. Phylogenetic perspectivesin innate immunity. Science284, l3l3-1318. Janeway, C. A. and Medzhitov R. (2002). Innate immune recognition. Annu. Rev.Immunol.20, t97-2t6.
T Ce[[Receptors AreRetated to Immunogtobutins Reviews Davis,M. M. (1990).T-cellreceptorgenediversity and selection.Annu Rey.Biochem. 59, 475-496.
r ch Resea Asai, T., Tena, G., Plotnikova, J., Willmann, M. R., Chiu. W. L, Gomez-Gomez,L., Boller, T., Ausubel, F. M., and Sheen,J. (2002). MAP kinase signalling cascadein Arabidopsis innate immunity. Nature 415, 97 7-98).
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Hoffmann, J. A. (2003). The immune response of Drosophila.Nature 426, JJ-)8. Ip, Y. T., Reach, M., Engstrom, Y., Kadalayil, L., Cai, H., Gonzdlez-Crespo, S., Tatei, K., and Levine, M. (1993). Dif, a dorsal-relatedgene that mediates an immune response in Drosophila.Cell 7 5, 7 53-7 63. Lemaitre, B., Nicolas, E., Michaut, L., Reichhart, J. M., and Hoffmann, J. A. (1996). The dorsoventral regulatory gene cassettespAtzle/ Toll/cactus controls the potent antifungal response in Drosophila adults. Cell 86, 973-983. Medzhitov R., Preston-Hurlburt, P., and Janeway, C. A. (1997). A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature )88, 394-)97.
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CHAPTER 23 ImmuneDiversitv
Poltorak,A., He, X., Smirnova,I., Liu, M. Y.,Huffel, C. V.,Du, X., Birdwell, D., Alejos,E., Silva, M., Galanos,C., Freudenberg,M., RicciardiCastagnoli,P.,Layton,B., and Beutler,B. (1998).DefectiveLPSsignalingin C3H/HeJ and C57BL/IOScCrmice:mutationsin Tlr4 gelre.Science 282, 2085-2088. Rutschmann,S.,Jung,A. C., Hetru, C.,Reichhart, J. M.. Hoffmann.J. A.. and Ferrandon.D. (2000).The Rel protein DIF mediatesthe antifungal but not the antibacterialhost defense in Drosophila. Immunity12, 569-580. Williams,M. J., Rodriguez.A., Kimbrell, D. A., and Eldon,E. D. (L997\.The l8-wheeler mutation revealscomplex antibacterialgene regulation in Drosophilahost defense.EMBOJ. 16, 6t20-6130.
Promoters andEnhancers C H A P T EO RU T L I N E Introduction Eukaryotic RNAPolymerases Consistof ManySubunits . RNApolymerase I synthesizes rRNAin the nucteolus. r RNApotymerase II synthesizes mRNA in the nucleoplasm. o RNApotymerase III synthesizes smatlRNAs in the nucteoo[asm. r A[[eukaryotic RNApotymerases have-12 subunits andare aggregates of >500kD. o Somesubunits arecommon to a[[threeRNApolymerases. . Thelargest subunitin RNApotymerase II hasa CTD (carboxy-terminaI domain) consisting of multiplerepeats of a heptamer. PromoterEtements Are Definedby Mutations and Footprinting o Promoters aredefinedby theirabil.ityto causetranscription of an attached sequence in an appropriate testsystem in vitro or in vivo. RNAPolymerase I Hasa BipartitePromoter r TheRNApolymerase I promoter consists of a corepromoter (UPE). andan upstream controletement r ThefactorUBFlwrapsDNAarounda proteinstructure to bringthe coreandUPEinto proximity. o SL1includes thefactorTBPthat is involved in initiationby a[[threeRNApotymerases. . RNApotymerase bindsto the UBFl-SL1 comptex at the core promoter. RNAPotymerase III UsesBoth Downstream and Upstream Promoters r RNApotymerase III hastwo typesof promoters. o InternaIpromoters haveshortconsensus sequences located withinthetranscription unitandcause initiationto occur a fixeddjstance upstream. . Upstream promoters contajnthreeshortconsensus sequences upstream of the startpoint that areboundby transcription factors. TFInBIs the CommitmentFactorfor Po[III Promoters . TFruA andTFxIC bindto the consensus sequences and enabte TFgBto bindat the startpoint. . TFuBhasTBPasonesubunitandenabtes RNApotymerase to bind.
II TheStartpointfor RNAPolymerase . RNApotymerase general factors II requires transcription (cattedTF11X) to initjatetranscription. r RNApotymerase havea shortconserved II promoters (theinitiatorInR)at the startpoint. Py2CAPy5 sequence r TheTATA II component of RNApotymerase boxis a common of an A-T-richoctamerlocated oromoters andconsists -25 bp upstream ofthe startpoint. . TheDPEis a common II proof RNApotymerase component box. motersthat do not containa TATA r A corepromoter II inctudes the InRand for RNApotymerase boxor a DPE. eithera TATA TBPIs a UniversalFactor . TBPis a component factorthat is of the positioning to bindits required for eachtypeof RNApotymerase promorer. . Thefactorfor RNApotymerase whichconsists II is TF11D, with a tota[mass-800 kD. of TBPand11TAFs, T B PB i n d sD N Ai n a n U n u s u aW l ay . TBPbindsto theTATA of DNA. boxin the minorgroove . It formsa saddle the DNAandbendsit by -80'. around . Someof theTAFs resemble histones andmayforma structureresembting a histoneoctamer. at the Promoter The BasaIApparatusAssembtes r Bindingof TFnDto the TATA boxis the first stepin initiation. . 0thertranscription in a defined factorsbindto the comptex regionon DNA. the lengthofthe protected order,extending 'it initiates . WhenRNApotymerase II bindsto the comptex, transcription. Initiation Is Fotlowedby PromoterCtearance o TFuE to mettDNAto a[[owpotymerase andTFnHarerequired movemenr. . Phosphorytation for etongation of the CTDmaybe required to begin. . Further is required at someprophosphorylation of the CTD initiation. moters to endabort'ive r TheCTDmaycoordinate processing of RNAwith transcriotion. Continued on nextpoge
609
?Ed
A ConnectionbetweenTranscription andReoair r Transcribed genesarepreferentialty repaired whenDNAdamage occurs. r TFnHprovides the Linkto a complex of reparr enzymes. e Mutations in theXPDcomponent of TFnH cause threetypesof humandiseases
?rlI/3| ShortSequenceEtementsBindActivators . Shortconserved seouence elements are in the regionpreceding dispersed the startooint. o Theupstream etements increase the frequency of initiation. r Thefactorsthat bindto themto stimulate transcriotion arecatledactivators.
@
PromoterConstruction Is Ftexibte but ContextCanBe Important . NoindividuaI upstream is essene[ement tial for promoter function,atthough oneor moreetements mustbe presentfor efficientinitiation. . Somee[ements arerecoqnized by muttipte factors, andthe factorthat is usedat any particular promoter maybe determined by the contextof the otherfactorsthat arebound.
i21tg{ EnhancersContainBidirectionaIElements ThatAssistInitiation r An enhancer proactivates the nearest moterto it, andcanbeanydistance eitheruostream or downstream of the pr0m0Ier. e A UAS(upstream activator in sequence) yeastbehaves [ikean enhancer butworks ontyupstream of the promoter. r Similarsequence etements arefoundin enhancers andoromoters. r Enhancers formcomolexes of activators that interactdirectty or indirectty withthe promoter.
Introduction Initiation of transcription requires the enzyme RNA polymerase and transcription factors.Any protein that is needed for the initiation of transcription, but which is not itself part of RNA polymerase, is defined as a transcription factor. Many transcription factors act by recognizing cis-actingsites on DNA. Binding to DNA, however, is not the only means of action for a transcription factor. A factor may recognize another factor, or may recognize RNA polymerase, or may be incorporated into an initiation complex only in the presence of several other proteins. The ultimate test for membership of the tran-
610
CHAPTER 24 Promoters andEnhancers
Enhancers Containthe SameElements ThatAre Foundat Promoters o Enhancers aremadeofthe sameshort sequence etements that arefoundin Dr0mote15. r Thedensity of sequence components is greater in the enhancer thanin the promoter. Enhancers Workby Increasingthe Concentration of ActivatorsNearthe Promoter o Enhancers usualty workontyin crsconfigurationwith a targetpromoter. . Enhancers canbe madeto workin trans configuration by tinkingthe DNAthat containsthetargetpromoter to the DNAthat viaa proteinbridge contains the enhancer or by catenating thetwo motecules. . Theprinciple is that an enhancer worksin in whichit is constrained anysituation to bein thesameproximity asthe promoter. Is Associated GeneExpression with Demethytation r Demethytation at the 5' endof the geneis necessary for transcription. CpGIs[andsAre Regu[atory Targets r CpGislands surround the promoters of geneswherethey constitutive[y expressed areunmethylated. . CpGistands alsoarefoundat the promotgenes. ersof sometissue-regulated o Thereare-29,000CpGislands in the numag nenome. o Methytation of a CpGislandprevents activationof a promoter wjthjnit. r Repression is caused by proteins that bind to methytated CpGdoubLets. Summarv
scription apparatus is functional: A protein must be needed for transcription to occur at a specific promoter or set of promoters. A significant difference between the transcription of eukaryotic and prokaryotic mRNAs is that initiation at a eukaryotic promoter involves alarge number of factors that bind to a variety of cli-acting elements. The promoter is defined as the region containing all these binding sites, that is, all the binding sites that can support transcription at the normal efficiency and with the proper control. Thus the major feature defining the promoter for a eukaryotic mRNA is the location of binding sites for transcription factors. RNA polymerase itself binds
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SurnsaqtuLs ro; alqrsuodsJr sr pue fula,rpe re1 -n11arSurureuJJ eqt Jo tsoru sluasardartI '(snl -oJIJnu aqt Surpnpxe snalf,nu aqt yo ued aqr) ruseldoalrnu eql ul pJtplol sr qJrqM 'II aspJaru -,{1od ypg sr aru.,{zua rolBru reqto eql '(,illruenb Jo srurel ur) srsaqluls VNU JelnllJl tsoru ro; stunoJf,p tI 'yNUJ.roy Surpor sauaSaqt SurqrJJsueJt JoJalqrsuodsarsrpup snlo '1 asereru,{.1od -epnu qlrqM Jqt ur saprsJJ VNU arur{zuaaql sr.dtr.r.rlf,e luaururord lsolu eqJ 'JqrJf,supndaqt reqr saua8 aqt qryn puods -aJJoJleql snallnu eql ur suorleJol lueJeJJrp elpq saspJeu.{1odVNU rlto,{rB>lna aJrql JqI raueldaqe;o sleader L11nu Lural-fxoqlec) a1d 1o6uqsrsuor(ureuop1eu 0ll e seq11aserauflod VNUurlrunqnslsabrelaql r 'saserar.ur{1od arPslrunqns ouJo$r vNUaalqlllPol uoururol '01 puP aie 009<,osalebar66e ylx rqo&e1na slrunqns zI- a^pqsasereurtlod 11yr 'ursPl0o0llnu ylg r oql ur svNUllpulssazrsaqlur\s 111asereuflod 'ursPlooallnu ypX r aql ur VNUtU sazLsaqlufis 11asetauf1od 'snl00lJnu ypX r oql ur VNUrsazrsaqlufs 1 esereufi1od
qrunqnsAueyrl 1o lsrsuolsasPjau^lod
vNur!]o&e1n3 '(Surssaro;4pue Sunr1dg '92 raldeq3 vNU aas)ldrnsuerl .,{.rerurrdeql ur uollJear a8e,teap e ruoJJsllnseJpeJlsul lnq'JIJSll IuJAJ uortPurruJel
('xaldtuor uLalord a6relp uuojo] ]telalurloluequeeql lp puelalouotdaql lp slolreJuorldursuetl asrMiaqlo ro palrolaqr\euy16) 'luplsrpql lpla^ospaleroloqfeu srol 1eq1os pabuelear -lej uorldulsupll pu[qoslp]eql sluaulola etoue 6urureluor .lalupquo Jofeuepalredr{1eso1r u V ' d q 0 0 2 ) surPluol ralouro.lo 'pelerlrut aql sr uorJdulsuell eieqM elrs eql uotJupallsdn ]loqslPle^as p spqII aserau[1od spualxo ralourotd ypg fiq peqursuerl y i.'!f ]*il*?j auab1errdfi1 1eq1 uollecolur pgxrlare uorloucsueJl to1lutodpelsaql 1o(dq g9>) flrurcrn alerpaululorll ur sluouroloaqt AuO sJolcel uorloucsuejlpu!qleql sluauolo ocuonbespesredslpsuteluoC
-_______-------JOCUeqUf
Relatedto bacterialsubunitB' BindsDNA Has CTD = (YSPTSPS)n lyeast n =26; mousen = 52] Relatedto bacterialsubunitB Bindsnucleotides
Relatedto bacterialsubunita
15*#{Cg J4.ii Somesubunits arecommon to atl classes of eukaryotic RNApotymerases andsomearerelated to bacteriaIRNApotymerase.
genomes are much smaller, the resident polymerase needs to transcribe relatively few genes, and the control of transcription is likely to be very much simpler (if it exists at all). Thus these enzymes are analogous to the phage enzymes that do not need the ability to respond to a more c o m p l e xe n v i r o n m e n t . A major practical distinction between the eukaryotic enzymes is drawn from their response to the bicyclic octapeptide d amanitin. In basically all eukaryotic cells, the activity of RNA polymerase II is rapidly inhibited by low concentrations of q, amanitin. RNA polymerase I is not inhibited. The response of RNA polymerase III to u amanitin is less well conserved;in animal cellsit is inhibited by high levels, but in yeast and insects it is not inhibited.
Promoter ELements AreDefined by Mutations andFootprinting o Promoters aredefinedby theirabitityto cause transcription of an attached sequence in an appropriate test systemin vitroor in vivo.
The first step in characterizing a promoter is to define the overall length of DNA that contains all the necessarysequenceelements. To do this, we need a test system in which the promoter is r e s p o n s i b l ef o r t h e p r o d u c t i o n o f a n e a s i l y assayed product. Historically, several types of systemshave been used:
. In the llcyte system,a DNA template is injected into the nucleus of tLreXenopus laevis oocyte. The RNA transcript can be recovered and analyzed. The main limitation of this system is that it is restricted to the conditions that prevail in the oocyte. It allows characterization of DNA sequences,but not of the factors that normally bind them. . Transfectionsystemsallow exogenous DNA to be introduced into a cultured cell and expressed.The system is genuinely ir vivo in the sense that transcription is accomplished by the same apparatus responsiblefor expressingthe cell'sown genome. It differs from the natural situation, though. because the template consistsof a gene that would not usually be transcribed in the host cell. The usefulness of the system maY be extended by using a variety of host cells. . Transgenicsystemsinvolve the addition of a gene to the germline of an animal. Expression of the transgene can be followed in any or all of the tissues of the animal. Some common limitations apply to transgenic systemsand to transfection: The additional gene often is present in multiple copies, and is integrated at a different location from the endogenous gene. Discrepancies between the expression of a gene in vitro and its expression as a transgene can yield important information about the role of the genomic context of the gene. . The in vitro system takes the classic approach of purifying all the components and manipulating conditions until fairhful initiation is seen. "Faithful" initiation is defined as production of an RNA starting at the site corresponding to the 5'end of mRNA (or IRNA or IRNA precursors). Ultimately this allows us to characterize the individual sequence elements in the promoter and the transcription factors that bind to them. When a promoter is analyzed, it is important that only t]nepromoter sequence changes. {jIS#ftil**"} shows that the same long upstream sequenceis always placed next to the promoter to ensure that it is always in the same context. Termination does not occur properly in t}jle tn vitro sysrems,and as a result the template is cut at some distance from the promoter (usually - 5 0 0 b p d o w n s t r e a m ) . T h i s e n s u r e st h a t a l l polymerases "run off" at the same point, thus generating an identifiable transcript. Are Definedby Mutationsand Footprinting 24.3 PromoterEtements
613
Upstreamsequence is alwaysthe same
Only the test promoterdiffers
Run-offtranscript is definedlength
f ltti*fl I+.-!iA promoter is testedby modifying thesequence thatis connectedto a constant upstream sequence anda constant downstream transcriotion unit.
Transcription
I s* \,
es\,
our\-
RNA stillmade:
either end, until at some point it ceasesto be active, as illustrated in FlStitrilHq.r+. The boundary upstream can be identified by progressively removing material from this end untilpromoter function is lost. To test the boundary downstream, it is necessary to reconnect the shortened promoter to the sequenceto be transcribed (sinceotherwise there is no product to assay). Once the boundaries of the promoter have been defined, the importance of particular bases within it can be determined by introducing point mutations or other rearrangements in the sequence. As with bacterial RNA polymerase, these can be characterized as up or down mutations. Some of these rearrangements affect only rhe rate of initiation; others influence ll:resiteat which initiation occurs, as seen in a change oI the startpoint. To be sure that we are dealing with comparableproducts, in each caseit is necessary to characterize the 5' end of the RNA. We can apply several criteria in identifying the sequence components of a promoter (or any other site in DNA): . Mutations in the site prevent function invitro or invivo. (Many techniques now exist for introducing point mutations at particular base pairs, and in principle every position in a promoter can be mutated and the mutant sequencetested in vitro or in vivo.) . Proteins that act by binding to a site may be footprinted on it. There should be a correlation between the ability of mutations to prevent promoter function and to prevent binding of the factor. . When a site recognized by a particular factor is present at multiple promoters, it should be possibleto derive a consensus sequencethat is bound by the factor. A new promoter should become responsive to this factor when an appropriate copy of the element is introduced.
No RNA made:deletion has enteredpromoter
Upstream boundary of promoter lies between endsof deletions li*tJith t+.'t Promoter boundaries canbedetermined by makingdetetions that progressively remove morematerialfromoneside.Whenonedeletion faitsto prevent RNA synthesis butthe nextstopstranscription, the boundary of the promoter mustlie between them. We start with a particular fragment of DNA that can initiate transcription in one of these systems.The boundaries of the sequenceconstituting the promoter then can be determined by reducing the length of the fragment from
674
C H A P T E2R4 P r o m o t e rasn d E n h a n c e r s
RNAPolymerase I Has a BipartitePromoter o TheRNApolymerase I promoter consists of a core promoter andan upstream (UPE). controIetement . ThefactorUBFlwrapsDNAarounda protein structure to bringthe coreandUPEinto proximity. . 511inctudes thefactorTBPthat is invotved in initjatjonby a[[threeRNApotymerases. o RNApotymerase bindsto the UBF1-SL1 complex at the coreoromoter.
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The detailed interactions are different at the two types of internal promoter, but the principle is the same. TFnICbinds downstream of the startpoint, either independently (type 2 promoters) or in conjunction with TFrrrA (type I promoters). The presenceof TFgC enablesthe positioning factor TFnIB to bind at the startpoint. RNA polymerase is then recruited. ft{-iiJ{tfIi'Fi summarizes the stagesof reaction at type 2 internal promoters. TFnICbinds to both boxA and boxB. This enablesTFnIBto bind at the startpoint. At this point RNA polymerase III can bind. The difference at type I internal promoters is that TFgA must bind at boxA to enable TF111C to bind at boxC. ilI{ri"iRf" i+"$ shows that, once TFnIChas bound, events follow the same course as at type 2 promoters, with TFnIBbinding at the startpoint, and RNA polymerase III joining the complex. T\,pe I promoters are found only in the genesfor 5S rRNA. TF111A and TFl1yCare assembly factors, whose sole role is to assistthe binding of TF11B at the right location. Once TF111B has bound, TFnIA and TFnICcan be removed from the promoter (by high salt concentrationinvitro) without affecting the initiation reaction. TFpB remainsbound in the vicinity of thestartpoint and its presenceis sufficient to allow RNA polymeraseIII to bind at thestartpoint.Thus TFynBis the only true initiation factor required by RNA polymerase IIL This sequence of events explains how the promoter boxes downstream can cause RNA polymerase to bind at the startpoint, farther upstream. Although the ability to transcribe these genes is conferred by the internal promoter, changes in the region immediately upstream of the startpoint can alter the efficiency of transcription. TF6C is a largeprotein complex (>500kD), which is comparable in sizeto RNA polymerase itself and contains six subunits. TFnIAis a member of an interesting classof proteins containing a nucleic acid-binding motif called a zinc finger (see Section 25.9, A Zinc Finger Motif Is a DNA-Binding Domain). The positioning factor, TFrrrlB,consistsof three subunits. It includes the same protein, TBP, that is present in the core-binding factor for pol I promoters, and also in the correspondingtranscription factor (TFnD) for RNA polymerase II. It also contains Brf, which is related to the factor TF1B that is used by RNA polymerase II. The third subunit is called B"; it is dispensableif the DNA duplex is partially melted, which suggeststhat its function is to initiate the transcrintion bubble. The role of B"
Startpoint
usebinding ilIfir"Jf{i: type2 potIII promoters il:i.ii InternaI to recruitthepositionofTllnCto boxAandboxBsequences IIL RNApolymerase ing factorTFn1B, whichrecruits
usethe ffiliJfii ;lr;.{i Internaltype 1 pot III promoters at boxAandboxC.to andTFn1C, factorsTFrrrA assl'mbty RNA whichrecruits factorTFn1B. recrujtthe positioning po['ymerase IIL
for Po[III Promoters 677 Factor 24.6IF1B Is the Commitment
may be comparable to the role played by sigma factor in bacterial RNA polymerase (seeSection I 1.I6, Substitution of SigmaFactorsMay Control Initiation). The upstream region has a conventional role in the third classof polymerase III promoters.In the example shown in Figure 24.8,there are three upstream elements.Theseelementsare also found in promoters {or snRNA genesthat are transcribedby RNA polymerase II. (Genes for some snRNAs are transcribed by RNA polymerase II, whereas others are transcribed by RNA polymerase III.) The upstream elements function in a similar manner in promoters for both polymerasesII and III. Initiation at an upstream promoter for RNA polymerase III can occur on a short region that immediately precedes the startpoint and contains only the TATA element. Efficiency of transcription, however, is much increasedby the presenceof the PSEand OCT elements.The factors that bind at these elements interact cooperatively. (The PSEelement may be essentialat promoters used by RNA polymerase II, whereas it is stimulatory in promoters used by RNA polymerase III; its name stands for proximal sequenceelement.) The TATA element confers specificity for the type of polymerase (II or III) that is recognized by an snRNA promoter. It is bound by a factor that includes the TBP,which actually recognizesthe sequencein DNA. The TBP is associated with other proteins, which are specific for the type of promoter. The function of TBP and its associatedproteins is to position the RNA polymerase correctly at the startpoint. We discussthis in more detail for RNA polymerase II (seeSection24.8,T8P Is a Universal Factor). The factors work in the same way for both types of promoters for RNA polymerase III. The factorsbind at thepromoterbeforeRNApolymerase itselfcan bind They form a preinitiation complex that directs binding of the RNA polymerase.RNA polymerase III does not itself recognize the promoter sequence, but binds adjacent to factors that are themselves bound just upstream of the startpoint. For the type I
ill#::i [l"^T#,ffi Ji::T,lil Hi,::il,'*:
is bound just upstream of the startpoint, thereby providing the positioning information. For the upstream promoters, TFnyBbinds directly to the region including the TATA box. This means that irrespective of the location of the promoter sequences,factor(s) are bound closeto the startpoinr in order ro direct binding of RNA polymerase III.
618
C H A P T E2R4 P r o m o t e rasn d E n h a n c e r s
TheStartpointfor RNA Polymerase II o RNApotymerase generaI II requires transcription factors(ca[[edTFsX)to init'iatetranscription. r RNApotymerase II promoters commonty havea shortconserved Py2CAPy5 (theinitiator sequence InrR)at the startpoint. r TheTATA boxis a common component of RNA potymerase II promoters andconsists of an A-T-richoctamerlocated-25 bp upstream of the startpoint. r TheDPE is a common component of RNA potymerase II promoters that do not containa TATA box. o A corepromoter for RNApotymerase II generalty includes the InRandeithera TATA boxor a DPE.
The basicorganization of the apparatusfor transcribing protein-coding genes was revealed by the discovery that purified RNA polymerase II can catalyzesynthesisof mRNA, but cannot initiate transcription unless an additional extract is added. The purification of this extract led to the definition of the general transcription factors-a group of proteins that are needed for initiation by RNA polymerase II at all promoters. RNA polymerase II in conjunction with these factors constitutes the basal transcription apparatus that is needed to transcribe any promoter. The general factors are described as TFnX, where "X" is aletter that identifies the individual factor. The subunits of RNA polymerase II and the general transcription factors are conserved among eukaryotes. Our starting point for considering promoter organization is to define the core promoter as the shortest sequence at which RNA polymerase II can initiate transcdption. A core promoter can in principle be expressedin any cell. It comprises the minimum sequence that enables the general transcription factors to assembleat the startpoint. Core promoters are involved in the mechanics of binding to DNA and enable RNA polymerase II to initiate transcription. A core promoter functions at only a low efficiency. Other proteins, called activators, are required for a proper level of function (seeSection 24.13, Short SequenceElements Bind Activators). The activators are not described systematically, but have casual names reflecting their histories of identification. We may expect any sequence components involved in the binding of RNA polymerase and general transcription factors to be conserved at most or all promoters. As with bacterial pro-
TATAbox Inr Coreoromoter -tnTn-contarnrno
DPE
-TATA-less coreDromoler FIfiURfI4"t* TheminimaIpolII promoter mayhavea TATA box-25 bp upstream ofthe InR.TheTATA boxhas theconsensus sequence of TATAA. TheInr haspyrimidines (Y)surrounding theCAat thestartpoint. TheDEP is downstream of thestartpoint. Thesequence shows the coding strand. moters, when promoters for RNA polymerase II are compared, homologies in the regions near the startpoint are restricted to rather short sequences.These elements conespond with the sequencesimplicated in promoter function by mutation. FISTJRE I4":* shows the construction of a typical pol II core promoter. At the startpoint, there is no extensive homology of sequence,but there is a tendency for the first base of mRNA to be A, flanked on either side by pyrimidines. (This description is also valid for the CAT start sequence of bacterial promoters. ) This region is called the initiator (Inr), and may be describedin the general form Py2CAPy5.The Inr is contained between positions -3 and +5. Many promoters have a sequence called the TATA box, usually lo cared -25 bp upstream of the startpoint in higher eukaryotes. It constitutes the only upstream promoter element that has a relatively fixed location with respect to the startpoint. The core sequence is TAIAA, usually followed by three more A-T basepairs. The TATA box tends to be surrounded by G-Crich sequences, which could be a factor in its function. It is almost identical with the -10 sequence found in bacterial promoters; in fact, it could pass for one except lor the difference in its location at -25 instead of -10. Single-basesubstitutions in the TATA box act as strong down mutations. Some mutations reverse the orientation of an A-T pair, so base composition alone is not sufficient for its function. Thus the TAIA box comprises an element whose behavior is analogous to our concept of the bacterial promoter: a short, well-defined sequencejust upstream of the startpoint, which is necessaryfor transcription. Promoters that do not contain a TATA element are called TATA-less promoters. Surveys of promoter sequencessuggest Ihat 50o/"
or rnore of promoters may be TATA-less.When a promoter doesnot contain a TAIAbox, it usually'contains another element, the DPE (downstr€rampromoter element), which is located at +28-+32. Most core promoters consist either of a TATA bor: plus InR or of an InR plus DPE.
Factor TBPIs a Universal o TBPis a component factorthat of the positioning to is required for eachtypeof RNApotymerase bindits Dromoter. o Thefactorfor RNApotymerase which II is TFnD, witha total mass consists ofTBPand11TAFs, .-800kD. The first step in complex formation at a promoter containing a TATA box is binding of the factor TFID to a region that extends upstream from the TATA sequence. TFnD contains two types of component. Recognition of the TAIA bo>r is conferred by the TATA-binding protein (TBP), a small protein of -30 kD. The other sub,units are called TAFs (for TBP-associated fact.ors).Some TA-Fsare stoichiometric with TBP; others are present in lesseramounts. TFnDscontaining different TAFs could recognize different promoters. Some (substoichiometric) TAFs are tissue-specific.The total mass of TF1D typically is -800 kD, contains TBP and I I TAFs, varying in rnassfrom 30 rc 2r0 kD. The TAFsin TFnD are narned in the form TAF1O0,where "00" gives the molecular mass of the subunit. Positioning factors that consist of TBP associated with a set of TAFs are responsible for identifying all classesof promoters. TFnIB (for pol III promoters) and SLI (for pol I promoters) may both be viewed as consisting of TBP associatedwith a particular group of proteins that substitute for the TAFs that are found in TF1D. TBP is the key component, and is incorporated at each type of promoter by a different mechanism. In the case of promoters for RNA polymerase II, a key feature in positioning is the fixed distance of the TATA box from the startpoint. FlSilRf,fi4.1:.shows that the positioning factor recognizes the promoter in a different way in each case.At promoters for RNA polymerase III, TFrrB binds adjacent to TFn1C.At promoters for RNA polymerase I, SLI binds in conjunction with UBF. TF1D is solely responsible for recognizing promoters for RNA polymerase II' At a promoter that has a TAIA element, TBP binLdsspecifically to DNA, but at other promoters it may be incorporated by association with
Factor 24.8 TBPIs a Universal
679
/^*oporymerase rl Q} Pol I promoters .
IBP
rl.r;!jiii: i:.:lI il A viewin cross-sectjon showsthat TBP surrounds DNAfromthesideofthenanowgroove. TBPconsistsof two related(40%identicat) conserved domains, whichareshownjn tightanddarkbtue.TheN-terminal. regionvariesextensivety andis shownin green.Thetwo strands oftheDNAdoubte hetjxarein lightanddarkgray. Photocourtesy of Stephen K. Burtey.
1\5 L 1
\
I /^*oporymerase
TBPBindsDNA
C
in an UnusuaI Way Startpoint
TBPbindsto the TATA boxin the minorqrooveof DNA. It formsa sadd[e around the DNAandbendsit bv -800.
Someof the TAFs resembte histones andmayform a structure resemblinq a histoneoctamer.
' RNApolymerases ,r:' arepositioned at atlpromotersby a factorthat contains TBP.
other proteins that bind to DNA. Whatever irs means of entry into the initiation complex, it has the common purpose of interaction with the RNA polymerase. TFnDis ubiquitous, but not unique. All multicellular eukaryotesalso expressan alternative complex, which has TLF (TBP-like factor) instead of TBP.A TLF is typically -60% similar to TBP.It probably initiates complex formation by the usual set of TF11factors. TLF does not, however, bind to the TATA box, and we do not yet know how it works. Drosophilaalso has a third factor, TRFI, which behavesin the same way as TBP and binds its own set of TAFs ro form a complex that functions as an alternative to TF1IDat i specificset of promoters. C H A P T E2R4 P r o m o t e rasn d E n h a n c e r s
TBP has the unusual property of binding to DNA in the minor groove. (Virtually all known DNAbinding proteins bind in the major groove.) The crystalstructure of TBP suggestsa detailedmodel for its binding to DNA. i:li.rl.j*i:,t", "rii shows that it surrounds one face of DNA, forming a "saddle" around the double helix. In effect,the inner surface of TBP binds to DNA, and the larger outer surfaceis availableto extend contactsto other proteins. The DNA-binding site consistsof a C-terminal domain that is conservedbetween s p e c i e s ,a n d t h e v a r i a b l e N - t e r m i n a l t a i l i s exposed to interact with other proteins. It is a measure of the conservation of mechanism in transcriptional initiation that the DNA-binding s e q u e n c eo f T B P i s 8 0 % c o n s e r v e db e t w e e n yeast and human beings. Binding of TBP may be inconsistent with the presenceof nucleosomes.Nucleosomesfonn preferentially by placing A-T-rich sequences with the minor grooves facing inward; as a result. they could prevent binding of TBP.This may explain why the presenceof nucleosomes p r e v e n t si n i t i a t i o no I t r a n s c r i p r i o n .
tz9
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tacts the factors TF1E, TFrrR and TFnH, which may align them in the basal factor complex. The factor TFnF is a heterotetramer consisting of two tlpes of subunit. The larger subunit (RAP74) has an AIP-dependent DNA helicase actMtythat couldbe involvedinmelting the DNA at initiation. The smaller subunit (RAP38) has some homology to the regions of bacterial sigma factor that contact the core polymerase; it binds tightly to RNA polymerase II. TFrrFmay bring RNA polymerase II to the assemblingtranscription complex and provide the means by which it binds. The complex of TBP and TAFs may interact with the CTD tail of RNA polymerase, and interaction with TFnB may also be important when TFnF/polymerasejoins the complex. Polymerase binding extends the sites that are protected downstream to +I5 on the template strand and +20 on the nontemplate strand. The enzyme extends the full length of the complex becauseadditional protection is seen at the upstream boundary. What happens at TAIA-less promoters? The same general transcription factors, including TFnD, are needed. The Inr provides the positioning element; TFID binds to it via an ability of one or more of the TAFs to recognize the Inr directly. Other TAFs in TFnD also recognize the DPE element downstream from the startpoint. The function of TBP at these promoters is more like that at promoters for RNA polymerase I and at internal promoters for RNA polymerase III. Assembly of the RNA polymerase II initiation complex provides an interesting contrast with prokaryotic transcription. Bacterial RNA polymerase is essentially a coherent aggregarc with intrinsic ability to bind DNA; the sigma factor, needed for initiation but not for elongation, becomes part of the enzyme before DNA is bound, although it is later released.RNA polymerase II can bind to the promoter, but only after separatetranscription factors have bound. The factorsplay a role analogousto that of bacterial sigma factor-to allow the basic polymerase to recognize DNA specifically at promoter sequences-but have evolved more independence.Indeed, the factors are primarily responsible for the specificity of promoter recognition. Only some of the factors participate in protein-DNA contacts (and only TBP makes sequence-specificcontacts); thus protein-protein interactions are important in the assemblyof the complex. When a TATA box is present, it determines the location of the startpoint. Its deletion causes the site of initiation to become erratic, although
RNApotyS;,+-J#.il :i',;"iii TfuBbindsto DNAandcontacts and mer,ase nearthe RNAexitsiteandat theactivecenter, 24.17, whichshows withFigure it on DNA. Compare orients in transcription. engaged structure the potymerase
anlr overall reduction in transcription is relatively small. Indeed, some TAIA-less promoters Iacl
InitiationIs Followed Clearance by Promoter I TFnEandTFnHarerequired to mettDNAto atlow po[ymerase movement. . Phosphorytation for of the CTDmaybe required to begin. etongation o Furtherphosphoryl.ation at of the CTDis required initiation. to endabortive somepromoters r TheCTDmaycoordinate processing of RNAwith transcri otion.
Ctearance 623 by Promoter 24.11InitiationIs Fottowed
Most of the general transcription factors are required solely to bind RNA polymeraseto the promoter, but some act at a later stage.Binding of TFnEcausesthe boundary of the region protected downstream to be extended by another turn of the double helix, to +30. Two further factors, TFnH and TFnJ,join the complex after TF1E. They do not change the pattern of binding ro DNA. TF11His the only general transcription factor that has multiple independent enzymatic activities.Its severalactivitiesinclude an AIPase, helicasesof both polarities,and a kinase activity that can phosphorylate the CTD tail of RNA polymerase II. TFrrHis an exceptional factor that may also play a role in elongation. Its interaction with DNA downstream of the startpoint is required for RNA polymerase to escapefrom the promoter. TFnH is also involved in repair of
I
o [YSPTSPS]n ilfiiJiiil il,i"t i" Phosphorytation of the CTDby the kinase activity ofTFllHmaybe neededto releaseRNApotymerase to start transcription.
624
CHAPTER 24 Promoters andEnhancers
damageto DNA (seeSection24.I2, A Connection between Tfanscription and Repair). The initiation reaction, as defined by formation of the first phosphodiester bond, occurs once RNA polymerase has bound. iliSl"{fi{: :4..ti= proposes a model in which phosphorylation of the tail is needed to releaseRNA polymerase II from the transcription factors so that it can make the transition to the elongating form. Most of the transcription factors are released from the promoter at this stage. On a iinear template, ATP hydrolysis, TFnE, and the helicase activity of TFIH (provided by the XPB subunit) are required for polymerase movement. This requirement is bypassedwith a supercoiled template. This suggeststhat TFrrE and TF1IHare required to melt DNA to allow polymerase movement to begin. The helicase activity of the XPB subunit of TFnH is responsible for the actual melting of DNA. RNA polymerase II stutters at some genes when it starts transcription. (The result is not dissimilar to the abortive initiation of bacterial R N A p o l y m e r a s e d i s c u s s e di n S e c t i o n I l . l l , Sigma Factor Controls Binding ro DNA, although the mechanism is different.) Ar many genes, RNA polymerase II terminates after a short distance. The short RNA product is degradedrapidly. To extend elongation into the gene, a kinase called P-TEFb is required. This kinase is a member of the cdk family that controls the cell cycle. P-TEFb acts on the CTD to phosphorylate it further. We do not yet understand why this effect is required at some promoters but not others or how it is regulated. The CTD may also be involved, directly or indirectly, in processing RNA after it has been synthesizedby RNA polymerase II. ilIirrtft.il f 4.1* summarizes processing reactions in which the CTD may be involved. The capping enzyme (guanylyl transferase),which addsthe G residue to the 5'end of newly synthesized mRNA, binds to the phosphorylated CTD: This maybe important in enabling it to modify the 5'end as soon as it is synthesized.A set of proteins called SCAFs bind to the CTD, and they may in turn bind to splicing factors. This may be a means of coordinating transcription and splicing. Some components of the cleavage/polyadenylation apparatus also bind to the CTD. Oddly enough, they do so at the time of initiarion, so that RNA polymerase is ready for the 3' end processing reactions as soon as it setsout! AII of this suggests that the CTD may be a general focus for connecting other processeswith transcription. In the casesof capping and splicing, the CTD
between A Connection andRepair Transcription 'Iranscribed genesarepreferentiatty repaired when DNAdamage occurs. 'fFnH provides of repair the tinkto a comptex Lrnzymes. of TFnHcause lMutations in theXPDcomponent lthree typesof humandiseases
In troth bacteria and eukaryotes, there is a direct linl< from RNA polymerase to the activation of repair. The basic phenomenon was first observed because transcribed genes are preferentially repaired. It was then discovered that it is only the template strand of DNA that is the targetthe nontemplate strand is repaired at the same rate as bulk DNA. In bacteria.the repair activity is provided by the zw excision-repair system (seeSection20.3, Excision Repair Systemsin E . coli). Preferential repair is abolished by mutations inthe gene mfd, whose product provides the link from RNA polymerase to the Uvr enzymes. l:i:!*fi:li:lii.li'i,lshows a model for the link between transcription and repair. When RNA polymerase encounters DNA damage in the template strand, it stalls because it cannot use the damaged sequencesas a template to direct cornplementary base pairing. This explains the specificity of the effect for the template strand (damage in the nontemplate strand does not impede progressof the RNA polymerase). FiilliftS,ir+.1$TheCTD The Mfd protein has two roles. First, it disis importantin recruiting enzymes that modifvRNA. placesthe ternary complex of RNA polymerase from DNA. Second, it causes the UvTABC en,ryme to bind to the damagedDNA. This leads to r:epair of DNA by the excision-repair mechfunctions indirectly to promote formation of a n i s m ( s e eF i g u r e 2 0 . I 1 ) . A f t e r t h e D N A h a s the protein complexes that undertake the reacbeen repaired, the next RNA polymerase to traverse the gene is able to produce a normal tions. In the caseof 3' end generation, it may participate directly in the reaction. transcript. A similar mechanism, albeit one that relies The general process of initiation is simiiar on different components, is used in eukaryotes. to that catalyzed by bacterial RNA polymerase. Binding of RNA polymerase generates a closed The template strand of a transcribed gene is prerferentially repaired following UV-induced complex, which is converted at a later stage to damage. The general transcription factor TFnH an open complex in which the DNA strands is i:nvolved. TFIH is found in alternative forms, have been separated.In the bacterial reaction. which consist of a core associated with other formation of the open complex completes the necessary structural change to DNA; a differsutrunits. TFnH has a common function in both inience in the eukaryotic reaction is that further tiating transcription and repairing damage.The unwinding of the template is needed after this sarne helicase subunit (XPD) createsthe initial stage.
andRepair Transcription between 24.1,2A Connection
625
transcription bubble and melts DNA at a damaged site.Its other functions differ between transcription and repair, as provided by the appropriate form of the complex. F I G U R ?n 4 . 2 0 s h o w s t h a t t h e b a s i c f a c t o r involved in transcription consists of a core (of five subunits) associatedwith other subunits that have a kinase activity; this complex also includes a repair subunit. The kinase catalytic subunit that phosphorylates the CTD of RNA polymerase belongs to a group of kinases that are involved in cell cycle control. It is possible that this connection influences transcription in responseto the stageof the cell cycle. The alternative complex consistsof the core associatedwith a large group of proteins that
are codedby repair genes.(The basicmodel for repair is shown in Figure 20.25.\ The repair proteins include a subunit (XPC) that recognizes damaged DNA, which provides the coupling function that enables a template strand to be preferentially repaired when RNA polymerase becomes stalled at damaged DNA. Other proteins associated with the complex include endonucleases(XPG,XPF,and ERCCI ). Homologous proteins are found in the complexes in yeast (where they are often identified by rad mutations that are defective in repair) and in the human being (where they are identified by mutations that cause diseasesresulting from deficiencies in repairing damaged DNA). Subunits with the name XP are coded by genes in which mutations cause the diseasexeroderma pigmentosum (see Section 20.1I, Eukaryotic Cells Have ConservedRepair Systems). The kinase complex and the repair complex can associateand dissociatereversibly from the core TFnH. This suggestsa model in which the first form of TF11His required for initiation, but may be replaced by the other form (perhaps in responseto encountering DNA damage). TF11H dissociates from RNA polymerase at an early stageof elongation (after transcription of -50 bp); its reassociationat a site of damaged DNA may require additional coupling components.
fI6{J*[ ?1+.8*TheTFrrH coremayassociate witha kinase i:E*iJRS f;4.1# Mfdrecognizes a staltedRNApotymerase at jnitiationandassociate witha repaircomptex whendamanddirectsDNArepairto the damaged temptate strand. agedDNAis encountered.
626
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nize. Common elements recognized by ubiquitous activatorsinclude the CAAT box, GC box, and the octamer. All promoters probably require one or more of these elementsin order to function efficiently. An activator typically binds a consensussequenceof < I 0 bp, but actually covers a length of -20 bp of DNA. Given the sizes of the activators, and the length of DNA each covers, we expect that the various proteins will together cover the entire region upstream of the startpoint in which the elements reside. In general, a particular consensussequence is recognizedby a correspondingactivator (or by a member of a family of factors). Sometimes, though, a particular promoter sequencecan be recognized by one of several activators. A ubiquitous activator,Oct- l, binds to the octamer to activatethe histone H2B (and presumably also other) genes.Oct- I is the only octamer-binding factor in nonlymphoid cells. In lymphoid cells, however, a different activator, Oct-2, binds to the octamer to activate the immunoglobulin r Iight gene. Thus Oct-2 is a tissue-specificactivator, whereas Oct-l is ubiquitous. The exact details of recognition are not so important to know as the fact that a variety of activators recognize CAAT boxes. The use of the same octamer in the ubiquitously expressedH2B gene and the lymphoidspecificimmunoglobulin genesposesa paradox. Why does the ubiquitous Oct- I fail to activate the immunoglobulin genesin nonlymphoid tissues?The clntextmustbe important: Oct-2 rather than Oct- I may be needed to interact with other proteins that bind at the promoter. Theseresults mean that we cannot predict whether a gene will be activated by a particular activator simply on the basis of the presence of particular elements in its promoter.
@
Until now, we have considered the promoter an isolated region responsible for binding RNA pol'ymerase.Eukaryotic promoters do not necessarilyfunction alone, though. In at least some casr:s,the activity of a promoter is enormously increasedby the presenceof an enhancer,which conLsists of another group of elements but is Iocilted at a variable distance from those regarded as comprising part of the promoter itself. The concept that the enhancer is distinct frorl the promoter reflects two characteristics. The position of the enhancer relative to the promoter need not be fixed, but can vary substantialty.i,i,'i,'i : ,, r shows that it can be either upstream or downstream. In addition, it can function in either orientation (that is, it can be inverted) relative to the promoter. Manipulations of DNA show that an enhancer can stimulate any promoter placed in its vicinity. In natural genomes, enhancerscan be located within genes (that is, just downstream of the promoter) or tens of kilobasesaway in either direction. For operational purposes. it is sometimes 0T use'ful to define the promoter as a sequence of DNA that must be in a (relatively)fixed sequences loccrtionwith regardto the startpoirzl.By this definition, the TATA box and other upstream elements are included, but the enhancer is excluded. This is, however. a working definition rather than a rigid classification. Elements analogous to enhancers, called upstream activator sequences (UAS), are
Enhancers Contain Bidirectiona LElements ThatAssistInitiation
o An enhancer promoter to it, activates the nearest or eitherupstream andcanbeanydistance of the promoter. downstream . A UAS(upstream in yeast activator sequence) [ikean enhancer but worksontyupstream behaves of the promoter. . Simitar etements arefoundin enhancers sequence andpromoters. o Enhancers that formcomplexes of activators interactdirecttyor indirectlywith the promoter.
or downi jl. Jiiir::'.,,rl An enhancer canactivatea promoterfromupstream canbe invertedrelativeto the promoter. andits sequence strelam [ocations,
ThatAssistInitiation Etements containBidirectional 24.15Enhancers
found in yeast. They can function in either orientation at variable distances upstream of the promoter, but cannot function when located downstream. They have a regulatory role: In several casesthe UAS is bound by the regulatory protein(s) that activates the genes downstream. Reconstruction experiments in which the enhancer sequence is removed from the DNA and then is inserted elsewhere show that normal transcription can be sustained so long as it is present anywhereon the DNA molecule. If a B-globin gene is placed on a DNA molecule that contains an enhancer, its transcription is increased in vivo more than 200-fold, even when the enhancer is several kb upstream or downstream of the startpoint, in either orientation. We have yet to discover at what distance the enhancer fails to work.
Contain the 7@ Enhancers SameElements That AreFound at Promoters r Enhancers aremadeof the sameshortsequence etements that arefoundjn promoters. o Thedensjty of sequence components is greater in the enhancer thanin the promorer.
A difference between the enhancer and a typical promoter is presented by the density of regulatory elements. Flfiilftf E4"?r+ summarizes the susceptibility of the SV40 enhancer to damage by mutation, and we see that a much greater proportion of its sitesdirectly influences its function than is the case with the Dromoter ana-
lyzed in the same way in Figure 24.21. There is a corresponding increase in the density of protein-binding sites. Many of these sites are common elements in promoters; for example, API and the octamer. The specificity of transcription may be controlled by either a promoter or an enhancer. A promoter may be specifically regulated and a nearby enhancer used to increasethe efficiency of initiation, or a promoter may lack specific regulation but become active only when a nearby enhancer is specifically activated. An example is provided by immunoglobulin genes, which carry enhancers within the transcription unit. The immunoglobulin enhancers appear to be active only in the B lymphocytes in which the immunoglobulin genesare expressed.Such enhancers provide part of the regulatory network by which gene expression is controlled. A difference between enhancers and promoters may be that an enhancer shows greater cooperativity between the binding of factors. A complex that assembles at the enhancer that respondsto IFN (interferon) yassemblescooperatively to form a functional structure called the enhanceosome. Binding of the nonhistone protein HMGI(Y) bends rhe DNA inro a structure that then binds severalactivators (NFKB, IRF, and ATF-Jun). In contrast with rhe "mix and match" construction of promoters, all of these components are required to create an active structure at the enhancer. These components do not themselves directly bind to RNA polymerase, but they create a surfacethat binds a coactivatingcomplex.The complex helps the preinitiation complex of basal transcription factors that is assemblingat the promoter to recruit RNA polymerase. We discuss the function of
b80
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f:G!JRft4-*4 An enhancer contains several structural motifs.Thehistogram pl"ots the effectof atl mutations that reduce enhancer functionto <75olo of witdtype.Bindingsjtesfor proteins are indicated belowthe histoqram.
630
CHAPTER 24 Promoters and Enhancers
coactivatorsin more detail in Section 25 .5, Activators Interact with the Basal Apparatus.
Enhancers Work by Increasing the Concentration Near of Activators the Promoter . Enhancers usuatly workontyin crsconfiguration with a targetpromoter. . Enhancers canbe madeto workin trans configuration by tinkingthe DNAthat contains thetargetpromoter to the DNAthat contains the viaa proteinbridgeor by catenating enhancer the two molecutes. . Theprincip[e is that an enhancer worksin any situation in whichit is constrained to bein the asthe promoter. sameproximity
How can an enhancer stimulate initiation at a promoter that can be located any distanceaway on either side of it? When enhancers were first discovered, several possibilitieswere considered for their action as elements distinctly dif ferent from promoters: . An enhancer could change the overall structure of the template-for example, by influencing the density of supercoiling. . It could be responsible for locating the template at a particular place within the cell-for example, attaching it to the nuclear matrix. . An enhancer could provide an "entry site"-a point at which RNA polymerase (or some other essential protein) initially associateswith chromatin. Now we take the view that enhancer function involves the same sort of interaction with the basal apparatus as the interactions sponsored by upstream promoter elements. Enhancers are modular, like promoters. Some elements are found in both enhancers and promoters. Some individual elements found in promoters share with enhancers the ability to function at variable distanceand in either orientation. Thus the distinction between enhancers and promoters is blurred: Enhancers might be viewed as containing promoter elements, which are grouped closely together, and as having the ability to function at increased distancesfrom the startDoint.
The essential role of the enhancer may be to increasethe concentration of activator in the vicinity of the promoter (vicinity in this sense being a relative term). TWotypes of experiment :l'i.;;i.isuggestthat this is the illustrated in 1:l:,ri.ii;li: case. A fragment of DNA that contains an enhancer at one end and a promoter at the other is not effectively transcribed, but the enhancer can stimulate transcription from the promoter when they are connected by a protein bridge. Structural effects,such as changesin supercoili n g , c o u l d n o t b e t r a n s m i t t e d a c r o s ss u c h a bridge; this suggeststhat the critical feature is bringing the enhancer and promoter into close proximity. A bacterial enhancer provides a binding site for the regulator NtrC, which acts upon RNA polymerase using promoters recognized by o5a. When the enhancer is placed upon a circle of DNA that is catenated (interlocked) with a circle that contains the promoter, initiation is almost as effective as when the enhancer and promoter are on the same circular molecule. There is, however, no initiation when the enhancer and promoter are on separatedcircles. Again, this suggeststhat the critical feature is localization of the protein bound at the enhancer, which increases the enhancer's chance of contacting a protein bound at the Dromoter.
Promoter
Enhancer
+
lnterlockedcircles
proteins intothevicinmayfunctionbybringing i:tr*t-iirii: l{r,-l,r'r Anenhancer at the opposite doesnot acton a promoter An enhancer ity of the promoter. whenthe DNAisjoinedinto effective endofa [ong[inearDNA,but becomes circutar on separate andpromoter An enhancer a circleby a proteinbridge. arecatenated. whenthetwo molecutes butcaninteract DNAs donotinteract,
Nearthe Promoter 6 3 1 of Activators the Concentration WorkbyIncreasing 24.i,7Enhancers
If proteins bound at an enhancer several kb distant from a promoter interact directly with proteins bound in the vicinity of the startpoint, the organization of DNA must be flexible enough to allow the enhancer and promoter to be closely located. This requires the intervening DNA to be extruded as a large "loop." Such loops have been directly observedin the caseof the bacterial enhancer. There is an interesting exception to the rule that enhancers are cli-acting in natural situations. This is seenin the phenomenon of transvection. Pairing of somatic chromosomes allows an enhancer on one chromosome to activate a promoter on the partner chromosome. This reinforces the view that enhancers work by proximity. What limits the activity of an enhancer? \pically it works upon the nearest promoter. There are situations in which an enhancer is located between two promoters, but activates only one of them on the basis of specificprotein-protein contacts between the complexes bound at the two elements. The action of an enhancer may be limited by an insulator-an element in DNA that prevents the enhancer from acting on promoters beyond the insulat o r ( s e e S e c t i o n2 9 . I 4 , I n s u l a t o r sB l o c k t h e Actions of Enhancers and Heterochromatin). The generality of enhancement is not yet clear.We do not know what proportion of cellular promoters require an enhancer to achieve their usual level of expression,nor do we know how often an enhancer provides a target for regulation. Some enhancersare activatedonly in the tissuesin which their genesfunction, but others could be active in all cells.
effect, methylation is a reversible regulatory event. It is triggered by modifications to histones that include deacetylation and protein methylation (seeSection 30.9, Methylation of Histonesand DNA Is Connected). Methylation also occurs as an epigenetic event. In this case, modification may occur specifically in sperm or oocyte, with the result that there may be a difference between two alleles in the next generation. This can result in differences in the expression of the paternal a n d m a t e r n a l a l l e l e s ( s e eS e c t i o n 3 1 . 8 , D N A Methylation Is Responsible for Imprinting). In this chapter we are concerned with the means by which methylation influences transcription, which is the same whether the methyl groups were added or removed as a local regulatory event or as an epigenetic event. Methylation at promoters for RNA polymerase II occurs at CG doublets. The distribution of methyl groups can be examined by taking advantage of restriction enzymes that cleave target sitescontaining the CG doublet. TVvotlpes of restriction activity are compared in ilt*iiRil li4.F*s. These isoschizorners are enzymes that cleave the same target sequence in DNA, but have a different response to its state of methylation. The enzyme HpaII cleaves the sequence CCGG (writing the sequenceof only one strand of DNA). If the second C is methylated, though, the enzyme can no longer recognize the site. The enzyme MspI, however, cleaves the same
Sitesare cleavedirrespectiveof methvlation
Expression l@ Gene Is Associated with Demethylation
N/ethyiated CCGG
MsPl
Nonmethylated CCGG
e Demethylation at the 5'endof the geneis necessary for transcription. Methylation of DNA is one of several regulatory events that influence the activity of a promoter. Methylation at the promoter prevents transcription, and the methyl groups must be removed in order to activate a promoter. This effect is well characterizedat promoters for both RNA polymerase I and RNA polymerase II. In
632
CHAPTER 24 Promoters andEnhancers
UUUU lvle
Methylatedsite is not cleaved
Nonmethylated site is cleaved
fi{;ii*t- Jri.;:$ Therestriction enzyme MspIcteaves at[ CCGG sequences whetheror not theyaremethytated at thesecond C.butHpaIIcteaves onlynonmethytated CCGG tetramers.
target site irrespective of the state of methylation at this C. Thus MspI can be used to identify all the CCGG sequences,and HpaII can be used to determine whether they are methylated. With a substrate of nonmethylated DNA, the two enzymes would generate the same restriction bands. In methylated DNA, however, the modified positions are not cleavedby HpaIL For every such position, one larger HpaII fragment replacestwo MspI fragments. fI*iift{ :il+"i{'; gives an example. Many genes show a pattern in which the state of methylation is constant at most sitesbut variesat others. Some of the sitesare methylated in all tissuesexamined; some sitesare unmethylated in all tissues. A minority of sitesare methylated in tissuesin which thegeneis not expressed, but are nlt methylatedin tissuesin which the gene is active.Thus an active gene may be described as undermethylated. Experiments with the drug 5-azacytidine produce indirect evidence that demethylation can result in gene expression.The drug is incorporated into DNA in place of cytidine and cannot be methylated, becausethe 5'position is blocked. This leads to the appearance of demethylated sitesin DNA as the consequence of replication (following the scheme on the right o f F i g u r eI 5 . 7 ) . The phenotypic effects of 5-azacytidine include the induction of changes in the state of cellular differentiation. For example, muscle cells are induced to develop from nonmuscle cell precursors. The drug also activates genes on a silent X chromosome, which raisesthe possibility that the state of methylation could be connected with chromosomal inactivity. As well as examining the state of methylation of resident genes, we can compare the
Mspl digest
Hpall digest Banduniqueto Hpall replacesMspl bands
Bandsuniqueto Mspl = methvlatedsites Band at same oosition = nonmethylatedsite fIfii-lF.gii4.t? The resuttsof MspI and HpaII cteavage are comparedby gel etectrophoresis of the fragments.
results of introducing methylated or nonmethylated DNA into new host cells. Such experiments show a clear correlation; The methylated geneis inactive,but the nonmethylatedgeneis active. What is the extent of the undermethylated region? In the chicken a-globin gene cluster in adult erythroid cells,the undermethylation is confined to sitesthat extend from -500 bp upstream of the first of the two adult cr genes to -500 bp downstream of the second.Sitesof undermethylation are present in the entire region, including the spacerbetween the genes. The region of undermethylation coincideswith the region of maximum sensitivity to DNAase I. This argues that undermethylation is a feature of a domain that contains a transcribed gene or genes.As with other changesin chromatin, it seems likely that the absence of methyl groups is associatedwith the ability to be transcribedrather than with the act of transcription itself. Our problem in interpreting the general association between undermethylation and gene activation is that only a minority (sometimes a small minority) of the methylated sites are involved. It is likely that the state of methylation is critical at specific sites or in a restricted region. It is also possible that a reduction in the level of methylation (or even the complete removal of methyl groups from some stretch of DNA) is part of some structural change needed to permit transcription to proceed. In particular, demethylation at the promoter may be necessaryto make it available for the initiation of transcription. In the y-globin gene, for example, the presence of methyl groups in the region around the startpoint. between -200 and +90, suppressestranscription. Removal of the three methyl groups located upstream of the startpoint, or of the three methyl groups located downstream, doesnot relieve the suppression. Removal of all methyl groups, though, allows the promoter to function. Transcription may therefore require a methyl-free region at the promoter (see Section 24.I9, CpG Islands Are Regulatory Targets).There are exceptions to t h i s g e n e r a lr e l a t i o n s h i p . Some genes can be expressedeven when they are extensively methylated. Any connection between methylation and expression thus is not universal in an organism, but the general rule is that methylation prevents gene expression and demethylation is required for expression.
with Demethytation 633 Is Associated 24.1,8GeneExpression
Are l@ CpGIslands Targets Regulatory . CpGislands surround the promoters of geneswheretheyare constitutivety expressed unmethytated. . CpGistands alsoarefoundat the promoters of genes, sometissue-regulated r There are-29,000CpGis[ands in the human gen0me. o Methytatjon of a CpGistandprevents activation of a promoter withinit. o Repression is caused by proteins that bindto methylated CpGdoubtets. The presenceof CpG islands in the 5'regions of some genes is connected with the effect of methylation on gene expression.Theseislands are detected by the presence of an increased d e n s i t y o f t h e d i n u c l e o t i d e s e q u e n c e ,C p G . (CpG= 5'-CG-3'l The CpG doublet occursin vertebrateDNA at only -20oh of the frequency that would be expectedfrom the proportion of G-C basepairs. (This may be becauseCpG doublets are methylated on C, and spontaneousdeamination of methyl-C converts it to T, which introduces a mutation that removes the doublet.) In certain regions,however, the density of CpG doublets reachesthe predictedvalue; in fact, it is increased by l0x relative to the rest of the genome. The
y-globin
lritiltlil]il 5'**&d
ffi
Exon Exon 12 500
1000
bp
APRT
5'r*-y Exon Exon 12
i:i--*i :i+..-:iJ ThetypicaIdensityof CpGdoubtets in mammatian DNAis -1/100bp,asseenfora y-gtobin gene. In a CpG-rich island,thedensity is increased to >10doubtets/100 bp.Theistandjn theAPRT genestarts-100 bp -400 bp into the upstream of the promoter andextends gene.EachverticalLinerepresents a CpGdoubtet.
CHAPTER 24 Promoters andEnhancers
CpG doublets in these regions are generally unmethylated. These CpG-rich islands have an average G-C content of -60"/", compared with the 40% average in bulk DNA. They take the form of stretchesof DNA typically I to 2 kb long. There are -45,000 such islandsin the human genome. Some of the islands are present in repeated Alu elements,and may just be the consequenceof their high G-C-content. The human genome sequence confirms that, excluding these, there a r e - 2 9 , 0 0 0 i s l a n d s .T h e r e a r e f e w e r i n t h e m o u s e g e n o m e ,- 1 5 , 5 0 0 . A b o u t 1 0 , 0 0 0o f t h e predicted islandsin both speciesappear to reside in a context of sequencesthat are conserved between the species,suggestingthat these may be the islands with regulatory significance.The structure of chromatin in these regions has changes associatedwith gene expression (see Section 30.1 I, Promoter Activation Involves an Ordered Seriesof Events); there is a reduced content of histone Hi (which probably means that the structure is less compact), the other histones are extensively acetylated (a feature that tends to be associatedwith gene express i o n ) , a n d t h e r e a r e h y p e r s e n s i t i v es i t e s ( a s would be expectedof active promoters). I n s e v e r a l c a s e s .C p G - r i c h i s l a n d s b e g i n just upstream of a promoter and extend downstream into the transcribed region before petering out. I?iltitiil;lri.#*icompares the density of CpG doublets in a "general" region of the genome with a CpG island identified from the DNA sequence.The CpG island surrounds the 5'region of the APRT gene. which is constitutively expressed. All of the "housekeeping" genes that are constitutively expressedhave CpG islands; this accounts for about half of the islands.The other half of the islandsoccur at the promoters of tissue-regulatedgenes; only a minority (<40%) of these geneshave islands.In these cases,the islands are unmethylated irrespective of the stateof expressionof the gene.The presenceof unmethylated CpG-rich islandsmay be necessary, but therefore is not sufficient, for transcription. Thus the presence of unmethylated CpG islands may be taken as an indication that a gene is potentially active rather than inevitably transcribed. Many islands that are nonmethylated in the animal become methylated in cell lines in tissue culture, and this could be connected with the inability of these lines to express all of the functions typical of the tissue from which they were derived.
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TBPBindsDNAin an Unusual Wav KCVICWS Burley, S. K. and Roeder,R. G. (1996). Biochemistry and structural biology oITFIID. Annu. Rev.Biochem.6, , 7 69-7 99 . Lee, T. I. and Young, R. A. (1998). Regulation of gene expression by TBP-associatedproteins. GenesDev.12, l)98-1408. Orphanides, G., Lagrange, T., and Reinberg, D. ( I 996). The general transcription factors of RNA polymerasell. GenesDev.10,2657-268J. R e s e ar ch Horikoshi, M. et al. ( I 988). Transcription factor ATD interacts with a TATA factor to facilitate establishment of a preinitiation complex. Cel/ 54, rolJ-t042. Ifim, J. L,, Nikolov D. B., and Burley, S. K. (I99rl. Cocrystal structure of TBP recognizing the minor groove of a TATA element Nature 365, 520-527.
ICm, Y. et al. (t 993 ). Crystal structure of a yeast TBP/TATA box complex. Nature 365, 5t2-520. Liu, D. et al. (1998). Solution structure oI a TBPTAFII230 complex: protein mimicry of the minor groove surface of the TATA box unwound by TBP. Cell 94, 57 3-583. Martinez, E. et al. (1994). TATA-binding proteinassociatedfactors in TFIID function through the initiator to direct basal transcription from a TATA-lessciassII promoter. EMBO J 13, 3),r5-3t26. Nikolov D. B. et al. 11992).Crystal structure of TFIID TATA-box binding protein. Nature )60, 40-46. Ogryzko, V. V. et al. (1998). Histone-like TAFs within the PCAF histone acetylase complex. Cell 94, j5-44. Verrijzer,C. P. et al. (1995). Binding of TAFs to core elements directs promoter selectivity by R N A p o l y m e r a s e l l .C e l l 8 l , I I l 5 - l 1 2 5 . Wu. J., Parkhurst. I(. M., Powell, R. M., Brenowitz, M., and Parkhurst, L. J. (2001). DNA bends in TATA-binding protein-TATA complexes in solution are DNA sequencedependent. J Biol. Chem.27 6, 14614-14622.
Assembles TheBasaI Apparatus at the Promoter Reviews N i k o l o v D . B . a n d B u r l e y , S . K . ( 1 9 9 7 ) .R N A p o l y merase II transcription initiation: a structural view. Proc Natl. Acad. Sci.USA94, 15-22. . . ( l 9 9 3 ) . I n i t i a t i o no I Z a w e l ,L . a n d R e i n b e r gD transcription by RNA polymerase II: a multistep process.Prog.NucleicAcid ResMol Biol. 44, 67-t08. Research Buratowski, S., Hahn, S., Guarente, L., and Sharp, P. A. (1989). Five intermediate complexesin transcription initiation by RNA polymerase II. C e l l5 6 . 5 4 9 - 5 6 1 . Burke, T. W and I(adonaga, J.T. \1996). Drosophila TFIID binds to a conserved downstream basal promoter element that is present in many TATA-box-deficient promoters. GenesDev. 10, 7tt-724. Bushnell, D. A, Westover,K. D., Davis, R. E., and I(ornberg, R. D. (2004). Structural basisof transcription: an RNA polymerase II-TFIIB cocrystal at 4.5 Angstroms. SciencejO), 983-988. Littlefield, O., I(orkhin, Y., and Sigler, P. B. (I999). The structural basis for the oriented assembly of a TBP/TFB/promoter complex. Proc.Natl. Acad. Sci.USA 96, l)668-l)67). Nikolov D. B. et al. (1995). Crystal structure of a TFIIB -TB P-TATA-element ternary complex. Nature )77, ll9-128.
References 637
lB
InitiationIs Fotlowed by Promoter Clearance
Reviews Calvo, O. and Manley, J. L. (2003). Strangebedfellows: polyadenylation factors at the promoter. GenesDev. 17, l32l-l)27. Hirose, Y. and Manley, J. L. (2000). RNA polymerase II and the integration of nuclear events. GenesDev.14, l4l5-1429. Price,D. H. (2000). P-TEFb,a cyclin dependent kinase controlling elongation by RNA polymerase lI. Mol CellBiol.20,2629-284. Proudfoot, N. J., Furger, A., and Dye, M. J. (2OO2l. Integrating mRNA processing with transcription. Cell 108, 501-5 12. Shilatifard, A., Conaway, R. C., and Conaway, J. W. (2003). The RNA polymerase II elongation complex. Annu. Rev.Biochem 72, 69)-7 15. Woychik, N. A. and Hampsey,M. (2002). The RNA polymerase II machinery: structure illuminates function. Cell 108. 45j-463. Resea r ch Douziech, M., Coin, F., Chipoulet, J. M., Arai, Y., Ohkuma, Y.,Egly, J. M., and Coulombe, B. (2000). Mechanism of promoter melting by the xeroderma pigmentosum complementation group B helicase of transcription factor IIH revealedby protein-DNA photo-crosslinking. Mol. CellBiol 20, 8168-8177. Fong, N. and Bentley, D. L. (2001). Capping, splicing, and 3'processingare independently stimulated by RNA polymerase II: different functions for different segments of the CTD. GenesDev.15, 178)-1795. Goodrich, J. A. and TJian, R. (1994). Transcription factors IIE and IIH and ATP hydrolysis direct promoter clearance by RNA polymerase II. Cel/ 77, 145-156. Holstege, F. C., van der Vliet, P. C., and Timmers, H. T. (1996). Opening of an RNA polymerase II promoter occurs in two distinct steps and requires the basal transcription factors IIE and tIH. EMBO J t5, 1666-1677. I(m, T I(., Ebright, R. H., and Reinberg,D. (2000). Mechanism of ATP-dependent promoter melting by transcription factor IIH Science288, t418-1422. Spangler, L., Wang, X., Conaway, J. W., Conaway, R. C., and Dvir, A. (2001). TFIIH action in transcription initiation and promoter escape requires distinct regions of downstream promoter DNA. Proc.Natl Acad. Sci-USA 98, 5544-5549.
638
CHAPTER 24 Promoters andEnhancers
A Connection between Transcriotion andRepair Reviews Lehmann, A. R. (2001). The xeroderma pigmentosum group D (XPD) gene: one gene, two functions, three diseases.GenesDev. 15,l5-2). Selby, C. P. and Sancar, A. (1994). Mechanisms of transcription-repair coupling and mutation frequency decline. Microbiol.Rev.58, )17-329. Resea rch Bregman, D. et al. (19961. UV-induced ubiquitination of RNA polymerase II: a novel modification deficient in Cockayne syndrome cells. Proc.Natl. Acad. Sci.USA 9J, I 1586-1 1590. Schaeffer,L. et al. (19931. DNA repair helicase: a component of BTF2 (TFIIH) basic transcription factor. Science 260,58-63. Selby,C. P.and Sancar,A. (1991). Molecular mechanism of transcription-repair coupling. Science 260, 5j-58. Svejstrup, J. Q. et al. ( I 995 ). Different forms of TFIIH for transcription and DNA repair: holoTFIIH and a nucleotide excision reDairosome. Cell 80,21-28.
Enhancers Contain BidirectionaI Etements That AssistInitiation Review Muller, M. M., Gerster,T., and Schaffner,W. (I988). Enhancersequences and the regulation of genetranscripion. Eur.J Biochem. 176, 485495. rch Resea Banerji,J., Rusconi,S.,and Schaffner, W. (1981). Expressionof B-globingeneis enhancedby remote sv40 DNA sequences. Cell27, 299-)08.
Enhancers Contain theSameElements That Are Found at Promoters Reviews Maniatis, T., Falvo, J. V., I(im, T. H., ICm, T. I(., Lin, C. H., Parekh, B. S., and Wathelet, M. G. ( I 998) . Structure and function of the interferon-beta enhanceosome. ColdSpringHarbor Symp Quant.Biol 63, 609-620. Munshi, N., Yie, Y., Merika, M., Senger, I(., Lomvardas, S., Agalioti, T., and Thanos, D. (1999). The IFN-beta enhancer: a paradigm for understanding activation and repression of inducible gene expression. ColdSpringHarbor Symp.Quant.Biol 64, 149-159.
Enhancers Workby Increasing the Concentration of Activators Near the Promoter Review Blackwood, E. M. and I(adonaga,J. T. (1998). Going the distance: a current view of enhancer action. Science281, 6O-6j.
rch Resea Mueller-Storm, H. P., Sogo, J. M., and Schaffner, W. ( 1989). An enhancer stimulates transcription in trans when attached to the promoter via a protein bridge. CeIl 58, 7 67-777 . Zenke, M. et al. ( 1986). Multiple sequence motifs are involved in SV40 enhancer function. EMBO J. 5. 387-397.
Targets AreRegulatory CpGIslands Review Bird, A. (20021.DNA methylationpatternsand Dev.16, 6-21. epigeneticmemory.Genes Research Antequera,F. and Bird, A. (I99)l . Number of CpG islandsand genesin human and mouse.Proc. Natl.Acad.Sci.USA90, 11995-11999. Bird,A. et al. (1985).A fractionof the mouse genomethat is derivedfrom islandsof nonmethylated,Cp-G-richDNA. Cel/40, 9l-99. Boyes,J. and Bird,A. (1991).DNA methylation inhibits transcriptionindirectlyvia a methylCpGbinding protein. Cell64, ll23-lli4.
References 639
Activating Transcripti on C H A P T EO RU T L I N E Introduction 'is r Eukaryotic controtted at the [eve[ aeneexpressionusually of initiationof transcription. ThereAreSeveraITypesof Transcription Factors r ThebasaIapparatus determines thestartpoint for transcription. o Activators determine thefrequency of transcription. o Activators workby makingprotein-protein contacts with the basaIfactors. r Activatorsmayworkvia coactivators. r Somecomponents of the transcriptionaI apparatus workby changing chromatin structure. I n d e p e n d e nDt o m a i n B s i n dD N Aa n dA c t i v a t e Transcriotio n o DNA-binding activityandtranscription-activation arecarriedby independent domains of an activator. . Thero[eof the DNA-binding domain is to bringthe transcription-activation domain into thevicinityof the promoter. TheTwo HybridAssayDetectsProtein-Protein Interactions o Thetwo hybridassay worksby requiring an interaction between two proteins, whereonehasa DNA-binding domain andthe otherhasa transcription-activation domain. ActivatorsInteractwith the BasaIApparatus r Theprinciple that governs the functionof atl activators is that a DNA-binding domaindetermines specificity for the targetpromoter or enhancer. o TheDNA-binding domain is responsible for localizing a transcription-activating domainin the proximity of the L^--l ---^--r..uq)dL dPPdrdLu5.
r An actjvator that worksdirectlyhasa DNA-binding domain andan activating domain. r An activator that doesnot havean activating domainmay workby bindinga coactivator that hasan activating domain. o Several factorsjn the basaIapparatus aretargetswithwhich activators or coactivators interact. r RNApolymerase maybeassociated withvarious atternative setsof transcription factorsin theformof a holoenzyme complex.
640
ProteinsAre Repressors SomePromoter-Binding r Repression is usuatly achieved by affecting chromatin structure,buttherearerepressors that actby bindingto specific promorers. Response E[ements Are Recognized by Activators . Response maybe [ocated etements in promoters or ennancers. o Eachresponse element is recognized by a specific activator. o A promoter mayhavemanyresponse elements, whichin turn mayactivate transcription independently or in certajn combi nations. ThereAre ManyTypesof DNA-Binding Domains r Activators areclassified according to the type o f D N A - b i n d idnogm a i n . r Members of the samegrouphavesequence variations of a specificmotifthat conferspecificityfor individuaI target sites. A Z i n cF i n g e M r o t i fI s a D N A - B i n d i nDgo m a i n r A zincfingeris a loopof -23 aminoacidsthat protrudes froma zinc-binding siteformedby HisandCysaminoacids. r A zincfingerproteinusua[[y hasmultipte zincfingers. o TheC-terminal partof eachfingerformsan cr,-heUx that bindsoneturn of the majorgroove of DNA. . Somezincfingerproteins bindRNAinsteadof. or aswetl a s ,D N A . SteroidReceptorsAre Activators . Steroid receptors areexamptes of ligand-responsive activatorsthat areactjvated by bindinga steroid(or otherrelated molecules). o There areseparate DNA-binding andligand-binding domains. SteroidReceptors HaveZinc Fingers r TheDNAbindingdomainof a steroidreceptor is a typeof zincfingerthat hasCysbut not Hisresidues. . Gtucocorticoid andestrogen receptors eachhavetwo zinc fingers, the firstof whichdetermines the DNAtarget sequence. r Steroid receDtors bindto DNAasdimers.
jfrd
B i n d i n gt o t h e R e s p o n sEet e m e n t Is Activatedby Ligand-Binding r Bindingof tigandto the C-terminal domain increases the affinitvof the DNAbindingdomainfor its speciiic targetsite in DNA. SteroidReceptors Recognize Response Etements by a CombinatoriaI Code o A steroidresponse etementconsists of two shorthalfsitesthat maybe pa[indrom'ic or directlyrepeated. r Thereareontytwo typesof halfsites. o A receptor recognizes its response etement by the orientation andspacing of the hatf sites. . Thesequence of the hatfsjteis recognized by the first zincfinger. o Thesecond zincfingeris responsibte for dimerization, whichdetermines the distancebetween the subunits. o Subunit separation in the receptor determinesthe recognition of spacing in the response e[ement, . Somesteroidreceptors functionashomodimers, whereas othersformheterodimers. o Homodimers patindromic recognize response etements; heterodimers recognizeresponse etements with directty repeated hatfsites.
@
Homeodomains Bind RetatedTarqets in DNA r Thehomeodomain is a DNA-bindinq domainof 60 aminoacidsthat hasihree cr-he[ices. r TheC-terminaI a-hetix-3 is 17 aminoacids andbindsin the majorgrooveof DNA. o TheN-terminal armof the homeodomain projects into the minorgrooveof DNA.
Introduction
r Proteins homeodomains maybe containing eitheractivators or reDressors of transcription. Hetix-Loop-Helix ProteinsInteractby CombinatoriaIAssociation o Helix-toop-helix proteins havea motifof 40 to 50 aminoacidsthat comprises q-helices of 15to two amphipathic L6 residues by a Loop. separated o Thehetices for dimer areresDonsibte formation. . bHLHproteins havea basicsequence adjacentto the HLHmotifthat is responsible for bindingto DNA. r Ctass A bHLHproteins areubiquitously B bHLHproteins are expressed. Ctass tissue-soecific. . A classB proteinusuatty formsa heterodimer with a classA orotein. r HLHproteins that lackthe basicregion prevent in a heterodimer a bHLHpartner frombindingto DNA. o HLHproteins formcombinatoriaI associaduringdevettionsthat maybechanged of opmentby the additionor removal proteins. specific LeucineZippersAre Involvedin Dimer Formation helix Theleucine zipperis an amphipathic that dimerizes. to a basicregion Thezipperis adjacent that bindsDNA. formsthe bZIPmotifin Dimerization whichthetwo basicregions symmetricatly in DNA. bindinvertedrepeats
Summary
Processingthe transcript Transport to cytoplasm
r Eukaryotic geneexpression is usualtycontrolted at the [eve[of initiationof transcriotion.
The phenotypic differences that distinguish the various kinds of cells in a higher eukaryote are Iargely due to differences in the expression of genesthat code for proteins, that is, those transcribed by RNA polymerase II. In principle, the expression of these genesmight be regulated at any one of several stages.We can distinguish (at least) five potential control points, which [orm the following series: Activation of gene structure
J Initiation of transcrintion
Translation it -**o 25.1,gene expressionin As we seein FIGURE eukaryotes is largely controlled at the initiation of transcription. For most genes,this is the major control point in their expression. It involves changes in the structure of chromatin at the promoter (seeSection30.11,PromoterActivat i o n I n v o l v e s a n O r d e r e d S e r i e so f E v e n t s ) , accompanied by the binding of the basal transcription apparatus (including RNA polymerase II) to the promoter. (Regulation at subsequent stages of transcription is rare in eukaryotic cells.Premature termination occurs at some genes and is counteracted by a kinase, P-TEFb, but otherwise antitermination does not seem to be employed.)
25.1 Introduction 641
Controlof transcription initiation: usedfor mostgenes Localstructure of thegeneis changed
Generaltranscriptionapparatusbindsto promoter
RNA is modifiedand processed: can controlexpressionof alternativeproductslrom gene
Finally, the translation of an mRNA in the cytoplasm can be specifically controlled. While employment of this mechanism is uncommon adult somatic cells,it does occur in some embryonic situations. This can involve localization of the mRNA to specificsiteswhere it is expressed and/or the blocking of initiation of translation by specificprotein factors. Regulation of tissue-specificgene transcription lies at the heart of eukaryotic differentiation. A regulatory transcription factor servesto provide common control of a large number of target genes,and we seekto answer two questions about this mode of regulation: How does the transcription factor identify its group of target genes, and how is the activity of the transcription factor itself regulated in responseto intrinsic or extrinsic signals?
mRNAis exportedfromnucleusto cytoplasm: notregulated
ThereAreSeveral Types ptionFactors of Transcri mRNAis translated: regulatedin amphibiandevelopment
principal.l"y i:i=,::iii;::. -i Gene expression is controtled at the initiationof transcriotion. It is rarefor the subsequentstages of geneexpression to be usedto determine whether a geneis expressed, aLthough controI of processingmaybeusedto determine whichformof a geneis repr e s e n t ei nd m R N A .
The primary transcript is modified by capping at the 5'end, and in general also is modified by polyadenylation at the 3' end. Introns must be excised from the transcripts of interrupted genes. The mature RNA must be exported from the nucleus to the cytoplasm. Regulation of gene expressionby selection of sequencesat the level of nuclear RNA might involve any or all of these stages,but the one for which we have most evidence concerns changesin splicing; some genesare expressed by means of alternative splicing patterns whose regulation controls the type of protein product (seeSection26.12, Alternative SplicingInvolves Differential Use of SpliceJunctions).
CHAPTER 25 Activating Transcription
Thebasa[apparatus determines the startpoint for transcription. thefrequency Activators determine of ption. transcri o Activators protein-protein workby making withthe basalfactors. contacts e Activators mayworkvia coactivators. . Somecomponents of thetranscriptional apparatus workby changing chromatin structure. Initiation of transcription involves many protein-protein interactions among transcrip tion factors bound at the promoter or at an enhancer, as well as with RNA polymerase. We can divide the factors required for transcription into several classes,which are described in the following 1is1.;jdiifil J:,;.: summarizes their properties. . Basal factors, together with RNA polymerase,bind at the startpoint and TAIA box (seeSection24.10,The BasalApparatus Assemblesat the Promoter). . Activators are transcription factors that recognize specific short consensus elements. They bind to sites in the promoter or in enhancers (see Section 24.11, Short Sequence Elements Bind Activators). They act by increasing the efficiency with which the basal apparatus binds to the promoter. They therefore increasethe frecuencv of tran-
scription, and are required for a promoter to function at an adequatelevel. Some activators act constitutively (they are ubiquitous), whereas others have a regulatory role and are synthesized or activated at specific times or in specific tissues. These factors are therefore responsible for the control of transcription patterns in time and space. The s e q u e n c e st h a t t h e y b i n d a r e c a l l e d response elements. Members of another group of factors necessaryfor efficient transcription do not themselves bind DNA. Coactivators provide a connection between activators and the basal apparatus (see Section 25.5, Aclivators Interact with the Basal Apparatus). They work by protein-protein interactions, forming bridgesbetween activatorsand the basal transcription apparatus. Some coactivators and other regulators act to make changesin chromatin (see Section30.7, AcetylasesAre Associated with Activators). The diversity of elements from which a functional promoter may be constructed, and the variations in their locations relative to the startpoint, argues that the activators have an ability to interact with one another by protein-protein interactions in multiple ways. There appear to be no constraints on the potential relationships between the elements. The modular nature of the promoter is illustrated by experiments in which equivalent regions of different promoters have been exchanged. Hybrid promoters, for example, between the thymidine kinase and p-globin genes,work well. This suggeststhat the main purpose of the elements is to bring the activators they bind into the vicinity of the initiation complex, where protein-protein interactions determine the efficiency of the initiation reaction. The organization of RNA polymerase II promoters contrastswith that of bacterial promoters, where all the transcription factors must interact directly with RNA polymerase. In the eukaryotic system, only the basal factors interact directly with the enzyme. Activators may interact with the basalfactors,or they may interact with coactivatorsthat in tum interact with the basal factors. The construction of the apparatus through layers of interactions explains the flexibility with which elements may be arranged and the distance over which thev can be disnersed.
Enhancer
RNA polymerase and basalfactors bind at promoter
Activators bind at promoter
Activators bindto distal sitesin promoteror to enhancers
Coactivators connect activatorsto basalfactors
Coactivators/ regulators act on local structure of gene
i !*liit[: 'l:1."]Factors include involved in geneexpression activators that RNApolymerase andthe basaIapparatus, coacbinddirectLy orat enhancers, to DNAat thepromoter andthe basalappatjvators that bindto bothactivators structure. ratus,andrequlators that acton chromatin
Domains Independent BindDNAandActivate Transcription r DNA-binding activityandtranscription-activation domains of an arecarried byindependent activator. r Theroleof the DNA-binding is to bringthe domain n i nto the vicinity transcription-activationdoma'i of the oromoter.
Activators and other regulatory proteins require two types of ability: o They recognizespecifictarget sequences located in enhancers,promoters, or
Transcription BindDNAandActivate Domains 25.3 Independent
other regulatory elements that affect a particular target gene. . Having bound to DNA, an activator exercisesits function by binding to other components of the transcription apparatus. Can we characterize domains in the activator that are responsible for these activities? Often an activator has separatedomains that bind DNA and activate transcription. Each domain behavesas a separatemodule that functions independently when it is linked to a domain of the other type. The geometry of the overall transcription complex must allow the activating domain to contact the basalapparatus irrespective of the exact location and orientation of the DNA-bindine domain.
Connectingdomain
DNA-binding domain
if*LJfr{I5"3 DNA-binding functions in a andactivating transcription factormaycomprise independent domains of the protein.
Upstream promoter elements may be an appreciable distance from the startpoint, and in many casesmay be oriented in either direction. Enhancers may be even farther away and always show orientation independence. This organization has implications for both the DNA and proteins. The DNA may be looped or condensed in some way to allow the formation of the transcription complex. In addition, the domains of the activator may be connected in a flexible way, as illustrated diagrammatically in ilESi.Jftf 85.3. The main point here is that the DNA-binding and activating domains are independent, and are connected in a way that allows the activating domain to interact with the basal apparatus irrespective of the orientation and exact location of the DNA-binding domain. Binding to DNA is necessaryfor activating transcription, but does activation depend on the p articular DNA-binding domain? FIfri.lRil E$.,{,illustrates an experiment to answer this question. The activator Ga14has a DNA-binding domain that recognizes a UAS and an activating domain that stimulates initiation at the target promoter. The bacterial repressor LexA has an N-terminal DNAbinding domain that recognizesa specificoperator. When LexA binds to this operator, it repressesthe adjacent promoter. In a "swap" experiment, the DNA-binding domain of LexA can be substituted for the DNA-binding domain of Gal4. The hybrid gene can then be introduced
Gal4 activator Bindingand transcription
@ No binding
Gal4 DNA-binding
Bindingand transcription
No binding
I
LexA DNA-binding
i:Ifil.Jft{fh.r"i Theabitityof Gat4to activatetranscription is independent of its specificity for bindingDNA.WhentheGal.4 DNA-binding is reptaced domain bytheLexADNA-binding domain, the hybridproteincanactivatetranscription whena LexAoperator is ptacedneara promoter.
644
CHAPTER 25 ActivatingTranscription
into yeast together with a target gene that contains either the U1S or a LexA operator. An authentic Gal4 protein can activate a target gene only if it has a rJAS.The LexA repressor by itself of course lacks the ability to activate either sort of target. The LexA-Gal4 hybrid can no longer activate a gene with a UAS,but it can now activate a gene that has a LexA operatorl This result fits the modular view of transcription activators. The DNA-binding domain serves to bring the protein into the right Iocation. Preciselyhow or where it is bound to DNA is irrelevant, but once it is there, the transcription-activatingdomain can play its role. According to this view it does not matter whether the transcription-activating domain is brought to the vicinity of the promoter by recognition of a U,4Svia the DNA-binding domain of Gal4 or by recognition of a LexA operator via the LexA specificity module. The ability of the two types of module to function in hybrid proteins suggeststhat each domain of the protein folds independently into an active structure that is not influenced by the rest of the protein. The idea that activators have independent domains that bind DNA and that activate transcription is reinforced by the ability of rhe rar protein of HIV to stimulate initiation without binding DNA at all. The tat prorein binds to a region of secondary structure in the RNA product; the part of the RNA required for tat action is called rhe tar sequence. A model for the role of the taI-tar interaction in stimulating transcription is shown in i:,r-'iJil!,: tiij.lr. The /ar sequenceis locatedjust downstream of the startpoint, so that when tat binds to /ar, it is brought into the vicinity of the initiation complex. This is sufficient to ensure that its activation domain is in close enough proximity to the initiation complex. The activation domain interacts with one or more of the transcription factors bound at the complex in the same way as an activator. (Of course, the first transcript must be made in the absence of tat in order to provide the binding site.) An extreme demonstration of the independence of the localizing and activating domains is indicated by some constructsin which tat was engineered so that the activating domain was connected to a DNA-binding domain instead of to the usual /ar-binding sequence. When an appropriate target site was placed into the promoter, the tat activating-domain could activate transcription. This suggeststhat we should think of the DNA-binding (or in this case the RNAbinding) domain as providing a "tethering" func-
tion, whosemain purposeis to ensurethat the activating domain is in the vicinity of the initiation complex. The notion of tethering is a more specific example of the general idea that initiation requires a high concentration of transcription factors in the vicinity of the promoter. This may be achieved when activators bind to enhancers in the general vicinity, when they bind to upstream promoter components, or, in an extreme case, when they bind by tethering to the RNA product. The common requirement of all these situations is flexibility in the exact three-dimensional arrangement of DNA and proteins. The principle of independent domains is common in transcriptional activators. We might view the function of the DNAbinding domain as bringing theactivatingdomain into thevicinity0f thertarryoint.This explains why the exact locations of DNA-binding sites can varv within the oromoter.
