Molecular Biology of the Gene, Fifth Edition

J .. Molecular Biology of the Gene ~~~~· FIFTH EDITION J . 1 I • l l\ I } BAKER BELL GANN LEViNE LOSICK ' \...

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Molecular Biology of the Gene ~~~~· FIFTH EDITION

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NOT FOR SALE

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Molecular Biology of the Gene F

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E D I TI

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James D. Watson Cold Spring Hiitbot I..aboratory

Tania A. Bake r Massachusetts lnstitutP of Teclmology

Stephen P. Bell Massachusetts Institute of Technology

Alexander Gann Cold Spring Harbor Laboratory Pross

Michael Levine University of California. Berkeley

Richard Losick Harvard Universit y

--PE:ARSON

Ht"f\iamin

Cummim.!.S

NOT FOR SALE

Bet1jamin C..ummin,gs Publisher: Jim Smith As.~iflte

Project Editors: Alexand ffl F11Howes,

Jeanne ~les"ky Senior Production Editor: Corinne Benson Manufacturing Manager; Pmn Augspwger Senior Marketing Manager: Josh Frost Production Management and Text Design~ Elm Stl'eet Publishing Services, lnc. Mt!dia Development and Production: Scienr..e Tochnologies and David Marcey, CLU Questions for Website; Peter Follette Art Studio: Dragonfly Media Group Olmpositor: Progressive lofonnation 1echnologies Cover Image; Tomo Narashima

Cold Spring Harbor Laoo.-atory Press Publisher and Sponsoring &litor; John Inglis Editorial Director; Alexander Gann Editorial Development Manag*'r: ]11n Argentinft Project Manager and flcwdopmentall-::ditor; Kaaren Janssen Project Coordinntor: Maryliz Dir.ke£Son Editorial Oevelopment Assistant: Nora Rke Crystal structure images: leemor joshua-Tor Cover concept sketch: Erica Baade, MDC Grc1phics Cover Designers: Denise Weiss, Ed Alkeson

JSBN 0-321-22368-3

t..opyright © 2004 Pearson Educal iort, rnc.. publishing as Benjamin Cummings. t30t SansorneStroot. San Francisco, CA 94111 . AH rights reserved. Man ufactured in the United StAtes of America. This publication is protected by Copyr·tght anu permission should be obtained from the publisher prior to any prohibited rep1·oduction. storage in a retrieval system, or transmission in any form or by any means, electronic, mechanical, photocopying, recotding, or like wise. To obtain permission(s) to uSP. m aterial from this w ork. please submit a written request to Pearson Edur.ation, Inc., Permissions Departlllent. 1900 E. Lake Ave nu e . Glenview, IL 60025. For infon natton regarding permissions, call647/486/2635. Many of the designations used by manufacturers and sellers to distinguish their products are claimed <-ts trademarks. Where those designations appear in this book. and the publisher was aware of a trademark claim, the designations have been printed in initial caps or all caps.

If you purchased this book within the United States or Canada you should be aware thilt it has been wrongfu lly imported without the approval of the Publisher o.r the Author.

---------Pf ,\ RSON

1 2 3 4 56 7 8 9 10-VHP- 07 06 05 04 03 www.aw-bc.com

Ht~ruamin

Cummings

"rww~cshlpress. com

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Preface s the fifth edition of Molecular Biology of tile Gene goes to press. completion of the human genome sequence is no longer nows. This was not something that could safely have been anticipated w h en the first ed ition appeared in 1965; even when the fourth editiou came out in 1987. few if any foresaw how quickly we would move into a world where whole genomes, not just ind ividual genes, could be visualized and compared. Tbere has boon a comparable leap in the e lucidation of protein structures as well. Tbus, in tho last few years. the structures of t he huge molecular mechines that drive the basic proces.t~es discussed in this book-DNA transcription, replication, prote in synthesis, and so forth - h ;;,ve Jargely been solved a t the atomic lovel. and many details of their iuncr workings revealed. T h e new edition of Molecular Biology of the Gene reflects thase advances, and many others besides. But when we sat down to plan this latest version. we wure all of a atind that much of the organization and scope of the original book sh ould be retained. Th is was not a matter of C(lnVenience-inevitably, in light of the dramatic c hanges that had taken place si nce the last edition. the vast bulk of the text had to be oompJclely rewritten .f1nyway, and all the art rendered afresh. No. the reasoning was simpJy that, more than ever in this genon•ic era, t hP.ro soemed a need for a book that explwncd what gtmes are and how they work, and this was exactly what Mnlecolar Biology of the Gene had originally been designed to do. Thus, we have r~i sted the temptation to become encyclopedic or to delve into allied dim:iplines. suc h as cell biology. Alo;o, we WWlted tho new edition to retain tt focus on principles and concepts. another feulure of its predecessors. And so we illustrate Out discussion svaringly \\oith experiments, whk.h eipJlear ma inly in boxes. These C'Onsiderations ensured the book dM not become unwieldy. As stated by its author in the profl-tcc lo the first edition: "Often 1 present ~ fact, and, be-cause of lack of space, t cannot outline the experiments that demonstrate its validity. Gi ven the choice between dcletiog an important principle or giving an experimental detail. I am ioclined to state the principle." 'fhe current incarnation of Moleculur IJiology of the Gene adheres unapologetically to this philosophy. An outline of this rww edition wiJJ thus be familiar to anyone who has used the book before. We begin (in Part t) with a series of chapters (modifted in the current edition) that place the field of molecular biology in oontext. These chapters summarize the history of genetics and molecu~ar biology and also present the ti meless chemical pri nciples that dctrum ine the structure and function of macromolecules. The text thereafter is organized to follow a familiar flow of topics. The nature of lhe genetic materiaJ. its organization and its maintenance, are discussed in Part 2: in add ition to cha pleTs on DNA structure, replication, rocornbinAtion. and repair. new to this part of the book is a chapter on chrom~omes , chromatin, and the nucloosomt:l. This addition reflects current <:~pp rec iation of how the context in which a given gene is found influences its function ancl regulation.

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The passage of information from gene to protein - so-ca1led ~ene ell pression -is r:overed in Part 3; and in P;-trt 4 we dc~scrihe the. regulation of thai process. As well as chapters on rhe basic fllflchan isms o1 geoe reguiHtion, Part 4 has chapters on lhe regulittion uf gene expression in animaJ development and in tho e volution of anima l diversl ty. These chapters again conform to a trad ilion esta blished by earlier editions: a lways there has been a cha pter or two lin'king basic anedutni sou:; of molecul11r Liology to pressing LiologJooJ questions. In the c u rrent edition, these chapters investigate perhaps the most striking re velation to come from comparing the complete genome sequenc69 of various animals: different animals including humans-contain largely the same genes a nd so differences between those a nimals must resul1 largely from changes in how those gttncs are exprnsscd. New to the current edHiun is the t'inal part - Part 5-compl'ising chHpters on experim em~:~l methods -the teclmilJues of molecular biology, genomics, and bioinforrnatics- and on the model Ol'ganisms whose study bas revealed many of the nndedyjog principles of molecular biology. We a lluded to th~ explosion in tl1e numbers of atomic stTtJctures solved in the last few years. These include not only many of the enzymes that mediate the basic processes of molecular biology. and many of the proteins that regulate those processes, but the nucleosome as well. While it remains true that many of the basic concepts in molt!c talar biology c.an be understood \1\rithoHt reliance 011 structural detail - indeed it is one of the strengths of the field th~t this is the case-nevurth oltls~. meiny rnech~nis tic insights come only from seeing these details. Accordingly, where structures shed light on how the molecules in ques1ion work, we present them; and we do so in a consistent style throughout the book. Each p flrt opener includ&8 a short text. outli ning what will be covered i11 the coming chapters, and a few photogfflphs. These pictures, from the Cold Spring Harbor Laboratory Archive. were all taken at the Laboratory on Long Island, the g.reat majority at the Symposiwn hosted there almost every summer since 1933. Captions identify who is in each pictw·e and when il was lttken. Wo thank Claro Bunce and tha (:,."HL Archive for help with these. Parts of tlae current edition grew out of an introductory r..ourse on molecular biology tC:I ught by one of us (RL) at Harvard lJniversity. and this author is g.roteful to Stevo Harrison and Jim Wang who contr ibuted to this course in past )•ears and whose influence is rcllocterl in Cha)Jter 6 anrl clsewhecc. We have shown sections of the manuscript to various colleagues and their comments have been most valuabl.,, greatly improving the accuracy and accessibility of the text and figures. Specifically we thcmk: Jamie Cate. Richard liliright. Mike Eisen. Chris Fromme, Ira l lall. Adrian Krainer. Karolin Luger. BiJ1 McGinnis. Matt Micha~l. Uly Mirels. Nipam Patel. Craig Peterson. Mark Ptashne. Uttam RajBhandury. 8Jld Bruce Stillman. ln addition, Craig Hunter draftea:l the section on tht1 worn• for Omvter 21 . We wso thank those who provided us with figures, or the wht•rewithall to create them, including: Scan Carroll, Seth Thirst, Edward Egelman, C..eorg Halder, Stuart Kim, Bi11 McGinnis, Steve Paddock, Phoebe Rice, Matt Scou, Peter Sorgec, Andr2:ej Stasiak, Thn1 Steitz, Dan Voytas. a nd Steve West. We are most grateful to Leemor Joshua-Tor whn rendered all rhe structw·e flgurf1S, often produdng multiple versions 8IId vatiently helping us see which best ~5'-Ff~~Al~ needed. We

are a lso grateful to those w ho provided their softwBre 1: Per Kraulis, Robert Esnouf, Ethan Menj tt, <mtl Barry Honig. Coordinates were obta ined from the Protei n J)ata Bank (www.rcsb.org/pdb/); and citations to those who solverl each structure are included in tlte figu re legonds. Our art program WAS developed a nd rendered by a talented and enthusiast ic team from the Dragonfly Media Cmup . led by Mike Demruay and Craig Duront. Renate HelJmjss helped to develop some of our initial sketch~ and provided ero-ly renderings of a n umber of figures. The cover image was rendered by Tomo Niuashima from an author conr..ept sketch by Erica Beade (MBC Grap hir~q ) , We thank those C:!t Cold Spring Harbor Laboratory Press who handled de velopment of this hook. fan Argentine. despite having t o eflforr:e the deadlines. wes throughout less cajoling than she wats tirelessly e ngaged in heJp;ng us solve the problems these presented. Maryliz Dickerson kept organ ized the mass of materia l we gen erated and Nora Rice helped coordinate a utho r meetings and o ther aspects o f tbe pro ject. Ocnisv Weiss and Ed Atkcson produced the c o ver design; antl Johu Jngli,:;, who in iti ~J ted this collubora tion, wats 011 hand with adv ice at critkal points in the process. Most of aJI , Kaaren Janssen, our editor, kept everything lifloal with t~fl energy. e nthusiasm , and acti. vily far b eyond anylhing we could reasonabJy have asked for, t hings simply wo uld not have got to this point without her. We a lso wish to acknowlfldge the work of thost> (It Ben jamin Cummings who coordinated production of the book. Frank Ruggirello ovocsaw the process carried o ut by Jim Smith, Kay Ueno. Corinne BPnson, Alf~xandra F£>1Jo wes t Jeanne ZaJf!sky, and Donna Kalal. Ingrid Mount at Elm Street Publ ishing Servict>..s co ped cheerfully with th e m~-tny rounds of changes to art and text oven very 1ale in the process. Michele Sordi, while part of the Ben jamin Cummings tei:llTl, helperl bring u s all together in the first place. And finally we gratefully acknowledge our families and friends who, throughout this period. provjded such strong support, despite having to put up with our frequent 11bsences and distractions. fames D. Watson Tania A. Baker Stephen P. BeD Alexander Gann Michael Levine Richard Losick

• Per KrauJis granted pen mssmn to lisE> Mo!Scripf {Kmuli!i P 1. 1991 . MOLSCt
Appliffi Cry:;tallowaphy 24: 946-950). Robert Esnouf gavt~ pcnnission to Uh'tl BobScripe (Esnotd' RM. 1997 journal of Moleculac Grophics t 5: 1 32 134). In additiOft. Ethan Merritt save us use ofH
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About the Authors JAMES D. 'W ATSON was Director of Col d Spring Harbor Laboratory from 1968 to 199.3 and i-; now its President. He spent his undergraduate years at tho UnivOISity o£ Chicago and received his Ph.D. in t!l50 from Indiana University. Between 1950 and 1953, he did postdoctoral research in C..openhttgen and Cambridge. England. While at Cambridge, .htt began the rollaboration that resulted in Lho elucidHtion of the double-helical structure of DNA in t 953. (For this discovery, Watson, Francis Crick, and Maurice Wilkins were awarded the Nobel Ptize 1962.) l..ator in 1953, he went to the Californialnstitute of Technology. He moved to Harvard in 1955, where he taught and did research on RNA synthesis and pl'ole in synthesis until 1976. He was too first Director of the Natiunal Center for Genome Researm of the National Institutes of f reatth from 1989 to 1992. Dr. Watson was sole author of the first, second, and thud editions of Molecular Biology of tl1e Gene, and a co-author of the fourth edition. These were published in 1965, 1970, 1976, and 1987 rvspectively. Wal<;On has a lso been involved in two other te>~:tbooks= he was one of the oriAinal a uthors of Molc.-cular Biology of the Cell. and is illso an author of Recombinant DNA: a Sllorl Course.

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TA"''IA A. BAKER is the Whitehead Professor ofBioJogy at the Massachusetts Institute of Techno logy and an Investigator of the Howard HuAhes Mediut Institute. She received a B.S. in biochemistry from the University of Wisconsin. Madison. and a Ph.D. in biochemistry frorn Stanfom University in 1988. Her grarluare research w-as carried out in the laboratory of Professor Arthur Kornberg and focused on m echanisms of initiation of DNA replication. She did postdoctoral researc.h in the laboratory uf Or. Kiyoshi Mizuuchi at the National Institutes o f Health, studying the mechani'>m and regulation of DNA tmnspos ition. Ha current research explores mechanisms v.nd regulation of genetic recombination, enzyme-catalyzed protein unfolding, and ATP-dupendent protein degrttdation. Professor Baker received the 2001 Eli lilly Research Award from the American Society of Mictobiology and the 2000 MIT School of Scien ce Teaching Prize for llndcrgradu(lte Education. She is co--author (with Arthur Kornberg) of the book VNA Replication, Second Edition. STEPHEN P. BI:.,.L is a Professor of Biology at the Massachusetts Jns1 itutc of Technology and an Assistant InvestigAtor of the Howard Hughes Medica) Institute. He received B.A. degrees from the Department of Biochemistry, Molecular Biology. and CeJJ Biology and the Integrated Scicnc:es Program at Northwestern Univ~ty and a Ph.D. in biochemistry at the University of California, Herke1ey in 1991. His graduate reseaxch was carried out in the labordtory of n.-. Robert Tjian and focused on euk.acyotic transcription. He did postdoctoral research in the laboratmy of Or. Bruce Stillman at ('.old Spriog Harbor La.b oratory, working on the initiation of cukaryotic DNA replication. f-lis current resB"&ch focuses on the mechanisms controlling the duplication of eukaryotic chromosomes. Professor BcH roreived the 2001 ASBMB-Sr.hering Plough Scientific Achievement Award and the Everett Moore .Baker Mornorial Award for EXet!llCtJce in Undergraduate Thaching at MIT in 1998.

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ALEXANDER GANN is Editorial Director of Cold Spring Harbor Laboratory Press, and a facu lty member of the WaUiou School of Biological Sciences at Cold Spring Harbor Laboratory. He received h is B.~c in microbiology from University College London and a Ph.D. m molecu lar biology from The Universiry of Edinburgh in 1989. His graduate research was carried out in the laboratory of Noreen Murray a nd focused on DNA recognition by restriction enzymes. He did postdoctoral research in the Jaboratory of Mark Ptashne at Harvard, working on transcriptional reguJation. and that of Jeremy Drockes at the ludwig Inst itute of Cancer Research at University C.ollcge London , where he .worked on newt Jimb regeneration. He was a Lecturer at lancaster University. England. from 1996 to 1999, before moving to Cold Spring Harbor Lciboratory. He is co--tmthor (wilh Mark Ptashne) of the book Genes & Signals {2002). MICHAEL LEVINE is a ProfOSS(lr of Molecular and CeJ I Biology at the University of Ca1 ifarnia, Berkeley. and is also Co-Din~ctor of the Center for lntegrntivc Genomi cs. He received his B.A. from the Department of Genetics Cll the Uuivefl)lty of Ollifomia. Berkeley, and his Ph.D. with Alan Garen in the Department of Molecular Biophysics and Biochernistt)1from Yalo University in 1981. A'i a postdoctoral feUow with Walter Gehring and Garry Rubin from 1982- 1984. he studied the molecular genetir.s of lJrosophilo development. Professor Levine's research group currently studies the gene notworks responsible for the gastru lation of the Drosophila and Cionn (sea squirt) embryos. H e holds the F. WiHUt.ms Chair in CcnetiQ. and Ocvelopmertt at the University of California. Beikcley. He was awarded the Monsanto Prize in Molecular Biology nom Lhe National Academy of Sciences in 1996. and was elected to the Amorican Acarlemy of Arts and Sciences in 1996 ant.l tht! NRlional AClidemy of Sciences io t 998.

RICHARD M. LOSICK is the Maria Moors Cabot Professor of Biology. a, Harvard ColJege Professor. and a Howard Hughes Medical [nstitute Profr.ssoc it~ tho Faculty o f Arts & Scienoos at Harvard lluiv&sity. Tfe

received his A.B. in cbemistiy ~t Princaton Univen;ity and his Ph.D. in biot:htnuistry at the Massochusetts Institute of 1echnology. Upou completion of his graduate work. Professor Losick was named a Junior Fellow of the Harv-drd Society of Fellows when he began his studies on RNA polymerase nnd the reguwlion of gene transcription in bacteria. ProfussoT Losiclc is e pust Ota irman of the Departments of Cellular end Developmental Hiology and Molecu1ar and CeJi ular Biology ~t Harvard University. He received the Camille and Henry Dreyfuss Te
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Brief Contents

PART

1

PART

2 7

PAR T

3

ix

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PART

P ART

Brief Omtents

4

5

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Detailed Contents P A R

CHA PT E R

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1 CHEMISTRY AND GENETICS 1

}

The Mendelian View of the World

5

MENDECS DISCOVERIES 6 The Principle of Independent Segr~aunn 6 Box /..- 1 Mendeliafl Latvs 6 Some Alleles Are Nei{hcr Dorrunant Ncr Reccsstve 8 Principle of Independent Assortment 8 CHROMOSOMAL THEORY OF HEREDITY 8 GENE LINKAGE AND CROSSING OVER 9 Box l -2 Genes Are Linked tv Chrvmosomes 10 CHROMOSOME MAPPING 12

CHAP T £R

THE ORIGIN OF GENETIC VARIABILITY THROUGH MUTATIONS 15 EARLY SPECULATIONS ABOUT WHAT GENES ARE AND HOW THEY ACf 16 PRELIMINARY ATTEMPTS TO FIND

A GEN&PROTEIN RELATIONSHIP 16 S..Cmmary l7 .Bibliograpey l B

2

Nucleic Acids Convey Genetic lnformarion AVERYtS BOMBSHELL: DNA CAN CARRY GENETIC SPECIFICITY ZO Vtral Genes Are Al.so Nud c1c Acills 2J THE DOUBLE HELIX Zl Box 2,f Chargaffs Ruk:s 23 Finding the Polyrnerases rhftt Make DNA Z4 Experimental Evidence Favors Str.md Separatmn during DNA Replication 26 THE GENETIC INFORMATION WlTHIN DNA 18 CONVEYED BY ntE SEQUENCE OF lTS FOUR NUCLEOTIDE BU lLDING BLOCKS 28 DNA Cannot Be rh~ Template that Direct ly OrJt..n Amino Acids during Prorein Synthesis 28 Box 2-2 Evidence that Genes C{mr:rol Amino Aod Sequence in Protems. 29 RNA Is Cheuucally Very Similar to DNA 30

19 THE CENTRAL DOGMA 3 J The Adaptor Hypo[ht.-sts of C rick J I

The Test-Tuh~ Synthesis of Pt(l{eins JZ Th(' Paradox of the Nons{'Cdfic-Appcari.ng RibosomL'S 32 Discovery ofMessen~r RN A (m RNA} 33

EI.1.Z)1rnatic Symhesis of RNA upon DNA Temrlarcs 3J Establish ing the Genttic Code J S ESTABLISHiNG THE DIRECTION OF PROTEIN SYNTHESIS 37 Start anJ S top S ignals Are AJso Encoded within DNA 38

THE ERA OF GENOMICS 38 Summary

39

Bibliography 4{)

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xii

Detailed ContonL<:

CHA P T ER

J

41

T11e Importance ufWeak Chemical Interactaons

Snmc lnnic Bonds Arc· Hydrogen Bonds

CHARACTERJSTICS OF CHEMICAL BONDS 41 Chemic<JLBonds Are Explainable an Quantum-Mechanical Terrns 42

Weak Interactions

in rhe 1-'orm of Ener~')' 43 &,uilibrium between Bond Making and Brealdng 4l

TI-lE CONCEPT OF FREE ENERGY 44 K...... b Exponentially RelareJ to aG 44 G.walcnt Boruh Are Very Stmng 44 WEAK BONDS lN BIOLOGICAL SYSTEMS 45 W~k &rtds Have Energies 45

Weal Bonds Arc Consra.ndy MaJe and Broken at Physiological Tt:mperatures 45

C HA P T ER

m Aquet)Wl SoiLitinns 49 Box 3~ I The l rruque,leSS vf Molecular Shapes

and the Ccmcet}t of Srlccl.i~~e Stickmess 50 Orgamc MoJt"Cl.Jle:s that Tend h > f ,mn Hydrogen Bonds Are W~ter Soluble 51 Hydrophobic .. Bon~ • Srabilize Macromolecules The AJvant~es ('If !l.G hetwecn 2 anJ 5 kcal/mol Weak &mds Auach Enzyn't::S to Substrates 53 Weak Bonds Mt..-diale Mosr Prore1n:DNA and Prorem:Pn."'(ein lnrcr~tions 53 1

S'ID'1lmtll)'

The Oisunct\<>tt between Polar anJ Nonpolar Molecules 4 5 Van drr Waals Forc(.'S 46

Hydrogen Bonds

Com plementary

Molecular Surfac~ 48 Water Muk-cules Fonn Hydrogen Bonds 49 Weak Bonds between Molecules

Cht:nm.:al-PronJ fom1
between 1 and 7 kcal/mol

l.Xm~nJ

53

DlhlitWapf\)· 54

47

4

The Importance of High-Energy 13onds

55

MOLECULES THAT DONATE ENERGY ARE THERMODYNAMICAlLY UNSTABLE 55 ENZYMES LOWER ACTIVATION ENERGIES IN BIOCHEMICAL REACTIONS 57 FREE ENERGY IN BlOMOLECULES 58 lli¢1-En~rgy Bonds Hydrolyze with LaJRe Nega( i \le D.U 58 HIGH-ENERGY BONDS IN BIOSYNTHETIC REACTIONS 60 Peptide Bonds Hydrolyze Spontaneously

60

Aclivarion uf Amino Addc; by Attachment of AMP 63 Nudeic Acid Precursors Are AcrivateJ hy the Presence of 0 - G 64 'fht> Vaf,~e of 0 -0 Rd~se in Nudt:te Ac;:ul Synfhesis 64

Q -0 Spliu, Char.:tctetize Most Biosynthelic Reactions 65 Summary 67 Bibliography 6 7

Pos iriv~

llG 6 1 ACTIVATION OF PREClJRSORS IN GROlW TRANSFER REACTIONS 61 ATP Versatiliry in G roup Transfer 62 O

Coupling of Neg.ltive wirh

47

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xiii

Detailed Contents

C HA P T ER

5

Weal< and Strong Bonds Determine Macromol~cular Structure 69 HIGHER..ORDER STRUCTURES ARE DETERMINED BY INTRA# AND

Different Protein Functions Arise frnm

INTERMOLECULAR INTERACTIONS 69 DNA Can Fom1 ;;J Regular Helix 69 RNA Form:s a Wide Varic£y of S tructutts

71

Chemical features ofPrOtt"tn Bvilding Bloch

71

The Pepti~ Bond 71 There A re Four Levels of Protein Structure 71 ex llclices an\.t J3 Sheers Are the Cumnton ronn~ of Seccln\.l<:try Structure 74 Box 5- J rktt.'1'minacion of Pi'Otein S£mcture 75

THE SPECIFIC CONFORMATION OF A PROTEIN RESULTS FROM ITS PATTERN OF HYDROGEN BONDS 78 ex Hdices C-ome Tngethcr to Form Co1leJ-Coils 80 MOST PROTEINS ARE MODULAR, CONTAINING TWO OR THREE DOMAINS 81 Pmte ins Are Composed of a Surprisingly Small Number of Sm1ctural Motif~ Rl Box 5-2 Large Proteius Are Ofc.m Om.sm.Kted of ~'fal Smaller Polypeptide Clwiru Bl

r A Rr

CHAPTER

2

Vanous Dnmain Combinations 82 \VEAK BONDS CORRECTLY POSITION PRO~ TEINS ALONG DNA AND RNA MOL& CULES 84 Protetns Scan alon~ DNA to Locate a Spedfic DNA-Bindi~ Site 85 Div~::rse Stnuegies for Prt:ltc in Recognition o f RNA

86 ALWSTERY: REGULATION OF A PROTEIN'S FUNCTION BY CHANGING ITS SHAPE 87 The Structural fhsis of A llosteric Rcgl,lation Is

Known for Examples Involving Small ligands,

Pmtein-Pn.ltem lnrer3Ctions. and Protein Modification 88 Nut AU Regulation of Proteins 15 Mediated by Allosteric Evem:s 91 Summary 91 Bibliography 92

MAINTENANCE OF TI-IE GENOME 93

6

The Srructures of DNA and RNA 97 DNA STRUCfURE 98 ON A 1~ Compo~ct! of Polynudcotide Chains

98

The Majnr Groove b Rich in C.herrtical Inf(lllTI:\tion 103

Eoch Base Has lts PreferreJ Tauwmeric fonu The Two S uanJs nf the Duuhk Helix An: HdJ Together by Rase Pairing in an Antiparall ~l Orwntation I 00

I 00

Bvx6~1

DNA Has 10.5Base PairsperTum of the Hdtx in Solut.Um: The Mica Experiment

I04 Tht Double Hdix Exists in Mulrirle C'..onfonnarinns 106

rum a Left-HanJ£...J Helix

The Twn l':hHiru uf the Duublt= Helix Ha ve

DNA Can Sometimes

Complemental)' Sequences 101 l Iydrogen Bonding Is lmportant for the Srccafictt~' of Base Pairing

ONA Strands Can Separate (Denawre) and Reassociate 108

102

Snmc DNA Molecules Are Circles

107

11 1

liascsCan Flip Out fmm the Double Helix 102 ONA TOPOLOGY 111 DNA Is Usually a Right-Handed l.Xluble Helix lO) Linking Numkr Is an lnvarianr Topological Pm~rty The U>uhle Helix H~ Minor cmd Major Grooves Im of Covalently Closed, Circulru- DNA 112 NOT FOR SP.LE

xi11

Detailed Conte11ts

Lmkmg Number Is Com~d of Twist and Writhe 11 Z Lit Is the Linl
114

DN A tn CeJis Is Negatively Supercoile.J

114

Echnlium lons Cause DNA to Unwind 120 Box 6~2 Proving that DNA Ha.s a Helical Periudiary ufalxm.( 10.5 Base Parrs per Tw11 frum the Tvpulvgtcal Prupa ues of DNA Rings 12 r RNA STRUCil.JRE 122 RNA Contams R1bose and Uracil

Nuclcosomes Introduce Negattve Supercoiling m Eukaryotcs I l 5 1opotsomt:rasc:s C..<~n Relax Supcrcoded DNA J15 Prokaryorcs 1-lave a Special TCl,roisorucrasc rhar: lntrOt.luces Su~rcClils into DNA 11 6

and l~ Usually Single-Stranded 122 RNA Chains Fo ld Back on Themselves t(')Fnnn Local Regtons ofDouhlc Helix S imilar to A,fonn DNA t l3

TCipoisom~rdses also

RNA Can Fold U p tnto Complex Terti9ty Stmctures

Unknot

and Discm:a~le DNA Molecules 11 7 Tor oi50fllerases Use a Covalent Protein-DNA Ltnkage w Cl~ve and ReJoin DNA Srrarnls 118 Topoisumemses f flrm an Enzyme Bridge anJ Pass DNA SL'f!mcnts [hwugh Each Other 118 DNA Topoisomm Can Be Separate-d hy Elcctrophorcsi~

CHAPTER

120

124

Some RNAs Arc Enzymes 125 1l1C Hammerhead RilX~Zyme C leaves RNA by the Fonnation ({ «\ Z', 3' C yclic Phosphare

I 25

Did Life Evolve from an RNA World? 126 Summary 126 f3ibl~h)'

r 27

7

C...hromosomes, Cf:uom,uut, and the Nudeosome CHROMOSOME SEQUENCE AND DIVERSITY 130

C b(omosomes Can Be C 1rcular or Linear 0 0 Every Cel1 Maintains a Chru-acteristic Numhcr of Chromosomes 131 Gen umc Size Is RdMed to the G-Jmplcxity

119 Ch romosome Stn.~cture Changes a~ Eul<ary(1tic Cdb Divide 143 Sister Chromatid Cobl">Sion and Chromosome Condensation Arc Mediated ~y SMC Proteins Mitosis Ma mr~ins the Parental C hromnsome Number

144

146

The Gap Phases of the C'..cll C ycle A lluw lime to Prepare fClr the Next Cell C ycle St
of the Organic;m 111 The E. coli Gen01ne Is Comrosed almost Entirely

netai1P.d Contents

Many DNA &!QtJ~n<:e- 1ndcpenJenr C...ontacrs MeJiate cite lntt:raction between rhe O>rc llistoOt$ and DNA l56 The Hisrone N~Tenninal Tails Stabilize DNA Wrapping around the Octamct 159

Nuckosome Remodcllng Complexes Facilit ate Nudc<'lSomc Movement 166

Somt: Nud cosomes Are f~,und in Spcofic Positions in vrvo: Nudeosome Positioning

l68

MoJUica.u on of the N -Terrnanal Tails of the H isrones Alters Chromatin Accessibility 169

HIGHER-ORDER CHROMATIN STRUCTURE 160 Histone H 1 BinJs to the Linker DNA between Nudwsomes 160

Bux 7-2 Detrrn1inmg Nudrowme Positum in the Cell 170

Nudeosome Arrays Can Form More C omplcK S u uctuTes: the 30-n m Fiher 161

Specific Enzymes Are Responsible for Histone Modification 17 1

The Histone N-Terminal Tails Are Rct.Juired for rhe formation of the 30-nm fiber 162 FurrhcrCornpaction ofDNA Involves Large Loops of Nudeosomal DNA 162

Nuclt...'OSOO'le Modtfication anJ Rert'lodding Work TogethtT to lncrei!St! DNA Acccssil->ilny 174 NUCl.EOSOME ASSEMBLY 175

Nude()SOffiOi Are Assembled rmmcdi;:uely after DNA Replication 175

Histone Variants A lter Nudeosome Function 163

As:semhly cJ Nuc:IL'(,..;omes Req uires Histone "Chaperones" 176

REGULATION OF CHROMATIN STRUCTURE 165 The lnrentetion of DNA wi.rh the Hist()lle Octamet Is Dynamic

C H APT~R

Surnmmy

I19

Bibli()~fll>h)'

180

16S

8

The Replication of DNA

181

THE CHEMISTRY OF DNA SYNTHESIS DNA Synthesis Requires Deoxynudcoside

182

T riphosphates and a Primer:Template Junction

182

DNA ls SynrhesizeJ by Extend ing the 3' End of rhe Primer I 83

Hydrolysis of Pyrophosphates ls the Dnving f orce for DNA SymhesL~

183

THE MECHANlSM OF DNA POLYMERASE 184 DNA Polymerases U:sc a S ingle Ac tive Site ro Catalyze DNA Synthesis 184

DNA Polymernses Resemble a Hand {bat Grips rhe Primer.Temrlare Junction 186 DNA Polymerases Are Prncesstve Enzymes 188 Exonudeascs Pr<x:trcad Newly Synthcsa.red DNA 191 THE REPLICATION FORK 192 Rorh Stronds of DNA Arc Synthesized Together at the Replication Fork

RNA Primers Must Be Remov<..J to Complete ON A Rt.•plicarion 194 DNA Hdlcases UnwinJ the Double Helax m AJv:mce of the Replkation FoTI< 194 Singk -StranJal Binding Pmteins Stabilize Singl.,..... Strnnded DN"A Prior to Replication 195 Bo.>. 8, l Determining the Pvlarity

of a DNA Helicasc 196 Toroisomerast::s Remove Supcrcoils Produced by DNA Unwmdn.1.g at the Rt•pbcarion Fork 198 Replication Fork Enzymes E~tend rhe Rttn~ o f DNA PolymcrrtSC Substr.nes lW

THE SPEClALIZATION OF DNA POLYMERASES 200 DNA Polyrr•ernses Are Spe.cializeJ for Different Roles in the Cell 200

Sliding Cl~m~ Dratrutti.c.:ally ln.crease DNA Polymerase Proces5ivity

192

l11e Jniriarion of a New Strant.l of DNA Require; an R NA Primer 19 3

201

Sliding C lamps Are Opened
NOT FOR SALE

xvi

Detailed Contents

DNA SYNTHESIS AT THE REPLICATION FORK 205 Box B~l ATP Cot~trol uf l'rlJfein Function: l..orutng P Sliding Clamp 206

Eukaryotk Chromosomes Are Replicated Exactly Once per ( :ell Cyclt- 223

lnteracrions berwt:en Replicanon Fork Proteins Form the E. coli Replisome 110

Pre-RC Fo nnarion ami ActivatiClfl 1~ Regulated to Allow only a Single Round of Replication during Each Cell Cycle 225

INITIATION OF DNA REPLICATION

Prc, Rcrlicativc Complex Fonnation Directs the lnitiation of Replication in Eukacyot:es 223

212

Spec ific Genomic DNA &-qucnces Direcr the (nihation uf ONA Rrplicatton 212

Stmilari[ies between Euka ry<Jtic and. Prokaryork ONA Rcplicatiun lniti:-ttiun Z28

The Replicon Model of R~.::plication lniriation l l l Rcplicatur Seq~nces Include lniriaror Binding Sites and &lsi)y Unwound DNA 211

FINISHING REPLICATION 228 Typt' II TuJX.liSUmt.·mses A rt:: Requued tn Separarc Daughtet DNA Molecules 228

BINDING AND UNWINDING: ORIGIN SELECTION AND ACTIVATION BY THE INITIATOR PROTEIN 214 Bux 8-3 The Identification ofOrigins of Replication and Replicawrs 2 I 4

l aggint; Strand Synthesis Is UnaHe to Copy rhe Extreme Ends of Linear Chromosomes

Protdn,Pr()(cin and Protcin,DNA Interact ions Direct the Initiation Process 217

Bo.xB-4 £.coli DNA Replicaticm Is Regulated by DNA· ATP Let!els and SeqA Z17 Box 8-5 The Replicacivn Factary HypodteStS 221

C H APT£R

1Clomerase ls a Novel DNA Polymernst." th
229

:no

Telomerase Solves the End Replicarion Problem by Extending the 3' End of the Chromosome 132 Summary 132

Bihlillgl"aph) 23-3

9

The Mutal...,ility and Repair of DNA 235 REPLICATION ERRORS AND WEIR REPAlR 236

REPAIR OF DNA DAMAGE 246 Direct Reversal of DNA Damage 147

The Nature of Mutations

Ba.~e

236

Some Replication Errors Esc.:pansiDn of Tritlle Repwt5

Excision Repair Enzymes Retr10ve Oanu:tgeJ

Bases by a Base·Ftipping Mechanism 248

Nudet)tide Excision Repair Enzymes deavt Damaged

Clluses Diseo.se 237

ON A on Either S i"d e of the lesion

250

M~match

Recombtnarion Repa irs DNA Breaks by Retriev1~ St-qucnce Information from Undamaged DNA 253

DNA DAMAGE 242

Translesion ONA Symhe;as Enables Rcplication

DNA Undergoes llamagc Spont
w Proceed across DNA Damage 254 Bux 9-3 TheY-FamiLy of DNA Polymerases 25(1

Box 9,2 The Ames Test 243 DNA Is Damaged by A lkylation, Oxidation,

Summury 257

Rep
and Radiation Z44 Mutations Are also Caused l:ly Base A~logs and lnt~.::rcalating Agents 24S

Bib~Wgra~

NOT FOR SALE

258

Detailed Contents

CHA P I E R

xvii

}Q

Homolog()U.S Recombination at the Molecuhu Level 259 MODELS FOR HOMOLOGOUS RECOMBINATION 259 The Holliday Model Illustrates K~·y Steps in Homologou~ Rl.>combination

260 Tht:> Double~trand Break Repair Model More Accurately Describes Many

Recombination Evencs 264 Bux l 0-l Huw w Resolt.e a RocumhmatiJJn httermediate ~lith T~·o I fulliday ]unctiuns 266 Douhlc,Suaml~i DNA Breaks A rise b}· Numcruus Means and lnitiare Homologous Rccombinarion 267

HOMOLOGOUS RECOMBJNATlON PROTEIN MACHINES 268 The Red3CD l lelkase/Nudease Processes Broken DNA Molecules fo.- Rccombinatlon 269 RccA Protein Assembles on Single·Strancled DNA and Prom('l{esSmmd 1nvasion 272

Newly Base,Paired P~mners Are Established within rhe RecA Filament

Ret:A

Homol~

274

Are Prcscm

in All Ormmisms

275

RuvAB Complex Spedfically Recogn izts 1 JollidC'ly Junctions Rnd Promotes Branch Migration 276 RuvC C leaves Specific DNA Strands at the Holliday Juncrion ro hnish RL-comhination 276

C H AP 'I !-R

HOMOLOGOUS RECOMBINATION IN EUKARYOTES 278 Homologous Recombinat ion Has Additional Functions in Eukacyotes 278 Homo logous Recombination Is Required for Chrotnosorne Segregation Juring Meiosis 279 Programmed Generation of Double-Stranded DNA Rrcaks Occurs duri n~ Mci<.'lSis 179

MRX Protein Processes the Cleaved DNA Endsfor Assembly of the Rec:A,like Srrand-Exchange Proteins 282 Dmc r Is a RccA-1ikc Protein that Spcdfically Functions in Meit-,ric Rccoml:linat.ion 182

Many Proteins Function Togerher to Promote

Mdotic Recombination 284 MATINGMTYPE SWlTCHING 285 Maring~Type Switching ls lnitiatcd by a Site-Specific 'D oub(c,Strand Break

Mating-Type Switching Is a Gene C o nversion Event, Not Assodat~d with Crossing Over 286

GENETIC CONSEQUENCES OF THE MECHANISM OF HOMOLOGOUS RECOMBINATlON 288 Gene Conversion Occurs because DNA Is. Repaired during Recombination

289

Summary 290 l:3ibliogrctph;J1

2Y1

11

Site-Specific Recombination and Transposition of DNA CONSERVATIVE SlT&SPECiflC RECOMBlNATJON 294 Sit~,Speci6c Recombination Occurs at SpL.ocific DNA Sequ~nccs

286

in the T~ct DNA 294

Site-Spec inc Recomhm ases C lcavt:• and Rejoin ONA Using a l'..t.walt>nl Protein;DNA lnterrt:lt!diare 296

Serine Recombinases Introduce Doublc ...Stranded Breaks in DNA and then Swap Strands ttJ Promntt: Rt:COmhinalion 2.98

T,rrosine Recomhina.scs Brc-.\k anJ Re j0in One PaiT of DNA Str
293

Structures of Tyrosine Recombinases BounJ to DNA Reveal the Mechanism of DNA Exchange 300

Box Il -l Applicauun of Site-Specific RerombiMticm to Genetic Enf!.ineeriltl! 302 BIOLOGICAL ROLES OF SIT£.SPEClFIC RECOMBINATiON 302 ~ lnu.wasc Promotes rhc Integration and Excision of a Vital Genome into the Hoo- CeH C hromosome Phage 'II. Excision Requires a New DNA-Beruli~ Protein

NOT FOR SALJ:

304

303

xviii

Detailed Contents

Th~ Hin Rccomhinase lnverts a Segment nfDNA AI·

DNA Transposaset; and Retmvtrnl l ntegtases Are

lowing E.xprc.ssmn of AltcrotittVe Gt:nes 305 Hin Recombination Requires a DNA Enhancer

Mt:mbcrs of a Prote-in Superfamily 321 Box I 1~2 The Path4Aiay vf Rer.rt.Mtul

306

eDNA Formation 322

Recombinases Convert Mulrimeric C ircular DNA Molecules into Monomen- 307

Poly~A

There Arc Orh~r Mechani5ms to Direct R~comh1.11.a tion ro ~cific Se~mc:mts of DN A 110

TRANSPOSITION 310 Some Gt!ne« ic Elements Muv-e to New Chromosomal Locations by Tra.nspositaon .31 0 Then: Al:e Three rrincip;.,) C lasses t,t Trnnspo5<11"1te El~ emcnts

311

EX.fu\-fPLES OF TRANSPOSABLE ELEMENTS AND THEIR REGULATION 327 IS4~Famlly Transrosons Are 01mpact Elc:ments

with Multiple Mechanisms for Copy Numher Conrrol 327 Box I I ~3 M aize Elements (lnd the Dascovery

ro Celluhlr DNA RcpUcatiCin 329

Tran.sposon." & ist as 11nth A uwnurnno~ and N(1ftaUtOOOffiOU5 Elements 3

n

Viral-lik~ RetrNrnn~-posoos and Retroviruses Carry

Terminal Repeat Sequences and Two Genes Important tu:r Rccomhination 31J

PoJy-A Ret rotransposorus Look Lrke GC'Ilf:s 314 DNA Tr.m~irion by a C'ul· 314

The lnteJtnl'\.Hatc in C ut-and-Paste TransJX!5ttion Is Finished. 11y Gap Repair 316 Tl1ere Are M ultiple M~ch
DNA Tmnsrosit ion bv a Repliamve Mt.'CI1~msm

314

v[Turnspowns 328 T nW TrartSJX'I'Siti.o n Is C'...oopled

DNA Transposons Carry a Transpnsase G ene, R an l ed hy Recomb nation Sires J J2

~nd~Pasre Ml:'chAnism

Rerronansposons Move

by a «Rcvef':)t;' Splicing" Mechanism

118

Vintl~ lik~ R etrotrClnSfol1SOOS and Retro viruses Move

Phage Mu Is an Extremely Robust Transposon 311 Mu Uses Target Immunity r:o Avoid Transposing into Irs Own DNA l3 1 Tc 1/Muriucr Element:, Are Extremely SucceR~ful DNA Elements in Eul<aryotes 334 Yeast Ty Ekmcnts Transpnse inm Safe Havet'lS in the Gen ome 33'i LINEs Promote Their Own Transposition and Even Transpose Cellular RNAs JJ6 V(O)J RECOMBINATION 337 The Early Events in V(D}] Recombination Occur by Mechanism Similar ro Traruposon Exciston 3 39 Stmtmary 342

BiMiograph:y 342

Usmg an RNA lnram(.....li
p A R T

C H A PTE. R

3

EXPRESSION OF THE GENOME 343

12

Mechanisms of Transcription

347

RNA POL\'MERASES AND THE TRANSCRIIlTJON CYCLE 348

Transcription Initiation Involves

RNA Polymerascs Come in DJfferenr Forms, but Share Many f"eaturc.~ '348

Three Defined Steps 351 THE TRANSCRIPTION CYCLE lN BACTERIA 353

TranscriptiOfl hy RNA Polyme•-a ~ Pn.ceeds in a Series of Steps 1 SO

&dCterial Promoters Vary in Snength and Sequence, hut 1{~ve C ertain Denning Features 353

NOT FOR SALE

Oetailed Contents

The
Tr..nsitioo to

TBP Binds to and Otstorts DNA Using a f3 Sheet Inserted into rhe Minor Groove

the Promoter 354 Box 12, 1 Consensus Sequences 355 to

th~ Open

C'...omplcx Involves Srn,telural Cha.nt,rcs in RNA Pt1,ymtr.tSe aO<.t in the Pr01'11C'ttt DNA .156

366

The O ther G eneral Transcription F~tors also ~ve Spec16c Roles in Initiation 367 In Vivo, Transcription Initiation Requires Addittonal Pmteins, Including the McdiarorCotnplex 368

Transcription Is Jmtaated bv RNA Polymerc'lse without the Nt.'t."<.i for a Primer 358

Mediator Consists ci Many Subunits. Some Conserved frClm Yeast to Human

RNA Polymerase Synthesizes Scvcml Short RNAs hefCltl' Entering the Elongation Phase 358 The Elongating PolymcrdSC l s a PfOCes:$ive Mat:hine

A New Ser of Factors Stimulate Pol 11 Elongauon t1nd RNA Proofreadin~ 370 Elo ngruing p~..,Jymetase ls Associatl.'1.1 with a New Set of Protein Factors Rcqutrt::d for Various Types uf RNA Processing 371

that Synrhe~izes and Proofreads RNA 3 59 Bvx 11-2 The SinJ,-le-Stdnmit RNA Polymero.ses 360 Tran..o;criptit'ln Is Tcrrnimned by Si~~als within the RNA Sequence 361

TRANSCRIPTlON IN EUKARYO'I'ES 363 RNA Polymerase ll Co.-c Promoters Are Mlldc up of Com11inations of Fo ur Different Sequence Elements 363 RNA Pdyrncrasc U Forms a Prc-ln it;ari.oo Complex

369

RNA Pnlymernses I and 111 Recognize Oi~tinct Promoters, Using Distinct Set., of1ranscriptic.)n Factors. but srill Require TBP 37-4 Samunary .176 Biblw~h1

377

wtth Gcncr.tl Transcript ion facwrs at The Prom()[er 364

CHA P T E R

}3

RNA Splicing 379 THE CHEMISTRY OF RNA SPLICING 380

Group l Introns Release a Linear lntron Rad1er

St.'quence~

than a Lariat 388 Box 13- I CQTlvercrng GnAtP l lntrons

within d1e RNA Determine Where Splicing Occurs 380

The lnrron Is Removed in a Fcnn Called a l ariat as the Flanki~ Ex<Jns Are Joined 381

Exons f:.U.n Differenf RNA Molecule:, Can Be Fust.••d by Traru;-Sphc ing 383

THE SPLICEOSOME MACHINERY 383 RNA SplK:ing r~ C.arrieJ Out by a Large COOlPlrx

Cal\cd the Splic~mc 383 SPLICING PATHWAYS 385

Can Catalyze RNA Splicing

187

inw Ribuzymes 389 How Docs the Spliceosomc Find rhe Splicl' Sires Reliahly? 39J ALTERNATIVE SPLlClNG 394 S ingle Gent'S Can Produce Multiple Producrs by Alrern;nivc Splicing 394 A ltt:rnariw Srl icin~ U; Regulated by Activ~tors and RcpressoTS 396

Box I3~2 Adenvvims amt the Disc{)4Jery v[ Splicing 398 A Small Group of lnt:rons Are Spliced by an A ltetnatiw Splicea;ome Composed NOT FOR SALE uf a Different Set of snRNPs 400

Assembtr. Rearrangements, and Catalysis Within the Spltt;:eosome: the Spiking P::~rhway lRS Sdf,SpJtc ing lntrons Reveal that RNA

xix

EXON SHUFFLING 401 E:xons Arc ShuftlcJ hy Rtt:t.lfTlhination

mRNA TRANSPORT 406 Once Proce..f.St'l1. mRNA J.s Packaf!eJ anJ Exported from rhe 1\;ud eus illl
to Produce Genes Encndmg New PrCitems 400

406

RNA EDITING 404

Summary

RNA Ediung Is Another Way of A lrermg the.: Sequence of an mRNA 404

CHAP TER

4{)8

Biblif.IBTOPI'l' 409

14

Transl~tion 41 1 MESSENGER RNA 412 Po lypeptide Chams Arc Spc..-cificJ by Oren,Reahng Framl!S 412 Prokarymic mRNAs Have a Ribosome Binding Sire that Recruits the Transl~tion<JI MachineJ)' 413 Eukaryot1c mRNAs Are Mo1.Hfied at Their 5' and V EnJs ro Facilitate Translation 414

TRANSFERRNA 4 15 tRN As Are Adaf'(ors between Coc.Jono; and Amino Ac ids 4 15 tRNAs Share a Common Secondary S nucwre that Rc-st:mhlcs a C loverleaf 4 16 tRNAs ll::t vc an L,Shaped Three·Di mensiona I Structure 417 ATTACHMENT OF AMINO ACIDS

INITIATION OF TRANSLATION 432 Prok~ry,lt ic mRN As Are Initially Rt.-cruirro w rhc

TOtRNA 417 tRNAs Ate C hargcJ hy the A ttachment t::rf an A mino Add to the l' Tenuinal Adenosine Nudcoride via a lligh· Enefl!Y Aql Lmkf.,'n ize Umquc StruccurCtl Fe-cttures of Cognate lRNA~ 420 Aminoacyl·tRNA f onnation ]s Very Accurntc 421 Some Ami.noocyl tRNA Symhet
THERlBOSOME 423 The RiJx,somt> h. C.omposcJ of a Largt. and a Sm~ll Subunit 425 Tiu:~ Larr,JC and Smr~ll Subun1ts Undergo Associ
each Cycle ofTranslatino

425

NO~

New Amino Acids Are Attach~.J to the (:.Terminus 0f tht:: Growm~ Polypeptide Chain 4l 7 Per tide Bonds All: Fmmt..-d 11y Tmn~fer of the Growing Polyperr:ide Cht~in from One tRNA to Another 428 Rahu..<.omal RN As Arc Both S tructural and Catalytic Detcnnmant~ of the Ribosome 428 The Ribc:J5om~ Has Thrt!C Binding S;res for tRNA 429 Channel ~ rhrough the R1bosome All0w the mRNA anJ On.,wmg Polypcptid~ to En ter <md/m Exir the Rabmome 430

Sm'(lll Sul-1unit hy 8~-Pairing to rRNA 433 A Srec•allZcd r.RNA Charged with a Moc.lifieJ Me, rhaonme BinJs Oirt.-ctly ro the Prokaryotic Small Suhumt 433 Three ln1riatioo Faaots Direct rhe Assembly nf an In itiation Comrlcx th~t Cont3it"\S mRNA and the Initiator tRNA 433 Eubryotic Ribosomes Are Recruit~ to rht: mRNA by rhc: S' Cap 435 The S ran Couon Ls Foum1 ~y Scanntng Oownsrr~~m fmm t he 5' End of the mRNA 437 Translation Jnuiatim Facturs I lold Eukaryonc mR, NAs in Circles 438 Bux 14~2 uORf.s Cttld IRESs · £ xceptwns thac Prm•e the Rule 439

TRANSLATION EWNGATlON 440 Aminmtcy(,tRNA~ Are- Od ivcreJ ro the A S ite

by Elongatton Fa... tur 8~-Tu 441 The Rabosnmc Uses Mulnple Mechanisms to Select ~ainst lncorrect Ami.noacyl~ tRNAs 441 The Ril'I()S(llnc Is a Ribozymc 442 Pepnde BonJ Fonnatton anJ the Elongat ion Faccor EF~ Drive Translocarion Llf the rRNAs and the mRNA 444

FOR SALE

f>etailed Contents

EF-G Dr;vf'S Tnm:;location by Displacing the tRNA Bound to the A Site 445 EF-Tu ~C DP

GDP/GTP Exchange and GTP Hydrolysis Contro l the Function of the Cl~s 1l Rele-~se f actor 450 Tile Ribosome Recycling Factor Mimics a IRNA 450

aru.l Ef-G,QDP Must Exchang~ GDr

for GTP Prio£ to Partici{'atl~ in a New Rt1Und

of Elongauon 446 A Cycle o( Pepti<.k Bond Fon ni.'\tion l ..()nsumes lwo

Molecuk"S of GTP and One Moltt:uie of ATP 446 Box 14,3 OTP-Binding Proteins, Conformational St!!itch~. and t11e Fidelity o.nd OrdeiiJlR of the Evcr1ts ofTl-an.<~latwn

447

TERMINATION OF TRANSLATION

448

Release F"delOrs Te nninatt' Transh-•uon tn Respon~ lO

Smp CoJons 448

CHAP T [ R

TRANSLATION,DEPENDENT REGULATION OF mRNA AND PROTElN STABlLITY 452 The S..,T A RNA Rescues Ribol,(Jtues that Tmnslate Broken mRNAs 451 Box 14-4 Anrihiorics Anesf C.eiL Divi..c:i(m hy Bloding Specifo: Seeps in Tmnslnnon 453 Eukaryotie Cel1s Degrade mRNAs that Are Incomplete or rhat Have J"rematurt:' Stop t:OOons 456 Scanmary 458

Short Rt.oogions of Class I Release Factors Recc:Jgnizt Stop Ct~1ns and Tn~er Rdc:-dllt: of tht> Pertidyl Ch~ln

xxi

BihtWgrctph.) 459

449

15

The o~nctic Code 461 THE CODE IS DEGENERATE

461

THREERULESGOVCRN THE GENETIC CODE 469

Perceiving Ordcc in the Makeup

of the CoJe 46Z W~ble tn

the Ant1coJon

Three Kinds ofPoinr MtJttttaons Alter 463

Three Codons Dire(:t Chain Termination 461 How the Cod ~ Was Cracked 464 Stimulation of Amino Acid lncorporatlOn by Synrhetic- mRNAs 465 Poly,U O:xks for Polyphenylal~ine 466 Mixed Copolymers Allowed AJdi& iClrLCIL Codo n Assi.gnmenrs 467 Transfer RNA Binding to DenneJ T rinudt'
p A R T CHAPTtR

4

the Generic Code 4 70 Genetic P£<.'l0f th~t the Code Is ReaJ in Units of Three 47 1 SUPPRESSOR MUTATIONS CAN RESIDE JN THE SAME OR A DIFFERENT GENE 471 lntergenic Suppres.<;iotl Involves Mutant tRNAs 472 Nonsense Suppre.ssoJS also ReaJ Nom1al Terminauon Signals 474 ProvifiR the Validity of rhe Geneuc Code

474

THE CODE IS NEARLY UNIVERSAL 475 Summary 477 Riblivgruphy 477

REGULATION

479

16

Gene ReguJation in Prokaryoteb

483

&•me Activator5 Work by A llostery and Regulate PRINCIPLES OF TRANSCRIPTIONAL Sfers ~ frer RNA Polytucta:se BinJing 485 REGUlATION 483 G ene Expresston ls Conn olleJ Action at a Distance and DNA Looping 486 by Regulatory Proteins 483 UX'l~rative Binding and A llosrery Have Man y Roles Many Promoters A re Re~ula ted by Acri\IHWrs 1ha[ &tl Gene Regulation 487 Hd.p RNA Polymet
xxii

Detailed Conte11ts

REGULAT ION OF TRANSCRIPTION INITIATION: EXAMPLES FROM BACTERIA 488

Rihosom~l Ptoteins Are Tmnslational Repre$SOfS

An Activator and
THE CASE OF PHAGE~

ofThetr Own Synthesis 506 Box 164 Ribos1wicches 509

CAP and Lac Repressor Have Opposing Effects on RNA Polymerase Binding to the lac Promoter 489 Box 16, 1 Decocting DNA-Rinding Sites 490 CAP Hcts Separate Activating

and DNA,f\inuing Surfac.es 492 CAP and l ac Repressor Bind DNA Using a Common Structural Mmif 493 Dox 16--2 Acrivar.or Bypass Experiments 493

The Act ivt ties of Lac Repressor and CAP Are Controlled Allostencally ~y l11eir SiJ:!,Jlals 496

Box 16-3 ]acoh. Monod, c.tn4 the Ideas Behmd Gc>m' Regulation 497 Cornbinatoricel Connd: CAP C'..ontrnb

LAYERS OF REGULATION 512 Alternative f>Bnerns of Gene Expression Control Lytic and LySL'>genic Growth 513 Regulatory Pro tetns and Their Binding SiLes }.. Repressor Binds to Oper.~tot Sir~ Cooperatively 515 Bro. 16,5 O:mcemrarion, Affinity,

and Coope-rlltive Binding 516 Repressor and C ro Bind in Different Patrerns to Gmrrol Lytic and Lysogenic Growth 517 Lysogenic lnuuc1ion Rel.)uires Proreolytic Cleavage o f>.. Repressor

518

Negauve Auroregulation d Repressnr Requirdi Lur~.g- Oistance lnr~tinns and a

Other Genes As Well 499 A ltemalive u Factors Oitt:<,t RNA PolymerdSe to A lternative Sets ofPr0motcrs 499 NnC and MerR Transcriptional Actavators that Work hy A llostery Rather than hy Recruitment 500

NtrC Has ATPa.se Acuvity and Works from DNA

Lart;e

DNA Loop 51 9 Another Anlvator. Adl, Controls the Dec~ion between Lyric ar1d Lysogenic Crowrh upun Infection of::~ New Host SZO &x l (),6 Generic Approaches rlutt ldema/ietl Gene~ fnwltJCd in the Lyticfl.y~">genic Choice 52 1

MetR Acrivate:s Tr.anscripti.Dn by Twisting Promoter DNA 501

Growth Omdition:; of£_ coli C onrrol r.he Stabilety of C.U Protein and thus rhe Lytic/Lysogenic Chnic.e 5ZZ

Some Repressors Hold RNA Polymerase at the Promoter Rather rhan Excluding It

T ran~criptional Amat~nnination in }.. Development 523

Sit.es Far fiom the Gene

500

S02

AraC aru.l Contro l iJf the araBAO C)peton hl· Antiactivation 503 EXAMPLES OF GENE REGULATION AT STEPS AFTER TRANSCRIPTION INITIATION 504 Amino Acid Biosynrhetk Opemns Are O.Jnttolloo by Premature Transcriptio n Termirutti.on )().4 LHAP II: t<

514

Retrore~ulation:

An Interplay (lf Comrols on RNA Synthesis anu Stability Derennanes in Gene Expression 524

Summary -~25 Bib&gTClphy 526

17

G~ne Regulation in

Eukaryotcs CONSERVED MECHANISMS

SZ9 Eubrvotic

Use a Range of ONA-Bitli.ling Domains.

OF TRANSCRlPTIONAL REGULATION FROM YEAST TO MAMMALS 531 Acttvawrs Have Sepamte DNA Binding and Activating Functions 511 Box 17, l The T
Regul~rors

but· DNA Recognition Involve; rhe Same Principles as Found m Bacteria

534

Activatms-; Regit.IOS Are Nct Well-Defined Stn~t:tures

NOT FOR SALE

536

Detwled Con rents

RECRUlTMF..NT OF PROTEIN COMPLEXES TO GENES BY EUKARYOTIC ACTIVATORS 537 Activatu:s Recruit the Tran~r iptional Machiner y to lhe Gene 537

Activator:, .md Repressors Somet imes Come in rieces 555

GENE "SILENCING'' BY MODIFICATION OF HISTONES AND DNA 556 Sil~ndng in Yeast Js Media ted by Deacerylatio n

Box 17-2 Chmma[Jn lmnumopreapitation 539 Activators ~l so RecTl.Jit N udeosome Modifiers th:c~.t Heir the Transcnption Machinery Bind «''t the Promoter 540 Action at a Distance: Luot>S attd Insulators 540 Appropriate Regulation ct Some <3roups <..l Genes Requi£es Locus C'..omrol Regiuns 543 SIGNAL INTEGRATION AND COMB1NATORIAL CONTROL 544 Activators Work 'together Syn ergistically to Integrate Signals 544 Signal Integration: cl'lc HO Uene Is Controlled by Two Regull:\tors; One Recruits Nudcosome Modifiers and the O ther Recruits Mediator 546 Sagnallntegrnt ion: Coopenttive Binding of Activators at the Human ~Interferon Gene 546 Combinatorial G.)ntrol Lies a t the Hean ofthe Complexity ami Diversity ufEukaryotes 547 Combinatorial Control of the Mating~Type Genes from Saccharomyce$ (:er~ 548 TRANSCRIPTIONAL REPRESSORS 549 SIGNAL TRANSDUCTION AND THE CONTROL OF

TRANSCRIPTIONAL REGULATORS 551 Signals Are Often Communicated to T ranscriptiooal Regulators lhrough Signal Trans, duction Path wc:1ys 55 I Signals Control the Activities ofF...ukaryotic Transcnptional Regulators in a Van cty of Ways 552 C H APTER

anJ Methylation of Histooes

556

Histone Mooitications and rhe H istone Code Hypothesis 558 ONA Methylation Is Associated with Silenced Genes an Mammalian Cells 558 Some Stares of Gene Expression Are Inhelire.d through Cell Division even when rhe Initiating Signal Is No Longer Present 560

Box 17-3 A Lysogens atuf rh.e Epigenetic Switch 562 EUKARYOTIC GENE REGULATION AT STEPS At1ER TRANSCRIPTION INITIATION 562 Some Activawrs Control Transcripuonal Elnngatton rather than Initiation 562 The Regul~tion of Alternative mRNA Splicing Can Produce Di«erent Prote in Ptodocts in Different Cell Types 56] Expression of the Ye-&t Tran.<;Criptiorutl A( tivawr Gcn4 Is Controlled at rhe Level ofTranslation 565 RNAS IN GENE REGULATION 567 Double~Srr.mded RNA Inhibits E."'rression of Genes Hmndogous lo rha1 RNA 568 Short Jmetfering RNA:i (siRNAs) Are ProduceJ from JsRNA and Direct Machinery thar S witches Off Genes in Varit 1us Ways 568 MicroRNAs Controlrhe Expression of some Genes during Development 570 Summary 571 Bibl.K>gTaph.)l 572

}8

Gene Regulation Juring Development 575 THREE STRATEGIES BY \VHICH CElLS ARE INSTRUCfED TO EXPRESS SPEClFIC SETS OF GENES DURING DEVELOPMENT 576 Sonae mRNAs Become l ocalized withi.n Eg~s and Embryos due ro an lmrinsil: Polarity in the C ytoskelet
xx1li

576

Cell ~ to--Cell Contac{ aru.l Secreted \~II S ignahnJ!

Molecules both Eliot O.anges in Gen(' Expression in Neighboring Cells 576

Box 18-I Microarray Assays: TIU!Or)' and Practice 577 Gradients of Secreted Sign::tlmg Molecules Can Instruct Celb. to Follow Different Pathways of Development Med on ll1eir Loca•ion 578 EXAMPLES OF THE THREE STRATEGIES FOR ESTABLISHlNG DIFFERENTIAL GENE EXPRESSION 580 l11e l ocalized Ash 1 Repressor Omrrols Muting Type in Yeast by Silenc ing rhe HO Gene 580

NOT FOR SALE

100\'

Detailed Contents

Segrnetttation Is Initiated by Loc~lized RNAs

Box 18.-2 Review of Cytoskeleton: Arymme['ry ami Gmwth

582

<:tt the Anterior a nd PO$terior P<.,les of the

A LocalizeJ mRNA In itiates Musde Differe m iatinn in the Sea Squirt Embryo 5t<4

Unfertilized Egg

599

The Bicmd G radaenr Regulates the Expn.>sSton

Cell-to--Cell Contact EHdts Oifferem.~ l Gene Expression in the Sporulating Bacterium.

of Segmentation Genes m a Concenrr<~rion~Oependenl Fashion

60 I

B. Slwrilis 584 Box 18---3 Ot'cn.~v of Ciona Development 585

Hunchback Expression Is also Regulated

A Skin-Nerve Rcgul~tory Switch Is Conrrolled hy Notch S igtmling m the Insect CNS 'i87

The Gradient of Hunchback Repressor Establishes Different ltmit:. of Gap Gene Expression 603

A Gradient of the Sonk Hedgehog Morrhogen Comrols rhe Fomtau oo of D1fferem Neurons 1n the Vertcbn:~te Neural Tube 58$

Hunchback arul G ap Proteins ProJuce Segmentation Stripes of G ene Expression 604

THE MOLECULAR BIOLOGY OF DROSOPHILA EMBRYOGENESIS 590 An Overview ri Drosophila Embryogenesis 590 A Morphogen Gradient Controll:i Dorsal-Venual Patterning of the Drosophila Embryo 590 Box l B-4 Overview (If

Drosophila De'-dopment 592 Box 18--5 The Role of Actwator Synetgy irr Developmem 597 CHAPTER

~t

rhe Level ofTransl~tion 60Z

Box 18-6 Bioin{mmaciw Merhod.s for ldennflcruion nf Complex Enhancers 605 Gap Repressor Grndients Produce many Snipes of Gene Exrre~ion 607 Shott-Range Tmns~:riptionC\1 Repressors Permi• Different Enhancers to Work Independently of ot1e Anoth er wirhin the Complex etre Regulatory Regiort 608 Su.nmary 609 Bil~QJ>hy

610

}9

Comparative Genomics anJ the Evolution of Animal Diversity 613 MOST ANIMALS HAVE ESSENTIALLY

THE SAME GENES 614 How Does Gene Dut)licatlon Give Rtse to Btological Diversity?

Suhde ~nge.s in an EnhancQr Sequence Can Produce New Ptttrems d Gene Exrression 6l..1 The Mlsexpression of Ubx Changes the Morphology of the fruit Fly 624

616

Box 19- 1 Gene Duplication and rhe lmpo11ance of Re~lazory Evolution 616 Box 19~2 DtqilicmKm of Globm Genc.s Produces New Expression Patterns and Di~ Protein Functions 618 Box 19-3 Creation of New Genes Drives Bacterial Eookaian 618 THREE \VAYS GENE EXPRESSION JS CHANGED DURlNG EVOl..U TION 619 EXPERIMENTAL MANIPULATIONS THAT ALTER ANIMAL MORPHOLOGY 620 01anges in Pax6 Exc:>ressiot\ Create Ectopic: Eves 621 Changes in Antp Expression Transfotm Arltennae into Legs 622 ltnporr .me c of Pro retn f unction: Im crconverston of frz an d Antp 62l

Changes in Ubx FunctiCln Mooify the Morphnlogy of Fnut Fly Embryos 626

C hanges in Ubx Targer &tchancets Can A h er Pan e ms of Gene Expression 627 Bo-" 19-4 T he Homeoric Genes of Dro&:>phila

Ate Organized in Specwl Chromosome Clusrets 627 MORPHOLOOICAL CHANGES IN CRUSTACEANS AND INSECTS 630 Arthropods Are Remarkably Diverse 630 Olanges in Vbx Exp~essiotl Explain Modifications in limbs aanong the Crustnceans 6l0 Why Insects Lade Ah:Jominallimhs 63 t Modification d Fltght Limb~ Might Arise from the Evt.llution of Regul<~tory DNA &.»quer-.ces Bo~

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19-5 C..;()-option. of Gene Networks far Ewlutimuuy lnnm.'ation 633

631

xxv

Detailed Contents

The Evolut•onal)' O rigins of H uman Speedl 637 How FOXP2 Fosters S~h in Humans 637 The Furore of Comparati.ve Genome Analysis 638

GENOME EVOLUTION

AND HUMAN ORIGINS 635 Humans Connun Surrrismgly Few Genes 6 35 The Human Genome Is very Simaar Lo lhat of the Mouse eind VinuaUy ldenucal to t he Chimp 636

p A R T

CHAPT l:R

5

Summary 639 Dihliogycphy 640

METHODS 643

20 647

Techniques of Molecular Biology INTRODUCTION 647 NUCLEIC ACIDS 648

Electrophoresas thro uf,!h a Gel Separntes DNA and RNA Molecules According to Sue 648 Restriction EnJonucl~ Cleave DNA Molecules at Panicular S ites 649

HylxKJil:::luon Probes Can ldenttfy Elc\:trophoretic~IIY'Separnted DNAs and RNAs 652 1$t'll~ti on of Specific Segments of DNA 653 ONA Cloning 654

Plasmid Vectors 654 Vector DNA Can Be 1ntroduced into Host O rganisms tn

by Transfonna tion 655 libr.mes of DNA Molecules Can Be Cre<3ted by C loning 656 H ybriJuatl£ln Can Be Used to Identify a Specific Cion£' an a DNA Library 657 C hemically Symhes•ied Oligonud~ides

Througllflut Sequencm.g 665 fhe PaireJ~End Str" tegy Permits the Assembly of Lu"8e Genumt Scaffoh1s

DNA HybnJazation C
C loning DNA

Shorgun Sequencing a Bacteria l Genome 663 Tlk Sh()(gun Strctttgy Permirs a l'arrial As..c;embly of l arge Genome Sequences 664 Box 10,2 &quetl{l(UrS Are U.scdfar High

666

Genome-Wiul' AnAlyses 667 OJmparative Genome Analysis 669

PROTEINS 672 Specific Proteins C..an Be Purilied

from C ell Extmcts 671 Purilic~ tion of~ Prot<-in Requires

a Spcc,fic Assay 673 Preparation of C..ell Extracts Containing Acuve Protems

673

Prote•ns Can Be Separated from One Another Usmg Column C h romatography 6 73

Affinity Oucmlatography Can Facilit
657

674

TI-ie Polymer~ Ch~ t n Rt:ru.:.ti.on (PCR) Amplines DNAs by Repeated Rounds of DNA Rcphcatio n in viu o 658 NesteJ Sets of ONA Fragments Reveal NudecxJ<.:le Sequences 660

Sep;muion of PrO£cins on P~.."1lyacrylanude Gels 67S Amihodies Visualize Electrophoretically -Separated

Box l O-l FvrcnStes and. the Polymerase Chain Re~tinn 661

Bibliogroplty

Proteins 676 Protein Molecules Can Be Directly Set.luent:ed Pro te<.)(nics 677

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679

676

xxvi

Defailt-d ContentB

CHAPTER

21

Model Organisms

681 THE NEMATODE WORM,

BACTERIOPHAGE 682 Assays of Phage Growth

C.aenorhabdins elegans 696

684

C . elegrms Has a Very Rapid Life Cycle

The Single--Step Gmw{h C'...urve 685 Phage Crossses and Complernentatton Tests TransJu~tiun and Recombinam DNA

C. elegans Is Composed of Relatively Few,

685

Well Studied CelllineL1J;.oe5 697 The Cell Death Pathway Was Discovere<.l

686

BACTERlA 687 Assays of Bacterial Growth 687

in C . elegan.~

Bacterial Pla~mi.Js C'.an Be Used

as Clon(ng Vectors 689 Transpos()ns Can Be Used to Cenerdtt' lnserttonal 689

Studies on the Mole<:u l~r Biok~y ofBa~teria Have Been Enhanced hy Recombinant DNA Technology, Whole-Genome Sequencing, anJ Tr.mscriptk1rull Profiling 690 Cells wuh Well-Developed Tools of Tradirional and Molecular Genetit.:S 691 691

693

The Exi.!;tence d Haploid. aru.l Diploid C ells Facilitate Generic Analysis of S. ceret•iswe 693 C'..enerating Precise Mutarions in Yeast ls Easy

THE HOUSE MOUSE, Mus musculus 705

h Js b~>)' to Inm.Juce Foceign DNA into the Mouse Embryo 707 Homologous Recombination Permn~ the Selective Ablation of individual G~nes Mice Exhibit Epigenetic Inheritance Bibliography 711

694

S. cet'evisiae Has a Small, Well~Oultacterized

Genome

694

S. cert?visiae Cells ChattJ;e She~pe as They Grow

Index

703

Mouse Embryonic Development Depends on Stem Cells 706

Phage and Bacteria To ld Us Most of the Fundamental Things about the Gene 692

BAKER'S YEAST, Sacchdromyce.~ cerevi«ae

698

THE FR\J IT FLY, DrosOJ)hila melanogASrer 699 Drosophila H as a RapiJ Lafe Cycle 699 The First Genome Maps We re ProduceJ in Dros(lphila 700 Genetic Mosaic~ Permit the Analysi& of lethal Genes in AJult Flies 702 1be Yt!asf FLP Recombtnase Pertnits the Efficient PT(l(luction of Oenel ic Mosaics 703 It Is Ea$y to Creare Transgenic Fruit Flies that Carry Foretgn DNA

Biochen"'ical Analysis Is E.spedally Powerful in Sit'nple

&cteria Are Accessihle to C ymlogtcal Analysis

698

RNA1 Was Disq>vercd in C . d t."gllns

Bacteria Exchange DNA by Sexual C--OnJugation, f'hage,Mediated Transductit'ln, a nd DNA-Mediated Transformatt~..m 688

Mut<~tlons ::md Gene and Operon Fusions

096

695

713

NOT FOR SALE

709

707

Class Testers and Reviewers We wish to thank all of the instruc tors for their thoughtful suggestions

and comments. including; Chapter Reviewers Ann Aguan no. Marymount Manhattan College Charles F. Austerberry. Creighton University David G. Bear, University of New MexiC"o Health Sciences Center Margaret E. Beard. J{o /y Cross Gail S. Begley, Northeastern University Sanford Bernstein, Scm Dir.go State Untver.sity Michael Blaber. Florida State University Nicole Bournias, C.alifomia State Uni versity. San Bernardino John Boyle. Missi:>sippi StaiR Unive.rGity Suzanne Bradshaw, University of Cincinnati Jolm G. Burr. University of Texas at Dallas Michael A. Campbell , Pennsylvania State llniversity. b'rie, Tbe Behrend College Shirley Coombe~-. /<•.."ings College. Utliversity of London Anne Cordon. llniversity of Toronto Sumana Datta, Texas A&M Universit}' Jeff Dejong, Umversity of Texa6 at Dallas furgen Denecke, University of Leeds Susan M. DiBartolomeis, Millen.·ville I Tniven;ity Santos h R. D'Mello, University of Texas at Da11os Robert j , Duronio. University of North Ccrolina, CJwpel Hill Steve.n W. Edwards. UniVersity of LivP.rpoo/ AJlcn Gathman, Southeost Missouri State University Anthony D. M. Glass. Uni flersity of British Columbia Elliott S. Co1dsteira. Ati2ono State University Ann Grens. Indiano Univorsity. South Bend Gregory 8. Hecht . Rowon University Robert B. Herling, University of Michigan David C. Higgs. UniveTsity of Wisconsin, Porkside Mark Kainz, Colgnte Unitrersity Gregory M. KPJly, Unh·ersity of We~tem Ontario Ann Klein schmidt, Alleglteny College Dan Krane. Wright State UviversilJ' Mark LevinthaJ, Purdue University Gary }. Lindqucster. Rhodes College

Curtis Loer, University of San Diegv Virginia lvtcDonough, Hope College Michael J. McPherson, University of Leeds Victoria Meller, Tufts University William L Miller, North Carolina S tate University Oragana Miskovk , University of ltFater/()0 David Mullin. Tulane Uni versity Jeffrey D. Newman . Lycoming Collc~e James 13. Olesen. Doll State University A nthony J. Otsuka, Illinois State Unjversity Karen P;:llter, Temple Univf.m;ity james G. Patton, Vanderbilt University Ian R. Phillips. Queen Mary. University of London Steve Picksley, Unil,ersity of Bradford Todd P. Primm, University of Texas at El Paso Eva Sapi, University of New Haven jon n. Scales, Mid western State University Michael Schultze, University oj' York Venkat Sharma. Universify of West Florida Erica L. Shelley. University of Toronto at Mississauso Elizabeth A. Shephard. Vnittersity College. London Margaret E. Stevens, Ripon College Akif Uzman, University of llotu;ton. DotAntown Quinn Vega, Montc-lair State University Jeffrey M. Voight, A lbany Gollege of Pharmocy Robert Wiggers. Stephen F. Austin State. University Bruce C. Wightman. Muhlenberg College Class Testers Charles F. AustcrbelT}'. Cmighton University Christine E. Bezotte. Elmira College Astrid Helfant, Hamilton Collq;e Gerald Joyce, The Scripp$ Research Institute Jocelyn Krebs, University of Alaska, Anchorage Cran Lucas. l.nuisiOna State University in Shreveport Anthony ). Otsuka, 1/Jinois Stot6 Uni~'e.rsity Charles Polson. Florida Institute of Technology Ming-Chc Shih, Univnsity of Juwa nvii

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About the CD and Website The student CD-ROM for Molecular Biology of the Gene provides resourcus to help students visualize difficult concepts. explore complex processes, and review their understanding of the m ost challenging ma terial presented in this course. This easy to use electronic resource provides students with rapid access to twenty interactive tutorials, thirteen structural animations. and criti£:31 thinking exercist'S that can be assignoo by instructors. The tutorials contain anima~ lions that are broken out step by step. so that students can focus on mastering one clement at a time. Every tutorial concludes with an "Apply Your Knowledge'' activity, where students are presented with a problem ano then guided through to the solution with interactive animations anr1 multiple choice questions. The structumJ animations run in CJ trME, an application that automatically converts the infonna· tion n(.>eded lo dd1ne the lhret.l-dimensional struL1.UfllS of many molecules into accurate molecular models and presents it in a window in your Nctscape Navigator browser. Finally, thP critical thinking activities ask students to actively engage with the material. The student website for Molecular Biology of the Gone :a lso J•rovides lhH twenty inter.u.:tive tutorials. fifteen structur.tl animations. and critical thinking exercises found on the Cn..ROM. but also contains additional msearch tools and web resources that are outstanding tools for students wishing to explore a chapter's concepts or extend their lmowlerl.ge beyond the scope of the text. ln combination with thr. StUdent CD, the student website provides a valuable SCt Of N'..SOUfCeS lO help students deveJo1J the skills they need to sm.:ceed in class.

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P A R T

CHEMISTRY A N D GENETICS

PART 1 ClwmiBtry and Ge11eUcs

P ART

OU TliNE

• Chapter l

The Mendetoo \hew of the World

ChaptEf 2

Nudetc Acids Convey Genetic

lnfoonabon Chapter 3

The Importance o1 Weak

Chemc:a11nterac'!JOr1S Olaptr-r 4 Chop«:~

s

The Importance of High Energy Boods weak atld Strong~ Deten-n10e Maaorndecular ~

U

nlike the rest of this book. the five chapters that make up Part 1 contain material largely unchanged from earlier editions. This is because the material remains as important as ever- even l.n these days of genome sequencing. Specifically, Chapters 1 and 2 provide an historical account of how the field of genetics and the molecular hasis of genetics was established. Key ideas and ·e xperiments are described. Chapters 3, 4. and 5 present the chemistry lhal lias at tha heart of molecular biology. We will discuss th~ fundamental chemical principles that underlie the structures of the macromol~culcs that figure so pron1inenLly throughout the rest of the book- DNA. RNA, and protein-and the interactions between those molecules. While the bulk of the material is retained from carJicr editions. some of it has been reorganized and more recent examples have been included. Chapter 1 addresses the founding events in the history of genetic-s from the classic work of Gregor Mendel up to that of Oswald T. Avery. We will discuss everything from Mendel's famous experiments on p eas, which uncovered the basic laws ot h eredity. to Avery's shocking (al the time) revelatio n thc\t DNA is tbe genetic material. Chi:iJJter 2. covers the subsequent revolution of molecular bjology, from Watson and Crick·s proposal that the structure of DNA is a double helix. through the elucidation of the genetic CO
PART 1 Chemistry and Genetics

thJee-dimensional shape. Likewise, it is weak bonds that hold together the two chains of the DNA molecule. We also consider. in ChapteT 5, bow the functi(ln of a protein can be regulated. One way is by changing the shape of t he protein. a mechanism called allosteric regu lat ion. Thus. in one conformation. a given protein may perform a specific enzymatic function, or bind a specific target molecule. 1n another con forma tion, however, it may lose that ability. Such a change in shape can be ttiggemd by the binding of another protein or a srnalJ moleCule E>uch as a s ugar. In other csses, an al-

l()steric effect can be induced by a covalent modification. For example, attaching one or more phosphate groups to a protein can trigger a change in t he shape of that protein. Another way a protein can be controlled is by regulating whe n it is brought into contact with a target molecule. In this way a given protein can be recruited to work on dif· ferent target proteins in re.sponse to different signals.

PHOTOS FROM THE COLD SPRING HARBOR LABORATORY ARCHIVES Vernon lnplm. Marshall Niterlbecg. and Matthias Staehelin. 1963 Symposium on Synthesis and Structure of Maaomcle-

wles. fngram demonstrated that genes antral the amino acid sequence of proteins; the mutation cat.ISing sldde-<el anemia produces a

si1gle arrino acid chalge in the hemoglobin protein (Chapter 2). Niretf>erg was key 10 unrav eling the genetic code. using protem synthesis directed by artificial RNA templates in vitro (Chapcers 2 and r4). Fa this achievement. he shared in 1tle 1968 Ndlel Prize for Medione.

Staeheln \NO~ed on the small RNA mclec..Jes, IRNA.s, which translate the genetic code irto arrino acid seque.·•ces of proteim (Chaptess 2 and 14).

Raymond Appleyard. George~ and Matha 0tas@, 19.5J Symposium on Viroses. Appleyard and BaNefl. both phage geneticists, are here sllov.t1 Wlltl
MelwWI Calvin. Frands ~ CeorJe G.anKM~. and James wa~scn.

1963 Sympo-

sium
their proposed structure of DNA,. Crick and \Nat· son shared in the 1962 Nobel Prize fat MedCcine (Chapter 2). Gamow, a physlast attracted to the problem of the getJeti< rode ( Chclpters 2 and l 4), fOtJflded an normal group of lilr.emirlded scientists called the RNA Tte Club. (He ts weamg the dw 6e- whi
MaX P~ 1971 symposium on StnldUre a11d Function of Proteins at the nveeDtmensional Level. Perutz shared, with .Johrl Kendrew. the 1962 Nobei Prize for"Olemistry; using X-ray oystaUography. and aftet' 25 years of effort, they l.\l€1e the first to solve the alorT1fC stri..IC.ll.xes of proteins, hemoglobin at1d myoglobin respediveJy (Chapter 5).

CaMn Bridges. 1934 Symposium on ~ pects of Crow1tL Bridges (stnwl reading lhe newspap€f) was part of T.H fv1ofgan's famous '11y gtoup- til at pioneered the development of fJe fMt fly Drosophila as a model geneoc organism (Chaptas 1 a"td 21). Vlfittl him is Or. t Buclctdt.L.

Joan Steitz and Friht.ipmann. 1969 Symposium on The Medtanism of Protein Syn-

thesis. StEitz's research focused on the struc· ture and function of RNA moleaJies, particularly those molved in RNA spicing (~er 13} and she was an autmr of the previous edition of tllis book lipmann shOIM!d that the tligh energy phosphate group in ATP is the source of EflEfgy

that ooves ~Y blologic.al processes (dlapte1 4). FOf this he shared in the 1953 Nobel Prize (Q( Medicine..

CHAP T ER

The Mendelian View of the World t is easy to comider human beings unique among living mganisms. We alone have developed compl-icated languages that allow meaningful and complex iotel'play of ideas and emotions_ Great civiHzations have developed and changed our world's environment in ways inconceivable for any other form of life. There has always boon a tendency. therefore. to think that something special differentiates hwnans from every other species. This belief hns found exvressiun in the many forms of religion through wbicb w e seek the origin and e:x"J)lore the reasons for our mdsteru.:e and. in so doing. try to create workable rules for c:onducting our lives. Litt le more than a century ago, it seemed natura) to think that, just as every human life begins and ends at a fixerl time. thE' human species and all other fun:ns of life must also have been created at a fixed moment. This belief was first seriously questionerl 140 years ago, when Charles Oatwin and Alfred R. WaJJa~;e proposed their theori\38 of evolution, based on the selection of the most fiL They slated that the vari· Olls forms of life ate not constant but continually give rise to slightly diffewnt anima ls and plants, some of which adapt to survive and multiply more effectively. At the time of this theory. they did not know the origin of this continuous variation, but they d id correctly real ize that these new ch.&acteristics must persist in the progeny if such vari· ati()JlS are to [orm the basis of evolution. At first, there was a great fmor against Darwin, most of it coming from people who did not like to believe that hwnans and the l'8ther obscene-looking apes could have a common ancestor. even if this ancestor had Jived some 10 million years ago. There was also initial opposition from many biologists who failed to find Thuwin's evidence convincing. Among these was the famous naturalist Jean L. Agassiz. tben at Hal'V3}"d. who spent many years writing against Darwin and Darwin's champion, Thomas H. Huxley, the most successful of the popularizers of evolution. Bu1 by the end of the nineteenth century, the scientific argument was almost complete; both the current geographic <listr.ibution of plants and animals and their selective occurrence in thfr fossil records of the geologic past were explicable only by postulating that continuously evolving groups of organisms had descended from a common ancestor. Today, evolution is an accepted fact for everyone except a fundamentalist minority. whose objections are based not on reasoning but on doctrinaire adherence to religious principles. An immediate consequence of Darwinian theory is the reaJization that life first existed on our Earth more t.bBn 4 billion years ago ill a simple form. possibly rese.mbling the bacteria-the simplest variety of life known today. The existeJlce of such small bacteria t~Us us that the essence of the living state is found in very smaU organisms. Evolutionary theory further suggests that the basic principles of Hfe apply to all living forms.

I

OUTL I NE

Menders Dlscovenes (p. 6)

•

Olrormsomallheory ol Heredrty (p. 8)

• Gene I.Jlkdge and Oossing Over (p. 9)

• Chromosome Mapping (p. 12)

• lhe Clfig1n of Genetic Variabl.ty through Mutations {p. •s) Eatly Sperulatjoos about What Genes Ne and How They Act (p. 16)

• Prelimfl1ary Attempts to rtrld a Gene-Protein Relationslip (p. '6)

5

MENDEL'S DISCOVERIES Gregor Mendel's experiments traced the results of breeding experiments (genetic crosses) between strains <>f peas differing in welldefined characteristics, like seed shape (round or wrinkled), seed color (yellow or green), pod shape (inflated or w rinkled), and stem length (tong or short). His conctmtmtiun on we ll-defined differences was of great importance; many breeders bad previously tried to follow t he inheritance of more gross qualities. like body weight. and were unablP. to ctiscover aily simple tulP-
The P rinciple of Independent Segregation After ascertaining that each type of parental strain bred true- that is, produced prog•my with particular qualities identical to those of the parents-Mendel performed a number of crosses between parents (P) differing in single characteristics (sm.:h as seed shape or seed color).

Box ·-' Mendelian taws

The most strling atmbute of a livlng eel is its .tility to bat1501it herecftafy properties from one eel genetatJon to another. The eostencc of heredity must h~ beef! notiCed by early humans, who witnessEd the passing of dlaracterisbcs, like eye or halt oolor, fran parents to offspnng. Its physical basis. however, was not !XIderstood unti the first ye.Ys of the twentJeth century, when. duong a remarkable period of creative activity, the chromosomal theory of heredity WdS estdblished. Hereditary trilr15mtSSIOn thr~ the spenn am egg became knO\I\IIl by 1SbO, and in 1868 Ernst Hacd<el. roting that sperm ronsistS largely of nuclear material, ~ that the nucleus es responsible for heredity. Almost 20 years passed before tt>e chromosomes were Slrlgled out as the actve ractors.. because the details of m1tosis, meoSis, aro fertilization haO to be wc:>rred ot.t first When lt1is WdS aa:om~, it could be seen that, Lrlifce other cellular constituents, the chromosomes are equaly dMded between daughter cells. Moreover. the com~r cared cflromosomal charlges that reduce the sperm and egg dlrocnosome m.rnber to the haploid number during metOSr.> became ~dabte as necessary fOf' kcepiflg the d'lromosome numbe' COf"'Stant. These facts, however, ffiffely ~ gested that dlroolosomes UJfry heredity. Proof came at the tum of the century With the discovery of the basic rules of heredity. The ooncepts were first pfOposed by C-reger Mendel in 1865 in a paper entrtted "Experiments on Plant H~ds· given to the Natual Sdence Sooety at Bmo. lrt his presentatiOn, Mendel descrlled in great dt'tail the pattems of transmsston c:l traits 1n pea plants (~ICh we disruss 11 detail below). hiS conckls!ons ot the principles of heredny, and their reievance to the controversial theories of twlutJon. The di,.,._e of saenti1ic opinQ"I, however, was not ravorablc, ood these tde.!IS were mmpletdy ignored, d€5ptte some eaffy efforts on Mendel"s part to interest the prominent biologists ot his time. In 1900, )6 years after Mendel's death, three plant breedei'S working lf!depeodently on afferent systems conhrrred lhe SlgNftcance cl fv'lcndel's forgotten work. Hugo De vries, Karl Corr€fl5, aod £rich Tschermak,. al doing expeflmems related to Mendel's, reached sim~ar condUSions before they knew of Mendel's work

Mendel s Discoveries

All the progeny lF1 = first filial generation} had the appearance of one parent only. For example, in a cross between peas having yellow seeds and peas having gf'ef'JI seedq, ~ll the progeny had yellow s~ds. The trait that i.!ppears in the F 1 progeny js called dominant, whereas the trait that does not appear in F 1 is called recessive. Tl1e meaning of these results became d ear when Mendel set up genetic crosses between F1 offspring. These crosses gave the important result that the recessive trait reappeared in approximately 25% of the F~ prot;eily, whereas the dominant t.L'ait appeared 1n 75% of thEW? offspring. For each of the sevtm tffiits he fo l1owed, the ratio in f'~ o f dominant to recessiv~ traits was always approximately 3:1. When these experiments were carried to a third (f.1) progeny generation. all the F'2 peas with rect>.ssive tra its bred true (produced progeny with the COCP.ssive traits). Those with dominant traits fell into two groups: one-third bred tme (produced only progeny with the dominant trail}; the remaining two-thirds again produced mi x~d progeny in a 3:1 ratio of dominant to recessive. Mendel correctly interpreted his re~lts as follows (Figme 1-1}; the various traits are con troUed by pairs of factocs {which we now a.ll genes). one factor derived from the male parent, the other from the fp.ma}e.. For example, pure-brooding strains of round pms contain two versions (or alleles) of the roundness gen e {RR}. where-~ pl.J..rehreeding wrinl-.led strains have two copies of the wrinklednt>.ss (rr) a1Jele. The round-strain gametes each have one gene for roundness lR}; the wrinkh-1d-stra!n gametes each have one gene for wrinldedness {r). In a cross between RR and rr. fertilization 1-'roduces an F 1 plant with botb alteles (Rrl The seeds look rotmd because R is dominant over r. We refet to the appearance or physical structure of an individual as its phenotype. and to its genetic composition as its genntype. Individuals with identical phenotypes may possess different genotypes; thus. to determine lhe genotype of an ~an ism. it is frequently necessary to perfonn genetic cros~.s for several generations. The term homozygous refers to a gene pair in which both the maternal ilnd paterna} genP.S are identical (for example, RR or rr). In con trast, those gene pairs in which paternaJ and maternal genes are different (for example, Rt) at'f' called heterozygous. One or several letters or symbols may be n sed to represent a partic:u)ar gene. The dominant a llele of the gene may be indicated by a capital letter (R). by a superscript i (r·· ), or by a + standing alone. ln our discussions here, we use the first conv(lntion in w hic h tht> dotninant allele is represented by t1 capital let t~r ancl the r(.-ccssivf" nJle1e by the lowercase lett~r. It is important to notice that ~gi ven gamete contains only one of the nvo copies (one a llele} of the genes p resent in the organism it comes from (foe example, either R Of' r, but never both) and that lht:l two t}'pes of gametes ace produced in equal numbers. Thus . there is a 5()-.50 chant.:e that a given gamete from an F 1 p£'.a will contain a particular gene (R or r}. This choice is purely .random. We do not expect to fillC:l exoct 3:1 rati o~ when wa examine a limited n umber ofF2 progeny. TbP. ratio will sometimes be sl ightly higher and <Jther t imea slightly lower. But as we look at increasingly larg~r samples. we expect that the ratio of peas with the dominant trait to peas with the recessive trait will approximate the 3:1 ratio more and more closely. The reappearance of the recessive characteristic in the F2 generation inrlicates that recessive alleles are neither morlifled nor lost in the F 1 (Rri gen eration , but that the dominant and reces.-dvf:l genes are

parenlal genf31'ation RR

rr

t

t r

R

Rr 1

r

(

,.