TheTwoHybridAssay DetectsProtein- Protein Interactions r Thetwo hybridassay worksby requiring an interaction whereonehas between two oroteins, a DNA-binding domainandthe otherhasa n. transcriotion-activation domai
i : r . t i i . i l,illl i " : :Ti h e a c t i v a t i n g d o m a i n o f t h e t a t p r o H t eliVncoaf n s t i m u t a t e t r a n s c r i p of a previous round in thevicinityby bindingto the RNAproduct tion if it is tethered asshownbythe of the means of tethering, Actjvation is independent of transcription. for the RNA-bindinq domain. domain substitution of a DNA-bindinq
Interactions 25.4 lhe TwoHybridAssayDetectsProtein-Protein
The model of domain independence is the basis f o r a n e x t r e m e l y u s e f u l a s s a yf o r d e t e c t i n g protein interactions. In effect, we replace the connecting domain in Figure 25.3 wiLh' a protein-protein interaction. The principle is '.i1.t-:. illustrated in i:ii.r-:i.t: We fuse one of the proteins to be tested to a DNA-binding domain. We fuse the other protein to a transcriptionactivating domain. (This is done by linking the appropriate coding sequencesin each caseand making synthetic proteins by expressing each hybrid gene.) I{ the two proteins that are being tested can interact with one another, the two hybrid proteins will interact. This is reflected in the name of the technique: the two hybrid assay.The protein wirh the DNA-binding domain binds to a reporter gene that has a simple promoter containing its target site. It cannot, however, activate the gene by itself. Activation occurs only if the second hybrid binds to the first hybrid to bring the activation domain to the promoter. Any reporter gene can be used where the product is readily assayed,and this technique has
given rise to several automated procedures for rapidly testing protein-protein interactions. The effectivenessof the technique dramatically illustrates the modular nature of proteins. Even when fused to another protein, the DNAbinding domain can bind to DNA and the transcription-activating domain can activate transcription. Correspondingly, the interaction ability of the two proteins being tested is not inhibited by the attachment of the DNAbinding or transcription-activating domains. (Of course,there are some exceptions for which these simple rules do not apply and interference between the domains of the hybrid protein prevents the technique from working.) The power of this assay is that it requires only that the two proteins being tested can interact with each other. They need not have anything to do with transcription. As a result of the independence of the DNA-binding and transcription-activating domains, all we require is that they are brought together. This will happen so long as the two proteins being tested can interact in the environment of the nucleus.
Activators Interactwith
Apparatus theBasaI The players Proteinl fused to DNA-binding domain
Protein2 fused to activation domain
The reportingsystem P r o t e i n - b i n d i n g s r t eR e p o r t e r g e n e
=\'ffi*.ffi
I
Y product CATorotherreporter proteins:no expression Noninteracting
Interactingproteinsactivateexpression
i i,.,,,ii1rr i:;.i. Thetwo-hybrid technique teststheabil.ity of two proteins to interactby incorporating theminto hybridproteins where onehasa DNA-binding domain and the otherhasa transcription-activating domain.
646
CHAPTER 25 ActivatingTranscription
that governs thefunctjonof a[[ Theprincipte is that a DNA-binding domain activators for the targetpromoter determines specificity or ennancer. is responsible for TheDNA-binding domain jn the localizing a transcription-activating domain proximity of the basaIapparatus. An activator that worksdirecttyhasa DNA-binding domain andan activating domain. An act'ivator that doesnot havean activating domainmayworkby bindinga coactivator that hasan activating domain. factorsin the basalapparatus Several aretargets with whichactivators or coactivators interact. RNApolymerase maybeassociated wjthvarious atternative setsof transcriotion factorsin the form of a holoenzyme complex.
An activator may work directly when it consistsof a DNA-binding domain linked to a transcription-activating domain, as illustrated in Figure 25.3. ln other cases,the activator does not itself have a transcription-activating domain, but binds another protein-a coactivator-that has the transcription-activating domain. liirti!{[ l1ii.,'"shows the action of such an activator. We may regard coactivators as transcription factors whose specificityis conferred by the
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(asin TFxyA)to providing just one small domain consisting of two fingers (as in the Drosophila regulator ADRI ). The activator SpI has a DNAbinding domain that consists of three zinc fingers. The crystal structure of DNA bound by a protein with three fingers suggeststhe structure illustrated schematically in FIi:il-{&L i.l:r.i4. The C-terminal part of each finger forms s helices that bind DNA; the N-terminal part forms a B sheet. (For simplicity. the p sheet and the location of the zinc ion are not shown in the lower part of the figure.) The three a-helical stretches fit into one turn of the major groove; each o helix (and thus each finger) makes two sequence-specificcontactswith DNA (indicated by the arrows). We expect that the nonconserved amino acids in the C-terminal side of each finger are responsible for recognizing specific target sites. I(nowing that zinc fingers are found in authentic activators that assistboth RNA polymerasesII and III, we may view finger proteins from the reverse perspective.When a protein is found to have multiple zinc fingers, there is at least a prima faciecasefor investigating a possible role as a transcription factor. This type of identification suggeststhat severalloci involved in embryonic development of D. melanogaster a r e r e g u l a t o r so I t r a n s c r i p t i o n . It is necessaryto be cautious about interpreting the presence of (putative) zinc fingers, though, especially when the protein contains only a single finger motif. Fingers may be involved in binding RNA rather than DNA, or may not even be connected with any nucleic acid binding activity. For example, the prototype zinc finger protein, TF11A,binds both to the 55 gene and to the product, 55 rRNA. A translation initiation factor, eIF2B, has a zinc finger, and mutations in the finger influence the recognition of initiation codons. Retroviral capsid proteins have a motif related to the finger that may be involved in binding the viral RNA.
SteroidReceptors AreActivators Steroidreceptors areexamples of ligandresponsive activators that areactivatedby bindinga steroid(or otherretated molecutes). Thereareseparate DNA-binding andtigand-binding d o m anis ,
t,
\.i
@
*iJ.#
Zn+*
Zn++
Cvs
His
Zn**
tl Phe ,J Leu
factor5PLhasa seriesof f:f*l,i*t*;:i": ;i Transcription patternof threezincfingers, eachwith a characteristic the zincresidues that constitute cysteine andhjstidine b i n d i n so i t e .
that inseft :1q.Zincfingersmayformcrhelices ftfi#Fi$li.1!, withB sheets intothe majorgroove, whichis assocjated on the otherside.
Steroid hormones are synthesized in response to a variety of neuroendocrine activities and exert major effects on growth, tissue development, and body homeostasis in the animal world. The major groups of steroids and some other compounds with related (molecular) activili.tfi. ities are classifiedin Ftr..iii[tI
AreActivators 6 5 3 25.10SteroidReceptors
Glucocorticoids increase bloodsugar;alsohave anti-inflammatory action
CH,OH |
Estrogensare involved in femalesex develooment
Androgensare required for male sex development
VitaminD is reouired for bone develooment
Retinoicacid is a morphogen tl u^
|
|
i?"^7---,y'cu" -HC F'l eU
I
tl CH
c-cH"
ilHC Thyroidhormones Thyroidhormonescontrolbasal metabolicrate II
,oz\o4.",-8:o' 'ilH \_/ v
I
(T.) triiodothyronine
CH tl HC
c-cH.
il" HC
cooH (trans)retinoicacid
!:i,i,q:i:1.11 l::. SeveraItypes of hydrophobicsmat[motecutesactivatetranscriptionfactors.
The adrenal gland secretes>30 steroids,the two major groups being the glucocorticoidsand mineralocorticoids. Steroidsprovide the reproductive hormones (androgen male sex hormones and estrogenfemale sex hormones). Vitamin D is required for bone development. Other hormones, which have unrelated structures and physiological purposes, function at the molecular level in a similar way to the steroid hormones. Thyroid hormones, which are based on iodinated forms of tyrosine, control basalmetabolic rate in animals. Steroid and thyroid hormones also may be important in metamorphosis (ecdysteroidsin insectsand thyroid hormones in frogs).
CHAPTER 25 Activating Transcription
Retinoic acid (vitamin A) is a morphogen responsible for development of the anterior-posterior axis in the developing chick limb bud. Its metabolite, 9-cisretinoic acid, is found in tissuesthat are major sitesfor storage and metabolism of vitamin A. Wemay accountfor thesevariousactionsin terms of pathwaysfor regulatinggene expression. These diverse compounds share a common mode of action: Each is a small moleculethat binds t0 a specific receptor that activatesgene transcription. ("Receptor" may be a misnomer: The protein is a receptor for steroid or thyroid hormone in the same sense that /ac repressor is a receptor for a p galactoside,i.e., it is not a receptor in the senseof comprising a membrane-bound protein that is exposedto the cell surface.) Receptors for the diverse groups of steroid hormones, thyroid hormones, and retinoic acid represent a new "superfamily" of gene regu.tators, the ligand-responsiveactivators. AII the receptors have independent domains for DNAbinding and hormone binding that are in the same relative locations. Their general organization is summarized in f:{j*fti tli,: {i. The central part of the protein is the DNAbinding domain. These regions are closely related for the various steroid receptors (from the most closely related pair, wilh94oh sequence identity, to the least well related pair, at 42"/o identity). The act of binding DNA cannot be disconnected from the ability to activate transcription, because mutations in this domain affect both activities. The N-terminal regions of the receptors show the least conservation of sequence.They include other regions that are needed to activate transcription. The C-terminal domains bind the hormones. Those in the steroid receptor family show identities ranging from 30oh ro 57oh, reflecting specificity for individual hormones. Their relationships with the other receptors are minimal and reflect specificity for a variety of compounds-thyroid hormones. vitamin D, retinoic acid, and so forth. This domain also has the motifs responsible for dimerization and a region involved in transcriptional activation. Some ligands have multiple receptors that are closely related, such as the three retinoic acid receptors (RARo, p, and y) and the three receptors for 9-cis-retinoic acid (RXRa, B, and y).
Hormone-binding regions and dimerization (identity variesfrom 57or"-15q") transcription) Glucocorticoid 57
Mineralocorticoid
65
Pr^daaiar^na
Androgen Estrogen Triiodothyronine VitaminD
:S DNAbinding
@Spacing
i',ir.:i-jiii con:jl l : Thefirstfingerof a steroidreceptor shownin is bound(positions trotswhjchDNAsequence purpl.e); between the spacing the second fingercontro[s (positions sequences shownin b[ue).
Retinoicacid 9-cls Retinoicacid
l:.ir::,i+ii ,r-1,i;l Receptors for manysteroidandthyroidhorm o n e sh a v ea s i m i l a ro r g a n i z a t i o w n ,i t h a n j n d i v i d u a I N - t e r m j n ar eI g i o nc, o n s e r v eDdN A - b i n d i nr egg i o na, n d a C-terminal hormone-binding region. Identities arerelative to GR.
SteroidReceptors HaveTincFingers . TheDNAbindingdomainof a steroidreceptor is a typeof zincfingerthat hasCysbut not His residues. . Glucocorticoid andestrogen receptors eachhave two zincfingers, the firstof whichdetermines the DNAtargetsequence. r Steroid receptors bindto DNAasdimers. Steroid receptors(and some other proteins) have another type of zinc finger that is different from Cys2/His2fingers.The structure is based on a sequencewith the zinc-binding consensus: C y s - X 2 - C y s - X31- C y s - X 2C- y s These sequencesare called Cys2/Cys2fingers.Proteins with Cys2/Cys2fingers often have nonrepetitive fingers, in contrast with the tandem repetition of the Cys2/Hisztype. Binding sites in DNA (where known) are short and palindromic. The glucocorticoid and estrogen receptors each have two fingers, each with a zinc atom at the center of a tetrahedron of cysteines.The two fingers form o,-helicesthat fold together to
form a large globular domain. The aromatic sides of the cr-helicesform a hydrophobic center together with a p sheet that connects the two helices. One side of the N-terminal helix makes contacts in the major groove of DNA. TVvoglucocorticoid receptors dimerize upon binding to DNA, and each engagesa successive turn of the major groove. This fits with the palindromic nature of the response element (see S e c t i o n 2 5 . I 3 , S t e r o i d R e c e p t o r sR e c o g n i z e ResponseElements by a Combinatorial Code). Each finger controls one important property of the receptor. i'ir.iitfiiriiir.i i' identifies the relevant amino acids.Those on the right side of the first finger determine the sequenceof the target in DNA; those on the left side of the second finger control the spacingbetween the target sitesrecognizedby each subunit in the dimer (see Section 25.1), Steroid Receptors Recognize Response Elements by a Combinatorial Code). Direct evidence that the first finger binds DNA was obtained by a "specificityswap" experiment. The finger of the estrogen receptor was deleted and replaced by the sequence of the glucocorticoid receptor. The new protein recognized the GRE sequence (the usual target of the glucocorticoid receptor) instead of the ERE (the usual target of the estrogen receptor. This region therefore establishesthe specificity with which DNA is recognized. The differences between the sequencesof the glucocorticoid receptor and estrogen receptor fingers lie mostly at the base of the finger. The substitution at two positions shown in
HaveZincFingers 6 5 5 25.11SteroidReceptors
Samesequence in both receptors Differentsequence in each receptor ttG Ln
^^s GRE icity specif
ERE sPecif icitY
i : a l - = ! i i . t * D i s c r i m i n a tbi oent w e eGn R a En dE R tEa r get sequences is determined by two aminoacjdsat the baseof the firstzincfingerin the receptor.
GRE/EnhancerPromoter
+:Ii;.= +:;::li. .t* Glucocorticoids regutategenetranscript i o n b y c a u s i ntgh e i rr e c e p t ot ro b i n dt o a n e n h a n c e r whoseactionis needed for oromoter function. ir:,i.iitI lil..rt: allows the glucocorticoid receptor to bind at an ERE instead of a GRE.
@
Binding to the Response Element Is Activated by Ligand-Binding
. Bindingof Ligand to the C-terminaI domain increases the affinityof the DNA-binding domain for its specific targetsitein DNA.
656
CHAPTER 25 Activating Transcription
We know the most about the interaction of glucocorticoids with their receptor, whose action is illustrated in Fii:ii.:trF. itir-3*.A steroid hormone can pass through the cell membrane to enter the cell by simple diffusion. Within the cell, a glucocorticoid binds the glucocorticoid receptor. (Work on the glucocorticoid receptor has relied on the synthetic steroid hormone, dexamethasone.) The Iocalization of free receptorsis not entirely clear; they may be in equilibrium between the nucleus and cytoplasm.When hormone binds to the receptor. though, the protein is converted into an activated form that has an increased affinity for DNA, so the hormonereceptor complex is always localized in the nucleus. The activated receptor recognizesa specific consensus sequence that identifies the GRE. The GRE is typically located in an enhancer that may be several kb upstream or downstream of the promoter. When the steroid-receptorcomplex binds to the enhancer, the nearby promoter is activated and transcription initiates there. Enhancer activation provides the general mechanism by which steroids regulate a wide set of target genes. The C-terminal region regulates the activity of the receptor in a way that varies for the individual receptor. If the C-terminal domain of the glucocorticoid receptor is deleted, the remaining N-terminal protein is constitutively active: It no longer requires steroids for activity. This suggeststhat, in the absenceof steroid. the steroid-binding domain prevents the receptor from recognizing the GRE; it functions as an internal negative regulator. The addition of steroid inactivates the inhibition, releasing the receptor's ability to bind the GRE and activate transcription. The basisfor the repression could be internal, relying on interactions with another part of the receptor, or it could result from an interaction with some other protein that is displaced when steroid binds. The interaction between the domains is different in the estrogen receptor. If the hormonebinding domain is deleted, the protein is unable to activate transcription, although it continues to bind to the ERE. This region is therefore required to activate rather than to repress activity.
SteroidReceptors Recognize Response Elements by a CombinatoriaI Code . A steroidresponse element consists of two short halfsitesthat maybe palindromic or directly repeated. r Thereareontytwo typesof halfsites. r A receptor recognizes its response etement bythe orientation andspacing of the ha[fsites. . Thesequence of the halfsiteis recognized by the first zincfinger. r Thesecond zincfingeris responsible for dimerization, whichdetermines the distance between the subunits. r Subunit separation in the receptor determines the recognition of spacing in the response etement. r Somesteroidreceptors functionashomodimers. whereas othersformheterodimers. r Homodimers pa[indromic recognize response heterodimers etements; recognize response etements with directlyrepeated hatfsjtes.
lii:ijiii-: i:.'r,,l,ri: formedfromthepatinetements ftg5p6n5e by severaldifferdromichatfsiteTGTTCT arerecognized between the hatf onthespacing entreceptors depending sites.
RXR
Each receptor recognizesa responseelement that consistsof two short repeats(or half sites). This immediately suggeststhat the receptor binds as a dimer, so that each half of the consensus is contacted by one subunit (reminiscent of the l" operator-repressor interaction described in Section l4.ll, RepressorUses a Helix-Ti-rrn-Helix Motif to Bind DNA). The half sites may be arranged either as palindromes or as repeatsin the same orientation. They are separatedby zero to four base pairs whose sequence is irrelevant. Only two types of half site are used by the various receptors. Their orientation and spacing determine which receptor recognizes the response element. This behavior allows response elements that have restrictedconsensussequencesto be recognized specificallyby a variety of receptors. The rules that govern recognition are not absolute,but may be modified by context, and t h e r e a r e a l s o c a s e si n w h i c h p a l i n d r o m i c responseelements are recognized permissively by more than one receptor. The receptors fall into two groups: . Glucocorticoid (GR), mineralocorticoid (MR), androgen (AR), and progesterone (PR) receptors all form homodimers. They recognize response elements
1 bp 3bp 4bp 5bp
withthedirectrepeat ii{,;Lii,;l: ,iii..,: etements i Response of whichone by heterodimers, TGACCT arerecognized m e m b ei sr R X R . whose half sites have the consensus sequenceTGTTCT.fl{iiiiiiJ;:*,ili-lshows that the half sitesare arranged as palindromes, and that the spacing between the sitesdetermines the tlpe of element. The estrogen (ER) receptor functions in the same way, but has the half site sequenceTGACCT. . The 9-crs-retinoicacid (RXR) receptor forms homodimers, and also forms heterodimers with -15 other receptors, including thyroid (TlR), vitamin D (VDR), and retinoic acid (RAR). r.;I ;,f ..i,ishows that the dimers reci llt,1.r ognize half elements with the sequence TGACCT. The half sites are arranged as direct repeats,and recognition is controlled by spacingbetween them. Some of the heterodimeric receptors are activated when the ligandbinds to the partner for RXR; others can be activated by ligand binding either to this subunit or to the RXR subunit. Thesereceptorscan also form homodimers. which recognize palindromic sequences.
Code Elements by a Combinatorial Response Recognize 25.13SteroidReceptors
657
Now we are in a position to understand the basis for specificity of recognition. Recall that Figure 25.17 shows how recognition of the sequence of the half site is conferred by t h e a m i n o a c i d s e q u e n c ei n t h e f i r s t f i n g e r . Specificity for the spacing between half sites is carried by amino acids in the second finger. The structure of the dimer determines the distance between the subunits that sit in successive turns of the major groove, and thus c o n t r o l s t h e r e s p o n s et o t h e s p a c i n g o f h a l f sites.The exact positions of the residuesresponsible for dimerization differ in individual pairwise combinations. How do the steroid receptorsactivatetranscription? They do not act directly on the basal apparatus, but rather function via a coactivating complex. The coactivator includes various activities, including the common component CBP/p300, one of whose functions is to modify the structure of chromatin by acetylating h i s t o n e s( s e eF i g u r e 3 0 . I 4 ) . AII receptors in the superfamily are liganddependent activators of transcription, but some are also able to represstranscription. The TR and RAR receptors,in the form of heterodimers with RXR, bind to certain loci in the absenceof ligand and repress transcription by means of their ability to interact with a corepressorprotein. The corepressorfunctions by the reverse o f t h e m e c h a n i s m u s e d b y c o a c t i v a t o r s :I t inhibits the function of the basal transcriotion
Corepressor
Steroidreceotor
Ligand
--:i,UliIi:'!.1]!'Thesteroid receptors TRandRAR bindthe SMRT corepressor in the absence of ligand.Thepromoter is notexpressed. WhenSMRT is disptaced bybindingof tigand.thereceptor bindsa coactivator comptex. Thisleads to activation of transcription bythe basaIapparatus.
CHAPTER 25 ActivatingTranscription
apparatus, one of its actions being the deacetyIation of histones (seeFigure 30.16). We do not know the relative importance of the repressor activity vis-d-visthe ligand-dependent activation in the physiologicalresponseto hormone. The effect of ligand binding on the receptor is to convert it from a repressingcomplex to an activating complex, as shown in FlSlJft[:S.I*. In the absenceof ligand, the receptor is bound to a corepressorcomplex. The component of the corepressor that binds to the receptor is SMRT. Binding of ligand causesa conformational change that displacesSMRT. This allows the coactivator to bind.
Homeodomains Bind Targets Related in DNA . Thehomeodomain is a DNA-binding domainof 60 aminoacidsthat hasthreecr-helices. r TheC-terminaI q-helix-3is 17 aminoacidsand bindsin the majorgrooveof DNA. o TheN-terminal projects armof the homeodomain into the minorgrooveof DNA. o Proteins containing homeodomains maybe either activators or repressors of transcription.
The homeobox is a sequencethat codes for a domain of 60 amino acidspresent in proteins of many or even all eukaryotes. Its name derives from its original identification in Drosophila homeotic loci (whose genesdetermine the identity of body structures).It is present in many of the genes that regulate early development in Drosophila,and a related motif is found in genes in a wide range of higher eukaryotes. The homeodomain is found in many genes concerned with developmental regulation. Sequences related to the homeodomain are found in several types of animal transcription factors. In Drosophilahomeotic genes, the homeodomain often (but not always) occurs close to the C-terminal end. Some examples of genes containing homeoboxes are summarized in F:fu{jqg ;}.5.f3. Often the genes have little conservation of sequenceexcept in the homeobox. The conservation of the homeobox sequence varies. A major group of homeobox-containing genes in Drosophila h'as a well conserved sequence,with 80% Io 90o/osimilarity in pairwise comparisons.Other geneshave lessclosely related homeoboxes. The homeodomain is sometimes combined with other motifs in animal transcription factors. One example is pre-
sented by the Oct (octamer-binding) proteins, in which a conserved stretch of 75 amino acids called the Pou region is located closeto a region resemblingthe homeodomain. The homeoboxes of the Pou group of proteins are the least closely related to the original group, and thus comprise the farthest extension of the family. The homeodomain is responsible for binding to DNA, and experiments to swap homeodomains between proteins suggestthat the specificity of DNA recognition lies within the homeodomain. As with phage repressors, though, no simple code relating protein and DNA sequences can be deduced. The C-terminal region of the homeodomain shows homology with the helix-turn-helix motif of prokaryotic repressors. We recall from Section 14.I l, RepressorUsesa Helix-Turn-Helix Motif to Bind DNA, that the l, repressor has a "recognition helix" (a-helix-3) that makes contacts in the major groove of DNA, whereas the other helix (u-helix-2) lies at an angle across the DNA. The homeodomain can be organized into three potential helical regions; the sequences of three examples are compared in f;.ii-in-ilf :1.i'...The best conserved part of the sequence lies in the third helix. The difference between these structures and the prokaryotic repressor structures lies in the length of the helix that recognizesDNA, helix-3, which is l7 amino acids long in the homeodomain, compared to nine residueslong in the l, repressor. The structure of the homeodomain of the D. melanogasler engrailed protein is represented
En Antp Oct-2
;15"i":fl. Helix 3 binds in schematically in Fll"irJlti: groove makes the majorthe major of DNA and protein ity of the contacts between and nucleic acid. Many of the contacts that orient the helix in the major groove are made with the phosphate backbone, so they are not specific for DNA sequence.They lie largely on one face of the double helix, and flank the bases with which specific contacts are made. The remaining contacts are made by the N-terminal arm of the homeodomain, the sequencethat just precedes
400
763 -'i:'
II
467
I
Pou region
t:T{-,iiltt maybethe so[eDNA;ilii.il:iThehomeodomain regulator. or maybe bindingmotifin a transcriptionaI c o m b i n ew d i t h o t h e rm o t i f s I. t r e p r e s e nat sd i s c r e t e (60residue) partof the protein.
N-terminal arm 1 GluLysArgProArgThr Aia ArgLysArg GlyArg GlnThr Tyr Thr ArgArgLysLysArgThrSerlle Glu
En Antp
AsnArgTyrLeu AsnArgTyrLeu
Oct-2
AsnGluLysProThr
En Antp Oct-2
Flii'js.[]i.;i;; Thehomeodomain generepresents themajorgroupof genes of theAntennapedio containing homeoboxes (en) represents in Drosophila; anothertypeof homeotic engroiled gene;andthe mammatian groupof transcription factor0ct-2represents a distantlyretated factors. Thehomeodomain is conventionatly numbered from1 to 60.It startswiththe N-terminaIarm,andthe threehelicaIregions occupyresidues 70-22,28-38,and42-58.Amino acidsin btueareconserved in atlthreeexamotes.
in DNA Targets BindRetated 25.14Homeodomains
659
Helices1 and 2 lie abovethe DNA
N-terminal arm lies In mrnorgroove
Helix3 lies in the majorgroove
. r t : : i : L : . : : H e t i x3 o f t h e h o m e o d o m abiinn d si n t h e majorgroove of DNA,wjth helices 1 and2 lyingoutside the doublehetjx.Hetix3 contacts boththe phosphate backbone a n ds p e c i f i b c a s e sT. h eN - t e r m i n a Ir mh e si n t h e m i n o r groove, andmakes additionaI contacts.
the first helix. It projects into the minor groove. Thus the N-terminal and C-terminal regions of the homeodomain are primarily responsiblefor contacting DNA. A striking demonstration of the generality of this model derives from a comparison of the crystal structure of the homeodomain of engrailed protein with that o{ the o2 mating protein of yeast. The DNA-binding domain of this protein resemblesa homeodomain and can form three similar helices:Its structure in the DNA groove can be superimposedalmost exactly on that of the engrailed homeodomain. These similarities suggestthat all homeodomains bind to DNA in the same manner. This means that a relatively small number of residuesin helix-3 and in the N-terminal arm are responsiblefor specificityof contactswith DNA. One group of homeodomain-containing proteins is the set of Hox proteins. They bind to DNA with rather low sequence specificity, and it has been puzzling how these proteins can have different specificities.It turns out that Hox proteins often bind to DNA as heterodimers with a partner (called Exd in flies and Pbx in vertebrates). The heterodimer has a more restricted specificity in vitro than an individual Hox protein; typically it binds the l0 bp sequenceTGATNNATNN. This still is not enough to account for the differencesin the specificities of Hox proteins. A third protein, Hth, which is necessaryto localize Exd in the nucleus, also forms part of the complex that binds DNA, and may restrict the binding sitesfurther. The same partners (Exd and Hth) are present together
CHAPTER 25 ActivatingTranscription
with each Hox protein in the trimeric complex, though, so it remains puzzling how each Hox protein has sufficient specificity. Homeodomain proteins can be either transcriptional activators or repressors.The nature of the factor dependson the other domain(s)the homeodomain is responsiblesolely for binding to DNA. The activator or repressor domains both act by influencing the basal apparatus. Activator domains may interact with coactivators that in turn bind to components of the basal apparatus.Repressordomains also interact with the transcription apparatus (that is, they do not a c t b y b l o c k i n g a c c e s st o D N A a s s u c h ) . T h e repressor Eve, for example, interacts directly with TF'D.
HelixProtei He[ixLoopns Interactby CombinatoriaL Association . Hetix-toop-helix proteins havea motifof 40 to 50 aminoacidsthat comprises two amphipathic c helices of 15to 16 residues separated by a [oop. . Thehetices for dimerformation. areresponsibte . bHLHproteins havea basicsequence adjacent to the HLHmotifthat is responsible for bindingto DNA. . Class A bHLHproteins areubiquitousty expressed. Ctass B bHLHproteins aretissue-specific. r A classB proteinusualty formsa heterodimer with a ctassA protein. r HLHproteins that lackthe basicregionprevent a bHLHpartner in a heterodimer frombindingto DNA. r HLHproteins formcombinatorial associations that maybechanged duringdevelopment bythe proteins. additionor removaI of specific
TWo common features in DNA-binding proteins are the presence of helical regions that bind DNA and the ability of the protein to dimerize. Both features are representedin the group of helix-loop-helix (HLH) proteins that s h a r e a c o m m o n t y p e o f s e q u e n c em o t i f : A stretch of 40 to 50 amino acids contains two amphipathic o helices separated by a linker region (the loop) of varying length. (An amphipathic helix forms two faces, one presenting hydrophobic amino acids, and the other presenting charged amino acids.)The proteins in this group form both homodimers and heterodimers by means of interactions between the hydrophobic residues on the corresponding facesof the two helices.The helical regions
MyoD
nta nsp nrg Arg LysAlaAlaThr l\4etArg cln ArgArgArg
ld
Arg LeuProAla LeuLeuAspGlnGluGluValAsnValLe-
MyoD
LeuSer Lys :ValfuriGlnAla Phecln Thr LeuLysArgCysThr
ld
L e u T y r A s p M e t A S t c l y C y s T ySr e r A r g t d t l i : y f G t nL e u v a t
Basic region Six conservedresidues are absentfrom ld Helix 1 Conservedresiduesare Joundin both MyoD and ld
Myo D rye Vaic tn,llii:ler;l nrg nsn AiLir,ilr! Arg lii r|ecrn Gty -LeuGtll ld Lis Va!,:etn lle'iie-u, ctu His val rli€tAsp li4ili4:Arg Asp lieu Glu
Helix2
iiirijEi. :i:,i:ir At[HLHproteins havereg'ions corresponding to hetixl andhelix2, whichare separated by a toopof 10 to 24 residues. BasicHLHproteins havea regionwith conserved positivecharges immediatety adjacent to helixL.
are l5 to l6 amino acids long, and each contains several conserved residues. Ttvo examples are compared in ljL:;i,iiiiill.ii{:. The ability to form dimers resideswith these amphipathic helices and is common to all HLH proteins. The loop is probably important only for allowing the freedom for the two helical regions to interact independently of one another. Most HLH proteins contain a region adjacent to the HLH motif itself that is highly basic, and which is needed for binding to DNA. There a r e - 6 c o n s e r v e d r e s i d u e si n a s t r e t c h o f I 5 amino acids (seeFigure 25.26). Members of the group with such a region are called bHLH proteins. A dimer in which both subunits have the basicregion can bind to DNA. The HLH domains probably correctly orient the two basic regions contributed by the individual subunits. The bHLH proteins fall into two general groups. ClassA consistsof proteins that are ubiquitously expressed, including mammalian Ef2lE47. ClassB consistsof proteins that are expressedin a tissue-specificmanner, including mammalian MyoD, myogenin, and Myf-5 (a group of activators that are involved in myogenesis [muscle formation] ). A common modus operandifor a tissue-specificbHLH protein is to form a heterodimer with a ubiquitous partner. There is also a group of gene products that specify development of the nervous system in (where Ac-Sis the tissue-specific D. melanogaster component and da is the ubiquitous componenr). The Myc proteins (which are the cellular counterparts of oncogene products and are involved in growth regulation) form a separate classof bHLH proteins, whose partners and targets are different. Dimers formed from bHLH proteins differ in their abilities to bind to DNA. For example, E47 homodimers, EI2-E47 heterodimers, and
MyoD-E47 heterodimers all form efficiently and bind strongly to DNA; El2 homodimerizes well but binds DNA poorly, whereas MyoD homodimerizes only poorly. Thus both dimer formation and DNA binding may represent important regulatory points. At this juncture, it is possible to define groups of HLH proteins whose members form various pairwise combinations, but it is not possible to predict from the sequences the strengths of dimer formation or DNAbinding. All of the dimers in this group that bind DNA recognizethe same consensussequence, but we do not know yet whether different homodimers and heterodimers have preferences for slightly different target sites that are related to their functions. Differences in DNA-binding result from properties of the region in or close to the HLH motif; f or example, E I2 differs fromB4T in possessingan inhibitory region just by the basic region, which prevents DNA binding by homodimers. Some HLH proteins lack the basicregion and/or contain proline residuesthat appear to disrupt its function. The example of the protein Id is shown in Figure 25.26. Proteins o{ this type have the same capacity to dimerize as bHLH proteins, but a dimer that contains one subunit of this type can no longer bind to DNA specifically. This is a forceful demonstration of the importance of doubling the DNA-binding motif i n D N A - b i n d i n gp r o t e i n s . The importance of the distinction between the nonbasic HLH and bHLH proteins is suggested by the properties of two pairs of HLH proteins: tineda-Ac-S/emcpairand the MyoD/Id pair. A model for their functions in forming a ;l*-ll;. regulatory network is illustrated in i:i{;t"::-}* In D. melanlgaster, the gene emc (extra' is required to establish the normacrochaetae) pattern of adult sensory organs. It mal spatial
Loop-HetixProteins Interactby CombinatoriaIAssociation 25.15 HeLix-
661
binding to DNA when it is sequestered by an HLH partner such as Id. It can activate transcription when bound to a bHLH partner such as El2 orE47.It can alsoact as a site-specific repressor when bound to another partner; the bHLH protein MyoR forms a MyoD-MyoR dimer in proliferating myoblasts that repressestranscription (at the same target loci at which MyoDEl2 lE47 activate transcription). The behavior of the HLH proteins therefore illustrates two general principles of transcriptional regulation. A small number of proteins form combinatorial associations.Particular combinations have different functions with regard to DNA binding and transcriptional regulation. Differentiation may depend either on the presence or on the removal of particular partners. ii,;:-i*.i: ili:.J."rAn HLHdimerin whichbothsubunits are o f t h eb H L H t y p ec a nb i n dD N Ab, u ta d i m eirn w h i c ho n e subunitlacksthe basicreqioncannotbindDNA.
functions by suppressing the functions of several genes, including da (daughterless) and the achaetescute(Ac-S)complex. ,4c-Sand da are genes of the bHLH type. The suppressoremccodesf.oran HLH protein that lacks the basicregion. We suppose that, in the absence of.emc function, the da and Ac-Sproteins form dimers that activate transcription of appropriate target genes, but the production of emc proLein causes the formation of heterodimers that cannot bind to DNA. Thus production of.emcprotein in the appropriate cells is necessaryto suppressthe function oI Ac-S/da. The formation of muscle cells is triggered by a change in the transcriptional program that requires severalbHLH proteins, including MyoD. MyoD is produced specificallyin myogenic cells and, indeed, overexpression of MyoD in certain other cells can induce them to commence a myogenic program. The trigger for muscle differentiation is probably a heterodimer consisting of MyoD -El2 or MyoD-847 rather than a MyoD homodimer. Before myogenesisbegins, a member of the nonbasic HLH type, the Id protein, may bind to MyoD and/or E l2 and E47 to form heterodimers that cannot bind to DNA. It binds to EI2lE47 beter than ro MyoD, and so might function by sequesteringthe ubiquitous bHLH partner. Overexpression of Id can prevent myogenesis.Thus the removal of Id could be the trigger that releasesMyoD to initiate myogenesls. AbHLH activator such as MyoD can be controlled in several wavs. It is prevented from
662
CHAPTER 25 ActivatingTranscription
Leucine Zippers AreInvolved in Dimer Formation . Theleucine zipperis an amphipathic hetixthat dimerizes. r Thezipperis adjacent to a basicregionthat binds DNA. r Dimerization formsthe bZIPmotifin whichthe two basicregions symmetricatty bindinverted repeats in DNA.
Interactions between proteins are a common theme in building a transcription complex, and a motif found in several activators (and other proteins) is involved in both homo- and heteromeric interactions. The leucine zipper is a stretch of amino acids rich in leucine residues that provides a dimerization motif. Dimer formation itself has emerged as a common principle in the action of proteins that recognize specific DNA sequences,and in the case of the leucine zipper, its relationship to DNA binding is especially clear, because we can see how dimerization juxtaposes the DNA-binding regions of each subunit. The reaction is depicted diagrammatically in i:ftui,JFt* i:*. Jis. An amphipathic cxhelix has a structure in which the hydrophobic groups (including leucine) face one side while charged groups face the other side.A leucine zipper forms an amphipathic helix in which the leucines of the zipper on one protein could protrude from the cr-helix and interdigitate with the leucines of the zipper of another protein in parallel to form a coiled coil. The two right-handed heliceswind around
each other, with 3.5 residuesper turn, so the pattern repeatsintegrally every seven residues. How is this structure related to DNA binding? The region adjacentto the leucine repeats is highly basic in each of the zipper proteins, and could comprise a DNA-binding site. The two leucine zippers in effect form a Y-shaped structure, in which the zippers comprise the stem and the two basicregions stick out to form the arms that bind to DNA. This is known as the bZIP structural motif. It explains why the target sequencesfor such proteins are inverted repeats with no separation. Zippers may be used to sponsor formation of homodimers or heterodimers. They are lengthy motifs. Leucine (or another hydrophobic amino acid) occupiesevery seventh residue in the potential zipper. There are four repeats of the zipper (Leu-X6) in the protein C/EBP (a factor that binds as a dimer to both the CAAT box and the SV40 core enhancer) and five repeats in the factors Jun and Fos (which form the heterodimeric activator,APt ). API was originally identified by its binding to a DNA sequencein the SV40 enhancer (see Figure 24.24it. The active preparation of API includes severalpolypeptides.A major component is Jun, the product of the gene c-jun,which was identified by its relationship with the oncogene v-jun carried by an avian sarcoma virus. The mouse genome contains a family of related genes, c-jun (the original isolate), junB, and junD (identified by sequence homology with jun). There are considerable sequence similarities in the three Jun proteins; they have leucine zippers that can interact to form homodimers or heterodimers. The other major component of API is the product of another gene with an oncogenic counterpart. The c-fos gene is the cellular homolog to the oncogene v-los carried by a murine sarcoma virus. Expression of c-fosacrivates geneswhose promoters or enhancerspossessan API target site. The c-losproduct is a nuclear phosphoprotein that is one of a group of proteins. The others are described as Fosrelated antigens (FRA); they constitute a family of Fos-like proteins. Fos also has a leucine zipper. Fos cannot form homodimers, but can form a heterodimer with Jun. A leucine zipper in each protein is required for the reaction. The ability to form dimers is a crucial part of the interaction of these factors with DNA. Fos cannot by itself bind to DNA, possibly because of its failure to form a dimer. The Jun-Fos heterodimer can, however,
@@@@
of the bZIPmotifare Thebasicregions irlt.;iiliii: :::'r.;l:iri zipper at the adjacent heldtogether by thedimerization zippers facesof twoleucine regionwhenthehydrophobic 'interact in oara[[eI orientation.
bind to DNA with same target specificity as the Jun-Jun dimmer, and this heterodimer binds to the API site with an affinity -10x that of the Jun homodimer.
Summary Transcription factors include basal factors, activators, and coactivators. Basal factors interact with RNA polymerase at the startpoint. Activators bind specificshort responseelements (REs) located in promoters or enhancers. Activators function by making protein-protein interactions with the basal apparatus. Some activators interact directly with the basal apparatus; others require coactivatorsto mediate the interaction. Activators often have a modular construction, in which there are independent domains responsible for binding to DNA and for activating transcription. The main function of the DNA-binding domain may be to tether the activating domain in the vicinity of the initiation complex. Some responseelementsare present in many genes and are recognized by ubiquitous factors; others are present in a few genes and are recognizedby tissue-specific factors. Promoters for RNA polymerase II contain a variety of short czs-actingelements, each of which is recognizedby a trans-actingfactor. The crs-actingelements are located upstream of the TATA box and may be present in either orientation and at a variety of distanceswith regard to the startpoint. The upstream elements are recognized by activators that interact with the
25.17Summary 6 6 3
basal transcription complex to determine the efficiency with which the promoter is used. Some activators interact directly with components of the basal apparatus; others interact via intermediaries called coactivators. The targets in the basalapparatusare the TAFs of TF11D, or TFnB or TFnA.The interaction stimulatesassembly of the basal apparatus. Another motif involved in DNA-binding is the zinc finger, which is found in proteins that bind DNA or RNA (or sometimesboth). A finger has cysteine residues that bind zinc. One type of finger is found in multiple repears in some transcription factors; another is found in single or double repeatsin others. Severalgroups of transcription factors have been identified by sequencehomologies. The homeodomain is a 60-residuesequencefound in genesthat regulatesdevelopment in insects and worms and in mammalian transcription factors. It is related to the prokaryotic helixturn-helix motif and provides the motif by which the factors bind to lNA. The leucine zipper contains a stretch of amino acids rich in leucine that are involved in dimerization of transcription factors.An adjacentbasic region is responsiblefor binding to DNA. Steroid receptors were the first members identified of a group of transcription factors in which the protein is activated by binding a small hydrophobic hormone. The activated factor becomes localized in the nucleus and binds to its specific response element, where it activates transcription. The DNA-binding domain has zinc fingers. The receptors are homodimers or heterodimers. The homodimers all recognize palindromic response elements with the same consensussequence;the difference between the responseelements is the spacingbetween the inverted repeats.The heterodimers recognize direct repeats, again being distinguished by the spacing berween the repeats.The DNA-binding motif of these receptors includes two zinc fingers; the first determines which consensussequenceis recognized, and the second responds to the spacing between the repeats. HLH (helix-loop-helix) proreins have a m p h i p a t h i c h e l i c e s t h a t a r e r e s p o n s i b l ef o r dimerization, which are adjacent to basic regions that bind to DNA. bHLH proteins have a basic region that binds to DNA and fall into two groups: ubiquitously expressed a n d t i s s u e - s p e c i f i cA . n active protein is usually a heterodimer between two subunits, one from each group. When a dimer has one
664
CHAPTER 25 ActivatingTranscription
subunit that does not have the basic region it fails to bind DNA, so such subunits can p r e v e n t g e n e e x p r e s s i o n .C o m b i n a t o r i a l a s sociations of subunits form regulatory networks. Many transcription factors function as dimers, and it is common for there to be multiple members of a family that form homodimers and heterodimers. This creates tne potential for complex combinations to govern gene expression.In some cases,a family includes inhibitory members, whose participation in dimer formation prevents the partner from activating transcription.
A?
Kererences ThereAreSeveral Types of Transcription Factors Reviews Lee, T. I. and Young, R. A. (2000). Ttanscriprion of eukaryotic protein-coding genes.Annu. Rev. G e n e L) 4 , 7 7 - 1 3 7 . Lemon, B. and Tjian, R. (2000). Orchestrated response: a symphony of transcription factors for gene control. Genes Dey.14,2551-2569.
Independent Domains BindDNA andActivate Transcription Reviews Guarente, L. (19871.Regulatory proteins in yeast. Annu. Rev.Genet 21,425-452. Ptashne, M. ( I 988). How eukaryotic transcriptional activators work. Nature )35, 681-689.
TheTwo-Hybrid AssayDetects Protei n- Protei n Interactions Resea rch Fields,S. and Song, O. (1989). A novel genetic system to detect protein-protein interactions. Nature 340,245-246.
Activators Interactwiththe Basal Apparatus Reviews Lemon, B. and Tjian, R. (2000). Orchestrated response: a symphony of transcription factors for gene control. GenesDev.14,2551-2569. Maniatis, T., Goodbourn, S., and Fischer, J. A. (1987ll. Regulation of inducible and tissuespecific gene expression. Science2j6, t237-1245. Mitchell, P., and TJian, R. (19S9). Ttanscriprional regulation in mammalian cells by sequencespecific DNA-binding proteins. Science245, 371-j78.
Myers, L. C. and I(ornberg, R. D. (2000). Mediator of transcriptional regulation . Annu. Rev. Biochem.69, 7 29-7 49 rch Resea Asturias, F. J., Jiang, Y. W., Myers, L. C., Gustafsson, C M., and I(ornberg, R. D. (1999). Conserved structures of me diator and RNA polymerase II holoenzyme. Science283, 985-987. Chen, J.-L. et al. (1994). Assembly of recombinant TFIID reveals differential coactivator requirements for distinct transcriotional activators. Cell79,93-105. Dotson, M. R., Yuan, C. X., Roeder,R. G., Myers, L. C., Gustafsson,C. M., Jiang, Y. W., Li, Y., I(ornberg, R. D., and Asturias, F. J. (2000). Structural organization of yeast and mammalian mediator complexes. Proc Natl. Acad. Sci USA 97, 14307 -l43l0. Dynlacht, B. D., Hoey, T., and Tjian, R. (I991). Isolation of coactivators associatedwith the TATA-binding protein that mediate transcriptional activation. Cell 66, 563-576. I(m, Y. J., Bjorklund, S., Li, Y., Sayre, M. H., and I(ornberg, R. D. (1994). Amultiproteinmediator of transcriptional activation and its interaction with the C-terminal repeat domain of RNA polymerase Il. Cell 77, 599-608. Ma, J. and Ptashne, M. ( I 987 ) . A new classof yeast transcriptional activators Cell 51, I I 3-I 19. Pugh, B. F. and Tjian, R. (I990). Mechanism of transcriptional activation by Sp I : evidence for coactivators.Cell 61, I 187-l I97.
SomePromoter-Binding Proteins AreReoressors Resea r ch Goppelt, A., Stelzer, G., Lottspeich, F., and Meisterernst, M. (1996). A mechanism for repression of classII gene transcription through specific binding of NC2 to TBP-promoter complexes via heterodimeric histone fold domains. EMBO J 15,3t05-)tt6. Inostroza,J. A., Mermelstein, F. H., Ha, I.,Lane, W. S., and Reinberg,D. (1992). Drl, a TATAbinding protein-associated phosphoprotein and inhibitor of classII gene transcription. Cel/ 70,477-489. I(im, T. K., Zhao, Y., Ge, H , Bernstein, R., and Roeder,R. G. (1995) TATA-bindingprotein residues implicated in a functional interplay between negative cofactor NC2 (Drl ) and general factors TFIIA and TFIIB. J. Biol. Chem. 270,t0976-t098r.
ThereAreManyTypes of DNA-Binding Domains Reviews Harrison,S. C. (1991).A structuraltaxonomyof DNA-bindingproteins.Nature35), 7 l5-7 19.
Pabo,C. T. and Sauer,R. T. (1992). Tlanscription factors: structural families and principles of DNA recognition. Annu. Rev.Biochem.61, 1053-1095. Research Miller, J. et al. (1985). Repetitive zinc binding domains in the protein transcription factor IIIA from Xenopus oocytes. EMBO J. 4, t609-16t4. Murre, C., McCaw, P. S., and Baltimore, D. (1989). A new DNA binding and dimerization motif in immunoglobulin enhancer binding, daughterless,MyoD, and myc proteins. Cell 56, 777-7 83.
MotifIs a DNA-Binding A ZincFinger Domain Resea rch I(adonaga,J. et al. (1987). Isolation of cDNA encoding transcription factor Spl and functional analysis of the DNA binding domain. C e l l5 1 , 1 0 7 9 - 1 0 9 0 . Miller, J. et al. (1985). Repetitive zinc binding domains in the protein transcription factor ILIAfrom Xenopus oocytes. EMBO J. 4, t609-t614. Pavletich,N. P. and Pabo, C. O. (1991). Zinc fingerDNA recognition: crystal structure oI a Zif268' D N A c o m o l e xa t 2 I A . S c i e n c2e5 2 , 8 0 9 - 8 17 .
AreActivators SteroidReceptors Reviews Evans, R. M. ( 1988) . The steroid and thyroid hormone receptor superfamily. Science240, 889-89 5. Mangelsdorf,D. J. and Evans, R. (1995). The RXR heterodimers and orphan receptors. Cell 83, 841-850.
HaveZincFingers SteroidReceptors Review Tsai, M J, and O'Malley, B. W. (1994r. Molecular mechanisms of action of steroid/thyroid receptor superfamily members. Annu. Rev. Biochem6),451-486. rch Resea Umesono, I(. and Evans, R. M. (1989). Determinants of target gene specificity for steroid/ thyroid hormone receptors.Cell57, 1139-1146.
Response Recognize SteroidReceptors Code Etements by a Combinatorial Reviews Mangelsdorf,D. J. and Evans,R. \1995).The RXR heterodimersand orphan receptors.Cell83' 8 4l - 8 50 . Yamamoto,K. R. (1985).SteroidreceptorreguIatedtranscriptionof specificgenesand gene networks.Annu Rev.Genet.19,209-252.
References 665
Resea rch Hurlein,A. J. et al. (1995).Ligand-independent repressionby the thyroid hormone receptor mediatedby a nuclearreceptorcorepressor. Nature)77, i97404. Rastinejad,F.,Perlmann,T.,Evans,R. M., and Sigler,P.B. (1995).Structuraldeterminants of nuclearreceptorassemblyon DNA direct repeats.Nature)7 5, 20)-21 l. Umesono,K., Murakami, K. K., Thompson,C. C., and Evans,R. M. ( l99l ) . Directrepeatsas selectiveresponseelementsfor the thyroid hormone, retinoic acid,and vitamin D3 receptors.Cell65, 1255-1266. @
Homeodomains Bind RelatedTargets in DNA
Review Gehring,W. J. et al. (1994).Homeodomain-DNA recognition.Cell78, 2l l-223. R e s erac h Han,I(., Levine,M. S.,and Manley,J. L. (1989). Synergisticactivationand repressionof transcriptionby Drosophila homeoboxproteins. Cell56, 571-58). Wolberger, C. et al. (199I). Crystalstructureof a MATc,2homeodomain-operatorcomplexsuggestsa generalmodel for homeodomain-DNA interactions.Cell67, 517-528.
@
Hetix-Loop-Helix Proteins Interact by CombinatoriaI Association
Review Weintraub, H. ( l99l ). The MyoD gene family: nodal point during specification of the muscle cell lineage. Science 251, 7 6l-7 66.
666
CHAPTER 25 Activating Transcription
Research Benezra,R. et al. (1990). The protein Id: a negative regulator of helix-loop-helix DNAbinding proteins. Cell 61,49-59. Davis, R. L. et al. (1987 | . Expression of a single transfected cDNA converts fibroblasts to myoblasts.Cell 5L, 987-1000. Davis, R. L. et al. (I990). The MyoD DNA binding domain contains a recognition code for muscle-specific gene activation. Cell 60, 7JJ-746. Lassar,A. B. et al. (1991). Functional activity of myogenic HLH proteins requires heterooligomerization with El2 /E47 -like proteins in vitro.Cell66, 305-315. Murre, C., McCaw P. S., and Baltimore, D. (1989). A new DNA binding and dimerization motif in immunoglobulin enhancer binding, daughterless, MyoD, and myc proteins. Cell 56, 777-783.
Leucine Zippers AreInvolved in DimerFormation Review Vinson,C. R., SigleaP.B., and McKnight, S.L. (1989).Scissors-grip modelfor DNA recognition by a family of leucinezipperproteins. 246, 9lI-916. Science Research Landschulz,W. H., Johnson,P.F.,and McKnight, S.L. (1988).The leucinezipper:a hypothetical structurecommon to a new classof DNA binding proteins.Science 240, 1759-1764.
199 aDDd lrau uo panulluoJ .luepuedep-71 6 elelPql auospupluapuaoap-In aleleql auos 'lV-nVsuorllunIerLlds 6urpnlruL aq]anpq auos -suollur 'suorpunr 9v-ngaluPs 'suoLlrunI aql epnlrurAllensn arrlds aq11eseruenbas 1nq snsuesuol ta6uol[q peulJep olpsuollur1e6re1 aq1 'auosoarrtds ZIn aql asuduor1eq1 sdNUus 6uLrrlds aArlpula]lp u! r JolesreqlouesasnAeMqlpd sdNUustueraJJL6 sasnsnlelpddV6uortdSa^qeulellv uV 'a1sa^tllp rr{1e1eraql uloJ ol Zn ql$ VNUUs gn uolj salpDosstp ueq| . lrPouPl vNUus9n 'dNUus tn 'auosoalrlos zg eql 0l euosoelrlds IE aql sile^uolpup'olrsalrlds,9 aql qllMllPrelul0l vNUus9n sMollp ln Joespalau dNUus 'spaaro.rd 6urrrlds se saxald -urol raqilnj qEnorql souas e sassed auosoerLlds aq1 Jo '6upLlds f-resse .ro1 'oruosoarLldseq1 -rausluauoduoleql llp surpluoltllrr.lM 1g o1xalduorV aql sile^uots61r1Xus pupEnJo 6urpurg 91-1/7i1
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p ulpluol911 peruesuol ldarxasl[[us a!] ll! r 1eq1aruanbas 'ourosoalrl0s aql auosqltMtaq1a6o1 o aql'su1e1ord rlloJsdNUus lPuorlrppe 9n puP 'rn'gn'zn':'|| ale6uDudsur pa^lo^ut s/[rl!usoA4a{] o
arvsVNUus 6urrrlds roJparrnbau .laqlouP0l uotlPlolouolllolj pallo}suPll st rtqtnrro suotllPat aqf . puoqp qlrqMut'suoqelguelsasupll 'raqla6ol 'altsalnds,E pele6queqlo.lpsuoxa lqbu pueUeleql puP st uollutaqf . stlt uoqMleuPle sPpasPalal eql }P pa^Pelr 'u0r1ur aql ur alrsqluPlq 'a]ts aqtle V ue1euorlrsod ,7 e o1peutols! pua,g aql pue st |PUel! r sr uollutaql uaqMpoulloJ elrlcls ,9 eqllp pa^Poll 'salortelna raqbrquLparues -uol qruelqaql . st aluanbas ssallnq lseefut paruasuol llem elrsarrlds,€ eql Jo urpallsdn lsn[ olrsqluplqp pueselrsarqds,g pue,g eql sallnbal6uLrqd5r lPllel e q6norqlspaalold6unt1d5vNUl.u-ard 'lualenLnba aiesollsarttds,€ {11euoL1run; 'tualp^Lnbe r\lleuoqrunJ olPsaltsalttds,9 llV . lle pup 'suotpunI 6utrtld5o uo r{1uo spuedap erqds1osrted;ouoqLuboral sltPdul peaualv suotpunl e]tlds 'svNUu-alo ul suo.llul jo suorlr.socl oM]lsPlpueoMl]slg oql le sopqoallnutp y16 (aruenbas aql sequlsap luPlsuolosaqlroJluaual!nba.l alil gv-n9 aq1 . Josuuolur alnrgv-lg aql pallerr{11eur6uo) 'gv aluanDes aql snsuasu0r sopnllur uollulaql1opua(1q6u),€ eql lP alrsaltlds,€ egl . 'ng aluanbes snsuasuol aql sepnlluruollureqlJopua(ge1),9 aql le ellsaltlds,9 aul r 'uollul aql ol a^tlPlar uollut-uoxa raql roj peuPUaie[eqI 'sauepunoq suollrsod aql ale sallsaltlds . AlalerpauruL saluanbes eql 6urpunollns alv suotpunI a]tlds lPallnN soluenbas iloqs uo!pnpollul
3NI]INO U]IdVH3
o
?frtill
SpticingIs Connected to Exportof mRNA . TheREFproteins juncbindto splicing tionsby associating withthe spliceosome. . AfterspLicing, theyremainattached to junction. the RNAat theexon-exon . Theyinteractwith the transportprotein TAP/Mex that exports the RNAthrough the nuctear oore.
?EM
GroupIi IntronsAutospticevia Larjat Formation . GrouD II intronsexcise from themselves RNAby an autocatalytic spticing event. . Thesplice junctions andmechanism of spticing of groupII intronsaresimilarto spticing of nuctear introns. r A groupII intronfoldsinto a secondary structure that generates a catalyticsite resemb[ing the structure of U6-U2-nuctear intron.
?frd
AtternativeSpticingInvotvesDifferentiaI Useof SpticeJunctions . Specific exonsmaybeexctuded or jn the RNAproduct inctuded by usingor junctions. faitingto usea pairof spticing o Exons maybeextended by changing one junctions of the sptice to usean alternativejunction. o Sexdetermination in Drosophila involves a series of atternative sp[ic'ing events in genescodingfor successive products of a pathway. . P etements of Drosophila showgerm[inespecifi c atternative spticing.
?r13arfrans-SplicingReactionsUseSmatlRNAs . Spticing reactions usuatly occurontyin crs junctions between splice on the same molecule of RNA. e trons-spticing occurs in trypanosomes and wormswherea shortsequence (SLRNA)is spliced to the 5'endsof manyprecursor mRNAs. . SLRNAhasa structure resemb[ing the Sm-binding siteof U snRNAs andmayptay an anatogous rolein the reaction. YeastIRNASpticingInvolvesCuttingand Rejoining o IRNAspl.icing occurs by successive cteavageandLigation reactions. TheSpl.icing Endonuclease Recognizes tRNA o An endonuclease cteaves theIRNAorecursorsat bothendsof theintron. e Theyeastendonuctease is a heterotetramerwith two (retated)catatytic subunits.
668
CHAPTER 26 RNASpticing andProcessing
. It usesa measuring mechanism to determinethe sitesof cleavage bytheirpositionsretative to a pointin theIRNA structure. . Thearchaeal nuctease hasa simpter structureandrecognizes a buLge-helix-bulge structuralmotifin the substrate. IRNACleavage and LigationAre Separate Reactions o Release of the introngenerates two halftRNAs that pairto formthe mature structure. o Thehatves havethe unusual ends 5' hydroxyland2'-3' cycticphosphate. r The5'-0Hendis phosphorytated by a polynucleotide kinase, the cycticphosphategroupis opened by phosphodiesterase to generate a 2'-phosphate terminus and3'-0Hgroup,exonendsare joinedby an RNAligase, andthe 2'phosphate is removed by a phosphatase. The UnfotdedProteinResponse Is Relatedto IRNASpticing . Irelp is an innernuclear promembrane tein withits N-terminal domain in the ER lumen,andits C-terminal domain in the nucteus. o Bindingof an unfolded proteinto the N-terminal domain activates theC-terminaI nuctease by autophosphorytation. o Theactivated nuclease cteaves Hac]. mRNA to release an intronandgenerate exonsthat areligatedby a IRNAtigase. . ThesplicedHaclmRNA codes for a trangenescodscription factorthat activates ing for chaperones that hetpto fotd proteins. unfotded The3' Endsof pof and polIII Transcripts Are Generated by Termination r RNApotymerase I terminates transcription at an L8-base terminator seouence. o RNApoLymerase III terminates transcription in poty(U)a seQU€nce embedded in a G-C-rich seouence. The3' Endsof mRNAs Are Generated by Cteavage and Potyadenytation r Thesequence AAUAAA is a signaIfor cleavage to generate a 3'endof mRNA that is pol.yadenylated. . Thereactionrequires a proteincomplex that containsa specificityfactor,an endonuclease, andpoty(A)polymerase. r Thespecificity factorandendonuctease cteave RNAdownstream of AAUAAA. . Thespecificity factorandpoty(A)potymerase processivety add-200 A residues t o t h e3 ' e n d .
o A - U - r i cshe q u e n cienst h e3 ' t a i Ic o n t r o l potyadenytation cytoplasmic or deadeny[ation duringXenopus embryonic deve[ooment. C t e a v a goef t h e 3 ' E n d o f H i s t o n em R N A M a yR e q u i r a e S m a tR l NA . Histone poLyadenylated; mRNAs arenot their 3'endsaregenerated by a cteavage reaction that depends on the structure of the mRNA. . Thecteavage reaction requires the SLBP to bindto a stem-Loop structure andthe U7snRNA to pair with an adjacent singte-stranded region.
for rRNA SmattRNAsAre Required ng Processi . TheC/Dgroupof snoRNAs for modifying is required the 2' positionof ribosewitha methylgroup. . TheH/ACA for groupof snoRNAs is required converting uridineto pseudouridine. o In eachcasethe snoRNA basepairswith a thetargetbaseto that contains sequence of rRNA for generate that is the substrate a typicaIstructure modification. Summarv
Productionof rRNARequires Cleavage Events o The[argeandsmat[rRNAs arereleased by cteavage froma common orecursor RNA.
Introduction I n t e r r u p t e d g e n e s a r e f o u n d i n a l l c l a s s e so f organisms.They representa minor proportion of the genesof the very lowest eukaryotes,but the vast majority of genes in higher eukaryotic genomes. Genesvary widely according to the numbers and lengths of introns, but a typical mammalian gene has seven to eight exons spreadout over - I 6 kb. The exons are relatively short (-100 to 200 bp) and the introns are relatively long (>l kb) (seeSection 3.6, Genes Show a Wide Distribution of Sizes). The discrepancybetween the interrupted organization of the gene and the uninterrupted organization of its nRNA requires processing of the primary transcription product. The primary transcript has the same organization as the gene and is sometimes called the premRNA. Removal of the introns from premRNA leaves a typical messengerof -2.2 kb. The processby which the introns are removed is called RNA splicing. Removal of introns is a major part of the production of RNA in all eukaryotes. (Although interrupted genes are relatively rare in lower eukaryotessuch as yeast, the overall proportion underestimates the importance of introns becausemost of the genes that are interrupted code for relatively abundant proteins. Splicingis therefore involved in the production of a greater proportion of total nRNA than would be apparent from analysis of the genome, perhaps as much as 50%.) One of the first clues about the nature of the discrepancyin size between nuclear genes and their products in higher eukaryotes was provided by the properties of nuclear RNA. Its average size is much larger than nRNA, it is very unstable, and it has a much greater sequence complexity. Taking its name from its
particte r .,, : , ' asa ribonucteoprotein hnRNA exists of beads. oroanized asa series
broad size distribution. it was called heterogeneous nuclear RNA (hnRNA). It includes pre-mRNA, but could also include other transcripts (that is, transcriptsthat are not ultimately processedto nRNA). The physical form of hnRNA is a ribonucleoprotein particle (hnRNP), in which the hnRNA is bound by proteins. As characterized in vitro, an hnRNP particle takes the form of beadsconnectedby a fiber. The structure is summarized in t i'.r,;lrr,;{ i. The most abundant proteins in the particle are the core proteins, but other proteins are present at lower stoichiometry, making a total of -20 proteins. The proteins typically are presentat - I 08 copiesper nucleus, compared with -l 06moleculesof hnRNA. Some of the proteins may have a structural role in packaging the hnRNA; several are known to shuttle between the nucleus and cytoplasm and play roles in exporting the RNA or otherwise controlling its activity.
26.1 Introduction 669
Exon 1 lntron 1
Exon 2 Intron2 Exon 3 Intron3
Exon4
lntron4 Exon 5
I Transcription f Cap at 5' end
I End modification V
exon
PolY(A)al 3' end tAzoo
I ^ .. . 5pilong + intron exon I Exon-introntunctronsare broken I
eron.arejoined I tu=
I
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Transporl
NUCLEUS
CYTOPLASM Azoo R N Ai s m o d i f i e di n t h e n u c L e ubsy a d d i t i o n st o t h e 5 ' a n d 3 ' e n d sa n d b y splicingto removethe introns.The splicingevent requiresbreakageof the exon-jntron j u n c t i o n sa n dj o i n i n g o f t h e e n d so f t h e e x o n s .M a t u r em R N Ai s t r a n s p o r t e tdn r o u g n nuc[earporesto the cytop[asm,whereit is trans[ated.
Splicingoccursin the nucleus, together with the other modificationsthat are made to newly synthesizedRNAs.The processof expressingan interrupted gene is reviewed in . The transcriptis cappedat the 5'end (seeSection7.9, The 5'End of EukaryoticnRNA Is Capped),has the introns removed, and is polyadenylated at t h e l ' e n d ( s e eS e c t i o n7 . 1 0 ,T h e J ' T e r m i n u sI s Polyadenylated).The RNA is then transported through nuclear pores to the cyroplasm,where it is availableto be translated. With regard to the various processingreactions that occur in the nucleus. we should like to kncrw at what point splicing occurs vis-a-vis the other modifications of RNA. Does splicing occur at a particularlocation in the nucleus, and is it connectedwith other events-for example, nucleocytoplasmictransport?Doesthe lack of splicingmake an important differencein the expressionof uninterrupted genes? With regard to the splicing reaction itself, one of the main questionsis how its specificity
670
CHAPTER 26 RNASpticing andProcessing
i s c o n t r o l l e d . W h a t e n s u r e st h a t t h e e n d s o f each intron are recognizedin pairs so that the correct sequence is removed from the RNA? Are introns excisedfrom a precursor in a particular order? Is the maturation of RNA used to regulate gene expression by discriminating among the availableprecursorsor by changing the pattern of splicing? We can identify several types of splicing systems: . Introns are removed from the nuclear pre-mRNAs of higher eukaryotes by a system that recognizesonly shclrtcons e n s u ss e q u e n c e sc o n s e r v e da t e x o n intron boundaries and within the intron. This reaction requires a large splicing apparatus,which takes the form of an array of proteins and ribonucleoproteins that functions as a large particulate complex (the spliceosome).The mechanism of splicing involves transesterifications, and the catalytic center includes RNA a sw e l l a sp r o l e i n s . . Certain RNAs have the ability to excise their introns autonomously. Introns of this type fall into two groups, as distinguished by secondary/tertiary structure. Both groups use transesterificaticlnreactions in which the RNA is the catalytic agent (seeChapter 27, CatalyticRNA). . The removal of introns from yeast nuclear IRNA precursorsinvolves enzymatic activitiesthat handle the substrate in a way resembling the IRNA processing enzymes,in which a critical feature is the conformation of the IRNA precursor.Thesesplicingreactionsare accomplished by enzymes that use cleavage and ligation.
Nuclear Splice Junctions AreShortSequences . Splicesitesarethe sequences immediatety surrounding the exon-intron boundaries. Theyare named fortheirpositions relative to theintron. r T h e5 ' s p l i c e s i t ea t t h e5 ' ( t e f t )e n do f t h e ' i n t r o n j n c t u d et h s ec o n s e n ssuesq u e n cGeU . r The3' spLice siteat the 3' (right)endof theintron i n c l u d et sh ec o n s e n ssuesq u e n A c eG . r TheGU-AG ruLe(origina[Ly catledthe GT-AG ru[ein termsof DNAsequence) describes the requirement for theseconstant dinucteotides at the firsttwo andlasttwo positions of intronsin pre-mRNAs.
Left (5') site
lntron
i:SlJg{[f$"] Theendsof nuclear intronsaredefined rul.e. bv the GU-AG
To focus on the molecular events involved in nuclear intron splicing, we must consider the nature of the splice sites, the two exon-intron boundaries that include the sites of breakase and reunion. By comparing the nucleotide sequenceof nRNA with that of the structural gene, the junctions between exons and introns can be assigned.There is no extensive homology or complementarity between the two ends of an intron. The junctions, however, have well conserved,though rather short, consensus sequences. It is possibleto assigna specificend to every intron by relying on the conservation of exon-intron junctions. They can all be aligned to conform to the consensussequencegiven in rI**Rf f$.:. The subscripts indicate the percent occurrence of the specifiedbase at each consensus position. High conservation is found only immediately within the intron at the presumed junctions. This identifies the sequenceof a generic intron as:
Junctions SpLice AreReadin Pairs . Spticing of pairsof ontyon recognition depends junctions. sptice . A[[5'splicesitesarefunctiona[[y and equivalent, equivalent. sjtesarefunctionatly at[ 3' spl.ice A typical mammalian nRNA has many introns. The basicproblem of pre-nRNA splicing results from the simplicity of the splice sitesand is illus:lfi.'i:What ensuresthat the cortrated in Ft+|iftf;. rect pairs of sites are spliced together? The corresponding GU-AG pairs must be connected acrossgreat distances(some introns are >10 kb long). We can imagine two types of principle that might be responsiblefor pairing the appropriate 5'and 3'sites: . Itcouldbe anintrinsicproperf of theRNA to connect the sitesat the ends of a particular intron. This would require matching of specific sequencesor structures.
GU......AG The intron defined in this way starts with the dinucleotide GU and ends with the dinucleotideAG; as a result, the junctions are often describedas conforming to the GT-AG rule. (This reflects the fact that the sequenceswere originally analyzed in terms of DNA, but of course the GT in the coding strand sequence of DNA becomesa GU in the RNA.) Note that the two sites have different sequencesand so they define the ends of the intron directionally.They are named proceeding from left to right along the intron as the 5'splice site (sometimes called the left or donor site) and the 3' splicesite (also called the right or acceptor site). The consensussequencesare implicated as the sitesrecognized in splicing by point mutations that prevent splicing in vivo and in vitro.
Pairingof wrong juncttonsr totilclrenroveexons
junctions are recognizedonty in the correct pairwise fI*$frf t{i.+ SpLicing combinations.
AreReadin Pairs 26.3 Sptice Junctions
677
. It could be that all 5'sites may be functionally equivalent and all 3' sites may be similarly indistinguishable, but splicing could follow rules that ensure a 5' site is always connected to the 3'site that comes next in the RNA. Neither the splice sitesnor the surrounding regions have any sequence complementarity, which excludesmodels for complementary base pairing between intron ends.Experiments using hybrid RNA precursors show that any 5' splice site can in principle be connected to any 3'splice site. For example, when the first exon of the early SV40 transcription unit is linked to the third exon of mouse B globin, the hybrid intron can be excised to generate a perfect connection between the SV40 exon and the B-globinexon. Indeed, this interchangeability is the basisfor the exon-trapping technique describedpreviously in Figure 4.11. Such experimentsmake two general points: . Splicesitesare generic:They do not have specificity for individual RNA precursors, and individual precursors do not convey specificinformation (such as secondary structure) that is needed for splicing. . The apparatusfor splicing is not tissuespecific: An RNA can usually be properly spliced by any cell, whether or not it is usually synthesizedin that cell. (We discussexceptionsin which there are tissue-specificalternative splicing patterns in Section 26.12, Alternative Splicing Involves Differential Use of Splice Junctions.) Here is a paradox. Ir is likely that all 5'splice sites look similar to the splicing apparatus, and that all 3' splice sites look similar to it. In principle,any 5' splicesite may be able to reactwith any 3' splicesite.In the usual circumstances,though, splicingoccurs only between the 5'and i'sites of. tll'e same intron. What rules ensurethat recognition of splicesitesis restricteds0thLltlnly the 5' and 3' sitesof the sameintron are spliced? Are introns removed in a specificorderfrom a particular RNA? Using RNA blotting, we can identify nuclear RNAs that represent intermediates from which some introns have been removed. l:,:iJRi ts"5.shows a blot of the precursors to ovomucoid mRNA. There is a discrete series of bands, which suggeststhat splicing occurs via definite pathways. (If the seven introns were removed in an entirely random order, there would be more than 300 precursors with different combinations of introns, and we would not see discretebands.)
CHAPTER 26 RNASpticingand Processing
(5.5kb) Primary transcript
Lacksintrons5 and 6
Lacksintrons4, 5, 6, and 7
Containsonly intron3
m R N A ( 1 . k1 b )
Fffitifif,f *.$ Northernbl.ottingof nuctearRNAwith an probeidentifies precursors ovomucojd discrete to mRNA. Thecontents of the moreprominent bandsareindicated. Photocourtesyof BertW.O'Mattey, BaylorCotlege of Medicine.
There does not seem to b e a unique pathway, because intermediates can be found in which different combinations of introns have been removed. There is, however, evidence ior apreferred pathway or pathways. When only one intron has been lost, it is virtually always 5 or 6. Either can be lost first. When two introns have been lost, 5 and 6 are again the most frequent, but there are other combinations. Intron 3 is never (or at leastvery rarely) Iost at one of the first three splicing steps.From this pattern, we see that there is a preferred pathway in which introns are removed in the order 516, 7 | 4, 2l | , 3. There are other pathways. though, because (for example) there are some molecules in which 4 or 7 is lost last. A caveat in interpreting these results is that we do not have proof that all these intermediates actually lead to mature nRNA.
The general conclusion suggestedby this analysis is that the conformation of the RNA influences the accessibilityof the splice sites.As particular introns are removed, the conformation changes,and new pairs of splice sites become available. The ability of the precursor to remove its introns in more than one order, though, suggeststhat alternative conformations are available at each stage.Of course,the longer the molecule, the more structural options become available, and when we consider larger genes,it becomes difficult to seehow specificsecondary structures could control the reaction. One important conclusion of this analysisis that the reactiondoesnot proceedsequentiallyalong the precurs0r. A simple model to control recognition of splice sites would be for the splicing apparatus to act in a processivemanner. Having recognized a 5' site, the apparatus might scan the RNA in the appropriate direction until it meets the next f'site. This would restrict splicing to adjacent sites.This model, however, is excluded by experiments that show that splicing can occur intrans as an intermolecular reaction under spec i a l c i r c u m s t a n c e s( s e e S e c t i o n 2 6 . 1 ) , t r a n s splicing Reactions Use Small RNAs) or in RNA molecules in which part of the nucleotide chain is replacedby a chemical linker. This means that there cannot be a requirement for strict scanning along the RNA from the 5' splice site to the 3' splice site. Another problem with the scanning model is that it cannot explain the existenceof alternative splicing patterns, where (for example) a common 5' site is spliced to more than one 3'site. The basisfor proper recognition of correct splice site pairs remains incompletely defined.
Pre-mRNA Spticing Proceeds througha Lariat . Splicing requires the 5'and3'splicesitesanda sitejust upstream of the 3' spticesite. branch o Thebranchsequence is conserved in yeastbut less in highereukaryotes. wetlconserved r A lariatis formedwhentheintronis cteaved at the 5'splice sitea , n dt h e5 ' e n di s j o i n e dt o a 2 ' oositionat an A at the branch sitein the intron. o Theintronis reteased asa lariatwhenit is cleaved at the 3'spticesite,andthe left andrightexons arethenligatedtogether. r Thereactions occurbytransesterifications, in fromonelocation to whicha bondis transferred another.
3',site GU UACUAACAG
5' site
!t
Pyro N Pyro Pyu, Puru A Py* Animalconsensus Cut at 5' siteand form lariatby 5'-2' bondconnecting the intron5'-G to the 2' of A at the branchsite
5'@
3/ z
Cut at 3' site and join exons;intronreleasedas lariat
s'-
3, 3/5'@ UACUAACAG
3'
tI -
v 5',
3',
Debranchintron 5'GU UACUAACAG3'
rlii.ilii i:ii.lr Spticing Firstthe 5'exon in twostages. occurs is cleaved off.andthenit isjoinedto the 3' exon.
The mechanism of splicing has been characterized in vitro using systems in which introns can b e r e m o v e d f r o m R N A p r e c u r s o r s .N u c l e a r extracts can splicepurified RNA precursors;this shows that the action of splicing is not linked to the processof transcription. Splicing can occur in RNAs that are neither cappednor polyadenylated. The splicing reaction as such is independent of transcription or modification to the RNA; these events, however, normally occur in a coordinated manner, and the efficiency of splicing may be influenced by other processingevents. The stagesof splicing invitro are illustrated in the pathway of i'i{.;lifif ;:ti"i"t.We discuss the reaction in terms of the individual RNA species that can be identified, but rememb er Ihat in v:o the species containing exons are not released as free molecules, but remain held together by the splicing apparatus. The first step is to make a cut at the 5'splice site, separating the left exon and the right intron-exon molecule. The left exon takes the form of a linear molecule. The right intron-exon molecule forms a lariat, in which the 5'terminus senerated at the end of the intron becomes
Proceeds througha Lariat Spl,icing 26.4 Pre-mRNA
673
linked by a 5'-2' bond to a base within the intron. The target base is an A in a sequence that is called the branch site. Cutting at the 3'splice site releasesthe free intron in lariat form; the right exon is ligated (spliced)to the left exon. The cleavageand ligation reactions are shown separately in the figure for illustrative purposes,but actually occur as one coordinated transfer. The lariat is then "debranched" to give a linear excisedintron, which is rapidly degraded. The sequences neededfor splicingare the short clnsensussequences at the 5' and 3' splicesitesand at the branch sire Together with the knowledge that most of the sequenceof an intron can be deleted without impeding splicing, this indicatesthat there is no demand for specificconformation in the intron (or exon). The branch site plays an important role in identifying the 3'splice site. The branch site in yeast is highly conservedand has the consensus sequence UACUAAC. The branch site in higher eukaryotes is not well conserved, but has a preference for purines or pyrimidines at each position and retains the target A nucleotide ( s e eF i g u r e 2 6 . 6 ) . The branch site lies l8 to 40 nucleotides upstreamof the 3'splicesite.Mutations or deletions of the branch site in yeast prevent splic-
ing. In higher eukaryotes, the relaxed constraints in its sequenceresult in the ability to use related sequences (called cryptic sites) when the authentic branch is deleted. Proximity to the 3' splice site appears to be important, becausethe cryptic site is always close to the authentic site. A cryptic site is used only when the branch site has been inactivated. When a cryptic branch sequence is used in this manner, splicing otherwise appearsto be normal, and the exons give the same products as wild type. The role of the branch sitethereforeis to identify the nearest3' splice siteas the targetfor connectionto the 5' splice site. This can be explained by the fact that an interaction occurs between protein complexes that b i n d t o t h e s et w o s i t e s . The bond that forms the lariat goesfrom the 5'position of the invariant G that was at the 5' end of the intron to the 2'position of the invariant A in the branch site. This correspondsto the third A residue in the yeast UACUAAC box. The chemical reactions proceed by transesterification: A bond is in effect transferred from one location to another. iiiitii..;i': iti".r shows that the first step is a nucleophilic attack by the 2'-OH of the invariant A of the UACUAAC sequence on the 5' splice site. In the second step, the free J'-OH of the exon that was released by the first reaction now attacks the bond at the 3' splice site. Note that the number of phosphodiester bonds is conserved. There were originally two 5'-3'bonds at the exon-intron splice sites;one has been replaced by the 5'-3'bond between the exons, and the other has been replacedby the 5'-2'bond that forms the lariat.
AreRequired snRNAs for Splicing E x o n1 - O H
ThefivesnRNPs invotved in splicing areU1,U2, U 5 ,U 4 ,a n dU 6 . Together proteins, with someadditionaI the snRNPs formthe soticeosome. At[the snRNPs exceptU6containa conserved sequence that bindstheSmproteins that are recognized generated by antibodies in autoimmune disease.
The 5' and J' splice sites and the branch sequence are recognized by components of the irji.1!i:rii:iiti;,I Nuclear spl.icing occursby two transesteri- splicing apparatus that assembleto form a large jn whichan0Hgroupattacks ficationreactions a phospho- complex. This complex brings together the 5' diesterbond. and 3' splice sites before any reaction occurs,
674
C H A P T E2R6 R N AS p t i c i n ga n d P r o c e s s i n g
which explains why a deficiency in any one of the sitesmay prevent the reaction from initiating. The complex assemblessequentially on the pre-nRNA, and several intermediates can be recognized by fractionating complexes of different sizes.Splicing occurs only after all the components have assembled. The splicing apparatus contains both proteins and RNAs (in addition to the pre-mRNA). The RNAs take the form of small molecules that exist as ribonucleoprotein particles. Both the nucleus and cytoplasm of eukaryotic cellscont a i n m a n y d i s c r e t e s m a l l R N A s p e c i e s .T h e y range in size from 100 to 300 basesin higher eukaryotesand extend in length to -1000 bases in yeast.They vary considerablyin abundance, from 105 to 106 molecules per cell to concentrations too low to be detected directly. Those restricted to the nucleus are called small nuclear RNAs (snRNAs); those found in the cytoplasm are called small cytoplasmic RNAs (scRNAs). In their natural state, they exist as ribonucleoprotein particles (snRNPand scRNP).Colloquially,they are sometimesknown as snurps and scyrps. There is also a class of small RNAs found in the nucleolus, called snoRNAs,which are involved in processingribosomal RNA (seeSection26.22, Small RNAs Are Required for rRNA Processing). The snRNPsinvolved in splicing. together with many additional proteins, form a large particulate complex called the spliceosome. Isolated from the in vitro splicing systems, it c o m p r i s e sa 5 0 S t o 6 0 S r i b o n u c l e o p r o t e i n p a r t i c l e . T h e s p l i c e o s o m em a y b e f o r m e d i n stagesas the snRNPsjoin, proceeding through s e v e r a l" p r e s p l i c i n gc o m p l e x e s . "T h e s p l i c e o some is a large body, greater in mass than the ribosome. :::.:,rri ,:iSummarizesthe cOmponentsof the spliceosome.The 5 snRNAsaccount for more than a quarter of the mass;together with their 4l associatedproteins, they account for almost half of the mass.Some 70 other proteins found in the spliceosomeare describedas splicingfactors. They include proteins required for assembly of the spliceosome,proteins required for it t o b i n d t o t h e R N A s u b s t r a t e ,a n d p r o t e i n s involved in the catalyticprocess.In addition to theseproteins, another -30 proteins associated with the spliceosomehave been implicated in acting at other stagesof gene expression,which suggeststhat the spliceosomemay serve as a coordinating apparatus. The spliceosomeforms on the intact precursor RNA and passesthrough an intermedi-
ate state in which it contains the individual 5' exon linear molecule and the right lariatintron-exon. Little spliced product is found in the complex, which suggeststhat it is usually releasedimmediately following the cleavageof the 3' site and ligation of the exons. We may think of the snRNP particles as being involved in building the structure of the spliceosome. Like the ribosome, the spliceosome depends on RNA-RNA interactions as well as protein-RNA and protein-protein interactions. Some of the reactions involving the snRNPsrequire their RNAs to basepair directly with sequencesin the RNA being spliced; other reactions require recognition between snRNPs or between their proteins and other components of the spliceosome. The importance of snRNA molecules can be tested directly in yeast by making mutations in their genes.Mutations in 5 snRNA genesare lethal and prevent splicing.All of the snRNAsinvolved in splicing can be recognizedin conservedforms in animal, bird, and insect cells.The corresponding RNAs in yeast are often rather larger, but conservedregions include featuresthat are similar to the snRNAs of higher eukaryotes. The snRNPsinvolved in splicing areUl,U2, \15,lJ4, and U6. They are named according to the snRNAs that are present. Each snRNP contains a singlesnRNA and several(<20) proteins. The U4 and U6 snRNPsare usually found as a single lU4lU6) particle. A common structural core for each snRNPconsistsof a group of eight proteins, all of which are recognized by an autoimmune antiserum called anti-Sm;
r : i , : r ! : i l r l r r T h es p t i c e o s o mi se- 1 2 M D a .F i v es n R N P s accountfor almosthaLfof the mass.The remainingproteins inc[udeknownspticingfactors,as wetl as proteins that areinvotvedin other stagesof geneexpression.
for Spticing 675 AreRequired 26.5 snRNAs
conservedsequencesin the proteins form the target for the antibodies.The other proteins in each snRNP are unique to it. The Sm proteins bind to the conservedsequencePuAU3-6Gpu, which is present in all snRNAs except U6. The U6 snRNP instead contains a set of Sm-like (Lsm) proteins. The Sm proteins must be involved in the autoimmune reaction, although their relationship to the phenotype of the autoimmune diseaseis not clear. Some of the proteins in the snRNPsmay be involved directly in splicing; others may be required in structural roles or just for assembly or interactions between the snRNP particles.About one third of the proteins involved in splicing are components of the snRNPs. Increasing evidence for a direct role of RNA in the splicing reaction suggeststhat relatively few of the splicing factors play a direct role in c a t a l y s i s ;m o s t a r e i n v o l v e d i n s t r u c t u r a l o r a s s e m b l yr o l e s .
DomainD
UG UA U UA
Intronpairing
88
,, ^ U A,G G"
4A n r l Gcnc" DomainA fliliJ$ll;;]i:.t U1 snRNAhasa base-paired structurethat createsseveraIdomains. The5' end remainssing[estrandedand can basepair wjth the 5' spticingsite.
676
CHAPTER 26 RNASpticing andProcessing
Initiates U1snRNP Spl.icing . U1snRNP initiatessplicing by bindingto the pairing 5'spticesiteby means of an RNA-RNA reaction. o TheEcomptex contains ULsnRNP boundat the 5'splicesite,the proteinU2AFboundto a pyrimidine tractbetween the branchsiteandthe 3'splicesite.andSRproteins connecting U1 s n R NtPo U 2 A F .
Splicing can be broadly divided into two stages: ' F i r s t t h e c o n s e n s u ss e q u e n c e sa t t h e 5'splice site,branch sequence,and adjacent pyrimidine tract are recognized. A complex assemblesthat contains all of the splicing components. . The cleavageand ligation reactionsthen change the structure of the substrate RNA. Components of the complex are released or reorganized as it proceeds through the splicing reactions. The important point is that all of the splicing components are assembled and haveensuredthat the splicesitesare availablebeforeany irreversiblechange is made to the RNA Recognition of the consensus sequences involves both RNAs and proteins. Certain snRNAs have sequencesthat are complement a r y t o t h e c o n s e n s u s s e q u e n c e so r t o o n e another, and base pairing between snRNA and pre-mRNA, or between snRNAs, plays an important role in splicing. The human Ul snRNP contains eight proteins as well as the RNA. The secondarystructure of the Ui snRNA is drawn in ill{itiit* fii.ll. It contains several domains. The Sm-binding site is required for interaction with the common snRNPproteins. Domains identified by the individual stem-loop structures provide binding sites for proteins that are unique to UI snRNP. Binding of Ul snRNPto the 5'splice site is the first step in splicing. The recruitment of Ul snRNP involves an interaction between one of its proteins (Ul -70k) and the protein ASF/SF2 (a general splicing factor in the SR class: see below) . U I snRNA basepairs with the 5' site by means of a single-strandedregion at its 5'terminus, which usually includes a stretch of four to six bases that is complementary with the splice site. Mutations in the 5'splice site and UI snRNA can be used to test directly whether pairing between them is necessary.The results of such
iri..1{.i. an experiment are illustrated in !ri{";i"tqf The wild-type sequence of the splice site of the 12S adenovirus pre-nRNA pairs at five out of six positions with UI snRNA. A mutant in the 12S RNA that cannot be spliced has two sequence changes;the GG residues at positions 5 Lo 6 in the intron are changed to AU. The mutation changes the pattern of base pairing between Ul snRNA and the 5' splice site, although it does not alter the overall extent of pairing (becausecomplementarity is lost at one position and gained at the other). The effect on splicing suggeststhat the base-pairinginteraction is important. When a mutation is introduced into UI snRNA that restores pairing at position 5, normal splicing is regained. Other casesin which correspondingmutations are made in UI snRNA to seewhether they can suppressthe mutation in the splice site suggeststhe general rule: Complementarity between Ul snRNA and the 5' splice site is necessaryfor splicing, but the efficiency of splicing is not determined solely by the number of base pairs that can form. The pairing reaction is stabilized by the proteins of the Ul snRNP. iii,r-iiii .,:'.i.ri i shows the early stagesof splicing. The first complex formed during splicing is the E (early presplicing) complex, which contains Ul snRNP.the splicing factor U2AF, and members of a family called SR proteins, which comprise an important group of splicing factors and regulators.They take their name from the presenceof an Arg-Ser-rich region that is variable in length. SR proteins interact with one another via their Arg-Ser-rich regions. They also bind to RNA. They are an essentialcomponent of the spliceosome,forming a framework on the RNA substrate.They connect U2AF to Ul (iiii:ii:ii:.:.;.lii). The E complex is sometimes called the commitment complex, because its formation identifies a pre-nRNA as a substrate for formation of the splicing complex. In the E complex, U2AF is bound to the region between the branch site and the 3'splice site. The name of U2AF reflectsits original isolation as the U2 auxiliary factor. In most organisms, it has a large subunit (U2AF65) that contacts a pyrimidine tract downstream of the branch site; a small subunit (U2AF35) directly contactsthe dinucleotide AG at the l'splice site. cerevisiae, this function is filled In Saccharomyces by the protein Mud2, which is a counterpart of U2AF6r, andbinds only to the pyrimidine tract. This marks a difference in the mechanism of and other organsplicing between S. cerevisiae
isms. In yeast, the 3' splice site is not involved in the early stagesof forming the splicing complex, but in all other known casesit is required. Another splicing factor, called SFI in mammals and BBP in yeast, connects U2AF/Mud2 to the UI snRNP bound at the 5' splice site. Complex formation is enhanced by the cooperative reactions of the two proteins; SFI and U2AF (or BBP and Mud2) bind together to the RNA substrate-l0x more effectivelythan either alone. This interaction is probably responsible for making the first connection between the two splice sitesacrossthe intron. The E complex is converted to the A complex when U2 snRNP binds to the branch site. Both Ul snRNP and U2AF/Mud2 are needed f.or lJ2 binding. The U2 snRNA includes sequences complementary to the branch site'
; r l l i i i i i l :: :l , ; r , ,M u t a t i o ntsh a t a b o t i s hf u n c t i o no f t h e mutaby compensating 5'spticingsitecanbe suppressed basepairing. that restore tionsin U1snRNA
InitiatesSpticing 677 26.6 U1.snRNP
A sequencenear the 5' end of the snRNA base pairs with the branch sequence in the intron. In yeast this typically involves formation of a duplex with the UACUAAC box (see Figure 26.14). Severalproteins of the U2 snRNP are bound to the substrate RNA just upstream of the branch site. The addition of U2 snRNP to the E complex generatesthe A presplicing complex. The binding of U2 snRNP requires ATP hydrolysis, and commits a pre-nRNA to the splicing pathway.