Rr /

,.

Rr /

rr

/

FIGURE 1- 1 How Menders first Jaw

(ll1depeftclent MS~l ~$ lhe l!l fat"IO of dominanliD recessive phenotypes CII'I\Oftllhe F:t progeny. R reptesa1CS ~ domnant gene: a"'f3 r the recesSIVe gene The ftU'1d seed repre>eflts the domi~ phenotype.. the wnnlded seed ~ recESSive phenotype

8

7'/ie Mendelian View of the World

independently transmit1ed and so are able to segregate independently during the formation of sex cells.. This principle of independent segregation is frequeptly referred to as Mende t•s first law.

parental gooeralion

X

Some Allel~ Are Nt!ither Dominant Nor Recessive

A

gametes

ln the crosses reported by Mendet on e member of each gene pair was deady dominan t to the other. Such behavior, however, is not universaJ. Sometim~ the heterozygous phenotype is intermediate between the two homozygous p h enotypes. For example, the cross between a pure-breeding red sn apdragon ( Anti1rhinum) and a purebreeding wh ite variety gives F1 progeny of the intBJ"mediate pink color. Tf these F1 progeny are crossed among themselves, the resulting F2 progeny contain red. phik, and white flowers in the pro portion of 1:2:1 (Figure 1-2). Thus, it is possible here to distinguish heterozygolcs from homozygd cs by their phenotype. We aJso see lhat Mendel's laws do not depmd on wh~ther one allele of a gene pair is dominant over the other.

a

(

F1 genernliorl

f A

l

Aa 1 gametes

t a )

A

Principle of Independent Assortment

>::male

f I CURE 1-2 1be inheritance of flower color in tfle snapdragon. One parent is homozygous b red flowas (AA) and the

other- homozygous for white floll\"efS (oa). No domoance IS present and the heterozygous F\ flowers ae pink. The l :2 : 1 ratio of red, p1nk, and white l"lowef5111 the r, j:Yogeny lS shown by ~ropnate colonng..

Mendel extended his breeding experiments to peas differing by more than on e characteristic. As before. he started with two strains of peas, each of whic..h bred pure when mated with itself. One of the strains had round yeHow seeds; the other. wrinkled green seeds. Since round and yellow are dominant over wrinkled and green. lbe entire F1 gene.rJ ation produced rormd yellow seeds. The f 1 generation was then crossed within itself to produce a number of F 2 progeny. which were examlned for seed appearance (phenotype). In addition to the two original phenotypes (.round yellow: wrinkled gree n). two new types (recombinants} emerged: wrinkled yellow and rouTJd green. Again Mendel found he could interpret the results by the postulate of genes. if h e assumed that each gene pair was indepcndtmtly trans· mitted to the gamete during se.x-cell for-mation. This interpretation is shown i n Figure 1-3. Any one gamete contains only one type of allele from e.Bch gene pair. Thus , the gametes p roduced by an F1 (RrYy} will have the composition Rl~ Ry, rY. or ty, but never Rr, Yy. l'Y. or RR. Furthermore, in this example. all four possible gametes are produced with equal frequency. There is no tendency of genes arising from one parent to stay together. As a result, the Fz progeny ph enotypes appear in the ratio nine round yellow, three round green, thn~e w rinlded yellow, and one wrinkled green as depicted in the Punnett square, named after the Rcitish mathematician who introduced it, in the lower part of Figme 1-3. This principle of independent assortment is frequently called Mendet-s second law.

CHROMOSOMAL THEORY OF HEREDlTY A principal reason foe the original failure to appreciate Mendel's di~ covery was the absence of firm facts about the behavior of chromosomes duri.ng meiosis and mitosis. This knowledge was available, however. when Mendel's lows were confirmed in 1900 and was seized upon in 1903 by American biologist Walter S. Sutton. Jn his dassic paper ..ThP Chromosomes .in Heredity," Sutton emphasized the importance of the fact tbat the diploid chromosome group consists of two

GenB Linlroge cmd Crossing Over

parental gereralloo

f IGURE 1-3 How Mendel's second

><

law(~ ~rtment) operates.

rrw

RRYY

~

In 1h1s examp4e. the inheittaoce of yellow (Y) and green (y) seed color is follovoied together with the inheritance of round (R) and~ (r) seed shape>. The Rand Y aleles are dam· nart <Wef r arld y. "fue genotypeS of the vanous parents and proge!ly are m.cated by letter ~.and four diHerent phenotypes are disllnguJShed by wnprtate shadirlJ.

~

.PY

"'

ry

gametes

l

)

J

F1 genemlion

Rr'ly

' '

RV

l

F2 generation gametes

•

Ry

'

rY

gametes

l

9

'

~ ry

J

morphoJogica11y similar sets anrl th at, during meiosis, every gamete .reeeives only one ch romosome of each homologo us pair. He then used this fact to explain Mendel's results by assuming that genes are parts of the chromosome. He postulated that th e yellow- an d green-seed genes are carried on a certain pair of chrom osomes a n d tha t the round- and wrinkled-seed genes are carried on a different pair. This h ypothesis immediately explains the expeTimentally ohservP.d 9:3:3:1 segregation :ratios. Although Sutton's paper did not prove the chromosomal theory of heredity. it wa s immensely important. for it brought together for the first time the independent disciplines of genetics (the study of breeding experiments) and cytology (the stt.tdy of cell structure).

GENE LINKAGE AND CROSSING OVER Men der s principle of independent assortment is based on the fact that genes located on different chromosomes behave in dep endently during meiosis. Often. however, two gp.nes do not assort independently because they are located on the same chromosome {linked genes: see Box 1-2, Genes Ar~ Linked to Chromosomes). Many

Box 1-J ~Ale linked to Chromosomes

lnitialy, ~ breeding experiments used genetic dfferences a..eady ergan and his ~ng colabaatO"S, geneticists Calvin B. Bridges. 1-lerrnam 1 Muler, and Alfred H. Stutevant. They wotked ~ the tW-ty fly Drosophi1o me/anogaster. The r«st 1Tlt.Jt3nt found WdS a male with Vvtlite eyes instead of lhe normal red eyes.. The V'hte-eyed varian£ appeared spontaneously in a Cl.Jinxe bottle of

a

red-eyed flies. Because essertlaly dJ Drosophila foood ., nature have red eyes, the gene leadng to red eyes was referred to as the wi1d-type gene; the gene leadilg to white eyes 11\'tJS caled a mlMlt gene (aUele). The Vutute-eye mul'i.lnt gene was immedidtely used in breeding expesiments (8oK 1-2 f.g~e 1), wifh the stribng result that the behavior of the aneJe completely paralleled the cistribution of an X chromosome (that 1s,. was sex..Jinked). This finding immediately suggested that this gene might be located on the X chromosome. together with those genes controlling sex. This hypothesis was q.ndfy confirmed by addruoncA genetic crosses using newly ISOLated mutant genes. Many of these adrlitiONI mu~t genes also were sec-linked b

parental getlef
ted

9

parental generation

~0

pheno(ype

~e

2

..I

gametes

IW

WY

wY

genotype

l

1

+

+

lf

1

red2

n w

"'

ww

red

9

Ww

red

0

WY

/

v

\\flite

wY

~'0

white

-

0

X

n

wY

n w.

F2 geneation

~

J

F1 geoeration

Ww

(r=h rr il

red

y

f+

""'·

WY

+

w

t

X

n .w

gametes

red~

redO

1

t

w

y

.w

f~ F genE!fation

Ww

0

X

X

ww

red

phenotype

w

w

(rt~,rt

F2generatioo red

2

Ww

vmite

~ red

0

WY

y

il white

0

wY

Box 1·2 f 1«SOrT)e cooespclflding 10 the w or W gene on the X chromosome

Gene Unkage uno Crossmg Over

examp les of nonrandom assortment were found as soon as a large number of mutant genes became available for breeding analysis. Jn every well-stUdied ca~>e. the number of linked groups was identical with lhe haploid chromosom e number. Foe example, there ar.e four groups of linked genes in Drosophiltl anti fuur morphologically dis~ tinct chromosomes in a haploid cell. Linkage. h owever. is in effect never complete. The p.robability that two genes on the same chromosome wil l remain together during meios is 1'8fl8P.S from just less that! 100% to nearly 50%. This variation in linkage suggests that there must be a mechanism for exchanging genes on homologous chromosomes. This mech anism is called crossing over. Its cytological basis was first described by Belgian cytologist F. A. Ja.n.sstlllS. At the start of meiosis, thwugh the process of synapsis. the homologous chromosomef'i form pairs with theic long axes parallel. At this stage, each chromosome has duplicated to form h .vo chromatids. Thus. synapsis brings together four t;hromatids (a tetrad), wbich coil about one another. Janssens postulated that. possibly because of tension resulting from this coiling, two of the chromatids might sometimes break at a COITcsponding place on each. These events could create four broken ends, which might rejoin crossways, so that a section of each of the two chtomatids woulrl be joined to a section of the other (Figure 1-4). ln this manner, raoombinant clwomatids might be produced that contain a segment derived from each of the original homologous chromosomes. formal proof of fanssens·s hypothesis that chromosomes physically interchange m aterial during synapsis carne more than 20 ye<.~n:: later, when in 1931, Barbara McClintock and Harriet B. Creighton, working at Co.rnell Unive.rsity with the corn plant Zeo mays, devised an elegant cytological derrmnstration of chromosome breakage and rejoining (Figure 1·5}.

parflltal genotypes

extrachranosomal

c

INK

c

wx

knob

malarial

I ~

non-crossover progeny

1

c

WJc

~

crossowr progeny

c

W1!

c

W)(

c

WK

c

Wlr

c

W:~t

c

WK

c

~

c

Wx

c

WJ(

j_

W.l(

c

Wx

c

WK

c

WK

c

Wx

::.4

c

c

J1

synapsis ol duplicaled

chomosornes to fonn tetrads

~~~tlreaks

at p<W\t ol corotact end

lu$es with aP0f110r1 of

theo1ter

FI "URE

1-4 Janssens"s llypolhesisof

crossing OWL

FIGU RE 1-5 Ch!rnonsbation ofphpkal ekCbanges between homologous chromosomes. 1n most cxganisms, pairs of tmmologous chromosomes have ideflfical

sh
c. wx progeny had to arise by crossmg 011er be'l\Neefl the c arJd wx 100. When Sl.ldl £;; wx offspoog were cytclog!Qlly eX&l'lined. ~<nob ctuomoscmes were seen, shOWiflg thai a knobless Wx rE!glOO had been pflystcally replaced by
12

The MMdeban View of the World

CHROMOSOME MAPPING Thomas Hunt Morgan and his students, however, did not await forma l c.:ytological proof of crossing over before exploiting the implication of Jansse11s's hypothesis. They reasoned that genes located close together on a c.ln:ornosome would assort with (>JU~ another much more regularly (close linkage) than genes located far apart on a chromosome. They immediate ly saw this as a way to locate (map) the relative positions of ~lH::s on cluomut;omes aud thus tu produc..:e CJ genetic map. The way they used the frequencies of the various recombinant classes is very straightfurward. Consider the segregation of three genes a1llocated on the same chromosome.. The arrangement of the genes can be determinL-d by means of three crosses. in each of which two genes are foUowed (two-factor crosses}. A cross between All and ob yields four progeny types: the two parental genotypes (AB and ob) and two recombinant genotypes (Ab and oB). A cross between AC and ac similarly give::; two parental combinations as well as the Ac and oC recombinants, whereas a cross between BC and be produces the parental types and the recombinants Be and bC, Each cross will producf: a spP.cific ratio of parental to recombinant progeny. Consider, foe example. the fact that the first cross gives 30% recombinants. the second cross 10%. and the third cross 25% . Th is tells us that genE'!S o and c are doser tugetber than o and b or b and c and that the genetic distances between a and band band care mo-re si milar. The gene arrangement that best fits thE-se data is o-c-b (Figure 1--ti}. The correctness of gene ocder suggested by crosses of two gene factors cao usually he unambiguously r:onfirmed by three-factor crosses. When the three genes used in the preceding example are followed in the cross ABC >< abc. six recombinant genotypes are found (Figura 1-7}. They fall into three groups of reciprocal pairs. The rarest of these groups arises from a double crossover. By looking for the least frequent class, it is often possible to insta ntly confirm (or deny) a postulated arrangeme nt. The results in Figure 1·7 inunediately confirm the ocder hinted at by the two-factor crosses. Only if the ord er is a-c-b d oes the fact that the rare recombinants are AcB and oCb make sense. The existence of m ultiple crossovers means that the amount of recombination between the outside markers a and b (ab) is usually less than the sum of the recombinAtion frequencies between a and c (cw) anrl c and b (c o). To obtain a more accurate approximation of the distance between t he outside markers. we caJculate the probability (oc X cb) that when a crossover occurs between c and b. a crossover also oc:x:utS between a and c, and vice versa (cb X ac). This probability subtracted from the sum of the frequ encifls expresses more occmatPJy the amount of recombination. The sU:nple formula ab:::::: or

~-

cb - 2{ac)(cb)

is applicable in all cases where the occurrence of one crossover does not affect the probability of anoth(),r crossover. Unfortunately,

1-6 Assignment of h tentative order of thtee g8\es on the basi.s of three two-factor crosses.

30%

f I c; U It l

25%

a

c

b

ChromOS(lme Mapping A

c

B X

tJ

A

c

a

c

A

c

a

c

l ~

67 5%

B

A

c

b

a

c

B

..

A

c

b

a

c

' l

b

c

1 B

A

c

b

a

c

b

A

c

B

a

e

~

!

B

A

c

tJ

a

c

b

A

c

B

a

c

7.5%

22.5%

lhi:> otass anses 'from a ~:~rea~<. belWeen

frorrl a break belween

aandc

c...C b

·this dass arises

~

B

b

l

B

b

2..5% this class anses from a break between a and c and c and b

accurate mapping is often disturbed by interference phenomena. which can either increase or ducrease the probability of correlated crossovers. tJsiog such reasoning. tbe Columbia University group hr.aded by Morgan had by 1915 assigned locations to more than 85 mutant genes in Drosophila (Table 1-1). placing each of them at distinct spots on one of the four linkage groups. or chromosomes. Most importantly, all the genes on a given chromosome were located on a line. The gene arrangement was strictly linear and never branchoo. The genetic map of one of the chromosomes of Drosophila is s hown in Figure 1-8. Distances between genes on such a map are measu:red in map unils, which are roh1ted to the frequency of recombination between the genes. Thus, ir the frequency of recombination between two genes is found to be 5o/o, the genes are said to be separated by fi ve map units. BecauRe of the high probability of double crossovers between widely spaced genf!s, such assignments of map units can be considered accurate only if recombination between closely spncoo genes is followed. Even wbe.n two genes are at the far ends ofa very Jons chromosome, they assort togetJ1er at least 50 % of th£~ time bocause of multiple crossovers. The two genes will be separated i£ an odd number of crossovers occurs between them, but they will and up together if ~n even number occurs between them. Thus, in the. beginning of the genetic analysis of Drosophilo, it was often impossible to determine whether two genes were on different chromosomes or at the opposite ends of one long chromosome. Only after large munbers of genes had been rnappoo was it poRsibln to dmnon~ttate convindngly that the munber of linkage groups equaUcd the number of cytologically visible chromosomes.ln 1915, Morgan, with his f!tudents Alfred H. Sturtevant, Hermann J. Muller. and Calvin B. Bridges. published their definitive book The 1\lfecllanism of Mendelian Heredity, which first announced the general validity of the chromosomal basis of h eredity. We now rank this c..oncept. along with the theories of flVolution anrl the cell. as a major achievement in our
13

FIGURE 1-7 The use of three-feet« cr055fi to assign gene 01dtlf. The least frequem pair of reopr001l ~ants must ariSe frilin .;~ doc..Jble otJssovet The percentages ~sted for the vacJOUS clas.s6 are the lheoretiG~ values ~ed for an infw-ntely large sample When fintte f"U'l'1befs of progeny are recorded, 11-Je exact vallles wil be st.bjea to random statistical tluduaboos.

TASU 1- 1111e 85 Mutanl ~enes Reported in Drosophilo 1Mkmogoster"i61 t9ts•

Name

Region Affected

Name

~nAftected

Abdomel1 Eye

Lethal. 13 Miniature Nctd"t

Body. death W1ng Veoaliorl Eyecolot

Group 1 Abnormal

Bar Bifid Bow Cherry Chrome

Venation W1ng Eye Color Body color

Redup~ted

RUby RudiiT1€f"ltafy

Leg

Cleft

Venation

s~

C1W

Wlfl9

Depressed Dotted

Wtng

Shifted Short

Bodycoor Venat100

lhora.x

Skee

Wing Wing

Eye eoior Ommatidia

Spoon

Wing Bodycolot

EOSin

Gc'een Jaunry

Body color

Spot Tan Trwcate Vermt.on Vvhllf:

Wtng

Yeteow

Lemon

Body color

Facet FOfked FurrOINed

Fused

Spoe Eye Venattoo

Wing

Amems

Wing Eye color Eye color Body cclor

Group2 Antlered

Wing

Ap&erous

Wing

Arc

Wmg Vcnauon Body color Wmg 1t'lorax mark

6alloon Black Blistered

Corrmr:l Confluer~

Cream II

Venation E:ye 00101

JalJrl(y L1mited I ittle CIOSSOVef Mon;la Ohve

Pte>< us Purple Speck

Dachs E.xtra vein

Wing Leg \lenat100

Frtnged

Wing

Strap Streah Trefoil Truncate Vest.glal

Curved

Wli'Q Abdorrlnal band Ctmmosome2

Omrr.at.Oa Body color VenatiOn Eye color Thorax n-ark

W1119 Pattem Panern Wing

Wtng

Group3

Band

Panern

flank

Eye colOr

Beaded Cream Ill Oef01med

Wing Eye cob

Floug)

Eye

Safrann

Eye

Sep.a

Dwarl El:lcrly

&ze of t:xxty Bodyeo40r

Giant

Sazeof body

Sooty Spirefess Spead Tndent

Eye color Eye color Body color Sptne Wrng

Pattern

K1dney

Eye

Lovv Ct0SS1n9 0\lef

Truncate

Maroon

Chl'omosome3 Eye color

Peach

Eye COlor

~te ocelli

WKlQ Pattern S.mpleeye

Wing

Eyeless

Eye

Wtitehe&:l

Groop4 Bent

*TI1e mAal ens laU 111to kxxmkage IJOIJPS Smcetourchrornosomeswere cy101ogicai!Yobsefved h s lnQcaled that !he geo"eS &e silueted on thtl m arrosomes Notice thai nua~ 11'1 vant1t1s yenes can acllo alter a srrve chw"acto. 5\Jch a:. 1>00y COlor tn difterenl ways.

The Origin of Gtmelic \/Driubility through Mutation~

~5

nonnal

Ted eyes

StraJgt\t

Sf~ht

wings

wings

long wings

tong

red e yes

1egs

104

99.2

~

~

B

long ~rae

'• ,

• •

_;;t:;;

long wings

75.5

-======= =t="" C

61

.t

vg

54.5

48.5

•

b

pt'

-t.-

3i 13 ~ ==== -l d dp

0

~

al

' 1.,

black

dad'ls

body

(shofl tegs)

mutant

THE ORIGIN OF GENETIC VARIABILITY THROUGH MUTATIONS It now became possible to understand the hereditary variation that is found throughout the biological world and that fonns the basis of the theory of evolution. Genes at'c normally copied ~xactly during chromosome duplication. Rarely. however. changPS (mutations) occur in genes to givfJ rjse to altered forms~ most- but not oil- of which ftmction less well than the wUiHype alldPS. This process is necessarily rare; otherwise, many genes would be changed during every cell cycle, and offspring would not ordinarily resemble their parents. ThHe is, instead, a strong advantage in the.re being a smaJI but finite mutation rate; it provides a constant source of new variability, nocessMy to allow plants and animals to ndapt to a constantly c ha11ginp. ph}' sical and biological cnvironmont. Surprisingly. h owever, the results of the Mendelian geneticis ts were not avidly seized upon by ihe classical biologists. then the authorities on tbe evolutionary relations between the various forms of life. Ooubts were rajsed ahout whether genetic chsng~ of thP. type studied by Morgan a nd his students wr.re sufficiP.nt to permit the evolution of radically new structures, like wings or eyes. ln~t£'ad, these biologists believed that there must also occur more powerful .. macromutations," and that it was these events that .allowed great evolutionary advances. Gradually, however, doubt!; vanished. largely as a result of the efforts of the mathematic:a) geneticists Sew all Wright, Ronald A. Fisher, and John Burden Sanclernon Haldane. ThP.y shownd that, consid~ring the great .age of Earth, the relatively low mutation rates found for Drosophila genes. together with only mild selective advantages, would be sufficient to allow the gradual act-'Umulation of new favorable attributes. By the 1930s, biologists began to reevaluate their knowledge on the origin of species and to understand tbe work of the mathematical gen~licil)ts. Among th~se new Darnrinians were biologist Julian 1luxley

dumpy wings

aristatess {shOO Sfistae)

(a grandson of Darwin's original publicist, Thomas Huxley). geneticist Theodosius Dobzbansky. paleontologist George Gaylord Simpson, nnd ornithologist Ernc;t Ma}'T. ln the 1940s all four wrotP. major work.c;. each showing from his speciAl viewpoint how Mendelianism and Oarwinism were indeed c.:ompatible.

EARLY SPECULATIONS ABOUT WHAT GENES ARE AND HOW THEY ACT Almost immediately after the rediscovery of Mendel's laws, geneticists began to speculate about both the chemical structure of the gene and the way it acts. No real progress could be made. however. because the chemicnl identity of the genetic material remained unknown. Even the realization that both nuclek acids and proteins are present in chromosomes did not really help, since lhe sttuclure of neither was at aU understood. The most fruitful speculations focused attention on the fact that genes must be, in some sense, self-duplicating. Their structure must be. exactly copied every time one chromosome becomos two. This f.\cl immediately raised the profound chentica l question of how a complicated molc:culr~ could oo precisely {X)pied to yield e act replicas. Some physicists also beaune intrigued with the gene. and when quantum mechanics i"l•trSt on the scene in the )ate 1920s, the possibility arose that in order to understand the gene. it wou ld first be necessary to roast'U" the suhtleties of the most advanced theoreliral phys i ~. Such tbottghts. however, never really took root. since it was obvious lhat even the best physicists or theoretit:al chemists would not concern themselves with a substance whose structure KtiJJ awaited elucidation. There was only one fact that they might ponder- Muller and L J. Stadler·s independent 192 7 c:liscoverlcs that X-rays induce mutations. Since there is e greater possibility that an X~ray will hit a Jaeger gene than a smaOer gene, the frequency of mutations induced in a given gene by a given X-ray dose yidds an estimate of the size of this gene. But even here, so many special assumptions were required that virtually no one. not even Mullf!f and Stadler themselves, took thfl estimates ve ry seriously.

PRELIMINARY ATIEMPTS TO FIND A GEN&PROTEIN RELATIONSHIP The most fruitfu l early endeavors to find a relationship between genes and proteins examined the ways in which gene changes affect which proteins are present it1 the cell. At first these studies were difficult, since no one knew anything about the proteins thai were present in structures such as the eye or tl1e wing. ft soon becaTJ'I(! clear that ge nes wilh simple metabolic functions would be easier to study than genes affecting gross stntctures. One o f the first usefl.t] examples camts from a study of a hereditary disease affecting amino acid metabolism. Sponta~ neous mutations occur in humans affecting the ability to metaboJize the amino acid phenylaJanine. When individuals homozygous for the mutant trait eat food containing phenylalanine. their inability to convert the ~unino acid to tyrosine causes a toxir. levcJ of phenylpyruvic acid to build up in the bloodstream. Such diseases, examples of "iuborn errors of metabolism," suggested to English physician Archibald

SUITlJTlOry

17

E. Garrod, as early as 1.909, that the wHd-type gene is responsible for the presence of a parth::ul;:tr enzyme. nnd that in a homoeygous mutant . the enzyme is congenitall )' ~bsent. Gal-rod's general hypothesis of a gene-enzyme relationship was extendfld in thfl 1930s by work on flower pigments by Haldane and Rose Scolt-Moncrjeff in England. studies on the hair pigrnenl or Jhe

guinea pig by Wright in the United Stales, and research on the pigments of insect eyes by A. Kuhn in Germany and by Boris Ephrussi and GeoJge W. BeadJe, working first in France and then in California In all cases, the evidence 1-evealcd that a particular gene affec1ed a particular step in the formation or the rer-.pective pigment whose ~ence changed, say, the color of a fly's eyes frool red to ruby. However, the lack of fundamental knowledge about the structures of the relevant enzymes r uled out deeper examination of the gene-en z.yme relation· ship~ and no assurance could be given either that most genes control tbe synthesis of proteins (by then it was suspected thai all enzymes were proteins ) or that all proteins am undnr gene control. As early as 1936, it became apparent to the Mend elian gen eticists

that future experiments of the sort successful in elucidating the basic features of Mendelian genetics were unli.k ely to yield productive evidence about how genes act. lnstead. it would be necessary to find biological ob}e<:ts more suitable for chemical analysis. They were aware, moreover. Ihat contemporary knowledge of nucleic acid and protein chemistry was completely inadequate for a fundamental chemical attack on even the most suitable biological systems. Fortunately. however, the limitations in chemistry did ool deter them from learning how to do genetic experiments with chemkaUy simple molds , bacteria. and viruses. As we s hall soo, the necessary chemical facts became available almost as soon as the geneticists were ready to use them,

S~1ARY Heredity is controlled by chromosomes. which are the cellular carriers of genes. Hereditary factors were first di~ ~overed and described by MendEl in 1865, but their importance was not J'e alized until the start of the twentieth (;entury. :Each gene can exist in a variety of different forms called alleles. Mend el prnpoS{.-d that a hentditary factor {nO\V lnown to be a gene) fur eae;h hereditary trait is given by each parent to each of its offspring, The physical basis for this llehavior is the d istribution of homologous chromosom£>.s duriJtg meiosis: one (ranflmnl)' chosen) of each pair of homologous chromoscmes is distributed to each haploid cell. When two genes are on tho same chromorome, they tend to be inh.mited tQ8€the.r (linked). Genes affecting different characlcristics are somr:timcs inherited Independently of each otller, Lecause they are located on diiterent chromosomes. lo any case, linkagA is sclrlom complete because homologous chromosomes attach to each other duri ng m(liosis and often break at identical s pots and N;1join Ct'ossways (t:.Tossing overt. O'Ossing over transfers genes initially Jornted on a paternally ticrived ch romosome onto gene groups <>riginating from the matemnl parenL

Different a lleles from the same gene arise by inheritable changes (mutations) in the gene itself. Norm~U y, genes are extremely stable and are copied exactly during chromosome duplic:af ion; mutation CJCCurs only I'H.I'ely and usually has harmful con.qequencos. Mutation d oes, however. play a positive role. si nce lhe accumulation of rare favorable mutalitms provides the basis Jor genetic variability that is presupposed by the theory of evolution. For many years. the stmcllll'e o f genes and Che chetlliC
18

The Mendelion \rlew of the w.,rlrl

BIBLIOGRAPHY General References Ayala F.J. and Kiger J.A. Jr. 1964. Modern genetics. 2nd edition. Eenjamin Cummings. Menlo Park, California.

Beadle G.W. and Ephrussi B. 1937. Develop moot of eye color in Drosopllila: Diffusible substances and their interrelafiolls. Genetics 22: 76-86. Carlson E.). 1006. Tht> gene theory: A critical history. -

Saundet's, Philadelphia. 1901. Ctmes, rodiation. and society: The life and wor.l: of H.]. Mu/Je.-r. C".ornell University Priiss, Ithaca,

New York. Caspari E. 1948- Cyroplasmi c inheritance. Adv, Genetics 2:1 - 66.

Correns C. 1937. Nicht MPtlde/nde verubung (ed. f . von Wettstein). Borntraegel', Berlin. Dobzhansky T. 1941. Genetics and the origin of species, 2nd edition. Columbia Uni\letsity Press, New York.

Moore J. 1972a Heredity and developmrmt. 2nd edilion. Oxford University Press, Oxfor-d, England. - - 1.972b . Readings in heredit}' oncl development

Oxford University Press, Oxford. England. Morgan T.H. 1910. Sex-linked inherit.a noo in DrosoplUla. Scil'!nce 32: 120- 122.

Morgan T.H.• Sturtevant A.H., Muller H.J.. an d Bndges C..B. 1915. The mee:hlUJism uf MPndelian heredity. Holt. RinehBrt & Winston, New York. Muller H.J. 1927. Artificial transmutation of the gone.. Science 46: 84-87 Olby R.C 1966. Origins of Mendelism. Constable and Company Ltd., London. Peters J.A. 1959. Classic papers in genetics. PrenttceHRil. £nglewoorl Cliff.._, New }ersP.y. Rhoades M.M. 1946. Plastid mutatiops. Co/C Sprmg liarbar Symp. Quant Bioi. 11: 202-207.

R. 1972. Cytoplasmic genes and organelles.. Academic Press. New York

Stiger

Fisher R.A. 1930. Tl7e genetical theory of naturol selection. C larenrlo11 Press, Oxford, England. C..arrod A.E. 190B. .Inborn errors of metabolism. Lancet 2: 1-i'. 73-79, 142- 148, 214-220.

Scoft-Moncrieff R 1936. A biochemical survey of some Mendelian fadocs for flower colo& f. Genetics 32:

Haldane J.B.S. 1932. The courses of el'olution . Harper lit Row. New York.

Simpson G.G. 1944. Tempo and nzotle Columbia Univr:rsjty Press. New Ynrk.

Huxlfly j. Ul43. Evolution; The modem synthesis. I farper & Row, New York.

Sonneborn T.M. 1950. The cytoplasm in heredity. Heredity

Lea O.E. 1947.

Act.inns

of

rodiolinns on

lis~ing

cells.

Macmillan, New York.. Mayr E. 1942. Systematics and the origin of species. Columbia University Pr~.ss. New York. 1982. The wowth of biologicnl thought: Diver,5ily, evolutwn. and inhe1itance. .Harvard Hnivasity Press, Cambridge, Massachusetts. McCli ntock B. 1951 . chromo••ome .. organization and gene expression. C'..olcJ Spring 1fa1'bor Symp. Qt~QTil. Bioi.

-

16: 13-57.

- - - 1984. The Significance of responses of genome to challenge. Science 226: 792-800.

M<..Ciintocl< B. and Creighton H.B. 1931. A correlation o f cytological and genetical £.TOSSing over in Zeo Moys. Proc. Nat!. Acod. Sci. 11: 492-497.

117-170~

4;

Jll

ovolvtlon.

11-36.

Stadler Lf. 1926. Mutations in barley induced by X-rnys and radium. SciP.nce 110: 543-548. Sturtevant A.H. 1913. Th.e Hoear -arrangemenl of six sexLinked hctors in Drosophila as shown by mode of association. /. Exp. Zoo/. 14: 39-45. Sturtevant A.H. and Beadle G.W. 1962. A n inlrodc.Jcticm to genetiC::s. Dover, New Ynrk. Suuon W.S. 1903. TILe chn.unQsunte ill heredity. Bioi. Bull, 4: 231 -251. Wilson E.B. 1925. The cell m deve/opmmlt 3cd edition. Macmi.Uan, New Yock.

u~td

hnedit}'.

Wright S. 1931. Evolul ion in Mendelian populations, Genetics 1 fl: 97-159. - - - 1941. The physiology o f the gene. Physiol. Rev

21: 487-527.

C H APTER

Nucleic Acids Convey Genetic Information

hat special molecules might cmry genetic information was appreciated b)' geneticistc; long befure the problem claimed the attention of chemists. By the 1930s, geneticis ts Legan s peculflling as to what sort of molecules could l!ave the kind of stability 1hat the gene demanded. yet be capable of permanent, s'Udden cha.nge lo the mutan t forms tbat must p(ovide the bas~._ of evoh1lion. Unti) the mid-1940s, there appearP.d to bf' no drrect way to attack the chemical t>.ssence of the gene. It was .known that chromosomes possessed a unique molecular constituent, deoxyribonucleic acid (DNA), but there was no va~ay to show that this constituent carried genetic in(onnation. as opposed to seTving merely as a molecular scaffold for a stiU undiscovered class of proteins especially tailorP.d to carry genP.tic information. lt was generaUy assumed that genes would be composed of amino acids because, at tlu!t time, they appeared to be the only biomolecules with sufficient complexity. It therefore made sense to approach the nature of the gene by as"king how genes function within cells. (n the early 1940s, research on the mold N eurospora, spearheaded by George- W. Beadle and Edward Tatum. w as gen erating increasingly strong evidence supporting thA 30-year-old hypothesis of Archibald E. Garrod that genes wurk by controlling the synthes is of specific enzymes (the one gene-one enzyme hypothesis}. Thus . given that all known enzymes had, by this time. been shown to I.Je proteins, the key problem was the way genes participaiP. in the synthesis of prote ins. From the vAry start of serious speculation, the simplest hypothes is w as that ~netic information within genes determines the o.rdet of the 2 0 rlifferent antillo acids within th() polypeptide c hains of proteins. Jn attemp ting to tom this proposal, intuition was of little help eveJ1 to the ~1 biochemists, since tbere is no Jogical w11y to use enzymes as tools to determine the orrler of each amino acid added to a poJypeptide chain. Such schemes would require. for the synthesis of a singJe type of protein. as mMy ordering enzymes as there are amino acids in the respective protein. But since all enzymes knolvn at that time were themsel~.res proteins (we now know that RNA can also act as an. enzyme in a few instances), still additiona l ordering enzymes would be necessary to synthesize the oroering enzymes. This situation clearly poses a paradox, unless we assume a fantastically interrelated series uf syntheses in which a given protein has many different enzymatic specificities. With such an assumption, U might be possible (and then only with great difficulty) to visualize a workable cell. It did not seem likely. however, that most proteins would be found to carry out multiple tasks. ln fact. all the current knowledge pointed to the opposite conclusion of one protein. one function.

T

O UTLINE

Averys SOmbsheU: DNA C<~n urry GenetiC Speafiaty (p. 20) The Double Helix ( p. 2 1)

• The Ge1eOC lnfOfTT1
• The Certral Dogma (p. 3 1)

• Establishing the Oirecton d Protein Synthests (p. 3

n

• The Era of (ienolrJics (p 38)

19

Nudeic Adds Cor1wy Geneli'c Jnformvtion

AVERY•s BOMBSHELL: DNA CAN CARRY GENETIC SPECIFICITY That DNA might be the key genetic molecule emerged most unexpectedly from studies on pneumoni~causing bacteria In 1928 English microbiologist Frederick Griffith made the startling observation that nonviruhmt strains of the bacteria became virulent when mixed with their heat-killed pathogenic counterparts. That such transformations from nonvirulence to vindence represented hereditary cbanges was shown by using descendants of the Jlmvly pathogenic strains to trBJ1sform still other nonpathogenic bacteria. This raised the possibility that when pathogenic cells are killed by heat. their genetic components ri!main undamaged. Mo-reover, once liberated from the heal-killed cells, these components can pass through the cell waU of the living recipient cells and undergo subsequenl genetic recombination with the recipient's genetic apparatus (Figure 2-1). Subsequent research has confirmed this genetic interpretation. Pathogenicity reflects the action of the capsule ge ne. which codes for a Jcey enzyme involved in the synt11esis of the carbohydrate-containing capsule I hat surrounds most pneumonia-causing bacteria. When the S (smooth) aJJele of the capsule gene is present. then a capsule is formed around the cell that is neces~ry for pathogenesis (the formation of a CRpsule also gives a smooth appearance to the colonies fomted from these cells}. When the R trough) allele of this gene is presentJ no capsule is formed and the respective cells are not pathogenic. Within soveraJ years after Criffi1h's original observation. extracts of the killed bacteria were found capable of inducing hereditary transformations, and a search began for the <:hernical identity of the transforming agent. At that time. the vast majority of biochemists stlJJ bf.lieved that genes were proteins. It therefore camP as a great surprise when in 1944, after some ten years of research. U .S. microbiologist Oswald T. Avery and his colleagues at the Rockefeller Institute

heat to kill caps fragneot released

p8lhogernc

S (smooth) cetl recombnanon and cell di\..i sion

entry Of chrornosome fragment bearing csp5 into~ cell F 1GtJ R l 2-1 Transformcstion of a genetic characteristic of a bacterial eel ~(U.S pneumonioe) by addition of ~-Idled eels of a Je~M!ticaly d"IHEn!nt m-ain. Here we show an Reel receNing a chomosomal fragment conti:iiring tl'le c.
Sed

The Double Helix

21

in New York . Colin M. MacLeod and Madyn McCarty, marle the

momentous announcement that the active genetic principle was DNA (FibJUfe Z·Z). Supporting their conclusion were key experiments showing tbat t·he transforming activity of thftir highly purified active fractions was destroyed by pancreaUc deoxyribonuclease. a recently purified enzyme that specifically degrades DNA molecules to their nucleotide building blocks and has no effect on the integrity of protein molecules or RNA. The addition of either pancreatic ribonuclease (which degrades RNA) or various proteolytic enzymes (protoindestroying) bad no influenc.e on th~ uan1;forming activity.

pathogene

s (smooth) ee.ll

Viral Genes Ar:c Also Nucleic Acids l::ven more important contl-rmatory evidence came from chemical studies w ith viruses and virus-infected cells. By 1950 it was possible to obtain a number of essentially pure viruses and to determine which types of molecules were present in them. This work led to the very important generalization that all viruses contain nucleic acid. Since there was at that ti.me a growing realization that viruses contain genetic material, the question immediately arose as to whether the nucleic acid component was the carrjer of viTal genes. A crucial test of the lJUestion came from isotopic study of the multiplication of T2, a bacterial virus (baf;teriopbage. or phage) containing a ONA core and a protective s hell buill up b)' the aggregation of a number of different protein molecuJes. ln these experiments. performed in 1952 by Alfred D. Hershey and Martha Chase working at COld Spring Harbor Laboratory on Long Island, the protein coat was labeled with the rndioactive isotope 35S and the DNA core with the radioactive isotope 32P. The labeled virus was tben used to fullow the fates of the phage protein and nucleic acid as phage multiplication proceeded. pm1icuIarly to see w hich labeled atoms from tho parental virus ente red the bosl ceJl and later appeared in the p1ogeny phage. Clear-cut resuhs emerged from these experiments; much of the parental nucleic acid and none of the parental protein was detected in the progeny phage (Figure 2-3). Moreover, it was possible to show that liHie of the pamntal protein even enters the baGteria; instead. it s tays attached to the outside of the bacterial cell, pefforming no fw1ction after the DNA component has passed inside. This point was neatly shown by violently agitating infected bacteria after the entrance of the DNA: the protdn coats were sbakcn off without affecting the ability of the bacteria lo fonn new phage particles. With some viruses it is now possible to do an even more convincing ex{)f'riment. For example, purifiod DNA from the mouse virus polyoma can enter mouse cells and initiate a cycle of viral multiplication p roducing many thousands of new poJyoma particles. Th~ primary function of viral protein is thus lo protect its genetic nucleic acid component in its movement from one cell to another. Thus no reason exists for proteins to play any part in the structure of a gene.

capS

~ '- ---. ~

l iSOiafe DNA caps

~

(

~

!

add DNA toR eels

THE DOUBLE HELIX fiGURE

While work was proceeding on the X-ray analysis of protein sttucture, a smaller number of sdenti:;ts were trying to solve the X-ray diffraction pa.ttern of DNA. The first diffraction patterns were taken in 1938 by William Aslhury using DNA supplied by Ola Hammarsten and Torbjorn Caspersson. II was nol until tbe early 1950s that high-quality

2-2

Isolation of C1 chemiully

pure traasfonning •&4!nL (Source: Adapted from Stohl f W. 1964 The medton~cs of mhe~itCJf1CfJ, Fig. 2.:3 Copyright © l964. ~cpnnt€d by perrrisSfOfl of Pearscn Educ..loon.

Inc.,

Ower Saddle Rlv6: NJ.)

Nudeic Acids Convey Cenulic Jnfo1T1TCJiion

~ mixing of virus

'I wiftt ttost eels

I

• \

violent

I agitation

32p

-

protein ·gll
l) ~~labeled

mJt~of

VIral cllromosom and produc1ion of fleW phage

release of new proget~y parudes

FIGURE "1-3 Oenwnsttation that only the DNA component of n anies the genetic: inf~ation ~nd lf\"" h pi'Otein c:oat sen1es onty as -il pe'Otective sh~tl

X-ray diffrdction photographs were taken by Maurice Wilkl nE and Rosalind Franklin (Figure 2-4). These photographs suggested not only that. tbe underlying ONA sh"UCturn was helical but that it was com~ posed of more than one polynucleotide chain -either two or tluee. At the same time, the covaJent bonds of ONA were being unambi guously established 1n 1952 a group of organic chemists wodcing in the laboratory of Alexander Todd sh owed that 3• - 5' phosphodiester bonds regularly link together the nudeot ides of DNA (Figure 2-51. In 1951, bccau,;e of inror~t in Linus Pauling's a helix protein m otif (whicJJ we shall consider in Chapter 5). an elegant theory of diffraction of helita1 molecules was developed b)l William Cochran. Francis H. Crick, and Vladimir Van rl. This theory made it easy tu test possible DNA structures on a trial-and-error basis. The correct solution, a complomentary double helix (see Chapter 6}, was found in 1953 by Crick and James U. Watson, then working in the laboratory of Max Perutz and John Kcndrew. Their aniva1 at the correct answer depended largely on finding the steroochemically most favorable configuration compatible With Ute X-ray dilfrection data of Wilkins BJld Franklin. ln the double beli.x, the two DNA chains are beld together hy hydrogen bonds {a w ook noncovalent chemical bond; see Chapter 3) between pairs of bases on the opposing strands (Figure 2-6). This base pairing is very specific: The purine adenine only base-pairs to the pyrimidine thymine. w hile the pmine guanine onJy base-pairs to the pyrimidine cytos1ne. In double-helical DNA, the number of A residues must be equal to the number ofT residues, while the number of G and C residu~ must Ukc"vise be et]ual (see Box 2-1 , Chargafrs Rules). As a re..wlt, the sequenr:e of the bases of the two chains of a given double heJix h ave a complementary .relationship and the sequence of any DNA strand exactly defines that of its pa rtner strand. The discovery of the double helix initiated a profound revolution in the way many geneticists analyzed their data. The ~me was no longet: a mysterious entity, the behavior of which could be investigated only by genetic experiment-s. Instead. it quir:kly became a real molr.cular object about whic.h chemists could think objectively, as they di.d about sruaJler molecules such as pvruvate and ATP. Most of the ~xcitement. however, came not meroly from the fact that the structure was solved. but also from the nature of the structure. Before the answer was known, there had always been the worry that it would turn out to be dull, revealing nothing ahout how genr s w.p.Licate and function. Fortunately. however, the an~cr was immensely exciting. ThP two intertwined strands of

F 1G u R E 2-4 The key x-ray pftotopaph involved in tlleehaadaucm of the ONA strucblre. This phologrc!Ph. taken by Rtls
With penntSSICn)

The Double Helix

5'end

I

0

I

5'

O= P-o-CH:i

I

0

o

0

phosphodiester [ linkage

I

s-

o= p-o..:.....~

I

o

0

3' end

2-S A portion of a DNA polynucleotide chain. showing lM J ' - 5 ' phospbodiester linbges that conned the nudeotides. Phosphate groops CUYlect 1he 3' c.arbon of ooe nucleotide with the 5' carbon of the next FIGURE

complemtmtary structures suggested that one strand serves as the specific surface (template) upon which the other strand is made (Figure 2-6}. lf this hypothesis were true, then the fundamental problem of gene replication, about which geneticists had puzzled for so many years, was. in fact. conceptually solved.

Biochemist E.rwW1 Chargaff used a tedtlique called "paper chrcmatography" to analyze the nucledide composition of IJl\lA By 19 49 his data showed not only that d1e four dfferent nudeotides are not present in equal amou-rts, but also that the exact ratios of the four nudeotK:Ies vary from me species to another (Box 2 l Table 1). These f.-dngs opened l4l the possibi~ty that it JS the precise anangement of oodeotides within a DNA molecule that ronfers its genet1c specifiCity. Chargaff's expenments also showed that the relative ratios of the foor bases were not random. The numbet of adenine {A) residues in all DNA samples was equal to the number of thymine (T) resklues, wtJije the number of guanine (G} residues equaled the nurrber of cytosine (C) residues. In addition, regardless of the DNA source. the ratio of purines to pyrimidines was always approximately one (pumes = pyrimidi~). The fl.lldamental signif~Cat"lCe of the A = T and G = C relatirnships (Chargaffs rules) could not emerge. hoNever, until serious attennon ~s given to the

three-dinensiona1structure of DNA

FIGURE 2-6 The replicatjon of DNA. The newly synthesized srrands are shown lrl aaflRe.

Box 2-1 (Continued)

BOX

1·1

TABLE

1

Data Leading to the Fonnutation of Charpff's RaJes

Pyrimkinee

1 .04 1 00 1.06

1.00 1 00 0 .9 1

1.92

1.()3

0 .97 1.70

1.54

1.07

091

11 1.0 0.99 1 02 0.99 1.0 10

105

0.95

1 09

0 99

1.0

0.4

OA

1.09

1.08

1.1

0.7

0.7

0.95

0.86

0.9

07

0.6

112

089

10

Cytosine

Ox

1.29

Human Hen

1 56 1 45

Salmon

143

Wheat

1 22 1.67 1 7~

Hemoptntus

Cytosine

1.43 175 1.29 1.43 118

Guanine

Yeast

P&ftlea to

Guanine

Thymine to

Adenine

Source

to

Adenine to Thymine

10

102 1 00

1.02

mfluenzae Escherichia colit<2 Avian tubercle baCillus

Serratia f"riJ¥cescens Bacillus schatz

Souc:e.· Mer Dwgalf E. e1 a 1949. .J Bioi Chern. 1n 'IC5.

Finding the Polymerases that Make DNA Rigorous proof that a single DNA chain is the template that directs the synthesis of a comp lt:~mentary DNA chain had to await the deve l~ opment of test-tube (in vitro) systems for DNA synthesis. These came much faster· than anticipated by molecular geneticists. whose world until then had been far removed from that of' the biochem ist well versed in the procedures needed for enzyme isolation. Leading th is biochemical assault on DNA replication was U.S. biochemist Arthur Kornberg, who by 195 6 had demonstrated ONA synthesis in csll-froo ffi\i.racts of bacteria. Over the next several years, Kornberg went on to show that a specific polymerizing enzyme was needed lo cat al yz~ the linking together of the building-block precursors of DNA. Kornberg's studies revealed that th~ nucleotide bu ilding blocks for DNA are energy-rich precursors (dATP, dGTP, dCfP. and d1TP; Figure 2-7). Further stud ies identified a single polypeptide, DNA polymerase ({DNA Pol r), that was capable of catalyzing the synthesis of new DNA strdnds.. Jt links the nudeotide precursors by 3' - 5' phosphodiester bonds (Figure 2..S). FurthermoTe. it works on ly in the presence of DNA. which is needed to order the four nucleotides in the polynucleotide product. DNA Pnl J depends on a DNA template to cl ete1'1l1irle the sequence of the DNA it is synthesizing. This was first demonstmted by aJlowing the enzyme to work in the presence of DNA molecules that containe
deoxythymidine

nucleoside:

frf""'"'''"" h;T&c

thjlrnine (DNA)

cytos4ne (DNA)

H"'- /H N

H~A ~~d N

H

0

Cl-1 deoXyribose

augar: nudeotide:

deoxycyti(!jne-5'- ptlOSf)hate

nudeoside:

cleoxyadeooSfl"le

purine base:

aderine (ONA)

HV H

H-4::C;t

~~~

N

H

OH de~e

a.gar: nudeobde: FIGuRE 2·7

deoxyadenosine -5'-phosphate

n. nudeotides of DNA.

The structures of the different component'S of each cl the

foor nudeotides are shOINTl.

5'

5'

3'

3'

HO

T

OH 3' \.:

T

. -~ ~

"'1 OH

3'

ONA po!ymerase

')

..

pyrophosphate

G-O

lpy-.. . . .

.

phosphate

5'

G

FIGURE 2 -8 Enzymcstic S)'nthesis of~ ONA dtam Qtalyz.ed by DHA polymerase L

. I

·- ---- -- --·- -- ~- -

3'

5'

26

N1.1cleic Acids Cunvey Cef!fllit: lnformnUon

TAB Lf. 2-1 A Comparison of the- Base CCmposition ot Enzymatically Synthesbed DNA and their DNA Template-s

Base Composition of the Enzymatic Product Source of DNA Template MiCfOCDCdJS

A+T

A+T

G+C

G+C

Adenine

Thymine

Guanine

Cytosine

In ProdUct

In Template

0.15

015

0.35

0.35

041

0.39

0 .22

0.22

0.?8

0.28

080

0.82

0 .25

0.25 0.28 0.32

0.25

0 .25 0.22 0.18

1.00 1 .32

0.97 1.35 184

Jysode;Kticus (a bacterUT1) Aerobacter aerogeoos (a bacterium) Escherichia coli Caat rhyrnus

{)29

ftlage T2

0.32

0.2 1 0.18

1.78

specificity. Thus there is no doubt that DNA is the direct template for its own fonnation.

Experimental Evidence Favors Strand Separation During DNA Replication Simultaneously w ith Kornberg's research, in 1956 l\·1 atthew Meselson and Franl W. Stahl, then at the California Institute of Technology, carried out an elegant experiment in wl1ich they separated d aughter DNA molocules, and in so d oing, showed that the two strands of the double helix permanently separate from each other during DNA re plication (Figure 2-9). Their success was due in part to the use of the heavy isotope 1 ~N as a tag to difffltentially label the parental and daughter DNA s trands. Bacteria grown in a med ium containing the heavy isotope 25N have denser DNA than bacteria grown under no~ ma l conditions with 1qN. Also contributing to the success of tho experiment was the development of procedures for separating heavy from light DNA in density gradients of heavy salts like cesium chloride. When high centrifuga l forces are applied, the solution becomes more dense at the bottom of the centrifuge tube {whic:h. when spinning, is the farthest [rom the axis or rotation). When the correct in itial solution d ensity is ch osen, the individual DNA molecules will move to the centtal region of the centrifuge tube where their density equals that of th€ salt solution. In this situation, the hea-vy molecules wiU form a band at a higher density (closer to the bottom of the tube) than the lighter molecules. lf bacteria cOntaining he~wy DNA are trans· ferred to a light metliwn {containing 14N) and allowed to grow, the precursor nud eotides available for use in DNA synthesis will be light; het1ce, DNA synthesized after transfer will be distinguishable from DNA made before transfe£. [f fJNA replic.ation involves strand sepru-ation, definitE- predictions cat! be made about the density of the DNA molecules found aftet· lo-arious growth intervals in a light medium. After one generation of growth, all the DNA molecules should contain one heavy strand and one light strand and thus be of intm:mediate hybrid d ensity. This result is exactly what Meselson and Stahl observet-L Likewise, after two generations of growtll. half the DNA molecules were light and half hybrid, just as strand separation predicts.

The Double Helix ~cteria growklg in ~: all DNA iS heavy

transfer to 14t\l medium

27

FIGURE 2•9 Use ofa cesium chloride (CsO) density gradient to demonstrate ~ separation of complementary striMds during DNA replication.

ONA ISCtafed rrom ttle 0!15 ts mll<ed IMUl csa ~ (6M, p (OOflS!t.y) -1. 7g/ml} and r~ace(Jiil1At/8cattrifuge

p=1.65

p= 1 .80

~

~)

I

»ght ~

sdution centrifuged at 140.000 l( g fCf -48 hr

p

=1.65

p:: 1.80 onec~

two genetations

generation afttlf" transfer to 14N

~tter transfer

to 1"N

lhe locabon Of DNA rJ1(lleaJeS 1wtm 1he centrifUge cal cefl be determined by I.At.ravicJtet Oplics

DNA was thus shown to be a semiconservative process in which the single strands of the double helix remain intact (are conserved) during ~ repJicatJon proc.css that distributes one parental strand into each of the two daughter molecules (thus, the ··semi.. in semi-conservative). These experiments r uled out two other models at the time: the conservative and the dispersive replication schemes {Figure 2-10).. ln the oonsen. .ative m<XIel, both of the parental strands were proposed to remain together and the two new strands of DNA would form an entirely new DNA molecule. ln this model, light DNA would be formed after <>ntt cell generation. In the dispersh;e model, whlch was

favored by many at the time, the DNA strands were proposed to be broken as fl-eqmmtly as every len base pairs and used to prime the synthesis of similarly short :regions of DNA. These short DNA fragments would subsequently be joined to form complete DNA strands. This complex model would lead to DNA strands that would be composed of both old and new DNA (thus non-conservative) and would only approach fully light DNA after many generations of growth.

Nucleic Acic!s Convey Genetic: InformaCion

2-10 Three possible medtanisms for DNA replkation. Vvtlen the stll.Jcture of DNA was disc~ several modeJs wete proposed to eJ
distributive

THE GENETIC INFORMATION WITHIN DNA IS CONVEYED BY THE SEQUENCE OF ITS FOUR NUCLEOTIDE BUILDING BLOCKS The finding of the double helix had effectively ended any controversy about whethtir DNA was the primary genetic substance. Evtin before strand separation during ONA replication was experimentally verified, the main concern of molecular geneticists had turned to how the genetic infonruttion of DNA functions to order amino acids during protein synthesis (see Box 2-2, Evidence that Genes Control Amino Acid Sequences in Protoins}. Wtth all DNA chains capable of forming double hHlices, the essence of their genet.ic specificity had to reside in the linear sequences of their four nucleotide building blocks. Thus. as information-containing entities. DNA molecules were by then properly reganled as very Jong words (AS we shall see later, they are now best considered very long sentences] built up from a fom-letter alphabet (A, G, C, and T). Even with only four letters, the number of potential DNA sequences (4N, where N is the number of letters in the seqHence) is very, very large for even tl1e smallest o f DNA molecules; a virtually infinite number of c.liffenmt ge11etic messages can exist. Now we know that a typical bacterial gene is made up of approximately 1.000 base pairs. The numbet of potential genes of lhis size is 4 1000, a number that is orders of magnitude larger than the number of known genes in every organism.

DNA Cannot Be the Template that Directly Orders Amino Acids during Protein Synthesis Although DNA must t;arry tbe information for ordering amino acids. it was quite clear that the double helix itself could not be the template for protein synthesis. Ruling out a direct role for DNA were experiments showing that protein synthesis occurs at sites where DNA is absent. Protein synthesis in aU eukacyotic cells occurs in the cytoplasm. which is separated by dle nudeer memLrane from the chromosomal DNA

The CenP-tk Injormatio11 w;lhin DNA 1~ Omveyed by thtJ St;quf'.IICf! of Its Four NudttotidP Buildin8 Bloch

Box 2-2 Eviden<e that
The iirst experimental evidence that genes (DNA) COillTOI amino acid sequences arose from the study of the hemoglobin pr'esent in tunans sufferilg from the genetic cis.ease sidde-cell anemia. If an individual has the 5 allele of the ~ gene (\1\hch

encodes ooe cl the two polypeptides that together foon hemog'obin) present tn both homologous chromosomes, a severe anemia results, chatacteri2ed by the red blood ~ haiAng a sldde<el ~ If only one cl the two aleles of the 13-giOOin gene are of the S form, ltle anerria is less severe and 1he red blood cells ~ear almost 11CfJTlal in shape. The type of hemoglobin in red bklod celts Is llevvlse correlated wtth the genetic pattem. In the SS case, the llernogtoOO IS abnormal, character· IZed by a solubiity dfferent from that d rt()l1'l")CC hemoglobin, YAlereas in the t 5 condttion. half the hemoglobin is normal artd half sidde. Wild-type hemoglobin molecules; are r.:oostructed from two kll'lds ot polypeptide chains: a chains and ~ chains (see £:lox 2 -2 FtgtJ"e 1 ) . Each d1ain has a molc
two~

cha'ns are presert

in each moleclJe, giving hemoglobin a mdecu!N waght of about 64,400 daltons.. lt.e a chains and 13 c:hoins are tontroled by astinct genes so that a s\ngJe mutation will affect either the a chain or the 13 chain, but not both. In 1957, Vemoo M. Ingram at Carrbidge University showed that sickle

hemogSobtn differs from oormol hemoglcbln by the change of in the 13 chain: at position 6, the glutamic aod residue found &n v..iki-type hcmogJobin is r€placed by valine Except for 1his ooe ch
Me arnno acid

1-2 fIGURE 1 Fcmnation of wild-type and sid4~l hemoglobin. (Sol.lrO!: l lustratiOf\ I!VIflg Geis. R1ght5 o.Nned by Howard Hushes Medical Institute. N~ ro be reprodt.Ja!d wfthout permssion.) 8 OX

Hit

~

Val

HtlG t-D1CUJ

H>C

~...,

H>G

~ Noffoll

!

San Jose

HbN 5oslon

F IGURE

~

Lys

~

2-2

i

Tyr

HbG

BOX

l ys

2

Asumrnowyofsonle

estaWished amino add substitutions in human hemotfobin v.nants.

H>E

~ H>M Seskaloon H>

ZfRt1 HbN ~1

H>O

Lys Tyr Arg

30

Nucle1c Acids Comrey Gene lie l nfCJrmDIJon

A second information-containing molecule therefore had to exist that obtains its genetic specificity from DNA. after which it moves to the cytoplru>m to function as the template for protein synthesis. Attention from the start fiJCused on the sti11 functionaUy obscure second class of nucleic acids, RNA. Thrbjom Caspersson and Jean Brachet had found RNA to reside largely in the qFtoplasm; and it was easy to imagine single

DNA strands. w hen not serving as templates for complementary DNA strands, acting as templates for complementary RNA chains.

RNA Is Chemically Very Similar to DNA More inspection of RNA strudure shows how it can be exactly synthesized on a ONA template. Chemically, it is very similar to DNA. It too. is a long. tm branched moJecuJe containjn.g four types of nudeotid~ linked 1ogethar by 3' - 5 ' phosphodiester bonds (figure 2-n). Two diff-eren<.-es in its chemical groups distinguish RNA from DNA. The first is a minor modification of the sugar component (Figure 2-12). The SUgiU' of DNA is dfl<»\yribose, whereas RNA contains ribose. identical tu deoxyribose except for t he presence of an additiona1 OH (hydroxyl) group. The second difference is that RNA contains no thymine, but instead contains the closely relsted pyrimidine uracil. Despite these differences, however, poJyribonudeolides have the potential for fonning complementary helices of the DNA type. Neither the additional hydroxyl group. nor the absence of the methyl group found in thymine but not in uridine, affects RNNs ability to form double-helical structures held together by base pairing. Unlike

FIGURE 2-11 Aportionofa polyribonudeoti (RNA) chain. flements .n red are dtstlnct from DNA

guanine

_....H

The Cen tral Dogma

31

l-12 Distinc:tit:lns ~ the nudeolides of RNA and DNA... A nucleotide of DNA IS sl'lowrl next to a nucleotide d RNA. PI RNA nt..Jdeo!ldes have the StJgar nbose (tnsteac!

fIGURE thymine (DNA)

uraci (RNA} 0

II

H:(N}(H I s

...

· -<¥,HO

'

3

2

r

0

Q

OH OH ribose

DNA, however. RNA is typically found in the cell as a single-stranded

molecule. lf double--stranded RNA helices are fonned, they most often are composed of hvo p arts of the same single-stranded RNA molecul~.

THE CENTRAL DOGMA .By the fall of 1953, the V~-'Orking hypothesis was adopted tlaat chromosomal DNA functions as tlJe template for RNA molecules, which

subsequently move to the cytoplasm. where they determine the arrangement of amino acids within proteins. In 1956, Francjs Crick referred to this pathway for the flow of genetic information as the ccnlral dogma.

r:;:, DupJication

Transcript ion Translation VNA -RNA Protein

Here the arrows indicate the directions proposed for the transfer of genetic infonnation. The arrow encircling DNA signifies that DNA is the tmnplate for its self-replicaUoiL The rurow bet ween DNA and RNA indicates that RNA synthesis (lranscriplion} is di.rected by a DNA template. Correspondingly, the synthesis of proteins (translation) i.s dimcted by an RNA tell'Jplate. M(lst importantly, the last two arrows were presooted as unidirectional; that 1s, RNA scqnences are n ever determined by protein templates. nor was DNA then imagined ever to be made on RNA tern· plilles- Thai proteins never serve as templ.ates for RNA has stood the test of time. However, as we will se.e in Chapter 11, RNA c:hai ns sometimes do act as temphfles for DNA chains of complementary sequcm.:e. Such reversals of tbe normal flow of in formation are very .rare e vents com pared with the enormous number of RNA molecules made on DNA templates. Thus, the central dogma as origmalJy proclaimed appxoximately 50 years ago still remains essentially v~id.

The Adaptor Hypothesis of Crick At first it seemed shnplest to believe that the RNA templates for protein synlhesis were £olded up to create cavities on their outer surfaces specific for the 20 different amiuo acids. Tlte cavities would be so

ot deoxyribose for ON/\), v.tlidl has a ~oxyl groop on catt:>oo 2 ( shown 10 red) In OOdltior~ RNA has the pynmidine base tJ
32

Nur(eif: l ll:ids Ccmwy C.enrJtic Iriforn'lnlron

shaped that only one given amino acid would fit. and in this way RNA would provide the information to order amino acids during protein synthesis. By 1955, however, Crick became disenchanted wfth this conventional wisdom. arguing that il wo uld never work. In the first place. the specific chemical groups on the four bases of RNA (A, U, C~ and q should mostly interact with water-soluble groups. Yet, the sp&cific side groups of many amino acids (for example, leucine, valine, and phenylalanine) strongly prefer intet'actions with water-insoluble (hydrophobic) groups. In tha second place. even if somehow RNA could be folded so as to d1spla}" some hydrophobic surfaces, it seemed at the time unlikely that an RNA template would be used to discriminate accurately between chemically very similar amino acids like glycine and alanine or valine and isoleucine, both pair-s differing only by the presence of single methyl {CH:~) groups. Crick thus proposed that prior to incorporation into proteins. amino acids are first attached to specific adaptor molecules, which in turn possess unique surfaces that can bind specificaHy to bases on the RNA templates.

The Test-Tube Synthesis of Proteins FIGURE 2-13 Bedron micrcJsraph of

ribosomes attached to thee~ retkubMn. This electron mcrograph {lOS,OOOx) shovvs a port!oo ol a panaeetic cell. The upper fight pot1A:Jn .sllows a porl'ton of the the tower .lef1 51-;ows a lcwge ~r of ribosomes attached to the endoplasmiC rellel.Atlm. Some rt>osomes exist free in the cytoplasm; others <~re dttadled to the mernbfa. nous endoplasnli<: reticufum. (Source: Counesy ot KR Porter) m~tex:hoodoon and

The discovery of how proteins are synthesized required the det.·eJopment of cell-free extracts capable of cru'Tying on the essential synthetic steps. These wf!re first effective! y developed beginning in 1953 by Paul C. 7..amecnik and his collaborators. Kev to their success were the recently available radioacth:ely-tagged amin~ acids. which they used to mark the tTaCe amounts of newly made proteins. as well as high-quality. easy-to-use. preparative ultracentrifuges for fmctionation of their cellular extracts. Early on, the cellular site of protein synthesis was pinpointed Lo be tlle ribosomes. small RNA-containing particles in the cytoplasm of all ceHs activeJy engaged in protein synthesis (Figure 2-13). Several years later. Zamecnik. by then collaborating with Mahlon B. Hr\8glarJd , went on to make the seminal discovery that prior to their incorporation into proteins. amino acids are first attached to w hat we now call transfer RNA (tRNA) molecules by a class of enzymes called aminoacyJ synthetases. Transfer RNA accounts for some 10°,b of all cellular RNA (Figure 2-14). To l:'learly everyone except Crick, this discovery waS totaU y unexpected. He had . of course. previously speculated that his proposed .. adaptors" might be short RNA chains. since their bases would be able t o base-pair with appropriate groups on the RNA m olocules that served as the templates for protein synthesis. As we shiiH relate later in greater detail (Chapter 14}. the transfer RNA molecules of Zamecnik and Hoogland are in fact the adaptor molecules postulated by Crick. Each transfer RNA contains a sequence of adjac.cnt bases (the anticodon) that bind specifically during protein synthesis ro successive groups of bases (codons} along the RNA templates.

The Paradox of the Nonspedfic#Appearing Ribosomes About 85% of cellular RNA is found itt ribosomes. and since its absolute amount is greatly increased in cells engaged in large.scale protein synthesis (tor example. pancreas and liver ceHs and rapidly growing bacteria) , ribosomal RNA (rRNA) was initially thought to be the template for ordering amino acids. But once the ribosomes of E. coli were carefully analyzed. several disquieting features emerged. First, aH E. coli ribosomes, as well as those from all other organisms.

The Ce11tral Dogma

are comp05ed of two unequally-sized subunits. each containing RNA. lhat either stick together or fall apart in a reversible manner, depend· ing on the surrounding ion concentration. Second, all the rRNA chains within the small subunits are i'lf similar chain lengths (about 1 ,500 bases in E. colll, as are the rRNA chains of the latge subunits (about 3,000 bases). Third. the base composition ofbolh the small and large rRNA chains is approximately the same {high in C and C) in all known bar.teria, plants, and animals, despite wide variations in the AT/GC ratios of their respective DNA. This was not to be expected. if the rR..I\JA chains w~re in fact a large collectio'rl of different RNA tern· plates made of a large number of different genes. Thus , neither tlle small nor large class of rRNA had th~ feel of template RNA.

3'

Discovery of Messenger RNA (mRNA) Cells infecte,J with phage T4 provided Lhe ideal system to find the true template. Following infection by this virus, cells stop synthesizing E. coli RNA; tl1e only RNA synthesized is transcribed off the T4 DNA. Most strildngJy, not only does T4 RNA have a base composition very similar to T4 DNA, but it does not bind to the I'ibosomal proteins that normally associate with rRNA to form ribosomes. Tnstead. after first attaching to previously exist ing ribosomes, T4 RNA moves across their surface to bring its bases into p ositions whel'c they can biod to the appropriate tRNA·amino acid precursors for protein synthesis (11gwe Z-15). In so acting. T4 RNA orders the Binino acids and i$ thus the long~sought-for RNA template for protein synthesis. Because it carries the information from DNA to the ribosomal sites of protein synthesis, it is caUed messenger RNA (mRNA}. The observation of T4 RNA binding to E. coli ribosomes. first made in the spring of 1960, was soon followed with evidence for a separate messengp.r dass of RNA within uninfected E. coli cells, thereby definitively rulh'J8 out a templale role for any rRNA. Instead, in ways that we shalt discuss more extensive)y in Chapter 14, the rRNA components of ribosomes, together with some 50 different ribosomal protejns that bind to them , serve as lhe factories for protein synthesis, functioning to bring together the lRNA -amino acid precursors in•o positions where they can read off the information ptovided by the messenger RNA templates. Only some 4% of total cellular RNA is mRNA. This RNA shows the expected large variations in length. depending on the polypeptides for which they code. Hence, it is easy to understand why mRNA was frrst overlooked. Because only a small segment of mRl\JA is attached at a given moment to a ribosome. a single mRNA molecule cnn simultaneously be read by several ribosomes. Most ribosomes are found as parts of polyribosomes {g.roups of ribosomes translating the same mRNA), which can include more than 50 members (Figure 2-16).

Enzymatic Synthesis of RNA upon DNA Templates As messenger RNA was being discovered, lhe first of the enzymes that transcribe RNA off DNA templates was being independently isolated in the Jabs of biochemists jerard Hurwitz and Sam B. Weiss. Called RNA polymerases, these enzymes function only in the presence of DNA, which serves as the template upon which single-stranded RNA chains are made. and use lhe nucleotides ATP. CTP. CTP, and UTP as precursors

OJRNA

3'1

lJ

c

G =:J s~

L__j

codon FIGURE

1-14

Veast alilnine tRNA

~e.

as determined by Robetl W. Halley and his asSC)Ciates. T'ne drllicodon .., d1is tRNA recognu:es the cvdcn fur aJ.mine ifl the mRNA. Several rrx:>difted l'llJcieostdes exiSt 11"1 the sttl.JCll..n>· ~ ; pseudouridine. T - tix>thymidlf'lE:, DHU = 5,6-0lhydr~, l = ~. m'G = l-rnethylguanosme. m' - 1~10sine. and m7G -= N,Ndmettm~

34

fI G U R IE

Nucleic A.c ids Convey Genetic Information

2•15

Transcription and

translation. The nucJeotidt:>s of mRNA are a5Sef1lbled to fonn a canplement&y cq>y of ooe strand of DNA Each group or three 15 a codoo that is complernel1tary to a grot..p (lf 1htee nudeol:ldE:s in the anticodon region (lf a specific tRNA rno~ec~Ae. When bdse pairtng OCOJTS, an amino arid canied at tlle other end (lf tt1e tRNA molea.Ae IS added to the growing protein chain.

•., CGic~ cc iiOC~ cc Uu

13'

mRNA

l

8fT1inO acid

(rigure 2-17}. In bacteria, the same enzyme makes each of the maj{)r RNA classes {ribosomal. transfer, and messenger). u sing appropriate segments of chromosomal DNA as their templates. Direct evidence that DNA lines up the corrnct ribonucleotide precursons came from seeing how tbe RNA base composition varied with the addition of DNA molecules of different AT/GC ratios. ln every enzymatic synthesis. the RNA AU/GC ratio was roughly similar to the DNA AT/GC ratio (Table 2~2).

F I GU RE

2-16 Diagram oh

polyribosome. Eadl fibosome .maches a1 a start signar at the s· end of an mRNA chain end synthesizes a pclypeJl(icle as it proceeds along me molerule. Several nbosomes f"lla'Y be a~ to OCle rnRNA

molecute at one time;

1he emire assembly is caled a polyrbJsome

ril:!osoo"e subunits

released

The Celllf'al Dogmo

site of nt.lCiectide addiliof\ to growing RNA strand

Ft G U R E

35

2-17 Etuymatic synthesis of

RNA upon it DNA temp~. Qdalyred by

RNA polymerase-

During transcription, only one of the two strands of DNA is used as a template to make R""lA. This makes sense, because the messages carried by the two stranrls, being complementary blJt not identk.al, are expected to code for completely different polypeptides. The synthesis of RNA always proceeds in a fixed direction, beginning at the 5' end and concluding "'rith the 3'-end nucleotide (see Figure 2-17}. By this time, there was firm evidence for the postulAted movemenl of RNA from the DNA-containing nucleus to the ribosome-containing cyt
Establishing the Genetic Code Given the existence of 20 ami.no acids but only four bases, groups of several nucleotides must somehow specify a given amino add. Groups of t wo. however. would spocify only 1fi (4 X 4) arnino adds. So from 1954, the start of serious thinking about what the genetic code might be like. most attention was given to how triplets (groups of three) might work. even though they obviously would provide moce permutations (4 x 4 x ~) than needed if each amino acid was specified by only a single triplet. The assumption of colinearity was then

TA 8 ._I:

2-2 Comparison of the Base Composition of Enzymatically Synthesiled RNAs with the Base Composition of their Double-Helical DNA Jemplates Composalion Of lhe RNA Bases

Source of DNA Template

12 Calf thymus Eschencflia coli Micrococcus /yscxletkticus (a bactenum)

A+U G+C

G+C

A 't'T

Adenine

Uracil

Guanine

Cytosine

Observed

In DNA

0.31 0 .3 1 0.24 0.17

0 .34 0 .29 0.24

0 .18

0.17 0.21 0 .26 0 .34

1.86

1 .84

tso

L35 0.97

0 16

0.19 0.26 0.33

092 049

0.39

Nucleic Acids Convey Genetic JnfOITIICiboll

very important- It held that successive groups of n ucleotides along a DNA chain code for successive amino acids along a given polypeptide chain. That colinearity does in facl exist was shown by elegant mutational analysis on bacterial proteins. c.arried out in the early 1960s by Charles Yanofsky and Sydney Brenner. Equally important were the genetic analyses by Brenner and Crick, which in 1961 first established that groups of three nucleotides are used to specify individual amino acids. But which s pecific groups of three baRes (coclons} delP.nnine which specific amino acids could on1y be learned by biochemical analysis. The major b1·eakthrough came when Marshall Nirenberg and Heinrich Matthaei, then working together. observed in 1961, that the addition of the synthetic polynucl~de poly U (UUUUU . . .} to a cell-free system capable of making proteins leads to the synthesis of polypeptide chajns containing cnly lhe amino acid phenylalanine. The nucleotide groups UUU thus must specify phenylalanine. Use of increasingly more complex, defined polynudeotides as synthetic messenger RNAs rapidly led t o the tdentification of more and more coclons. Par1iclllarly important in completing the code was the use

of polynudeotides like AGUAGU, put together by organic chemist Har Gobind Kho.rnna. These further defined polynucleotides were cri1i· cal to test more specific sets of codvns. Completion of the code in 1966 revealed that 61 out of the 64 possible permuted groups <'..on-esponded to amino acids. with most amino acids being encoded by more than one nuclootide triplet (Table 2-3).

FIGURE 2-18 Demonstration that RNA is 5)1rtllesized in the nudeus and moves to the qtoplasm. (a) Autoraciograr.h cJ a ceiJ (Tellahymeno) ellp<>Sed to radioactive cytidine for 15 mllllltes.. Supe~ Clrl a photograph of a then section of the cell is a photograph of an exposed silver emulsiorL Each dad< spot represefltS the path of an electron emitl€d rrom a 3f-l (trrtJum) atom lhat has been 111ccrpcrated into RNA. Almost all the nevJy made RNA rs found within the nudeus.. (b) AutocaOograph of a similar cd exposed to radioactive cytdine fOf 1:2 ~tes and the!l allowed to 8f01t1 fo( 88 minutes lf1 the presef1Ce of nonrodioactJVe cytdne PTacriuRy all the labciW1CfXJ)Oiated into RNA lfl the first 12 milii.JteS has left the nucleus and moved into the q'tq>lasm. (Source: Coortf'sy of O.M. Prescott. U~rSity of Cdorado Medici!! School: reproduced from 1.964. Ptogr. Nucleic Acid Res. Ill: .35, With perrrjsroo.)

TABU

2-3 The Genetic~ second position

uuu we WA UUG

F'tle

leu

cuu

cue ~

0

= r.o

-

CUA

ucu ucc

Ser

UAC

UCG

1m I!1S

ccu

CAU

UCA

CCC Lev

UAU

CCA

CAC

Pro

CAA

ax;

CCG

CAG

AlJU

PO)

MU

Tyr

lJGU

Cys

UGC

$lcp

1!1!.1

stop

UGG

His

an

CGU CGC

CGA

Alg

()

~

E

I;:

AlJC

le

PC-A

NJA AUG

Met

GUU

G\JG

Tk

Val

APe AAA

/lCG

A./>13

GCU

GAU GAC

GCC

GUC

GtlA

N:.C

GCA

GCG

Ala

AGU Asll

lys

~

/1.00

Ser

Arg

GGU Asp

GGC

GAA

GAA

GAG

AGC

G1u

GGG

-a

::t'

eGO

Gty

Eswhlishing the Direction of Prorein Synthesi&

37

ESTABLISlUNG THE DIREC"'TION OF PROTEIN SYNTHESIS The nature of the genetic code. once determined. led to further questions about how a polynucleotide chain directs the synthesis of a polypeptide. As we hi!Ve seen here and shaH discuss in more detail in Chapter 6, polynucleotide chains (both DNA and RNA) are synthesized in a 5' - 3' direction. But what about the growing polypeptide chain? Is it assembled in an amino-tenninal to carboxyl-terminal direction. or the opposite? This question was ;mswered in a classic experiment 'in which a ceJl-free system was u sed for carrying out protein synthesis. The call-free system was cceated u sing an extract from immature ted blood cells (known as reticulocytes) from a rabbit. which are efficient factories for the synthesis of the n-and {:\-globin subunits of hemoglobin. The cell-free system was treated with a radioactive an1ino acid for a very few seconds {less than the time required to synthesize a comple te globin chain) after which (Jrotein synthesis was immediately stopped. A brief radioactive labeling regime of this kind is known as a pulse or puke-labeling. Nex1. globin chains that had completed their growth during the period of the pulse-labeling were separated from incomplete chains by gel electrophoresis (Chapler 20). The full-length polypeptides were then treated with an enzyme. the protease trypsin, that cleaves proteins on partic ular sites in the polypetide cha in ~ thereby generating a series of peptide fragements. ln the final step of the e:xpe.riment, the amount of radioactivity that had bP.en incorporntad into each peptide fragment was measured (Figwe 2 -1 9).

a

•

~

NH2

:=

(

FtGU RE

COOH

)

2·19

into • growing MPePtide main. lhe expern-nental detaits Me described lf"' the text {a) DrstfilxJtloo of racioac:bvity amoog ~ted chains after a short peood of labeling. (b) lncorptXation of label plotted as a func1ion of postioo of the peptide Wthin the c~ed chaifl.

b

• • ~

•

·;;;:

'5

"'

•

0

'6 ~

• • NH2

COOH position of peptide

lnmfpOritlion of label

Keep in mind that the globin chains were at various stages of completion during the period of t he pulse (Figure 2-19a). Thus, nascent chains that had only just started to be synthesized would be unlikely to have reac hed cmnplatiun during the period of the pulse because the time of the pulse-Labeling was less than the time required to synthesize a complete globin chain. On the other hand, globin chains that we.r e almost full length would be highly likely to have reached completion during the pulse. Also, keep in mind that only chains that had reached full length during the time of the pulse were isolated and subiected to trypsin treatment. lt. therefore, fol lows that the b'ypsin-generated peptides w.i th the leas t amou.nt uf radioacrive amino acid (normalized to the ~e of the peptide) should have derived from r~ions of the globin protein that w ere the first to be synthesized. Conversely. peptides with the g.reatest amount or raclioac• ivily BhouJd hat•e derived from regions of the protein that were the last to be synthesized. The results of the experiment are shown in .f'igure 2· 19b. As you can see. radioactive rnbeling was lowest foc peptides from the aminotennina l region of globin and greatest for peptides from the c.a.rboxylterminal region. We. therefore. conclude that the direction of protein synthesis is from the amino-terminus to the Girhoxyl-tenninus. rn other words. during protein synthesis the first amino add to Le incorporated into the nascent chain is the amino acid at the amino terminal end of the protein and tbe last to be incorporated is at the carboX)•l-terrninus.

Stan and Stop Signals Are Also Encoded within DNA Initially, it w as guessed thttt transll'! tion of an mRNA molecule w ould

commence a t one end and finish when the e ntire mRNI\. message had been read into amino acids. But, in fact. translatjon both starts and stops at internal positions. Thus. signals must be pTcsent wilhin DNA (and its mRNA products) to initiate and tenninate tra nslation. First to be workHd out w ere the stop signals. Three seperete coclons (UAA. UAG. and UGA). first .known as nonsense l:odons, do not d ired the addition of a varticular amjno acid. Instead, those codons serve as translational stop signals (sometimes called stop codons). More cornplicared is the way translational start signals arc encoded. The amino acid methionine starts all polypeptide cltains. but the trlpl(lt (AUCJ th~r c..od es for thP.se initialing mP.thionjnes a lso codfls for methionine residues that have internal locations. The AUG codons. at which polypeptide chains start, are p.rf1(Jeded by specific purine-rich blocks of nucleolides that serve to attach mRNA to ribosomes (see Chapter 14}.

THE ERA OF GENOMlCS With the elucidation of the central dogma, it became clt>.ar by the mid-Hl60s how the gerwtic blueprint contained in the nudeotlde

sequence could determine phenotype. This meant that profound insights into the nature of living things and their evo)uti.on would be rever~ led

from DNA SefJUencP.s. Jn recent yr.ars the Rdvent of .rapid. automated DNA sequencing methods has led to the dett'rmi nation of

Summary

39

complete genome sequences for a wide variety of organisms. Even the human genome, a s ingle copy of which is composed of more them 3 bi Ilion base pairs, has been elucidated and shown to contain more than 30,000 genes. During the upcoming years. many more complete genome assemblies will be available from a broad spectrum of organisms, including puplars. sponges, jellyfish. crustacflans, stta urchins, frogs, and dogs. ln the future it should be possible to extend the interpretation of genome sequences beyond the identification of genes and their encoded proteins. Other classes of ONA sequences mediate replication. chromosome pairing, recombination. and gene regulation. rt is possible to envision a day when comparative DNA sequem!e analysis will reveal basic 1nsights into the origins of complex behavior if1 humans, such as the acquisition of language, as well as the mechm1isms Wlderlying the evolutionary diversification of animal body plans. The purpose of the forthcoming Ghapters is to provicle a firm foundati o n for unde.rstandl.ng how DNA functions as the template for biological comple.xity. The remaining chapters in Part t , review the basic chemistry and biol<Jgy relevant to the main themes of this book. Parl 2, Maintenance of the Genome, describt'ls the structure of the genetic material and its faithful duplication. Part 3, Expression of the Genome, shows how the genetic instructions contained in DNA is converted into proteins. Part 4, Regulation. describes strategies for differential gene activity that are used to gone.rate complexity within organisms (for example, embryogenesis) a11d diversity among organisms {for example. evolution). Finally, Port 5, Methods, describes Vaiious laboratory techniquP.s, bioinformatics approaches, anrl mode) systems that are commonly u~ed to inv.-. stigate biological problem.<~ .

SU~1.MARY

The disco\lery that DNA is the genetic material can be traced to experirntmts performed by Griffith, who showed thilt nonvirulent strairu> uf badttria could be gt~t tetlaiUy tr'~nsformcrl 'With a f'ubstance derived from a hoot-killed pathogenic .stta.in. Avery. McC~rty. a11d Macleod subRequently demonstrated that the transforming substam·e was DNA. Furtl1er evidenc~ tha.t DNA is the genetic material Who<; obtained by liershey and Chase in ex~rlmeuts with radio·labelerl bacteriophltgE>~ Building on Chargaff's ruJ~ and franklin and Wilk.iru;' X-ray di.ffrac:tiot1 studies, Warson and Crick proposerl a douhle.h,elkal structure of DNA. In this mode), two polytJucle(Jtide chair~s are twisted around each other to form a regular cloubltl helix. The two ch~int~ within the double helix are bPlcl togp,ther by hydrogen bond~ between pairs of ~~~es. Adeni.ne is alw~ys joinc~d to chymine. and guanine is aJways bondoo to cylo8ine. The e~isteoce of the base pairs means that the sequence of nucleotide5 a long the two mains are not identit;aL but t:omp i~MT~entary. 'fhfl finding of lhi..c; relatiunshtp suggested a mechanism for the replication of DNA in whir:h each ruanrl sl:li'Ves as a tern plate foe its complement. Proal for Uti!' hvpothesls Cl:ln~ from (~) the ol>servation of

MeseJ~n

and Stahl that the two strands of each double he-

Ux separate during each round of DNA replication, <~nd (b) Kornberg·s discovery of an enzyme that uses liinglestrand ed DNA as a template for the synthesis of fl cnmplermmtary slnmd. As we htwe soon, aoc.ording to the ..centmJ dogma '' informa.tioo flows from DNA loRNA to prorein. This transfunnat\un is 1:n:hieved in two steps. First. DNA is t raoscrihed inw an RNA inteouediatti (mes.-.eeager RNA), J~Jld se<'.<Mld the mRNA j5 tran....tllted into protein. Translation nt the mRl\JA •·equiws RNA adaptor molecules C~:t1led lRNAs. 1'he ~ey rharac.terisHc of lhe genetic code is t!Uil wch [tiplet codon is rer.ognized hy a tRNA. which is a.<;60· dated with a cognate ~mino ucid, Out of 64 (4 X 4 ><. 4J potenli!il <:odons: 61 are used to spt:K:ify the 20 ar11ino add l>uiding ulo<;ks of proteins. whereas 3 are used tn provide cham-lermitltlting segnals. Knowledge of the genetic code allows us tQ predict protein CfJdhlg sequences from ONA SE?{Jllet:lce& The advent of rapid DNA sequendng methods has ushered in a new era of geoutnics, in whit:h wmplete genome sequeuces are heing detennirtf!d ror a wide variely nf organisms. induding humans.

40

Nuc#Pic Acids Omvey Genetic lnforrnulion

BIBLIOGRAPHY Genern1 References Brenner S .• Strelton AO.W.• and Kstplan S. 1965. Genelic code~ The nonsense triplets for chain termination and Lhei.r supprest'ion. Natum206: 994- 998. B~rmtlr S .. JocoL F.. and M~lson M. 19£1. An un~table intt!1111ediatl' carrying information from genes to ribosomes for prole in synlh~is. Noture 100: 576-581.

Cairns J., Stent C.S.. and Wat~on U l .• tidS- 1966. Phage and ll1e origins of moleculat biology. Cnld Spong l larbor Lab<.Jnttory Press. Cold Spri n~ ffurbor. N~w Yorl\.. Owrgaff E. 1951. Structure and function of mtcleit: acid s fit' cell con,;tilu~nts . Fed. Proc. 10! 65 4 -659_

Cold Spring Harbor Sympotria em Quantitative Biology. 1966. Volume31: The gcmeticcode. Cold Spring Hachor Laboralory Press, GJid Spring Ha rbor, New York..

Crick F.H.C. and Watsvu J.D. 1954. The complen~aotary s h·uc:h•re of rleoxyribonucleic acid. Proc. Roy. Soc. (A) 223:8096.

Crick EH.C. 1955. On cl~erale template and ttae adaptor hypothesis. A note for the RNA Tie flub, unpublished. Mentioned in Crick's 1957 discussion, pp. 25-26, in The sl.cudll1'8 uf nucleic: ar..ids and their role in protein syntllflsis, Biochem. Soc. Symp. no. 14. Cambridge Univt:ll'sity Press, Camhr.idge. EnglHnd . - - 1958. On protein synthesis. Symp. Soc. £.xp Bioi. 12: 548-555.

- - - 1963, Tlw rect~nt ~xr;item~nt in the lem. Prog Nucleic A c id Res. 1: 164-217.

codin~ prob~

- --1988. What Mad Pursuit : A PersDnal View of Scientific:Discovery·. Basi(; l3i)'(}ks, New Yor'k.

Echols H. and Gross C.A., eds. 2001 . Operators and

Promoters: Tile Story of Molecular Biology a nd its Creators. University of California Press, Berkeley. CaJifomia. Franklin R.E. and CosJin.g R.G. 1953. Mul~J a.r conf;guration irJ sodium thyrnonucleasc. Nature .171: 740 - 741 . He£Sh#y .t\.(.). and Chase M . 1952. lndependt!nt function of viral protein and nudeic add on growlh of bacteriopha,ge. / . Gen. Physiol. 36: 39-56 . Hoagla11d M.B., Stuphenson M.l.... Scott J.J·:, Htlr.nt 1..1.. and Zamt'Ulik P.C. 1958. A nolubl ~ ribonud~ic lldd intermediate in ptnlein synt~is . {. Bioi. Chern. 231: 241 - 257.

HoUt1y R.W., Apgar J., Everett G.A.. Madison J.T.• Ma.rqul$&e M .• Merrill S.H., Ptlnswil:k ).R, and Zamir A. 1965. a ribonucleic <1cid. Science 147:

Strm:ture of 1462-1465.

lngrem V.M. 1957. C'..ent~ mut~tions iu numan lwmnglobin; The d1ernica} ditierence between normal und sid
lacob f. and Monod J. 1961. Genetic regulatory n'lf:l('ha· ni5rns in the syn thesis of pmWn.s. / . Mol. 13iol. 3: 318 - 356.

Judson H.F. 1996. 1'he eighth day of creation, Expanded ediliu n. Co)d Spring Harbor Ll:shoratnry Press. Cold Spting J:larhur, New YorkKor nbet-g A. 1960. Biological synthesis of deox~Tibon u cleic acid. Science 131: 1503- 1508.

Kornberg A. und Baker T.A. 1992. DN" Replication. W.H. Freeman, New York McCarl}' M . 1985. The tronsformin;; princ ipltJ: Discoveril1fS t11ot gtmffl> ore made of DNA. Noctou. New York.

Meselson M. and Stahl P.W. 1958. The replication of ONA in Escherichia coli. Proc. Nat. Acad. Sci. 44: 671 -68Z.

Nirenberg M.W. M d Matthooi J.H. 1961. The dependence uf <:ell-free prvt~in synthesis in E. coli u pon na tura lly oc.curcing or xynth~li c po l)1·ibonudeulides. Proc. Not Aced. Sci. 4 7: 158 6- 1602. O lby R 1975. The potl1 to I he douhle helix. l 'nive..sity of Washington Press. Saattle. .Portugal F.fl. and Cohen J.S. 1960. A centwy of DNA: A his-tocy of the discovezy of the structure and fr.mr:lion of the genetic sui»>tance. MlT Press, ('..arnl:xidge, Massachusetts. Sarabhai A.S., Stretton A.O. W.. Rrenn er S., and Bolte A. 1964. Co-linearity of the gene w itb the polypeptide r.hain. Noture ZOJ : 13-17. St<....lt G.S. and CaJendar R 1978 . .Molecular genetics; An mlroductory narmtlve. 2ntf edition. F'n:!emaQ, San Francisco. Volli.in E. and A
WiU..i.JJs M.H.F., Stokes A.R. ami Wilson H.R. 1953. Moler.u1ar slruc~ ure of' dooxyt:lf!ntose oucf<>ic ctdd. Nature 171: 7:-38 -740.

Yartofsky C.. Carlton B.C. , Cuest f.R.. Helinski D.R.. and Henni.ng U. 1964- Ou the coli.neari1y of gtirt strudure. Proc. NaL /\cod Sci. 51: 266-272.