Intron 5' splicesite
F2 U1snRNPand factorASF/SF2-: bind5' splicesite
Branch site
Pytract
l +
G U2AF65U2AF35
tl
U1snRNP U2AFbinds Py tract and 3' splicesite
3' splicesite
:!::
UACUAAC
Py-AG
| ,.',ru, SF1/BBP connects U1 snRNPto U2AF
Ft{ii1ftil t*.1 i Thecommitment (E)comptex formsbythe successive addition of U1snRNP to the 5' splicesite,U2AFto the pyrimidine tract/3'splice site, andthe bridging proteinSF1/BBP.
TheEComplex Can BeFormed by Intron Definition or Exon Definition r Thedirectwayof formingan Ecomplex is for U1 snRNP to bindat the 5'splicesiteandU2AF to bindat a pyrimidine tractbetween the branch site andthe 3'spticesite. o Anotherpossibitity is for the complex to form between U2AF at the pyrimidine tractandU1 snRNP at a downstream 5'spticesite. o TheEcomplex is converted to theA comptex when U2snRNP bindsat the branch site. o If an Ecomptex formsusinga downstream 5'spLice site,this splicesiteis reptaced by the appropriate upstream 5'spticesitewhenthe Ecomptex is converted to the A comotex. o Weak 3'splicesitesmayrequire a spticing enhancer [ocated in theexondownstream to bind SRproteinsdirectly.
There is more than one way to form the E complex. Figure 26.I2 illustratessome possibilities. The most direct reaction is for both splice sites to be recognized acrossthe intron. The presence of UI snRNP at the 5'splice site is necessary for U2AF to bind at the pyrimidine tract downstream of the branch site, making it possible that the 5' and 3' ends of the intron are brought together in this complex. The E complex is converted to the A complex when U2 snRNP binds at the branch site. Thebasicfeature of this routefor splicing is that the two splicesitesare
ilt#*HL*{t".tl Theremaybe multipteroutesfor initial"recognition of 5'and3'spticesites.
678
CHAPTER 26 RNASpticing andprocessing
with1ut requiring any sequences 7utside rec7gnized of the intron This process is called intron definition. In an extreme case,the SR proteins may enable U2AFlU2 snRNP to bind invitro in the absenceof Ul, raising the possibilitythat there could be a UI -independent pathway for splicing. An alternative route to form the spliceosome may be followed when the introns are long and the splice sitesare weak. As shown on the right of the figure, rhre5' splice site is recognized by Ul snRNA in the usual way. The l' splice site is recognizedas part of a complex that forms acrossrl:,enextexon,though, in which the next 5' splice site is also bound by Ul snRNA. This Ul snRNA is connected by SR proteins to the U2AF at the pyrimidine tract. When U2 snRNP joins to generate the A complex, there is a rearrangement in which the correct (leftmost) 5'splice site displacesthe downstream 5' splice site in the complex. The important feature of this route for splicing is that sequencesdownstream of the intron itself are required. Usually these sequencesinclude the next 5'splice site. This process is called exon definition. This mechanism is not universal: Neither SRproteins nor exon definition are found in S. cerevisiae. "Weak" 3'splicesitesdo notbind U2AF and U2 snRNP effectively. Additional sequencesare needed to bind the SR proteins, which assist U2AF in binding to the pyrimidine tract. Such sequencesare called "splicing enhancers,"and they are most commonly found in the exon downstream of the 3'splice site.
@
Form 5 snRNPs theSpLiceosome
the A converts Bindingof U5andU4/U6snRNPs atl whichcontains comptex to the B1spliceosome. necessary for spticing. the components passes of further througha series Thespticeosome proceeds. asspticing comptexes atlowsU6snRNA to interact Release of U1snRNP the 81 withthe 5'splicesjte,andconverts to the B2spliceosome. spliceosome can fromU6snRNP, U6snRNA WhenU4dissociates pairwith U2snRNA active to formthe catatytic site. Following formation of the E complex, the other snRNPsand factorsinvolved in splicingassociate with the complex in a defined order. i:i'iri"iiiff ii.:ri-t shows the components of the complexes that can be identified as the reaction proceeds.
The B I complex is formed when a trimer containing the U5 andIJ4ltJ6 snRNPsbinds to the A complex containing UI and U2 snRNPs. This complex is regarded as a spliceosome becauseit containsthe componentsneededfor the splicing reaction. It is converted to the 82 complex after Ul is released.The dissociation of Ul is necessaryto allow other components to come into juxtaposition with the 5' splice site, most notably U6 snRNA. At this point U5 snRNA changes its position; initially it is close to exon sequencesat the 5' splice site, but it shifts to the vicinity of the intron sequences.
U5 U6 )J2AF / t'Y Au :-: ,/
vY
Au -'-
Bl complex trimerbinds USlUAluB U5bindsexonat 5' site U6bindsU2
82 comPlex U1 is released U5 shiftsirom exon to intron U6 bindsat 5' splcesite
C1 complex U4 is released U6lU2 catalyzes transesterification U5 bindsexonat 3' sPlicesite 5' site cleavedand lariatformed
I nre hydrolyzed C2 complex remainboundto lariat UZ|US|UG 3' site cleavedand exons ligated SplicedRNA is released Lariatdebranched
in which stages proceeds throughdiscrete reaction ,rr...i1 Thespticing i ii.t'rjiiii that recognize of components the interaction invotves formation sotjceosome sequences. the consensus
Formthe SPticeosome 679 26.8 5 snRNPs
Thus several pairing reactions between snRNAs and the substrate RNA occur in the course of splicing.They are summarized in f3{rltSiit{i-i5. The snRNPshave sequencesthat pair with the substrateand with one another. They also have single-strandedregions in loops that are in close proximity to sequencesin the substrate, and which play an important role, as judged by the ability of mutations in the loops to block splicing. The basepairing between U2 and the branch point, and between U2 and U6, createsa structure that resembles the active center of group II self-splicingintrons (seeFigure 2 6.20) . This suggeststhe possibility that the catalytic component could comprise an RNA structure generated by the U2-U6 interaction. U6 is paired with the 5' splice site, and crosslinking experiments show that a loop in U5 snRNA is immediately adjacent to the first basepositions in both exons. Although we can define the proximities of the substrate(5' splicesite and branch site) and snurps (U2 and U6) at the catalyticcenter (asshown in Figure 26.14\, the components that undertake the transesterifications have not been directly identified. ' ::' ' U 6 - U 4p a i r i n gi s i n c o m p a t i bwt ei t h U 6 - U 2p a i r i n g . The formation of the lariat at the branch site WhenU6joinsthe spticeosome it is pairedwjth U4.Retease of U4 is responsible for determining the use of the 3' a[[ows a conformationaI change in U6;onepartofthereteased sequence splicesite,becausethe 3'consensussequencenearformsa hairpin(darkgray).andthe otherpart(btack)pairswith U2. estto the 3'side of the branch becomesthe target Anadjacent regionof U2is atready paired withthebranch site,which bringsU6intojuxtaposition for the secondtransesterification.The secondsplicwiththebranch. Notethatthesubstrate RNAis reversed fromthe usuaIorientation andis shown3'to 5'. ing reaction follows rapidly. Binding of U5 snRNp to the 3'splice site is needed{or this reaction,but there is no evidence for a base-pairing reaction. The catalytic reaction is triggered by the The important conclusion suggested by release of U4; this requires hydrolysis of ATp. these results is that the snRNAcomponents of the The role of U4 snRNA may be to sequesterU6 splicing apparatus interact both among themselves snRNA until it is needed. i:i;i"iiil,,:r+:* shows and with the substrateRNA by meansof basepairthe changesthat occur in the basepairing intering interactions,and theseinteractionsallow for actions between snRNAs during splicing. In the changesin structure that may bring reactinggrlups U6lU4 snRNP,a continuous length of 26 bases into appositionand may evencreatecatalyticcenters. of U6 is paired with two separatedregions of Furthermore, the conformational changes in U4. When U4 dissociates,the region in U6 that the snRNAs are reversible; for example, U6 i s r e l e a s e db e c o m e s f r e e t o t a k e u p a n o t h e r snRNA is not used up in a splicing reaction and structure. The first part of it pairs with U2; the at completion must be releasedfrom U2 so that second part forms an intramolecular hairpin. it can reform the duplex structure with U4 to The interaction between U4 and U6 is mutuundertake another cycle of splicing. ally incompatible with the interaction between We have described individual reactions in U2 and U6, so the release of U4 controls the which each snRNP participates,but as might be ability of the spliceosometo proceed. expected from a complex series of reactions, For clarity, the figure shows the RNA subany particular snRNP may play more than one strate in extended form, but the 5'splice site is role in splicing. Thus the ability of Ul snRNp to actually closeto the U6 sequenceimmediately promote binding of U2 snRNPto the branch site on the 5' side of the srretch bound ro U2. This is independent of its ability to bind ro the 5' sequence in U6 snRNA pairs with sequencesin splice site. Similarly, different regions of U2 the intron just downstream of the conserved snRNA can be defined that are needed to bind GU at the 5'splice site (mutations that enhance to the branch site and to interact with other such pairing improve the efficiency of splicing). splicing components.
680
C H A P T E2R6 R N AS p l . i c i nagn d P r o c e s s i n g
Some of the PRPproteins are components of snRNP particles, but others function as independent factors. One interesting example is PRPl6, a helicasethat hydrolyzes ATP and associatestransiently with the spliceosometo participate in the second catalytic step. Another example is PRP22,another ATP-dependenthelicase,which is required to releasethe mature mRNA from the spliceosome. The conservation of bonds during the splicing reaction means that input of energy is not required to drive bond formation per se,which implies that the ATP hydrolysis is required for other purposes. The use of ATP by PRPI6 and PRP22 may be examples of a more general phenomenon: the use of ATP hydrolysis to drive conformational changes that are needed to proceed through splicing.
An Alternative Uses Apparatus SpLicing snRNPs Different set usesanother splicingpathway An alternative the U12sp[iceosome' that comprise of snRNPs by longerconsensus Thetargetintronsaredefined junctions, but usuatly sequences at the sptice junctions. inctude the sameGU-AG junctions AU-AC. Someintronshavethe splice andsome including somethat areU1-dependent that areU12-dependent.
ilii,=;* Spl.icing utitizesa seriesof basepairing :i,,"Lit.rsnRNAs andsplicesites. reactions between An extensive mutational analysis has been undertaken in yeast to identify both the RNA and protein components of the spliceosome. Mutations in genesneeded for splicing are identified by the accumulation of unspliced precursors.A seriesof loci that identify genespotentially coding for proteins involved in splicing were originally called RNA, but are now known as PRPmutants (for pre-RNA processing).Several of the products of these genes have motifs that identily them as RNA-binding proteins, and some appear to be related to a family of ATPdependent RNA helicases.We suppose that, in addition to RNA-RNA interactions, protein-RNA interactions are important in creating or releasing structures in the pre-mRNA or snRNA components of the spliceosomes.
GU-AG introns comprise the vast majority (>98% of splicing junctions in the human genome). Lessthan 17o use the related junctions GC-AG, and then there is a minor classof introns marked by the ends AU-AC (comprising 0.17o of introns). The first of these introns to be discovered required an alternative splicing apparatus,called the UI2 spliceosome, which consistedof Ul1 and Ul2 (relatedto Ul and U2, respectively), a U5 variant, and the U4uru.and U6u,u.snRNAs. The splicing reaction is essentially similar to that at GU-AG introns, and the snRNAs play analogous roles. Whether there are differencesin the protein components of this apparatus is not known. It now turns out that the dependence on the type of spliceosomeis also influenced by sequencesin the intron, so that there are some AU-AC introns splicedby U2-t1pe spliceosomes, and some GU-AG introns splicedby Ul2-type spliceosomes.A strong consensussequenceat the left end definesthe Ul2-dependent type of
snRNPs 6 8 1 UsesDifferent Apparatus Spticing 26.9AnAlternative
intron: 5'GAUAUCCUUU.. .pyAG63'. In fact, most Ul2-dependent introns have the GU. . . .AG termini. In addition, they have a highly conserved branch point, UCCUUPuAPy, which pairs with U I 2. For this reason, the term Ul2-dependent intron is used instead of AUAC intron. The two types of introns coexist in a variety of genomes,and in some casesare found in the same gene. UI2-dependent introns tend to be flanked by U2-dependent inrrons. What is known about the phylogeny of these introns s u g g e s t st h a t A U - A C U l 2 - d e p e n d e n t i n t r o n s may once have been more common, but tend ro be converted to GU-AG termini, and to U2dependence, in the course of evolution. The common evolution of the systems is emphasizedby the fact that they use analogoussetsof base pairing between the snRNAs and with the substratepre-nRNA. The involvement of snRNPsin splicing is only one example of their involvement in RNA processingreactions. snRNPsare required for several reactions in the processingof nuclear RNA to mature rRNAs. Especiallyin view of the demonstration that group I introns are selfsplicing, and that the RNA of ribonucleaseP has catalytic activity (as discussedin Chapter 27, Catalytic RNA), it is plausible to think that RNA-RNA reactions are important in many RNA processingevents.
@
been completed. There may, however, also be a direct connection between splicing and export. Introns may prevent export of mRNA becausethey are associatedwith the splicing apparatus. The spliceosome also may provide the initial point of contact for the export apparatus. FIGURE t6-ie shows a model in which a protein complex binds to the RNA via the splicing apparatus.The complex consistsof >9 proteins and is called the EJC (exon junction complex). The EJC is involved in several functions of splicedmRNAs. Some of the proteins of the EJC are directly involved in these functions, and others recruit additional proteins for particular functions. The first contact in assembling the EJC is made with one of the splicing factors. After splicing, the EJC remains attached to the mRNA just upstream of the exon-exon junction. The EJC is not associatedwith RNAs transcribed from genes that lack introns, so its involvement in the processis unique for spliced products. If introns are deleted from a gene, its RNA product is exported much more slowly to the cytoplasm. This suggeststhat the intron may provide a signal for attachment of the export
Splicing Is Connected to Exportof mRNA
r TheREF proteins junctions bindto splicing by associating withthe sp[iceosome. . Afterspticing, theyremainattached to the RNAat junction. the exon-exon o Theyinteractwith the transportproteinTAP/Mex that exports pore. the RNAthroughthe nuctear After it has been synthesized and processed, nRNA is exported from the nucleus to the cytoplasm in the form of a ribonucleoprotein complex. The proteins that are responsiblefor transport "shuttle" between the nucleus and cytoplasm, remain in the compartment only briefly. TWoimportant questions are how these proteins recognize their RNA substrates,and what ensuresthat only fully processedmRNAs are exported. The answers in part may lie in the relative timing of events: Spliceosomesmay form to remove introns before transcription has
C H A P T E2R 6 R N AS p L i c i nagn d P r o c e s s i n g
; Proteinbinds splicing complex I
at I Proteinremains junction V exon-exon
on-exon
I
V EJC binds proteinsinvolved in RNA export,localization, decay
Fn$[.sftf fS.ld TheEJC (exonjunction comptex) bindsto RNAby recognizing the spticing comp[ex.
rl.l-;:ili;,:i 1: A REFproteinbindsto a splicingfactor with the spticed andremains RNAproduct. REFbindsto pore. an exportfactorthat bjndsto the nuctear
apparatus.We can now account for this phenomenon in terms of a seriesof protein intera c t i o n s ,a s s h o w n i n i i i ; : i i i i . , i 1 , . : , 'T. h e E J C includes a group of proteins called the REF family (the best characterizedmember is calledAly). The REF proteins in turn interact with a transport protein (variously called TAP and Mex), which has direct responsibility for interaction w i t h t h e n u c l e a rp o r e . A similar system may be used to identify a spliced RNA so that nonsense mutations prior to the last exon trigger its degradation in the cytoplasm (see Section 7.14, NonsenseMutations Trigger a SurveillanceSystem).
@
II Introns Group Autosplice via Lariat Formation
II intronsexcjse themsetves fromRNAby an Group splicing event. autocatatytic junctions Thesplice andmechanism of of spLicing groupII intronsaresjmitar to spLicing of nuctear i ntrons. structure A groupII intronfoldsinto a secondary a catatytic siteresembting the that generates intron. structure of U6-U2-nuctear
Introns in protein-coding genes (in fact, in all genes except nuclear IRNA-coding genes) can be divided into three general classes.Nuclear pre-nRNA introns are identified only by the possessionof the GU . . . AG dinucleotides at the 5' and 3' ends and the branch site/pyrimidine tract near the 3' end. They do not show any common features of secondary structure. Group I and group II introns are found in organellesand in bacteria. (Group I introns are found alsoin the nucleus in lower eukaryotes.) Group I and group II introns are classified according to their internal organization. Each can be folded into a typical type of secondary structure. The group I and group II introns have the remarkable ability to excise themselves from an RNA. This is called autosplicing. Group I introns are more common than group II introns. There is little relationship between the two classes.but in each casethe RNA can perform the splicing reaction in vitro by itself, without requiring enzymatic activities provided by proteins; however, proteins are almost certainly required invivo ro assistwith folding (seeChapt e r 2 7 , C a t a l y t i cR N A ) . r : i r , rr.i;i r ' I i r r 5 [ 6 * t t h a t t h r e e c l a s s e so f introns are excisedby two successivetransesterifications (shown previously for nuclear introns in Figure 26.61.It the first reaction,the 5' exon-intron junction is attacked by a free hydroxyl group (provided by an internal 2'-OH position in nuclear and group II introns, and by a free guanine nucleotide in group I introns). In the secondreaction, the free l'-OH at the end of the releasedexon in turn attacks the 3' intron-exon junction. There are parallelsbetween group II introns and pre-mRNA splicing. Group II mitochondrial introns are excisedby the same mechanism as nuclear pre-mRNAs via a lariat that is held togetherby a 5'-2'bond. An example of a lariat produced by splicing a group II intron is s h o w n i n i : : , r 1 r ,,,' i r .i .i . W h e n a n i s o l a t e dg r o u p II RNA is incubated in vitro in the absenceof additional components, it is able to perform the splicing reaction. This means that the two transesterificationreactions shown in Figure 26.18 can be performed by the group II intron RNA sequence itself . The number of phosphodiester bonds is conserved in the reaction, and as a result an external supply of energy is not required; this could have been an important feature in the evolution of splicing. A group II intron forms into a secondary structure that contains several domains formed
via LariatFormation Ii IntronsAutosptice 26.11Group
', S p l i c i nrge t e a s ea sm i t o c h o n d rgi ar o l up iI intronin the formof a stable[ariat.Reproduced from VanderVeen,R.,et aL.EMB]J. 1,987. 6: L079-1,084. Photocourtesy of LeslieA. Grivelt,European Molecular Biol.ogy 0rganisation.
Threec[asses proceed of spticing reactions by two transesterifications. First,a free0H groupattacks the exon1-intronjunction.Second, the 0H created at the endof exon1 attacks theintron-exon 2 iunction.
by base-pairedstemsand single-strandedloops. Domain 5 is separatedby twct basesfrom domain 6, which containsan A residuethat donatesthe 2'-OEgroup lor the first transesterification.This constitutes a catalytic domain in the RNA. comparesthis secondarystructure with the structure formed by the cclmbinarion of U6 with U2 and of U2 with the branch site. The similarity suggeststhat U6 may have a catalyticrole. The featuresof group II splicingsuggestthat splicingevcllvedfrom an autocatalyticreacrion undertaken by an individual RNA molecule, in which it accomplisheda controlled deletion of a n i n t e r n a l s e q u e n c e .I t i s l i k e l y t h a t s u c h a
684
CHAPTER 26 RNASpticing andProcessing
reaction requiresthe RNA to fold into a specific conlormation, or seriesof conformations, and would occur exclusively in cisconformation. The ability of group II introns to remove themselves by an autocatalytic splicing event stands in great contrast to the requirement oi nuclear introns for a complex splicing apparatus. We may regard the snRNAs of the spliceosome as compensating for the lack of s e q u e n c ei n f o r m a t i o n i n t h e i n t r o n , a n d p r o viding the information required to form particular structuresin RNA. The functions of the snRNAs may have evolved from the original autocatalytic system. These snRNAs actiri trans upon the substratepre-mRNA; we might imagine that the ability of Ul to pair with the 5' splicesite, or of U2 to pair with the branch s e q u e n c e ,r e p l a c e d a s i m i l a r r e a c t i o n t h a t r e q u i r e d t h e r e l e v a n t s e q u e n c et o b e c a r r i e d by the intron. Thus the snRNAs may undergo r e a c t i o n sw i t h t h e p r e - m R N A s u b s t r a t e ,a n d with one another, that have substitutedfor the seriesof conformational changesthat occur in RNAs that splice by group II mechanisms. In effect.these changeshave relieved the substrate pre-nRNA of the obiigation to carry the s e q u e n c e sn e e d e dt o s p o n s o rt h e r e a c t i o n .A s the splicing apparatus has become more complex (and as the number of potential substrates h a s i n c r e a s e d ) ,p r o t e i n s h a v e p l a y e d a m o r e important role.
Nuclearsplicingconstructsan activesite from pairingbetweenU6-U2and U2-intron
3'Exon2GA
ACAAUCAU OH
II
v Exon1---G U Group ll splicingconstructsan activecenter from the base pairedregronsof domains5 and 6
Domain5
nnnn
3 ' E x o n 2Y A
nn
YYYYAYY OH Domain6
II
v
Exon 1--G U
i:lil:..i+ir ii:..ii: Nuclear spticingand groupII spticing invotve theformation of similarsecondary structures. The groupII sequences aremorespecific in nuclear spl.icing; spticingusespositions that maybe occupied by either purine(R)or pyrimidine (Y).
Alternative Spl'icing InvolvesDifferentiaL Use of Sp[ice Junctions . Specific exonsmaybeexcluded or inctuded in the RNAproduct by usingor faitingto usea pairof junctions. spticing r Exons maybeextended by changing oneof the junctions junction. to usean alternative splice . Sexdetermination in Drosonhilo invotvesa series spLicing eventsin genescodingfor of atternative products successive of a pathway. r P etements of Drosophila showgermtine-specific atternative splicing.
When an interrupted gene is transcribed into an RNA that gives rise to a single type of spliced mRNA, there is no ambiguity in assignment of exons and introns. The RNAs of some genes, however, follow patterns of alternative splicing, which occurs when a single gene gives rise to more than one mRNA sequence. In some cases,the ultimate pattern of expression is dictated by the primary transcript, becausethe use of different startpoints or the generation of alternative 3'ends alters the pattern of splicing.In other cases,a single primary transcript is spliced in more than one way, and internal exons are substituted. added, or deleted. In some cases, the multiple products all are made in the same cell, but in others the processis regulated so that particular splicing pattems occur only under particular conditions. One of the most pressing questions in splicing is to determine what controls the use of such alternative pathways. Proteins that intervene to bias the use of alternative splice sites have been identified in two ways. In some mammalian systems,it has been possible to characterize alternative splicing invitro, and to identify proteins that are required for the process.In D. melanogaster, aberrations in alternative splicing may be causedeither by mutations in the genes that are alternatively spliced or in the genes whose products are necessaryfor the reaction. :ri.:.il shows examples of alternative irit,i,i{;;r splicing in which one splice site remains constant, but the other varies. The large T/small t antigens of SV40 and the products of the adenovirus EIA region are generatedby connecting a varying 5' site to a constant 3' site. In the caseof the T/t antigens, the 5' site used for T antigen removes a termination codon that is present in the t antigen mRNA, so that T antigen is larger than t antigen. In the case of the EIA transcripts, one of the 5' sites connects to the last exon in a different reading frame, again making a significant change in the C-terminal part of the protein. In these examples, all the relevant splicing events take place in every cell in which the gene is expressed,so that all the protein products are made. There are differences in the ratios of T/t antigens in different cell types. A protein extracted from cells that produce relatively more small t antigen can cause preferential production of small t RNA in extracts from other cell types.This protein, which was calledASF (alternative splicing factor), turns out to be the same
Useof SpliceJunctions 685 Differential Invotves Splicing 26.12ALternative
, ';-:r:!: : i: . ; Atternative formsof spticing maygenerate a varietyof proteinproducts gene.Changfromanindividuat ingthesptice sitesmayintroduce termination (shown codons by asterisks) or change reading frames.
as the splicing factor SF2, which is required for early stepsin spliceosomeassembly and for the f i r s t c l e a v a g e - l i g a t i o nr e a c t i o n ( s e e F i g u r e 26.13). ASF/SF2 is an RNA-binding prorein in the SR family. When a pre-mRNA has more than one 5'splice site preceding a single 3'splice site, increasedconcentrationsof ASF/SF2promote use of the 5'site nearest to the 3'site at the expense of the other site. This effect of ASF/SF2can be counteractedby another splicing factor, SF5. The exact molecular roles of the factors in controlling splice utilization are not yet known, but we see in general terms that alternative splicing involving different 5'sites may be influenced by proteins involved in spliceosome assembly.In the caseof T/t antigens, the effect probably rests on increasedbinding of the SR proteins to the site that is preferentially used. Alternative splicing also may be influenced by repression of one site. Exons 2 and 3 of the mouse troponin T gene are mutually exclusive;
686
CHAPTER 26 RNASpLicing andProcessing
exon 2 is used in smooth muscle, whereas exon 3 is used in other tissues.Smooth muscle contains proteins that bind to repeated elements Iocated on either side of exon 3, and which prevent use of the 3'and 5'sites that are needed to include it. The pathway of sex determination in D. melanogasler involves interactions between a genes series of in which alternative splicing events distinguish male and female. The pathway takes the form illustrated in F3*l"i$til 3*.If, in which the ratio of X chromosomes to autosomes determines the expression of sxl, and changes in expression are passed sequentially through the other genes to dsx, tll'e last in the pathway. The pathway starts with sex-specific splicing of sxl. Exon 3 of the sxl gene contains a termination codon that prevents synthesis of functional protein. This exon is included in the mRNA produced in males, but is skipped in females. (Exon skipping is illustrated for another e x a m p l e i n F I f i $ R {; S . : r i . ) A s a r e s u l t , o n l y females produce Sxl protein. The protein has a concentration of basic amino acids that resembles other RNA-binding proteins. The presence of Sxl protein changes the splicing of lhe transformer(tra) gene. Figure 26.21 shows that this involves splicing a constant 5' site to alternative J' sites. One splicing pattern occurs in both males and females, and results in an RNA that has an early termination codon. The presence of Sxl protein inhibits usage of the normal 3' splice site by binding to the polypyrimidine tract at its branch site. When this site is skipped,the next 3'site is used. This generates a female-specific mRNA that codes for a protein. Thus /ra produces a protein only in females; this protein is a splicing regulator. tra2 has a similar function in females (but is also expressed in the male germline). The TIa and Tra2 proteins are SR splicing factors that act directly upon the target transcripts. TIa and TIa2 cooperate (in females) to affect the splicing of dsx. Figure 26.23 shows examples of casesin which splice sites are used to add or to substitute exons or introns, again with the consequence that different protein products are generated. In the doublesex(dsx) gene, females splice the 5' site of intron 3 to the 3'site of that intron; as a result translation terminates at the end of exon 4. Males splice the 5'site of intron 3 directly to the 3'site of intron 4, thus omitting exon 4 from the mRNA and allowing trans-
lation to continue through exon 6. The result of the alternative splicing is that different proteins are produced in each sex: The male product blocks female sexual differentiation, whereas the female product repressesexpressionof malespecificgenes. Alternative splicing of dsxRNA is controlled by competition between 3' splice sites./sx RNA has an element downstream of the leftmost 3' splice site that is bound by Tra2; Tfa and SRproteins associate with Tra2 at the site, which becomes an enhancer that assistsbinding of U2AF at the adjacentpyrimidine tract. This commits the formation of the spliceosometo use this 3' site in females rather than the alternative 3' site. The proteins recognize the enhancer cooperatively, possibly relying on formation of some secondary structure as well as sequenceper se. Sex determination therefore has a pleasing symmetry: The pathway starts with a femalespecificsplicing event that causesomission of an exon that has a termination codon, and ends with a female-specificsplicing event that causes inclusion of an exon that has a termination codon. The events have different molecurar bases.At the first control point, Sxl inhibits the default splicingpattern. At the last control point. Tta and Tra2 cooperateto promote the femalespecificsplice. The Tra and Tra2 proteins are not needed for normal splicing, because in their absence flies develop normally (as males). As specific regulators, they need not necessarilyparticipate in the mechanics of the splicing reaction; in this respectthey differ from SF2, which is a factor required for general splicing, but can also influence choice of alternative splice sites. P elements of D. melanogaster show a tissuespecificsplicing pattern. In somatic cells there are two splicing events,but in germline an additional splicing event removes another intron. A termination codon lies in the germlinespecificintron; as a result, a longer protein (with different properties) is produced in germline. We discussthe consequencesfor control of transposition in Section2l .I5 , P ElementsAre Activated in the Germline, and note for now that the tissue specificity results from differences in t h e s p l i c i n ga p p ar a t u s . The default splicing pathway of the P element pre-mRNA when the RNA is subjected to a heterologous (human) splicing extract is the germline pattern, in which intron 3 is excised. Extracts of somatic cells of D. melanogaster,however, contain a protein that inhibits excision of
Low
\6
<-
"ehun.o0/tcinn ^ v\
No product
<-
Defaultsplicing + no product
+
Tra protein
I -splicing promotes t
Defaultsplicing no product
n
-
I
V Blocksfemale differentiation (andpromotes maledevelopment)
I
V Male in a pathway invotves in D. melonogoster {:lili:iiil;i,:].::;.:' Sexdetermination at anystageof the Btocks whichdifferentspticingeventsoccurin females. pathway resu[tin ma[edevelopment.
Alternativesplicingeventsthat involve ::ii.''i.iitir ":+ ,i t both sitesmaycauseexonsto be addedor substituted.
Junctions 687 Useof Sptice DifferentiaI Invotves Spticing 26.L2 Alternative
this intron. The protein binds to sequencesin exon 3; if these sequencesare deleted,the intron is excised.The function of the protein is therefore probably to repress associationof the spliceosomewith the 5'site of intron 3.
@
frans-Splicing Reactions UseSmall. RNAs
. Spticing reactions usuatly occurontyin crs junctions between splice on the samemotecule of RNA. o frons-splicing in trypanosomes occurs andworms wherea shortsequence (SLRNA)is spticed to the 5'endsof manyprecursor mRNAs. o SLRNAhasa structure resembting theSm-binding siteof U snRNAs andmayplayan anatogous rote in the reaction. In both mechanistic and evolutionary terms, splicing has been viewed as an intramolecular reaction, essentiallyamounting to a controlled deletion of the intron sequencesat the level of RNA. In genetic terms, splicing occurs only in cis. This means that only sequences on the same moleculeof RNA can besplicedtogether.The upper
part of FISU*E 3s"t4shows the normal situation. The introns can be removed from each RNA molecule, allowing the exons of that RNA molecule to be splicedtogether, but there isno intermolecular splicing of exons between different RNA molecules. We cannot say that trans splicing never occurs between pre-mRNA transcripts of the same gene, but we know that it must be exceedingly rare, becauseif it were prevalent the exons of a gene would be able to complement one another genetically instead of belonging to a single complementation group. Some manipulations can generate transsplicing. In the example illustrated in the lower part of Figure 26.24, complementary sequences were introduced into the introns of two RNAs. Base pairing between the complements should create an H-shaped molecule. This molecule could be spliced in cli, to connect exons that are covalently connectedby an intron, or it could be spliced in trans, to connect exons of the juxtaposed RNA molecules. Both reactions occur ir vitro Another situation in which trans-splicingis possibleinvitro occurs when substrateRNAs are provided in the form of one containing a 5'splice site and the other containing a 3' splice site
fllStJftsfr*.f*i.Spticing usuatly occurs ontyin os between exonscarriedon the samephysical. RNAmolecute. but frons-spticing canoccurwhenspeciaI constructs aremadethat supportbase pairingbetween introns.
688
C H A P T E2R 6 R N AS p t i c i n ga n d P r o c e s s i n g
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r*tfl*tit llrl.iii;':The3' and5' cleavages in S. cerevisiae pre-tRNA subunits oftheendonuarecatatyzed bydifferent ctease. Anothersubunitmaydetermine locationof the fromthe mature cleavage distance sitesby measuring TheAI baseoairis alsoimportant. structure. Fl*LJfrfi.:{i.t.rSpticing of yeasttRNAin yifrocanbefol lowedbyassaying the RNAprecursor andproducts by gel etectrophoresis.
TheSplicing Endonuclease Recognizes tRNA o An endonuctease cteaves theIRNAprecursors at bothendsof theintrono Theyeastendonuclease is a heterotetramer with two (reLated) catatyticsubunits. r It usesa measuring mechanism to determine the sitesof cteavage by their positionsretativeto a oointin theIRNAstructure. r Thearchaeal nuctease hasa simpter structure and recognizes a butge-hetix-butge structuraI motifin the substrate. The endonucleaseis responsiblefor the specificity of intron recognition. It cleaves the precursor at both ends of the intron. The yeast endonucleaseis a heterotetramericprotein. Its activities are illustrated in fl{iLiH[ t*, ;|*. The related subunits Sen34 and Sen2 cleavethe 3' and 5'splice sites,respectively.Subunit Sen54 may determine the sites of cleavageby "measuring" distancefrom a point in the IRNA structure. This point is in the elbow of the (mature) L-shaped structure. The role of subunit SenI 5 is not known, but its gene is essentialin yeast. The basepair that forms between the first base in the anticodon loop and the base preceding the 3' splice site is required for 3' splice site cleavage.
flI ii iJFlr i:l:::. i.'i,i Archaeat t RNA sp[i ci ng endonuclease motif. cleaves eachstrandat a bulgein a bulge-helix-butge An interesting insight into the evolution of IRNA splicing is provided by the endonucleases of archaea. These are homodimers or homotetramers, in which each subunit has an active site (although only two of the sites function in the tetramer) that cleavesone of the splice sites. The subunit has sequences related to the sequencesof the active sites in the Sen34 and Sen2 subunits of the yeast enzyme. The archaeal enzymes recognize their substratesin a different way. though. Instead of measuring distance from particular sequences,they recognize a structural feature called the bulge-helix-bulge. l,I:;li"illtr :,:,.i.iiishows that cleavage occurs in the two bulges. Thus the origin of splicing of IRNA precedes the separation of the archaea and the
Recognizes IRNA Endonuclease 26.15TheSpticing
69t
Each 5'terminus ends in a hydroxyl group; each 3' terminus ends in a 2',3'-cyclic phosphate group. (All other known RNA splicing enzymes cleaveon the other side of the phosphate bond.) The two half-tRNAs base pair to form a tRNA-like structure. When AIP is added. the second reaction occurs. Both of the unusual ends generated by the endonucleasemust be altered. The cyclic phosphate group is opened to . Release of theintrongenerates two half-tRNAs generate a 2'-phosphate terminus. This reacthat pairto formthe maturestructure. tion requires cyclic phosphodiesteraseactivity. . Thehalves havethe unusual ends5' hydroxyl and product has a 2'-phosphate group and a The 2'-3' cycticphosphate. group. 3'-OH . The5'-0Hendjs phosphorytated by a The 5'-OH group generatedby the nuclease potynucteotide group kinase, the cyclicphosphate phosphorylated phosphodiesterase generate must be to give a 5'-phosphate. is opened by to a 2'-phosphate terminus and3'-0Hgroup,exon This generatesa site in which the 3'-OH is next endsarejoinedby an RNAligase, andthe to the 5'-phosphate. Covalent integrity of the 2'-phosphate is removed by a phosphatase. polynucleotide chain is then restored by ligase activity. AII three activities-phosphodiesterase, The overall IRNA splicing reaction is summapollnucleotide kinase, and adenylate synthetase rized in ' ,r,,:ii, .r.-:.-:ir. The products of cleavage (which provides the ligase function)-are are a linear intron and two half-tRNA molearranged in different functional domains on a cules. These intermediates have unicue ends. single protein. They act sequentially to join the two IRNA halves. The splicedmolecule is now uninterrupted, with a 5'-3'phosphate linkage at the site of splicing, but it also has a 2'-phosphate group marking the event. The surplus group must be removed by a phosphatase. Generation of a2',3'-cyclic phosphatealso occurs during the IRNA-splicing reaction in plants and mammals. The reaction in plants seemsto be the same asin yeast,but the detailed chemical reactions are different in mammals. The yeast IRNA precursors also can be spliced in an extract obtained from the germiPhosphodiesterase opens phosphatering nal vesicle (nucleus) of.Xenopusoocytes. This tRNA IRNA shows that the reaction is not species-specific. Base Base chain chain Xenopusmust have enzymes able to recognize the introns in the yeast tRNAs. The ability to splice the products of IRNA genes is therefore well conserved, but is likely to have a different origin from the other splicing reactions (such as that of nuclear premRNA). The IRNA-splicing reaction uses cleavageand synthesisof bonds and is determined by sequencesthat are external to the itl,,i::,; S p t i c i no g f t R N Ar e q u i r esse p a r a tneu c l e a saen dl i g a s ea c t j v i t i e sT. h e intron. Other splicing reactions use transesterexon-intron boundarjes arecteaved by the nuclease to generate 2'to 3'cycticphosphate ification, in which bonds are transferred directly, a n d5 ' 0 H t e r m i n iT. h ec y c l i cp h o s p h a itseo p e n e tdo g e n e r a t3e' - 0 Ha n d2 ' p h o s p h a t e groups. The5'-0Hjs phosphorylated. Afterreleasing the intron,the IRNAhalfmotecules and the sequencesrequired for the reaction lie fotdinto a tRNA-Like structure that nowhasa 3'-0H.5'-Pbreak. Thisis seated by a ligase. within the intron. eukaryotes. If it originated by insertion of the intron into tRNAs, this must have been a very ancient event.
and l@ IRNACleavage LigationAreSeparate Reactions
692
CHAPTER 26 RNASpticing andProcessing
TheUnfolded Protein Response Is Related to IRNASplicing o Irelp is an innernuctear proteinwith membrane its N-terminaI domain in the ER[umen,andits Cterminaldomain in the nucteus. . Binding proteinto the N-terminaI of an unfotded domainactivates the C-termjnal nuclease by autophosp horytation. o Theactivated nuclease cleaves HaclmRNA to retease an intronandgenerate exons that are tigatedby a IRNAligase. . Thespticed HaclmRNA codes for a transcription factorthat activates genes codingfor chaperones proteins. that hetpto fotdunfolded
An unusual splicing system that is related to IRNA splicingmediatesthe responseto unfolded proteins in yeast.The accumulation of unfolded proteins in the lumen of the endoplasmicreticulum (ER) triggers a response pathway that Ieadsto increasedtranscription of genescoding for chaperonesthat assistprotein folding in the ER. A signal must therefore be transmitted from the lumen of the ER to the nucleus. The sensorthat activatesthe pathway is the protein Irelp. It is an integral membrane protein (Ser/Thr) kinase that has domains on each side of the ER membrane. The N-terminal domain in the lumen of the ER detectsthe presence of unfolded proteins, presumably by binding to exposedmotifs. This causesaggregation of monomers and activatesthe C-terminal domain on the other side of the membrane by autophosphorylation. Genes that are activated by this pathway have a common promoter element called the UPRE (unfolded protein responseelement). The transcription factor Haclp binds to the UPRE, and is produced in responseto accumulation ol unfolded proteins. The trigger for production of Haclp is the action of Irelp on Hacl nRNA. The operation of the pathway is summarized in ; li l;'::i: . .: t. Under normal conditions, when the pathway is not activated,Hacl mRNA is translated into a protein rhat is rapidly degraded.The activation of Irelp resultsin the splicing of the Hacl mRNA to change the sequenceof the protein to a more stableform. This form provides the functional transcription factor that activatesgeneswith the UPRE.
Unusual splicing components are involved in this reaction.IrelP has an endonucleaseactivity that acts directly on Hacl mRNA to cleave the two splicing junctions. The two junctions are ligated by the IRNA ligase that acts in the IRNA splicing pathway. The endonucleasereaction resembles the cleavage of IRNA during splicing. Where doesthe modification of Hacl nRNA occur? Irelp is probably located in the inner nuclear membrane, with the N-terminal sensor domain in the ER lumen, and the C-terminal kinase/nuclease domain in the nucleus. This would enable it to act directly on Hacl RNA before it is exported to the cytoplasm. It also would allow easy accessby the IRNA ligase. There is no apparent relationship between the Irelp nuclease activity and the IRNA splicing endonuclease,so it is not obvious how this specializedsystem would have evolved.
proteinresponse occurs by i ir,ritir ,,,:,-,'r Theunfotded a to produce spticingof HAClmRNA activating speciaI the UPRE. factorthat recognizes transcription
Is Related to IRNASpticing 6 9 3 ProteinResponse 26.1.7lhe Unfotded
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as shown in FI*[Jftile*.3*. Other RNA polyThe3' Endsof po[Iand merases do not show discrete termination, but pol.IIITranscri ptsAre continue past the site corresponding to the 3' end, which is generated by cleavageof the RNA Generated by Termination by 3S.33. an endonuclease,as shown in FfSLiRE
Information about the termination reaction for eukaryotic RNA polymerases is less detailed than our knowledge of initiation. RNA polymerases I and III have discrete termination events (like bacterial RNA polymerase), but it is not clear whether RNA polymerase II usually terminates in this way. J'ends of RNAs can be generatedin two ways. For RNA polymerase I, the sole product of Some RNA polymerases terminate transcriptranscription is a large precursor that contains tion at a defined (terminator) sequencein DNA, the sequencesof the major rRNA. The precursor is subjected to extensive processing.Termination occurs at a discrete site >1000 bp Promoter Terminator downstream of the mature f'end, which is generated by cleavage.Termination involves recognition of an 18-baseterminator seouencebv an ancillary factor. With RNA polymerase III, transcription in vitro generatesmolecules with the same 5'and 3' ends as those synthesized in vivo.The termination reaction resemblesintrinsic termination by bacterialRNA polymerase (seeSection I I.2I, There Are Two \pes of Terminators in E. coli). Termination usually occurs at the second U within a run of four U bases,but there is heterogeneity, with some molecules ending in three or even four U bases.The same heterogeneity is seen in molecules synthesized in vivo, so it seems to be a bonafide feature of the termination reaction. Just like the prokaryotic terminators, the i:*tJ*f J*-.iF Whena 3' endis generated by termina- U r u n i s e m b e d d e d i n a G - C - r i c h r e g i o n . tion, RNApotymerase andRNAarereleased at a discrete Although sequencesof dyad symmetry are pres(terminator) sequence in DNA. ent, they are not needed for termination, because mutations that abolish the symmetry do not prevent the normal completion of RNA synthesis.Nor are any sequencesbeyond the U run necessarybecauseall distal sequencescan be replaced without any effect on termination. The U run itself is not sufficient for termination, because regions of four successive U residues exist within transcription units read by RNA polymerase III. (There are no internal U5 runs, though, which fits with the greater efficiency of termination when the terminator is a U5 rather than a Ua seeuence.) The critical feature in termination must therefore be the recognition of a Ua sequence in a context that is rich in G-C basepairs. How does the termination reaction occur? F;*liftf.iri"3t When a 3'endis generated bycteavage, RNA polymerase continues transcription whilean endonucte- It cannot rely on the weakness of the rU-dA asecteaves at a defined sequence in the RNA. RNA-DNA hybrid region that lies at the end of . RNApolymerase I terminates transcription at an 18-base terminator sequence. r RNApotymerase III terminates transcription in poty(U)a sequence embedded in a G-C-rich Seouence.
694
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(the CPE) in the 3'tail. This is another AU-rich sequence,UUUUUAU. In Xenopusembryos at least two type of clsacting sequencesfound in the 3'tail can trigger deadenylation. EDEN (embryonic deadenylation element) is a I7-nucleotide sequence.ARE elements are AU-rich and usually contain tandem repeatsof AUUUA. There is a poly(A)-specific RNAase (PARN) that could be involved in the degradation. Of course, deadenylation is not always triggered by specific elements; in some situations (including the normal degradation of mRNA as it ages),poly(A) is degraded unless it is specifically stabilized.
Cleavage of the 3' End of Histone mRNA May Require a Sma[[ RNA o Histone mRNAs arenot potyadenytated; their3' endsaregenerated by a cleavage reaction that depends on the structure ofthe mRNA. . Thecleavage reaction requires theSLBP to bjndto a stem-toop structure andthe U7snRNA to pair with an adjacent single-stranded region.
Some mRNAs are not polyadenylated.The formation of their 3' ends is therefore different f rom the coordinated cleavage/polyadenylation reaction. The most prominent members of this mRNA classare the mRNAs coding for histones that are synthesized during DNA replication. Formation of their 3' ends depends upon secondary structure. The structure at the 3'terminus is a highly conservedstem-loop structure, with a stem of 6 bp and a loop of four nucleotides.Cleavageoccursfour to five bases downstream of the stem-loop. TWo factors are required for the cleavagereaction: The steml o o p b i n d i n g p r o t e i n ( S L B P ) r e c o g n i z e sr h e structure, and the U7 snRNA pairs with a purine-rich sequence (the histone downstream element, or HDE) located-10 nucleotidesdownstream of the cleavagesite. Mutations that prevent formation of the duplex stem of the stem-loop prevent formation of the end of the RNA. Secondary mutations that restore duplex structure (though not necessarilythe original sequence)behave as revertants. This suggeststhal formationof thesecondary structureis moreimportant than the exactsequence. The SLBPbinds to the stem-loop and then interacts with U7 snRNP to enhance its interaction with the downstream bindine site for U7 snRNA.
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i ir'l':rrir ,, ; I ": Generation of the 3' endof histoneH3mRNA thatbasepairs depends ona conserved hairpinanda sequence w i t hU 7s n R N A . U7 snRNP is a minor snRNP consisting of the 63 nucleotide U7 snRNA and a set of severalproteins (including Sm proteins; see Section26.5, snRNAs Are Required for Splicing). The reaction between histone H3 mRNA , .h e a n d U 7 S n R N A i S d r a w n i n : , , , t t i i r :, , i . .r , , , T upstream hairpin and the HDE that pairs with U7 snRNA are conserved in histone H3 mRNAs of several species.The U7 snRNA has sequences toward its 5' end that pair with the histone m R N A c o n s e n s u ss e q u e n c e s 3 . ' p r o c e s s i n gi s inhibited by mutations in the HDE that reduce ability to pair with U7 snRNA. Compensatory mutations in U7 snRNA that restore complementarity also restore 3'processing. This suggeststhat U7 snRNA functions by base pairing with the histone mRNA. The sequence of the HDE varies among the various histone mRNAs, with the result that binding of snRNA is not by itself necessarilystable, but requires also the interaction with SLBP. Cleavageto generatea J'terminus occurs at a fixed distancefrom the site recognizedby U7 snRNA, which suggeststhat the snRNA is involved in defining the cleavagesite. The factor(s) actually responsiblefor cleavage,however, have not yet been identified.
of rRNA Production Events Cleavage Requires o ThelargeandsmatlrRNAs by cleavage arereteased precursor froma common RNA, The major rRNAs are synthesized as part of a single primary transcript that is processedto
Events Cteavage of rRNARequires 26.2I Production
i : i: i i i.:i: .t'i-::..;' MatureeukaryoticrRNAsare generatedby "i cleavage and trimmingeventsfrom a primarytranscript.
: i,.i;:;ri.::, :::. Thern operons in E. colicontaingenesfor both rRNAand tRNA.Theexact[engths (P)and ofthetranscripts depend on whichpromoters (t) areused.EachRNAproductmustbe released terminators fromthe transcriptby cutson eitherside.
698
CHAPTER 26 RNASpLicing andProcessing
generate the mature products. The precursor contains the sequencesof the 185, 5.8S,and 28S rRNAs. In higher eukaryotes, the precursor is named for its sedimentation rate as 45S RNA. In lower eukaryotesit is smaller (35S in yeast). The mature rRNAs are released from the precursor by a combination of cleavage events and trimming reactions. $ifi#ftEjili!.:T shows the general pathway in yeast. There can be variations in the order of events, but basically similar reactions are involved in all eukaryotes. Most of the 5'ends are generateddirectly by a cleavage event. Most of the 3'ends are generatedby cleavagefollowed by a 3'-5' trimming reaction. Many ribonucleases have been implicated in processing rRNA, including the exosome, which is an assembly of several exonucleases that also participatesin nRNA degradation (see Section 7.13, mRNA Degradation Involves Multiple Activities). Mutations in individual enzymes usually do not prevent processing, which suggeststhat their activities are redundant and that different combinations of cleavages can be used to generate the mature molecules. There are always multiple copies of the transcription unit for the rRNAs. The copies are organized as tandem repeats (see Section 6.9, The Repeated Genes for rRNA Maintain Constant Sequence). 5S RNA is transcribed from separategenes by RNA polymerase III. In general, the 5S genes are clustered, but are separate from the genes for the major rRNAs. (In the caseof yeast, a 5S gene is associated with each major transcription unit, but is transcribed independently.) There is a difference in the organization of the precursor in bacteria. The sequence corresponding to 5.8S rRNA forms the 5'end of the Iarge (23S) rRNA, that is, there is no processing between thesesequences.Fli;ilrt*tti-i3*shows that the precursor also contains the 5S rRNA and one or two tRNAs. In E. coli, the seven rrn operons are dispersedaround the genome; four rrn Loci contain one tRNA gene between the 165 and 23S rRNA sequences,and the other rrnloci contain two IRNA genes in this region. Additional IRNA genesmay or may not be present between the 55 sequenceand the 3' end. Thus the processing reactions required to release the products depend on the content of the particular rrnlocus. In both prokaryotic and eukaryotic rRNA processing, ribosomal proteins (and possibly other proteins) bind to the precursor, so that
669
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3urlre-srr e aJoJeJaqlsr pue 'elnJaloru VNU Ienprnrpur up urqlrM uortezrue8ro ;o aSueqr r P s a A I o A UU r O r l J e e Jq J B g ' S U O J l u V NUI pue 'suorlur 'suortur JpJIJnu 1 dnor8 pue 1 dno;8 rrloLrelna JpnlJur srualsLs aq1 'aleraua8 .{.aqr sJlplpeuJelur Jql pue lJilt Lt7 sluaru reqr -arrnbar laql [q paqsrnSurlsrpsp 'uortf,eeJ Jo sadLl rnoy lseal te are JraqI 'VNU Io aruanbas eJnleru eqt otur suoxa yo Sururof Jqt pup suoJlul Jo leloruer aqt saqsrldruo>e Suorldg
AreuunS
eral proteins; the U4lU6 snRNP contains two snRNAs and several proteins. Some proteins are common to all snRNP particles.The snRNPs recognize consensussequences.Ul snRNA base pairs with the 5' splice site, U2 snRNA base pairs with the branch sequence,and U5 snRNP acts at the 5' splice site. When U4 releasesU6, the U6 snRNAbase pairs with U2; this may create the catalytic center for splicing. An alternative set of snRNPs provides analogous functions for splicing the U12-dependent subclass of introns. The snRNA molecules may have catalytic-like roles in splicing and other processingreactions. In the nucleolus, two groups of snoRNAs are responsible for pairing with rRNAs at sites that are modified; group C/D snoRNAs indicate target sites for methylation, and group ACA snoRNAs identify sites where uridine is converted to pseudouridine. Splicing is usually intramolecular, but transsplicing (intermolecular splicing) occurs in trypanosomes and nematodes. It involves a reaction between a small SL RNA and the premRNA. The SL RNA resembles Ul snRNA and may combine the role of providing the exon and the functions of UI. In worms there are two types of SL RNA: One is used for splicing to the 5'end of an mRNA, and the other is used for splicing to an internal site. Group II introns share with nuclear introns the use of a lariat as intermediate, but are able to perform the reaction as a self-catalyzedproperty of the RNA. These introns follow the GTAG rule, but form a characteristic secondary structure that holds the reacting splice sites in the appropriate apposition. YeastIRNA splicing involves separateendonucleaseand ligasereactions.The endonucleaserecognizes the secondary (or tertiary) structure of the precursor and cleavesboth ends of the intron. The two half-tRNAs releasedbylossof the intron can be ligated in the presence of ATP. The termination capacity of RNA polymerase II has not been characterized, and 3' ends of its transcripts are generated by cleavage.The sequenceAAUAAA, Iocated I I to 30 basesupstream of the cleavage site, provides the signal for both cleavage and polyadenyIation. An endonuclease and the poly(A) polymerase are associatedin a complex with other factors that confer specificity for the AAUAAA signal. Transcription is terminated when an exonuclease,which binds to the 5'end of the nascent RNA chain created by the cleavage, catches up to RNA polymerase.
Kererences Introduct'on Reviews Dreyfuss,G., I(m, V. N., and I(ataoka, N. (2002). Messenger-RNA-binding proteins and the messagesthey carry. Nat. Rev.Mol. CellBiol.3 t95-205. Dreyfuss, G. et al. (1993). hnRNP proteins and the biogenesis of rrRNA. Annu. Rev.Biochem.62, 289-J2t Lewin, B . ll97 5l . Ilnits of transcription and translation: sequence components of hnRNA and mRNA. Cell4,77-93.
AreShort Nuctear SpliceJunctions Sequence:; Reviews Padgefi,R. A. (1986). Splicingof messengerRNA precursors.Annu Rev.Biochem55, Ill9-1150. Sharp, P.A. (1987). Splicingof mRNA precursors. Science 2)5, 766-771.
Proceeds through Pre-mRNA Spticing a Lariat Reviews Sharp, P. A. (t 994). Split genes and RNA splicing. Cell77, 805-8 t 5. Weiner, A. (1993). mRNA splicing and autocatalytic introns: distant cousins or the products of chemical determinism. Cell 72, 16l-164. Research Reed, R. and Mani,rtis,T. (1985). Intron sequences involved in lariat formation during pre-mRNA splicing.Cell4l, 95-105. Reed, R. and Mani,rtis,T. (1986). A role for exon sequencesand splice-site proximity in splicesite selection. 'iell 46, 681-690. Zhtang, Y. A., Goklstein, A. M., andWeiner, A. M. ( 1989) . UACUAAC is the preferred branch site for mammalian mRNA splicing. Proc.Natl. Acad. Sci.USA 56,2752-2756.
for Splicing AreRequired snRNAs Reviews Guthrie, C. (1991) MessengerRNA splicingin yeast: clues to why the spliceosome is a ribonucleoprotein. Science25), 157-163. B. (1988). Spliceosomal Guthrie, C. and Par.terson, snRNAs. Annu. Rev.Genet.22, 387-419. Maniatis, T. and Rt'ed,R. (1987). The role of small nuclear ribonrLcleoprotein particles in premRNA splicinl;.Nature )25, 67)-678. Research Grabowski, P. J., St:iler, S. R., and Sharp, P. A. ( I 985 ). A multicomponent complex is
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U1snRNP Initiates Spticing
KEVlEW Brow, D. A. (2002). Allosteric cascadeof spiiceosome activation. Annu Rev.Genet.36, 33)-360. rch Resea Abovich, N. and Rosbash,M. (I997). Cross-intron bridging interactions in the yeast commitment complex are conserved in mammals Cell 89, 40)-4),2. Berglund, J. A., Chua, I(., Abovich, N., Reed, R., and Rosbash,M. (1997). The splicing factor BBP interacts specifically with the pre-mRNA branchpoint sequence UACUAAC. Cell 89, 781-787. B u r g e s s ,S . , C o u t o , J . R . , a n d G u t h r i e , C . ( 1 9 9 0 ) .A putative ATP binding protein influences the fidelity of branchpoint recognition in yeast splicing.Cell 60, 7 O5-717. Parker,R., Siliciano,P. G., and Guthrie, C. (1987). Recognition of the TACTAAC box during mRNA splicing in yeast involves base pairing to the U2-like snRNA. Cell49, 229-2]9. Singh, R., Valcdrcel,J., and Green, M. R. (1995). Distinct binding specificitiesand functions of higher eukaryotic polypyrimidine tract-binding proteins. Science 268, llT j-\176. Wu, S., Romfo, C. M., Nilsen, T. W., and Green, M. R (1999). Functional recognition of the 3' splice site AG by the splicing factor U2AF35. Nature 402,8)2-835. Zamore, P. D. and Green, M. R. (1989).Identification, purification, and biochemical characterization of U2 small nuclear ribonucleoprotein auxiliary f.actor.Proc.Natl. Acad. Sci.USA 86, 924)-9247. Zhang, D. and Rosbash,M. (1999). Identification of eight proteins that cross-link to pre -mRNA in the yeast commitment complex. GenesDev. r), t8t-592. Zhuang, Y. and Weiner, A. M. (1986). A compensatory base change in UI snRNA suppressesa 5 splicesite mutation. Cell46, 827-B)5.
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TheEComptex CanBeFormed by Intron Definitionor ExonDefinition
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CHAPTER 26 RNASpticing andProcessing
FormtheSoliceosome 5 snRNPs Reviews Kramer, A. (I996). The structure and function of proteins involved in mammalian premRNA splicing.Annu. Rev.Biochem.65, 367409. Madhani, H. D. and Guthrie, C. (1994). Dynamic RNA-RNA interactions in the spliceosome. Annu Rev.Genet 28, l-26. Resea rch Lamond, A. I. (1988). Spliceosomeassembly involves the binding and release of U4 small nuclear ribonucleoprotein. Proc.Natl. Acad Sci.
usA85,4tt4r5. Lesser, C.F.andGuthrie, C. (1993). Mutations in U6 snRNA that alter splice site specificity: implications for the active site. Science 262, l 982-r 988. Madhani, H. D. and Guthrie, C. (19921.A novel base-pairing interaction between U2 and U6 snRNAs suggestsa mechanism for the catalytic activation of the spliceosome. CellTI, 80 3-8 17. Newman, A. and Norman, C. (1991). Mutations in yeast U5 snRNA alter the specificity of 5' splice site cleavage. Cell 65, ll5-123. Sontheimer, E. J. and Sreirz,J. A. (1993). The U5 and U6 small nuclear RNAs as active site components of the spliceosome. Science 262, 1989-r996.
AnAtternative Spticing Apparatus Uses Different snRNPs rch Resea Burge, C. B., Padgett,R. A., and Sharp, P. A. (1998). Evolutionary fates and origins oI Ul2type introns. Mol. Cell2, 77)-7 85 . Dietrich, R. C., Incorvaia, R., and Padgett, R. A. '1997). Terminal intron dinucleotide sequencesdo not distinguish between U2- and Ul2-dependent introns. MoL Cell1, I 5l-I60. Tarn, W-Y. and Steitz, J. (1996). A novel spliceosome containing Ul l, Ul2, and U5 snRNPs excisesa minor classAT-AC intron in vitro. CelI 84,801-811.
Is Connected Spticing to Export of mRNA Reviews Dreyfuss,G., I(m, V. N., and Kataoka, N. (2002). Messenger-RNA-binding proteins and the messagesthey carry. Nat Rev.Mol. CellBioL 3, 195-205. Reed, R. and Hurt, E. (2OO2l. A conservedmRNA export machinery coupled to pre-mRNA splicing. Cell 108, 523-5j1.
Resea r ch I(ataoka, N., Yong, J., I(im, V. N, Velazquez, F., Perkinson, R. A., Wang, F,, and Dreyfuss, G. (2000). Pre-mRNA splicing imprints mRNA in the nucleus with a novel RNA-binding protein that persists in the cytoplasm. Mol Cell 6, 673-682. Le Hir, H., Gatfield, D., Izaurralde, E., and Moore, M. J. (2001). The exon-exon junction complex provides a binding platform for factors involved in mRNA export and nonsensemediated mRNA decav.EMBO J.20. 49874997. Le Hir, H., Izaurralde,E., Maquat, L. E., and Moore, M. J. (2000). The spliceosomedeposits multiple proteins 20-24 nucleotides upstream of mRNA exon-exon iunctions. EMBO J.19. 6860-6869. Luo, M. J. and Reed, R. (1999). Splicing is required for rapid and efficient mRNA export in metazoans. Proc Natl. Acad. Sci.USA 96, t49)7-14942. Luo, M. L., Zhou,2., Magm,I(., Christoforides,C., Rappsilber,J., Mann, M., and Reed, R. (2001). Pre-mRNA splicing and mRNA export linked by direct interactions between UAP56 and Aly. Nature 4l), 644=647 Reichert, V. L., Le Hir, H., Jurica, M. S., and Moore, M. J. (2002). 5'exon interactions within the human spliceosome establish a framework for exon junction complex structure and assembly. GenesDev. 16, 277 8-27 9 1. Rodrigues,J. P.,Rode, M., Gatfield,D., Blencowe, B., Blencowe, M., and Izaurralde,E. (2001). REF proteins mediate the export of spliced and unspliced mRNAs from the nucleus. Proc. Natl. Acad. Sci USA 98, t010-1035. Strasser,I(. and Hurt, E. (2001). Splicing factor Sub2p is required for nuclear mRNA export through its interaction wrthYralp. Nature 4t),648-652. Zhou,2., Luo, M. J., Straesser,I(., I(atahira, J., Hurt, E., and Reed, R. (2000). The protein AIy links pre-messenger-RNA splicing to nuclear export in metazoans. Nature 407, 4Ol-405.
GroupII IntronsAutosptice via Lariat Formation Review Michel, F. and Ferat, J.-L. (1995). Structure and activities of group II introns. Annu Rev. Biochem.64, 4)5-461.
Alternative SpLicing Invotves Differential. U s eo f S o l i c eJ u n c t i o n s Review Green,M. R. ( I 99I ). Biochemical mechanisms of constitutiveand regulatedpre-mRNAsplicing. Annu.Rev. CellBiol.7. 559-599.
Research Handa. N.. Nureki, O., I(urimoto, I(., ICm, I., Sakamoto, H., Shimura, Y., Muto, Y., and Yokoyama, S. (1999). Structural basisfor recognition of the tra mRNA precursor by the Sex-lethalprotein. Nature )98, 579-585, Lynch, I(. W. and X{aniatis, T. (1996). Assembly of specific SR prc,tein complexes on distinct regulatory elements of the Drosophiladoublesex splicingenhancer. GenesDev.10, 2089-2101. Sun, Q., Mayeda, l\., Hampson, R. K., I(rainer, . eneral A . R . ,a n d R o t t m a n ,F . M . ( 1 9 9 3 1 G splicing factor SF2/ASF promotes alternative splicing by binding to an exonic splicing enhancer. GenesDev.7, 2598-2608. Tian, M. and Mani,rtis,T. (19931.A splicing enhancer complex controls alternative splicing of doubles:x pre-mRNA. Cell74, 105-l14. Wu, J. Y. and Maniatis , T. (1993). Specific interactions between proteins implicated in splice site selection and leguiated alternative splicing. Cell75,l06l-t070.
UseSmat[RNAs Reactions trans-Sp[icing Review Nilsen, T. (1993). l?ans-splicingof nematodepre' mRNA. Annu. Rev.Immunol. 47, 413-440. Resea r ch Blumenthal, T., Evans, D., Link, C. D., Guffanti, A., Lawson, D., Thierry-Mieg, J., Thierry-Mieg, D., Chiu, W. L., Duke, I(., ICralY, M., and I(m, S. K. (2002). A global analysisof C elegans operons.Natu"e 417, 85 l-854. Hannon, G. J. et al. (1990). trans-splictngof nematode pre-mRN A in vitro Cell 61, 1247-1255. Huang, X. Y. and Hirsh, D. (1989). Asecondtransspliced RNA le ader sequence in the nematode C elegans.Proc Natl. Acad Sci. USA 86, 8640-8644. Krause, M. and Hilsh, D. (1987). Atrans-spliced leader sequen,:eon actin mRNA tn C elegans. Cell49 , 7 5)-7 '51. Murphy, W. J., Watkins, K. P., and Agabian, N. ( l936). Identilication of a novel Y branch structure as art intermediate in trypanosome mRNA proce sr;ing: evidence for tr ans- splicing. Cell47, 517-525. Sutton, R. and Boc'throyd, J. C. (1986). Evidence f.or trans-splicingin trypanosomes. Cell47, 527-535.
Recognizes Endonuctease TheSpticing tRNA rch Resea Baldi, I. M. et al. | 9921. Participation of the intron in the reaction catalyzed by the Xenopur IRNA splicing endorruclease. Science255, t404-1408.
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TheUnfolded ProteinResponse Is Retated to IRNASpticing
R e s e ahr c Gonzalez, T. N., Sidrauski, C., Dorfler, S , and Walter, P. (1999). Mechanism of non-spliceosomal mRNA splicing in the unfolded protein responsepathway. EMBO J.18, )l19-382. Sidrauski,C., Cox, J. S., and Walter, P. (1996). IRNA ligase is required for regulated mRNA splicing in the unfolded protein response. Cel/ 87, 405-4t). Sidrauski, C. and Walter, P. (1997). The transmembrane kinase Irelp is a site-specificendonuciease that initiates mRNA splicing in the unfolded protein response. Cell 90, I 0 3l - r 0 3 9 .
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The3' Endsof mRNAs AreGenerated by Cteavage and Potyadenylation
Review Wahle,E. and I(eller,W. (1992).The biochemistry of l'-end cleavageand polyadenylationof messenger RNA precursors. Annu.Rev. Biochem61,419-440. R e s erac h Bouvet,P.,Omilli, F.,Arlot-Bonnemains,Y., Legagneux,V.,Roghi,C.,Bassez,T., and Osborne,H. B. (1994).The deadenylation
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AreRequired Smal.L RNAs for rRNA Processi ng
Resea rch
Bousquet-Antonelli,C.,Henry,Y., G'elugne,J. P., M., and Kiss,T. (1997).A Caizergues-Ferrer, smallnucleolarRNPprotein is requiredfor pseudouridylationof eukaryoticribosomal RNAs.EMBOJ. 16,4770-4776. Ganot,P.,Bortolin,M. L., and I{ss, T. (I997). Sitespecificpseudouridineformation in preribosomal RNA is guidedby smallnucleolarRNAs Cell89, 799-81)9. M., and Kiss,T. Ganot,P.,Caizergues-Ferrer, (19971. The family of box ACA smallnucleolar RNAsis definedby an evolutionarilyconservedsecondarystructureand ubiquitous sequenceelerrrentsessentialfor RNA accumuDev.ll,94l-956. Iation. Genes I(ass,S.et al. (1990).TheU3 smallnucleolar ribonucleoproteinfunctionsin the first step of preribosomalRNA processing.Cell60, 897-908, ICss-Laszlo, Z.,Henry,Y.,and Kiss,T. (1998). Sequenceand structuralelementsof methylation guide snoRNAsessentialfor site-specific ribosemethylation of pre-rRNA.EMBOJ. I7, 797-807. ribose I(iss-Laszlo, Z. et al. (19961. Site-specific methylation oI preribosomalRNA: a novel function for srnallnucleolarRNAs.Cel/85, 1077-t068. Ni, J.,Tien,A. L., and Fournier,M. J. (1997). synSmallnucleolarRNAsdirectsite-specific in rRNA. Cell89, thesisof pseuctouridine 565-57i.
Balakin, A. G., Smith, L., and Fournier, M. J. (1996\. The RNA world of the nucleolus: two major families of small RNAs defined by different box elements with related functions. Cell 86, 82)-814.
References 705
CatalyticRNA C H A P T EO RU T L I N E
I
Introduction GroupI Introns Undertake SeLf-SpLicing by Transesterification o Theonlyfactorsrequired for autospticing in vitroby groupI intronsarea monovatent cation,a divalentcation,anda guanine nucteotjde. o SpLicing occurs by twotransesterifications, withoutrequiring inputof energy. r The3'-0Hendof the guanine cofactor attacks the 5'endof the intronin the first transesterificationr The3'-0Hendgenerated at the endof thefirstexonattacks thejunctionbetween theintronandsecond exonin the second transesterifi cation. . Theintronis reteased asa [inearmotecu[e that circutarizes whenits 3'-0Hterminus attacks a bondat oneoftwo internaIoositions. . TheG414-A16 internalbondof theintroncanalsobe attackedby othernucteotides in a trans-splicing reaction. GroupI Introns Forma Characteristic Secondary Structure r Group I intronsforma secondary structure with nineduplex reglons. r Thecoresof regions P3,P4,P6,andP7havecatalytic activity. . Regions P4andP7arebothformedby pairingbetween conserved consensus sequences. e A sequence adjacent to P7basepairswiththe sequence that contains the reactive G. RibozymesHaveVariousCatatyticActivities o Bychanging the substrate binding-site of a groupI intron, it is possible to introduce alternative sequences that jnteract with the reactiveG. o Thereactions followctassical enzvme kinetics with a low catatyticrate. o Reactions using2'-0Hbondscoutdhavebeenthe basjsfor evolving the originaI catalytic activjties in RNA. S o m eG r o u pI I n t r o n sC o d ef o r E n d o n u c l e a sTehsa t SponsorMobil.ity . Mobite intronsareabteto insertthemselves into newsites. o Mobi[e groupI intronscodefor an endonuctease that makes a double-strand breakat a targetsite. o Theintrontransposes intothe siteof the doubte-strand breakby a DNA-mediated repticative mechanism. GroupIi IntronsMayCodefor MuttifunctionProteins o Group II intronscanautosplice in vitro,but areusualty assisted by proteinactivities codedwithinthe intron. 706
@
. A singtecodingframespecifies a proteinwith reverse transcriptase activity,maturase activity,DNA-binding motif,and a DNAendonuclease. . Thereverse generates transcriptase a DNAcopyof the RNA sequence thattransposes by a retroposon-tike mechanism. o Theendonuclease cleaves targetDNAto a[[owinsertion of thetransposon at a newsite. SomeAutospticing Introns RequireMaturases o Autospticing intronsmayrequirematurase activities encoded withintheintronto assistfotdingintothe active catatyticstructure. The CatatyticActivity of RNAaseP Is Dueto RNA r Ribonuctease Pis a ribonucteoprotein in whichthe RNAhas catalyticactivity. ViroidsHaveCatatyticActivity . Viroids andvirusoids forma hammerhead structure that has a setf-cleaving activity. o Simitar structures canbegenerated by pairinga substrate strandthat is cteaved by an enzyme strand. . Whenan enzyme strandis introduced into a cett,it canpair with a substrate strandtargetthat is thencteaved. RNAEditing0ccursat Individua[Bases o Apo[ipoprotein-B andglutamate receptors havesite-specific deaminations catatyzed by cytidineandadenosine deaminases that change the codingsequence. RNAEditingCanBe Directedby GuideRNAs . Extensive RNAeditingin trypanosome mitochondria occurs byinsertions or deletions of uridine. o Thesubstrate RNAbasepairswith a guideRNAon both sidesof the regionto be edited. r TheguideRNAprovides thetemptate for addition(or Less often,deletion) of uridines. r Editingis catalyzed by a complex of endonuclease, terminal uridyttransferase activity,andRNAligase. ProteinSpticingIs Autocatalytic o An intein hasthe abitityto catatyze its ownremoval from a proteinin sucha waythat theflankingexteins are connected. r Protein spticing is catatyzed by theintein. o Mostinteinshavetwo independent proteinsplicactivities: i n ga n da h o m i n eg n d o n u c l e a s e . Summary
169
slua^la6p^pallserrnbau vNUrJouoqlnpoldlz'gz
ol pJsseJoJdsr 1eq] lduJsueJt Lrerurrd a18urs e 1o ged se pezrseqluz(sare syNUr roferu aq1 'vNU roslnJalo uourrrol e uroj] a0eneep fiq pasealar elesVNUI aq1 e llputspuea6te1
qua^l a6enea'13 sarrnbau vNUrJo uor+lnpold 'pJlJrluepr uaaq 'JJAJ la,{.lou e^eq -uoq 'aBeneJIJro; alqrsuodsarLllenpe (s)ro1 -JPJaqJ 'Jlls eSe^eelr aql Sururyapur pJAIOAUI sI yNUus Jqt leqt stseSSnsqrlqM 'vN5us 4n z{.qpazruSorJJ etrs eq1 uolJ Jf,uelsrp pJXrJp tp sJnJJosnururJJl,€ p JleJeue8o1 a8eneal3 agfs qllM uoIllPJJluI Jql oslp sarrnbar tnq 'Jlqpls .dpressarau11asl1 z(q sr yo Surpurq rqt qlr^{ tou leqt tlnsar VNUUs 'syN5ru Juolsrq snorJEAaql Suoure sJrre^ A(H aqt Jo aJurnbas aq1 'VNUr.u Juotsrq eql qllm SurrrBdaseq Lq suorlJunl VNUus /n tpqt stse8 -3ns srql 'Surssarord eJolseJosle dlrreluaur ,€ -aldruor eJotsartpql vNuus Zn ur suortptnu ,{rolesuadruol 'VNUus Zn qtlM rred ot,{tq1qe JJnpJr teql g(H rqt ur suorlptnru ,{q pJtlqlqul sr Surssarord,g 'saruanbas snsuJsuoJVNUtu Juolsrq aql qll,lr rrBd teql puJ ,S slr pJplnrol saruanbasseq vN5us 4n aqJ 'sar;adsIeJJAesJo svNuru €H Juolsrq ur pJ^resuoJ aJevNuus /o qlltzr srted lPql iI(H aqt pue urdrreq ruearlsdn e{I
',,r'rr r rtr.iiriir I UI UMptp
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'vNUus /n qlm slredaspq1eq1aruanbese pueurdtLeq pa^lasuole uo spuadep ',.; r:!{i1-.l,iri vNUtx€H auolsrqJ0 puo /t aql Jo uorlPreue9r.;
wvnn9cncn
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'vNuus /nroJ Jlrs durpurqueJrlsuMop Jql qlrM uorlJeJalur slr aJupque ol dNuus zn qlIM sltp -rJlur uJql pue dool-ruals eq101spurqdg'IS eqJ 'atuanbas paxa aqi uaLfi ruuuldu,fi alu,Ltst alnpnus [npuons atlllo uo4awoltpqt s1se33ns srqJ 'stue] -ralJr se Jleqaq (aruanbas put3rro aqt ^lupsse -JJu lou q8noqt) aJnDnJls xaldnp Jrolsar ler{l suortptnur .drepuora5 'VNU aql Jo pue eq1 Jo uoppruJoJ luanard dool-rua1seql Jo rrrels xaldnp Jql uorleuroJ luarra.rd leql suorlelnw JO 'a1rsa8errealJJqr Jo urpJrls -rrmop sJprloelJnu0I- pJlprol (gOg ro 'luJruelr rueeJtsumopauolslq aqt) aruenbas qlr-aur,rnd e qlrm srred yggus 4n eql pup 'eJnlJnJls aqt sazruSorar (4919) uralord Surpurq dool -tuals aqJ :uorlJeeJ a8erreap eqt roJ palnbar eJp sJolJeJo.141'doo1-urals aql Jo upeJlsuMop sJspqJArJot rnoJ srnJJo a8e,{ea13'sJprtoJllnu rnol Jo dool e pue dq 9 Jo ruals P qlrM 'aJntJnJls dool-rua1spe^resuo) .dp8tq p sr snu -rrurJt,€ Jqt tp eJnDnJls JqJ 'JJnlJnJls ,{repuo -ras uodn spuadap spuJ ,€ JrJr{l Jo uorteruJo{ 'uorlerrldar y1qq Suunp pazrsaqluz(serp lpql sJuolsrq JoJSurpor svNgru aqt Jrp ssep VNUrU slql Jo sraqruJru luauluord tsoru aqJ 'uorlJeJJ uope1.{uapedlodTa8errealJpateurpJoo) Jqt rrroJJ lueJeJJrpaJoJaraql sr spuJ ,€ Jreql Jo uorlPu -roJ eql 'patelduape,(1odtou Jrp sVNUru JruoS 'uorbar pepuerls-e16uts luarelpeup qlrm rredo1y11Xus e /n eql pupalnllnllsdool-ue1s uolpeorabenealr aq1 o ol purqol d€ls aq] sarrnbar 'VNUur eql Joalnllnllseq1uo spuadep e r\qpaleraueb arpspuo leql uoqleara6eneelr 1ouares!fl!ur ouolstll o ,€ rraql:palelAuapeAlod
e arrnbau vNUllPLus ,teWVruUuauolsr.H Jo
pul ,t aql Joe6enea';3 'pazrrqpts .{gerryrads sl tr ssepn paper8ap sr (y)z(1od'(sa8e1rse VNUIU Jo uortep -er8ap Ieurou aql Supnlrur) suortenlrs auos ur islueruelJ rrynads Lq paraSStrl sziBmlBlou sr uorlelz(uapPJp'Jsrnol Jo 'uorleperSap aqt ur pa^Io^u eq plnoJ ]Eql (Nuvd) JseYNu rrJrr -ads-(y)L1od p sr rrrql 'vnnnv Jo sleadarruap -uel ureluoJ dllensn pue qJIJ-nV JJe sluJruele Egy'aruanbas eprtoJpnu-4I p sl (tuarualauoq -e1.{uapeaprruo,{rqrua) Nfqg'uor1e1z(uapeap ,ra33r:1upJ Iret,€ eqt ut punoJ saruanbas SutpB -rr;o ad,{t oml tspJl 1esozhqurasndouayu1 'aJuenDas 'nYnnnnn qrrr-nv reqloup sr slqJ 'llpt ,€ aql q (saf aqt)
lpql os tosrnrard eql 01 purq (suralord.raqlo .{lqrssodpue) suralord leurosoqrr'Surssarord ygg.r rr1o,{.re>lne pue rr1o,{relord qloq uI 'sn)olutJ JelDIl -red aqt Io turluo) Jqt uo puadap snnpord aql aseJIJJo1parmbar suortJealSurssarordaql snqJ 'pue ,€ Jql pue aruanbas SE Jql uJJMlJq tue -sard aq lou,{eru.ro z(erusaua8VNUI Ipuoltrppv 'uor8ar srql ur saua8 VNUI oMl ureluoJ ooluJJ JJqlo rqt pue /sJ)uJnbas y51gr S€Z pup 59I eql ueJMlJq aua8 y551 Juo uIPJuoJ rJol UJJ rnoJ lJruouJ8 aqt punore pasradsrpare suorado UJJuJ^es ertrl'un'a uI 'svNul oMl ro auo pue yNUJ SS Jql sureluoJ oslp rosJnJJrd aqt teqt 'saruanbasasaqtuaaulaq 3ur sMoqsFr'{,'{,iii l;*:ji.i_i:{ -ssarord ou sr rrJql 'sr lpql 'VNdr (gE7) a8rel er{t Jo puJ ,E aql srurol VNUr Sg'E o1 Surpuods -JJJoJaruanbas JqJ 'erJJDequr rosrntard aql Jo uollpzlue8ro aqt ur J)uJreJlrp p sr JrJqI ('r{puapuadapur paqrrJsueJl sr tnq 'lrun uorl -dtnsuBrl rolBru qJee qlr,rvrpelerJossesr aua8 ']sea,{ ese) eql q) 'syNur roleur Jql loJ ss e Jo saua8 aqt ruorl aleredJs JJe tnq'pJJelsnp eJe saua85g aqt 'praua8 uI 'III Jserrrx,{1odygg Lq saua8 aleredJs uoJJ pJqrrtsueJl sl VNU SE ' ( J ) u J n D J Sl u P l S -uo) ureturpw VNUJ JoJ sJUJC paleadag aq1 '6'9 uoIDaS aas) sleadar uapupt se paztue8ro are sardor eqJ 'sVNUr Jqt JoJ lrun uorldrrrs -ueJl eql Jo satdor aldqlnru sLerr,rpJJe JJJqJ 'sJIn)elolu
pue6utrqd5 6urssa:or6 VNU9ZUljdVHl
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VNU} VNHI 59I
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reopens the primary circle by reacting with the G4r4-A16bond. The UUU (which resemblesthe 3' end of the l5-mer releasedby the primary ryclization) becomesthe 5'end of the linearmolecule that is formed. This is an intefinolerularreaction, and thus demonstrates the ability to connect together two different RNA molecules. This seriesof reactionsdemonstratesvividly that the autocatalytic activity reflects a generalized ability of the RNA molecule to form an active center that can bind guanine cofactors, recognize oligonucleotides, and bring together the reacting groups in a conformation that allows bonds to be broken and rejoined. Other group I introns have not been investigated in as much detail astlrreTetrahymenaintron,but their properties are generally similar. The autosplicing reaction is an intrinsic property of RNA invitro, but to what degree are proteins involved invivo? Some indications for the involvement of proteins are provided by mitochondrial systems,where splicing of group I introns requires lhe trans-acting products of other genes. One striking caseis presentedby t}re qttl8 mutant of Neurosporacrassa,which is defective in splicing several mitochondrial group I introns. The product of this gene turns out to be the mitochondrial tyrosyl-IRNA synthetase! This is explained by the fact that the intron can take up a tRNA-like tertiary structure that is stabilizedby the synthetaseand which promotes the catalytic reaction. This relationship between the synthetase and splicingis consistentwith the idea that splicing originated as an RNA-mediated reaction. subsequently assistedby RNA-binding proteins that originally had other functions. The in vitro self-splicing ability may represent the basicbiochemical interaction. The RNA structure creates the active site, but is able to function efficiently in vivo only when assistedby a protein complex.
@
GroupI IntronsForm a Characteristic Secondary Structure
I intronsforma secondary Group structure with nineduplexregions. Thecoresof regions P3,P4,P6,andP7have catatyticactivity. P4andP7arebothformedby pairing Regions between conserved consensus seouences. A sequence adjacent to P7basepairswiththe that contains sequence the reactive G.
Firsttransfer 3'-OH end of G attacks 5' end of intron .
s', 3', pG-OH
Y
3',
pNpNpNpNpNpNpXpXFXpXpXpX
pNpNpNpNpNpN-OH + pGpXpXpXpXpXpX
Secondtransfer 3'-OH of exon A attacks 5' end of exon B
f. tla. tt
Third transfer 3'-OH end of intronattacks bond15 basesfrom5' end
I
V ---oH
G-P €"
+
in whichbonds reactions ilili;liii. i' ,: SeLf-spticing occursby transesterification at eachstageareindiThebondsthat havebeengenerated areexchanged direct:ty. boxes. catedby the shaded
All group I introrrs can be organized into a characteristic second,rrystructure with nine helices ( P l - P 9 ) . i i , ' , i " i ' , . ;s h o w sa m o d e l f o r t h e s e c ondary structure of the Tetr ahymena inlr on. \W o of the base-pairerlregions are generatedby pairing between con:;ervedsequenceelements that are common to g|oup I introns. P4 is constructed from the sequencesP and Q; P7 is formed from sequencesR and ^i.The other base-pairedregions vary in sequenc(l in individual introns. Mutational analysisic entifies an intron "core" containing P3, P4, P6, and P7, which provides the minimal region that can undertake a catalytic reaction. The leogths of group I introns vary widely, and the cr)nsensussequencesare located a considerable distance from the actual splice junctions. Some of the pairing reactions are directly involved in brinl;ing the splice junctions into a conformation that supports the enzymatic Structure Secondary I Introns Forma Characteristic 27.3 C;roup
attacks pArb or
3 ' - O Ho f
6CCUpU2oUG 5'G..UUUpA1
eronr feoH
5'G..UUUpA
5, CUCUCU 3'GGGAGG IGS
t
First. 5, lransrer3,
o
at+6gg
I
P1
Cyclization
P8 J
5 ' U A G U C3 ' 3 ' A U C A G5 '
Reversecvclization
H
I H,O
-.^e
ZrY"-
*
o'\
Linearization products 414 L - 1 5R N A
2 bp form at 3' end of intron
f,tililFqil I intronshavea common fF,4 Group secondary structure thatis formedby ninebase-paired regions. The sequences of regions P4andP7areconserved, andidentify the jndividuat sequence elements P,Q, R,andS. P1 js created by pairingbetween theendofthe left exonand the IGSof the intron;a regionbetween P7andP9pairs withthe 3'endof theintron.
1 . 1 9R N A lrans reaction
GalapAl6CCUpUzog6
uuupAl6ccupU20uG
F:{iiigI i]"3 Theexcised introncanformcircles by usingeitherof two internaIsjtesfor reaction with the 5'end,andcanreopen the circtes by reaction withwateror otigo-nucteotides. reaction.Pl includesthe 3'end of the left exon. The sequence within the intron that pairs with the exon is called the IGS, or internal guide sequence.(Its name reflectsthe fact that originally the region immediately 3' to the IGS sequence shown in the figure was thought to pair with the 3'splice junction, thus bringing the two junctions together. This interaction may occur, but does not seem to be essential.)A very short sequence-sometimes as short as two bases-between P7 and P9 base pairs with the sequencethat immediately precedesthe reactive G (position 4I4 in Tetrahymena)at the 3' end of the intron. The importance of base pairing in creating the necessary core structure in the RNA is emphasizedby the properties of czi-actingmutations that prevent splicing of group I introns.
770
CHAPTER 27 CatatyticRNA
Such mutations have been isolatedfor the mitochondrial introns through mutants that cannot remove an intron in vivo, and they have been isolated for the Tetrahymenaintron by transferring the splicing reaction into a bacterial environment. The construct shown in Fl{ilt*[ t3.* allows the splicing reaction to be followed in E. coli. Tl;reself-splicing intron is placed at a location that interrupts the tenth codon of the B galactosidasecoding sequence.The protein can therefore be successfully translated from an RNA only after the intron has been removed. The synthesis of B galactosidasein this system indicates that splicing can occur in conditions quite distant from those prevailing in Tetrahymenaor even invitro. One interpretation of this result is that self-splicingcan occur in the bacterial cell. Another possibility is that there are bacterial proteins that assist the reaction. Using this assay,we can introduce mutations into the intron to see whether they prevent the reaction. Mutations in the group I consensus sequences that disrupt their base pairing stop splicing. The mutations can be reverted by making compensating changesthat restore base pairing. Mutations in the corresponding consensus sequencesin mitochondrial group I introns have
Introninsertedin codon10
site and CatalyticRNA has a guanosine-binding substrate-binding site
3', Guanosinebindingsite-
5' ,'CUCUCU
Substratebindinosite-
I
Translation I B galactosidase
I
Blue plaquesgenerated by stainingfor,' o B galactosidase
V :
*
uuuAuu
UUUACCU
I
lncludes416un6g2o
FirsttransferG-OF occupiesG-bindingsite;5' exon site occupiessubstrate-binding
q.oH \ s', eilffiJr/
GGGAGG
@
::i;.il: il i; it .r.r; Ptaci ng the Tetrahy mena intronwithinthe coding sequence creates anassay forse[fB-gatactosidase spticingin E. coLi.Synthesis of B galactosidase canbe testedby addinga compound that is turnedblueby the js carriedby a bacteriophage, Thesequence enzyme. so (containing thepresence of blueplaques infectedbacteria)indicates successfuI splicing.
SecondtransferG't1ais in G-bindingsite;5' exonis in s;ite substrate-binding
G
/: GGGAGG
@3' 5"..,cucucu similar effects.A mutation in one consensus sequence may be reverted by a mutation in the complementary consensussequenceto restore pairing; for example, mutations in the R consensuscan be compensatedby mutations in the S consensus. Togetherthese resultssuggestthat the group I splicing reaction depends on the formation of secondarystructure between pairs of consensus sequenceswithin the intron. The principle established by this work is that sequences distantfrom the splicejunctions themselves are requiredto form the activesite that makesself-splicingpossible.