CHAPTER

The Importance of Weak Chemical Interactions he macromolecuJes that w ill preoccupy us throughm•t this book-and those of most concern to molet."'Ular biolvgists-are proteins and nucleic HC:ids. These are made of umino acids and nuclcotides l'espet:..1ively, and in both cases the constituents are joined by covalent bonds to make polypeptide (protein) and polynucleotide {nucleic acid) chains. Covalent bonds are stTong. stable bonds, and essentiaHy Tlf'ver brP.ak spont.a.neous1y within biological syslflms. Buf weaker bonds also exist. and indeed are ,,ital for life. parUy because they Ull1 form and break under the physiological conditions pr~mt within cells. Weak bonds mediate the interactions between enzymes and their substrates, and between macromolecules-m~1 strikingly, as we shaU see in later chapter:s, between proteins and DNA, or proteins and other proteins. But equally important, weak bonds also mediate interactions between different parts of individual macromolecul~. detennining the shape of those molecules and hen~ their biological function. Thus, although a protein is a linear chain of covalently-linked amino acids. its shape and function are detcnnined by the stable throedimensional structure it adopts. TI1at shape is determined by a large collection of individually weak interactions that fOI'm between ami11o acids that do n ot need to be adjacent in the primary sequence. likewise, it is the weak. noncovalent borcds that hold the two chains of a DNA double helix together. In this chapter we consider the nature of c hemical bonds. concen~ trnt\ng in large parr on the weak bonds so vital to the proper function of all biological macromolecules. 1n parlicu1ar we describe what it is that gives weak bonds their weak character. These bonds include van der Waals bonds, hydrophobic bonds. hydrogen bonds, and ionic bonds.

T

OU TLIN E

• Characteristics of ChemiCal Bonds (p. 4 1)

Tht:! G:Jr>cept ~Free Ene.gy (p

44)

• Weak Bonds in Biological

~ems

(p. 45)

CHARACTERISTICS OF CHEMICAL BONDS A chemical bond is an attractive force thai holds atoms together.

Aggregates of finite size are called molecules. Originally. it was thought that only covalent bond s hold atoms together in molecu les; now, weake r attractive forces are known to Le imporwnt in holding together DU111Y macromolecules. Foe e xample, the four Jl(llypeptide chains of hemoglobin are held together by the combined action o( several weak bonds. It i s thus now customary also to calJ weak positive interactions chemical bonds. even though they are not strong enough, when present singly. to effectively bind two atoms tvgetheT. Chemical bonds are characterized in several ways. All obvious charac.terisik of a bond is its strength. Strong bonds almost oever faJJ apart at physioJogkal temperntt..ttes. This is why atoms unitoo by covalent 41

42

The l mpDnonce af l'tW Chemieol lh l erocUons

-- -- - -- - - -- - - -3-1

F ICO U R £

Rotation ilbout ~

~-<&

bond in glucose_ 1il5 Cilfboo~ bond IS a sangle Wnd, and so arr.; of the three

a

b

(;

H

Of.(

Ho.. . ._ I_,H

H ......_ I

c

coofiguratJorn;, {a), {b), or (c), may occur.

l

c-

o

c I

H

,.,.ti

H.. . ._ l

c-'

OM

I ~----'\H

K~c.._.c -~oo HOK~-~OH H0~~)10H I I tt

H

H

HO

H

0

II

'..,.....c N I

H

FIGURE

l-2

The planar 51lape of the

peptide bond. Shown here is a pol1ion of an

extended poly~ chatn. Aarosl no mtatim 6 possible about the pepltde bond because of its por1Jal doubl~bond dlalaCte (see rrnddle pancl) M the atm'IS lfl the '(fay area must 6e 10 the same plare Rotation 1s possible, h~ver.. .around the r~ two bonds, \llhcn make up the polypepbde conflgufat100s. (Soufce: Adapted froln Pa.Jkng L 1960. ThE norure ofthe chemtcal bond and the srrur:ture of molecules ond aysroJs· An lfJlroduction ro modem srrucrural cl1emtsey. 3rd edition, p. qgs. Copyn~t e 1960 Cornell tJruversit:y Used by IJE'IITliSSIOfl of the ptblisha:)

I

()H

~

I

OH

H

I

OH

bonds always belong to the same molecule. Weak bonds are easily broken. and when t hey exist singly, they exist fleetingly. Only when present in ordered groups do weak bonds last a long time. The strength of a bond is c orrelated with its length. so that two atoms connected by a strong bond are always closer togethe•· than the same two atoms held together by a weak bond. For example. two h ydrogen atoms bound covalently to form a h ydrogen molecu le (I·HO aro 0.74 A apart, whereas the same two atoms held together by van der Waals forces are 1 .2 A apart. Another impot'tant characteristic is the maxi mum number of bonds thai a given atom can make. The number of covalent bonds that an atom can form is called its valence. Oxygen , for cxampte. has a valence of two: It can never form more than two covalent bonds. There i.<;; more variability in the case of van cler Waals bonds, in which the limiting factor is purely steric. The number of possible bvnds is limited only by the number vf atoms that can touch each other simultaneousl y. The formation uf hydrogen bonds is subject to more restrictivns . A covalently-bonded hydrogen atom usually participates in only one hydrogen bond, whereas an oxygen atom seldom participates in more than two hydrogen bonds. The angle between tw o bonds originating rrom a single atom is called the bond angle. The angle between two specific covalent bonds is always approxi.mately the same. For example, when a carbon atom has four single covalent bonds. t hey are directed tetrahedrally (bond angle = 1(J9° ). In contrast. the angles between weak bonds are much more variable. Bonds differ also in the freedom of rotation they allow. Single covalent bonds permit free rotation of bound atoms (Figure 3~ 1), whereas double and triple bonds a:re quite rigid. Bonds with parliaJ doublebond cbarac te,-, suc h as the peptide bond, are also quite rigid. For that reason , the carbonyl (C= O) and imino (N=C) groups bounrl togP.ther by th.e peptide bond must lie in the same plane (Figure 3·2). Much weaker, ionic bonds, on the other b~nd. impose no restrictions on the relative orientations of bonded atoms.

Chemical Bonds Are Explainable in Quantum-Mechanica1 Terms The nature of the forces , both strong and w eak. that give rise to chemical bonds remained a mystery to chemist'i until the quantum theory of the atom (quantum mechanics] was developed in the 1920s . Then. for the first time. the va:rious empirical laws about how chemical bonds are fonned were put on a fum theoretical basis. fr was realized that all chemical bonds, weak as well as strong. are based on electrostatic forces. Quantum mechanics providOO explanations for coWtlent

bonding by the sharing of electrons and also for the formation of weaker bonds.

ChemicaJ,Bond Fonnation Involves a Change in the Fonn of Energy The spontaneous formation of a bond between two atoms a lw~ys involves the release of some of the internal energy of the unbonded atoms and its conversion to another ene.rgy form. The stronger the bond , the greater the amount of energy released upon its formation. The bonding reaction between two atoms A and B is thus described by

A+ B - AB +energy

(Equation 3-tl

where A8 represents the bonded aggregate. The rate of the reaction is directly pro.vort.ional to the ~uency of collision between A and B. The unit most often used to measure eneiiD' is the calorie, the amount of energy required to raise the temperature of 1 gram of water from 14.5 11C to 15.5 OC. Since thousands of calories are usually involved in the breaking of a mole of chemica] hcmds , most energy changes w ithin chemical reactions a re expressed in kilocalories per mole. However. atoms joined by chemical bonds do not remain together forever, since there a lso exist forces thnt break chem ical b onds. By far the most important of these forces arises from heat enc~gy. Collisions with fast-moving molecules or atoms can break chemicJiJ bonds. During a collision. some of the kinetic energy of a rnoviflg moh'!cule js given up as i1 pushes apart two bon ded a toms. The faster a molecule is moving (t he h igher the temperature), the graater the probability that, upon collision, it will break a bond. Hence, as the temperature of a collection of molecules is lncrease lL the stability of their bonds decreases. The breaking of a bond i s thus always indicated by th e formula AB +energy- A+ H

(Equation 3-2]

The amount of energy that m ust be added to break a bon d is exactly equal to the amount that was released upon formation of the bond. Thjs equiva len c;e follows frorn tho first law of thermodynamics, which states that energy (except as it is inter<.:onvertible with mass) can be neither made nor destroyed .

Equilihrium between Bond Making and Breaking Every bond is thus a result of the combined actions of bond-malcing and bond-bi'tiaking forces. When an equilibriwn is reoched in a d osed system, the number of bonds forming per unit time will equal tbe number of bonds breaking. Then the p roportion of bonded atoms is descrjhed by the foll owing mass action formula: con cAB

where 1
(EqtJatioo 3-3) COJ1<.: 8,

and

con~

are the concentrations of A, B. end AB. respectively, in moles per liter. Whether we start w ith onty free A and B. wit h only the molecule AB, or with a combination of AB and free A and B, at equi1ibrium the proporHons of A, B. and AB v.riU rear.h the concentrations given by

Keq.

44

1'he Importance of Weak Chemical fnleroclions

THE CONCEPT OF FREE ENERGY There is always a change in the form of energy as the proportion of bonded atoms moves toward the equilibrium concentration . Biologically, the most useful way to express this energy c.hange is through the physical chemist's concept of free energy, denoted by the symbol G. which honors the great nineteenth-century physicist fosiah Gibbs. We shall not give a rigorous description of free energy in this text nor 'Show how it differs from 1he other fom1s of f!nergy. For this, the Teacler must refer to a t:hemistry text that discusses thP. second law of thermodynamics. It must suffice to say here that free tmergy is energy tltat has the ability to do wo.rk. The secontl law of thermodynamics tells us that a decrease in free enelp;y (.AG is negative) always occ\ll'S in s pontaneous reactions. When equilibrium is reached. however, there is no furtheT change in the amount of free energy (AG = 0). The equilibrium state for a closed col· Jection of atoms is thus the slate that contai ns the lf".ast amoW11 of free energy. The free energy los1 as equilibriwn is approach ed is eitller transformed into heat or used to in(.;IBase the amount of entropy. We shall not attempt to define entropy here except ki say that the amount of entropy is a m~asure of the amount of disorder. The greater the disorder, the greater the amount of entropy. The existence of entropy means that many spontaneous chem1cal reactio.ns (those with a net decrease in free energyl need not proceed with an evolution of heat. For example, when sodium c.:hlm·ide {NaCl) is dissolved in water. heat is absorbed rather than released. There is, nonetheless, a net decrease in free energy because of the increase in disorrler of the sodium and chlorine ions as thf1y move from a solid to a dissolved state. ~ Is Exponentially Related to llG

CIP.arly, the st£Onger the bond , and hence the greater the change in free energy (~G) that accompanies its formation. the gw.ater the proportion of atoms that must exist i.n the bonded form. This commonsense idea is quantitatively expressed by lhe physical-ch emical formula .iG = - RT ln ~

I A 8 L E :1-1 lbe Numerical R8ationsl!ip be~

the Equ;Jibriwn

or

K.,q = e

~CJKr

(Equation 3-41

where R is the univerSal gas constant. T is the absolute temperature. In js the logarithm (of Keq) to the base e, ~ is the equilibrium oonstant. and e = 2.718. Insertion of the appropriate values of R (1.987 cal/deg-rnol) and T (298 at 25 '.!(;) tells us that AG values as low as 2 kcaJ/mol can drive a bond-forming reaction to virtual completion if alJ reactants are present at molar concP.ntrations (Table 3-1 ).

Constant and .1(; at lS"' c

l(.q

AG. keallmoJ

0001

4.009

0 .0 1 0.1 10

2.726 1363 0

10.0

- 1 363

100.0 lOCO.U

- 2.726 - 4.009

Covalent Bonds Are V«y Strong The AG values accompanying the for mation of covalent bonds from free atoms, such as hydrogen or oxygen. are very large and negative in

fiign, usua11y - !10 to - 110 kcallmol. Equation 3-4 tells us that ~ of the bonding reaction will be correspondingly large. and so the concentratiun of hydrogen or oxygen atoms existing unbound will be very smalL For example, with a A.G value of - 100 kcal/mol, if w e start with 1 mol/L of the reacting atoms, only one in lOw atoms wHI remain unbound when equilibrium is reached.

WEAK BONDS IN BIOLOGICAL SYSTEMS The main types of weak bonds important in biological systems are the van der Waals bonds. hydrophobic bonds, hydrogen bonds, and ionic bonds. Sometimes, as we shall soon see, the distinction between a hydrogen bond and an ionic bond is arbitrary.

Weak Bonds Have Energies between 1 and 7 kcal/mol The weakest bonds are the van der Waals bonds. These have energies (1 to 2 kcaf/mo!) only slightly greater than the lctnetic energy of heat motioh. The energies of hydrogen and ionit: bonds range between 3 s..otl 7 kcal/mot In liquid solutions, almost aiJ molecules fOrm a number of weak bonds to neru:by atoms. All molecules are able to form van der Waals bonds, whereas hydrogen and ionic bonds can form only betwef'n molecules that have a net charge (ions) or in which the charge is unequally distributed. Some n10lecules thus have the capacity to f01·m several types of weak bonds. Energy consideration.-,, however, tel1 us that molecules always have a greater tendency to form the stronger bond.

Weak Bonds Are Constantly Made and Broken at Physiological Tcmpel1ltures The energy of th.e stronge:;t weak bond is only about ten times larger than the average energy of kinetic motion (heat) at 25 ~ (0.6 kcal/mo]}. As th~re is a significant sprP.ad in thA energies of kinetic motiOII, many molecules with sufficient kinetit: energy to break the strongest weak bond always exist at pbysiological temperatures.

The Distinction between Polar and Nonpolar Molecules All forms of weak interactions are based on attractions betwoon electric charges. The separation of e let:tric ~ harges can be permanent or temporary, depending on the atoms involved. For example, the oxygen molecule (0:0) has a symmetric distribution of electrons between its two oxygen atoms, so each of its two atoms is Wlcharged. In contrast. there js a nonuniform distribution of charge in water (H:O:H). in whlch the bond electrons are unevenly shared lFigure 3-3). They are held more strongly by the oxygen atom, which thus carries a considt>rable negative charge, whereas the two hydrogen atoms together h(lve an equal amotmt of positive charge. The center of the positive charge is on one side of the center of the negative charge. A combination of separdted positive and negative charges is <:aUed an electric dipclle moment. Unequal electron sl1aring reflects dissimilar affinities of the bonding atoms for electrons. Atoms that bave a tendency to gain electrons ate called electrohegativ~ atoms. Electropositive atoms hmre a tendency to give up electrons. Mo1ecules (such as H:.tO l that have a dipole moment are called polar molecules-. Nonpolar nroli!Cules are those with no efffdive dipole moruAnts. ln methane (CH,). for example, the carbon and hydrogen atoms have similar affinities for their shared electron pairs. so neither lhe carbon nor the hydrogen atom is noticeably charged. The distribution of chruge in a molecule can also be affected by the p~.nt:e of nearby molecules, particularly if the affcded molecule

\1M der Waals radius of~

/ .-!"~ -V'

covateot bood length

/

van derWass

radius of oxygen

-~

/ \A A

... .--direCtion of dipole movement fIG U R £

molewte.

3-l lhe stnKture of a walet

46

The Importance of Weok Chemical Interactions

1oA

F I c;; U R E 3-4 \lariation of van del Waals

forces with distance. The atoms shown tn thrs diagrdiTI are atoms of the lflt'rt tare gas
•

permissim)

sA

•

•

a bQut4A

van derwaals at1FctCtion jUst balanced ~ ~e

rorces. OWII'l9

1o irtefJ)elletrallOn ot outer e1ec1ron shells

acetate

•

•

is polar. The effect may cause a nonpolar molecule tu acquire a slightly polar character. lf the second molecule is not fJOlar, its presence will still alter the nonpolar molecule. establishing a fluctuating chruge distri· bution. Such induced effects. however. give rise to a much smaller separntion of charge than is found in polar molecules. resulting in smaller interaction energifls and correspondingly weaker chemical oond.s.

Van der Waals Forces Van der Waals bonding arises from a nonspecific attractive foroo originating when two atoms come close to each other. U is based not on the existence of p ermanent charge separations . but rather on the induced

fluctuati ng charges caused b.v the nearness of molecules. It therefore operates between all types of molecules. nonpolar as well as polar. It depends heavily on the distance between the interacting grotJps. since the bond energ_y is inversely propor1ional to the sixth power of distance (Figure 3-4). There also exists a more powerful van der Waals repulsive force, which comes into play at even shorter distances. This repulsion is caused by the overlapping of the outer electron shells of the atoms involved. The van de r Waa)s a ttractive and repulsi ve fo rces balance at guanine

a certain distance specific for each type (If atom. This distanc.:e is the so-called \'an der Waals radill8 {Table 3-2 a.rul Figure 3-5). The van der Waals bonding energy between two atoms separated hy the sum of

F. c;; U R E 3-5 Drawings of several molecules with the van der Waah radii of ttle Cl'tom5 shown in ~. blue. and

their van der Waals rndii int:reases with the size of the respective atoms. For two average atoms. it is only about 1 kcal/mol, which is just slightly more than the average thermal energy of moleeules at

orange.

room temper ature (0.6 kcaUmo)}.

Week Bonds lll Bml~ic:al SJ'siP.ms

This means that van der Waals forces are an efft.>ctive binding force at physiologk..al temperatures only whell several atoms in a given molecule are bound to several atoms in another molecule. Then the en~rgy of interaction is mm;h greattH' than the diSS<X:iating tendf'.ncy resulting from random thermal movements. For several atoms to inl~r at:t effectively, the molecular fit must be precise, since the distance separating any two interacting atoms must not bo much greater than the sum of their van dcr Waals radii (Figure 3-6). The strength of interaction rapidly approaches zet'O when this distance is only slightly exc~ded. Thus, thP. strongest type of van der Waals contact arises when a molecule t:ontains a cavity exactly compfemcntary in shape to a protruding group of another molecule, as is the case with an antigen and its specific ;mtibody (Figure 3~7 ) . In this instance, the binding energies sometimes can be as large as 20 to 30 kcal/mol , so that antigen-antibody complexes seldom faiJ apart. The bonding pattern of polar molecules is rarely dominated by van dar Waal5 intf!ractions. sinue such molecult>s can acqv ire a lower energy state (lose more free energy) bv forming other types of bonds.

I ABLE

47

3-2 \WI dft waals Radii of the Atoms in Biologiall Molec:ules der Waals radius (A)

tl8fl

Atom H

12

tv

1.5

()

1.4

p

19

s

1 85

CH3 group

20

~If

17

tflid
aromattc rT\OieC.ule

•

,·~

e.O a o,.

Hydrogen Bonds A hydrogen bond is formed be1ween a covalently bound donor hydrogen atom with some positive c.:harge a nd a negatively char-ged, c:ovalently bound acceptor atom (Figure 3·8}. For example. the hydrogtm atoms ·Of l:he amino {- NH.l) group are attracted by the negatively charged keto ( - C= O) o"'ygen atoms. Sometimes. the hydrogen -bonded atoms belong to gruups with a u nit of cha rge (such as NH3 .. or COO ). ln other cases, both the donor hydrogen atoms and the negative au..-eptor atoms have less than a unit of charge. Thfl biologically most important hydrogen bonds involve hydrogen atoms covalently bound lo oxygen atoms (0-H) or nitrogen atoms {N-H). Likewise, the r\egative acceptor atoms are usually nitrogen or oxygen. Tahie 3-3 lists some of the most important hydrogen bonds. Jn the absence of surrounding water molecuh:1s, bond energies rcmge between 3 and 7 kt:a11mol. the stronger bonds involving the greatei dlarge differences between donor and acceptor atoms. Hydrogen bonds m-e thus weaker than covalent bonds. yet considerably stronger than van dcr Waals bonds. A hydrogen bond, therefore, will hold two atoms closer together than the sum of their van der Waals radii, hul nCJt so close together as a covalenr bond would bold them. Hydrogen bonds. unlike van der Waals bonds. are highly directional In the strongest hyrlrogen bonds, the hydroge.n atrun po)nts directly at the occeptor atom (Figure 3 -9). If it points more than 30c away, the bond enr:rgy is much less. Hyrlrogen bonds are also much more spedfic: than V6n der WaaJs bonds, s ince they demand the exi!;teru.;e of molecules with complementary donor h ydi'ogen and aa:eptor groups.

• • 0

FIGURE ]-6 thear•angementof

moleades in a ~et of a aystal formed by the amino arid &fyrine. The ~ of the moteClie1. IS derew med by the \lc.J der Waas oo CJf the groups. ~ f01 the 1\1--11 o conlaets. v,h,th a«- shortened b( the farq<Jbon of hydtogm bonc:!s. (Source- Ad;,pterl from p,uumg L 1960_The nai1Jfe of the chern.col bond and the structure of molecules ond crystals. An l(lrroduc.tKJn to modem Slnxtuml chemJstrt 3r" e&uoo. p 26'2. Copynght c 1960 Corneli i.Jntllffi!ly Used l:1y' proni!.SlCltl of rhe ptblisher.)

T"BU

of fliologicatly Important Hydrogen Bonds

Some Ionic Bonds Are Hydrogen Bonds Many organic mo l~u les pos~s ionic gruups that contain one or more units of net positive or negative charge. The negatively charged mononudeotidcs. for example, contain phosphate groups, which are n egatively c harged. whereas each a mino acid {exrept proline) has a negative carboxyl gro"Up (COO ) and a positive amino group (NH3 ~ ) .both of which carry a unit of charge. These c.hurged groups are usuRlly neutralized b y nearby. oppositely charged groups.

J-] App.-oxirnate Bood Lengths

Approximate H bond length {A)

Bond

o

H .......

o

0- H ~··..• O

0-H ....... N N-H ....... o N - H -·u 0

N

H ...... N

2 70 :!: 0 10

2 .63 + 0 10 2.88 ... 0 l 3 3 04:!: 013

2 93

+

0 t (;

3 10 :!: 0.13

48

Th8 l mportof?Df' of Wttok C:hernicullnteroctivns

b

hy
.,. . . . o

tl)'~ boocJ between a ~ged ~ QfOUJ)4lf'Jd

lhe t)ydroxyl gl'(q) of tyrOS!tle

hydrogen bond betwc!el'l a cha'ged amiOO group and a d'larged c:arboxyl group

F t GU R IE

3-8 Example of hydrogen

bOlwSs in biological moleaAes.

f t G u R E ~-7 Antibody....antigen interaction. Tne structure shows the complex between Fab D 1.3 and lysozyrne. (FiS<:hmann T.O.., l3entley G.A., Bhat T.N., BoiJiot G~ ~ RA. PhiClips S.E_. Tello D•• and Pcljok R! 1991. J. Biof. Chern 266: 129 l5.)

The electrostatic fon.:es a<.:ting between the oppositely c harged groups an~ called ionic bonds. Their average bond energy in an aqueous soJu-tion is about 5 kca)/mol. In many cases. either an inorganic cation like Nai , K , or Mg+ or an inorganic anion like Cl or SO/ neutrRlizes the charge of ionized organic ntolet:ultlS. When this happens in aqueous solution, the neutralizing cations and anions do not carry fixed positions because inorganic ions are usually surrounded by shells of water molecules and so do not directly bind to oppositely charged groups. Thus, in watt..solulions, elt~ctrostatit: honrls to surrounding inorganic cations o c anions are usually not of primary importance in determining thP molecular shapes of organic molecules. On the other hand, highly directional bonds Tesult if the opposite1y charged groups can form hydrogen bonds to each other. For example. coer and NH3 .... groups are often held together by hydrogen bonds. Since these bonds are stronger than those that involve groups wilh less than a unit of charge, they 81'E' correspondingly shorter. A strong hydrogen bond can also fonn between a group with a unit chatge and a group having less than a unit charge. For example, a hydrog•m atom belonging to an amino group (NH:t} bonds strongly to an oxygen atom of a carboxyl group (Coo - ).

Weak I nteractions Demand Complementary Molecular Surfaces Weak binding forces are effective only wh en the interacting surfaces are close. This proXimity is possible only when the molecular surfaces h ave oomplemental"y struch.IJ't:Sy so that a protruding group for positive charge) on one surfAce is matched by a cavity (or negative charge) on another. That is, tbe interat.:ting molecules m ust have a lock~and-

lit.rok Bonds in Biolo8ical Systems

key re)ationshi p. ln cells, this requiremen t often means that some molecules hardly ever bond to other molec:ules of lhe same kind. b3cause such molcculs.co do not have the properties of sym rnel.ry necessary for self-interaction. For example, some pohu molecules contain donor hydrogen atoms and no suitable acceptor atoms. whereas other molecules can accept hydrogen bonds but b ave no hydrogen atoms to donate. On the other hand, there are many molecules with the necessary symmetry to permit strong self-interaction in celJs. WateT is the roost important example of this.

49

a

b

Water Molecules Form Hydrogen Bonds Under physiological conditions. water molecules rarely ionize to form H"" and OH ions. Instead , they exist as polar H-0-H molecules with both the hydrogen and oxygen atoms forming strong hydrogen bonds. In each water molecule, the oxygen atom l .'a n bind to two external hydrogen atoms, whereas each hydrogen atom can bind to one adjacent oxygen atom. These bonds are directed tetrahedrally (f'\gure 3-10}, so in its solid and liqu id forms, each water molecule tends to have four nearest n eighbors, one in each of the four directions of a tetrahedron. In ice~ the bonds to these neighbors are very rigid and the arrangement of molecules .fixed. Above the melting temperature (0 OC}, the energy of thermal motion is sufficient to break the hydrogen bonds and to allow the water rnolecuJes to change their nearest neighbors continually. Even in the liquid form, however, at any given instant most w ater molecules are botuu1 by four strong hydrogen bonds.

F J CUR ( 3-9 DirectioMI properties of

hydrogen bonds. (a) The vector aloP€ the <.owlent 0-H bond points dltectly at lt.e. aca:ptor oxygen. thereby Cotmlr18 a strong bond. (b) The wctor points away frOCT11he DXYget'l

atom, resulting in a much wealcer bood

Weak Bends between Molecules in Aqueous Solutions The aver age energy of a secondary bond, though small compared to that of a covalent bond, is nonetheless strong enough com pared to heat energy to ensure that most molecules in aqueous solution will form secondary bonds to other molocules. The proportion of bonded J- J0 l>iagtam of a lattice formed by water molecules. The ent'fgy

Ft G U R E

by farmr~ spedfic hyaogen bonds befvveen wat.el' rnclerules fiMX'S the affi31'lgemert of the mdecules i1 adjocem 1etri.'lhedrons. Oxygen aklms are indcated by ~
thE pu:tlJred structure ss nevroheless

predommt in water as wei as in ICe. (Source; AclaiJted from Pauling L 1960- The nature of the chemtrol bond ond the srrucrure ol moleo.Jes and crystols: An lillloduction to modem structural chemistry, 3rd editioo. p. 262. Copynght <e 1960 Cornel Unwersity. Used by permiSSIOn of the pOO!is.her.)

50

ThP-lmportonce of ~k Chemical Jnteroctions

to nonlxmded arrangements is given by Equation 3-4. corrected to take into ac.:count the high concentration of molecules in a liquid. lt tells us that interaction energies as low as 2 to 3 kcal/mol are sufficient at physiological temperatures to force most mole<.:ules to form the maximum numb er of strong secondary bonds. The specitk structure of a solution at a given instant is markedly influenood by which solute molecules are present. not only bet:ause molecules h ave specific shapes. but also because molecules diller in which types of secondary bonds they can form. Thus. a :moJP.Cule wHI tend to move until it is next to a molecule with wruc.:h it can form the strongest possible bond. Solutions, of course, are not static. Because of the disruptive influence of heat. the spedfic <.:onfiguration of a solution is constantly

- -- - - - - - - - - - - - - - - - - - - - -

Box 3 -1 lbe Urliqllec1ess of Mo1ec:ular Shapes and the Concept of SeJec:tive Stickiness

Even thoogh most celulclr f'l'ldecutes are built 4=1 from only a smal number of chernK.al groups, such as OH, NHlt and CHI< there es great speaicity as to which rrdecules tend to lie next to each othet 1lis is bec~e each rnok'o.Jle has Ulique booding properbes. One vefY dear demonsb'ation comes from lhe specificity d stereoisomers. Fa example, proteins are at.Nays

constructed from L-amino ac;js, never nom thew min()' wnageS) the D-afl'W10 actds ( Box 3- 1 Figure 1). AJthot.tgh the £>- IYld L-amino aods have id€f1tic.at COViRnt ~ thet- bindi-lg p-opetties to asymmetric molecUes .are often very affererlt. Thus.

most enzymes are spoofic for L-ciD1ino acids. If ell L-amino acid is able to attach to a specifiC enzyme, the .:ramno acid is

tmble to bind Most mclecutes lf1 cells can make good ~ bonds v.Ath only a smatl ntmbef of dher molecu&es. partly because most molea-'es in biol<:@cal systems exist in an aqueoos ern.ironment The fonnation of a bond in a eel therefore depends nd only on v.tlether 1INO I'Tl()ierules bind vvell to each other. but also on \1\Alether bond folmation IS OJ6
o •I BOX J-1 fIG U R ~ I 1be CWO ste.reoisomers of the amino acid alanine. (Sauce: Adapted from Pauflng L 1960_The natUfe ofthe chemical bond and the structure of molecufes and aysrols: An tntro<Juction to modem strucr.tJrol ch~ 3rd ed4bcrl. p. 465. Copyrrght ~ 1960 comel1 Unwerstty. Used by permss4oo of the publisher. Afld from Patl~118 L- 19~3. General d1emisrFy. 2nd edition, p. 49R Cqlyright 1953 by W H. freeman Used IMth ~.)

changing from one arnmgement to another of approximately the same energy content. Equally important in biological systems is the fact that metabolism is continua lly transforming one mole<.:ule into another and so automatically changing the nature of the secondary bonds lhAt can be formed. The soluti on structure of cells is thus constantly disrupted not only by heat motion. b ut a)so by the metabolic transformations of the cell's solute molecules.

Organic Molecules Titat Tend to Form Hydrogen Bonds Are Water Soluble The energy of hydrogen bonds per atomic group is much greater than that of van der Waals contacts; thus, molecules will form hydrogen bondJ> in preference to van der Waals contactc;. for example, if we try to mix water with a compound that cannot foon hydrogen bonds, such as benzene, thtJ water and ttHnzene molecules rapidly separett~ from each other, the water molncules Imming hydrogen bonds Among themselves while the benzene molecules attach lo one another by van der Waals bonds. It i" therefore impossible to insert a nonhydrogenbonding organic molecule into water. On the other ]land, polar molecuJes such as glucose and pyruvatfl. which contain a large number of groups that fonn excellent hydrogen bonds (such as = 0 or OH), are soluble in water (that is, they are hydrophilic as opposed to hydrophobic). While the insertion of such groups into a water lattice breaks water~wnteJ' hydrogen bonds. il results simullaooously in the f\'P.efl the polar organic molecule and water. These alternative arrangements, however. are not usually as entrrgetically satisfat.:tury as the waterwater arrangements, so that even the most polar molecules ordinarily have only limited solubility. Thus. almost all the molecules that cells acquire , either through food intake or through biosynthesis. are sornftwhat insoluble in water. These moltJCules, by their thermal movements, randomly collide with other rnoJocul es until they find complrlmentary molecular sw·faces em whir.h to attach and thereby release water molecules for waterwater intfJract ions.

Hydwphobic "Bonds" Stabilize Macromolecules The strong tendency of water to exclude nonpol ar groups is frequently referred to as hydn1phobic bonding. Some t:hemists like to call all the bonds between nonpolar groups J·n a water solutjon hydrophobic bonds (Figure 3~11). ln a sense this term is a misnomer, for the phenomenon that H seeks to emphasize is Ute absenve, not the presence. of bonds. (The bonds that tend to form between the nonpo1BT groups are due to van der Waals attractive forces.} On the other hand, the term hydruphobic bond .is often useful. since it emphasizes t.hr. fact that oonpolar gl'OU~ will tty 10 arrange themselves so that they are not in contact with wate1' molecules. Hydrophobic bonds are lm.{JOrtant both in the stabiUzation of proteins and complexes of proteins with other molecules and in the partitioning of proteins into membranes. They rna}' aCCOW1t for as much as <me-half the total ftee enP.rgy of protein folding. Consider. for example, the different amounts of energy generated when the amino acids alanine and glycine are bound, in water. to a

52

The lmportanct:! of ~k Chemico.l lllferactJoru>

..

b

L...J

1A

l-11 f)Wnple$ of \'an~ Waals (hydrophobic) bonds between the nonpolill" side lf0Cip5 of amino adds. The hydrogens are not thcfecated lf"ldMdt..raty_For the sk of clarity, the van d€f Waals 001 are reduced by l()Ofo. The strlJOural fO!i'l'lulas adJ(!Cef'lt to each space-filling dra\Ning indicate the Clllaflgemenlof lhe atoms (a) Phenylalanc~euane bond. (b) Pheflylafanl,-,ephen~tfle bond (Soun:e· Adapted frorn Sd1eraga HA. T1le proreins, 2nd e&t.on p. 527. Copyright © Harold Schffilga. U'X'd W1 permiSSIOn.) fIG U R l

third molecule that h as a surface complementary to alanine. A methyl group is present in alanine but not in glycine. When alanine is bound to the third molecule, the \an der Waals contacts around the methyl group yield 1 kcal/mol of energy. whkh is not released when gJycine is bound instead. From Equation 3-~. we know that this smaJJ energy difference alone would give only a factor of 6 between the binding of alanine and glycine. However. this
interior of cells ll1an under nonagueous conditions. Frequently, the energy difference is 3 to 4 kcal/mol, since the molecules involvoo oftf!n contain polar groups that can form hydrogen bonds.

The Advantage of ~G between 2 anJ 5 kcal/mo( We have seen that the energy of just one secondary bond (2 to 5 kcaJ/mol) is often sufficient to ensure that a mola:ule preferentially binds to a selected group of molecules. Moreover, these energy differences are not so large that rigid lattice arrangements develop within a cell; that is. the interior of a cell never crystallizes, as it would if the energy of secondary bonds were several times greater. Luger energy differences would mean that the secondary bonds seldom break. resulting in low diffusion rates incompBtibla with ce11ular existence.

SummaI}

Weak Bondtii Attach Enzymes to Substrates Secondary forces are necessarily the basis by which enzymes and thejr substrates initia lly combine with each other. Enzymes do nCJt indiscriminately bind all molecules, having noticeable affinlty only for their own substrates. Since enzymes catalyze both directions of a chemical reaction, they must have specific affinities for both setJ; of reacting molecules. In some cases. it is possiblr. to measure an equilibrium COilstant for the binding of an enzyme to one of its subslrdtes (F..quation 3-4), which t:."'nsequently enables us to calculate the !:J.G upon binding. Tiris calculation in turn hints at which types of bonds may be involved. For 6G values between 5 and 10 kcal/mol. several stror1g secondary bonds are the basis of specific enzyme-substrate int~ractions. Also worth n oting is that the ~G of binding is nover exceptionally high; thus. eneyme-substrate complexes can be both made and broken apart rapitlly as a result ofrandom thennal movement. This explains why enzymes can function quickly, sometimes as often as 1Qfl times per second. JI enzymes were bound to their substrates, or more importantly tc;> their products. by mom powerful bcm(ls, they woulcl act much mom s1ow1y.

Weak Bonds Mediate Most Protein:DNA and Protein:Protein Interactions As we wiU see throughout the book. interaeticms bernreen proteins and DNA, and between proteins and other proteins, lie at the h<'art of how r.ells detect and respond to signals, express genes, replicate, repair. and recombine their DNA, and so on-as well as how those processes are regulated. Again. these interactions are mediated by weak chemical bon ds of the sort we have described in this ehapter. Despite the low e11ergy of each individual bond. affinity in these interactions, and specificity as welJ, resu1tr:; from the combinAd effeds of many such bonds between any 1wo interacting molecules. In Chapter 5 we return to these matters wjth a detaHed look at how pwteins are built. how they adopt particular structures. and how they bind DNA and each o ther.

SUMMARY Many important chemic-al events in cells do not involve the making or breaking of covalent bonds. The cellular location of most moJecules depends on weak, or secondary. at.tn"lctive or repulsive fOrces. (n addition. weak bonds are important in determining the shape of many molecules. especially very large ones. The m0$t important of these weok forces are hydrogen bonds, van der Waals interactions. hvdrophobic bonds. and ionic bor1ds. Even though these forces are relatively weak. they are still large enough to ensure that the rip,ht .molec;ules (or atomic group6) interact with each other. For example, the surface of an enryme is uniquely shaped to a Uow spt!dfic attraction ofits RubstJ·a tes. The fum1ali.on of all cbemicall>onds, woak intr.ractions as weH as l'ltrong OO\Ialenl hondo;, pr-oceP.ds according to tbe laws of thermodynamics. A bond tends to fonn when

the rAsult would be a release of free energy (negative .6G}. f"or the bond to be broken, this ,;arne amount of free energy must be supplied. Bocause the formation of cova~ lent bonds bet"voon atoms us ually involves a very large r~ega tive QC, covalently bound atoms a lmost never sep arate spontaneously. In contrast. the .6C values- accompanying the formation ofwE!Clk bonds are only several times lerger than the average thermal energy of molecules at physiologi('.al te mpera tures. Single weak bonds are thus frequently being made and broken jn living cells. Molecules having polnr (charged) groups interact quite differently from noupolar molecules (in which the charge is s_ymmetrically distributed). Polar m.olecules can farm good hydrogen bonds, whereas nonpolar molecules can form only van dec Waals bonds. The must imporlanl polar molecule is watf3r. Each water molecule

Tlu~

lmportonceof Wrok Chemirol lnleroctious

can form (ow- hydrogen bonds to other water molecules. Although f-'IOlar molecules tend to be soluble in water (to various degree~) . nonpolar molecules are insotuble because they cannot form hydrogen bonds with water molecules. [very distinct molecule has 2 unique molecular shape that .restricts the numbt:r of molt:eules wilh wlaich it can folTTI strong secondary bonds. Strong secondary interac-

lions demand both a complementary (loc);~nd~key} relat,onship between the two bonding surfaces and the involvement of many atoms. Although molecules bound to~ethel' by only one Ol' two secondary bonds freq«ently ran apart. a rollection of tht>.Se weak bonds can .~:esull in " stable aggll!gate. The (act that doubl&-helical DNA never falls apart spontaneously demonstrates the extrerne stability possible in such an aggregate.

BIBLIOGRAPHY General References Brandon C. and Tooze J. 1999. Introduction to proLflin strl!cture. \.,arlaud Publishing. New York. Creighton T.E. 1992. Proteins: Struc.ft.jfP. (,md mnlecJJlur properties. 2nd edition. W. H. FreP.rnan. New York. - - - 1983. Pmtoins. heeman" San Francisco. Donohue J. 1968. Selected topics in hydrogen bonding. In Structural chemistry ond molcculw b1ol~·· (ed_ A. Rlch and N. IJavirlson). pp. 443- 465. Freeman, San Francisoo. Fersht A. 1999. S tt·ucture cuuf mcchUI11sm in prolein science· II RUide to enzyme rotolysis und protein {oldir1g. W.H. freeman, New York. Cray lLB. 1964. Electrons afld chemical bondillg_ EenJ810in Cummings, Menlo Jlaik. California. Klotz f M . 1 oo:;. Enorgy dwn~s in biuchelmCul r eUcfl()llS. AcarlefTiic Pre$, New York. Kyle }. 1995. MecllUilism in protein chemif>try. Carland Publishing, New York.

- - - 1995. Struct1.1re in protein chemistry. Carland

Publishing. New York.

LehtJinger A.L. 197 1. Bioenergetics, 3rd edition. Benjamin Cummings, Menlu Park. California. Lesk A. 2000. Introduction Eo prol t::itl orchilec.ture: The slnJClural biology of proleins. Oxfurd Univorsity Press. New York.

Marsh R.E. 1966. Some comments on hydrogen bonding in purine and pyrimidine bases. In Structural chemistry ond molecular biology (ed. A. Rich and N. Davidson). pp. 485- 4a9. Freeman. San Francisco.

Morowitz H.J. 1 970. Entropy fot biologists. Academic Press, New York Pauling L. 1960. The flature of the ch,.~mical bond, 3rd edition. Cornell Univen;ity ~-.s.1thaca. New York Tinoco r. (ed.). Sau E'.r I<., Wang j.C., p,~lisi J.O. 2001. Physir:ol chemistry: principles ood uppliwtions ill bjl' sciences, 4th edition. PIEmtice Hall Cnllege Division. Upper Saddle River, New JE'.rsey.

CHAPTE R

The Importance of High~Energy Bonds

n the previous chapter we looked at the formation of weak bonds from the thermodynamic viewpoint. F..ach time a p o,entia l weak bond was considered, the question was posed. Does its formation involve a gain or a loss of free energy? Only when l:lG is negative does tlm thermodynamic equilibrium favor a reaction. This same approach is equaHy valid for covalen1 bond::;. The fact that enzymes are usually involved in the making or breaking of a covalent bonci doos not in any sense alter the requirement of a negative llG. On superficial examinatjon, however, many of the important covalent bonds in cells appear to be formed in violation of the laws of thermodynamics, particularly those bonds joining small molecules togethor to form large polymeric rnolecu)P.s. Tbo fonnatiou of such bonds involves an increase in free energy. Originally, this fact suggested to some people that cells had the unique abilit y to work in violation of thermodynamics and that lhis property was, in fact, the rea l "sectet of life... Now, however, i( is clear that t.h~~e biosynthetic processes do not ";olate thermodynamics but rather are based on different reactions from those originally postulated. NuclP.Ic acids, for example. do not form by t he condensation of nudooside phosphat(}s; glycogen is not forn1ed directly from glucose resid ues; proteins are not formed by the union of amino acids. l nstead, the monomerio precursors, using energy present in .ATP. are first converted to high·energy ..activated " precursors, which then spontaneou Rly (with the help of ~ pecific enzymesj unite to form larger molecules. In this chapter, we shall illustrate these ideas by concentrating on the thermodynamics of peptirle (protein) and phosphodicster [nuclelc acid) bonds. First. however. we m ust briefly look at 90me gencrnJ thermodynamic properties of covalent bonds.

I

OUTLINE

• Molecules that Donate ~'Y N e ThennodynClfntt:ally Lnstct>le (p. 55)

Enzymes Lower Adivation Energies 111

8iodlerrncaf Re«tiOfls (p. 57)

• Free f.nergy m Biomolerules (p. 58)

• High·Ene-gy Bonds tn 8~lic Reactions (p. 60) Act1vat1011 of Prea.trSOfS in Group lfansfer Reactions {p. 61)

l\tfOLECULES THAT DONATE ENERGY ARE THERMODYNAMICALLY UNSTABLE There is great variation in the amount of free energy possessed by specific molecules. This is bocause covalent bonds clo not all have the :;arne bond energy. As an example. the cov
ss

Tltfl/mporlcmct> of High-Enmgy Boni!s

bonds. This idea seems almost paradoxical at first glance since it means that the stronger the bond, tbe less energy it can give off. But the notion automatically makes sense when we realize that an atom that has formed a very strong bond has already lost a large amount of free energy in this process. Therefore, the best food molecules (molecules that donate energy) are those molecules that contain weak covalent bonds and are therefore tllermod}namlcally unstable. For example. glucose is an excellent food molecule since there is a great decrease in free energy when it is oxidized by o~ygen to yield carbon dioxide and water. On the other hand. carbon dioxide, composed of strong covalent dollhie bonds between carbon and o:xygen, known as carbonyl bonds, is not a food molecule in animalsIn the absence of the energy donor ATP, carbon dio:xide cannot be transformed spontaneously into more complex organic molecules. even with the help of specific enzymes_ Carbon dio:xide can be used as a primary source of carbon in plants only because the energy supplie-d by light quanta during photosyn1hesis results in the formation ofATP~ The chemical reactions. by which molecules are transformed into other molecules containing less free energy, do not occw at significant rates at physiological temperatures in the absence of a catalyst- This is because even a weak covalent bond is. in reality, very stron~ and is only rarely hroken by thermal motion within a cell. For a covalent bond to be brol:.en in the absenc.e of a catalyst, energy must be supplied to push apart the bonded atoms. When the atotns are partiall}' apart. Ihey can J"CCombinc with new partners to form stronger bonds. 1n the process of r~ombination. the energy releasoo i11 the .sum of the fr~ energy suppHed to break the old bond plus the difference in free energy between the old and the new bond (Figure 4-1 ). The energy that must be supplied to break the old covalent bond in a molecular transformation is called the adivation energy. The acthration c11e rgy is usually less thar. the energy of the original bond becat\sc molecular rearrangements generally do not involve the prorluction or completely free atoms. Instead, a collision between the two reacting molecu,es is required, followed by the temp orary forma tion of a molecular complex called the activated slate. rn the activated state. the dose proxitnit)' of the two molocules makes each other's bonds more labile. so thar less energy is neederl to break a bond than when the b<md is present in a free molecule. Most reactions of covalent bonds in ceHs are therefore described by (A-B)

f I (i U ft E

+ (C-D) _.. (A-

D)

+ (C-B)

IEqual.ion 4-tl

4-1 '11M! energy of activation of

a memkal reedion: (A- B) + (C-D)- (A-D) + (C-B) This reaction is~ by d deaease in free

activation energy

energy.

reactiOn

progress of react10n

Enzymes Lower Activatio n Enngies in Bitx:hf!lT!ic:ul Reaction.«

The mass action expression for such a reaction is

llG =

-

RT In Keq or

~

= e- .lOfflT

4-1 The Relittioaship between~

I ABl f

concA 0 X concC B K = ------,,.--.,.---=--=[f.quation 4-Z] ~at concA- 11 X concc - o where concA 8, oon cc 0• and so on, are the concentration~ of lhe several reactants in moles per liter. Here. also. the value of 1<..q is related to .l.C by Eqn~tion 4-3 (see ulso Table 4-1) . !Equati
Because energies of activation are generaJly between 20 and 30 kcallmol, activated states practically never occur a t physiological temperatur£.-s. High adivation energies are thus barriers prcventiug spontaneous rearrangements of cellular-covalent bonds. these barriers me enormously important. Life would be impossible if they did not exist , for aU atoms would be in the state of least possible energy. There would be no way to temporarily store energy for futura work. On the other nand, life would also be impossible if means were n ot found to sclcclivcly lower the activation energies of certain reactions. This also must happen if cell grow1h is to occur at a rate sufficiently fast so as not to be seriously impeded by random destructive forces. such as ionization or ultraviolet radiation.

57

cmd ~C (.l.C ::- - R1 in K..J ~

10 · i l Q-:0.

1Cr-4 10 a

10

&

10

I

1Cf1

10' IIY

103

AG (kcallmol) 8.2 6.8

51 41 2.7 14 0.0 - 1.4

- 2.7 - 4.1

ENZYMES LOWER ACTIVATION ENERGIES IN BIOCHEMICAL REACTIONS Enzymes are absolutely necessary for life. The function of enzymes is to speed up the rate of the chemical reactions mquisile to cellular existence by lowering the activation energies of molecular rearrangements to values that can be supplied by the heat of motion (Figure 4-2). When a specific eneyme is present, there is n o longer an e ffective barrier preventing the rapid formation of the reactants possessing the lowest amounts of free energy. Enzymes never affect tbe nature of an equilibrium: They merely speed up the rate at whjch it is reached. Thus, if the thermodynamic equilibrium is uufavo.rablc for the formation of a mo1ccule, the presonce of an enzyme can in no way br1ng about the molect.Jle's accumulation . Because enzymes must catalyze L-ssenlially every cellular n1olecular rearrangement knowing the free energy of various molecules cannot by itself tell us whether an energetically feasible rearrangement will, in fact, occur. The rate of the reactions must always be considered. Only if a cell possesses a suitable enzyme will the reaction be important.

F I Ci U R t activation

energy of t.tneatalyzed

reacticn

progress of reaction

4-2 Enqmes (£01ot wrve)

lower activation energia atd thus speed up the rate of die readiOIL Note \hat !J.G rerr.cnns the same because the equilibrium posit1on rema10~ unaltered.

FREE ENERGY IN BIOMOLECULES Thermodynamics te lls us that aH biochemical pathways must be characterized by a decrease in free energy. This is d early the case for degradative pathways, in which thermodynamically unstable food molecules are converted to more stable compounds, such as carbon dioxide and wa t(ff, with the evolution of heat. All degratlative pathways have two primary purposes: (1} to produce the small organic fragments necessary as buil~ing blocks for larger organic m olecules and (2) to conserve a significant fraction of the free energy of the original food molecule in a form that can do work. This latter purpose is accomplished by coupling some of the steps in dcgradative pathways with the simultaneous format ion ot· high-energy molecules such as ATP, which can store free energy. Not all the free energy of a food molecllle is converted into the free ~ncrgy of high-energy Jnolecules_ lf 1his were the case , a degradative pathway would not be c haraclorized by a decrease in free energy, and there would be no driving force to favor the breakdown of (ood molccnles. lnstP.ad , we find that all degradative pathways are characterizet! by a con\lersion of at least one-half the hee energy of the food molecule i'llto heat or entropy. f"or example, it is estimated thai in cells, apvroxlmately 4.0% of the free energy of glucose is used l o make new nigh-energy compounds, lhP. rnmaindP.r bci ng diSl"ipated into heat energy a nd entropy.

High-Energy Bond" Hydrolyze with Large Negative /J.G A high-energy molecule contains one or more bon ds whose breakdown by water, called hydrolysis. is accompanied by a large decrease in free energy (5 kcaJ/rooU. The specific bonds whose hydrolysis yiolds these lflrgA negt1tive ac; values are called high-energy bonds, a somewhat misleading term, since it is not the bond energy but the free energy of hydrolysis that is high. Nonetheless, the term high-energy bond is generally employed. and for convenience, we shall continue this usage by marking high-energy bonds with the symbol "'· The energy of hydrolysis of the average hlglrenergy bond (7 kcaUmol) is very much smaller than the ammmt of energy that would be released if a glucose molecule were to be completely degraded in one step (688 kcallmol). A one-step breakdown of glucose would be inefficient in making high-energy bonds. This is undoubtedly the reason why biological glucose dp,gradatiro .requires so many steps. ln this way. the amount of eneigy released per degradalive step is of the same order of magnitude as the free energy of hydrolysis of a high-energy bond. The most important high-energy compound is ATP. It is formed from in01:ganic phosphate G and ADP, using energy obtained either from degradative reactions or from the sun. a process known as photosynthesis. There are, howevet, many other important high-eneigy compounds. Some ar~ rlim<:t:ly forme1d clurlng dagmdative rp:action.s; others ore fol'ffi(:tl using so me of the fmc nnergy of ATP. Table 4-2 lists the most important types of h.igh-<me.l'RY bonds. All involve either phosphate or sulfur a toms. The high-energy pyrophosphate bondfl of ATP arise from the union of phosphate gtoups. The pyrophosphate linkage (0-0) is not, howovcr. the only kind of high-enP..rgy ph~phate bond: The attachment of a phORphatP. group to the o>.-ygcn atom of e carboxyl group creates a high-energy acyl bond. ll is now clear that high-energy bonds involving sulfur atoms play almost as imJxutant a role in ener:gy

Free Energy in Bivmoler:ulet>

TABU.

4-l

lmpclftant Oasses of Higti-Energy Bonds

t.G of Reactioo, kcat/mol t.lG= - 6

Nucleoside dtphosphates

aderJOSine-G-0

Nucleo.<>ide

adenosine---G- O-O

triphosphates

6G = -6

(AOP)

(ATP)

ATP ~M:Jf'+ Q - 0

.t\G==-8

l:not phosphates

Amlnoacyl adenylates

fio

H~ - c~

I

Guanidin\lm phosphates

N

H 3C

/'-..

C-

' o-

H

crealire - P ~ creallne

N-G

II

NH creatine phosphate

Ttlloosters

metabolism ac; those involving phosphorus. The most important molerule containing a high'i3ncrgy sulfur bond is ace tyl-CoA This bond is th
+P

f)G : -8

Tht' lmportom:e of Hlgh-Einergy Bonds

60

HIGH-ENERGY BONDS IN BIOSYNTHETIC REACTIONS

E

(I)

u; ,., Ul

0

21

Ullfavorable:

AB ~ c-o ~ VefY favorable:

Cll

.:: B-

e

D-E progress of reaction

FIGUR£ 4-3 Ff~gy dlaPJes ic1 a ndti-slep metalxllic pathway. A - B C -+ 0 - E. Two steps (A- 8 and C-. D) do not f.lvcr the A - E cli'OOlon of thE reaction, SlllCe they have small ~

6Gvalues. they are 111stgnfficant OW11"lg to t:t-.e very large pegative AG v<*JE:s pn:Mded in Slf!p S B - C and 0 -• E Therefore. the 0\lf?ra• reacron fa~ the A - E coo~ . Howelte~

Th e construction of a large molecule from smaller building blocks often requires the input of free energy. Yet, a biosynthetic pathway. like a degradative pathway, would not exist if it w ere not characterized by a net decrease in free energy. This means that many b iosyn· thetic p athways demand an external source of free energy. These free-energy sources are the higb-energy compounds. The making of many biosynthetic bonds is cot1plcd with the brN~ kdown of a highenergy bond. so that the net change of free energy is always negative. Thus. high-enCI'gy bonds in ceUs generall}f have a very short life. Almost as soon as they are formed d uring a degradative reaction . they are enzymatically broken down to y ield the energy n eeded to drive another reaction to completion. Not all the steps in a biosyn thetic pathway requjre the breakdown of a h igh-energy bond. Often, on1y one or two steps involve l'Uc h a bond. Sometimes this is because the llG. even in the absence of an externa lly added high-energy bond. favors the biosynthetic direction. In other cases. ~G js effectively zero or may even be slightly positive. These sm all positive f:lG values. however. are not sign ificant so long as they are followed by a reaction characterized by tho h yrlroJysis of a high-energy bon d . Rather. it is the s um of all the free.energy changes in a pathway that is significant. as shown in Figure 4-3. ll docs not really matter that the Keq of a specific biosynthetic step is slightly (80~20) in favor of degradation if the K.eq of the succeeding step is 100:1 in favor of the forward biosynth etic direction . Likewise. not aU the steps in a degradftlive pathway genf'.rate highenergy bonds. For example~ only two steps in tho lengthy glycolytic (Emhdcn-Meyerhof) breakdown of glucose generate ATP. Moreover, there are many degmdative pathways that have one or more steps I'C(]Uiring the breakdown of a high-energy bond. The glycolytic b rea'kdown of glucose is again an example. lt uses up two molecuJes of ATP fur every four that it generates. Here, of cow se, as in every energy-yielding rlegradative process, more high-energy bonds must be mat.le than cons umed.

Peptide Bonds Hydrolyze Spontaneously The formation of a dipeptide a nd a watf'.r molecule from two amino acicls requires a llG of 1 to 4 kr.al/mo1. depcnd~ng on which amano acids are being joined. These positive ~Gval ues by themselves tcJJ us that polypeptide chains cannot form from free amino acids. (n addition. we m ust ta ke into account f be fact that w ater molecules have a m uch, much high or conamtration than any other cellular molecules (generally more than 100 times higher). All equilibrium reactions in w hich water participates are thus strongly pushed in tbe direction that consumes water molecules. This is easily seen in the definition of equilibrium constants. For example, the reaction forming a dipeptide, amino acid(A) + ammo add(B) -

dipcptirle{A-.B) + HzO 1Equation 4-31

has the following

c~lli libJ'ium

K eq

constant;

concA- Il x conc"P :; - - - - - - cond' X coneD

(Eqllatio.n 4-4}

where con ce ntrations are given in moles per liter. Thus, for a given Keq value (related to 6G by the formula tlG = - RT In ~). a much greater concentration of w ater means a correspondingly smaller concentration of the clipeptid~. The relative concentrations are. the.re fore. very important. In fact, a simple calc ulation shows that hydrolysis may often proceed spontaneously even when the tlG for the nonhydrolytic reaction is - 3 kcaUmoL Thus. in theory, proteins are u nstable and. give n sufficient time . will spontaneously degrade to free amino acids. On the oth ~r hand. i n the absenm of specific en zymes, these spontaneous rates aro too slow to hcwe a significant t:ffect on cellular metabolism. That is, once a protein is made, it remains stab le unless its degradation is cata lyzed by ~ specific enzyme.

Coupling of Negative with Positive 6-G Free energy must be adrled to amino acids before they can be united to form pl'oteins. How this happens became clear with the discovery of the fundamen tal role nf ATP as an P.nergy donor. ATP contains thrf'e pho.~ phato groups attac:tmd to an adenosine molecule (adenosine-0G -G ). When one or two of the terminal -G groups a re broken off by hydrolysis , there is a significru"tt decrease offree energy.

Adenosine-0--G-G-G + H~O - Adenosine-0--G-G + G (Equatioo 4-51 (tlG = - 7l'Callmol) Adenosine--0-G+ G-G Adcnosine---0-0 - 0-G + HlO (Equation 4.fi) (A G = - 8 kcal/ruo])

Adenosine---G-O- G + H 20 -

Adenosinc-0--G + 0 (bC = - 6 kcal/mol)

IF.quatW,n +7)

AH these breakdown reaL1ions have negative tlG values con~ider11bly greater in absolute value (numerical value w ithout regard to sign) than the posilive I::J.G \7aJues accompanying the formation of polymeric molecules from their monomeric building blocks. Tite essential trick underlying these biosynthetic reaction s. which by themselves have a positive f).G, is that they are coupled with 1he breakage of high-energy bonds, characterized by negative b.C of greater absolute value. Thus, durin~ protein synlh~is, the formation of each peptide bond rae = +0.5 kcatlmol) is coupled with the breakdown of ATP to AMP and pyrophosphate. which bas a ac of - 8 kcallmol {see Equation 4-6). This results in a net 6G of - 7.5 kcallmol, more than sufficient to ensure that the equilibrium favors protein synthesis rather than breakdown.

ACTIVATION OF PRECURSORS IN GROUP 1RANSFER REACTIONS When ATP is hydrolyzed to ADP a nd phosphate, most of the &ee energy is Jiber-dted ac; heat. Because heat energy cannot be used to make covalent bonds. a coupled reaction cannot be the result of two COlll(Jletely sevar·ate reactions, one with a pol'>itive IJ.G, the other \1\tith a negative AG. Instead. a coupled reaction is achieved by two or

more successive reactions. These are aJways group-transfer reactions: reactions. not involving oxidations or reductions, in which molecules exchange functional groups. The enzymes that catalyze these reactions are called transferases. Consider the reaction (A- X) ·f- (B- Y) - - (A- B)

+ (X-

Y).

(Equatioo 4-8]

In this example. group X is exchanged with component B. Crouptransfe r reactions are arbitrarily defined to exclude water as a partacipant. When wa ter is involved , (A-B) + (J 1-GH ) -

( A-QJ-I)

+ (B-H).

(Equation 4-91

This reaction is called a hyd rolysis, and the enzymes involved are called hydrolases. The group-transfer .reactions that interest us here are those involvi ng groups attache«l by high-energy lxmds. WhP.n such a high~en ergy group h: lronsf~rrcd to a.n a ppropriate acceptor molec'.lle, it becomes attached to the occoptor by a high-energy bond. Croup transfer thus aJlows the trans fer of high-energy bonds from one molecule to dn othc r. For example, Eqttations 4·10 and 4·11 show how energy present in ATP is transferred to form GTP, one of the prccwsors ~d in RNA synthesis:

AdP.nosine-0-Q ... @ + Guanosine-&~ Adenosine-0-0 + Guanosine-0 - 0 Adenosine-0-&-~ + Guanosine - G - O Adenosine- 0-G + Cuanosine-0-G-0.

~uation

4-101

JEq. 4-ttl

The high-energy 0- group on GTP a llow s it to 11nite spontaneously with a nother molecule. t."TP if; thus an example of what is called an aclivated molecuJe; corr~ pond ingl y. the process of transferring a high· energy group i'l CR11ed group activation.

ATP Versatility in Group Transfer ATP synthesis has a k ey cole in the controlled trappins of the energy of molecules that serve as energy donors. ln both oxidative and photosynthetic phosphorylations, energy is used to synt hesize ATP from A OP am] phosphate: Adenosine--G-O +

0 + energy -

Adenosine-0-0-0 IEqualion 4-12)

Because ATP is the original biological recipient of h igh-energy groups, it m uf;t bo thr: 1\tarting point or a variety of w..actions in which highenergy groups are transferred to low~nergy molecu1cs to give them the potentia l to react spontoneou.c;;ly. ATP·s contra! role u tilizes the fact that it conta ins two high-<morgy bonds whose splitting releases specific groups. T1tis is seen in Fi~ 4-4, which shows three important groups arising from ATP: 0-G. a pyrophosp hate group: - AMP, a n adenosyl m onophosphate group: a nd -0. a phospha te group. ft is important to notice that theso high-energy groups reta in their highenergy quality only when transferred to an appropriate acceptor group to a molecule. For example, a lthough the transfer of a COO group yields a high -energy COCJ-9 acylphosphate group, the tra.nsfer of the same group to a sugar hyd•·o:xyl group (- C- OH), as in the fonnation of g lucose-6~ph os phatc. gives rise to a low-ene rgy bond (less than 5 kcal!mol decrease in ~G upon hydrolysis).

Ac;ti~·olion

fl/ Frecum1rs m Croup Tronsfrr Re(Jdions

ATP

R- C

0

4-4 inwofma ATP. F I (i U R f

A

A

• - o- Q-Cil + ~

' o-Q + e-~

AMP

PDP 0

o-e + •- c,o-~ - AMP

Activation of Amino Acids by Attachment of AMP The activation of an amino acid is achieved by transfer of a n AMP group from ATP to the COO group of the amino aci d . as sh own in EqURtion 4-13: H

R

H

H

I I ~o H - ~- c-c + Adenosine--G- O - O I I "-o-

H

R

O

1 J ~ H- N·- c-c +c -o I I 'o- Q-Adenosine H H IEquation 4-131

(In the equation, R represents the specific side group of the amino acid ) The enzymes that catalyze this type of reaction are caJled aminoal:yl syntbelases. Upon activation, an mnino acid (AA) is thermodynamically capable o f being efficiently used for prote in synthesis. Nonethelnss, the AA-AMP r.omplexes ara not the direct pm<:ursocs of protnins. Instead. for a reason we shall explain in Chapter 14, a second group tran sfer must oocur to t ransfer the amino acid, still ar;tivated at its carboxyl group, to the end of a tRNA molecule: AA-AMP + tRNA ~ AA-tRNA + AMP.

!Equation 4-141

A peptide bond then fo1't1ls by the con densation of 1hc AA-tRNA ruoleculr. onto the (md of a growing polypuptide chain: AA-tRNA + grow ing polypeptide chain (of n amino acidsj tRNA + growing polypeptide chain (of n + 1 amino acidsl IF.quataon 4-15)

Thus. the final step of this ..coupled react ion." l ike that ot all other coupled reactions. necessarily inllolves the removal of the activaUng grou.p and the convursion of a high-energy bond into one with a lower

til

Important zroup msosfen

free a nergy of hydrolysis. This is the source of the negative t::.C lhat drivf'.s the roAction in the direction of protein synthesis.

Nucleic Acid Precursors Are Activated by the Presence of G-0 :Roth types of nur:lr.ir; add , DNA and RNA, are huilt up from mononucleotidn monom.Prs. atso called nucleoside phosphate. Mononucleotides. however. are thermodynamically even less )ilcely to r:ombinc than mnino acids. This is because the phosphod.iestcr bond s that link the former together release considerable free energy upon hydrolysis ( - 6 kca!/rnol). This means that nucleic acids wilJ spontaneously hydrolyze. at a slow rate. to mononudeotides. Thus, it is evan more irnpor1ant thclt activated pmcursors be used in the synUmsis of nuclcir. acids than i n the synthesis of proteins. Tho immediate precursors for both DNA and RNA oro tho nucleos.ide-5'-triphosphatos. For DNA, t hese precursors arc d ATP. dGTP. dCI'P, and dTTP (d stands for deoxy}~ for RNA, the precu rsors are ATP, GTP, CT.P. and UTP. ATP thus, not only serves as tho m ain sou rce of high-energy groups in group·trilllsfer reactions. but it is itsel f a direct precursor for RNA. The other three RNA precursors all arise by group-transfer reactions like those described in Equations 4-10 and 4-11. The deoxytriphosphates are fmm cd in basically the same way: Aftrr the deoxymononudootides h ave been synthesi;ood , thHy are transformed to the triphosphate form by group t ransfer from ATP: Oooxynucleosidc-G + ATP -

Dooxynucleoside-G-G + ADV, U~qu al ion 4-16)

Dcoxynuclooside-0-0 + ATP Dcoxynudeoside-0-G-0 + ADP.

(Equation

4 -11)

Tbesn triphosphah1s can then uniln to form polymu-;]ootidcs held together by phosphodiestur bonds. Jn this group-transfer reaction. a pyrophosphate bond is btoke n and a pyrophosphate Rroup released:

Deoxynucleoside-0-G - G + growing polynucleotide chain (of n nuclcotirles)

0 - G + growing polynucleotide chain (n + 1 nu.clootidcs).

IEqt..oiP, 4-181

This reaction , unlike that which forms peptide bonds, does not have a negative /lG. 1n fact, the t::.G is s ligbtly posithre (ahoul 0 .5 kcal/mol) . This situation immediately poses the question-as polynud eot idcs obviously form- What is the source of tho necessary free energy?

The Value of 0-0 Release in Nudeic Acid Synthesis The needed free energy comes from the splitting of the high-energy pyrophosphate group that is formed simultaneously with the highenergy phospbodicstcr bond. All cells contain a powerful enzyme, pyrophosphatase, which breaks d own pyrophospha te molecules a lmost as soon as they a re for·met.l:

0 - G----. 2 ·

{t::.G = - 7 kcal/mol}.

lf.quation 4 -191

The large negative 6.G means that the reaction is effectively irreversible. This means that once G -G is broken down. it nevor reforms. TilE: union of the nucleoside monophospbate group (Equation 4-16), roupled with the splitting of the pyrophosphate groups (Equation 4-19}, has an equilibrium constant determined by tlw combined .1G valu£•s of the two reactions: (0.5 kcal/mol) + (- 7 kca1Jmol). The resulting value {6.G = - 6 .5 kcal/mol) tells us that nucleic e~cids almost never break down to reform their nucloosidc triphosphate precursors. Here we see a poworful example of the fact that oftun it is thu freeen£:rgy change accompanyin~ a group of reactions that dotermincs whether 2 reaction in the group will take place. Reactions with smaJl, positive AC valuos. which by themselves would ncvor take pJace, are often part of important metabolic pathways in which they aro followed by reactions with large n egative t:J.G values. At all times we must rcmem~ her that a single reaction (or· even a single pathway} never OC'Curs .in isolation: rather, I he nature of the equilibrium is constantly being c.han~EJd through the addition and removal of metabolites.

G- G Splits Characterize Most Biosynthetic Reactions The synthesis of nudeic acids is not the only reaction where direction is determined by the releas e and splitting of G-0. In fact, essentially all biosynthetic reactions are characterized by one or more steps th01t release pyrophosph ate groups. Consider. for example, tbe activation of an amino acid by the attachmf:nt of AMP. By itself, th~: transfAr of a Wgh-encrgy bond from ATP to the AA- AMf' complex has a slightly positive !:J.G. Thernforo. it is the release and splitting o f ATP's lct111inal pyrophosphate group that provides tho negative !J.G that is necessary to drive the reaction. The great utility of the pyrophosphate split is neatly demonstrated whon we considCT the problems that would orise if a cdJ attempted to synthesize nucleic acid from nucleoside diphosphatcs rather than triphosphatcs (Figure 4-5). Phosphate, rathm than pyrophosphate. would be liberated as the backbone phospho-diester JinkagQs woro made. The phosphodiester linkages, however, are not &table in the presence o f significant quan1ilies of phosphalo. hPCause they arc fonned w ithout a significant release of fmc energy. T hus, the biosynthetic reaction would be easily reversible: if phosphate were to accu· mulate, the roaclion would begin to move in the direction of n ucleic acid breakdown acconling to the law of mass action. Moreover, it is not feasiblv for a cell to remove the phosphate groups as soon as they arc generated (thereby preventing this reverse rear:tionl. as all cells require a toigni6cant internal level of phosphate to grow. In contrast, a seQUP.ncc of reactions that libernto pyrophosphate and then rapidly break it down into two phosphates disconnects the lihcrntion of phosphate from the nucleic acid biosynthesis reaction, and thereby prevents the possibility of reversing the hiosynthetic reaction (see Figure 4-5). In consequonco. it would be very difficult to accumulate enou gh phosphate in the ce ll to drive both reactions in the reverse, or break· down. direction . II is dear that the use of nucleoside triphospbates t1S precursorS of nucleic acids is not a matter of chance. This same type of argument tells us why ATP, and not ADP. is the lc('y donor of high--energy groups in aU cells. A t firs l this preference secmerl arbitrary to biochemists. Now, however, we see that many ruactions using AUP as an energy donor would occur equally well in both directions.

fi6

The ImportancE! of High-EnP.T8Y Bondf'

a

+

G phosphate

grow!flQ dlain

(n+ 11ong)

b

G -G-ovr

o-., -

+

pyrophosphate

gro\Wlg chaan (nlong) growing ctJain (n+ 1 1ong)

G-& -ovr

+

+

nucleoside tfllhosphale

-

G + O

pyroprwsphate

G- &

phosphate

growiog chain (rr+ 1long) growjng Chaln

(n+ 21ong) F I f;U R E 4-5 Two s cenarios for nudeic add biosyntbe5is.. (a) Synthesis ol nudoc aods USIJl8 nucleoside diphosphates. (b) Synthesis of nudeic: acids using nucleoside ttiphosphates.

SUMMARY The biosynthesis of many molecules appears, at a superficial glance. to violate the thermodynamic law that spontaneous reactions always i.Ilvolve a decrease in free enecgy (LlG is negative). Foe example, lhe formation of pmteins from amino acids has a positive ~G. 1'his paradox is removed when we realize that the biosynthetic reactions do not proceed as initially postulated. Proteins. for example, are not formed from free amiuo acids. lnslt::ad,

the precursors are first enzymatically converted lo highenergy activated molecules, which. iJ1 tl1e presence of a specific enzyme. spontaneously unite to form the desired biosynthetic product. Many biosynthetic processes ate thus the result of "coupled" read ions. the rust of which supplies the energy that allO\vs the spontaneous occtl.frence of the secoml reaction. The primary enel'gy source in cells is 1\TP. It

Bibliogrqpby is formed from .A11P and it)Org
during d egradative reactions bmch as fermentation or respiration) or during photosynthesis. ATP contains sevcnl high·evergy bonds whose hydrolysis has a large negative AG. Croups link«! by h igh-energy bonds ere called higb enf'rgy groups. High-energy groups can be transferred to other 111Q(ecules by group-traosfflr reactions. thereby rreeling new high energy compounds. T hese derivative

high-energy molecules are then the immediate precursors foe many biosynthetic stQps.

Amino adds are activated by tho addition of an AMP group, originating from ATP. to form an AA- AMP molecule. The energy of the high-enetgy bor.d iu the AA- Al'v1P mo1ecule is simil6l' to that of a high-en ergy

67

bond of ATP. Nonetheless. the gmu(rlmnsfer rea(;tion proceeds to rompiE>tion ~use the high-energy G ~G molecl.lle , creatal when the M - AMP moleculE' is

formed. is broken down by the enzyme pyrophosr.hatase to low-energy groups. Thus. the reverse reaction, G -G + AA-AMP- ATP + AA. cannot occur. Almost e~ II biosyflthP.lic reactions result in the relem>e of G-G. Almost as soon as il (s mAde. it is enzymatically broken down to two p hosphate molecules. thereby making a ~ve.-sal of the biosyf!thetic reactiQn \mpossible. 'l'hc great u tility of the 0 - 0 split provides an explanation fO£ why ATP, not ADP. is the primary energy donor. ADP cannot llat.isfer a h igh-energy group and at the same time produce 0 -G groups as a by-product.

BIBLIOGRAPHY Genet'al References Kornberg A 1962. On th e metabolic significan ce of phosphorolytic and pyr,ophosphotolytic reactions. In Hori· 2ons in biochemistry (ed. M. l<.asha and B. Pullman), pp. 251 - 264. AcademicPress, New Yock.

Krebs H.A and Kornbe.rg H.L. 1957. A rutvey of the el'lergy t ransformation in living matf'rial. l?rgeb. Physio/. IJiol. Chem. Exp. Pharmakol. 4 9:212..

Ne-Json O.L. and Cox M .M. ZOOO. L.ehninger principles of biochemistry, Jrd edition. Worth Publishing. New York

tion of ~mzyme reactJon illtermediotes. Methods i.n Enzymology, vol. 354. Academic Press, San Diego, California. Silverman RB. l002. The organic cllernist.ry of enzyme. ootalyzed reactions.. Acac\P~ic Press, San Diego. California.

Strye.r L. 1995. Biochemistry. 4th edition. Freeman. New York.

1\.'icholls D.G. and Ferguson S.J. 2002. BioenergetiCIJ 3 . Academic P ress, San Diego, california.

1'inoco 1. (ed.), Sauer K., Wang ).C. and Puglisi J.D. 20()1 . Physical chemistry. Principles and applicutionB in life sdences, 4lh edilinn. PrenH~ Hall CoJJege Division, Upper Sacldle River, New Jersey.

Puticb D.L. (ed.) 2002. Mt>J.bods in enzymology: Enzyme .kinP.tit.•s and mechanism: Detfll'tion ond characteriza-

Voet D., Voet J.G., and Pratt C. 2002. Fundamentals of bio. chemistry. John Wiley & Sons, New York..

CHAPlER

Weak and Strong Bonds Determine 1Aacrounolecular Structure NA. RNA. and protein are aU polymers of simple building blocks. As we learned in Chapter 4, synthesis of these polymel'S depends on the controlled, cat aly~ed linkage oJ activated building blor.ks. For DNA and RNA, these building blocks are nuclcotides (sec Figure 2-11 ). For prolt~ins. the lruilding blocks arc the 20 ami no acids donated from thoir activa led intermediates. the clonor tRNAs. Assemhly or thesP. chains requires bTeakage of multiple high-energy bonds for the. addition of each building block. For all these molecules , the order of the constih.tenl bu ilding blocks d(ltermines their genetic and biochemical function. Weak bonds play a critical role in determining the structure and function of theso polymers. The primary information of RNA, DNA. and proteins is the order ot' their covalently-linked building blocks. NrverthPJess, it is only after they have formed extensive additional weak bonds between their different parts that these polymers adopt characteristic shapes that allow them to carry out theil' functions. The hydrogen bonds and ionic, hydrophobic:, and van dcr Waals interaf:tions described in Chapter 3 direct proteins to form critical binding sites and ONA to assume its dotsble helical stn•cture. lndood, the disruption of these interactions (by heat or detergent, for example) without disruption of coval ent bonds completely destroys the activity of all but a few biological polymers. In this chapter we briefly describe tho structua-..: of biological macromolecules and the forces that control thflir shape. DNA and RNA are discussed br.iefly here and morn thoroughly in Chapter 6. We then focus on the diverse structures of proteins. The final sections of the chapter focus on the interactions between proteins and nucleic acids. an activity central to many of the processes we will c.ncounteT in this bo ok, and the control of protflin fnnction by allostery.

D

OU T L INE

• Higher-order 5tructutes h e Oetermmed by Intra- and lntermolecoli:lr lnteredkJns (p. 69)

• The Specific Conformatim of a Protein ResUts from Its Pattem of Hydrogen Bonds (p. 18)

• Mosl Prot@ins Are MOOolar, Conlall"'lng Two ()(Three Dorru~ms (p 81)

• weak Bonds Cooectty POSitiOO Proteins .;!1011g DNA ard RNA Molecules {p. 84)

• Alostery: Regulation of a Protetn's F\mctim by Cl1ang111g Its Shape {p. 87)

HIGHER..QRD~R STRUCTURES ARE DETERMINED BY INTRA.. AND INTERMOLECULAR INTERACTIONS DNA Can Form a Regular Helix DNA molecules usually have regular helical configurations. This is because most DNA molecules contain two antipata1ld polynucleotide strands that have complementary structures (see Chapter 6 for more details). Both in1ernal and external noncovalent bonds stabilize the structure. The t\.-vo stranflS are .held togflthcr by hydrogen bonds beh.-voon pairs of complementary purines and pyrimidines (Figure 5-1). Advnine is Always hydrogen-bonded to thymine, whfilroas guanine is 69

70

Weuk a111l Stroug Bands flt>l£-.rmine Macromoler.vfar Structure

FtGU RIE 5-1 The hydrogen--bonded

base pails of DNA. The flguc shows the position and leng1h of the hydrogen bonds betvveen the base pairs_n,ec0\.31ent boods between 1he atorns within eadl base are shown, but double ilrld s~ bonds
11.1 A

1o.sA

I

\

f'IGUR£ 5~2 11te breaiUngoftenninal

base pairs in ONA by riitndom ltlerma1 motion. The figure shoiiVS that once some bond!> have broken at the t~. they can refoml {luwer left) or addfttonal borlds can breaJ;.

hydrogen-bonded to cytosine. In addition. virtually all the surface atoms in the sugar and phosphate groups form bonds to water molecules. Tbe purine-pyrimidine base pairs are found in the center of the DNA molecule. This arrangement allows their flat s.urfaces to stack on lop of each other. creating shared ('1T - 1T) electrons between the bases and limiting their contret with water. This arrangemt-mt, known as base stacking. would be much less satisfactory if only one polynucleotide chain were present. Because pyrimidines are smaller than the purinns. single-stranded DNA would result in the unfavorable exposure of hydrophobic surface between adjacent bases. Thr. presence of r.omplementary base pairs in double-helical DNA makes a regular strur.turo possible, since ear.h base pair is of the same si7:e. The double-helical DNA molecule is very stable for two reasons. First, disruption of the double helix would bring the hydrophobic purines ano pyrimidines into greater contact with water, which is very unfavorable. Second. rloublc-stranded DNA molecules contain a v·ery large number of wook bonds, arranged so that most of them cannot break without simultaneously breaking many others. Thus, for example, even though thermal motion is constantly breaking apart the purine-pyrimidine pairs at the ends of each molecule, the two chains do not usually full apart because other hydrogrm bonds 1n the molecule nre stilt intact (.Figure 5-2). Once a given bond is broken. th~ rnost likely next event is thfl rcfonning of the same hydrogen bonds to restore the original molecular configuration. rathur than the breaking of additional bond-;. Sometimes. of course, the first breakage is followed by a second. and so forth. Such multiple breaks, however. are quite rare. so that double helices held togethct' by ffi{)J'C than ten base pairs arc very stable at room temperature. When ONA stranns do comn apart without reforming. this typically stm-ts at on£! end of th~ mo)e}{:ule. and proceeds inward. This is hecause

the interactions between the bases aJ thE> end of the DNA are the least supported by adjacent interactions. That is, they have only one n eighboring OOse pa ir to bn]p sect.J.re the infP.rnction. As df.>SCrlbed in ffiOre det11il below, U1e sam e principle-the use of multiple Wflak bondsgoverns the stability of proteins. Ordered collections of secondary bonds become less anrl less s table as their temperature is raised above physio logical tcmporaturos. At elevated temperatures, the simultaneous breakage of several weak bonds is more frcqunnt. After a s ignjficant numbfir h :~~ve broken, a molecule usually loses its original fonn (the pTocess of denaturation) and nssumes an inaclive. or den atured. configuration. Thus, as the temperature rises, more interactions are required to maintain the double-stranded natura of' DNA.

RNA Forms a Wide Variety of Structures In oontl'ast to the highly regular stmcture of the DNA double helix. RNA is usually found as a single-stranded molecule. Some RNA molecules (such as messenger RNAs) function as transient carriers of genetic information and arc constantly associated w ith proteins and thus do not have an indetJc n ncnt, slable, tert iary l'old . Othe r RNA moleculES fold into uniql.!t1 te rtiary structures. For these RNAs, intramolecular interactions between distinct regions load to the formation of specific elements of sCQJndarv structum. These interactions are prindpally between the bases of the RNA and include traditional Watson-Crick base pairing, unusual base pairing found only in RNA, and hydrophobic base stacking. RNA differs from DNA in that the ribose sugar of the backbone carril:lS a 2'-hydroxyl group. ln the fo lded structure of RNA molecu les, these 2'-hydroxy] groups often partici· pate in interactions that stabilize the structure. The binding of divalent m etal ions {such as Mg2 1 • Mn2 + , and Ca2 ' ) to the RNA is often critir.ftl. to the formntion of a stable. folded conformation a1CC
Chemical Features of Protein Building Blocks In contrast to tho f'our nucleotide building blocks used for RNA or DNA, tbP 20 amino add huilding blor.ks used for p mtP.in synthesis are highly

cliYcrse. The r.orrunon structural features of the amino acids are tho

72

t-\ffik tmd Strot'8 Bonds Delerroir~e Ma cmmoTP.Culor Slruclm~

fi(;UilE 5~3 The common struc:tura! feaWres cf amino adds.

central carbon ((~) linked 1o a hydrogen. a primary amino group. and a carboxylic acid group (Figure 5-3). The fourth linkage is to a variab le side chain calloo the R group. The R groups of the 20 amino acids can be catt."gorized by their si?c. shape, and chentical composiHon (Figure 5-4). The R groups fall into four categories: neutral-nonpolar, n~mt raJ JX>lar. acidic, and basic. The neutral~nonpolar side chains are composed of simple carbon chains ur aromatic rings and make principally hydrophobic contacts. The neutral-polar side chains include hydroxyl, sulfhydryl , amide. and itnidm>".ole moitics and ntake primarily hydrogen bond interactions. The charged {acidic and basic) side chains include primary and secondary amines and carboxylates and mal-e ionic ami hydrogen bonding interactions. All four types of side chains participate in van der Waals contacts, as these associations are only depcndE:nt on the proximity of atoms. rather than thoir specific; r:hontical Irulk.cup.

The Peptide Bond The primary covalent llnkage between amino

acid~

in proteins is the

peptide bond (Figure 5-5). This bond is made. when 1he primttry amino group of one amino acid is covalently joined to the carboxylic acid group of a second amino acid. Thls linkage has a partiall_y doublebonded charactet. Because this type of bond involves more than one pair of ehdrons , rotation around this link~gu is limited; completely free rotation about a bond is only possible when atoms are attached by single bonds. (For example, the methyl groups of ethane. H:~C-CH3 , rotate about the carbon-carbon bond.) In contrast to the pepti
There Are Four Levels of Protein Structure The final three-rlimensional structure or shape of a protein is formed through the sequential association of increasingly distant amino acids. The types of interaction s observed within a protein can be divided into four classes (Figure 5~7)_ The linear sequence of ammo acids in the polypeptide chain is the primary structure. Nearby amino acids associate w ith one another to form regions of secondary structure. The clements of socondary structure are usually formed through interactions between those parts of the amino acids that make up the polypeptidf! backbone rather than the side chains. As we will see below, ~ helices and J3 sheets are the elements of secondary structure. These clen1cnts pi!O~ together in n d ofined mnnner to generate e given polypcptido•s tertiary structure, which is ilio overall conformation of a single polypeptide chain. Many protejns are composed of multiple polypeptide chains known as protein subunits. The manner in which these subunits associate with one another is referred to as the protein•s quarternary structure. The informat ion contained within the primary s tructure is nearly always sufficient to determine the eventual tertiary structure of a polypeptide. This was demonstrated in a classic experiment in

neutraklOnpolar amno adds

glyrine (Giy. G) li

alanine (Ala, A) ti

li

basic amino acids

ac:fdic amino acids aspartic acid

gMamicacid

lysine

(Asp. D)

{Glu. E)

(lys. K} H H

H

~o I I -?0 K- t<-c-c H-N"-c-c

I

l

I

'\r

I

H

tl

I

H

I ~

'

I

I

Qo

I

I c~

'

H-N·-c-c:r

0

H

0

I

CH:,

I

,~ c~

I

NH;

•••• • ~·· ••• neutral-polar amino acids valine (Val. V) H H

I

I

0

?'

H-tt-c-c I I~ K

Qi

'-'

/\.

H,C

CH,

serine

tkeonine

(Ser, S )

(lN. T)

H

I I ti

H

l #'o I Ctft

H-N"-c-c

'o-

I

H

f

H

J

~o

1

'

H- N"- c -c

J

H

tyrosine (Tyr. Y)

(){,_

J ' ~0 r-r-c:-c::

1-1-

I

I f

'o-

at

H

U

I

{Gin. Q) H

ti H-

I

I

-?'0

l H

I ~

' o-

H"'-C-C

1

2

c

c-~

OH

glutamine

asparagine (Asn, N) H H

H/ 1

0

OH

jz

#'-NH.t

c

0 ~'~

•

• methionine {Mel. M)

lryptopharl

(Trp, W) H H

I

I

HI

I ~

H

-?0 ' o-

I I

H- N" -c-c

1 ~f.

H

I I

~0

1-1-N"-C-C tt

r~ s

~,CH H H

I

JJ• •

~

'o-

leucine

cysteine (Cys, C)

(Leu.l) tl

H

I

I

~0

I H

I c::H,

'

H-t<-c-c

I

Ctl

~c!'~

0

H

ti

I I /J 0 1-1-N·-c-c:r I CHa I ' U H I

SH

74

Weak rmd StronR Bonds Del~rmine Mocromolecular Structure

e Il

which the single-polypeptide enzyme ribonuclease was subjected to harsh conditions that interfere with hydrogen llonding and other weak chemical interactions leadillg to the complete denaturation (or unfolding) of the ()Qlypeplide. When the denatured ribonuclease was restored to conditions that allow the formation of weak c hemica) bonds. the enzyme rapidly regained both its normal three-dimensional structure and RNA cleaving activity. For a description of how protein :;lt.r\lctures are worked out experimentally. see Box 5·1, DelerminRtion of Protein Structure.

a Helices and p Sheets Are the Common Forms of Secondary Structure

f' I Ci U R IE S-5 Peptide bond.

The brod;-

ets •ndicate the two ilCT!IIlO aad resdles that are

pned by a pej:Cl(!e bond

ft Ci u R E 5-6 cJ> ilnd \l1 angles of rotation about the Ca-N end c.rc bonds. The shaded «eas represent the pknes of the peptide bonds. (Swrce: lllustratia\11Ving Ceis. Rights OWned b; Howard Hughes Medical Institute_Not to be reproduced v..oithout per11'4s~.)

The most stable arrangement of a polypeptide backbone is the a helix. This is a rlght-handecl helix, repeating every 5.4 A along the helical axis (Figure 5-8). This structure is preferred because the peptide backbone has favorable c!> and \jJ angles that accommodate a regular pattern of hydrogen bondjllg between carbonyl and imino groups on the same chain. The hydrogen-bonding potential of the peptide bac.kbone is fully utilized to stablize the !\trudure. As a consequence of the precise gerr roetry of the polypeptide chain, each tum of the a helix has 3.6 amino acids. lf. for examplf', four amino acids were used per tum. the hydrogen bonds would not be so neatly formed. nor would the individual backbone atoms fit together so well. Many amino acid sequences can adopt an a helical secondary structure. This is because the structure of then: helix is stabilized by contacts between the nearly universal backbone atoms of the carbonyJ <.Jmf imino groups in the polypeptide chain. The only amino acid that Jacks these atoms is proline. which cannot participate as a donor in tbe hydrogen bonding that stabilizes the helix because of its cydic chemic.al structure. Thu.~. proline is a helix-breaking residue. Although their structures do not prevenl it, glycine, tyrosine, and serine are also rarely fuund in a helices. Another consequence of the fact that a helices are constructed through exclusively backbone contacts is that the side chains project away from the helix. This puts these side chain:; in an ideal position to interact with another region of the prot~in or another macromolecule, such as DNA The second common secondary structural element is the I) sheet (F~gure 5-9}. In contrast to the a helix, the J> sheet is a highly extended form of tlte polypeptide hacltbone. Stablization of the I} sheet structure comes from alignment of regions of polypeptide in this extended

secondary

,'

,

=0·'

,.-·",'

.

~·,''

•

5 ...7 Four levels of protein stJUctUJe. (Souce: Adapted from Branden C and b
J 1cuR f

BoX 5-1 Determinatiol1 of Protein SlnletVre

There are two prinq,al methods to determine the three-dintension.:!l structure of proteinS. The first to be developed was X-ray aystallograptw- This method ~es on the fonnation of highty crdeied crystals of PIJC proteil_As with the original affraction studes rJ DNA fibers, the ill'adiation rJ protein aystats ~ h1gh-energy X-rays results in dffraction patterns lhat are related tu the structUre of the protein Mae recentty, nuclear magnetic resonance techriques have been developed to elucidate the conformation of smaller pmteins. This techniqJe e>Je to predct a protein's three-.:lmens1oM struaure from its primary .amno .aad seQIJ€nce, because, after aM, lhat information is sufficient for a ,:n>tein to adopt a uni~e conformaoon. Although progress l!i being made in the prediction d protcin stJtJcUe based on amil'\o acid sequence, the WI detemlination of the energetic constraints of a partirular seqt~ence is still beyond the most powerful computaticnll approoches. Nevet1heless, prediction of ceftain secondary structt.Sal elements (such as the common a. heix structLfe introduced below) &s beeoo1ing increasingly reliable. The ina~ large number of available ~entaly-detennined s1ructures has provided an important resource for making protein stJucttre predctions based on amino acid 5e9Jence These atcrnic stn..ldUres have helped to define fam~ies of ai'T'lif10 acid Seq.JeflCeS 1Mt share reklted thr~ shapes By companrlg the seqJences of protans d unkncP.rvn strumne with those lhat have been defer-mined, it is often pa55ible to mal<e stnJctLfcj predictions based on the identified sintLanty. Co~ this lnformat10n ~lh computer algorithms that predict secondary structlxes is prcMog to be a powerllJ method fer pre&ting how protens fold The ~erm ootW is 1M lh€se approaches wit allow at least an approxrn.ate struc· ttre to be predicted for any r:mtein fran tts primary sequ6iee alone

quarternary

W~k

011d Stro1Jg &ncft; Detru-mi lle Mac:romoiP.CtJlar Structure

F f Ci U R f 5-8 A pclypeptide main folded into a helical CCf'lfiguration calted the a helix. (Soorce: MoleoJar structure a~ed from PaUing L 1960_ The llOture of the chemical bond and rhe strudl.lfe of mofeaJies and crystals: An tntroductJOO to ~structural~ 3rd e6lit..n, p, SOO. Copyrigl1t © 1960 Cornell University. Used by permLSsion of d-.e publisher.)

0

5.4A (3.6 rcsidtles}

conformation such that hydrogen bonds can fonn betwe<m carbonyl groups of one (3 strand and NH groups on the adjacent strand. 'I)'pically. a region of I) sheet is composed of four to six separate stretches of poly(lflptide (each forming ao individual ~ strand). each eight to ten amino acids in length in the~ sheet, adjacent amino acids are related by a rotation of 180° and thus their respective side groups emerge from opposite sides of the

~

sheet (see Figure 5~9b).