@
HaveVarious Ribozymes Activities Catalytic
Bychanging the substrate binding-site of a group to introduce alternative I intron.it is possible that jnteractwiththe reactive G. sequences reactions foltow kinetics with The ctassicaI enzyme a lowcatalyticrate. Reactions using2'-0Hbondscoutdhavebeenthe basisfor evotvingthe originalcatalyticactivitjes in RNA.
Third transferGala is in G-bindingsite; 5' end of intron site is in substrate-bincling u
of the groupI intronin Tetrahymena lil{:i.jltt i:,r',ir.Excision of theoccupants between reactions rRNAoccurs bysuccessive site.The siteandthesubstrate-binding theguanosine-binding [eftexonis pink,andthe rightexonis purpte. The catalytic activity of group I introns was discovered by virtue of their ability to autosplice, but they are abl,: to undertake other catalytic reactions invitro. All of these reactions are based on transesterific;rtions.We analyze these reactions in terms of their relationship to the splicing reaction itself. The catalytic activity of a group I intron is conferred by its ability to generate particular secondary and lertiary structures that create active sitesthat are equivalent to the active sites o f a c o n v e n t i o n a l ( p r o t e i n a c e o u s )e n z y m e . illustrates the splicing reaction in l:;i;iji:l{:l:-i.,:terms of these sites (this is the same seriesof reactionsshown previously in Figue 27 .2).
Activities 7lt Catatytic HaveVarious 27.4 Ribozymes
Contactsfound beforesubstratebindino Cs pairswith IGS site near 5' end of RNA
G-OH attacks CpC bond Contactstound after substratebindino
C is transferredto 3'-G; Ca is released
F:GUftt27.? Theposjtion oftheIGSin thetertiarystructurechanges whenP1is formed by substrate binding.
The substrate-binding site is formed from the PI helix, in which the J' end of the first intron base pairs with the IGS in an intermolecular reaction. A guanosine-binding site is formed by sequencesin P7. This site may be occupied either by a free guanosine nucleotide or by the G residue in position 4l4.In the first transfer reaction, it is used by free guanosine nucleotide;it is subsequentlyoccupiedby (+t+. The secondtransfer releasesthe joined exons. The third transfer creates the circular intron. Binding to the substrate involves a change of conformation. Before substrate binding, the 5' end of the IGS is close to P2 and P8; after binding, when it forms the Pl helix, it is close to conservedbasesthat lie between P4 and P5. The reaction is visualized by contacts that are detected in the secondary structure in FiSlJftgt?.7. In the tertiary structure, the two sitesalternativelyconractedby Pl are 37 A apart, which implies a substantial movement in the p o s i t i o no f P l . The L- l9 RNA is generated by opening the circular intron (shown as the last stage of the intramolecular rearrangements in Figure 2 7. 3 ) . lt still retains enzymatic abilities, which resemble the activities involved in the original splicing reaction. We may consider ribozyme lunction in terms of the ability to bind an intramolecular sequencecomplementary to the
772
CHAPTER 27 CatalvticRNA
AnotherC, binds; transferreaction is reversed
C5 is released, regeneratingL-19 RNA
F t G t l S t? ? . & T h eL - 1 9l i n e a rR N Ac a nb i n dC i n t n e substrate-binding site;thereactive G-0H3'endjs located in theG-binding site,andcatalyzes transferreactions that convedtwoC5otigo-nucleotides into a Caanda C6otigonucteotide.
IGS in the substrate-binding site while binding either the terminal G414or a free G-nucleotide in the G-binding site. FI6U*f ?7.8 illustrates the mechanism by which the oligonucleotide C5 is extended to generate a C6 chain. The C5 oligonucleotide binds in the substrate-bindingsite,whereas Gala occupies the G-binding site. By transesterification reactions, a C is transferred from C5to the 3'-terminal G, and then back to a new C5molecule. Further transfer reactions lead to the accumulation of longer cytosine oligonucleotides. The reaction is a true catalysis,because the L- l9 RNA remains unchanged and is available to catalyze multiple cycles. The ribozyme is behaving as a nucleotidyl transferase.
Sequence-specific endoribonuclease 3', G-OH
Substrate
19-basevirusoid
24-baseRNA 0.0006
cccccc pre-tRNA RNAaseP completr:pre{RNA RNAaseT1 GpA galactosidase lactose B L-19lntron RNAaseP RNA
5'
3', - . ; 5 ' * Q g C U - O H GGGAGG5' 5'G @3'
Ky (mM)
Enzyme
Turnover (/min)
0.04 0.00003 0.00003 0 05 4.0
0.5
1.7 0.4 29 5,700 12,500
RNAligase
5'@Y*"3' +G
Phosphatase
\r-un
v
5' UCUp3' uuuAuu
+
s', UCU-OH3',
3
il,:+LlitL :i1".:.1 Catatytic reactions involve of the ribozyme betweena groupin the substratetransesterifications bindingsiteanda groupin the G-binding site.
Some further enzymatic reactions are char-::,!.t.The ribozyme can funcacterizedin :rTi:,qiiti tion as a sequence-specificendoribonuclease by utilizing the ability of the IGS to bind complementary sequences.In this example, it binds an external substrate containing the sequence CUCU, instead of binding the analogous sequencethat is usually contained at the end of the left exon. A guanine-containing nucleotide is present in the G-binding site,and attacksthe CUCU sequencein precisely the same way that the exon is usually attacked in the first transfer reaction. This cleavesthe target sequence into a 5'molecule that resemblesthe left exon, and a l' molecule that bears a terminal G residue. By mutating the IGS element, it is possible to change the specificity of the ribozyme so that it recognizessequencescomplementary to the new sequenceat the IGS region. Altering the IGS so that the specificity of the substrate-binding site is changed to enable other RNA targetsto enter can be used to gen-
+rISLiiqil by RNAhavethesame ,ill:.1i]Reac.:ions catatyzed the rate although by proteins, features asthosecatatyzed of substrate is stower.TheKMgivesthe concentration measthisis aninverse vetocity; required for half-maximum Theturnover for substrate. ureof theaffinityof t heenzyme e o l e c u l et rsa n s n u m b egr i v e st h e n u m b eor f s u b s t r a tm site. formedin unittimelly a sing[ecatatytic
erate a ligase activity. An RNA terminating in a l'-OH is bound in the substrate site, and an RNA terminating in a 5'-G residue is bound in the G-binding sit.e.An attack by the hydroxyl on the phosphat,: bond connects the two RNA molecules,with the loss of the G residue. The phosphltase reaction is not directly related to the str'licingtransfer reactions. An oligonucleotide sequence that is complementary to the IGS and terminates in a 3'-phosphate can be lttacked by the G4r4. The phosphate is transferred to the Gaia, and an oligonucleotide ',,vitha free 3'-OH end is then released.The phc,sphatecan then be transferred either to an oli;lonucleotide terminating in 3'-OH (effectively reversing the reaction) or indeed to water (releasinginorganic phosphate and completing, an authentic phosphatase reaction). The reactions catalyzed by RNA can be characterizedin the sirmeway as classicalenzymatic reactions in terrrLsof Michaelis-Menten kinet;f,: i ii analyzesthe reactions catalyzed ics. trli.;Lil'dt: by RNA. The I(y'ralues for RNA-catalyzed reactions are low, anrl therefore imply that the RNA can bind its subs:rate with high specificity.The turnover numbers are low, which reflects a low catalytic rate. Lt effect, the RNA molecules behave in the s,rme general manner as traditionally defined Ior enzymes, although they are relatively slow <:omparedto protein catalysts (where a typical range of turnover numbers is 1 0 3r o 1 0 6 ) . A powerful extension of the activities of ribozymes has been made with the discovery that they can be regulated by ligands (seeSection 13.7, Small RNA Molecules Can Regulate
CatalyticActivities HaveVarious 27.4 Ribozymes
773
::iti;&i i:..1 t A ribozyme is contained withinthe 5'untranstated regionof the mRNA codingfor the gtucosamine-6-phosphate. enzyme that produces WhenG[c-6-P bindsto the ribozyme, it cteaves offthe 5'endofthe mRNA, thereby inactivating it andpreventing furtherproduction ofthe enzyme. Translation).ij'L:ij$iii:,:: summarizesthe regulation of a riboswitch. The small metabolite GlcN6P binds to a ribozyme and activates its ability to cleave the RNA in an intramolecular reaction. The purpose of the system is to regulate production of GlcN6P; the ribozyme is located in the 5' untranslated region of the nRNA that codes for the enzyme involved in producing GlcN6P, and the cleavage prevents translation. How does RNA provide a catalytic center? Its ability seems reasonableif we think of an active center as a surface that exposes a series of active groups in a fixed relationship. In a protein, the active groups are provided by the side chains of the amino acids, which have appreciable variety, including positive and negative ionic groups and hydrophobic groups. In an RNA, the available moieties are more restricted, consistingprimarily of the exposed groups of bases.Short regions are held in a particular
interaction between the RNA catalyst and the RNA substrate will rely on base pairing to create the environment. Divalent cations (typically Mg2+) play an important role in structure, typically being present at the active site where they coordinate the positions of the various groups. They play a direct role in the endonucleolytic activity of virusoid ribozymes (seeSection 27.9, Viroids Have Catalytic Activity). The evolutionary implications of these discoveries are intriguing. The split personality of the genetic apparatus-in which RNA is present in all components. but proteins undertake catalytic reactions-has always been puzzling. It seems unlikely that the very first replicating systemscould have contained both nucleic acid and protein. Suppose,though, that the first systemscontained only a self-replicating nucleic acid with primitive catalytic activities-just those needed to make and break phosphodiesrer bonds. If we suppose that the involvement of.2'-OH bonds in current splicing reactions is derived from these primitive catalytic activities, we may argue that the original nucleic acid was RNA, because DNA lacks the 2'-OH group and therefore could
**.1.'dffitril*,':1"i{ii;:rrur in which bonds can be broken and made in another molecule.It seemsinevitablethat the 714
CHAPTER 27 CataLytic RNA
not undertake such reactions. Proteins could have been added for their ability to stabilizethe RNA structure. The greater versatility of proteins then could have allowed them to take over catalytic reactions, leading eventually to the complex and sophisticatedapparatusof modern gene expression.
SomeGroup I Introns Codefor Endonucleases ThatSponsor MobiLity o Mobite intronsareableto insertthemselves into newsites. . MobilegroupI intronscodefor an endonuctease that makes a doubte-strand breakat a targetsite. o Theintrontransposes intothe siteof thedoubtestrandbreakby a DNA-mediated replicative mechanism.
Certain introns of both the group I and group II classescontain open reading frames that are translatedinto proteins. Expressionof the proteins allows the intron (either in its original DNA form or as a DNA copy of the RNA) to be mobile:It is able to insert itself into a new genomic site. Introns of both groups I and II are extremely widespread, being found in both prokaryotes and eukaryotes. Group I introns migrate by DNA-mediated mechanisms, whereas group II introns migrate by RNA-mediated mechanisms. Intron mobility was first detectedby crosses in which the alleles for the relevant gene differ with regard to their possessionof the intron. Polymorphisms for the presenceor absenceof introns are common in fungal mitochondria. This is consistentwith the view that these introns originated by insertion into the gene. Some light on the processthat could be involved is castby an analysisof recombination in crosses involving the large rRNA gene of the yeast mitochondrion. This gene has a group I intron that contains a coding sequence.The intron is present in some strains of yeast (called ol+)but absent in others (trl-). Genetic crossesbetween ro+and al- are polar:The progeny are usually
The introrris replicated and then inserted
1 1 1 1 i ; il:i i. r; ' . : f A n i n t r o nc o d e fso r a n e n d o n u c t e at hsaet m a k eas oftheintronis duplicated breakin DNA.Thesequence double-strand andtheninserted at the break.
Mutations can occur in either parent to abolish the polarLty.Mutants show normal segregation, with eq ual numbers of o+ and o- progeny. The mutations indicate the nature of the process. Mutations in the ro- strain occur close to the site where the intron would be inserted. Mutations in the ot+strain lie in the reading frame of the intron and prevent production of the protein. Tris suggests the model of ]'iii.riti:,ri'. i"r,in v,zhichthe protein codedby the intron in an o+ slrain recognizesthe site where the intron should be inserted in an co-strain and causesit to be preferentially inherited. What is the action of the protein? The product of the o intron is an endonu cleasethat recognizesthe o- ger'eas a targetfor a double-strand break.The endonucleaserecognizesan 18 bp target sequencethat contains the site where the intron is inserted The target sequenceis cleaved on each strand of DNA two basesto the l' side of the insertion site.Thus the cleavagesitesare 4 bp apart and generate overhanging single strands. This type of rleavage is related to the cleavage characteristic of transposons when they migrate to new sites (see Chapter 21, Transbreak probably iniposons).The dorLble-strand tiates a gene colrversion processin which the sequence of the rrt+gene is copied to replace the sequenceof the o- gene. The reaction involves transposition by a duplicative mechanism, and occurs solely at the level of DNA. Insertion of
That sponsorMobil.ity 27.5 SomeGroupI IntronsCodefor Endonucleases
775
the intron interrupts the sequencerecognized by the endonuclease,thus ensuring stability. Many group I introns code for endonucleasesthat make them mobile. Severaldifferent families of endonucleasesare found; one common feature is the presenceof the amino acid sequenceLAGLIDADG near the active site. Similar introns often carry quite different endonucleases.There are differencesin the details of insertion; for example, the endonucleasecoded by the phage T4 td intron cleaves a target site that is 24 bp upstream of the site at which the intron is itself inserted.The dissociationbetween the intron sequenceand the endonuclease sequence is emphasized by the fact that the s a m e e n d o n u c l e a s es e q u e n c e sa r e f o u n d i n inteins (sequencesthat code for sel{-splicing proteins; see Section 27.12, Protein SplicingIs Autocatalytic).
The variation in the endonucleasesmeans that there is no homology between the sequencesof their target sites. The target sites are among the Iongest and therefore the most specific known for any endonucleases (with a range of 14to 40 bp). The specificity ensures that the intron perpetuates itself only by insertion into a single target site and not elsewhere in the genome. This is called intron homing. Introns carrying sequencesthat code for endonucleasesare found in a variety of bacteria and lower eukaryotes. These results strengthen the view that introns carrying coding sequences originated as independent elements.
GroupII IntronsMay Codefor Multifunction Proteins GroupII intronscanautosptice in vitro,but are usua[[y assisted by proteinactivities codedwithin theintron. A singlecodingframespecifies a proteinwith reverse transcriptaseactivity,maturase activity, DNA-binding motif,anda DNAendonuctease. generates Thereverse transcriptase a DNAcopyof the RNAsequence that transposes by a retroposon[ i k em e c h a n i s m . Theendonuctease cteaves targetDNAto altow insertion of thetransposon at a newsite.
i-1.i.:i.tl.li: ;ir'.ii: Reverse transcriptase codedby an intronallowsa copyof the RNAto beinserted at a targetsitegenerated by a doubte-strand break.
776
CHAPTER 27 CatatyticRNA
The best characterized mobile group II introns code for a singleprotein in a region of the intron beyond its catalytic core. The typical protein contains an N-terminal reverse transcriptase activity, a central domain associatedwith an ancillary activity that assistsfolding of the intron into its active structure (called the maturase; see Section 27 .7, Some Autosplicing Introns Require Maturases), a DNA-binding domain, and a C-terminal endonuclease domain. The endonucleaseinitiates the transposition reaction, and plays the same role in homing as its counterpart in a group I intron. The reverse transcriptase generates a DNA copy of the intron that is inserted at the homing site. The endonuclease also cleaves target sites that resemble, but are not identical to, the homing site at much lower frequency, leading to insertion of the intron at new locations. t:;ii:i.il;:h ir;i. :i:1illustrates the transposition reaction for a typical group II intron. The endonucleasemakes a double-strand break at the target
site.The endonucleaseactivity requires both the domain of the protein and the intron RNA. The protein domain cleavesthe antisense strand of DNA, and the intron RNA actually cleavesthe sense strand. This reaction directly inserts the intron into the DNA target site. The intron RNA provides the template for the synthesisof cDNA. Almost all group II introns have a reversetranscriptaseactivity that is specificfor the intron. The reversetranscriptase generatesa DNA copy of the intron. the result being the insertion of the intron into the target site as a duplex DNA. The mechanism resemblesthe transposition of retroviruses,in which the RNA is an obligatory intermediate (see Section 22.2, TlrreRetrovirus Life Cycle Involves Transposition-LikeEvents). The type of retrotranspositioninvolved in this caseresembles that of a group of retroposons that lack LIRs, and which generate the 3'-OH needed for priming by making a nick in the target (see F i g u r e 2 2 . 2 0 i n S e c t i o n2 2 . I 2 , L I N E S U s e a n Endonucleaseto Generatea Priming End).
to the two cataly.ic amino acids. The maturase activity is locaterl some distance away on the surfaceof the protein. I n t r o n s t h a t c o d e f o r m a t u r a s e sm a y b e unable to splice themselves effectively in the absenceof the prctein activity. The maturase is in effect a splicinl;Iactor that is required specifically for splicing of the sequencethat codesfor it. It functions tc, assistthe folding of the catalytic core to forrn an active site. The coexistenceof endonucleaseand maturase activities in the same protein suggests a route for tht: evolution of the intron. irirli:iir,.;' I suggeststhat the intron originated in an independent autosplicing element. The insertion into th s element of a sequencecoding for an endonuclease gave it mobility. The insertion, howe ver, might well disrupt the ability of the R|trA sequence to fold into the active structure, This would create pressure for assistancefrom proteins that could restore 'fhe incorporation of such a folding ability. sequence into tlLe intron would maintain its independence. Some group II introns that do not code for maturase activit es may use comparable proteins that are coled by sequencesin the host genome. This sul;gestsa possibleroute for the evolution of gent:ral splicing factors. The factor may have originirted as a maturase that specifically assistedthe splicing of a particular intron. The coding sequcncebecameisolatedfrom the intron in the host genome, and then it evolved to function with ir wider range of substratesthat the original intron sequence.The catalytic core of the intron could have evolved into an snRNA.
SomeAutospLicing IntronsRequire Maturases . Autospticing intronsmayrequire maturase activities encoded withinthe intronto assist fotdinginto the activecatalyticstructure.
Although group I and group II introns both have the capacity to autosplice in vitro, under physiological conditions they usually require assistance from proteins. Both types of intron may code for maturase activities that are required to assistthe splicing reaction. The maturase activity is part of the single open reading frame coded by the intron. In the example of introns that code for homing endonucleases,the single protein product has both endonucleaseand maturase activity. Mutational analysis shows that the two activities are independent. Structural analysis shows that the endonuclease and maturase activities are provided by different active sites in the protein, each coded by a separatedomain. The endonuclease site binds to DNA, but the maturase site binds to t h e i n t r o n R N A . , ' . . r , . ' .: : : , ' s h o w s t h e s t r u c ture of one such protein bound to DNA. A chara c t e r i s t i cf e a t u r e o f t h e e n d o n u c l e a s ei s t h e presence of parallel cr helices, which contain the hallmark LAGLIDADG sequences,Ieading
: i ' ; . 1 r t .' 1' . i A h o m i n ign t r o nc o d efso ra ne n d o n u c t e activfamitythatatsohasmaturase aseoftheLAGLIDADTi arepaftofthetwoa helices sequences ity.TheLAGLIDADG amjnoacidscloseto the in tl-ecatalytic that terminate by an activesite'isidentified Thematurase DNAduplex. of the protein. on thesurface etstrwhere arginine residue
Maturases 777 IntronsRequire 27.7SomeAutospticing
\/\t/\t\f\r'
\/\/\t\/\t\/
II
v InIron
gene Endonuclease is insertedintointron
Introncarries endonuclease
component superfluous, though. The RNA alone can catalyzethe reaction! Analyzing the results as though the RNA were an enzyme, each "enzyme" catalyzes the cleavage of multiple substrates.Although the catalytic activity resides in the RNA, the protein component greatly increasesthe speed of the reaction, as seen in the increase in turnover number (see Figure 27.I0). Mutations in either the gene for the RNA or the gene for protein can inactivate RNAase P invivo, so we know that both components are necessaryfor natural enzyme activity. Odginally it had been assumed that the protein provided the catalytic activity, whereas the RNA filled some subsidiary role-for example, assisting in the binding of substrate(it has some short sequencescomplementary to exposed regions of IRNA). Theseroles, however, are reversed!
Maturasegene is insertedinto intron
ViroidsHaveCatalytic Activity lntroncarries endonuclease and maturase
i i ' ; U * [ t i . 1 5 T h ei n t r o no r i g i n a t eads i n d e p e n d e n t sequence codingfor a self-splicing RNA.Theinsertjon of theendonuclease sequence created a homing intronthat wasmobi[e. Theinsertion of the maturase sequence then enhanced theabitityof theintronsequences to foldinto the activestructure for splicing.
@
TheCataLytic Activityof RNAaseP Is Dueto RNA
o Ribonuctease Pis a ribonucteoprote'in in whichthe RNAhascatalyticactivity.
One of the first demonstrationsof the capabilities of RNA was provided by the dissection of ribonuclease P, an E. coli tRNA-processing endonuclease.RibonucleaseP can be dissociated into its two components, the 37 5 baseRNA and the 20 kD polypeptide. Under the conditions initially used to characterize the enzyme activity in vitro, both components were necessary to cleave the IRNA substrate. A change in ionic conditions (an increasein the concentration of Mg2+) renders the protein
778
CHAPTER 27 CatatyticRNA
Viroids andvirusoids forma hammerhead structure that hasa self-cleaving activity. Simitar structures canbegenerated by pairinga substrate strandthat is cteaved by an enzyme strand. Whenan enzyme strandis introduced into a cet[,it canpairwitha substrate strandtargetthat is then cteaved.
Another example of the ability of RNA ro function as an endonucleaseis provided by some small plant RNAs (-350 bases)that undeftake a self-cleavagereaction. As with the caseof the Tetrahymenagroup I intron, however, it is possible to engineer constructs that can function on external substrates. These small plant RNAs fall into two general groups: viroids and virusoids. The viroids are infectious RNA molecules that function independently without encapsidation by any protein coat. The virusoids are similar in organizalion but are encapsidated by plant viruses, being packaged together with a viral genome. The virusoids cannot replicate independently. but require assistancefrom the virus. The virusoids are sometimes called satellite RNAs. Viroids and virusoids both replicate via rolling circles (seeFigure I6.6). The srrand of RNA that is packagedinto the virus is called the plus strand. The complementary strand, gen-
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Stem3! A t/u GA UA
. , ' . : i .:. - ' - . i : A h a m m e r h e rai db o z y mfeo r m sa V shaped tertiarystructure in whichstem2 is stacked upon stem3. Thecatatytic centerliesbetween stems2 and3 andstem1. It contains a maqnesium ionthatinitiates the hydroLytic reaction.
reaction to occur in vivo. A ribozyme designed in this way essentiallyprovides a highly specific restriction-like activity directed against an RNA target. By placing the ribozyme under control of a regulated promoter, it can be used in the same way as (for example) antisenseconstructs specificallyto turn off expression of a target gene under defined circumstances.
fer, though, in which the actual coding sequence in DNA remains unchanged. Changesin the information coded by DNA occur in some exceptional circumstances,most notably in the generation of new sequencescoding for immunoglobulins in mammals and birds. These changes occur specifically in the somatic cells (B lymphocytes) in which immunoglobuIins are synthesized(seeChapter 23, Immune Diversity). New information is generated in the DNA of an individual during the process of reconstructing an immunoglobulin gene, and information coded in the DNA is changed by somaticmutation. The information in DNA continues to be faithfully transcribed into RNA. RNA editing is a processin which information changesat the levelof mRN,4.It is revealed by situations in which the coding sequence in an RNA differs from the sequence of DNA from which it was transcribed. RNA editing occurs in two different situations, each with different causes.In mammalian cells there are casesin which a substitution occurs in an individual basein mRNA, causing a change in the sequence of the protein that is coded. In trypanosome mitochondria, more widespread changesoccur in transcripts of several genes. when basesare systematicallyadded or deleted. t t;;;i3 i:S ;' r-.i ij summarizes the sequences of the apolipoprotein-B gene and mRNA in mam-
Apolipoprotein B genehas 29 exons
RNAEditing Occurs at IndividuaL Bases . Apotipoprotein-B andglutamate receptors have site-specific deamjnations catalyzed by cytidine a n da d e n o s i n d e a m i n a st e h sa tc h a n gteh ec o d i n g sequence.
A prime axiom of molecular biology is that the sequenceof an nRNA can only representwhat is coded in the DNA. The central dogma envisaged a linear relationship in which a continuous sequence of DNA is transcribed into a sequenceof mRNA that is in turn directly translated into protein. The occurrenceof interrupted genesand the removal of introns by RNA splicing introduces an additional stepinto the process of gene expression: The coding sequences (exons) in DNA must be reconnectedin RNA. The process remains one of information trans-
720
C H A P T E2R7 C a t a t y t i R c NA
I
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UAA
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oftry
IntestinemRNA has UAA codon that terminates synthesisat 2153
i:l{i,i!'ii.'r i.li i* The sequenceof the apo-B gene is the samein intestineand Uver,but the sequence ofthe mRNA is modjfiedby a basechangethat createsa terminatjon codonin intestine.
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gene.The sequencesof the gene andprotein given i n i i i , i : * l : . ; . * * a r e c o n s e r v e di n s e v e r a l t r y panosome species.How doesthis gene function? The coxll mRNA has an insert of an additional four nucleotides (all uridines) around the site of frameshift. The insertion restores the proper reading frame; it inserts an extra amino acid and changesthe amino acidson either side. No second gene with this sequence can be discovered, and we are forced to conclude that the extra basesare inserted during or after transcription. A similar discrepancybetween nRNA and genomic sequencesis found in genesof the SV5 and measlesparamyxoviruses, in these cases involving the addition of G residuesin the mRNA. Similar editing of RNA sequencesoccurs for other genes,and includes deletions as well as additions of uridine. The extraordinary case of the coxIII gene of Trypanosomabrucei is summ a r i z e di n a i * : i R l i I . I : . More than half of the residuesin the wRNA consistof uridinesthat are not codedin thegene Comparison between the genomic DNA and the mRNA shows that no stretch longer than seven nucleotides is representedin the mRNA without alteration, and runs of uridine up to seven baseslong are inserted. What provides the information for the specific insertion of uridines? A guide RNA contains a sequence that is complementary to the correctly edited mRNA. l'i*ijFlt i,r.ii-i shows a
model for its action in the cytochrome b gene of Leishmania. The sequenceat the top of the figure shows the original transcript, or preedited RNA. Gaps show where baseswill be inserted in the editing process.Eight uridines must be inserted into this region to create the valid mRNA sequence. The guide RNA is complementary to the mRNA for a significant distance, including and surrounding the edited region. T\zpicallythe complementarity is more extensive on the 3'side of the edited region and is rather shon on the 5'side. Pairing between the guide RNA and the preedited RNA leaves gapswhere unpaired A residues in the guide RNA do not find complements in the preedited RNA. The guide RNA provides a template that allows the missing U residues to be inserted at these positions.When the reaction is completed the guide RNA separates from the mRNA, which becomes available for translation. Specification of the final edited sequence can be quite complex. In this example, a lengthy stretch of the transcript is edited by the insertion of a total of l9 U residues, which appears to require two guide RNAs that act at adjacent sites. The first guide RNA pairs at the 3'-most site, and the edited sequencethen becomesa substratefor further editing by the next guide RNA. The guide RNAs are encoded as independent transcription units. fg*LlFtfl;,-:.i:: shows a map of the relevant region of the Leishmania
ISSLGIKVE AUA UCAAGUUUAGGUAUA AAA GUAGAG A frameshift
A U A U C AA G U U U AG G UA U A A A A G U AG A U U G UA U A C C UG G U A G G U G U A A U R N A S E O U E N C E I S S L G I K V D C I P G R C N Proternseouence
r:'i:r-iii{il,!li: ThemRNAfor the trypanosome coxll genehasa frameshiftretativeto the DNA;the correctreadingframeis createdby the insertionof four uridines.
UAUAUGUUUUGUUGUUUAUUAUGUGAUUAUGGUUUUGUUUUUUAu,J,o,,,,,UAGAUUUAUUUAAUUUGUUGA A
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722
CHAPTER 27 CatatyticRNA
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uteuS
Inteins have characteristicleatures. They are found as in-frame insertions into coding s e q u e n c e s .T h e y c a n b e r e c o g n i z e d a s s u c h becauseof the existenceof homologous genes that lack the insertion. They have an N-terminal serine or cysteine (to provide the -XH side chain) and a C-terminal asparagine.A typical intein has a sequence of -I50 amino acids at the N-terminal end and -50 amino acidsat the C-terminal end that are involved in catalyzing the protein splicing reaction. The sequence in the center of the intein can have other functions. An extraordinary feature of many inteins is that they have homing endonuclease activity. A homing endonuclease cleaves a target DNA to create a site into which the DNA sequencecoding for the intein can be inserted ( s e eF i g u r e2 7 . l 2 i n S e c t i o n2 7 . 5 , S o m eG r o u p I Introns Code for EndonucleasesThat Sponsor Mobility). The protein splicing and homing endonuclease activities of an intein are independent. We do not really understand the connection between the presence of both these activities in an intein, but two types of model have been suggested.One is to supposethat there was originally some sort oI connection between the activities,but that they have sincebecome independent and some inteins have lost the homing endonuclease.The other is to supposethat inteins may have originated as protein splicing units, most of which (for unknown reasons) were subsequentlyinvaded by homing endonucleases.This is consistentwith the fact that homing endonucleasesappear to have invaded other types of units as well, including, most notably, group I introns.
l@ Summary S e l f - s p l i c i n gi s a p r o p e r t y o f t w o g r o u p s o f introns, which are widely dispersedin lower eukaryotes,prokaryotic systems,and mitochondria. The information necessaryfor the reaction residesin the intron sequence(although the reaction is actually assistedby proteins in vivo). For both group I and group II introns, the reaction requires formation of a specificsecondary/ tertiary structure involving shott consensus sequences.Group I intron RNA createsa structure in which the substrate sequence is held by the IGS region of the intron, and other conservedsequencesgeneratea guanine nucleotide binding site. It occurs by a transesterification involving a guanosine residue as cofactor.No input of energy is required. The guanosine
X=SorO
o
tl
c-NH2
j.-
trx
H CHz
tl
\
I
/' Exteinl /
oftransesterifications i:i{rilli:ii:lr.rl Bonlsarerearranged througha series orthe-SHgroupof cysteine orthreonine invotving the-0H g'oupsof serine bondandtheinteinis reteased by a peptide ar€connected untjLtheexteins (-terminus. witha circularized
breaksthe bond at the 5'exon-intron junction and becomes linked to the intron; the hydroxyl at the free end of the exon then attacks the 3'exon-intron jtrnction. The intron cyclizesand losesthe guanot;ineand the terminal l5 bases' A seriesof relaled reactions can be catalyzed via attacks by the terminal G-OH residue of the intron on intental phosphodiester bonds. By providing appropriate substrates,it has been possible to engitreer ribozymes that perform a variety of catalytic reactions, including nucleotidyl transferase activities. Some group I and group II mitochondrial introns have opt:n reading frames. The proteins coded by group J introns ate endonucleasesthat make double-strandedcleavagesin target sites in DNA; the cleavageinitiates a gene converslon process in whi<:h the sequence of the intron itself is copied ilrto the target site.The proteins
27.13Summarv 725
coded by group II introns include an endonucleaseactivity that initiates the transposition process,and a reversetranscriptasethat enables an RNA copy of the intron to be copied into the target site. Thesetypes of introns probably originated by insertion events. The proteins coded by both groups of introns may include maturase activities that assistsplicing of the intron by stabilizing the formation of the secondary/tertiary structure of the active site. Catalytic reactions are undertaken by fhe RNA component of the RNAase P ribonucleoprotein. Virusoid RNAs can undertake s e l f - c l e a v a g ea t a " h a m m e r h e a d " s t r u c t u r e . Hammerhead structures can form between a substrate RNA and a ribozyme RNA, which allows cleavageto be directed at highly specific sequences.These reactions support the view that RNA can form specificactive sitesthat have catalytic activity. RNA editing changes the sequence of an RNA after or during its transcription. The changes are required to create a meaningful coding sequence. Substitutions of individual basesoccur in mammalian systems;they take the form of deaminations in which C is converted to U or A is converted to L A catalytic subunit related to cytidine or adenosine deaminase functions as part of a larger complex that has specificity for a particular target sequence. Additions and deletions (most often of uridine) occur in trypanosome mitochondria and in paramyxoviruses. Extensive editing reactions occur in trypanosomes in which as many as half of the basesin an mRNA are derived from editing. The editing reaction uses a template consisting of a guide RNA that is complementary to the nRNA sequence.The reaction is catalyzed by an enzyme complex that includes an endonuclease,terminal uridyltransferase,and RNA ligase, using free nucleotides as the source for additions, or releasingcleavednucleotidesfolIowing deletion. Protein splicing is an autocatalytic reaction that occursby bond transfer reactions and input of energy is not required. The intein catalyzes its own splicing out of the flanking exteins. Many inteins have a homing endonuclease activity that is independent of the protein splicing activity.
726
CHAPTER 27 Catal.yticRNA
References GroupI IntronsUndertake Setf-Spticing by Transesterification Reviews Cech, T. R. (1985). Self-splicingRNA: implicarions for evolution. Int. Rev.Cytol 91, 3-22. Cech, T. R. ( 1987) . The chemistry of self-splicing RNA and RNA enzymes. Science236, t532-t5j9. Resea rch Been, M. D. and Cech, T. R. (1986). One binding site determines sequence specificity of Tetr ahymenapre -rRNA self- splicing, /ranssplicing, and RNA enzyme activity. Cell 47, 207-216. Belfort, M., Pedersen-Lane,J., West, D., Ehrenman, K., Maley, G., Chu, F., and Maley, F. (1985). Processingof the intron-containing thymidylate synthase (td) gene of phage T4 is at the RNA level. Cell 41, )75-J82. Cech, T. R. et al. ( I 981 ). In vitro splicing of the rRNA precursor of Tetrahymena'.involvement of a guanosine nucleotide in the excision of the intervening sequence. Cell 27, 487-496. I(ruger, I(., Grabowski, P. J., Zatg, A. J., Sands, J., Gottschling,D. E., and Cech, T. R. (1982). Self-splicing RNA: autoexcision and autocyclization of the ribosomal RNA intervening sequenceof TetrahymenaCell 11, 147-1i7. Myers, C. A., I(uhla, B., Cusack,S., and Lambowitz, A. M. (2002). tRNA-like recognirion of group I introns by a tyrosyl-tRNA synthetase. Proc Natl. Acad. Sci.\JSA 99. 2630-26)5.
GroupI IntronsForma Characteristic SecondaryStructure Research Burke, J. M. et al. (1986). Role of conserved sequence elements 9L and 2 in self-splicing of the Tetrahymenaribosomal RNA precursor. Cell 45,167-176. Michel, F. and Wetshof, E. ( I 990). Modeting of the three-dimensional architecture of group I catalytic introns based on comparative sequence analysis. J. Mol Biol. 216, iB5-610.
Ribozymes HaveVarious Catatytic Activities Review Cech, T. R. (1990). Self-splicingof group I introns. Annu Rev.Biochem 59,54)-568.
Research Winkler, W. C., Nahvi, A., Roth, A., Collins,J. A. and Breaker,R. R. (2004). Controlof gene expressionby a natural metabolite-responsive ribozyme. Nature 428, 281-286.
@
SomeGroupI IntronsCodefor Endonucleases ThatSponsorMobil.ity
Review Belfort,M. and Roberts,R. J. (1997). Homing endonucleases: keepingthe housein order. Nucleic AcidsRes.25. J)79-)388.
GroupII IntronsMayCode for Multifunction Proteins Reviews Lambowitz, A. M. and Belfort, M.(1993). Introns as mobile genetic elements. Annu- Rev. Biochem.62, 587-622. Lambowitz, A. M. and Zimmerly, S. (2004). Mobile group II introns. Annu. Rev.Genet 38, l-35. r ch Resea Dickson, L., Huang, H. R., Liu, L., Matsuura, M., Lambowitz, A. M., and Perlman, P. S. (2001). Retrotransposition of a yeast group II intron occurs by reverse splicing directly into ectopic DNA sites. Proc Natl. Acad. Sci USA 98, r)207-rj2r2. Zimmerly, S. et al. (1995). Group II intron mobility occurs by target DNA-primed reverse transcription. Cell 82, 545-554. Zimmerly, S. et al. (1995). A group II intron is a catalytic component of a DNA endonuclease involved in intron mobility. Cell 8j, 529-5)8.
IntronsRequire SomeAutosplicing Maturases Resea rch Bolduc et al. (2003). Structural and biochemical analyses of DNA and RNA binding by a bifunctional homing endonuclease and group I splicing factor. Genes.Dev. 17, 2875-2888. Carignani, G. et al. (1983). An RNA maturase is encoded by the first intron of the mitochondrial gene for the subunit I of cytochrome oxidase in S. cerevisiae.Cell ]5, 7j3-7 42. Henke, R. M., Butow, R. A., and Perlman, P. S. ( 1995). Maturase and endonucleasefunctions depend on separate conserved domains of the bifunctional protein encoded by the group I intron aI4 alpha of yeast mitochondrial DNA. EMBO J. 14, 5094-5099.
Matsuura, M., No;rh, J. W., and Lambowitz, A. M. ( 2 0 0l ) . M e c l r a n i s mo [ m a l u r a s e - p r o m o t e d group II intron splicing. EMBO J 20, 7259-7270. Viroids Have Catatytic ActivitY Reviews Doherty, E. A. ani Doudna, J. A. (2000). Ribozyme strlrctures and mechanisms. Annu Rev.Biochem.69,597-615. Symons, R. H. ( I 992 ). Small catalytic RNAs. Annu Rev.Biochem.61,641-671. Research Forster,A. C. and Symons, R. H. (1987). Selfcleavage of virusoid RNA is performed by the proposed 55-nucleotide active site. Cell 50, 9-t6. Guerrier-Takada, tl., Gardiner, K., Marsh, T., Pace, N., andAltman, S. (1983). The RNAmoiety of ribonuclear;eP is the catalytic subunit of the enzyme. Cell )5, 849-857. Scott,W. G., FinclL,J. T., and ICug, A. (I995). The crystal structrtre of an all-RNA hammerhead ribozyme: a proposed mechanism for RNA catalytic cleavage. Cell 81, 991-1002.
Bases at IndividuaI RNAEditingOccurs r ch Resea Higuchi, M. et al. (19931. RNA editing of AMPA receptor subunit GluR-B: a base-paired intron-exon structure determines position and efficiency. CeU 75, l)61-l)70. Navaratnam, N e:.al. ( I 995 ). Evolutionary origins of apoB mRNA editing: catalysisby a cytidine deaminase that has acquired a novel RNAbinding motit at its active site. Cell 81, r87-195. Powell. L. M.. Wattis, S. C., Pease,R. J., Edwards, 'f. J., and Scott,J. (1987). A Y. H., Knott, novel form ol tissue-specificRNA processing produces apolipoprotein-B48 in intestine. Cell 50,83I-840. S o m m e r ,B . e t a l . ( 1 9 9 1 ) . R N A e d i t i n g i n b r a i n controls a delerminant of ion flow in glutamate-gated c.eannels. Cell 67 , ll-19.
RNAEditingCanBeDirected RNAs bv Guide R e s e a hr c Aphasizhev R., Slticego,S., Peris,M., Jang, S. H., Aphasizheva, I., Simpson, A. M., Rivlin, A', and Simpson , L. l2OO2).Ttlpanosome mitochondrial 3' lerminal uridylyl transferase (TUTase):thekey enzyme in U-insertion/ deletion RN,a.editine. Cell 108, 637-648.
References 727
Benne, R., Van den Burg J., Brakenhoff, J. P., Sloof, P.,Van Boom, J. H., and Tfomp, M. C. (I986). Major transcript of the frameshifted coxll gene from trypanosome mitochondria contains four nucleotides that are not encoded in the DNA. CeU46,819-826. Blum, B., Bakalara,N., and Simpson, L. (1990). A model for RNA editing in kinetoplastid mitochondria: "guide" RNA molecules transcribed from maxicircle DNA provide the edited information. Cell 60, 189-198. Feagin, J. E., Abraham, J. M., and Stuart, K. (1988). Extensive editing of the cytochrome c oxidase III transcript in Trypanosomabrucei. Cell 51,4I)422. Seiwert, S. D., Heidmann, S. and Sruarr, K. (1996). Direct visualization of uridylate deletion in vilro suggestsa mechanism for kinetoplastid editing. Cell 84,831-841.
728
CHAPTER 27 CatatyticRNA
ProteinSpticingIs Autocatalytic Review Paulus, H. (2000).Proteinsplicingandrelated forms of protein autoprocessing.Annu Rev. Biochem. 69,447496. Research Derbyshire,V.,Wood,D. W., Wu, W., Dansereau, J. T.,Dalgaard, J.2., andBelfort,M. (1997). Geneticdefinition of a protein-splicing domain:functionalmini-inteins support structurepredictionsand a model for intein evolution. Proc.Natl.Acad.Sci.USA 94. tt466-tt47t. Perler,F. B. et al. (L992). Interveningsequences in an ArchaeaDNA polymerasegene. Proc.Natl. Acad Sct.USA 89, 5577-5581. Xu, M. Q., Southworth,M. W., Mersha,F.B., Hornstra,L. J., and Perler,F.B. (19931. in vitro prorcin splicingof purified precursor and the identificationof a branchedintermediate. C e l l7 5 , l i T l - 1 3 7 7 .
6ZL aODd lrau uopanuquoJ 'solnqnlorrrru 0l uorllauuolaql Aeusaxaqduor apr^old oMlasaqllrouuol1eq1suralold eq1 r 'uorllunJluoruorluor r0J s! III-lCl ol spurq1eq1xaldLuor urelordgg93aq1 r lprtuassa 'II-l0l sralnllnrlsurlPurolql ]P pau.loJ lPnsn y o upsr1eq1xaldLuor uralordpazrlerrads aql ol anrlpurallp xalduol uralordp spur.€alauorluel eqf '11-363 uorbar Llrp-I-Vaql )uplJtpq] III-I0l pueI-l0l pa^rasuol soluonbos iloqsoqllo lslsuolsluaualaNll o 'srsolrru [1e1elnrre ale6erEas o1pruselde rvrolle 1e eq1f,qaostaatals u! pagquepra.lpsluouala[!]l r o1fi1L1Lqe a^eHsalauollual anLst^dil's ur saluanbas iloqs vNo 'uMoul oql aLlf o lou sryp6 enrlLladar Jouor]lunJ 'yp6 enqLladar slunoueabrel lo rrlofile4na raq6rquLse]euor]uolo uleluolseuosouro.lql
salau.lorlual vN0a^qqaoauureluolAPhl
'salu0n00s vN0alrtleles ur qlu sr.leql urleur0rql0ralaq a^PquolJos0.r0ruo.llu0l o 'uorDal lueuorlualslr ur suuoJ aql ol salnqnlo.llrur lPql eroqrolauq JoluauqrPlleoql y o r\qelpuLds lrlolru aql uo plaqsrauosorxo.lql rrlofire1na alnao uoqe6ar6ase sI aurosoruolql lqo^rPln3 aql e,rLb o1puedxe sauos ,,'sgnd,. -ourolqleuefioduo uoLssardxa auab1osalrsale]p{} spueflr uorssaldxl
eual^lod auagJo salrsle puedxl sauosourolLll 'deu pasn e se oq upl 1eq1 1err6o1o1Ar ouolnlod. spupq souese eneqsuereldLpsauosoruo.lql lo Jo spuPgrulol saurosoruorql aual,{lod 'srxp popualxa aql ulorJ ale leql s00ol leu0s0ul0rql qsnlqduel MOqS seuosouroiqr uo uoLsserdxa auabJosolr$r papuaul arv sauosouorql LlsnlqouPl 'spueqlolut polellualuol qlP-l-9 aql ut alP sOUO! r 'spuPqlalut oql r aql ueql}uolu0ll-! ut iaMolelPspuPq 'spueq-9 pallpraleqlrqM'suorlpuls aql Josauasp Jooluereadde aq] asnPlsanbruqlal 6ururels ur.P|.lol a^pqol sauosoruo.lql I
sulailed6uLpueg a^eHsauosorxorql
'asPq0lelut ln0 -qbnorqlpalredAlasuap suor6eX . utPualugeuolqlolaloq;o 's0ruosou0rql llloltul sselst qrtqM'utlsuolqlnaJo ulloJ upqlpelredA11q6q 6uun6 r aql 'asPqdlalut aql ur sr.urleuorlll sseu lelauab Jo 'srsolrur seuosouto.l!r r oq uPl r{1uo uaes 6uunp lPnpLnLpul utlPtuoJLllolalaH pue utlPtx0lqlnl olut papl^]osI ullPtu0lql '0luenDos sns -uesuolllJDadsfiueaneqlou op]nq ql!-.l-Voresl!fi otlf r 'suvsro suvt/\l pollPl eql 01poqrPnes! vNo r llJt]edslP xllleu .lPallnu sesuanbes xulew aseqdralulue o1 vNo qlPnv saluanbasllJllads 'peqrellPaie vN0 paltoriadns 1osdooleq1 ose{oP}e}rl o qrtq/v\ 01ploljelsutaloldPaAeqsauosotllo.ltll 'qI luepuadoput 98- JosutPulop asPqdra]ut [1art1e6au st utlPtuo.lq] olurpalrolradns J0vN0 . plo#Prs e o] lqo,{rP)nl paqlP]lv suteuo0puPsdool sPH vN0 'dq gg1/rorredns aq1 r I- sr.butttor.redns ;o flrsuapa6erane 'sureu0p polrolladns [1anL1e6au ool- sPqptoellnu0q1 . ]uapuedaput paltolredns{ auiouo!leuape8 aqt 'polJquapt e^eq uaoq lou aql r alelPql suralotd loJalqtsuodsal VN6eql 6uLsuepuor 'uraloldlo VNUuollP leql sluabe^q paploJun aLll o oq uel puessPunq vNo%08-st ptoallnulPua]leQ ptoallnNe sI auoua9lPuellP8 otll 'llaqsut010lo polquasseald P olutvNooql uesulsasnlt^ vNolsluoqds. '1rpunole aql alquassP llaqspeeq sasur^VNUsno]ueulelll r AeqlseeuouabVNUaqt asuapuol 'pasuapuol aql uttlltMplle ltelrnI o filaualyastlleqspPaq 'lloqspPaq eq]Joalnllnlls oql fq paltultl eql r eq uPllPql vNo;o q16ua1 srsnlh P olut polPlod.lolu! sleol ltaql olut pabellPdarv sauoua! lPlL^ u0qlnpollul
3 N r t r n 0u 3 l - d v H 3
rflfir
wm
@
Telomeres HaveSimpteRepeating Sequences o Thete[omere is required for the stabitity of the chromosome end. r A tetomere consists of a simpterepeat wherea C+ A-richstrandhasthe seouence cr(A/r)F4. Te[omeres Sea[the Chromosome Ends . TheproteinTRF2 catalyzes a reactjon in whichthe 3' repeating unit of the G+ Trichstrandformsa loopby disptacing its homotog in an upstream regionof the telomere.
Introduction
A general principle is evident in the organization of all cellular genetic material. It exists as a compact massthat is confined to a limited volume, and its various activities, such as replication and transcription, must be accomplished within this space.The organization of this material must accommodate transitions between inactive and active states. The condensed state of nucleic acid results from its binding to basic proteins. The positive chargesof these proteins neutralize the negative charges of the nucleic acid. The structure of the nucleoprotein complex is determined by the interactions of the proteins with the DNA (or RNA). A common problem is presentedby the packaging of DNA into phages, viruses, bacrerial cells, and eukaryotic nuclei. The length of the DNA as an extended molecule would vastly exceedthe dimensions of the compartment that contains it. the DNA (or in the case of some viruses,the RNA) must be compressedexceedingly tightly to fit into the space avallable. Thus
Telomeres Are Synthesized by a Ribonucteoprotein Enzyme o Tetomerase usesthe 3'-0Hof the G+ T telomeric strandto primesynthesis of tanreoeats. demTTGGGG . TheRNAcomponent of telomerase hasa sequence that pairswiththe C+ A-rich repeats. r Oneof the proteinsubunits is a reverse transcriptase that usesthe RNAastemptate to synthesize the G+ T-richsequence. Te[omeres Are EssentiaI for SurvivaI Summarv in contrastwith the customarypicture of DNA as an extendeddoubk helix, structural deformationof DNA to bendor fold it into a mlre clmpactform is the rule rather than exceotion. The magnitude of the discrepancybetween the length of the nucleic acid and the size of its compartment is evident ftom the examples summarized in FIGUHt eS.1.For bacteriophagesand for eukaryotic viruses, the nucleic acid genome, whether single-stranded or double-stranded DNA or RNA, effectively fills the container (which can be rodlike or spherical). For bacteria or for eukaryotic cell compartments, the discrepancy is hard to calculate exactly,becausethe DNA is contained in a compact area that occupies only part of the compartment. The genetic material is seen in the form of the nucleoid in bacteria and as the mass of chromatin in eukaryotic nuclei at interphase (between divisions). The density of DNA in these compartments is high. In a bacterium it is -10 mg/ml, in a eukaryotic nucleus it is -100 mg/ml, and in the phage T4 head it is >5O0mg/ml. Such a concentration in solution would be equivalent to a gel
Compartment Shape
Dimensions
Type of NucleicAcid
Length
TMV
filament
0.008x 0.3pm
One single-stranded RNA
2pm=6.4kb
Phagefd
filament
0.006x 0.85pLm One single-stranded DNA
2pm=6.0kb
Adenovirus
icosahedron 0.07pmdiameterOne double-stranded DNA 11pm = 35.0 kb
PhageT4
icosahedron 0 . 0 6 5 x 0 . 1 0p m
E. coli
cylinder
One double-stranded DNA 55 pm = 170.0kb
1 . 7x 0 . 6 5p m
One double-stranded DNA 1 . 3 m m = 4 . 2 x 1 0 3 k b
Mitochondrionoblate (human) spheroid
3.0x 0.5pm
-10 identical double-stranded DNAs
Nucleus (human)
6 pm diameter
46 chromosomesof double-stranded DNA
spheroid
50 pm = 16.916 1 . 8m = 6 x 1 O ok b
f t f i ' - l H H f ; ST . th e L e n g t h o f n u c l e i c a c i d i s m u c h g r e a t e r t h a n t h e d j m e n s i o n s o f t h e s u r r o u n d i n g c o m Dartment.
730
CHAPTER 28 Chromosomes
of great viscosity. We do not entirely understand the physiological implications, such as the effect this has upon the ability of proteins to find their binding sites on DNA. The packaging of chromatin is flexible; it changes during the eukaryotic cell cycle. At the time of division (mitosis or meiosis), the genetic material becomes even more tightly packaged, and individual chromosomes become recognizable. The overall compression of the DNA can be described by the packing ratio, which is the Iength of the DNA divided by the length of the unit that contains it. For example, the smallest human chromosome contains -4.6 x I07 bp of DNA (-10 times the genome size of the bacterium E. coli).This is equivalent to 14,000 pm (= 1.4 cm) of extended DNA. At the most condensed moment of mitosis, the chromosome is -2 pm long. Thus the packing ratio of DNA in the chromosome can be as great as 7000. Packing ratios cannot be established with such certainty for the more amorphous overall structures of the bacterial nucleoid or eukaryotic chromatin. The usual reckoning, however, is that mitotic chromosomes are likely to be five to ten times more tightly packaged than interphase chromatin, which indicates a tlpical packing ratio of 1000 to 2000. A major unanswered question concernsthe specificityof packaging. Is the DNA folded into a particular pattern, or is it different in each individual copy of the genome? How does the pattern of packaging change when a segment of DNA is renlicated or transcribed?
@
ViralGenomes Are Packaged into TheirCoats
o Thelengthof DNAthat canbeincorporated into a virusis [imitedbythe structure of the headshell. r Nucteic is extremety acidwithinthe headshetl condensed. r Fitamentous RNAviruses condense the RNA genome it. astheyassembte the headshetl around . SphericaI DNAviruses insertthe DNAinto a preassembted proteinshelt.
From the perspective of packaging the individual sequence,there is an important difference between a cellular genome and a virus. The cellular genome is essentiallyindefinite in size;the number and location of individual sequences can be changed by duplication, deletion, and
rearrangement. Thus it requires a generalized method for packagingits DNA, one that is insensitive to the total content or distribution of sequences.By cc,ntrast,two restrictions define the needs oI a virus. The amount of nucleic acid to be packaged is predeterminedbytl:,esize of the genome, and it nrust all fit within a coat assembled from a protein or proteins coded by the viral genes. A virus particle is deceptively simple in its superficialappealance.The nucleic acid genome is contained within a capsid, which is a symmetrical or quasisymmetrical structure assembled from one or only a few proteins. Attached to the capsid (or incorporated into it) are other structures; these structures are assembledfrom distinct proteins and are necessaryfor infection of the host cell. The virus palticle is tightly constructed.The internal volume of the capsid is rarely much greater than the volume of the nucleic acid it must hold. The ,lifference is usually less than twofold, and often the internal volume is barely larger than the rLucleicacid. In its most cxtreme form, the restriction that the capsid rnust be assembledfrom proteins coded by the virus means that the entire shell is constructed from a single type of subunit. The rules lor assembly of identical subunits into closec structures restrict the capsid to one of two tylles. For the first type, the protein subunits stack sequentially in a helical array to f.orm a fillmentlus or rodlike shape. For the second type, they form a pseudospherical shell-a type of structure that conforms to a polyhedron with icosahedral symmetry. Some viral capsidsare assembledfrom more than a single type of protein subunit, but although this extends the exact types of structures that can br: formed, viral capsids still all conform to the general classesof quasicrystalline filaments or icosahedrons. There are two types of solution to the problem of how to construct a capsid that contains nucleic acid: . The protein shell can be assembled around the nucleic acid, thereby condensing the DNA or RNA bY Proteinnucleic acid interactions dudng the process c'f assembly. . The capsid can be constructed from its componr:nt(s) in the form of an empty shell, into which the nucleic acid must be insertt:d,being condensedasit enters. The capsidis assembledaround the genome for single-stranded RNA viruses. The principle
into TheirCoats ArePackaged 28.2 ViralGenomes
731
: : ,.':-ii .: A heticalpathfor TMVRNAis created bythe stacking of proteinsubunjts in thevirion. of assembly is that thepositionof the RNA within the capsidis determineddirectly by its binding to the proteinsof theshell.The best characterizedexample is TMV (tobacco mosaic virus). Assembly starts at a duplex hairpin that lies within the RNA sequence.From this nucleation center, it proceedsbidirectionally along the RNA until it reachesthe ends. The unit of the capsidis a two-layer disk, with each layer containing 17 identical protein subunits. The disk is a circular structure, which forms a helix as it interacts with the RNA. At the nucleation center, the RNA hairpin inserts into the central hole in the disk, and the disk changesconformation into a helical structure that surrounds the RNA. Additional disks are added, with each new disk pulling a new stretch of RNA into its central hole. The RNA becomes coiled in a helical array on the inside of the protein shell. as illustrated i n i t r , l . , , r ' , l : : ',,.
The spherical capsidsof DNA viruses are assembledin a different way, as best characterized for the phageslambda and T4. In each case, an empty headshell is assembledfrom a small set of proteins. Theduplexgenlme then is inserted into the head,accompanied by a structural change in the capsid. ; i . i . : . : :, i . - r S u m m a r i z e st h e a s s e m b l y o f lambda. It startswith a small headshellthat contains a protein "core." This is converted to an empty headshellof more distinct shape.At this point the DNA packagingbegins,the headshell expands in size though remaining the same shape, and finally the full head is sealedby the addition of the tail.
732
C H A P T E2R8 C h r o m o s o m e s
r:'i;i,Jir! passes ;$.:t Maturation of phage [ambda through several Theemptyheadchanges stages. shape andexpands whenit becomes fittedwithDNA. Theetectron micrographs showthe partictes at the startandthe endof the maturation pathway. Topphotoreproduced fromCue,D.andFeiss, M. 1993.Proc.Natl.Acad.Sci.USA.90:9290-9294. Copyright1993National Academy ofScience, U.S.A. Photo courtesyof Michael G.Feiss, University of Iowa.Bottomphoto courtesy of RobertDuda,University of Pittsburgh. A double-strandedDNA that spansshort distances is a fairly rigid rod, yet it must be compressedinto a compact structure to fit within the capsid. We should like to know whether packaging involves a smooth coiling of the DNA into the head or whether it requires abrupt bends. Inserting DNA into a phage head involves two types of reaction: translocation and condensation. Both are energetically unfavorable. Tlanslocation is an active processin which the DNA is driven into the head by an ATPdependent mechanism. A common mechanism is used for many viruses that replicate by a rolling circle mechanism to generatelong tails that contain multimers of the viral genome. The best characterizedexample is phage lambda. The genome is packaged into the empty capsid by the terminase enz).rne.il{illlit i:rii.isummarizesthe process.
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r e c o m b i n a t i o n r e a c t i o n s .N u l l m u t a t i o n s i n either of the genes coding for the subunits of HU (hupA and -B) have little effect,but loss of both functions causesa cold-sensitivephenotype and some loss of superhelicity in DNA. Theseresultsraisethe possibilitythat HU plays some general role in nucleoid condensation. Protein HI (also known as H-NS) binds DNA, interacting preferentially with sequences that are bent. Mutations in its gene have turned up in a variety of guises (osmZ,bglY,pilG), each of which is identified as an apparent regulator of a different system. These results probably reflect the effect that Hl has on the local topology of DNA, with effectsupon gene expression that depend upon the particular promoter. We might expect that the absenceof a protein required for nucleoid structure would have serious effects upon viability. Why, then, are the effectsof deletionsin the genesfor proteins HU and Hl relatively restricted?One explanation is that these proteins are redundant,and that any one can substitute for the others so that deletions of.all of them would be necessary to interfere seriouslywith nucleoid structure. Another possibilityis that we have yet to identify the proteins responsiblefor the major f e a t u r e so f n u c l e o i d i n t e g r i t y . The nucleoid can be isolateddirectly in the form of a very rapidly sedimenting complex, w h i c h c o n s i s t so f - 8 0 % D N A b y m a s s . ( T h e analogous complexes in eukaryotes have -507o DNA by mass; see Section 28.4, ThreBacterial Genome Is Supercoiled.)It can be unfolded by treatment with reagents that act on RNA or on protein. The possiblerole of proteins in stabilizing its structure is evident. The role of RNA has been quite refractory to analysis.
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integrity. (In "op:n" circular molecules,which contain a nick in r)ne strdr-Id,or with linear molecules,the DNA can rotate freely in responseto the intercalation, thus relieving the tension.) In a natural closed DNA that is negatively supercoiled,the . ntercalation of ethidium bromide first remov,3sthe negative supercoilsand then introduces positive supercoils.The amount of ethidium brornide needed to achieve zero supercoiling is a rneasureof the original density of negative supelcoils. Some nicks occur in the compact nucleoid during its isolation; they can also be generated by limited treatnent with DNAase. This does not, however, abolish the ability of ethidium bromide to introduce positive supercoils. This capacityof the gr nome to retain its responseto ethidium bromide in the face of nicking means that it must havc many independent chromosomal domains, and that thesupercoilingin each domqinis not affeaedby eventsin theotherdomains. This autonolny suggeststhat the structure of the bacterial r:hromosome has the general organization depicted diagrammatically in iii,,ii:ii:,r::r.'. puar, domain consistsof a loop of DNA, the ends c,f which are secured in some (unknown) way that doesnot allow rotational events to prop,igate from one domain to another. Early data suggestedthat each domain consistsof -40 kb of DNA, but more recent analysis suggeststhat the domains may be smaller, at - 1 0 k b e a c h . T h i s w o u l d c o r r e s p o n dt o - 4 0 0
TheBacterial Genome Is SupercoiLed
Thenucteoid has-100 independent negativety suoerco'ited domains. Theaverage densityof supercoi[ing is - 1 t u r n / 1 0 0b o .
The DNA of the bacterial nucleoid isolated in vitro behaves as a closed duplex structure, as judged by its response to ethidium bromide. This small molecule intercalates between base pairs to generate positivesuperhelical turns in "closed" circular DNA molecules, that is, moleculesin which both strandshave covalent
; I 11:,:: 'r'i : TheLacterial genome consists of a large number of loopsof luplexDNA(in the formof a fiber), at thebaseto formanindependeachof whichis secured e n ts t r u c t u rdaoI m a i n .
Is Supercoited 735 Genome 28.4TheBacterial
Duplex DNA Unconstrained path is supercoiled in soace and createstension Constrained path is supercoiled aroundproteinbut createsno tension
fIGURf28.8 An unrestrained supercoil in the DNApath creates tension, but notensionis transmitted alongDNA whena supercoiI is restrained by proteinbinding.
domains in the E. coli genome. The ends of the domains appear to be randomly distributed instead of located at predetermined siteson the chromosome. The existence of separate domains could permit different degrees of supercoiling to be maintained in different regions of the genome. This could be relevant in considering the different susceptibilitiesof particular bacterial prom o t e r s t o s u p e r c o i l i n g( s e e S e c t i o n l l . l 5 , Supercoiling Is an Important Feature of Transcription). As shown in FI$URI2&.*,supercoiling in the genome can in principle take either of two forms: . If a supercoiled DNA is free its path is unconstrained,and negative supercoils generate a state of torsional tension that is transmitted freely along the DNA within a domain. It can be relieved by unwinding the double helix, as describedin Section 19.12, Supercoiling Affects the Structure of DNA. The DNA is in a dynamic equilibrium between the states of tension and unwinding. . Supercoiling can be constrainedif proteins are bound to the DNA to hold it in a pafiicular three-dimensional configuration. In this case,the supercoils are represenredby the path the DNA follows in its fixed association with the proteins. The energy of interaction between the proteins and the supercoiled DNA stabilizesthe nucleic acid, so that no tension is transmitted along the molecule. Are the supercoils in E. coliDNA constrained in vivo or is the double helix subject to the torsional tension characteristicof free DNA? Measurements of supercoiling in vitro encounter the
736
C H A P T E2R8 C h r o m o s o m e s
difficulty that constraining proteins may have been lost during isolation. Various approaches suggestthat DNA is under torsional slressinvivo. One approach is to measure the effect of nicking the DNA. Unconstrained supercoils are releasedby nicking, whereas constrained supercoils are unaffected. Nicking releases-5oo/o of the overall supercoiling. This suggeststhat about half of the supercoiling is transmitted as tension along DNA, with the other half beino absorbed by protein binding. Another approach uses the crosslinking reagent psoralen, which binds more readily to DNA when it is under torsional tension. The reaction of psoralen with E. coli DNA in vivo corresponds to an average density of one negative superhelicalturn/200 bp (o = -0.05). We can also examine the ability of cells to form alternative DNA structures; for example, to generate cruciforms at palindromic sequences. From the change in linking number that is required to drive such reactions, it is possible to calculate the original supercoiling density. This approach suggestsan average density of o = -0.025 , or one negative superhelicalturn/ I 00 basepairs. Thus supercoils /o create torsional tension in vivo. There may be variation about an average Ievel, and the precise range of densities is difficult to measure. It is, however, clear that the level is sufficient to exert significant effectson DNA structure-for example, in assistingmelting in particular regions such as origins or promoters. Many of the important features of the structure of the compact nucleoid remain to be estabIished. What is the specificity with which domains are constructed? Do the same sequencesalways lie at the same relative locations, or can the contents of individual domains shift? How is the integrity of the domain maintained? Biochemical analysis by itself is unable to answer these questions fully, but if it is possible to devise suitable selectivetechniques, the properties of structural mutants should lead to a molecular analysis of nucleoid construction.
Eukaryotic DNAHas Loops andDomains Attached to a Scaffold e DNAofinterphase chromatin is negativety supercoi[ed into independent domains of -85 kb. r Metaphase chromosomes havea proteinscaffotdto whichthe loopsof supercoited DNAareattached.
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Band p22.3 p22.2 p22.1 p21 p11.4 p 1 1. 3 p11.2 cenlromere q12 q13 1
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f iiri.ifii:ili:l.trr Thehuman X chromosome canbedivided into distinctregionsby its bandingpattern.Theshort armjs p andthe longarmis q,'eacharmis djvjdedinto regions larger thatarefurthersubdivided. Thismapshows a [ow resotution structure; at higherresolution, some bands arefurthersubdivided intosmatter bands andinterbands, e.9.,p21is dividedintop21.1., p27.2,andp21,.3.
Iower G-C content than the interbands. This is a peculiar result. If there are -10 bands on a large chromosome with a total content of -100 Mb, this means that the chromosome is divided into regions of -5 Mb in length that alternate between low G-C (band) and high G-C (interband) content. There is a tendency for genes (as identified by hybridization with mRNAs) to be located in the interband regions. All of this arguesfor some long-range sequencedependent organization. The human genome sequenceconfirms the basic observation. :'i*r-i"!ti; ;*.::.i shows that there are distinct fluctuations in G-C content when the genome is divided into small tranches (DNA segmentsor lengths). The average of 4lo/o G-C is common to mammalian genomes. There are regionsas low as 30o/oor as high as 650/o.When longer tranches are examined, there is lessvariation. The averagelength of regions with>4joh G-C is 200 to 250 kb. This makes it clear that tne band/interband structure does not represent homogeneous segmentsthat alternate in G-C content, although the bands do contain a higher content of low G-C segments.Genes are concentrated in regions of higher G-C content. We have yet to understand how the G-C content affects chromosome structure.
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Lampbrush Chromosomes AreExtended r Sitesof geneexpression on lampbrush from chromosomes shc,w loopsthat areextended the chromosomaI axis.
It would be extremely useful to visualize gene expression in its natural state in order to see what structural chrangesare associatedwith transcription. The couLpressionof DNAin chromatin. coupled with the difficulty of identifying particular geneswithin it, makes it impossibleto visualize the transcription of individual active genes. Gene expres:;ion can be visualized directly in certain unusual situations in which the chromosomes are found in a highly extended form that allows indivjdual loci (or groups of loci) to be distinguished. Lateraldifferentiation of structure is evident h many chromosomes when they first appear for meiosis. At this stage, the chromosomes resemble a series of beads on a string. The beadsare densely staining granules, properly known;rs chromomeres. In general, though, there is little gene expression at meiosis, and it is not practical to use this material to identify the activities of individual genes. An exceptional situation that allows the material to be examined is presented by lampbrush chromosomes, which have been best characterized in certain amphibians. Lampbrush chromosomes are formed during an unusually extended meiosis, which can last up to several months! During this period, the chromosomc's are held in a stretched-out form in which thr:y can be visualized in the light microscope. At a later point during meiosis, the chromosomesre\.ert to their usual compact size.
AreExtended 741 28.9 Lampbrush Chromosomes
A L a m p b r u schh r o m o s o mies a m e i o t i c bivaLentin which the two Dairsof sisterchromatidsare heLdtogetherat chiasmata(indicatedby arrows).Photo courtesyof JosephG. Gatt,Carnegie Institution.
The lampbrush chromosomes take their name from the lateral loops that extrude from the chromomeres at certain positions. (These resemble a lampbrush, which is an extinct object.)The loops extend in pairs, one from each sisterchromatid. The loops are continuous with the axial thread, which suggeststhat they represent chromosomal material extruded from its more compact organization in the chromomere. The loops are surrounded by a matrix of ribonucleoproteins that contain nascent RNA chains. Often, a transcription unit can be defined by the increase in the length of the RNP moving around the loop. An example is shown in j,l,ri,ijl
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Thus the loop is an extruded segment of DNA that is being actively transcribed. In some cases,loops correspondingto particular genes have been identified. For these cases,the structure of the transcribed gene-and the nature of the product-can be scrutinized in situ.
Polytene Chromosomes FormBands . Polytene havea series chromosomes of dipterans map. of bandsthat canbe usedasa cytotogicaI A [ a m p b r u schh r o m o s o m l oeo pi s s u r r o u n d ebdy a m a t r i xo f r i b o n u c t e o p r o t eRi en p. r o d u c e d The interphase nuclei of some tissuesof the larfromGatL, BioL. 1999.10: J. G.,et al.MoL. Cell.December, 4385-4402. Photocourtesy of JosephG. GaLt, Carnegie vae oI dipteran flies contain chromosomes that Institution. are greatly enlarged relative to their usual condition. They possessboth increased diameter r.; :r showsan examThus the extended stateessentiallyproffers an and greaterlength.;i,r.ri-ri;: ple of a chromosome set from the salivary gland unfolded version of the normal condition of the The members of this set are chromosome. of.D. melanogaster. The lampbrush chromosomes are meiotic called polytene chromosomes. bivalents, each consistingof two pairs of sister Each member of the polytene set consists chromatids. shows an example in of a visible seriesof bands (more properly, but which the sister chromatid pairs have mostly rarely, describedas chromomeres). The bands separatedso that they are held together only range in size from the largest,with a breadth of -0.5 pm, to the smallest,at -0.05 pm. (The smallby chiasmata.Each sisterchromatid pair forms a seriesof ellipsoidalchromomeres,-l to 2 pm est can be distinguished only under an electron in diameter, which are connected by a very fine microscope.)The bands contain most of the mass thread. This thread contains the two sister of DNA and stain intensely with appropriate duplexes of DNA and runs continuously along reagents.The regions between them stain more the chromosome, through the chromomeres. lightly and are called interbands. There are -5000 bands in the D. melanlgasterset. The iengths of the individual lampbrush chromosomes in the newt NotophthalmusviriThe centromeres of all four chromosomes descens range from 400 to 800 pm, compared of.D melanogasteraggregateto form a chromowith the range of l5 to 20 pm seenlater in meiocenter that consistslargely of heterochromatin. (In the male it includes the entire Y chromosis.Thus the lampbrush chromosomesare -30 times lesstightly packed. The total length of the some.) Allowing for this, -75o/oof the haploid entire lampbrush chromosome set is 5 to 6 mm DNA set is organized into alternating bands and is organizedinto -5000 chromomeres. and interbands. The length of the chromosome
742
C H A P T E2R8 C h r o m o s o m e s
. Freez:ein dry ice r Wasl-with ethanol . Dip ir agarsolution o DenatureDNA . Add radioactiveprobe o Wasl- ofi unreactedprobe . Autoradiography
iri{il-tfti: lii.lri: Thepolytene chromosomes of D.melonogasterform anatternating series ofbands andinterbands. Photocourtesy ofJos6Bonner, IndianaUniversity. set is -2000 pm. The DNA in extended form would stretch for -40,000 pm, so the packing ratio is -20. This demonstrates vividly the extension of the genetic material relative to t h e u s u a l s t a t e so f i n t e r p h a s e c h r o m a t i n o r mitotic chromosomes. What is the structure of these giant chromosomes?Each is produced by the successive replications of a synapseddiploid pair. The replicasdo not separate,but instead remain attached to each other in their extended state. At the start of the process,each synapsedpair has a DNA content of 2C (where C represents the DNA content of the individual chromosome). This amount then doubles up to nine times, at its maximum giving a content of 1024C. The number of doublings is different in the various tissues of the D. melanogasterlarva. Each chromosome can be visualized as a large number of parallel fibers running longitudinally that are tightly condensed in the bands and lessso in the interbands. It is likely that each fiber representsa single (C) haploid chromosome. This gives rise to the name polytene: The degree of polyteny is the number of haploid chromosomes contained in the giant chromosome. The banding pattern is characteristic for each strain of Drosophila.The constant number and linear arrangement of the bands was first noted in the I930s, when it was realized that they form aqttologicalmapolthe chromosomes. Rearrangements-such as deletions,inversions, or duplications-result in alterations of the order of bands. The linear array of bands can be equated with the linear array of genes. Thus genetic rearrangements, as seen in a linkage map, can be correlated with structural rearrangements of the cytological map. Ultimately, a particular mutation can be located in a particular band. The total number of genes in D. melanogaster
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ll:ii:i.J fif ,,il:i,,: ir IndividuaIbandscontaining particutar genescanbeidentified by in situhybridization. exceedsthe numller of bands, so there are probably multiple gerles in most or all bands. The positions of particular genes on the cytological map ( an be determined directly by the technique oI in situ hybridization. The protocol is summarized in 4iiii!.i*{ i:*:.51i}. A radioactive probe representing a gene (most often a labeled cDNA clone derived from the mRNA) is hybridized with the denatured DNA of the polytene (hromosomes in situ. Autoradiography identilies the position or positions of the correspondirg genes by the superimposition of grains at a particular band or bands. An More recently, example is shown in iit,l,f'rtii;:*",iJi:l. fluorescent probes have replaced radioactive probes. With this type of technique at hand, it is possibleto determine directly the band within which a particuler sequencelies.
Polytene Chromosomes Expand at Sitesof Gene Expres;sion . Bands of geneexpression on polytene that aresil.es to give"puffs." chromosomes expand One of the intriguing features of the polytene chromosomes is t hat active sites can be visualized. Some of the rands passtransiently through
Expand at Sitesof GeneExpression 743 28.11Potvtene Chromosomes
! iilii Hl-;i*. ll I Chromosome IV of thei nsectC.tentans has . eproduced t h r e eB a t b j a nr i n g s ' i nt h e s a l i v a rgyt a n dR fromCell,vo[.4, Danehott, 8., et at.,Transciption in poLy1975.with tenechromosomes , pp.1-9. Copyright permission Photocourtesy fromEtsevier. of BertiIDaneNobeIInstitute. hoLt,MedicaI ;'.: ::i.:i .::..:.. A magnified viewof bands87Aand87C rnsifuwithLabeted RNAextracted shows theirhybridization iromheat-shocked ce[ts.Photocourtesv ofJos6Bonner, IndianaUniversitv.
an expanded state in which they appear like a puff on the chromosome,when chromosomal material is extruded from the axis. Examples of some very large puffs (called Balbiani rings) are shown in :::i:r;iii'li::j j ' What is the nature of the puff? It consists of a region in which the chromosome fibers unwind from their usual stateof packing in the band. The fibers remain continuous with those in the chromosome axis. Puffs usually emanate from singlebands, although when they are very .arge, as typified by the Balbiani rings, the swelling may be so extensive as to obscure the underlying array of bands. The pattern of puffs is related to gene expression. During larval development, puffs appear pattern. and regressin a definite, tissue-specific A characteristicpattern of puffs is found in each tissue at any given time. Puffs are induced by the hormone ecdysone that controls Drosophila development. Some puffs are induced directly by the hormone; others are induced indirectly by the products of earlier puffs. The puffs are siteswhere RNA is beingsynthesized The accepted view of puffing has been that expansion of the band is a consequence of the need to relax its structure in order to synthesize RNA. Puffing has therefore been viewed as a consequenceof transcription. A puff can be generated by a single active gene. The sitesof puffing differ
744
C H A P T E2R 8 Chromosomes
from ordinary bands in accumulating additional proteins, which include RNA polymerase II and other proteins associatedwith transcription. The features displayed by lampbrush and polytene chromosomessuggesta general conclusion. In order to be transcribed, the genetic material is dispersedfrom its usual, more tightly packed state. The question to keep in mind is whether this dispersion at the grosslevel of the chromosome mimics the events that occur at the molecular level within the mass of ordinary interphase euchromatin. Do the bands of a polytene chromosome have a functional significance,that is, does each band correspond to some type of genetic unit? You might thinkthat the answerwouldbe immediately evident from the sequence of the fly genome, becauseby mapping interbands to the sequence it should be possible to determine whether a band has any fixed type of identity. Thus far, though, no pattern has been found that identifies a functional significancefor the bands.
TheEukaryotic Is a Chromosome Device Segregation js hetdon the mitotic A eukaryotic chromosome spindte by the attachment of mjcrotubules to the kinetochore that formsin its centromeric region. Centromeres oftenhaveheterochromatin that is richin sateltite DNAseouences.
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Centromeres HaveShort DNASequences in S. cerevisiae r CENetements areidentifiedin 5. cerevisiae bu the abitityto al.l.ow a ptasmid to segregate accuritety at mitosis. o CEN etements consistof the shortconserved sequences CDE-I andCDE-III that ftanktheA-TrichregionCDE-II.
If a centromeric sequenceof DNA is responsible for segregation,any molecule of DNA possessingthis sequenceshould move properly at cell division, whereas any DNA lacking it should fail to segregate.This prediction has been used to isolate centromeric DNA in the yeast, S. cerevisiae.Yeast chromosomes do not display visible kinetochores comparable to those of higher eukaryotes, but otherwise divide at mitosis and segregateat meiosis by the same mechanisms. Genetic engineering has produced plasmids of yeast that are replicated like chromosomal sequences(see Section 15.8, Replication Origins Can Be Isolatedin Yeast).They are unstable at mitosis and meiosis, though, and disappear from a majority of the cells becausethey segregate erratically. Fragments of chromosomal DNA containing centromeres have been isolated by their ability to confer mitotic stability on these plasmids. A centromeric DNA regions (CEN) fragment is identified as the minimal sequencethat can confer stability upon such a plasmid. Another way to characterize the function of such sequences is to modify tlnelriLin vitro and then reintroduce them into the yeast cell, where they replace the corresponding centromere on the chromosome. This allows the sequences required for CENfunction to be defined directly in the context of the chromosome.
A CENfragmt:nt derived from one chromosome can replact: the centromere of another chromosome with no apparent consequence. This result suggeststhat centromeres are interchangeable . Thel are usedsimply to attach the chromosometo the spindle, and play no role in distinguishingonechromosome from another. The sequences required for centromeric function fall within a stretch of -I20 bp. The centromeric region is packagedinto a nucleaseresistant structure and binds a single microtubule. We may ttrerefore look to the S.cerevisiae centromeric regio.c to identify proteins that bind centromeric DNA and proteins that connect the chromosome to the spindle. 'J*.JS, As summarizr:d in i'3{*:i"ifif,:, three types of sequence elenrent can be distinguished in the CEN region: . cell cycle-dependent element (CDE)-I is a sequence of 9 bp that is conserved with minc,r variations at the left boundary of all centromeres. o CDE-II is ,t >90o/oA-T-rich sequence of 80 to 90 bp found in all centromeres; its function could depend on its Iength rather than exact sequence.Its constitution is reminiscent of some short tandemly repeated(satellite)DNAs (seeSection6.I2, Arthropod SatellitesHave Very Short Identical Repe;rts).Its base composition may CduseSorrLecharacteristic distortions of the DNA oouble helical structure. . CDE-III isan I I bp sequencehighly conserved at the right boundary of all centromeres. Sequenceson either side of the element are lesswell conserved,and may also be needed for centromeric function. :CDE-III could be longer than I I bp if il turns out that the flanking sequencesare essential.) Mutations in CDE-I or CDE-II reduce, but do not inactivatt:, centromere function, but point mutations in the central CCG of CDE-III completely inactivate the centromere.
in S. cerevisioe 747 HaveShortDNASequences 28.14Centromeres
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. A specialized proteincomptex that is an is structure atternative to the usuaIchromatin formedat CDE-IL r TheCBF3 proteincomptex is that bindsto CDE-III function. for centromeric essentiaI . Theproteinsthat connectthesetwo comptexes may providethe connection to microtubules.
Can we identify proteins that are necessaryfor the function of CEN sequences?There are several genes in which mutations affect chromosome segregation, and whose proteins are localized at centromeres. The contributions of these proteins to the centromeric structure are I8.tS. summarized in FISUftE A specialized chromatin structure is built by binding the CDE-II region to a protein called Cse4,which resemblesone of the histone proteins that comprise the basic subunits of chromatin (see Section 3I.3, Heterochromatin Depends on Interactions with Histones). A protein called Mif2 may also be part of this complex, or at least connectedto it. Cse4and Mif2 have counterparts that are localized at higher eukaryotic centromeres, called CENP-A and CENP-C, which suggeststhat this interaction may be a universal aspect of centromere construction. The basicinteraction consistsof bending the DNA of the CDE-II region arounci a protein aggregate; the reaction is probably assistedby the occurrence of intrinsic bending in the CDE-II sequence. CDE-I is bound by the homodimer CBFI; this interaction is not essential for centromere function, but in its absencethe fidelity of chromosome segregationis reduced-10x. A 240 kD complex of four proteins, called CBF3,binds to
CDE-III. This interaction is essentialfor centromeric function. The proteins bound at CDE-I and CDE-III are connected to each other and also to the protein structure bound at CDE-II by another group o f p r o t e i n s ( C t f1 9 , M c m 2 l , O k p l ) . T h e c o n nection to the microtubule mav be made bv this complex. The overall model suggeststhat the complex is localizedat the centromere by a protein structure that resembles the normal building block of chromatin (the nucleosome). The bending of DNA at this structure allows proteins bound to the flanking elements to become part of a single complex. Some components of the complex (possibly not those that bind directly to DNA) link the centromere to the microtubule. The construction of kinetochores probably follows a similar pattern, and usesrelated components, in a wide variety of organisms.
HaveSimple Telomeres Sequences Repeating o Thetelomere for the stabilityof the is required end. chromosome r A tetomere of a simplerepeatwherea consists C1(A/I)L4. C+A-rich strandhasthe sequence
Another essentialfeature in all chromosomes is the telomere, which "seals"the end. We know that the telomere must be a special structure, becausechromosome ends generated by breakage are "sticky" and tend to react with other chromosomes, whereas natural ends are stable. We can apply two criteria in identifying a telomeric sequence: . It must lie at the end of a chromosome (or, at least at the end of an authentic linear DNA molecule). . It must confer stability on a linear molecule. The problem of finding a system that offers an assay for function again has been brought CDE-I CBFl Microtubules to the molecular level by using yeast. All the ctf19 plasmids that survive in yeast (by virtue of possessingARS and CEN elements) are circular cDE-rl Cse4 Okpl DNA molecules. Linear plasmids are unstable (becausethey are degraded). Could an authencDE-il1 tic telomeric DNA sequence confer stability on a Iinear plasmid? Fragments from yeast DNA 'iswoundaround that prove to be located at chromosome ends f3t:iifi[tii, t$ TheDNAat CDE-II a proteinaggregate including is Cse4p, CDE-III boundto CBF3, can be identified by such an assay,and a region andCDE-I is boundto CBF1. These oroteins areconnected from the end of a known natural linear DNA by the groupof Ctf19,Mcm21, andOkp1.
748
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spulauosoruorql oql lPassarauolal 'lsee^ uI suollf,unJ eJeuolJt Jtprlr) aql ,lroq sureldxa srqJ 'uoll -rppp roJ alertsqns elqellns e se pazruSo)eJeq 01 puJ aql ro; st parmbal sI lpqt IIV 'tuelelaJJl Jq ,{eru aruanbastJexe rtaqr '(paual.roqspue) paua -er8ual Sutaq Lllenulluo) ale sJrJruolJt JI 'runrrqllnba rtueuLp ut aq plnoM Suruagoqs pue uorsuelxg'eruosouorql eql Jo pua Jqt o1 dn alerqdJJ ol aJnlIeJ uoJJ Surlpsar sleadar Jo ssol eql t)eJJtunoJ plnoM srsaqlur(s0^0u ap dq sleadar io uolllppP JqJ 'uor1er11dagJoJ tuJlqord e arv vNO reaulf Jo spug JqJ'z'9I uolDes uI pJssnlslp selnJelolu VN( reJurl 3u1terr1da.ryo z(rlntlyytp Jql anlos plnor apLr uorle)qdeJ z(ranaur auosoruoJqJ eql Jo pua aql ot slpedeJJlrJruolal Jo uouppy 's10aaaJ 0u 2u1nt4t2 Jl2J 2tl1l0 spua a41 01u0pappa 2fi svadat )uau.I)lal $a a[' lseatr ur uorlerrldal JeqV'tsea^ olut pa)np -oJlur saJeurolel elpIIIJ Jo eleJ Jqt sI SuIIeJ^er e J o r r ru J A f ' u o r t e r a u a 8 J a d ( s l p J d e JZ o t I ) dq g1 or 1 .{.qra8uol sa,ror8Jreruolet rqt 'prmol -lo; sr JuolJ IIJr Ienpl^rpur up uJqM 'qlSuJI ur Lre.t spue aql 'uorlelndod aruosouedz(rl e uI 'sJlnJaloru vN(I TPJUIIJo spue aql Jo sall -radord Iensnun atuos Lq uaat8 are suollJunJ JJJruOIelP A\oq lnoqe suolleJlpul aluos 'puens q)Ir-V-) eql Io uoueper8ap palnu11rr;o -ads e st JJeqt JSneJaqpaleraua8 Llqeqord st pet -g rql 'pupJts a13urse .{.1ensnsI seseq9I ot VI roJ qJrqM'pueJls q)lr-J-D aql Io uolsusxe eql sr aluanbas JrJaruolal aqr Jo .drradordlensnun '7 aug 'aldruexa rtraua8 e sMoqs J-,-y'fifr ii4ll{i3;l ot I slLupue l
What feature of the telomere is responsible for the stability of the chromosome end? FIGURE 28.29shows that a loop of DNA forms at the telomere. The absenceof any free end may be the crucial feature that stabilizesthe end of the chromosome. The average length of the loop in animal cells is 5 to l0 kb. 28,30shows that the loop is formed FIGURE when the 3' single-stranded end of the telomere (TTAGGG)" displacesthe same sequence in an upstream region of the telomere. This converts the duplex region into a structure like a D-loop, where a series of TTAGGG repeats are FIGURE 28.29 A loopformsat the endof chromosomal displacedto form a single-stranded region, and DNA.Photocourtesy ofJackGriffith,University of North the tail of the telomere is naired with the homolCarolina at Chaoel Hilt. ogous strand. The reaction is catalyzed by the telomerebinding protein TRF2, which together with other proteins forms a complex that stabilizes the chromosome ends. Its importance in protecting the ends is indicated by the fact the deletion of TRF2 causeschromosome rearrangements to occur.
TRF2
Te[omeres Are Synthesized by a Ribonucleoprotein Enzyme o Telomerase usesthe 3'-0H ofthe G+Ttelomeric strandto primesynthesis of tandemTTGGGG reDeats. o TheRNAcomponent of tetomerase hasa sequence that pairswith the C+A-rich repeats. r Oneof the proteinsubunitsis a reverse transcriptase that usesthe RNAastemptate to synthesis the G+T-rich sequence.
FIGURF ?8.30 The3'single-stranded endofthetetomere (TTAGGG)" disptaces the homologous repeats fromduplex DNAto forma t-[oop.Thereaction is catatyzed by TRF2.
formed from the second guanine in each repeating unit. A series of quartets could be stacked Iike this in a helical manner. Although the formation of this structure attests to the unusual properties of the G-rich sequenceinvitro, it does not of course demonstrate whether the ouartet forms in vivo.
750
CHAPTER 28 C h r o m o s o m e s
The telomere has two functions: . One is to protect the chromosome end. Any other DNA end-for example, the end generated by a double-strand break-becomes a target for repair systems. The cell has to be able to distinguish the telomere. . The second is to allow the telomere to be extended. If it is not extended, it becomes shorter with each replication cycle (because replication cannot start at the very end). Proteins that bind to the telomere provide the solution for both problems. In yeast, different setsof proteins solve each problem, but both
are bound to the telomere via the same Drotein, Cdcl3: . T h e S t n l p r o t e i n p r o t e c t s a g a i n s td e gradation (specifically, against ,any extension of the degradation of the C-A-strand that generatesthe G-tail). . A telomerase enzyme extends the CA-rich strand. Its activity is influenced by two proteins that have ancillary roles, s u c h a s c o n t r o l l i n g t h e l e n g t t Lo f t h e extension. The telomeraseusesthe 3'-OH of the G+T telomeric strand as a primer for synthesir;of tandem TTGGGGrepeats.Only dGTP and dTTPare needed for the activity. The telomeraseirsa large ribonucleoprotein that consistsof a templating RNA (coded by TLCl) and a protein with catalytic activity (EST2) The short RNA ,romponent (159 baseslong in Tetrahymena, and 192 baseslong in Euplotes)includes a sequenceof 15 to 22 basesthat is identical to two repeatsof the C-rich repeating sequence.This RNA F,rovides the template for synthesizing the G-rich repeating sequence.The protein component of the telomerase is a catalytic subunit that can act only upon the RNA template provideclby the nucleic acid component. i'ri.r:i::i:'.:.i ;i i shows the action of telomerase. The enzyme progressesdiscontinuou:;ly: The template RNA is positioned on the DNA primer, severalnucleotides are added to the prin:rer,and then the enzyme translocatesto begin again. The telomerase is a specializedexample of a reverse transcriptase,an enzyme that synthesizes a DNA sequence using an RNA template (see Section 22.4, Yiral DNA Is Generated by ReverseTranscription).We do not know how the complementary (C-A-rich) stran
Binding:RNAtemplatepairswith DNA primer D N Ap r i m e r
3'W 3',
5:
RNA templal RNA templatedirec Polymerization: of nucleotidesto 3'end of DNA
Polymerizationcontinues I to end of templateregion Y
J
3', Enzymemovesto Translocalio;r:
5',..TTGGGGTTGGGGTTGC 3', .,.AACCCC 5',
MCCC 3',
positions i:Li:li-:iii. itsetfby basepairiii .i i Tetomerase singleandtheprotruding ingbetween theRNAtemplate oneata time It addsGandT bases DNAprimer. stranded Thecyclestarts to the primer, asdirectedbythetemplate. a g a i nw h e no n er e p e a t i nugn j th a sb e e na d d e d .
(typically 5 to l5 kb in human beings) and short The in yeast (typically -300 bp in S. cerevisiae). probability basic control mechanism is that the that a telomere will be a substrate for telomeraseincreasesas the length of the telomere shortens;we do not know if this is a continuous effect or if it depends on the length falling below some critical value. When telomerase acts on a telomere, it may add severalrepeating units. The enzyme's intrinsic mode of action is to dissociateafter adding one repeat; addition of severalrepeating units dependson other proteins that cause telomerase to undertake more than one round of extension. The number of repeats that is added is not influenced by the length of the telomere itself,but insteadis controlled by ancillary proteins that associatewith telomerase.
Enzyme 757 by a Ribonucleoprotein AreSynthesized 28.18Tetomeres
The minimum features required for existence as a chromosome are: . Telomeres to ensure survival. o A centromere to support segregation. . An origin to initiate replication. All of these elementshave been put together to construct a yeastartificial chromosome (YAC). This is a useful method for perpetuating foreign sequences.It turns out that the synthetic chromosome is stable only if it is longer than 20 to 50 kb. We do not know the basisfor this effect, but the ability to construct a synthetic chromosome allows us to investigate the nature of the segregationdevice in a controlled environment.
@
Telomeres AreEssential for SurvivaI
Telomeraseactivity is found in all dividing cells and is generally turned off in terminally differentiated cells that do not divide. F{{;i.ffts I*.3f shows that if telomerase is mutated in a dividing cell, the telomeres become gradually shorter with each cell division. An example of the effects of such a mutation in yeast are shown in lii;LsgttIS.i3, where the telomere length shortens over - I 20 generations from 400 bp to zero. Lossof telomereshas very bad effects.When the telomere length reaches zero, it becomes difficult for the cells to divide successfully. Attempts to divide typically generate chromosome breaks and translocations.This causesan increasedrate of mutation. In yeastthis is associated with a loss of viability and the culture becomespredominantly'occupied by senescent
f,ttwild-tvpe
Divisions 40
frtT-deficient
120 40
80 120
ijil'tjEtil f S.S.3Tetomere lengthjs maintained at -350bp in wiLd-type yeast,but a mutantin the frfl genecoding forthe RNAcomponent oftelomerase rapidty shortens its telomeres to zeroLength. Reproduced with permission fromNakamura, T.M.,et a|.1,997. Science. 277:955-959. o 1997AAAS. Photocourtesy of Thomas R.Cech andToru Nakamura, University of Co[orado.
cells (which are elongated and nondividing, and eventually die). Some cells grow out of the senescingculture. They have acquired the ability to extend their telomeres by an alternative to telomerase activity. The survivors fall into two groups. The members of one group have circularized their chromosomes: They now have no telomeres, and as a result they have become independent of telomerase.The other group uses unequal crossing-over to extend their telomeres Telomere Telomere (ft**ftf f *-34). The telomere is a repeating structure, so it is possible for two telomeres to misalign when chromosomes pair. Recombination between the mispaired regions generates an unequal crossing-over, as shown previously in Figure 6.1: When the length of one recombinant chromosome increases.the length of the other decreases. Cells usually suppressunequal crossingover because of its potentially deleterious consequences. Two systems are responsible for suppressingcrossing-overbetween telomeres. One is provided by telomere-binding proteins. yeast, the frequency of recombination In i:i$11*il ;.18.-iii Mutation in tetomerase causes telomeres to shoden in eachcetldivision. Eventual lossofthetelom- between telomeres is increased by deletion of erecauses chromosome breaks andrearranqements. the gene tazl, w};'ich codes for a protein that
+ + I + +
752
C H A P T E2R 8 Chromosomes
regulates telomerase activity. The second is a general system that undertakes mismatch repair. In addition to correcting mismatched basepairs that may arise in DNA, this system suppresses recombination between mispaired regions. As shown in Figure 28.34, this includes telomeres. When it is mutated, a greater proportion of telomerase-deficientyeast survive the loss of telomeres because recombination between telomeres generatessome chromosomes with longer telomeres. When eukaryotic cells are placed in culture, they usually divide for a fixed number of generationsand then enter senescence.The reason appears to be a decline in telomere length because of the absenceof telomerase expression. Cells enter a crisis from which some emerge, but typically the cellsthat emerge have chromosome rearrangementsthat have resulted from lack of protection of chromosome ends. These rearrangements may cause mutations that contribute to the tumorigenic state. The absenceof telomeraseexpressionin this situation is due to failure to expressthe gene, and reactivation of telomerase is one of the mechanisms by which these cells then survive continued culture. (This of coursewas not an option in the yeast experiments in which the gene had been deleted.)
mary Sum The geneticmaterial of all organismsand viruses takes the form of tightly packaged nucleoprotein. Some virus genomes are inserted into preformed virions, whereas others assemble a protein coat around the nucleic acid. The bacterial genome forms a dense nucleoid, with -20o/o protein by mass,but details of the interaction of the proteins with DNA are not known. The DNA is organized into -100 domains that maintain independent supercoiling,with a density of unrestrained supercoils corresponding to -llI00 to 200 bp. In eukaryotes,interphase chromatin and metaphase chromosomes both appear to be organized into large loops. Each loop may be an independently supercoiled domain. The basesof the loops are connected to a metaphase scaffoldor to the nuclear matrix by specificDNA sites. Transcriptionally active sequencesreside within the euchromatin that comprises the majority of interphase chromatin. The regions of heterochromatin are packaged-5 to l0x more compactly, and are transcriptionally inert. AII
Crossing-over occurswhen mismatch repairis absent
\,/ n
I
Y
{-it-'{Jii 11.,i:.;.r. ng-over i n telo mericregionsis usu-'-l+ Crossi systems, butcanoccur bymismatch-repair a[[ysuppressed event crossing-over An unequal whentheyaremutated. the a[[owing extends of oneof theproducts. thetetomere of telomerase. in the absence chromosome to survive
chromatin becomes densely packaged during cell division, when the individual chromosomes can be distinguished. The existence of a reproducible ultrastructure in chromosomes is indicated by the production of G-bands by treatment with Giemsa stain. The bands are very large regions (-10? bp) that can be used to map chromosomal translocations or other large changes in structure. Lampbrush chromosomes of amphibians and polytene chromosomes of insects have unusually extended structures, with packing ratios < I 00. Polytene chromosomes of D. melanogasteraredivided into -5000 bands. Thesebands vary in size by an order of magnitude, with an average of -25 kb. Transcriptionally active regions can be visualized in even more unfolded ("puffed") structures, in which material is extruded from the axis of the chromosome. This may resemble the changes that occur on a smaller scalewhen a sequence in euchromatin is transcribed. The centromeric region contains the kinetochore, which is responsible for attaching a chromosome to the mitotic spindle. The centromere often is surrounded by heterochromatin. Centromeric sequenceshave been where they identified only in yeast S.cerevisiae, consistof short conservedelements.TheseelelnCNTS, CDE-I ANdCDE-III, biNd CBFI ANdthc CBF3 complex, respectively,and a long A-Trich region called CDE-II binds Cse4to form a specialized structure in chromatin. Another group of proteins that binds to this assembly provides the connection to microtubules. Telomeres make the ends of chromosomes stable. Almost all known telomeres consist of multiple repeats in which one strand has the
28.20Summarv 753
g e n e r a l s e q u e n c e C " ( A , 1 T ) * ,w h e r e n > l a n d m = | to 4. The other strand, Gn(TlA\*, has a single protruding end that provides a template f o r a d d i t i o n o f i n d i v i d u a l b a s e si n d e f i n e d order. The enzyme telomerase is a ribonucleoprotein whose RNA component provides the template for synthesizing the G-rich strand. This overcomes the problem of the inability to replicate at the very end of a duplex. The telomere stabilizes the chromosome end because the overhanging single strand G " ( T l A ) m d i s p l a c e si t s h o m o l o g i n e a r l i e r repeating units in the telomere to form a loop, so there are no free ends.
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CHAPTER 28 Chromosomes
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756
CHAPTER 28 Chromosomes
tion rate and genomic instability. Cell lO6, 275-286. Nakamura, T. M., Cooper, J. P., and Cech, T. R. (1998). TWo modes of survival o{ fission yeast without telomerase. Science282, 493496. Nakamura, T. M., Morin, G. B., Chapman, I(. B., Weinrich, S. L., Andrews, W. H., Lingner, J., Harley, C. B., and Cech,T. R. (1997). Telomerase catalytic subunit homologs from fission yeast and human. Science277, 955-959. Rizki, A. and Lundblad, V. (2001). Defects in mismatch repair promote telomerase-independent proliferation. Nature 4ll , 7l j-7 16.
Nucleosome C H A P T EO RU T L I N E Introduction The Nucleosome Is the Subunitof A[[ Chromatin o MicrococcaI nuclease from releases individuaI nucleosomes chromatin as115oartictes. o A nucteosome -200 bp of DNA,two copies contains of each , Z BH c o r eh i s t o n (eH 2 AH , 3 ,a n dH 4 ) . . DNAis wrapped around surface of the protein the outside 0cramer. DNAIs Coiledin Arraysof Nucleosomes . >95%of the DNAis recovered in nucleosomes or multimers whenmicrococcal nuclease DNAof chromatin. cleaves r Thelengthof DNApernucteosome variesfor individuat tissuesin a rangefrom154to 260bp. Nucleosomes Havea CommonStructure . NucteosomaI DNAis dividedintothe coreDNAand[inker DNAdepending on its susceptibitity to micrococca[ nuctease. . ThecoreDNAis the lengthof 146bpthat is foundon the coreparticlesproduced by prolonged digestionwith micrococcaI nuctease. o LinkerDNAis the regionof 8 to 114bp that is susceptibte to earlycleavage by the enzyme. r Changes in the lengthof [inkerDNAaccount forthe variaDNA. tion in total lengthof nucleosomal . H1is associated with linkerDNAandmaylie at the point whereDNAentersand[eaves the nucleosome. DNAStructureVarieson the Nucleosomal Surface . DNAis wrapped 1.65timesaround the histoneoctamer. . Thestructure of the DNAis alteredsothat it hasan increased in the middl.e, buta number of basepairs/turn number decreased at the ends. The Periodicityof DNAChanges on the Nucleosome r -0.6 negative in turnsof DNAareabsorbed by the change bp/turnfrom10.5in solution to an average of 10.2on the nucleosomaI whichexplains the [inking-number surface. parao0x. of the Histone0ctamer Organization r Thehistoneoctamer hasa kernelof an H3z-H4z tetramer withtwo H2A-H2B dimers. associated . Eachhistone is extensively interdigitated withits partner. . A[[corehistones havethe structural motifof the histone fold.N-terminaI taitsextendout of the nucteosome.
in the ChromatinFiber The Pathof Nucleosomes . L0 nmchromat'in from30 nmfibersand fibersareunfolded consjstof a stringof nucleosomes. . 30 nmfibershaves'ixnucleosomes/turn, whichareorganizedinto a sotenoid. . Histone for formation ofthe 30 nmfiber. H1is required Assemb[y of ChromatinRequires Reproduction of Nucteosomes r Histone but duringreplication, arenot conserved octamers tetramers areconserved' dimersandH32-H42 H2A-H2B . Therearedifferentpathways of nucteofor the assembty of reptication. andindependentty duringreplication somes . Accessory of proteinsarerequired to assistthe assembly nucte050mes. . CAF-1 proteinthat is Linked to the PCNA subis an assembty of for deposition it is required unit of the rept'isome; foltowing reptication. H3z-H4z tetramers r A different proteinanda variantof histoneH3 assemb[y assembty. maybe usedfor reptication-independent Lieat SpecificPositions? Do Nucleosomes r Nucteosomes positions asthe resutt mayformat specific eitherof the [oca[structureof DNAor of proteinsthat intersequences. actwith specific o Themostcommon positioning is when causeof nucteosome a boundary. proteins bindingto DNAestablish o Positioning of DNAarein the mayaffectwhichregions on the nucleosome finkerandwhichfaceof DNAis exposed surface. Genes0rganizedin Nucleosomes? AreTranscribed . Nucleosomes whentranarefoundat the samefrequency genesaredigested with genes or nontranscribed scribed nuclease. micrococcaI . Someheavily genes to beexcept'ionaI appear transcribed cases that aredevoidof nucteosomes. by Transcription Histone0ctamersAre Disptaced o RNApotymerase duringtranhistoneoctamers disptaces with reassociate but octamers in a modelsystem. scription haspassed. DNAassoonasthe potymerase r Nucleosomes passes whentranscription arereorganized througha gene. Continued on nextpage
757
?DM
N u c t e o s o mDei s p l a c e m eannt d Reassembly RequireSpeciaIFactors r Ancitlary factorsarerequired bothfor RNA potymerase to disptace octamers during transcription andfor the histones to reassemb[e into nucleosomes after ption. transcri
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InsulatorsBtockthe Actionsof Enhancers and Heterochromatin Insutators areableto btockpassage of any activating or inactivating effectsfrom enhancers, silencers, andLCRs. Insulators mayprovide barriers against the spread of heterochromatin.
?EfB
InsulatorC s a nD e f i n ea D o m a i n . Insulators arespeciatized chromatin structuresthat havehypersensitive sites.Two insulators canprotectthe regionbetween themfroma[[ externaleffects.
JEETI InsutatorsMayAct in OneDirection o Someinsutators havedirectionatity. and maystoppassage of effectsin onedirection but notthe other.
InsutatorsCanVaryin Strength o Insulators candifferin howeffectively passage theybl"ock of an activating signat. DNAase Hypersensitive SitesReftect Changes in ChromatinStructure . Hypersensitive sitesarefoundat the progenes. motersof expressed o Theyaregenerated by the bindingof transcription factors that displace histone octamers. D o m a i nD s e f i n eR e g i o nTs h a tC o n t a i n ActiveGenes r A domaincontaining geneis a transcribed defined by increased sensitivity to degradationby DNAase I. An LCRMayControIa Domain o An LCR is located at the 5'endofthedomain andconsists of several hypersensitive sites. WhatConstitutesa Regutatory Domain? . A domainmayhaveaninsulator. an LCR. a matrixattachment site.andtranscription unit(s). Summarv
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Introduction
Chromatin has a compact organization in which most DNA sequencesare structurally inaccessible and functionally inactive. Within this mass is the minority of active sequences.What is the general structure of chromatin, and what is the difference between active and inactive sequences?The high overall packing ratio of the genetic material immediately suggeststhat DNA cannot be directly packaged into the final structure of chromatin. There must be hierarchies of organization. Thefundamentalsubunit of chromatinhas the sqmetype of designin all eukaryofes.The nucleosome contains -200 bp of DNA, organized by an octamer of small, basic proteins into a beadlike structure. The protein components are histones. They form an interior core; the DNA lies on the surface of the particle. Nucleosomes are an invariant component of euchromatin and heterochromatin in the interphase nucleus and of mitotic chromosomes.The nucleosomeprovides the first level of organization, giving a packing ratio of -6. Its components and structure are well characterized. The second level of organization is the coiling of the series of nucleosomes into a helical
il:l1JffiTi"[1':,',f :i:ffi,tlil.'"'ff if#: m i t o t i cc h r o m o s o m elss e eF i g u r e2 8 . 1 1 ) . I n chromatinthis bringsthe packingratio of DNA
758
CHAPTER 29 Nucteosomes
to -40. The structure of this fiber requires additional proteins, but is not well defined. The final packing ratio is determined by the third level of organization, the packaging of the 30 nm fiber itself. This gives an overall packing ratio of -I000 in euchromatin, cyclically interchangeable with packing into mitotic chromosomes to achieve an overall ratio of -10,000. Heterochromatin generally has a packing ratio of -10,000 in both interphase and mitosis. We need to work through these levels of organization to characterize the events involved in cyclical packaging, replication, and transcription. We assumethat associationwith additional proteins. or modifications of existing chromosomal proteins, are involved in changing the structure of chromatin. We do not know the individual targets for controlling cyclical packaging. Both replication and transcription require unwinding of DNA, and thus must involve an unfolding of the structure that allows the relevant enzymes to manipulate the DNA. This is likely to involve changes in all levels of organization. When chromatin is replicated, the nucleosomes must be reproduced on both daughter duplex molecules. In addition to asking how the nucleosome itself is assembled,we must inquire what happens to other proteins present in chromatin. Replication disrupts the structure of chromatin, which indicates that it both poses a problem for maintaining regions with
specific structure and offers an opportunity to change the structure. The mass of chromatin contains up to twice as much protein as DNA. Approximately half of the protein mass is accounted for by the nucleosomes.The massof RNA is < l0% of the mass of DNA. Much of the RNA consists of nascent transcripts still associatedwith the template DNA. The nonhistones include all the proteins of chromatin except the histones.They are more variable between tissuesand species,and they comprise a smaller proportion of the mass than the histones.They also comprisea much larger number of proteins, so that any individual protein is present in amounts much smaller than any histone. The functions of nonhistone proteins include control of gene expressionand higherorder structure. Thus RNA polymerase may be consideredto be a prominent nonhistone. The HMG (high-mobility group) proteins comprise a discreteand well-defined subclassof nonhistones (at least some of which are transcription factors).A major problem in working with other nonhistones is that they tend to be contaminated with other nuclear proteins, and so far it has proved difficult to obtain those nonhistone proteins responsiblefor higher-order structures.
nuctei consists i:iriiii'lLiill. "l Chromatin outof tysed spitting Thebaris seriesof particles. of a compactly organized fron Cell,vot.4, Oudet.P.,etal.,ELec100nm.Reproduced 1975,wlth Copyright . . . . pp.281-300. tronmicroscopic permission Chambon. of Pierre Photocourtesy fromElsevier.
TheNucleosome Is the Subunit of Atl.Chromatin r MicrococcaI nuclease releases individual. nucteosomes fromchromatin as11Sparticles. -200 bp of DNA,two . A nucteosome contains (H24,H2B.H3. histone copiesof eachcore a n dH 4 ) . e DNAis wrapped around the outside surface of the proteinoctamer.
When interphasenuclei are suspendedin a solution of low ionic strength, they swell and rup:li;.1, ture to release fibers of chromatin. $'i{;i"J{":*: shows a lysed nucleus in which fibers are streaming out. In some regions, the fibers consist of tightly packed material, but in regions that have become stretched, they can be seen to consist of discrete particles. These are the nucleosomes.In especiallyextended regions, individual nucleosomesare connected by a fine thread, which is a free duplex of DNA. A continuousduplexthread of DNA runs through the series of particles.
by Fiili,jiii:j':;r,.,IndividuaInuc[eosomes are reteased Thebar nuclease. wjthmicrococcal digestion of chromatin js 100nm.Reproduced P.,et at., vo[.4, Oudet. fromCelL, 1975, microscopic . . . , pp.281-300.Copyright Electron of Pierre Photocourtesy fromEtsevier. with permission Chambon. Individual nucleosomes can be obtained by treating chromatin with the endonuclease micrococcal nuclease, which cuts the DNA thread at the junction between nucleosomes. First it releasesgroups of particles, and then, it releasessingle nucleosomes.Individual nucle;1F"fas compact osomes can be seen in i,l{,i.iSil -I lS. at particles.They sediment contains-200 bp of DNA assoThenucleosome of two copies that consists octamer ciatedwith a histone known are These H4. H3, and eachof H2A, H2B, is association Their as the core histones.
of AttChromatin 759 Is theSubunit 29.2Ihe Nucteosome
G G H 2 Ax 2 = z 8 k D @@Hzex2=28kD (lfi)ttsx2 =3okD @ @ H 4x 2 = 2 2 k D 2 0 0 b p D N A = 1 3 0k D Length= 67 nm
Axisof symmetry = 3.2nm! Protein <--! Radiusof gyration !
Totalprotein = 108 kD
6nm @
H1=z4kD
-___-------____occupymost of height(6 nm)
1 1n m
iliuRI tF.3 Thenucteosome IS.5 Thetwo turnsof DNAon the nucleosome consists of approximately ruSeiffE e q u am I a s s eosf D N Aa n dh i s t o n e(si n c t u d i nHg1 ) .T h e lie ctosetogether. predicted massof the nucleosome is 262kD.
DNA "leaves"
DNA"enters" Sites80 bp apart on linearDNA are closetogetheron nucleosome
760
FiStiRg *9.4 Thenucteosome maybea cylinder withDNA organized into twoturnsaround the surface.
tlfi{JBfit9-$ Sequences onthe DNAthatlieon djfferent turnsaround the nucleosome mavbe ctosetoqether.
illustrated diagrammatically in f.gGiJRf *S"i. This model explains the stoichiometry of the core histones in chromatirL; H2A, H2B, H3, and H4 are present in equimolar amounts, with two molecules of each per -200 bp of DNA. Histones H3 and H4 are among the most conserved proteins known. This suggeststhat their functions are identical in all eukaryotes. The types of H2A and H2B can be recognized in all eukaryotes, but show appreciable speciesspecificvariation in sequence. Histone Hl comprisesa set of closelyrelated proteins that show appreciable variation between tissuesand between species.The role of Hl is different from that of the core histones. It is present in half the amount of a core histone and can be extracted more readily from chromatin (typically wirh dilure salr [0.5 M] solution). TheH1 canberemovedwithoutfficting the structureof the nucleosome, which suggests that its location is external to the particle The shape of the nucleosome corresponds to a flat disk or cylinder of diameter 11 nm and
height 6 nm. The length of the DNA is roughly twice the -34nm circumference of the particle. The DNA follows a symmetrical path around the octamer. i:ISugEt$.4 shows the DNA path diagrammatically as a helical coil that makes two turns around the cylindrical octamer. Note that the DNA "enters" and "leaves" the nucleosome at points close to one another. Histone Hl may be locatedin this region (seeSection29.4, Nucleosomes Have a Common Structure). Considering this model in terms of a crosssection through the nucleosome, in t'gfiti&flt$-S we seethat the two circumferences made by the DNA lie close to one another. The height of the cylinder is 6 nm, of which 4 nm is occupied by the two turns of DNA (each of diameter 2 nm). The pattern of the two turns has a possible functional consequence. One turn around the nucleosome takes -80 bp of DNA, so two points separatedby 80 bp in the free double helix may actually be close on the nucleosome surface, as illustrated in $3SEJffifr f S.{i.
C H A P T E2R9 N u c t e o s o m e s
ili.i;3lf ]lS-li MicrococcaI nuctease digests chromatin in nucleiinto a multimeric series of DNAbandsthat canbe separated bygeletectrophoresis. Photocourtesy of Markus Nott,Urniversitdt Ziirich.
ExtractDNA and electrophorese
DNAIs Coited in Arrays of Nucleosomes c
>95%of the DNAis recovered in nucleosomes or muttimers whenmicrococcaI nuctease DNA cteaves of chromatin. TheLength of DNApernucteosome variesfor jn a rangefrom154to 260bp. individual, tissues
When chromatin is digested with the enzyme micrococcalnuclease,the DNA is cleavedinto integral multiples of a unit length. Fractionation by gel electrophoresisrevealsthe "ladder" presentedin Fitil"jfi*Jt3"f. Such laddersextend for -I0 steps,and the unit length. determined by t h e i n c r e m e n t s b e t w e e n s u c c e s s i v es t e o s .i s -200 bp. i:i.r1ifiii!r.it shows that the ladder is generated by groups of nucleosomes.When nucleosomes are fractionated on a sucrosegradient, they give a seriesof discretepeaks that correspond to monomers, dimers, trimers, and so on. When the DNA is extracted from the individual fractions and electrophoresed,each fraction yields a band of DNA whose size corresponds with a step on the micrococcal nuclease ladder. The monomeric nucleosome contains DNA of the unit length, the nucleosome dimer contains DNA of twice the unit length, and so on. Each step on the ladder representsthe DNA derived from a discretenumber of nucleosomes. We thereforetake the existenceof the 200 bp ladder in any chromatin to indicate that the DNA is orga-
z o
P rooo
o J
800 600 400
the contains ;ti].;i Eachmultimer of nucteosomes il?i.rll[1I of DNA.In thephoto, number of unit[engths appropriate Theimagewascona DNAladder. artjficiaI bands simulate to withsizescorresponding fragments structed usingPCR Wyeth ofJanKieteczawa, actuaIbandsizes.Photocourtesy Research.
The micrococcal ladder is nizedinto nucleosomes generated when only -2o/o of the DNA in the nucleus is rendered acid-soluble (degradedto small fragments) by the enzyme. Thusa small proportion of the DNA is specificallyattacked;itmust representespeciallysusceptibleregions. When chromatin is spilled out of nuclei, we often seea seriesof nucleosomesconnectedby a thread of free DNA (the beads on a string). The need for tight packaging of DNA in vivo, however, suggeststhat probably there is usually little (if any) free DNA.
jn Arravs of Nuc[eosomes 767 29.3 DNAIs Coiled
This view is confirmed by the fact that>95yo 0f the DNA of chromatin can be recoveredin theform of the200 bp ladder.Almost all DNA must therefore be organizedin nucleosomes.In their natural state,nucleosomesare likely to be closely packed,with DNA passingdirectly from one to the next. Free DNA is probably generated by the loss of some histone octamers during isolation. The length of DNA present in the nucleosome varies somewhat from the "typical" value of 200 bp. The chromatin of any particular cell type has a characteristicaveragevalue (+5 bp). The averagemost often is between 180 and 200, but there are extremes as low as 154 bp (in a fungus) or as high as 260 bp (in a sea urchin sperm). The averagevalue may be different in individual tissues of the adult organism, and there can be differencesbetween different parts of the genome in a single cell type. Variations from the genome average include tandemly repeatedsequences,such as clustersof 5S RNA senes.
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Nucteosomes Have
a Common Structure
r Nucleosomal DNAis divided into the coreDNAano tinkerDNAdepending on its susceptibiLity to micrococcaI nuc[ease. r ThecoreDNAis the [engthof 146bp that is found on the coreparticles produced by prolonged digestion with micrococcaI nuclease. . LinkerDNAis the regionof 8 to 71,4bpthat is susceptibte to earlycleavage bythe enzyme. o Changes in the lengthof [inkerDNAaccount for jn totallengthof nucleosomal the variation DNA. r H1is associated with linkerDNAandmaylie at the pointwhereDNAentersandleaves the nucteos0me. A common structure underlies the varying amount of DNA that is contained in nucleosomes of different sources.The associationof DNA with the histone octamer forms a core particle containing 146 bp of DNA, irrespective of the total length of DNA in the nucleosome.The variation in total length of DNA per nucleosome is superimposed on this basic core structure. The core particle is defined by the effects of micrococcal nuclease on the nucleosome monomer. The initial reaction of the enzyme is to cut between nucleosomes,but if it is allowed to continue after monomers have been sener-
C H A P T E2R9 N u c t e o s o m e s
Base parrs 180 160 140
Time of dioestion -.>
tgliilftl t:1.* Micrococcal nuclease reduces the lenqth of nucteosome monomers in discrete steps.Photocourtesyof RogerKornberg. StanfordUniversity Schoolof Medicine. ated, then it proceedsto digestsome of the DNA of the individual nucleosome.This occursby a reaction in which DNA is "trimmed" from the ends of the nucleosome. The length of the DNA is reduced in discrete steps,as shown in !:ttjljftf ;i*.t. With rat liver nuclei, the nucleosome monomers initially have 205 bp of DNA. After the first step. some monomers are found in which the length of DNA has been reduced to -I65 bp. Finally, this is reduced to the length of the DNA of the core particle, 146bp. (The core is reasonably stable, but continued digestion generatesa "limit digest." In the limit digest,the longest fragments are the 146 bp DNA of the core, whereas the shortestare as small as 20 bp.) This analysis suggeststhat the nucleosomal DNA can be divided into two regions: o Core DNA has an invariant length of 146 bp, and is relatively resistant to digestionby nucleases. o Linker DNA comprises the rest of the repeating unit. Its length varies from as little as 8 bp to as much as 114 bp per nucleosome. The sharp size of the band of DNA generated by the initial cleavagewith micrococcal nuclease suggeststhat the region immediately available to the enzyme is restricted. It represents only part of each linker. (If the entire linker DNA were susceptible, the band would range
from 146 bp to >200 bp.) Once a cut has been made in the iinker DNA, though, the rest of this r e g i o n b e c o m e s s u s c e p t i b l e ,a n d i t c a n b e removed relatively rapidly by further enzyme action. The connection between nucleosomes is represented in iili":,i-ilill ,a;i.'iii. Core particles have properties similar to those of the nucleosomes themselves,although they are smaller. Their shape and size are similar to those of nucleosomes;this suggeststhat the essentialgeometry of the particle is established by the interactions between DNA and the protein octamer in the core particle. Core particlesare more readily obtained as a homogeneous population, and as a result they are often used for structural studies in preference to nucleosomepreparations. (Nucleosomestend to vary because it is difficult to obtain a preparation in which there has been no endtrimming of the DNA.) What is the physicalnature of the core and the linker regions? Thesetermswereintroducedas operationaldefinitionsthat describethe regionsin termsof their relativesusceptibility to nucleasetreatment.This description does not make any implication about their actual structure.It turns out, though, that the major part of the core DNA is tightly curved on the nucleosome, whereas the terminal regions of the core and the linker regions are more extended (see Section 29.5, DNA Structure Varies on the Nucleosomal Surface). The existence of linker DNA depends on factors extraneous to the four core histones. Reconstitution experimenls in vitro show that histones have an intrinsic ability to organize DNA into core particles,but do not form nucieosomeswith the proper unit length. The degree of supercoiling of the DNA is an important factor. Histone Hl and/or nonhistone proteins influence the length of linker DNA associated with the histone octamer in a natural seriesol nucleosomes."Assemblyproteins" that are not part of the nucleosome structure are involved in vivo in constructing nucleosomes from hist o n e s a n d D N A ( s e e S e c t i o n2 9 . 9 , R e p r o d u c tion of Chromatin Reouires Assemblv of Nucleosomes). Where is histone Hl located?The Hl is lost during the degradation of nucleosome monomers. It can be retained on monomers that still have 165 bp of DNA, but is always lost with the final reduction to the 146 bp core particle. This suggeststhat HI could be located in the region of the linker DNA immediately adjacent to the core DNA.
+
200bp *
165bp +
146bp
Core particles Trimmed Mononucleosomes nucleosomes
nucteinitiatlvcleaves between nuctease MicrococcaI t yepsi c a t t yh a v e- 2 0 0 b p D N A .E n d o s o m e sM . ononucteosom of DNAfirstto -165 bp,andthengentrimmingreduces the Length particles with 146 bp. eratescore
If Hl is located at the linker, it could "seal" the DNA in the nucleosome by binding at the point where the nucleic acid enters and leaves ( s e eF i g u r e 2 9 . 4 ) . T h e i d e a t h a t H l l i e s i n t h e region joining adjacentnucleosomesis consistent with old results that Hl is removed the most readily from chromatin, and that Hldepletedchromatin is more readily "solubilized." In addition, it is easier to obtain a stretched-out fiber of beads on a string when the Hl has been removed.
Varies on DNAStructure Surface the NucleosomaI o DNAis wrapped the histone 1.65timesaround octamer. o Thestructure of the DNAis alteredsothat it has in the number of basepairs/turn anjncreased number at the ends. middte. buta decreased
The exposure of DNA on the surfaceof the nucleosome explains why it is accessibleto cleavageby certain nucleases.The reaction with nucleasesthat attack single strands has been especiallyinformative. The enzymes DNAase I and DNAaseII make single-strandnicks in DNA; they cleavea bond in one strand, but the other strand remains intact at this point. Thus no effect is visible in the double-stranded DNA. Upon denaturation, though, short fragments are releasedinstead of full-length single strands.If the DNA has been labeled at its ends, the end fragments can be identified by autoradiography, as summarized in r lilijltf, i:ii.t :. When DNA is free in solution, it is nicked (relatively) at rand o m . T h e D N A o n n u c l e o s o m e sa l s o c a n b e nicked by the enzymes, but only at regular intervals. When the points of cutting are
Surface 763 on the Nucleosoma[ Varies 29.5 DNAStructure
Electrophoresis
Labeledfragment
Labeled fragment+
i::+ijiiFl:*..11 Njcksin double-stranded DNAarerevealed by fragments whenthe DNAis denatured to givesingtestrands. If the DNAis labeledat (say)5'ends.on[ythe 5'fragments arevisibleby autoradiography. Thesizeof thefragment identifies the distance of the nickfromthe [abeled end.
S l i s - I 0 b a s e sf r o m t h e l a b e l e d 5 ' e n d , 5 2 i s -20 basesfrom it, and so on). Their positions
s12 s11 s10 S9 S8
s7 S6 JC
irii;t-:*.ir ;-::"i: Sitesfor nickinglie at regutar interva[s atongcoreDNA,asseenin a DNAase I digestof nuclei. Photocourtesy of Leonard C.Lutter,HenryFordHospita[, Detroit,ML
determined by using radioactivelyend-labeled DNA and then DNA is denatured and electrophoresed, a Iadder of the sort displayed in i:ii.;F:al:ij..1:is obtained. The interval between successivesteps on the ladder is l0 to I L bases.The ladder extends for the full distance of core DNA. The cleavage sitesare numbered as Sl through Sl3 (where
764
C H A P T E2R9 N u c t e o s o m e s
relative to the DNA superhelix are illustrated in F{*i.iR[:*"13. Not all sites are cut with equal frequency: Some are cut rather efficiently, whereas others are cut scarcelyat all. The enzymes DNAase I and DNAase II generate the same ladder, although with some differences in the intensities of the bands. This shows that the pattern of cutting representsa unique seriesof targets in DNA. determined by its organization, with only some slight preference for particular sitesimposed by the individual enzyme. The same cutting pattern is obtained by cleaving with a hydroxyl radical, which argues that the pattern reflects the structure of the DNA itself rather than any sequencepreference. The sensitivity of nucleosomal DNA to nucleasesis analogous to a footprinting experiment. Thus we can assign the lack of reaction at particular target sites to the structure of the nucleosome, in which certain positions on DNA are rendered inaccessible. There are two strands of DNA in the core particle, so in an end-labeling experiment both of the 5' (or 3') ends are labeled, one on each strand. Thus the cutting pattern includes fragments derived from both strands.This is implied in Figure 29.I1, where each labeled fragment is derived from a different strand. The corollary is that, in an experiment, each labeled band in fact can represent two fragments that are generated by cutting lhe samedistance hotli. either of the labeled ends.
How, then, should we interpret discrete preferencesat particular sites?One view is that the path of DNA on the particle is symmetrical (about a horizontal axis through the nucleosome, as drawn in Figure 29.4). If.,for example, no S0-base fragment is generated by DNAase I, this must mean that the position at 80 basesfrom the 5'end of.eitherstrand is not susceptibleto the enzyme. The second numbering schemeused in Figure 29.13 reflectsthis view, and identifies 57 = site 0 as the center of symmetry. When DNA is immobilized on a flat surface, sites are cut with a regular separation. lii suggeststhat this reflectsthe recurijltii.ii:l:;.:i:r. rence of the exposedsite with the helical periodicity of B-form DNA. The cutting periodicity (the spacingbetween cleavagepoints) coincides with-indeed, is a reflection of-the structural periodicity (the number of basepairs per turn of the double helix). Thus the distancebetween the sitescorrespondsto the number of basepairs per turn. Measurements of this type suggest that the averagevalue for double-helical B-type D N A i s 1 0 . 5b p / t u r n . What is the nature of the target siteson the nucleosome? riir\i+ii-:;i'.i.:ir: shows that each site has three to four positions at which cutting occurs; that is, the cutting site is defined t2 bp. Thus a cutting site represents a short stretch of bonds on both strandsthat is exposedto nucleaseaction over three to four basepairs. The relative intensities indicate that some sites are preferred to others. From this pattern, we can calculate the "average" point that is cut. At the ends of the DNA, pairs of sitesfrom Sl to 54 or from Sl0 to SI 3 lie apart a distanceof 10.0baseseach.In the center of the particle, the separation from sites54 to SI 0 is I 0.7 bases.(This analysisdeals with averagepositions, so sites need not lie at an integral number of basesapail.) The variation in cutting periodicity along the core DNA (10.0 at the ends, 10.7in the middle) means that there is variation in the structural periodicity of core DNA. The DNA has more bp/turn than its solution value in the middle, but has fewer bp/turn at the ends. The average periodicity over the nucleosome is only 10.17 bp/turn, which is significantly less than the 10.5 bp/turn of DNA in solution. The crystal structure of the core particle suggeststhat DNA is organized as a flat superhelix, with 1.65 turns wound around the histone octamer. The pitch of the superhelix varies and has a discontinuity in the middle. Regions
Side view
Top view
DNA schemes dividecoreparticle i'li:tii:il'l:i,i :; Twonumbering S1to 513fromone into 1.0bp segments. Sitesmaybe numbered 0 of thedyadsymmetry, end;or taking57to identifycoordinate -7 to +7. thevmavbe numbered
positions on DNArecur ii.ii:iiili::i:i;j, i n Themostexposed with a periodicity that reflectsthe structureof the doubtehetix.(Forctarity,sitesareshownfor ontyonestrand.)
thateach shows anatysis ;:i;,, ! -, Highresotution ;iIi:i,ii-li: susceptibte adjacent of severaI I consists sitefor DNAase of sites phosphodiester bonds,as seenin this exampte 5 4 a n d S 5 a n a t y z e idn e n d - t a b e t ecdo r e p a r t i c l e s . C.Lutter.HenryFordHospitat. of Leonard Photocourtesy Detroit,MI.
Surface 7 6 5 on the Nucteosomal Varies 29.5 DNAStructure
of high curvature are arranged symmetrically, and occur at positions +l and +4. These corres p o n dt o 5 6 a n d S 8 ,a n d t o 5 3 a n d S I l , r e s p e c tively, which are the sites least sensitiveto DNAase I. A high-resolution structure of the nucleosome core shows in detail how the structure of DNA is distorted.Most of the supercoilingoccurs in the central 129 bp, which are coiledinto 1.59 left-handed superhelicalturns with a diameter of 80 A (only four times the diameter of the DNA duplex itself). The terminal sequenceson either end make only a very small contribution to the overall curvature. The central 129 bp are in the form of BDNA. but with a substantial curvature that is needed to form the superhelix. The major groove is smoothly bent, but the minor groove has abrupt kinks. Theseconformational changes may explain why the central part of nucleosomal DNA is not usually a target for binding by reguiatory proteins, which typically bind to the terminal parts of the core DNA or to the linker sequences.
@
ThePeriodicity of DNA Changes on the Nuc[eosome
that nucleosome strings can take more than one f.orminvitro, depending on the conditions. The degree of supercoiling on the individual nucleosomes of the minichromosome can be measured as illustrated in ft{ilJft*!l$.11 *. First, the free supercoils of the minichromosome itself are relaxed, so that the nucleosomes form a circular string with a superhelical density of 0. Next, the histone octamers are extracted. This releasesthe DNA to follow a free path. Every supercoil that was present but constrained in the minichromosome wili appear in the deproteinized DNA as -l turn. Now the total number of supercoils in the SV40 DNA is measured. The observed value is close to the number of nucleosomes.The reverseresult is seenwhen nucleosomesare assembledinvitroonto a supercoiled SV40 DNA: The formation of each nucleosome removes -l negative supercoil. Thus the DNA follows a path on the nucleosomal surfacethat generates-l negative supercoiled turn when the restraining protein is removed. The path that DNA follows on the n u c l e o s o m e , t h o u g h , c o r r e s p o n d st o - 1 . 6 7 superhelicalturns (see Figure 29.4). This discrepancy is sometimes called the linking number paradox.
o -0.6 negative turnsof DNAareabsorbed by the change in bp/turnfrom10.5in solutjonto an average of 10.2on the nucteosomaI surface. which explains paradox. the [inking-number
I Treatwithtopoisomerase
Some insights into the structure of nucleosomal DNA emerge when we compare predictions for supercoilingin the path that DNA follows with actual measurements of supercoiling of nucleosomal DNA. Much work on the structure of setsof nucleosomeshas been carried out with the virus SV40.The DNA of SV40 is a circular molecule of 5200 bp, with a contour length -1500 nm. In both the virion and infected nucleus, it is packagedinto a seriesof nucleosomes, which together are called a minichromosome. As usually isolated,the contour length of the minichromosome is -2 l0 nm, which correspondsto a packing ratio of ^-7 (essentiallythe same as the -6 of the nucleosome itself). Changesin the salt concentration can convert it to a flexible string o{ beadswith a much lower overall packing ratio. This emphasizesthe point
C H A P T E2R9 N u c t e o s o m e s
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FJ{itJ}Jfl tg".'r{:: Thesupercoils of the SV40mjnichromosomecanberetaxed to generate a cjrcutar structure, whose lossof hjstones thengenerates supercoi[s in thefreeDNA.
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state unwound FI#ilf{ilJ#"f* The10nmfiberinpartiatty Photo of a stringof nucteosomes. canbeseento consist of Catifornja, University Hamkato. courtesy of Barbara Irvine. FI*i.!fiiliit"ti Thegtobutar bodiesof the histones are in the histoneoctamer The locatized of thecoreoarticte. locations oftheN-terminal taits,whichcarrythesitesfor modification, arenot known, though,andcoutdbe more ftexibte.
ThePathof Nucleosomes 'tnthe Chromatin Fiber from30 nm fibersareunfotded 10 nmchromatin fibersandconsistof a stringof nucleosomes. whichare 30 nmfibershavesix nucteosomes/turn, into a sotenoid. organized of the 30 nm for formation H1is required Histone fiber.
f,:*tifril3*.I1 TheN-terminaI histonetaitsaredisorderedandexit fromthe nucleosome betweenturnsof t h eD N A . ible N-terminal tail, which has sites for modification that may be important in chromatin function. The positions of the tails, which account for about one quarter of the protein mass, are not so well defined, as indicated in FftltiftSf $.f . The tails of both Hl and H2B, however. can be s e e n t o p a s sb e t w e e n t h e t u r n s o f t h e D N A superhelix and extend out of the nucleosome, as shown in ill*u$i* ;l*.f 1. When histone tails are crosslinked to DNA by UV irradiation, more products are obtained with nucleosomes compared to core particles, which could mean that the tails contact the linker DNA. The tail of H4 appears to contact anH2A-H2B dimer in an adjacent nucleosome; this could be an important feature in the overall structure.
When chromatin is examined in the electron microscope, two types of fibers are seen: the l0 nm fiber and f 0 nm fiber. They are described by the approximate diameter of the thread (that of the 30 nm fiber actually varies from -25-30 nm). The l0 nm fiber is essentially a continuous string of nucleosomes.In fact, at times it runs continuously into a more stretched-out region in which nucleosomesare seenas a string of beads, as indicated in the example of F5*ljtrilt+.;i;]. The I0 nm fibril structure is obtained under conditions of low ionic strength and does not require the presence of histone HI. This means that it is a function strictly of the nucleosomes themselves. It may be visualized essentially as a continuous series of nucleosomes, as shown in flI{i{JF'til'S.!.$.It is not clear whether such a structure existsinvivo or is simply a consequence of unfolding during extraction in vitro When chromatin is visualized in conditions of greater ionic strength, the 30 nm fiber is
Fiber in the Chromatin 29.8 ThePathof Nucleosomes
769
iji,l;iti i;":i.::::: The10 nm fiber is a continuousstrinqof nuc[eosomes.
i*l*tJF.[i]$"il:iThe30 nmfiberis a heticaI ribbonconsistingof two parattel rowsof nucteosomes coitedjnto a solenoid.
i:iiiiii:i .il.f + The30 nm fiberhasa coitedstructure. Photocourtesy of Barbara Hamka[0. University of Catifornia,Irvine.
of both interphase chromatin and mitotic chromosomes. The most likely arrangement for packing n u c l e o s o m e si n t o t h e f i b e r i s a s o l e n o i d , i n which the nucleosomes turn in a helical array that is coiled around a central cavity. The two main forrns of a solenoid are a single-start, which forms from a single linear array, and a two-stan, which in effect consistsof a double row of nucleosomes. eI*{.:ff*;S.il$ shows a two-start model suggestedby recent crosslinking data identifying a double stack of nucleosomes in the 30 nm fiber. This is supported by the crystal structure of a tetranucleosome complex. The 30 nm and l0 nm fibers can be reversibly converted by changing the ionic strength. This suggeststhat the linear array of nucleosomesin the l0 nm fiber is coiled into the 30 nm structure at higher ionic strength and in the presenceof Hl. Although the presenceof Hl is necessaryfor the formation of the l0 nm fiber, information about its location is conflicting. Its relative ease of extraction from chromatin seems to argue that it is present on the outside of the superhelical fiber axis. Diffraction dara, though, and the fact that it is harder to find in 30 nm fibers than in l0 nm fibers that retain it, would argue for an interior location.
;l'J',ir"s; *T:l?T ;t',""i':i ff:Li;,il;
coiled structure. It has -6 nucleosomesfor every turn, which correspondsto a packing ratio of 40 (that is, each pm along the axis of the fiber cont a i n s 4 0 p m o f D N A ) . T h e p r e s e n c eo f H l i s required. This fiber is the basic constituenr
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C H A P T E2R9 N u c t e o s o m e s
How do we get from the 30 nm fiber to the specificstructures displayed in mitotic chromosomes? Is there any further specificity in the arrangement of interphase chromatin? Do particular regions of 30 nm fibers bear a fixed relationship to one another, or is their arrangement random?
Reproduction of Chromatin Requires Assemb[y of Nucleosomes o Histone octamers arenot conserved during reptication, but H2A-H28 dimersandH32-H4z tetramers areconserved. o Therearedifferentpathways for the assembly of nucleosomes duringreplication andindependentty of reoUcation. o Accessory proteinsarerequired to assistthe assembty of nucleosomes. . CAF-1 proteinthat is linkedto the is an assembly PCNA subunitof the replisome; it is required for deposition of H3z-H4z tetramers fotlowing reptication. r A different proteinanda variantof assembty histoneH3maybe usedfor replicationindependent assemb[y.
Replication separatesthe strands of DNA and therefore must inevitably disrupt the structure of the nucleosome.The transienceof the replication event is a major difficulty in analyzing the structure of a particular region while it is being replicated. The structure of the replication fork is distinctive. It is more resistant to micrococcal nucleaseand is digestedinto bands that differ in size from nucleosomal DNA. The region that shows this altered structure is confined to the immediate vicinity of the replication fork. This suggeststhat a large protein complex is engaged in replicating the DNA, but the nucleosomes re-form more or less immediately behind it as it moves along. Reproduction of chromatin does not involve any protracted period during which the DNA is free of histones. Once DNA has been replicated, nucleosomes are quickly generated on both the duplicates. This point is illustrated by ;lrri.!j*,which the electron micrograph of ttri.{.i.iiii, shows a recently replicated stretch of DNA that is already packagedinto nucleosomeson both daughter duplex segments. Both biochemical analysis and visualization of the replication fork therefore suggest that the disruption of nucleosome structure is
ri: Replicated i ncorporated DNAis immediately i:is;ti i;rt r:r,1..,-l L.McKnight, ofSteven intonucteosomes. Photocourtesy at Dat[as. Center MedicaI UTSouthwestern Iimited to a short region immediately around the fork. Progressof the fork disrupts nucleosomes,but they form very rapidly on the daughter duplexes as the fork moves forward. In fact, the assembly of nucleosomes is directly linked to the replisome that is replicating DNA. How do histonesassociatewith DNA to generate nucleosomes? Do the histones preform a protein octamer around which the DNA is subsequently wrapped? Or does the histone octamer assembleon DNA from free histones? iritiiririi:,ri: .ii shows that two pathways can be used ir vitr7 Io assemblenucleosomes, depending on the conditions that are employed. In one pathway, a preformed octamer binds to DNA. In the other pathway, a tetramer of.H3z-H4z binds first, and then twoH2A-H2B dimers are added. Both these pathways are related to reactions that occur in vivo The first reflects the capacity of chromatin to be remodeled by moving histone octamers along DNA (see Section 10.3, Chromatin Remodeling Is an Active Process).The second representsthe pathway t h a t i s u s e di n r e p l i c a t i o n . Accessoryproteins are involved in assisting histones to associatewith DNA. candidates for this role can be identified by using extracts that assemblehistonesand exogenousDNA into nucleosomes. Accessory proteins may act as "molecular chaperones" that bind to the histones in order to releaseeither individual histones or complexes (H32-H42or H2A-H2B) to the DNA in a controlled manner. This could be necessarybecause the histones, as basic proteins, have a general high affinity for DNA. Szcft withinteractionsallow histonestoform nucleosomes out becomingtrappedin other kineticintermediates (that is, other complexesresultingfrom indiscreet binding of histonesto DNA).
Assembty of Nucleosomes of ChromatinRequires 29.9 Reproduction
Octamerassembles Preformedoctamer binds on DNA
I uzn-Hza
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=*i"l*[ ,?i].1.:In vifro,DNAcaneitherinteractdirectty withanjntact(crossl.inked) histoneoctamer or canassembtewiththe H32-H42 tetramer, afterwhichtwo H2A-H2B dimersareadded. Attempts to produce nucleosomesin vitro b e g a n b y c o n s i d e r i n g a p r o c e s so f a s s e m b l y between Iree DNA and histones.Nucleosomes Iorm in vivo, though, only when DNA is replicated. A system that mimics this requirement has been developed by using extracts of human cellsthat replicateSV40 DNA and assemblethe products into chromatin. The assembly reaction occurs preferentially on replicating DNA. It requires an ancillary factor, chromatin assembly factor (CAF)- 1, that consisrsof >5 subunits, with a total mass of 238 kD. CAF- I is recruited to the replication fork by proliferating cell nuclear antigen (PCNA), the processivityfactor for DNA polymerase.This provides the link between replicationand nucleosomeassembly,
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CAF-1 acts stoichiometrically, and functions by binding to newly synrhesizedH3 and FI4.This suggeststhat new nucleosomesform by assembling first the H32-H42 tetramer, and then adding the H2A-H2B dimers. The nucle-
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C H A P T E2R9 N u c t e o s o m e s
F{filJltE tS.** If histoneoctamers wereconserved, old andnewoctamers woutdbandat different densities when replication of heavy octamers occurs in lightaminoacids.
osomes that are formed in vitro have a repeat length of 200 bp. They do not have any Hl histone. though, which suggeststhat proper spacing can be accomplishedwithout HI. When chromatin is reproduced, a stretch oIDNA alreadyassociated with nucleosomes is replicated, giving rise to two daughter duplexes. What happens to the preexisting nucleosomes at this point? Are the histone octamers dissociated into free histones for reuse, or do they remain assembled?The integrity of the octamer can be tested by crosslinking the histones. The next two figures compare the possibleoutcomes from an experiment in which cells are grown in the presence of heavy amino acids to identify the histones before replication. Replication is then allowed to occur in the presence of light amino acids.At this point the histone octamers are crosslinked and centrifuged on a density gradient. fl3fiLlSil JS.ffSshows that if the original octamers have been conserved, they will be found at a position of high density, and new octamers will occupy a low density position.
Replicationfork advancestoward nucleosome
Histonetetramer is displacedand disassembles
3. H3-H4tetramersbind to daughter duplexes
4. H2A-H2Bdimersbind
fromDNA. F.i{;{Jlr.[ histoneoctamers disptaces forkpassage iiii,-t* RepLication synthesized Newty dimers. andH2A-H2B tetramers into H3-H4 Thevdisassembl.e Theotdand dimers. andH2A-H2B tetramers into H3-H4 areassembted histones into withthe aidof cAF-1at random anddimersareassembled newtetramers fork. reptication the behind immediatety nucteosomes new
in arereplicated f,itlitre i*" i:F Whenheavyoctamers between banddiffuseLy thenewoctamers lightaminoacids, whichsuggests thatdisassemheavyand[ightdensities, tetramers and dimers, which are assembledfrom hasoccurred. btyandreassembty newly synthesized histones. Nucleosomes assemble-600 bp behind the replication fork. This does not happen, though. Little material is Assembly is initiated when H32-H42 tetramers found at the high density position, which sugbind to each of the daughter duplexes, assisted geststhat histone octamers are not conserved. by CAF-1. TWo H2A-H2B dimers then bind to The octamershave an intermediate density,and each H32-H42tetramer to complete the histone ${{:;.:ft{: *+.:li.lshows that this is the expectedresult octamer. The assembly of tetramers and dimers if the old histoneshave been releasedand then is random with respectto "old" and "new" subreassembledwith newly synthesized histones. units, which explains the resultsof Figure 29 .29The pattern of disassemblyand reassembly The "old" H32-H42tetramer could have an abilhas been difficult to characterize in detail, but t*.3*. ity to be transiently associatedwith a single a working model is illustrated in FEGiisf, strand of DNA during replication; it may in fact The replication fork displaceshistone octamers, have an increased chance of remaining with which then dissociate into H32-H42 tetramers the Ieading strand for reuse. It is possible that and H2A-H2B dimers. These "old" tetramers nucleosomes are disrupted and reassembledin and dimers enter a Dool that also includes "Dew"
of Nucleosomes 773 Assembly Requires of chromatin 2g.g Reproduction
a similar way during transcription (see Section 29.I l, Are Tlanscribed Genes Organized in Nucleosomes?). During S phase (the period of DNA replication) in a eukaryotic cell, the duplication of chromatin requires synthesis of sufficient histone proteins to package an entire genomebasicallythe same quantity of histonesmust be synthesizedthat are already contained in nucleosomes.The synthesisof histone mRNAs is controlled as part of the cell cycle, and increases enormously in S phase.The pathway for assembling chromatin from this equal mix of old and new histones during S phase is called the replication-coupled (RC) pathway. Another pathway, called the replicationindependent (RI) pathway, exists for assembling nucleosomesduring other phasesof cell cycle,when DNA is not being synthesized.This may become necessaryas the result of damage to DNA or becausenucleosomesare displaced during transcription. The assemblyprocessmust necessarilyhave some differencesfrom the replication-coupled pathway, becauseit cannot be Iinked to the replication apparatus.One of the most interesting features of the replicationindependent pathway is that it uses different variants of some of the histones from those used during replicarion. The histone H3.3 variant differs from the highly conserved H3 histone at four amino acid positions.H3.3 slowly replacesHl in differentiating cells that do not have replication cycles. This happens as the result of assemblyof new histone octamers to replace those that have been displaced from DNA for whatever reason. The mechanism that is used to ensure the use of H3.3 in the replication-independent pathway is different in two casesthat have been investigated. In the protoz oan Tetrahymena,histone usage is determined exclusively by availabiliry. Histone H3 is synthesizedonly during the cell cycle; the variant replacement histone is synthesized only in nonreplicating cells.ln Drosophila,however, there is an active pathway that ensures the usage of H3.3 by the replicationindependent pathway. New nucleosomescontaining H3.3 assembleat sitesof transcription, presumably replacing nucleosomesthat were displaced by RNA polymerase. The assembly processdiscriminatesbetween H3 and H3.3 on the basisof their sequences,specificallyexcluding H3 from being utilized. By contrast, replication-coupledassemblyusesboth types of H3 (although H3.3 is availableat much lower levels than Hl, and therefore enters only a small proportion of nucleosomes).
774
CHAPTER 29 Nuc[eosomes
CAF-l is probably not involved in replication-independent assembly. (There also are organisms such as yeast and Arabidopsis for which its gene is not essential, implying that alternative assembly processesmay be used in replication-coupled assembly.) A protein that may be involved in replication-independent assembly is called HIRA. Depletion of HIRA from in vitro systemsfor nucleosome assembly inhibits the formation of nucleosomeson nonreplicated DNA, but not on replicating DNA, which indicatesthat the pathways do indeed use different assemblymechanisms. HIRA functions as a chaperone to assistthe incorporation of histones into nucleosomes. This pathway appears to be generally responsible for replication-independent assembly;for example, HIRA is required for the decondensation of the sperm nucleus, when protamines are replaced by histones, in order to generate chromatin that is competent to be replicated following fertilization. Assembly of nucleosomes containing an alternative to H3 also occursat centromeres (see Section 31.3, Heterochromatin Depends on Interactions with Histones). Centromeric DNA replicates early during the replication phase of the cell cycle (in contrast with the surrounding heterochromatic sequencesthat replicate later; seeSection 15.7, Each Eukaryotic Chromosome Contains Many Replicons). The incorporation of H3 at the centromeres is inhibited, and instead a protein called CENP-A is incorporated in higher eukaryotic cells (in Drosophila it is called Cid, and in yeast it is called Cse4).This occurs by the replication-independent assembly pathway, apparently becausethe replication-coupled pathway is inhibited for a brief period while centromeric DNA replicates.
DoNucleosomes Lie at SpecificPositions? r Nucteosomes mayformat specificpositions asthe resutteitherof the [oca[structureof DNAor of proteinsthat interactwith specificsequences. o Themostcommon cause of nucteosome positioning is whenproteins bindingto DNA estab[ish a boundary. r Positioning mayaffectwhichregionsof DNAarein the linkerandwhichfaceof DNAis exposed on the nucleosome surface. We know that nucleosomes can be reconstituled in vitro wilhout regard to DNA sequence, but this does not mean that their formation in tzivois independent of sequence.Does a partic-
ular DNA sequencealways lie in a certain position in vivo w\th regard to the topography of the nucleosome? Or are nucleosomesarranged randomly on DNA, so that a particular sequence may occur at any location, for example, in the core region in one copy of the genome and in the linker region in another? To investigatethis question, it is necessary to use a defined sequenceof DNA; more precisely, we need to determine the position relative to the nucleosome of a defined point in the l illustratesthe principle of a DNA. :-;rl,r:tii,i1:.'r procedure used to achieve this. Suppose that the DNA sequence is organized into nucleosomes in only one particular configuration, so that each site on the DNA always is located at a particular position on the nucleosome. This type of organization is called (or sometimes nucleosome positioning nucleosome phasing).In a seriesof positioned nucleosomes,the linker regions of DNA comprise unique sites. Consider the consequencesfor just a sing l e n u c l e o s o m e . C l e a v a g ew i t h m i c r o c o c c a l nuclease generatesa monomeric fragment that constitutes a specificsequenceIf. the DNA is isolated and cleavedwith a restriction enzyme that has only one target site in this fragment, it should be cut at a unique point. This produces two fragments, each of unique size. The products of the micrococcal/restriction double digestare separatedby gel electrophoresis.A probe representing the sequenceon one side of the restriction site is used to identify the corresponding fragment in the double digest. This technique is called indirect end labeling. Reversing the argument, the identification a of single sharp band demonstrates that the position of the restriction site is uniquely defined with respectto the end of the nucleosomal DNA (as defined by the micrococcal nuclease cut). Thus the nucleosome has a unique sequenceof DNA. What happensif the nucleosomesdo rollie at a single position? Now the linkers consist of differentDNA sequences in each copy of the genome. Thus the restriction site lies at a different position each time; in fact, it lies at all possible locations relative to the ends of the jrl: ,r1ir, mOnomeriC nuCIeOSOmalDNA. .i;ji,i,flil, generates shows that the double cleavagethen a broad smear, ranging from the smallest detectablefragment (-20 bases)to the length ot the monomeric DNA. In discussing these experiments, we have treated micrococcal nuclease as an enzyme that
Positioningplacestargetsequence(red)at uniqueposition
Micrococcalnucleasereleasesmonomers
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II
i
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sites ptaces restriction positioning f:iili.iiii: ;,lil.I I Nucteosome bymicroto the[inkersitescteaved positions retative at unique nuctease. coccaI
cleavesDNA at the exposedlinker regions without any sort of sequencespecificity.The enzyme actually does have some sequence specificity, though, which is biased toward selection of A-T-rich sequences.Thus we cannot assume that the existenceof a specificband in the indirect end-labelingtechnique representsthe distance from a restriction cut to the linker region. It could instead represent the distance from the restriction cut to a preferred micrococcal nuclease cleavaqesite!
Positions? 7 7 5 Lieat Specific 29.L0DoNucteosomes
. It is extrinsic: Theftrst nucleosome in a region is preferentiallyassembledat a particular site. A preferential starting point for nucleosome positioning resultsfrom the presence of a region from which nucleosomes are excluded. The excluded region provides a boundarythat restricts the positions available to the adjacent nucleosome. A series of nucleosornes may then be assembled sequentially, with a defined repeat length. It is now clear that the deposition of histone octamers on DNA is not random with regard to sequence. The pattern is intrinsic in some cases,in which it is determined by structural features in DNA. It is extrinsic in other cases.in which it results from the interactions of other proteins with the DNA and/or histones. Certain structural features of DNA affect placement of histone octamers.DNA has intrinsic tendencies to bend in one direction ratner than another; thus A-T-rich regions locate so that the minor groove faces in toward the octamer, whereas G-C-rich regions are arranged so that the minor groove points out. Long runs .!" .' In the absence of nucleosome Dosition- of dA-dT (>8 bp) avoid positioning in rhe cening,a restriction site[iesat at[possibte Locations in djftral superhelical turn of the core. It is yet posnot ferentcopiesof the genome. Fragments of at[ possib[e sible to sum all of the relevant sizesareproduced structural effects whena restriction enzyme cutsat a targetsite (red)and micrococcaI nuctease and thus entirely predict the location of a parcutsat the junctions between nucleosomes (green). ticular DNA sequencewith regard to the nucleosome. Sequencesthat cause DNA to take up more extreme structures may have effectssuch This possibility is controlled by treating the as the exclusion of nucleosomes,and thus could naked DNA in exactly the same way as the chrocauseboundary effects. matin. If there are preferred sites for micrococPositioning of nucleosomes near boundcal nucleasein the particular region, specificbands aries is common. If there is some variability in are found. This pattern of bandscan then be comthe construction of nucleosomes-for exampared with the pattem generatedfrom chromatin. ple, if the length of the linker can vary by, say, A differencebetween the control DNA band l0 bp-the specificity of locarion would decline pattern and the chromatin pattern provides eviproceeding away from the first, defined nucledence for nucleosomepositioning. Some of the osome at the boundary. In this case,we might bands present in the control DNA digest may expect the positioning to be maintained rigordisappearfrom the nucleosomedigest,indicatously only relatively near the boundary. i n g t h a t p r e f e r e n t i a l l y c l e a v e dp o s i t i o n s a r e The location of DNA on nucleosomescan unavailable. New bands may appear in the be describedin two ways. l.iL,;tii'.::t;i,i.:shows nucleosomedigestwhen new sitesare rendered that translational positioning describesthe p r e f e r e n t i a l l y a c c e s s i b l eb y t h e n u c l e o s o m a l position of DNA with regard to the boundaries organization. of the nucleosome.In particular, it determines Nucleosome positioning might be accomwhich sequencesare found in the linker regions. plished in either of two ways: Shifting the DNA by l0 bp brings the nexr turn . It is intrinsic: Everynucleosome is deposited into a linker region. Thus translational positionspecificallyat a particular DNA sequence. ing determines which regions are more accesThis modifies our view of the nucleosible (at least as judged by sensitivity ro some as a subunit able to form between micrococcalnuclease) . any sequenceof DNA and a histone DNA lies on the outside of the historre octamer. octamer. As a result, one face of anv Darticular
776
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RNA is compactedby proteins, but we know (from the sequenceof the rRNA) how long the transcript must be. The length of the transcribed DNA segment. which is measured by the length of the axis of the "Christmastree," is -85% of the length of the rRNA. This means that the DNAis almost completely extended. On the other hand, transcription complexes of SV40 minichromosomes can be extracted from infected cells.They contain the usual complement of histones and display a beaded structure. Chains of RNA can be seen to extend from the minichromosome, as in the example of Fi{;|ifiilt*"}*. This argues that transcription can ii*l-iFi I*"-l= Theisolatednucleolar genesrDNAtranproceed while the SV40 DNA is organized into scriptionunjtsatternate with nontranscribed DNAsegnucleosomes. Of course, the SV40 minichroments.Reproduced fromMitter, 0. L.andBeatty, B.R.1969. mosome is transcribed lessintensively than the Science. 1.64:955-957. Photocourtesv of 0scarMitter. rRNA genes. Transcription involves the unwinding of DNA, and may require the fiber ro unfold in restricted regions of chromatin. A simplistic view suggeststhat some "elbow-room" must be neededfor the process.The featuresof polytene and lampbrush chromosomes describedin Chapter 28, Chromosomes, offer hints that a more expansive structural organization is assoc i a t e dw i t h g e n e e x p r e s s i o n . In thinking about transcription, we must bear in mind the relative sizes of RNA polymerase and the nucleosome. The eukaryotic enzymes are large multisubunit proteins, typically >500 kD. Compare this wirh the -260 kD of the nucleosome. $t{#ftf,t1;.}? illustratesthe approach of RNA polymerase to nucleosomal DNA. Even without detailed knowledge of the interaction, it is evident that it involves the approach of two comparable bodies. Consider the two turns that DNA makes around the nucleosome. Would RNApolynerase have sufficient accessto DNA if the nucleic acid were confined to this path? During transcription, as RNA polymerase moves along the template, it i I S l i F I f $ . s + A n S V 4 0m i n i c h r o m o s ocm eb e t r a n an binds tightly to a region of -50 bp, including a scribed. Reproduced fromJ. Mol.Bio.,vol.131.GarigLio, Iocally unwound segmentol-l2bp. The need to P.,et at.,Thetemplate of theisoLated . . . , p.131.Copyphotocourtesv unwind DNA makes it seem unlikely that the right1.979, with permission fromElsevier. of Pierre Chambon. segment engaged by RNA polymerase could remain on the surfaceof the histone octamer. Attempts to visualize genes during transcripIt therefore seemsinevitable that transcription have produced conflicting results.The next tion must involve a structural change. Thus the two figures show each extreme. first question to ask about the structure of active Heavily transcribed chromatin can be seento genesis whether DNA being transcribed remains be rather extended (too extended to be covered in organized in nucleosomes. If the histone nucleosomes).In the intensively transcribedgenes octamersare displaced,do they remain attached coding for rRNA shown in FItil.tfr$: t'"+.jt$,the in some way to the transcribed DNA? extreme packing of RNA polymerases makes it One experimental approach is to digest hard to seethe DNA. We cannot directly measure chromatin with micrococcal nuclease,and then the lengths of the rRNA transcripts because the to use a probe to some specific gene or genes to
778
CHAPTER 29 Nucteosomes
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l':iil"!*l :i;.-i-i:RNApolymerase disptaces DNAfromthe histoneoctamer asit advances. TheDNAloonsbackand attaches(to polymerase or to the octamer) to forma closed[oop.Asthe pol.ymerase proceeds, posit generates itivesupercoiling ahead. Thisdisp[aces theoctamer, whjch keeps contactwith DNAand/orpolymerase. andis inserted behjndthe RNApolymerase. n u c l e o s o m e ( t h e n e x t b a s e st o b e a d d e d a r e essentiallyat the axis of dyad symmetry), pausrng ceases,and the polymerase advancesrapidly. This suggeststhat the midpoint of the nucleosome marks the point at which the octamer is displaced(possiblybecausepositive supercoiling has reached some critical level that expels the octamer from DNA). This releasestension ahead of the polymerase and allows it to proceed.The octamer then binds to the DNA behind the polymerase and no longer presentsan obstac l e t o p r o g r e s s .I t i s l i k e l y t h a t t h e o c t a m e r changesposition without ever completely losrng contact with the DNA. Is the octamer releasedas an intact unit? Crosslinking the octamer's proteins does not create an obstacleto transcription. Transcription can continue even when crosslinking is extensive enough to ensure that the central regions of the core histones have been linked. Ihis implies that transcription does not require Cissociationof the octamer into its component
780
C H A P T E2R9 N u c t e o s o m e s
FT$#fii:, tL;-+* URl3genesequences arefusedto a regupromoter latedGALL andto a ribosomaI DNAsequence. The positioned UR43 hastransitionatty nucleosomes before transcription. Whentranscription is inducedunderthe control promoter, of aninducibte positions nucleosome arerandomized.Whentranscription is repressed, the nucteosomes resume theirpaticu[arpositions. Reproduced fromSuter.B., et at.L997.EMBO J. 1.6:21,50-21,60. Copyright O 0xfordUniversityPress. Photos courtesy of FritzThomas, ETHZi.irich.
histones, nor is it likely to require any major unfolding of the central structure. The addition of histone Hl to this system, however, causes a rapid decline in transcription. This suggests two conclusions:The histone octamer (whether remaining present or displaced)functions as an intact unit, and it may be necessaryto remove Hl from active chromatin or to modify its interactions in some way. Thus a small RNA polymerase can displace a single nucleosome, which reforms behind it, during transcription. Of course, the situation is more complex in a eukaryotic nucleus. RNA polymerase is much larger, and the impediment to progressis a string of connectednucleosomes. Overcoming this obstacle requires additional factors that act on chromatin (seeChapter 30, Controlling Chromatin Structure). The organization of nucleosomesmay be changed by transcription. i1i*i:fiil?+.4t]shows what happens to the yeast URA3 gene when it is transcribed under the control of an inducible
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an anri-BEAF-32 antibody srains-50% of rhe interbands of the polytene chromosomes.This suggeststhat there are many insulators in the genome, and that BEAF-32 is a common part of the insulating apparatus. It would imply that the band is a functional unit, and that interbands often have insulators that block the propagation of activating or inactivating effects. Another example of an insulator that defines a domain is found in the chick p-globin LCR (the group of hypersensitive sitesthat controls expressionof all B-globin genes;see Seclion 29.20, An LCR May Control a Domain). The leftmost hypersensitive site of the chick p-globin LCR (HS4) is an insulator rhar marks the 5'end of the functional domain. This restricts the LCR to acting only on the globin genes in the domain. A gene that is surrounded by insulators is usually protected against the propagation of inactivating effectsfrom the surrounding regions. The test is to insert DNA into a genome at random locationsby transfection.The expressionof a gene in the inserted sequence is often erratic; in some instances it is properly expressed,but in others it is extinguished.When insulatorsthat have a barrier function are placed on either side of the gene in the inserted DNA, however, its expression typically is uniform in every case.
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Insulators MayAct in 0neDirection
. Someinsutators havedirectionatity, andmaystop passage of effectsin onedirectionbut not the other.
Insulators may have directional properties. Insertions of the transposon gypsyinto the yellow (y) locus of D. melanogaster causeloss of gene function in some tissues,but not in others. The reason is that the y locus is regulated by four enhancers,as shown in tISl,JRf PS.4S. Wherever gypsy is inserted, it blocks expression of all enhancers that it separatesfrom the promoter, but not those that lie on the other side. The sequenceresponsiblefor this effect is an insulator that lies at one end of the transposon.The insulator works irrespective of its orientation of insertion. Some of the enhancers are upstream of the promoter and others are downstream, so the effect cannot depend on position with regard to the promoter, nor can it require transcrip-
784
C H A P T E2R9 N u c t e o s o m e s
Positionsof enhancersfor soecifictissues wing body bristles tarsal blade cuticle claws
Exon 1
Exon 2
A Insertionof insulatorand expressionpattern
FIGURil t$.46 Theinsutator ofthegypsy transposon blocks the actionof an enhancer whenit is placed between tne enhancer andthepromoter.
tion to occur through the insulator. This is difficult to explain in terms of looping models for enhancer-promoter interaction, which essentially predict the irrelevance of the intervening DNA. The obvious model to invoke is a tracking mechanism, in which some component must move unidirectionally from the enhancer to the promoter, but this is difficult to reconcile with previous characterizationsof the independence of enhancers from such effects. Proteins that act upon the insulator have been identified through the existence of two other loci that affect insulator function in a trans-acring manner. Mutations in su(Hw) abolish insulation: y is expressedin all tissues in spite of the presenceof the insulator. This suggests that su(Hw) codes for a protein that recognizes the insulator and is necessaryfor its action. Su(Hw) has a zinc finger DNA-motif; mapping to polytene chromosomes shows that it is bound at a large number of sites.The insulator contains twelve copiesof a26 bp sequence that is bound by Su(Hw). Manipularions show that the strength of the insulator is determined by the number of copies of the binding sequence. The second locus is mod(mdg4),in which mutations have the opposite effect. This is observed by the loss of directionality. These mutations increasethe effectivenessof the insulator by extending its effects so that it blocks utilization of enhancers on both sides.su(Hw) is epistatic to mod(mdg4);this means rhat in a double mutant we seeonly the effectof su(Hw). This implies that mod(mdg4) acts through
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lli.,;.j$if. ':l:i,i:: In adutterythroid cetts.the aduttB-gtobin geneis highLy I digestion; the embrysensitjve to DNAase geneis partiatly (probabty sensitive dueto onicB-gtobin js not sensitjve. Photos spreading effects), but ovalbumin c o u r t e so y f H a r o t dW e i n t r a u bF,r e dH u t c h i n s oCna n c e r Research of MarkT.Groudine. Center. Usedwithpermission sitivity to DNAase I extends over a considerable distance.Often we think of regulation as residing in events that occur at a discrete site in DNA-for example, in the ability to initiate transcription at the promoter. Even if this is true, such regulation must determine, or must be accompanied by, a more wide-ranging change in structure. This is a difference between eukaryotes and prokaryotes.
U'.i4;11.:i. iF.:-!; Sensitivity to DNAase I canbemeasured by d e t e r m i n i ntgh e r a t eo f d i s a p p e a r a nocfet h e m a t e r i a I hybridizing probe. witha particutar
actually engaged in transcription at any moment; this implies that the sensitivity to DNAase I does not result from the act of transcription, but instead is a feature of genesthat are able to be transcribed. What is the extent of the preferentially sensitive region? This can be determined by using a series of probes representing the flanking regions as well as the transcription unit itself. The sensitive region always extends over the entire transcribed region; an additional region of severalkb on either side may show an intermediate level of sensitivity (probably as the result of spreadingeffects). The critical concept implicit in the description of the domain is that a region of high sen-
An LCRMayControl a Domain r An LCR is located at the 5' endof the domainand hypersensitive sites. consistsof severaI
Every gene is controlled by its promoter, and some genes also respond to enhancers (containing similar control elements but located farther away), as discussed in Chapter 24, Promotersand Enhancers.Theselocal controls are not sufficient for all genes,though. In some cases,a gene lies within a domain of several genes,all of which are influenced by regulatory elements that act on the whole domain. The existence of these elements was identified by the inability of a region of DNA including a gene and all its known regulatory elements to be
29.20An LCR MavControl a Domain
cific controls. Some of these controls are autonomous: Expression oI the e and y genes 5' hypersensitive sites 3' hypersensitive site appears intrinsic to those loci in conjunction with the LCR. Other controls appear to rely upon position in the cluster, which provides a HS4 HS1 Globingenes suggestion IhaI geneorderin a cluster is important for regulation. 60 80 The entire region containing the globin F I * I J RiE9 . 5 4 A g t o b i nd o m a i ins m a r k ebdy h y p e r s e n - genes,and extending well beyond them, consitivesitesat eitherend.Thegroupof sitesat the 5' side stitutes a chromosomal domain. It shows constitutes the LCR andis essentiaI forthefunctionof atl increased sensitivity to digestion by DNAase I oenes in the cluster. ( s e eF i g u r e 2 9 . 5 2 \ . D e l e t i o n o f t h e 5 ' L C R restores normal resistanceto DNAase over the whole region. TWo models for how an LCR works propose that its action is required in properly expressedwhen introduced into an order to activate the promoter, or alternatively, animal as a transgene. is required to increase the rate of transcripThe best characterizedexample of a regution from the promoter. The exact nature of Iated gene cluster is provided by the mammalian the interactions between the LCR and the indiB-globin genes.Recallfrom Figure 6.1 that the vidual promoters has not yet been fully o- and B-globin genes in mammals each exist defined. as clusters of related genesthat are expressedat Does this model apply to other gene clusdifferent times during embryonic and adult ters? The cr-globin locus has a similar organidevelopment. These genes are provided with a zation of genes that are expressed at different large number of regulatory elements, which times, with a group of hypersensitive sites at h a v e b e e n a n a l y z e d i n d e t a i l . I n t h e c a s eo f one end of the cluster, and increased sensitivthe adult human B-globin gene, regulatory ity to DNAase I throughout the region. Only a s e q u e n c e sa r e l o c a t e db o t h 5 ' a n d 3 ' t o t h e small number of other casesare known in which gene.The regulatory sequencesinclude both an LCR controls a group of genes. positive and negative elements in the promoter One of these casesinvolves an LCR that region, as well as additional positive elements controls geneson more than one chromosome. within and downstream of the gene. The Ts2 LCR coordinately regulates a group of A human B-globin gene containing all of genesthat are spreadout over I20 kb on chrothese control regions, however, is never mosome I I by interacting with their promotexpressedin a transgenicmouse within an order ers. It also interacts with the promoter of the of magnitude of wild-type levels.Some further IFNygene on chromosome 10. The two types of regulatory sequenceis required. Regions that interaction are alternatives that comprise two provide the additional regulatory function are different cell fates, that is, in one group of cells identified by DNAase I hypersensitive sitesthat the LCR causesexpressionof the geneson chroare found at the ends of the cluster. The map of mosome I l, whereas in the other group it causes FI**qr f *.54 shows that the 20 kb upstream of the gene on chromosome l0 to be expressed. the e gene contains a group of five sites, and that there is a single site 30 kb downstream of the p gene. Ttansfecting various constructs into mouse erythroleukemia cells shows that sequencesbetween the individual hypersensitive sitesin the 5'region can be removed without much effect, but that removal of any of the r A domainmayhavean insutator, an LCR, a matrix sitesreducesthe overall level of expression. attachment site,andtranscription unit(s). The 5'regulatory sitesare the primary regulators, and the cluster of hypersensitive sites is called the locus control region (LCR). We If we put together the various types of strucdo not know if the 3'site has any function. The tures that have been found in different sysLCR is absolutely required for expression of tems, we can think about the possible nature each of the globin genes in the cluster. Each of a chromosomal domain. The basic feature gene is then further regulated by its own speof a regulatory domain is that regulatory ele-
WhatConstitutes
a Regulatory Domain?
CHAPTER 29 Nucleosomes
ments can act only on transcription units within the same domain. A domain might contain more than one transcription unit and/or enhancer. FiGUR[8S"55summarizes the structures that might be involved in defining a domain. An insulator stops activating or repressing effects from passing. In its simplest form, an insulator blocks either type of effect from passing acrossit, but there can be more complex relationships in which the insulator blocks only one type of effect and/or acts directionally. We assume that insulators act by affecting higher-order chromatin structure, but we do not know the details and varieties of such effects. A matrix attachment site (MAR) may [re responsible for attaching chromatin to a site on the nuclear periphery (see Section 28.6, Specific Sequences Attach DNA to an Interphase Matrix). These are likely to be responsible for creating physical domains of DNA that take the form of loops extending out from the attachment sites.This looks very much like one model for insulator action. In fact, some MAR elements b e h a v e a s i n s u l a t o r s i n a s s a y si n v i t r o , b u t i l seems that their ability to atrach DNA to the matrix can be separated from the insulator function, so there is not a simple cause and effect. It would not be surprising if insulator and MAR elements were associatedto maintain a relationship between regulatory effects and physical structure. An LCR functions at a distance and may be required for any and all genes in a domain to be expressed(see Section 29.20, An LCR May Control a Domain). When a domain has an LCR, its function is essential for all genes in the domain, but LCRs do not seem to be common. Several types of cis-actingstructures could be required for function. As defined originally, the property of the LCR restswith an enhancer-like hypersensitive site that is needed for the full activity of promoter(s) within the domain. The organization of domains may help to explain the large size of the genome. A certain amount of space could be required for such a structure to operate,for example, to allow chromatin to become decondensed and to beconte accessible.Although the exact sequences of much of the unit might be irrelevant, there might be selection for the overall amount of DNA within it, or at least selection might prevent the various transcription units from becoming too closely spaced.
nsulator MAR LCR
Enhancer
t-r{;7 Transcription units flGllR*?*"55 Domains maypossess threetypesof sites:insulators to preventeffectsfromspreading MARs betweendomains, to attach the domain matrix.andLCRs for inito the nuclear that arerequired tiationoftranscription. An enhancer mayacton morethanonepromoterwithinthe domain.
Summary All eukaryotic chromatin consists of nucleosomes. A nucleosome contains a characteristic length of DNA, usually -200 bp, which is wrapped around an octamer containing two copieseach of histonesH2A,H2B, H3, and H4. A single Hl protein is associated with each nucleosome. Virtually all genomic DNA is organized into nucleosomes.Tfeatment with micrococcal nuclease shows that the DNA packaged into each nucleosome can be divided operationally into two regions. The linker region is digested rapidly by the nuclease; the core region of 146 bp is resistant to digestion. Histones H3 and H4 are the most highly conserved, and an H32-H42tetramer accounts for the diameter of the particle. The H2A and H2B histones are organized as two H2A-H2B dimers. Octamers are assembledby the successiveaddition of two H2A-H2B dimers to the H3z-H42 kernel. The path of DNA around the histone o c t a m e r c r e a t e s- 1 . 6 5 s u p e r c o i l s .T h e D N A "enters" and "leaves" the nucleosome in tne same vicinity, and could be "sealed" by histone Hl. Removal of the core histonesreleases-L0 supercoils. The difference can be largely explained by a change in the helical pitch of DNA, from an averageof 10.2 bp/turn in nucleosomal form to 10.5bp/turn when free in solution. There is variation in the structure of DNA from a periodicity of 10.0bp/turn at the nucleosome ends to 10.7 bp/turn in the center. There are kinks in the path of DNA on the nucleosome. Nucleosomes are organized into a fiber of 30 nm diameter that has six nucleosomes per turn and a packing ratio of 40. Removal of Hl allows this fiber to unfold into a l0 nm fiber that consists of a linear string of nucleosomes. The l0 nm fiber probably consistsof the l0 nm fiber wound into a 2-start solenoid. The l0 nm fiber is the basic constituent of both euchromatin and heterochromatin; nonhistone
29.22Summary 7 9 1
proteins are responsible for further organization of the fiber into chromatin or chromosome ultrastructure. There are two pathways for nucleosome assembly.In the replication-coupledpathway, the PCNA processivity subunit of the replisome recruits CAF-1, which is a nucleosomeassembly factor. CAF- I assiststhe deposition of H32-H42tetramers onto the daughter duplexes resulting from replication. The tetramers may be produced either by disruption of existing nucleosomesby the replication fork or as the result of assemblyfrom newly synthesizedhistones. Similar sourcesprovide the H2A-H2B dimers that then assemblewith the H32-H42tetramerto complete the nucleosome. The H3z-H4ztetramer and the H2A-H2B dimers assembleat random, so the new nucleosomesmay include both preexisting and newly synthesizedhistones. RNA polymerase displaceshistone octamers during transcription. Nucleosomesreform on DNA after the polymerase has passed,unless transcription is very intensive (such as in rDNA) when they may be displacedcompletely. The replication-independent pathway for nucleosome assembly is responsible for replacing hist o n e o c t a m e r s t h a t h a v e b e e n d i s p l a c e db y transcription. It uses the histone variant H3.l instead of Hl. A similar pathway, with another alternative to H3, is used for assembling nucleosomesat centromeric DNA sequencesfollowing replication. An insulator blocks the transmission of activating or inactivating effects in chromatin. An insulator that is located between an enhancer and a promoter prevents the enhancer from activating the promoter. T\ryoinsulators define the region between them as a regulatory domain; regulatory interactions within the domain are limited to it, and the domain is insulated from outside effects.Most insulators block regulatory effectsfrom passingin either direction, but some are directional. Insulators usually can block both activating effects(enhancerpromoter interactions) and inactivating effects (mediated by spread of heterochromatin), but some are limited to one or the other. Insulators are thought to act via changing higher-order chromatin structure, but the details are not certaln. TWotlpes of changesin sensitivity to nucleases are associatedwith gene activity. Chromatin capable of being transcribed has a generally increased sensitivity to DNAase I, reflecting a change in structure over an exten-
792
CHAPTER 29 Nucleosomes
sive region that can be defined as a domain containing active or potentially active genes. Hlpersensitivesitesin DNA occur at discretelocations, and are identified by greatly increased sensitivity to DNAase L A hypersensitive site consists of a sequenceof -200 bp from which nucleosomes are excluded by the presence of other proteins. A hypersensitive site forms a boundary that may causeadjacent nucleosomes to be restricted in position. Nucleosome positioning may be important in controlling accessof regulatory proteins to DNA. Hypersensitive sites occur at several types of regulators. Those that regulate transcription include promoters, enhancers,and LCRs. Other sites include origins for replication and centromeres. A promoter or enhancer actson a single gene, but an LCR contains a group of hypersensitive sitesand may regulate a domain containing several genes.
References TheNucleosome Is theSubunit of A[[Chromatin Reviews I(ornberg, R. D. (1977). Structure of chromatin. Annu. Rev.Biochem.46,9)l-954. McGhee, J. D., and Felsenfeld,G (1980). Nucleosome structure. Annu. Rev.Biochem 49, ltlS-t156. Research I(ornberg, R. D. (1974). Chromatin structure: a repeating unit of histones and DNA. Science 184, 868-87 r. Richmond, T. J., Finch, J. T., Rushton, B., Rhodes,D., and Klug, A. (1984). Structure ot t h e n u c l e o s o m ec o r e p a r t i c l ea t 7 A r e s o l u tion. Nature Jll, 5j2-5)7 .
DNAIs Coited in Arravs of Nucteosomes Rese arch Finch, J. T. et al. .1977). Structure of nucleosome core particles of chromatin . Nature 269, 29-36.
Nucteosomes Havea Common Structure rch Resea Shen, X. et al. (I995). Linker histones are not essential and affect chromatin condensation in vitro. Cell 82, 47-56.
DNAStructure Varies on the NucteosomaI Surface Review Wang,J. (1982\.The path of DNA in rhe nucleosome.Cell29, 724-726. Research Richmond,T.J. and Davey,C. A. (2003).The structureof DNA in the nucleosomecore. Nature42j, 145-150.
ThePeriodicity of DNAChanges on the Nucteosome Review Travers,A. A. and Klug, A. (19871.The bending of DNA in nucleosomesand its wider implications.PhilosTrans.R. SocLond B Biol. Sci.317. 5)7-561. 0rganizationof the HistoneOctamer Resea rch Angelov,D., Vitolo,J. M., Mutskov V.,Dimitrov S., and Hayes,J. J. (2001). Preferential interaction of the corehistonetail domainswith Iinker DNA. ProcNatl Acad.Sci.USA98, 6599-6604. Arents,G.,Burlingame,R. W.,Wang,B.-C.,Love, W. E., and Moudrianakis,E. N. (1991).The nucleosomalcorehistoneoctamerat 3I A resolution: a tripartiteprotein assemblyand a left-handedsuperhelix.ProcNatl.Acad.Sci u s . 48 8 , r 0 1 4 8 - 1 0 r 5 2 . Luger,I(. et al. (1997).Crystalstructureof the n u c l e o s o mceo r ep a r r i c l ea t 2 8 A r e s o l u t i o n . Nature389,251-260.