13 sheets L'Ome i.n predominantly one of two forms. These diHer in the relative orientations of their chains (Figure 5-1 0). In one, the adja~ cent chains run in the same amino-to-carboxyl direction to produce a parallel 13 sheet. In the other. the adjacent chains run in opposite directions to yield an antiparallel 13 sheet Although less common, there are also f3 sheets that have both parallel and antiparallel compo~ nents. In both parallel and antiparallelp sheets, all the peptide groups lie approximately i11 the plane of the sheet. Strucrural studies have -revealed that in most cases the inrlividual sb:11nds o f p sheets tend to be twisted along their length in a right-handed manner (Figure 5-11). Thus, instead of flat sheets of protein. regions of~ sheet tend to curve to generate a compact protein module. For a protein to fold properly, both the backbone and the side chains must adopt conformations that maximize favorable interactions. The a helix and f3 sheet are both very stable c onfoTmatlons of the polypeptide backhone. But for each side chain to make the maxi· mum number of weak bonds, proteins have to adopt more varied

HiF/Ier-Ordet· S trudures A re Determmed by Intra- and l.ntermolecwar Interactions

a top-vtew

77

b stdeview

~sheets are held ~ by hydrogen bonds. (a) A J3 sheet~ shown from above Note that the oxyger5 and Mrogens of the backbone are fdly hydrogeclxlnded. (b) A ~ sheet stlown from a side \llt'W. ll'ris illustrates the locatiOI'I of the side 8JOt4)S. v.hct1 alternate between emergtr~g from atxNe « be4ow the plane of the (> shee-t (Source: MoltnJar strucllxe adilpled from Pauling l 1960. The nature of the chemiroJ bond and the SlnJCture of mcieaJies and uystols: An introducrion to modern structural chemistty. 3rd e
FrCUR IE 5-9

a

N

N

N

N

\

\

\

\

O= C

O= C

fa

fa

O=C

\

N-

O= C

\

/

H

c~

\

' H- NI

bo

I

o=c

,.....

N- H

\

N-

'

H-

··..

ba

I o= c _,,.~ \

H

H-

c

I H-N \

c= o

O= C

bo

I

o= c

~····

N- H

I

c= O

I H- N \ Cu I

"?..

c

\

"···

H-

N- H

c\ C= O

NI \ C0 I c

C=

fa

' H- NI

I c~

C= O

NI \ Cu I

NI

I

c\

\ N-H I

c~

c= o

I c~

C= O

c

c~

c~o

I c~

I H- N \ C0 I

.-.

N

fa

\ / N-H I

c~

c =o

~ / o=c

~

I

I

H- NI

N-

fa

b

H-

\ NI Cu \ C= I N \ C(J I

C

\ c.,. 1 O ..··H-N \ C= I

c\

N-

H ..·.O=

0

O ·..·H -

O= C

\N -

I C \ C I N \ C= I C0 \

H·..O =

I C

I C

N-

\r

\

/ 'tt.

C

N

f I CU RE 5 -10 Two types of p 5beets. (a) Parallel ~ sheet: schematic diagram show¥1g hydrogen bond pattern; note lhe ch
N

C

I

\ pa

~ C = Q .... H -

N

I \ Q·..·H-N C=O \ I c'\ 1ea H ·...O= C N- H \ I N - H "·· O==: C I \ Cu C., \ I C=O ·"· H- N I \ O·-..H - N C= O \ I Ca C0 I \

H····O= C

\N -

I H .... O=C

N-

I

\

\C /a

C

N

C

H

78

WPOk and Strong Bond& Determine MCJCromolPculrlr Structure

c FIGUJt£ 5-11 p sheets twist in a

rjgbt·handed mannef along Oleir length. lhe schemlllle shows dle mixed stnx:ture of Itt f. colt Pfotein ltiloredoxin p st\'ands are drawn as e~rrows from the i!l'ilino to the cafboxyl end of lhe protew~ (Soutc.e: Adapted from &anden C a-.d Tooze J. 1999. tntroducrion ro protem Slflid'ure,. 2nd edition, p. 20. fig 2 7 a,)

shapes_ The three-dimensional structures of the polypeptide chains of proteins are thus compromises between the tendency of the backbones to fonn either a helices or (3 sheets and the tendency of the side groups to twist the backbone into less regula!' configtn-ations that maximize the strength of th~ secondary bond~ formed hy those side groups {Figure 5-12). As we discuss in more detail below. one of the strongest influences on protein folding can be attributed to the burial of hydrophobic (nonpolar) amino acid side groups into the core o{ the protein's s tructure. This leads to the prediction that in aqueous solutions, proteins conhtining very large numbers of nonpolar side groups wiU tend to intefnalize the nonpolar residues and be more stable than proteins containing mostly polar groups. If we disrup t a polar molecule held tcrgether by a large number of internal hydrogen bonds, the decrease in free energy is often s mall since the polar groups can then hydrogenbond to water instead. On the other hand. when we disrupt molecules having many nonpolar groups. there is usually a much greater Joss in free enet-gy because the dismption necessarily inse11s nonpolar groups into water.

THE SPECIFIC CONFORMATION OF A PROTEIN RESULTS FROM ITS PATTERN

OF HYDROGEN BONDS

FtCUR£

5-12

Reg-and~

features of protein .5buctures. Jnegular ronfiglmloos tn the backbone (green) allovv the n1i1Xlmum formaticn of ser.oodary $I~OVre {13 4leet ., pur~ and n hdix sn turqucnse) by otJ'!er regions of ttJe prote~n lhe struct\Jre stlown IS that ol the [ 1 prote10 of adenovlflJS. (fnetri8rk U . Chen G.. Vaughn O.L. Steflll.Kld A., <Jnd Joshua Tot L 2<XX). Mol Cell 6. l49.) trnage ~ed 1Mth MoiSnipt. BobSrnpt. illld Raster 30 .

Whereas a portion of the energy stnbilizing a protHin is provided by hydrophobic interat.1ions, the specific conformation of a protein structure is largely determined b) hydrogen bonrls. The energy associated with the hydrophobic stabilization of proteins has no directional c.omponent. whereas hydrogen bonds requi..-e precise clifrtances ancl angles (see Figure 3-9 and Table 3-3). In general, fl11 hydrogen-bond donors and acc,.eptors within a protein's inte..-ior have suitable mates. Failure to make a hydrogen bond in the protein interior is entngelically costly, at the rate of a few kilocalories per hydrogen bond. The vitally important role ofhydrogen bonds in proteins is to destabilize incorrect structures as much as to stabilize the correct one. The necessity of satisfying all the hyd entirely of., sbeets or (l( helices adopt structures s panning a wirle range {Figure 5-'1 3). Of rourse. some polypeptide sections must be less regular to ~now their chains to tum at the ends of
Tl1e Specific Ccmformotion ofa Protein Results from Its Patta·n

of Hydrogen Bonds

a

b

c

~

-- -- -- - - - - -

5- Jl Polypeptide chain totting. {a) Pr(){etnS c~ of ( l hekes: myoglcblf1 and the N-temmal domain of }.. repressor. (b) Proteins composed of fl sheets: the Green Fluores. cent Protein (CEP) and gamma oystaltine (c) Q)rr1panson of the N- teml1nal domalfl of l\ repressa, ~of n tnces Wldl the C terminal dcKnaU1 of}. represSOf. c~ of P sheets. ((a) \qtechovsS;y J.. &.>fendzen J., Ou K , Schlichtwlg 1.• and Sweet RM. 5lbntted cmd Beamer U and P
0

0

-

hydrogen bonds that accompany their formation (for example. see Figure 5-14).

In addition. the less regular structures of these loops
j3strand 1

f1 strand2

fIG U R £ 5-14 Acfiacent antipar
hairpin lcqJ. ThE- b<:xKh within the ~ loq:l (111 ~area at top of ~re) C!fe green

f 1 cuR 1 5-15 The leucine z:q,per from the yeasttransaiptioo factof Gal4. ~ leucn! lJJ:fler IS en example ol a coleO col (see text) liffe we shew-~ tvvo Vrf!W'> of the leuone zipper: from the side (on the left) and lrom ckNe (C11 the llltf). (Fllenberger TE, Brard CJ~ sm..dll K.. and Hartison s.c 1992 Cell71 1223.) Images prepa!'ed v.11h MoiScrl)l. BobSCJl't and Rasler 3D.

a Helices Come Together to Form C<Jiled-Coils Many pnlypeptioP.:;; interact with nne anothflr through the supercoil· ing of a helices around each other. Typically. this can only occv.r when the nonpolar side chains along each a heJix are arranged so that their side groups contact the other helix. The twi sting of the helices around each other reflec(s the nonintegral (3,6 residues per h.rrn) nature of the o h elix, which allows the side gTOups to pack neatly together only when the a. helices interact at an angl fl of lH" from parallel. If the o helices remained perfectly rigid. they could totay in contact for only a few residues. But by supercoiling in a Le fthanded direction. neatly p acked. highly stable. coiled-coils are created {Figure S..1 5). One example of a coiled-coil is found in the leucine zipper fami ly Clf DNA-binding proteins. The~e DNA-binding factors havP. two subunits that come together to form a dimer through the use of a coiled"(;()il region. This coiled-coil region is called a leucine zipper due to the repeating appearance of leucine or other amino acids with an aliphatic side group, such as valine or isoleucine. These leucines appP-ar in a regular pattP.rn a,<; follows. If you oon.
Most Proteins Are Modular. Containing Two or Three Domains

I!

MOST PROTEINS ARE MODULAR, CONTAINING TWO OR THREE DO~WNS The subunits of soluble proteins vary in size from less than llKJ to larger than 2.000 amino acid residues. The smallest polypeptides that form folded proteins have molecular weights of about 11.000 daltons (approximately 100 residues), but most are between 20,000 and 70,000 daltons for a ~ing)e subunit. Proteins larger than about 20,000 daltons are often formed from two or more domains (Figure 5-lfi; r-oee also Box 5~2. Large Proteiru; Are Of~ ten Constructed of Several Smaller Polypeptide Chains). The term do· main is used to describe a part of the structure that appears separatu from the rest, as if it would be stabla in solution on its own, which is often the case. Typically, a single domain is fonned from a continuous amino acid sequence and not portions of sequence scattE>red throughout the polypeptirle. 1'his i.~ an important point wh~n considAring how multidomain proteins have evolved.

Proteins Are Composed of a Surprisingly Small Nwnber of Structural Motifs Determination of the first half-dozen protein structur~ showed a bewildering variety of protein folding motifs, implying thft existence of

an infinite number of protein structures. Now that we know the thToo~nsionaJ structures of thousands of proteins, however, it appears that a Telatively small numbel' of different domains account for most of the large variety of protein structures. Although an aL-curate estimate is not possible, the number of truly unique domain motifs will be orders of magnitude smaller than the number of unique proteins . Specific kinds of domain motifs are often associated with particular kinds of activities. One frequently observed motif has been termed the dinucleotide fold because it is frequently found In enzymes that bind

FIG U R E S-16 Pyruvate lcinase is composed of distinct domains. The predominant cbnalns of the enzyme ate shown 111 tUf~. ptJiple, and red (Allen s.c and Mufliead H. 1996. Acto Oysto/Jogr. D. &of. OystoliQI. 52:499.) Image prepi.!fed with MdSrnpt. BobScr9, and Raster 30

Box 5-~ Large Proteins Ale Often Constn.Jdecl of ~era& Smaller

Polypeptid• Chains Most large protms are regtjar aggregates of several srn*r polypepbde dlains. The ~ among the pclypepnde chains mablg up Slld1 a protein ts termed its q..~artcmary structtxe. Fa excrnpe, the macn::Jn1C.ltea.A mnpexes re.sponsi:lfe for the ~thesis of RNA (RNA~) cnj prcJte¥l (rhlsome) ae eadl ~ies of multiple subunrts. The complexes are about 500,000 cmd 2,500,000 dahoos, respedive~. but do not Wlclode any inc:i\.1dual subtrits greatB tl-a1 200,000 da&tons. 1he ribosome ts c~ of both protein and RNA subu~ This type of fac::tJY t5 ca~ a nbon.Jde.ar prote1n (RNP}. Why are &arge protetn COO'lplcxes composed of ml.~tipfe subtrits rather lha" a sin~ lafge SL.lbu1it7 lhe use of mulbple SLlbl.rli!s to build large prote11 c.orrcple><es reflects a buici~ pn'lciple applicable to ~I~ structures. nonfn.ing as well as livrtg. Thcs pnndple states that 11: is rntXh easier to redJce the tnpac1 of constructton mstakes if fatJty 5l.hJrlts can be dtscarded before they are incorpaatcd into the mal product. For ecample. ~ us consider two alternative ways of rons1n.lctTng a rnderule \vrth a miUtOn atoms. In sd'leme 1, we buad the structure atom by atom; in scheme 2, we frn buid a thousand smaller LMs. each Vv\th a thooSiYld atoms, but subseq..Jently JX4 ~ Sliulits together into the miliorratom product Now calSider that our buiding process randa'nty rncies mistakes,.I1SE'rtlng the wroog atom once every 100.000 times. Let us assune that each ~ reUis in a nonftxlc101al product Under scheme I, each moleaJe wil contain, on 1he average, ten wong atoms, cr1d so ai'nost no good jXodJCls will be syntl-esized. Under scheme 2, hoNeva, mistakes wil oo:u U1 oriy 1% of the sul:ulits. If there is a device to re.;ea the bad sul:vrwts, then 8CJOd products can be made easiy, and the eel \Mil hardty be both· ered by the oca.Jrence of the~ nonfl.flcliooal stbun1t 'llis is the same constructxln suategy that foons the basis the asseml::iy line. 1n which rofTll*ated lldustrial prodoos, st..dl as radiOS cr1d autanC!bites, il'e C«<Stn..lded M each stage or assembly, there are devices to throw i!tNCI( bad subuoo. In industlial asserrbly lines. mi:stal:es vvere initiany removed by human hands; now, automation often replaces maouat control. rn cds, mistakes are sometimes controfled by the speofidty of enzymes. If a 1'11Cln01Tleric suburit is wrongly put together, it USll"ly will not be recognaed by the polymer-making enzyme and hence wil not be incorporated into a macromolea.Je. In cxher cases, faulry sl.bstances aJe rejected because they are unable to ~become part of stable molecutar aggregates.

a

ATP (Figure S.17). This domain binds ATP through a central, parallel J3 sheet with a helices on both sides. The nucleotide binding site is on the carboxyl end of the 13 strands. \Vhat varies is the numbet and detailed arrangement of the a helices and. to a Jesser extent, the o rder of the 13 strands. Related domains of similar structure serve the same function in many different proteins.

Different Protein Functions Arise from Various Domain Combinations The various functional properties of proteins appear to arise from their modular construction in much the same way as computen; with different specifk.ations can be assembled from the appropriate moduJar components. Numerous examples can be given. There are, for example. many dehydrogenase enzymes, each working on a specific substrate. Each enzyrHt'\ consists of two domains, one a coutlt1on dinucleoHL1e

bJosl Proteins Are Modular, Ccm.tcll.rling Two or 'fhree Domams

83

f I C l1 RE 5-17 Emymes that bind AI'P. lhe red arrows point to the- ATP 'I1"'IOeoJes bound w1thn ead1 51!U:ttn- (a) RecA (b) l.JnaA_ ((a) Story Rlvt and Stettz. TA 1992. NcnJre l 55: 374. (b) Er2'.belger J.P., Pirruc.ceUo MM. and Berger J.M 2002. EM80 J. 2 I: 4763 - 73 ) lrnages prepared with MolSapt, BobSaipt,

and Raster 3D.

binding domain that binds the coenzyme NAD+. the other a domain that binds substrate and has the catalytic site. The :otmcture of the latter domain v&ies arnnng different dehydrogcnases. The gene regulatory repressor and activator proteins provicle another example of modular consln tction . The Lac repressor and the r..atabolitc gene activator protein (CAP} of E. coli both contain multi ple domains. T he crystal structure of CAP shows two domains; A larger domain binds a molecule of cyclic AMP in its interior. while the smaller domain recognizes specific DNA sequences (Figure 5-18 ). There are significant amino acid sequence similarities between the cAMP-binding domain of CAP and the regulatory subunit of cAMP-dependent protein kinase. suggesting that the cAMP-bin ding domain of both proteins evolved from the same f I C l1 Itt 5-18 CAJt complex with cAMP intefadihg with bent DNA. The larger domain of CAP. shown in turQuoise, OOds c:yck

AMP. shown 1r1 red and yeicm in tlle centef of that t ClOd Rastef 30.

84

Wea.k and Strong &nds Determine Mucromol~ular Sto.Jctwoe

precursor. In CAP. this cAMP-binding domain is attached to the DNA-binding domain, so that changes in cAMP levels control transcription levels. In the kinase, the cAMP-binding domain regulates the activity of the fust enzyme in a cascade of enzymes that result in the breakdown of stored glycogen.

WEAK BONDS CORREcrLY POSITION PROTEINS AWNG DNA AND RNA MOLECULES DNA-binding proteins mediate many of the centraJ processes in biology. The bonds that hold these proteins onto DNA are the same coHection of weak bonds that give proteins. DNA, and RNA their own specific tlu-eedimensionaJ configlll"ations. The most abundant DNA-binding proteins have a structural role in pac:.kaging and compacting the huge amount of DNA that must be fitted into the cell. For example. the nucleus or a human cell is only 10 f.Llll (lo- s mE'.ter) across but contains roughly 2 meters of double-stranded DNA. There are many ways that proteins can recognize DNA~ Some protein-DNA interactions are specific for particular sequenct's of DNA, whereas others are more specific for DNA in specific conformations. For example, when DNA is unwound in the cell during DNA replication or recombination, the single strands are rapidly oouod by single-stranded DNA-binding proteins (SSBs). These proteins bind with little sequence specificity but are highly specific; for singleversus double-stranded DNA To accomplish this specificity. the ptimary interactions between SSBs and the single--strandru:l DNA are through ionic or hydrogen bond interactions with the phosphate backbone or through intercalation of bulky ring-shaped si
a helix. 2. The major groove is .rich in hydrogen bond acceptors and clonal'S located on the edges of the bases (see .F igure 6 -10). Mote

importantly. the pattern of hydrogen bonding elements is distinct for each of t\1e base pairs. Thls allows the pattern of hydrogen bond donors and acceptors to act as a code for the sequence of the DNA. ~n the same way that hydrogen banding between the base pairs ensures the appropriatP. recognition of complementary DNA

Wrok Bonrlf'. Correctly Pt1$ilion Protems al ong DNA and RNA Moloc1118s

85

sequences during DNA hybridization. A diagram of the pattern of hydrogen-bon ding donor and acceptor r~idues in the major groove for eac;h base p air illustrates tb e distinct pattern for each base pair (see Figure 6-10). Note that not only can a G:C base prur be easil y llistinguishec.J from an A :T base pair, but A:T and T:A, and C:C and C:G base pairs can a lso be distinguished. (n contrast, the p attero of basf' pairs in the minor groove bas significantly less information and generally only allows the distinction of A:T and G:C. 3. n helices have a thpo)e moment that lt>.arls to their N·t~rminal end being positively charged. This positively..c;harged end frequently makes weak internctions with the phosphate backbone adjacent to the major groove. The heHx·turn ~helix motif was the first protein motif involved in sequence-specific DNA binding to be identified. This motif is composed of two adjacent o. helices that are separated by a short turn (Figure 5-21]. One a helix, C'.allod the recognition helix. is responsib)c for DNA recognition. The second a helix is located approximately perpendicular to I he first n helix. Although these two helices form the core of the DNA recognilion motif, other nearby regions of helix-tum-helix DNA-binding proteins freqtmntly stabilize the arrangement of these two Clt hfllices and contact the DNA. Other DNA-binding motifs also insert n helices into the major groove, such as the zinc finger and leucine zipper DNA· binding motifs (as we shall discuss in Chapter 17). Whereas the use of an o helix is the predominant fonn of specific DNA recognition. some proteins do use different strategies. An extTt:lme example of this is seen with the TATA-binding protein lTBP). which determines the site of transcriptional initiation at many e ukaryotic promoters (see Chapter 12). TBP uses an extensive Tegion of 13 sheet to recognize the minor groove of the so-caJJoo TATA-box (Figure 5-22). So, in this case. we see the use of 13 shed instead of o hel ix and interact ions with the minor groove rather than the major groove (for a detailoo discussion of this maller, see ChaJlter 12).

5-20 SchematX of intetitdion between the recognition ltetix of~ reptessor monomel' and maju groove of operatot DNA. (Soorce: Adapted from Jordan SR aod Pabo CO. l988. Stfucttre of lhe larrbda «Dffl>lex at 2..5 Aresolooon. ~ :242 693-899. Cop~fl~t cr' 1988 Amencan Assoc:laOC('I klr the~ or soeoce F I (i U R E

~ Vv'dl pen11isslon )

Proteins Scan along DNA to Locate a Specific DNA, Binding Site Many DNA-binding proteins make substantiaJ contacts with t he DNA backbone as well a.o; with the specific base pairs of their recognit ion sites. Mediating these backbone contacts are patches of positivelycharged amino acids located at sites very dose to those that bind to the base pairs. These associations rely primarily on electrostatic attraction between these positive patches and the negatively-cllarged phosphate backbone of the DNA. Because the backbone has a similar negatively-charged surface, regardless of the sequence, these proteinDNA backbone contacts contribute substant ially both the specific and nonspecific affinity of a protEJin for DNA. Thus. even a highly spocific DNA-binding protein wiiJ have a substantial affinity for nonspccilk DNA sites as well. For example, thfl affinity of some well-charac:tm-ized regulators of gene ex-pression (such as fhe lactose repressor) for t heir recognition sequences is about 1l1i-fold greater than their affinity for nonspecific DNA. As a consequence. in the cell these proteins are typkally bound at a number of nonspecific sites as weJl as at their specific target sequence. This is due to the m uch larger nu.mber of nonspecific sites r.ompared to the sp«:ific: sites. Indeed, ewtty nucleotide in thn genome

FaGtJRE

5-21 Ceomettyoh.

..~~ lhesdlematic shows t'NO rronomers of A repressor tlound to the q>erator. The helices 1n eadl monomer are la-

be4ed

t lO :;. II IS heliJI 3 vh::t11nset15 ..liD l ht!

groove as ~ itl r!fSUre 5-10. (Source Jldclp~ frcm 10rdiY'I S.R 1'JI'ld P.ixJ C.O. 1988 Strudure o1 the tambda ClJI11I)IeX at 2 5 Aresolu~

bcnSocnce242' 893-899, f 2b, page89S.

COpynght ~ 1988 American Assooanon for the Adv
86

Weak or1d Stmns Bonds Determine .Mncmmolecvlm Strucfol"f'

fIGURE 5-ll Structure of the TBP~TATA box c.omplex. The badbofle of TBP ts shown in purple at the top of the fJgi.Jre; the DNA helix below is shown irl gray N'od rose. (Nikolov D-B..

Oi6l H,

~ ED~ U~hevc1

A.A. His
Lee OK Roeder R.G., and Bu-ley SJ<. 1995

No!Lrre ?;n· 1 19.) Image prepared YJilh

MoiScript Bob~ dnd Rasler 30. Extended DNA on a ltle.-side of ~modeled by Leemor Joshua-lOr.

can be considered thP- beginning of a potential (and almost always nonspecifi c) binding site. Thus in e. coli, which has - 5 X 106 bp in its circular genome. there would be - 5 X 10'1 nonspecific· binding sites. So. although the ratio of specill:c to nonspecific DNA bind ing affinity is high (lOS-fold}. the ratio of nonspecific~to-specific sites is even higher (5 x tO!i-fold}. This comparison expla ins why the cell would have to contain rnuJttplP. copies of the rtjpressor protein to ensure continued occupancy of the specific regulato ry DNA-binding site. Under these co-nditions. most of the repressor protein molecules will be bound to nonspecific sites. Nonspecific protein-DNA interactions are not just an unavoida ble consequence of proteins using the charge of the DNA backbone in DNA recognition. These interactions are believed to speed up the rate at which a given regulatory protein finds its appropriate target. Nonsp ecifically-bound proteins are constrained , by their charge interaction, to diffuse linearly a Long DNA, rather than simply hopp ing on and off the DNA. This diffusion a llows a DNA-binding protein to sample si tf'.s at random in their "se~r:h" for a specific binding site. By being restricted to linear movements, proteins will reaeh their targets faster than if they were free to diffuse throughout tne cell. A smaH subset of DNA-binding proteins do not merely diffuse on DNA, but instead, actively track along the DNA. These proteins use

directional movement on DNA to perfo rm key fun ctions during DNA replication. repair, and recombination (see Chapters 8, 9, and 10). Because this movement is directional, it requires energy. Th us, these proteins hydrolyze ATP to direct changes in their binding to DNA.

Diverse Strategies for Protein Recognition of RNA 1\s introduced aoove, RNA is structurally more diverse than DNA RNA~ bjnding proteins have various roles in RNA function, from stabilizing the RNA to enzymatically processing the RNA. The structures of several RNA-binding proteins bound to their target molecules reve-,a] various strategies for protein-RNA recognilion.

Allosrery: Regulation of a Protem's Function by ChOJJBing Its Shape

Some RNA-binding proteins interact specifically with doublestranded RNA In these r.ases. the proteins recognize feAtures that distin~uish the RNA from the DNA double heHx. For t>.xam.ple, the presence of the 2'-hyclm.>.-yl group is clearly a distinguishing feat:uw of RNA. as is the fact t hat RNA forms predominantly an A-form helix (see Chapte£ 6). which has both deeper and narrower grooves than the B-form helix. In contrast to the DNA-binding proteins discussed above. these proteins do not engage the nucleic acid by inserting ot helical regioi)S into the RNA grooves. Many important RNA-binding proteins bind to RNA molecules that are nol in a regular helica.J conformnliun. lndudlld are proteins that interact with messenger RNA molecules dw-ing transcription and RNA processing. Likewise. machineries lhat splice and translate RNA co11tai.n subunits consisting of RNA complexed with protein. The ribonudear protein (RNP) motif is one of the most common protein sequence m otifs that is dedicated to making specific RNA contacts. This 80 residue domain has a mixed n - J3 fold (Figure 5-23). It b i11ds to s tem-loop structures in RNA, as illusbated by the complex of the spliceosomal protein UtA (see Chapter 13) w ith Ut snRNA (see Figure 5-23). Clearly the shape of the RNA binding smface is specific for this structural motif in RNA.

ALLOSTERY: REGULATION OF A PROTEIN'S FUNCTION BY CHANGING ITS SHAPE The binding of either small or large molecuJes {ligands) to a protein can cause a substantial chauge in the conformation of that protein. Such ligaod-jnduced conformational changes can have a variety of

FIG U R l 5~2J Structure of spliuosomill protein· RNA compt.x: U1A binds hairpin I of Ul snRHA. The prot€1n IS shown in gray; lhe U I snRNA IS shCMJn in green. (Oul:ll'ldge C MO Ill.., Evan> PR., Teo CH,.

and Ndgai K l 994. Nature !in: 432.) Image prepared With MoiScnpt. Bob~ and Raster 30.

88

l4~k and St.rong Bonds

Determine Macromolecular Structure

effects, from increasing the affinity of tbe protein for a second ligand, to switching the enzymatic activity of a protein on or otT. This is known as allosterir;: regulation and is a prevalent control mechanism in biological systems. ''AIJostery" means "other shape;• and the basic mechanism is as follows: A ligand binding at one site on a protein changes the shape of that protein. As a result of that change. an active site, or another binding site, elsewhere on the protein is altered in a way that increases or decreases its activity (Figure 5-24]. Examples of proteins controlled in this w
The Structural Basis of Allosteric Regulation Is Known for Examples Involving Small Ligands, Protein..Protein Interactions, and Protein Modification Here we consider the detailed structural basis for three cases of allosteric regulation. Ln one. the DNA-binding activity of a transcriptional regulator is (;()(!trolled by the binding of a small molecule to that protein. ln another. we see how a protein-protein interar.fion, and a protein phosphorylation event, can mediate allosteric regulation of an enzyme involved in cell division.

Small Molecule E1feclor: Lac Repressor Regulation by Allo/aclose The /ad gtme of E. coli encodes the lactose repressor (Lac}. This protein (about whlch we will learn more in Chapter 16) is controlled allosterically-indeed, it was one of the earliest cllaracterizOO examples of an allostericaUy controlled DNA-binding protein. The protein is involved in gene reguJation. and, when bound to DNA. it prevents transcriptinn of the genes required for the ceU to use the sugar lactose as a catbon

FIGURE

5-24 Schematic'\liewofhow

a reg\Jiator

the binding of an end-produ
I

allosteric transfonnalion..

b

substrate

I

A llostcrr

~gulauon

ofo Protein ·s Functmn by Changlflg It!I Shape

source. .However, when lactose is present in the environment, a specifi c form of litis sugar ((3-1-6-allolactose) induces expression of the lactose genas. TbP. allolactose inducer functions by directly binding to the Lac repressor protein aDd destabilizing its interaction with DNA. Strut..·twal. analysis revoo.ls that the Lac repressor changes shape upon indue« binding. (Those structmal studies used the artificial indocermolecule isopropyl-P..D-thiogalactoside [IPTGI.) This change in shape, in lum, explains how the DNA-binding activity of the protein is weakened. Lac repressor is a large protein (a tetramer o f 15 5 kDaJ and contains distinct domains involved in DNA binding, prote in m ultimeriza. Uon , and inducer binding. The vel)' N-terminal region of the protein (amino acids 1 to 49) is a h€!lix-tum-heJix motif that s pecitlcally binds the ONA major groove within the control region of the promoter, as we have seen in the c-.ase of ~ repressor. Adjactm1 to this region is aJl additional helix. known as the hinge helix, that makElS minor groove contacts. The inducer· binding pocket, in contrast, is in the middle of the large core domain (composed of residues 62-333). Comparing the DNA-bound structure of Lacl wjth that of the protein free from DNA (and bound to inducer) provides a picture of why these two states are essentially mutually exclusive. Bin
89

b

~25).

The a llosteric modification of the enzyme aspartate transcarbamoylase by its ligand, LIP, pro vides another example of a small molecule e ffector (figure 5-26). In that c ase the ligand induces a well-c haract erized change in protein tertiary structure.

Protein Effector: Cdk Activation by Cyclin We now turn to a case of allosteric regulation of an enzyme by the interaction betwP.en that em;yme and a regulatory protein. The enzyme (called Cclk2) is a member of a fami ly of kineses known as cyclin-dependent kinases (Cdks} that regulate progression through the cell cycle. It is inactive until compJexed with a regulaJory protein r.elled a r.yclin. Binding o f that second protein induces a confonnat ional change that alters the structure of Cdk2 around its aclive site, partially activating its ftmction . Further canformational changes induced by phosphorylation of a speCific threonine residue n earby activate the enzyme further (see belowJ_ The structural details of the allosteric event mediatoo by cycl in binding have been establis hed. The structure ofetlk2, free from cyclin, l ooks vmy like that of other kinases. 1Wo el ements of Cdk2 structure are critica.J for its regulation: an a helix, called the PSfAIRE helix, and a flexible loop. called lh e T loop. These are both locatt:Ki n ear the kinase active site. Cyc1in binding induces allo::neric c hanges in the location of the T Joop and f'STA rR.G helix of Cdk2 (Figure- 5-~7J . In the a bsence of a bound cydin. the loop is tocated at the ent s:ance to the active site and the helix is well away from that site. In this confonnation, a glutamate residue critical to catalysis is held outside the active site. Binding of the cyclin results in the movement of the helix into the active site , allowing the critical glu tamate residue to take part in catalysis. Cyclin binding also moves the loop away from the entrance of the a r.tive site , allowing ace~ of the protein suh;trate.

FIG U ~IE 5-25 Allosteric chanles of 1M repressor. Eoch part of the ftgure shows a

M1ef ot Lac repressor. (a) The left Side of the ftgt..re shows thf dimerof the U1Wc.er-lac. repressor complex. Binang of inducer causes a chaflge 1n the struct\Jre that red\.K:es i!ffnty of rep~esscr fa rhe qJerator. (b) The rigtt Side of the fi~ shows the dime- in the absence or 10docet lr1 this case-. tile hmge hclice:s form and the N~IJl'llnill da'r:laln mcllies contact 1Mth the ope~atot seqJenCe. (Soorce: Adapted from lewiS et al 1996 5aence 271 · 6, fig 12. Copvntftt C. IS96 ArnEncan Assooaborl for theM ~11Cefl"e11l of Soetlce. Used Wlth petmiSS«ln.)

90

t-t~k and Stron,g Elonds Determine Mor.romolectJklr Struc:ttJre

FlGVR£ 5-26 lheaUosteri<modific.ation

0

catalytic polypeptide

substrate

ofaspartale~(~)

•

regulatQ(}' polypeptide

CTP (inhibitor)

by

CJP.

active sites

Phosphorylation as Effector: Cdk Activation b_ v CAK As we have just seen, Cclks are activated by binding cyclins. Full activation of Cdk requires a second aJlosteric change in that enzyme, mediated by phos.phorylation. This phosphorylation takes place on a threonine residue wilhin the T loop mentioned above. This modification leads to further

FICUR£ 5-27 Cydin-induc:eclconfonnationalchangesirsCdk. (a)

ThE> mooomenc tcinase stll.laure, shown in t\llquolse. is irlaaive. The positlon of 1he PSTAlRE l1eJix holds a critical residue otu of the catdlytic c.enter; v.tlere ATP is locdted, and the T loq> blocks access to the Pf(l[ein substrate (not stlo\t\'0)- (b) The structure shows 1he repositloffing or the helix upon binding of qdn (sho\Nn in purple) ClOd the removal of the loop from the op€11ing of the catalytic center. Ths ~is partially active. (c) ~phosphorylation of the T loop (show! in red), the Ccl:-cydin complex becomes ILly active (Schutze-Gahrnen U., De Bendt HL. arid Kim 5.H.. 1996. J Med Chern 39: 4540: Jeffrey P.D~ Russo A.A. Polyak 1<., Gibbs E-. Htli'VI,'itz l. Massague J., and Pavletldl NP. 1995. Norure 376: 3 , 3. RtJsso A.A.. Jeffrey P.O.. and P(jVeltich N.P.. 1996. NotSUud Bio/3:696.) II"Mges prepared with~ SobSaipt. and Raster 30.

Summozy

91

reorganization of the active s ite of the Cdk. Ouce added, the phosphate group is bmmd by three arginines, each from a £lifferent region around the catalytic deft. These interactions fix the C'~talytic cleft in a conformation favorable for high activity. The phosphorylation i.~ performed by another kinase (ca1led CAK). Many kinases are activated by a similar phosphorylation event. The two events that togethat activate Cdks-bii1ding of cydin •nu3 phosphorylation -OCCUI:' in that o.rclel:'. This is because cyclin binding not only increases the activity of the enzyme. but also makes the T loop accessible for phosphoryla1ion by CAK.

Not AU Regulation of Proteins Is Mediated bv Allosteric Events Some proteins are controlled in ways that do not involve allostE'.ry. For example, one protein can recruit anotht->T to particular locations or substrates and in that way control what that protein acts on. 11Vhen we discuss regulation of RNA polymerase (the enzyme that ttan..r.;cribes genes into mRNAJ. we will see that what (i.n that case) is usually meant by regulation is the c.hoice of which gene is transcribed at any given time~ This is clone by regulatory proteins. which bind the DNA with one sudace and the RNA polymerase w ith another. These intE'..ractions bring the enzyme to the gene {or genes) that bear appropriate binding sites for that particular regulator. This is an tlxampJe of cooperalive binding of proteins to DNA.

SUMMARY DNA . RNA. and prote•ns are all polymers, eacb composed of a defined set of subunits }o.ined by covalt-'flt bonds. For example, DNA is made up of chains of nucleotides, and proteins are chains of amino acids. The three-dimensional shape of each s uc.h polymer is further detcrminetl by rnultiJ>Ie weak. 01' secondary. interactions between those s ubwtits. Thus. in the case of DNA, hydrogen bonds a nd stacking interactions between the bases of nudootides account for the dmlble-he)ical character of that molecule. Likewise, the s table three-dimensional structure of a given protein requires multiple weak intera.cl ion between (nonadjacent) amino acids within the polypeptide chain. We discussed the natllre of lbE'..se weak Lands in Cllapler 3; io th is chapter we Looked at how lhose weak interactiorl$ determine the shapes of mulecuJes and the inlerat.1 ioru; between and among tbern. particularly proteins. (We sha ll consider the structures of DNA and RNA in more detail in

O.apter 6 .) There are multiple levels to the struclurat organinltion of a pmteiiL The initial covalent linkage of rhe am.i,no acids as the pnmary strUcture. Each amino acid is linked to lhe nexl by a peptide bond. Sec.:Qndary structure is formed hy interactions belwtlt'.n amino ~cids typ ically found ralher near each other in the primary structure of the proleiiJ.. The a helix and ~ sheet are examples of secoooruy structural elements. The tertiary structure o f fl p_ro.. teio i£ the final three-di meru;ional shape of a polypeptide chain. and i s determined by the arrangement of the various el ements of seoonc:lary structure in an energetically favorable way. for many proteins thme is another level of structural organization-the quarternary stn.\C'ture. This

refers to m ultimerization of individual polypep~ide chains into dimer or higher-order structures. Many pr-oteins work a1> multimers- hernoglobio.is a teti'Bmer, for example, and m any DNA-bjnding proteins wodc as dimel'S. Many nalive proteins cootaill several iliscrete fotrled sectioos (domains) that are stable hy themselves IUld which arise from a continUous amino acid sequence. Combinations of such domains account fur a large variety of alJ known p.rotems. The number- of tru.ly uniqt~e d omains i:l probably only a few hundred. Each domain is often associated with a specific functiollfll ar.C.ivity. for example, DNA binding. The specific shape of each mac.ron10lecule restricls the number of other molecules y,.rith which it Gall inteJ:Cmt. Strong secondary interacrions bt'!lween moleculef; den18Itd both a COrrT(JJementary (lock-and-key) .relationship bclween the two bonding surfaces and the 1nvolvement of many atoms. Although molecules bound together by only one or two seamdary bonds frequently full apart. a coJlection of these weak bonds can resuJt in a quite stable romplex. The fact that double"helical DNA does not fall tipart spontaneously shm-,;s just how stable such complex~ can be. Although cmnplexes held together by multiple weak bonds are nut observed to fall apart spontaneously, their assembly can occur spontaneously, with the correct bonds fonning in a step-by-step mnnner (the principle of ~f-assembly). The hinrung of specific proteins to specific sequences along DNA molecules also involves lhe fonnation of w~).:' bonds, usually hydrogen bonds botwoon groups on DNA baSf>.S and approptiBte acceplot' or donor groups on proteins. Most regul~tory proteins use an a helix to recognize speeific DNA sequences. That ..recognition helix"' fits into the ffiijjor

weak and SJron,g Bonds Determine Mo.cromolecular StructJ:Jre

groove of DNA. and the amino acids in the helix contact the edges ofbsses in a 5eqUtlllce-5pecific manner. Tht!tw. copl!idS are ~;t.abilized by t he binding enel'gy of the t:pecific interactions. DNA bi nding proteins also amtain regioru; that allow nonspecific bonding to the DNA backbone- These nonspecific backbone interactions permit Hncar ditfusion along DNA. allowing proteins to reach their specific targef sequence,; more qujcl
tions. often J·egulate protein funcliun through a llile on the protein separate from the .region of that protein that mediates its main function (the _active sile o f an aoeyrne, DNA-bind ing domain , etc). This binding or modification tMggers a change i n the sha pe of the protein which in· c.reases or uecre~s the ac1ivity of the active site , or DNAbinding domain, esf;entially switc-.hing lhe activ ity ClJl o r

off. ln uthcr cases, a prolein may be controlled by moditlcatjon or bjnd.ing of a ~ond protein. in ways that do not involve allostery. For example, modification can create a site on a prote in lhat is recogoi2ed by a se.cood protP.in . Such protein -protein interactions can recruit proteins lo particular locations or substrates, and in that way control their activity.

BIBLIOGRAPHY Books Brandon C. and Tooze f. 1999. lnlrodur t.ion lo protein structure. 2nd edition. Garland Publishing, Now York. Pauling L. 1960. The nature oftJJe cllemical hond, 3rd edition. Cornell University Press. Uhaca. New York.

Weak Bonds Correctly Position Proteins along DNA and RNA Molecules De Guzman R.N.. 'l\Jmer R.B., and Sommers M.F. 1999. Protein-~A :recognition. Biopolym crs 48: 181 - 195. Sperling R and Wachtel E.). 1981 . The histones. Adv. Prot. Chern. 34: 1 - 52.

The Specific Conformation of a Protein Results from Its Pattern of Hydrogen Bonds Omthia C. 198 4. P rinciples that determine the slruclures of proleins. Arm. Rev. Biocl1em. 53: 537- 572.

Allostery: Regulation of a ProteinJs Function by Changing Its Shape

Rose C.E. 1979. Hiererarchical orguni7.ation of domains in globular proteins. f. Mol. Biol. 134: 447- 470.

BeU C.E. and Lmvis M. 2001. The Lac repressor: A second generation of structural and fundionw studies. Cur. Opin. inStruct. Bio. 11:19 - 25. Paoo H.C.. Kercher M.A.. Lu P., Markiewicz P. , Miller J.H., Chang G .• and Lewis rvl 1997. Lac repressor genetic map m real space. T1DS 22: 33 4.- 338.

Steitz T. A., Weber J.T., and MBUhew J.B. 1982. Catabolite gene activa.or protein : Structure. homology. with other proteins. and. cyclic AMP and DNA binding. Colcl SprinB Hornor Symp. in 0.J(lnt. 8 10, 47: 419- 426.

PavJeticb N.P. 1999. Mechanisms of cyclin-dependent kinase regulatioll; Structures of cdks, their cydin activators, and cip and INK4 inhibitors. f. Mol. Bini. 287; 821 - 828.

Most Proteins Are ModularJ Containing Two or Three Domains

P A R T

MAINTENANCE OF THE GENOME

Port 2 Mainltmorrr;e of the Genome

PAR1

O UT l i NE

• Chaptet 6

fhe Structures ot DNA and RNA

Chapter 7 Qlrorno-.:>Omes. Chromar•n, and the Nucleosome

Chapter 8 0 1apter 9

The Replication of DNA

The Mutability and Repa1r of DNA

Chapter 1o Homdogous Rec.ombinatxm at the Moleallar Level

Chapte1 11 Site-Speof1c Recombtnatioo and lfansposition of DNA

art 2 i.s dedicated to the stmcturo of DNA and tho proccs!>~ that p ropagCcite, mainl~n, and ~Iter it from l1ne c:.eU generation lo the next. Ln Chapters 6 through 1 1, w e will examine DNA and its close relative, RNA, and address the following questions:

P

• How do tho structures of DNA and RNA account for their func· tions? • How are DNA molecules.. which are extraordin.arily lung coo1pared to the size of the cell. packaged within the nudeus? • How is DNA replicated accurately and completely during the cell cycle. and how is this .achieved with high fidelity? • How is DNA protecrerl ftom spontanrous and environmPJ\tttl dam~ age, and how is damage, once inflicted . reversed? • How aro DNA sequences exchanged hetw~en DNA chromosomes if1 processes known as recornb ina1 ion and transposition?

ln answering these questions, we will see that the DNA molecule is subject both to conservative processes that act to n1~intain U unaltered from generation to generation, and to other processes that bring about profound c hanges in the genetic materiaJ that help drive evolution. In the cell, DNA is subjected to foi'Ces that peel apart its strands. twist it into topologically constrained structures, v.rrap it around and through pmtein as.<;ernblics, anr1 break and reseal its backbone. These m~mipu latioos are mediated by myriad enzymes and molecular machines that p ropagate. maintain. and alter the genetic material. Chapter 6 explores the structure of DNA in atomic d etail. from the chemistry of its bases and backbone, to the base~pairi ng inte ractions and other forces that hold the two strands together. DNA is often topologically constrained. and Chapter 6 considers the biological effects of such constraints, together with enzymes that alter topology. This chapter also explores the structure of RNA. Despite the close similarity of its chemistry to that of DNA, RNA has its own distinctive stn.lctural features and properties, including the remarkable capacity to act a.s a cetalyst in severaJ eellular processes. As we will learn in Chapter 7. ONA is not naked in the r.eH. Rather. it is pAckaged with specialized proteins in a structure called c hromatin. This packaging allows exCEedingly long molecules to l>t3 ac~ commod.ated in tbe cell and to be accurately segregated to daughter cells during cell division. Chromatin can be modified to increase or decrease the ac()essibility of the DNA. These changes contribute to ensuring it is replicated, recombined, and transcribed at the right time and in the right plal.-e. Chapter 7 introduces us to the histone and nonhistone components of chromatin , to the struct ure of chromatin_. and to the enzymes that mediate chromatin modification. The structure of DNA offered a likeJy mechanism for how genetic material is duplicatecL Chapter 8 desr:ribes this copying mechanism in detail. We describe the seroiconE>ervative nature of DNA replication, and the elaborate collection of enzymes and other proteins required to call)' it out. But the replication machinery is not infallible. Each round of replication results in errors, which~ if left uncorrected. would become mutations in daughter DNA molecules. In addition. DNA is a fragile molecule that undergoes damage spontaneously and £rom chem icals and radiatioH. Such damog~ must be detected and meuded if the geuetic material is to avoid rapidly accumulating an rmacceptable load of

Pm1 2 Mointenonr:e of the Cef!Dme

95

mutations. Chapter 9 is devoted to the mechanisms that detect and re~ pair damage in DNA. Organisms from bacteria to humans rely on similar, and often highly conserved, mechanisms for preserving the in· tegrity of their DNA. Failure of these systems bas catastrophic consequences, such as canceT. The final two chapters of Part 2 reveal a complementary aspect of DNA metabolism. Jn contrast to the conservative processes of replication and repair, which seek to preserve the genet ic material with m.inimal alteration, the processes considered in these chapters are designed to bring about new arrangements of ONA sequences. Chapter 10 covers the topic of homologous recombination-the p1ocess of breakage and reunion by which very similar chromosomes (homologs) exchange equivalent segments of DNA. Homologous re· combination aJlows the generation of genetic diversity, and ~ lso re· placement of missing or damaged sequences. Two mod~ls fnr pathwiiys of homologous l13(;()mbination are described. as well as the fascinating set of molecular motors that search for homologous sequences betv.•een DNA molecules and then create and resolve the intermediates predicted by the pathway models. Finall}'. Chapter 11 brings us to two specialized kinds of rocombination known as sile·specific recombination and traru;position. These p rocesses I~ to the vast accumulation of snme fiequtmces within the gonomes of m~ny organisms. including humans. We will discuss the molecular mechanisms and biological consequences of these forms of genetic exchange.

PHOTOS f"'ROM THE COLD SPRING HARBOR LABORATORY ARCHIVES

Barbara McC1intoc:k and Robin Hdlid.-;.. 1984 Symposium on Recombination at the ONA Level. McCiintod proposed the exis· tence of ttansposons to account for the restJit5 r::1 t1er geneoc sttJdies with maize. C
96

Port 2 Mmntencmr:e of the Gename

Arthur Kornberg. 1978 Symposium on DNk Replication and R«ambination. Kombefg's extenstve contributions to the study of DNA repflca... han (Chapter 8} began With punfylng the f~ emyme that could ~thesiz.e DNA, a DNA pcty.. rnet(!Se from E. coli. t-is experiments sh®.red that a DNA template was required lor nevv DNA synthesis, coohlming a pretfiCliln of the model for DNA replication prq::>osed by Watson and end:. f Of this \AA:Jik t
Meciclne.

Reiji Obza~ 1968 Sfi11P05ium on Repli. utian of DNA in Miaoor'ganisms. Olczaki had at this time Jt1Sl shown how. dlrog DNA replicarK>rl, one of the new strcnds is synthesized in short fragments that are only later jcjt1ed together. The existence of these "aazal
Matthew Mesetscln. 1968 Symposiunl on Rep1icalicln of DNA in Microot-gcrisnws. Meselson, With frank Stahl. derronstrated that DNA IS replicared by a semt-eonservative mechlr nisrn. fus V'.0S once F<~mOl.r.ily called "the most beootlftA e xpeome11t in btotog1 (Ol
PiAII Modrid\, 199~ Syhlposiurn 0&\ DNA

and Chromosomes. A poneer .n the DNA repair ficld (CI"laptef 9). llt1odid1 wOO<ed out much of \tle med1Mlisuc basis of rrCsma1th repair.

Fra.nklin Stahl and Max Delbriick. 1958 S,.posium on Exchange of Genetic Material: Mechanism and Con~uences.. Stahl wa<; Meselson's partfler 10 the~ described above. i.lfld sd>sequEI"lf.ly cootribtlted much to wt undefstandlr1g of homologous tecombirlatiort (Chapter 10). Debuck was the iruii.Jefltial cofolfl1def of the so-called "Phage Group" - a g~oup of stienhsts tt>.at ~ bacteriophage as lhe 1irst model system of molec:tJiar biology (Chaplet 2 1}.

CH A PTER

The Structures of DNA and RNA

T

he discovery that DNA is the prime genetic molecule. carrying all the he~ditary information within chroro.osomes, immediately focused attention on its structure. It was hoped th~t knowledgP. of the structure would reveal how DNA carries the genetic messages that are replicated when chromosomes divide to produce two identit~ copies of themselves. During the late 1940s and early 1950s. several re:>earch group5 in the United States and. in E\lrupe engaged •n serious efforts-both cooperative and rival-to understand bow the atoms of ONA are linked together by covalent bonds and how the resulting molecules ru~ arranged in three-dimtmsionaJ space. Not surprisingly, there irutially were (ears that DNA might have very complit:aterl and perhaps b\za.rre structures that differed radically fmm o ne gene to another. Great relief, if not general elation, was thus expressed when the fundamental ONA stnu,i ure was found to be the double helix. It told us that all genes have roughJy the same throo-dimensi onc:~l form and th4-1.f the differenL'eS between two genfJS res.ide in the un.ler and number of their four nucleotide building blocks along the complemental'y strands. Now. some 50 :years after the discovery of the double helix, this simple description of the genetic material remains true and has not bad to be apprnciab1y altered to accommodate new findings. Nevertheless, we have come to realize that the structure of DNA is not quite as unifocm as was fi.rsl thought. Fnr e:xample, the chromosonm of some small viruses have single-stranded. not doubl&stranded, molecules. Moreover, the precise orientation of the base pairs varies slightly from base pair to base pair in a manner that is influenced by the local DNA ~qucnco. Some DNA seql..tellces even permit the doub lo heli x tn twist in the )eft-handed sense, as opposed to the right-handed sense originally fonnuJated for DNKs general structure. And while some DNA mole<.:ules are linear. others are circular. Still additional com plexity comes from the supercoiling (further twisting) of the double helix. often around cores of DNA-binding proteins. Likewise, we now realize that RNA. which at first glance appears to be very similar to DNA, has it.~ own distinctive structural features. lt is principalty fottn41 as a single-stranded mo lecule. Yet by means of intra~strand base pmring, RNA exhibits exlentsive double-heJical character and is capable of folding into a wealth of diverse terti~ structures. These structures are full of surprises. s uch as nonclassical base pairs, base-backbone interactions, and knot-like configurations. Most remarkable of all, and of profound evolutionary sigpificance, some RNA molecules are enzymes that carry out reactions that are at the core of information transfer from nucleic acid to protein, Clearly, the structures of DNA and RNA are richer and more intricate than w as at first appreciated. Indeed. there is no one generic structure for DNA and RNA. As we shall see in this chapter, there are in fact variations un commo11 th emes of structure t hat arise from the unique physical, chemical. and topological properties of the polynucleotlcl.e chain.

OUTLINE DNA Structure (p. 98 )

• IJNA k1pology ( p. 111} RNA StrudUre (p. 122)

97

The Strucfure.s vf DNA anrl RNA

98

DNA Is Composed of Polynucleotide Chains

:r

-s

20 A(2 nm)

b

The most important Witure of DNA is that it is usually compmed of two polynucleotide chains b\risted around each other in lhe fnrm of~ double helix (Figure 6-1). The upper part of the figure (a) presents the structure of the double helix shown in a schematic foiD1- Note that if inverted 1800 {for example, by turning this book upside-down). the double helix loob superficially the same, due to the complemenlary nature of the two DNA strands. The space-filling model of the double helix. in the lower part of U\e figure (b), shows the component.c; of the DNA molecule and their relative positions in the helical structure. The backbone of each strand. of the helix is composed of altem ating sugar and phosphate residues; the bases project inward but are accessible through the major and minor grooves. L.et u s begin by considering the nature of the nucleotid.e. the fundamental building block o r DNA. The nucleotide consists of a phosphate joined to a sugar, known as 2'-deoxyribose. to which a base is a ttachedThe phosphate and the sugar have the structures shown in Figure 6 -2. The sugar is called 2' -deoxyribose because there is no hyd roxyl tlt position 2' {just two hydrogens). Note that the positions on the ribose are designated with primes to distinguish them from positions on the bases (see the dis<.russion below}. We om think of how the base is joined to 2 '-deoxyribose by inmgining the removal uf a molecule of water between the hydroxyl on the 1' carbon of the sugar and the base to form a glycosidic bond (Figure 6-2). The sugar and base alone are called a nucleoside. Likewise, w e can imagine linking the phosphate to 2 '-deoxyribose by removing a water molecule from between the phosphate and the hydroxyl on the 5' carbon to make a 5' phosphomo noester. Adding a phosphate (or more than one phosplllite} to a nucleoside creates a nucleotide. Thus. by making a glycosidic bond between the base and the sugar, and by making a phosphoeste.r bond between the sugar and the phosphoric acid. we have created a nucleotide (Table 6 -1 ).

0

II I

-o- P- O.H

0 phosphoric acid

FIGURE 6-1 The helKal &tructure of DNA. (a) Schemahc modeJ of the ~ hel[l( One turn of the trl1t(34 Aor 3A nm) spans approl
nucleclide (dAMP)

FIGURE 6-2 Formation of nudeotide by removal ot watec. carbon atOmS in 2'-rleoxyribo<;e are labeled 1(1 red.

The l"llJil'lbers of lfle

DNA Structure

99

TABU 6-1 Adenil\~ arut R~ed Compounds

Base Adenine

Nucleoside 2'-deoxyadenosine

Nudeolide 2'--deoxyadenosine 5'~phate

Structure

M-Ia

&> Molecular wsigl1t

135.1

•:r) ~"'

1-4

~ ._

H

H

H

OH H

251.2

·-r- h-.. H

OH

H

H

....

OHM

331.2

Nudeotides are. in turn , joined to each other in polynucleotide chains through the 3'-hydroxyl of 2'-deoxyribose of one nucleotide and the phosphate attached to the 5 -hydroxyl of another nucleotide (Figure 6·3). This is a phosphodiester linkage in which tl1e phosph oryl grou p between the two nucleotides has one su~ar esterified to il 1

6-3 Detailed structure of poiVJ1Udeotide polymer. The st:ruc'tUfe F I CURE

shows base pairing ~een purines (10 l:iue) ~ pyrimidines (in y~}.

ao:! the

pllosphOOe;te linkages of the bad:bone. {Source: Adapted fiom Otc:kerson RE. 1983. Scientific Ament;cn 249: 94. lflustrotion. Irving

GetS. tmage from Irving Ge.s Coledion/How.mS H~es Medcaltostitutioo. Not ro be teptOdcJCed without penrissioo.)

100

T rw Strucfures of ON/t ond RNA

adenine

guanine

through a 3 '-hydro>."}' I and a second sugar esterified lo it through a 5'-hydroxyl. Phosphod.iestcr linkages c reate the repeating, sugarphosph~te backbone of the polynucleotide chain, which is a regular feature of DNA. In contrast, the order of the bases along the polynucleotide chain is irregular, This irregularity as well as the Long length is. as we s.hall see, the b:lsis for the enormous information content of DNA. The phosphoiliester linkages impart an inherent J:>Olarity tn the DNA chain. This polarity U. defined by the asymmetry of the oucleotidcs and the way they are joined. DNA chains have a free 5'-pho.'\phate or 5'·hydroxyl at one end and a free 3'-phosphate or 3'-hydroxyl at the othe.r end. The convention is to write DNA sequences from the 5' end (on the le ft) to the 3' end. generall)' with t1 5'-phosphate and a 3' ·hydroxyl.

Each Base Has Its Preferred Tautomeric Fotm

f!IGUR£ 6-4 Purines 11J1d pyrimidines. The dotted lifies irldic:Clt(: tfle SiteS oJ attadvnent ot the bases tD the 5U83fS. For s~mpkity. hyd.ogens tJre emitted frorn 1he sugars and bases ., subsequmt figures, eJ(Cept where

ThP- bases in DNA faU into two classes, purines and pyrlmidi~ The purines are adenine and guanine, and the pyrimidines are t::ytosine and thymine. The purines are derived from the double-ringed structure shown in Figure 6-4. Adenine and guanine share this essentiw structure but with different groups attached. Likewise. cytosine and thymine are variations on the single-ringed structure shown in Figure &4. The figure also shows t.he numbming of tlm positions in the purine and pyrimidine rings. The bases are attached to the deoxyribose by glycosidic linkages at Nl of tbe pyrimidines or at N9 of the purines. F.ach uf the bases exists in two ~ltemative tautomeric stales, which are in equilibrium with each other. The equilibrium lies far to the side of the conventional structures shtJwn in Figure 6-4. which aro the pw.dominant states and the ones impurtant for base pairing. The nitrogen atoms attached to the purine and pyrimidine rings are in the amino form in the predominant state and o nly rarely assume the imino configuration. Lilev.-ise. the oxygen atoms attached to lhe guanine and thymine norma lly have the "keto form and only rarely take pn the enol configuration. As examples, Figure 6-5 shows tautumeri7.ation of cytosine into the imino form (a) ancl guanine into the enol form {bJ. As we shall see. the capacity to form an alternative tautome r is a frequent source of errors during DNA synthesis.

pertinent to tilt':' itustralloo.

The Two Strands of the Double Helix Are Held Together by Base Pairing in an Antiparallel Orientation The double helix is composed of two poJynudf'otide chains that are held together by weak. noncovalent bonds between pairs of bases, as shown in Figure 6-3_ Adenine on one chain is alw11ys paired with thymine on the other chain a nd. likewise, guanine is always paired. with cytosine. The two strands have the same helical geometry but base pairing holds them together with the opposite polarity. That is, the base at the 5' end of one strand is paired with the base at the 3' end of the other strand. The strands are said to have an antiparaUeJ orientation. This antipitl'allel orientation is a stereochemical consequence of the way that adenine and thymine, and guanjne and cytvsine, pair with each together.

DNA Structure

a

imflo

aminO

' H'-N/

b

..._. / H

H

,i~ 0

101

N

I

,:r: N

Ill&

' H

keto

'o

~

I

I

enol

'- / H 0

FIGURE 6-5 Base tautomen. Amino~ imioo and keto ~erol tautomerisrr. (a) Cytois usually ir1 the arni'1o foon but rarely lams the irrjno coofigurarion. (b) Guarttne IS usually 1n the keto form btJt r; r<~rely foU'ld 1r1 the enol mnfi~ranon Sine

The Two Chains of the Double Helix Have Complementary Sequences The pairing helween adenine and thymine, and between guanine and cytosine. results in a complementary relationship between the ~uence of bases on the two intertwined chains and gives DNA its self·enroding cha'fOCter. For example. if we have the sequence 5'-ATGTC-3' on t.me chain, the opposite chain must have the complementary ~ence 3'~TAC..AG-5'.

The strictness of the rules for this "Watson-Crick" pairjng derives from the complementarity both of shape and of hydrogen bonding properties between adetline and thymine and between guanin~ and cytosine (Figu:re 6-6). Ad enine and thymine mHtch up so that a hydrogen bond can form between the exocyclic amino group <:tt C6 on adenine and the cmbonyl at C4 in thymine; and likewise, a hydrogen bo.(u:.l can form between N l of adenine and N3 of thymine. A corresponding arrangement can be drawn between a guanine and a cytosine, so that there is both hytirogen honding and shape complementarity in this ba1>e pair as well. A G:C base pair has three hydrogen bonds, oocause the exocycJic NH2 Ht C2 on guanine ties opposite to, and can hydrogen bond with, a carbonyl at C2 on cytosine . Likewise. a hydrogen bond can fonn between N1 of guanine and N3 of cytosine and between the carbonyl at O:i of guanine and the exocyclic NHl at C4 of cytosine. Watson-Crick base pairing ~ quires that the bases are in their preferred tautomeric states. An important feature of the double helix is that the two base pairs have exactly Ihe same gcomelryi having an A:T base pair or a G:C base pair between the two sugars does not perturb the arrangement of the sugars because the distance between the sugar attachment points are the same for both base pairs. Neither does T~A or C:G. In other words.

f t Ci U R E 6-6 A:T and G:£ base paiBlhe flguce shows hydr~ bonang between

the bases.

102

The Structures of nNA onr.J RN/1

th ere Is an approximately twofold axis of symmetry that relates the two sLJgars and all four base pai rs can be accommodated within the same arr-.mgemtmt without any distortion of lhe overall structure of the DNA. In adtlition. the base pairs can slack neatly on top of each other between U1e two be lica) sugar-phosphate bac:kbones.

Hydrogen Bonding Is Important {Ql" the Specificity of Base Pairing

f I (; U R IE

fi. 7

A:C inc.ompatibility.

1he

strc.Jt1llfe shows the tnabifity of cldcnine to f0«11

lhe proper tlydfosen bortds wilt1 cyt:owle

The

base pan IS the(elore unstable.

The hydrogen bonds between compleme.ntacy bases are a fundclment~l feature of the double helix, contributing to the thermodynamic stability of the helix and the spcc1ficity of base pairing. Hydrogen bond; ng might not, at first glance, appear to conlrlhule importantly to the stability o f DNA for the following reason. An organic molecule in &JUBOUS solution has all of its hydrogen bonding properties satisfied by water molecules that come on aJIO o ff very r~pidly. A~ a result. for ev~ry hydrogen bond that is made when a base p uir forms, a llydrogen bond with water is broken that was there before lhe base pair formed. Thus* the net energetic contr~bution of hydrogen bonds to the stability of the double hellx would appear to be modest. However, when polynucleotide strands are separate~ water m olecules are lined up on the bases. When stronds come togetber in the d ouble helix. the water molecules i:lle displaced from the bases. This creates disorder and inc reases entropy, thereby stabilizing the double helix. Hydrogen bonds are not the only force that stabilizes the double helix. A second important contribution comes from stacking inlerac.tions between the b-dSes. The bases are flat. relatively water-insoluble m olecules. and they tend to stack above each other roughly perpendicular to the direction of the helicaJ axis. Electron cloud interactions (1t-7r} between bases in the helical stacks contn1rute significantly to the stability of the double he lix. Hydrogen bond ing is also important for the specificity of base pairing. Suppose we tried to pair rui adenine with a cytnsine. Then we would have a hydrogen bond acceptor (N1 of a denine) lying opposite a hydrogen bond t:tcccptor (N3 of cytosine) wUh no room to put a water molecule in between to satisfy the two acceptors (Figure 6-7}. Likewise, two h ydrogen bond donors. the NH 2 groups at C6 of adenine and C4 of cylosinc. would lie op posite each other. Thus, an A:C base pair would be unstHble because water woulrl have to bo stripped off the donor and acceptor groups without restoring the hydrogen bond formed within the baSP. pair.

Bases Can Flip Out from ·t he Double Helix

fIG U R E 6-8 Base flipping. StiUCture of tsOialed DNA. ~the ffipped cytosine ~dte ilnd ~t-oe

smal di~ns to the adjac~ b3se pairs. QCJif11QSdt.ISk.as S., ~ S~ ROCei'\S RJ.. and Cheog X. \994 Celf76- 357 fnlage prepared \ll/lftl &t>Scnpt, MdScrlpt. and Raster 3D)

As we have seen, the energetics of the double bel i_x favor the pairing of each base on one polynucleotide stra nd with tha complementary base on the othor str.and. Sometimes. however. individual bases ca.n protrude from the d ouble helix in a remarkable phenomenon known as base Hipping shown in Figure 6-8 . As we shall see in Chapter 9, certain enzymes that m ethylate bases or remove damaged bases do so with the base in om extra-heUcal cunfigwatiun in which it Is flivped out from the double helix, enabling the base to sit in the catalytic cavity o£ the enz}'ffie. Furthermore. enzymes involved in homologous recombination and DNA repair are believed to scan DNA for h omolog}' or Jesions by flipping out one base after another. This is not energetically expensive because only one base is Aipped out at a time. Clemly. DNA is more flexible than might be asswned at first glance.

DNA Is Usually a Right-Handed Double Helix Applying the handedness ml e from pllysics. we can see that each of the polynud ootide chains in lhe double helix is rigbl-handed . ln your mind 's eye. hold yo ur rigllt hand up to the DNA molecule in Figure 6-9 with your thumb pointing up and along the long axis nf thP. helix and your fingers following the grooves in the helix. Trace a long om~ strand of the heHx in the direction in which your thumb is pointing. Notice that yo u go arounn the helix in the same direction as your fingers are pointing. This does not wurl< if you u~ your le ft .hcmrl . Thy it! A ronsequttnco of tho helical na ture of DNA is its periodicity. Each base pair is displaced (twisted) from th e pre\'ious one by about ~6c. Thus. in lim X-ray crystal structure of DNA it takes a stack of about 10 base p airs to go complete ly aro und the helix (360' ) (see Figure 6-la). That is. tht:l helical periodicity is generally 10 base pairs per turn of U1e he lix. For further discussion, see Box 6-1, DNA Has 10.5 Oase Pairs per Turn of the Helix in Solution: The Mica Experiment.

The D()uble Helix Has Minor and Major Grooves As a result of lhe double-helical stn.cture of the two chains, the DN A molecule is a lung extended polymer with two grooves that are not equal in size to each o ther. Wh y are there a minor groove and a major groove? II is a simple con._<;equence of the geometry of the base plfir. The angle at whicb the two s ugars protrude frorn the base pairs (that is, the angle between the glycosidic bonds) is about 120c (for t he n arrow angle or 240u for the w ide angle) (see Figures 6-lb and 6-6). As a result, as more and more base pa1rs stack on top of each other, the narrow angle between the sugars on one Pdge of the base pairs generates a minor groo,•e and the large angJe on the 01 her edge generates a major groo\'e. (If th e sugars pointed away from each o ther in a straight lin~. that is, at an anglo of 180'"' . then the two grooves wouJd be of equal dimensio ns and thttre would be n o minor and major g.rooves.)

The Major Gro()ve Is Rich in Chemical Information The edges of each base pair are exposed in the major a mi minor grooves, creating a pattern of 'hydrogen bond donors and acceptors a nd of \lan der Waals surf11t:eS thut idenlifi('S the base pair (see Figure 6-10). The edge o f an A:T base pair displays the foJicnving chemical groups in the following order in the major groove: a hydrogen bond acceptor (the N7 of adenine). a 'hydrogen bond donor (the exocyclic amino group un C6 of adeni ne), a hydrogen bund f'\Cceptor (lhe carbonyl group on C4 uf

3'

5'

3'

right-handed

s

3" feft-handed

6-9 l.eft- and righl--h.andecl helices. The two polynucleotide chains 10 lhe tb.ble tleb !Map arot.J1d one arother .n a nght tlanded m&lner. F l Ci U R E

Box 6-1 DNA tta5 10.5 Base Pairs per Twn of the Helix in Solution:

The Mica EXperiment

vaue of 10 base pais pet tLm vatles 50'l'le'Mlat under cifferert C1JC1dill0r'6. A dasgc experirner'l that was caTied oot in the 1970S defnolstreted that DNA ibsabed on a sufac:e has sunevdla& greater ltlcYI 10 base pairs per tun. Short

This

segments d 0\ll\ were ~ to bind to a mica suface. The presence of 5'-termlnc'll phosphates on the ONAs held them in a fixed mentabon on the mica The mi<:a-botxld DNAs were then exposed to DNAse I, an enzyme (a deoxymonudease) that deaves the pros~er hoods in the ONA badbooe. Becau;e the en zyme is bUky, it IS roy able to deave ~er bonds on the DNA su~e furthest from 1he mea (think d the ~ ilS a cylinder lytng doNn on a flat St.rlace) c1Je to the stenc diffecuky cJ reacloi~ the sides or OOttt.m surface of the DNA /ls a rest.b, the length of the restJting fragments sroud feflect the periodiCity of the DNA. the

nt.lli>er of base pasrs per tum. After the rnKil-bound DNA was exposed to DNAse the resulflng fragments were separated by electrqfloresis in a pclyaaylamde gel, a jelly-like matrix (Box 6- I F"l8lJre 1, see also Chapter 20 for an explanabon of seJ electrophorestS) Because DNA is negatnt~ ch.Yged, it mlgl(ltes through the gel toward the posetive pole of the electric field The get matrix impedes movement of the fragments in a mannef that IS proportional to their length such that larger fragments m~grate more slovvty than smallef fragments. When the experiment IS carried out, we see dusters of DNA fragments of average sizes 10 and 11, 2 1, 31, c'Yld 32 base pairs and so foot\ that .s. 1n multiples d 10.5, vhch is the number of base pairs per n.m This value of 105 base pairs per turn is ck:~ to thai d DNA 10 sducion as infened by other methods (see the secfun titled The Double Helix Bctsts 10 Mu1ttple Conformations, bda.-v). The strategy of using DNAse to probe the structured DNA is ro.N used to analyze the interactioo of DNA with (X(lleins (see Chapter 17).

0

~

-

22 21 20

-

11

1()

80 X

6-1

f I c; U f< E

J The mka e.periment.

thymine) and a bulky hydrophobic surface {the methyl group on C5 uf fhymjne). Similarly, the edge of a G:C base p air displays the tdlowing groups in the major groove: a hydrogen bond acceptor (at N 7 of guanine). a hydroge n bond acceptor (the carbonyl on C6 of guanine), a hydrogen l:xmd donor (lhe exocyclic amino group on C4 of cytosine), a small nonpolar ltydrugen (the hydrogen at C.5 of cy{osine).

DNA Structure major groove

m~groove

0 H

H

A

\

A

k . ; -H......,~ O N

(~:~~~~H

/

N-l\ -

o ....... ..._N

A

\

\

A

H

0

A

rfinof groove

mnorgroove

major groove

mapgrOO\Ie

H

A

A

H

A

Thl,S, there are charactflristic patterns of hydrogen bonding and of overall shape that are exposed in the maiot groove that distinguish an A:T base pair from a G:C base pair, and, for that maHer, A:T from T:A. and C:C from C;G. We car. think of these features as a code in which A represents a hydrogen bond acceptor. D a hydrogen bond donor, M a methyl group, and H a nonpolar hydrogen. In such a code. A 0 A M in the major groove signifies an A:T base p~ir. and A A D H s tands fur- a G:(: base pair, Likewise. M A D A stands k11· a T:A base pair and H D A A is characteristic of a C:C base pair. 1n all cases, 111 is code of chemical groups in the major groove specifies the identity of the base pair. These patterns are import;mt becau se they allow proteins to unambiguously teeognize DNA sequnnces wilhout having lu open and thereby disrHpt the double helix. Indeed. as we shal l see, a princi pal dooodlng mechanism relies upon the abitity of amino acid side chains to protrude into the ma~)r groove and to recognize and bind to specific DNA sequences. The minor groove is n ot as rich in chemical informa tion and what information is available is less useful for distinguishing between base pairs. The small size of the minor groove is Less able to atXOmmodate amino acid side chains. Also. A:T and T:A base pairs and G:C antl C:G base pairs look similar to one another in the minor groove. Au A:T base pair has a hydrogen bond acceptor (at N3 of adenine). a nonpolar hydrogen (at N2 of adenine) and a hydr~en bond acceptor (the carbonyl on C2 of thymine). Thus. its code is A II A. But this code is the same if read

105

FIGURE 6-10 CMmkal groups 6posed in the maio' and minor grooves ft"Om th@ edges of the base pairs. Tre letters in red Identify hydrog€1\ bond a<X:eptors (A). hydrogen bond dcr.ors (D), 11011polar hydrogens (H), ald ITlCtl-¥ groups (M).

tOft

Tflrt StrucltJres of DNA and RNA

in the opposite direction, and hence an A:T bnse pair does not look very different frum a T:A base pair from the point of view of l11e hydrogenbonding properties of a protein poking its side cnains inlo the minor groove. Ukev.ise, a C:C base pair exhibits a hydrogen bond acceptor (at N3 of guanine), a hydrogen bond donor (the wrocyclic amino group on C2 of guanine), and a hydrogen bond acceptor (the carbonyl on C2 of (.....vtosine), representing the code A D A. Thus. from the point of view of hydrogen bonding. C:G and G:C base pairs du not (ook very different from each othet either. The minor gtoove docs look different ".vhen

comparing an A:T base pair with a C:C base pair, but C:C and C:G, or A:T and T:A. cannot be easily d istinguished (see Figure & 10).

The Double Helix Exists in Multiple Conformations Early X-ray diffroclion studies of DNA, which were carrifld out usang concentrated solutions of DNA that had been drawn out into thin fibers. revealed two kinds of structures, the t; and the A forms of DNA (Figure 6-11}. The B form. which is observed at high humidity, most closely corresponds to the average struchtre of ONA lmder physiological oonditions. lr has 10 base pairs per turn, and a wide major groove and a narrow minor groove. The A form , which is !)bserved under conditions of luw humidity. has 11 base pairs per tum. Its major groove is narrower and much deepE".r than that of the B form, and its minor groove is broader and shallower. The vast majority of the DNA in lhe cell is in the B form. but DNA does adopt the A struc:ture in certain DNA-protein complexes. Also, 1'\S we shall see, lhe A form is similar to the structure that RNA adopts when double helical The B form of DNA represents an ideal structure that deviates in two respa.1s from the DNA in ceUs. First. ONA in solution. as we have seen, is somewhat more fl.\risted on average than the B form, having on

fiGURE 6-11 ~odelsofthe~A,andZ fot~nS of DNA. The sogar-ptlosphate bad< booe of eocf1 dlatn as m the outside 111 al S1nJCtureS {ooe ~te Md Me green) witt! the bases (silver) ooer.ted inward. Side views a~ shown at r:he top. and VIeWS along the helical ans at the bottom (a} lhe B fom1 of DNA. the usual form found in c:etls, is dlaracterized by a l1elic:at turn e1.<ery 10 base patrs {3.4 nm); ad)aCent stacked base P<*>~e 0..34 nm apart 1he major ~e_ (b) The more compact A form of DNA has

I I base ~ per run and exhibits a Cz,ge tilt ()f the base J>iliiS with respect to the fldlx am. In CldditJon, the Aform has a central hcle (bottom). fus h€lical form is adopted by RNA- DNA Mel RNA- RNA ~'aces. (c) Z DNA lS a left-h.vlded helix dnd has a zigz.ag (hence 7) ~ (Soutce: Courtesy of C ~and P. B. Dervan.)

a BONA

b A ()IJA

c ZONA

nN" Structur f' a

b

average 10.5 base p<:~irs per tum of the helix. Second. the E form is an average structure whereas real DNA is not perfectly regtJlar. Rather. it exhibits variations in its precise structure from base pair to base pair. This was revealed by comparison of the Q"ystal structures of individual DNAs of different sequences. For example, the two members of each base pair do nor always lie exactly in the same plane. Rather. they can display a ..propeller twist" arrangement in which the two flat Lases counler rotate relative to each other along the long axis of the base pair, giving the base pclir a propeller-like character (Figure 6-12). Moreover, the precise rotation per base pair is not a constant As a result. the width of the major and minor grooves varies locally. Thus. DNA molecules are never pedectly regular double helices. Instead. their exact cunformation depends on which base pair (A:T. T~. G:C, or C:G) is present at each position along the doubl€! helix and on the identity of neighboring base pairs. Still. tlle E fonn is fur many purposes a good first a pproximation of the structure of DNA in cells.

101

f I & U R E ~12 The i)ropeftet l\Wst between the purine and pyrimidine base pairs of a right-handed helix. (a) The slnJCti.Xe shows a seq.Jence of thlee consecutive A:I base pairs Wllh normal \Natsoo-oidc bonOOg. (b) Propeler M'b"t ca..>ses rotation of the bases cbout lheif long axis. (Source: Adopted from A,ggaarwd et aL 1988. Soence 242: 899-907. figt.re Sb. Cq:>yright © 1988 American Assooation fct the Advancet'nerlt of Soeoce. Used by permiSSfOn}

• dec»cyguancsif'le as in B DNA

!

syn position

•

•

orguanrne

DNA Can Sometimes Fonn a Left,Handed Helix DNA containing alternative purine and pyrimidine residues can fold into left-handed as well as right-handed helices. To understand huw DNA can form a left~handed helix, we need to consider the glyoosidic bond that connects the base to the 1' position of 2 ' -deoxyribose. This boncl can be in one of 1:\vo conformations called syn and onti (Figure 6-13). In right-handed DNA. th~ glycosidic bond is always in the anti conformation. In the left-hande d helix. the fundamental repeating unit

fl Ci u R f & 13 Syn and onti positions of guanine in B and l DNA. In rigtrt·harlded E! CNA, the glycosyl bond (colored red) c.onnect.-,g the base to the deoxyt'l:.ose group 1s ~ in the anti posi1011, W"ttle tn left-handed Z ONA

usually is a purine-pyrimidine dinucleotide. wiU, the glycosidic bonrl

Jt rotqtes lf1 the direction d the arrow, fum1!ng

in the anti conformation at pyrimidine residues and in the ·'o/TI c(lnfnrrnatiun at purine residues. It is this syn conformation at the purine nucleotides that is responsible for the left-handedness of the helix. The change to the syn position in the purine residues to alternating unti-S,}'l1 conformations gives the backbone of left-handed DNA a zigzag Jook (hence its designation of Z DNA; see Figure 6-11) , which riistinguislles it &om right-handed forms. The rotation that effects the

1he syn corfumation a 1fle purine (here 81~ nme) residues btJt remafr1s Ill the regtAar anti pOSition (no rotation) Jn the pyrirri
The Structures ofDNA and RNA

108

change from anti to s}'n also t:auses tlle ribose group to undergo a change in its pucker. Note, as sh own in Figure 6-13, that C3' and cz· can switch locations. In solution alternating purine-pyrimid ine residues assume the }eft-handed conformation only in the presence of high concentrations of positi\'ely charged ions (for example, Na 1 J that shield the negatively charged phosphate groups. At lowe r sall concentrations, they form typical l'ight-handed confolJTlations.. Tha physlologjcal significance uf Z DNA is uncertain and left- handed helicfls probably account at most fur only a small pToportiun of a cell's DNA. Further details of the A. B. and Z forms of ONA. are presented in Tab)e 6-2.

DNA Strands Can Separate (Denature) and Reassociate Because the two strands of the double helix are held together by relativ('ly weak (noncovalent) forces, you m ight expect t hat the two strands could come apart easily. lndeed. the original structure for tne double h elix suggested that DNA replication wou ld occur in just thi.s manner. The complementary strands of the double helix can also be made to come a part when a solution of DNA iS heated above phy~io lugkal temperatures (to near 1 00e C) or under conditions of hlgh pH, ~ process known as denaturation. However, this complete separation of DNA strant.ls by denaturation is revers ible. When heated snlutions of denatured DNA are slowly cooled. single strands often meet their complementary strands and reform regular double helices (Figure 6-14). The capacity to renature denatured DNA molecules permits artificial h ybrid DNA mulecnlcs to be formoci by slowly cooling mixtures uf denatured DNA frtJrn two different sources. Likewise, hybrids can Le formed between cmnplemenlary strands of ONA and RNA. As we shall see in Chapter 2 0 , the ability to form hybrtds between 1wo single-stranded nucleic acids, called hybridi7.ation, is tlle basis

TA 8 l E

6-2

A Comparison of the Stnlc.'bal Properties of A. B. itl1d Z DNAs as Derived from Single-crystal X..f(ay Analysis

Helix Type

Overall proportions Rise per base P&f

Hem<-packing dcameter Helix rotation sense Base paJrs per helix repeat Base pc1irs per tum of hetil(

Short a.nd broad 2.3A 25.5A Right-handed 1 - 11

Rotation per base~..Pitctt pet Mn of helix

Hcllx axis location Major-grOO\Ie propclffions

Longf!r and thinner 3.32A

ZHA Right~

33.6° 24 6A + 19°

Ttlt of basfl nOfmals to heb ax•s Base-pair mean pi(Jp€!1er twist

z

B

A

ElongaJ.ed and sim 3.8A 18 .4 A I eft-handed

1 - 10 35.9" 33.2A

- 60' per 2. bp 456A

- 1.2"

- 9"

2 12

Ma;or groove

-f-16" Through baSe paws

- o<' tviro glf.JO\Ie

Extremely na«D\111 but

Wide and of 1ntem1adtate

Flattened out oo heliX

·t- 18"

very deep

dP.ptl1

surtace

Minor-groove Pfoportioos

Vet)! broad but shallow

NarrOIN and of intermediate depth

Extremely flafi'OW bUL

Glycosyl-tJond corJ1ormatiorJ

anti

anti

anti at C syn at G

very deep

DNA Structure

~~ IJ1'U"\

/ DNA moleCIJes denatured t>y heating

I

-

_

DNA molecule

·-·--;---·---·--·-.·----·--·- ·---·--· --·--·-·.-._- _-_-_(--< sI

cool slowly and start to refiature

mssiog region a

I

a

I

COI1tinue to renatl.l'e :

----- - - - -

-

--

FIGURE 6-14 Reanneafing 4lnd hybridlutioo. A mx\tJte of two othe!wtse IdentiCal double-slrande:l DNA rrdec.lles, ooe flOrmal vvdcHype Ctit\ and the other a mAant nissing a short SITetch of nucJeotides (matted as region • WI red), afe denatu-ed by healillg. The denaued DNA molecules are dowed to renature

t7,t ilobation just below the melting ~1.11e- This ueatment rest-Cls in two types of reflat~Xed molet.Ue5. ~Of CCJfll>l€tely ret1a1:1.Jre d molecules lfl W1ICh two ~tary wjd-type strands ftfum a l'e1lx and tvvo axnplementary m.Jl.xll strands reform a llefil< 1he Oil ter type are ~!d m~. compO&ed of .1 w4d-type and a mutn strand, ~a short u~ Coop of DNA (regKJO a)

Ole type 6

tO.. sevet·a l i ndispensab le techniques in molecular biology, such .t1S &ruthem blot hybridization {see Chapter 20) and DNA mic:roarray analysis (see Chapter 18. Bux 18-1}. Important insights into the properties of the double helix were obtained from classic experiments carried out it1 the 1950s in which the denaturation of UNA was studied under a variety of con ditions. In these experiments, DNA denalurntion was monitored by measuring the absorbance of ultraviolet light passed through a solution of DNA. DNA

- ~-

109

110

Th~ Stnmtu~ of nN,A

ami RNA

maximally absorbs ultraviolet light at a wavelength of about 260 run. It is the bases that are principally responsible fur this absorption. When the temperature of a solution of DNA is raised to near the boiling point of water. the optica1 density, called absorbance. at 2fi0 nrn markedly increases, a phenomenon kno·w n as hyperchromicity. The explanation for this increase is that duplex DNA absorbs less u ltraviolet light by about 40% than do individual DNA chains. This hypochromicity is due to base s tacking. which diminishes the c.apacity of the bases in duplex DNA to ~bsorb ultraviolet light. lf we p!ot tl1e uptic·dl de11sity of DNA as a function of temperature. Wfl observe that u.e im.:rease in absurptiuo ou;urs ClbJ'uptly over a relatively narrow temperature ra11ge. The midpoint of this transition is the melting point or Tm(Figure 6-15). Like ice.. DNA melt~; it undergoes a transition from a highly ordered double-helical structure to a much less ordered structure of individual strands. The !'harpness of the increase in absorbance al the m elting temperature tel ls us that the denaturation and renaturation of complementary DNA strands is a highly cooperative. zippering-lU..e process. Renaturation, fur example, probably occurs by means of a slow nucleation process in wJ1ich a relati"ely small s tretch of bases on one strand find and pair with their complement on the co mplementary strand (midrlle panel of Figure 6· 14}. The remainder nf the two strands th en rapidly z jpper-ttp from the nu cleation site to refo,.m an extended double he1ix (lower panel of Figure 6-14). The melting temperature o f ONA is a characteristic of each ONA that is largely d etennined by the G:C content of the ONA and the ionic strength of the solution. The higher the pe~nt of G:C base pairs in the ONA (and hence thP. lower the content of A:T base pairs). the higher the melting point (Figure 6-16). Like\.vise, the higher the saiL ooncentralion of the solution, the greater the temp erature at which the DNA denatures. How do we explain this behavior? G:C base pairs contribute more to the stability of DNA than do A:T b.T!se pairs because of the greater number of hydrogell bonds for tbe fonner (three in a G.:C base pair \1ersus two for A:T) but a lso importantly. because the stCJcking interactions of G:C base pairs with adiacent base pairs are mote favorable than the corresponding interactions of A:T base pairs with their neighboring base pairs. The effect of ion if.:: sfJWgth reflects another fundamental feature of the double helix. The backbones of the two DNA strands contain phosphoryl

l

FIGURE 6-15 DNA~c:urve.

single siTanded

~

<

double stranded

DNA Topology

. .. · ..•

100

•

80

..•·· .•

~

.•

•• . ...•· .· ..··

•

..

Ill

~ 1%1

c:

60

(j) 0

~ +

I»

c

40

c

..

(lJ

::1

C)

20

.. . . ..

. . ..

..

0 +---~~·-------------------,------~ 100 £0 70 00 00 TJC)

grou ps which carry a negative charge. These negative charges are close enough across the two strands that if not shielded. they tend to cause the strands to repel each other, fad litating their separation. At h igh ionic strength. the negative charges are sllieldc>..d by cations, thereby stabilizing the helix. Conversely. at low ionic strength the unshielded negative charges render the helix less stable.

Some DNA Molecules Are Circles It was initially believed that all DNA molecules are linear and have nvo free end s. lndeed, the chromosomes of eulcaryotic cells eacl1 contain o single (extremely long) DNA molecule. But now we know that some DNAs are circles. For example, the chromosome of tl1e small monkey DNA virus SV40 is a circular. double-helical DNA molecule of about 5,000 base pai.J's. Also. most (but not all) bacterial chromosomes are circular; E. coli has a circular chromosome of alxmt 5 million base pairs. Additionally. many bacteria have small autonomously replicating genetic elements known as plasmids. which are generally circular DNA m olecules. lntereslirlgly, some DNA molecules are sometimes linear and sometimes circular. The most well-known example is that o f the bacteriophage .A. a DNA virus of E. coli. The phagp, A genome is a linear double-stranded molecule in the v irion pCU'ticle. However. when the A genome js jnjectoo in to an E. coli cell dm j ng infecliun. the DNA circularizes. This occurs by base--pairi ng between single-stranded regions that protrude from the ends of the DNA and that have complementary seqmmces. also known as ''sticky encls. ••

DNA TOPOLOGY As DNA is a flexible structure. its exact molecular parameters are a function of both the sunuunding ionic environment anfl the nature of the DNA-binding prote.ins w ith which it is complexed. Because their ends are free, linear DNA molecules can freely rotate to acconunodate

tt1

fiGURE 6- 16 DependenceofDNA denabHation on G + C content and on salt

a!Clc:enhation. The greater the G + C content. lhe higher the lerl1Jlefature must be Ia denature the DNA strarld. DNA from differerw SOLXces was dissot.ted 111 solttiOnS of low (red lif)e) and ll1gh (green line) conc€1'\lJ;;ttions of sd1 at pH 7.0. The poots represent the l~ture at v.hdl the DNA denatuted, grct*ted agairt>t the G + c c.oment. (Sourc.e: Data from Marf11Uf J. and Ooty P. 1962. Journal of Molectr lor Brology 5. 120. Copyright ~ l%2, \...nh perm iSSion fTom Elsevier Soence.)

changes in the number of times the two chaiJ1s of the double helix twist ah(mt oach other. But if t'hc two ends are covalently linked to form a circular DNA molecule and if there are no interruptinns in the sugar-phosphate backbones <>f the two strands, t.h en the absolute n umber of times the chains can twist about each other cannot change. Such a covalently closed. cin:u1ar DNA is said to be topologica lly oonstrained. Even the linear DNA molecules of eukaryotic chromosomes are subject to topological constraints d ue to their extreme length, en lrainmonl in c hromatin, and interaction with other cellular components (see Chapter 7). Despite these constraints, DNA particip<stes in n umerous tly namic processes iJl the cell. For exampl11, the two strands of the double helix, which are twisted around each other, must rapidly separnto in order for DNA lo b e duplicated and tu be transcrjbed into RNA. Th us, understanding the topology of DNA and how the cell ll<.•th CJCCOmmndates and exploits lnpntogical constr::\ints during ONA replication, transcription , and utl1e.r chromosomal transactions is of fundamt.tntal importance in molecular biology.

Linking Number Is an lflvariant Topological Property of Covalently Closed, C;rcular DNA Let us considPr the topo logical properties of covalently dosed, circu· lar DNA, which is referred to as cccUNA. BecHuse lllere are nu interruptions in either polynuclflotide chain . the lwo strands of et:e.ONA cannot be separated from ~ach other without the breaking of a cova· lent bond. Jf we wished to separate the two circular strands '"'ithout perma nently breaking any bonds in the sugar-phosphate bnckbooes. we wouJ d have to pass one strand through the o ther strand repeatedly {we will encounter ~n enzyme that can perform just this feat!). The number of times one strnnd would have to be passed through the other strand in orc.Jer for the t wo strands to be entire ly separated from each other is calJed the linking number (Figure 6-17). Th<J linking number. which is always an integer, is an invariant topological prop· erty of cccDNA, no matter how much the shape of the ONA molecule is distorted.

Linking Number Is Composed of Twist and Writhe The Linking n umber is the sum of two goometric components called the twist and lhe writhe. Let us consider twist first. Tw ist is s imply the n umber of helical rums of one strand about the other. that is, the number of times one strand completely wrdps around the other strand. Consider a cccDNA that is lying flill on a plane. In tbis flat confom taJion. the li nking number is fully composP.d of twist Indeed, tbe twist can be eat~~i ly determined by counting the nwnber of times the two strands c ross each other (see Figure 6-17a). The h elical crossovers (twist) in a right-handed helix are df'fiocd as pusitivo such that the linking number of DNA will have a posit ivP value. But cccDNA is generally not lying flat on i t pl:tne. ~~ther, il i ~ u~uruly torsionally stressed such that the lung axis of the double helix crosses over itself. often repeatedly. in three-dimensional space (Figure 6-17b). TI1is is called writhe. To visualize the distortions caused by torsional stress. think of the coiling of a telephone curd that has been overtwisted. Writhe can take two forms. One form is 1he jnterwound or p ledonemic writhe, in which the long axis is twisted around itself, as depicted in Figure 6-1 7u and Figure 6-18a. The other form Clf \"ltithe is

DNA Topology

a

113

c

b

t~se

20

bp: 360

bfJ : 360

bp: 360

36

Uc 32

Lk: 32

Tw: 36 0

Tw: 36 Wr: - 4

Wr:

Lk·

Wr:

Tw: 32

o

fiCURE 6-11 Topo1ogical states of covalently dosed, drallar (ccc) ONA. lhe ftgure stJO.NS

of the retaxed (a) to the negatively super-coiled (b) form of ONA. The strain in the supercoiled form may be taka1 up by s~ (b) cr by local d•sruptioo of base~(<). !Adapted from a d!agrarn pro~ by Dr M Gellert ] (Soorte. MOOfied fmm Kornberg A. and Baker TA l 992. DN/1 n:plicotion, t I - 2 I , p. ~'2 C 1992 by W. H. Fteernan at~d Company. Used v.llh permission)

CQrlVerSKJO

a toroid or spiral in which tlw loQg axis is wound in a cylit1d.rical manner. as often occurs when DNA wraps around protein {Figure 6-18b). The writhing number (Wr) is the total number of interwound and/or spiral writhes in cccDNA. For example, the molecule shown in Figure 6-17b has a writhing number of four. lnterwound writhe and spiral writhe are topologically equivalent to each other and are readily interconvertible geometric properties o f cccDNA Also. twist and writhe me intarconvertible. A molecule of b

FIG U It l 6-18 Two forms of wn.he ot ~oiled DNA. The figue shovvs tnterwound {a) m toroidal (b) writhe of ccd)NA of the~ length. {a) The IC1tBWOUnd a pled.onemic wrtdle IS formed by twis11ng of the double hei•Cdl DNA moleaJe 0\lef ItSelf as depicted in the eJ
114

The Structure5ofDNA ond RNA

occDNA can readily unde~o distortions that convert some of its twist to writhe or some of its writhe to twist without the breakage of any covalent bonds. The only constraint is that the sum of the twist number (7W) and fhP. writhing number (Wr) m ust remain oqual to the linking number {Lk). This constraint is described by the equation: Lk = 711·· + 14r.

l l0 Is the Linking Numbel" of Fully Relaxed cccDNA under Physiological Conditions Consider cccDNA that is free of supercoiling (that is. it is said to be relaxed) and whose twisl corrosponds to that of the B form of DNA in solution un der phys iological conditions (about 1 0 .5 base pairs pE'J' turn of the helix). 'The linking ntJ.mOOr (J,k) of such cccUNA under physiological conditions is assigned the symbol Lk0 • Lk0 for such a molecule is the number of base pairs divided by 10.5. For a cccDNA of 10.500 base pairs. Lk = i 1.000. (The sign 1s positive because the twists of DNA are right-handed.) One way to see this is to inlagine pulling one strand of the 10,500 base pair cccDNA out into a flat circle. If we did this. then the other strand would cross the flat circular strand 1,000 times. How can we remove supercoils from cccDNA if it is 11ot already relaxed? One procedure is to treat the DNA mildly with the enzyme DNasc (, so as to break on aveTagc one phospbodiester bond (or a small number of bonds ) in each DNA molecule. Once the DNA has been "'nicked., in this manner. it is no longer topologically constrained and the strands can rotate freely, allowing writhe to dissipate (Figure 6-19). lf the nick is then repah'oo, the rest~lting cccDNA m olecules will be relaxed and will have on average an Lk that is equal to Lk 0 . (Due lo rotational fluctuation &t the time the nick is repaired. some of the resulting cccDNAs will have an Lk that is somewhat greater than Lk0 and others will have an Lk that is somewhat lower. Thus, the relaxation procedure will generate a nBIToW spectrum of cr.cDNAs whose average Lk is equal to Lk0 ) .

DNA in Cells Is Negatively Supercoiled The extent of supercoiling is measured by the difference between Lk and Lk". which is called the linking difference: AJ...k = Lk - Lk0 fIGURE DNAHL

6-19 Relaxing OWl With

_..,pivot

•

DNA Topolewy

t 15

If the flU of a cccDNA is sjgnificantly different from zero~ then the DNA .is torsionally streinecl and hence it is supercoiled. If Lk < Lk0 and !J.Lk < 0, then the DNA is said to be "11egativcly su~rcoiled." CoJlversely. if Lk > Lk0 and J1Lk > 0, then the DNA is "positively supercoi.led... f or example. the molecule shown jn Figure 6-17b is negatively supercoiled and has a linking dift'trrencc of 4 because its Lk (32) i.q four less than that {36) for the relaxed fonn of the molecule shown in Figure 6-17a. Because tll.k: and Lk 0 ru-e dependent upon the length of the DNA molecule, it is more convenient to re fer to a normalized measure of supercoiling. This is the superhelicaJ density, which is assigned the symbol u and is defined as: a = llLk/Lk0 . Orcular DNA moleculP.s purified both from bacteria and eukaryotes aro usually negatively supercoi:lod. having values of u of aboul - 0.06. The electwn micrograph shown in Figure 6-20 compares the structures ofbacteriophage DNA in its l'elaxed form with its supercolled form. What doos su perl:'teli£'.al density mean biologi£'.ally? Negative superroils can be thought of as a store of free energy that aids in p.rocASS(l..S that .require strand separation, such as DNA replication and t:rnnscription. Because Lk = 7W + Wr. negative supercoils can be converted into untwisting of the rlouble helix (compare Figure 6-17a with 6-17b). Regions of negatively supercoilRd DNA., therefore. have a tendency to partially unwind Thus, strand separation can be accomplished more easily inTiegatively superet1ilcd DNA than in .relaxed DNA. The only organisms that have been found to have posilively supercoiled DNA are certain thennophiles, m icroorganisms that live under eonditions of extreme high tempP.ratures, such as in hot springs. In this case, ths pc.-sitjve supcrcoils can be thougbt of as a store of free energy that helps keep the DNA from denat uring at the elevated temperatures. In so far as positive supcrcoils can be converted into more twist {positivPly supercoiled DNA can be thought of as being overwound), strand separation require.s more energy in thetmophiles than in ocganisms whose DNA is negatively suparcoiled.

Nucleosomes Introduce Negative Supercoiling in Eukaryotes As w e shall see in the next chapter. DNA in the nucleus of eukaryotic is packaged in small partides known as nucloosomes in which the double helix is wrapped almost two tiroP.s around the outsi de circumfurence of a pmtein core. You will he able to recognize this wrapping as the toroid or spiral form of writhe. Importantly. it occurs in a left-handed manner. (Convince yourself of this by applying the handedness rule in your mind's eye to DNA wrapped around the nucleosome in Chapter 7, Figure 7-18). It tw·ns out t:bat writhe in the fonn of leftbanded spirals is equivalent to negathre superr:oils. Thus. the packaging of DNA into nucloosom~ introduces negative supcrhelical density. rell~

Topoisomerases Can Relax Supercoiled DNA As we have soon. the linking number is aa jnvariant propel'ly of DNA that is topologically constrained. [t can only be changed by introducing interruptions into the sugar-phosphate backborm. A remarkable class of enzymes known as topoisomerases are able to do just this by introducing transient s ingle-stranded or double-stranded breaks into the DNA

FIGURE

6-20 Electron microgw-aph of

superroiled DNA. The upper e.lec:trofl microgfapb is a rel.axed (noosupercoited) DNA ~ a1e of baeleriopllage PM2, The rovrc, eectron rnacrog.aph shows the P1age in •ts supertwiSted form. (Source: Electron rnicr~hs coutesy atWang l C. 1982. SoentJic Amertean 247. 97)

116

The S trur.tures of DNA ond RN A

FIGURE 6-21 SchefNtk for c:baflging

the lndng number il'! DNA with lopoisomerase ft. Topmsometase I binds to DNA. aea~es a dot..ble-straodecl br8c'lk, passes t.r!CtA DNA tl-fctJgh ~ gap then reseals tile break.

pass back duplex

reseal

through break

break

Topoisomerases all! of two general types. Type ll topoisommases change tho linkirJg number in steps of two. They makP transient d oublestranded bmaks in the DNA through which they pass a segment of uncut duplex DNA before resealing the break. This ty pe of reaction is shown schematically in Figure 6-21. Type 11 topoisomerases require the energy of ATP hydrolysis ror their action. Type I topoisomerases, in contrast. change the linking number of DNA in steps of one. They make transienl single-stranded breaks in rhe DNA, allowing lhe uncut stmnd to pass thmugh the break before resealing the n•ck (Figure 6-22). [n contrast to the type II topo isomerascs, type [ lopoisorncrases do not require ATP How to poisomerases relax DNA and promote other related reaction-; in a controlfp.(J and conrerted mannt:>l' is explained below.

Prokaryotes Have a Special Topoisornerase that Introduces

Supercoils into DNA Both prokaryotes and eukarytoes have type rand type n topoisomerases 1hat are c-a pable of removing supercoils from DNA. In addition. however, prokaryotes have a special type H topoisomcrase known as DNA gyrase that i.ntroduQJS. rather than removes. negative supercoils. DNA gyrase is responsible for the negative suporcoiling of chromosomes in prd.a.ryotcs. 'fhis n egative su perooiling facilitates the unwinding of thfl DNA duplex, which stimulates many reactions of DNA including initiation of bot h transcription and DNA repJication.

FIGURE 6-22 Schematic mechanism ot action fm topoisomerase f. The enzyme cus a ~gle Slrand ol.re PNA du~ passes lhe u11wt svand th~ the break. then reseals the break. The process 1'K1eases tht> tinklf'lg ntJI'l'ltEr by + 1. ~

e ~

Ll< =n

pass strand

niek

§

thl~break

and ligate

(

•

€ ~

U<=n+1

DNA Topology

117

Topoisornerctses also Unknot and Di~ntangle DNA Molecules ln addition to relaxing supcrooiled DNA, topoisomeraSf>.s promote several otlw.r reactions important to maintaining the proper DNA st:ructul'e within cells. The enzymes use the same transient DNA break and strand pas:;age reaction lhat they use to relax DNA to carry out these reactions. Topoisomerases can both catenate and decatenate circular DNA molecules. CireulaT DNA m oloculf'.s are said to be catenated if they are linked together 1ike two rings of a r;ha.in (Figure 6-23a). Of these two activities. the ability of topoisomcrases to decatenate DNA is of clear biological importance. As we 'INiiJ see in Chapter 6, catenated DNA rnolecules are conunonly produced as a round of DNA replication is finished {see Figure 8-33), Topoisomerases play the essential role of nnlinking these DNA molecules to allow them to s~parate into the two daughter cells for coil division . Decatenation of two covalently closed circular DNA molecules requires passage of the two DNA strands of one nlolecuJe through a double-stranded l:lw.ak in the second DNA molecule. Tltis reaction thorefore depends on a type 11 topoisomerasc. The requirement for decatenation explains why type n topoisomerases are essential ce11ular proteins. However, if at least one of the two catenAtP.d DNA molecules carries a nick or a gap, then a type I enzyme may also unlink the two molecules (Figure 6-23b). Although we often focus on circuJar DNA molecules when oonside~: ing topological issues, the long Jinear chromosomes of cukaryotic organisms also P.xperience topologic.al problems. For exampiP, during a round a type 11 topoisomerase

0

F I GURE

calenalioo ,

"decalenatioo

b type I topoisomernse

catenatioo , 'decatenation

c type It topoisomerase

d t~ nropcisomerase

6-23

T~s

decatenate. disentilngte. and \W\knot OHA.. (a) Type" topO\SOfT1efase ca11 catenate and decalenate covalently dosed, ciro.Jal DNA moleetlt.-5 by ~troduong a dotble-stftmded break "' ooe ONA a11d pass.ng the otha DNA rnolt1:ult ltvoogh the beak (b) Type I tcpcliSOmerases can my cater)ate i'll1d decateoate molecules if one DNA sband ha$ a nd or a saP· 1hts tS because these enzyrres dedve amy 011€0NA stlafld~t a time. (c) &rtangledlong lnear rnA molecules, f,eneraled, !Of exanple. during the replic.Jtioo of etJcaryotic du'OITtOSOfl'll:S. can be disentangled by a topoc SOfr1eii!Se. (d) rnA 1-.ncts can als-o be ur*notted by topOISanerase action,

of DNA replication. the two double-stranded daughter DNA molecules will often become entangled (Figure &23c~ These sites of entanglement, jusl like the links between m tenated DNA molecules. block the ~para lion of the daughlm chromosomes during mitosis. Tbf'.refow., DNA disantanglelllont. generally cataly~P.d by a type n lopoisomcrase, is also required for a successful round of DNA replication and ceU d ivision in

eukaryotE?S. On occasioo, a DNA molecule becomes knotted (Figure &23d). Por example , some s1te-spocific recombination reactions. which we shall discuss in Uf>lail in Chapter 1 t . give rise to knotted DNA products. OncE' again, a type n topoisomerase can "untie" a knot in d uplex DNA. If the DNA m olecule is nicked or gapped, theh a lype J enzyme cun also do this job.

Topoisomerase1i Use a Covalent Protein-DNA Linkage to Cleave and Rejoin DNA Strands To perform thoir fundions, topoisomerases must cleave a DNA slmnd (or two s trands} and then rejoin the cleaved s trend {or strands). 1bpoisomerases aro able to promote both DNA cleavage and rejoining without the assistance of other p roteins or high -energy co-factors (for example. ATP; also see btllow) because thPy use a cnvalent-intermediate mochanism. DNA cleavage oa: w~ whtm a tyrosin~ residue in the active site of the topoisomerase attacks a ph osph.odiester bond in the backbone of the target DNA (Figure 6-24). This attack generates a break in the DNA. wh.(?t'eby the topoisomc~BSE: is covalently joined to one of the broken ends vi a a phospho-tyrosine linkage. The other end of the DNA termiMtes with a free UH group. This e nd is also held tightly by the enzyme, as we will see below. The phospho-tyrosine linkage con serves the en ergy of the pJtosphodiester bond that was cleaved. Therefore, the DNA ca11 be re-sealed simpJy by reversing the Ol'iginal reaction: the OH group from 011e broken DNA end attacks the phospho-tyrosine bond reforming tbe DNA p hosphodiASte r bond. This reaction rejoins the DNA strand and releases th e topni~ somerase. which can th en go on to catalyze another reaction cycle. Although as noted abo' 'e, type 0 topoisomerases requite ATP-hydrolysis for activity. the energy releo.c;cd by this hydrolysis is used to promote conformational changes in the topoisomerase--DNA complex mtber than to deave or rejo in CNA.

Topoisomerases Form an Enzyme Bridge and Pass DNA

Segments through Each Other Between the steps of DNA cleavage and DNA rejoining, tbe topoisomerase promotes passage of a second segment of DNA through the break. Topoisomecase function thus requires that DNA cleavage, strAnd passage. and DNA rejoin ing all occur in a highly coordinated manner. Structures o( Sfllleral different topoisomei'oses have provided .insight into h ow the reaction cyde oreurs. Here w e will explain a rnorle1 for huw a type f topoison'lora~ relaxos DNA. To initiate a relaxation cycle, the topoisomerase binds to a segment o f duplex DNA in which the two strands are melted (Figure 6-25a). Melting o f the DNA sll"and.s is favored in highly negatively supercoiled DNA (soe above}. ma.king this DNA an exaillent substrate for relaxation. One ()t the DNA strands binds in a cleft ln the enzyme that plaCt'!S it lll'.str tJw

DNA Topology

a

b

s------------------------------------~ ~

s·c.._____________,>J'OH

_ ________

s·~·

>:r

d

(?

5"-·_ __

__,)3'C!:!

lA:'--w~-----"---">:r :r--- 0~

FIGURE

6-24

TopoisofMfMes deaw DNA using a

awatent tyrosine-DNA inlermediirte.

(a) SdlematJc of the c1eavage af1d rejOI(llflg react lOr\. For Slmpltc.ty, only a ~ strand of ONA IS sf1wm. See Figtl"e 6 25 lor a IT\OI'l! reaflstlc p tcttxe. The same ~ es used by lype fl topoisomerases, a~ two enz.ymf' Stbu'uts are requre:Q ooe to cleave each of lhe two ONA strand$ Topotson' emses wrnetunes M 10 lhe 5 · Side: and somelmes to the 3 • Side. (b) Close-up view of the phospho-tyroone

ro.ralent irteiTI"leliate.

tyrosine intermediate (Figure 6-25b). The success ofthe reaction requires that the othor end of the newly cleaved DNA is also tightly bound by the enzyme. After cleevaga, the topoisortlf!rase undergoes a larga conlonnational change to open up a gap in the cleaved strand . with the enzyme bridging the gap. The second tuncleaved) DNA strand then pa.ssas though the gap, and binds to a DNA-binding site in an internal ·•donutshaped., hole in the protein (Figure 6-25c}. After strand passage occws, a second oonformationa1 change in the topoisomernse-DNA complex brings the cleavf!d. DNA ends back together (Figure 6-25 d)~ rejoi.ning of the DNA strand occurs by attack of the OH enrl on th e phosophotyrosine bond (see above). After rejoining. the enzyme must open up one final time to release the DNA (Figure 6-25e). This product DNA is identical to the starting ONA m olecule. except that the linking numbec h as

boon increased by one. This general n1eehanism, in which the enzyme providP.s a " protein bridge" during the 6tmnd passage reaction can also be applied to the type J( topoisomerases. The ty pe IJ enzymes, however, are dimeric (or in som e cas~..s tetrarneric). Two topoisomerase subunits, with their active s ite tyrosine residues. are requ ired to cleave the two DNA strands and mal-e the rloubJo-stranded ONA bmnk that is an essential feature of the type lf topoisom£>.rase n 1echanism.

"120

The Structures of DNA nnd RNI\

cleavage and

opening of gate

a

c

b

DNA releaw

e

d

f 1c; u R£ 6-25 Model for the reaction cyde catalyzed by a type I topoisomerase. The f1gtre shows a SEries of proposed steps for the relaxation of one turn of a negativEly ~coded paasm.d ONA. The 1\'110 strands of DNA are shown as datk gray (and not dra\'\.'0 to scale) The four domains of the prulem are labeled in patle1 (a). Domain I1S shown 1r1 red, I is blue. lit is grei!n, and N is orange. (SO\xce: Adapted

from (h.}mpoux J. 200L DNA lclp<JisomE;fases. Annual Review of Btochemstry 70: 369-413. Copyright f) 2001 by ~I RelnEMIS.. '1/WJW.annu.""reviews.org.)

A B C D

--FIG U R l

6-26 Sdlematic of

elecbophoretK separation of DNA topoisornels. l ane A represents relaxed a tlldt.'d orcular DNA; lane B, linea~ ONA; lane C, highly supe«:Qiled ca::DNA; and lane D, a ladder of tClpOISOr11ei'S.

DNA Topoisomers Can Be Separated by Electrophoresis Covalently closed. circular DNA molecules of the same length but of different linking numoors are called DNA topoisomers. Even though topoisoroers have the same molecular weight, they can be separated fron1 each other by electrophoh"$iS through a gel of agarose (see Chapter 20 for an explanation of gel electrophoresis). The basis for this separation is that the gre-rller the writhe, the more compact the shape of a cccDNA Once again. think of how supercoiling a telephone cord causes it to become more compact. The more compact the UNA. the more easily (up to a point} it is able to migrate through the gel matrix (Figure 6-26). Thus. a fully relaxed creDNA migrates more slowly than a highly supercoiled topoisomer of the same circular DNA. Figure &27 shows a ladder of DNA topoisomers resolved by gel electrophoresis. Molecules in acljacent rungs of the ladder differ from each other by a linking number differenre of just one. Obviously. electrophoretic mobility is highly sensitive to the topological state of DNA (see Box &2. Proving that DNA Has a Helical Periodicity of abo ut 1 0.5 Base Pairs pBr Turn from the Topologic-dl Properties of DNA Rings).

Ethidiurn Ions Cause DNA to Unwind Ethidium is a large, flat. multi-ringed cation. Its planar shape enables ethidium to slip. oc intercalate, between the ~1acked base pairs of DNA

DNA Topology

1Z1

Box 6-~ Proving that DNA Hou a Helical Periodicity of about to.s Base Pairs per Tum froln lhe Topological Properties of DNA Rings

The observation that DNA tq:>otsomers can be separated from each Olher etec~bcafty IS the basis for a SlfTlJie e>q:>emtent that ptlM!S lhat DNA has a helical pencdcity of about 10.5 base pairs per tum If"\ so\ADn. Consider three cccONAs of sezec; 3,990, 3,995, and 4,011 base pairs that were relaxed to CDJT1)1ell00 by treatment with type I tqxiscrnerase. ~ subtected to ~ through agarose, the 3,990- and 4,01 1-base-pair DNAs echibit essenbaUy idef"lticat

Due to thermal fluouaOon. tqxllsomerase treatment iKtUally generates a naJTON spec1rum of topotsomers, bu: for ~ let us consider lhe rnd:>iity of onfy ttle most abmdant topoisomer (tmt correspondf"€ to the ce£:00A ., its most re1axed state). The rnobnlties of the rmst abl.l1dartt t~ for the 3,990and 4,01 1-base-pair DNAs are in&stingutShable b&(Jllse lhe 2 1-base-pa.r difference between them tS negfigible ccrnpared to the stzes of the rings. The most abundant topcisomer for lhe 3,995-base-pair ring, hcAA'ever, tS found to mgrat.e slighlty more f"?t)idly than the other tv.to nngs even though it is only 5 base pails larger than the 3,990-base-paer ring. How are~ to explain this anomaly? The 3,990- and 4,0 11 base-par rings i1 their most retaxed states are expected to ha..-e linmg numbers equal to Lk?, that cs, 300 tn the case of the 3.990-base-pair ring (dtviang lhe sll£ by 10.5 base piW>) and 382 in the case of ti-e 4,ot 1-base-pair ring_BecdJse Ll< 15 eers of 380 01 381. However, Uf' for the :3_995-base-pair nng is 380.5. Thus, even 11 tts most relaxed state, a c~tiy dosed circle of 3,995 base pairs would necessarily have alloot hC!lf a Ufllt of wnthe {ds lin~.Klg
{Figure 6-28). Because it fluorP..scvs when exposed to ultraviole t light, and because its fluorescence increases dramatically after intercalation. ethi dium is used as a stain to visuali~ DNA When an eth:idium ion intercalates between two base pairs. it causes th•~ DNJ\ to unwind by 26° , reducing the nonnal rotation ~:~er base pair from - 36° to - 10°_ In other words, ct:hidium decreases the twist of ONA. Imagine the extreme case of a DNA molecule that has an et.hidium ion between every base pair. Instead of 10 base pairs per tum it would have 36! Wbrm cthidium binds to linear DNA or to a nicked circle, it simply causes the helical pitch to increase. But consider what happens when ethidium binds to covalently dosed, circular DNA. The Linking number of the ca:DNA does not change {no covalent bonds arc broken and J"E)sealed), but the twist decrea~ by 26" for each molecuJe of ethidium thAt has bound to the ONA. Eecause Lk = Tw+ Wr, this decrease in Twmust be compensated for by a corresponding increase in Wr. If the circular ONA is initiaJly negativcJy supercoi led (as is normally the case for circular DNAs isolated from cells), then the addition of Plhldium will increase Wr. In other words, the addition of ethidium wiU relax the DNA. H enough ethidium is added, the negative supercoiling will be brought to zero, and if even more ethid ium is added, Wr will increase ahove ~ro and th~ DNA will become positively supercoiled.

--- --- --------

F1c;uu. 6-27 sep.rationotretaxedand supercoiled ~by gel eledrophor~s. Relaxed and ~ed DNA topoiSClmerS m-e resolvt:d by gEl eledrq:hort'SlS. The speed 'Mitl IM-Mdl1he DNA mdeaJes rrigrale increases as the number of~ uns l()(fea5eS. (Sol-ICe. CO\J1esy of J. c wa~)

1Z2

The Strucfttres of DNA and RNA

FIGURE 6-28 Intercalation ofethidUn into DNA. Bhidit..m IOCI'easE!S the spaong of successrue base paars. distorts the regular sugarphosphate backbone. and decreases the twist of the helix.

backbone

Because the binding of ethidium increases Wr, its presence greatly affects the migration of cccONA during gel electrophoresis. In the presence of nonsaturating amounts of ethidium. negatively supercoiled circular UNA-. a re more relaxed and migrate more slowly. whereas relax.e d creDNAs become positively supercoiled and migrate more rapidly.

RNA STRUCTURE RNA Contains Ribose and Uracil and Is Usually Single,Stranded We now tum our attention to RNA, which differs from DNA in three respects (f'igum 6-29). First, the backbone of RNA contains ribose rather than 2' ·deoxyribose. That is. ribose has a h ydroxyl group at the 2' position. Second, RNA contains uracil in place of thymine. Uracil HGURE 6-29 Structural features of

RNA. The figure sh<Jo..vs the struCtUre of the bad
Send

oJ

o=P-O- CH2

I

o0

I

OH

O= P - O- CJ:12

!-

0

Q 0

U OH

J

0

O = P -O- CH 2

~-

Q 0

I

A

OH

o - P - 0 -CH 2

0

C

b-Q OH Ott l"end

RNA Structure

has the same ~ingl e-ringed. !;t ructure as thymine, except that il l.ar.ks the 5 methyl group. Thymine is in effect 5 methyl-uracil. Third, RNA is usually found as a single polynucleotide chai11. Except for the case of certain viruses, RNA is not the genetic material and does not need to be capable of serving as a template for its own replication. Rather. RNA functions as the intermediate. the mRNA, between the gene and the protein-synthesizing machinery. Another function of RNA is as an adaptor, the tRNA. between the codons in the rnRNA and amino adds. RNA can also play a structural cole, as in the case of the RNA components of the ribosome. Yet another role for RNA is as a regulatory molecule. which through sequence complementarity bin ds to, and interferes with the translation of, certain mRNAs. Finally, some RNAs (including one of the structural RNAs of the ribosome} are enzymes that catalyze essential reactions in the ceiL In all of these cases, the RNA is copied as a single strand off onJy one of the two strands of the DNA template. and its complementary strand does not fD'jst. Rs'\IA is (;apable of fonn1ng long double helices, but these are

tZ3

a

b

unusual in n i:!1 Ufi>u

RNA Chains Fold Back on Themselves to Form Local Regions of Double Helix Similar to A-Form DNA Despite being single-stranded, RNA molecules often exhibit a great deal of double-helic-dl charac:ter (Figure 6-30). This is because RNA chains frequently fold back on themselves to fom1 base-paired segments between short stretches of complementary sequences. If the two stretches of complementary sequence are near each other, the RNA may adopt one of various stem-loop struclures in which the intervening RNA is looped out Irom the end of the double-helical segment as in a hairpin~ a bulge, or a simple loop. The stability of sud1 stem-loop structu~ is in some instances enhanced by the special properties of the loop. For- example, a $tern-loop with the "tetraloop" sequence is unexpectedly stable due to special base-stacking interactions in the loop (Figure & 31). Base pairing can also take place between sequences that are not contiguous to form complex structures aptly named pseudoknots (Figure &32). The regions of base pairing in RNA can be a regular double helix or they can contain disconlinuities. such as noncomplementary nucleotides that bulge out from the helix.

f Ui U R E 6-30 Double helical d1araderistics of RNA In on RNA O'lOicaAe !laving regions of ccmpltrnentary sequences, the lfltef\lffia'1g (noncc:xrplementaJy} stretches of RNA may berome "looped our to form one of the stn.!cttJres llustroted an the figure (a) hawpn (b) bulge (c) loop

wee

6-3) Tebaloop. Base stac.klng tnteri1dims promote and stabthe the tetfaloop structure. The &rJY cirdes betvveeCI the riboses shown tn p!Jrple represent the phosphate rror eties ot tre RNA backbone. HO(ll.Orltallines ~ f I (i U R E

resent base stacking tnteraclions.

C(Ul.JCG)G Tetraloop

124

The Slroctures CJf DNA and RNA

6-32 PseudolmoL lhepseudol<not strt.JC:'!Ure IS fooned by base pairing between 1101"1CDntlgUOUS ~wy sequence. FIGURE

, ;'

,I '

J

, + I

..~. H-0 ~N

- H· •0

\

ribose

FICUR£

6-13

C:U base pair.. The

s1rUct\Jre shows~ bonds that aWow base

-~

J,.. ,'' I

0

..

.,..~---

,.i "

. I

I

I

t

A feature of RNA that adds to its propensity to fonn double-helical structures is an additional, non-Watson-Crick base pair. This is the G:U base pair, which has hydrogen bonds between NJ of ura<:il and the carbonyl ou C6 of guanine and between the carbonyl on C2 of uracil and Nt of guanine {f'igure 6-33}. Because G:U base pa1rs can ocr.ur as well as the fuur conventim1al. Watson-Crick base paics. RNA chains have an enhanced capacity for se)f-complementarily. Thus, RNA frequently exhibits local regions of base pairing but not the longftrange, rf'gl.llar h elicity ofDNA The presence of 2'-hydtox)'ls in the RNA backbone prevents RNA from adopting a B-fonn helix. Rather, double-hel ical RNA resembles the A-form structure of UNA. As such. the minor groove is wide and shallow. and hence accessible, but recall rbat the minor groove offers little sequence--specific information. Meanwhile. the major groove is so narrow and deep that it is n ot very accessible to amino acid s ide chains from interacting proteins. Thu.s. the RNA double helix is quite distinct from the DNA double helix in its detailed atomk structure and less well suited for sequence-specific interactions w ith proteins (although some proteins do bind to RNA in a sequeurespecific manner).

paifing to occur between guanine and I.Jf<JCil

RNA Can Fold Up into Complex Tertiary Structures Freed of the cunstraint of forming long-range regular hel ices, RNA can adopt a wealth of tertiary structures. This is bec'duse RNA has enormous rotational freedom in the backbone of it8 non-base-paired regions. Thus, RNA can fold up into complex tertiary structures frequently involving unconventional base pairing, such as the base triples and base-backbone intcractious seerJ in tRNAs (see. fur example, the illustration of the li:A:U base triple in f-igure 6-34). Protetns can assist the formation of tertiary ~1ructures by large RNA molecules, such as those found in the ribosome. Proteins shield the nt!gative charges of backbone phosphates. whose eJectrostatic repulsive fon;cs would otherwise destabilize the structure.

Researchers have takcu advantage of the potential structural comple.xity of ~1\jA to 8enerate novel RNA species (not found in nature) that FICiURE

6-34

~A"iJ ~triple.

fhe

struct1.re shows one example of hydrogEr~ bonding that alovv-s ontJStJal triple base pairing

U:A:Ubase~

RNA Structure

125

have s pecific:. desirable properties. By synthesizing RNA molecules with :randomize{] sequences. it is possible to generate mixtures of oligonucleotides representing enormous sequence diversity. For example, a mixture or oligoribonucleotides of length 20 and having tOur possible nucleotides al each position would have a PQtf'lnl.,.i.al complexity of 4zn seque nces or 1012 sequences! From mixtures of diverse oHgorihonudeotides, RNA molecules can be selected biochemically that have particular properties, sw::b as an ~ffinity for a specifir: small molecule.

Some RNAs Are Enzymes It was widely l.Jelieved for many years that only proteins could be enzymes. An enzyme must be able to bind a substrdte, carry out a chemical reaction. release the pioduct and repeat this sequence of events many times. Proteins are welJ,suited to this task because they are composed of many diffP.rent kinds of amino acids (20} and they can fuld into complex tertiary structures with binding pockets for the substrate and smaU molecule co-factors and an active site foe catalysis. Now we know that RNAs. which as we have seen can similarly adopt complex tertiary ::;tructures. can also be biological catalysts. Such RNA enzymes are known as ribozymes, and 1hey exhibit many of the fea tures of a dnssir..al enzyme, such as an act.ive site, a binding site for a substrate, and a binding site ror a CO'factor. such as a metal ion. One of the first ribozymes to be discovered was RNAsc P. a rlbonu, clease that is involved in generating tRNA molocules from larger, prec~ sor RNAs. RNAse P is cOmposed of both RNA and protein; however. the RNA moiety alone is the catalyst. The protein moiety of RNAse P facilitates the reaction by shielding the negative chazges on the RNA so that it can bind effectively lo its negatively-charged substrate. The RNA moiety is able to catalyze cleavage of the tRNA precurror in the absence of the protein if a small. positively--charged cmmter ion. such as the pt:lptide spetmiclioe, is used to shield the repult";ive, negative charges. Oth£.-r ribozymes carry out trans-esterification readions involved in the removal of intervening sequences known as introns from precW'sors to certain mRNAs, tRNAs, and ribosomal RNAs in a process known as RNA splicing (see Chapter t 31.

The Hammerhead Ribozyme Cleaves RNA by the Formation of a 2 ', 3' Cyclic Phosphate Before concluding our discussion of RNA. let us look in more detail at the structure and function of one particular ribozyme. the hammerhead. The hammerhcarl is a sequence-specific ribonudease that is found in ~ain infectious RNA agents of plants known as viroids. which depend on self-cleavage to propagate. When the viro\d replinates, it produces multiple copies of itseJf in one continuous RNA chain. Single viroids arise by cleavage. and this cleavage reaction is carried out by the RNA sequence armmd the junction. One s uch self-cleaving sequence is called the hammerhead because of the shape of its secondary structure, which consists of three base·paired stems (1, U, and W) sucrounding a core or noncomJllementary nudeotides required for catalysis (Figure 6-35). The tertiary structlll'e of the hammerhead, however, looks more like a w ishbone (Figwe 6-36). To understand how the hammerhead works, let us first look at how RNA undergoes hydiolysis w1der alkaline conditions. At high

NH2

b

I ~.

o

':p··

01

6-35

\

o·

- H. OH

o

0~~I

structure cf ""' ~ riboxyme. 1he molecule is shown with ttle l'M:> halves of ead1 st£m coo~ with a loop, b4Jt none of the three stems need be a IO<:f)· 1n fact. m the \III'Oid. the two halves of stem mare not joined v.cth a loqJ. (a) lhe figure shows tht.'~cted ~ndaty structures of tl<e hammemead ribozyme. Watsoo·Crick ~interactions are shown In red; the SOSSile bond !!> shown by a red F l CURE

Secondary

C!fTOW; approxlfTli')te mllii'Tlal S\Jbstrate strands

are labeled in bk.le; (U) uracil: {A) adert4ne; {C) c.y10Slne; (G) BU8f\lfle (b) 1lle hammerhead ribol.yme deavage reactton 1rwolves an •ntermediary state &.lnng whrll Mg(OH) m complex with lhe ribozyrrE (shown in green) am as a general base catillyst to ren10Vt.' a protor, from tt.e 2•-11<,-droxyt of the acti~ site cytosine (si'10Wtl at pcsltm 17 ICl p.v1 (~)). and kl lri6ale h deawlge reaction at the sossile pho~ster bond at the acbve sete. (Soorce· (a) Redrnwn fmm McKaym 0. B and Wedekind J E l 999 In The RNA IM:Jfkt 2fld edtioo {ed. R. f. Gestdilnd et i:!l.) r· 267, f18ure 1, p.-1rt A Cold 5p~ H.1fbor. NY. (b) R~~
126

fiGURE

The Structure:. vf DNI\ and RNI\

6-36

~~oflbe

hammemead ribo~ Thts \liew of 1t1e

refined hamm.ert1eild 11bozyme structure shows

the c<>I"1SefVe!Scnpt. BobSOl'(. and Raster 30.)

pH. tho 2'-hydroxyl of the ribose in the RNA backbone can become deprotonated. and the resulting negatively-charged oxygen can attack the scjJ;s ile phosphate at the 3 ' positioJl of the SBJTie tibose. This rflar.tion breaks the RNA chain, producing a 2'. 3' cyclic phosphate and a frt>..e 5'-hydroxyl. Each ribose in an RNA ch ain call undergo this reaction. completely cleaving the parent molecule into nucleotides. (Why is DNA not similarly susceptible to alkaline hydrolysis?) Many protein ribonu.deases also cleave their RNA substrates via the fo.,mation of a 2'. 3 1 c ydic phosphate. Working a t normal cellulae pH. these protein enzym~ use a metal ion, bound at their active site, lo activate the 2'-hydroxyl of the RNA. The hammerhead is a sequence-specific ribon uclease, but it too cleaves RNA via the formation of a 2'. 3' cyclic phosphate. Hammerhead-mediated cleavage involves a ribozyme-bound Mg-t+ ion that rleprotonates the 2 ' -hyrirm<J11 at nP.utral pH, resuJting in nucleophilic attack on the scissilo phosphate (Figure 6-35b}. Because the normal reaction of the hammerhead is self-cleavage, it i.s not really a cata lyst; each molecule normally promotes a £eaction one time only. thus having a turnover number of one. But the hammerhead can be engineered to function as a true ribozyme by dividing the molecule into two portjons-one. the riboz}'mc , that contains 1he catalytic core and the other, the substrate. that contains the cleavage site. The substrate hinds to the ribozyme at stems I and m (Figure f3- 35a) . Afte r cleavage, the su bstrate is released and replaced by a fresh uncut substrate, thereby allowing repeated rounds of cleavage.

Did Life Evolve from an RNA \Vorld? The discovery of ribozymes has profoundl_y altered our view of how life might have evolved. We can now imagine that there was a primitive form of life based entirely on RNA. l n this world. RNA would have funct ioned as th~ genetic m aterial anrl as the enzymatic machinery. This RNA world would have proceded life as we know it today, i n which information trans fer is based on DNA, RNA, and protein . A hint that the protein world might have arisen from an RNA world is the discovery that the component in the ribosome that is respon sible for the formation of the peptide bon d . the peptidyl transfora~. is an RNA molecule (see Cbapter 14). Unlike RNAse P. the hamme rhead, and other previous ly known ribozymes which ad on phosphorous centers. the pcptidyl trans~ fcrase acts o n a carbon center to create the peptide bond. II thus links RNA chemistry to the m ost fundamental reaction i n t he prote in wor1d. peptide bond formation. Perh aps then the ribosome r ibozyme as a relic of an earlier form of tHe in which ~n enzymes were RNAs.

SUMMARY ONA is usually in the form of a right-handed d ouble helix. The helix consists of two polvdooxynudeotide chains. Ear:h chain is an a lternating polymer- of deoxyribose sugw-s

ph0$phate backbone is regular. the order ofba...-.es is inegular and this is responsjb te for the in formation oontent of

phosphodieste~~

of the double helix are orlenled i n an antiparelleJ manner- that is, they run in opposite directioJJS.

and phosphates that are joioed together via

linkages. One of four bases protrudes from each sugar: adenine and guanine, which are purines, and thymine and cytosine, w hich are pyrimidines. While thP. .sugar-

DNA Ear:h chain has a 5' to 3' polarity, and the two cllliins

Pairing between the bases holds the chains together. Pairing is media ted by hyd~n bonds ant! is specific::

Bibliography

adfmine on one chain is a lways paired with thymine on the othet chain, whP.reas guanine is always paired ,>\lith cytosine. This strict base pai1ing reflects the hxed locations of hydrogen atoms in the purine and pyrimidine bases in the forms of those bases found in DNA. Adenine and cytosine :almost always exist in the amino as opposed to tht> imino tautomeric: forms, w h ereas guanine and thymine a lmost always exist in the keto as opposed to enol {()rms. The complementarity between the bases on the two s trands gives DNA it:s self-coding character. The two slr.mds of the d ouhle heliK fall apart (denaturel upon exposure to high temperature. extremes of pH. Of' any agent thaf causes the breakag.t of hydrogen bonds. Upon sJow return to normal cellular conditiom. the dena r:ured s ingle strands can s pecificalJy reassociate to bipJ~ · cally active d ouble helices (renature or anneal). ONA in solution has a helical periodicity of about 10.5 base pairs per tl1m of the fu!lix. The stacl-Jng of base pairs upon each ethel' creates a llelix with two grooves. Decause the sugars protrude from the bases at an angJe of about 120<. the grooves are unequal in size. The edges of eacl1 base pair are exposed in the grooves. creating a pattern of hydrogen bond do nors and at:ceptOI'S a nd of wn «..er Wa<1ls surfuces that identifies the ~se pair. The v.,jdt;~r- or mojol'-groove is richer in cbern[ca.J infOrtnation than the nan·o w- or minor-groove and is more important f~ recogoition by nudectide sequellce-spedfic bind ing proteins. Almost all cellular DNAs are extremely long molecules. with 011ly one ONA molecule within a given ohl'omosomt>. Eukaryotic cells accommodalt! this- tHd reme length in part by wrapping the DNA atound protein particles known as nur.leosomes. Most ONA molecules are linear but $Orne DNAB are cirdes, as is often lhe case fOI' the chmmosomes or ptokaryotes a nd ror certain viruses. DJ\o'A is A~ ib lt:!. Unlt:!SS tltti molel:ul1:1 is topologically cnnstrairu:d. i t can l'ret:ly rotate to acC'ommodate changes in the number of times U1<• two stt-ands twist about each other. DNA Is topologically ooo.straiDBd wh~n il is in the form of a covalently closed circle, or whtm it i~ entrained in chromatin. The linking numbt::r is an invariant topofogkal prOperty ut covJ~lently clost:d. circular DNA . It is tl1E> number of times one strand would have to be passed lhro·ugh the Olh er strand in order to separate the two circular strands. The linking number is the sum of two interconvertibJ~ geometric properties: twist. which is the number of times the two strands are wn~ppet.t around each othm; and 1h~ writhing number, whkh is the number of times the long axis of the DNA crosses over i l.sr.lt' in spat:e. DNA is relaxed under physiological conditions when it has about

t27

1<J.5 base pairs pet turn and is free of writhe. If t11e linking number is docreased, then the DNA becomes to.-siooally stressed, and it ~s said to be negatively supercoilect DNA in cells is usually negatively supercoiled by about 6% . Th.e }eft-handed wrapping of DNA around nudeosomes introduces neg-otive supen:oiting in cul..a.ryotes. ln prokaryoles, w hich tack histooos. the enzyme nNA gyrase is responsible for generating negative super-coils. DNA gyrase i s a member of the type IT family of t()poisomerases. These en.Z)•m es r:hange the linking number of DNA in s teps of two by making a transient break in the double helix and passing a reg ion of duplex DNA through thtl break. Some lype rr topoisomerns~ relax sup~rr.oiled DNA. whereas DNA gyrase genl?.('ates negative supercoils. 'i)rpe I topoisomerases also relax supetcoiJed DNAs. but d o so in sreps of one in which one DNA strand is passed through a tra)lsient nick in the other st.rarul RNA diffe1s from DNA in the foll ow ing way&; its bad.lxme contains ribose rather than 2' ·deoxyri~: it oontains the pytimidine \lra(:il in place of thyntine: and i l usually exists as a singll:l p olynudeoticle chain, without a ('omplementary chain. As a consequence of being a single strantl, RNA can fold bad;, on itself to f()rm shorl stretches of douLie hefix between regions that are oompl~ montary to Pach othE:!t. RNA allows a greater range of base pairing than doos DNA. Thus. as we ll as A:U and C:G pairing. \J c~an also pait Wilh C. This <:a pacity to form a non-Wutson·Crick base prut adds to the pt'opcnsity of RNA to form doubl~ helicaJ segments. Freed o f the constraint of foi1Tli.ng tong-range .regular h~tices. RNA ( :at! fonn complex tertiary structures, which ar~ oftf:n MsP.tl on unconventional interactions bclween bases and the su~ phosphate backbone. Some RNAs act as cw.ymes- they catalyze themical reactions in the ce ll aud in vitro. These RNA enzymes are known as ribozymes, Most ribozymes ~t el on phosphol'Ous centers, as in ttle case of lhc ribonuclease RNAse P. RNAse P is com posed of protein and RNA, but it is the RNA moiety that is the catalyst. The hammerhead is a svlf-cfc.aving RNA , which cuts tha RNA bar.kbone via the fOt-mation of a 2 ' . 3' cyclic phosphate in a reac&ioo thai involves a n HNA-bound MgH ion. Pe ptidyt trat:tsft}rase is an e>
BIBLIOGRAPHY Books OJJd Spnng Harbor Symposwm on Quontilal.ive Biology. 1982. VoJume 47: Structures of DNA. Cold Spring

Harbor Laboratory Press. Cold Sptiog Harbor. N. Y. Gesleland. R.F., cech. T.R., and A1kios. J.P.. 005. 1999. The RNA WoJ"lcl. 2ntl edition. Cold Sp1ing Harbor Lai>Oratory Press, C.old Sp.ring Harbor. N~ Y.

Kombel'g. A. an~ Baker·, T.A. 1992. DNA Replirolion. W. H. Freeman, N.Y. Sa.~nge.r,

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Springer-Verl&g, N.Y.

Sarma, R.H ., ed. 19t!1 . Bimolec:ulor StP.reodynomiCs, Vols. 1 and 2. Adenloe Press, Guilderland. N. Y

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T M Structut"f',; of DNA and RNA

DNA Structure Dickerson, RF.. )~.The DNA hcbx and how it is read. Sci. Amer. 249: 94- 11 1.. Franldin. R.E. and Gosling. R.G. 1953. Molecular conJiguratiun jn sodium thymonudt=ale. Nature 171: 740- NJ

Rich, A., Nordheim, A.• a nd Wang , A1tf. 1984, Till! r:hemish)' and biology of left-handed Z DNA. Annu. Rev. llioch em. 53: 791-846. Roberts, R.J. 1995 On base flipping. CeJ/ 82(1 ): 9-lZ.

Wang. A H. , Fujii, S., van Boom. J.H .. and Rich, A . 1983. Right-handed and left-hant.letl double-helical DNA: Struc tural studies. Cold Spn'ng Hori>. Symp. Quant Biol. 47 Pt 1 : 33-44. Watson, J.O and Crick. F.H.C. 1 953. Molooular structure of nucl~ic acids: A stmct\lre for deoxyribonucleic adds. Nature 171: 737-738. -

- . 1953 . Geoeti,:
Wilkins, M.H.F., Stokes, A.R., and Wilson. HR. Hl53. Molecular structure of d~oxypentose nucleic acids.. Nolure 171: 738-740 .

DNA Topology Bauer, W.R.. Crick. F.H.C.• and White, J.H. 1900. Supercoiled DNA. Sci. Amcr. 243: 118- 133. Bolt:s, T.C.., While, f.H .. and Cozzart::Ui, N.R. 1900. Structure of p leclonemically supercoiled DNA /. Mol. Bio l. 213:931-951.

Cha mpoux. }.J. 2001 . ONA Topotsomerases: Stru<;h..u·e. Function, and Mechanism. An nu. Rev. H;oehem. 70: 369- 413.

Cric k. F..H.C. 1976. LinUng numbet-s a nd nudeosomes. Proc:. Nat/. Acad Sci. 73: 2639- 2643. D~.

Rand Cozzan~lli. N.R. 1992. 1bpologicaf structure of' DNA k nots and catenanes. l'v1ethods Enzymol 212: 120 - 130.

Gelle.rt, G.H. 19tn . DNA Biochcm. 50: H79-910 .

topoisometas~. Anm~.

R011.

Wang. ].C. 2002. Cellu lar rol e~; of DNA topoisornerases: A mnlt:r.ufat perspt~Ctiv e. Not. Rev. Mol . Cell Bioi. 3: 4 30 - 440. Wasserman. S.A. and CozzaroUi, N.R. 1986. Biochemical topology: Applications to ONl\ NlCOmbinatton and replicalioTL Science 232: 951 - 960~

RNA Structure Doherty, E.A. and Doudna. j.A. 2001 . Rihoeymtt structllres and mechanisms.. Ann. Rw. Biophys. Biomol. Struct. 30: 4 57 - 475 .

McKay, U.B. aJld Wetlt1khul, J.E. 1999. Small rihozymes. In ·1ne RNA World, 2nd edition (ed. Gastoland. R.R et comes of agt~. Cel/90: 833- 840 .

CHAPTER

Chromosomes, Chromatin, and the Nucleosome n Chapter fl, we considered the structure of DNA in isolation. Within lhe ceU, however, DNA is associated with proteins and each DNA and its associated protein is called a chromosome. This organization holds true for prokaryotic and eukaryotic cells anrl even for viruses. Packaging of the DNA into chromosomes serves several impmtant functions. First, 1hc chromosome is a compact form af the DNA that readily nts inside the ceU, Second, packaging the DNA into chromosomes serves to protect the DNA from damage. Completely naS...ed DNA mQ}ecules are relatively unstabla in cells. 1n contrast, chromosomal DNA is ext remely stable. allowing ihe in[ommtiQn encoded by the DNA to be .reliably passed on. Third. onJy DNA packaged into a clrromosome can be transmitted efficiently to bclth daughter ceJls each time a cell divides. Finally. the chromoS()rne confers an overall organization to each molecule of DNA. This organization facilitates gene expression as weU as the recombination between parental chromosomes that generates the diversity observed among different indlvirluals of any organism. Half of the molecular mass of a eukaryotic chromosome is protein. ln eukaryQtic cells, a given region of DNA with its associated proteins is oiHed chromatin and the maiority of the associated proteins are small, basic proteins callad bic;tones. Although not nearly as abwulant. other proteins, frequently referred to as the non-histone proteins, are assQci· ated witb the chromosome. These proteins indude the numerous DNAhinding proteins that regulnte the transcription, replication. repair, and recombination of cellular DNA. Each of these topir:s will be discussed in more detail in the next five c.hapters. The proteins in chromatin perform another essential function: they compact the DNA. The following calculation makes 1he importance of this function clear. A human cell c ontains 3 X 109 bp per haploid set of chromosomes. The thickness of ea.c h base pair (the .. rise") is 3.4 A. Therefore, i( the DNA moJ(.-cules in a haploid set Qf chromosomes were laid out end-to-end, the total length of DNA would be apptoximately 1010 A, or 1 meter! For a d ipl oid cell (as human cell5 typic,;aJiy are}, this length is doubled to 2 meters. Since the diameter of a typi!=al human cell nucleus is only 10-15 J.I.ITleters, it is obvious that the DNA must be compacted by several orders of magnitude to fit in such a small space. Aow is tbis achieved? Most UJmpaction in human cells (and all other eukaryotic cells) is the result of the regular association of DNA with histones to fonn structures called nucleosomes. The formation of nucleosomes is the first step in a process that allows the DNA to be folded into much more compac:t strur:tures that .reduc::e the linear length by as much as 10,000-fold. Compacting the DNA d oes not come without a cost. Asso-

I

O UTL.IN E

• Ctlr()fi)()SOOle Sequence and Diversfty

• The Nudeosorne (p. 151)

• Higher-Oc:der G-romatin Structure (p. 160)

• Regutatron of Chromatin StructtJre (p_165)

• Nudeosane Assembly (p. 175)

ciation of the DNA with bistonfls and other packaging proteins reduces the accessibility of the DNA. This reduced accessibility can interfere with the proteins that mediate wplication. repair, recombination, and - perhaps most significantly-transcription of the DNA. Indeed, parlrenl regions of the chromosom~ at different times. Understanding the structure of nudf!QSOmes anrl tbe regulation of their association with DNA is therefore critical to understanding the .regulation uf most events involving DNA in eukaryotic cells. Although prokaryotic cells typically have smaller genomes. the need to compact their DNA is still substantial. E. coli must pad its approximately I mm chr omosome into a cell that is only 1 p.m in length. It is les.~ clear how prokaryotic DNA is compacted. Bacteria ha\-e no histones or nucleosumes, for example, but they do have other small basic proteins that may serve similar functions. In thls chapter we will focus on the better understood chromosomes and chromatin of eukaryotic cells. We will first consider the underlying DNA sequences of chromosomes from different organisms~ focusing in particular on the change in proLein coding content. We will then discuss the overall mechanisms that ensure that chromosomes are accuratel.Y transmitted a~ cells divide. The remainder of the chapter "\'Vill locus on the struclwe and regulation of eukaryolit chromatin and its fundamental building block. the nudeosome.

CHROMOSOME SEQUENCE AND DIVERSITY Before we discuss the structure of chromosomes in detail, it is important to understand the features of the DNA molecules that fonn their foundation. The recent sequencing of the genomes of numerous organisms has provided a wealth of information concerning the makeup of chromosomal ONAs and how their characteristics have changed as organisms have increased in complexity.

Chromosomes Can Be Circular or Linear The traditional view is that prokaryotic cells have a single. circular chromosome and eukaryotic ce11s have multiple, linear c-hromosomes ffa.blt? 7-1 }. As t.no re prokaryotic: O"rganisrns have bootJ s tudied, tbi~ view has been challenged. Although the most studied prokaryotcs (such as E. coli and B. subtilis) do indeed have single circular chromosomes. there are now numerous examples of prokaryotic cells that have multiple chromosomes) linear chromosomes. or even both. 1n oontr.alit. aU eukaryotk ce.Us have multiple linear chromosomes. Depending o n lbe eukaryotir. organism. the number of chromosomes typically varies from 2 to less than 50, but in rare instances can rea,:h

Chmmv:-;vm~ SeqtJP.ttCt' unrl

TAB LE 7- 1

Oiversily

131

variation ih Owomosome Makeup in Different Orgar!isms

SpeCies

Nurnbacl

Clwomosome

ctwomosomes

CGP)'I"'Umber

Form of chromosome(s)

Genome stze (Mb)

PROKAAYOTES

M)'coplasma genltalium Escherichia cot; K~f2 Agroi::J8c.1ettum turne.faciens Sinomhizroium mBiilob

0.58

1

CirClllar

t

Circular

46

4

3 Circl.llar 1 Linear

5.67

3

Circular

6.7

1

EUKARYOTE$ ~romycet; cerevlslae

16

l or2

lil"lf>.at

'12.1

3

la- 2

L~near

12.5

6 5

2 2

4

2

linear linear Linear

Micl'of)udet.R5 5

Micronucleus 2

Linear

Macronucleus 225 22 19 +X andY 22+XafldY

Macronucleus 10- 10.000

(budding yeast) Schi.tosaccharomyces pombB (f'8Sion yeast)

C. elegans (rOIJldwOITT)) ArabtdopsiS ttnliana (weed) Drosophila me1anogaster (frllit fty} Tetiahymena thermophtlus (protozoa) Fugv rvbripes (fish) Mus musculus (l'l"'IU$e) Homo sspqlefJS

linear tu•ear

2

Ltnear

thousands {for example, in the mac-.ronucleus of the protozoa Tetrahymena. Table 7-1). Circular and linear chromosomes ead1 pose specific challenges that must be overcome for maintenance and repJication of the genome. Circular d rromosomes require topoisomerases to sepa.rate the daughter molecules after they are replicated. Without these enzymes, the two daughter molecules would remain interlocked, or catenated. with one another after replicaHon. In oontrast, the DNA ends of the linear eukaryotic chromosomes have to be protected from enzymes that normally degrade DNA end s and present a different set of difficulties during DNA replication. as we shall see in Chapter 8.

Every Cell Maintains a Characteristic Number of Chromosomes Prokaryo1ic cells typically have only one complete copy of their chromosome(s) that is packaged into a structurn called the nucleoid (Figure 7-lb). When prokaryotic c:::eUs are dividing rapidly. however. portions of the chromosome in the process of replicating are present in two and sometimes even four copies. Prokaryotes also frequently carry one or more smaller independent circular DNAs. called plasmids. Unlike the la.-ger chromosomal D NA. plasmids typically are n ot ~n tial fur bacterial growth. instead, they cnrry genes that confer desirable

traits to the bacteria, such as antibiotic resistance. Also distinct from chromosomal DNA. plasmids can be present in many complete copies per cell. The majority of cukaryotic cells are diploid; thnt is. they contain two copies of ear.h r.hrornosome {see F•gure 7·1c)~ The two copies of a given chromosome are called homologs; one is derived from each

180 220 (MicrootlCieus)

2

2

97 125

365 2.500 2.900

ta:t

ChromDSCJmes. Cl!romotin, and tht! Nuc:lP.OStJme

FIGURE 7-1 Comparison of typical prokaryotic and ~cceU. (a) lhe &meter of a typiCal eukaryotJc reH ts - 10 JJ.ffi. The typjc:al ~tic C£1 is - 1 JI.ITI tong. (b) Prol<aryotic chwmosomal ONA is

b haplOid bacteria

lo<:ated m the l'll.ldecld and otet.Jpies a substCiflti.11pation of the internal fe8ic.n of the eel. Unlike the aiaryotic nucleus, the ntJdeold is not sepemted fmm the rE.'milinder of the cell by a membrane. Plasmid D.NA is .sho.rm ~n red. (c) E.t.Jkat}'Qtic chromosomes are locate
haploid C@fl

parent. But. not all cells in a eukacyotic organism are diploid: a subset of eukaryotic cells are either haploid or polyploid. Haploid ceJis conlain a single copy of each chromosome and are involved in sexual reproduction (for example. 1!;perm and eggs are haploid cells}. Polyploid cells have more than two copies of each chromosome. Indeed. some organisms maintain the majority of their adult cells in a polyploid state. In extreme cases there can be hundreds or even thousands of copies of each chromosome. This type of global genome ampliflcatiol) allows a cell to generate larger amounts of RNA and, in turn. protein. For example, megakaryocytes are specialized polyploid cells (-12.8 copies of each chromosome) that produce thousands uf platelets which lack chromosomes but are i;ln essential component of human blood {there are - 200,000 platelets per milliliter of blood). By becoming polyploid. megaka.ryocytes can maintain the very high level-; of metabolism necessary to produce la..ge nwnbers of platelets. The segregation of such a large number of chromosomes is difficult, therefore polyploid cells have almost always stopped dividing. No matter the number, eukaryotic chromosomes are always contained within a membrane-bound organelle called the nucleus (see Figure 7-lc).

Genome Size Is Related to the Complexity of the Organism Genome size (the length of DNA associated with one haploid complemen t of chromosom(>..s) varies substantially between d ifferent organisms (Table 7-2}. Because more genes arc required to direct the fonnatjon of more compl~x organjsms lat JP.aSt wben comparing

TAB Lf.

7-l

~ of the Gene Oetlsity in Different Orgat~iSIIJS' Getlomes

Genome size (Mb)

Species

Approximate nt.ltlber genes•

Gene density

500

860

or

(~b).

PROK.ARYO'Il:S (bacteria) Mycoplasma

0.58

genlt~JitUm

Streptococcvs

22

2,300

1,060

pneumoniiJ Escherichia c oli

46

4 ,400

950

K- 12 Agrobactenum

5.7

5.400

960

67

6 ,200

930

12

5,800

1100

12

4,900

4\0

220

> 20,000

> 90

97

19.000

200

180

13 .700

00

845

-22.000

- 26

5.000

nd

nd

Fugu rubtJpes

365

2 .900 2.500

> 31.000 27.000

> 85

Homo saprens

29.000

12

25.500 > 45.000 > 45,000

200 > 100 > 20

nd

nd

rumefacierts &nom1Zoblum

melilotl EUKARVOTES (anfmals) Fungi SaccfJ~s

cereviSlae Schizosaceharomyces pombe

Pmrozoa Tetrahymena

thermophila /I'Nertebrares

Caenorhabdit•s

eJegerrs Drosophila melanogaSter StrongyfOC6!1trOfUS

purpUrstus Locust8 rrngrstona Vertebrates

Mus rrnscultJs

93

Plants

A.rabKJopsrS thaliana Oryza sat1V8 (riCe)

Zeamays Fritillafla assynaca l(ll~ip)

•ncJ

~

not deteml!fl()d

125 430 2,200 120000

134

Chmn10t>omes. Chmmabn. end the NuckN>I'OirlB

bacteria, single-cell eukaryotes, and multicellular cukaryotes-sec Chapter 19). it is not surprising that genome size is roughly correlated with m1 organism's Bpparcnt complnxity. Thus. prokaryotic cells typicaUy havo gcnomes smaller than 10 megabases (Mb). The genomcs of singJ&ccH cukaryolcs are typically less than 50Mb, although the m()f() L-omplex protozoans c-.m have genomes greater than 200 Mb. Mu]ticcJluJar organisms have even larger genorncs that can reach sizes grouter rhan 100,000 Mb. Although thRI'P- is a Coh'tl)at ion between genome size and organism complexity, it is far from perfect Many organisms of apparently similar complexitie s have very different genome sizes: a fruit fly has a genome approximately 25 times smaJler than a locust and the rice genome ~about 40 times smaJJcr than wheat (S£le Table 7-2}. ln these ('.Xamples, the munber of gnnes rather than the expansion in genome size appears to be more closely related to organism complexity. This becomes df'.ar when we examine the rf'Jativc gene densities of dilfcrent genomes.

The E. coli Genome Is Composed almost Entirely of Genes The great majority of the single chromosome of the bacteria E. coli encodes proteins o:r structural RNAs (Figure 7-2). The majority of the nona...-oding sequences are d{ldicate
More Comp£ex Organisms Have Decreased Gene Density What explains the dramatically different genome sizes of organisms o( apparentJy simi1Ar complexity (such as the fruit fly and Jocust}'? The

I ...&;;ii;lm•••--•-------i•••ll••• •~ ••n••••l•••m•-•·--• ·~.,. genes

•

inlrons

repeated sequences

RNA polymerase gene

•

U inte.-genetic sequences

Escherichia coli (57 genes)

saccharomyces cere\IISJ8e (31 genes)

••

•t l 8 •

I

DrOSOphila melanogaster (9 genes)

HUman (2 genes) _

0 f I (i u R E

10000

• . _ . . . •• •

30000

. ... .

•

•

•

. .. _ . t

40000

7- l Cotnparison of the dwomosomaJ gene density b different organisms.

A representative 65 kb reg10n of DNA IS iltlJSIJatecl for each organism. lhe region thot encodes the li:lfg~

sol:mtl of RNA pdymefase (RNA Pclll for lhe ~die c.els) es indtc..ated in rf'd. Note ha..v the oomber of genes encoded w thlll the samt' leng\tl of ONA dE:aeases as orgar.ism complexity inae.ases.

~·

I

......__ • • •

50000

•

60000

1

Jl

Chromosome Sequrtnt."f' 11111:1 Diver$i1J.'

135

differences are largely related to gene density. One simple measure of gene densHy is the average number of genes per Mb of genomic DNA. Thus, if an organism has 5,000 genes and a genome size of 50 Mb, then the gene dP.nsity fur that organism is tOO genes/Mh. When the gene densities of different organisms are comparP.dr it becomes clear that different org<misrns use the Jjene-cncoding pc:1tentia.l of DNA with varying efficiencies. There is a rough inverse correlation between organism complexity and gene density; the less complex the organism, the higher the gene density. For example, the highest gene densities are found for virusf!S that in some instances usc both strands of the DNA to encode overlapping genes. Although overlapping genes are r-.ue. bacterial gene density is coru:;istcntly near 1,000 genes/Mb. Gene density in eularyotic o~:ganjstn~ is consistently tower and more variable than in their prol<;aryotic counterparts (see Table 7-2). Among eukaryoles, there is still a genera) trend Jor gene density to decrE,ase with jnr.r~sing nrganism complexity. The simplP. m liccllnlar eukarvot~ S . cerevisioe has a gene density very dose lo prokaryotcs (- 500 gcncs/ML}. In c.-ontrast, the human scnome iis estimated to

have a 50-fold lower gene density. In Figure 7-2 the amount of DNA sequence devoted to the cxpt-ession of a related gcnP. l.onscrved across nll organisms (the large su.b\1nit of RNA polymerase) is compared. iJJustnning the vast differences in gene densit)T. Organisms with much larger gf'.nomes than humans are likely to have rn.uch lower gene densities. What is responsible for this reduction in gene density?

Genes Make Up Only a SmaU Proportion of the Eukaryotic

Chromosomal DNA Two fuctoTS contributfl to the decreased gene density obsP.rved in eukaryotic cells~ inLTeases in gen(l size and increases in the ONA

betwoon genes, callfld inlergenic sequences. lncli vidual genes me longer for two re.asons. First. ns organisms become increasingly COh1p1ex. there is a signifirnnt increase in regions of DNA required to direct and regulate lranscription. called regulatory sequences. Second, proteinencoding gcnrs in eukaryotcs frc.d introns, rue removed from the RN/\ aftCT transcription in a process called R..l\IA splicing (Figure 7-3); we shall consider RNA spJiclnf, in detail in Chapter 13. The presence of intruns can il)crcase url:mlaticalJy

2

f l (iURl 7·3 SmematicofRNAsplicing.

ONA exon 1

primary

RNA transcript

sriced rnRNA

1

2

~

2

3

Trensmp1100 of pre-mRNA t> ""'liated Clt the arrow shown above eJ
3

scnpt IS then processed (by spliong) to remove n
s .-.c========~~-.c===========~--- 3

5'---·

ChmmosnmL>s- Chromatin, and the Nuclcosonw

TA 11 L £

7- 3 ContribvtiOn cf lnttcns and Repeated sequences to Different Gencmes Oenedenaty

Species

(genesJMb)

Avarage nUillbet ol lntrons per gene•

Percentage of DNA thai is repelit~

PROKARVOTES (bacteti&)

ESCf7enchJB coti K--12

<1

950

0

480

004

200 fl()

3

12

5 6

46

EUKARYOTES (~s) Fungi

S8cct'.a.romyces cerevtSr cse

3.4

lnvertebr:Jtes

Caenorhabditis B/egans Drosophila metanogaslBr

~

6.3

Vertebrates

Fugu rutlftfJBS Homo sap~ens

lS 8 .5

2.7

Plants

Arclt:JICJopsis tflaiiMEl Oryza sativa (rice)

t25

470

3 nd

nd 42

the length of DNA rcqu.ired to encode a gene (Table 7-3). For example, the average t.ranst:ribEJd regions of a hwmm gene is about 2 7 kb (this should not be confused with the gene density). whereas the aver-dge protein-coding region of a human gene is 1 .3 kb. A simple calcu lation reveals rhaL only 5% of the average human pl'o tein-cncoding gene diroctJy encodes lhe desin:>.d protejn. The remaining '95% js made u p of inlrons. Consistent ·w ith their higher gene density, simpler eukaryotes h ave far fewer introns. For exHmple, in the yeast S. cerevisiae, only 3 .5% of genes have introns. none of which is greater than 1 kb (see Table 7-3). An exp losion in the amount of intergcnic sequences in more complex organisms is responsib Je for the remain ing decreases in gene density. lntorgcnic DNA is the portion of n genome thnt is not associated wi th the exprf'SSion of proteins or structuTa l RNAs. More than 60% of the human genome is composed oi intergenic.: sequences and most of this DNA has no known function (Figure 7-4]. 'fhere are two kinds of in tergcnic DNA ~ unique and repeated. About a quarter of the intcrg£Jnic IJNA is unique. These regions comprise many apparently nonfunc:Honal rclic..-s. lnduding nonfunctional mutant genes. w-nc fragments, ~ nd pscudogenes. The mutant ~ncs and gEJne fragm~nts arise from simple random mutagem1Sis or mistakes in DNA recombination . Pseudogenes a rise from the ac:tion of an cn?.ymP. caiJrd reverse transcriptase (Figure 7-5 and ChnptEJr 1 t }. This enzyme copies RNA into double-stranded DNA (referred to as copy DNA or c l>NA) uut is only expressed by certain types of viruses that require this en zyme to reproduce. But, ns a side effect of infe<;tion by s uch a virus, the <..'CII ular mRNAs can be copied into DNA, and the resulting DNA fragments reintegrated into tbe genome at a low ratf!. The$ copies nrc not expressed. however. because they lack the correct sequences to di~ rect theil' expression (such sequences are generally not pnrt of n gene's RNA product, see Chapter 12).

CIJromosvme Sequence CJnd lJiversity

I other jnlergenec regions 000 M> ~~Jilo: ~~

1-

1n1crosatelltes

.""}

fii~

"J

~

;:

90Mb

r genome-wide -

l

repeals 1,400Mb

137

FIGURE 7-4 The otganization at1d content of the human genome. The hum
of each of the vanous types or sequences. (SoUice: Adapted from &own TA 2002. Genomes, 2nd edition. p. 23. box 1.4. ~ 2002 BIOS Scientific Ptt>ishers. Used by pembssion.

WV\1\N.t:.andf.com.)

...,

inlrons.

UTRs

gene fragments

c eMs 48Mb

The Majority of Human lntergenic Sequences Are Composed of Repetitive DNA Almost half of the human genome is composed of DNA sequences that are repeated many times in the genome. There are two general classes of repeated ONA: microsatcJlitc DNA and genome-wide repoots. Microsatellite DNA is composed of very short (less than 13 bp), tandemJy-repE>.ated sequences. The most common microsatcUite sequences are dinucleotide repeats (for exampJa. CACACACACACACACA). These repe-~ts a.ri~ from difficulties in accurotcly duplicating the DNA and represent nearly 3% of 1he human genome.