ThePathof Nucteosomes in the Chromatin Fiber Review Felsenfeld,G. and McGhee, J. D. (1986). Srrucrure of the 30 nm chromatin fiber. Cell44, 375-]77. Resea rch Dorigo, B., Schalch,T., I(ulangara, A., Duda, S., Schroeder,R. R., and Richmond, T. J. (2004). Nucleosome arrays reveal the two-start organization of the chromatin fiber. SciencejO6. t57 t-l573. Schalch, T., Duda, S., Sargent, D. F., and Richmond, T. J. (2005). X-ray structure of a tetranucleosome and its implications for the chromatin Libre.Nature 4)6, lJ8-141.
Reproduction of Chromatin Requires Assembty of Nucteosomes Reviews Osley,M. A. (1991). The regulation of histone synthesis in the cell cycle. Annu. Rev.Biochem 60, 827-86t.
Verreault, A. (2000). De novo nucleosome assembly: new pieces in an old puzzle. GenesDev. 14, t4)o-14]8. Resea r ch Ahmad, I(. and Henikoff, S. (2001). Centromeres are specialized replication domains in heterochromatin. J CellBiol l5 3. l0l-l 10. Ahmad, I(. and Henikoff, S. (2002). The histone variant H3.3 marks active chromatin by replication-independent nucleosome assembly.Mol. Cell 9, ll9l-I200. Gruss,C., Wu, J., I(oller, T., and Sogo,J. M. (19931. Disruption of the nucleosomes at the replication fork. EMBO J. 12, 453)-4545. Loppin, B., Bonnefoy, E., Anselme, C., Laurencon, A., I(arr, T. L., and Couble, P. (2005). The histone H3.3 chaperone HIRA is essential for chromatin assembly in the male pronucleus. N a t u r e4 3 7 , l j 8 6 - l ) 9 0 . Ray-Gallet, D., Quivy, J. P., Scamps, C., Martini, E. M., Lipinski, M., and Almouzni, G. (2002). HIRA is critical for a nucleosome assembly pathway independent of DNA synthesis. Mol. Cell9, 109l-1100. Shibahara,I(., and Stillman, B. (19991. Replication-dependent marking of DNA by PCNA facilitates CAF- I -coupled inheritance of chromatin. Cell 96, 57 5-585. Smith, S. and Stillman, B. (1989). Purification and characterization of CAF-I, a human cell factor required lor chromatin assembly during DNA replication in vitro. Cell 58, 15-25. Smith, S. and Stillman, B. (1991). Stepwiseassembly of chromatin during DNA replication in vitro. EMBO J. lO, 97 1-980. Tagami, H., Ray-Gallet, D., Almouzni, G., and Nakatani, Y. (2004). Histone Hl.I and Hl.l complexes mediate nucleosome assembly pathways dependent or independent of DNA synthesis.Cell ll6, 5l-61. Yu, L. and Gorovsky, M. A. (1997). Constitutive expression, not a particular primary sequence, is the important feature of the H3 replacement variant Llv2 in Tetrahymenathermophila. Mol. Cell.Biol 17, 6]03-6310.
AreTranscribed 0rganized Genes in Nucteosomes? Review I(ornberg,R. D. and Lorch,Y. (1992).Chromatin structureand transcriprion.Annu. Rev.Cell Biol 8, 56)-587. HistoneOctamers Are Disptaced by Transcription R e s e a hr c Cavalli,G. and Thoma,F. ( 1993) . Chromatintransitionsduring activationand repressionof galactose-regulated genesin yeast.EMBOJ. 12,460)46r).
References 793
Studitsky,V. M., Clark, D. J., and Felsenfeld,G. (1994). A histone octamer can step around a transcribing polymerase without leaving the remolare. cell 7 6. 37 l-382.
@
Nucleosome Disp[acement and Reassembty RequireSpeciaIFactors
R e s erach Belotserkovskaya, R., Oh, S.,Bondarenko,V.A., Orphanides,G., Studitsky,V. M., and Reinberg,D. (2001).FACTfacilitates transcriptiondependentnucleosomealteration.Science 3Ol, 1090-1093. Saunders, A., Werner,J.,Andrulis,E. D., Nakayama,T.,Hirose,S.,Reinberg,D., and Lis,J. T (2003).TrackingFACTand the RNA polymeraseII elongationcomplexthrough chromatinin vivo. Science )Ol, 1094-1096.
@
Insulators Btock theActions of Enhancers andHeterochromatin
Reviews Gerasimova,T. I. and Corces,V. G. (2001). Chromatin insulators and boundaries: effects on tra nscription and nu clear or ganizalion. An n u. Rev.Genet.35, 19)-208. West, A. G., Gaszner,M., and Felsenfeld,G. (20021. Insulators: many functions, many mechanisms. GenesDev. 16. 27 l-288.
?W
Insulators CanDefinea Domain
rch Resea Chung, J. H., Whiteley, M., and Felsenfeld,G. (19%l . A 5' element of the chicken p-globin domain serves as an insulator in human erythroid cells and protects against position effect rn Drosophila Cell 74, 505-514. Cuvier, O., Hart, C. M., and Laemmli, U. K. (1998). Identification of a classof chromatin boundary elements.Mol. CellBiol 18,7478-7486. Gaszner,M.,Yazquez, J., and Schedl,P. (1999). The Zw5 protein, a component of the scs chromatin domain boundary, is able to block enhancer-promoter interaction. GenesDev. l), 2098-2107. I(ellum, R. and Schedl, P. ( I 991 ) . A position-effect assayfor boundaries of higher order chromosomal domains. Cell64,941-950. Pikaart, M. J., Recillas-Targa,F., and Felsenfeld, G. ( I 998 ) . Loss of transcriptional activity of a transgene is accompanied by DNA methylation and histone deacetylation and is prevented by insulators. GenesDev. 12, 2852-2862. Zhao,I(, Hart, C. M., and Laemmli, U. K. (1995). Visualization of chromosomal domains with boundary element-associatedfactor BEAF-12. Cell 81, 879-889.
794
CHAPTER 29 Nucteosomes
MayActin OneDirection Insutators Research Gerasimova, T. I., Byrd, I(., and Corces, V. G. (2000). A chromatin insulator determines the nuclear localization of DNA. Mol. Cell 6, r025-to)5. Harrison, D. A., Gdula, D. A., Cyne, R. S., and Corces,V. G. (1993). A leucine zipper domain of the suppressor of hairy-wing protein mediates its repressive effect on enhancer function. GenesDev.7, 1966-197 8. Roseman, R. R., Pirlotta, V., and Geyer, P. K. (19931.The su(Hw) protein insulatesexpression of the D melanogasterwhite gene from chromosomal position-effecrs. EMB) J. 12, 435-442.
Insulators CanVaryin Strength Research Hagstrom, I(., Muller, M., and Schedl,P. (1996). Fab-7 functions as a chromatin domain boundary to ensure proper segment speci{ication by the Drosophila bithorax complex. Genes D e v .1 0 , 3 2 0 2 - ) 2 1 5 . Mihaly, J. et al. \1997). In situ drssectionof the Fab-7 region of the bithorax complex into a chromatin domain boundary and a polycombresponse element. Development124, l 809-l 820. Zhou, J. and Levine, M. (1999). A novel cziregulatory element, the PTS, mediates an antiinsulator activity inthe Drosophilaembryo. Cell99,567-575.
DNAase Hypersensitive SitesReflect Changesin Chromatin Structure Review Gross,D. S. and Garrard, W T. (1988). Nuclease hypersensitive sites in chromatin. Annu Rev. Biochem.57, 159-197. rch Resea Groudine, M., and Weintraub, H. (1982). Propagation of globin DNAase I-hypersensitive sites in absence of factors required for induction: a possible mechanism for determination. Cell 30, t3t-t39. Moyne, G., Harper, F., Saragosti,S., and Yaniv M. ( I 982 ). Absence of nucleosomes in a histonecontaining nucleoprotein complex obtained by dissociation of purified SV40 virions. Cel/
30,r23-r30. Scott,W. A. and Wigmore, D. J. (1978). Sitesin SV40 chromatin which are preferentially cleavedby endonucleases.Cell 15, l5l l-1518. Varshavsky, A. J., Sundin, O., and Bohn, M. J. (1978). SV40 viral minichromosome: preferential exposure of the origin of replication as probed by restriction endonucleases.Nucleic AcidsRes.5, )469-)479.
Domains DefineRegions ThatContain ActiveGenes Research Stalder,J. et al. (1980). Tissue-specific DNA cleavage in the globin chromatin domain introduced by DNAase I. Cell20, 45t-460.
An LCRMayControl a Domain Reviews Bulger, M. and Groudine, M. (1999). Looping versus linking: toward a model for long-distance gene activation. GenesDev.Lj^,2465-2477. Grosveld, F., Antoniou, M., Berry, M., De Boer, E., Dillon, N., Ellis, J., Fraser, P., Hanscombe, O.. Hurst, J., and Imam, A. (1993). The regulation of human globin gene switching. Philos. Trans. R. SocLond.B Biol. Sci.)39, t8)-t9t.
Research Gribnau, J., de Boer, E., Tfimborn, T., Wijgerde, M., Milot, E., Grosveld,F., and Fraser,P. (I998). Chromatin interaction mechanism of transcriptional control in vitro. EMBO J. 17, 6020-6027. Spilianakis, C. G., Lalioti, M. D., Town, T., Lee, G. R., and Flavell, R. A. (2005). Interchromosomal associationsbetween alternatively expressedloci. Nature 475, 637-645. van Assendelft, G. B., Hanscombe, O., Grosveld, F., and Greaves,D. R. (I989). The B-globin dominant control region activates homologous and heterologous promoters in a tissue-specific manner. Cell 56, 969-977.
WhatConstitutes Domain? a Regulatory Review West,A. G., Gaszner,M., and Felsenfeld,G. (2OO2l .Insulators:many functions/many mechanisms.Genes Dev.16, 27 1-288.
References 795
Chromatin Controlting Structure C H A P T EO RU T L I N E Introduction ChromatinCanHaveAtternativeStates . Chromatin structure is stableandcannotbe changed by alteringthe equitibrium of transcription factorsand histones. ChromatinRemodeting Is an ActiveProcess r Thereareseveral remodeling chromatin complexes that use energyprovided by hydrotysis of ATP. r TheSWI/SNF, RSC. andNURF comptexes atlareverylarge, andtheysharesomecommon subunits. . A remodeling for comptex doesnotitselfhavespecificity anyparticular targetsite,but mustbe recruited by a componentof thetranscription apparatus. N u c t e o s o m0er g a n i z a t i o M n a yB e C h a n g e d at the Promoter o Remodeting comptexes arerecrujted to promoters by sequence-specjfi c activators. r Thefactormaybe released oncethe remodeting complex h a sb o u n d . r TheMMTV promoter positionrequires in rotational a change ing of a nucteosome to a[[owan activator to bindto DNAon the nucteosome. HistoneModificationIs a KeyEvent r Histones aremodified by methylation, acetylation, and phosphory[ation. HistoneAcetytation0ccursin TwoCircumstances o Histone acetytation occurs transjentlyat replication. o Histone acetylation is associated withactivation of gene expresslon. AcetytasesAre Associatedwith Activators r Deacetytated chromatin mayhavea morecondensed structure. r Transcription activators areassociated with histoneacetylaseactivities in [argecomplexes. o Histone acetylases varyin theirtargetspecificity. o Acetylation coutdaffecttranscription in a quantitative or quatitative wa;.
796
AreAssociated with Repressors Deacetylases o Deacetytation is associated with repression of geneactivity. r Deacetylases arepresentin comptexes with repressor activity. Methytationof Histonesand DNAIs Connected . Methytation of both DNAandhistonesis a featureof inactive chromatin. o Thetwo typesof methytation eventmaybe connected. by Modification ChromatinStatesAre Interconverted o Acetytation with geneactivation. of histones is associated . Methylation is associated with of DNAandof histones heteroch romatin. PromoterActivationInvolvesan 0rderedSeriesof Events . Theremodeting mayrecruitthe acetytating comptex complex. . Acetytation maybethe eventthat maintains of histones the jn the actjvated state. complex AffectsChromatin Structure Histone Phosphorylation o At leasttwo histonesaretargetsfor phosphorylation, possibtywith opposing effects. SomeCommonMotifsAre Foundin ProteinsThat Modifv Chromatin . Thechromo domain is foundin several chromatin oroteins that haveejtheractivating or repressing effectson gene expressron. . TheSETdomainis partof the catatyticsite of protein methyttra nsferases. . Thebromodomainis foundin a varietyof proteins that interactwith chromatin andis usedto recognize acetylated siteson histones.
Summary
Introduction When transcription is treated in terms of interactions involving DNA and individual transcription factors and RNA polymerases, we get an accurate description of the events that occur ilt vitro,but this lacks an important feature of transcription in vivo. The cellular genome is organized as nucleosomes, but initiation of transcription generally is prevented if the promoter region is packagedinto nucleosomes.In t h i s s e n s e ,h i s t o n e s f u n c t i o n a s g e n e r a l i z e d repressorsof transcription (a rather old idea), although we see in this chapter that they are also involved in more specificinteractions. Activation of a gene requires changesin the state of chromatin: The essentialissue is how the transcription factors gain accessto the promoter DNA. Local chromatin structure is an integral part o{ controlling gene expression.Genesmay exist in either of two structural conditions. Genesare found in an "active" state only in the cells in which they are expressed.The change of structure precedesthe act of transcription, and indicates that the gene is "transcribable."This suggeststhat acquisition of the "active" structure must be the first step in gene expression. Active genes are found in domains of euchromatin with a preferential susceptibilityto nucleases(seeSection29.l9,Domains Define Regions That Contain Active Genes).Hypersensitivesites are created at promoters before a gene is activated (seeSection 29.I8, DNAase Hypersensitive SitesChange Chromatin Structure). More recently it has turned out that there is an intimate and continuing connection between initiation of transcription and chromatin structure. Some activators of gene transcription directly modify histones; in particular, acetylation of histones is associatedwith gene activation. Conversely,some repressorsof transcription function by deacetylating histones. Thus a reversible change in histone structure
in the vicinity of the promoter is involved in the control of gene expression.This may be part of the mechanism by which a gene is maintained in an active or inactive state. The mechanisms by which local regions of chromatin are maintained in an inactive (silent) state are related to the means by which an indiv i d u a l p r o m o t e r i s r e p r e s s e d .T h e p r o t e i n s involved in the formation of heterochromatin act on chromatin via the histones, and modifications of the histones may be an important feature in the interaction. Once established, such changesin chromatin may persistthrough cell divisions, creating an epigenetic state in which the properties of a gene are determined by the self-perpetuatingstructure of chromatin. The name epigeneticreflectsthe fact that a gene may have an inherited condition (it may be active or may be inactive) that does not depend on its sequence. Yet a further insight into epigenetic properties is given by the self-perpetuating structures of prions (proteinaceous infectious agents).
CanHave Chromatin States Alternative . Chromatin is stableandcannotbe structure of the equitibrium changed by altering factorsandhistones. transcription
Two types of models have been proposed to explain how the state of expression of DNA is changed: equilibrium and discontinuous change-of-state. ',r ., shows the equilibrium model. Here the only pertinent factor is the concentration of the repressoror activator protein, which drives an equilibrium between free form and DNA-bound form. When the concentration of the protein is high enough, its DNA-binding site is occupied, and the state of expression of the
ontheconIn anequitibrium model. thestateofa bindingsiteon DNAdepends centratjon of the proteinthat bjndsto it.
States CanHaveAlternative 30.2 Chromatin
797
DNA is affected. (Binding might either repress or activate any particular target sequence.)This type of model explains the regulation of transcription in bacterial cells, where gene expression is determined exclusively by the actions of individual repressor and activator proteins (see Chapter I2, The Operon). Whether a bacterial gene is transcribed can be predicted from the sum of the concentrations of the various factors that either activate or repress the individual gene. Changes in these concentrations a/ any time will change the state of expression accordingly. In most cases,the protein binding is cooperative, so that once the concentration becomeshigh enough, there is a rapid association with DNA, resulting in a switch in gene expressron. A different situation applies with eukaryotic chromatin. Early in vitro experiments showed that either an active or inactive state can be established,but this is not affected by the subsequentaddition of other components. The transcription factor TFnyA,which is required for RNA polymerase III to transcribe 5S rRNA genes, cannot activate its target genesin vitro if, they are complexed with histones. If the factor is presented with free DNA, though, it forms a transcription complex, and then the addition of histones does not prevent the gene from
RNA polymeraseand factors cannotget accessto DNA
Histoneoctamerscannot ,f, get accessto DNA 0
,r!i:.iiitii Jil..i If nucteosomes format a promoter. transcription factors(andRNApotymerase) cannotbind.If tran; c r i p t i o nf a c t o r s( a n dR N Ap o t y m e r a sbei)n d t o t h e promoter to establish a stabte comptex forinitiation,histonesareexcluded.
798
CHAPTER 30 Controtting ChromatinStructure
remaining active. Once the factor has bound, it remains at the site; this allows a successionof RNA polymerase molecules to initiate transcription. Whether the factor or histones get to the control site first may be the critical factor. l:*{.!ft1,.l,J.Jillustrates the two types of condition that can exist at a eukaryotic promoter. In the inactive state, nucleosomes are present, and they prevent basal factors and RNA polymerase from binding. In the active state. the basalapparatus occupiesthe promoter, and histone octamers cannot bind to it. Each tvne of state is stable. A similar situation is seen with the TF1D complex at promoters for RNA polymerase II. A plasmid containing an adenovirus promoter can be transcribed in vitro by RNA polymerase II in a reaction that requires TF11Dand other transcription factors. The template can be assembled into nucleosomes by the addition of histones. If the histones are added beforetl:'eTF1D, transcription cannot be initiated. If the TF1D is added first, though, the template still can be transcribed in its chromatin form. Thus TF11D can recognize free DNA, but either cannot recognize or cannot function on nucleosomal DNA. Only the TFnD must be added before the histones; the other transcription factors and RNA polymerase can be added later. This suggests that binding of TFnD to the promoter creates a structure to which the other components of the transcription apparatus can bind. It is important to note that these in vitro systemsuse disproportionate quantities of components, which may create unnatural situations. The major importance of these results, therefore, is not that they demonstrate the mechanism used iz vivo,bul that they establish the principle Lhat transcriptionfactorsor nucleosomesmay form stablestructuresthat cannot be changed merely by changing the equi lib rium with fr ee cjmpjnents.
Chromatin RemodeLing Is an ActiveProcess There areseveral chromatin remodeling complexes that useenergyprovided by hydrotysis of ATP. TheSWI/SNF, RSC, andNURF comptexes a[[arevery [arge, andtheyshareretated subunits. A remodeling comptex doesnotitsetfhave specificityfor anyparticutar targetsite,but must be recruited by a component of thetranscription appararus.
The general processof inducing changes in chromatin structure is called chromatin remodeling. This consistsof mechanisms for displacing histones that depend on the input of energy. Many protein-protein and protein-DNA contacts need to be disrupted to releasehistones from chromatin. There is no free ride: The energy must be provided to disrupt these contacts. fii-:i":{il.:!{.i,::l illustrates the principle of a dynamicmodelby a factor that hydrolyzes ATP. W h e n t h e h i s t o n e o c t a m e r i s r e l e a s e df r o m DNA, other proteins (in this casetranscription factors and RNA polymerase) can bind. i"5i;i,Ji'ti .:1,.ii..:.' 51m-".izes the types of remodeling changes in chromatin that can be characterized in vitro: . Histone octamers may slide along DNA, changing the relationship between the nucleic acid and protein. This alters the p o s i t i o n o f a p a r t i c u l a r s e q u e n c eo n the nucleosomal surface. . The spacing between histone octamers may be changed, again with the result that the positions of individual sequences are altered relative to protein. . The most extensive change is that an octamer(s) may be displaced entirely from DNA to senerate a nucleosomefree gap. The most common use of chromatin remodeling is to change the organization of nucleosomes at the promoter of a gene that is to be transcribed. This is required to allow the transcription apparatus to gain accessto the promoter. Remodeling is also required, however, to enable other manipulations of chromatin, including repair reactions to damaged DNA. Remodeling most often takes the form of displacing one or more histone octamers. This can be detected by a change in the micrococcal nucleaseladder where protection againstcleavage has been lost. It often results in the creation of a site that is hypersensitive to cleavagewith DNAase I (see Section 29.I8, DNAase Hypersensitive Sites Change Chromatin Structure). Sometimes there are lessdramatic changes,for example, involving a change in rotational positioning of a single nucleosome; this may be detected by loss of the DNAase I I0 base ladder. Thus changes in chromatin structure may extend from altering the positions of nucleosomes to removing them altogether. Chromatin remodeling is undertaken by large complexes that use ATP hydrolysis to provide the energy for remodeling. The heart of the remodeling complex is its ATPasesubunit.
Remodeling complexes are usually classified according to the type of ATPasesubunit-those with related ATPasesubunits are considered to belong to the same family (usually some other subunits are common as well). | lillii]$ ::l+.iikeeps the names straight.The two major tlpes of complex are SWI/SNF and ISWI (ISWI stands for
of chroii{.:i.:iii:ii.'!.-iThedynamic modelfortranscription provided by thatcanuseenergy matinreties uponfactors fromspecific nucteosomes hydrotysis of ATPto disp[ace D N As e o u e n c e s .
Nucleosome is displaced , r .r
'i
!l
II
v
V Spacing becomes
,i,'\
rl.;
i
IT I
v
Gap of free DNA
to nucleosomes cancause +'iiltJitF comptexes :!Li.+Remodeling fromDNA.or canreornucleosomes slidealongDNA,candisplace ganize nucleosomes. between thespacing
Is an ActiveProcess 799 Remodelinq 30.3 Chromatin
of a (remodeled) nucleosome on the original DNA or to displacement of the histone octamer Type of Complex SWI/SNF lSWl Other to a different DNA molecule. The S\M/SNF comYeast SWYSNF lSWl lNO80complex plex alters nucleosomal sensitivity to DNAase RSC ISW2 SWRI I at the target site, and induces changes in protein-DNA contacts that persist after it has Fly dSW|/SNF NURF been releasedfrom the nucleosomes. The Swi2 (brahma) CHRAC ACF subunit is the ATPasethat provides the energy for remodeling by SWI/SNF. Human hSW|/SNF RSF NuRD There are many contacts between DNA and hACFAIVCFR lNO80complex a histone octamer; fourteen are identified in hCHRAC SRCAP the crystal structure. All of these contacts must WICH be broken for an octamer to be released or for Frog WICH Mi-2 it to move to a new position. How is this CHRAC achieved? Some obvious mechanisms can be ACF excluded becausewe know that single-stranded DNA is not generated during remodeling (and t I G i J R Ei i 0 . 5 Remodeling complexes canbe classified bytheirATPase there are no helicase activities associatedwith subunits. the complexes).Presentthinking is that remodimitation SWI). Yeasthas two SWI/SNF comeling complexes in the SWI and ISWI classes plexes and three ISWI complexes. Complexes use the hydrolysis of ATP to twist DNA on the of both types are also found in fly and in the nucleosomal surface.Indirect evidence suggests human being. Each type of complex may underthat this createsa mechanical force that allows take a different range of remodeling activities. a small region of DNA to be released from the SWI/SNF was the first remodeling comsurface and then repositioned. plex to be identified. Its name reflects the fact One important reaction catalyzedby remodthat many of its subunits are coded by genes eling complexes involves nucleosome sliding. It originally identified by SWI or SNF mutations in was first observedthat the IS\M family affectsnudeSaccharomyces cerevisiae.Mutations in these loci osome positioning without displacing octamers. are pleiotropic, and the range of defects is simThis is achieved by a sliding reaction, in which the ilar to those shown by mutants that have lost octamer moves along DNA. Sliding is prevented if the carboxyl-terminaldomain (CTD) tail of RNA the N-terminal tail of histone H4 is removed, but polymerase II. These mutations also show we do not know exactly how the tail functions in genetic interactions with mutations in genes this regard. SWI/SNF complexes have the same that code for components of chromatin, in parcapacity; the reaction is prevented by the introducticular SINI, which codesfor a nonhistone protion of a barrier in the DNA" which suggeststhat a tein, and SIN2,which codesfor histone H3. The sliding reaction is involved, in which the histone SI4'1and SNF genes are required for expression octamer moves more or less continuously along of a variety of individual loci (-120, or 2"h, of. DNA without ever losing contact with it. S. cerevisiaegenes are affected). Expression of One puzzle about the action of the S\M/SNF these loci may require the SWI/SNF complex complex is its sheer size. It has eleven subunits to remodel chromatin at their promoters. with a combined molecular weight -2 x 106. It SWI/SNF acts catalytically invitro, and there dwarfs RNA polymerase and the nucleosome, are only -150 complexesper yeast cell. All of making it difficult to understand how all of these the genes encoding the SWI/SNF subunits are components could interact with DNA retained on nonessential,which implies that yeast must aiso the nucleosomal surface.A transcription complex have other ways of remodeling chromatin. The with full activity, howeve(, called RNA polymerase RSC (remodelsthe structure of chromatin) comII holoenzyme, can be found that contains the plex is more abundant and also is essential. It RNA polymerase itself, all the TFn factors except acts at - 700 target loci. TBP and TFnA,and the S\M/SNF complex, which SWI/SNF complexes can remodel chrois associatedwith the CTD tail of the polymerase. matin in vitro without overall loss of histones In fact, virtually all of the S\M/SNF complex may or can displace histone octamers. Both types of be present in holoenzyrne preparations. This sugreaction may pass through the same intermegests*rat the remodeling of chromatin and recogdiate in which the structure of the target nucienition of promoters is undertaken in a coordinated osome is altered, leading either to reformation manner by a single complex.
800
CHAPTER 30 Controtting Chromatin Structure
NucLeosome 0rganization MayBeChanged at the Promoter r Remodeling complexes arerecruited to promoters by sequence-specifi c activators. . Thefactormaybe reteased oncethe remodeling c o m p t ehxa sb o u n d . r TheMMTV promoter requires a change in rotational positioning of a nucleosome to attowan activator to bindto DNAon the nucleosome.
How are remodeling complexes targeted to specific sites on chromatin? They do not themselves contain subunits that bind specific DNA sequences.This suggeststhe model shown in !.1.-",r-ri,:i: :;;:.1,, in which they are recruited by activators or (sometimes)by repressors. The interaction between transcription factors and remodeling complexes gives a key insight into their modus operandi. The transcription factor Swi5 activates the HO locus in yeast. (Note that Swi5 is not a member of the SWI/SNF complex.) Swi5 entersnuclei toward the end of mitosis and binds to the IIO promoter. It then recruits SWI/SNF to the promoter. Swi5 is then released,Ieaving SWI/SNF at the promoter. This means that a transcription factor can activate a promoter by a "hit and run" mechanism, in which its function is fulfilled once the remodeling complex has bound. The involvement of remodeling complexes in gene activation was discovered because the complexes are necessaryfor the ability of certain transcription factors to activate their target genes. One of the first examples was the GAGA factor, which activates the hsp70 Drosophilapromoter invitro. Binding of GAGA to four (CT),-rich siteson the promoter disrupts the nucleosomes, creates a hypersensitive region, and causesthe adjacent nucleosomes to be rearranged so that they occupy preferential instead of random positions. Disruption is an energy-dependentprocessthat requiresthe NURF remodeling complex. The organization of nucleosomesis altered so as to createa boundary that determines the positions of the adjacent nucleosomes. During this process,GAGA binds to its target sitesand DNA, and its presencefixes the remodeled state. The PHO system was one of the first in which it was shown that a change in nucleosome organization is involved in gene activation. At the PHO5 promoter, the bHLH regulator
complex bindsto chromatin A remodeting (or repressor). viaan activator
PHO4 responds to phosphate starvation by inducing the disruption of four precisely positioned nucleosomes. This event is independent of transcription (it occurs in a TATA- mutant) and independent of replication. There are two binding sites for PHO4 at the promoter. One is located between nucleosomes, which can be bound by the isolated DNA-binding domain of PHO4, and the other lies within a nucleosome, which cannot be recognized. Disruption of the nucleosome to allow DNA binding at the second site is necessary for gene activation. This action requires the presence of the transcription-activating domain. The activator sequence of VPl6 can substitute for the PHO4 activator sequence in nucleosome disruption. This suggeststhat disruption occurs by protein-protein interactions that involve the same region that makes protein-protein contacts to activate
at the Promoter MayBeChanged Organization 30.4 Nucleosome
transcription. In this case,it is not known which remodeling complex is involved in executing the effects. A survey of nucleosome positions in a large region of the yeast genome showed that most sites that bind transcription factors are free of nucleosomes.Promoters for RNA polymerase II typically have a nucleosome-free region -200 bp upstream of the startpoint, which is flanked by positioned nucleosomeson either side. It is not always the case, however, that nucleosomesmust be excluded in order to permit initiation of transcription. Some activators can bind to DNA on a nucleosomal surface. Nucleosomesappear to be preciselypositioned at some steroid hormone responseelementsin such a way that receptors can bind. Receptor binding may alter the interaction of DNA with histones,and may even lead to exposure of new binding sites.The exact positioning of nucleos o m e s c o u l d b e r e q u i r e d e i t h e r b e c a u s et h e nucleosome "presents"DNA in a particular rotational phase or becausethere are proteinprotein interactions between the activators and histones or other components of chromatin. Thus we have now moved some way from viewing chromatin exclusively as a repressivestructure to considering which interactions between activators and chromatin can be recuired for activation.
The MMTV promoter presents an example of the need for specific nucleosomal organization. It contains an array of six partly palindromic sites, each bound by one dimer of hormone receptor (HR), which constitute the HRE. It also has a single binding site for the factor NFl, and two adjacent sites for the factor OTF. HR and NFI cannot bind simultaneously to their sites in free DNA. i:I{li,iS*;iii.!"shows how the nucleosomal structure controls binding of the factors. The HR protects its binding siresat the promoter when hormone is added, but does not affect the micrococcal nuclease-sensitive sites that mark either side of the nucleosome. This suggeststhat HR is binding to the DNA on the nucleosomal surface; however, the rotational positioning of DNA on the nucleosome prior to hormone addition allows accessto only two of the four sites. Binding to the other two sites requires a change in rotational positioning on the nucleosome. This can be detected by the appearanceof a sensitive site at the axis of dyad symmetry (which is in the center of the binding sitesthat constitute the HRE). NFI can be footprinted on the nucleosome after hormone induction, so these structural changes may be necessaryto allow NFI to bind, perhaps because they expose DNA and abolish the steric hindrance by which HR blocks NFI binding to free DNA.
Histone Modification Is a KeyEvent r Histones aremodifiedby methytation, acety[ation, andphosphorylation.
i : i r ' i l i ; ; i;.i r . i H o r m o nree c e p t oarn dN F 1c a n n o bt i n d jn theformof [insimuttaneously promoter to the MMTV earDNA,but canbindwhenthe DNAis oresented on a nuc[eosomaI surface,
CHAPTER 30 Controtting ChromatinStructure
Whether a gene is expressed depends on the structure of chromatin both locally (at the promoter) and in the surrounding domain. Chromatin structure correspondingly can be regulated by individual activation events or by changesthat affect a wide chromosomal region. The most localized events concern an individual target gene, where changes in nucleosomal structure and organization occur in the immediate vicinity of the promoter. More general changes may affect regions as large as a whole chromosome. Changes that affect large regions control the potential of a gene to be expressed.The term silencing is used to refer to repression of gene
activity in a local chromosomal region. The term heterochromatin is used to describechromosomal regions that are large enough to be seen to have a physically more compact structure in the microscope. The basis for both types of change is the same: Additional proteins bind to chromatin and either directly or indirectly prevent transcription factors and RNA polymerase from activating promoters in the region. Changesat an individual promoter control whether transcription is initiated for a particular gene. These changesmay be either activatrng or represslng. All of these events depend on interactions with histones. Changes in chromatin structure are initiated by modifying the N-terminal tails of the histones, especiallyH3 and H4. The histone tails consist of 15-30 amino acids at the N-termini of all four core histones and the C-terminus of H2A. The tails of H2B and H 3 p a s s b e t w e e n t h e t u r n s o f D N A ( s e eF i g tre 29.21 in Section 29.7, Organization of the Histone Octamer). : 1i,,i.if !: :', ;, shows that they can be modified at several sites by methylation. acetylation, or phosphorylation. Other modifications, such as mono-ubiquitylation or sumoylation, also occur but are less well characterized. Acetylation and methylation occur on the free (e) amino group of lysine. As seen in I i i ', i, acetylation removes the positive chargethat resideson the NHI form of the group. Methylation also occurs on arginine. Phosphorylation occurs on the hydroxyl group of serine and also on threonine. This introduces a negative charge in the form of the phosphate group. Lysine can be mono-, di-, or trimethylated (all still positively charged), and arginine can be mono- or dimethylated (symmetrically or asymmetrically). Thesemodifications are transient. They can change the charge of the protein molecule, and as a result they are potentially able to change the functional properties of the octamers. Modification of histones is associatedwith structural changes that occur in chromatin at replication and transcription. Phosphorylations on specific positions and on different histones may be required for particular processes,for example, the Serr0position of Hl is phosphorylated when chromosomes condenseat mitosis. In synchronized cells in culture, both the preexisting and newly synthesizedcore histones appear to be acetylated and methylated during S phase (when DNA is replicated and the histones also are synthesized).During the cell rycle, the modifying groups are later removed.
The coincidence of modification and replication suggeststhat acetylation (and methylation) could be connected with nucleosome assembly. One speculation has been that the reduction of positive chargeson histones might lower their affinity for DNA, thus allowing the reaction to be better controlled. The idea has lost some ground in view of the observation that nucleosomes can be reconstituted, at least invitro, with unmodified histones. Histone acetylation is essentialfor nucleosome assemblyin yeast, and is probably required for some of the protein-protein interactions that occur during later stagesof the reaction (seeSection 10.6, Histone Acetylation Occursin TWoCircumstances).
Sites of modificationin H3
1
2
3
4
5
6
7
I
I
1011 121314
8
I
1011 121314
in H4 Sitesof modiiication
2
3
4
5
6
7
T h eN - t e r m i ntaaLi [ so f h i s t o n eHs 3a n dH 4 at severaI methylated, or phosphorylated canbeacetytated, positions.
Lysine
Serine
CH, t-
of or phosphorylation Acetylation of Lysine positive of a protein. charge serinereduces the overatl
Is a KevEvent 30.5 HistoneModification
803
A cycle of phosphorylation and dephosphorylation occurs with HI, but its timing is different from the modification cycle of the other histones. With cultured mammalian cells, one or two phosphate groups are introduced at S phase. The major phosphorylation event is the later addition of more groups at mitosis, though, which brings the total number up to as many as six. AII the phosphate groups are removed at the end of the processof division. The phosphorylation of HI is catalyzedby the M-phase kinase that provides an essentialtrigger for mitosis. In fact, this enzyme is now often assayedin terms of its Hl kinase activity. Nor m u c h i s k n o w n a b o u t p h o s p h a t a s e ( s )t h a t remove the groups later. The timing of the major Hl phosphorylation has prompted speculationthat it is involved in mitotic condensation. In Tetrahymena(a pro-
tozoarrl, however, it is possible to delete all the genes for Hl without significantly affecting the overall properties of chromatin. There is a relatively small effect on the ability of chromatin to condense at mitosis. Some genes are activated and others are repressedby this change, which suggeststhat there are alterations in local structure. Mutations that eliminate sitesof phosphorylation in Hl have no effect,but mutations that mimic the effects of phosphorylation produce a phenotype that resembles the deletion. This suggeststhat the effect of phosphorylating HI is to eliminate its effects on local chromatin structure. Do histone modifications affect nucleosome structure directly, or is their effect upon chromatin indirect? There is not in fact much evidence for any difference in the properties of nucleosomes depending on the state of the modification of the histones. Severalcaseshave now been characterized, though, in which histone modification createsbinding sites for the attachment of nonhistone proteins that change the properties oI chroma tin. The range of nucleosomes that is targeted for modification can vary. Modification can be a local event, for example, restricted to nucleosomes at the promoter. It also can be a general event, extending, for example, to an entire chromosome. FSli#R* 3*,:!* shows that there is a general correlation in which acetylation is associated with active chromatin, whereas methylation is associated with inactive chromatin. This is not, however, a simple rule, and f,:Slifif -:*.1* Acetylation of H3 and H4is associated withactivechromatin, whereas methvlation is associated the particular sitesthat are modified (as well as withinactivechromatin. combinations of specificmodifications) may be important, so there are certainly exceptions in which (for example) histones methylated at a certain position are found in active chromatin. Histone Site Modilication Function Mutations in one of the histone acetylasecomH3 Lys-4 Methylation Activation plexes of yeast have the opposite effect from H3 Lys-9 l,4ethylation Chromatincondensation usual (they prevent silencing of some genes); Lys-9 Methylation Requiredfor DNA methylation this emphasizes the lack of a uniform effect of Lys-9 Acetylation Activation acetylation. H3 Ser-10 Phosphorylation Activation H3 The specificity of the modifications is indiLys-14 Acetylation Preventsmethylationat Lys-g H3 Lys-79 Methylation Telomericsilencing cated by the fact that many of the modifying enzymes have individual target sitesin specific H4 Arg-3 Methylation histones. fg$ilftil3*"?'i summarizes the effectsof H4 Lys-S Acetylation Assembly some of the modifications. Most modified sites H4 Lys-12 Acetylation Assembly are subject to only a single type of modification. H4 Lys-16 Acetylation Nucleosomeassembly In some cases,modification of one site may actiLys-16 Acetylation Fly X activation vate or inhibit modification of another site. The idea that combinations of signals may be used f?fiijR$ i*".! 1 Mostmodifieds'itesin histoneshavea single.specifictype of to define chromatin types has sometimes been modification,but somesitescan havemorethan one type of modification. IndividuaIfunctionscan be associated with someof the modifications. called the histonecode.
804
CHAPTER 30 Controllinq Chromatin Structure
Histone Acetylation 0ccursin Two Circumstances . Histoneacetytation occurstransiently at rentication. r Histoneacetylation is associated with activation of geneexpression.
All the core histones can be acetylated. The major targets for acetylation are lysines in the N-terminal tails of histones H3 and H4. Acetylation occurs in two different circumstances: . during DNA replication, and . when genesare activated. When chromosomes are replicated. which occurs during the S phase of the cell cycle, histones are transiently acetylated. i:: t:i,iijiI ;,{:.r*-i;i shows that this acetylation occurs before the histones are incorporated into nucleosomes. We know that histones Hl and H4 are acetylated at the stagewhen they are associatedwith one another in the H)2-H42 tetramer. The tetramer is then incorporated into nucleosomes. Quite soon after, the acetyl groups are removed. The importance of the acetylation is indicated by the fact that preventing acetylation of both histones H3 and H4 during replication causesloss of viability in yeast. The two histones are redundant as substrates.becauseyeast can manage perfectly well so long as they can acetylate either one of these histones during S phase. There are two possible roles for the acetylation: It could be needed for the histones to be recognized by factors that incorporate them into nucleosomes,or it could be required for the assembly and/or structure of the new nucleosome. The factors that are known to be involved in chromatin assembly do not distinguish between acetylatedand nonacetylated histones, which suggeststhat the modification is more likely to be required for subsequent interactions. It has been thought for a long time that acetylation might be needed to help control protein-protein interactions that occur as histones are incorporated into nucleosomes.Some evidence for such a role is that the yeast SAS histone acetylase complex binds to chromatin assembly complexes at the replication fork, where it acetylates r6lys of histone H4. This may be part of the system that establishesthe histone acetylation patterns after replication.
Outside of S phase,acetylation of histones in chromatin is generally correlated with the state of gene expression.The correlation was first noticed because histone acetylation is increased in a domain containing active genes, and acetylated chromatin is more sensitive to DNAase I and (possibly) to micrococcal nucleii.r.:lrishows that this involves the ase. i.i*:ritiS: acetylation of histone tails in nucleosomes. We now know that this occurs largely because of acetylation of the nucleosomes in the vicinity of the promoter when a gene is activated.
occurs on hisirI{.il,lHf at rep[ication i+-i.i.iAcetytation into nucleosomes. tonesbefore theyareincorporated
lI{,ilfiii ;ii,t.1.:iAcetytationassociatedwith geneactivatjon occursby directtymodifoinghistonesin nucleosomes.
in TwoCircumstances Occurs Acetytation 30.6 Histone
In addition to eventsat individual promoters, widescalechangesin acetylation occur on sex chromosomes. This is part of the mechanism by which the activities of genes on the X chromosome are altered to compensate for the p r e s e n c eo f t w o X c h r o m o s o m e si n o n e s p e c i e s but only one X chromosome (in addition to the Y chromosome) in other species(see Section 31.5, X Chromosomes Undergo Global Changes).The inactive X chromosome in female mammals has underacetylatedH4. The superactive X chromosome in Drosophilamales has increasedacetylation of H4. This suggeststhat the presenceof acetyl groups may be a prerequisite for a lesscondensed,active structure. In male Drosophila, the X chromosome is acetyIated specificallyat r6lys of histone H4. The histone acetyltranoferase(HAI) that is responsible is an enzyme calied MOF that is recruited to the chromosome as part of a large protein complex. This "dosagecompensation" complex is responsible for introducing general changes in the X chromosome that enable it to be more highly expressed.The increasedacetylation is only one of its activities.
@
o a
Acetylases AreAssociated with Activators
Deacetytated chromatin mayhavea more condensed structure. Transcription activators areassociated with histoneacetytase activities in [argecomptexes. Histoneacetytases varyin theirtargetspecificity. Acetytation couldaffecttranscription in a quantitative or quatitative way.
sion; in fact, the ability of butyric acid to cause changes in chromatin resembling those found upon gene activation was one of the first indications of the connection between acetylation and gene activity. The breakthrough in analyzing the role of histone acetylation was provided by the characterization of the acetylating and deacetylating enzymes, and their association with other proteins that are involved in specific events of activation and repression.A basic change in our view of histone acetylation was caused by the discovery that HATs are not necessarily dedicated enzymes associated with chromatin: rather, it turns out that known activators of transcription have HAT activity. The connection was established when the catalytic subunit of a group A HAI was identified as a homolog of the yeastregulator protein Gcn5. It then was shonm that Gcn5 itself has HAI activity (with histonesH3 and H4 as substrates).Gcn5 is part of an adaptor complex that is necessary for the interaction between certain enhancers and their target promoters. Its HAT activity is required for activation of the target gene. This enables us to redraw our picture for the .:., action of coactivatorsas shown in f],{-iiit,Ji: il{"}.11 where RNA polymerase is bound at a hypersensitive site and coactivatorsare acetylating histones on the nucleosomesin the vicinity. Many examples are now known of interactions of this t1pe. Gcn5 leads us into one of the most important acetylasecomplexes. In yeast, Gcn5 is part
Coactivators
Basalapparatus
Activators
Acetylation is reversible.Each direction of the reaction is catalyzedby a specifictype of enzyme. Enzymes that can acetylate histones are called histone acetyltransferases or HATs; the acetyi groups are removed by histone deacetylases or HDACs. There are two groups of HAT enzymes: those in group A act on histones in chromatin and are involved with the control of transcription; those in group B act on newly s y n t h e s i z e dh i s t o n e s i n t h e c y t o s o l , a n d a r e involved with nucleosome assembly. lWo inhibitors have been useful in analyzing acetylation. Trichosratin and butyric acid inhibit histone deacetylases,and causeacetylated nucleosomes to accumulate. The use of these inhibitors has supported the general view that acetylationis associatedwith gene expres-
806
CHAPTER 30 ControtlingChromatinStructure
I 5r;iili!:.;tl,,.i+ Coactivators mayhaveHATactivitiesthat acetylate the tai[s of nucleosomaI histones.
of the 1.8 MDa Spt-Ada-Gcn5-acetyltransferase ( SAGA) complex, which contains severalproteins that are involved in transcription. Among these proteins are several TAF1s. In addition, the TAFtr145subunit of TFnD is an acetylase.(Yeast TAF1l4S is the homolog of mammalian TAF1250; both are known as TAFI.) There are some functional overlaps between TFnD and SAGA, most notably that yeast can manage with either TAFrrl45 or Gcn5,but is damagedby the deletion of both. This suggeststhat an acetylaseactivity is essentialfor gene expression,but can be provided by either TFnD or SAGA. As might be expected from the size of the SAGA complex, acetylation is only one of its functions, although its other functions in gene activation are lesswell characterized. One of the first general activatorsto be characterizedas HAT was p300/CREB-bindingprotein (CBP). (Actually,p300 and CBPare different proteins, but they are so closely related that they are often referred to as a single type of activity.) p300/CBP is a coactivator that links a n a c t i v a t o r t o t h e b a s a l a p p a r a t u s ( s e eF i g ure 25.7).p300/CBPinteractswith various activators, including horrnone receptors,AP- I (c-Jun and c-Fos),and MyoD. The interaction is inhibited by the viral regulator proteins adenovirus EIA and SV40 T antigen, which bind to p300/CBP to prevent the interaction with transcription factors; this explains how these viral proteins inhibit cellular transcription. (Thisinhibition is important for the ability of the viral proteins to contribute to the tumorigenic state.) p300/CBP acetylatesthe N-terminal tails of H4 in nucleosomes. Another coactivator, PCAF, preferentially acetylates H3 in nucleosomes. p300/CBPand PCAFform a complex that functions in transcriptional activation. In some cases yet another HAT is involved: the coactivator ACTR, which functions with hormone receptors, is itself an HAT that acts on H3 and H4, and aiso recruitsboth p300/CBP and PCAF to form a coactivating complex. One explanation for the presenceof multiple HAT activitiesin a coactivating complex is that each HAT has a different specificity, and that multiple different acetylation events are required for activation. A general feature oI acetylation is that a group A HAT is part of a large complex. ,''!r.ri:r,: 1r..: : shows a simplified model for their behavior. HAT complexes can be targeted to DNA by interactions with DNA-binding factors. This determinesthe target for the HAT. The complex also contains effector subunits that affect chromatin structure or act directly on transcription. It is likely that at least some of the effec-
tors require the acetylation event in order to act. Deacetylation,catalyzedby an HDAC, may w o r k i n a s i m i l a rw a y . Acetylation occursat both replication (when it is transient) and at transcription (when it is maintained while the gene is active). Is it playing the same role in each case?One possibility is that the important effect is on nucleosome structure. Acetylation may be necessary to "loosen" the nucleosome core. At replication, acetylation of histones could be necessaryto allow them to be incorporated into new cores more easily. At transcription, a similar effect could be necessaryto allow a related change in structure, possiblyeven to allow the histone core to be displacedfrom DNA. Alternatively, acetyIation could generatebinding sitesfor other proteins that are required for transcription. In either case,deacetylation would reverse the effect. Is the effect of acetylation quantitative or qualitative? One possibility is that a certain number of acetyl groups are required to have an effect, and the exact positions at which they occur are largely irrelevant. An alternative is that individual acetylation events have specific effects.We might interpret the existenceof complexes containing multiple HAT activities in either way-if individual enzymes have different specificities, we may need multiple activities either to acetylate a sufficient number of different positions or because the individual events are necessaryfor different effects upon transcription.At replication,it appears(at least with respectto histone H4) that acetylation at any two of three availablepositions is adequate, favoring a quantitative model in this case.Where chromatin structure is changed to affect transcription, acetylation at specific positions is important (seeSection 31.3, Heterochromatin Depends on Interactions with Histones).
r ii-ii jrri ii.,.r 1, Comptexes or structure that modifychromatin theirsitesof that determine activityhavetargetingsubunits hisor deacetytate thatacetytate enzymes action,HATor HDAC that haveotheractionson chrotones,andeffectorsubunits matinor DNA.
with Activators AreAssociated 30.7 Acetytases
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Deacetylases Are Associated with Repressors
Deacetytation is associated with repression of gene activity. Deacetytases arepresent in complexes with repressor activity.
In yeast,mutations in SlNf and RPD3behave as though these loci repressa variety of genes.The proteins form a complex with the DNA-binding protein Ume6, which binds to the URS/ element. The complex repressestranscription at the promoters containing URSl, as illustrated ';i.i.ii. Rpd3 has histone deacetylase in :.:i='"titi.: activity; we do not know whether the function of SinS is just to bring Rpd3 to the promoter, or whether it has an additional role in repression. A similar system for repressionis found in mammalian cells.The bHLH family of transcription regulators includes activators that function as heterodimers, including MyoD (see Section 25. I 5, Helix-Loop-Helix Proteins Interact by Combinatorial Association).It also includes repressors, in particular the heterodimer Mad:Max, where Mad can be any one of a group of closely related proteins. The Mad:Max heterodimer (which binds to specific DNA sites) interactswith a homolog of Sinl (calledmSin3 in mouse and hSinS in human beings).mSin3 is part of a repressivecomplex that includes his-
iriiltliii; "i;;.-i+ A repressor comptexcontainsthree components:a DNAbindingsubunit,a corepressor, and a histone deacety[ase.
CHAPTER 30 Controlting Chromatin Structure
tone binding proteins and the histone deacetyIasesHDAC I and HDAC2. Deacetylaseactivity is required for repression. The modular nature of this system is emphasized by other means of e m p l o y m e n t : A c o r e p r e s s o r( S M R T ) , w h i c h enables retinoid hormone receptors to repress certain target genes,functions by binding mSin3, which in turns brings the HDAC activities to the site. Another means of bringing HDAC activities to the site may be a connection with MeCP2, a protein that binds to methylated cytosines (see Section 24.19, CpG Islands Are Regulatory Targets). Absence of histone acetylation is also a feature of heterochromatin. This is true of both constitutive heterochromatin (typically involving regions of centromeres or telomeres) and facultative heterochromatin (regions that are inactivated in one cell although they may be active in another). T\upicallythe N-terminal tails of histones H3 and H4 are not acetylated in heterochromatic regions.
Methylation of Histones andDNAIs Connected o Methytation of both DNAandhistonesis a feature of inactivechromatin. o Thetwo typesof methytation eventmaybe connected.
Methylation of both histones and DNA is associated with inactivity. Sitesthat are methylated in histones include two lysines in the tail of H3 and an arginine in the tail of H4. Methylation of H3 elys is a feature of condensed regions of chromatin, including heterochromatin as seen in bulk and also smaller regions that are known not to be expressed.The histone methyltransferase enzyme that targets this lysine is called SUV39Hl. (We seerhe origin of this peculiar name in Section 30.13, Some Common Motifs Are Found in Proteins That Modify Chromatin).Its catalyticsite has a region called the SET domain. Other histone methyltransferasesact on arginine. In addition, methylation may occur on Telys in the globular core region of H3; this may be necessaryfor the formation of heterochromatin at telomeres. Until recently, it was thought that histone methylation was irreversible. Histone demethylaseshave now been identified, though, including a lysine-specificdemethylase (LSDI) that
acts on I(4 of histone H3, and an enzyme that demethylatesarginine on histonesHl and H4. We do not yet know how demethylation is regulated. Most of the methylation sites in DNA are C p G i s l a n d s ( s e e S e c t i o n2 4 . 1 9 , C p G I s l a n d s Are Regulatory Targets).CpG sequencesin heterochromatin are usually methylated. Conv e r s e l y , i t i s n e c e s s a r yf o r t h e C p G i s l a n d s located in promoter regions to be unmethylated in order for a gene to be expressed(see Section 24.18, Gene Expression Is Associated with Demethylation). Methylation of DNA and methylation of histones is connected in a mutually reinforcing circuit. Methylation of H3 is the signal that recruits the DNA methylase to chromatin. The order of events is that H3 el,ys is deacetylated to create the substrate for methylation. H3 is then converted to the Meelys or the Me2elys condition, which provides a binding site for the DNA methylase. Some histone methyltransferase enzymes contain potential binding sites for the methylated CpG doublet, so the DNA methylation reinforces the circuit by providing a large| for the histone methyltransferase to bind. The important point is that one type of modification can be the trigger for another. These systemsare widespread,as seen by evidence for these connections in fungi, plants, and animal cells, and for regulating transcription at promoters used by both RNA polymerases I and II, as well as maintaining heterochromatin in an inert state.
The reversetypes of events occur if we compare the activation of a promoter with the generation of heterochromatin. The actions of the enzymes that modify chromatin ensure that activating events are mutually exclusive with inactivating events. Methylation of H3 el-ysand acetylation of H3 lalys are mutually antagonistic. Acetylasesand deacetylasesmay trigger the initiating events. Deacetylation allows methylation to occur, which causesformation of a heterochromatic complex (see Section 31.3, Heterochromatin Depends on Interactions with Histones).Acetylation marks a region as active (seeSection10.I l, Promoter Activation Involves an Ordered Seriesof Events).
Activation Promoter Involves an 0rdered of Events Series o Theremode[ing mayrecruitthe comptex acetylating comp[ex. o Acetytation of histonesmaybethe eventthat state. in the activated maintajns the complex
How are acetylases(or deacetylases)recruited to their specific targets? As we have seen with remodeling complexes, the process is likely to
Chromatin States AreInterconverted by Modification Acetytation of histones is associated with gene activation. Methytation of DNAandof histones is associated with heterochromatin. irii:i:ltir::ij.i ;' summarizesthree types of differencesthat are found between active chromatin and inactive chromatin: . Active chromatin is acetylated on the tails of histones H3 and H4. . Inactive chromatin is methylated on el-ys of histone H3. . Inactive chromatin is methylated on cytosinesof CpG doublets.
chroactivates i l.tiiiii:t: of histones ..rii.i : Acetylation inactivates of DNAandhistones matin,andmethylation chromatin.
Series of Events Involves an 0rdered Activation 30.11Promoter
be indirect. A sequence-specificactivator (or repressor) may interact with a component of the acetylase(or deacetylase)complex to recruit lt to a promoter. There may also be direct interactions between remodeling complexes and histonemodifying complexes. Binding by the SWI/SNF remodeling complex may lead in turn to binding by the SAGA acetylasecomplex. Acetylation of histones may then in fact stabilizethe associationwith the SWI/SNF complex, making a mutual reinforcement of the changes in the components at the promoter.
We can connect all of the events at the promoter into the seriessummarized in ftf:#gtil3{,i.ti.*. The initiating event is binding of a sequencespecificcomponent (which is able to find its target DNA sequencein the context of chromatin). This recruits a remodeling complex. Changes occur in nucleosome structure. An acetylating complex binds, and the acetylation of target histones provides a covalent mark that the locus has been activated. Modification of DNA also occurs at the promoter. Methylation of cytosine at CpG doublets is associated with gene inactivity (see SecIion24.I8, Gene Expression Is Associatedwith Demethylation). The basis for recognition of DNA as atargel for methylation is not very well established (see Section 31.8, DNA Methylation Is Responsible for Imprinting). It is clear that chromatin remodeling at the promoter requires a variety of changes that affect nucleosomes, including acetylation, but what changes are required within the gene to allow an RNA polymerase to traverse it? We know that RNApolymerase can transcribe DNA in vitro at rates comparable to the in vivo rare (-25 nucleotidesper second) only with a template of free DNA. Severalproteins have been charactedzed for their abilities to improve the speed with which RNA polymerase transcribes chromatin in vivo. The common feature is that they act on chromatin. A current model for their action is that they associatewith RNA polymerase and travel with it along the template, modifying nucleosome structure by acting on histones.Among these factorsare histone acetyIases.One possibility is that the first RNA polymerase to transcribe a gene is a pioneer polymerase carrying factors that change the structure of the transcription unit so as to make it easierfor subsequentpolymerases.
HistonePhosphorylation
AftectsChromatin Structure r At leasttwo histonesaretargetsfor phosphorylation, possibty with opposing effects. I i * i j R l - : t i i . : +P: r o m o t earc t i v a t i o inn v o t v e bs i n d i n go f a sequence-specific actjvator, recruitment andactjonof a remooelingcomptex, andrecruitment andactionof an acetytating comptex. Theorderofeventscandifferor canevenbesjmuttaneous depending on the gene.
810
CHAPTER 30 Controtting Chromatin Structure
Histones are phosphorylated in two circumstances: . cyclically during the cell cycle, and . in association with chromatin remodeling.
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with chromatin assembly factor ASFI. Genes D e v .1 5 , l l 5 5 - 1 I 6 8 .
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Acetytases AreAssociated with Activators r ch Resea histone Brownell, J. E. et al. (1996). Tetrahymena acetyltransferaseA: a homologue to yeast Gcn5p linking histone acetylation to gene activation. Cell 84, 843-851. Chen, H. etal,. (1997). Nuclear receptor coactivator ACTR is a novel histone acetyltransferase and forms a multimeric activation complex with P/CAF and CP/p300. Cell90, 569-580.
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816
CHAPTER 30 Controlling Chromatin Structure
(20041.Human PAD4regulateshistonearginine methylationlevelsvia demethylimination. Science )06, 279-283.
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SomeCommon MotifsAreFound in Proteins ThatModifvChromatin R e s erach D h a l l u i n , C . , C a r l s o n ,J . E . , Z e n g , L . , H e , C . , A g g a r wal, A. I(., and Zhou, M. M. (1999). Srructure and ligand of a histone acetyltransferase bromo domain. Nature 399, 491-496.
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EpigeneticEffectsCanBe Inherited . Epigenetic effectscanresultfrommodificationof a nucleic acidafterit hasbeen synthesized or by the perpetuation of protein structures. Y e a s tP r i o n sS h o wU n u s u aI ln h e r i t a n c e o The5up35proteinin its wild-type sotubte formis a termination factorfor transtati on, . It canalsoexistin an atternative formof otigomeric aggregates, in whichit is not actjvein proteinsynthesis. o Thepresence form of the oligomeric proteinto newtysynthesized causes theinactivestructure. acquire . Conversion between thetwo formsis inftuenced by chaperones. o Thewitd-type formhasthe recessive genetic statepsr andthe mutantformhas genetic the dominant statePSf+.
Introduction . Ep'igenetic effectscanresultfrommodification of a nucteic acidafterit hasbeensynthesized or by the perpetuation of proteinstructures.
Epigenetic inheritance describesthe ability of different states,which may have different phenotypic consequences,to be inherited without any changein the sequenceof DNA. This means that two individuals with the same DNA sequence at the locus that controls the effect may show different phenotypes.The basiccause of this phenomenon is the existence of a selfperpetuating structure in one of the individuals that does not depend on DNA sequence. Several different types of structures have the ability to sustain epigenetic effects: . A covalent modification of DNA (methyIation of a base). . A proteinaceous structure that assembies on DNA. . A protein aggregare that controls the conformation of new subunits as they are synthesized. In each casethe epigeneticstateresultsfrom a difference in function (typically inactivation) that is determined by the structure. In the case of DNA methylation, a methylated DNA sequencemay fail to be transcribed, whereas the nonmethylated sequencewill be 'r expressed., .i: . 'r : shows how this situation is inherited. One allele has a sequencethat is methylated on both strands of DNA, whereas the other allele has an unmethvlated secuence.
P r i o n sC a u s eD i s e a s ei ns M a m m a [ s . Theproteinresponsibte exists for scrapie jn twoforms:the witd-type noninfectious to proformPrPc, whichis susceptibte form andthe disease-causing teases, to proteases. PrPs., whichis resistant r TheneurotogicaI canbetransmitdisease tedto miceby injectingthe purifiedPrPsc proteininto mice. . Therecipient mouse musthavea copyof thePrPgenecodingfor the mouse protein. r ThePrPsc itsetfby proteincanperpetuate PrPprotein the newlysynthesized causing forminsteadof the to takeupthe PrPsc form. PrPc r Multipte mayhavedifferstrainsof PrPsc of the protein. entconformations Summary
Replication of the methylated allele creates hemimethylated daughters that are restored to the methylated state by a constitutively active methylase enzyme. Replication does not affect the state of the nonmethylated allele. If the state of methylation affectstranscription, the two alleles differ in their state of gene
site produces of a methylated r il.iiiii :r,I , i Replication strandis theparental DNA, in whichonl"y hemimethyiated hemimethrecognizes methytase methyl.ated. A perpetuation ytatedsitesandaddsa methylgroupto the baseon the in the originaIsituation. daughter strand.Thisrestores A nonmethyon bothstrands' whichthesiteis methylated afterreptication. nonmethytated Lated siteremains
31.1Introduction 819
expression, even though their sequencesare identical. Self-perpetuating structures that assemble on DNA usually have a repressiveeffect by forming heterochromatic regions that prevent the expression of geneswithin them. Their perpetuation depends on the ability of proteins in a heterochromatic region to remain bound to those regions after replication, and then to recruit more protein subunits to sustain the complex. If individual subunits are distributed at random to each daughter duplex at replication, the two daughters will continue to be marked by the protein, although its density will be reduced to half of the level before replication. shows that the existenceof enigeneticeffectsforcesus to the view that a protein responsiblefor such a situation must have some sort of self-templating or self-assemblingcapacity to restore the original complex. It can be the state of protein modification, rather than the presenceof the protein per se, that is responsiblefor an epigeneticeffect.Usually the tails of histones H3 and H4 arenot acerylated in constitutive heterochromatin. If centromeric heterochromatin is acetylated, though, silencedgenesmay become active. The effect may be perpetuatedthrough mitosis and
meiosis,which suggeststhat an epigeneticeffect has been created by changing the state of histone acetylation. Independent protein aggregatesthat cause epigenetic effects (called prions) work by sequestering the protein in a form in which its normal function cannot be displayed. Once the protein aggregale has formed, it forces newly synthesized protein subunits to join it in the inactive conformation.
Heterochromatin Propagates froma Nucleation Event r Heterochromatin is nucteated at a specific propagates sequence andthe inactivestructure alongthe chromatin fiber. . Genes withinregions of heterochromatin are inactivated. r Thelengthof the inactiveregionvariesfromce[[ to cet[ asa resutt,inactivation of genes in this positioneffectvariegation. vicinitycauses . Simitarspreading effectsoccurat tetomeres andat the sitentcassettes in yeastmatingtype.
An interphase nucleus contains both euchromatin and heterochromatin. The condensatit_rn state of heterochromatin is close to that of mitotic chromosomes.Heterochromatin is inert. It remains condensedin interphase, is transcriptionally repressed,replicates late in S phase, and may be localized to the nuclear periphery. Centromeric heterochromatin typically consists of satellite DNAs; however, the formation of heterochromatin is not rigorously defined by sequence.When a gene is transferred, either by a chromosomal translocation or by transfection and integration, into a position adjacent to heterochromatin, it maybecome inactive as the result of its new location, implying that it has become heterochromatic. Such inactivation is the result of an epigenetic effect (see Section 31.10, Epigenetic EffectsCan Be Inherited). It may differ between individual cells in an animal, and results in the phenomenon of position effect variegation (PEV), in which genetically identical cellshave ']r.r':' Heterochromatin is createdby proteins that different phenotypes. This has been well charassociate with histones. Perpetuation throughdivisionrequires acterized in Drosophila. ijl",L.rr'.-!i. ,:;!,:. shows an t h a t t h e p r o t e i n sa s s o c i a tw e j t h e a c hd a u g h t edr u p t e x andthenrecruitnewsubunits to reassembte the repressive example of position effect variegation in the fly c0mplexes. eye. Some of the regions in the eye lack color,
820
CHAPTER 31 Epigenetic Effects AreInherited
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inactive region may then be extended by the ability of further HPI molecules to interact with one another. The existence of a common basisfor silencing in yeast is suggestedby its reliance on a common set of genetic loci. Mutations in any one of a number of genes cause HML and HMR to become activated, and also relieve the inactivation of genes that have been integrated near telomeric heterochromatin. The products of these loci therefore function to maintain the inactive state of both types of heterochromatin. il{;il+ii:,';li .ii:proposes a model for actions of these proteins. Only one of them is a sequencespecific DNA-binding protein. This is RAPl, which binds to the C1-3Arepeats at the telomeres, and also binds to the crs-actingsilencer elements that are needed for repression of HML and HMR. The proteins Sir3 and Sir4 interact with RapI and also with one another (they may function as a heteromultimer). Sir3/Sir4 interact with the N-terminal tails of the histones H3 and H4. (In fact, the first evidence that histones might be involved directly in formation of heterochromatin was provided by the discovery that mutations abolishing silencing at HML/HMR map to genes coding for H3 and H4.) Rapl has the crucial role of identifying the DNA sequences at which heterochromatin forms. It recruits Sir3/Sir4, and they interact directly with the histonesH3lH4. Once SirS/Sir4 have bound to histones HllH4, the complex may polymerize further and spread along the chromatin fiber. This may inactivate the region, either becausecoating with SirS/Sir4itself has an inhibitory effect, or becausebinding to histones Hf /H4 induces some further change in structure. We do not know what limits the spreading of the complex. The C-terminus of Sirl has a similarity to nuclear lamin proteins (constituents of the nuclear matrix) and may be responsiblefor tethering heterochromatin to the nuclear periphery. A similar seriesof events forms the silenced regionsatHMRandHML (seealsoSection19.22, Silent Cassettes aL HML and HMR Are factorsare Repressed).Three sequence-specific involved in triggering formation of the complex: Rapl, Abf I (a transcription factor), and ORC (the origin replication complex). In this case, Sirl binds to a sequence-specificfactor and recruits Sir2, -3, and -4 to form the repressive structure. Sir2 is a histone deacetylase.The deacetylation reaction is necessaryto maintain binding of the Sir complex to chromatin.
histoneH3 i::i:ll!ii.:l i" I Bindingof HP1to methytated of furthermolecutes because formsa triggerfor sitencing H P 1a g g r e g aot en t h en u c t e o s o cmhea i n .
is initiated of heterochromatin .r Formation i':,fiq.l$i :1.1 w h e nR a p lb i n d st o D N A . 5 i r 3 /b4i n dt o R a p la n da t s ot o potymerizes a[ongchromatin comptex H3/H4.Ihe histones matrix. to the nuctear telomeres andmayconnect
with Histones 823 on Interactions Depends 31.3 Heterochromatin
Formation of heterochromatin in the yeast S. pombe depends on a complex that contains severalRNAi molecules (seeSection ll.I0, RNA Interference Is Related to Gene Silencing). These RNAi molecules are produced by transcription of centromeric repeats to give RNAs that are cleaved into smaller units. The complex also contains proteins that are homologs of those involved in heterochromatin formation in other organisms, including Argonaute, which is involved in targeting RNAinduced silencing complex (RISC) remodeling complexes to chromatin. The RNAi components are responsible for localizing the complex at centromeres. The complex then promotes dimethylation of histone H3 by a histone methyltransferase. How does a silencing complex represschromatin activity? It could condense chromatin so that regulator proteins cannot find their targets.The simplest casewould be to supposethat the presence of a silencing complex is mutually incompatible with the presenceof transcription factors and RNA polymerase.The causecould be that silencing complexes block remodeling (and thus indirectly prevent factors from binding) or that they directly obscure the binding sites on DNA for the transcription factors. The situation may not be this simple, though, because transcription factors and RNA porymerase can be found at promoters in silenced chromatin. This could mean that the silencing complex prevents the factors from working rather than from binding as such. In fact, there may be competition between gene activators and the repressing effects of chromatin, so that activation of a promoter inhibits spread of the silencing complex. Another specializedchromatin structure forms at the centromere. Its nature is suggested by the properties oI an Saccharlmycescerevisiae mutation, cse4,that disrupts the structure of the centromere. Cse4is a protein that is related to histone H3. A mammalian centromeric protein, centromere protein A (CENP-A), has a related sequence. Genetic interactions between cse4and CDE-II, and between cse4anda mutation in the H4 histone gene, suggestthat a histone octamer may form around a core of Cse4-H4,and then the centromeric complexes (core binding factors) CBFI and CBF3 may attach to form the centromere. The centromere may then be associatedwith the formation of heterochromatin in the region. In human cells,the centromere-specificprotein CENP-B is required to initiate modifications of
824
CHAPTER 31 Epigenetic EffectsAre Inherited
histone H3 (deacetylation of elys and lalys, followed by methylation of elys) thar trigger an association with the protein Swi6 that leads to the formation of heterochromatin in the resion.
Polycomb andTrithorax AreAntagonistic Repressors andActivators o Polycomb groupproteins(Pc-G)perpetuate a state of repression throughce[[divisions. r ThePRE is a DNAsequence that is required for the actionof Pc-G. r ThePREprovides a nucteation centerfromwhich Pc-Gproteinspropagate an inactivestructure. . Noindividual Pc-Gproteinhasyet beenfoundthat canbindthe PRE. o Trithorax groupproteinsantagonize the actionsof the Pc-G. Heterochromatin provides one example of the specificrepressionof chromatin. Another is provided by the genetics of homeotic genes in Drosophila,which have led to the identification of a protein complex that may maintain certain genes in a repressed stale. Pc mutants show transformations of cell type that are equivalent to gain-of-function mutations in the genes Antennapedia (Antp) or Ultrab ithorax, becatse these genes are expressedin tissuesin which usually they are repressed.This implicates pc in regulating transcription. Furthermore, Pcis the prototype for a class of -I5 loci called the pcgroup (Pc-G);mutations in these genes generally have the same result of derepressing homeotic genes, which suggeststhe possibility that the group of proteins has some common regulatory role. The Pc proteins function in large complexes. The PRCI (Polycomb-repressivecomplex) contains Pc itself, several other Pc-G proteins, and five general transcription factors. The Esc-E(z) complex contains Esc,E(z), other Pc-Gproteins, a histone-binding protein, and a histone deacetylase.Pc itself has a chromodomain that binds to methylated H3, and E(z) is a merhyltransferasethat acts on H3. These properties directly support the connection between chromatin remodeling and repression that was initially suggestedby the properties of brahma, a fly counterpartlo SW2. brahmacodesfor a component of the SWI/SNF remodeling complex, and loss of brahma function suppressesmutations in Polycomb.
Consistentwith the pleiotropy of Pcmutations, Pc is a nuclear protein that can be visualized at -80 sites on polytene chromosomes. These sites include rhreAntp gene. Another member of the Pc-G,polyhomeotic,is visualized at a set of polytene chromosome bands that are identical with those bound by Pc.The two proteins coimmunoprecipitate in a complex of -2.5 x 106D thatcontains l0 to 15 polypeptides.The relationship between these proteins and the products of the 40 Pc-Ggenes remains to be established.One possibility is that some of these gene products form a general repressivecomplex, and then some of the other proteins associate with it to determine its specificity. The Pc-G proteins are not conventional repressors.They are not responsiblefor determining the initial pattern of expression of the geneson which they act. In the absenceof PcG proteins, these genes are initially repressed as usual, but later in development the repression is lost without Pc-G group functions. This suggeststhat the Pc-Gproteinsin someway rec1gand nizethestateof repressionwhenitis established, perpetuate division of then act t0 it thrlugh cell they r ;:::t,:':,:t,, in cells. I shows a model the daughter which Pc-G proteins bind in conjunction with a repressor,but the Pc-G proteins remain bound after the repressor is no longer available. This is necessaryto maintain repression,so that if P c - G p r o t e i n s a r e a b s e n t ,t h e g e n e b e c o m e s activated. A region of DNA that is sufficient to enable the responseto the Pc-Ggenes is called a PRE (Polycomb responseelement). It can be defined operationally by the property that it maintains repressionin its vicinity throughout development. The assayfor a PRE is to insert it closeto a reporter gene that is controlled by an enhancer that is repressedin early development, and then to determine whether the reporter becomes expressedsubsequentlyin the descendants.An effective PRE will prevent such reexpression. The PRE is a complex structure that measures - I 0 kb. TWoproteins, Pho and Pho I , with DNA-binding activity for sites within the PRE have been identified, but there could be others. When a locus is repressedby Pc-G, however, the Pc-G proteins occupy a much larger Iength of DNA than the PRE itself. Pc is found locally over a few kilobasesof DNA surrounding a PRE. This suggeststhat the PRE may provide a nucleation center, from which a structural state depending on Pc-G proteins may propagate. This model is supported by the observation of
t i r r r i , t 'I,:,, r P c - G p r o t e i n s d o n o t i n i t i a t e r e p r e s s i o n . b u t a r e r e s p o n s i b [ e f o r m a i n t a i n i ni tg.
effectsrelated to position effect variegation (see Figure 3I.4), that is, a gene near to a locus whose repressionis maintained by Pc-G may become heritably inactivated in some cells but not others. In one typical situation, crosslinking experiments in vivo showed that Pc protein is found over large regions of the bithorax complex that are inactive, but the protein is excluded from regions that contain active genes.The idea that this could be due to cooperative interactions within a multimeric complex is supported by the existence of mutations in Pcthat change its nuclear distribution and abolish the ability of other Pc-Gmembers to localize in the nucleus. The role of Pc-G proteins in maintaining, as opposed to establishing, repression must mean that the formation of the complex at the PRE also depends on the local state of gene expressron. A working model for Pc-G binding at a PRE is suggestedby the properties of the individual proteins. First Pho and PhoI bind to specific sequenceswithin the PRE.Esc-E(z)is recruited to Pho/Phol; it then usesits methyltransferase activity to methylate 27Lysof histone Hl. This createsthe binding site for the PRC, because the chromo domain of Pc binds to the methyIated lysine. The Polycomb complex induces a more compact structure in chromatin; each
and Activators and TrithoraxAre AntagonisticRepressors 31.4 Potycomb
825
PRCI complex causesabout three nucleosomes to become lessaccessible. In fact, the chromo domain was first identified as a region of homology between Pc and the protein HPI found in heterochromatin. Binding of the chromo domain of Pc to 27lys on H3 is analogous to HPI's use of its chromo domain to bind to elys. Variegation is caused by the spreadingof inactivity from constitutive heterochromatin, and as a result it is Iikely that the chromo domain is used by Pc and HPI in a similar way to induce the formation of heterochromatic or inactive structures (see Section 10.13, Some Common Motifs Are Found in ProteinsThat Modify Chromatin). This model implies that similar mechanisms are used to repressindividual loci or to createheterochromatin. The trithorax group (trxG) of proteins have the opposite effect to the Pc-G proteins: They act to maintain genes in an active state. There may be some similarities in the actions of the two groups: Mutations in some loci prevent both Pc-G and trx from functioning, suggestingthat they could rely on common components. The GAGA factor, which is coded by rhe trithorax-like gene, has binding sitesin the PRE. In fact, the siteswhere Pc binds to DNA coincide with the sites where GAGA factor binds. What does this mean? GAGA is probably needed for activating factors, including trxG members, to bind to DNA. Is it also needed for Pc-G proteins to bind and exerciserepression? This is not yet clear, but such a model would demand that something other than GAGA determines which of the alternative types of complex subsequentlyassembleat the site.
X Chromosomes Undergo Gl.obaI Changes o Oneof thetwo X chromosomes is inactivated at random in eachceltduringembryogenesis of eutherian mammats. o In exceptional cases wherethereare>2 X chromosomes, at[but oneareinactivated. o TheXrc(Xinactivationcenter)is a crs-acting regionon the X chromosome that is necessary and sufficient to ensure that onLyoneX chromosome remains active. r Xrcinctudes theXrsfgene.whichcodes for an RNA that is foundontyon inactive X chromosomes. o Themechanjsm that is responsible for preventing XtsfRNAfromaccumutating on the active c h r o m o s o im se unknown.
826
CHAPTER 31 Epigenetic Effects AreInherited
Inactivate one9X X
r-j
x
i._.,-_.ij...)
Double expression ix
Halve expression two I X
r: |
Xo
+.1*iJ** .itt.1{i Different means of dosage compensation areusedto equatize X chromosome expression in mare andfemale. Sex presents an interesting problem for gene regulation, becauseof the variation in the number of X chromosomes.If X-linked geneswere expressed equally well in each sex, females would have twice as much of each product as males. The importance of avoiding this situation is shown by the existence of dosage compensation, which equalizes the level of expressionof X-linked genesin the two sexes. Mechanisms used in different speciesare summarized in !:lil*qil "!.i.:*: . In mammals, one of the two female X chromosomes is inactivated completely. The result is that females have only one active X chromosome, which is the same situation found in males. The active X chromosome of females and the single X chromosome of males are expressed at the same level. . In Drosophila,tll'e expression of the single male X chromosome is doubled relative to the expression of each female X chromosome. . In Caenorhabditis elegans,the expression of each female X chromosome is halved relative to the expression of the single male X chromosome. The common feature in all these mechanisms of dosage compensation is that the entire chromosomeis the targetfor regulation. A global change occurs that quantitatively affects all of the promoters on the chromosome. We know most about the inactivation of the X chromosome in mammalian females, where the entire chromosome becomes heterochromatic. The twin properties of heterochromatin are its condensed state and associatedinactivity. It can be divided into two types: . Constitutiveheterochromatin contains specific sequencesthat have no coding function. In general these include
satelliteDNAs, and they are often found at the centromeres. These regions are invariably heterochromatic because of their intrinsic nature. . Facultative heterochromatin takes the form of entire chromosomes that are inactive in one cell lineage, although they can be expressedin other lineages. The example par excellence is the mammalian X chromosome. The inactive X chromosome is perpetuated in a heterochromatic state,whereas the active X chromosome is part of the euchromatin. Thtrs identicalDNA sequences are involved in bothstates.Once the inactive state has b e e n e s t a b l i s h e d ,i t i s i n h e r i t e d b y descendant cells. This is an example of epigeneticinheritance, becauseit does not depend on the DNA sequence. Our basicview of the situation of the female mammalian X chromosomes was formedby the single X hypothesis in 1961. Female mice that are heterozygous for X-linked coat color mutations have a variegated phenotype in which some areasof the coat are wild-type, but others are mutant. shows that this can ts be explaine d if one of the two X chromosomes inactivatedat random in eachcell of a small precursorpopulation Cellsin which the X chromosome carrying the wild-type gene is inactivated give rise to progeny that express only the mutant allele on the active chromosome. Cellsderived from a precursor where the other chromosome was inactivated have an active wild-type gene. In the caseof coat color, cells descendedfrom a particular precursor stay together and thus form a patch of the same color, creating the pattern of visible variegation. In other cases,individual cells in a population will express one or the other of X-linked alleles; for example, in heterozygotesfor the X-linked locus G6PD, any particular red blood cell will express only one of the two allelic forms. (Random inactivation of one X chromosome occursin eutherian marrrmals. In marsupials, the choice is directed: It is always the X chromosome inherited from the father that is inactivated.) Inactivation of the X chromosome in females is governed by the n-l rule: However many X chromosomes are present, all but one will be inactivated. In normal females there are of course two X chromosomes, but in rare cases where nondisjunction has generated a 3X or greater genotype, only one X chromosome remains active. This suggestsa general model in which a specific event is limited to one X chro-
are BothX chromosomes activein precursorcell Wild-typecoatcolor Mutantcoatcolorgene
.
/ n:2,:,:z:"n:'""Jl:"\ activeallele .,--..,:l
activeallele
.- -i?--J
is caused bytheran, ' X-tinked variegation in eachprecursor of oneX chromosome dominactivation in whichthe + atteteis on the activechromoce[t.Cetts cetlsin whichthe - altetejs somehavewil"dphenotype; have on the activechromosome mutantphenotype.
mosome and protects it from an inactivation mechanism that applies to all the others. A single locus on the X chromosome is sufficient for inactivation. When a translocation occursbetween the X chromosome and an autosome, this iocus is present on only one of the reciprocal products, and only that product can be inactivated. By comparing different translocations, it is possible to map this locus, which is calledthe Xic (X-inactivation center). A cloned region of 450 kb contains all the properties of tlne Xic. When this sequence is inserted as a transgene on to an autosome, the autosome becomes subject to inactivation (in a cell culture system). Xic is a cis-actinglocus that contains the information necessaryto count X chromosomes and inactivate all copies but one. Inactivation spreads from Xic along the entire X chromosome.When Xlc is presenton an X chromosomeautosome translocation, inactivation spreads into the autosomal regions (although the effect is not always complete). Xic conrains a gene, called Xist, that is expressed only on the inactiveX chromosome. The behavior of this gene is effectively the opposite from all other loci on the chromosome, which are turned off . Deletion of Xlsl prevents
Changes 827 GlobaI Undergo 31.5 X Chromosomes
24.19, CpG IslandsAre Regulatory Targets).presumably occur later as part of the mechanism of inactivation. The n-l rule suggeststhat stabilization of Xrsl RNA is the "default," and that some blocking mechanism prevents stabilization at one X chromosome (which will be the active X). This means that, although Xic is necessaryand sufficient for a chromosome to be inactivated,the products of other loci may be necessaryfor the establishment of an activeX chromosome. Silencing of Xlsl expression is necessaryfor the active X. Deletion of the gene for DNA methyltransferase prevents silencing of.Xist, probably because methylation at Ihe Xist promoter is necessaryfor cessationof transcription.
X-inactivation invo[ves stabitization of XrstRNA,whichcoatstheinactivechromosome.
an X chromosome from being inactivated. It does not, however, interfere with the counting mechanism (becauseother X chromosomes can be inactivated). Thus we can distinguish two f e a t u r e s o f .X i c : a n u n i d e n t i f i e d e l e m e n t ( s ) required for counting, and the Xist gene required for inactivation. i ir,, ,::i: , . . . illustrates the role of Xlsf RNA in X-inactivalion. Xist codes for an RNA that Iacksopen reading frames. The XzslRNA "coaIs" the X chromosome from which it is synthesized, which suggeststhat it has a structural role. Prior to X-inactivation, it is synthesizedby both female X chromosomes. Following inactivation, the RNA is found only on the inactive X chromosome. The transcription rate remains the same before and after inactivation, so the transition depends on posttranscriptionalevents. Prior to X-inactivation, Xist RNA decays with a half life of -2 hours. X-inacrivation is mediated by stabilizingthe XrilRNA on the inactive X chromosome.The XlslRNA shows a punctate distribution along the X chromosome, which suggeststhat associationwith proteins to form particulate structures may be the means of stabilization. We do not know yet what other factors may be involved in this reaction and how the Xlsl RNA is limited to spreading in cli along the chromosome. The characteristicfeatures of the inactive X chromosome, which include a lack of acetylation of histone H4, and m e t h y l a t i o n o f C p G s e q u e n c e s( s e e S e c t i o n
CHAPTER 31 EpigeneticEffectsAre Inherited
Chromosome Condensation Is Caused by Condensins . SMCproteins areATPases that inctude the condensins andthe cohesins. r A heterodimer proteins of SMC associates with othersubunits. r Thecondensins causechromatin to be moretightty positive coitedby introducing supercoits into DNA. o Condensins areresponsibte for condensing chromosomes at mitosis. . Chromosome-specific condensins arerespons'ibte for condensing inactive X chromosomes in C.elegons. The structures of entire chromosomes are influenced by interactions with proteins of the SMC (structural maintenance of chromosome) family. They are ATPasesthat fall into two functional groups. Condensins are involved with the control of overall structure, and are responsible for the condensation into compact chromosomes at mitosis. Cohesins are concerned with connections between sister chromatids that must be releasedat mitosis. Both consist of dimers formed by SMC proteins. Condensins form complexes that have a core of the heterodimer SMC2-SMC4 associatedwith other (non-SMC) proteins. Cohesinshave a similar organization basedon the heterodimeric core of SMCI-SMC3. i li,i.;l; I :. i ,: shows that an SMC protein has a coiled-coil structure in its center that is interrupted by a flexible hinge region. Borh the amino and carboxyl termini have ATP- and DNA-binding motifs. Different models have been proposed for the actions of these proteins
ATP- and DNAbindingsite
Coiled-coil Hinge
Coiled-coil
ATP- and DNAbindingsite
C-terminus
interactions between by antiparatteI dimerjze F..I$*i{{i:.::i SMCproteins regions of eachsubunithaveATP-and the centraI coitedcoits.BothterminaI thatatlows two structure mayformanextended DNA-binding motifs.Cohesins to be [inked. differentDNAmotecutes
il5iitlftE: ;i -i.i:i An SMPproteinhasa "Walker modute" with an ATP-binding motifandDNA-binding siteat each end,whichareconnected bv coiledcoi[sthat are[inked by a hingeregion.
Condensin ATP ATP -
86"
ATP ATP
Cohesin
Thetwo halves Flt]t"!l-l{ -:;1.:r-J of a condensin arefotded havea moreopenconbackat an angteof 6o.Cohesins formation withan angleof 86obetween thetwo halves. depending on whether they dimerize by intraor intermolecular interactions. Experiments with the bacterial homologs of the SMC proteins suggestthat a dimer is formed by an antiparallel interaction between the coiled coils, so that the N-terminus of one subunit bonds to the C-terminus of the other subunit. The existence of a flexible hinge region could allow cohesins and condensins to depend on a different mode of action by the dimer. f:*it-i*i i.1.1.! shows that cohesins have a Vshaped structure, with the arms separatedby an 86'angle, whereas condensinsare more sharply bent back, with only 6o between the arms. This enables cohesins to hold sister chromatids together, whereas condensins instead condense an individual chromosome. *:.i*li*L::'*1":.*shows that a cohesin could take the form of an extended dimer that crosslinks two DNA molecules. Ft*L{**3::. .r+ shows that a condensin could take the form of a V-shaped dimer-essentially bent at the hinge-that pulls together distant
structure mayforma compact ilItiLigfi.11""'itlCondensins compacted' DNAto become bybending atthehinge,causing sites on the same DNA molecule, causing it to condense. An alternative model is suggestedby experiments to suggestthat the yeast proteins dimerize by intramolecular interactions, that is, a homodimer is formed solely by interaction between two identical subunits. Dimers of two different proteins (in this case,SMC I and SMCS) may then interact at both their head and hinge regions to form a circular structure as illustrated in ru*i.:El-: iri.,l ,r. Instead of binding directly to DNA, a structure of this type could hold DNA molecules together by encircling them. Visualization of mitotic chromosomes shows that condensins are located all along the length of the chromosome, as can be seen in If.{;Ltit{:1 i.t*i. (By contrast, cohesinsare found at discrete locations.) The condensin complex was named for its ability to cause chromatin to
byCondensins 829 Is Caused Condensation 31.6 Chromosome
on the X chromosome. In C. elegans,a protein complex associateswith both X chromosomes in XX embryos, but the protein components remain diffusely distributed in the nuclei of XO embryos. The protein complex contains an SMC core, and is similar to the condensin complexes that are associatedwith mitotic chromosomes in other species.This suggeststhat it has a structural role in causing the chromosome to take up a more condensed, inactive state. Multiple sites on the X chromosome may be needed for the complex to be fully distributed along it. The complex binds to these sites,and then spreadsalong the chromosome to cover it more thoroughly. Changes affecting all the genes on a chroi'ii:.iRi --:.i.:...r Cohesins maydimerize byintramotecutar mosome/ either negatively (mammals and connections, andthenformmuttimers thatareconnected C. elegans)or positively (Drosophila),are thereat theheads andat thehinge.Sucha structure coutdho[d fore a common feature of dosage compentwo motecutes of DNAtogether by surrounding them. sation. The components of the dosage compensation apparatus may vary, however, as well as the means by which it is localized to the chromosome, and of course its mechanism of action is different in each case.
DNAMethylation Is Perpetuated by a Maintenance Methylase o Mostmethylgroups in DNAarefoundon cytosine on bothstrands of the CpGdoublet. r RepUcation convertsa fuLtymethytated siteto a hemimethylated site. r Hemimethytated sitesareconverted to futty methytated s'itesby a maintenance methylase. I.iiL:liI i:i.]S Condensins arelocatedatonqthe entire Length of a mitoticchromosome. DNAis red;condensins Methylation of DNA occurs at specific sites. In areyetlow. Photocourtesy ofAnaLosada andTatsuya Hirano. bacteria, it is associatedwith identifying the bacterial restriction-methylation system used for phage defense, and also with distinguishing condensein vitro.It has an ability to introduce replicated and nonreplicated DNA (seeSection positive supercoils into DNA in an action that 20.7, Controlling the Direction of Mismatch useshydrolysis of AIP and depends on the presRepair). In eukaryotes, its principal knor,rmfuncence of topoisomeraseI. This ability is controlled tion is connected with the control of transcripby the phosphorylation of the non-SMC subtion; methylation is associated with gene units. which occurs at mitosis. We do not know inactivation (see Section 24.18, Gene Expresyet how this connects with other modifications sion Is Associated with Demethylation). of chromatin-for example, the phosphorylaFrom 2o/oto 7 o/oof the cytosines of animal rion of histones.The acrivation of the condensin cell DNA are methylated (the value varies with the species).Most of the methyl groups are found in CG "doublets," and, in fact, the majority of the CG sequences are methylated. UsuGlobal changes occur in other types of ally the C residues on both strands of this short dosagecompensation.In Drosophila,a complex palindromic sequence are methylated, giving of proteins is found in males, where it localizes the structure.
ilkffffi*1iL1t*"J",#iisH*T;
830
CHAPTER 31 EpigeneticEffectsAre Inherited
Fullymethylatedsites
Me
Me I JV Replication
Hemimethylated sites
Perpetuation methylase
I
Demethylase
I t-tc'>X1"
\rve by is controlted F5{.i1.1fiil li .Ii=:Thestateof methylation "": methyl Denovoandperpetuation threetypesofenzyme. h sa v en o t b e e n a s e sa r e k n o w n ,b u t d e m e t h y t a s e identified.