fl..nctional gene

DNA

~~~ /t,_'- - - -----=---_...·J

\

tnwlsuiptkwl/splictng

'---....RNA ~~ DNA

F IG1J R £ 7-5 Processed pseildogenes arise from integration of reverse transcnbed mRNAs. W1ffl reversetra~e IS Jlfesenl 111 a eel. mRNA moleoJes ca11 be Cllpied Into dsONA In rare .nstalceS, these DNA mderues can inlegrate ltlto the genome creatiqg pset.dogenes.. Because rrtrons are rapdy removed fran ~ crao-scribed RNA!;, these pseudogenes have tl'Ecommon charactenstic: d lacktrg IO'trons. This distil· guisheslhe~from the cqJy of lhe gene tram v.t-.ich it was derived In a&iticn ~lack the app(Qpllate J>f~

·---functional gene

sequences to drea theirlm1saiplioo as these ate not part cl the rrRNA fran v..hK:h they are pseudogene

derived.

Genome-wide repeats are much la.~Ecr than their rnicrosatcl.Lito t.:ountmpBl'ts. Eoc.h genome-wide repeat unit is greater than 100 bp i11 lflng1h and many are grealer then 1 kb. These sequences can be found either as single copies dispersed throughout the grnome. or as closely-spuccd dusters. Although there ore n~rous classes of such repeats, their oommon feature is that aU arc- fonns of transposable elements. Transposable clemf'nts arc sequences that can .. movo" from one place in tho genome to another. In 1.-ansposition, as this movement is caHed. the e1cm(11l.l moves toe new position in the genome. often leaving tho original copy bohind. Thus, these Se<Jllencr:s multiply and accumulate throughout the genome. Movement of transposable elP..mcnts is a rdativeJy rare event in human cells. Nevertheless, over long periods of time, these clements have boon so successful at propagating t;opies of themselves that they now c.:omprise approximately 45'% of the human gcnoma ln Chapter 11 we wiJl consider the mechanism by which transposable elemtmts move a.rom1d the genome and how their movement is controllfld to prevent chromosome damage. Although we ha:we discussed tlw nature of intergr-nic St;quence iu lhc oontexl of thP human wmorne, many of t}}{l same features am found in other organisms. For exe~mplc, comparison of the known sequences of portions of several plants with very largo gcnomcs (such as maize) im:u . c-dtes that transposable clements are lilre.ly to comprise an even larger percentage of these genomcs. Similarly. evPn in the compact gcnomes of E. coli and S cerevisiae. there art! examples of transposabJo elements and microsate llitr. repeats (see 1-igure 7·2). The diffurence is that tboSf! elements have been Jess successful at occupying the gcnomcs of these simpler organisms. This lack of success is likely a oomuimition of inefficient duplication and/or more efficient elintination (either by repair events or by elim-ination of organisms in which duplla~tion has occurred). Although it is tempting to refer to repeated DNA as junk DNA. the stabJe maintenance of these sequences over hundr~ds to thousnnds of germratioos suggests that 1ntergenh.: DNA confers a positive value (or selective advantage) to tho host organism.

CHROMOSOME DUPLICATION AND SEGREGATION Eukaryotic Chromosomes Require Centromeres, Telomeres, and Origins of Replication to Be Maintained During Cell Division There are several important DNA clements in cukacyotic t:h.romosomes that are not genes and are not involved in regulating the expression of genes (Figure 7-6). Thf'.Se elements indude origins of replication that di· rect the d uplication of the chromosomal DNA, ccntromeres thai act as "'handles., for the movement of chromosomes ioto daughter cetls, and le lomeros that prote<.:t and replicate the ends of linear chromosomes. All 1hcst> frBiuros are critical for the proper duplication and segregation of l.hn ch.J·omosomflS during C(ltl division. We now lcok at each of these clf"lrocnts in more detail. Origins of replication are the sitos at which the DNA replication machinery assembles to initiate replication. They are {ound some 3o-40 kb apart throughout lhe length of each eulwryotic dttomosoroo. Prolaryotlc chromosomes also require origins of replication. Unlike their euka.ryotic cou nterparts, prokar)rotic chromosomes typically have

Chromosomp VvpJication and

r;

~

tek>mere

\

ong~nof

repliCation

nuclear - . .

membrane

.-.

I

centJomere-

•

\

DNA replication

I

I

~tion

• :;-

-

~ spincle

mitosis

F I CURE 7-6 c.entrolneres, origins of the ~ ..-d telornen!s are required for eukaryotic ckomosome mainteoance. Each a.layobc chromosome flCiudes tv.<> lelor1"6es. one certromere, Cl"ld I'T1
only a single site of repliw ti on initiation. Tn general, origins of replica· tiun are found in noru.:odins regions. The DNA sequences that arc recognized as origins of replication 8l'fl discussed in detail in Chapter 8. Centro~ B.fC tcquircd for the t:orrect ~cegation of the ch romo-

somes after DNA replication. The two copies of each replicated c hromosome are called daughtCJ' chromosomes and they must be separated with on~ copy going to cac:h o f the two daughter cells. Like origins of replic.:~:ttion. centromeres direct the formation of an elaborate protein comp1r>.x, in this t.-ase, ca.Hed a kinetoc.hore. The Jdnctochore interacts with the machinery that pulls the daughtct' chromosomes away from one another and into the two daughter cells. In contrast to the many origins of replication found o.n each cukaryotic chromosome, il is critical that each chromosome h as one and only one ~ntromore (ngl.lre 77a). In the absencv of a ccntromnre, the replicated chromosomes segregate randomly, leading to fre<1uent loss or duplication of chromosomes (F i,;uro 7-7b). If prmicnt in multipJc copi«.!S, oontromeres can caUSP. a single chl'Omosome to be pulled into both daughter cells, leading lo dnomosomc breakage (Figure 7-7c). Ccntromcrcs vary greatly in size. In lhe yeast S. cerevisiae. centromeres are less than 200 llp. In oontrast, in the majority of cukaryotP.s, centromew.s arc > 40 kb Wld are composed of Jargdy .repetitive DNA scquent..-es (Figure 7-6). Telonwres arr located at the lwo ends of a li nnnr chromosCime. Like origins of replication and centromcres. tclomeres arc: bound by a number of proteins. In this case. the proteins perform two important functions. First, te1omeric proteins distinguisl1 the naturnl ends of tbc chromosome from sites of chromosnmc breakage and other UNA breaks in

I \ t

Chromosomes, c..'J~romutin, und th f' Nuclf'Osome

140

a one centromere

v\~~--

~~

..__

one cr.rnmos<~me for eacfl call

b oo centromeres

~I

:(? ~~

'

it-' :/}~_: II'

random segregation of ckomosorne

c two C<rornetes

dlrtJJnosome breakage (rue to more than one centromere)

---------- --fl cuR 7·7 More less than one centromer9 leads to dlrontosonH! loss or breakage. f

OC'

(a) Nama! dlromosomes h.we one centromere. After r~catial of a d1r()I'T1()S()ffl€ each copy of the cefr tromere directs the foon/Jt10n of a kinetochore. These two kinetodlores then bsnd to oppoSde poles of the mitotic Spincte and are puned ifllo lhe opposite ~ides of the eel plior to cell diviSion. (b) Ovomosomes lacking centrome.es are rilpldly lost from cells. In the absence of the c:entrt:lfTleJe, the dvomosomes do not attach to the sp111dle and are randvmly distrib.Jted to the two daughter cells. Ths leads to frequent events ih Vllflidl Ol"le drlughler ~s two copies of a chranosome and the other daugt1e- ceUIs rrns.sllg the same chromosome. (c) Ollomosomes with~ or mOte centromeres are ftequentiy broken dunng segregalm If a chromosome has fOOre than one cen!fomere. il can be bound simlitaneousJy lo bcKh poles ot lhe ~olic spndJe. When segregal!On is inilic3ted. tt1e opposing forces of ltle mit01ic spindle freqtiently break chiOmosornes attached to both poles.

the celL On1inarHy, ONA ends are the sites of frequent recombi•1ation and DNA degradation. The proteins that assembln at tclomcrcs form a structure that is resistant to both of these events. &>cond. telomeres act as a specialized origin of replication that allows the ce.U to replicate th~ ends of the Ghrom osomes. For reasons that wiH be dP.SCribed in detail in Chapb~r B. the standard DNA repli-

cation machinery cannot completely replicate the ends of a linear chromosome. Tclomeres facilitate end replication throug h the recrui tm ent of an unusual DNA polymerase called telomet"ase. In contrast ao .most of the chromosome, a substantia! portion of the telomere is maintaim~d in a singlc-strandoo form {Figure 7-9). Most tciomeres have a simp!n rr.pcating sequence that vari{ls from otgan-

1251?p

t

(C f•a S carevisiae

- -...

I

II___, IP

CDE 1-111 · ·-._ ,--

----~

b S.pombe

l )

- - ------....

- - -cen1

40-100kb ~

F 1G u R E 7- 8 Centromere size and com· positim vanes drarnatic;.;llly bd:ween different OfgO!lisms_ 5. cereVJSiae cenlf0rl1(:fes de sm~l and c.omposed of non--repetitlW sequences. In

contrast the cenuorneres of other~ such as the frutt fly, Drosophila melonogoster; and the fiss1oo yeast. Sdlizosoccharomyces pombe. are rTli.JCh larger and aft' largdy composed of repetitive seQllenc::es. ~Y me central 4-7 l' of the DrosophiJo ood ~ c:entromeres are repetitive

D melanogastar -400kb

d t-luman

0

11- -

]

)

240 kb to several tva>

ism to organism. This repeat is typically cnmposcd of a shnrt TC-rich repeat. For example. human telorneres have the repeating sequence of 5'-TfAGGG-3'. As wo will see in f'J1apter 8, the repetitive nature of teJomeres is a consequence of their unique method of replication.

Eukaryotic Chromosome Duplication and Segregation Occur io Separate Phases of the Cell Cycle During cell division. the chromosomes must be duplicated and segregated into the daughter ceUs. In bacterial cells thnse events CX.'CUT simultaneously. That is, as the DNA is replicated, the ff'..Su tring two copies are soparatP.d into opposite sidP.S of t he cell. Althmlgh it is clear tha t these events are tightly n-gulated in bacteria, the details of how lhis regulation is achieved are poorly understood. ln contrast. eukaryotic cells duplicate and segregate their chromosomes at distinct times dm-ing cell division. We will focus on these event s for the remainder of our discussion of chromosomes. The events tocjlJired for a single round of Cf!Jl divi.s ion are collectively known as the cell cycle. Most eu.karyotic ceJl divisions maintain the number of drromosomcs in the daughter cells that were present in the parental l:ell. This type of division is called mitotic cell division. The mitotic ce11 cycle can be divided into four phases; Gl, S, G2, and M (Hguro 7-10). The key events involved in chromosome

F 1G u R E 7-9 The structure of a typical telomere. lhe repczlled sequence (from hlJJ11011 cefls) ts shown 1n a representative box. Note that the regton of ssDNA allhe 3' end o! the d'lro~ome can be hl.rl&-eds of bases long.

DNA.

142

Chromt)SDilJP...<;. <:hromotin. u n tf the Nl.(clcnsomc>

FIGURl 7~10 lhe eukaryoticmitotic

all qde. There are four stages of ltle ~ cell cyde. Chlornosornal ~tion omxs ru10g S phase and rnromosome segregation oco...s dJring M phase. The G 1 and G2 gi¥) phases alow 1he c.eU to prepare for

the next events 1n the cell cyde. For~ tTlaf'IY eli<xyollc eels use the G I phase of the cdl cycle to establish thai the level of nutrients 1s StJffioently high to allolv the complebon of celt di\lisson,

DNA

prepafe for

r6pllcation

cell drvi.sion

p.ropa.gation oecur at dis tinct times during the cell cycle. DNA synthosis occurs during the synthesis. or S phw.e. of the cell cycle. resulting in the d upbcation of each chromosome (Figure 7-11}. Each chromosom e of the duplicated pair is caUed a chromatid. and the two chromatirls o f a given pair are r.aJicd si,;ter chromatids. Sister chromatid s are hf:)d together after duplication through the action of a molecu le ca lled cohesin. which we describe below. The process tbal holds them together is called sister c::hromatid cohesion and this tethered s tate is maintained until the chromosomes segregate from one another. ChromOSome segregation nccurs during mitosis or the M phase of tho cflH cycle. We will consider the ovm-all p rocess of mitosis below. but fin;t we fucus on three key steps in the process (Figure 7~12). First. eac.;h pair of sister chromatids is bound to a stru<.1 ure called the mitotic spindle. This structure is oomposed of long, protein fibets called microtubuJcs that are attacherllo one of the two microtubule organizing

FICUR£

7-11

The events ofS pbas~

key events in S phase

Two IT'la!()f dvomosomal events occur dllflOg S phase. DNA r€pltcabon ccpes ead1

chromosome completely, and shortly after replic.alion has oa:urred. sister c.hrOf'rlatid a:>hesin i.s estctflshed by placing nng-shaped cohestn moloo.des around the two <-OJ:l1€S d the rElC6\IIy replicated DNA Each blue or red "tube~ re!J(esents an ssONA molea.Jie_

i~

ofONA replication

more repfi(;ation

es1abltshment of cohesion

stster chromatids

au-omosotrlf.• Duplic;al iDil U(IQ Scgregutioll

key events in M phase

F 1G u R E

7-12 The events of mitosis (M phase). fu-~ maJOr events <XaJr d\lflng mrtosts. FirSt, the two kifletoc:hores of each hnked siste-chrOI"Tla'tid pair att~ch 10 opposite poles of the rnttotic spnde.

Once ;,II I<Jnetodlores
a ct-emilotic spmdle centers (also called wntrosomes in animal cells or spindle pole bodies in ywsts ant! uthar flmgi). The microtubule organi:"dng centers are locaterl on opposite sides uf tht: cdl forming "pules" tuwcrrd which the microtubules pull the chromatids. Chromatid attachment is mediated by the ldnetOl:hOI'c assembled at each centmmere (Figure 7-6). Second. the ruhesion between the chromatids is dissolved. BefOTC cohesion is dissolved, it l'e sists the pulling foro!S of the mitotit: ~pindJe. After mhP.Sion is dissolved , the third major event in mitosis can occur: sister chromatid separation. fu th~ abs~nl:e uf the cuunte:rbalandng fun:e of chromatid cohesion, the chromatids are capidly pulled towa('d op posite poles of the mitotic spindJe. Thus. oohesion between the sister chmmatids and attacllment of sister chromatid ki11etochOL'f:'.S to opJl(lSitf:l poles of the m itotic spindle play opposing rol~s th
Chromosome Structure Changes as Eukaryotic Cells Divide As chromosomes proceed through a round of cell division. their structure is altered n umerous time~ however, there are two main states for the chrumusomes (Figure 7-13). The chromosomes are in their most compad form as cells proceed through mitosis or meiosis. The process th at resu Its in this compact form is ailled chromosome oondensation. In this condensoo s tate the chromosomes are completely d isf,lntangled from one another, greatly facilitaling the SHgrogation process. During the Gl. S, and G2 phases (wllectively referred to as interphase), the chromosomes are significantly less compad. Jndeed , at thrnre stages of the ~u cyde, the chmmosomes are likely to be highly intertwined. resembling more of a plate of spaghetti than the organized view of chromosomes during m itosis. Nevertheless. even during these stages the struet\u'e uf the chromosomP.S change. DNA £eplication

:143

1M

Chromosomes. Chromuflll. and the Nvdeosvme

M phase

lntsrphase

FIG U R E 7-13 Changes in dwomatin stnx:ture.. 0uClrTKJ50mes are rrnOOmally cor.detised 1n M phase and decondensed throogflout the rest of the cell cycle (G 1. 5. ~d G2 m mtotle celts) Togethet these decondense:l stages ae referred to as inletphase

requires the nearly compl(!te disassembly and reassembly of the proassociatoo with each r.;hromOS<Jme. Immediately after DNA replication. sister-chromatid coh~..sion is established. linking the newly replicated chmmatids to one another. As transcription of individual genes is turned on and off or up and duwn. there are assuciated changes in the structure of the chromosomes )n thostl ~ions occun ing throughout the ceiJ cycle. Thus. the chromosome is a constantly changing structure that is mor~ like an organelle than a simple string of DNA. t~ios

SisteT Chromatid Cohesion and Chromosome Conden..~tion

Are Mediated by SMC Proteins The key proteins thai mediate sister chromatid cohesion and chromosome condP.nsation are related to une another. The structural maintenancf:l of chn;mosome (SMC) protein~ are extended proteins that form defined p airs by interdcting through lengthy ooilud-coH domains (soo Chapter 5). Together witb non-SMC proteins they form multiprulein complexes that ad to link two ONA helicr:>,s together. An SMC-protein containing complex caUe.d t:ohesin is required to link the two daughter DNA duplexes (sister chromatids) together after DNA replication. It is this Hnkage that i s the basis fur sjst& chromatid coh esion. The strudtu e of cohesin is tho ugl1t tu be a large ring composed of two SMC proteins and a third non-SlviC proteu1. Indeed. there is growing evidenre that the mechanism of s ister chromatid cohesion is that both daughter chromosomes pass through the center of the cohesin protein r.ing (Figw·e 7-14). In this model, proteolytic cleavl'-age of the nun-SMC subunit of cohesin Tesu lts in the opening of the ring and the luss of cohesio. The chromosome condensation that accompanies chromosome segregation Hlso require s a related SMC-containing-complex coiled condensin. Although less is known about the structure and fundiuJl of this oomplex, it sh ares many of tbe features uf the t.;ohesin cumplex~ s uggesting that it too is a ring--shaped complex. If so. it may use its ring-like nature to induce chromosom e condensation. For example, by linking different regions of the same chromosome together condensin could readily red uce the overall linear length of the chroroosom~ (Figure 7-14).

ChrotnOSDme Dvplicotion and SF-grt!gol ioll

14-5

7-14 A speculative rnodeJfDI' lhe strudure of cohesins and (ondi!Min$.

fIGURE

c!Yomatln

Collesin!:. .,nd condensins are ~IS of the nuclear scalfc~. Both play important roles

in brmg~ng distant or difierent regioos of DNA l0get11ef. The proposed nng-sh
anaphase

~ Nucleosome

Mitcsis Maintains the Parental Chromosome Number We now return to the overaJ I proet:£s of mitosis. Mitosis occurs in Se\1· eral stages (figure 7-1!1). Duling prnphaSE, the chromosomt;JS condt!nse into the highly oornpact form required for segregation. At the P.:nd o f prophase, the nuchJar envdope breaks down and the cell fmters metaphase. During metaphase, t h ~ mitutic: spindle forms and the k1netochores of sisl~ c:hrumahcls ;dlach to the microtubui~. Proper chromeitid attachment is only achieved when tht~ two kinctochores of a sisterchromatid pair ru-e attached to microtubules emanating from opposite microtubule mganizing centers. This type of attachment is calloo bivalent altadunent (see Figure 7-15) ami results in the microtuhules exerting t~nsion on the chromatid pair by pulling the sisters in opposite c.lir&:tiom;. .Attachment of both t.:hromatids to microtubules emanating from the same microtubule Oiganizing center or attachmtmt of only ont'! chromatid of the pair, called monovalent attachment. d<Jt.:os not a·csult in ttms ion and eventually leads to cllrurnosome loss. The tension exerted by bivalent attachment is opposed by siste•· chromatid cohesion and resu lts h1 all the chromosomes aligning in tl•e middle of the cell bet ween tlae two micrulubule organ i7Jng centers [this position is called the metaphase plate). At this point. each sist•~r d u·omatid i~ ptt3pared to be segrcg
The Chlp Phases of the CeJJ Cycle Allow Time to Pl'epare for the Next CeU Cycle Stage while alsc Checking that the Pre\'icus Stage Is Finished Correcdy The remaining two phases uf tbe eukaryotic r:ellt:yde are gap phases. G1 oocurs priur to ONA synthes is and G2 behveen S ph
Cltmmnsome lJvplication unCI Sewegafion

147

7-15 Mitosis in detaiL Prior to mitOSis. lhe dvomosomes are in a decondense6 state called intefphase. During pmphase chromosomes are condensed and de-r.aogted ill preparaOOn for~ and the nuclear membrane surrOUldalg the F Ui URI

dvornosomes breaks do\MI '' ~ eukaryotes. Outing metaphase, each sister-dllomarid pawa~ 10 opposite poles of the mitolic spindle. .Anaphase IS Ulitiated by the loss of sistef· chrOITiillid coheslOf'l resulting tn the separaliO(l of sistet chromatids. Te~ is distinguished by the loss of d1rot'Tl0501TlE' ~ and Lhe reforrnaWn of the nudear rnembtane around the two pqx.~latioos of segregated dlromosomes. CytOktneSIS IS the fii1CI1 event of the eel cycle dUMg v.f1tdl the celu~ membrane surrounding the two nudet constricts and ever~tuaGy C<Xr(>1etely separates into twO dautflter eels. AI DNA mofecules
prophase

stranded mon0\13Jent attacl'vnent

bivalent attathment

-~~- broken

coheSiOn

rings

telophase

cytokinesis

da\Jghter cell

daughter~

bufore mitosis to prevent either event from occurring with damaged chromosomes. This de lay allows time for the damage to be repaired before the cell cycle continues.

Meiosis Reduces the Parental Chromosome Number A second type of eukaryotic cell division is specialized to produce celts that have ha lf the t1u mbe.- of chromosomes tha11 the paren t al nell Lilr..e the mitotic cell cyde. the m eiotic uell cyde includf:IS a G1 . S, and an el ung~teU G2 phas~ (Figure 7·16). During tha ffiMiotic s vh ast:, €Hch chromosome is replicated •md the daughter chromatids r:emain a.-,sodated as in the mil otic S ph~se. Cells that enter mt!iosis must be dipJoid and thus contain two copies of e..ach chromosome. one defived from each parent. Aft~o~r DNA replication, the.s('? related siste.r-chromatid pairs, caHed homologs. pHir wilh o ntl another and recombine. Recom· bination ~tween th(• humulogs creatt:s a physical linkage betwoon the two l10mologs thHt is required to connect the two related sistef*chromatid pairs during chromosome segregation. We will discuss the details of m eiotic recombination in Chapter 10. The most significant difference between tht! m ilotit: and meiotic ct~ll cydt}S occurs d uring ch romosome st:gregation. Unlike mitosis, during w hich there is a single round of chromosome segregation, chromosomes participating in meiosis go through two m unds of segregation known as mciosjs I and fl. Like mitosis, each of the.se segregation ev~nts includ ~ a propha~. metaphase. and anaphase stage. During the metaphase of meiosis L a lso caHed metaphase I, the homo logs attach to opposite poles of the microtubule-based spindle. This attachment is mediated by the kinetochore. Because both kinetochores of each sisterchromatid pair are attachoo to the samo pulE: of the rnicrotullUJe spindlt~, thls m teraclion is 11lfen~d to as monovalent atladuneot (in contrast to the bivalent a ttachment se.:>..n in m itosis, in which the kinetocbores of each sister-chromatid pair bind to opposite poles of the spin d l~). As in mitosis. the paired homulogs initially resist the tension of the spin dle pulling them apart. In the case of meiosis I, this is m ediated through the physical ronnt:ctions betwBP..n the laomologs, or crossovers, that ate induced by recombination. This resistance also requirt!S sistP.I.'-Chrornatid oohf:lsiafl aJong thP. arms uf the sistt~r l.:hromalids. When cohesion along the arms is eHmina!OO d uTing anaphase I, the homologs are reles:tSE>..d from one anutluw and segregate to opposite pules of the uell. lmpodantly. the crihesion between th e sisters is maintdinerl ncai' the centromere. resulting in the sister chromatids retnaining paired . The second round of segregation during meiosis, meiosis n, is very similar to mitosis. The major difference is that a round of DNA replication does not precede th is segt-egatiun event. Jm;tead, a spin dle is fmm~ in asooci~ti()n with uach of the two newly S4.lparated sjster r..:hromaUd pairs. As in mit05is, d uring metaphase U, these s pindles attach in a bivalfmt manntrr to the kinetochores of e.ach sist~chromulid pairThe coht'$ion tbat remains at the centromei"E!S af1er meiosis I is critical to oppose the pull of tho. spindle. As in mitosis, anaphase fi is initiated by the elimination of (Je ntrumtlrtt cohasion. At this point there are four sets of chromosomes in the ooU, eal:b of whit:h contains only one r:opy of each chromosome. A nucleus forms around each set of chromosomes, and then llie t.:ytuplasm is divided to form four haploid cells. These cells ~ now n>ady to mate to form new diploid cells.

Chmmnsnnrc Duplirotion and Segregorion

•

DNA replic:alion

•

149

FIGURE 7-16 Meiosis in detail. Uke mnosis, meiosis V3l1 be di\11ded n o disaete stages After DNA replication, hcmologous sister chroniatids pa~r with one another to form struc:1UTes w th fOI.a re4ated chrom~. for sirJl:>lu:ity, onJy a stngle d-romosome as

shown segregating with the blue copy beiflg from ooe parent and the yelfoi.'V copy from the othet. Outing p«~r"ing. d"'I{Jfl"l(l(ids fran the different 56\er chromatids reccml:>ifie lo form a link betvveen the homologous c:hromoscrnes called a c::h!asma. IJunng metaphase ~ the two lcinetochaes of each 545tet-d1romalid pair attach to one pole of the metOtic spncle. H~ sister-dlromatid kinetcx:hores attdch to ~e poles creatmg tensiOn that. is tesisted by t.he c.onnection betvveen the tromologs. Entry 11110 CJnaphase I rs correlated wth two ~nts wlidl together result 1n lhe separatJon of the homologous chromosomes from one another. The sislffsiS. £luring metotic met~ II. two meiolic: 'Spindles are formed As an mitotic metaphase. lhe kil"le(Oc'hores associated Wlth eadJ sester-chromatJd pa.r attach to opposite p:lles of the I"T"ledlC sptncles. OlJnre anapr.ase II. the remamltlg cohesion between mesisters is lost and the sister d uornatids separete from Of'le another. The lour separate sets CJf ch0f110501'l"1€S ~e then padaged lflto nudet and ~ated mto four eels to aeate foor spores or ~ AI DNA moJe.. ctJes are docble-strilllded (Source: Adapted from Murray A. and Hunt T. 1993. The <.-ell cycle: The inttoductJOn, fJtl. 10.2. Copyright © 1993 by Oxford l.lniversiy Press, Inc. Used by periTISSlOtl of Oxford Urwefsity Press, Inc)

metaphase I

"'0

u;

1

anaphase I

metaphase II

=en

·;;; 0

'ii

E

four gametes (or spores)

150

r.hromosnmes. ChromoUn, cmd the N ucl f!OSOITIE'

Mphase

10-f'lm fiber

30-nm fiber

FlfitJ RE 7-17 Forms of dwomatin structuresetm in the £M (a) Electfon rncrographs of M phase and irllerpha!.e DNA show the changes 1r1 the stnJct\Jfe of chromatJn. (b) Ek.<1ron r-niaograpl-6 or diffetent loons d ~ in IC\terphase cells 4lo.N the 30nm and 10-nm chromatlr'l ftbers (beads on a string). (Sa.rce: (a) Courtesy of VICtoria Foe. e 2002 fmn Alberts B. et al 2002.1v1nfecu/ar bio!O!JI allhe ceil. 'llh ed"ltion. R€piOO.Jced by permssioo of Routledge file~ part of The Taytrx & Francis Group. (b) Cou't6y of 8aJbara l-lamkalo; e 2002 fran Alberts B. et al. 2001. MoleQJ/or biology of r~ cell. 4th edtion. R~:pcoduced by perm5S!C11 of Rl.;utledge lr\c., p3rt
Different Levels of Chromosome Structure Can Be Observed by Microscopy Micr<J5copy has long been used tG observe chromosome strtJdure and function. lndP.ed. long before it was clear that chromosomes were the sow-ce of the genetic information in the eel). their movements and changes d uring ceJI division w erP well understood. The compact nature of condensed mitotic chromosomHS also makes them relativt?ly easy to visualize even by simple light micl'oscopy (Ftgure 7-17a). 1ndee.d, H was io this form that chromosomes were first identified. Condensed chl'omosomes are also used to d etermine the chromosomal make-up of human c ells to detect such abnormalities as chromosomal deletiuns or individuals with extra copies uf a single cllromowme. Chromosomal DNA not in mitosis (that is, in interphase) is Jess compact (Figure 7-1 7a). In the e lectron microscope two states of ch roma tin ar e rea.dily observed: fibers w ith a diameter of either 30-nm ur 10 t~ m (Figure 7-17b). The 30-nm £he1· is a more compact vet-sion of chromatin that is frequently folded into Jarge loops reachjng out from a protein oore or scaffold. In contrast. the 10-nm fiber is a less compa«.:t form of chromatin that rosemble.s a regular scri~s of "bt:ads on a string." These beads are nucloosGmes. We will fi1·st focus nn the nature of the nud oosome. indtJding how they are formed, and then describe h ow nur.leosome-dependent stmctures control gil tb=tl effects on the acoossibility of nudHar DNA.

fhe Nvc:IP.fiSOf11f'

THE N UCLEOSOME

15 1

------ -

Nucleosomes Are the Building Blocks of Chromosomes The majm·ity of the DNA in eukaryotic coils is packaged into n ud eosomes. The nucloosome is compo~d of a c-tJre of eight histone pmteins and the DNA wrapp&l around them. The DNA between each nucltlOsome (the "s tring" in the "beads un li string") is t;alled linker UNA. By assembling in to n ucleOSOmes, t he DNA is compacted approxinuttely sixfold. T his is fu r short of the 1.ooo- to 10,0()0-fold DNA compaction obSP.rved in eukaryotic cHUs. Nevertheless, this first stage of DNA packaging is essential fo r all the rP-maining levels of DNA compat..1 ion. The DNA most tightly MSsociated with the n ucleoso rn(1. called !he cote ONA, is w-ound app roximately 1.65 ti m es around th~ outs id e uf

th e histone octamer like thread around a spool (Figure 7'~ 18). The l ength of DNA associated w ith each n udeosome can be determined us ing n uc:l ease h ec-Jtment (Box 7-1 , Micrococcal Nucl ~se and the DNA Ass ociated wath the Nudeosome). T he - 14 7' base pair length of thi:s DNA is an invariant feature of nucl~osomes in all eukaryutic cells. Jn conu·a:::t. the length of the linker UNA between n ud ~:~osome~ is variable . TypicaJiy thi.s di:::tance is 20- 60 bp and p_,a(:h eukaryo te has a characteristic average lin ke r DNA length (Table 7-4)_ The differen c..'P in avf'rBgP. linker DNA length is likely to (eflect the different:es in the nature of largt!r st nu.:t un~s formed by n ucloosomal DNA

a

nudeosome

.....,_

I 1

core

'

<>~one

F I(; ll R E 7-18 DNA packagH into

nudeasomes. (a) Schematic of the packag-

J ]core DNA '

(146 bp)

)0'~ ~ON~ .)f

(20-60 bp)

b

ing «ld Of'g
wrapped atound the histone protem core H2A is shovvn m red, HW m yeblv. H3 in ptnple, and H4 tn green. Note that the colors of the dif· ferer1t t:istooe protffl.s f1efe and 1n f~ ~n.x:tures «e l.he same (Luger 1\., Mader A.W~ IOdmond Rl<., Sargent O.f . af1d RidTnood TJ. 1997. Nature 3B9: 251-160.) ltr\c3ge prepared with BobSoipt. MoiScnpt.. and RastEf 3D

Box 7-1 Mkrocoaal Nuclease and the DNA Associated with the Nudeosotne

Nudeosc.rnes INef"e ftrst purified by treating chromosoo"les

With a sequence nunspecific nuclease called mia'ococal 11udeitse. lhe ility of this enzyme to deave DNA is primarily govemed by the accessbility d lhe DNA. Thus, miCTO('.O(".cal nuclease cleaves protein-free DNA sequences rapidy and poteln-assooated DNA sequences (JOO'ly. Lmited treatment of chnxnosanes \\1th this enzyme results in a rudease-reSIStant pq>ulation of DNA molerules that are assodated with histones. These DNA ll""decules are between 160-220 base pai-s in length and ere associated with two copies e&h of h1stoncs H2A. H2B, H3, and 1-14. On average, these particles iidude the £X\IA tightly assooated with the nucleoscrne as well as one l$lil of Tiri<er DNA More extensive micrococC
-unm[® Q

The avetage length of ONA associated with each nudeosome be measured i1 a sirr.ple experiment (Box 7-1 Figlxe 1) . Orunatin is treated with the enzyme microcnc:cal ru:lease but INs tiTle trly gently.This resUts in sing'le ruts in scrne but not all of the linker DNA After ..-ukase treatment the ONA is extracted from al p otefis (inducirlg the htstooes) and subjected to gel electrophoresis to separate the DNA by si2e. Electn:~:h:111~sis GJ1

reveals a nladdel" of ~ents that are

mtl~

ts rnly partiaUy Cigested Thus, sometimes mumpte nudeosomes W1ll remai1 unseparated by Qgestion, leadtlg to Cf\JA fragrnercs equivatmt to aUthe DNA bound by 'hese nucleosomes. Futhef digestirn INOI.lld result in al linker DNA bffig deaved and the f00"11atim of nucleosorne m-e pcl"'ides and a sl'1gle - 147 bp fragment

bp

800 000

•--

-

400

released

nudeosome core partiCle 200

---

of the averege

nudec::&rne-to-nudeosome
®

-----

BOX 7•1 FIGU Rl 1 Progressive dige$tiOn of nudeosonwtl DNA with Mnase. (Sowce:Courtesy of R.D. I
7-4 At.'efa~e le.ngths of Linker DNA in Various Oflanisms

TA 8 L E

Nucfeosome rweat

$pedes

lengUl (bp) S.cerevrs~ae

160- 165

- 260 - 180

Sea urchin (sperm) D me/anogasler

185- 200

Ht.lmafl

Avetage lf1Qr DNA tengttl (tp) 13- 18 - 110

- 33 38- 53

in e.ach organism 1-ather than diffeTences in the nucleosomes \hem· selves {see section on Higher~Order Chromatin Structure). In any celt thero are stretc:hes of DNA that are not packaged into nucleooomes. Typically these are regions of DNA engaged in gene expression. rnplication, or recombination. Although not bound by nucleosomes, these sites are typically associated with non-histone proteins that are either regulating or participating in these events. We will discuss the mechanisms that remove nucleosomes from DNA and maintain such .regions of UNA in a nucleosome-free state below and in Chapter 17.

Histones Are Small, Positively,Charged Proteins HisLoncs are by far the most abundant proteins associated w ith eukaryotic DNA. Eukaryotic cells rommonly contain five abundant histones: H1 , H2A, H2B. H3, and H4. Histones H2A. H2B. H3. and H4 are the core histones and form the p10tein core around which nucleosomal DNA is wrapped. Histone Ht is n ot part of the nucleosome core partide. Instead, it binds to the linker DNA and is referred to as a linker histone. The four core histones are present in equal amounts in the cell. whereas Hl is half as abundant as the other histones. ~Chis is consistent with the fin ding that only one molecule of Hl is associated with each nucleosome lwh ich contains two copies of each core histone}. Consiste-nt w ith their close association with the negatively-c harged DNA molecule, the histones have a high conte11t of positively-cha-rged amino acids (Table 7-5). Greater than 20% of the residues in each histone are either lysine or arginine. The core histones are also relatively small proteins ranging in size from 11 to 15 kilo deltons {kd). whereas histone H1 is about 20 kd. The protein coro of the nucleosome ls a disc-shaped structure that assembles in an ordered fashion only in the presence of DNA. Without DNA. tbe core .histones form intermediate assemblie.s in solution. A conserved region found in every core histone, called the histone-fold domain, mediates the assembly of these histone-only T A 81 f

7-S
Core hstones

~('4)

H?A

14.000 13<900

Y k

H2B

lilker histone

"-of Lysine arid Arginine

Hlsfone

22%

H3 H4

15 ,400

23%

11,400

H1

20.800

24% 32%

154

Chromosomes. Chromatin. rrnd tile Nucleosome

f 1G u R E

7-19 The core IH.stones shan • common stnl(turaf fold. (a) The foUl' histones are diagramed as ~neu molecules. The regions of the histone fold motif that f(l(m a helices eWe !l'lOO!ed as cyfrndas. Note that there are adtacent regiOns d ead1 hstone that are struc:tufalty c:istw'tct includmg additional 0 hehc.al regJOOS. (b) The relical regions of two l'nstones (het"e H2A and H2S) rome together

a N-terrrifla1 tail

histone fotd

H2A

c

H2B

c

H3

N

to form a dimer. H3 and H4 also use a si!T'.Iar 11'1terec:•xm to form H3 2 ·H~ tetrClfl1efS. (~Ufce:

Adapted frorn Alber1s a et al 2.002. 1vfo/ecular

b

biology ofthe cell, 4th edroon, p. 209, jjg 4-26o

N

Ccpyoghf 0 2.002 l<ej:Xoduced by pcrms.sion

t

cJ Routledge/Tayb &Frcmcis Books, Inc.}

intermediates (Figure 7-19). The histone fold is composed of three a helical regions separated by two short um;t:ructured loops. In each case the histone fold mediates the formation of head to tail heterodi.mers of specific pairs of h istonP.s. H3 and H4 histones first form heterodimers that then rome together to form a tetramer with two molecules each of H3 and H4. In t.:Ontrast. H2A and H2B form hcterod.imers in solution but not tetranlers. The assembly of a nuclE>.osome involves the ordered association of lbese building blocks with DNA (Figure 7-20). FU:st. the H3•H4 tetramer binds to DNA; then two H2A·H2B dimers join the H3·H4DNA complex to torm the final nucleosome (see figure 7-18). We will discuss bow this assembly proc.;ess is accomplished in the cell later in the chaptet. T he cow histones each have an N-terminaf extension , called a "tail, .. because it lacks a defined structure and is accessible with1n the intact nudeosoroe. T his accessibility can be deter.ted hy treatment of nucleosomes with the pro tease trypsin (which specillcally cleaves pro teins a f1er positively-charged amino acids). Treatment of nuclem;omes with trypsin rapidly removes the accessible N-terminal tails of the histones but cannot cleave the tightly packed histone-fold regions (Figure 7-21). The expased N-terminal tails are not rflquired for the association of DNA with the histone octamer, as the DNA is still tightly associated with the nucleosome after protease treatment. Instead, the tails are the sites of extensive modifications that alter the fundion o[ individual nudeosomes. These modifications include phosphorylation. acetylation. and methylation on serine and lysine residues. We will .return to the mle of hi.:;tone tail modification in nucleosome function lattrr. Now. we turn to the detailed structure of the nt1cloosome.

The Atomic Structure of the Nucleosome The higb-resolution three-dimensional structure of the nucleosome core particle (Figure 7-18b. 147 bp of DNA plus ·an intact histone

Fa (i u R E

H2B

H4

7-20

The assembty of a

nudeosome. The assemi:Jiy of a nxleosome IS iNtlared by lhe klm\ation of a H31+14:~ tetra mer. The tetrnrner then binds to dsONA. The H3.l-H42 tctramer bound lo DNA reouts l'A'O copies of the H2A•H2B otmer to cOfTl'lere the dSSefT"bly ot the nudeosome. (Source: Adapted from .Alberts B. et al. 2002. MoleculGt biologyoftht?cell, 4th edilior\ p. 2 10 , fig. 4-27. Copynght ~ 2002. RcpJodlxed by pemlisSIOO of Rou~ 8 FranciS Books, Inc.)

H2A•H2B d imer

N

N

N

N

155

156

Cftrvmr:>snmes. Chrol11l11in. and tile Nuclf'oSom~

octamer} has re\'ealed much about how it functions. The high affi n-

. H2B

FICiU~E

hiStone COJe

7•21 lheN-tenninaltails

of the awe histones are accessible to proteaes. Treatment of 1'\Udeosomes with lim1t111g amounts of proteases that deall1:! afh~ basic ammo .acids (tat e.)(ilmple, IJWS~n) ~f!Ca!Jy removes the N-terfl"'II

ity of the ouc:;leosome for DNA, the distortion of the DNA when bound to the nudeosome. and th~ l~ck of DNA sequenca specifkily, can each he explained by the nature of the mteractioos between the

hi.stones and the DNA. The struuture also sheds light on the function and location of theN-terminal taHs. Finally. the interaction between the DNA and the histone octarner allows an understanding of the dynamic nature of the o ucleosome and the process of nucleosome assembly. Although not perfectly symmetrical, the nudeosome has an approximate twofold axis of symmetry, caUcd the dyad axis. This r:an be visualized by thjnking of the face of the octamer disc as a clock with the midpoint of the 147 bp of DNA located at the 12 o'clock position (Figure 7· 2.2). This places the ends of the DNA just short of 11 and 1 o'r.lock. A line drawn from 12 to 6 o' clock through the middle of the disc defines the dyad axis. Rotation of the nucleosome around this axis by 180.. reveals a nearly identical view of the nucleosome to that observed prior to rotation. The H3·H4 tetramers and H2A·H2B dimers each interact with a particular region of the DNA within the nucleosome (Figure 7-23}. Of the 147 base p airs of DNA induded in the structure, the histonefold regions of the H3•H4 tetramer interact with the central 60 base pairs. The N-terminal region of H3 most proximal to the histone-fold region forms a fuurth o helix that interacts with the final1 3 bp at each end of the bound DNA (this region is distinct from the unstructured H3 N-terminal tail described above). If we pichue the nucleosome with a clock face as described above, the H3·H4 tet:ramer forms the top half of the histone octamer. Importantly, histone H3•H4 tetramers occupy a key position in the nudeosome by binding the middJe and both ends of the DNA. The IWo HzA·HZB dimeiS each associate w ith approximately 30 bp of DNA on either side of the central 60 bp of DNA bound by H3 and H4. Using the clock analogy again, the DNA associated with HZA·HZB is located from approximately 5 to 9 o'clt.x:l< on either face of thn nudeosome disc. Together. the two H2A·H2B dlmers form the bottom part of the histone ocblmer located ac:ross the disc from the DNA ends. T be extensive interactions between the H3· H4 tetramer and the DNA help to explain the ordered assembly of the n\ldeosome (Fig\LJ'EI 7-24). H3•H4 te usmer association wit h the middle and ends uf the bound DNA would .-esult In the DNA being extensively bent and constrained making the association of H2A·H2B dimers relatively easy. In contrast, the relatively short length of DNA bound by H2A· H2E dimers is not sufficient to prepare the DNA for H3•H4 tetramer bindi ng~ This more limited association of H2A•H2B dimeJS bas been hypothesized to facilitate lheir release as n ucleosomaJ DNA js 1ranscrjbed. Such a mechanism would allow RNA polymerase increased access to nucleosomal DNA during transcription.

Many DNA Sequence,fndependcnt Contacts Mediate the Interaction between the Core Histones and DNA A closer look at the interactions between the histones and the nucleosomal DNA reveals the structural basis for the binding and bending of the DNA within the nucleosome. Fourteen distinct sites of contact are observed, one for each time the minor groove of the DNA fa ces the

The Nuckosome

8

b

3

9

6

-

6

FIGuRE' 7-22 the nudeosome 11ft an~ twofold axis of symmetry. Three vtews of the ~tomic struaure of the ll.Jdeos()IT(l are shown Each shows a 90' rotatior1 arOO"ld the axis between the 12 and 6 o'dock positions of the wcw shown in Figure 7-Zh. Ncie that a l8Cf rotabOn reveals i!l struc::tu'e neariy tdentical to the ortgl(lC!I WNJ. The diagram below each sboct\Je illustrates the rotations. (a) Oystaf structt.J'e. (Luger K, Mader A.W. Richmond RK., Sargent O.F., and Richmond t J. 1997 Nature 389: 151-260.) Images l)'cpared With BobSoipt. MoiScnpt. and Ras1er 30 (b) Cartoon schernahc

a

7-2.3

lntentdtons of the histones wittl nudeosomal DNA. (a) H.S· H4 bnd the m&:ie and the ends of the DNA The DNA t-oone! by the H3•H4 telr
FIGURE

orange. (luger K. Madet AW. Richmond RK, Sargent D.F. and Richmond TJ. 1997. Narure 300: 251 260.) Images prepared wllt1 BobScnpt MoiScripl, and Raster 30

3

enlry

exit

'

' 9

ts

Qnumosvme.s, ChromCJttn. and the Nucleosumf"

158

7-24 Nudeosome lacking H2A The H2A Nld H28 hisfooes have been artifitialy removed fron' thas VteW of the nudeosome. This structure is likely to resemtie the ONA•H~oH4;> tetrC!Inefimermediate in the

F f(; tJ lU and tUB.

asserrbly of a n\Jde050f'1'1e (SE'e f'JgUre 7·20). (luger K, Mader A.W~ Richmond R.I.<, Sargent D.F., and f?.ichmond TJ. 1997. Nature 389: 251- 260.) Image prepared wrth ~t MolScnpt. ood Raster 30

h istone octamer (Figure 7-25). The association of DNA with the n ucleosome is mediated by a large number ( - 140} of hydrogen bonds between the h istones and the DNA. The majority of these hydrogen hoods arc between the proteins and the oxygen atoms in the phosphodiester backbone near the minor groove of the DNA. Only seven hydrogen bonds are mad~ between the protein side chains and the bases in the minor groove of the DNA. The Ia~ mJmber of these hydrogen bonds (a typical ~uem:.-e·spe cific DNA-binding protein only h as about 20 hydrogttn bonds with DNA) provides Lhe driving force to bend the DNA. The highly ba,..ic nature of the histones also serves to mask the negative charge of the phosphates that would ordinarily resist DNA bending. which brings the phosphates on the inside of tho bend into unfavornbly close proximity. The basic nature of the histones also facilitates the close juxtapositio n of the two adjacent DNA helices ner.essary to wrap the DNA more than once around the histone octamer. The finding that all the sites of contact between tht~ histones and the DNA involve either the minor groove or the phosphate backbone is t;onsistent with the non-sequence-specific nature of the assol:iation

fiGURE

7-25 The sites of contact

between the histone,; and the DNA. IU danty, only the •nteroc1Jons betvveen d single

a

H3· H4 mer are shwn. A subset ol the pilfts ol the htstooes that interact With the DNA are htghligtrted In red Note that these re&10f15 dus· ter around the mnor groove of the DNA. (luger K, Madc"f A.W, Richmond RK. Salgent D.F.• .:~nd RIChmond TJ ' 991. Nature 389: 251 - 260 ) Image JX'E!P
Raster 3D.

Tht:: Nuclt::osome

of the histone octamer with DNA. Neither the phosphate backbone nor the minur groove is ric.h in base-specific information. Moreov-er. of tht-"! mwen hydrogen bonds formed with the bases in the minor groove. nvne are with elements that distinguish between a G:C and A:T base pairs (see Chapter 6, Figure 6-10).

The Histone N -Tenninal Tails Stabili%e DNA Wrapping around the Octamer

The structure of the nucleosome also tells us something about the histone N-terminal tails. The four H2B and H3 tails emerge from between the two DNA h elices. Their path of exit is formed by two adjacent minor grooves, making a .. gap'' between the two DNA helic$ ju~ big enough for a polypeptide chain (Figure 7-26a). Strikingly, the H2D and H3 tails emerge at approximately equal distances from one another around the oc:tamer disc (at approximate ly 1 o'clock and 11 o'clock for the H3 tail~ and 4 o'clock and B o'clock for H2B). The H4 and H2A tails e merge from either tbe "top" or "bottom·· face of the octamer and are located at 3 o'd uck and 9 u·dock for H4 and 5 o·clocl< an d 7 o'clock for H2A (FigtJre 7-26b). By emerging both betwoon and on either side of the DNA helicE!S. the histone tails serve as the grooves of a SCI'f1W, directing the DNA to l.vrap around lhe histone octamer disc in a lef1-handed manner. As we discussed in Chapter 6 , the left-handed nature of the ONA v.-Tapping introduces negative supercoi1s in the DNA The parts of the tails most proximal to the histone disc (and therefore not subject to the protease cleavage discussed above) also make some of the many hydrogen bonds between the hi~toncs and the

DNA as they pass by the DNA.

b

a

H4

H4 FIGuRE 7-26 lbe h~e tails eme~p fnMn the core of ttle nudeosome at specific

(a) The~ \/lew ~lu5tretes thai the H3 and H2B falls emerge fr0111 between the two ONA below both DNA helices. (Luger K.. Mader AW, F poiiliotl5.

hei~ In cOI"tt:rast, U1e H4 and l12A tails emerge eilher above or

Script. Mo!Solpt, and Rastes 30.

100

Cbromosom~s. Chr'OlfiOIIn . and

the Nud~som e

HIGHER..QRDER CHROMATIN STRUCTURE Histone H l Binds to the Linker DNA between Nucleosomes

H1 bot.md !fiGURE 7·27 H;str,me Hl binds two DNA hei"N:es. Upon mteraet~ wah a nudeosome. histone HI bros to the ~nker DNA <11 one er.d cl the nudeoson-e and the central

DNA hettx of !he nudeosome bound DNA (the rr.iddle ot the 147 bp bound bylhe core

hiStOne oaarner).

F I G U R IE 7-28 1'he addition oUtl leads to more compact nudeosomaf DNA. l he twO images show an efectron mtcrogrC!Jlh of 11uc:leo<;cmal DNA in the presence (<~) and at>sen:e (b} of hlstor.e t-Il Note the rrore rot"fllOC1 and defined structure of the DNA rn the presence of hrstone Hl (Soorce-lhoma et

al. lnvolvement of tustone Hl II'\ the orga~·u~a t101'1 cf the 11U(k050r1'1E:.1 een Biology. 63: 410. ~4&6)

Once nucleosornes are formed, tho next step in the packaging of DNA is the binding af histone Hl. Like the core histones, Hl is a small, positively-charged protein (Table 7-5}. Hl interacts wUh the linker DNA between nucleosomes. further tightening the association of the DNA with the nucluosome. This can be de tected by lhe ina-eased protection of nudeosome) DNA from micrococcal nuclease diges1ioo_ Thus. in contrast to the 147 bp protected by the core histone.s. addition of rustone Hl to a nucleosome protects an additional 20 bp of DNA from rnicrocoa::al n uclease dige&1ion . Histone H1 has the unusual property of binding two distinct regions of the DNA duplex. Typically. these two regions are part of the same ONA molecule associated with a nudeosome (Figure 7-27). The sites of Hl binding are locatod asymmetricalLy relative to the nucleosome. One of the two regions bound by H1 is the linker DNA at one end of the nucleosome. The second site of DNA binding is in the middle ol the associated 147 bp (the only DNA duplex present a1 the dyad axis). Thus, the additional DNA, protected from nuclease digestion dc..q;ribed above, is restticted to linker DNA on only one side of the nucleosome. By bringing these two regions of DNA into close proximity, H1 binding increnses the length of the DNA wrapped tightly around the histoneoctamer. H1 binding produces a more defined angle of DNA entry and exit from the nudP.Osome (Figure 7·Z8)~ This effect, which can be visualized in the electron microscope, results in the nucleosomat DNA taking on & distinctly zigzag appearance. The angles of entry and exit vary substantially depending on conditions (including salt concentration, pH, and the presence of other proteins)_ If we assume these angles are

1-ligher-OnlerChrnmaUn Structure

1.61

FIGURE 7-29 Histone HI induces fighter DNA wrapping around tile nudeosome. 1he two int.'Strcltions sho.N a comparison of lhe ~ng of Ct-JA around lhe nudeosome in the ~esef1Ce and iDsence of histone H 1. Clrle histone Hl can associdte with

each nudeosome Histone HI biflds to both inke.f DNA and the DNA ~dx located m the middle of the R..Jdeosome-bound DNA.

approximate ly 20° relative to the dyad a.xis. this wouW result in a pattern in which nucleosomes would alternate on eithe.- side of a central region of linker DNA bow1d by historte H1 (Figure 7-29).

Nucleosome Arrays Can Fonn More Complex Structures: the JO..nrn Fiber Binding of l-11 stabilizes highel"-order chromatin structures. In the test tube. as sall concentrations are increased. the addition of histone H1 results in the nudeosomal DNA forming a »nm fiber. Thjs structure. which can also be obsetved in vivo, represents tltc next level of DNA compaction. More importantly. the incorporation of DNA into this fiber makes the DNA less accessible to many DNA-dependent enzymes {such as RNA polymemses). There are two models for the structure of the 30~nm fiber_ ln the solenoid model, the nucleosomal DNA forms a superhelix containing approximately six nucleosomes per tum (see Figure 7-18a). This structure is supported by both EM and X-ray diffraction studies, which indicate that the 30-nm fiber has a h elical pitch uf approximately 11 nm. This is also the approximate diameter of the nucleosome disc, s uggesting that the 30.nm tiber is composed of nucleosome discs stacked on edge iu the form of a helix (Figvre 7-30a). lu this model, the flat surfaces on either face of lhe histone octamer disc are adjacent to each other and the DNA surface uf the nucleosomes forms the outside accessible surface of the superhelix. The linker DNA is buried in the center of the superhelix. but it never passes through the axis of the fiber_ Rather. the linker DNA circles around t.he central axis as the DNA moves from one 11 urJeosome to the next

16Z

ChromosolllBS., Chrumolin. cmd the Nudeosome

7-30 Twomodefsfortlle »nm dvomatin fiber. (a) The solenoed model. Note that the linker DNA does not pass thfoogh the centrdf axJS of the Sl..lpefhelix and fl(iUitE

a solenoid

e»t

that the sides and entry ar.d poil'\ts of the nudeosomes are relatively ir.accessible. (b) The "zjgzil( model. In this ~l the ~nker DNA frequentty passes lhrot.lgh the central aJOS of the fiber and the sides and even the entry and E')(lt points are more accessiJie. (Source: Pollard T: ar.d Earnshaw W 2002. Cell biology, 1st edtion. p. 202, f1 3-6. Copyright © 2002. Reproduced by pes-mission of WB. S.u1ders Inc.)

bzigzag

',_Jtk- ~ s41 ~ tJ linker DNA

An alternative model for the 3D-run fiber is the uzigzag,. model (Figure 7-30b). This model is based on the zigzag pattern of nucleosomes forn1ed upon Hl additio11. In this case. the JQ-nm fiber is a compacted form of these zigzag nucleosome arrays. Analysis o f lhe spring-like nature of isolated 3(}-nm fibers supports this zigzag model. Unlike the solenoid model, the zigzag conformation requires the linker DNA to pass through the central axis o f the fiber in a relatively straight form (see Figure 7-30b}. Thus, longer linker DNA favors this coiuormation. Because the average linker DNA varies between d ifferent species (see Table 7-4), the form of tho 3o-nm fiber may not alw ays be the t>ame.

The Histone N-Terminal Tails Are Required fer the Formation of the 30-nm Fiber Core histones lacking their N-terminal tails are incapable of forming the 30-nm fiber. The most likely role of the tails is to stabilize tbe 3Q-nm fiber by interacting with adjacent nucleosomes. This model is

supported by the

thr~dimenslonal

structure of the nucleooomc,

which shows that the amino terminal tails of H2A, H3, and H4 each interact with adlacent nucleosomes in the crystal lattice (Figure 7-31). For example. the histone H4 N-terminus makes multiple hydrogen bonds with H2A and HzB on the surface of an adjacent nucleosome in the crystal The residues of H2A and H 213 that interact 1Arith the J I4 tail are conserved across many eukaryotic organisms but are not invol\Ted in DNA binding or formation of the histone octamer. One pos:,ibility i~ that these regions of H2A and H2B are conserved to mediate inter· nucleosomal interactions with the H4 tail. As we shall see below. the histone tails are frequent targets for modification in the cell. lt is likely that these modifications influence the ability to form the 3o-nm fiber and other highftr-order nucleosome structures.

Further Compaction of DNA Involves Large Loops of Nucleosomal DNA Together. the packaging o f DNA into nucleosomes and the 30-nm fiber results in the compaction of the Jine.ar length of DNA by approxi-

Higher-Order Chromatin S tructure

163

FIG U R E 7-31 A speculative model

for the~ of the »nm fiber by histone N-.t~ tails. In ttls lllOdel the 30-nm fibef ts ilustrated using the "ztgzag"

30-nm fiber

modEl Several different tail-histone core intetac\lons are possil:k Here the interadions are shown as between every alternate hstooe tu they could also be IIVIth aq~nt cot' more distatlt histores.

mately 40-.fold. This is stiU insufficient to fit 1 - 2 meters of DNA into a nucleus approximately to-s meters across. Additional folding of the 30-nm fiber is required to compact the DNA further. Although the exact nature of thls folded structure remains unclear, one popular model proposes that the 30.nm fiber tbrms loops of 40- 90 kb that are held together at their ba...c:es by a proteinacious structure referred to as the nuclear scaffold (Figure 7-32). A variety of methods have been developed to identify proteiJJs that are part of this structure although the true nature of the nuclear scaffold remains mysterious. Two classes of proteins that contribute to the nuclear scaffold have been identified. One of these is topoisomerase U (Topo fl). which is abundant in both scaffold preparations and purified mitotic chromosomes. Treating cells with drugs that result in DNA breaks at the sites of Topo 11 DNA binding generates DNA fragments that ate about 50 kb in size. 'fhis is similar to the size range observed for limited nuclease digestion of chromosomes and suggests that Topo ll may be part of the mechanism that bolds the DNA at the base of these loops. The SMC proteins are also abundant cmnponents of the nuclear scaffuJd As we discussed uarlier (see section on Chromosome Duplication and Segregation). these proteins are key components of the machinery that condenses and holds daughter chron1osomes together after chromosome dup]it:ation. The associations of these proteins with the nucleru scaffoJd may serve to enhance tJ1cir functions by providing an underlying foundation for their interactions with chromosomal DNA.

Histone Variants Alter Nudeosoroe Function The core histones are among the most conserved ~ukaryotic proteins; the nucleosomes formed by these proteins are very similar In aJl eukaryotes (Figure 7-33a). But there are several h istone variants found in eukaryotic cells. Such unorthodox histones can replace one of the four standard histones to form alternate nucleosomes. Such nudeosomes may serve to demarcate particular regions of chromosomes or confer spedalized functions to the n udeosomes into which they are incmporated. For exrunple, H2A.z is a variant of H2A that is widely distributed in eukaryotic nucleosomes and is generally associth~fore.

~64

Chromosomes, Chmmatin. and the N ucltKJt;ome

a

b

------ - - - -- -

- - - - - - - --

FIGURE 7-32 Tbe higtlet-ctdet structure of dmnnatin. (a) A transmission electron mcrogr~ sllows chromatm emerg~ from a central sti\.!Ct\Jre of a chromosome. The electJoo-dense regions are the ru:.lear sCAffold that acts to organze the large amounts of DNA foond in et.byotic duomosomes. The bar represents 200 nm (b) A model for the struct\.Jre ol a eukai)'Ohc chomosome shows that the majonty d the rnA IS padaged into large loqls of 30·nrn fibeJ that ~e tethered to the nuclear~ at their~ Sttes of actiVe [l\IA rnatllplllatlon {for example, sites c4 transcnption ~ ONA rephcat100) are m the lorm of 1Q.nrn flbel or even OQked DNA (Source: (a) Courtesy of J.R Pat.Asoo and U.K laemmli.)

ated with tmnscrib€d regions of DNA. There is little change in the overall structure of a nucleosome containing this variant histone. Instead. the presence of the H2A.z histone inhibits nuclcosomes from forming repressive chromatin structures. creating regions of easily accessible chromatin that are more compatible with transcription.

a noonal (nonvanant) hlstones

b with CENP-A .........-

...

irlteraciiOn With kinetochore protein

\

FIC#UR E

7•33 Alteration of chromatin by in(orpoRiion of histone variants. (a) Transition befQ sti.lndafd hestones. (b) ~n of CENP-A 10 J:,lace of hlstooe H3 as

tv.~ 1(}-mt u.d 30-r!m fibers

proposed to act as a binding Site fcc one cc- more CCfll'lOnenlS of 1he klne4oehora

A second histone varian t, CENP-~ is associated with nucleosomes that include centromeric DNA. In this chromosomal region. CENP-A replaces the histone H3 subunits in nucleosomes. These nucleosomes are incorporated into the kinetochore which mediates attachment of the chromosome to the mitotic spindle (see l-'igure 7-12}. Compared to H3, CENP-A includes a substantiaJ extension of the N-term.inal tail region. Thus. l ike nucleosomes with H2A.z. it is unlikely 1hat incorporation of CENP-A changes the core structure of the nucleosome. Instead, the extended tail of CENP-A may generate novel binding s ites

for other protein components of the kinetochore (Figure 7-33b}. Given the critical role of the histone N-tennini in the formation of higher-order chromatin stt·uctures. these changes may alter the interactions between uucleosom6s at the centromare/kinatochore as well.

REGULATION OF CHROMATIN STRUCTURE The Interaction of DNA with the Histone Octamer Is Dynamic As we will learn in detail in Chapter 17. the incorporation of DNA into oucleosomes can have a profnund impact on the expression of the genome. In many instances it ;s critical that nudoosomes can he moved or that their grip on lhc DNA can he loosened to allow access to particular regions of DN.A. Consistent with this .requirement, the association of the hJstone octamer with the DNA is in herently dynamic. In addition, there are factors that act on the nucleosome to increase or decrease the dynamic nature of this association. Together, these properties allow changes .in nucloosome position and DNA association in response to the frequently <.;hanging needs for DNA accessibility. Like all interactions mediated by noncovalent bonds, the association of any particular region of DNA with the histone octamer is not permanent: any individual region of the DNA will transiently be released from

1.00

Chromnsomm;. Chcomntin. ond the N11deoson!fl

7-34 A model for gaining access to nudeosom*"assoc.i21ted ONA.

fiGURE

Studies of the ability of ~fiC DNA· bulaity of the DNA. Thus, DNA sttes dosest to the entty and e>al points are the most dCO:Sslble and sites closest ro the midpoint of the bound DNA are least access~ble

tight interaction with the octamer now and then. This release is analogous to the occasional opening of the VNA double helix (as we discussed in U.apter 6). The dynamic nature of DNA bindmg to the histone core structure is important. because many DNA-binding proteins strongly prefer histone-free DNA. Such proteins can only recognize their bincling site when it is rele~ from the Mstone octamer or is contained in linker or nucloosome-free DNA. As .a result of inlerwltlent, spontancuus unwrapping of DNA frum lhe nucleosarne, a protein Gan gain actxlSS to its DNA-binding sites with a probability of 1 in 1,000 to 1 in 100.000 , depending on where t·he binding s ite is within the nudcosome. Tho rnors central the binding site. the l.ess freq uently it is accessible. Thus, a binding site near position 73 of the 147 base pairs tightly associated with a n udeosome is least frequently a~ssible. whereas binding sites near the ends (positions 1 or 147} of the nucleosomal DNA are most frequently accessible. These findings indicate that tho mechanism of e~ posuro is due to unwrapping of the DNA from the nudeosomc, rather than to the DNA brieOy comi11g ofT the surface of the histone octamer (Figure 7-34). It is importanl to note that these studies were periormed un a population of lndi\lidu al nu cleosomes in a test tube: the a bility of DNA to unwrap from the nucleo-.o:;ome may be different for the large nucleosomal arrays in tl1e celt

Nucleosome Remodeling Complexes Facilitate Nudeosome Movement The stability of \he histone octamer-DNA interaction is influenced by large protein complexes referred to as nucleosome remodeling c~m:pl~ 'Tb.ese multi-\)rotein com{l1P.xes facilitate chan~es in nudeosome location or interaction with the DNA using the energy of ATP h ydrolysis. These changes can come in three flavors: (11 "sliding"" of thtJ histone octamer along the DNA (f'i gure 7-35a}, (2} the complete " transfer" of a histone octamcr from one DNA molccuJe to another (Figure 7·35b}, or (3) the "remodeljng" of the nudeosome to allow increased access to the DNA (Figure 7-35c).

Regule1tion of Chmmntin Structure

a sliding

c

b transfer

FIGURE

7-35

16 7

1\ktdeosome movement

utalyud by nudeosome remodeling actMties. (a) Nudeosome movement by siding along a DNA rooko.Jie exposes sites for DNA-binding proteins. (b) Nudeosome IT'()Vernent Cdn altemati\iely occur by tJanSfer of the nudeosare from one strand of DNA lO another. (c) Remodeling akt.Ns association of a ONA~cing protei~'~ Without altefilll its posrtion on DNA.

+

All nucleosome remodeling complexes can facilitate nucleosome sliding. however, onJy a subset have the ability to transfer or remodel nudeosomes without altering their position on the DNA. The exact structural alterations of tl1e nudeosomc that lead to remodeling are not clear. Nevertheless. it is clear that the DNA associated with the~ ..r~ modeled.. nucleosomcs is more accessible. There are multiple types of nudcosome remodeling complexes in any given cell (Table 7-6j. They can have as few as two subunits OT more than 1 0 subunits. Although the ATP hydrolyzing subunit is relatively well-conserved among these different complexes, the addition of different subunits can modulate functiDn. For example. these complexes can include subunits that target them ro particular chromosomal locations. In some instances, this targeting is mediated by interactions between subunits of the remodeling cornple.x and DNA bound tranScription fac._ tors (Figure 7-36). In other instances, l~lization can be mediated through intm-actions with specific modifications of the histone subunits themseh•es (via chromo- o.r bromodomains, as we shall see beJow ).

TABlE 7-6 Nudeosome Remoclet. . and Modifying complexes

AJ'P...oependen\ ctwomatin RfltK)deling Complexes

Tvpe

Number of subunits

Bromodomainla.omodomain

Slide

TranSfer

Restructure

8 - 11 2- 4

8romodoma1n

Yes

Yes

Yos

fSWI

No

Mi2JNuRD

8 10

Chf0mcdorna1n

Yes Yes

No No

No

swt/SNF

No

1118

Chro1Tl0$nnw.s. Cl1ramatin, ond the Nucleasome

FIGtJRE 7•36 Twomodesof DNA-binding protein-dependent nudeo$orne positioning. (a} Association of

a

c:::- ff - C )_ -

many DNA·binding proteins IMth DNA is 'ncompatible With the assoootion of the s~.e ONA vith the t1istone octamer. Because a nudeo

0 -

:.:::::==.J/f ==-

130bp

some requites rnxe than 147 bp of DNA to forrn, if tvvo such fac1ors btnd to the OtJA le.s than ths distance apart. the inte.Nerm~ (.'filA camot assemble 1nto a nudeosome. (b) A subset of 01\JA.bindif]g proteins hove lhe ability to bind to 11\Jdeosomes. Once bound lo DNA. such pr~ Will faolitate the ~obly of n\.1deosomes .rnmediately adjacent to 1tle proterfs

nucleosome-free

tNA-hJnOOg site. b

Some Nucleosomes Are Found in Specific Positions in vivo: Nudeosome Positioning Because of their dynamic interactions with DNA. most nudeosomes are not fixed in their locations. But there are occasions when restricting nudeosorne location, or positioning nucleusomes as it is called. is beneficial. Typically. positioning a nudcosome allows the ONA binding site for a regulatory protein to remain in the accessible linker DNA region. In many instances, such nucleosome-free regions w·e latgcr to allow extensive regulatory regions to remain accessible. Nudcosome pobitioning can be directed by DNA-binding pToteins or particular DNA sequences. In the c:ell, the most frequent method involves competition between nud eosomes and DNA-binding proteins. Just as many proteins cannot bind to DNA within a nucleosome, prior binding of a protein to a site on DNA can prevent association of the core histones with that stretch of DNA. [f two such DNA-binding proteins are

bound to sites positioned closer than the minimal region of DNA re-quired to assemble a nucleosome (- 150 bp), the DNA between the proteins will remain nucleosome-free [Figure 7-36a). Binding of additional proteins to adjacent DNA can further increase the size of a nucleosom~ free region. In addition to this inhibitory mechanism of proteindependent nucloosome positioning. some DNA-binding proteins

Plislone octamer

- - - -- --- -

7-37

Nudeosomes prefs IO .,;nd bent DNA. Speafic DNA seqoeores c.an positiOn rlvcleosornes.. Because the ONA IS bent severefv dwing assoc.iaoon ~ the nudeosome, DNA ~ that position nucleos0111t'S ~ mtrmcatly bent I"~T base pars have an lnmosic tendency to bend toWard the m nor groove and G.C base paifs have the OppOSit£ tendency. Sequences rh
interact tightly w ith adjacent nucleosOlnesr leading to nucJeosomes preferentiu /ly assembling hnmedjately adjacent to these proteins (Figure 7-36b). A second method of nucleosome positioning involves particular DNA sequences that have a high affinity for the nudeosome. Becai.JSi} DNA bound in a nucleosome is bent, nuclC050mes preferentially form on DNA that bends easily. A:T-rich DNA has an intrim,i c tendency to bend toward the minor groove. Thus. A:T-rich DNA is favored in positions in which the minor groove faces the histone octamer. G:C-rich [)N:A h as the opposite tendency and, therefore. is fuvored when the minor groove is facing away from the histone octamer (Figure 7-37}. Each nucleosome will try to maximize this arrangement of A:T-rich IUld G:Crich sequences. It is important to note that such a lternating stretches of A:T-rich and G:C-rich DNA a re rare. More importantly. despite ~ing favored . ,;uch unusual sequences are not required for nucleosome assembly. These mechanisms of nucleosome positioning innu ence the organization of nucleosomes in the genome. Despite this, the majority of nucleosomes are not tightly posit ioned. As } ' Ou will learn in the chapters on euktuyotic transcription (Chapters 12 and 17), tightly positioned nucleosomes are most often found at s ites directing the initia tion of transcription. Although we have discussed positioning primarily as a method to ensure that a regulatory DNA sequence is accessible. a positioned nud eosome can just as easily prevent access to specific DNA sites by being positioned in a manner that overlaps the same sequen ce. Thus, positioned nu cleosomes can have both positive and negative effects on the accessibility of nearby DNA sequences. An apprnach to mapp ing ntJcloosome locations is rlru:crihed ill Box 7-2. Determin ing Nuclcosome Position in the Cell.

Modification of the N ..Teminal Tails of the Histones Alters ChTomatin Accessibility When histo11es are isolated froiil cells , 1heic N-terminal tails are typically modified with a variety of small molecules (Figure 7-38). Lysi nes

170

Clu'Omosomes, Chromctin . ruW the Nuclea;ome

in the tails are frequently modified with acetyl groups or methyl groups and serines are subject lo modification with phosphate. Typically, acetylated nudeosomes arc associated with regions of the chromosomes that are transcriptionally .active and deacetylated nucleosomes are associated with transcriptionally-repressed chromatin. Unlike acetylation. methylation of different parts of the N-tenninat tails is associated with both repressed and active chromatin, depending on the particular amino acid that is modified in the histone tail. Phosphorylation of the N-tenninal tail of histone H3 is commonly observed in the Wghly-condensvd chromatin of mitotic chromosomes. It has been proposed that these modifications result in a ..code" that can be read by the proteins involved in gene expression and other DNA transactions (Figure 7-38). How does histone modification alter nucleosome function? One obvious change is that acetylation and phosphorylation each act to reduce the overall positive charge of the histone tails; acetylation oflysine neutralizes its positive chruge {Figure 7-39}. This loss of positive charge reduces the affinity of the tails for the negativtJiy-clu1rged Lac:kLone of the DNA Equally important, modification of the histone tails affects the ability of nncleosome aiTays to form more repressive higher-order chromatin structure. N3 we described above. histone N-terminal tails are required to fonn the 30.nm fiber, and modification of the tails modulates this function. For example, amsi.stent with the association of acetylatcd Wstones with expressed regions of the genome, nudeosomes with this roodifi0:1tion are significantly less likely to participate in the formation of the repressive 30-nm fiber.

The signifiCance of the. location of nudeosomes adjacent to important regulatOJy sequences has led to lhP developrnen1 of methods to monitor the location of nudeosomes in cells. Many of these methods e>qJiort the ability cA nudeosornes to protect ll'JA fran digestjon by miOUCoo:al rnJdease. M. described in Box 7- 1, micrococcal nuclease has a strong preference to 001vc DNA bervveen nudeosomes rather than DNA tigl1ly associated with nudeosornes. TllS property can be used to map ni..Jdeosomes that are asso~ \o\.Jjth the same position throughout a eel pq>ulation (Box 7-2 figure t ). To map nucleosome locaftln accurately, It cs Important to ISOlate the ceCiular chfornabrl and treat it with the appropriate amol.Wlt of micrococ.c:al nuclease with minimal &sruption cA the ~rctD chruna6n structure Uis is ~pically achieved by gently lysing eels vvhle leaving the fluclei intact The nudet are then briefly treated (typtcally tor 1 rnilute) With several dff~ coocentraoons of miaococcal nuclease. a proteio smal enough to rapidy diffuse into the nucleus. lhe goal of the titratioo is f{)( rOCrocow!l nuclease to cleave the region of interest only once in each cell. Once 1he DNA has beEn digested, the nuclei can be lysed an
To Identify the sites of deavage in a particiJar regiof\ it is oec€:5SafY to create a defined end point for allhe cleaved fragments and exploit the specificity of DNA hybridization. To create a defined end pant, the punfied DNA from each sample as cut Wlth a msm~n r.nzyme kOCMIIl to cleave adjacef)t to 1he site of interest. After separation by StZe using agarose geJ electrophoresis, the DNA IS denatured and transhmed to a nit:roceiUose membrane. This allows a labeled DNA JXObe of specific ~ to hybridize to lhe DNA (this is called a Southern blot aod is descmed in more detail in Chapter 20). In this case, the OOA probe IS carefully d105etl to hybndlze immediately adjacent to the restric00r1 enzyme cleavage ste at the site of interest Plter hybridz.ation and wast.~ the lJNA probe will shoN the size of the flagrnerts genetated by microtDC.CCll nuclease in the region of intefest How do the fra6'f>ent SJZes reveal the loc
appear as a latge gap •n the laclc!cr of DNA bands detected oo the Soothem blot Frequently. there are arrays of positioned nlde«:>soo1es ~ding to a stnilar 160- 200 bp perioddty to sites of deavage aod protection.

~~~~

7-2 f IGURIE 1 Analy5is of n&JCieosoene positioning in the c.ell The experimental 51E;>S in determning nudeosome postbOning in me eel are illustrated See box text for det:ils. BOX

l

isolate oodei

c. o~ggo 0

induce dotA:lle-slrand bc'eal<.s in linl<er DNA with

1

micrococcal nJdease

'"~:\

• MNase etA sMs [ ' RE deavage sfles

~v ' ~ I ~. . 1cut

isolate cleaved DNA and with res4riction enzyme

~

0

I~ 1111111111 11111111111 111111 L___lL__ ____JL____j

31'd

2nd

1st

cleavage at specific regions between positOned

nudeosomes

171

Cl1romosom£'l;, Duvmatin. and the NuclrfOSOnlf1

FIGUR£ 7-38 Modificationsofthe histone H-terminal tails alters the function of duomatin. lhe Sites of knoiM1 histone moaf~eations atf' ilt.Jstrated on eadl histone The ITia)
histone-fold domain

=::;;f===:8/I~ c

H2A

N 8 :1 H28

c

7 9 K · --· NI-----t-------~//N~----------~~- gene silencing

...,..t________,.,I,,_ -

N

N! ......................

gene expression Jistone

deposttioo

get"'esiler1cing/ ~,I~--~~00

N •---------r l / ' 1 N ----tt- --.t . . .~'1f

T

!

1r

~e

condensatioll

gene

expression

..........................//---?

N~

N

,.

I

tJ'an~

·------------~~~-- ~~

H4

N

N _ _ _ _ _ _ _ ___,, _

~

172

histone

N -------~----~-----r~- deposltioo N ....c=-11!""""!!!!~~-~...a-t't'

gene

.

exp"es81011

In addition to direct effects on nucleosomal funr.tinn. modifi('.ation of his tone tails also generates binding sites for proteins (Figure 7-39b). Spocific protein d omains ca11ed brotnodotnains and chromodomains mediate these interactions. Bromodomain-containing proteins interact with a cctylatt:rl bit---tone tai ls and chromorloma.in-containing proteins interac;t with methylated hi~1one tails. Many of the proteins that contain bromodomains are themselves associated with histone tail-specific acetyl transferases (Table 7-7}. Such complexes can facilitate the ma\ntenance of acetylated chromatin hy further modifying regions that are already acetylated (as we shall discuss below). The association of chromodomain-containing proteins with histone tail-specific methyl-

RfJguloti<m of Chromatin Structure

unmodified

methytated

H2A

+ .....

+y

1 --

-t----

173

--'J

+ +H3 ~ If + 0

+'J ( .. H4....

.,

+~ ~' ++ ~ ~

H2A

l

~+ -!~ H4 -=~' ~~+• • Q --... ++

H28 H4

H3

+

+ ' +

chromo

- -

)l

+ L+ n

+I,·-.

++ H4

+

chromOdomain protein

+. n

+ + H4

a: 1G tJ R E 7-19 Effects of histooe tail modifications. (a) The elfect on the associ.:ilion IMth trud~d DNA. Unmodified and methyiated hslooe I¥ are thought to assoc:iat:e mOfe tigJ1tly 1Mth nudeoscmal rxvA than ~ed htStone ta.ls. (b) ModificatiOn of histone taifs aeates bu-dmg SiteS for

dlromdtin-modifying enzymes.

ating enzymes suggP.Sts a similar mechanism for the maintenanc.e of methylated nudensomes (Table 7~7}. Other bromodomain- and chromodomain..:ontaining proteins are not histone modifying proteins but instead are proteins jnvolved in regulating transcription or the fonnation of heterochromatin. For example. a key component of the transcription machinery called TFIID also includes a bmmodomain. This domain dire~ts the Iranscription machinery to siles of nudeosome ac.:etylation. which con-

tributes to the increased tra nscriptional activity of th e DNA assod aled with acetylated nucleosorues. Similarl)'• nucleosome-remndeling complexes ftequently include subunits with bromodomains (Table 7-7).

Specific Enzymes Are Responsihle for Histone Mooification The histone modifications we have just de&:ribed are dynamic and are mediated by specific enzymes. Histone acetyl transferases catalyze the addition of acetyl groups to the lysines of the histone N-termini. whP.reas histone deacetylases remove thP.Se modifir.ations. Similarly.

histone methyl transferases add methyl groups to histones (histone demethylases have yet to be identifi.etl). A number of different histone acetyl transferases have been identified and are distinguished by their abilities to target different h istones or even different lysines in the same histone taiL Similarly. each histone methyl transferase targets

174

TABl.E

Chmmm;omes. Chromolm, ond U1e N1Jr.feosvme

7-7 N&ldeosome Modifying Enl)'mes

Type

Number of subunits

SAGA

PCAF NuA3

Catalytic subunit

Bromodomaini'C11l'omodomairl

15

GenS

Brorr~odomain

11

PCAF Sas3

Bromodomaln Nfllther

Fsa1 P300/CBP

Ouornodomaln

Catalytic subunit{s)

Bromodomaifl/Chromodomain

NuA4

3 6

P300/CBP

1

Type S~comp~x

NvRD SIR2 Com pie><

Number of sWt!nits

7 9 3

HOAC 1/HOAC2

Neit~er

Chromodomain

Slr2

Nerther

Name

BromodomainiChromodomain

Target histone

SlN39/CLR4

Chromodoma.tn Neith<.>r Neithef

H3 (lysine 9) H3 (Lysine 4) H3 (Argirune 3)

Str1 PRMr

H3 and H2B H3endH4

H3 H4

H2A. H2B. H3. and H4

Bromodom&Jn

HDAC 1/HDAC2

Target histones

specific lysine or arginine on spedfic histones (Table 7-7). Because d ifferent modific:ations have different effects on nudeosome function, the modification of a nucleosorne with different h istone aC€tyl transferases or methyl transferases can result in various effects on chromatin structure and function (see figure 7-3tH. Like their nucleosome remodeling complro<: r,(lunterparts. these mod ifying enzymes are part of large muJtipmtein complexes . Addilional subunits play important roles in recruiUn~ these enzymes to specific regions of the DNA. Similar to the nudeosome-remodeling complexes, the~e interactions can be with transcription factors bound to DNA nr directly with mndified nudensotnP.s. The recruitment of the~ enzymes lo partic:ular DNA regions is responsible for the distinct patterns of histone modification observed along the chromatin and is a major mechanism for modulating the levels of gem~ expression along the eukaryotic chromosome (see Chapter 17),

Nucleosome Modification and Remodeling Work Together to Increase DNA Accessibility TI1e combination of N-tenninal tail modifications and n ucleosome remodeling can dramatically change the accessibilily of the DNA. As we

will learn in Chnpters 12 and 17. the protein complexes involved in these modifications am frequently recruited to sites nf active transc:ription. Although

th~ order nf theirfunr.lion

is not always the same. the

combined action can result in a profound, but localized, change in DNA accessibility. Modification of N-terminal tails can reclure the ability ot'

Nvcleo.o;onTP 1\SSfmlbly

7-40 Chromatin remodeling complexes and histone mocifying enzymes work together to alter ctnomatin 51:ructure. Sequence-speOfi IJNA..blrlding protetns typi c4y reaut these enzymes to speofic ff!StOnS of a chiOI110scJ"I'Ie. fn the llustl"i!liJCn, the first DNAbndtng proten recruts a dYOCT'Iatln rernodeirls CClfl1'lex that modiiies the adjacent nucJeosome. ~the cnes~ry of the assooated ~ lh.s, allows the btndlllg ol a second I:X\IA· bndng prater~ that tecrutts a histone acetyl transfefase. Bo; modfymg the N term.nal r.,lls of the ~ nudeosomEs, this enzyme chal\5es the c:orformatKlll of the chrtvnatm ffOITl the 30-nm form ro the more acce5Sible 11-nm foor Although we shew the order cJ as.500cltloo as d110Ft Gu R£

1 75

JO.fVn

chromatin

tibet'

ordefs are observed and CMI be that rea\lllmeflt of a histone methyl transfcrilSe Jn51edd of ~ htstone acetyltJaosJerase co.Jd res~t IIi lhe fCJI'ITlClOOn of I"'1Clre c.ompact and ~ dvomalln.

mahn remodeling complex then h!Stooe ac:etyf transferase, bah eq._~aly effec.twe. It IS also true

nudensomA :nrays tn form ropre;sive slrur.tlli'P,s, <:raating sites th.<'lt c:an recruit other proteins, including nudeosome remodelers. Remodeling of too nudeosomes c..:a n then further increase the accessibility nf the nucleOS{)(TI.8]. DNA to allow DNA-binding proteins access to their binding sites. In addition. these complexes can cause the sliding. or release. of the nur:leosomes. In combination with the appropriate DNA-binding proteins or DNA sequences, these changes can result in the positioning or relPASe nf nudee>Mmes at specific siles nn the DNA (Figure 7-40).

NUCLEOSOME ASSEMBLY Nucleosomes Are Assembled Immediately after DNA Replication The duplication of a chromosome requires replication of the DNA and the reAssembly of the associated proteins on each dAughter DNA molecule. The latter process i.s tightly Jinked to DNA replication to ens ure that the newJy replicated DNA is rapidly packaged in1o nudeosome s. Jn Chapter 8 we will discuss the mechanisms of DNA replication in detail. Here we discuss the mechanisms that dirAct the assembly nf m.tdeosomes after the DNA is rep1icated. Altho ugh the replit;ation of DNA requires lhe partial disassembly of the n ucleosome, the DNA is rapidl~ repackaged in an ordered sertes of events. As discussed earlier. the firsl step in the assembly of nucleosomes on the DNA is the binding of an H3·H4 te ll'amAr. Once the letramer is bound , two H2A·H2B dimers associate to for m th e fina l nucleosome. Hl joins this complex last. presumably during the formation of higher-order chromatin assemblies. To duplicate a chrornnsome. al Jess1 half of the nudeosomf'S on the daughter chromosomes must be newly synthesized. Are all the o ld histones lost and only new histones assembled in to n ucleosomes? rr not, how i!re the nld histones distributed between the two daughter chromosomes? The fote of the old histo nes is a particularly important issue given the effect modification of the hi~1.ones c.an have on the accessibUity of the resulting chromatin. 1f the nld histones were lost completely. then chromosome duplication would erase any .. memory" nf the previously modified nucleo~mes. In contrasc, if the old histones were retained o n a single chromosome~ that chromosome would havP a distinc;t set of modifications relative to the other eopy of thn chromosome.

r::) histone acetyl lransferase

ONA binding pr~ 2 rea\lits histone acetyl transferase

176

ChromosomFs. Chmma1.iv. and tlw Nuclcosomtl

f'IGU RE 7-41 The inheritance of

hKtones after DNA replkation. As the dtrornosome is repltcated. hJStones thai w~ asscx:>ated wth the parental ch1'01"11060me are differently distributed. The ht!.tone t13-H4 tetrcrners are randomly transferred to one Of the two daughtef strands btJt do not enter rnto the soll.i>le pool or HJ.H4 tetrar'lEfS. Newly synthesized H3414 tetramm foon the basis of the nudeoscrnes on the strand ttwl does not rilent the pa-ent:ll tetramer. In contcdSt, H2A and H2B dimers ~re refeased into the soluble pool and ~e fOf f-l3.ti4 association with newly synthesized H2A Qnd H26. As a c.onseqoence of ttis type of distnbution. m ~. every sea:nd H3-H4 tetlamef on nevdy syntheSized DI\!AWI be denved frcxn the pacen,al chr~ These terramers ..we ll'ldude 1he MOOflcations added to the parent~ ni..ICieosoma 'The H2A·H~B &nets are ITlO(e l!kety (0 be
new lhisklnes:

H.2A

0

H2B

r::_ H3

_H4

parental

nudeosorne DNA replication machinery "

aa

H2A•H28 dimer

H 3+14 !etmmer

1n experiments that differentially labeled old and new histones, it was fo und that the old histones ere present on both of the claughter chromosomes {Figure 7-41). Mixing is not entirely rnndom, howe"er. H3•H4 te\nuners and H2A•H2B dirners are composed nf either aU new or all old histones. Thus. a$ the replication fork pilsses. nucleosomes are broken down into their component subassemblies. H3•H4 tetramers appear to remain bound to one of the two daught~r duplexes at random and are never released from ONA into the free pool. In contrast. the H2A•H 2B dimers are released and enter the local pool available for new nudeosome assembly. The distributive inheritance of old histones dw'ing chromosome duplication provides a mechanism for the accurate propRgation of the parental pattern of histone modification. By this mechanism. o ld his-

tones. no matter on which daughter- chromosome they end up. lend tn be f(lund close, in .location. to their position on the parental chromosome (Figure 7-42). This localized inheritance of modified histones 11rovides a limited number of modifications in similar positions on each daughter chromosome. The ability of these modifications to recruit enzymes that add similar modifiGfltions lo adjacent nudeosomes {see the discussion of bromodomains and chromodomains above} provides a simple mechanism to maintain similar states of modification after DNA replication has occurred. Such mechanisms

are likP.1y to play a c.r:itk~ J role in the inheritance of chromalin states from one generation to another.

Assembly of Nudeosomes Requires Histone "Chaperones" The assembly of nudOOS
Nucleoson~e AssPmbly

_, H2A •

ok:l histones; new htstOl'lEIG:

H2S •

H3 •

H2A 0 H2S (' HJ

H4

C H4

~

replication f

okl H3•H4 tetramer

old H3-H4

tetramer

aeety1 lransfecase binds

acetylated hlslone tails

1

177

7·42 Jnheritance of pah!ntal HUt4 tetramers facilitates the inheritance

FIG U R £

of d!romatW! states.. As a chr()&TIC60me is replic:aled. the distrihrtlon o f the pctrental H3·H4 tetramers r:esUts 1n the daugtlter chro1"10501TleS reretvW'Ig the same ~fKOOons as the parent The ability or these modifications to teauit enzymes tllat pefocm the same modifications faolltates the c.arect prc:lpclgdtioo of the ScYne state of~ to the two daugllt€1' chromosomes. ~t1011es shown on the core regi(lrl!. of the histooes for ~· In reaUry, this modif~CU~ ~ geneealy on the N -termnal tais.

"118

Cbromosomes. Chromatin. aruJ the N ucleoSOll'll!

resulted in little or no nudeoSDmc formation. Instead . the majority of the histnnes aggregate in a nonproductive form. For correct nudoosome assembly, it was necessary to rnise salt r.oncentrations to very high levels (> 1 M NaCl) and then slowly rf!duce the concentration over many hours. Although useful fur 8$Scmbling nucleosomes for in vitro studies (such as for the structural studies of the n udeosome described earlier), elevated salt concentratlons are not involved in nudeosome assembly in vivo. Studies of n lldP,o,;ome assembly under p hysiological salt concentrations identified facturs required to direct the assembly of hlstonP.S (Jnto the DNA. These factors are negatively-charged proteins t b~t form complexes with either H3•H4 tetramers or H2A·1 £2B dimers (see Table 7-8) and escort them to sites of nucleosome assembly. Because they act to keep h istones from interacting with the DNA nonproductively, tbesP. factors have been referred to as histone chaperones (see Figure 7-43). How do the histone chaperones direct nucleosome assembl y lo sites of new DNA synthesis? Studies of the histone H3•H4 tetramer chaperone CAF-1 reveal a likely answer. Nucleosome assembly directed by CAF-1 requires that the target DNA is replicating. Thus, replicating DNA is marked in snme way for nudeosome assembly. Interestingly1 this mark is gradually lost after replication is complP.ted. Studies of CAF-1-dcpendent assembly have determined that the marie. is a ring-shaped s liding damp protein called PCNA. As we will discuss in deta il in Chapter 8, this factor forms a ring around the DNA duplex and is responsible for holding ONA polymerase on lhe DNA d uring DNA synttu:~is. After the polymerase is finished, PCNA is released ft•om tho DNA polymerase but is still linked to the DNA. In this condition. PCNA is availabJc to interact with other p roteins. CAF1 associates with the released PCNA and assembles H3•H4 tet111mers (.)referentially nn the PCNA·boond DNA. Thus. by associating -with a component of the DNA replication machinery. CAF·J is directed to assemble nudeosomes at sites of recent DNA re plication.

old histones:

H2A .

new histones: ~ H2A

0

H2EI . H2B

H3 .

H4

C H3 l- H4

7-43 Owomatin assembly~ tors facilitate the assembly of nudeosornes. Mer the rephcation fodl has passtxl, chromatin assembly factOfs chaperone free H3-H4 tetracn€fS (CAH) and H2A-H28 &ners (NAP-I) to the site of neiNfy replicated DNA. Once at rhE- nev.ty repllcat.ed DNA. l.h ese f"ICURE

factors tf
H3-H4 tetramer

sf!ding c:l.:!mp.s T~ nng-shaped auxilially repli cdlion factCM'S erri'de the DNA and are released

......._ H3-H4 tetramer

fra-n !he replicarion machinefy as the replication toe\< moves, A more de\al.led desmp110n of ~ ~damps ar~d !heir function in DNArepkation 15 presented in Otapter 8.

Summary

t79

TA Bl E 7-8 Prcpertiu of Jfistone Owlperones

Name

CAF·l

RCAF NAP-J

NtJmberof subunits

Histones bound

Interaction with s5ding damp

4 1 1

H3•H4 H3•H4' H2A•H2B

Yes No No

SUMMARY Wtthitl the cell. DNA is organized into large structures caUed chromosomes. Alth ough the DNA forms the foundations fur each chromosome, as much as half of each c::!J.romosOOle is composed of protein. Chromosomes can be either circular or linear. however, each ceU has a cbarnclcristic numlw.r and composition of chromosomes.. WE now know the sequence of the entire genome of ·n umerous organisms. Thf5e sequences have revealed that the llJld~r lying DNA of aac;h organism•s chromo~'Ome.s is u..-;ed more or IP.ss effidootly to encorle protcins. Simple organisms tend to nse the majority of DNA to encode protein; bowever, more comple" organisms use only a smalt portion of their DNA to actually e.ocode proteins or RNAs. ('..ells must cat6ful1y maintain their complement of chromosomes as they divide. Each chromost.Jflle must have ONA elements that direcl chromosome maintenance during cell division. All chromosomes must have one ot more Ol'igins of replicahon. Jn cukaryotic cells, Centrorneres play a critical role in I he ser.,regarion of r;hromosomeb and telomt>re$ help to protec:t and replicate the ends of lintmr chromosomes. Eukatyotic cells carefully separate the events thai duplicatt~ and segregate chromosomes as cell division proceeds. Chromosome segregation can OCClt{ in one of lwo manners. During mHosis, a highly specialized apparatus ensures tha• on e copy of each duplicated chr~ mosome i s delivert:d to each daughter ceU. During meiosis, an addilionaJ round nf chromosome segyegatio.o (wilh-

oot ONA replication) further reduca; the number of c::hromosomes in the resulting daughter cells. The combination of euknryotic DNA and its associated proteins is referred lQ ilS r:hromatin. The fundamental unit of duomatin is the nud~ome. which is mad e up of t·wo copies each of the cote hislonas (H2A. H2fl. H3. and H4) and approxirna.leSy 147 bp of DNA. This protein-DNA canplex serves two important t1mctions in lhe cell: it compacts the DNA to allow it to fit inlo the nucleus and it -restricts the acce.~ibility of the DNA. This l atttll" function is extensively exploilfld by the cell 10 regulate many different DNA transactions including gene expression. The atomic structure of the nucleosome shows that the DNA is wrapped about 1.7 tirrms around the outside of a disc-sbaperl. hislone protein core. The interactions between the DNA and lhe histones are extensive but unifonnly ba$e nonspecific. The .oature of these interactions explain both the b endh1g of the DNA around the h istone

od:amer and lhe· ability of virtually aU DNA sequences to be incorporated into a nucleorome. This structure also rev~ls the location of the N~t enninaJ tails of the histones and their rol£l in di.rflcting the pHth o{ the DNA around the histones. Onco DNA is packaged tnto nudeosomes. it bas the ability to form more complex structures that a llow arlditiooal compocti.Ofl of the ONA. This process is lncilitaled by a fifth histonu called Hl. By binding the DNA associated with the nucleosome, H1 et~uses the DNA to wrap more lightly around the octamer. A more compaut £orm of r:hromatin, the 30-nm fther. is readily fonned by arrays of Hl -bouncl nudeosomes. This structure is more reprcssive than DN A packaged into .oucleosome$ alorJe.. Current evidence suggests thai the incorporation of DNA into this structure results in a dramatic ~uclion in its aCCessibility to the enzymes and proteiru; involved .i n transcriplioo of the ONA. The interaction of the DNA with lhe bistones in the nuclcosome is dynamic, allowing DNA~binding prot·eins intel'flli"enl access to the DNA. Nucleosome-rerHodeJing complexes increase t he accessibility of DNA inoorporntcd into nucleosomes by in~sing the mobility of nucroosomes. 111ree forms of mobilily can be observed: sliding of the histone CICtamer along the DNA, complete lt"ansfer or the histQne octamer from one DNA molecule to another. and more subtle remodeling of the protein~DNA intetaclions within t.h(' nucleosomes. These complexes are localized to partic::ular regions of the genome to facilitala a lterations in chromatin accessibility. A subset of .oucleosomes is restricted to fixed positions in the genome and are sa\d t(l be "positioned ... NucJeosome positioning can be di-

ret-:ted by ONA-bindiog proteins Or particular DNA Sequences. Modification of tbe lljstone N-lerminal tails also a lters the aucessibility of chromatin. The lypes of modifications include Aeetylatiun and melhylation of lysines ~ tid phos()horylaHon of serioes. Acetylation of N-tcrmin2l tails is frequently associated with regions of active. gene expression. These modifications ruter both tbe properties of lne nucleosome itself as well as acting as binding sites for proteins that influenr..e the accessibility of the chromatiJL These modifications also recruit enzymes that perform the same modification. leading to similar modification of adjacent nudeosomes. It is likely that this leads to the stable

180

Chromosomes. Chromalfn. and tl1e NuclP.mome

propagation of regions o f modified nucleosorneslchromatin as the chromosomes are duplicated. Nucleosomes are assembled immediately aftP.r lhe DNA is replicated. leaving little time during whic h the DNA is unpackaged . This involves the funr.tion of specialized histone chaperones that escort the H3·1 14 tetrdJl'lers and .H2A·.H2D dimers to the replicatioo fork. OurinR the replia dian of the DNA nucleosomes are tran-

sicntly disassembled. Histone H3·H4 tctramers and HZA•H2B dimers are randomly distributed to ooe or thP. othez- daughter molecules. On average, e2ch new DNA moleclJie receives half o ld and half new histones. Thus, both r.hromosomes inherit modified histo(ICS which can then act as "seeds" for the similar modification of adjar.enl bistones.

BIBLIOORAPHY Books A lberts B., Johnson A ., L&wia J., RafT M .• Robt:rts K. , 81ld Waltor P. 2002. Mo/eculllr biology of the cell. 4th edi-

tion. Carland Science, New \'ortc Brown T.A. 2002. Genomes 2, 2nd edition. }ohn Wiley & Sons. New York, N ew York with BIOS Sclerttilie PubUs hers Umitcd, Oxfom, United Kingdom. Elgin S.C.R. and Workman J., cds. 2000. Chromolin slructu ro Olld gene expro.c;.<>ion, 2nd edition. Oxford University Press, Loodon, llnited Kingdom. Murray A. and Hunt T. 1993. The cell cycle: An introduction . WJ I. Freeman and Co.• New York. Weaver RF. 2002. Molecular biology. 2nd edition. Mc:-Graw-Hill ffigheT Educ.stion, New York. Wolffe A. 1998. Chromatin: Structure and function. 3n:1 edition, Academic Press, San Diego. California

Chroftl01i0mes Bendich A.J. and Drlir:a K. 2000. Prol-aryotic and e ukaryotic cll1'ornosomes: What's the difference? flioessay6 22: 481-486.

lntematio.na1 Hwnan Genome Sequencing Consortium 2001. lnitial sequencing BJl(l analysis of the human genome. Nature 409: 860- 921.

Nucleosomes Annunziato A.T. and l l.ansen J.C. 2000. Role of histone a<:etylatiun in the assembly and modulation of chromatin strur.tures. CJtP.nP. Kxpr S: 3 7-61. Belmont A.K, Oielw } S•. Nye A.C., Strukov Y.G.. and Tumbat T. 1999. Large-sm lc r.hromatin structure and function. Cu.u. Opin. Cell Bioi. U: 307-311.

Eberharter A.. and Becker P.B. 2002. Histone acetylation: A switch botwACn I'Cprcssive and permissive t:hro-malin. EMBO Reports 31: 224- 229. Gregory P.O.• Wagner 1C., and Horz W. 2001. Histon~ ecflt:Y· lation and c:hromali,n remodeling. f.'xp. CeU. lle.<;. 265: 195-202. Hayes J.J. and Hansen J.C. 2001 . Nucleosomes and the c hromatin fiber. Curr. Opin. Genet. Dev. 11: 124- 129. Jenuwrin T. and Allis C.D. 2001 . Translating the histone code. Scien ce 293:1074-1080. Luger K., Madev A.W., Richmond R.K.• 1997. Crystal structure of tlm nuclaosome core pa.rticle at 2.8 A resolution . Nature 389: 251 - 260. Luger K. and Richmond T.J. 1 998a DNA binding witbin tbe nucleosome core. Curr. Opin. Struct. Bioi. 8: 33 - 40. Luger K. and Ric-hmond T.J. 199Bb. The histone tails of the nudeosome. Cun: Opin. C.enet. Dev. 8! 140- 146. Narliker C.f., Fen H-Y. and Kingston R.E.. 2002. Cooperation betwE~en complexes that regulate chromatin sh:ucture and transcription. Ccll108: 475-4B7. Roth S. Y.• Denu J.M.. and Allis 0. 2001. Histone acetyl transfereses- Arm. Rev. Biochem . 70:61 - 1.20. Thomas J,O. 1999. Histone Hl: Location and role. cu,... Opin. CelL Rio/. 11: 31 2- 317. WOQdr:oc:k C.L and Oimitrov S. 2001. Highet>-ordar s bucture of chromatin and chrom()f;omes. Curt: Opin. Ge11et. Dev. n: 13n- t 35.

CHAP T ER

The Replication of DNA

hen the DNA double helix was disc..overed. lhe feRture that most exdted biologists was the complementary rehttionship bt!tween the bases on its intertwined polynucleotide r.hAins. lt seemed unimaginable lhat such o compiPmentary struclme would nnt be utilized as the basis for DNA replication. In far.t , it was the $elfcomplementary nature revealed by the DNA structure that fma lly Led most biologists to accept Oswald T. Avery's conclusion that DNA. nflt some fnm1 nf protein. was the carrier of genetir. information (Chapter Z). Jn our discussifln of hetw templates act, we emphasi1ed lhat two identicaJ su rfac~ will not Attract enc:h ot her (Outp ter 6}. Instead. it is mucb easier to visualize the a ttraction of groups with opposite shape or (:harge. Thus, without any delailed structwal knowledge. we might guess that a molecule as romplicated as the gene could not be copied ciire<:tly. lnstead, replication would involve the formation of a molncule complem entary in sh ape. and this, in tum, would serve as a template to make a replica of the original molecule. So, in the days betore detailed knowledge of protein o r nucleic. acid structure, some geneticists wondered whether DNA served as a template for a specific. protein that. in tum, served as a template fen a oorresponding DNA mulec.ule. But ns !loon as the self-oJmplementary nature of 11NA bPCame k.nnwn, thtt idea that protein templates might play a role in DN/\ replication was d iscard ed. It was immensely simpler to postulate that each of the two strands of e\•ery paroutal DNA molecule served as a. template for the formatinn nf a complementary daughter strand. Although frnm the start this hypnl hP..sis sP.P.mf*l ton gond not to he true, experimental support nonetheless had to be generated. 1-lappily. withu1 five years of thP discovery of the double helix, decisive

W

evidenr.c

emeTgE~d

OUT L INE

• The ChemiStry of Of\JA Synthests (p. 182) The-Mech.m1s.n o f DNA Pntyrn\l1dS€ (p. 184)

• The Replication fork (p. 192) The Speciah~ of ONA Pofymerases (p 200)

• DNA Synthesis at the Rephcatioo Fotk (p. 205)

• lnbatton of DNA Replicatiocl (p. 212)

• Blflding and UflWlOdlng. Ongtn SeJection and Actl\latton by the Initiator Prolan (p. ;214)

Fentsh1ng Replic.afion (p 2211)

for the separation of the complementaTy s trands

during DNA replkation h>ee discussion ofMeselson and Stah l experiment jn C:hapter 2} and firm enzymnlogical proof thai DNA a lone CR n function as the templnte fm the synthesis of new DNA strands. With th ese results, the problem of how genes re plicate was in o ne sense sol\fed. But in another sense. tbe study of DNA replicaHon had onJy begun. As we vtill see in this r;hapler. the replir:atio n of even the simplest DNA molecuJe is a complex. multi-stt>p pmr.ess, involving m any more enzymes than was initiaJly anticipated fnllowine the discoYcry of th e first DNA polymet'U;ng en~me. The replication of the large, linear r:hromosomes of eukeryotes is still more r;omplex. These chromosomes require many s tart sites of replication to synthesize lhe entiro c hromosome in a timely fashion, and the initiation of replication must be carefully coordinated to ensure that a ll sequenc;cs are replicated exactly once. Jn this chapter, we will first describe the basic chemistry nf DNA ~ynthesi s and the fuflr:lion nf the enzymes that catalyze this reaction. 1111

Tl1e RepUcoticm of DNA

lliZ

We wUJ then discuss how the synthesis of ONA occurs in the context of an intact chromosome at structures caHed replication forks. An array of additionilJ proteins are required to prepare the ONA for replication at thP.!>e :;ites. The last part nf the chapter focuses on the initiation and termination of DNA replication. DNA replication is tightly eontrollerl in a ll cells and initiation is the s tep that is rcgu h'lted. We wiH desr:ribe h ow replication initiation prnteins unwind the DNA duplex at specific sites in the genome caJled origins of re plicA~ tion. TogP.ther. the pmteins involverl itl DNA Te ptlr.ation represent an inh·ic:ate machine tlu~t performs this r.rjtir.a1 process wilh astounding .speed, accuriJr.y. and cnmJ.l)P.Ieness. 1

THE CHEMISTRY OF DNA SYNTHESIS DNA Synthesis Requires Deoxynucleoside Triphosphates and a Primer:Template )uoction For the synthesis of DNA to proceed , two key substrates must be present. First, new synthesis requires the four deoxynucleoside triphosphates- dGTP. dCf'P. dATP, and dTIP (Figure 8-lal. NucleosidR triphospbates have three phosphoryl groups which are aUa<.;hed via the 5' hydroxyl of the 2'-deoxyribose. The innermost phosphoryl group {tha t is, the group proximal to the deoxyribose) i.s called the ex-phosphate w hereas the middle and outermost gro ups ~reca lled the J3- end )!-phosphates, respectively. The second important substrate for DNA synth{-ts is is a particular armngemP.nt of ssDNA and dsDNA called a primer:template jun£tion (Figure 8-lb}. As suggested by its name, the primcr:template junction has two key components. The template provides the ssDNA that wiU direct the additiun of each complementary deoxynucleotide. The primer is complementary to, but shortP.r than. the template. The primer must also have an flX pnSt-!d 3'0H adjacent to thP. ~ingle str.mded region of the template. It is this 3'0H that will be extended as llP.W nucleotides are added. Formally. only the primer pori ion of the pri.rner:template junction is a substrate fo.- DNA synthesis since only the primer is chemically modified d uring DNA synthesis. The template only provides the information necessary to pick w hich nucleotidns are added. Nevertheless, both a primer and a template are essent ial for aH DNA synthesis.

8

0

0

0

' o-

I o-

I o-

J~ (A.G. C. orT)

HO-~-o-~-o- "-o-0120 ·

ssONA

FIG U R E 8-1 Salb.strales .-equired for DftA synthesi$. (a) rhe gent:1al Sfl'tlCtlre of the 2'~eoxynudeoslhospl1ates.. The positions of the o. • Jh ;,nd y-phospllates are labeled {b) The struc

ture of a generalized pn111er:ternpfate Jlll1(tloo The shorter J){ifner strar'.d IS completely an~ to the longer DNA ~ and must have a free 3' OH ad;acet1t to a 5SONA regton of the t~ l he longer DNA stfClfld Includes ~ ~on i311nealed to the pnrner and an a
The Clwmistry of nNA Synrhesis

DNA Is Synthesized by Extending the 3 ' End of the Primer The chemistry of DNA synthesis requires that the new t:hain grows by extending the 3' end of the primer (Figure 8-2). lndeed, this is a \illivorsi\l feature of tho synthesis of both RNA anrl DNA. Tho phosphndiestcr bond is formed in an Sc-;2 reaction in which the hyclroxyl group at the 3' end of the primer strand attacks the a-phosphoryl grm.tp of the incoming nucleoside triphosphate. The leaving group for the reaction is pyrophosphate, which arises from th e roleaso of the 13- and y-phosphatcs of the nucleotide su bstrate. The template strand direcls which nf tho four nur:Jeoside triphosphates is added. The nucleoside triphosphate that base-pairs with tht1 template strand is highly favored for addition to the p1imcr strand. Recall that the two strands of the double helix have an antiparaJJel orientation. This arrangement means that the template strnnd for DNA synthesis hac; the opposite orientation of the growing DNA strand.

Hydrolysis of Pyrophosphate Is the Driving Force for DNA Synthesis The addition of a nucleotide to a growing polynucleotide chain of length n is indicated by the following reaction: XTP

+ (XMP)n- {XMP), +l + · -G

OH

primer

5'

template 3' HO

~

F t ~u R ~

_ PY _ ro_ph-osphata __ se_

8-2 Diagram of the methanism of ONA sy11Ulesis.. ONA synthesis t5 nt~ated by the rtudeopt'ijic attack of the u phosptlate of the mcomang d\ITP. llis results '" the exteliSIOn of the i1'1aJrnmg 3 · end of ~ primer by ooe nucteotide an<1 the release of one molecule of pyrophosphate Pyrophosphitase rapdy llydrolyzes the pyrophosphate uito two phosphate mdecules.

2

Q

183

But thCl free energy for this reaction is rather small (6C = - 3.5 kcallmolej. What then is the driving forco for the po lymerization of nudeotidfl1) lnto DNA? Additional &ee energy is p rovided by the rapid hyd.-olys js of the pyrophosphate into two phospl1ate groups by an eneyme known as pyrophosphaliise:

The net result of nucleotide addition and pyrophosphate hydrolysis is

the breaking of two high-(}fl()tgy phnsphate bonds. Therefore, UNA synthesis is a couplod procPss, with an overall real:tion of XTP + (XMP),.

lXMP)11 ,

,

+ 20.

This is a highly fa vorable :roat:.1ion with a AG of - 7 kcal/mola which corresP<Jnd$ lo an equilibrium CfJnstant (~ of about lOS. Such a high Keq means that the DNA synthesis reaction is effoclively irreversible.

THE MECHANISM OF DNA POLYMERASE DNA Polymerases Use a Single Active Site to Catallze DNA Synthesis The synthesis of ONA is catalyzed by an enzyme called DNA polymerase. Unlike most enzynms. which have an active site dedicated to a single reaction, DNA polymerase uses a single active site to catalyze the addition of any of the four deo.xynucleosid<' triphosphates. DNA polymerase accomplishes thif> Cfitalytic tlexibility by P..xploiting the nearly jdentiual geometry of lhe A:T and C:C base pairs {rem ember that the d imensiom; of the DNA hrdix arc largely independent of the DNA sequonccj. The DNA po1ymerase mon;tors the ability of the incoming nucleotide lo form an A:T or G:C base pair rathor than detecting the exar.t n uclentide that enters the active site (Figure 8-3}. Only when a correct base pair is formed are the 3'0H of the primer and the a-phosphate of the incoming nucleo:.idc tript10sphate in the optimum position for catalysis to occur. lncorrcct ba~pairing loacJs to dramatically low
1'he Meclmt1ism

a cooect baSe pair

of f>NA Polymera.<;e

ltiS

b inoorrect base p¥

--FICURE 8-J Coffectly paired bases owe requit'ed for DNA polymerase cata~ nucleotide ad&tion. (a) Schemaic. ~am of the attack ot a primer 3'0H end on a correctly base-paired dNTP. (b) 5

b FI C V R E -('.c~o

~ '>lo

'

8-4 Sc:hernatk ihstra-

tiOQ of 111e steri~ constr.aints preventing utalpis usinz rNTPs by

DNA pclymerase. (a) Bindi!ll of a correctly bose-paired di\'TP to the Cf'.JA polyfY1elC!Se. Under these coodltions, the 3 'OH of the primer tlOd the a phosphate cl the c1'NTP are in dose pro»mty. (b) Addition of a 2'0H restAts in a s1el1c dash with amino aOds (the OISairrinator ilmino aods) an the rndeolide llindLng pocket. TI"Ifs rest1ts 111 the a-phosphate of the dN1P beil'£ d!Spfaced
186

The RepJicolivn of DNA

DNA Polymerases Resemble a Hand that G.-ips the Primer:Template Junction A molecular understanding of how the DN A polymerase <;atalyzes DNA synthesis has emerged from studies of the atomic structure of various DNA polymcJ1lscs bound lo prirner.template junctions. Theso slructures reveal that the ONA substrate ~its in a large cleft that resembJes a partially closed right hand (Figure 8-5). BasRd on the analogy to a hand. Ihe thron
gers, and palm. The palm domain is composed of a ~ sheet and contains the primary elements o f the catalytic site. In particular. this region of DNA polymerase binds two divalent m etal ions (typically Mg •z o t· Zn u J that nlter the chemical environment around the correctly base-paired dNTP and the 3'0B of the primer (Figure 8-6}. One F I c; U R E 8-5 The thfee.dhnensiona1 strudure of DNA po1ymerase resembles a rigtlt hand. (a)~ of DNA

polymerase bound to a ()limer.template junction. The fingers, thunb. and patm art= roted. "Ole recently synthesized DNA IS assoc.jon dagram. lhe fingers and the thurrb are composed ot u helices. The palm domain is obsrured by the DNA lhe i~ dNlP IS shown 1n red (b the base and the deoxyribose) and yellow (for the triphosphate fTlOiety). The template slrand of the DNA IS stlown 10 dad< gri!fY and the primer strond is shol.....n in rtght gray. (Dc:d>lie S., Tabor Sv Loog ANi~ Richardson CC. and Elenberger l 1998. fiJoture 391: 15 l .) Image prepared With BobSa!pt. MdSCJl)t, and Raster 3D

a fingers

\

v

lhumb

, . /palm

The Mtichonism of DNA Pol}wemse

187

F I Ci U R E 8-6 Two metal ions bound to

a

0

;?c-o-~-o c-o- ~e-o u 0 II 0

0

c 0

/

0

c

-ai)

DNA po~yme,ase catalyle nucl~ add~ tion. (a) llllstration of the active sate of a DNA polymerase. The two metal lOriS (shoon in green) Ne held in pace by interactions lMth two hgNy conserved ~rtdte re5idues. Metal ion A prirnarify tnteracts With the 3'0H r~ m reduced associarion between the 0 and the H. ThiS teaves a nudeq:hltc 3'0 . Nletal m B intefaas with the lript'.osphates of the lflComing d'ffP to newahze theft negative chatge. After c.ata!Y-;is, lhe pyrophosphate prodtxt is s1a,llired

thO\Jgh sMiar interactiOnS wth metal ion B (ootshOIM\). (b) Three-O~onot struct~ of

the act»ve Stte rretal 10ns assooaled wth the DNA polymerase, the 3 'OH eod of the primer and the incomng nudeotlde. The metal ior6 are shovom in green and the remaong elet'nents are 5hown in the same
the polymerase shown here IS roughly

equwler.t to rot:ltmg the image sho\'Vn m Ftgu~ 8-Sb - 180" around the aJ
metal ion reduces the affiniq; of the 3'0H for its hydrogen . This generates a 3'0- that is primed for the nucleophilic attack of the a-pho~ phatll of tho incoming dNTP. The second meta] ion coordinates lhe negative charges of the ~and -y-phosphat~ of the dNTP a nd sta biLizes the p)'rophosphate produced by joining th~ primer and the incoming nucleotide. In addition to its role in catalysis, the pabn domain also monitors thfl accuracy of base-pairing fm tho most recently added nudeotides. This region of the polymeraoo m akes oxtensivfl h}drogon bon d contact.o.; w ith basn pairs in the minor groove of tho newly synthesized DNA. Those contacts am n ot base-specific but only form if the recently added nudeoti des (whichever they m ay bo) arc cClJ'nlelly base-paired. Mismatched DNA in this region dramatically slows cat~lysis. The combination of tho slowed catalysis and reduced affinity for tho newlJ• synthesized DNA allows the release of the primer:templatP. from thP polymeraoo active site and binding 1o a separalr. proofreading nuclea..c,e active site on lhe polymerase. What are the roles of the fingers and the thumb? 7'he £ ngcTS are also important fnr catalysis. Several rosidues located w ithin thn fingers bind to the incoming dNTP. More importantly, once a corret.'t Lase pair is formed botween the incoming dNTP an d the template. the finge.r domain moves to f!ndnse the dNTP (Figure 8-7}. This closed form of the polymerase hand stimulates catalysis by moving the incom ing nucleotide in close contact with the catalytic m()la) ions. The fingor domain also associa tes with the template region, leading to a nearly 90c tum of the phosphodiester back.bone of the template imrnedia lf!ly aftC". r the active s ite. This bend serves to expose only the firs t templ.ato b&e after th e primer at the catalytic si te. This confomlation of the template avoids any confusion concerning which templale base is rnady to pair with the next nucleotide to bo added (Figure 8-8). ln contrast to the fingers and the palm. the thumb domain is not intimately involved in catalysis. Instead , the thumb inleracl') w ilh th e DNA that has been mo:>t I'OCWltly synthesized (see Figure 8~9). This serv~s two purposes. First, it maintains tho cortect position of the primer and the active site. Second, the thumb helps to maintain a strong ru:sociation botwoon the DNA poJymerase and its substrate. This association cnntributos to the ability of thR DNA polymernse to add many dNTPs each time it binds a pri mer:ternplatP. junction (see below)_ To summarize, an ordered series of event<> occurs ei.it:h time the DNA polymerase adds a nud eotida to the growing DNA cb ain. The incoming nucleotide base-pairs with the next available template ba.~. This intt>l'action causes the ..fingers" of th~ polymerase to close around tho base-paired dNTP. This conformation of Jhe enzyme pfacr_c: the criticcil catalytic metal ions in a position to uataJyzP formation of the noxt phosphodiester bond. Attachment of the base-paired n ucl~tid f! to I he primer l<>.ads to the re-opening of the fingers and the movement of the primer:template junction by onf! ba.;se pair. The polymcl'ase is then l-eady for tht: next cycle of addition. lmporlRntly. each of these events is strongly slimuJatod b·y correct base-pairing between the incom ing dNTP and the template.