Hemimethylated sites
Fi{;l-i€i--ti.tt Thestateof methylated sitescou[dbe perpetuated byanenzyme thatrecognizes ontyhemimethylatedsitesassubstrates. Such a site is described as fully methylated. Consider,though, the consequencesof replicating this site. F:ii*.ql3 t"'i:: shows that each daughter duplex has one methylated strand and one unmethylated sgand. Such a site is called hemimethylated. The perpetuation of the methylated site now depends on what happens to hemimethylated DNA. If methylation of the unmethylated strand occurs, the site is restored to the fully methylated condition. If replication occurs first, though, the hemimethylated condition will be perpetuated on one daughter duplex, but the site will become unmethylated on the other daugh-
ter duplex. fi:*ilFiil.i .i"t* shows that the state of methylation of DNA is controlled by methylases, which add methyl groups to the 5 position of cytosine, and demethylases, which remove the methyl groups. (The more formal name for the enzymes usesmethyltransferase as the description.) There are two types of DNA methylase, whose actions are distinguished by the state of the methylated DNA. To modify DNA at a new position requires the action of the de novo methylase, which recognizes DNA by virtue of a specific sequence.Ir actsonly on nonmethylated DNA, to add a methyl group to one strand. There are two denovomethylases (Dnmt3A and Dnmt3B) in mouse; they have different target sites,and both are essentialfor development. A maintenance methylase acts constitutively lnly on hemimethylatedsitesto convert them to fully methylated sites. Its existence means that any methylated site is perpetuated after replication. There is one maintenance methylase (Dnmtt ) in mouse, and it is essential:mouse embryos in which its gene has been disrupted do not survive past early embryogenesis. Maintenance methylation is virtually 100% efficient, ensuring that the situation shown on
Methytase 831 by a Maintenance Is Perpetuated 31.7 DNAMethytation
the left of Figure 31.19 usually prevails in vivo. The result is that, if.a denovomethylation occurs on one allele but not on the other, this difference will be perpetuated through ensuing cell divisions, maintaining a difference between the allelesthat does not depend on their sequences. Methylation has various types of rargets. Gene promoters are the most common target. The promoters are methylated when the gene is inactive, but unmethylated when it is active. The absenceof DnmtI in mouse causeswidespreaddemethylation at promoters, and we assumethis is lethal becauseof the uncontrolled gene expression.SatelliteDNA is another target. Mutations in Dnmt38 prevent methylation of satelliteDNA, which causescentromere instability at the cellular level. Mutations in the corresponding human gene causea diseasecalled ICF (immunodeficiency/centromere instability, facial anomalies). The importance of methyIation is emphasizedby another human disease, Rhett syndrome, which is causedby mutation of the gene for the protein MeCP2 that binds methylated CpG sequences. The methylases are conventional enzymes that act on a DNA target. There may, however, also be a methylation system that usesa short RNA sequenceto target a correspondingDNA s e q u e n c ef o r m e t h y l a t i o n ( s e e S e c t i o n 1 3 . 6 , Antisense RNA Can Be Used to Inactivate Gene Expression) Nothing is known about the mechanism of operation of this system. How are demethylated regions established and maintained? If a DNA site has not been methylated, a protein that recognizes the unmethylated sequencecould protect it against methylation. Once a site has been methylated, there are two possible ways to generate demethylated sites. One is to block the maintenance methylase from acting on the site when it is replicated. After a second replication cycle, one of the daughter duplexes will be unmethylated (as shown on the right side of Figure 31.l9). The orher is acrivelyto demethylate t h e s i t e , a s s h o w n i n i : l i . i , : * I" i i " . ;lt, e i t h e r b y removing the methyl group directly from cytosine, or by excisingthe methylated cytosine or cytidine from DNA for replacement by a repair system. We know that active demethylation can occur to the paternal genome soon after fertilization, but we do not know what mechanism is used. One interesting possibility is that the cytidine deaminaseAID may be involved; it can deaminate methylated C residues, creating a mismatched base pair that a repair system might then correct to a standard (unmethylated) C-G pair.
832
CHAPTER 31 EpigeneticEffectsAre Inherited
?,
?,
.fl -\,1 / -\ \ /'-
|
kHl/-.;
n
rw-
-1l\
( ,n/
l-
1-l
.{ ---v
}#U 3',
5'
/\\ Removal of Removal of Removal ol group S-Me-C methyl base nucleotide
::i**itf, -::;i.i:liDNAcoutdbe demethylated by removing the methylgroup.the base.or the nucleotide. Removal of the baseor nucteotide woutdreouire its replacement by a repairsystem.
DNAMethylation Is Responsible for Imprinting . Paternal andmaternal altetes mayhavedifferent patternsof methytation at fertilization. r Methylation is usua[[y associated with inactivation of the gene. r Whengenes aredifferentiatty imprinted, survival of the embryo mayrequire that thefunctional altele is provided by the parentwith the unmethytated attete. o Survivalof heterozygotes for imprintedgenesis different, depending on the direction of the cross. r Imprinted genesoccurin clusters andmaydepend on a [oca[controlsite wheredenorzo methylation prevented. occursunlessspecifica[[y
The pattern of methylation of germ cells is estabIished in each sex during gametogenesis by a two-stage process: First the existing pattern is erased by a genome-wide demethylation, and then the pattem specificfor each sex is imposed.
All allelic differences are losr when primordial germ cells develop in the embryo; irrespective of sex, the previous patterns of methylation are erased,and a typical gene is then unmethylated. In males, the pattern develops in two stages.The methylation pattern that is characteristic of mature sperm is established in the spermatocyte, but further changes are made in this pattern after fertilization. In females, the maternal pattern is imposed during oogenesis, when oocytes mature through meiosis after birth. As may be expected from the inactivity of genesin gametes,the typical stateis to be methylated. There are casesof differences between the two sexes, though, for which a locus is unmethylated in one sex. A major question is how the specificityof methylation is determined in the male and female gametes. Systematicchanges occur in early embryogenesis.Some siteswill continue to be methylated, whereas others will be specifically unmethylated in cells in which a gene is expressed.From the pattern of changes,we may infer that individual sequence-specificdemethylation events occur during somatic development of the organism asparticular genesare activated. The specific pattern of methyl groups in germ cells is responsible for the phenomenon of imprinting, which describesa difference in behavior between the allelesinherited from each parent. The expressionof certain genesin mouse embryos depends upon the sex of the parent from which they were inherited. For example, the allele coding for IGF-II (insulin-like growth factor II) that is inherited from the father is expressed,but the allele that is inherited from the mother is not expressed.The IGF-II gene of oocytes is methylated, but the IGF-II gene of sperm is not methylated, so that the two alleies behave differently in the zygote.This is the most common pattern, but the dependence on sex is reversed for some genes.In fact, the opposite pattern (expression of maternal copy) is shown for IGF-IIR, the receptor for IGF-II. This sex-specificmode of inheritance requires that the pattern of methylation is established specificallyduring each gametogenesis.The fate of a hypothetical locus in a mouse is illustrated in liiji.iiii: :1i..:1.j.In the early embryo, the paternal allele is nonmethylated and expressed,and the matemal allele is methylated and silent. What happens when this mouse itself forms gametes? If it is a male, the allele contributed to the sperm must be nonmethylated, irrespectiveof whether it was originally methylated or not. Thus when the maternal allele finds itself in a sDerm.it must
be demethylated. If the mouse is a female, the allele contributed to the egg must be methylated; if it was originally the paternal allele, methyl groups must be added. The consequence of imprinting is that an embryo requires a paternal allele for this gene. Thus in the caseof a heterozygous crosswhere the allele of one parent has an inactivating mutation, the embryo will survive if the wild-type allele comes from the father, but will die if the wild-type allele is from the mother. This type of dependence on the directionality of the cross (in contrast with Mendelian genetics) is an example of epigeneticinheritance, where some factor other than the sequencesof the genes themselvesinfluencestheir effects(seeSection l l . l 0 . E p i g e n e t i cE f f e c t s C a n B e I n h e r i t e d ) . Although the paternal and maternal alleles have identical sequences,they display different properties, depending on which parent provided them. These properties are inherited through meiosis and the subsequentsomatic mitoses. Imprinted genes are sometimes clustered. More than half of the l7 known imprinted genes in mouse are contained in two particular regions, each containing both maternally and paternally expressedgenes. This suggeststhe possibility that imprinting mechanisms may function over long distances.Some insights into
is that llt:{l!'ll :i 1",:i ThetypicaIpatternforimprinting a methylated [ocusis inactive.If this is the maternaI andwit[beessenalteleis active, attete, on[ythepaternal patternis resetwhen tia[ for viabitity.Themethytation gametes areformed, sothat a[[spermhavethe paternal type. typeanda[[oocyteshavethe maternal
for imprinting Is Responsibte 31.8 DNAMethytation
833
this possibility come from deletions in the human population that causethe Prader-Willi and Angelman diseases.Most casesare causedby the same 4 Mb deletion, but the syndromes are different, depending on which parent contributed the deletion. The reason is that the deleted region contains at least one gene that is paternally imprinted and at least one that is maternally imprinted. There are some rare cases.however. with much smaller deletions.Prader-Willi syndrome can be causedby a 20 kb deletion that silencesgenes that are distant on either side of it. The basic effect of the deletion is to prevent a father from resetting the paternal mode to a chromosome inherited from his mother. The result is that these genesremain in matemal mode, so that the paternal as well as maternal alleles are silent in the offspring. The inverse effect is found in some small deletions that causeAngelman's syndrome. The implication is that this region comprisessome sort of "imprint center" that acts at a distance to switch one parental tlpe to the other. Methylation is also responsiblefor epigenetic effectsthat control the expressionof rRNA genes. The phenomenon of nucleolar dominance describesthe transcription of only one set of parental rRNA genes.It results from methylation at cytosinesin the promoters for the genes inherited from one Darent and not the other-
Imprinted 0ppositeLy Genes CanBeControlled by a SingLe Center r Imprinted genesarecontrolted by methylation of crs-acting sites. o Methylation maybe responsibte for either inactivating or activating a gene.
Imprinting is determined by the state of methylation of a cli-acting site near a target gene or genes.Theseregulatory sitesare known as differentially methylated domains (DMDs) or imprinting control regions (ICRs). Deletion of these sitesremoves imprinting, and the target loci then behave the same in both maternal and paternal genomes. The behavior of a region containing two genes, Igp and I119,illustrates the ways in which methylation can control gene activity. ::ii,i.:::i:i..i*i shows that these two genes react oppositely to the state of methylation at the ICR located between them. The ICR is methylated on the paternal allele. I1.19shows the typical
834
CHAPTER 31 EpigeneticEffectsAre Inherited
lgt2
ICR
H79 Enhancer
Paternalallele Me ACTIVE
ME INACTIVE Maternalallele
INACTIVE
ACTIVE
f,i{**F .i:.":ii ICRis methytated on the paternaI atlete, wherelgf2 is activeandH19is inactjve.ICRis unmethylatedon the maternalatlele,whereIgF2is inactiveand H19is active.
Maternalallele CTCF bindsto unmethylatedICR tgf2
Enhanceris blocked
+:3*LiRf 3 i.t* TheICRis an insulator that prevents an enhancer fromactjvating lgf2. Ihe insulator functions ontywhenit bindsCTCF to unmethytated DNA.
response of inactivation. Note. however, that Igf2 is expressed.The reverse situation is found on a maternal allele, where the ICR is not methylated. H19 now becomesexpressed,but Igf2 is inactivated. The control of lgp is exercised by an insulator function of the ICR. fl**it$. -q1.il4shows that when the ICR is unmethylated, it binds the protein CTCF.This createsan insulator function that blocks an enhancer from activating Ihrelgp promoter. This is an unusual effect in which methylation indirectly activates a gene by blocking an insulator. The regulation of I1l9 shows the more usual direction of control in which methylation creates an inactive imprinted state. This could reflect a direct effect of methvlation on Dromoter activity.
@
Epigenetic Effects CanBeInherited
. Epigenetic effectscanresuttfrommodification of a nucleic acidafterjt hasbeensynthesized or by the perpetuation of proteinstructures. Epigenetic inheritance describesthe ability of different states,which may have different phenotypic consequences,to be inherited without any change in the sequence of DNA. How can this occur? We can divide epigenetic mechanisms into two general classes: . DNA may be modified by the covalent attachment of a moiety that is then perpetuated. TWo alleles with the same sequence may have different states of methylation that confer different properties. . A self perpetuating protein statemay be established. This might involve assembly of a protein complex, modification of specificprotein(s), or establishment of an alternative protein conformation. Methylation establishesepigenetic inheritance so long as the maintenance methylase acts constitutively to restore the methylated state after each cycle of replication, as shown in Figure 31.19.A stateof methylation can be perpetuated through an indefinite seriesof somatic mitoses.This is probably the "default" siruarion. Methylation can also be perpetuated through meiosis: for example, in the fungus Ascobolus there are epigenetic effectsthat can be transmitted through both mitosis and meiosis by maintaining the state of methylation. In mammalian cells, epigenetic effects are created by resetting the state of methylation differently in male and female meioses. Situations in which epigenetic effectsappear to be maintained by means of protein statesare lesswell understood in molecular terms. Position effect variegation shows that constitutive heterochromatin may extend for a variable distance, and the structure is then perpetuated through somatic divisions. There is no methyIation of DNA in Saccharomyces and a vanishingly small amount inDrosophila,and as a result the inheritance of epigenetic states of position effect variegation or telomeric silencing in these organismsis likely to be due to the perpetuation of protein structures. i:iiiliF.lI i.;t:; considerstwo extreme possibilities for the fate of a protein complex at replication.
. A complex could perpetuate itself if it splits symmetrically, so that half complexes associate with each daughter duplex. If the half complexes have the capacity to nucleate formation of full complexes, the original state will be restored. This is basically analogous to the maintenance of methylation. The problem with this model is that there is no evident reason why protein complexes should behave in this way. . A complex could be maintained as a unit and segregateto one of the two daughter duplexes. The problem with this model is that it requires a new complex to be assembled de novl orr the other daughter duplex, and it is not evident why this should happen. Consider now the need to perpetuate a heterochromatic structure consisting of protein complexes. Suppose that a protein is distributed more or less continuously along a stretch of heterochromatin, as implied in Figure 31.4. If individual subunits are distributed at random to each daughter duplex at replication, the two daughters will continue to be marked by the protein, although its density will be reduced to half of the level before replication. If the protein has a self-assemblingproperty that causes
on to proteincomptexes ll+Ljitl-.i{'1 ,il: Whathappens chromatin duringreplication?
Effects CanBeInherited 31.10Epigenetic
83s
iii-]..:i:i=:: I.iri-_: Acetytated coresareconserved anddjstributed at random to the daughter chromatin fibersat reptication. Eachdaughter fiberhasa mixtureofotd (acetytated) coresandnew(unacetylated) cores. new subunits to associatewith it, the original situation may be restored. Basically,the existence of epigeneticeffects flrces us t0 the view that a protein responsible such a situatiln must h6veslme slrt for of self- templating or self- assembling capacity. In some cases,it may be the state of protein modification, rather than the presence of the protein per se,rhar is responsible for an epigenetic effect. There is a general correlation between the activity of chromatin and the state of acetylation of the histones,in particular the acetylation of histones H3 and H4, which occurs on their N-terminal tails. Activation of transcription is associatedwith acetylation in the vicinity of the promoter; and repressionof transcription is associatedwith deacetylation (see Section 30.7, AcetylasesAre Associatedwith Activators). The most dramatic correlation is that the inactive X chromosome in mammalian female cellsis underacetylatedon histone H4. The inactivity of constitutive heterochromatin may require that the histones are not acetylated.If a histone acetyltransferaseis tethered to a region of telomeric heterochromatin in yeast, silenced genes become active. When yeast is exposed to trichostatin (an inhibitor of deacetylation), centromeric heterochromatin
836
CHAPTER 31 EpigeneticEffectsAre Inherited
becomes acetylated, and silenced genes in centromeric regions may become active. The effect may persistevenafter trichostatinhas beenremoved. In fact, it may be perpetuated through mitosis and meiosis. This suggeststhat an epigenetic effect has been created by changing the state of histone acetylation. How might the state of acetylation be perpetuated? Suppose that the H)2-H42 tetramer is distributed at random to the two daughter duplexes. This creates the situation shown in tiGi-iftil]1.1$, in which each daughter duplex contains some histone octamers that are fully acetylated on the H3 and H4 tails, whereas others are completely unacetylated. To account lor the epigenetic effect, we could supposethat the presence of some fully acetylated histone octamers provides a signal that causes the unacetylated octamersto be acetylated. (The actual situation is probably more complicated than the one shown in the figure, because transient acetylations occur during replication. If they are simply reversed following deposition of histones into nucleosomes, they may be irrelevant. An alternative possibility is that the usual deacetylation is prevented, instead of, or as well as, inducing acetylation.)
YeastPrions Show Inheritance UnusuaL o TheSup35proteinin its wild-type soluble formis a terminationfactorfor transtation. r It canalsoexistin an atternative formof in whichit is notactjvein otigomeric aggregates. proteinsynthesis. . Thepresence of the otigomeric formcauses newty proteinto acquirethe inactive synthesized structure. . Conversion between thetwo forms'is influenced bv cnaDer0nes. o Thewitd-typeformhasthe recessive geneticstate psr andthe mutantformhasthe dominant genetic stateP5.[+.
One of the clearestcasesof the dependenceof epigenetic inheritance on the condition of a protein is provided by the behavior of prions. They have been characterized in two circumstances:by genetic effectsin yeast, and as the causative agents of neurological diseasesin mammals, including human beings. A striking epigenetic effect is found in yeast, where two different states can be inherited that map to a single genetic locus, although the sequence of the
I Termination f
[PSl+]state:no termination
ai ---* Sup35 [psl-]
Sup35 [PS/.]
-.>
:ilii-jt:::r:i,.i:r Thestateof theSup35 protein determines whether termination of transtation occurs.
geneis the samein both states.The two different states are lpsi-l and [PSI+].A switch in condition occurs at a low frequency as the result of a spontaneoustransition between the states. The [ps{ genotype maps to the locus SUP35, which codes for a translation termination factor. l:l,i.i!iir-i: .':l'summarizesthe effectsof the Sup35 protein in yeast.In wild-type cells,which are characterizedas lpsi), the gene is active, and Sup35 protein terminates protein synthesis.In cells of the mutant [PSI+]type, the factor does not function, which causesa failure to terminate protein synthesis properly. (This was originally detected by the lethal effects of the enhanced efficiency of suppressorsof ochre codons in [PSI+]strains.) [PS1+]strains have unusual genetic properties. When a lpsi-l strain is crossed with a [PSI+]strain, all of the prlgeny are IPSF). This is a pattern of inheritance that would be expected of an extrachromosomal agent, but the [PSI+] trait cannot be mapped to any such nucleic acid. The [PSI+]trait is metastable, which means that, although it is inherited by most progeny, it is Iost at a higher rate than is consistent with mutation. Similar behavior is shown also by the locus URE2, which codes for a protein required for
fi{.;tiiii -i r.,:r::Newlysynthesized Sup35proteinis conof preexistvertedinto the IPSI+]stateby the presence ing IPSI+]protein.
nitrogen-mediated repression of certain catabolic enzymes. When a yeast strain is converted into an alternative state,called lURE3l, the Ure2 protein is no longer functional. The [PSI+]state is determined by the conformation of the Sup35 protein. In a wild-type [psi-] cell, the protein displays its normal function. In a [PSI+]cell, though, the protein is present in an alternative conformation in which its normal function has been lost. To explain the unilateral dominance of [PSI+]over [psi-] in genetic crosses,we must suppose that the presenceof protein in the lPSfl rtatu causesall the protein in the cell to enter this state.This requires an interaction between the [PSI+] protein and newly synthesized protein, which probably reflects the generation of an oligomeric state in which the [PSI+]protein has a nucleating role, -i i.iil-i. as illustrated in iii':i.iFlf; A feature common to both the Sup35 and Ure2 proteins is that each consistsof two domains that function independently. The C-terminal domain is sufficient for the activity of the protein. The N-terminal domain is
Inheritance 837 ShowUnusual 3L.11YeastPrions
sufficient for formation of the structures that make the protein inactive. Thus yeast in which the N-terminal domain of Sup35 has been deleted cannot acquire the [PS/+]state, and the presence of a [PS1+]N-terminal domain is sufficient to maintain Sup35 protein in the [PS1+] condition. The critical feature of the N-terminal domain is that it is rich in slutamine and asparagineresidues. Loss o{ function in the [PSI+]state is due to the sequestrationof the protein in an oligomeric complex. Sup35 protein in [PSI+]cells is clustered in discrete foci, whereas the protein in [psr] cellsis diffusedin the cytosol.Sup35 protein from [PS1+]cells forms amyloid fibers ir vitro-these have a characteristic hish content of B-sheetstructures. The involvement of protein conformation (rather than covalent modification) is suggested by the effects of conditions that affect protein structure. Denaturing treatments causeloss of the [PS1+]state. In particular, the chaperone Hsp 104 is involved in inheritance of [PSI+].Its effectsare paradoxical. Deletion of.HSPl04pre-
[psi-] protein [PSl'] Converl rn vitro
+
I n c o r n o r a l t ri n l o l r n n s o m e
ll
tv
Fuseliposomewith[psi ] yeast
vllv
ttl t e a s t r e m a i n s L p s i - ]i ' i ) i . i i l l - : t t t " : : : 1 , :i F l $1']
state'of u.rr, affid 838
protein canconvert the lpsrl
CHAPTER 31 Epigenetic Effects Areinherited
vents maintenance of the [PSI+]state,and overe x p r e s s i o no f H s p 1 0 4 a l s o c a u s e sl o s s o f t h e [PSI+]state.This suggeststhat Hspl04 is required for some change in the structure of Sup3 5 that is necessary for acquisition of the [PSI+]state, but that must be transitory. Using the ability of Sup35 to form the inactive structure in vitro, it is possible to provide biochemical proof for the role of the protein. i ji;i.iii:: :;.i..i:;rillustrates a striking experiment in which the protein was converted to the inactive form in vitro, put into liposomes (where in effect the protein is surrounded by an artificial membrane), and then introduced directly into cells by fusing the liposomes with [psr] yeast. The yeast cells were converted to [PS1+]! This experiment refutes all of the objections that were raised to the conclusion that the protein has the ability to confer the epigenetic state. Experiments in which cells are mated, or in which extracts are taken from one cell to treat another cell, always are susceptibleto the possibility that a nucleic acid has been transferred. When the protein by itself does not convert target cells,though (even though protein converted to the inactive state can do so), the only difference is the treatment of the proteinwhich must therefore be responsible for the converslon. The ability of yeast to form the [PSI+]prion state depends on the genetic background. The yeast must be [PIN+] in order for the [PS1+]state to form. The [PIN+] condition itself is an epigenetic state. It can be created by the formation of prions from any one of several different proteins. These proteins share the characteristic of Sup35 that they have Gln/Asn-rich domains. Overexpression of these domains in yeast stimulates formation of the [PSI+] state. This suggests that there is a common model for the formation of the prion state that involves aggregation of the Gln/Asn domains into selfpropagating amyloid structure. How does the presenceof one Gln/Asn protein influence the formation of prions by another? We know that the formation of Sup35 prions is specific to Sup35 protein. that is, it does not occur by cross-aggregationwith other proteins. This suggeststhat the yeast cell may contain soluble proteins that antagonize prion formation. These proteins are not specific for any one prion. As a result, the introduction of any Gln/Asn domain protein that interacts with these proteins will reduce the concentration. This will allow other Gln/Asn proteins to aggregate more easily.
Prions Cause Diseases 'tnMamma[s . Theproteinresponsibte for scrapie existsin two forms:the witd-type noninfectious formPrPc, whichis susceptible to proteases. andthe diseasecausing formPrPsc, whichis resistant to proteases. . Theneurotogical disease canbetransmitted to miceby injectingthe purifiedPrPsc proteininto mrce. o Therecipient mouse musthavea copyof the PrP genecodingfor the mouse protein. o ThePrPsc proteincanperpetuate itsetfby causing the newlysynthesized PrPproteinto takeupthe PrPsc forminsteadof the PrPc form. r Multiplestrainsof PrPs.mayhavedifferent conformations of the orotein. Prion diseaseshave been found in sheep, in human beings,and, more recently, in cows. The basicphenotype is an ataxia-a neurodegenerative disorder that is manifested by an inability to remain upright. The name of the diseasein sheep, scrapie, reflects the phenotype: The sheep rub against walls in order to stay upright. Scrapiecan be perpetuated by inoculating sheep with tissue extracts from infected animals. The diseasekuru was found in New Guinea, where it appearedto be perpetuated by cannibalism, in particular the eating of brains. Related diseases in Western populations with a pattern of genetic transmissioninclude Gerstmann-Strausslersyndrome and the related Creutzfeldt-Jakob disease (CJD), which occurs sporadically. Most recently, a diseaseresembling CJD appears to have been transmitted by consumption of meat from cows suffering from "mad cow" disease. When tissue from scrapie-infected sheep is inoculated into mice, the diseaseoccurs in a period ranging from 75 to I50 days.The active component is a protease-resistantprotein. The protein is coded by a gene that is normally expressedin the brain. The form of the protein in normal brain, called PrPc, is sensitive to proteases.Its conversion to the resistantform, called Prps', is associatedwith occurrence of the disease. The inf ectious preparation has no detectable nucleic acid, is sensitive to UV irradiation at wave lengths that damage protein, and has a low infectivity (l infectious unit / 105 PrPs'proteins). This correspondsto an epigenetic inheritance in which there is no change in genetic information (becausenormal and diseasedcellshave the same PrP gene sequence), but the PrPs'form of the protein is the infectious a g e n t ( w h e r e a s P r P c i s h a r m l e s s ) .T h e P r P s '
form has a high content of B sheets,which form an amyloid fibrillous structure that is absent from the PrPc form. The basis for the difference between the PrPs'and Prpc forms appearsto lie with a change in conformation rather than with any covalent alteration. Both proteins are glycosylated and Iinked to the membrane by a GPl-linkage. The assayfor infectivity in mice allows the dependenceon protein sequenceto be tested. i.1l:,t;l{t r;i. ii; illustratesthe results of some critical experiments. In the normal situation, PrPs' protein extracted from an infected mouse will induce disease (and ultimately kill) when it is injected into a recipient mouse. If the PrP gene is "knocked out, " a mouse becomesresistantto infection. This experiment demonstrates two things. First, the endogenousprotein is necessary for an infection, presumably becauseit provides the raw material that is converted into the infectious agent. Second.the causeof diseaseis not the removal of the PrPc form of the protein, becausea mouse with no PrPc survives normally: The diseaseis causedby a gain-offunction in PrPs'.If the PrPgene is alteredto prevent the GPl-linkage from occurring, mice infected with PrPs'do not develop disease, which suggeststhat the gain of function involves an altered signalling function for which the GPIlinkage is required. The existence of species barriers allows hybrid proteins to be constructed to delineate
irii;lJliri;i i,:,i.i A Prps'proteincan onty infect an animalthat has the same PrPcprotein. type of endogenous
in Mammats 839 Diseases Cause 31.12Prions
the features required for infectivity. The original preparations of scrapiewere perpetuated in severaltypes of animal, but these cannot always be transferred readily. For example, mice are resistantto infection from prions of hamsters. This means that hamster-PrPst cannot convert mouse-PrPc to PrPsc.The situation changes, though, if the mouse PrP gene is replaced by a hamster PrP gene. (This can be done by introducing the hamster PrP gene into the PrP knockout mouse.) A mouse with a hamster PrP gene is sensitive to infection by hamster PrPsc.This suggeststhat the conversion of cellular PrPc protein into the Sc state requires that the PrPsc and PrPcproteins have matched sequences. There are different "strains" of PrPs',which are distinguished by characteristic incubation periods upon inoculation into mice. This implies that the protein is not restricted solely to alternative states of PrPc and PrPsc,but rather that there may be multiple Sc states.These differences must depend on some self-propagating property of the protein other than its sequence. If conformation is the feature that distinguishes PrPs'from PrPc, then there must be multiple conformations, each of which has a selftemplating property when it converts PrPc. The probability of conversion from PrPc to PrPsc is affected by the sequence of PrP. G e r s t m a n n - S t r a u s s l e rs y n d r o m e i n h u m a n beings is caused by a single amino acid change in PrP.This is inherited as a dominant trait. If the same changeis made in the mouse PrP gene, mice develop the disease.This suggeststhat the mutant protein has an increased probability of spontaneousconversioninto the Sc state.Similarly, the sequence of the PrP gene determines the susceptibility of sheep to develop the diseasespontaneously; the combination of amino acids at three positions (codons I)6, I54, and l7l ) determines susceptibility. The prion offers an extreme case of epigenetic inheritance, in which the infectious agent is a protein that can adopt multiple conformations. each of which has a self-templating property. This propefiy is likely to involve rhe srare of aggregation of the protein.
@
Summary
Ihe formation of heterochromatin occurs by proteins that bind to specific chromosomal regions (such as telomeres) and that interact
thread from an initiation center. Similar events occur in silencing of the inactive yeast mating type loci. Repressivestructuresthat are required to maintain the inactive statesof particular genes are formed by the Pc-G protein complex in Drosophila.They share with heterochromatin the property of propagating from an initiation center. Formation of heterochromatin may be initiated at certain sites and then propagated for a distance that is not precisely determined. When a heterochromatic state has been estabIished. it is inherited through subsequent cell divisions. This gives dse to a pattern of epigenetic inheritance, in which two identical sequencesof DNA may be associatedwith different protein structures, and therefore have different abilities to be expressed.This explains the occurrence of position effect variegation in Drosophila. Modification of histone tails is a trigger for chromatin reorganization. Acetylation is generally associatedwith gene activation. Histone acetylasesare found in activating complexes, whereas histone deacetylasesare found in inactivating complexes.Histone methylation is associated with gene inactivation. Some histone modifications may be exclusive or synergistic with others. Inactive chromatin at yeast telomeres and silent mating type loci appears to have a common cause,and involves the interaction of certain proteins with the N-terminal tails of histones Hl and H4. Formation of the inactive complex may be initiated by binding of one protein to a specific sequence of DNA; the other components may then polymerize in a cooperative manner along the chromosome. Inactivation of one X chromosome in female (eutherian) mammals occurs at random. The Xrclocus is necessaryand sufficient to count the number of X chromosomes. The n-I rule ensuresthat allbut one X chromosome are inactivated. Xic contains the gene Xist, which codes for an RNA that is expressedonly on the inactive X chromosome. Stabilization of Xist RNA is the mechanism by which the inactive X chromosome is distinguished. Methylation of DNA is inherited epigenetically. Replication of DNA createshemimethylated products, and a maintenance methylase restores the fully methylated state. Some methylation events depend on parental origin. Sperm and eggscontain specificand different patterns of methylation, with the result that paternal and maternal allelesare differentlv exnressedin
xt:l,llT nTJffi:T:,H :iJ:,":il:T; CHAPTER 31 Epigenetic Effects AreInherited
the embryo. This is responsible for imprinting, in which the nonmethylated allele inherited from one parent is essentialbecause it is the only active allele; the allele inherited from the other parent is silent. Patterns of methylation are reset during gamete formation in every generation. Prions are proteinaceous infectious agents that are responsible for the diseaseof scrapie in sheep and for related diseasesin human beings. The infectious agent is a variant of a normal celIular protein. The PrPscform has an altered conformation that is self-templating: The normal PrPc form does not usually take up this conformation, but does so in the presence of PrPsc.A similar effect is responsible for inheritance of the [PS1] element in yeast.
References Heteroch gates romati n Propa from a NucleationEvent Resea rch Ahmad,K. and Henikoff,S. (2001).Modulationof a transcriptionfactor counteractsheterochromatic genesilencingin Drosophila. CelI104, 839-847.
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9re 'YNul eql asPq Jo Ieururrel aqt Jo dnoJSltirO-,2to -,€ eLItol pa>lur srprJEourue aql ;o dnorE uV HOO) eql'prrp ourrueue ot pe>lurT VNUI e sry11X11/[reoullup 'srsaqlu^sureloJctpua lpql suopo) uorteurruJetaerql aql Jo euo 'gvn ]a1drr1aqt sruopoJ taque JqI '11aserau.ri.lod VllU ,{11enadsa'saseraru,{.1od vNU rltozhe>lnourpua) z{quorldr,rrsuerl stlq1qul1l lsapto11a4d altuau,rvrnoorrlsnru snouosrodaqt ruor; panrr -ap aprldadelro rlpzbrq e sr (urlrueure-n ,{1n; aroru) uqlueuy '(aueu aqr aruaq) pua qtpe salrsa8eneapn1y lp anpq sreqruaru aql 'eruoua8 uerunq aqr u1 '3uo1dq Ienpr^rpur 'saruanbespeleler 'pasradsrp 00€- qJpa nlv eqJ Jo tes e sr/[11ure; 'YNU nlv 01palPlar JrP ]Pq] dUS aqt Io vNU S/ aql;o sged aqt sasrrdurorulpluop nlv eqJ 'suorpun[ 3ur -rqds ;o a8esnaql ur sa8ueqt .{q nnpord alEurse ruory strnpord VNU luara;JrpJo uort)npord aqr saqrrrsap6upgds e1lleu;ellv 'uraloJo aql uo ereqMeslapalpJol atrspuoJese ol alnJelou IlprusE Surpurq;o tlnser eql se alls auo re (,{rprlpe aroJareqtpue) uorleruroJuoJstr a8ueqr ot uralord e;o ,llgrqe aqt saqTJJSap uorlelnEarlyapollv 'eIuosoruoJqJ Jaqlo aql uo,{dor p Jo uorte^llrB sluaaard teql passardxaaq ol a1a1pulpqopounurur tsJrJaql ruorJ)ppqpaa;.dqpasnet fl srqJ 'ul1n -qolEoununurpassardxaaqt ro; SurpoJalelleauo,{.1uo;oar[roqd -ru.{.1 relnrrlred,{ue ur uorssa-rdxa aql saqrrJsapuoJsnllxelgallv 'eurosoruoJq)P uo snJol uaar8e Suddnrro aua8 B Jo sruroJe^rlpurallp lpreles Jo auo sr alolle uV ',{peaarp dllensn sJorunl aq1 'adfu aurdor8eaqt yo saurdoyo srsaqlu,{saql JoJSurpor saua8,{.rrBrspnuseldauldot6v 'dnor8 aleqdsoqd p erl palJauuoJ ere Surrre8ns aql Jo suorlrsod,gpue,E qJIqM ur firyy rqrdr aleraua8 ot elprtsqnse sp dIV sesntpqt aru,{.zuaue sr aselt/[r elelfuepv 'pruseld eq1 ssoluodn runrralreq eqt sllr>ltusrueq)eu eqJ Jo 'spnuseld,{q pasn usrueq)eur IeArAJnse sr uals/ls uolplppe uV 'zlroruaru sl 1I's11a) rot]eJJeeuoJaq lerrSolounrururro; alqrsuodsaJ pue aleralllord o1 palelmurts are srotdarar rryoads-ua8rlueq1r.tr sa/.roqdruLl seslep IpJaAas razrosdolanapasuodsarJunrutur alrl -depeaql 'ua8rlue qlru uorlJpralurrr;oads 4aqr.{q palelrDp are leqr sa{roqdu.{1 ,{q pereparu asuodsaraqt sr,l$1unuu! an$dppv
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'paurquor uaqu,{lleqtal JSneJalqprl eJesallas -ruaqr z(qleqt suortelnru oMl uaqM srnJJo /i1geq1a1)llaqlufs 'ad{louaqd Ipqtel Jrlaqtu,{se asner ot tleJelul suortel -nru eql eururrJlep 01sluetnru gggg .{.laleurxorddeyo uortelap Jr zlerreue ot passorJsl luetntu e ^,{.qaraq.tr lsee,{.Surppnqur anbru -q)at petptuolnp up sl (VgS) s1s/[leue /[eue rgaua6 r11aq1u/ig 'rapJo arupsaqt ur JnJ)o saua8snoSolouroqereqm sapadsluareJJrpJo p suorEar uaaMteq drqsuorlelar saqrrJsep /[ua1u/i5 leuosouorqJ 'uopoJ arupsaqt ot puodserpup pDe ouurp arups aql reaq syggl rntuoudg 'apor rrlaua8 aqt ur Euruearu erups eql a^pq suopoJ ru/[uoufi5 'sJruosouroJqtpasdeu,{s aJnl Jo -rnns lerrSoloqdroru aqt seqrJJsepxalduol leuauoldeufs aq1 'tuale^Iq e pallp) sI ernlJnrls 8ur11nsar eql lsrsorauJo tJpls eql (sauosoruorqr snoEoloruoq8urtuasarder)sprleru le srnr)o tpql -orqf, retsrsyo srredoMl eql uortprJosspJql saqrJlsapslsdpufs Jo 'saruosoelJnu uortezrue8roaql a8ueqJ ol dJV Jo Jo srs,{.1orpziq sasn1r lxaldruor SurraporuarurleruoJq) p sl INS/IMS 'asuodsare sra88rrlpup arnl -JnJlsJo aruanbasprlplur up sazruSorarurats,{saqt lpql sreJnlEaJ uoruruor aqJ 'xrlaq elqnop aql ur aEeurep01 sua1s,{s;o trear leqt tas eq1srraqtouv'suollplnur esuesuoualeq sVNUru saper8 lpql -ap ruatsz(saql sr aldruexaeuo 'stxaluoJ leql luaralJrp IeJaAasur pJsnsrural eql'sJorra roJsprJeJralJnupaqr sruats,{s aluelllaruns 'uoruasur ro uorlelap aspqe peq spq teql aua8 e ur aruerJSurpearleurErroaqt sJrolser teql aspqp Jo uorlelap ro uoluesur up srtossa.tddls glqsaruer; y 'uorlelnlu AJeruude sDeJJaaql Jo srJlle ro roy salesuadruo)lpql uollptnru puorJs e srrossetddnsV 'VNC ul a8ueqr tnoqum uorlelnru p Jo leur8rroaql Eursrazrar slJaJJaeqt seleururle luala puoJese uaqM srnrro uossarddn5 'uoIlpIJeAalqpreprsuoJEumoqs ,!rou lnq tolseJup uoruuro) p urorJlua)sepparunsard,{qpalplar11pseua8yotase sr/[lrue;ladns V 'srxeu/!to slr JeAosessorJ ll lpql os aredsur y1qq xaldnp pesolr p JoSuqrorrr{t saqrr)sap6ug1or.radn5 'prosnJr^e se q)ns ,snrr^ e ueql rellptus sr 1eq1lua8e snort)aJurue sr ue6oqled le.rgqns y 'uorlesuapuoJ auosotuorq) ur pello^ur ere qJrqM 'sursuJpuoJaql pue taqtaSot spqpruorq)retsrsplor{ qJrqu 'surseqoraql epnlrur teqt sulatord;o dnor8 e srqrrrsap(165) saurosoluolrll alueualuleu lunllnr1s +o 'JolPInbeJP uPqt raqto t)npord uralord ro VNU,{.uero; sapoJeua6 lprnpnt}s v 'xaldnp Ieurou aq1olur trJlou op teqt srpd aseqro saseqdq pesneJ VNq1 uorlpuroJuoJ aqt ur a8ueqr p sr uolpolslp lernpru15 V Jo 'xaldnp aqt puers (snoEoloru Jo -oq) snoraardaql Suoeldsrp,,(qs,norEpuerts VN61,lrau p qJrqm ur sesnrrl aruosJo uorlerlldar Jo rpou p sr lueuef,eldslp puptls
A tetrad describesthe four (haploid) sporesthat result from meiosis in yeast. (The term originally was used to describethe structure found at the beginning of meiosis,now known asa bivalent, that containsall four chromatids,producedby duplicationof a homologous chromosome pair.) TFrrDis the transcription factor that binds to the TATA sequence upstream of the startpoint of promoters for RNA polymeraseII. It consistsof TBP (TATAbinding protein) and the TAF subunits that bind to TBP. Thalassemiais diseaseof red blood cellsresulting from lack of eithersorBglobin. Third-basedegeneracydescribesthe lessereffecton codon meaning of the nucleotide present in the third codon position. tumeThe Ti plasmid is an episomeof the bacteriumAgrobacterium faciensthat carriesthe genesresponsiblefor the induction of crown gall diseasein infected plants. Tight binding of RNA polymeraseto DNA describesthe formation of an open complex (when the strandsof DNA have separated). The TIM complex residesin the inner membrane of mitochondria and is responsiblefor transporting proteins from the intermembrane spaceinto the interior of the organelle. Bacterial transposons carrying markers that are not related to their functior', e.g.,drug resistance,are named asTn followed by a number. Toteranceis the lack of an immune responseto an antigen (either self antigen or foreign antigen) due to clonal deletion. A Totl-tike receptor (TLR)is a plasma membrane receptor that is expressedon phagocytesand other cellsand is involved in signaling during the innate immune response.TLRs are related to IL-l receptors. The TOMcomplex residesin the outer membrane of the mitochondrion and is responsiblefor importing proteins from the cytosol into the spacebetween the membranes. A DNA topoisomeraseis an enzyme that changesthe number of times the two strandsin a closedDNA molecule crosseach other. It doesthis by cutting the DNA, passingDNA through the break, and resealingthe DNA. TopologicaIisomers are molecules of DNA that are identical except for a differencein linking number. A traiter is a nontranslated sequenceat the 3' end of an nRNA following the termination codon. trans configurationof two sitesrefersto their presenceon two different moleculesof DNA (chromosomes). A trans-acting product can function on any copy of its target DNA. This implies that it is a diffusible protein or RNA. A transcript is the RNA product produced by copying one strand of DNA. It may require processingto generatea mature RNA. Transcription describessynthesisof RNA on a DNA template. A transcription factor is required for RNA polymeraseto initiate transcription at specificpromoter(s), but is not itself part of the enzyme.
A transcription unit is the sequencebetween sitesof initiation and termination by RNA polymerase;it may include more than one gene. The transcriptome is the complete set of RNAs present in a cell, tissue,or organism. Its complexity is due mostly to mRNAs, but it also includes noncoding RNAs. A transducingvirus carriespart of the host genomein placeof part of its own sequence.The best known examples are retroviruses in eukaryotesand DNA phagestn E. coli. A transesterification reaction breaksand makes chemicalbonds in a coordinatedtransfer so that no energy is required. Transfectionof eukaryotic cellsis the acquisition of new genetic markers by incorporation of added DNA. The transfer region is a segmenton the F plasmid that is required for bacterial conjugation. TransferRNA(IRNA) is the intermediate in protein synthesisthat interprets the geneticcode.Each tRNA can be linked to an amino acid. The IRNA has an anticodon sequencethat is complementary to a triplet codon representingthe amino acid. Transformationof bacteriais the acquisition of new geneticmaterial by incorporation of added DNA. Transformationof eukaryotic cellsrefers to their conversionto a state of unrestrained growth in culture, resembling or identical with the tumorigenic condition. The transforming principle is DNA that is taken up by a bacterium and whose expressionthen changesthe properties of the recipient cell. A transgeneis a genethat is introduced into a cell or animal from an external source. A transition is a mutation in which one pyrimidine is replacedby the other, or in which one purine is replacedby the other. Transtationis synthesisof protein on an mRNA template. Translational positioning describesthe location of a histone octamer at successiveturns of the double helix, which determines which sequencesare located in linker regions' Translocationdescribesthe stageof nuclearimport or export when a protein or RNA substratemoves through the nuclear pore. Translocationis the movement of the ribosome one codon along mRNA after the addition of each amino acid to the polypeptide chain. Protein translocation describesthe movement of a protein across a membrane. This occurs acrossthe membranes of organellesin eukaryotes, or acrossthe plasma membrane in bacteria. Each membrane acrosswhich proteins are translocatedhas a channel specializedfor the purpose. Movement of a protein acrossa lipid bilayer usually requires a translocon, an integral membrane protein that provides a channel for displacementof polypeptide segmentsacrossthe membrane. A transmembrane protein extends acrossa lipid bilayer. A hydrophobic region (typically consistingof a stretch of 20 to 25 }llydrophobic and/or uncharged amino acids)or regionsof the protein
86s
residesin the membrane. Hydrophilic regionsare exposedon one or both sidesof the membrane. The transmembraneregionis the part of a protein that spansthe membrane bilayer. It is hydrophobic and in many casescontains approximately20 amino acidsthat form an cr-helix.It is alsocalled t h e t r a n s m e m b r a n de o m a i n . Transplantationantigen is protein codedby a major histocompatibility locus,present on all mammalian cells,involved in interactions between lymphocytes. A transposaseis the enzymeactivity involved in insertionof transposon at a new site. Transposition refers to the movement of a transposonto a new sitein the genome. A transposonis a DNA sequenceableto insert itself (or a copy of itself) at a new location in the genome without having any sequence relationship with the target locus. A transversion is a mutation in which a purine is replacedby a pyrimidine or vice versa. tRNAfMetis the specialRNA used to initiate protein synthesisin bacteria.It mostly usesAUG, but can also respondto GUG and CUG. tRNAiMet is the specialIRNA usedto respondto initiation codons in eukaryotes. tRNAmMet insertsmethionine at internal AUG codons. A true reversionis a mutation that restoresthe original sequence of the DNA. The twisting number of a DNA is the number of basepairs divided by the number of basepairs per turn of the double helix. A two hybrid assaydetectsinteraction between two proteins by meansof their ability to bring togethera DNA-binding domain and a transcription-activatingdomain. The assayis performed in yeast using a reportergenethat respondsto the interaction. Ty standsfor transposonyeast, the first transposableelement to be identified in yeast. A type I topoisomeraseis an enzyme that changesthe topology of DNA by nicking and resealingone strand of DNA. A type II topoisomeraseis an enzyme that changesthe topology of DNA by nicking and resealingboth strandsof DNA. U3 is the repeatedsequenceat the l'end of a retroviral RNA. U5 is the repeatedsequenceat the 5' end of a retroviralRNA. A stretch of underwoundDNA has fewer basepairs per turn than the usual average(10 bp = I turn). This meansthat the two strands of DNA are lesstightly wound around each other; ultimately this can lead to strandseparation. Unidirectional replication refers to rhe movement of a sinsle replication fork from a given origin. An uninduciblemutant is one where the affectedgene(s)cannot be expressed.
866
Glossary
The unit ce[[ describesthe stateof an E. colibacterium generated by a new division. It is 1.7 pm long and has a single replication origin. An up mutation in a promoter increasesthe rate of transcription. Upstreamidentifies sequencesin the oppositedirection from expression;for example, the bacterialpromoter is upstream of the transcription unit, and the initiation codon is upstream of the coding region. An upstream activator sequence(UAS)is the equivalent in yeast of the enhancer in higher eukaryotesand is bound by the GAL4 transcriptional activator proteins. A V gene is sequencecoding for the major part of the variable (N-terminal) region of an immunoglobulin chain. A variabte region (V region) is an anrigen-binding site of an immunoglobulin or T cell receptormolecule. V regionsare composedof the variable domains of the component chains.They are coded by V gene segmentsand vary extensively among antigen receptorsas the result of multiple, different genomic copiesand of changesintroduced during synthesis. Variegation of phenotype is produced by a changein genotlpe during somatic development. The vegetative phasedescribesthe period of normal growth and division of a bacterium. For a bacterium that can sporulate,this contrastswith the sporulation phase, when sporesare being formed. The viral superfamity comprisestransposonsthat are related to retroviruses.They are defined by sequencesthat codefor reverse transcriptaseor integrase. Virion is the physical virus particle (irrespectiveof its ability to infect cellsand reproduce). A viroid is a small infectious nucleic acid that does not have a protein coat. Virutent phage mutants are unable to establishlysogeny. A virusoid is a small infectious nucleic acid that is encapsidated by a plant virus together with its own genome. VNTR(variablenumber tandem repeat)regionsdescribevery short repeatedsequences,including microsatellitesand minisatellites. The wobbte hypothesis accountsfor the ability of a IRNA to recognize more than one codon by unusual (non-G-C, non-A-T) pairing with the third baseof a codon. The writhing number is the number of times a duplex axis crosses over itself in space. The zinc finger is a DNA-binding motif that typifiesa classof transcription factor. A zoo btot describesthe use of Southern blotting to test the ability of a DNA probe from one speciesto hybridize with rhe DNA from the genomesof a variety of other species.
Index Note: /= figuvg; 1 = 1261s
A AAG (alkyladenine DNA glycosylase), 506 A A U A A A s e q u e n c e .l o r m R N A 3 ' e n d c l e a v a g e . 695,695f A-Bl, 187 .48O blood group locus. 28, 28/ Abundance, 92-91 Accessory proteins, in nucleosome assembly, 770 Ac/Ds family, 541-542, 54If Acentric fragmen$, 5)9, 5j9f, 7 45 Acetosyringone, 404, 404f Acetylation, histone activation of gene expression and, 805-806, 805/
A domain, 184 ADPR (adenosine diphosphate ribosyl), 172 ADP-ribosylation, diphtheria toxin and, 172 cr-genes, 49 O49 2, 490f , 49 1f Agropine plasmids, 402 AID (activation-induced cytidine deaminase), 589-590 Alfalfa mosaic virus (AMV), 7))-734
AMV (alfalfa mosaic virus), 7)3-734 Anchor sequences charge distribution, "positive-inside" rule, 218
Alleles definition of, 24
protein orientation 833f
polymorphic wild-type, 57
modiiication, 8o), 8o1f,804, 804f transient at replication, 805, 805/ Acquired immunity (adaptive immunity), 594,
r e c e s s l v ez, ) , z ) _ I wild-type, 28,28f,57 Allelic exclusion, productive reartangement and,
5e4f,602 Acridines, frameshilt mutations and, I I Acrobacterium tumefaciens,crown gall disease, 40l-4O5, 402f-4o4f Actin gene, evolution, introns and, 52, 52l Activation-induced cytidine deaminase (AID),
582-584,58)f Allosteric control, 107 Alternative splicing in human genome. 84 RNA, splice junction differential use, 685-688, 686f,
5 8 9 - 59 0 Activators acetylasesand, 806-807 acidic, 647 binding. by short sequence elements, 627-628,627f coactivators S?sCoactivalors concentration of near promoter, enhancer increase of,6)I-612,6)lf CRP seeCRP activator functions, 642-641, 64)f histone acetyltrans{erasesand, 806-807, 806f, 807f homeodomain proteins, 660 inducible, regulation of , 65 I -652 interaction with basal apparatus, 646-648, 647f,648f ligant-responsive, steroid receptors as, 653-654,654f recognition of response elements, 649-651, 650f rranscription stimulation models, 648 ubiquitous, 628-629 without activating domains, 647 A c l i v e c d s s e t t e sf.o r y e a s t m a t i n g , 4 8 8 4 8 9 . 4 8 9 f A d a p t i v e i m m u n i t y ( a c q u i r e di m m u n i l y ) , 5 9 4 . 5e4f,602 Adaptor, IRNA, I30, ll0/ Addiction syslems, 42O, 42lf Adenine, 6 Adenosine diphosphate ribosyl (ADPR), 172 Adenovirus DNA replication, by strand displacement, )94, )9rf initiation, terminal proteins and, )9 4-)9 5, 795f Adenylate cyclase, 321 Adenylate synthase, in IRNA splicing, 692, 692f
687f Alternative splicing factor (ASF), 685-686 Alu family, 564-565 Amber codon (UAG termination codon), 172, 199 Amber suppressors,206, 208-209 Amino acids intrinsic discrimination, 203-206, 204f, 2o5f novel, insertion into stop codons, 199, l99f related. related codons and, l9o-l'92, l92f substitutions, recoding and, 212, 2l2f IRNA charging reaction, 200-201, 200f,2O9 Aminoacyl-IRNA amino acid component, 190 control,160 definition of, Il1 entry into A site, 169, l70f peptide bond synthesis, polypeptide chain transfer, t68-r69, r68f peptidyl transfer reaction, water in, 174, 174f in protein synthesis. 1.29,152 A silebinding. 154. 154[. t55f P sitebinding. 154. l14f. l55f selection, for A site insertion, 21o,210f Asite,211 entry into. 167 insertion.177-178 selection Ior insertion, 210, 2l0f structure, vJ puromycin, 168-169, l68f ternary complex structure, EF-G and, 171' lTlf Aminoacyl-tRNA synthetases in charging IRNA with amino acids, 200-201, 200f,20e c l a s sI , 2 0 1 - 2 0 2 , 2 o l f , 2 0 2 f crystal structure, 202, 2O2f nucleotide-binding f old, 202 signature sequences, 20 I -202
20)-206, 204f, 205f Amphibian globin genes, i04 Ampliconic s€gments, in Y chromosome male-specific rcgio\,87,87f
Algorithms, for gene identificaion, 65-66 Alkyladenine DNA glycosylase (AAG), 506
dominant,25,25f imprinting, DNA methylation and',8)2-8)4, multiple, 27-28, 28f . Seea/so Polymorphisms
c l a s sI I , 2 0 l l 2 0 2 , 2 O 2 f IRNA contact, 20),203f de{inition of, 131 recognition specilicity, proofreading mechanisms,
and, 276-2)8, 237f, 2)8f
Angelman's syndrome, 834 Animal cells genetic material in, 5, 5/ genomes, microRNAs, j42-343, j4)f Annealing, I3 Anthropod satellite DNA, short identical repeats, '119-120, ll9f Antibodies definition of, 572 interaction with antiger.s, 57 2, 57 2f structure, t />-) /6, ) /oJ Anticodons Seealso Codon-anticodon base pairing definition oI, 130, L)of mutations, 2Ol, 207 -20 8, 207f suppressorswith, 2o7-208, 207f IRNA specificity and, 1 31, I 3fl Antigenic determinant (epitope), 57 5, 591 Antrgens cfonal expansion and, 57 4-57 5, 574[ definition of, 572 interaction with antibodies, 57 2, 57 2f recognition, rT 4-57 5, 57 4f Anti-insulator elements, 786 Antiparallel orientation, oI polynucleotide chains, 7, T Antisense genes, 338-))9, j)9f Antisense RNA, for gene expression inactivation, ))8-139, j)9f Antisense slland,, I29, I29f Anti-Sm, 675-676 Antitermination control of transcription, definition oi, 287
29I, 29 If
phage lytic cascade and, )57-758, )58f,359f phages, 754-)55, 354f RNA polymerase and', 29)-295, 29 4f Antitermination protein binding sites, 292-29 ), 2931 extension of transcription unit, 29 l-292, 29 lf AntiTRAP, 734, ))4f Anucleate cells, 413, 4l3f APt,663 Apolipoprotein B gene, RNA editing, 720-721 ' 720f a2 prctern, 492 A protein, in phage genome replication, )97-J98,397f Aqueous channel formation, translocon and,
2)r-2)),2)2f TS Aquifex aeolicus,
867
Arabidopsis thaliana
Avirulent bacteria, l Axial elements
chromatin assembly, 774 DNA, centromeric, 746
definition of, 466 formation. double-strand breaks and, 468, 468f 5-Azacytidine,633
family size and, 81, 8l/ genome, 80-8f, 8U Archaea g e n o m e s i z e s ,g e n e n u m b e r a n d , 7 8 , 7 8 f IRNA splicing moldf, 691-692, 69tf ARE elemenrs, t42, I42f,697 Arms
B Bacillus anthracis, T9 Bacillus subtilis
definition of. 483 IS elemenrs, 525-526, 525f ARS elements (autonomously replicating sequence), )84-385. )85f, 49), 493f Ascomycete tetrads, spore formation, 47 5, 47 jf Ascus, 475 ASF (alternative splicing factor), 685-686 ASF/SF2 complex, 686 AsiA phage-encoded product, 3 5 5 aminoacyl-IRNA, t67, 177-t78, 2tO, 2tof function, l65 rRNA and, l8l
cotranslational translocation, 246-247, 247f culture, phage infection plaques, 360,360f daughter cell division, septum formarion, 4ll412,
41rf,4t2f
Assembly factors, 617, 6t7f,7)4 Ataxia telangiectasia, 5 l7
division, mutations, cell shape and, 412413, 417f EF-Tit. aminoacyl-tRNA entry into A site, 167-168,
ATMP kinase. 518 ATP hydrolysis
r67f
helicase DNA unwinding and, 4J5476, B6f in prepriming reaction, 449 for protein transport. 241 Attachment site (41, 487f
by translation, ))6-))8, 17f, ))Sf of trp operon, ))5-)36, tif, t6f definition of, 133 Attenuators. SeeIntrinsic terminators AUG initiation codon context and, 160, l6of in eukaryotes, l6)-164, 16Jf mutation, 21 I recognition during elongation, I 58-l 59 Shine-Dalgarmo sequence and, I6l-l62lf Autocatalytic splicing, vs nuclear splicing, 685, 685/ Autogenous regulation intrinsically self-limiring, 128 macromolecular assembly synthesis control, )27-)28, )28f of phage T4 p32, )26-j27, )26f, )27f by repressor, )19-J20, )2Of of r-protein synthesis, 325-)26, j25f, )26f Autoimmune disease,571 Autonomous controlling elements. 540, j4lf, j42 Autonomously replicating sequence (,4RSelements), 384-385, )85f, 49), 49)f Autoradiography. of replicon origin, 380, 380/ Autosplicing (self-splicing) definition of. 683 group I introns, transesredfication in, 7 07 -7 09, 7 o8f-7 tof group II introns, 683-684, 684f, 7 16-7 17, 7 I6f introns, maturases for, 7 I 6, 717, 717f,7 l8f Avian immunoglobulins, assembly f rom pseudogenes, 593-594,593f
cell cycle and, 4I04lI,4l0f chromosome segregation, site-specific recombination and 415417, 416f D constant, 41 0 initiation, 409, 4O9f multicopy control, 177 mutations, cell shape and, 41241), 4Bf septum formation, 411412, 411f, 412f, 4t5,415f ribosome s u D u n l r sr n , 1 > t , I ) t J translocation in, 169-170, 170f RNA polymerase, 262-26J, 262f activation of repair ar'd, 625-627, 626f subunits, 265-266, 266f termination at discrete sites, 286-287, 287f rRNAgenes, ll2 IRNA production, 698-699, 698f Sec system, 247 -249, 247 f, 248f segregation, mutations, cell shape and, 412-48,
4r)f septum formation FtsZ and, 413414,414f long filaments,4Il sigma factors, conserved regions, 280, 28Of signal recognition particle counterpafi, 2jO-2)l sporulation control, by sigma lactors, 28)-286, 284f-286f
enhancers, 6l I F-positive, 399 gene clusrers, 70]-)04 gene expression, j)-)4,
transformation in, )4, 4f vegetative phase, 283 vJ eukaryotic genomes, replication and, )77 Bacteriophages SeePhages Balbiani rings, 7 43-7 44, 7 44f B a m i s l a n d s ,1 1 4 , 1 1 4 f
r-proteins for, )25, i25f gene expression con1trol JO2, )32 via DNA SeeTranscription; Ttanslation via RNA SeeRegulator RNA genes, 38 genome
Band formation, on polytene chromosomes, 742-74),743f Banding patterns, of chromosomes, 7 40-7 41,
34f negative regulation of, 30), )jJf positive regulation of, )OJ,30)f
control
Index
SpoOJ protein, 420 sporulation, 418419 trp genes
conjugation, mediation by F plasmid, )98-399, )99f
occupation, 154, l54f translocation, | 69-17 0, 170f IRNA entry, 179, 179f a-specific genes, 490491, 49Of, 49If Asp-IRNA syntherase, IRNA conract. 2$,2jlf
868
sigma factors, 282 Soj protein, 420
control of, 1))-j34, ))4f termination of, 33) -)) 4, 33 4f Background level, {or mutations, l4 Back mutations, I6-17 Bacteria Seealso specific bacteria
A slte
de{inition of, 481 intasome in lambda recombin aIron, 486488, sequence requirements, 484, 484f Attenuation
catalytic subunit, 442 RNA polymerase, 282
replication, 409425 C constant, 4I0
74of,741f Basal apparatus assembly at promoteL 621-62), 622f, 6nf interaction with activators, 646-648, 647f, 64Sf interaction with enhancers, 63 I
nucleoid, 7 )4-7j5, 734f size, gene number and, 78. 78/ supercoiled. 7 J5-7 36, 7 j5f, 7 )6f initiation factors, I 58, 158f, 164 lysogenic,350 mating, )99, )99f
transcription startpoint and, 642 Basal factors (general factors), 6 I I
membrane layerc, 246-247, 247f mRNA
Basal level elements (BLEs), 629, 650 Basal level transcription, 106
enzymatic degradation, 140-I41, l4lf instability of, I l6 life cycle of, I)5-137, B5f, l36f number of proteins coded by, I 36 ribosomal protein synthesis, I34-l]5,
fJ5f mutation rates, l4-15, l5l operons, attenuation control oI, ll8 pathogenic, "pathogeniciry islands" in, 79 phages in, i5l-j52, JSlf plasmids in, )51-)52, 15lf polysome size, l)4 posttranslational translocation, 246-247, 247f promoters, conserved sequences,271 protein exporr, 247 protein synthesis, 180 initiation, 157-158, t57f, t58f, t62,162f initiator IRNA, 158-159, l59f termination codons, 172 RecBCD system. stimulation by cftr sequence, 47O,47rf r e g u l a r o rR N A , 3 4 | - J 4 2 , 3 4 2 1
localization of transcription-activating neaL 647, 647f
domain
Basal transcription apparatus, 6l I Base excision repaia 5001 50i Base mispairings (transitions), l5-16, I6f Base pairing codon-anticodon. SeeCodon-anticodon base pairing rnmnlampnr' ",
definirion of, 7, 7/ for mRNA synthesis, 31, I]/ in group I intron core formation, 709-710 inosine, 196, 196l RNA control, ll8 wobble, anticodon modifications and, t96-197, r96f Base pairs complementary,
7, 4 delinition of, 7, T distance on restriction map, 40 mismatched, I8-19 nucleic acid hybridization and. I2-I4, tJf, \4f in protein synthesis initiation, l6I-162, t6If, l62f
Bases modification, in IRNA, 194-196, I95f removal, excision repair in mammalian cells and. 504-505,505f B cell memory phenomenon, 594 B cell receptor (BcR), 595, 595/ B cells (B lymphocytes) antibodies on, 598 clonal expansion, ,7 4-57 5, 574f o e n n l t r o n 0 1 ,) / z development, 595,595f differentiation, 587, 594, 594f B C R ( B c e l l r e c e p t o r ) ,5 9 5 , 5 9 5 f
cap o, t)9 r, t)9 2, t)9 5' end oI mRNA and, 138-l 39, I 38/ Capsids,viral, 7)l-714, 7)2f, 7))f empty, 733 icosahedral symme:uy, 7 jl F.NA.in, 7)l-732, 7)2f Carboxy-terminal domain (CTD ) definition of, 612 phosphorylation, 624, 624f in RNA processing, 624-625, 625f
Chick p-globin LCR, 784 Chicken c-globin gene cluster, 631 Chimpanzee genome, vs human genoma, 89 Chip technology, 94, 94f chl sequence, stimulation of bacterial recBCD system, 470,471f Chloroplast division, ring formation, FtsZ and, 414, 4l4f DNA, circular, 69 evolution of, 72 genomes, 69
Cascade,regulatory, in phage lytic development, 75j-)54, j5)f, )54f
functions coded by,71,7 lf size, 7 | introns in, 72 N-terminal leader sequence, 24J, 24)f
Cassette model, of yeast mating, 488489, 489f Catalase,245 Catalytic activities, of ribozymes. 7 1'1-715, 7 llf-7 14f Catenation, of circular DNA, 480
protein leaders, 240 protein synthesis, 240 protein targeting sigrials, 24) role in photosYnthesis, 72
Caulo bacter ff escentus,420 C-bands, on centromeres, 745,746f CBP (CREB-binding protein), 807 cdc6 protein, j87-J88
transport receptors
B i P ,2 2 7 , 2 ) 4 , 2 ) 4 f Bivalents, in meiosis, 29,29f,461 B L E S ( b a s a l e v e l e l e m e n t s ) ,6 2 8 , 6 5 0 Blocked reading frcme, )2, )2J Bloom's syndrome, 5I7
CDE (cell cycle dependent elements), 747 ,747f, 748 C/D group, of snoRNAs, 699,699f
B lymphocytes SeeB cells Bovine growth hormone, signal sequence, 228,228f lox.4 sequence elements. of lambda RNA,293,294
cDNA (complementary DNA srrand) cloning approach nRNA purification and, 140 synthesis, intron RNA as template for, 717 CDP (CAAT-displacement protein), 649
BEAF-32, 78)-784,78)f p clamp, clamp loader and, 440441 p2 microglobulin, 601 p2 microglobulin gene, 601-602 B selection, 598 B-form of DNA, 8 bHLH proteins, 661
loxB sequence elements, of lambda RNA, 291, 294 Branch migration , 46J, 46)f Branch site, in RNA splicing, 674 Breakage and reunion at consensus sequences,in immunoglobulin gene generation, 582, r82f in homologous recombination, 461, 46l,f 533f in nonreplicative transposition, 5j]-5j4, RAG protein catalysis of, 584-586, 585f, 586f in recombination, 29, 29f Breakage-fusion-bridge cycle, 5)9, 540f Bromodomain, Sl2 Bromouracil, mutations induced by, I5-16, 16f Bulge-helix-bulge motif, for tRNA splicing, 69t-692,69If Buoyant density, I 18. ll8f. l2o Butyric acid, histone deacetylaseinhibition, 806 Bypassing recoding and,2l2,2l2f ribosome movement and, 214-215, 214f
CDI proteins, T cell recepror and,598-599,599f Cell cycle, bacterial replication and, 410--41i, 410/ Cell cycle dependent elements (CDE), 747 , 747f,748 Cell-mediated immunity (immune response), 571, 57jf,599 CENfragments, 747, 7 47f CENP-A,748,774,824 CENP-B,824 CENP-C, 748 Central dogma, I0-l 1, I t/ Centromere binding to microtubules, 7 45-746, 7 46f c-bands,7 45, 7 46f definition of, 745 DNA sequences, in S cerevisiae,7 47, 7 47f eukaryotic, 7)9, 719f protein complex binding,748, 748f repetitive DNA, 746 separation during mftos\s, 7 45, 7 45f
bZIP structure, 66), 663f
cFl,696
C
CFII, 696 c-fos ge\e, 663 C gene
CAAT boxes, 628, 628f, 629, 649 CAAT-displacement protein (CDP). 649 Caenorhabitis elegans essential genes, number of, 90, 90/ gene expression, systematic analysis by RNAi. 344 genome, 80 family size and, 81, 8l/ nonrepetive DNA content of, 62 s i z e ,6 l l 8 1 , 8 r / lin4 rcgl]lator gene, )42-)43, )4)f linl4target gene, )42-)4), )43f microRNAs, )42-)4), 34)f SL RNA (spliced leader RNA), 689 X chromosome condensins, 830 dosage compensal\on, 826, 826f CAF-l (Chromatin assembly factor-ll, 77 l, 774 Cancer susceptibility, DNA repair deficiency and, 5 I 7 Candida, genetic code changes, t98
immunoglobulin synthesis and, 57 6-577 single recombination in light chain assembly, 577, 57 8f, 579 Chaperones chaperonin system, 225-226, 225f, 226f denatured protein and, 224 Hsp70 system, 225-226, 225f, 226f mechanisms of. 225-226, 225f, 226f mitochondrial, 244 newly synthesized protein and, 224 in protein folding, 223-224, 22)f, 224f protein membrane transport and, 224-225 ' 225f SecA, 247-249, 248f SecB, 247-248, 248f Chaperonin system, 225-226, 225f, 226f Chemical proofre ading, 2O4, 204f Cs gene,588, 589 Chiasma,29,29f c h i a s m a t a ,4 6 I , 4 6 2 f
Trc,243 TOC,243 Chromatids defiaitio\
ot, 29, 29f initiating or recipient, 465
Chromatin active vs inactive, 809, 809f assembly replication-couPI ed P alhw aY, 7 7 4 replication-independent pathway, 774 fibers 1O nm, 7 69, 7 69f 30 rlm,769-770,770f nucleosome path it, 7 69-77 l, 7 69f' 770f inactivation, 822 packaging, 730-7)l protein mass, 759 release of nucleosomes from, 759,759f remodeling, 7 7 0, 7 9 8-800, 7 99f ' 8o0f definition oI, 799 dynamic model o1,799, 799f nucleosome organization changes in, 799, 799f at promotet 810 remodeling complexes, 799-800, 799f' 8O0f binding to chromatin via activators,
80r-802,80u in gene activation, 80 I replication, 758-759 reproduction,
nucleosome assembly for, 77 l-77 4,
77rf-77)f structure chromocentet 719 DNAase hypersensitive sites and', 7 86-7 88, 7 87f epigenetic state, 797 equilibrium mod el, 7 97 -7 98, 7 97f euchromatin, 719 heterochromatin, 7J9, 7 j9f histone phosphorylation and, 810-81 l' 8 I lf insulators and, 785, 785f interphase, 7)8-7)9 organization, levels of, 758-759 stabiliry of, 797 -798, 7 98f subunit, fundamental. SeeNucleosomes Chromatin assembly factor- 1 (cAF- I ), 77 | ' 77 4 chromocenter, 739 Chromodomains, 812, 822, 822f Chromomeres, 741 Chromo-shadow domain, 822 Chromosomal walking technique, {or gene identification, 6j-64, 6jf, 64f Chromosomes, 729-756 banding patterns, 740-741, 7 4of' 7 41f
Index
869
c e n t r o m e r e ,7 ) 9 , 7 j 9 f circular catenated, 409 site-speciiic recombination and, 416417, 416f condensation, condensins and, 828-810, S2gf, gl}f daughter, partitioning process and, 4l74lg, 4lgf o e l r n l t r o no I , / J 1 DNA,24,24f domains SesDomains, chromosomal end sealing, telomeres and, 749-7jO, 749f,75Of eukaryotic replicons on, )87-)84, )$f, )84f as segregation device, 744-746,745f, 746f individual, during mitosis, 7 )B-779 instability, DNA repair deficiency and, 5 I 7 lampbrush, 7 4l-7 42, 7 42f loop formation, ar end, telomeres and, 749-750,750f
Coding ends, 582, 582f Coding region definition of, )),34f
promoter recognition by sigma factors and, 279-280,280f Conservative transposition, 528, 528f
replacement sites, 104 silent sites, 105 Coding strand (sense strand), t29, 129f,258,255f Codon-anticodon bas€ pairing, l9l interaction, 154, l54f modified bases and,,1,96-t97, t96f
Conserved sequences,27J Constant region (C region), in immunoglobulin, 575-576,576f Constitutive gene, 9l
recognition specificity of. 209-210 third-base degeneracy and, t9t, 192-\93, t92f wild-type or mutanr, 207-2O8,207f wobble hypothesis and, 19 2-19 3, 19 1f rRNA interacrions, l8l-182, l8I/ simplification, 198 Codon families, 192, 198-199
multiforked, 41041 I, 4tOf pairing, independent from synaptonemal complex tormation,469 polytene, 7 42-7 4), 7 4)f, 7 44f band formation on, 742-7 4), 74)f p u f f f o r m a t i o n a t g e n c e x p r e s s i o ns i t e s . 74)-744,744f
formation, 7)6-737, 7j7 f MAR sequence and, 718 segregation, 4l l, 415417 , 4t6f s i t e - s p e c i f i cr e c o m b i n a t i o n a n d , 4 l j 4 I 7 , 416f synapsed, homologous recombination berween, 460462, 46tf, 462f cI ge^e, )60 cII gene, for lysogeny,368, J68f CIII gene, for lysogeny, )68, )6Bf
Codons Seealso specific cod.ons amber or UAG termination, 172, 199 AUG initiation SeeAUG initiation codon base pairing with anticodon, l9l definition of, 30
in Drosophila melanogaster,56I-562,
56lf reverse transcriplases, 563, 56jf Copy choice recombination, 459, 556,556f Copy number, 419 Cordycepin, 695 Core enzyme, of RNA polymerase, 267 Corepressors,306 Core promoters, 6l 5
interpretation, I 9l meanlngs recoding and, 2ll-212, 2t2f third-base degeneracy and, t9t, 192-1%, t92f IRNA and, 190, 192-193 number of, for each amino acid, l9l, l9tl opal or UGA termination, 172 related, related amino acids and, l9O-192, \92f synonyms,190 in bypassing, 214 termination, J2 triplet, 190.190/ Coevolution, II5-l16
cII prorein, 368, )68f,374 cr-acting DNA sequences ,4RSelements, 784-j85, )85f functions, 302-)03 crj-acting siIes, J5-)6, )5f ds-dominant mutation, 308-109, 108/ 9-crs-retinoicacid (RXR) receptor, 6j7-6j5, 6j7f cis/trans Iest (complementarion lestl, 25-26, 26f Cistron, 26 CJD (Creutzfeldt-Jakob disease),20, 839
Commitment factors for pol III promoters, 616-618, 6I7f RNA polymerase binding and, 619-620, 62Of Compatibility grovps, 42t, 42 I f Complement
440441
Classswitching by DNA recombinarion, 587-589, 588/ by novel recombination, 589-5gO, jS9f, 5g0f Clear plaques, )60, 36Of Cleavage reactions messenger RNA 3' end generation and, 695_697, 695f,696f rRNA producrion, 697-699, 698f Clonal deletion, 571 Clonal selection rheory,574-575, 174f, j77 Cloverleaf structure, of transfer RNA, ll0-lll, Clusters, 98-l 26 Cm gene, 588
Il0/
CMV (cytomegalovirus), 215 Coactivating complex, 630 Coactivators basal apparatus interactions, 646-647, 647f HAT activities, 806-807, 806/ histone acetyltransferases and, 806-807, SO6f, SOTf transcription and, 68, 64jf Cockayne's syndrome, 627
870
Index
definition of, 26 functions, 572-57) MHC locus coding for, 600 Complementarity measurement, by filter test, t)-14, t4f Complementary base pairing
replication and, II, t l/ Complementation |#oup, 26 Complementation, in vitro, 430 Complementation test, 25-26, 26f
526f c-oncgene,558 Condensins, 828-8)0, 829f, 8)Of Conjugation mediation by F plasmid, )98-399, )99f transfer of single-stranded DNA, 400-40 1 Consensus sequences definition of, 273 for immune recornbination, 53l, 58lf
cossites, 713 Cosuppression, 345 Cotranslational translocation, 22 1-22j, 2nf in bacteria, 246-247, 247f requirements, 23), 2)jf
Countertranscript, 422 coxll gene, RNA editing h, 72t-722,722f CpG doublets, 6)4,634f CpG islands definition of, 19 merhylation, 615 as regulatory targets, 6)4-6)5, 634f X chromosome inactivation and, 828 CREB-binding protein (CBp), 807 C region (constant region), in immunoglobulin, 575-576,576f Cre//ox recombination system, 485, 456, 486f Cre recombinase,4S4 Creutzfeldt-Jakob disease rcJDl, 20, $9 Cro protein
definition of, 7, 7/ for mRNA synthesis, )), )3f Complementary single strands, of nucleic acids duplex formation, l2-11, Bf
Complete enzyme (holoenzyme), 265 Composite transposons, IS modules, 52i-j26,
Core sequence. 483 Corticoids, as activators, 654l
SRP receptor, 230 translocons. SeeTranslocons signal sequences,227 -228, 228f
Cognate tRNAs, 200 Cohesins, 466467, 828-810, 829f, 83Of Cointegrate, in replicative transposition, 5fl-r36, 5)2f,535f Colinearity. 32-33, )jf
c-Jun gene, 66) Clamp loader, 439, 89f, ClassIII region, 600
autonomous, 540, 541f, 542 En, gene expression al]d, 542-544, 543f Spm, gene expression and, 542-i44, 54)f definition of, 518 formation of transposon families, 540-542, 54If nonautonomous, 5 40-542, 54 I f Coordinate regulation, 307 apia elements
GUG, in protein synrhesis. 158-160, 160/ initiation, l2
recombining, connected at synaptonemal complex, 465467, 466f replication cycle, 4l 0-41 l, 410/ scaffold
Constitutive heterochromatin, 826-827 Consritutive mutants, 108, 109 Controlling elements
525f,
DNA-binding specificity, helix-3 and, 362-)6),362f runctlons, J / I cro repressor for lytic infection, 370-j73, )72f mRNA, 168 Crossover control, 469 Crossover fixation model, maintenance of identical r e p e a t s ,I I 6 - 1 1 7 , I l T Crossovers, 468469 in meiosis, 29, 29l in nonreplicative transposition, 5j3, 533f in replicative transposition, 5j-I-sjt, 5j2f a! 4-strand sIage, 99, 99f unequal SesUnequal crossovers Crown gall disease, 401402, 402f T-DNA genes, 402405, 40)f, 404[ virulence getes, 403404, 404f CRP (cyclic AMP recepror protein), t63 CRP activator "activating" region, l2l
acrivation of transcription, l2l binding site locations, )22-323, )2)f consensus sequence, )22, )22f c y c l i cA M P a n d , ) 2 1 , ) 2 I f dimer, 12l DNA bending and, )22, )22f glucose and, J2I, 322f monomer. 12l mutations, 122 Cryptic satellite anthropod, l19, ll9f definition of, I I8
Denaturation, ofDNA, 13, 13/ De novo merhylase, 81 l, 8) lf Density gradient, 1 I 8, 1 t 8/
closed molecule, linking number, 477, 478 coding strand sequence, 190 complementarity, recombination process and,
Deoxyribonucleases SesDNAases Deoxyribonucleic acid SesDNA DHFR (dihydrofolate reductase), 240
conformation, )22, )22f
DHFR genes (dihydrofolate reductase genel, 42, 42f Dicentric chromosome, 539-5 40, 5 40f Differentially methylated domains (DMDs; imprinting control regions; IcRs), 834, 814/ Dihydrofolate reductase gene (DHFR genes), a2,42f
contact with sigma factors, 280-282, 281f, 282f damage methylation, 5O3,503f RecA protein activation, 5l l-514 repair systems. 502-50), 502f, 50)f
Dimer formation, by helix-loop-helix proteins,
CIDNA, 69
660-661, 662f Diphtheria toxin, ADP-ribosylation and, 172 Diploid organisms, 24
of homeodomain, 659 o f r e p r e s s o r ,l l 0 l l l I of RNA polymerase. 648 C-value,60-61, 60, 6ll C-value paradox, 61 Cyclic AMP adenylate cyclase synthesis. 121 C R P a c t i v a t o ra n d , ) 2 1 , ) 2 \ f glucose levels and, 12 I s t r u c t u r e ,) 2 1 , ) 2 l f Cyclic AMP receptor protein (CRP), 361 ryI mutations, 168 ryR mutations, 168
structural distortior]Ls, 502-501, 502f regions, 832, 8)2f
demethylated
denaturation, ll, 1T density,730-731 double-helix mod'el, 6-8, 7f, 8f base flipping, 506, 5O6f B-ring relationship in, 440-44I,
Dipoid phenotypes, 49 1492 Direct repair systems, 500-501. 500, 508 Direct repeats definition of, 524 g e n e r a t e db y i n s e r t i o n , 5 2 7 . 5 2 7 { reciprocal recombination between , 529, 529f in retroviral RNA, 554, 554f Diseasegenes, RFLPs associatedwith, 58-59, 59f
major groove in, 7-8, 8/ minor groove in,7-8, 8f
Dislocalion SeeReverse lran:location Displacement loops SeeD loops )9f, 40f Distances, gerelic,3840,
right-handed, 8 twisting number, 47 7 -47 I underwound, 8
definition of, 104 of DNA sequences, 105 globin gene, 105-106. 105f, l06f in globin genes, I03, 103/
Cytoplasmic domain, 601 Cytoplasmic inheritance, 68
rate of, 105-106, 105f,I06f of repeated sequences.rate of neutral substitution
cytosine deamination, ).5, l5f, 18, l8f,502, 502f flipping, by methylase, 506, 506/ Cytosol. protein synthesis, 22O, 220f cytotoxic T cells (killer T cells). 573
and, 107, l07f of replacement sites, 105-106, 105f, l06f o I s i l e n t s i t e s ,1 0 5 - 1 0 6 , 1 0 5 / D loops (displacement loops) mitochondrial origin maintenance , 388-)89, )89f
D
single-stranded, 464 DMDs (differentially methylated domains; imprinting control regions; ICRs), 834, 834/
Dam methylase. DNA methylation and, 380-181,
DNA
Cytotype, 546
380f,5)8 dam system, 518 Daughter nucleoids, recondensation, MukB and, 4r8,418f Daughter strands. in DNA replication, 8-9, 9f Deamination of cytosine, 15, l5f, 18, l8f of 5-methylcytosine, l8-19, l8/ in RNA editing, 72r, 72lf Decaping, mRNA degradation and, 143, 143/ Degradation bacteria mRNA, enzymes in, 140-I41, ).4\f mRNA, 141-144, l4)f, I44f Degradosome. in bacterial mRNA degradation.
r41,r4tf Delayed early genes, 292 Deletions definition of, 16 in DNA sequences, 529 frameshift mutations and. 11, I I/ generation of replication-defective viruses, 558,558f in identifying genes, 6), 6)f from immune recombination, 582, 582f i n t h a l a s s e m i a s I, l 0 - l 1 1 , I l 1 / delta elements, 5 59-5 60 D e m e t h y l a s e s .8 l l , 8 l U Demethylation, gene expression atd, 6)2-6J), 6)2f,6))f
amount, in genome SeeC-value attachment, to interphase malrix, 7)7-7i8,
overwound,
440f
8
double-strand breakage by type II topoisomerases,479 cleavage complex. 481, 48lf conversion to single-strand, 4354)7, 436f type II topoisomerases and, 479,481, 48lf
Divergence
591-593,592f Cytomegalovirus (CMV l, 2)5
Cytidine deaminase, induction, of somatic mutations,
bending by cRP activator and,
replication errors, 5o2, 502f replication stoppage, 45 l-452, 452f single-base changes, 502, 5O2f
Dihydrouridine, 195, 195f
C T D s e eC a r b o x y - t e r m i n a l d o m a i n C-terminal domains oi DNA, 654
29,2ef
738f
D a s e p a r r s ./ - 6 , / J . 6 J duplex formation and', l2-l3, llf point mutations of, 15 speci{icity of replication and , 8-9, 9f bases,modified, 18-19, l8f, l9f B-form, 8 binding contacr points. 27 5-277 , 27 5f, 27 6f leucine zippers and, 662-663 in minor groove by TBP, 620-621, 620f, 62lf to RNA polymerase, 27 l-27 2, 27 2f, 27 5-27 7, 275f, 27 6f sigma la(tor control ol. 271-272,272J centromeric, T46 in chromosome. 24, 24l circular in bacterial genome, 7 )5-7 )6, 7 )5f, 7 )6f catenation,480 minus strand, 197 plus strand, 197 reciprocal recombination between direct repeats,
5' end, initiation of transfet eukaryotic, I l8 in gene, 2-l genetic information in. 5l
400, 400f
as genetic material in animal cells, 5 iabacleia, 14,4f in viruses, ,l-5, 5/ i n g e n o m e s ,1 l hemimethylated, )80-382, )9of-)82f highlyrepetitive, I17 hybrid or heteroduplex, 475 in breakage and reunion, 462464, 46)f, 464f definition of, 462-463 Iormation, 29-30,29f formation ot, 29-30, 29f recombinants with, 99, l00f insertion into phage head, 732, 7)2f knors, 480,481 lagging strand, semi-discontinuous replication, 415, 4)5f leading strand, continuous replication, 435, 4)5f length in vints, 7)0f, 7)I vJ compartment
size, 7 )0, 7 3Of
linear integration of, 557, 557f recombination with circular DNA, 460' 460f mainband, ll8, l18/ methylation, )45, 538, 632 Dam methylase and, 380-18i, 380f,5)8 \n Drosophila melanogaster,6)5 epigenetic ef{ects, 819-820, 8l9f
recombination with linear DNA, 460, 460f supercoiling of, 47 6477 , 47 6f ft-acting sires,35-)6, )rf
of germ cells, 8)2-83j histone methylation and, 808-809 imprinting and, 8)2-8)4, 8))f maintenance of, 8)0-8)2, 81lf targeting for repair of mismatch error and,
cls cleavage, 485-486 cleavage, by restriction endonuclease, )9-4O' 40f
508-509, 5O9f targets, 812
529,529f
Index
877
minus strand, 554-55 5, 55 5f, 7 19 modification, at promoter, 810 nonrepetitive, I l6 nucleosomal, 7 58, 7 60, 7 6Of cleavage sites,7 64-7 65, 7 65f corc,762-76j l a d d e r o r g a n i z a roi n o f , 7 6 l - 7 6 2 , 7 6 | f l e n g t h o f , 76 l - 7 6 2 , 76 t f , 76 2 length reductions, 7 62, 7 62f linker,762,763 pcriodiciry. 7 66-7 67. 7 661 rotational posirioning, 777, 777f surface variatiun s, 7 6)-7 66, 7 64f, 7 6jf translational posirioning, 77 6-777 , 777f p a c K l n gr a u 0 , / ) 6 plus strand,554 555,555f polynucleotide chains, 6. 7, 7f antiparallel orientation of, 7, 7/ daughter strands, 8-9, 9f parental strands, 8-9, 9/ template strands, 8-9, 9/ protein coding, l2
binding, to oriC, 448449,448f replication at origin and, 382 DNA-arrest mutants, 447 DNAases (deoxyribonucleases) definition of, l0 I chromatin digestion, 7 88-7 89, 7 89f DNA cleavage sites, 7 65, 7 65f hypersensitive site creation, 786-788, 787f single-strand nicks in DNA, 7$-764,764f II, single-strand nicks in DNA, 76)-764 DNA-binding domains function,645 homeodomain, 65 8-660, 659f, 66Of independence from transcription activation, 64)-645, 644f localization, of transcription-activating domain, 647, 647f specificity, for target promoter or enhancer, 646-647 in steroid receptors, 654 tethering function, 645 transcription activation specificity and, 644-645,644f types, 65I-652, 652f
repair systems SeeRepair systems repetitive,117,746
DNA-binding sire, 109 DnaB protein
replication, 429454 centraldogma and, l0-l l, I l/ DNA polymcrases in, 10, 4t0-41I , 430f, Btf repression at silencers and, 49249j semlconservative, 8-9, 9f successiveactiviLies,448 replication fork, 9-10, l0/ retroviral, 554,554f s a t e l l i t eo r s i m p l e s e q u e n c e ,1 0 0 . I 1 7 , I I 8 , I l 8 l
in oric replicon, 419 prepriming, 448, 448f, 449 replication fork and,44l, a42f DnaC protein, prepriming, 448, 448f,449 DNA-delay mutanrs, 447 DNA-dependent prorein kinase (DNA-PKcs), 516-517, 5t7f DNA lingerprinting, I l8 DnaJ protein, 45 1
selfish,52l sequences
DnaK protein, 45 I DNA ligase
assembly site or paJ,45 I breakage by type I topoisomerases,479 conjuga tional transfer of . 400--40 l, 4OOf,40 | f replication, 4)54)7, 436f strand separation, 448449, 445f, 477, 477f structure, )0, 47 647 8, 47 6f, 477f sugar rn, 6 supercoiling SeeSupercoiling, DNA symmetry, ll4 synthests error-prone,507 repair,4)O,430f replication SseDNA replication semidiscontinuous, 4)441j, 435f telomeric, 7 49-7 50, 7 5Of topology m a n i p u l a r i o no l . 4 7 7 . 4 7 7f topoisomerases and, 47 847 9, 47 9f trans - acting siles, ) 5-3 6, j 6f trans cleavage, 486 transcription, central dogma and. l0-l l, I l/ transfer, between organelle and nucleus, 72-7f unwinding, gyrase and, 449 writhing number lW). 478 dnaA gene, )81 dnaA gene mutations, 449 DnaA protein availability, control of, 182
872
Index
433f,4j4f subcomplexes, 4J9440, 89f V, complement synthesis to damaged strand, i07 DNA repair deficiency diseases,5 l7-5 l8 DNA repair synthesis, 4)O,4)0f DNA replicases definition of, 430 functions, 441, 441f, 444445 DNA replication initiation, priming for, 4)7 4j9,
4)7 f, 438f
DNA restriction analysis. SaeDNA fingerprinting DNA sequences,protein coding, for more than one
zinc finger moiif, 651, 652-653,65)f DNA-binding proleins, 449
single-strand
pyrimidine dimer and, 510 replication at 5' end and, 394, )94f sliding clamp. 440441, 440f, 441f structure, commonality of, 43)4)4,
s e m i - c o n s e r v a t i v e 4, 3 0 , 4 3 0 1
rearrangements, 522, 522f transposons aru), 528-5)0, 529f recombination. class switching from. 587-589, 5gg/ renaturation, ll, 1y
changes, mutations and, l4 in genome mapping, 57 nonrepetitive, 6l-6), 62f repetttlve, 6 l-61
replicase activity of, 431 replication stall, DNA damage and, 507 IV, complement synthesis to damaged strand, 507 nuclease activities, 4)1432, $2f l'-OH end or primer, 4)74J8, 4)7f priming requirement, 188 p r o k a r y o t e ,4 ) 0 , 4 ) l f
votein, Domains
4547,
46f, 47f
chromosomal active genes and, 7 88-789,789f definition of, 790 DNA supercoiling in, 7]5, 7)5f in eukaryotes, 736-7)7, 7)7 f insulator sites, 7 90-7 9\, 7 91f LCR control of, 789-790,790f
AMP inrermediare, 444, 446f Okazaki fragments linking and, 443'444,446f DNA mapping techniques, restriction endonucleases and, 1940,4Of DNA methylases, 8)I, 83lf dna mutants, 4294)0 DNA-PKcs (DNA-dependenr prorein kinase), 516-517, 5t7f DNA polymerase a,444 a, 44044r,
locus control region, 790-791, 79lf matrix attachment sites,790-791, 79lf regulatory, 7 9O-7 9 I, 7 9 lf DNA-binding. SeeDNA-binding domains Dominant negative, I l0 Dosage compens ation, 826, 826f doublesex (dsx) gene, 686-687 Double-strand breaks (DSBs) generation, critical points, 589 in immune recombination, 582,582f initiation of homologous recombination, 464465, of mating type switching, 491494,494f repair systems, r00f, 501, 516-518, 517f
465f
synaptonemal complex formation and, 467469,468f target, w gene as, 7 I 5-7 | 6, 7 I 5f
44of, 44rf, 444 catalytic activity, 434 catalytic core, 44I, 441f,442f clamp loader, 4)9, Bgf 6, function of, 444445
Double-stranded RNA (dsRNA), degradation of mRNA, 343-145, )44f, )45f Doubling time, 410
in DNA replication, 43O4)t, 4)0f.43tf e, function of, 444 enzyme units lagging strand synthesis and, 442, 4Bf leading strand synthesis and. 442,443f error-prone,592
DP thymocytes, 598 Drosophila melanogaster
Down mutations, 274 Downstream, 258
errors during replication frameshifts, 412 substitutions. 432 e u k a r y o r i c ,4 j o 4 ) 1 ,
7 43f
embryonic development, 65 l eye color, position effect variegation, B2O-821, 82lf Fab - 7 region, 7 85-7 86, 7 85f gene expression map, 94 genes
4jtf
elongation, 444445 initiation, 444445 exonucleolytic activily, 4)24)4, 4)3f fidelity of replication and, 4J24r, 4iJf function of, I 0 r,4)t-4j2,4)2f
Antp,824 brahma,824 emc,66I-662
III holoenzyme subcomplexes, $9440,
bithoru locus, 7 85, 825 chromosome banding pattern, 7 42-7 $, centromeres, 742-743 DNA, centromeric, 746 DNA methylation, 635
4j9f
essential, number of, 90-91 Eu(var),81I
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sizes,differences irr, 4)45, 44f,4rf structural, j02. Seealso lac genes transcribed, 777 -77 8, 77 8f types, number of , 8I-83, llf, Genetic code, f89-2 I 5 anticodons SeeAnticodons
d
deletions in thalassemias, I I0-l I I, I I I/ during development, I0l-102, 102,f
82f
p
exons,5l,5l/ intron length, 4l
breaking, 190 codons. SeeCodons definition of, 30
cluster formation, 102-107, 103, 109 deletions in thalassemias, I I0-l I I, I I l/ during development, l0l-102, 102/
evolution, I92 fixed starting point for, l0
exons, )I, )rJ of goat, I09 intron length, 4l--42 pseudogenes, I09
reading frames, l1-12 sporadic alterations, 197-199, 197f, l98f Genetic information central dogma, 10-1 I, f l/ DNA base sequences and, 6
restriction map, 40, 4ll speciesdiflerences, 43, 4jf
Genetic linkage, 24 Genetic linkage maps, 56
clusters evolution of, I I t-I l2 formation by duplication, l0l-104, divergence, 105-106, lo5f, l06f evolution, 103-104, LO)f, 106, l06f organization, structural, 41,42f, l0l,
Genetic locus, 24
pseudogene, I08, I08/
expression, as unidirectional, I I positional,53
lo2f, l0)f
l02f
Genetic mapping minisatellites for, 123-125, 124f, l25f RFLPS for, 58-60, 59f Genetic recombination, gene conversion and, I l6
superfamily, 51, 5ll Globin protein, synthesis, l)3-134, lj4f B-Globulin gene DNAase I digestion, 7 88-7 89, 7 89f
cenetics, history of, 3l
LCR,789-790, 790f p-Globulin promoter, hlryersensitive sites, 787
Genome
for RNA, identification oI, 67
duplicated differences in, 100-l0I retention of function, I 15
number of
universality, 19l-192
Genes
as coding unit, 2, T colinearity with prcre\n, 32-)),
SeeInterrupted genes length of, j3-74,34f mispairing, I l6 nonallelic, exons in, I l6 nontranscrjbed, 7 7 7-7 7 8. 7 7 8[
overlapping, 4547, 46f,47f, 5) protein-coding, 70-7 1, 7Of, 82
Translation visualization, direct, 741
)L-')\
by sequence, 57 minisatellite vari abllity, 124, l24f number of genes in, 56
82rf
eukaryotic, control by of initiation of transcription, 64t-642,642f inactivation, by antisense RNA, 338-339, 339l
fnrnrntein
by linkage, 56 by restriction cleav age, 56-57
essential, number of, 89-92, 90f, 9lf expressed number, 93
bacterial, nucleoid, 7 ) 4-7 ) 5, 7 ) 4f contents ot, 2, 55, 57-58, 58J definition of, 56 DNA in, I I
Glucocorticoid receptors activation, by ligand binding, 656, 656f zinc fingers, 615-656, 656f Glucose, CRP activator and',721, j22f
extrachromosomal bacteriophages SeePhages plasmids SeaPlasmids gene distribution , 85-86. 85J
Glutamate receptors, mRNA editing, 721, 72lf Glycolases, base flipping, 506, 5o6f Glycosylases, excision repair in mammalian cells and,
genes in, 2
GMP-PCP, I68 cl phase,387,787f
human, number oI genes in, 83-86,84f individual variation in. 57-58, 58[ mapprng
504-50r,505f
Gratuitous inducers, 307 GRE (glucocorticoid response element), 649
Index
875
GT-AG rule, 671 GTPasecenter, 179 cTP-binding proteins, in protein synthesis. t79 GTP hydrolysis activation, of ribosomal translocation, I72 aminoacyl-IRNA entry into A site and, r67-168, t67f initiation factors and, 165-166, t65t', t66f mismatched aminoacyl-IRNA and, 2l I in SRP-receptor inreractin. 2]1, 8lf Guanine, 6 Guanine nucleotide, in self-splicing reaction, 7Og,7OSf GUG codon, in protein synrhesis, I 58-160, 160/ Guide RNA, 7 22-7 24, 7 23f, 7 24f Gyrase DNA unwinding and,,449 enzyme turnover, 482 signal inversion model, 481482,
482f
H H/ACA snoRNAs, 699-7OO,700f Haclp transcription factor. 693, 69}f H a en op h i I i u s i nJl u en zae ge nomt gene number in. 78 size, family size and, 8 I, 8l/ Hairpins at coding end, in immune recombination, jB4-jBj, 585f formalion, 59O,590f Hammerhead ribozyme Mg2+ initiation of catalysis, 7 t9-720, 72Of self-clevage,7 19, 7 19f H antigen (O antigen). 28 Haploid phenotypes, 49 t492 Haploid-specific f unctions, 49O, 49Of Haplotype, 59-60 H a p t e n s ,5 7 5 , 5 9 1 HATS SeeHistone acetyltranslerases Hbanti-Lepore, lll HbH (hemoglobin H), 1l I HbKenya, I11-112 H b L e p o r e ,l l l HDA (high-density oligonucleotide arrays), measurement of mRNAs, 94, 94l HDACs SeeHistone deacetylases Headshell, DNA Iength in virus and. 7)I-7l2,7)2f Heat-related gen es, 27 8-27 9 Heat shock genes, 649-650 Heat shock proteins hsp70 genes, scsand scs'elements,78J-784, 7S3f Hsp70 system, 225-226, 225f, 226f Hsp90 system. 225-226, 226f Heat shock transcription factor (HSTF), 650 Heavy chains, in antibody tetraner, 575-576, j76f Heavy strand, 120 HeLa/SV40 cells, replication appar atus, 447f Helicase-primase complex, 44 I Helicases DNA unwinding. 4)5-4)6. 4)6f h e x a m e r i c ,4 J 6 , 4 ) 6 f as priming requirement, 418, 418/ stbrnir, 625-626, 627 Helix-loop-helix motif (HLH). 651 Helix-loop-helix proteins (HLH proteins) basic or bHLH proteins,66l-662, 662f dime r formation, 660-66t, 662f interaction, by combinatorial association, 660-662, 661f,662f
876
Index
definition of, 651 f unctions/roles of , ) 62, 3 62f lambda repressor, 362, j62f Helix-turn-helix region, Cro and repressor in, 37 I Helper T cells, 572 Helper virus, 558 Hemimethylated sires,83 l, 83 l/ Hemoglobin adult vs embryonic, l0I-i02, synthesis, \)3-134, l)4f Hemoglobin anti-Lepore, I I 1 HemoglobinH(HbH), lll
102/
Hemoglobin Kenya, I I l-l 12 Hemoglobin Lepore, 1l I
structure, 48 I
nonbasic, 661-662, 662f sequence motif, 660-661, 66U
Helix-rurn-helix model, of repressor DNA-binding, )62,362f Helix-turn-helix motifs
Heterochromatin
modifications,
802-804, 80l, 80jf, 804, 804f of Hl tail, 812, 8l2f by methylation, 80), 803f,8O4, 804f by phosphorylation, 803, 801, 804,804f sites, 803, 803/ nRNA,, 697, 697f
octamers in chromatin remodeling, 799,799f conserveo, / /2. / lzJ disassembly of, 77), 77)f displacement by transcription, 779-7 81,, 779f,780f Iormation of , 7 67-7 69, 7 67f-7 69f placement of, 776 reassembly oI, 77 2-77 ), 77 )f phosphorylation, chromatin structure and, 8 I0-8 I I 8r 1f preformed, 770,77Lf
absence of histone acetylation, 808 constitutive, 7 40, 826-827 definition of, l19, 739,7)9f
Histone acetyltransferases (HATs) activators and, 806-807, 806f, 807f active ys inactive chromatin and, 809,809f
epigeneric inheritance, 820, 820f,821, 821f, 8)5-8)6,836f extension. gene inactivation and,, 821, 82lf facultative, 827 f ormation, in y e a{, 82)-824, 823f
Histone deacetylases(HDACs) activators and, 806-807, 807f active yJ inactive chromatin and, 809,809f repressors and, 808, 808/ Histone fold, 768
insulators and, 782 interacrions, with histone, 822-824, 822f, 82)f propagation, 820-821, 82lf
Histone methyltransferase SUV39Hl, 822, 822f HLA locus, 599 HLH (helix-loop-helix motif), 651
s a t e l l i r eD N A a n d , I t 7 - l 1 9 , t t 8 f , 1 t 9 f self-assembly. 835-8)6. 8t6f self-assemblying complexes, 820, 820f Heterogenous nuclear RNA (hnRNA), 669,669f Heterogenous ribonucleoprorein particle (hnRNP), 669,669f hflA gene murations, 174 HflA host protein, 368 hfl B gene mutations, f 74 Hfq prorein, 142 Hfr (high frequency recombinarion), 401 H)/H4,82) High-density oligonucleotide arrays (HDA), measurement of mRNAs, 94, 94l Highly repetitive DNA, t 17, I l8 him genes,486 Hinge region, of repressor, I I 0, I I I HIRA,774 Histone acetylation, 801-804, 80)f, 8O4f activation of gene expression and, 805-806,805/ epigenetic inheritance, 8j6, 8)6f HATs and, 806-807, 806f,8O7f HDACs and. 806-807, 807f replication and, 807 reversibiliry, 806 transient at replication, 805, 805/ cote. 7 59-7 60, 760f,7 67-7 69, 7 67f-769f oellnrnon ot, /)6 Hl, 760,763, 770 Hj,760,767 H4,760,767 H2 A-H28, disassembly,7 81, 7 82f H2A-H2B dimer, 7 67, 7 67f, 77 2, 77 ) H32-H42 tetramer, 7 67-7 68, 7 67f, 7 68f,772, 77 7 H) J varrant,774 interactions, with heterochromalin, 822-824, 822f,82)f methylation DNA merhylation and, 808-809 HPI binding and. 822,822[
HLH proteins
SeeHelix-loop-helix
proteins
H2 locus, 599 HMI locus telomeric silencing, 821, 82) unidirectional transposition and, 493, 494 yeast mating type and, 488489,489f I1MR locus telomeric silencing, 82I, 823 unidirectional transposition and, 49), 494 yeast mating type and, 488-489, 489f hnRNA (heterogenous nuclear RNA), 669, 669f hnRNP (heterogenous ribonucleoprotein particle), 669,669f HO endonuclease cleavage oI MAT Iocus, 4%-494, 494f production, regulation of, 494496, 49 5f transcription, 49 5-49 6, 49 5f Holliday junctions genelation, by RecA, 472 resolution, 46), 464f, 468469 by Ruv system, 472473,47)f Holoenzyme (complete enzyme\, 265 Homeobox-containing genes, 658 Homeodomain definition of, 65 I Drosophila melanogaster,658-660, 659f, 660f helical regions, 659, 659f Homeodomain-containing proteins, 660 Homologousend-joining (NHEJ).516-518, 517 Homologous recombination branch migration, 46), 463f breakage and reunion, 461, 46If,462-464, 46)f,464f hybrid DNA and, 461462,462f initiation double-strand breaks and, 464-465, 465f by endonuclease, 464 between synapsed chromosomes, 460462, 46rf,462f hop2 mutation, 469 Hotspots definition ol, I04,470 for mutations, 17-I8, l8f
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Locus control region (LCR), 789-790,790f,791 Long interspersed repeated segments SdeLINES Long patch pathway. for excision repair in mammalian cells, 505, 505/ L o n g t e r m i n a l r e p e a rr L T R ) , 5 5 5 Loss-of-function mutations, 27 LPS (lipopolysaccharide), 602 LSDI (lysine-specific demethylase), 808-809 LTR enhancer,557 U3 region,557 LTR-containing retroposons, 563 LtR-pol-LTR, 559 L T R S ,5 5 6 , 5 5 7 Luxury genes, 93 Lyases.excision repair in mammalian cells and, 504-505,505f Lymphocytes SeealsoB cells; T cells development, IgM synthesis and, 586-587 lmmalure, poot oL >/+) / ), >/+l target cell identification, 575 Lysine-specific demethylase (LSD I ), 808-809 Lysis, 150 Lysogeny balance with lytic cycle, critical stage, )7)-)74,37)f control circuit, 168 definition of.,35O, )51, )93 pathway, )68-)70, )69f repressor dimeric structure and. l6l repressors and, )65-366, 165f requirements, cII and CIII genes, )68,368f Lytic cycle, balance with lysogeny, critical stage, 17)-)74, )7)f
surveillance systems, 145, 145/ X chromosome, dosage compensation, 326, 826f MAPK (mitogen-activated protein kinase), 604
localization, 1 46-1 47, I 47f measurement of, 93-94, 94f monocistronic, I36
MARS (matdx attachment regionsl, 7)7-7)8,
nucleotide sequences coding region, ll, l4l
778f,79r
lead,er,33,74f
Maternal inheritance, 67-68, 69f Mating type switching by HO endonuclease expression regulation, 494-496,495f mother cells a.nd, 495, 495f M,4T locus cleavage.by HO endonuclease,493494,
494f
mate type switching, 493-94, 494f regulator protein coding, 490-492, 49Of, 49),f yeast mating type conversion and, 488489, 489f Matrix attachment regions (MARS). 7)7-7)8, 7)8f,791 Maturases a u l o s p l i c i n gi n t r o n s a n d . 7 16 . 7 1 7 . 7 1 7 f .7 I 8 J e n d o n u c l e a s easn d . 7 1 6 . 7 1 7 .7 1 7I . 7 l 8 f Mb (megabasepairs), 40 M C H p r o t e i n , T c e l l r e c e p t o ra n d , 5 9 7 - 5 9 9 , 5 9 8 f , 5 9 9 f MCM protein (minichromosome maintenance p r o t e i n ) . j 8 6 - ) 8 8 , ) 8 7f Mediators, 648 Megabase pairs (Mb), 40 Meiosis
crossing-over, 29, 29l extended, lampbrush chromosomes and, 741-742,742f harar^?!,d^ro
/7q
recombination events and, 468469,
468f
srages,homologous recombination during, 46t,46rf timing of events, 468, 468f
M Macromolecular assembly, synthesis, autogenous regulation oI, 327-)28, 328f Mad:Max heterodimer, 808 Maintenance methylase, 83 l-832 Maize clonal analysis, 5)8, 5)9f transposition, 5J8-540, 5i9f, 540f Major groove, of DNA double helix, 7-8, 8/ Major histocompatibility complex (MHC) classI antigens, 599 gene organization , 601, 60lf s t r u c t u r e ,6 0 1 , 6 0 U classII antigens, 600, 60I, 60I/ coding of immune system genes, 599-602, 600f,60rf definition of. 571 Mammals DHFR genes, 42, 42l DNA, mitochondria, 70, 70f excision-repair pathways, 504-505, 505f genes, exons, 43,44f genomes, ll7 Seealso Hnman genome endogenous retroviruses, 561 G-C content in, 741,74lf mitochondrial, 70-7I size of, 8l HPt , 822-82), 822f, 823f MHC regions, 600, 600/ nontranscribed spacer length, I l4 prion diseases,8)9-840, 8)9f repair systems, 5 1 5-516 replication. of mitochondrial DNA, 189. 189/ satellite DNA, hierchical repeats, I20-121,
r2rf-r23f
isolation of, 140, l40f removal, inhibition of translation and, l)9-140 poly(A)-fraction, 140 polycistronic, intercistronic regions ol, l)6, l)7f polysome and, l3?-1j4, l34J pre-nRNA processing, )4, )4f production, RNS splicing and, 669 purification, cloning approach for, 140 retroviral,552 RNA editing, 720-721, 72Of,72rf scarce,93 secondary structure changes, 332 size of, 128f 30S subunit, I79 stability protein synthesis and, I 16 sequence and, I4l-147, 142f structure and,, 141 -I 43, I 42f structure
bivalents, 29, 29f, 461 checkpoint system, 469
in
lrarler, )3,34f nucleotide triplet, 130 poly(A)+
Melting. SeeDenaturation Membranes, protein insertion mechanism of, 2)8-240, 2]9f posttranslational, leader sequences and, 240-24r, 240f Memory cells, 575, 594 Mendelian inheritance, restriction sites and, 58, 58/ Mendel's laws, 24 Meselson-Stahl experiment of 1958, 9, 9f Messenger RNA (nRNA), 127-148 abundant, 92-93 bacterial
a d i v e c e n r e r s ,1 7 7 - 1 7 8 , 1 7 8 f l'terminal stretch of A residues or poly(A)+, r)9-r40, r40f surveillance sysrems, I 44-l 45, l.45f synthesis, 73, 33f, )8, 38f, 618 3'terminal end, 670 transcription, I29, I29f in bacreria, lj5-136,135f, I)6f eukaryotic vJ prokaryotic, 610-61 I translation, I29, 1,29f,163 lj5f, 136f in bacteria, I)5-I)6, in eukaryotic cells, 135 protein-synthesizing system for, 130 l1)f by ribosomes, l)2-l)), transporr, 145-147, l45f 5' untranslated re gion, ) 40, 341f untranslated region or 5r UTR, ll6 Metallothionein gene response elements, 650-65t,650f Methanococrusj annaschii, 7 I Methionine, in protein synthesis, I 58-l 59, I 59/
life cycle of., 135-l)7 , 1)5f, l)6f protein synthesis in, l)4-1)5, I35f base pairing with IRNA, in protein synthesis
Methylases base flipping, 506, 506f
initiation, l6I-162, l6lf, I62f binding of p32, )27, )27f
Methylation capped on 5' end of mRNA, l)8-Ij9, of Cpc islands, 615
coding region, I 36 decapping, 14), l4)f 143f, 1.44f destabilizing elements, 141-l 43, l42f 3' end, generation of, 695-697 , 695f, 696f
degradation, I4)-145,
5' end,670 eukaryotic, 134, 695-696, 696f capped 5'end, 138-l)9, I38f life cycle ol. l)7-l)8. l38f poly(A)+, 179-140, 140f transcdpt modification, lj7, l37f export, RNA splicing and, 682-68), 682f,68)f functions, I28,696 l|,istor'e, 697, 697f hybridizarion with cDNA, 92-9), 92f initiation sequence, 2l I interactions, with rRNA, 180
DNA,8]I,83I/ l38f
DNA SeeDNA, methylation histone, DNA methylation and, 808-809 rRNA, snoRNA base pairs and, 699, 699f of transposons, in plants, 542 5-Methylcytosine, deamination, I8-I9, I 8/ Methyltransferases base flipping, 506 DNA.8lI, 83r/ Met-rRNAi, r 65-1 66, 165f, I 66f MFD protein, stalled RNA polymerase and, 625-626,626f MHC seeMajor histocompatibility complex Mice SeaMouse Michaelis-Menten kinetics, ribozyme catalytic readlons ano, / IJ, / rtl
Index
879
Micrococcal nuclease chromatin digestion. 759, 759f, 76t, 778-779 cleavage reactions, 77 5-77 6 reduction oI nucleosome monomer length, 7 62-763, 7 62f, 76)f transcription and, 77 8-779 MicroRNAs, )42-3 4), 34jf Microsatellites definition of, i24 replication slippage, 124-125, l25f unstability of, i24-125 Middle genes, 282,28) Min CiD, 415,4l5f Miniature interverted-repeat transposable element (MrTE). 561 Minicells, 4l l Minichromosome maintenance protein (MCM protein), )86-188, )87 f Minisatellites definition of, 100 for genetic mapping, 123-t25, t24f, t25f Minor groove, of DNA double helix, 7-8, 8/ Minus strandDNA, 554-555, 555f.7t9 Minus strong-stop DNA, 55,1-555 Mismatches ofbasepairs, l8-19 protein synthesis errors, I 56, I 56/ repair systems, 500, 501 base excision, 500, 501 directional control, 507-509, 508f, 509f nucleotide excision, 5001 501 Missense mutations definition of, 172 tRNA, 206-207 , 2O7f Missense suppressors,206-207, 207f, 2O9 MITE (miniature interverted-repeat transposable element), 561 Mitochondria chaperone activity, 244 daughter, 424425,424f division, dynamin and, 414 DNA, 68-69
number of genes in, 83,83f sequences,vs human genome sequences, 66-67, 67f innate immune response, 604 MHC regions, 600, 600/ pseudogenes, 66 satellire DNA hierarchical repeats in, 120-12), l2lf-12)f quarter-repeats in , 120-12I, l2lf
trans-actrLg s]te nrrtiiinnino 417-4lR
Mspl cleavage, 632-6)), 6j2f,63)f MIDNA, 69 MTOCs (microtubule organizing centers), 745 MUA protein, 530-5)l, 53lf MUA transposase, 5)O-5)l, 53lf MUB protein, 531 Mud2 protein, 677 mudrA gene, 541 mudrB gene, 541 muk gene mutarions, interruption of segregation, 4t8, 418f Multicopy inhibition, 5)7 -5) 8, 5J7f Multimeric proteins, 109-l 10, I l0/ Multiple alleles, 27 -28, 28f Multiple metal response element (MRE), 650-651 Mutagens acridines SeeAcridines induced mutations and, 14 point mutations and, l6 Mutants constitutive, 108 uninducible, 108 Mutations
mod(mdg4) gene, 7 84-7 85, 7 85f Monocistronic mRNAs, I l6 Monster particles, 7f 4 MotA phage-encoded product, 355 Mouse aj3-globulin gene, 109 centromeres, satellite DNA in, I I 9, I l9/ DNA, ll8, tt8/ Gapdh gene, 66 genome divergence of repeated sequences.rate o{ neutral substiturion from, IO7, IOTf
880
Index
n{
| 7
in prorein, 16, 16l regulator gene identification and, )09,3O9f t r u e r e v e r s i o no t , l 6 - 1 7 . l 7 J unfavorable,459 Mutator phenotype, 507 Mutator transposon, 54I mut gene, activities, 507-5O8, 508f MUISL system, 509,509f Mymplasma capricolum, genetic code changes, I 97 Myc proteins, 661, 662 Myoglobin, in globin superfamily, 51, 5 U
N NAD (nicotinamide adenine dinucleotide), 172 Nascent protein definition of, I 33 ribosome and, 179 translocation into ER, 2J2-233, 2Bf Nascent RNA, I 36 Natural selection, 89, 91-92, 459 Negative complementation, 310, )IOf Negative regulation, /ac gene control and,,305,705f Neurospora trassa, q,tl8 mutant, 709 Neutral sites, rate of substitution, lO7, l07f Neutral substitutions, 27
background level, l4 in bacterial division or segregation, cell shape and 4t24r),4rJf
NFI binding, nucleosome and, 8O2,802f NF-KB factor, 652 NF-xB-like pathway, 601 N-formyl-merhionyl-IRNA (rRNArMet), t58-ti9, NHEJ (homologous end-joining), 516-518, 517f NHEJ (nonhomologous end-joining), 586 Nicking
changes, neutral, 104 cts- acrrng, pailitioning, 41 74\ 8 in cls-actingsire, 108-109. 108/
replication, 424425, 424f segregation, 424425, 424f TIM complexes, 243, 244, 244f TOM complex, 243-244, 24)f Mitogen-activated protein kinase (MApK), 604 MMTV promoter, 802
spontaneous, 14, 567
advantageous, 104 back, I 6-17
c a u s i n gd e a f e c t s ,9 1 , 9 l l
somatic variation, 67, 68f origins, D loops and, 388-389, )89f protein leaders, 240 protein synthesis, 240
second-site reversion of, l7 silent,27,27f
mRNA
circular,69
distribution of genes in, 69-7O,70f stze of., 69
in replacement sites, 105 in samegene,25-26,26f
in silent sites, 105
SeeMessenger RNA MSH repair system, 5O9, 592
6l)-6]14, 614f
rates, reduction by repair systems, l8-19 recessive,gene function and, 25, 25f
somatic mutations, immune diversity and, 590-59r,59rf MRE (multiple metal response element). 650-651
human, 70, 70/ variable organiza tion of, 7O-7 l, 7 0f, 7 If evolution by endosymbiosis, 72-7j, 7 2f genetic code changes, 198, 198/ genomes, 49
point, l5 in promoter characterization, in pseudogenes, 108, 108/
t59f
i n c o n s e n s u ss e q u e n c e s 7 , l0-7 l\ in control sites, .rs-acting, 15, l5l deleterious, 104, 108, 108/
of DNA supercoils, 736 in immune recombination,
deletions, l6 in different genes, 26, 26f DNA sequence changes and, l4 effects of, 26, 27f
of nonreplicative transposition, 5)), 5)Jf, 534 of transposition, 570, 510f nucleosomal DNA, 763-764, 764f with phage A protein, conversion of double-strand
evolurion and, 89 exon, j9, 4) favorable,459 lorward, t6-17 frameshift, 31, 31/ gain-of-function,27 hotspots, 17-t9, t8f, t9f induced, l4 insertions, l6 inlron, )9,4j leaky,26 linear arrangement ol, 24 loss-of-function, 27 nonlethal, causing lethality in combination, 9r-92,9rf nonsense oellnllton ot, I /z nRNA degradation and, 144-145 |RNA, 206-207, 206f
584, 585f
initiation
to single-strand DNA, 4)6-4i7, B6f resolution of Holliday junction. 46), 464f retrotransposition oI non-lTR elements nad, ,66,566f in T-DNA transfe\ 405406,405f Nick translation, 4)2, 4)2f Nicotinamide adenine dinucleotide (NAD), I72 Nijmegan breakage syndrome, 5 17-5 | I Nitrogenous bases,in nucleic acids, 6 Nitrogen starvation, Escherichiacoli, 279 N nucleotides, 584-585, 585f Nonallelic copies, t02 Nonallelic genes, exons in. 1 l6 Nonautonomous controlling elements, 540-542, 54lf Nonhistone proteins, {unctions, 759 Nonhomologous end-joining (NHEJ), 586 Nonhomologous recombination, replication-def ective viruses and, 558, 558f Non-Mendalian inheritance, maternal, 67-68, 69f
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of phage genomes, rolling circles a\d,, )87f, )97-)98 phage T4, 445448,447f restarting, primosome for, 45 145), 452f, 45)f semidiscontinuous, 442 single-strand DNA, 415437, 436f speed, 381 unidirectional, 37 8-) 80, )7 8f , 37 9f ys cell cycle condition, 177 Replication-defective transforming viruses, 558, 558/ Replication errors definition of, 502, 502f repair systems, 5ll-5I), Replication eyes
5l2f,513f
bidirectional vs unidirectional, )7 8-)7 9, 37 8f in circularreplicon,j79, )791 definition of, )7 8, 37 8f measurement of replicon size and, 18l, 38lf Replication forks foci formation, 383-)84, )84f generation, of hemimethylated sequences, 5J8 initiation, 378, )78f movemenl detection of, 379-380, j79f, )8Of estimation of, l8l stalled disassembly,tus-ter complex and, 45245),451f from DNA damage, 451452, 452f reactivation. 45245), 45)f repair by recombination, 452, 4r2f replication-repair pathways for, 5 I 1-5 I l, 5r2f,517f ler elements and, 45245), 453f Replication forms, creation, at origin, 44845O, 448f.449f Replication slippage, | 2a-125, 125f Replicative transposition cointegrates and, 531-133, 532f definition of, 527, 527f TnA family, 5)4-i)6, 5jrf Replicons, 37 6-)91 Seealso specificreplicons circdar, 179, )79f definition of, 177 on eukaryotic chromosomes, 381-384, 387f, )84f extrachromosom al, ) 9 2407
delinition of, 463, 5)2 Holliday junction, 463, 464f Resolution site (/'eJsite), 535
blockage of lytic cycle, )65,365f competition with low-affinity sites, )\7-)t9, )t9f loop formation, )15-)16, jl,5f, jl6f rate of dissociation from, I I 4 release, inducer binding and, )14-j15, RNA polymerase binding and, 3 I 6
)I5f
simultaneous at two separate sites, )15-)t6, )t5f cleavage lytic cycle induction and,36l-32, )6lf RecA protein activation and, 5 14 conformation, DNA-binding and,,)12, )12f control of lac gene, 704-)05, )05f by small-molecular inducer, 306-308, )07f defective, constitutive expression and, 309, 309f definition of, 103 dimers cooperative binding with operator, 164, )65f cooperative inter acrions, )67, ) 67f DNA-binding, lll
binding of repressor to more than one operator and, 1t5-116
half-turn, )62,362f high-affinity, jl6-)17 low-affinity, )16-)17 )llf
N-terminal, )6I,36lf histone deacetylasesand, 808, 808/ homeodomain proteins, 660
in Drosophila melanogaster,561-562, 56lf LTR-containing, 563 in plants, 561
IPTG complex binding, I t 5 isolation, 3 I l
reverse transcriphses, 56j, 56jf T! elements in yeast, 559-561, 559f- 56lf
headpiece, )10, )12
/ac operon, )07-)08, monomers
budding, 551,553f copy choice recombination,
structure of, ll l, 3l l/ lllf
N-terminal domain, )61-)64, 164f
)65-)66,366f paftitioning on DNA, ll7 phage lambda lytic cascade and, 159-360 positive control mutations, 365-166, 366f as positive regulator, )65-)66, j6rf proteins, 648-649, 649f
direct model of, )14415,315f equilibrium model ol, ]14, )l5f
tetramenc core dimers in, )11,31lf, )I2f monomers in, ll l, lI U mutations, )I1, jllf
b i n d i n g t o o p e r a t o r ,S l S - 3 1 6 , ) 1 3 f , ) 1 4 f
556, 556f
definition of, I I DNA, 554, 554l J enos,))/ ) enos,))/ r n t e g r a n o n ,> ) t . ) ) / J integration into chromosome, 556-557, 557f gag gene, T! elements and, 559, 5591 gag terminariort codon, 552-553 genes, coding lor polyproteins, 552-553, r53f mRNA, 552 as plus strand viruses, 554 pol gene, Q elements and, 559,559f po1reading frame, 552-55)
recognition helix, 659 release
definition of, 106
autogenous control (autogenic circuit), 319-720, )20f, )65-)66, )65f
(RFLP)
Retrotranslocation. SedReverse translocation Retrotransposons. SeeRetroposons Retroviruses, 55O-568
)07f
domains of, 3 t0-3 t l, 3 l0/
promoter-binding
40f
Retroposons,550-568 classes.562-564, 5 62f-5 64f definition of, 55 I
in ligand absence,steroid receptors and, 658, 658/ methylated CpG doublets and, 615 polycomb group proteins and,824-826, 825f repressor-operator binding and, j 17-j I 8, 3 | 8f
Repressors
s t e t o l o ,n a l r s l l e s ,o ) / , o ) / _ r rdssite (resolution site), 515 Restriction endonucleases, DNA cleavage, )940, Restriction enzymes, at methylated sites,
Retinoic acid, 614, 654f
C-terminal, )61, )6lf DNA-binding, j12,3l2f
synthesis, )68, )68f c,', gene prevention of, l7 I lysogeny pathway and, )68-)70,
o I t r a n s c r i p l i o n ,b y h i s t o n e d e a c e t y l a t i o n . 808. 808/
specificity of, 658, 658f by steroid receptors, 657-658, 657f,658f recognition, by activators, 649-65 l, 650f
construction, 56-57 definition of, 40, 40/ tandem gene clusters, I l2-1 Il, I I3l Restriction site polymorphisms, 58, 58/ Reticuloendothelial system, 220
DNA-binding sites
octamer formation, )67-)68, )67f at OR2, interaction with RNA polyfll€rose at Pnu,
R e p l i s o m e ,4 2 9 , 4 4 7 Repression
recogniton
Restriction maps CDNA vs genomic DNA, 40, 4tl
5' end and, 393-394,194f vs circular replicons. 178-179. )781 origins. 377
rolling circles and, )96-)97 , )96f, 197f size, )8), )8)f Iermlnus, J / /
on metallothionein gene, 650, 650/ parrnoromrc, 6) / , o) /J
specificity, helix-turn-helix motifs and, )6)-364, )6jf, )64f
mutations in, )12-)l), domains, j6l, 36lf
656f
definition oI, 641
equilibrium equation and, )17, 317f form,36l-362, )6If helix-turn-helix motil a\d, )62, j62f
mutations, )09, )12-)\1,
regional controls, 184
Response elements binding, activation by Iigand binding,656,
632-6)),672f Restriction fragment length polymorphism delinition of, 58, 58/ diseasegenes and , 58-59. 59f 59f for genetic mapping,58-60,
in homologous recombination, 512 Iinear
autoradiography of, 380, 180/ electrophoresis ol, j7 9-3 80, 37 9f, )80f plasmid incompatibility and, 421, 42lf
Resolvases definition of, 527, 532 Holliday junctions a\d, 47 )47 4, 47 4f in TnA transposition, 534-5)6, 5)5f
)69f
as repression requirement, I I 0 translational, )24, j24f Rereplication, eukaryotic, licensing factor control of, )85-386, 786f, 787f Resolution
repf ication-defective transforming, 558, 558f reproductive cycle, 551-552, 551f, 552f retroposons, reverse transcriplases, 563, 563f RNA, 554, 554l RNA-dependent transposition and, 55 I RNA genomes, 555-556 transduction, of cellular sequences, 558-559, 558f translation, 2 I l Retrovoiruses, in human genome, 56)-564, Reverse transcriptases copy choice recombination, 556, ,56f
564f
definition ol, 552,554
breaking and rejoining bonds in, 536
Index
885
group II intron transposition reaction and, 7 16-7 r7, 7 I6f
bacterial, protein synthesis initiation in, 157-I58,
in retroposons, 56), 56)f RNAase H activity, 554-555
binding of elongation factors, 170-172, 171f regulator blockage ot, )24, )24f codon-anticodon base pairing and, l9l
telomerase, 7 5 l-7 52, 7 5 lf Reverse transcription, I0-I2, I l/ priming, in LINE elements, 566-567, 566f processedpseudogenes and, 565, 565f Reverse translocation, 2)4-235, 234f
1571,158f
substrate-binding site and, 7 I I-7 I 3, 7 | | f-7 llf change, with substrate binding, 712,
control, of RNA seacondary structrre, 337, ))7f, J)8 eukaryotic, scanning from 5' end, 163-164, l67f free, protein synthesis in, 222,222f
RF2, t7) RF3, l7l
functions, 177, 179-180, lSOf interaction wirh IRNA, I 521-155,154f, 155f
RF-C,445 RF gene mutations, I74 RFLP SeeRestriction fragment length polymorphism RFs SeeReleasefactors
Iarge subunit peptide bond formation in, 154-155,
definition of, 287 function, 288-291, 289f, 29Of. 695 gene mutations, 29O-29 | polariry, 289-290 rut srte, 288-289, 289f transcription termination, 287, 288-29I, 28ef,2e0[ Ribonuclease E, in bacterial mRNA degradation, l4t R i b o n u c l e a s eP ( R N A a s eP ] } , 7 0 7 , 7 1 8 Ribonucleases (RNAases). 10 Ribonucleic acid SeeRNA
L-tg,7t2,712f m u t a t i o n s , p l a s m i d i n c o m p a t i b i l i t ya n d , 42)424,421f
movement
nucleotide sequences
bypassing and, 214-215, 214f translational control ar'd,, )24-)25,
crystal structure, 2l I l' end of, I 80 in protein synthesis, 179-182,180,
l8t/ translation accuracy and, 2 I 0-2 I I 185,153 2lS, peptidyl transferase activiry, 182-183, l8t 285, I 5l size of, l28f structural conf ormation, I 78 structure, 17 5-177, 176f tandem repeats, Il2-113, lllf Ribosome active centers, 177-179, 178f, 179f aminoacyl-IRNA selection, for A site insertion, 210,210f
886
Index
324f
mRNA translation, 1 32-1 33, | 33f mutations, I5l organelle, I 5l position, tryptophan control of, )37-338, proteins or r-proteins, I 5l protein synthesis, 152-15), l53f rRNAand,179-180,180/
structural conformation, and, I8l subunits
conformational changes in protein synthesis, l8l, l8 r/
functions of, 128, I28f G-C rich region, 694 guide, 7 22-7 24, 7 2)f, 7 24f
5 . 8 SR N A , 1 7 5 l a r g e s u D u n l t s ,t 5 t , t J t J l i f e c y c l eo I , l ) 4 , 1 ) 4 f
regulation, )25-)26, )25f, )26f Ribosomal recycling factor (RRF), 174, 175f Ribosomal RNA (rRNA)
repeated genes, I 1.1-l I 5 in ribosomal subunits, 175-177, 176f r-protein autogenous control and, J26, )26f l6s
double-stranded, degradation of mRNA, 143-345, 144f,74rf l' end generation, 694, 694f,70O
hairpin structure, attenuation, ))), J))f I, replication of ColEI, 422424, 422f, 423f
sedimentation rarc oI, lj2-l)j sites for binding charged IRNA
interactions, 17 6-177, I77 f codon-anticodon pairing and, l8l-182, l8f/ with mRNA, 180 wirh IRNA, 180 length, 778 processing, small RNAS lor, 699-7 0O, 699f, TOOf production, cleavage events for, 697-699, 698f
7r2f definition of, l
r54f,r55f 55RNA,175
Ribonucleoprotein. l)), IJ3f Ribonucleoprotein enzyme, telomere synthesis, 75O-752,75tf Ribonucleoprotein particles, l5l, l5lf Ribosomal proteins (r-proteins), synthesis, autogenous
alternative genes, 49 base pairing with mRNA, in protein synthesis initiation, l6l-162, l6lf, l62f deiinition of, l4 functions, 128, 129, 180, 180/ genes, I l2
and, l2-l), llf catalytic activity Seea/so Ribozymes guanosine-binding site and, 7 l1-7 lj, 7 llf-7 l3f RNAase P and, 7I8 conformational
RFl, l7l
Rho-dependent terminators, 287, 288, 290, 29Of, 29 4 Rho factor
base paring, duplex formation
exons, 38, 38/ introns, 18, 38/ processlng CTD in,624-625,625f
))7f
P site, I 54, I 54l
early heavy chain expression and, 586-587, 587f in protein synthesis, 128-129, l28f reassociation, kinetic analysis of, 92-9), 92f replication, central dogma and, tO-l l, I l/ retroviral, 554, 554f reverse transcription, l0-12, I l/ 55, t75, t79,698 5 . 8 5 ,I 7 5
A sfte, 154, l54f
455,698
structural analysis, I 53
satellite. SeeVirusoids subunit complex formation
connections between, 17 6-17 7, 177f initiation factors and, I 57-I 58, 157f, l58f peptide bond formadon in, 154-155, 154f, l5rf, t75 protein synthesis initiadon in, 157-158, r57f, t58f rRNA in, 175-177, 176f )os,176, t76f 50s, t76, t76f 7 O S ,1 76 - 1 7 7 , 1 76 f , t 7 8 f size of, 152-15), l5)f small, 133, I33/ translation, initiation site and, 1,36-l)7 translation accuracy and, 209-211, 21Of translocation activation of, 172 hybrid state modeI, 169-17O, 170f,'178 Ribosome-bindin g siIe, | 57, l 17f Ribosome stalling, ll6 Riboswitch, )40, 341f, 7 | 4-7 I 5, 7 | 4f Ribozymes, 707-726 catalytic reactions, 7 ll-7 15, 7 llf-7 l4f Michaelis-Menten kinetics and, 7 13, 7 Bf riboswitch, 7 l4-7 | 5, 7 l4f definition of, 707 hammerhead Mg2+ initiation of catalysis, 719-720,720f self-clevage,7 19, 7 l9f Rickettsia,T2 RlD.4 (regulatory inactivation of DnaA), 382 Rifampcin, blockage of transcription by bacterial RNA polymerase, 266 Ri plasmids, 402 RISC (RNA-induced silencing complexl, 3M RNA
sequence changes, 105 7SL,564-565 small nucleolar, 699-700, 699f spliced leader, in t/afls-spicing reaction, 689-690,689f structure, at attenuator, ))7, 337f sugar in, 6 synthesis, 258. Seealso Tlanscription RNA polymerases and, l0 in transcription brbble, 259-260, 259f, 26Of transcribed from pseudogene, 109 translation, central dogma and, l0-I l, I I/ types of. SedMessenger RNA; Ribosomal RNA; Tfansfer RNA U run, 694 in viral capsid, 731-7)2,7j2f viroid, l9-2O,20f in viruses, 5 RNAase E, degradation of bacteria mRNA, 141, 14lf RNAase P (ribonuclease P), 707,718 RNAases (ribonucleases), l0 RNA-coding genes, in mitochondria, 70,70f RNA editing definition oI, 707 guide RNA and, 722-724, 72)f,724f at individual bases,7 2O-7 21, 7 20f, 7 2lf in trlpanosome mitochondria, 7 2l-7 22, 7 22f RNAi. SeeRNA interferenc€ RNA-inducing silencing complex (RISC), 824 RNA interference (RNAi) definition of, 143 generadon of siRNA, 344, 344f RNA silencing and, 345 systematic analysis of gene expression, 344 RNA ligase, 690, 7 13, 7l3f RNA polymerase activation of repair, 625-627, 626f active site, 263-264, 264f
antitermination protein and, 29I-292, 29lf arrested, restarting of , 269 -27 0 attenuation, 337, ))7f Bacillussubtilis,282 bacterial coding strands, 266, 266f
size, vs nucleosome size,78,779f specificity, control of, 267,267f in sporulating cells, 284 stalled, MFD protein and, 625-626, 626f structure, 262-265, 262f-265f, )37 , )J7 f
structure of , 262-26), 262f
with TFIB, 622-623, 62)f T3 phage,261-262,262f
subunits, 265-266, 266f
T7 phage,26I-262,262f
template slfiands, 266, 266f
transcription, 642, 64)f
binding, 323 to ancillary factors, 292-294, 293f, 294f DNA. 270, 27Of, 275-277, 275f,276f to PREpromoter, )68, )68f wirh promoter, repressor-operator binding and,
3r6 bond breaking and making, 264,264f classes,6l I conf ormation, 264-265, 265f contact, on one face of DNA, 275-277, 275f, 276f corc enzyme,267 degradation deficiency, 62 7 DNA binding, 27O,270f DNA binding contact points, 275-277, 27 5f, 276f DNA transcription, in vitro, 81,0 eukaryotic. subunits, 612-613, 6llf function, 10, 258 genes, J25, )25f helix-2 interaction, ) 65 -3 66, ) 66f holoenzyme complex, 648, 648f I,6tl as bipartite promoter, 614-615, 6lrf promoters, 6l I transacription termination, 694-695, 694f il,6rt,64) indirect trigger for termination, cleavage as,695
bubble formation, 259-260, 259f, 26of catalysis,260-261, 26If of delayed early genes,292 of immediate early genes, 292 of lambda gene, 292-29), 293f transcription units and, 292 yeasI,26),263f RNA regulator, control, of plasmid ColE I compatibility system, 422-424, 422f, 42)f RNA silencing, RNA interference and, )45 RNA splicing A complex formation, 677-679,679f alternative reactions in, 46-47, 46f, 47f splice junction differential use, 685-688, 686f,687f
abortive initiation, 268, 268f open complex, 268,268f phases,274-275,275f
777 , 777f rpos gene,342 r-proteins, I 5l RRF (ribosomal recycling factor), 174, 175f IRNA SeeRibosomal RNA RseA protein, sEfactor and, 279,279f R segments, 554, 554f R-UR 554 rut sile,288-289,289f RuvA,47)474 RuvAB complex, 47347 4, 47 4f RtrvB,473474 Ruv system, Holliday junction resolution,
RyhB, 341
C2 complex formation, 679f dJ-splicing reactions, 688, 688/
S
control, 670 definition of, 34,34f,38, 669 E complex conversion to A complex, 677-679 E complex formaion, 677, 678f, 679f exon definition process, 67 8-679, 67 8f
as intermolecular reaction, 688 junctions, 67 l-67), 672f
694-695, 69 4f
viroids and, 7 l8-7 19 virusoids and, 7 l8-7 19 Rom protein, 421 Rotational positioning, of DNA on nucleosome,
Cl complex formation, 679f
resilience to rearrangement, 647 startpoint, 618-619, 619f transcription, 624 in vllrd transcription, 781
transacription termination, r i r r r i n oi n i i i r r i ^ n
396-)97, 796f of T-DNA, 405406, 405f products, 387f, )96-)97, )96f replication of phage genomes, )87f, 397-798
472473,473f RXR receptor (9-crs-retinoicacid receptor), 657-658,657f
intron definition Vocess, 67 8-67 9, 67 8f exon junction complex, 682, 682f
downstream and upstream promoters for, 6t5-6r6,6t6f
of single-stranded multimers, 387,
apparatus, 674-675 Bl complex formation, 679,679f 82 complex formation, 67 9f
initiation complex assembly, 621 methylation at promoters, 612 promoters, 61,1,8O2
III, 6TI
RodA,4I2 Rolling circles generation
initiation, by Ul snRNA, 676-678,677f,678f
lariat {ormation, 680, 68)-684, location, 670,67Of
684f
mRNA export and,, 682-683, 682f,68)f nuclear, vs group II, 685,685f pathway, 672,672f pre-mRNA lariat formation, 673-674, 67)f sIages,67)-674,67)f sites,670-67 |, 67 lf
Sarcharomycescerevisiae chromatin remodeling complexes, 800, 800/ DNA centromeric, 746 short conserved sequences in, 7 47 , 7 47f genes cse4,824 exons,43,44f expressed, measurement of, 94, 94l repair,5l5 surveillance systems, I 44- I 45 genome, 79-80,79f mitochondrial, 69 nucleosome positioning, 777 size, family size and, 8I, 8U snoRNAs, 699 mating type, 488489, 489f meiosis, timing of events in, 468, 468f mitochondria
DNA,70-7r,7u
sigma factor release, 268-269, 268f size change, 268-269, 269f
SnRNAS pairing reactions, 680-681, 680f, 68lf spliceosome form aion, 67 4-67 6, 67 5f
MSA repair system, 509
ternary complex, 268, 268f
spliceosome SeeSpliceosome
musSl mutanat'474
tight binding, 268
-..ll^:..^ JP'rUrrE
initiation of transcription, 6 I0 loose binding site, 267
^--^-^+,,dupordrur
alternative, 681-682 snRNA components, 680
method for seeking binding sites, 27 0-27 l, 27 \f nucleosome displacement during transcription. 779-78t,780f positioning factors, 61 9-620, 620f
stages,676 systems, types ol, 670
p r o m o t e r b i n d i n g, r a t e o l , 2 7 0 - 2 7 l . 2 7 0 f promoter recognition, consensus sequences and,
trans-splicingreactions, 688-690, 688/
tissue specificity, absence of, 672 transesterilication, 67 4, 67 4f tRNA
272-274,274f reading into origin, 449450
cleavage stage,692, 692f endonuclease, 69 I-692, 69 lf, 692f
replication at 5'end and, )94, )94f sigma factor. 154 control of DNA binding, 27 l-272, 272f rcIease,268-269, 268f
ligation stage, 692, 692f unfolded protein response and, 69), 693f in yeast, 690, 690f,69lf
specificity control and, 267, 267f substition, promoter recognition and, 278,278f
evolution of, 691-692, 69lf
U6-U4 pairing, 680, 680f RNA splicing and processing, 669-7Ol RNA viruses, recombination, 459
genome, 69
replication origins, isolation of, 784-)85, RNA polymerase II, 612, 6l3f
)85f
RNA splicing, 677 IRNA splicing, 690, 690f,691f SAGA, 807 SAGE (serial analysis of gene expression), 94 sarellire DNA anthropod, short identical repeats, I l9-120, I l9l definition of, 100 on density gradient, I 18, t 18/ heterochromatin and, I l7-I I9, 118f, 1l9f mammalian, hierchical repeats, 120-12), 12lf-lzjf in mouse centromeres, 119, ll9f sequencing, I20 Satellite RNAS Seevirusoids Satellite tobacco necrosis virus (STNV), l2 Schizosacharomyces p ombe cir genes, S\2
Index
887
DNA, centromeric. 746 genomes, 79-80.79f
promoter recognition, consensus sequences and, recognition of heat shock gene promoters, 650 rercase, z / 2 of RNA polymerase, 267, 267f
Scyrps, 675
s Ef a c t o r ,2 7 9 , 2 7 9 f s7ofactor, 278, 280-281, 28lf
Secondary attachment sites. 483 Secondary immune response. 575, 594 Second-site reversion, of mutation, l7 Sec system, 247-249, 247f, 248f Sector,5lS-519 Sec6I translocons, 2))-234, 234f, 239 S e c Yc o m p l e x , 2 3 2 S E D Sf a m i l y , 4 1 2 , 4 1 4
sF factor, 284-285, 285f sG factor. 284-285, 285f
definition of, 675
structure
functions, 684 generation of l'end of histone nRNA,697,697f in RNA splicing pairing reactions, 680-681, 680f, 681f
conserved regions, 280, 280/ helix motif, 280-281 , 281[ substition, promoter recognition and, 278, 27 8f Signal ends, 582,582f Signal peptidase, 229 Signal recognition particle (SRP)
spliceosome f ormatio\,
reverse cyclization, 708, 7 l0f Semiconservative replicarion, of DNA. 8-9, 9/ Semidiscontinuous replicarion, 4)44)5, 4)5 f Sense strand (coding strand), 129, l29f Septal ring, 4I4 Septum formation (septarion) bacterial, FtsZ and. 4l)414,4t4f in bacterial daughter cell division, 411412,
41lf,4r2f FtsZ and, 4l?414,414f location, min gene and, 415,415f pedseptal annulus and, 4114I2, 41Lf Serial analysis oI gene expression (SAGE), 94 Severe combined immunodefiicency (SCID) mutation, 586 Sex steroid hormones, as activators, 654l SFI splicing factor, 677 SF2 splicing {actor, 686 SGA (synthetic genetic array analysj,sl,91, 91f Shine-Dalgarno sequence. 161-162, l6If, t80, )24 Short interspersed repeated segments (SINES), 56)-564,564f Short patch pathway, for excision repair in mammalian cells, 505, 505/ S h o r t s e q u e n c ee L e m e n r sa. c t i v a t o rb i n d i n g . 627-628,627f Sigma factors Bacillussubtilis.282 cascades, 282-28) configuration, at open complex formation, 280,28rf control of DNA binding. 27 l-27 2, 27 2l of sporulation, 28)-286, 284f-286f core enzyme, 28O,282f DNA contact, 280-282, 281f, 282f Escherichia rcli, 27 8-280, 279f lytic cascadeand, 354 phage developmeil and, 282-28j, 283f
888
Index
base-paired structure, 67 5-67 6, 67 6f initiation of splicing, 676-678, 677f,678f mutations, 676-677, 677f Small nucleolar RNAS (snoRNAS), 699-7OO, 699f SMC proteins (structural maintenance of
Alu domain. 2)0,2)0f
conformation, signal binding and, 230, 230f f u n c t i o n s ,2 2 8 , 2 ) 0
708f-7rof
binding with signal sequence. 228-229,229f components, 564-565
chromosome), 828,829f Sm proteins, 676 SMRT corepressoa 658, 6581 808 S N P s e eS i n g l e n u c l e o t i d e p o l y m o r p h i s m
S domain, 2)0,2)0f SRP receptor interaclion, 2)0-231, 23lf structure, 229-210, 230f Signal recognition particle receptor (SRP receptor), 230
snRNAs SaeSmall nuclear RNAs snRPs, 680 Snurps,675 Solo ds, 559-560
Signals, for protein synthesis, in free ribosomes, 222,222f
Somatic mutations
Signal sequences anchor sequences as, 2)7 of bovine growth hormone , 228, 228f for protein translocation initiation, 227-228, 227f,228f interaction with SRP,228-229, 229f Silencers, repression of silent cassettes, 492493, Silencing definition of, 802-801 telomeric, 821 Silent cassettes aI HML locus, repression oI, 49249), at HMR locus, repression of., 492493, for yeast mating, 488489, 489f
67 4-67 6, 67 5f
UI
418f mitochondrial, 424425, 424f S e l e n o - c y s - t R N A .1 9 9 , l 9 9 f Self-assembly,oI protein, 22), 224
intermolecular reaction, 709 p r i m a r y c y c l i T a l i o n .7 0 8 . 7 l 0 f
2rj-2r4,2t3f Slow-stop mutants, 4294)0 SL RNA (spliced leader RNA). rrars-rpicing reaction. 689-690,689f Small cytoplasmic RNAs (scRNAs), 675 Small nuclear RNAs (snRNAs)
bacterial counterpart, 2)0-2i I
group I introns, transesterification in, 707-709,
Slippery sequences,frameshift mutations and,
st4, 281-282
Segmental duplications, in human genome, 85-86,85f Segregation chromosomal, panitioning process and, 417-419,
Self-cleavage,of viroids and virusoids, 719,7 l9f Selfish DNA, 62 Self-splicing (autosplicing)
chromosome segregation
and, 415417,41.6f
SCID mutation (severe combined immunodefiicency mutation), 586 Scrapie, 20, 819 scRNAs (small cytoplasmic RNAs), 675 SecA,247-249, 248f SecB, 247-248, 248f Sec61complex,2)2
Site-specific recombination,
279-280,280f
immune diversity and, 590-591, 591f induced, by cytidine deaminase and uracil glycosylase. 591 -59 ), 5921 UNGand,590 Somatic recombination allelic exclusion and, 581 493f
of immunoglobulirs, 57 6-57 7 Somatic segregation, 67 S O Sb o x , 5 1 4 S O S r e s p o n s e ,5 l l Spacers,6I5
49)f 49)f
Silent mutations, 27 Silent sites definition of, 10,1-105 d i v e r g e n c eo f . | 0 5 - l 0 6 . I 0 5 / mutations in, I05 Simple sequence DNA (sarellireDNA). I l7 SINES (short interspersed repeated segments), 56)-564,564f S1Ngene, 495 Single copy replication control, 177 Single nucleotide polymorphism (SNP) definition of, 57 frequency per genome, 57-58, 59 for genetic mapping, 58-60 Single-strand binding prorein (sSB), 436-418, 4)6f,438f Single-strand exchange, 5 i0-5 1 l, 5 10/ Single-strand invasion, 464 Single-strand passagereaction, 480 Single-strand uptake (single-strand assimiliarion) catalyzed by strand-transfer proteins. 47t47),472f definition o{, 47 I Single X hypothesis, 827, 827f siRNA (short interfering RNA). )43, )44, j44f sIR proreins. 492,49), 49)f S[]/Sir4,823
Sparsomycin, inhibition Speciesdifferences
of peptidyl transferase, I 78
exons and, 42-4), 4)f in gene number, 78, 78l in gene organizatron, 5l-52, 52f in genome sequences,66-67, 67f i n g e n o m e s i z e ,6 0 - 6 1 , 6 0 f , 6 l f , 7 8 , 7 8 f in globin gene clusters, 1 1 l-1 I 2 introns, 4l in proteins, 104 S p h a s e ,3 8 3 , 4 9 J , 7 7 4 Spliced leader RNA(SL RNA), /rdnr-spicing reaction, 689-690,689f Splice junctions, in alternative RNA splicing, 685-688, 686f,687f Spliceosome components,68l o e n n r n o no t , 6 / t , 6 / ) J formation, 5 snRNPs and, 679-681, 679f- 68lf REF proteins binding to splicing junctions and, 682-68),683f U I 2 -type, 681-682 Splice recombinant DNA, 461 Splic€ sites )" 672 cvtting, 67jf,674 recogntllon oI/ 6 /+b / > 5" 672 binding to Ul snRNP. 674 cutlrng, 673-674,673f
mutations, 676-677, 677f recognition of, 67 4-67 5 branch, recognition of, 674-675 crypl\c, 67 4 definition oI, 67 0-67 l, 67 lf generic,672 recognition, 673 Spm element, gene expression and,, 542-544, 543f SpoIIIE protein, 419 Spontaneous mutarions definition of, 14 horspors, t7-).9, t8f, 19f frommodifiedbases, l8-19, I8l l9l SpoI 1 protein, generation oi double-strand breaks, 467468,467f Spore formation, in Ascomycete tetads, 47 5, 47 5f Sporulation cascade, 28)-284,
284f
control, by sigma factors, 283-286,284f-286f forespore, 284-286, 284f-286f mother cell, 284-286, 284f-286f Sporulation genes, 41 841 9 sr2 protein, 278
in nucleosome, 7 66-7 67 , 7 66f phage genome replication and, 198 positive, 477 structure and, 476478, 476f,477f torsional tension, 716 in transcription, 27 7-27 8, 27 7f Superfamily, 51, 574 Suppression definition of, l7 nonsense, readthrough and, 208-209, 2O8f Suppressor definition of, I 7 f r a m e s h i f t ,) 1 , 2 ) . ) - 2 1 4 , 2 l l f IRNAS, 206-207, 206f competition with wild-type IRNA, 208-209, 208/ missense, 206-207, 2O7f mutations, 209 nonsense, 20 6-208, 20 6f, 207f sup35 proreins, 816-8)8, 8)7f Su(HW) proteins, 784-785, 785f S u ( v a r ) p r o t e i n s ,S l 2 Surrogate light chain (SL chain), 595 Surveillance systems, 144-145, l45f
SRB loci, 648 SRE (serum response element), 649 S regions, 588, 588, 600
) u l v l v a f , t e l o m e r e sa n o , / > 2 - / > J . / 1 2 1 , / ) t J
sRNAS
Sv40 enhancer, 6)0, 63Of
E coli,34l-)42,342f
suvl9Hl,
808,822,822f
SV4O DNA, 77I
TATA box, 619, 621, 621f,623, 628, 628f TATA element, 618 TATA-lesspromoters, 6I9 tat protein, 644, 644f TBP (TATA-binding prorein), 615, 619-621, 62Of, 62lf T cell receptor (TCR) ab, 57 6, 595-596, 596f, 598 antibodies on, 598 cD3 proteins and, 598-599, 599f definition of, 57),574 development, 598,598f gd.,576, 595-596,596f gene, organizatio\ oI, 596-597 , 596f, 597f MCH protein and, 597-599, 598f, 599f proteins, 596 T cells (T lymphocytes) clonal expansion, 574-575, 574f cyloloxic,57l definition of, 572 development, 598,598f helper,572 killer or cytotoxic, 571 TCR SeeTcellreceptor T.DNA Acrobacterium tumefaciens,402406, 4OJf4O5f genes, crown gall disease and, 402405, 40)f, 404[ ldansfer, 405406, 405f Telomerase, in telomere synthesis, 7 50-7 52, 7 5 ll
SRP SeeSignal recognition particle SRP receptor (signal recognition particle
SV40 minichromosomes DNA supercoiling, 7 66-7 667 , 766f n u c f e o s o m eg a p . / 6 6 - / 6 / , / 6 / l
receptor ), 230 SR proteins, 677
transcription complexes, 77 8, 77 8f SWI gene,495
chromosome end sealing, 749-750, 749f,750f definition of, 748 extension by recombination, 7 52-7 5j, 7 52f
SSB (single-strand binding protein]l, 436-4)7 , 4364)8, 436f, 4)8f
SWI/SNF complex
{unctions,750
Startpoint,258 STE genes, 488 Stem-loop binding prorein (SLBP), 697 Steroid receptors activation, by ligand binding, 656, 656f as activators, 65)-654, 654f definition of, 65 I DNA binding as dimers, 655 recognirion of response elements, by combinatorial code, 657-658, 657f, 658f SMRT corepressor binding, 658, 658f zinc fingers, 65r-656, 655f,656f Steroid response elements, half sites, 657, 657f Stop codons
binding, 810, 810/
s_trortenrng,/ >2- / ) ), / J2J
definition of, 799-800, 800/ recruitment, 801
simple repeating sequences, 748-749, 7 49f survival and, 7 52-7 53, 7 52f, 7 53f
Swi5 transcription factor, 80 I SWl5 regulator,496 SxI protein, 686 Synapsis (chromosome pairingl, 46 | Synaptonemal complex axial element formation, double-strand breaks and, 468,468f connection, with recombining chromosomes, 465467, 466f definition of, 46 I formation, 465466 aiter double-strand breaks, 467469, 468f independent from chromosome parrng, 469
definition of, 172, 197, l97f f r:mechifrino
:r
) I d
novel amino acid insertion in, 199, l99f Stop-transfer signal, 216-2)7 , 2j7f Strand displacement, )94, )95f
assimiliation, 47 147 3, 47 2f, 47 3f o{ protein synthesis, 2I0-2Il Structural maintenance of chromosome proteions (sMC proteins), 828, 829f Substitutions, during DNA replication, 412 Subviral pathogens, 20 su(HW) gene, 7 84-7 85, 7 85f Suicide substrates, 484, 485 Supercoiling, DNA in bacterial genome, 7 35-7 36, 7 3rf, 7 )6f DNA structure and, 476478, 476f, 477f gyrase and, 481482, 482f introduction by topoisomerases, 47 8479, 479f inverted, release of, 482,482f negaIive,477
s y n t h e s i s ,b y r i b o n u c l e o p r o t e i ne n z y m e . 750-752,75tf Telomeric silencing, 821 Temperature, environmental, 278-279
heat-related genes and,
Template recognition, in transcriptio\, Template strands , 8-9, 9f, 258, 258f T4 endonuclease v (Ta-pdg), 506
Terminase, 7 )2-7 )), 7 33f Termination of DNA replication, 429 mode of elongation and, 295
structure axial elements, 466
pausing and, 288 prevention, by pQ, 294-295 in protein synthesis, I 55, I 55/
central elements, 466, 466f j a l e r a l e l e m e n t s ,4 6 6 , 4 6 6 1 nodes or recombination nodules, 466,466f proteins in, 466467, 466f Synonyms,190 Synteny, 66-67, 67f Synthetic genetic array analysis (SGA), 91, 9ll Synthetic lethal, 9 I
T TAF',619, 621 "Take-off" sites,in bypassing, 214-2I5, 214f Tandem gene clusters, restriction map, I l2-l 1 I, I I 3/ Tandem repetition, I l7-l l8 T antigen, 45 1 TAP/MEX transport protein, 68), 68)f rar-binding sequence, 644, 644f TATA-binding prorein (TBP), 615,619-621, 620f, 62lf
260, 26lf
Teratomas, 402 Terminal proteins, viral initiation and, 394-39r, 395f Terminal uridyltransfetase (TUTase), 723-724, 724f
recombination and, 467 468 mutations, 466467
Strand-exchange reactions, f or replication repail 512-51),5r2f,5r)f Strand-transfer proteins, catalysis, of single-strand Streptomycin inhibition,
Telomere
RNA polymerase and, 29j-29 5, 2e4f of transcription, 26I, 26lf rho factor and, 287 , 288-291, 2 8 9 f , 2 9 O f Termination codons resf alterations, sporadic, 197-198, re7f, definition of, 12 nonsense supressor tRNA, 207 -2O8, 2O7f for protein synthes$, 172 r e c o g n i l i o n b y p r o t e i n l a c t o r s .l T r - 1 7 4 , 1 7 3 f . 1 7 4 f Terminators bacterial, 286-287, 287f inE coli,287-288,287f inrrinsic, 287-288, 287f rho-dependent, 287, 288 delinition of, 258, 258f rho-dependent, 29O, 290f
Index
889
Tetrad. ascomycete, 47 5, 47 5f
independence from DNA-binding domain.
Tetr a hymena t lt er moph i I a genetic code changes, I 97
64)-645.644f basal level, 106 base pairing, in "transcription
group I intron inE coli,TlO-7ll,7llf
btbble,"
259-260,
259f,260f
secondary structu re, 7 09-7 10, 7 l0f self-splicing. 7 07-7 09, 7 0 8f-7 l]f
coding strand, 258, 258f constiutive, 109 control
histone modification, 804 n r S l o n el r S a g e ./ / +
by antitermination, 291, 29lf
TFs SeeTranscription tactors T h a l a s s e m i a sd, el c t i o n s i n , I l 0 - l I l , I I I / Thermts aquaticls. signra lactor, 280,280f Thermusthennop hl1ri.r,ribosome, strucrural
definition of, ll2
conlormarion, l8l Third-base degeneracy, l9l,
D N A s e q u e n c e ,2 5 8 in gene expression, ))-34,
e n h a n c e r s ,6 l l - 6 1 2 , 6 1 2 f promoters. 6Il,6l2f by translation. ))6, ))6f
192, l92f Thymidine dimers, excision repair system, 516,516f Thymine,6, l8-19, l8f Thyroid hormones, as activators, 654f TIM complexes, mitochondrial, 24', 244, 244f Ti plasmids
)4f,258-259 histone octamer displacement, 779-781, 779f, 780f initiation, 260-261, 261f, 610 eukaryotic gene expression and, 641-642,642f RNA polymerase and, 267-269, 268f transcription factors and, 621-622 m o d e l s y s t e m s ,2 6 1 - 2 6 2 , 2 6 2 f
definition of, 406 crown gall diseaseand, 401-402, 402f T-DNA. 402-405, 40)f, 4O4f 71aloci proteins, 600 TLF,620
nRNA. 129, 129f,156, l56f nucleosomes and. 777-77 9, 77 8f, 779f products. primary transcript, 258 promoter recognition, consensus sequences and,
T4 ligases,444 T L R S( t o l l - l i k e r e c e p t o r s ) ,6 0 2 - 6 0 4 , 6 o l f
272-274,274f promoters, 258,258f
T lymphocytes SseT cells TnA family. replicarive transposition, r4-r6, tnpA gene,5)5,535f
rDNA cluster, II), 535f
t n p R E e n e ,5 ) 5 , 5 ) 5 f T n l 0 t r a n s p o s i t i o n ,n o n r e p l i c a t i v em e c h a n i s m , 5 1 8 T n 5 t r a n q p ' ) \ ( ' nn. u r r r c p l i c ai rt e t r a n r p o s i t i o n , 5)4,5)4f T n 1 0 t r a n s p o s o n ,I S I 0 R a c r i v e m o d u l e , p r o m o t o r s i n , 5)6-5)7, 5J7f
ll4f
repair and, 625-627, 626f repair systems in yeast and, 5 i 5 repression, by histone deacetylarion, 808, 808/ restarting, arrested RNA polymerase and, 269-270 sldges,260-261, 26lf e l o n g a t i o n ,2 6 I , 2 6 l f initiation, 260-261, 26lf template recognition, 260, 26lf
in RNA splicing, 674, 674f in splicing reactions, 68), 684f Transfection,5 Transfection systems, f or promoter characterization, 6 I l Transfer genes, on F plasmid, 398-799, )99f Transfer region, 399 Transferrin nRNA, iron-responsive 142-14), r42f Transfer RNA (IRNA) ,ltern:tivP
oenPs
element,
49
anticodon, specilicity determination and, I I l, 1 lU base modifications, 194-196, l95f anticodon-codon pairing and, I96-197, 196f bases invariant or conserved, I I I semi-invariant or semi-conserved, I J I h i n r J i n o< i r e s 1 6 9 1 7 O f A,154, r54f,r60,177, t78f E, r54, r54f, 178f P, 154, 154f,160, 177, 178f charged SeeAminoacyl-IRNA charging process. 1)1, 20O-2O1, 2O0f, 209 cognate, 200 kinetic proof reading and, 2Q3-20 4, 20 4f conformational structure, active sires and, 205-206, 205f contact, with 2lS rRNA, 182 deacylated, I 54 delinition of, 14 3' end,194, l94f 5' end,194, l94f E site,182 function of, 128-129 identification, by synthetases,201 interactions
Tn 10 transposon, nonreplicative transposition, 53)-5)4, 5))f Tn10 transposon, transposirion, SS6-5J8, 537f
startpoint, basal apparatus and, 642 supercoiling, 27 7 -27 8, 27 7f
isoaccepting, 200-20l
tn transposons or composlte transposons, 525-526, 525f,526f
template strand, 258, 258f telmtnatron
modified bases, l9,t-197, ).95f, 1.96f mutations
l o l e r a n c e ,) / J Toll-like receptors (TLRS),602-604, 60jf TOM complex, mitochondrial, 24)-244, 24)f topA gene mutations, 4l8 Topoisomerases activiry, similarity wirh sire-speciificrecombination, 484486,485f introduction ol supercoiling. 47 8-479, 479f type 1, 277 , 479 single-strand DNA and, 479, 480, 48Of type ll, 479 Seealso Gyrase d o u b l e - s t r a n dD N A a n d , 4 7 9 , 4 8 1 , 4 8 I f Topological isomers, 477 TPA response element (TRE). 650 T3 phage RNA polymerases, 26l-262, 262f T 7 p h a g e R N A p o l y m e r a s e s ,2 6 I - 2 6 2 , 2 6 2 f tra gene, 686 Trarler, )), )4f TraM, 400 TRAM (translocating chain-associated membrane), 23),233f,239 Traur-acting DNA sequences, 302, )O), JO3f ?anj-acting mutations, 16, l6l Tranr-acting sites, j5-)6. 36f Transcribed genes, nucleosomes and, 777-778, 778f Transcript. l8 Transcription. 256-29 6 activation, 640-665 by CRP activatot l2l DNA binding and, 644
890
Index
attenuation by hairpin structure in RNA,
3)), )))f in bacteria RNA polymerase, 286-287, 287f rho factor and, 287, 288-291, 289f, 290f by RNA polymerase l, 694-695, 694f by RNA polymerase lII, 694-695, 694f t r a n s l a t i o n a lc o n t o l o f , ) ) 5 , 3 ) 5 f terminators, 258,258f twin domain model. 277. 2771 Transcription factors (TFs) definition of, l0l 'IF1r4,622 TFtrB, with RNA polymerase, 622-621, 621f T F 1 D ,6 t 9 - 6 2 0 , 6 2 t . 7 9 8 activator contact, 647, 647f binding to TATA box. 622 TFTE,624 TFff,62) TFIH, 624, 625-626, 626f TFil,624 TFtilA, 617, 798 TFDrB,6t6-6t8,6t7f types, 642-64), 64)f Transcription units. 258, 292 Transcriptome, 56 Transducing viruses, 558-559, 558/ Transesterilication in group I intron self-splicing, 7O7-7 09, 708f-7 Ilf in protein splicing, 7 24-725. 725[ i n r i b o T y m e c a t a l y t i cr e a c l i o n s .7 1 \ . 7 | \ f
with ribosome, 154-155,154f, l55f with IRNA, 180
missense, 206-207, 2O7f nonsense, 206-207, 206f nomenclature, I I I numbering system, 111 A site. protected regions, 181 size of, l28f s p e c i f i c i t y ,I l I , l l U 50S subunit, 182 structure, 128-129, 128f, 177-178, 178f a c c e p t o ra r m , 1 3 0 - 1 1 1 , l ) O f , l j 2 f anticodon arm,130f, l)1, l)2f arms, ll0, 130f,132f base pairing, 130f, I)l D arm, I 10, l)1, IJ2f enzyme-related changes, 202, 2O2l extra arm, llof, l3l l o o p s , 1 3 0 ,1 1 0 / s e c o n d a r yo r c l o v e r l e a f ,1 3 0 - 1 3 1 , 1 3 0 / s r e m s .I 1 0 . I J j l . l J 2 . l 3 2 f tertiary L-shaped, 13I-l)2, TyC arm, 130f,l)1, l32f
l32f
suppressor competition with wild-type tRNA, 208-209,208f nonsense codon recognition, 206-207, 2O6f synthesis. from longer precursors, 194, 194/ wild-type, competition with suppressor IRNA, 208-209,208f Ttansformation, 3-4 transformer(tru) gene, 686
Transgene, 6 I l Tfansgenic systems, for promoter characterization, 6l l Tfansitions (basemispairing), l5-16, l6f Translation accuracy, ribosomal influences on, 209-211, 2I0f bacterial, cistrons and, I 17 control of attenuation, )36-i)8,
3j7 f, ))8f by frameshifting, 213-214, 2l7f of transcription termination, )35, ))5f de{inition of, 211, }32
frequency,525 i n t e r m e d i a t e s .c o m m o n . 5 ] 0 - 5 ] l . 5 ) 0 f , 5 ) l f in maize, 538-540, 539f,540f nonreplicative, 527-528, 528f, 5))-r34, 5))f replicative, 527, 527f cointegrates and, 531-5)3, 532f TnA family, 5j4-536, 5)5f RNA-dependent, 55 I
UAS (upstream activator sequencel, 629-6)0 uBF,615,6l5f Ubiquitin, 214 UEP (unit evolutionary period), 105, 106
ofTnl0,536-578,5)7f Transposons (transposable elements), 522-547 classes,521
ending posttermination
composire or rn, 525-526, 525f,526f in conservative transposition, 528, 528f controlling elements SeeControlling elements
of eukaryotic nRNA, I 62-1 63 in gene expressiorr,))-)4, )4f mRNA, 129, l29f
definition of, 16, 62,52) DNA rearrangements, 522, 522f, 528-530, 529f
reaction, 17l termination reaction, l7l
regulation, repressors and, 32)-)25,
)24f
ribosomal, initation site and, I l6-1 l7 ribosome and, 14, 34l Ttanslational positioning, of DNA on nucleosome, 776-777,777f Ttanslocating chain-associatedmembrane (TRAM), 2)9 Translocation
diecr,52) indirect,52l in Drosophila melanlgaster, 561-562, 561,f families, formation of, 54O-542, 54lf in human genome, 85, 85f,561-564, 564f insertion sequences, 524-525, 524f "inside-out," 526 intermittent activities, 52 l
definition of, 22 I
inverted terminal repeats, r\A, 5)5, 5)5f nonfunctional, 567, 567f
duplication separation and, 99 protein
i n n o n r e p l i c a t i v et r a n s p o s j l i o n ,5 2 7 - 5 2 8 , 5 2 8 f reciprocal recombination, between inverted repeats,
control 232-233, 233f cotranslational, 22 1-223, 22)f default pathway, 227, 227f
529,529f in replicative transposition. 527. 5271,
Escherichia coli Sec-independent system, 249-250,
24ef initiation. signal sequences f or, 227 -228, 227f, 228f initiation signal sequences,interaction with SRP, 228-229,229f posttranslational, 222 reverce, 234-235, 2)4f signal recognition particle in, 228-229,229f in protein synthesis, 154 of random sequence with exon, 4849, 48f ribosomal activation of, 172 hybrid state model, 169-170, 1.70f,178 termlnase,/ 52- / JJ, / JJI IRNA structural changes, 178, 178/ Translocation factors, structural mimicry, 17)-17 4, 17)f Translocons aqueous channel formation, 2)l-2)),
2)2f
components, 2)),21)f deiinition of, 212 in protein translocation, 2ll protein transport and, 221, 221f routes to, Sec system and, 247-249, 247f, 248f srze,232 Transmembrane d omains, 235-276, 236f, 2)9, 2)9f Transmembrane prcteins, 235-236, 235f, 236f Transmembrane region, 601 Tfansplantation antigens (MHC classI proteins), 599 Transposable elements. SeeTransposons Transposases cri-preference, 5)7 -5) 8, 5)7 f definition of, 525 in TnA transposition, 53 4-53 6, 5) 5f
53r-53),5)2f target site selection, 518 Transposons, in hybrid dysgenesis, 544-545, 544f Transvection, 6l I Transversions, l5 Tra protein, 687 Tra2 protein, 687 TraY 400 TRE (TPA response element), 650 Tfichostatin, histone deacetylaseinhibition, 806 Tf ichothiodystro phy, 627 "Trigger factor," 248 IRNA. SeeTransfer RNA rRNArMet (N-formyl-methionyl-IRNA),
tlp operon, leader region, alternative base-paired conlormauons, St6-tJ /, )1 /| ,rp repressor, 319-)20 True reversion, of mutation, 16-1,7, 17f Trypanosomabrucei coxlll gene, RNA editing in, 722,722f Tt!?anosome, mitochondria,RNA editing in, 721-722,722f Tfyptophan control, of ribosome position, )37-)18,3)7f lrp operon attenuation conrrol and,,136,1)6f Tryptophan RNA-binding attenuation protein (TRAP) control of B subtilis trp opercn, 131-3)4, tRNArrrcontrol ot,334, )34J
)74f
T/t antigens, 685-686 Tumorogenisis, retroviral integration and, 557 Tus-ter complex, replication fork disassembly, 7 2)-7 24, 7 24f
composite transposons and, 525-526, 526f conservative, 528, r28f
Twisting number \Tl, 477478 Two hybrid assay,645-646, 646f 4/ elements rever6e transcriprases,5 63, 5 6)f
control of frequency. 5)7, 5)7f definition of, 459, 523, 560
i n y e a s t ,5 5 9 - 5 6 1 , 5 5 9 f - 5 6 1 f fYrosine. cr cleavage of DNA, 485--486
Transposition
I 5 8-159, | 591
tRNArMet, 164 tRNAmMet, 158
45245),453f TUTase (terminal uridyltransferase),
U UAA termination codon (ochre codon), 172 U2AF splicing factor, 677, 678f UAG termination codon (amber codonl, 172, 199
UGA codons, seleno-cys-IRNA insertion, 199, l99f UGA supressor,206-207 UGA termination codon (opal codon), 172 umuc gene mttations, 507 UmuD'2C complex (DNA polymerase vl, 5O7 umuD gene morations, 507 UmuD protein, cleavage, RecA protein and, 5 l4 Unequal crossovers causes,99-100, 100/ definition of, 99-100, 100/ in gene cluster qualitative effects, I 10, I I0/ quantitative effects, I I0, I l0/ rearrangement and, 109-1 12, t 101 I t 1/ minisatellite, I25 inthalassemia, II0-f Il, f Il/ Unfolded protein response element (UPRE), 69J,69Jf UNG (uracil DNA glycosylase), 590 Unidentified reading lrame (URF), l2 Uninducible mutants, 108 Unit cell, 409 Unit evolutionary period (UEP), 105, 106 Untranslated region (5r UTR), 136 UPE (upstream promoter element\, 615,615f Up mutarions, 274 UPRE (unfolded protein response element), 693, 693f Upstream, 258 Upstream activator sequence \UASl, 629-6)0 Upstream promoter element (UPE), 615, 615/ Upstream promoter elements, 644 ut-R,554 Uracil, 6 Uracil glycosylase, induction of somatic mutations, 591-593,592f Uracil triphosphate (UTP), 721 ure2 prorein, 8)6-838, 817f U residues, in RNA editing, 723-724,724f URF (unidentified reading frame), 32 Uridine modification, in IRNA, 195, l95f in pseudouridine synthesis, 699-7O0, 699f 724f in RNA ediring, 721-722,722f,72j-724, URSI gene,496 URS2 gene,495,496 u3-R-U5, 560 U run, 694 Ul snRNA, base-paired structure, 675-676f U4 snRNA, 680 UTF (uracil triph osphatel, 7 21 l'UTR (trailer),136 UUG codon mutant, 2I I in protein synthesis, I58-I59 U6lU4 snRNA, 680,680f uvr system of excision repair, 501, 504, 504f
V Variabledomain (V domain),576 Variablenumber tandem repeat (VNTR), t24 variable region (V region), in immunoglobulin, 575-576,576f Variegation,518
duplicate formation, 99
Index
891
V domain (variable domaur), 576 Vegetarive phas€, of bacteria, 281 v-fos gene, 66)
Virusoids (satellite RNAS) catalyric activiry, 7 l8-720,
AshI nRNA, 146-147, l47f cassette model, 488489, 489f centromere, protein complex bindir\g, 7 48, 7 48f chromatin remodeling complexes, 799-800, 800/ essential genes, number of, 89-90,90f
7 19f, 720f
hammerheads, 719,719f Vitamin D, 654,654f v-1un gene,663
V gene oellnlnonoI,>/o->/I recombination, with J-C genes, 582, 582f segments, 577, 581 in kappa family, 580, 580/
VNTR (variable number tandem repeatl, 124 v-onc gene, r58
in lambda family, 580, 580/ single recombination in light chain assembly, 577, 578f,579 s o m a t i cm u t a t i o n s , 5 9 0 - 5 9 1 , 5 9 1 / successiverecombinations in heavy chain assembly and, 579-580,579f V gene promoter, activation by recombination, 586,586f Viral superfamily, 5 62, 5 62f VirA-VirG system, 4O440r, 404f
genomes, 79-80,79f heat shock transcription factor. 650 leader sequence for cytochrome cl, 242, 242f
VPl6 protein,647 V region (variable region), in immunoglobulin, 575-576,576f V segment, 581
for cytochrome c oxidase, 24o,24of mating type, 488489, 489f M-4Tlocus, mate type switching, 49)494,494f
W
mitochondrial introns, 49, 7 15, 7 16f mRNA degradation, 14)-144,
Watsonand Crickdoublehelix model,6-8,7f,8f O gene,astargetfor double-strand break, 7 t5-7 t6, 7 trf Wobble hypothesis,192-19), 1.9)f,198 Writhing number (Wl, 47I
X
Viruses adenovirus DNA replication, by strand displacement, 194, J95f initiation, terminal proteins and, )94-)95, 395f
395f
replication, 393 during infective cycle, l2 by strand displacement. )94,395f replication-defective transf orming, 5 58, i 58f retroviruses Se€Retroviruses RNA, recombinalion, 459 RNA in, 5 rransoucrng, > t6-> t9, > >aJ Virus-like-particles (VLPs), generation by 7y elements, 560-56r,56tf
Index
splicing endonuclease, IRNA recognilion,
6erf,6e2f
facultative heterochromatin, 827 G - b a n d s ,7 4 1 , 7 4 1 f gene density, 86
surveillancesystems,l44l-l 45 temperature-sensitivemutants, 690 translation,frameshift control of, 2 I l tRNAiMet,I64 IRNA splicing, 690, 690f, 69tf IRNA tertiarystrxcl.ule,l)2, l32f D/ elements,559-561,559f-56tf upstreamactivatorsequence,629-630
histone acetylation, 806 inactivation, n- l rule, 827 -828, 828f single X hypothesis, 827,827f
Xenopus laevis
DNA in,4-5, 5/ genomes,11,12
892
consitutitve heterochromatin, 826-827 dosage compensation, 826, 826f
X-degenerate segments, in Y chromosome malespecific region, 86-87, 87f
assembly, 7 ) l-7 34, 7 )2f, 7 ))f capsids, 7l I-714, 732f.71)f
nerper, ))d initiation, terminal proreins and, )94a95, IRES elements, 164 mRNA translation, l6l plant, muhipartite. 7ll plus strand, 554
Schizosacharomycespombe genomes, 7 9-8O, 7 9f S/r,el-5 mutations, 146-147 , l47f
X chromosome
Virulence genes, in crown gall disease. 403404,404f Virulent mutations, 160
embryonic development, 69 6-697 globin gene, 104
7
nontranscribed spacer Iength, lI4, l l,4f oocytes, IRNA splicing, 692 rDNA cluster, I l5
Zellwegersyndrome,245 l7I ir ^L -
lfui -t !- r- .
r- r^r+v irfr r
definition of, 651 DNA-binding and, 652-653, 653f on steroid receptors, 655-656, 655f, 656f ZipA prorein, 414
snoRNAs, 699 55 RNA gene promoters, 616 Xeroderma pigmentosum, 5l 5-516 Xer site-specificrecombination system, 416416, 416f Xrc (X-inactivation center), 827 Xrt RNA, X chromosome inactivation, XRCCI/ligase-3,505
I44f
PabIp,165 prions, 836-838, 817 IPSI+/ strains, 836-818, 8J7f, 838f repair genes, 5 l4 RNA polymerase, 263, 26)f
Viroids catalytic activiry, 7 | 8-7 20, 7 l9f, 7 20f definition of, l9-2O, 20f h a m m e r h e a d s ,T 1 9 , T 1 9 f Virons, l9
l4)f,
mutants, 275,690
827-828, 828f
X-transposed sequences,in Y chromosome malespecific region, 86, 87/
Y Y chromosome, male-specific region, 86-87, 87f Yeast Seealso Saccharomyces cerevisiae: Schizosachar omyces p om be
Tikl
mnr^tian
447
zip2 mlrarion, 469 Zoo blot, 67-64, 64f Z-ringformation,413414, 414f
691-692,