DNA Polymeras~ Are Processive Enzymes Catalysis by DNA polymerase is rapid. DNA poJymerases are (;apable of adding as many as 1,000 nucleotides per second to a primer strand.

The lvfechonism of DNA Polymerase

a 0-helV
189

FIGURE 8-7 DHA~ ..grips... 0-heHx the template and the incoming l!lldeotide (dosed) w1tet\ i1 c()Jft(t base pair is meNte. (a) Atl ilh..straoon of the changes m DC\IA po~ 5tltJC\txe after the i~ r"'Udeotide ~s c:.otrectly to the ~e DNA. The pfimcly change .s a 40" rotation o1 one of the hetices in lhe finger domain c~!led the o-helix. In the open confoonation tt.s helill t>distant from the ir1comlllg nudeotlde W1t'n the pclymerase is If\ the dosed confc.-rrntion, this hefix moves and makes several ifrt!ortant 111teractJOns with the incoo1mg dNTP. A tyTosine nW:e.stac!<Jng lflleradions with lhe bose of the dr-JTP and two chatged residues associate v.1V1 the triphosphate. The
I

me

/ 3' b

•

If ,

.,

The speed of DNA synthesis is largely due to the proccssive nature of DNA polymerase. Proccssivity is a c haracteri.c;lic of enzymes thai operate on p ol:ymeric s ubstrates. In the case of DNA polymcrasos, the dP.gree of processivity is defined as I he average number of nucleotidP.s

added each time the enzyme binds o primec:templote junction. Each DNA polymerase has a charac teristic processivity that can range from only a few nuclootidf'.s lo moro than 50,00 0 bases added per binding event {Fig ure 8-9). The rate of DNA synthesis is dramatically increased by adding multiple nucleotides per binding event. lt is the initial binding of

Cyrosrne, !';sine. and Cllgirine can be seen belird the 0-neix in pink. The base and the deoxyroose of the tnCorring d\JTP are shoNn m red, lhe pnrner is sllOIM\ in 6ght grcrv. and the temp'ate S!Jand IS shov..n 111 daf\ grey The IWO catalytic metal ions Ne shovvn in green, and the phmphates 21e shovvn in yelow. (~ S.. labors. Long AM. Richardson CC. and EJenberger T. 1998.1\lort;(e 391: .25 1.) Image Pfepared with BobScript,.IVIotSo'!pt, and Raster 30

190

Thefleplicorrcm of DNA

f I (lURE 8-.8 •Uustration of the path of the~ DNA through the DNA pd)merase. The recently repkated DNA as assooated \Mfh the palm region of the VNA polymerase. f.J. the active site. the first base ol the S!f1gle-stranded reg1011 of the terwlote is in a postbOn ecpeaed for dooble-5tranded DNA /ls one follow; t11e templace strand roward ils 5' end, the phosphocieste' bdckbone abn..f!tly ~ 9£f. fus results m tt1e seoond and i!U Slh;equent ~e-stranded bases bemg placed m a posit:~ro 1M prevents any possbtity of base·pclirtng with o dNTP bound af the

fingers

y

\

thllmb

s· 5'

actvesire.

poly merase to the primer:template junction that is the rate-limiting step. In a typical D NA polymerase reaction. it takes approximately one second for the DNA polymerase to locate and bind a primar: templa tr. junction. Once bound. a ddition of a nucleotide is very fast (in the mi llisecond range). Thus. a completely nonprocessive DNA polymerase would a dd approximately 1 oose pair per second. In contrast, lhe fastest DNA polymerases a rld as many as 1.000 n ucleotidcs per second by remaining associated with the template for multiple rounds of dNTP addition. Consequently, a highly processive palymf'.t"ase increases the overall rate of DNA synthesis by

FIGURE

8-9

DNA~ases

syntflesUe DNA in a ~e manner. This bnaoon shovvs the dtferen<e between 3 procesSM' and a l1()flpfOCeS5I\ DNA pdymefase.. Both DNA pdymera.~ bird lhe pr..-ner:tefTlllate jllriC1lorl Upon boding. the nonprocE:SSive enzyme adds a single ~TP to the 3 ' €f'\d of the primer and then i.s r~ from the nevv primer.template ju1dJorl In contrasl. i! processive DNA polyll'lef'aSC adds many dNTPs ead1 t:rt-ne it btods to the template

DNA polymerase binds (sloW)

I

l 5'

I

many dNTPs added

:Juumu~.'''''n••••nm·5"

3' ,

I Ill lilt lllllllflllfllll i n•s·

5, II II lll!l U IIIII II II II II II CJH 3•

as .much as 1,000-fold compared to a completely nonprocessive enzyme. Increased processivity is facilita ted by the ability of DNA pol}nwra scs to sJic1o along the DNA tnmplatP.. Oncfl bo ond tn a primer:tcmpla te junction, ONA polymerase interacts tightly with much of the double-stranded portion of the DNA in a seque nce nonspecific manner. These interactions include electroslatic interactions betwee n the phosphate backbone and the .. thumb" domain, and intP.rac tions between the minor groove of the DNA a nd tho palm domain (c1P.scriood nbcwe). The scqucnce-indP.pendent nat ure of these inte ractions permits the easy movement of the ONA even after it b inds to polymnresc. Each time a n ud rotidfl is adde d to th o primer strand. tho DNA parliaJly .re lea~Ses from the polymerase (the hyd rogen bon ds with the minor groove are broken but the e lectrostatic interac tions with th(l thumb are maintained}. The DNA the n rapidly rP-binrls to th e polymerase in a pos ition that is shifted by ono base pair us ing thr. samo seque nce nons pecific mechanism. Fudher increases in p rooessivity a:re achiev ed through interactions

between Ute DNA pol_ymerasc and a .. sliding clamp" protein that completely encircles the DNA, a:s we shall discuss fw'thor below.

Exonucleases Proofread Newly Synthesized DNA A system based only on base-pair geometry and the complementarjty botween the bases is incapable of reaching tho extraord inarily high levels of accuracy dull are nbscn·cd For nN A synthasis jn the cell (a pproximately 1 mistake in every 1010 base pai rs added). A major lim.il to DNA polyme&usc accuracy is the occasional {approximatP.ly once in to-" Limes) flickering of the bases into the "wrong'' tautomoric fonn (imin o or enol; see Figure 6-5). These alternate forms of the bases allow incorrect base pairs tn be correctly positioned for- cata lysis. As wa now dpscribe. proofreading allows those mistakes to be cormctA
lions with the palm region. This altered geomotry reduces tha rate of nucleotide additi on in much the same way that addition of a n incorrectly paired dNTP reduces catalysis. Th us, when a mismatcht?d nucleotide is added, it both decrp.ases the rate of new n uclootid e addition and incrP.ases the rate of proofrf:ading exonuclease activily. As with DNA synthesis. proofreading can occur wilhoul releasing the DNA from the polymerase (F igure 8 -10). When a mismatcht.-d basr. pair i..s detectP.d by the ~olymerase, the p.rimer:t~mpl ate junction slid es away from the DNA polymerase active site and in1o the Pxonudeasc site. (This is because the mismatched DNA has a reduced affinity of the palm .region .) After the incorrect base pair is removed, the correctly p aired primor:tcmpla te junction slides back into the DNA poiyo)erase active site and DNA synthesis can continue. In essnnce, pmof.rea
THE REPLICATION FORK

I\

Both Strand.'> of DNA Are Svnthesized Together at the Replication Fork s· 3.

Fl c 0 ft f

8-10 Proofn!acfltlg

exonudeases re..-.oves bases from lt1e 3' erld of mismat(hed DNA. (o) When M illcooe<.t nudeo6de is lnt:OtpOrated lflto the DNA by a ~ the JOte ot ONA syflthesis 6 reduced end lhe effiruty of tnt: 3•t:n0 ol the priiTif'l' for the ONA polymerase aarve Site is &minished (b) When rnsrr~d, the 3' end of the ONA has 1M eased afltr~ for the proofrerldng ~ase acwe site Ortce bourd altus ac:tJVe site. the rrusmatched nudeobde IS removed (c) Oncetne mlstni.ltmed ruck?otDe as removed. the affinty vf lhe- ~ base-pa!t'ed 0NA lor t'oe DNApdymerase &1ive Site IS restOied ~ 0\!A synthe5is contlfil.e. (Source · Adapted from Bal<eiT.A_Clfld aelf SP. 1998. Polyrooases cYld the replisome. Modl1nes w(tm machines- Celf 92. 296, fig_ lb c~ @ 1998 With ~se.n from f.lsevle:)

Thus far we have discussed DNA synthesis in a reJatively artificial contexl That is, at a primer:template junction that is producing only nne new straud of DNA. ln the cell. both strands of the DNA duplex arc replicated al the same time. This n '!quarp,s separation of the two strands of the double helix to create two template DNAs. The junction betwor.n the nowly separated template strands and the unreplicated dup lox DNA is known a.(:j the replication fork (Figure 8·11). The repti· cation fork moves continuously toward tha d uplex region of unreplicated DNA. leaving in its wake two ssDNA templatP,s that direct the formation of two daughter ONA duplexes. The anti-paralle l nature of DNA creates a complication for the simultaneous replication of the two exposed templates at the replication fork. Because DNA is o nly synthesized by elongating a 3' end, on ly one of the n.vo exposed temp lates can be rr:plicated continuo usly as the replication fork moves. On this template strand , the polymerase sim ply '-'chases" the replication fork. The newly synthesized DNA strand directed by this template is known as the leading strand. Synthesis of the new ONA strand directed by t he oth er ssONA template is mom problematic. This template directs t he DNA polymense to move in the opposite direction of the repli cation forlL The new DNA strand di re~.;ted Ly this template is known as the lagging strand. A., shown in f igure 8-11 ~ this strand of DNA must be synthesized in a di~cnnti nuous fashion.

The RP.plication Fot*

tfwection of lead;ng strnnd polyrrle(ase movemetll Ollef'al directioo of DNA replicaCion

'

direc:li
F l «; u R l 8-11 The replication fork. Newly synthestzed DNA ~ icldicated in red and RNA primers 4!re llldicated lfl green The Okazaki fragments shown ae artfiaaWy si1€Jrt for IRustri!live puf)XlSeS- Ill the ca~ Okazaki ftr:~gments can vaty between 100 to gre
Although the leading strand DNA polymerase can re plic.atc its template as soon as it is exposed , synthesis of the lagging strand must wait for movement of the rep lication fork to expose a sum1antial length of templfltP. before it can be revlk.atcd. Each time a substantial length of new lagging strand template is exvosed. DNA synthct.is is initiated and continues until it reaches tbe 5' end of the previous newly synthesized stretch of lagging strand DNA. The resulting short fragments of n ew DNA funned on the lagging stmnd are called Okazaki fragments and can vary in length from 1,000 to 2,000 nucleotides in bacteria and 100 to 400 nucleotides in eukaryotos. Shortly after being synthesized, Okazaki fragments are covalently joined together to generate a continuous. intact strand of new DNA. Oka2aki fTagments are. therefore. transient intermediates in ONA replication.

The lniti~tion of a New Strand of DNA Requires an RNA Primer & described above. all DNA polymerases require a primer with a free 3·on They cannot initiate a new DNA strand de novo. How are new strands of DNA synthesis started? To accomplish this, the ceH lakes

advantnge of the ability of RNA polymernses to do what DNA polymerases cannot: start new RNA chains de novo. Primase is a specialized RNA polymerase dedicated to making short. RNA primers (5 - 10 nudeotides long) on an s::;DNA template. These primers are subsequently extended by DNA polymerase. Although DNA polymerases incorporate only dooxyribonud ootides into DNA. they can initiate synthesis using either an RNA primer or a DNA primer annealed to the DNA template. Although both the leading and lagging strands require prima.se to initiate DNA synthesis. the frequency of p rimase function on the two slrancls is dramatically different (see Figure 8-11). Each leading

llflreplicated DNA

193

194

Tile Replication of DNA

:Jil III IIi Dii ill IIIIIQIII;1111111::

lR.-H

:lfiDfUJIU[1''' n'WilliIUJI(:

! ~onoctease 5'

:Jnunnrrurn•••1ilnlnum:: I

I

prima:template ~

10NA pdymeraS<

:JnniJiiJijltUIIUIIIIIIIIUnif:: !ONA;ga,e

f I ~lJ R E

8-12 Removal of RNA primers

from newly~ DNA. The seq·

uential functiOn oH~NAse H. 5 • exorolldease, ONA pdymerase, and ONfl Igase duing the removal of RNA ~ tik.Jstrated. DNA ~ Pftor t(l RNA pnmer removal i!> $hown Ill

rnners

~ the RNA prlmEf rs shown in greer~. and the

ne-My ~DNA that rep~ the RNA Jll1l'l1Cf cs st10Ml in rEd

strand requires only a sing]e RNA primer. ln contrast, the discontinuous synfhesis ·of the lagging strand means that new primers rife needf!d for each Okazaki fragment. J!ec:ause a sjngle re plk.ation fork c an replicate millioi.'Js of base pairs. synthesis of the lagging strand can require h undreds to thousands uf Okazaki fragments and lbeir associated RNA primers. Unlike the RNA polymernses involved in mRNA, rRNA. and tRNA synthesis {see ChapleT 12), primase doe$ not require specific DNA sequenCeS to initiate synthesis of a new RNA primer. Instead, primase is activated only when it <:~ssochttes with other DNA replication pro-teins, such as DNA helicase. T hese proteins are considered in m ote detail below. Once activated, primase synthesizes a RNA primer u sing the most recently exposed lagging strand template, regardless of sequence.

RNA Primers Must Be Removed to Complete DNA Replication

To complete DNA replication. the RNA primers used for the initiation must be removed and replaatd with DNA {Figure 8-12.). Removal of the RNA primers can be thought of as a DNA rep~ir event and this process shares many of the properties of excision DNA repair, a process covered in dehtil in Chapter 9. To replace the .Rt\IA primers with DNA. an enzyme called RNAse H recognizes and remows most of each RNA primer. This en zyme spedfically degrades RNA that is base-paired with DNA (hence, the "H'' in its na:m.e. which stands for hybrid in RNA:DNA hybrid). RNAse H ren1,oves all of th~ RNA primer eXL'Cpt the rihonudeotide directly linked to the DNA end. This is because RNAse H can only cleave bonds between two ribonudeotid~. The final ribonucleotide is removed by an exonuclease tbat degrades RNA or DNA from their 5' end. Removal of the RNA primer leaves a gap in the douhJ.e.stranded DNA that is an idool substratu for DNA polymerase- a primer:template junction (see Figure 8-12). DNA polymerase fills this gap until every nucleotide is base-paired. lealling a DNA molecu le that is complete except for a break in the bclckbone between the 3'0H and 5' phosl-JhRte of the repaired strand. 11tis "nick" in the DNA a m be repaired by an enzyme called DNA ligase. DNA ligase uses a high-energy co-fact£Jr (S\Jch as A1P) to create a phosphodiester bond between an adjacent 5' phosphate and 3'0H. Only after all RNA primers are replaced and the associated nicks are sealed is DNA synthesis complete.

DNA Helicases Unwind the Double Helix in Advance of the Replication Fork DNA polymerases are gP.nerally poor at separating the two base-paired DNA. Therefore, at the replication fork. a second clas.cs of enzymes. called DNA helicases, catal)•ze (he sevaration of the two strands of duplex DNA. These enzymes bind to and move diredionally along ssDNA using the energy of nucleoside lriphmphate (usually ATP} hydrolysis t o disp)are any DNA strand that is annealed to the bound ssDNA. TypicaUy. DNA helicases that act at replicalil•n forks are hexa· meric proteins that asswne t:he shape of a ring {Figure 8·13). These ring· shaped protein oompl~xes encircle one of the two single strands at the replication fork near the single-stranded:double-strcmded junction. strdlld.c; of duplex

Thr lkplic.ntion For*

UIS

F I GU R £ 8·13 DNA helk.ases separ.te

lite two $hands of the dooble f,eliJL When ATP IS added to a ONA hel\case bou'ld 1o ~ the helicase moiiCS With a defined pola~ ity on the ssOI\!A. In the lflstance illusttated. the ONA tacase has a 5'-•3' polarity lhis polanty me(ll1s that the I)NA helitase v.oold be brund to the lawng~and tef'l'lllate at the ~~

-

mmi-

~~DPI+ ~

3'mJP--~~~ ~~l~

\\ ~-

s·• •!!1!!JaJJ~'

-

Like DNA polymerases, DNA h elicases act prucessivcly. Each time they associate with substrate, they unwind multiple base pairs of DNA. The ring-shaped hexameric DNA helicases found at replication forks exhibit high prOcCssivity because they encircle the DNA. Release of lhe helicase from its DNA substrate thereforn requires the opening of the bexarneric protein ring, which is a rare event. Alternatively. the helicase cc:m dissociate when it reachm; the end of the ON A s trand that it has encircled . Of course, this anangement of enzyme and ONA poses problems for the binding of the DNA helicase to the DNA substrate in the first place. Thus, there are ~pccinlized mechanisms that assemble DNA belicases around the DNA in cells (see .. Initiation of Replication" below). 1'hls topological linkage between proteins involl'ed in DNA revlication and their DNA subst.rates is a common mechcmism to increase processi~ity. Each DNA helicase moves a long ssDNA in a defined direction. This property i:.~ a. characteristic of eac:h DNA helicase called its polarity (see Box 8-1, Determining the Polarity of a DNA Helir..ase). DNA helicases can have a polarity of either 5'-3' or s·-s•. Tbis direction is always defined ttccording to the stnmd of DNA bound (or encircled foe a ring-shaped helicase} rather than the strand tbat is displaced. In lhe case of a DNA helicase tha1 func:::t ions on the lagging strand template of the replication fork, the polarity is 5'-3' to allow the DNA bAlicase to pror.eed toward the duplex region of the replication fork (see Figure 8·13}. As is nue for all enzymes that move a long DNA in a directional man ner. movement uf the belicase a)ong ssDNA fEl<JU iros the input of chemica.J energy. For helicases, this energy is provided by ATP hydrol)rsis.

Single-Stranded Biruling Proteins Stabilize Single--Stranded DNA Prior to Replication After the DNA helicase has passed . the newly generated single-stranded DNA must remain free of base-pairing until it can be used as a template fOf' DNA syntltesis. To stabiJize the separa1erl strands, single-stranded DNA binding protein~ (desiWlated SSBs) rapidly bind to the separated

fork

196

The Replicoti<m of DNA

strands. Binding of one SSB pmmoles the binding ofanother SSB to the immediately adja.cenl ssDNA {Figure 6-14). This is r..alled rooperative binding and occurs because SSB molecules boUDd to immediately adjacent regions of ssDNA can also bind to each other. Tlus strongly stabilizes the internction of the SSB with ssDNA making sites already occupied by one or more SSB molecules preferred over other sites. Cooperative binding ensures that ssDNA is rapidly coated by SSB as it emerg&; from the DNA helicase. {Cooperative binding is a prop-

BOx 8-1 Determining tbe Polarity of a DNA Hefiuse

The actillity d a DNA heicase can be detected by its ability to Qsplace one strand of a DNA duplex from another. In a typical DNA flelicase .assay, the Stbstrate IS composed of one short, labeled ssONA annealed to ooe long, uriabeled ssDNA (typicaly the label is radioactive l.2p iocorpa-ated into lhe

ssDNA drde by DNA helic.ase, it will migfate acc.ordirlg to its actual size, 200 bases. A modification of this simple expenment can be used to

detefrnine the polarity of a DNA heficase. Suppose there is

a restricbon enzyme cleavage site located

within the base-paired region (Box 8-1 Figure 2} When this site is deaved it witl generate a largely single-stranded, linear DNA with two regions of dsDNA of different lengths at each end Remember that DNA helicases btnd to ssDNA, not clsDNA. Thus, the only place that a DNA hel4case can bfnd th1s new linear substrate is between the two dsDNA regiOfls. Bealuse of the polarity of DNA her~tases, any given DNA heticase can· displace only one of the tvvo short ssDNAs. Because the two short ssONA regions are of different lengths. lhe size of the released tJagment wdl reveal v-klich directioo the DNA helicase moved along the ssDNA regiotl of 1he linear stb.>trate.

short ssONA). Consider a large circular ssDNA (for example.. 5,000 bases) ~
wtl displace the short linear ssONA from lhe la.-ge ssDNA cide. Separation of the slrancls can be detected by a change in electrophoretic mobility of the short. labeled ssDNA, in a noodenatuirlg agarose gel (see Chapter 20). After the gel is exposed fD x~ray f~m to detect oriy the radiolabeled DNA, the position in the gel that lhe short DNA occupies can be determned. V\otien it as hytxldlzed to the ssONA circle, the short ssDNA will cO-migrate with 1he large ssDNA ctrde. In

contrast, once the short ssONA has been displaced han lhe

a 200bases {radiolabeled) DNA

b

asymmetna~lly

0 0

0

0

0 0 ~ ffi ffi 0

0 & 0

(f)

0

boiled AT P

DNA helicase

0 ---1 --

x-ray film exposed to agarose gel

8-1 I I CURe I A IHodremical assay few DNA helcase activity. (21) DNA St.bstrate to cletectlleicaseact:Mty. A 5,000 f:p ~ ssONA ciro.!;lr CNA is~ to a 200-base raddabeled

80X

DNA. foe COI"\II'eruence rhe two moleo.Jies are not dlawn to scale. (b) To detect DNA hebcase activity. rhe DNA substrate is exposed~ 1he ONA helic.;se (lfl lhis cose IMth ili1CI without RPJ H'ter the ~ the resth1g DNA molecufes a e ~ by ~garose gel dec;:trophoresis (nandenatuing). When the sllort, raOOiatded DNA is base-paired wCh the lgrge SSONA drde, both I'Tlde<;u1es Wit co-migratf: as a !age moteaie.. In contrast after the a-lA hellcase has acted, the short rcdo1alleled sSOOA wit nngrate at a position ronsiSteflt with the teng1h of the short raOOI
Tilt! R~plicolion Fork

80 X 8-1 F1 G U R E 2 A biod1emic.al assay for DNA heliatse polarit)(. (~) Tne

a

restriction enzyme

deavage site

I

OOA~te. The same ONA~te

i•ustreted If\ t tgure 1 is cleaved With a re.sbi<;:OOo enzyme thclt leaves blunt ends.

The restnction enzyme is d10sef1 to cleave ~. leaving 125-base and 75-b;,se radiclabeJed ssDNA kagmeots annealed to the ends d a s,()()O-base wldt:ded ssDNA. The s· and ~· ends of the resulbng DNA molecules are ift
b

0

• 0

~

0

0

(f)

0

0 ®

ATP

5'- 3' 3 -+- 5' DNA hekase

. " -- - -- 125b -

boiled

-

75 b · --

x-ray film exposed to agarose gel

erty of many DNA-binding proteins. see Box 16-4, Concentration, Affinity. and Cooperative Binding.) Once covered with SSB. ssDNA is held in an elongated state lhat facilitat£.-'S its use as a template for DNA or RNA primer synthesis. SSB interaciB with ssDNA in a sequence-independent manner. SSBs primarily contact ssDNA through e lectrostatic interactions with a binding of additional SS8s ~

\

J I
1D7

198

The Replication of DNA

the phosphate backbone and stacl<.ing interactions wjth the DNA bases. Jn contrast to sequence specific DNA-binding proteins, SSBs make few, if any, hy drogen bonds to the ssDNA bases.

Topoisomerases Remove Supercoils Produced by DNA Unwinding at the Replication Fork As the strands of DNA are separated at the replication fork. the double-stranded DNA in front of the fork becomes increasingly positively supercoiled (Figure 8·15). This accumulation of supercoils is the Tesult of ONA helicase eliminating the base parls between the two strands. lf the DNA strands remain w1broken, there can be no reduction in linking number (the number of times the two ONA strands are intertwined) to accommodate this u nwinding of the DNA duplex (ooe Chapter 6}. Thus. as the DNA helicase proceeds, the DNA must accommodate the same linking number within a smaller and smaller number of base pairs. Indeed, for the superhelicity to remain the same, one DNA link must be removed approx-

f' I G U R E 8-15 Ac:tion of tnpoisomerase at the replication fork. As positive svperOJis acrumlkte in froot of the repication

fOOl, lopoisomerclses rapdlv ret'TlO\ie them In this d.agram, 1he actloo of bpo II refflOIIes positM> StJpercoil indured by a r~ICOOon bk By passf08 one ~rt d the unrt'p!icated

me

dsONA lhro~h ad~ ~ lf1 a 1"\e'Cllby ~d region, the po:;ttive supercods CQI'l be removed It IS worth notlng thet th.s ~ would reduce the ~nbJg OI..JI'l'1ber by tiNO and thus would onty heve to f.l<Xl.lr once every 20 bp replicatEd Although lhe adion nf a twe II topoisomease is •ll.IStlated here, type I topo\SOmerases can also remove the posi~ Sl.Jperojls generated by the replicabon fork.

topoisomerase

pass ONA

through the break

n

imately every ten base p airs of DNA unwound. If there were no 1necbanism to relieve the accumulation of these supercoi1s. the replication mal:h inery would grind to a halt in the face of mounting pressure. The problem is most clear for the circular chromosomes of bacteria (see Figure 8-15), but it also applies to eukaryotic chromosomes. Because oukaryotic chromosomes are not dosed circles. they r:ould. in principle rotate along their length to dissipate the introduced super-coils. This it> not the case. however: it is simply not possible to rotate a DNA muJocule that is millions of b:ise pairs long eat:h time one t um of the helix is unwound. The supercoils introduced hy the actio n of the DNA helicase are removed by lopoisomera::;es that acl on the unreplicaled doublestranded DNA in tront of t he replication fork (Figure 8-15 ). These enzymes do thi:; by breaking either one or both strands of the DNA without letting go of the DNA and passing rhe same number of DNA strands through the break {as we discussed in Chapter 6). This action relieves the accumulation of wpercoils. ln this way. to}JOisomerases act as a "swiwlase" that rapidly dissipatP.S the a<:eumul.ation of JC:upercoils induced b} DNA unwinding.

Replication Fork Enzymes Extend the Range of DNA Polymerase Substrates On its own, DNA polymerase can onJy efficiently extend 3'01 I prime.-s annealed to ssDNA templatt!S. The addition of primase, DNA helicase, and topoisomerase dramatically extends the possible substrates foe DNA polymerase. Primase provides the abiJHy to initiate new ONA strands on any piece of ssDNA. Of course. the use of primase also imposes a reqHiremenl for the rP.moval of the RNA primers to complete replkation. Similarly. strand separation by DNA helicase and dissipation of positive supercoils by topoisomerase

allow DNA polymerase to replicate dsDNA. Al though the names of tbe proteins change hom organism to organism (Table 8· 1), tho same set of enzymaric activities is used by organisms as diveTSe as bacteria. yeast. and hurmms to accomplish chromosom;~~l ONA replication. It is noteworthy that both DNA helicase and topeisometase perform their functions without permanently altering the c:h.P.mu:al structure of DNA or synthesizing wly new molecul.P.. DNA helicase breaks only the hydrogen bonds that hold tll~ two strands of ONA together \vithout breaking any covalent bonds. Altbough topoisomerases break one or more of DNA's covalent bonds, each bond broken is precisely reformed bdorc tho release of the DNA (see FiRure 6-25). Instead of altering the chemi01l structure of DNA, the action of these t~nzymes

TABll

8-1

E~ tf1.ac func:tion at~ Repliortion

Primase

fCRic

E. coli

S. cemvlsise

tkJman

OnaQ

Primase (PfU WAI 2)

Primase

PNAfmhcase

OnaB

f.(lcm complex

SSB

SS8

ffPA

Mom C()rrlple} flPA

Topoiscrnerast>S

Gyrase. Topo I

Topo 1. 11

Topa 1. 11

results in a DNA molecu le with an altered conformation. Importantly, the.se c:onformalional altPrations are essentia l for the duplir.ation of the large dsONA molecules that are the foundation of both bactt:trial Etnd eukoryotic chromosomes. The protciDs that act at the replication fork interact tightly but in a sequence-independent mannor with the DNA. These interactions exploit the features of DNA that are the same regardless of the particu lar base pair: the negative charge and structure of the phosphate backbone ( for El"-"31Tlpl e. tho thumb d omain of DNA polymerase]; the hydrogen bonding residues in the minor groove (fm example. the p&hn dumain of the ONA polymerase); the h ydrophobic stacking interadions between lhe bases (for example, SSB). m addition, many of these proteins have structures that allow them lo encircle (for example, DNA helicase} o r enc:ompa.ss Ifor example, DNA polymerase) the IJNA to remain assodated with the DNA.

THE SPECIALIZATION OF DNA POLYMERASES DNA Polymerases Are Specialized for Different Roles in the Cell The centraJ role of DNA polymerases in the effic:itmt a nd accurate replication of thE' genome requires that ceJJs have multiple specialized DNA po lymt~rases. f'or exampl e, E. coli bas at lt!ttst five DNA polymerases thal are distinguished by their enzymatic properties, subunit composition, and abundance (Table 8-2}. DNA poly merase lll (DNA Pol lllJ is tbe primaf)· enzyme involved in thP. replication of the chromosome. Rocau lie tl1e entire 4.6-Mh E. cnli genom e is replicated by two replication forks, DNA Pol ill must be highly processive. Consistent with these requirements. DNA Po1111 is generally fo und to be part of a larger complex that confers very hlgh pro-

cessivity-a complex known as the ONA Pol m holoenzyme. In contrast, DNA polymerase 1 (DNA Poll) is specialized for the removal of tlle RNA primers that are used to initiate UNA synthesis. For this reason , tbis DNA P<Jlymerc:1se h as a 5' exonuclease that allows DNA PoJ [ to remuve RNA or DNA immediately upstream of the site of UNA synthesis. Unlike UNA Pol m. DNA Pol 1 is not highly procef>sive, adding only 20- 100 nucleotides per binding event. The:;e properties are ideal fOT RNA primer removal and DNA

syJ1 the~is ACfOS8 the

resulting ssDNA gap. The 5' exonucleese o f DNA Pot 1 can remove the RNA-DNA linkage thai is resistant to RNAse H (see Figure 8 -12). The short extent of synthesis hy DNA Poll is ideal for relJiacing the short region previously occupied by the RNA primal'S (<10 nucleolides). Be<-.ause bot h DNA Pot I and DN A Pol tU arc involved in DNA replication. both of these enzymes must be highly accurate. Thu s, both proteins carry an associated proofreading exonucl ease. The remaining three DNA polymerases in E. coli are specialized for DNA repair and lack proofreading activities. These enzymes are discussed in Chapter 9 . Rukaryotic ceUs alc;o have multiple DNA polymerases. with a typical l.ell having mure than 15. Of rbese. three are essential to duplicate the genome: DNA Pol b, DNA Pol e, and DNA Pol wprima.se. Each of these eu'karyolic DNA polymerases is composed of multip le subunits (see Table 8-2). DNA Pol cdprimase is specifically involved in initiating new DNA strands. This four-subunit protein complex consists of

TheS

TA Bl f

8~ 2

Activities and functions of DNA Polymerases

Prokat'yotic (E. coli)

Pol ' Pol II (Din A)

NufNx!r of subunits

Rt-JA primer removal. DNA repatr 1

Pd 111 core Pol Ill holoeoz.yme POl IV (D!n B)

3 9

Pol V (UrnuC. UmuD'fC)

3

Eukaryoflc

Function

Number of

ONA repaw Dlromcsomc-fe plicnllOn

Ctm:wrosare replicatiOn IJNA w.paw, TrdflS Leslon Syn(flel)tS

(TlS) TLS FunctiOI1

subunits

Pd o

4

P1imef synl~ dllt~ng DNA repltcatlon

Base exastOn repair MitoctlondfiaJ DNA repli~ticfl

rot a

1

Pol-y

3

Pol ~

2- 3

Poll'

4

and repair

Pol O Pol(; PdA Pol p.

'

1

1

DNA replicaoon; nucleotide and base exosion repair DNA replication; I'1\JClOOtlOe and base exci&On repa1r DNA mpw of crosslmf<s TransJesion synttlc95 (TLS ) Meiosis-associated ONA rcpatr Somalic hypemlulatiOO

Pol ~

TlS

Poi TJ

Relatively accurate TLS past cis-syn cyclObutane d.-ners

Poll Revl

I

TLS. sornat1c hypet'mulallon

1

TLS

a two-subunit DNA Pol a and a two-subunit prirnase. After the primase sy11thesizes a RNA p rimer. the resulting RNA primer:template jundion is immediate!), handed off to the associated DNA Pol u fo iJ1itiate DNA synthesis. Due to its relat..ively low processivily. DNA Pol a/primase is rapidly replaced by the highly processiv e DNA polymer-rises o and s . The process of re placing DNA Pol tt/primase with DNA Po t B or e is called polym~::rase switching (Fig ure 6-16) and res u lts in thrtj6 different DNA po lym erases futJ ctioning at the e ukaryotic revlication fork. As in bacteri a l cells, the majo rity o f the rem aining eukaryotic DNA polymerases are invo lved in DNA repair.

Sliding Clamps Dramatically Increase DNA Polymerase Processivity High processivity at the replication fork ensmw rapid chromosome duplication. As we have discussed, DNA polymernscs at the replication fork synthesize thousands to millions of base pairs wilhout releasing from the template. DesJ.Jile thls. when looked at in the absence of other

202

Thfl Replirolion of DNA

FIG U R l 8-16 DNA polymerase swhd!ing during et~karyotjc DNA replication. The order of DNA J:clymerase function is ilustroted The length of the DNA synthesized &s shorter than 10 reeity lOt ilt.JStlative purposes. Typically the c:cxnbtned DNA Pol o./pnmase product is ~ 50- 100 bp and the ftJrttlef extension by Pol ~ or Pol ll ts ~'t.'er\ 100 and 10,000 nudeo6des. A!though bcih r.JNA Pol5 <'Jrrl ~can substirute for DNA Pol o/pnmase, 1t IS likely that they func:l.10n m the Ieplicauoo of spec 1f1c. DNA sltands (te~di ng o r

s·lt Ill mIIII II II 1111111 II 1111 ll1 Ufll iiUIIIIIIIIIII Ulllllllll111 m's-

s-' II llll If 1111111111 ti l t1,3'

I:XIIA Pol a./ primase

RNA primer synthesis by ptimase

5'

lagging). Curren~ studies ha'lle yet

to detennine II'Jhtch polymerase funct'OOs on v.hich stland, ~. DNA Pol ~ ()r e

s·

lltllllllflllllii!!lllllllll!llllllllllJJJl(3.

1 proteins. the DNA polyn1erases that act at the replication fork are only able to synthesize 20-100 base pairs before releasing from the template. How is the processivity of these enzymes increased so dramatically at lhe replication fork? The ke:r to the high p rocessivity of the DNA polymetases that act at replication forks is their association with protei ns called sliding DNA damps. These proteins are composed of multiple identical subunits that assemble in the shape of a "doughnut." The b()lo in the center of the clamp is large enough to encircle the DNA double he lix and leave room for a layer of one or two Wi'l.ter molecules between the DNA and the protein {Figure 8-1 7a). These clamp proteins slide along the DNA without dissociating from it. Sliding DNA damps also bind tightly to DNA polymerases al replication forks. 'fhus, the clamp encircles the n ewly synthesized d o uble-strand ed DNA and t he p olymerase associates with the primer:template jun~ tion (Figure 8-17b). This complex between the polymerase and the sliding clamJ.l moves efficienlly along the DNA template during DNA syntbesi~. How does the association with the sliding damp change the proccssivity of the DNA polymerase? In the absence of the sliding clamp , a ONA polymerase dissociates and rliffuses away from the

The Speciali-Lation of DNA Palym

b

di"ectioo of replication sliding damp

' f 1G u R E 8-17 structure of • sliding DNA damp. (a) Three-dimEnSIOnal structtxe of a sliding ONA

dar!'ll assooated with DNA. The opcr~ing lhrougtl the cetlter of the sidt~ dMnp IS about 35 ar~gsborns and the vM1th ol the DNA heltx IS approxirnatdy 20 aflgStroms. Thls provides enoogh space 1o allow a lhin tayer c.Janv and the DNA This is tnouftlt to allow the damp to slide ~ng the DNA easily. (l<.nshna l .S., Kong J<.P., Cary S., Blxgcrs. P.M., and KunyM l 1994. Cel/ 79' 1233.) Image pn:pared v.'ith BobS<:JJt>t. MoiSalpt, and R<~ster 30. (b) Sliding ONA dilmps ei1Ctrde the newly replicated ONA produced by an associated ~ polymerase. The slldingd.Ynp tntefacts With the port of the ONA pdymerase that is doses1 to the nev.-ly synthesized IJNA as it emf.18es ftom the DNA polymaase.

of one orl'No water rrdeaks between the sfiding

template DNA on average once every 20- 100 base pairs synthesized. In the presence of the sliding clamp, the DNA polymerase still disengages its active site from the 3'0H end of the DNA frequ ently, but the

association wHh the sliding clamp prevents the polymerase from diffusing away from the DNA (Figure 8-18). By keeping the ONA polymerase in close proximity to the DNA. the sliding clamp ens ures that the DNA polymerase rapidly rebinds the same primer:template junction . vastly increasing the processivity of the DNA poly merase. Once an ssDNA template is completely copied, the DNA polymerase must be released from this DNA and the sliding clamp to atl at a new prunet!ternplete junction. This release is a.x:omplished by a change in the affinity between the DNA polymerdse and the s1iuing clarop that depends on the bound DNA. DNA polymerase bound to a prjmer:template junction bas a high a.ffirUty for the clamp. Jn contrast. when the DNA polymerase reaches the end of an ssDNA template (for example, at the flnrl of an Okazakj fragment), a change in the conformation of the DNA polymerase reduces its affinity for the sliding d amp and the DNA

{see Figure 8-18}. Thus, when a polymerase completes the replication of a stretch of DNA, it is released by the sliding clamp so it can act at a oew primer:template junction. The clamp. on the other band. remains bound to the DNA and can bind other enzymes that act on the newly synthesized DNA (as w e descrjbe below).

21M

The Repli'r.oti0f1 of DNA

F Ul U It E 8-18 5Jiding DNA de~

inaease the~ of associaled DNA pofymemses.

~

oNA polymerase 1

: mr I- ONApdymeraserel.....,.l from primer·temptate

mnnnl111111111l~uumrrmnmm:: DNA polymerase• rebinds to thE!

same p1mer.tempfate aocl

1

COI'Itiflues DNA synthesis

in lhe absence of a primer: lernplate DNA polymefaSe is released from sliding clamp

1

Once released from a DNA polymerase, sliding clamps are not immediately removed from the replicatad DNA. Instead. othf>.r proteins that must function at the site of recent DNA synthesis to pe.rform their function interact with the clamp proteins. As described in Chapter 7, enzymes that assemble chromatin in eukaryoti.c cells are recruited to the sites of DNA replication by an interaction with the eukaryotic sliding DNA clamp (called PCNA). Similarly, eukaryotic proteins involved in Okazaki fragment repair also intfll'3Ct with sliding damp proteins. In each case, by interactin,g with sliding clamps, tht::se proteins accumulate at sites of new DNA synthesis where they are needed the most. Sliding clamp proteins are a conserved part of the DNA replication apparatus derived from organisms as diverse as viruses, bacteria, yeast, and humans. Consistent with th~ir conserved function, the structure of sliding clamps de.rived from these different organisms is also conserved (Figure 8-19). In each case, the clamp has the settne sixfold symmetry and the same diameter. :De!o;pite the similarity jn overall structure, however, the number of subunits that come together to form lhe clamp differs.

Sliding Clamps Are Opened and Placed on DNA hy Clamp Loaders The sliding clamp is a closed ring in solution and must open to encircle the DNA double helix- A special class of protein complexes. called 5liding damp loadel'5, catalyzes the opening and p lac:ement of sliding clamps on the DNA These £mZymes couple ATP binding and hydrolysis

DNA Synthesis ul the &plication Fork

a

b

f l GU RE 8 ..19 1be three-dimensional stiUCtUre of sfidinc DNA d~ iso1ated from clfferent OIJ'anisms. Slidng OOA d~ are footld across al 01ganisms and share a simitar SlruC'll.Jre. (a) lhe sltd~ 0NA &rnp f1001 E coli IS ~ of two Copi€S of lhe ~ protein. (Kong X.P., 0nrust R, ODonnel M~ and Kenyan J. 1992. Cell 69: 425.) (b) The T4 phage sli&ng £l\IA ~ is a tJimer of the gp45 protein. (Moarefi L Jeruz~\1 D., Turner J., ODe~ M,, and t
to the placement of the sliding clamp around primer:template junctions on the DNA (see Box 8-2. ATP Control of Protein Function). The damp loader also removes sliding damps from the DNA when they are no longer in use. Like DNA helicases and topoisomerases. these enzymes alter the conformation of their target (the sliding clamp) but not its chemical composition. What controls when sliding clamps are )oaded and removed from the DNA'? Loading of a sliding damp occurs anytime a prime~tem plate junction is present in the cell. These DNA structures are formed not onJy during DNA replication but also during several DNA repair even1.s (see C'..hapter 9). A sliding damp can only be removed from the DNA if it is not being used by another enzyme. Sliding clamp loaders and DNA polymerases cannot interact wilh a s1iding clamp at the same time because they have overlapping binding sites on the same face of the sliding damp. Thus, a sliding damp thai is tKJund to a DNA polymerase is not subiect to remnvaJ from the DNA. Similarly. nucleosome assembly factors. Okazaki fragment 1'epair proteins, and other DNA repair proteins aH interad with the same region of the sliding clamp as tho clamp loader. Thus. f>liding clamps are only mmovRd from the DNA onca all the enzymes that interact with them have com~ pleted their function.

DNA SYNTHESIS AT THE REPLICATION FORK At the replication fork the leading and lagging strands are synthe-sized s imultaneously. This has the important benefit of limiting the amount of ssDNA pTesent in the cell during DNA replication. When a ssDNA region of DNA is broken, there is a complete break in the chromosome that is much more difficult to repair than an ssDNA break in a dsDNA region. Moreover, repair of this type of lesion

c

frequently leads to mut~tion of the DNA (see Chapter 9 ). Thus, limiting the time the DNA is in this state is crudaL To coordinate tho. replication of both DNA strands, multiple DNA polymerases function at the replication for k. In E. coli the coordjnate action of these polymerases is facilitated by physically ]inking them together in a large muJt iprotein complex called the DNA Pol ITI holoenzyme {Figure 8-20)_ Holoenzyme is a general 11ame foro multiprotein complex in whjch a core enzyme activity is associated ·witb ad ditional compo.nents that enhance function_ The DNA Pol Ill holoenzyme includes two copies of the ..core" DNA I'ol Ill enzyme and one copy of the five protein -y-complex {the E. coli sliding clamp loader). Although present in only one copy, the ')'-complex binds to both copies of the core DNA l'ol HI and is essential to the formation of the holoenzyme (see Figure 8-20t.

8ox a-2 mP Control of Protein hlnction: Loacfins a slicHng clamp

ATP btndilg and hydrofysts coopted to sJicliflg clamp W1len bound to AfP, the daf'llJ loader GJ'1 bind and open the sliding damp ring by causing one d the sul:~tmit: St.Jtwit interfaces to come apart (Box 8-2 FtgUre 1). The now open sflding clarrp is brooght to the DNA through
IS

~?

the sliding ~ r.1g. ~ the DNA helicase, binding ssDNA is likely to be 1he key event I.Xl~ DNA. In these cases. bildlfl8 to ATP stcbliLes a ronfoonatton of Uie enzyme lh free in solutiOn), and this process wuJd be <:k#J if tlte ATP-stct>iiled complex is tigl-tly associated To €flS1.Jre that disasserrtxy oca.rs at the appopriate ~ pace, and rate, AlP hy<.tolysis is used to lObate dSc'.6Sernbly. For ~ ATP hyc:Yclysis Cc'l.lS€5 the damp loader to revert back to a state i.-l whkh it caiT10t bind ather the sicing damp or DNA Reverslon to this gromd state may ocru ~ the enzyme 5 stil bound to the ~ d AlP hydtiysis (PDP and PJ or may reqt.ire their release The fml key rrecharism to crup'.e AlP hydrolysis to a reaction pertains to the trigger for AJ P hydrolysis. It IS oitical that the fac1of not hyaolyze ATP unti a desired~ as ~led T}'picalty. foonaOOn of a paniojar complex biggers A1P hy&dysis. Jn the case ot the c!arr{> loader, this ro~ is che tErtiary complex of lhe sliOOg damp. the darrp loader, and the prima:tempfate jtrdim. Thus, ATP control of these molecular events IS most directly r~ted to controlli~ the timing of cootorrnational changes by the enz.yme. By requifing the enzyme 10 altenate between tiM:> confoonational states in order and requimg the formation ot a key ~r~termediate to trigger ATP hyd-olysis, the enzyme can acromplish wOf'k. In oontrast. it the er1zyme merely bourld and re4eased AlP (witholA hydrolysis). the reaction would rett.m 10 the lntial state as often as it would proceed forward and lmle, if any, VIIOik VVOI.id be accompllshed

DNA Synthe.'
Box 8--1 (Continved) BOX 8-l FICURE 1 ATPc:ontrot of sftding DNA damp~· (a) Sliding darr't> loaders ae fi'we Sllbunll proleln COfTl:lle~<eS INhose ac\ivity is controlled by ATP bi00ng and hyckdysts. In E.W, the~ loader is called the y<.omplex, and 1n eokaryotle cclls it IS CAlled repli loader must be bomd to ATP. (c) Grice l:x:U1d to ATP. the clamp loader binds the damp a1d opens the nng at one of the sd>unit:st!JU111t int€ffaces. (d) The resulbng COfTlllex con now bind to a-JA. DNA bmditlg is medialed by lhe damp loader. V'Alidl prefermtialy bnds to pnmer. ~junctions. Correct binding to the 0\lA has tv.o consequences: First the qJened sliding damp is positioned so that dsONA is in wflat wil be the 1de" of the ~-Second, OOA btnding ~tes AlP hydrotysis by the danp loader. (e) 8ecause only an AW-bound damp loaOO- CAn bind to the ~ afld to DNA. lhe loU' form of tile damp loader rapidly disassociates from the damp and the DNA. leaving behmd a dosed ~ positioned around the dsONA porton of the primer: ~e junction. {Source: Based on O'OonneU M. et al2001 Oamp looder S!JlJCttJre predicts the archtect\Jre of ONA polymease Ill holoenzyme and RfC. OJrret~t Biology ll: R942. fig 5. Copyright
a

b

c

1 ATP h}Uolysis 5'

Pol Ill core

I

j'~ -

(c:lan'l> loadEr)

f I CURl 8·10 T1Me compositio" of the DNA Pol HI holoenzyme. There are three enzymes 10 each copy of the DNA Pol Ill holoenzyrne· two COpteS of the DNA PoilU core Enl}1'11e and one CJpy o4 the y-compl~ n~e ~ex mdvde>two CQP!e5 d the T-~. each of wh!CM rdxles a (b'l1CW) that •ntetacts With one ~Pol lII c.o.c. Analysis d t!1e <mno aod ~1Ce of 1he 7-proter'l rdtates that d1e ~ Pol Ill binOOg lqliOO of the prctcin IS separated trorn the pdrt of the ptotm li1\IOtved 111 damp loading by an extended fbt>le lri.er. Th.s linker IS Pfoposed to alow the two pdymerases to f'1'lO\Ie 10 a relanvety l"ldepei !dent rnannEf that WOlJid be necessary foe one ~to rcpicatc flc IMOng str'anrl and the other to replicate the laggr,g S1ra"td (Soorce; Based on O'Or:xmdl tvt e1 aL 2001 CJarrp loader-s1.ructure ~the ate:htect~ of ONA ~ maase Ill hdoenzyme C!f'ld RFC Cutrenl BJOiogy 11 : R94.3, ftg 6. ~ © 2001 wrth pe1J1"CSSSCC'l from flsev!E'f.)

How do two DNA polymerases remain linked at the replication fork while synthesizing DNA on both the leading and lagging template s tr.mds? A moclel that explains this proposes that the replicA1ion machinery exploits the flexibility of DNA (Figure 8-21)_ As the helicase unwinds the DNA at the replication fork. the leadiflg strand is rapidly copied while the lagging strand is spooled out as ssUNA that is rapidly bound by SSB. Intennittently. a new RNA primer is synthesized on the lagging strand template. When the lagging strand DNA polymerase completes the previous Okazakj fragment. this polymerase is released from the template. Because this polymP.rase remains tethered to the leading strand DNA polymerase, it will bind to the primer:template junction nearest the replication fork - the one formed by the newly synthesized RNA primer on tbe lagging strand. By binding to this RNA primer, the lagging strand polymerase forms a new loop and inmates the next round of Obzaki fragment synthesis. This model is called the ..trombone model" in refP.renr.e to the changing size of the DNA loop formed by the lagging strand template. DNA replication in eukaryotic cells also requires rnuJtiple DNA polymerases. Three different DNA polymernses are present at P.ach replication fork: DNA Pol u/primase. DNA Pol ~. and DNA Pol ~ (see FiguNJ 8-1.6}. DNA Pol a/plimsse initiaw,s new strand::; and UNA Pol ~ antl e extend these strands. Although there is evidence that DNA Pol l5 and e synthesize opposite DNA strands. it remains unclear which polymerase is responsible for leading and which is responsible for lagging strand synthesis. s ;milarly, the proteins that recruit, maintain. and coordinate the action of thesE> three polymerases at tha eukaryotic DNA replicaHon fork remain unknown (thf! eukaryntic sliding damp loader, RF-C, does not perform this funetion).

DNA Synthe:is at the Replication For*

fIG UA E.

8•21 The "'trombone,. model

for coordinating replication by two DNA polymerases at the E.. coli replication fork. (a) The DNA hellc.ase at the E. colt l.)IIA repk.ation faa tJavCis on rhe lagging strand template in a S'-3' ciredlon The t:X\!A Pol • l'doenzyme irteracts YAth the DNA hckase thCA.Jgh the ,-.subunit, which also binds to both DI\IA pdymerases. One ONA Pd Ill core IS r~ cating the leading suand cn1 me other DNA Pcl Ill o;;~e ~ the lagg1ng SSB coots the ssONA regiOns of the DNA (for Sir11pLoly SSI3 oo ltle lagging strand IS only shown in part (a)). {b) P€fiocically. DNA pirnaseWIII associate with the DNA 11elicase dnd synth~e a re-N pnmer on the laggl1g strand t~ (c:) Wlen the lclgging rnv-.d DNA polymerase cOO"())etes lin ~ fragn'1ert. IllS released from the stdng clarryJ and tf1e DNA.

a / leading $~land "" DNA~

S'lr"

b

c

ONA~~

reteased from DNA siding~ i\ller ~of an Otlazald fragmer1t

210

The l feplicotion of DNA

d

e

~

~

3

• ,

5'

Ft GU ft E 8-21 (conmued} (d) 1he rt'Ce11tty prrned I~ strand ONA IS then a tafgd of the clcrr{J bader, wtich asserrbles a new siding ~at the pR"ner:!Em~ JllllC1'0n created by syntheslq a reN RNA pnmer (e) The~ ltlndlOO With its associated sidrB damp bt1ds to the lagg1ng strc!Od DNA paymease, which nlld1e5 DNA. S)'fltflesls on the next Okazaki tragrnrnt Althol.1gtl this ~ion has concen1Jated oo the rrore COC1'l*x ~ occurnng dt.ffig !he !>)'flthesis of the lagging strc!tld, dJmg hs ertre process, neN SSONA ~ b the tead'(lg strnnd has beEfl genooted and rapdy replicated by the leading strand DNA Polk

Interactions between Replication Fork Proteins F<>nn the E. coli Replisome The connections between the components of the DNA Pol Ill boloenzyme cue not the only interactions that occur between the components of the bacterial replication fork. Several protein-protein interactions, heyo11d those between lhe components of the P ol 1ll h oloe11zymP. facilitate rapid replication fork progression. The most important of these is an interaction between the DNA heJkase (the hexameric dnaB protein; see Table 8-t) and the DNA Pol ill holoenzyme (Figure 8-22j. This interaction . which is mediated by the clamp loader component of the holoonzyme, holds the helicase and the DNA Pol III holoenzyme together. In addition, this association stimulates the activity of the belic.ase by increasing the rate of helicase movement tenfold. Thus, the DNA helicase slows down if it becomes separated from the DNA polymerase (see Figure &-22). The coupling of he licase activity to the

presence of DNA Pol llJ prevents the helicase from "running away" from the DNA Pol ru hoiOP.nzyme and thus serves to coordinate these two key replication fork enzymes. A second important pr otein-p rotein interaction occurs between the DNA helicase and primase. Unli ke most proteins that act at thf, B. coli replication fork. primase is not tightly associated with the fork. Instead, at an interval of about once per second. primase

VNI't Synthesis at the Ueplirotion Fork

a

b

- - -- - -- -

8-ll 8in6tg of the DNA helkase to DNA PoliO hGioenzyme ~the rate of DNA str.mtf separation. The T~b.Jnit of the damp loader lflteracts with both the ONA t-dcase .and the DNA po¥nerase at the replicatioo fo1.;. (a) Yvnen thts •nleiaction IS made. lhe DNA tlek.ase OOWJnds the DNA at~ the same rate as the DNA polymesases replicate the ONA. (b) tf the DNA tdcase is not asscxiated With ONA Pot mholoenzyme, 0\lA lJr'1Wir'lOOg slows by tertfold Under these condtlons. the ONA pdymerases can replicate ta5$er than the DNA hekase can sepiYate lhe suands ct tneplicatcd I:X\!A. This ~ tl-e DNA Poi lU lloloenlyrr.e to "catch~· to the o-.JAhelic.ase and the f~ of a Ml repsorre FIGURE

assoclates with the helicasf' and SSB~coated ssUNA and synthesizes a new RNA primel". Although the interactjon between the DNA helicase and primase is relatively weak, this interaction strongly stimulates primase function (approximatel y 1.000-fold). After an RNA primer is synthesized, the primase is released from the DNA helicttse into solution. The relatively weai interaction between the R. coil primase anrl DNA belicase is important for regulating the length of Okazaki fragments. A tighter association would result in more frequent primer synthesis on the lagging strand and. therefore. shorter Okazaki fragments. Si milarly, a weaker interaction would w.sult in longer Okazaki fragments. The combination of all the pruteins that function at the n:plication fork is referred to as the replisome. Togethe:r these proteins form a finely tuned factory for DNA synthesis that contains muiUpJo intcractinR machines. Individually theso machines perform important specific functions. When brought together their activities arf> coordinated by the interactions bfltwoon them. Although these inlP.raclions are parliculurly wt".ll unden;tood in E. <..'Oli cells, sturues of bacteriophage and eukaryotic DNA replication machinery show that a similar coordination between multiple ma£".hines is involved in DNA replication in these organisms. Indeed , there are clear parallels between the proteins known lo be involved in replication in 6. coli and those functionfng in these other organisms. A table of the names of fa.-;tors performing analogous functions in phage, prokaryolic, and eukaryotic DNA replication is shown in Table 8-1.

21 t

212

ThH Ikplir:ation of DNA

1o fully appreciate the amazing capabilities of lhe enzymes that rep licate DNA, imagine a sit uation in which a DNA base is the size of your textbook. Under these con ditions double-stranded DNA w ould be approximately one meter in diameter and the E. coli genome would be a large circle about 500 miles (800 km) in circum~ feren ce. More importan tly, the replisome would be the size ot' a FedEx delivery truck and would be moving at over 600 kmlhr (375 mph)! Replicating the E. coli genome would be a 40 minute, 250 mile (400 km) trip for two such machiues, each leaving two 1 meter DNA cables in their wake. Impressive ly. during this trip tbe replication mat;hinery would, on average, make only a single error.

INITIATION OF DNA REPLICATION Specific Genomic DNA Sequences Direct the Initiation of DNA Replication The initial formation of a replication fork requires the separation of the two strands of the DNA duplex to provide a template for the synthesis of both the RNA primer and new DNA. Although :;ttand separation (also called DNA unwinding) is most easily accomplished at chromosome e nds, DNA synthesis genera1Jy initiates at intemaJ regions. Indeed for circular chromosomes. the lack of chromosome ends makes internal DNA unwinding essential to replication initiation. The specific sites at which DNA unwinding and initiation of r~plir.ation occur aro r:alled origins of replication. Depending on the organism. there may be as few as one or as many as thousands of origins per chromosome. ii'Mtiator

l

----------------The repf«on f'IOde1 FIGURE 8~23

Binding ol tile inibata to the Tepkata stimll" !ales tnitiatioo of replic-ation and the dc,Jpllcation of the associated DNA.

The Replicon Model of Replication Initiation In 1 963 f'ran9)is Jacob. Sydney Brenner, and Jacques Cuzin proposed a model to explain the events c ontrolling the initiation of replication in bacteria. They defined a fllhe DNA replicated frum a particular origin as 8 replicon. For example, bP,cause the single chromosome found in E. coli cells bas only one origju of replication, the entire chromo-some is 8 single replicon. h1 contrast, the presence of multiple origins of replication divides eoch eukaryotic chromMoma jnto mnhiple replicons-one for each origin of replication. The replicon model proposed two components that rontrolled the initiation of replication: the replicator and the initiator (Figure e-23). The replicalor is defined as the entire sel of cis-acting DNA sequences lhat is suffidP.nt to direct the initiation of DNA replication. This is in contrast to t he origin of replication which is rhe site on the ONA where the DNA is unwound and DNA synthesis initiates. Although the origin of replication is always part of the rep1icator, sometimes {partjcularly in eukaryotic ceJJs) the origin of replication is only a frad .ion of the DNA sequences reqtsirecl to direct the initiation of replication {the replicator). The same distinction Glll be made bP.twf!fm a transcriptional promotf.lf and the start site of ttanscription. as we will see in Chapter 12. The second component of the replicon model is the initiator protein. This prot-ein specifically recognizes a DNA element in the repiicator and ar:tivates the initiation of replication (see Figure 8-23). Initiator proteins have been identified in many difierent organisms, .including Lat~teria.

viruses, and eukaryotic ceUs. Although these proteins are not closely related, lht1y all t>t:fect the sitt:5 tilat will become origins of replication. As we will see beJow, the initiator protein is the only sequencespecific DNA-binding protein involved in the initiation of replication. The remaining prote ins required for repl ication initiation do not bmd to DNA SAquence specifically. Instead, these proteins are recruited to lhe re plicator through a combination of protein~protuin h1te.ractions and affmity fm· SJWCifiC UNA structures {for example, ssDNA or a primer:tempfat e junction).

Replicator Sequences Include Initiator Binding Sites and Easily Unwound DNA The DNA sequences of replicators share two common features (Figure 8-24). First. they include a binding site (or the initiator protein thai nucl ~.atcts the assembly of the replication initiation machinery. Second. they include a stretch of AT-rich DNA that unwinds readily but not spontammu sly. Unwinding of DNA a t replic..:atms is rontrolled by the replication initiation prote ins, and the action of these proteins is tightly regulated in most orgc:tnisms. The single replicalor required for E. coli chromosomal r~plication is called oriC. There are two repeated motifs that are critical for oriC function {Figure A-24n). The 9-mer m.obf is the binding s ite for the E. coli initia tor. On.aA, and is repeated five rimes at oriC. The 13-mer motif, repeated three times, is the initial site of ssDNA formation during initiation. Although the specific sequences are different, the overall structures of rep licators derived from many eukaryotic viruses and the singfecell eukarynte S. cem\.'isiae are similar (Figure 8-24b-c). Th~ methods

a OriC {E. coli}

'

--------~~~==~~~======~-----=~--

~

9 245bp

b SV40

EP EF' F' P P F' 65bp

c S. cerevisiae

B2

81 100bp

A

F I C U A f 8 -24 $t,n,ct"re of t"Cplicators. 1l1t? DNA ek:o"«lt!: th;,t ITlai<e t.tp 1hrc~ wekharacteriK~ cepflcators are sto.-.n The 1011:1ator ONA·birong sites &e shov.n tn gl'eet'l, efemef1s that faditate ONA t1l\Ni1dirlg 111 blue, and the s:re of the fr.iliJNA S}tltflesis ~ red (the site for eriC is outside the sequence shown). {a) otiC tS COf1lXlS'f'CI of IOlK "9-met OnaA bndlng SiteS and thfee pl3·met' repeated elements that are the Sl\e of tnitlal ONA Uf11Mf100g. {b) The origm ot the EUkdryotK 111M SV40 IS composed of q peotan\ef b!n&ng Sties (P) foe the ir~mttOt prolelf1 called r anf~ge11 and a 20 bp early palll'ldr£lme (EI') t11at rS the s11e d rnA \.Jfl\MrOng. (c) Three demet'lts are «:CCfYlmmr~y lvund at 5. c:ereii!Stae n:pk.ators. The A and BI eleme(l(s bind to lhe •nitiatof ORC The 82 ~ taoitatei ONA u ''"'nd'ng and btnd1ng of other n;pkalion tac\o!E

214

T.he Rt~plirotioll o{TJNA

" " "' i ONA bt'lding

DNA b;mling

5'~' 3~==~~=====e==~ I

I

~jymetterl

DNA

l DNA

CNAstrand

separation

I.JI1\I\IICiOing

s·~3·

3'~·

replication

8-25 Func:tions of the initiatot proteins d.mng the ntiatioo of DNA rrpfication. The tnee c.omrnoo fux1ions of lllitlator proteins are 1lustrated: ONI\ binding, DNA stran:l separatior1, and rep&icdlioo protein Fl
1eaulment. (Here the recruited prolan as

illustrated as.., DNA helicase. llowever, the reouted protCillS affet for each 1f1it0ta protem)

used to define origins of replication are described in Box 8 -3. The Identification of Origins of Rep lication and Replicators. Repficators founrl in multicellular eukaryotes are not well understood. Their identification and c haracterization has bmm hampered by the lack of gerwtic assays for stable propagation of small circular DNA comparable to those used to identify origins in singlt;-c;ell eukru:yotes and bacteria (see Box 8-3}. In the few instances in which replicators have h.ltm identified, they are found to be much larger than the replicators identified in S. cvrevisiao and bacterial chromosome&, generally en compassing more than 1,000 bp of DNA. Unli.krt their smaller cmmterparts, m utations that eliminaru the function of these replicaturs a.re n o l readily isolated, peihaps because i mportant elements within these sequences are redtmdant.

BINDING AND UNWINDING: ORIGIN SELECTION AND ACTIVATION BY THE INITIATOR PROTEIN lnitiator prot~ ns typically perfonn three different fundions d uring the initi
Box 8-J 11le kkmtification of Origins of ReplicatioQ and Replicators Replicata sequences are l:'ypiajy idertlfied ~ geneoc assays. fu ~E!t the first yeast repicatcrs were identified us"'S a DNA transf~ assay (Box 8-3 f'tgt.re I ) . ., these stucfleS, irivestigatnrs randanly cloned genomic DNA fragmems into ptasmids lading a r~'10r tut c.onta1ning a seJectable marker Fa the plasmid to be maintaned in a cell after transformation. the dmed DNA fragment had to contain a yeast replicator.

The identified DNA fragments were called autonomously replicating sequences (ARSs). Although these ~ences acted as repic.atCIS in the ri"~ coote.xt of a a cukY plasmid, fur1her evidence was r~ed to demonstrate that these sequences Wf'fe also replicators in their native d-Jomosornal

location To demonstrate that these DNA SEq.JerCes acted as rephcators 1n the chrornoscxne ft was necessary to develq:> ~ to Identify the locatioo of ~ ()( replication i1 the cell 01e appoach to identify origi1s takes advartage d the lltltJSual swcue d the DNA reptJcalion intermediates formed during repticatioo initiation. UrJike eithef ft.Aiy reptteated or fully unreplicated DNA.. DNA that IS in 1he Pf0Ct5s or being ~ IS ra klear. For ~e. a DNA fragment (generated by cleavage of the

DNA wrth a restnction enzyme) that does not cootaa1 an ongll of replication wil take on a variety of "¥-shaped" corforrnatloos as It is repticated (13ox 8-3 Fi~ 2, bkJe DNA ~). Sim~arly. irnrrte
or

or

or

Box 8-3 (Continued) are larger
mation ctxJut the tocatiof1 of a potential OOgi1 of replicanoo. How can the ~I gels identify the DNA rterrnedates assoaated \'\4th a replk.ation algin? lhe parrkular pattern of DNA migratioo can lead to uneqJivoc.al evtdence of an aigio of replicatioo. The I11£1St lfi.ISUal structLres migrate most s~ f) the first dirnens1m. for e.xcnple, a Y-shaped motea.J!e 1hat has ti"'Kee equlllength arms wil migrate the most sloMy of al such molerules derived from a p«ticular DNA frdgflltfi (Box 8-3, Figure 3b), a1d therefore wi be at !he top d an arc of DNA molecules that ae nonhnear. 111 contrast a ¥-shaped rnoteo.Ae vvith two very short replicated NITlS and a large repkated regtan wM ~every Slfl)JIaiy to the ~version of the Si1me DNA fragment Fmally. the Y~ed motecule that results from the .Ynost ccrT"II*teJy replicated fragmat IS Similar n shape to a 1llear mdecule 1:1/Vo tJTles the SIZe d the t..JnJePlicated fragment Thus, as a [X'lA rrdeosle is repicated by a si1gle replicDtion fm it wil migrate in post. t~ that vary from a spot that is we to the unrepllcated frag-

in an an: that evEntually reaches a locatioo that a li1ear mderule t\Mce the size of the unrepljcated DNA \NCXJd be e>tpeeted to mtgrate to. ThiS sl1ape is called a Y.arc and is ioc.fiCiltlve that a moleaje is in !he process of ~ replicated Because aU DNA fTICllecules are replicated durif18 each round of replicatOl. dle majMy of DNA fragnent.s ...vii sOO.N this typed pattern. Molecules that contain an ongin of replication form ~ ble- shaped replication intermediates !hat migrate even ITICI"e slowly in the first dimenstOn tllan Y-shaped molerules. The larger the bt.Vble, tlle more these molecules migrate differently from ~near DNA. Unfortunately, it is diffirutt to <\stin· glllsh the arc ci intermediates created by a bub~tain~ng fragment (caHed a bubble arc) from ooe created by ¥-shaped mer1t

intermediates (Box 8-3 Figure .3b and c). This difficulty can be

overcome lf the ongsn is located asymmetricaUy in the DNA fragment In thes instance, the 11termediates will start out as b~es but INhen the re(Jication fork closest to the end of the fragment completes replication. the buttie-shaped intermed"lcltes wiU become Y-shaped. This sCH:alted bubble-to-Y transition is easily detected as a discontinuity in the arc and is highly indicative of an ongin (Box &3 Figure 3d). Thus, ideally, the restric1ion enzymes chosen will asymmetrically flank the ongin of ~ic:ation to be deteded.

8-] FlCUR.E 1 Genetic idllntificalion of repliutoB. A plasmid BOJ(

(a smal oraJar DNA molerule) Wlltainirlg a selectable 11"\CP.er IS cut vWh a restoct10n eflZ}lTle lhat resuts In the exaslUl of the plasmids normal replicator. Tt11s leal.-6 a DNA fragment that lads a replicatot K> isolate a rep!icatorfrom a partnllar organism, the DNA from that aganism IS cut v.ittl the same restriction er1zyme ar~d ligated 1010 the cut plaSt'Tlld to reaeate circutar plasrnids each irdlding a single fragment denved from the test cwgaRsm. Ths DNA is then traflskmned 11to the host org«~isrn and the recombttlant plasmds are selected using a selectable ~r oo lhe pta:,rOO (tor example. if ltle m~er ronlerred antbotic restStance. the cells would be grown 11 the presence of 1l1e antlbtdic). ~els that grow are l!ble to mairllaln 1f1e ~id CYld its seledable rnatket indicating that the plasmid <.a.r1 replicate in the eel ClOd must cootam a repk.ator. lsclatioo of the plasm~ from the rost ceJ and sequenong vf the insened DNA allcms the identification of the sequence ot the tragmehl that C/Jfltdlns lhe replicator. fu1her mutagenesis ci the inserted DNA (suth as deletion oJ·spec:ific reglOOS at lhe inserted ONA), t~ by a repetition of the assay ~!lows a finer definel!OO of the

l

plale cells oo seledable media

cells that contain plasmjdONA £ic with replicator

\"\ ;:-- : -

gCY-N

l

isclate ONA from eels that grow

~

r~or.

215

Boa 8-3 (Continued)

2 DNA that is in tile process of repliudon has an unusual

BOX 8-] FIGUR£

strudure. Results at resbiction €llZ)Tl1E! de.wage of DNA in the process ot r~ are shov.n The iil.Jstratioo shows the grovvltl cf a •repkation lxbble" (aeate
. -===~~========~=;~:-~~ ... ... ~

'..

tion fori<s progres~ avJay from at1 origtl of repicatioo). The coosequences of rutting tllese repkatioo intetTnelA fragment contCl111111g an Dligin of replica-

~~=~i====~:~~== I • , •

I

t

.

:

c=======~====~ +====~======~ :--=:====

+ --<<:::>>----

~I

<

lion can ptoduce the p«rem ere the left

+

+ b

c:

d flfi

origin

- != -• p-olJc

Ill

t=a •

e~

.

f) ~.

. .

... .

.

e~ ~ bullble-arc

8·3 FtGU RE 3 Molewlar identification Gl an origin of replication. (a) By ele<.trqlhoreticaly separating DNA in two dirnet1sions, DNA in dle process of repkation can be separated from flMy repl!cat.ed or unrep1icated DNA. Taal DNA IS isolated from di\liOing (afld therefore, replia!ting) eels. The DNA IS sepacated first by size and shape (I.Mlg heft!~ eledrophoresis through relatively smaU pores). Then the clectricfteld is rotated by 9Cf and the DNA ts separa1ed predCJOllnanlly by Site (~esed with low \10\tBge in large pore agarose). SolAhem ~lysis is used to detect the ONA of intetest The three afferent patterns that cat\ be observed are ii..Jstrared 1he lafgest replicatiorl l::lt.ttlles migrate the slov>JeSt in the fi-st drnel1sion (c) and V-41aped rnoleaJes With nearly equalleog1h cmns rnigtate the next slowest (b). Because the "Y-arC' and "btlbble-arc" patterns are
....,

':-'- '·. .•

prote •

e .~

.

f) ~

.

Binding and Unwinding: Origin Selection and Activation by the Initiator Protem

21.7

in:teroctions resni~ i.n the sf!paration of the DNA strands over more than 20 bp within the 13-mer repeal region. This u nwormd DNA provides an ssDNA l€01platc for additional replication p.rott:ins to bt.>gin tbe RNA and DNA synthesis steps of replication (see below). The formation of ssDNA at a site in the chromostJm e is not sufficient for the DNA helicase and other replication proteins to assemble. Rather, DnaA recruits additional replication proteins to the ssDNA formed at the replicator including tho DNA hcficase {see below). The regulatio n of E. oo!i r~plication is lin ked to the control of DnaA acti vity and i.s discussed in Box 8-4, S. coli DNA Replication ls Regulated by DNA•ATP Levels and SeqA. In eukar.yotic ceUs. the initiator is a six protein complex called the origin l'ecognition complex (ORC). The function o{ ORC is b~t tmderstood in yeast cells. ORC recognizes a conserved sequence found in yeast replicators, called the A-element, as well as a second less conserved Bl~lement (see Figure 8-24). Like DnaA. ORC binds and hydrolyzes ATP. ATP binding is required for sequence-specific DNA hindiug a t the origh1. Llnlike DuaA. binding of ORC to ye-llllit .repVcators does not itself direct strand separation of the adiacent DNA ORC is, however. required to recruit all the remaining replication proteins to the replicator {see below). Thus. ORC performs two of the three functions common to initiators: binding to the repficator a nd recruiting other replication pro teins to the replicator.

Protein..Protein and Protein..DNA Interactions Direct the Initiation Proc5s Ooce the initiator binds to the replicator. the remaining steps in the initiation o f replication are largely driven by protein-pro tein interactions and protein-DNA interactio ns that are sequence independent. The end result is tht1 assembly o f t wo replication fork machines that wt:~ describud earlier_ To explore the events thai produce these protein machines. we fust turn to E. coli, in which they nre understood in the most d etail. After the initiator (DnaAJ has bound to oriC and unwound the 13-mer DNA, the combinatiop of ssDNA ctnd Dna A recr uits a complex of two proteins: the DNA h ulicase, DnaH. a nd helicase loade r

Box 8-.t £coli DNA Replication Is Regulabd by DNA•ATP Levels and SeqA In al Of~ISITJS i( IS o1tical tmJt replication initiation IS tightly c.ortroled to ensure 1hat chromosome number and eel nurrber remain apprqxiately balanced Although this balance is most tightly regtJated in eukaryotic: tclls (see beloN), E coli also prevent 11.11laWay ctnomosorr~e ~bon by fl1hib;ting recently 0. tJated origir1s frcrn re-M1~. Several <.flfferent fl'leChanisrns act to prevent relptd replication re-ilitlation from on'C. One method exploits changes in the methylated state of the DNA before and aftef DNA replication (Box 8-4 Figure 1). lrl £ cdl cells an enzyme caUed Dam methyl transfetase adds a meth)tt group to the A within evety GATC sequence (note that the sequence is a patfndrome). Typically the genpme is fully methylated at GATC sequences- 11i1S situation is challged after

re~icatect Because the A residies in the nevviy synd1esized DNA stJands are t.ro1ethylated, those sites that have been recently replicated wiAbe methylcted oo

each GATC sequence is

cfiy one strand (refared 1o as hemimethylated). The herntrnethylated state of the ne'AIIy replicated (}(iC 15 detected by a proiEin called SeqA. SeqA binds tightly to the GATC seqt~ence, but orJy wtlen it is flemimethylclted There is an ab.Jndanre of GAfC sequences imme&ately aOJaCffil to oriC. Once replicatiOO has initiated, SeqA binds to these sites before they can beoome fully methylated by the Dam methyl transfefase. Binding of SeqA has two conseqLJeflCes. First it dramatlcaUy reduces the rate at which the I:11:>Ul(:l GAK s.tes are

218

Ttm Fkplif:otion

of DNA

Box &-4 (Continued)

BOX 8-4 f i CURE 1 SeqAbound to hemimetf¥ated DNA inhibits re-initiation from recently replkated daughter origins. (a) Prior to DNA r«:pficalion, GATC sequeoces thr~ me f . roli

l cl

c:

genome are memy1ated on both strands ("fully" methylated). Note that throughout the figure. the l"''l(;thyt groops are reptesented by rt'd hexagons. (b) DNA repicaoon CDOVef'lS these sates to the hemmethylated state (orty one strand of the OW\ is methyiated) (c) Hernimelhylaled GATC sequ:nres ace rapidy boUld by~ (d) Bolni SeqA protein irtl;bits the ful metllylation of these sequences and 1t1e btndlf18 of one by DnaA protetrt (for s~. only cne of the two daughter moJroJes. is lustrated in parts d, e, ~nd f). (e) When SeqA irirequer~dy dsassociates from the GATC sites, 1he sequences can become fU1v methylated by Dam DNA methvf transferase, preventing reblnrling by SeqA. (f) When the GA1C !>ltes become flily methyiated, lXlaA Clfl bind and direct a new ratnd of

3 1

Pille

I I I 111 :

l

9 ;

GAIC

L [ ~3'

9 99

I r:m;tn]) f

r

initiate replicatiOn

i -3 5' 3 5'

repic:ation from the daughter oriC replicators.

OoaA

d

rr:

3

+=-

s~

e 5'

!

••

•

Bind;,~

ond Unwinding: Origi11 Sei#Jdi0f1 ond Activation by the lnilintor Protein

219

Box ... (Continued)

9-mer bOOing sites ootside of oriC (DnaA also acts as a

methylated. Second, when bound to these oriC proximal sites. SeqA prevents OnaA from associating wrth oriC and initiating a new cound of repication. Thus, the conversion of tfie eriCproximal GATC sites from methylated to hemimethylated (an event that rs a direct conseqllence d initiation of replication from oriC) leads to the inhibition of e>naA binding and, there-

transcriptimal regulator at a number of promoters), and as they are replicated, this number doubles. lhe i1crease \n DnaA binding sites acts to reduce the levels of avaitabte OnaA Together these methods rapidly and dramaticaRy reduce the abtlity of E coli to ritiate replication from new copies of one

fore, prevents rapid reinitiation of replication from ltle two

Althoogh lhese medlaoisms prevent rapid re-initiatior'l.

nevJy synthe9zed daughter copies of oriC. DnaA is targeted by other mechaniSJTlS that mibit rapid re-ilitiation at the ne,My synthesized oougtlter cq:>tes of oriC. As desoibed above. only DnaA bound to ATP ca; direct initiation d replicationi hoNever. this boufld AlP is converted to ADP dtlrq; the initiation process. lhus, the process of cirectiig a round of replication initiation 11CJCtivates DnaA preventing Its reuse. The process of exchanging the bound ADP for an ATP is a slow ooe. further de~ 1he aca..rnulation d r~-competent ATP bomd Dr1aA. The process of replicating nearby sequences also acts to reduce the amount or DnaA avaiW>Ie to bind at oriC. There ~e more thcYr 300 DnaA

imibiticrl does not necessarily last llntil cell Ovision is COI'llltete Indeed, for £ coli cells to dvide at the maxlnlt.lm rate. !he daughter copies of oriC must initiate repicativn prior to the rornpletioo of the previous round of replication. This is becallse E coli ceRs can d'n.ide every 20 mmutes but it t-*es mae man 40 minutes to replicate the E coli genome. Thus, under ~ groMh cooclitioos, £ coli cells re-iflitiare repk.ation ooce and sometimes tvvice prtor to the corT'lpletion of prev'ious rotlllds of replication (Boll 8-4 Figure 2). Even undef such

l

segregated

growth

conditions, initiation does not occur more than mce per roi.JI"'od of eel dMsim ~ for each round of cell division, there is only one fOUld of repication initiation rrom otiC.

8-4 FICO V R E 2 Origins of nplitatioo re-tnitiate ~ c.ation prior to eel cfrvisiotl in qpicly growing cells. To allow tile genome to be fully replcated Pf1DI' to eac;h round of ~~ di\iisiorJ, bacteoal cds frequMtly have to i11itiate ~ tep&ication fcom their single origin prior to lhe completion of cendivision. This means that ltle chromosomes that are segregated into the daugtlter eels Me being actively replicated This is 111 contrast to eokaryotic eels, which do not Slart chr()I'Tl()$Dfl1e segrt:gatioo prior to tile completiorl of all DNA rep1icatioo. 80 X

repCicat~ chromosomes

~

this

F ------- ----1 =ep\icated • replicated • res:>fiealed once tw1ee

DnaC (Figure 8-26). Both proteins are p resent in six copies within the complex. The DNA helicase is mai.utained in <2n in'='ctive st~te in the helicase/he[icase loader complex. Once bound to the ssDNA at the origin, the helicase loader directs the assembly of its associated DNA helicase around the ssDNA (recall that ssDNA passes through the middle of tht: hdicase's hexameric protein ring). This prQCess is analogous to the assembly of sliding ONA d amps around a primer:tempJate junction. Upon completion of this task. the helicase lo~dcr is re leased activating the helicase. One helicase is loaded onto each of t.he two separated ssDNA strands at the origin, and the orient ation of these two helicascs is such that they wiU proceed toward each other as they move with a 5'--+3' polarity along theu associated ssDNAs.

ihe. Rttplicntimr vf DNA

FIGURt

8-26 A model for E. coli

initiation of DNA replication. The major e\lef1ts 1n the£ roli wntiation of rep~c:ation are illislrated. (a) lvUtiple DnaA·ATP prcrteins bind to the repeated 9·mer sequences within otiC (b} BiPcfirll of OnaA·ATP to lhese sequeoces leads to straJld separation with!fl the 1.3-mef repeats. l his IS mediated by an s.sONA oodittg

a

r

·

OnaA•ATP 5'

3' 5'

:r

13-mer

:3'5'

b

:i

doman in DnaA·ATP. (c) I::J\!A t1eticase (OnaB) and the ()NA helicase loader (OoaC) assooate

with lhe IJnaA bound origirt An s.sOOA binding don1air1111 tl1e heliciJse loader as v..-eUas pratetn1'!~ 111tefactlor1s "~ OnaA are requ.recl to form this c.omplex. (d) DNA he\icase loadefs catalyze the openmg ot the DNA helicase pro· tein nng at1d placemerrt of~ ring arOl.J'ld tl1e ssDNA at 1he origirl. loading of the DNA ttelt case leads to lhe ~n of the hehc:ase loader from the repbcator and activates the DNA heiKases.. (e) The DNA helicases eadl recruit a DNA primase which synthesiz~ a11 RNA ~ on each tefll'latl?. The m011ement of the DNA helicases aCso refl'lClV€5 i!fl)l remai111ng DnaA boutld to ~ repltcotor. (f) 1lle newly S'f'llhesized priml'fs are rewgmed by the damp loader componelltS of two ONA Pol l! ~Sliding damps are assembled on each RNA primer. and leading sti?JI'ld synthesis is initiated by one d lhe two CDfe ONA PoiiH enzymes of el!dll1vloenzyrr.e. (g) Mer each DNA hekase has moved awrc:oomately I ,000 bases, a sewnd RNA pruner is synthesized oo each lagging srrand t@Ol'late cY1d a sldng darrp is loaded l1le restJtJng prirner:~ jurldion is recogrjzed by the serood IJWI Pol Dl core enzyme in each holoenzyme. resulbng tn the 1111batlon of lagging strand synthesis. {h) leading and tagg;ng strand s.,.-,thesis IS now W.ittated at each repkation f()rl( and cot~tinues to ~ end of the template or until anather repliccr tiCYl fork fran an ad,acent origin of replication IS

c

loa<1er {OnaC)

:~ d 3'.:::: .

III;Jt;

s·•

e

DNA polymerase Ill holoenzyme

f

reached.

s·

p'

•:t

Binding and Unwinding: Origin Selection and ACtiVCJtia1 by the Initiator Protein

221

The protein~protein interactions between the helicase and other components of the replication t'ork described above direct thtJ assembly of the rest of the replication machinery (see Figure 8-26}. llelicase recrttits DNA primase to the ()rigin DNA. resultin{?. in the synthesis of an RNA primer on each strand of the urigin. The DNA Pol 10 holoenzyme is brought to the origins through interactions with the primer:template junction and the helicase. Once the holoenzyme is present. sliding clamps are assembled on the RNA primers. and the leading strand polymerases are engaged. A s new ssDNA is exposed by the action of the helicase . it is bound by SSB and DNA primase synthesizes the first lagging strand primers. Tb~ new primer:template junctions are targeted by the clamp loaders. which place two additional sliding clamps on the lagging strands. These clamps are recognized by the remaining unengaged rore DNA Pol £II en7.ymf?S. resulting in the initiation of lagging strand DNA synthesis. At this point, two replication forks havtl been assembl od and initiation ot' replication is complete (t!xactly how the two replication forks ar~ assembled i.s a m aftttr of debate , see Box 8-5, The Rcplicalion Pactory Hypothesis}.

~ 8-5

The ~M Factory Hypothesis

There are two ways to think of the relative rnnt:ir:Yl of the DNA iYld the replicatJoo madunery (Eox 8-5 Figure l). One SIJ1lpte \/leoN is that the rep~attion machlllEJY ~ along the DNA Ln a f'l'li:ll"lllE! analogous to a train rn<:MI1g along its tracks. repticatng both strands ol the approachq DNA. In tlis traditional l.lievv, the DNA helicases pass by one another irrrnediately atter loading and subsequently act independently from one anocher at the tvvo new rep&ation ~ An alternative view suggests that the DNA moves wtile the r~ation rnacn.nery ts s~ simiar to fikn moving into a rTlOIIle prqector lv1e<:hanistic.ally, it has been proposed that the two DNA heicases do not pass by each other but lnst.ead "l'lln Into each othet' and remain assooated fu the reflWnder of the replicotion process. The VIeW d repicatkln occurring at statiC sites has become raeasingly favored Studies of bactenal DNA rep\ication dearty illoote that the rep~c.ation rt'lC3chmery reJ1'liltns In a 911ge location Vl.<'ithin the cell during DNA synthesi:s. lnstead of the replication rnadlinery mcMng, the DNA moves .n and out or this "replication factory" and in the process is duplicated Similarly, re{)lication in eukdtyotic cells ts obsetved to ocrur at discrete sites within the cell nudeus Stu~ of the helicases that functio'\ at replication forks also support a static replic:attoo

madune.y. Several hexameric DNA helc.ases fofm dclubtehexamers. This suggests that rather than the two hexameric helk.ases rapidly separating from earn other afte Initiation {as suggested by the "railroacr model), lhey ~ together throt.Jghout the replication process. These two VIeWS d the asserrbly of the replicabon forl< also have interest;,g cbnsequences concerning the ONA that is repliccted by each DNA Pollll holoenzyme. rf the DNA helicases pass by one another ~ety after lhey are loaded, then the dosest strands that car; be replicated slm.dtaneously by the fv.Jo polymerase; rl the DNA Pof Ill hotoenzyme Ml be the Watsm and Crick stsar;ds of the most recently unwound DNA (Box 8-5 Figure I, left panel). In cootrast.. tf the two helicases rema1n assooated aftef ribatlon, then it as possible that the lagg.ng straxi DNA polyrnerases of the DNA Pclullroloenzyme could associate with e~ther of tvvo primed tefTJ>Iates, ~ they are novv both nearby. By most e.5timations, irl thiS' scenario, the cho!ce will be for each DNA Pol Jll holoenzyme to have me same tefllllate strand for the lead·~ng and lagging wand synthesis. That IS, one core enzyme

vvill replicate the

"Crick• strand of the DNA and the other will repacate the soo" sband of the DNA (Box 8-5 F.gure 1, right panel)

'Wa~

Box 8-5 (Continued)

helicases associate with each other

G

DNA primase makes leading strand primef syntheses

~~:§§§§§§~~~·§§§§§§§§:~

G

DNA pc1mase makes first tagging slrand primer

secood DNA pclymefase II core eOZ)me f*tds neaest pn!'nef

5'

BOX S.S fl G U R £ 1 In the left panel, the tNo DNA llelicases function indepel"'dently. In the right panel. the two DNA llelicases remain associated witl'l one another. Note that in the right pane\ one DNA Poi nt holoenzyme uses only the ~tson strand as a tt'f11)1ate and the Olher uses c:Kiy the Crici strand as a template. For~ the DNA Pol II is not shown associated \Mth the ONA helicases.

t1mdi.ng ond Unwinding: Origin Selection ond Activation b)' th~ lnitintor Prolein

Eukaryotic Chromosomes Are Replicated Exactly Once per Cell Cycle As discusS€(} in Chapter 7 , the events required for eukaryvtic cell divi· sion occur at distinct times during cell cyde. Chwmosomal DNA replication occurs only during the S ph~ of the cell cycle. During this time. all the DNA in the cell must be duplicated exactly once. IncompJete replication of any part of a chromosome causes inappropriate links between daughter chromosomes. Segregation of linked chromosomes causes chromosome b~lt! or loss (Figure 8-27}. Rereplicalion of DNA can also have severe con~uences. increasing the number of copies of parricular regions of the genome. Addition of even one or two more copies of critical regulntory genes can lead to catasttophic defects in gene expression, ceU division, Ol' the response to environmental signals. Thus, lt is uitica1 that every base pair in each chromosome is replicated once and only om:t! each time a eukaryolic cell divides. The need to replicate the DNA once and only once is a particular challenge for eukaryotic chromosom es because they each have many (l{'igins of replication. First, enough origins mu.~t be activated to ensure that each chromosome is fully replicated during each S phase. Typically. not all potential origins need to be activated to complete replication hut. if too few are activated. £€€ions of the genome will escape replication {se.e Figure 8-27). Seco11d , although some potential origins may not be used in any given round of cell division. no origin of replication can initiate after it hss been Teplicated. Thus. whether an origin is activated to cause its own replication or replicated hy a replication fork derived from an ad jacent origin, it must be inoctivott.""Cl until the next round of (,'ell division (Figure 8-28). H these conditions were not true, the DNA associated with an origtn could be rcpJicated twice in the same ceiJ cycle.

Pre--Replicative Complex Formation Directs the Initiation of Replication in Eukaryotes The initiation of replication in e ulcnryotic cells requires Lwo steps to .at d istinct times in the t:eH cycle (see Chapter 7): replicator selection ~md origin activ.ation. RepHcator sell:lclion is the process of identifying sequences that will direct t he initiation of replication (X.:c ur

F I Gll R E 8•27 Chromosome bre
chromosome segregation

as a t'e5U1t of incomph!te DNA replication. This iBustratioo shows the coosequences of anootnplete replicotim followed by chrornosorne segregation. The top Clf each illl.lStlatlc:¥1 stlows the entire duomosome. The bottom .sho.Ns the details of the chromosome bJ&lbge at the DNA level. (For the daaiis of chromosome segregaticn, see Chapter 1.) As the

dlromosornes are pulled apart stress .s placed on the Lneplccated DNA. rest.Atng in the breakage ot the cbomosome.

The R Rplir:olion ofDNA

Z24

RWRE

8-28 Reptk.alors aa-e inilctivated

by DNA replication. A chromosome with &,e replicators ts shown The replkotors labeled 3 and 5 are the flrsl to be activ
2

3

4

5

I Ofigin 1 iriliales

t orign 2 is passwely repkated

the VNA o~~e~lappmg w.th the n1Xl1bef

2 and 4 repiiUllors. Wh6'l a replteator rs coped by .a bk dertved from an .adl«frl Oflgin pn
- -- - 1

•

en~e the

+

complete replicinion of

each chromosome

and occurs in Gl (prior t o S phase}. This process leads to I he assembly of a multiprotein complex nl each replicalor in the genome. Origin activation only occurs after cells enter S phase anci triggers the replicator-associated protein complex to initiate DNA unwinding and DNA polymerase recruitment. The separntion of replicator selection and origin octivation is different from the situation in prokaryotic cells, where the recognition of replicntor DNA is intrinsically coupled to DNA unwinding and polymerase recruitment. As we w ill see be1ow, the temporal separation of these two ev~nts in e ukaryotic ceUs ensures that each chromo-some is replicated onJy once during each cell cycle (bocterial ceJls solve Ibis prnblem differently. see Box 8-4, E. coli DNA Replication Is R~u lated by DnaA·ATP Levels and SeqA. Replicato.r selection i.<> mediated lJy the form;ltio n of pre-rephrntive complexes (pre-RCs) {Figure 8-29). The pre-RC is composed of four ~parate proteins that assemble in an OJ'dered fashion a l each replk atot. The first step in the tbTIIL:-'ttion of the pre-RC is the cecognitioo of' the rcplkator by the eukaryotic initiator. ORC. Once ORC is bound. it recruits two holicase loading proteins (Cdc6 and Cd.t1). Together, ORC and the loading proteins recruit a protein that is thought to be the eukaryotic replication fork helicase (the Mcm 2---7 complex}- Formation of the pre-RC doe.s not lead to the immediate w1winding of origin DNA or the recruitment o f DNA polymerases. Lnstcad thr pre-RCs that nee formed during G1 .are o nly activated to initiate replication after cells p (lss from the G1 to the S ph ase of tho cell cycle. Pre-RCs arc activa ted lo initiate replication by two protein kineses (Cdk and Ddk; Figure 8-30). Kinases are proteins that covalently attach phospha te groups to target proteins [see Chapter 5). Ea<:h of these kinases is inactive in Gl and is activated only wh en ccHs en ter S phase. Once
Binding cmd l lnwiruling: Origin SP-Ioction and J\cti'VfJt ion by the lnitictor Protein replica tor

origin and the initiat ion of replication (:;ee .Figure 8-30) . These new proteins inc h1de the th w e eukaryotic DNA po lymerases and a numhe.r of other proteins required for their recruitment. Interestingly, the polymcrases assemble at the origin in a particular order. ONA Pol 8 and e associate first, followed by DNA Pol o:/primase. This order ensures that all three DNA polymerases are pt·e sent a t the origin prior to the synthesis of t h e fust RNA primer (by DNA Pol a/primase). Onl}' a subset of the proteins that assemble at the origin go on to function as part of the eukaryotic replisome. 1n addition to the three DNA polymerases, the Mcm complex and marty of lhe factors required for DNA p olymecase recruitment bocom e part of the rep1ication fork machinery. Sim.ilar to the E. coli DNA .helicase Joade.r (OnaC), lbe other factors (such as Cdc6 and Cdll) are released or destroyed after their role is complete (see Figure 8-30).

Pre..RC Formation and Activation Is Regulated to Allow only a Single Round of Replication during Each Cell Cycle How do eukaryotic cells control the activity of hundreds or even thousands of origins of r eplication s u c h 1ha t not even one is a cti-

vated more than once during a cell cycle? The answer lies in the tight regulation of the formation and -activation of pre-RCs by cyclin-dependent k.inases (Cdks). Cdks play two seemingly contradictory roles in regulating pre-RC function (Figure 8-31.). First. as we described above, tbey are required tn activate pre-RCs lo initiate DNA repJicat ion. Second, Cdk activity inhibits the fonnation of new pre-RCs.

8-29 The steps ih the formation of the ~repliclltive complex (JWHK:)_ The assembly of the ,xe-RC is i!fl ordered proCESS that is rnibated by the tlSSOdation of the ongin recognition e-ornplex with the replicaklr. Once ba.rod 1o lhe replicalor, ORe re
FlGUR£

226

fiGURE

71lP. RP.plication of DNA

8-30 Activation ofthepre-RC

leads ao the assembly of the eubryotic repfecation fod<. As eels erter no tre S phase of the eel cyde, ~ and Ddk phosphorylate ~tion protens 10 trtgger ti-le intiat100 of re~ The events that lead to DNA I.Jl'1WIOdiog at the Ofigin are poorly U'lder· stood but are likely to require lhe actMty of lht> Man corrpex and resUI i1 the reaUtment of a rtJrrber of awciiclry repication factots and DNA Poll> and £ . DNA Pol o1prlmaSe IS ~ reotlfted after DNA Pd f> and e. Ch:e present at the 01"18'1\ DNA Pol o1pnmase synthesizes an RNA pomer e~nd OOefly extends ll The resubng prmer.template junction is recognized by the eukaryo4ic s!idng clamp loa<\er (RF-C), \1\ihch assembles a slid~ damp {PCNA) at these slles. Elther DNA fti 5 a £ recognzes ths ~mer and beglllS leading strand ~s. After a pericd of DNA unwlrdng. ClllA Pol ulprirnase synthes&les additicnal prrnEfS, I.A.t!dl aaow tile O!Jat10n of lagging strand ONA synthesis by eilher DJA Pol 6 or e.. Here we llllstrate Polli 01 the leading strand tn:l Pol ~ en the lagging strznd.

• p, awciliary factors

'"'Qt£ ~

I Cdc&

start lagging

strand synthesis

-

Bi111firJs ond Unwinding: Ongjn Selection orn:J -f1,ctivation by the Initial or Protein

Cdk activity low )

FIGUR.f

,.---~~--K,

".. _

pre-RC formation altowed

no pre-RC acnvalion

r,~

ZZ7

8-11 EffedofCdkadivityon

pre-RC formation and activation. High Cdk activity is requwed for exJSting pre-f(C ca-rpbes to •~ti
The tight connection between pre-RC function, Cdk levels, and the cell cycle ensures that the eukaryotic genome is replicated only once per cell c;yde (Figure 13-32). Ac:tive Cdk is absent during Gl. whereas elevated levels of C.dk are present during the remainder of the cell cycle

nopre-RC

pre-RC

activation

activation

G1

assembly phase Cdk levels low

c ellcyde

FlGU RE 8 -12 eel cycle regulation of <:& aaMty and pre-RC formation. In G 1. Cdr. levels are low and nevv pre·RC complexes can form but cannot be activated. lA.Jnng S phase, the elevated levets of Cdk activity trigger the initiation of DNA replication and prevent any new pre-RC CC~f1l)iel< fofmation on newly rcz:Aicated DNA. Onc.e a pre-RC is used for the o tiation of replication il IS nece.ssaady dlsm.anded (recall that at least one key c:ompooent of 1t>e pre-RC, the Mcm corT"{)Iel<, becomes part of the replication fed). Sim~My. replication of pre--RC associated ONA Cllso causes destJuctioo of the comj:kx (not stlO\Nn). Because Cdk ~ remain htgh ~I the end cf ,...-,ilOSts, no new pre-RC ~es can be formed tJntJl ctromosome segreg(lbofJ is cornpk:te. \Mthout new pre RC cCll1lplexes, refltlation is impossible.

228

'f'he P.eplimtintl nf DN A

(S, C2, and M phases). Thus. during each c:eH cycle there is only one opportunity for pre-RCs to form (during Gl) and only one opportunity for those pre-RCs to oo activated (during S. G2, and M- nlthough in pr..lctice all pre-RCs are activated or disruptEd by replication forks in S phase). Pre-RCs are disassem bled after tbey are activated or after the DNA to which they are bound is replicated. These exposed replicators are then available for new pre-RC formation and rapidly bind to ORC. Despite the presence of the initiator at these sites, the elevated levels of Cdk activity inS, G2, and M phase cells prevents the association of the other members of the pre-RC complex with ORC. It is only when ce1ls s~regate their chromosomes and cQmplete cell division that Cdk activity is eliminated a nd now pre-RC complexes can form.

Similarities between Eukaryotic and Prokaryotic DNA Replication Initiation Now that we bavc described initiation in eukacyotes and prokaryotes. it is clear that the general principles of replication illitiation are the same in hoth cases. The first step is the recognition of the replicator by the· initiator protein. The initiator protein in combination w ith one or more .belicnse loacllng pmteins. recruit the DNA helicase to the replicalor. The b e licase (ancl potentially otber proteins at the origin in enkaryotes} generate a region of ssDNJ\ that can act as a template for RNA primer synthesis. Once primers are synthesized, the remaining components of the replisom e assemble through interactions with the resulting primer;templale junction.

FINISHING REPLICATION Completion of DNA replication requires a set of sp ecific events. These evtir\ts are different fur circular versus linear chromosomes. For a circular chromosome, the conventional replication fork machinery can replicate the entire molecule, but the resulting daughter molecules arc topologically linked to one another. In contrast. replication ofthe very ends of linear chromosomes cannot be comp1eted by the replication fork machinery we have discussed so far. Therefore, organisms containing linear chromOsomes have devel oped novel strategies to overcome this end replication problem.

Type II Topcisomerases Are Required to Separate Daughter DNA Molecules

ft GU R £ 8•)) lopo$iomefase II

utaf)'leS the decatenation of replkation ptoducts.. M er a oralar IJNA molecule is repliCated, the restAttng complete dclughtef IJI\IA. molecules rema1n liliterl to one anothet Type II DNA lOfX'l$0f"I1Efases CQf\ efficf.entlv sep<ml\e (or decatenate) these 1)\JA cirdes.

After replication of a d n ..-ular chromosome is complete, the resulting daughter DNA mola:ules remain linked together as catena nes {Figure 6-33). Catemme is the general term for two circles that are linked (simi· lar to links in a chain ). To segregate these chromosomes into separate daughter cells, the two circuJBT DNA molecules must be disengaged from one another. This separation is accomplished by the adjon of type II topoisomemses. As we saw in Chapter 6, these enzymes have the ability to break a double-stranded DNA molecule and puss a second double--stranded DNA molecule through this break. Thus, type ll topoisomerases catalyze a break in one of the two daughter molecules and aHow the second daughter molecule to pass through the hreak. Thts reaction decatenates the two daughter chromosomes, allowing their segregiltion into separate cells.

Finishing Replication

229

Although the importance uf this activity for the separation of circular chromosomes is most clear. the activity of type ll topoisomerases is alsn a ·itical to the segregation oflarge linear molecules. Although there is no inherent topological linkage aft er the replication of a line-.ar molecule, the large size of eukaryotic chmmosom es necessitates the intricate folding of the DNA into loops attached to a protein scaffold. These attachments lead to many of the same problems that circular chromosomes have when the two daughter chromosomes must bQ separnted.

Lagging Strand Synthesis Is Unable to Copy the Extl"eme Ends of linear Chromosomes The requirement for an RNA primer to initiate alJ new DNA synthesis creates a diJerruna for the roplic.ation of the ends of linear chromosomes. This is called the end replication problem (Figure 8-34). This diffia.alt}l is not observed during the duplication of the leading strand template. ln that case, a single internal RNA prime.:r can direct the initiation of a DNA strand that can be extended to the extreme S' terminus of its t emplate. In contrast. the requirement for multiple primers to complete lagging sl.rond synthesis means that a complete copy of its templnte cannot be made. Even if the end of the l ast RNA primer for Okazaki fragment synthesis anneals to the final base pairs of the lagging strand template. once this RNA m olecule is removed . there will remoin .a short region of unreplicated ssDNA at the end of the chromosome. This means lhat each round of DNA replication would result in the shortuning of one of the two d<wghter DNA molecules. Obviously,

:s-~ s_ ~

s•

:

~

F f (i U R E 8~34

wl

s

3

s _.

~

2

+ ilK

'

/ last ~alai« ~s- rragment

~~~~~~~

~

5'

The end repic:alion problem As the Jaggmg stumd replication rnac:hinecy reaches tl'le end of the chrornosome. at some poa11 pnrnase no looger has sdfi<'Jeflt spoce lO ~hesize a new RNA primer. fus resulls in n:ornpSete replicatioo and a shm ssONA region c3t the 3' end of the laf:lgsng strand DNA product When lhis DNA product is repltcated 1n the next roundr one of the two products will be sha'tened «~
replication..

l

<eplicale again

~r•--~----------------------~-j~

s··---~~-I5JI--~-.:---~3' + ~:~ ~ .~ ~ ~ ::::::::::::::::::::::::~

+ +

..

2:to

Tht> Replirotinn of DNI\

fiGtJit£ 8-JS Proteinprimingasa solution to the end repficatian ptOblem. By tlnC!ing to the DNA pclymefase and to the 3' end of the template,_, ptote.-1 provtdes the

pnrni:r1g hydr~ group to lrihated ONA S)'T\lf'le-56. In tile exdmple shown. the pc01~in fffi1es all DNA synthesis as ts seen for many \llfl.JSeS. for b\ga DI'JA molerules, thiS method CQfT1b.nes lMih convenbOnal ortgln func:t10n to repticdte lhe

/

protein

5' ~0

3'

DNA polymerase

~

~-#

.P

chromosomes. !!J'

0

p

l l

3'

sn-o

+

3'

t his samario would disrupt the complete propagat ion of t he genetic material from generation to generation. Eventually, genes at the end of the chromosomes wo uld be IQSt. Cell<> solve the end replication problem in a variety of ways. On~ solution is to use a protein instead of an RNA as the primer for the lab1 Ol.nts are rlouble-stranded , the 3' end of ench chromosome extends beyond the 5' end as ssDNA. This unique structure acts ~s a novel ut1gin of replication th&t compensates for the end replication problem. This or)gin does not interact with the same proteins as the remainder of eukaryotic: origins. but it in ~1:ead recruits a specialized DN'A polylll€rCISe called telumerase.

Telomerase Is a Novel DNA Polymerase that Does Not Requjre an Exogenous Template Telomerasc is a remarkable enzyme that includes both protein and RNA components (and this is, therefore. an example of a ribonucleoprotein, see Chapter S). L..i.ke .,u uther DNA polymexases. telomerase acts tu extend the 3 ' end of its DNA substrate. BuJ unHkD most DNA polymera,ses, telomerase dt;>eS n ot need an exogenous DNA template to direct the addition of new dNTPs. lnstea.d, the RNA component of telomera~ serves as the template for adding the telomeric sequence to the 3' termin us at the end of the chrom osome. TelomerHse specifically dnngates thfl 3'0H of particular ss.DNA sequences using its own RNA as a template. The newly synthesized DNA is single-stranded.

The key to telomera.c;e function is revealed by the RNA component of the enzyme. Tbe sequence of the RNA includes 1.5 copies of the comple ment of th e telomere sequence {for humans, this Sf}(Juence is 5' -TAACCCTAA43'). This region ofthe RNA can anneal to the siugle--stranded DNA at the 3' end of the telomere (Figure 8-36). Annmling occurs in such a way that a part of the RNA template remains single-stranded, creating a J:f'imectemplate j\Jnction that Cil.I1 be acted on by telomerase. The protein component of te]omerase is related to a class of DNA polymerases that U8ft RNA templ<'ttes r.alled reverse tnlnscriptase& {As we Rhall see in 4

8-:J6 Replication of telomeres by teJomerase. Telomerase uses its RNA component 1o ~meal to the 3' erx1 of Che ssONA regi(JI'l of the telomere. Telomerase then uses 1ts reverse transcription actiVity to synthesize ONA to the end oC the RNA ternplclte Telomerase then displaces the RNA flom lhe DNA product and r€binds at ttJe end of the Fl GU R E

LRNA tebnefase

telornee ~d ~the PfOO$

s•.____ _

\

repeat

)

l=

-·

lelomerase extends 3' end of telomere

3'1:::=::::::=:==::=:=~ s·l ··s~·

~=.:::=::::::::.;1

I

addilionaJ 3' end DNA can act as template for new Ol
tmgnent

:

:::§'

FI(;UR E 8-37 Extension of the J' end of the telomere by ~erase $ohles the end replication problm\ Alth~ tdomerase only diredly extends the l ' end of the telomere. by ~«Mdtng an ad&tiorldltcmplate for ldgglng strend DNA synthesis, both eods of the
Chapter 11. these enzymes "re.verse trdllSCiibe'' R..\rA into DNA instead uf the more conventional transcription of DNA into RNA.) Tile telomerase synthesizes DNA to the end of the RNA templote but cannot continue to copy the RNA beyond that point. The RNA template disengages from the UNA produm. re-annenls to the last three nucleotides of the telomere. and then repeats this pt'OL--ess. The characteristics of tc)omerase are in some w~:~ys distinct nnd in other ways similar to those of otber DNA polymerases. The indusion of an RNA compOnent, the lock of a requirement for an exogenous template. find the ahiJity to use an entirely ssDNA substrate sets telomc:rase a part from other DNA polymerases. In addition .. teltJmerase must bave thf;3 ability to displace its RNA temphtte from the DNA product to allow mpeated rounds of template-directed synthesis. Fonnally, this means that telomcrase includes an RNA·DNA helicase activily. On the othe•· hand, like alt other DNA polymerases, telomerose requires a template to direct nucleotide addition, can only extend a 3' end ofDNA, uses the sam~ nucleotide pret..ursors. a nd acts in a proet3ssive manner, adding many S&JUence repeats each time it binds to a DNA .substrate.

Telornerase Solves the End Replication Problem by Extending

the 3' End of the Chromosome When telomer.tse acts on the 3' end of the telomere, it only extends this end of the chromosome. How is the 5' end extended? This is accomplished by the lagging strand DNA replication machinery {Figure 8~37). By providing an e xtended 3 ' end, tclomcrase provides additional tempJate for the ac«ion of the Jagging strand replication machinery wbic;h can then extend the 5' e nd of the DNA. It is important to note that there will still be an ssDNA region At the end of the chromosome. The action <>f teJomer-dSe and the lagging strand replication machinery, however, can ensw-e that the telomere is m aintained at suf6cient length to protect the end of the chromosome from becom· iog too short {and potfmtialJy deleting important genes). Although extension of telomeres by telomernse could theoretically go un indefinite ly~ proteins bound to the double-strnnded regions of the telomere carefully regulate telomere length. These proteins ad as weak inhibitors of telomerase n.(:tivity. \1\/hen there are only a few copies of the telomere sequence .repent. fev,t of these proteins will be bound to the telomere and telomerase activity '""ill be activated. As the telomere gets longer, these proteins wilt accumulate and inhibit the telomernw. Tlle repetitive nature of the te)omeric DNA sequence means that variations in the length of the telomere are readily tolaraled by the cell. Whether a chromosome has 200 or 400 repeats of the telomeric repeat. it wilJ he protected from recombination and degradation.

SUMMARY DNA synthesis is depellden\ tlpon the presence of two types of substrates: the four ciem.:ynucleoside triphospbates. d.ATP. dGTP. tlCrP. and dGTP; anrl tim template DNA structure, a primer.template junction. The template DNA detPnnines the sequencP. of in<JOl'pornted nudeotidos. The primer ~es as the substrate foe deoxynudeotide arlohioo, each being added successively to the OH at its 3' end.

DNA synthesis is ca~yzed by an enzyme caUed DNA polymerase that uses a single active site to add any of the four d.NTP pn!Clll'SQl"S. Structurol studies of DNA polymernses mveal that they resemble B hand that grips the catalytk site. This slnt<:ture l'Dt1.ltibutes to the extremely ac:curote nature of the ONA synthesis readinn. DNA polymernses are prQC.e58ive; ooch time they bind a substrate, they add many nucleotides.

Bibliosrophy

Proofrsading exonoclea<;{lS fu.rther enhances the accurt:tey of DNA synthesis by acti.ng lik~ a "delete key" thai fE'_IllOves incorp rectly added oudeotides. ln the cell. both strands of a DNA template are dupllcated sUT1ultaneously a t a Rtructure called the replication fork. BecaUi>'e rhe two strands of the DNA are <mtiparalleJ. only o.oe of the template DNA strands can be replicated in a cootit'\uous fashion (called the ~ing ~trend). 1'l.e other DNA stn:tnd (caiiEJd the lagging t;lrand) must be synthesized first as a series o r s ho11 DNA ~nts. called ~ fragmen ts. Each ONA strand is initiated with an RNA primer that is synthesiz.ed by an en?.yme called primase. These primers must be removed to complete the repHcation process. After the Teplacement. of. the RNA primers with DNA. iill of the separat~l.)' primed lagging strand DNA fragments are Joined «
233

Thus , the replicahon reaction works best when the entirt' array of replication proteins are pre$81t at the n>plicataon foTk Together th•s set of protei'ls fonns a comple'l called the replisome. The initiation of DNA replication is du·ected by specific U NA sequen£'.es caJled replir:ators T he physical site of replication initiation is called an origin of repli r2tio~ llte repHcator is spedfk..alty bound by a p t'Ole in ca1led the initiator which stimulates t11e um,..ti.ruling of the origin LJNA and the rec..rui~ent of other proteins required for the iniliation of repli.cation (st~ch a:1 ON.A helicase). T he s ubsequent events in the i nitiulion of DNA replication are largely drivtJn by ~ither prutei.&prolein ur non-specif:k protein-DNA inlernctions. In eukaryotic cells ~he initiation of UNA replication is ~ghtly regulated to llnstlre that every nucleotide of eVei)' ch.romosotne is replicated once and (mly once per round of ceU ilivision. ihis tight regulation is accomplished by controlling the (ormation and adivallon of 1:1 mul tiprotcin 815Sembly called thP. pl'e-repHcanve comptex (flT& HC). F01·n1ation of these complexes at rep licators is requited to recmit the proteins necessary to in itia•e DNA replicatio~ The abiJity to form f)nd activate pre-1<0; is contr(IUed by a cell cycle regulated kinase called cyclin-dependent kinase. During the G1 phase of the ceU eyr ie pre·RCs can be (bmted but ca.tmot dired the iniliation of ~pHt.alion. During the remaindeT of the cell cycle (the S, C2, and M phases), any existing pre-RCs can injtiate DNA replication but no nev.• pre-RCs «'.an be fonned Thus. any part.iwlar pre--RC ana only direct one round of initi~tion per ooll cycle. ensuriog that the ONA is replicated exactly ooc.e. F'mishing DNA repi.K;at:lon requires tJ-.e action of specific en zymes. For circular chromosomes. type II DNA topcls~ merases separate the topol~caUy linked circular products from one another. Linear r.hmmooomes also require special proteins to ensure their cornp1etc replication. Jn eukaryotic ce11s, a specialized DNA polymerase called telomerase allows tlte end., of the chromosome (called telomeres) l.o act as a llnique origin ofrepUcation. Dy exte nding the 3' ends of the telomere, tclonlefli.Se eHminates the progtessi\/e loss of cltroruosvme ends that conventionaJ synthesis by tlte replir.ation fodc nlaChinery would cause.

BIBLIOGRAPHY Books Brown T.A. 2002. Genom£>.s. 2nd edition. John Wile y. New York. end BIOS Scientific Publish ers Ltd., Oxford, United Kjngdom.

}ager J. and Pata J.D. 1999. Getting a grip: PolynJerases and t hei.f' s ubs trate complexes. Curr. Dpin. $ trttrl. Biol. 9: 21 - 28.

The Mechanism of DNA Polymerase

DeParnphilis M.l . 1996. DN A replication in eukmyolic fXIlls. Cold Spring HArbor Laboratory Press. Cold Spring HaJbor, New York. Kornberg A, ant1 B~ker 'T.A. 1992. DNA Replication. 2nd edition. W.H. Freeman, New York.

Doublie S. and Ellenberge'l" T. 1998. The mechanism of acti~m o f T7 DNA polymerase. Curr. Opin. Struct. B;ol. 8: 704- 712. Steitz T.A. 1998, A mer.hanism for all polymerases. Nature

Chemistry of DNA Synthesis

Sutton M.D. and Walker G.C. 2001 . Managing DNA poJymerases: Coordinating DNA replication, DNA repair, and DNA recombins tion. Proc. Nnll. Arod. Sci.

Brautigam C.A. and Ste itz T.A 1998. Struch.rralond funcp tiona) insigl1ts provided by crystal structures of DNA polymerases. Otrr. Opin. Stroct. Bioi. 8: 54 - 6 3.

391: 231- 232.

98: El342-8349.

234

The Rep1icati(ln of I)NA

The Replication Fork Baker T.A. and BeJI S.P.. 1W8. Polymerases and the replisome: Machines within machines. Cell 92.: 295- 305. Benkovi.c S.J. Valentine A.M. and Salinas F. 2001 . Replisome-mediated DNA replic.ation. Annu. Rev. Biochem. 70: 1R1- 208.

Patel P. H., Suzul.l M., Adman E.. Shinkai A., and Loeb L.A. 2001. Prol.aryotk DNA polymerase 1: Evolutioo. Structtll'e, and "base flipping" mechanism for nucleotide selection. /. Mol. Bioi. 308: 823- 837.

Initiation of DNA Replication

Wang J.C. 2002. Cellular roles of DNA topoisvmeruses. Nat. Rev. Mol. Cell Bioi. 3: 430- 440 .

Gilbert O.M. 2001. Mt~king sense of eularyotic replication origins. Scienef' 294: 96-100. Jacob F., Brenner S., and Cuzin F_ 1963. On the regt.~lati on of IJNA replication in bacteria. Col d Spri11g Hari;Jor Symp. Quant. Bioi. 28:329 - 348. Tye B.K. 1999. MCM proteins in DNA r eplication. Annu. Rev. Biochem. 68: 649-GB6.

The Specialization of DNA Polymerat;es

Finishing Replication

O'Donnell M .• Jeruzalmi D., and l
Kunkel T.A. and B~hflnek K zooo. DNA fi delity. Annu. Rtm Biochum. 69: 497- 529.

replia~t ion

Greid(}r C.W. 1900. Telomere length r~ulation. Annu. Rev. Hiocnem. 65: 337 - 365

CHAPTER

The Mutability and Repair of DNA

he perpetuation of the genetic material from generation to generation depends on maintaining rates ofmutation at low levels. High rates of mutat-ion in the germ line would destroy the species, and high rates of mutation in the- soma would destroy the individtmL Living cells require the correct functioning of thousands of genes. each of wrudt could be damaged by a mutation at many sites in its protein-coding sequence or in flanking sequences that govern its expression or the processing of its messenger RNA. (f progeny are to have a good chance at survival, DNA seque11ces musl be passed on largely unchanged io lhe germ-line. Likewise, the specialized cells of the adult organism could not carry out lheir lTlission if mutation ratElS in the soma were high_ Cancer, for example, arises from cells that have lost the capacity to grow and divide in n controUcd manner as a consequence of darru3ge to genes that govern the a..-eU cycle. lf ratf:lS of mutation in the soma were lligh, thE! incidence of cancer wouJd be catastrophic Bnd unsustainable. At the same time. if the genetic material were perpetuated with perfect fidelity, the genetic variation needed to drive evolution wouLd be lacking, and new species. indurHng humans, would not have arisen. Tbu.,, life and biodivmsity depend on a happy balance between mutation and its repair. In this chapter, we consider the causes of mutation and the systems that are responsible f"r reversmg or correcting, and thereby minimizing, damoge to tho genetic materia). Two important sources of mutation are inaccuracy in DNA replk.ation and chemical damage to the genetic material Replication errors arise fr<Jm tautomerization, which, as we have seen in Chapter 8. imposes an upper limit on the aCC\ll'BCy of base-pairing during DNA replication. The enzymatic machinery for replicating DNA attempts to <.'Ope with the mi&incorporation of incorrect nucleotides through a proofreading mechanism, but some errors escape detection. Also. DNA is a rornplex end fragile organic molecule of finite chemical stability. Not only does it su ffer spontaneous damage such as tho loss of bases. but it is also assaulted by natural and unnatwaJ chemicaJs and radiati.orl that break its backbone and chemically alter its bases. Simply put. errors in replication and damage to the genetic material from the rnvironment are unavoidable. A third impo11ant source of mutation is the class of insertions genoraled by DNA elements known es transposons. Transposition is a major topic in its own right. which we shall consider in detail in Otapter 11. Errors in replication and damage to DNA have two consequences. One is, {)f C()urse, permanent changes to the DNA (mutations), which can alter the coding sequence of a gene or its regulatory seqt1ences. The second consequence is that some c hemical alterations to the DNA prevent its use as a template for replication and transcription. The effect of mutati(lOS generally berome manifest only in the progeny of

T

OUT LI I\lE

• Replicatiorl £rrors and Th81 Repair (p. 236) DNA Damage (p 242)

23ti

The Mlllc#lity ond Repair ofDNII

the cell in which the sequence alteration bas occu.rrcd, but [esions that impede replication or transcription can have immediate eftects on ceU function and survival. The chaJlenge tor the c:eiJ is twofold. l'' irsr, it must sc.ar1 the genome to detect errors in synthesis and damage to the DNA Second. it must mend the lesions and do so in a way that, if possible, restores the original DNA sequence. llere we will discuss errol'S thai are generated during replication, lesions that arise from spontaneous damage lo DNA, .and damage that is wrought by che tnical agents anrl radiation. ln eac.h case we shall consider how the alteration to the genetic matE'rial is detocted ctnd how it is properly repaired. Among the questions we shall address are the following: how is the DNA mended rapidly enough to prevent errors from becolllillg set in the genetic m aterial as mutations? How does the cell distinguish the parental stmnd from the daughter strand in re pairing replication errors? How does tho ceJ1 restore the proper DNA sequence when. due to a break or severe lesion. the original soquenr..6 can no longer be road? How does tbe cell cope with lesions that block replication? The answers to these- questions depend on the kind of error or lesion that needs to be repaired. We begin by considering errors that occur during replication and how thfly are repaired. We then consider various kinds of lesions that arise spontaneously or from envinmmental assaults before turning to the. multiple repair mechanisms that allow the cell to mend rhis damage. We \1\rill see that multiple overlapping systems enable the cell to cope with a wide range of insults to DNA, underscoring the investment th~ living organisms make in the prese,rv(ltion of the genetic material.

REPLICATION ERRORS AND THEffi REPAIR The NatUre of Mutations Mutations indurle almost every conceivable change in DNA sequenceThe simplest mutations are switches of one base for another. There are two kinds~ transitions, which are pyrimidine-to-pyrimidine and purinetcrpurine substitutions, sudt as T lo C and A to G; and transversions. which are pyrim)di.ne-to-pwine and purine-to-pyrimhiine substitutions. such as T toG or A and A to Cor T lFigure 9-1}- Other simple mutations are insertions or deletions of a nucleotide or a small number of nudeotides. Mutations that alter a single nucleotide are called point mutations.

Other kinds of mutations cause more drastic changes in DNA. such as extensive insertions and deletions and gross rearrangements of chromosome structure. Such changes might be caused. for example, by the insertion of a transposon. which typically places many thousands of nucleotides of foreign DNA in the coding or regulatory sequences of a gene {see Chapter 1 t) or by the aberrant actions of cellular recombination processes. The overall rate at which new mutations arise

f I CURE

9-1

Base chaf1Ce substitutions.

(a) liansmons.. (b} liansversions.

a

b

u I ~

~

T -,

~

~

c

((~X')J T

C

spontaneously at any given site on the chromosome ranges from about 10 - 6 to 10- 11 per round of DNA replic:ation, with some sites OIJ the chromosome being ··hotspots" where mutations ariso at high frequency anct other sites und ergoing alterations at a comparativeJy low frequency. One kind of sequence that is parlicularly prone to mut ation merits special comment boc.ause of its importance in h uman genetics and d is-ease. These m utation-prone sequen ces are repeats of simple di-. tri- or letran udeotide sequences. which are Jmmvn as DNA microsatellites. One weiJ-knolA'Tl example involves rep eats of the dinucleotide sequence CA. Stretc.hes of CA repeats are found at many \1\rtdely scattered s ites i11 the chromosom es of hwnans and some other eukaryotes. The replication machinery has difficulty copying SQch repeats accurateJy, fre<Juently undergojng "s lippage." This slippnge increases or red uces the 1.m mber o f copies of the repeated sequence. As a result, the CA repeat length at a partir:ular site on the chromosome is often highly polymorphic in the pop\Jlation. This polymorphiso1 provides a cotwenient physical marker for mapping inherited mutations. such as mutations lhat increase the propensity to certain diseases in humans (see Box 9-1., Expans ion of Triple Repeats Causes Disease).

Some Replication Errors Escape Proofreading As we have seen, rhe replication machinery achieves a remarkably high degree of accurocy using a proofreading mechanism. lhe 3 ' - 5' exonuclease component of the replisome, which removeS wrongly incorporated nucleolides (as we discussed i n Chapter 6}. Proofreading improves the fidelity of DNA replication by a factor of about 100. The proofread ing ~onuclease is not, however, foolproof Some misincorporated nucleotides est.:apc detection and l'lel:Ome a mismak.:h between the newly syntltesized strand and the template strand. Three d iffarent nucleotidcs can be misincorporated opposite each of the four kinds of nucleoti(\es in the template strond (for example. T, C. or C o pposite a T in the templAte) for a total of 12 possible roisanatches (T;T, T:C, T:C'.. and so forth). H the m.isi.ncorpo:rated nucleotide is not su bseq uently d etected

Box 9-1 Expansion of Triple Repeats causes Disease

Ardher wei-known exaJ1l>le of error-prcfle sequences is repeats of the ~ nudeotide sequences CGG and CAG in certain genes.. In hLJmans such ~ repeats are often found to undergo ~ from one generation to the next. resulnng ., &eases that are progressiVely fT'IOfe severe fl the duk:ten and granddlidren of affl.cted tndMduals. Examples of diseases that are caused by tTiJ*.'I expansioo are ildt.At ITlUSCl.iar (myotrfn:e 1mpaa-s ~n lf'l neurms of the btain. indudl-lg the transcnption of the gene lor the receptOr of a neurotransrrJtter: Sl1lilar polyglutam11e strell:.hes from OG ~ in othEr genes may also exert rheir effects by disrupting rteracnons between tr
298

FIGURE

The Mulobilily anrl Rt~pair of DNII

9-2

A mutatioo can be

pennanendy incorporated by replication. A mutobon may be tntroduced by m~S~ncorpora lion of a base ill the first roond of repliCdlton. an the second 100nd of rt>pl«:.ation. the fl'li.JtDtiOn llecomes pemiOiielitfy tOCOfpoldtcd Ill the ONA sequence ftrstr~

replicatiorl (misincorpot'ation)

ond replaced, the sequence c hange wiU become pennanent in the genome: during a second round of replication, the misincorporated nucleotide. now part of the template strand. will direct the incorporation of its complementary nucleotide into the newly synthesized strand (Fig· ure 9-2). A t this point. the mismatch will no longer exist; instead it will have resulted in u permanent chaJJge (a mutation) in the DNA sequence..

Mismatch Repair Removes Errors that Escape Proofreading Fortunf'ltcly, a mechanism exists for dr.tocting mismatches and repairing them. Final responsibility for the fidelity of DNA replication rests with this mismatch repair system, which jncreases the accuracy of DNA synthesis by an additional two 10 three orders of mag11itude. The mismatch repair system faces two challenges. First, it must scan the genome for mismatches. Because mismatches are transient (they are eliminated following a second row1d of replication w hen r.hey result in mutations), the m ismatch repair system must rapidly find and 1-epair mismatches_ Second, the system must correct the mismatch accwately; that is, it must replace the rnisincorporatcd nucleotide in the newJy synthesiZed strand and not the correct nucleotide in the parental sb'Bnd. h1 E- coli, mismatches are detated by a dimer of the mismatch repair protein MutS (Figure 9-3). MutS scans the DNA. recognizing mismatches from the distortion they cause in the DNA backbone. MutS embraces the mismatch-containing DNA, inducing a pronounced kink in the DNA and a conforma• ional change in MutS itself (FJ,gure 9-4). A key to the specificity uf MutS is that DNA containing a mismatch is much more readily distorted rhan properly base-paired DNA. This complex of MutS and the mismatch-containillg DNA recruits MutL. a Sf':C(mcl protein romJ.lOilent of the repair systeln. MutL. in turn, activates MutH, an enzyme that causes an incision or nick on one strand near the site of the mismatch . Nicking is followed by the action of a specific heJicase (UvtO) and one of three cxonucleases (sec below). The hclicase unwinds the DNA, starting fro.:n the incision and moving in the direction of the site of the mismatch, and the exonud~se progressively digestt> the displaced singlP. strand. extending to and beyond the site of the mismatched nucleotide. This action produces n single-stranded gap. which is then fiUed i.n by DNA pol ymcrase ill (Pot ID) and sealed with ON A ligase. The overall affecr is to remove the mismatch and replace i t with the correctly base-paired nucleotide.

Replirotion Errors and Their RF.puir

239

f I c; U R E 9-l Mismatduepair pathway for the repair Clf repliutioo errors. (Sot.Jrr:e: Adapted from Junop MS • Obmolova G~ Rausch K. Hsieh P, and Yang W 2001. Composite active Site of an ABC ATPase. Mol Cell 7 : 10 . fig 6b.)

Mutl-£:)t £m ~

MutH -

T '-

·~------~--../"-_

!

DNA polymerase

mismatch repaired

240

Thtt MuttJhility nnd flEplnr of DNA

F l c; u R E 9-4 Crystahtructure of the MutS-DNA romplex. 1\.lolice the tonk in 1he OOA, p1esent near the bottom of the strucrure. Also, near the top of the structu~e of the enzyme, is ATP. shown in gteEO and red. (JI..In
Yall: W

200J. Composite ac.tNe site of
ArPase. Mol Celt7: 1- 12.) tmage prepared \'\itt~

BobSaipt. Mo1ScrlJt, and Raslet 30.

But how does the E. coli mismatch repair system know which of the two mi.smntched nucleotides to replace? If repair occurred ranrlomly, then half of the time the enor would become permanently established. in the DNA. The answer is that E. coli tags the paw.ntal strand by transient hemimethylation as we now describe. The E. coli enzyme Dam methylase methylates A residues on both strands of the sequence 5'-GATC-3'. The GATC sequence is widely distributed clong the entire genome (occurring Ht about once eve.ry 256 base pairs {411)}. and aU of these sites are methylated by the Dam methylase.

WhM a replication fork passes through DNA that is rncthylaled at GATG sjtes on both strands (futly methylated DNA), the resulting daughter UNA duplexes will be heroimcthylated (that is. methylated on onll the parental strand). Thus for a few minutes, W1tiJ the Dam methylase catches up and m ethylates the newly synthesized strand. daughter DNA duplexes will be methylated only on the s tranrl

that~

as a

tempfat~

(Figure 9-Sa). Thus. the new ly synthesized st.....-.md is marked lit locks a methyl group) and hence can be recognized as the strand for repair. The MutH protein binds at such hernimethylated sites, but its endonuclease activity is normally latent. Only when it is contacted by MutL and MutS located at a nearby mismatch (which is likely to be within a distance of a few hundrerl base pairs) rloes MutH ber.ome activated as we described above. Orlce activated. MutH selectively

Replirotion Errors anrl Their Repair

a

3"

nkks the unmcthyJated strand. so only m~wly synthesized DNA in the vicinity of the mismatch is removed and replaced (Figure 9-5b). Methylation is therefore a "memory" device that enables the E. coli repair system to retrieve the correct sequence from the p arental strand if an error has been made during replication. Different e:xonudease5 are used to remove single-stranded DNA between the nick created by MutH and the mismatch. depending on whether MutJI cuts the DNA on the 5' or the 3' side of the misincorpoi'ated nucleotide. lf the DNA is cleaved on the 5' side of the mismatch, then exonuclease VU or RecJ. which degrade DNA i.rJ a s'-.3' direction. remove the stretch of DNA from the MutH-induced cut through the misincorporated nucleotide. Conversely, if the .nick is on the 3' side of the mismatch, then the DNA is removed by exonuclease I, which degrades DNA in a 3'-5' direction. As we have seen, after removal of the mismatched base, DNA Pol Ill fills in the missing sequenre (Figure 9-6). Eukaryotic ce1ls aJso repair mismatches nnd do so using homologs lo MutS (called MSH proteins for Mut~ homoJogs) and MutL {called MLH and PMS). Indeed, eltkaryotes have mt~ ltiple MutS-like proteins with different specificities. For example. one is specific for simple mismatches, whf:lreas another recognizes small insertions or deletions resulting from "slippageJ· during UNA replication. Dramatic evjdence that mismatch repair plays a critical role in higher organisms came from the discovery that a generic predisposition to colon cancer (hereditaty nonpolyposis r:oloroctal cancer) is due to a mutation in the genes for human homologs of MutS {specifically the MSH2 hornolog) ancl Mull.

Z41

ftc:; u R E 9-5 Dam methyiatlon at repliation fmk. (d) Replic«
242

The Mutobzliry and Repuir of DNA

a

b

I

3'- - - - - --+-

=r-,

s·----

MutH/ n~

Exo Vh or Rec.J

(5'-3'exo)

3'1

1

MutL,MutS, helicase H, AlP

~-----,

9

s••..------ ==-->3' OH

•

)

3' -==----~~'------'--------, s· ...._ _ _ _ __ __,l3' OH I

1

1

)

3"0H

3'0H

----

fIGURE ~ Direction.aity in mismatch repair: exonuclease removal of mismatched DNA (a) Unmeth~ GAfC is s·

of m~abOn. (b) t..Jnmett¥ated GA.TC is 3' d mutation.

Even though eukaryotic cells have mismatch repair systems. they lack MutH and E. coli's clever trick of using hemimethylation to tag the parental strand. (Indeed, most bacteria Jack Dam methylase and are also unable to use heroiroethylation to mark the newly synthesized strand.) How then does the misffil!tch Yepair syste m know which of the two strands to corre<:t? Lagging s trand synthesis. as we saw in Olapter 8. takes place discontinuously with the formation of O kazaki fragmen ts that are joined to previously synthesizorl DNA by DNA ligase. Prior to the ligation step. the Okazaki fragment is separated from p reviously synthesized DNA by a nic~ whkh can be thought of as being equivalent to the nick created in E. coU by MutH on the newly synrhesized strand. Indeed, extracts of eukaryotic cells wHI repair mismatches in artificial templates that contain a nick and do so selectively on the strand that carries the nick. Recent results indicate that human homologs of MutS (MSH) interact with the sliding clamp component of the repHsome (PCNA, w hich we discussed in Chapter 8). and would thereby be recruited to t he site of discontinous DNA synthesis on the lagging s tnlnd. Interaction with the s liding clamp could also l'el:ruit mismatch repair proteins to the 3' (growing} end of the 1ead1ng strand.

DNA DAMAGE DNA Undergoes Damage Spontaneously from Hydrolysis and Deamination Mutations arise JlOt only from errors in replication but also from damage to the DNA. Some damage is caused. as we shall see. by environmental factors, such as radiation and so-called mutagens, which are chemical

DNA I>omoge

Z43

agents that increase the rate o[ mutation (see Box 9-2. The Ames Test}. But DNA al...c;o undergoes spontaneous damage from the action of water. {This is ironic since the proper structure of the double helix depends on an aqueous environment)

Figure l ). However, if the mutant ceOs are treated with a chemical that IS mutagenic (and henc.:e potentially carcinogenic), the chemical wil cause the misser1se or framestlift mutaOOrl (depending on the nature of the mutagen) to revert 1n a small number of the mutant cells. This reversal restores the capacity of the- cells to grow ~nd form co1onies (11 solid medium kldc.ing histidine. The more potent the mutagen, the greater the nt111ber of colonies. Sane chemicals that cause canc€fS are not mutagerlic to begin with, but rather a~ ~ verted into mutagens by the liver, which metabolizes toreign substances. To identify chemicals that are converted irto mutagens in the Wver, the Ames test trei~ts potentia~ mutagens with a mixtu-e of liver enzymes. OJemicals that are

Determining the potential OJronogenic effKI5 of cflerrricats in

animc:lts is time-consuming and eJ
I

f0lf1d to be mutagenic in the Ames test can then be tested for their potential cardnogenic effects in animats.

Salmone/18 bacteria culture

/reQl.iring histidtne to gaw

j 1oS cells added IQ agar \Mltl nutrients

but no rnti
::=~t irl agar

inc&Jbate 12 holn

-

suspe<:ted mutagen added 1o medl.ITI in agar

1

) /

cofOFites arising from spontaneous revertants

BOX 9-2 FICiURE 1 Thelbnestest.

colonies of revenaniS induced by the mutagen

244

The Mutobility ond Repair of DNI\

a 0

1~

0

N

J

b _

tizO ____,!~,............,

__ .. oti

~ ~ c

9-7

Mutation due to hydrolw1ic damage. (a) OearrilnallOn of cytosrne aei5les urzrl. (b) DcpumatJon or guaflWie by hydroly-.515 oeatf:s apurinic Oeo)()'l'ibose. (c) I.JeanW\ation of 5-methyt cyl.OSifle ~tes a Tldtural base in DNA, ltlyfrtlne F I C \J RI;

The most frequent and important kind of hydrolytic rtamagc is c;leamination {)r the base cytosine {Figu.re 9-7a). Under normal pbysiologicaJ tXIndilions, cytosine undergoes SpontaneOUS ueamination, thereby generating the unnatural lin DNA) base ucacil . Un1dl preferentially peirs with adenine and so introduces that base in the opposite strand upon replication , rather than the C that would have been directed b y C. Ad enine aod guanine are a lso subject. to spontaneo us deamination. Deamination converts adenine to hypoxanthine, which hydrogen bonds to cytosine rntheJ' than to thymine; guanine is c-onverted to xanthlne, which continues to pair witlJ cytosine. though with only n.vo hydrogen bonds. DNA also undergoes depurination by spontaneous hy drolysis of the N-glyoosyl linkage, and this produces an abasic site (tl1at is, deoxyribose IRcking a base) in the DNA {Figure 9-7b). Notice that, in contrast to the mplication errors discussed above, al l of these hydrolytic reactions result in alterations to the DNA that are tmnatural. Apurinic sites are, of course, unnatural and em:h of the clearoination reactions generates an unnatural base. This situation allows dumges to be recognized by the repair systems describetl below. This .situation aJso suggests an explanation for why DNA has thymine instead of uraciL If DNA naturally contained uracil instead of thyminP., then clearnination of cytosine would generate a n atural base. which the repair systems could not easily recognize. The hazard of having deamination generate a nalurally occurting ba~m is illu.c;troted by th~ problem caused by rhe presence of 5-methyJ cytosine. Vertebrate DNA frequently contains S·methyl cytosine in place of cytosine as a result of the action of me thyl transferases. This modified base plays a m le in the tTanscriprional silencing (see Chapter t 7). Deamination of 5-me thyl cytosine generates thymine (Figure 9-7c), which obviously wi1l not be recognized as an abnormal base and, following a round of DNA replication, can become fixed as a C to T transition. Indeed. mRlhylatcd Cs are hotspots for s pont aneous mutations in vertebrate DNA.

DNA Is Damaged by Alkylation. O.xidation.. and Radiation

f I(;UR£ 9-8 c:; modiftcation. The ftgure shows speofic sites oo guat'lln(' that are ~.able to damage by cheO'liC~ tre.3tmffll such as ~ or t»>idat!OC1, and by radiation. lhe p«rllcts of these modificelions in! ol\en higlty mutagenic.

DNA is vulnerable to damage from alkylation, oxidation, and radiation. In alkylation. methyl or el11yl groups are t ransferred to reactive sites on the bases and to phosphates in the DNA b ackbone. Alkylating chemicals include nitrosaminos and the v ery pote nt laboratory mutagen N-methyJ-N•-ni(ro-N-nitrosoguanidine. One of the most vulnerable sitP.s of alkylation is the oxygen of carbon ~tom 6 of guanine {f'igUI'P 9~6). The product of this methylation, c:,y;-methylguanine , of1en mispairs with thymintJ'" rosulting in the change of a G:C base pair into an A:T base pair when the damaged DNA is replicated. DNA is also s ubjact to attack from reactive oxygen species (for example. 0~ • H?.O~. and OH · }. These pote nt oxidizing agents rue generatet"l by ionizing radiation and by chemical agents that generate froo radicals. Oxidation of guanine, for ex:ample. generates 7,6-dihydTOS.oxoguanine or oxoG. The oxoG adduct is higllly mutagenic because it can base-pair ·with adenine as well as with cytosine. If it base-pairs with ade nine during wpli(;Biion, it gives rise to a G:C to T:A ttansvcrsion, which is one of the most c.ommon mu tations found in buman cancers. Thus, perhaps 1he carcinogenic effects of ionizing radiation and ox.idi7ing agants are partly caused by free radicals that convert guttnine to oxoG.

DNADnmo.ge

."

FIGURE

9-9 Thymine dime£ U

indices the forry~ation of a cydobt..~~ane between adjacent thymines.

{~~ N

•.( "-H

0

0

Yet another type of damage to bases is caused by ultraviolet light. Radiation with a wavelength of about ZOO nm is strongly absorbed by the bases. one consequence of which is the photochemical fus ion of two pyrimidines that occupy adjacent positions on the same polynucleotide chain. In tl1e case of two thymines. the fusion is called a thymine dirner {Figure 9-9), which comprises a cyclobutane ring generated by links between carbon atoms 5 and G of adjacent thymines. In the case of a thymine adjacent to a cytosine, the resulting fusion is thyrnin&cytosine adduct in which the thymine is linked via its carbon atom 6 to the carbon atom 4 of cytosine. These linked bases are incapable of lJasc..pairing aod cause the DNA polymcrase to stop during replication. FinaUy, gamma radiation and X-rays {ionizing radiation) are particularly hazardous because they cause double-strand breaks in the DNA. which are difficu lt to repair. Ionizing radiation can directly arrack (ionize) the deoxyribose in the DNA backbone. Alternatively, this radiation can attack indirectly by generating reactive oxygen species (described above}. wltich in tum react with the deoxyribose subunits. Because cells require intact chromosomes to replicate their DNA. ionizing radiation is w~ed therapeutica lly to kilJ rapirlly proliferating cells in cancer treatment. Certain anticancer drugs , such as bleomycin, also cause breaks in UNA. Ionizing radiation and agents like bleomycin that cause DNA to break arc said to be dastugenic (from the Greek klastos, which means "broken''). Mutations Are also Caused by Base Analogs

and Intercalating Agents Mutations are also caused by compounds that substitute for normal base-; (base analogs) or slip between the bases (intercalating agents) to cause errors in replication {Figlll'e 9-H l}. Base analogs are struc-

turally similar to proper bases but differ in ways that make them treacherous to the cell Thus, base analogs are similar enough to the proper bases to get taken up by cells. converted into nucleoside triphosphates, and incorporated into DNA duri ng replication. But, because of the structural differenc.es between these analogues and the proper bases. the analogues base-pair inaccurately, leading to frequent mistakes during the Teplicatiun process. One of the most mutagenic base analogs is 5-bromcUJ'acil, an analog of thymine. The pre:;ence of the bromo substituent allows th e base to mispair with guanine via the enol tauto:mer (see Figure 9-lOa). As we saw in

246

l 'he Murability ll(ld Repair of DNA

9-10 Base analogues and intercalating agwds dlillt cause mlJiations fIGURE.

in DNA. (a)

a -

H ·· ..

Base~ of thymtne,

5-brornouraOI.
-

H

(b) lnten:alatire agfflls. H 5-bromotJraal (keto tautomer)

5-bromOlr.JCII (f,JOOIIatJtomer)

guanine

b

Chapter 6, the keto tautomer is strongly favored over the enol tautamer, but more so foT thymine than for 5~bromouraci l. As we discussed for ethic1ium in Chapter 6, intcn:a.lating agents are flat molecules containing severn) polycyclic rings that bind to the equally tlat purine or pyrimidine bases of IJNA, just as the bases bind or stack with each other in the double heHx_ Intercalating agents, such as proflavin., aoidine, and et.hidium, cause the deletion or addjtio.n of a base pair or. even a few base paics. When suc:h deletions or additions arise in a gene, they can have profound consequences on the translation of its messenger RNA because they shift the coding sequence o ut of its proper reading frame. as we shall St::e when we consider the genetic code in Chapter 15. How do intercalating agents caUBe short insertions and deletions? One possibility in the case of insartions is that. by slipping between Ute bases in the template strand, these mutagens cause the DNA polymerase to insert an extra nucleotide opposite the intercalated molecule. {The intercalation of one of rhese structures approximately doubles t.he typicaJ dis lance between two base pairs.) Conversely. in the case of deletions, the distortion to the template caused by the presence of an intercalated molecule might cause the polymerase lo skip a n ucleotide.

REPAIR OF DNA DAMAGE As we h ave seen, damage to DNA can have two consequences. Some kinds of damage, such as thymine dimers or nicks and breaks in the DNA backbone, create impediments to replication or transcription. Other kinds of damage create altered bases that have no immediate

Repair of DNA Damage

247

structural consequence on replication but cause mispairing; these can Tesult in a permanent alteration to the DNA sequence after replication. For example, the conversion of cytosine to uTadl by deamination creates a U:G mismatch. whic.h. aft~r a round of replication. becomes a C:G to T:A trausWon mutation on one daughter chromosome. These consjderations explain why cells have evolved elaborate mechanisms to identify and repair damage before it blocks replication or causes a mutatioo. Cells would not endure long without such mechanisms. ln this se£1ion. we consider the systems that repair damage to DNA (Table 9- 1). In the most direcr of these sy1itcms (repmsenting lrufl repair), a repa ir enzyme simply reverses {undoes) the damage. One more e1aborate slHp involves excision repair systems, in which the damaged nucleotide is not repaired but removed from the DNA. ln excision repair sys tems~ the other, undamaged , strand serves as a template for reincorporation of rhe correct nucleotide by DNA polymerase. As we sl1alf sec, two kinds of P-xcision Tt"lpair mdst, one involving the removal of only the damaged nucleotide and the other. tha .removal of a s h ort stretch of single-stranded DNA that contains

the lesion . Yet more elaborate is recombinational repail'.. which is employed when both strAnds are damaged as when the DNA is broke n. fn such situatjons. one stTand c.annot serve as a template for the repair of thP. other. Hence in recombinational repair {known as double-.strand break repair). sequence in formation i.s retrieved from a second undamaged copy of the chromosome. Finally, when progression nf a tcphcating DNA polymerase is blocked by damaged bases, a special translesion polymerase copies across the sire of the damage in a manner rltat rloes not depend on b~se pairing between the remplate and newly synthesizec.l DNA strands. This mechanism is a system of last resort because translesion synthesis is inevitably h ighly error-prone (mutagenic}.

Direct Reversal of DNA Damage 1m example of repair by simple reversal of tlamage is photoreactivation. Photoreactivation directly rP.verscs the formation of pyrimidine dirners thar result from ultraviolet irradiation. In photoreactivation. the eflZ)•me DNA p botolyase captures energy from light and uses it to break the covalent bonds linldng ad jacent pyrimidines (Figure 9-11}. fn other words, the damaged bases are mended direcrly. Another example of direct reversal is the removal of thP methyl gt"oup from the methylated base 0"-rnethylguanine (see above). In this case,

TABlE 9-1 0NA Repair Systems

Msmatch repBir

Replication errors

MutS. rvlutt . aod MutH in E coli MSH, Mlli . and PMS in ht.xrlans

PtdOI'e activa lion

Base excrs101'1 repair

Pyrirridine d~s Di!maged b;!se

DNA photolyase DNA glycosytase

N'-X;levlide excisiOn repaif

Pytimidine dimer

UvrA. UvrS. UvrC , and UvrD in£ COli

BuiJ
XPC. XPA. xPD. ERCCI-XPf and

Double-strand tlleal<s Pynmdine dirner 0' apunnic site

RecA and RecBCD in E coli Y-family DNA poCym:Jrases, suet\ as UmUC in E. coli

XPG •n t'.unans

Oct.role-strand break repair l rar.slesioo DNA synthesrs

1'he Mutability one! Repair of DNA

uv

FIGURE 9-11 PhotOINdivation. lN inadi&lon causes formatton of thymine Omcr5. Upon exposure to light DNA photoly;,~ b-eaks the nng formed between the dimers to restpre the two lhymtne residue>.

a ntethyltransferase removes the methyl group from the guanine residue by transferring il to one of its own cysteine residues (Figure 9-12). This is very costly to the cell bet:ause the mathyltransfurase is not catalytic; having once accepted a methyl group, it cannot be used again.

Base Excision Repair En~mes Remove Damaged Bases by a Base~Flipping Mechanism The most prevalent way in which DNA is cleansed of damaged bases is by repair systems that remove and replace the altered bases. The two principal repair systems are base excision .repair and nucleotide excision ,-epair. ln the base excision repair. an enzyme called a glycosyl.ase recognizes and removes the damaged base by hydrolyzing the glycosidic bond (Figure 9-13). The .resulting abasic sugar is removed from the DNA backbone in a further endonucleolytic step. Endonucleolytic cleavage also removes apurinic and apyrimidinic sugars that arise by spontaneous hydrolysis. After the damaged nucleotide has been enlire!ly removed from the backbone. a repair DNA polymerase and DNA ligase restore an intact siJ'and using the undamaged strand a'> a template. DNA glycosyJases arc lesion-specific and cells have multiple DNA glycosylases with different specificities. Thus, a specific glycosylase recognizes uracil (generated as a consequence of deamination of cytosine}, and another is responsihle for removing oxoC (generated as a t;onsequenc.e or oxidation of guanine). A total of eight d iffurent DNA glycosylascs have been identified in the nuclei of human cells. OeallSin£ the genome of damaged bases is a formidable problem because each base is buried in the UNA helix. How do DNA glycosy1ases detect damaged bases while scanning the genome? Evidence indicates that these enzymes diffuse laterally along the minor groove of the DNA until a sp ecific kind of lesion is d etecte d . But how is the fiGU•E 9-12 Meu,yt group removal. Methyl tran~ catalyzes the tltvlsft:'l" of the mdhyt gr~ on <J6-methyt guanu-..e to

a cysteme residue on the enzyme, dlefeby restoring the flormal G 11 DNA.

Repair of nNA Domage

normal base

nttmal base

5'

249

I

~

O-P-0- ~

I

0

o q AP [ site

M> endo cleaves here (makes 3'0H JorPoC I)

/

o=P-o-~ 0 I

exonudease

0

0

r

APendofexo

O=P,- 0 - % 0

3'

3'

3'

FIGURE 9-ll Base excis.iof'l pathway. ltte c.lr.tdf gly(osylase reaction.. UfOCil glytosylase hyQnlyses the ~cosi& bond to release ...-a<.lt from the DNA bad site, lecMr1g a 5' phosphate 1he resulti1g 8
enzyme able to act on the base if it is btrried in the helix? The answer to this riddle highlights the remarkable tlexihility of DNA. X-ray crystallographic studies reveal that the damaged base is 11ippcd out so that it projects away from the double helix. where it sits il1 the specificity pocket of the glycosylase {Figure 9-14). Inte-restingly, the double helix

fIGURE 9-14 Stnlcture of a f>NA.., sfyc0$ylase c:omplelc. The enzyme is shown m Sf"Y
403: 859-866. lmGge prepared wi1h BOOScript, ~and

Raster 30.)

250

The MvluhiHty ond Repair ofDNA

f I cuRE 9·15 otto«;~ A repair.. Oodation d guanll'le pt'oduces oxoG The rno
is ablP. to allow base flipping with only modest distortion to its structure and hence the energetic cost of base flipping may not be great (see Otapter 6 and Figure 6--8). Nevertheless, it is unlikely that glycosylase.s tlip out every base to r.beck for abnormalities as they diffuse along DNA. Thus, the mechanism by whlch these enzymes scan for damaged bases remains mysterious. What if a damaged base is not removed by base excision before DNA replication'? Does this inevitably mean that the lesion will cause a mutation? ln the case of oxoG, which has the tendency to mispair with A. a fail-safe system exists (Figure 9-15}. A dedicated g)ycosylase recognizes oxoC:A base pairs generated by misincocpomtion of an A opposite an oxoG on the template strand. In this case, however, the g lyoosyla.se removes the A. Thus, the repair enzyme recognizes an A opposite an oxoC as a mutation and removes the undamaged but incorrect base. Another example of a fail-safe system is a glycosylase that Temoves T opposite a G. Such a T:C mismakll can arise, as we have seen, by spontaneous deamination of 5-methyl cytosine, which occurs frequently in the DNA of vertebrates. Because both T and G are normal bases. how can the ceU recognize whic.h is the incorrect base? The glycosylase system assumes, so to speak, that the T in a T:G mismatch arose from deatnination of 5-methyl--cytosine and selectively removes the T so that jl: can be replaced with a C

Nucleotide Excision Repair EnzyPles Cleave Damaged DNA on Either Side of the Lesion Unlike base excision repair. the nucleotide excision repair enzymes do not recognize any particular l~ion. Rather, this system works by recognizing distortions to the shape of the doublo helix, such as those caused by a thymine dimer or by ihe presence of a bulky chemical adduct on a base. Such distortions trigger a clmin of events that lead to the mmoval of a sbort single-stranded segment (or patd1} that includes tl1e lesion. This removal creates a single-stranded gap in t11e DNA, whir:h is filled in

by DNA polymerase using the undamaged st raml as a templAte and thereby restoring lhe original nucleotide sequence. Nucleotide excision repair in E. coli is largely accomplished by four proteins: UvrA. UvrB, UVJC, and UvrO (Figure 9-16). A complex of UvrA and UvrO sc.ans the DNA. with UvrA bejng responsible for detc:cting distortions to the helix. Upon encountering a cliSiortion. UvrA exits the complex and UVTB melts the DNA to create a singlestranded bubble around the lesion. Next. UvrB recruits UvrC. and UvrC creates two incisions: one located eight nucleotides away on the 5' side of the lesion and the other four or five nucleotides away on the 3' side of the lesion. These cleavages create a 12 to 13 msidue-long. single-stranded DNA segment, which is made accessible by the action of the DNA helicase UvrD. Finally, DNA polymerase I (Pol I) and DNA ligase fill in the resulting gap. The principle o f nucleotide excision repair in higher cells is much the same as in E. coli but the machinery for detecti ng, excising. and repairing tl1e damage is more complicated, in\lolving 25 or more polypeptides. Among these is XPC, which is responsible for detecting distortions to the helix. a function attributed to UvrA in E. coli. As in E. coli, the DNA is opened to create a bubble around the lesion. Formation of the bubble involves the helicase activities of the proteins XPA and XPD (the equivalent to UvrB in E. cD!Jl and the single-strancl binding protein RPA. The bubble creates cleavage s ites on the 5' side of the lesion for a nuclease known as ERCCI-XPF and on the 3' side for the nuclease XPC (representing the function of UvrC). Jn higher cells, the resulting single-stranded DNA segment is 24 to ~2 nucleotides long. A.o; in bacteria, the DNA segment is relP.ased to create a gap that is fi lled in by thfl ac:tion of UNA polymerase and ligase. As their names imply. the UVR proteins are needed to mend damage from ultraviolet light ; mutants of the uvr genes are sensitive to ultraviolet light and lack the capacity to remove thymine-thymine and thymine-cytosine adducts. In fact, these proteins broad ly recognize and rP.pair bulky adducts of many kinds. Nucleotide P.xdsion repair is important in humans, too. Huma ns can exhibit a genetic disease called xeroderma pigmentosum, which renders afflicted individuals high ly sensitive to sunlight and results in skin lesions, including skin cancer. Seven genes {referred to as XP genes) have been identified in which mutations give rise to xeroderma pigmentosum. These genes correspond to prote ins {such as XPA, XPC. XPD. XPF. and XPG. reterred to above) in the h uman pathway for nucleotide exdsion repair, underscoring tho importance of nucleotide excision repair in mending damage from ultraviolet light. Not only is nucleotide excisi on repair capable of mending damage throughout the genome. but it is also capable of rescuing RNA polymerase, the progre"sion of which has been arrested by the prese nce of a lesion in the transcribed (template} ~trnnd of a gene. This phenomenon. known as lransa-iptioo-coupled repair, invol\les recruitment to the stalled RNA polymerase of nucleotide e.xcil!lion repair protP.ins (Figure 9 -17). The significance o f transcription-coupled repair is that it focu.~cs repair enzymes on DNA (genes} bAing actively transcribed. In effect. RNA polymcrase serves as another damage-sensing protein in the cell. Central to transcription-coupled rep air in cukaryotcs is the

general transcription factor TFUH. As we wm see in Chapter 12, TFIIH unwinds the DNA template during the initiation of transcription. Subunits of TFIIH i nclude the DNA helix-opening proteins XPA and XPD discussed above. Thus, miH is responsiblp for two separate

·rhe Mutobility and Repmr of DNA

distortion

9--16 Nucleotide u
ment frorP tl'le dl..t>iell. and ONA Pol l ClOd ligase rep;w and SfMI tl'le 8C1> (Source. (parts a-d) AOOpte~ ffM1 lou Y and Van Hooten B 1999 SUand open•ng by the lMA, cortlf'lex alows dt n
\

UvrAB+ film

a s•

b

~

!i'

nick

Rt:!poir of DNA Dom0/1'

2!93

F f G U R £ 9-17 Transcription cot.lpled

DNA repair. (a) RNA pofymerase transcri:Jes DNA I'1ClfTl"laalv upstfeam of the lesicn (b) Upon encountering the lesioo In DNA RNA poly-

merase stals and ttaflSC.rip6on srops. (c) RNA polymerase re
.am transcription

3'

oodeotideexdsiOO repat proteins

functions: its strand-separdting helicases melt the DNA around a lesion during nudeotide ll.Xcision repair (including transcription-coupl ed repair) and also help to open the DNA template during the proc~s of gene h-anscription. S,ystems for coupling repair to trnnscription also e~'t in

p rokaryotes.

Recombination Repairs DNA Breaks by Retrieving Sequence Information from Undamaged DNA Excision repair oses the undamaged DNA strand as a template to replace a damaged segment of DNA on the other strand. How do cells repair double--strand breaks in DNA in which both strands of the d uplex are broken? This is accomplished by the double-strand break (DSB) repair pathway, w h ic h retrieves sequ ence infocmation from the sister chromosome. Because of its central ro.l£ in general, homologous terombination as well as in repair. the DSB-repair pathway is an important topic in its own right, which we shall oonsider in detail in Chapter 10. DNA recombination also l1clps to repair errors in DNA replication. Consider a replication fork that encounters a lesion in DNA (such as a thymine dimer} that has not been corrected by nucleotide excision repair. The DNA polymerase will sometimes stall attempting to replicate

over the lesion. Although the template strand cannot be used. the sequence information can be retrieved from the other daughter molecule of the replication fork by rE.'Combination (see Chapter 10). Once tthis recomhi ntttional repair is complete, the n ucleotidP. excision system hc\.
Translcsion DNA Synth~sis Enables Rt:plication to Proceed across DNA Damage In th~ examples we have considered so far. damage to the DNA is mended by excision followed by resynthesis using an undamaged remplate. But such repair systems do not operate with complete cf&iency and sometimes t1 replicating DNA polymerase encounters a le::;ion, such as a pyrimidine dimer or an apurinic site. that has not been repaired. Because such lesions are obstacles to progression of the DNA poly· mcrase. the repl ication machillery must attempt 1o copy across the lesion or be fnrr:ed to Ceast! replicating. Even if r.ell:> cannot repair these l ~ion s. there ls a tail-safe mechanism that allows the ll!plicalion machinel)' lo bypass these sites of d.smage. This mechanism is known as lranslesion synthesif!i. Although this mechanism is, as we shall see, highly errorprone and thus likely to introduce mutations, translesion synthesis spares lhe cell the worse fate of an incompletely rep.licated chromosome. TransJesion synthesis is catalyzed by a spet ializcd class of ONA volymer·a ses that synth~ize D.f\;A directly across the site of the damage (Figure 9-16). Translesion synthesis in E. coli is C«trried out by a oomplex of the proteins UmuC and UmuD'. UmuC is a member of a distinct family of DNA polymerases found in many organisms known as the Y-family of DNA polymerases (Figure ~19 flnd Box 9-3, The \'-Family of DNA Polymerases).

Repair of DNA nawoge DNA polymerase Itt

\

---

f I C \JR t

-----------.5.

~- ,. v --~----~----·

zss

9-18 Translesion DNA synthesis.

~encountering a !eYOO 11 the ~e

~

durif1g replication. DNA polymerase lll W1h its 5lidillg clamp dissociales from the DNA afld .s replaced by the trar~Siesaoo l:l\JA polyrnetase. w1ich extends DNA synthesis across the 1hymir.e

Pol IV ex Pol V (Oin B) (UITill£Y2C)

transleSIOI"l po¥nerese is then replaced by the

s

dimef on the templcte (~)strand. The

DNA polymerase Ill (SClLJre: R. Woodgate)

ONAPol Ill

\

F I Ci U R E 9·19 Clystal structure of a lranslesioo polymerase. ShoM1 here IS the strucll.lre ot a ~ (Y..farniy cw.) poly merase, in gray, ill compex with template DNA. in pupte. and an 10comtlg ....~. ., red. (Ling H, Brudsocq F~ Woodgate R, in1 \'afl8 W 2001.

Cel/107: 91 - 102. Image pepared with BobSaipt, MoiScripl ood Raster 30.)

An important feature of these polymerases is that. alt hough they are template dependent, they incorporate nucleotides in a manner that is in dependent of base pairing. This exp)~ ios how the enzymes ca.n synthesize DNA over a lesion on th~ template strand . Rut. ~ use rhe enzyme is not "reading.. sequence information from the template. tcanslcsion synthesis is often highJy error-prone. Consider the case of an apurinic or apynnidinic site in which the lesion contains no base-specific informati on. The translesion polymerase synthesizes across the lesion by ins erting n uclootides tn a manner that is not guided by base pairing. Nonetheless . the nucleotide incorport1ted ffii:!Y not btl rdndom -some tr.cwslesjon polymerdses incorporat e spe-

cific nucleoticlcs. For example. CJ human m ember of the Y-fam ily of translesion polymorases correctly inserts two A residues opposite a thymine dimer. liD 9-l The Y-Famity of DNA Polymerases DNA polymerases can be Bfoq>ed Vlto lamifies, shown in various cdcws tn the iigLre, based on lheir amino acid sequence SIITrilafilies to each othet Recently. UrntJC and certal'l other transleSIOO DNA pclymerases have been discovered to be fou1d.. tng members of a large and dlsmct famly of DNA polymerases kncJv.m as lhe Yfamily, vvtnch are fCU1d in alllhfee domains of life, Bacteria, Archaea, and Eukaryota Members of the Y..farnifv of DNA polymerases characteristlcally carry out DNA synthesis with km fidelity on undamaged DNA templates but have the capacity to bypass lesions •n DNA that ~ ~ by manbecs of the othef famiteS of DNA pdymerases. Ba< 9-3 F"tgt.re , stUNS a phylogenettc tree for the Y-famiy of tran51eslon DNA polymerases. VcoDinP

Mtu Dri'

Eoo Dlri3 PmuOrnP

PaeOirf'

MkJ DiriX Sco SC10A9.22c: OOh HaiYqjH

NmeOinP Ua Dlrl'

I

~Oirt>1

Bha&i1472 eauv~

Bhaf3H2741 Bsu YqjW 8Mfl()X269 Bsu SP8C2 UvrX ~Or113

uaumuc Efa pA01 UwJ\

PsyRUB R27 Wut.B '--~~=========== S1t1 Eoo R391 RIXI16

Sc REV1

Eoo~

SpREV1

Sty~

MmREV1h

stySamB Sty TP110~ Eco ~01 Mlc8

CmREV1h

Smat.Ue

Mm Rad3CB

Hs

IJmRadlre ScRad30

Sp Rad3Ut ES01

HsRad30A MmRad30A

DmRad30A NRad0300 LmUmuC

.BOX 9-3 FIGURE 1 lbepltylosenetictreeoftheY..fanMtyofONA polymereses. (Source; Adap1ed from Olvnon H. et al Lener to the editor The Y-faoity ot DNA polymerases. Mol Cell 8: 1. fJg 1.)

Su/TIJTlllly

251

Becnuse of its high error rate, trans)esion synthesis c.an be r.orutil lered a system of last resort. It enables the cell to survive what mjght othervvise he a catastrophic block to replication but the. price that is paid is a higher level of mutagenesis. For this reason, in E. coli the t:ranslesion polymerase is not present under normal cin;umstances, Rather, its synthesis is inducec:l only in response to DNA damage. Thus, the genes encoding the translesion polymerase are expresserl as part of a pathway known as the SOS response. Damage lr..ads to the protooJytic aestructjon of a transuipljonal reprru;sor [the LexA ..epres~ sor) which cAJntrols expression of genes involved in the SOS response indudlng those for UmuC and UmuD, the inactive precursor fur UmuD'. Interestingly. the same pathway is also responsible for the proteolytic conversion of UrnuD to UmuD' . Cleavage of LexA and UmuD are both stimulated by a protein caUed RecA. which is activated by single-stranded DNA resulting from DNA damage. RecA is a dual·func~ ticm protein that is also invo lved in DNA recombination as we shall see in Chapter 10. Finally. ~:ta.ru;lesion synthesis poses severn] fascil1C1tlng and as yet unanswered questions. How does the translesion polymerase recognize a ~•tailed replication fork? How docs the translesion enzyme replat::e the normal replicative polymerase in the DNA replication complex? Onrn DNA s.ynthesis is extended across the lesion, how does the nonnal replicative polymerase switch back to and replace the lronslesion enzyme at the replia:lfion foTk? Tranqlesion po)ymerases have )ow processivity. so perhHps they simply dissociate fmm the template shortly after oopying across a lesion. Nonetheless. this explanation still leaves us with the challenge of understanding how the normal processive enzyme is able to reenter the replication machinery.

SUMMARY Organisms can survive only if their DNA js replicated faithfuJly and is protected from chemic;aJ and physicaJ damage that woul(l change its aJding properties. The limits of oc:curate replication and repah of damage are revealed by the natural mulatioo rate. Thus, Htt avetage nucleotide is likely to be changed by mistal\.e <:mly about ooce every 1 0!1 times it is replicalt!d. altho~h error rates for individual bases can vary over a 10.000-fold range. Much of the accuracy of replfcation is Inherent in the way DNA polymerase copies a template. The initial selection of the correct base is guided by complementary pairing. Accuracy is increased by the proofreading acti vity of ONA poJymernse. Finally, in mismatch repair. the- newly ~;yo thesized DNA strand is S(.'311ned by an eneyme that initi· ales replacement of DNA containing incorrectly pain~d bases. Despite these safeguards. mistakes o( aU ty~ oc<:trr: baSP. suLstitutions. small and large addUions anrl deletions , and gross t-earrangements of DNA SE!quences. CeUs have a large repertoire of enzymes devoted to repairing DNA damage that would otherwise be lethal or would alter DNA so as to engooder damaging mulations. Some enzymes directly rever.;e DNA da.rnagtl. such as photolyases, which re"~'~ pyrimidine dimer formation. A more versatile strategy js excision repair, in which a damaged segment is removed and replaced through new ONA l>}'nlhesis for which the undamaged strand S@rves a5

a template. In base excision repair. DNA glyoosylases and endonucleases remove only the damaged nucleo1ide. whereas in nucleotide excision repair a short patch of single-st.Taoded DNA containing the lesion is removed. In E. coli. excision repart is initiated by the UvrABC endonuclea.-;e. which creates ~ bubble over the site of the damage and cuts out a 12-nocleotidc segment of the DNA strand that includes the lesion. Higher cells c-cUTy out nuclooUde exCision repair in a similar manner but a much lafE-er number of proteins is involved ;md the excised, singlestranded DNA is 24- to 32.-ces idues long. An alternative repair method, which is particularly impvrtant if no template for repair synthesis is available (as In the case of a double--strand break), is recombinational or double...strand break repair, in which an intact DNA strand is copied from a diffet-et1t but homologous duplex. Finally, traosle;ion 5ynthesis eJ'lables rep)i(.>ation to con tinue across damage that blocks the progression of a replicating DNA polymerase. Thanslesion synthesUi is mediated by a distind a11d widespread f.unily of DNA polymerases thai are able to c::arry out DNA synthesis in an error-prone manner that does not oopend on base pairing. Mutagenesis and its repair are of CQnetJm to us because they pennaoently affect the genes thaf organisms inherit and because cancet' iS often cauSP.(l by m utations in somalic cells.

258

The Mutability and Repair of DNA

BIBLIOGRAPHY Boob Friedberg E.C., Walker G.C.. and Siede W. 1995. DNA repair and mutagenesis. ASM Press, Washington, O.C. KombeJK A. and Baker T.A. 1992. DNA hlplicalion. 2nd ooition. W. H. freeman. N.Y.

Replicadon Errors and Their Repair Lindahl T. and Wood R.D. 1999. Quality t;outrol by DNA repair. Science 286: 1897 -1905.

DNA Damagt! Singer B. a nd Ko::;roierek J T. 195Z. Chf!mical rnut.:sgenB:>is. Annv. Rev. Biochem. 52:655- 693.

Repair of DNA Damage Bridges B.A. 1999. DNA repair. Polymerases for passing lesions. Curr_ Bioi. 9: R475- R477.

Citterio E., Vermeulen W., and H<;JEUjrnakers f.l-1. Transcriptional heaJing. Cel/101! 447- 450.

2fKl(J.

de Laat W,L., }aspers N.G.• and Hooijmalers j .H. 199!J. Molecular mechanism of excision nucleotide repair. Genes Dev..13: 768 - 785.

0rdpkin R.. Reardon }.T., Ans.ari A., Huang J.C.., Zawel L, J\hn K., Sa.ncar A •• and ReinbfJrg D. 1994. Dual role of

TFHH in DNA excision repair and in tran...criplion by RNA Polymerase Il Natllre 368: 7ti9-772. Klcczkowska H.E.. Marra G.• Lettieri T.• and Jiricny J. 2001. hMSH3 and hMSHn interact with PCNA and colocalize with it to 1·eplication foci. Genes and Development 15; 724-736.

C HAPT E R

Homologous Recombination at the Molecular Level

A

n DNA is recombinant DNA. Genetic exchange works constantly

to blend and rearrange chromosomes, most obviously during meiosis, when homologous chromosomes pair priur to tbe first nuclear division. During this pairing, genetic exchange between the c.:hromosomes occurs. This exchallge, c::lassir.ally tenned crossing over. is one of the results of homologous recombination. This recombination inVCllves the phylSl through DNA damage by replacing the damagcr! section with an undamaged DNA strand from a homologous chromosome. Rec...ombination also provides a mechanism to restart stalled (IT damaged replication forks. Fwthermore, special types of recombination rf:!glllate the expression of some genes. For e>.:aznple. by switching s pecific segmenL"> within chromosomes, cells can put otherwise dormant gem,'S into sites where

tlmy are m<prossed. In addition to providing an eXp]analion for genetic processes, elucidf:lting the molecuJar medlanisms of recombination has .led to the devel· opmont of methods to manipulate genes. lt is. for example. now routine to generate "knock-out" and .. transgenic .. variants in many different experimental organisms {see Chapter 21). These methods for deleting and introducing genes within the context of a whole orga.ni~m rely on recombination and are exceedingly powerful for detctmiuing ge..11c function.

MODELS FOR HOMOLOGOUS RECOMBINATION Elegant early experimfmts using heavy isotopes of atnms 1ncorpo.-ated into DNA provided the first molecular view of the pr(lcess of horovlogous recom bination. This is the s ame approach used by

O U TLINE

• Models fa' Homologous Recombtnat~on (p. 259)

Homologous Recornbw-.ation ProteJn Mac:hines (p, 26S)

• Homologous Recombtnateon 11'1 Eukai)UteS
• • Genetic ConsequetlCes of the Mechan1sm of Homologcus Recombinatioo (p_288)

Matthew Meselson and Fra nk W. Stahl to show that DNA replicates in a semiconservative manner (sec Chapter 2 ). ln their experiments, Mese)son and Stahl demonstrated tha t the products of replication contain one olrl And one new ly S)mthesized DNA strand. In contrast, tbis same expcrim.e n lal approach revealed that recombination is conservative. involving the dire<-1. breakage and rejoiniDg of DNA molecu les. As we wi ll see in the following sections. we now understand thal breakage and joining of DNA js a centra l aspect of homo I· ogous recomb inati on . Bu t reeomhinatio n a lso o ften i m., oJv~ b otlJ the limited d estruction and resynthesis of DNA strands. In the years since tllflse initial experiments. numerous models tv explctin the molecular n1echanism of genetic exchaJ;'lgc have been proposed. Kf.Jy steps of homologous recombination shared by these models include: 1. AHgnment of tv.,n homologous DNA molecul«S. By llomologou!i wn mean tbat the DNA set1uences are identical or nearly identical for

region or ut ) ~st a hundred base ~irs or so. [X)spite this high degree of similarity. DNA molecules can have small regions of sequence ilifference and may. for example. airry different st:quence varianrs, lmown as alleles, of the same gene. Introduction of breaks in the DNA. The breaks may occur 1n one DNA strc:md or involvH both DNA strands. The ne:~lure of these breaks is the feature that Jargely distinguishes the two models described below. For mation of initial short regions of base pairing betvveen the two recombining ONA moleculP.s. This pairing oa::urs when e1 singlestranded region of DNA o rigi~1ing from onEI parental molecuJe pairs with its cumplementary stnmd in the homologous duplex DNA molecule. This step is caUed strand invasion. As a result of strand invasion. the two DNA molecules become connected by aossing DNA strands. This croes structure is called a Holliday ;oncUon. Movcmen1 of the .Holliday junction. A Holliday jWJction can m ove along the DNA by the repeated melting and formation of base pairs. Each time the junction moves. base pai:rs are broken in the parental DNA molecules while identical base pairs are formed in the recombination intermediate. This process is called branc.h migration. Cleavage of the Holliday junction. Cutting the ONA strands wi thin rhe Holliday junc tion regenerates two separate duplex DNA molecules~ and there fore finishHs genetic exchange. T his process is called ..esolution. As we will see, whic h of the two pairs of DNA strands in the Holliday junction are cut during resolution has a large impact on the extent of DNA exchange that occurs between the two recombining m olocules. ii

2.

3.

4.

5.

The Holliday Model Illustrates Key Steps in Homologous Recornbina tion A s imple anrl h isloricully important model for homologous M(;ombinalion is the Holliday model (Figuce 10-1). Although it is now d ear that most recombination events involve some new DNA synthesi~-a fP.ature absent from this model-the Holliday model very well illustrates the DNA stTand invasion, branch migration, a11d Holliday junction rt~solulion proc.-esses centra} to homologous recombination.

Models for 1-Famologot!s Recombination

261

a 5' aligned tlomologous d\.4)1oxes

II

B

c

II

B

c

b

c

3'

3' 5'

a a

b

..... !/

:r sngle-straOOed breakS in each duplex

:r fl

A

c c

A

·----·-------- -----·----------------------

a

. . .. __ -... __

a

1

c fl

---- --------

___ ... --- # ------~ ---··

A

3' strand

c

--- ---- ... ~-- .... ~--- .... __ -.... _ --- ..

B

c

B

c

__

iwasion b

3'

!/

a

b

t:tanctl migation

_____ , _____... -_ .,.., -

1

d

~=

c

A

EA

•

A hybrid

3' 5'

d\Jplexes

a a

._b./ c --- ...... __ _ -. . ... ___ ... ....__ ... __ _ -- - -- __---...

....

nGU R E I G-1 Holliday modeJ through the steps of branch migration. lhe small arrOINheads oo the DNA sin&'e ~ po!flt in the s' to 3' direction Note that A afld o, B ilOd b, C and c specify difrer€11 al6es, N1d have sfightly afferent ~ seqt~enca Therefore, heteroduplex DNA corotarilg those genes

(sivJvn 111 the expanded sediOI"t 111 panel d) wi hi!ve some ~ches.

When illustrating the Holliday model. it is useful to picture the two homologous, double-stranded DNA molecules. aligned. as shown in Figure 10-ta. These molecules, although n early identical, carry ditlerent alleles of the same gene (as is denoted by the Ala, Bib, and Clc symbols in Figure to-1}. which are helpful for foJl owing the outcome of recombination. Recombination is initiated by the introduction of a nick in each DNA molecule at an identical location (figure 10-tb). DNA strands nt'~ar the nick site can then be "'peeled" away from their complementary str-. md s, freeing th~~ strands to invade, and ultimately ba~pair with, the homologous dupltlx (Figure 10-tc). In the structure shown in the figure, this invasjon is symmetrical: tha t is, the same region o f DNA fiequence is "swapJX!d'' between the two molecules. Strand invasion generates the HolJiday junction , the key recombination intermndiate. The Holliday junction generated by strand invasion am then move along the DNA by branch migration. This migrdtion increases the length of the DNA exchanged. Jf the two DNA molecules are not identical but. for example, carry a few small sequence differences, as is tme often between two alleles of the same gene-branch migrntion through th~ regions of sequence difference generates DNA duplexes CWTying one or a few sequence mismatches (see Band balleles in Figure 1Q-1d and the inset). Such regions are rolled heteroduplex DNA. Repair of these mismatches can have important genetic consequences. a point we return to at the end of the chapter. Finishing recombination requires resolution of the Holliday junction by cutting the DNA strands near the sile of the cross. Resolut ion occurs in one of two ways, and, therefore, gives rise lo two distjnct classes uf DNA products, as we now describe. Figure 10-2 illustrates where the alternative pairs of DNA cut sites occur on the branched DNA. To make these cut sites easier to visualize. the Holliday junction is "rotated .. to give a square-planner structure with no tmssing strands. The two strands w ith the same sequence and polarity must be d oaved; the two ~lternati ve choices for cleavage sites are marked 1 a nd 2 in Figure 10-2. The cut sites marked 1 occur in the two DNA strands that were not broken duri.rtg the ini tiation reaction (Figure 10-1h). If these st rands are now cut , and t.hen covalently joined (the second reaction catalyzed by DNA ligase as we discuss below), the result ing DNA nwl~ules will have the strut.:ture a nd sequenc e shown on the left in the bottom of the figure. These products are referred to as "splice.. recombination products, because the two original duplexes are now ..spliced together" such that regions from the parental DNA molec ules arc cova lently joined togF!ther by a region of hybrid duplex. As seen by fo11owing the a llele marke~. generation o f split.~ products results in reassortment of genes that flank the site of recombination. Therefore. thi s type of recombinant is also called the crossover product. as, within this DNA molecule, crossing over has occucred between the A and Cgenes. In contrast, the alternative pair of cut sites in the Holliday junction (marked 2 in figure 10-2) is in the two DNA strands that were broken to initiate recombination. After resolution and covalent joining of tltE: strand s at these sites, the resulting DNA molecules contain a region or "patch" of hybrid DNA. ThesP molecules arc thus known as the patch products. (n these proclucts, recombination does not result in reassortment of the genes flanking the sire of initial cl«*!vagt3

Motfpjs; for Homologous Recombi

site 1

!

DNA

deavage

·splice~

or crossover products

teassortmecrt of flool
-patch" or "OI'H:rOSSOVElr products no reassortment

~:.~~A~;;~;;8;;;;;;;;:::c:::: ~~A~~~~B~~~;;~C~~; A

b

c

~===a:~;;;~B~ ;;;;;~~cc~~~ b 8

A

C

..:.~ c

o-2 Holliday junc:tion deavage. Two alternative pairs of ONA s1tes can be an dUring resclulion. Cleavage at <:lfle par of sites generates the kspfice· or oossover prodUCiS. Cleavage at tl>e sec~ pail of siles yit::.Jds the •patch" or I"IClfH:fOSSOVe Pfoducts. The InSet shows a H~iday .flJJlCllon DNA stn.Jcrure. Notice that the DNA is c()Ollfetely base-paired ., this ~ure. n G u RE I

(see fate of the Ala and Clc allelP. markers in the figure). These molecules are, therefore , a lso known as the non-crossover products. Factors that influence the site and polarity of resolution will be rlisc.ussod below.

The DoubJe..Strand Break Repair Model More Accurately Describes Many Recombination Events I lomolO(loUS recomhination is often initiated by doubl~stranded breaks in UNA A common model describing this type of genetic exchange reaction is the double--stranded break-repair pathwa}' (Figure lo-3). As w ith the Holliday model, this pathway starts with aligm~d homologous chromosomes- But in this case. the initiating event is lhc introduf:.'tion of a double-stranded break (DSB) in one of the two DNA molecules (Figure 1U-3a). The otlmr DNA d uplex rP-mains illtact Because doublest::randed DNA breaks ou.:ur relat ivel}' frequently (as we shall soe below), this type of initiating event is attractive compared to the pair of aJ ignod nicks that l'IJ'O proposed to initiate re<..'Ol1tbi.n~tion by the Holliday model However, the asymmet ric initial breakage of the two DNA molecules in the OSB-rep air model necessitates that later stages in the recombination process are also ac;ymmetric, as we w ill seeAfter introduction of the DSB, a ONA-clea";ng e nzyme scquontia.Uy degrades lhc broken DNA mole<.:ule to generdte regions of singlo~tranded DNA (Figure 10~3bJ. This processing creates single-strand extensions, known as ssONA tails. on the broken DNA molPCt.Jlcs; these ssDNA tails le m\inate with 3'ends Jo some cases. both strands at a OSB are procesBflrl, whflrM~'> in other ca.c;es, only thr: 5'-terminating strand is degraded. The ssDNA tails generated Ly this pro<::ess then invade the unbroken homol ogow; DNA duplex (Figure 1Q-3c). This paneJ of the figure shows one strand invaston. as likely occurs initially, wh ereas the next panel shows the two invacling ~tran ds. In ~h case, the invading strand basepairs with its complementary ~trand jn the oth~.r DNA molecule. Because the invading slJ·a nds e nd with 3 ' tem1ini. they CW1 serve as primers for new DNA synthesis. Elongation from these DNA endsusing the oomplementary strand in the ho mologous duplex as a template-serves to regenerate the regions of DNA that were d estroyed during the prooossing of the strands at the break site (Figure 10·3 d,e). Jf the two original DNA duplexes were not identical in sequence near the site of the break (for example. having s ing le base-pair changes as described above). sequence information c...:ou1d be lost during recombination by the DSB--ropair pathway. ln the recombination event shown in Figure t o-3. ~uence infonnation lost fron1 the gray DNA molecule as a result of DNA processing is replaced by the :;equence present on the hlue duplex as a result of ONA synth ~is. This nonreciprocal ~p in DSJ3.-n!pair sometimes luaves a genetic trdue- giving rise to a gene conversion event-a point we will return to at the end of the chapterThe two Holliday jliDctions found in the recombination inlermediates gen erated by this modol m ove by branch migrotion and ultimately aro resolvoo to fi nish recomblnl'ltion. Once agai n. th~ strands that are d eaved during llJsolution of these Holliday junctions detennine whRther the product DNA molecules w ill «.:ontain reas.-,orted genes in the regions Banking the site of l'C(X>mbination (that is. result in crossing over} or not. Tl1e d1fferent ways to resolve a recombinatim1 intermedlate containing two Holliday junctions are explained in Box 10.1. I-Iow to Resolvo a Re-combination Intermediate with Two Holliday Junctions.

2 66

Homologous RP.COmi.Jination at fhR MoleculllT LP.vel

Box 1o-1 How to Resolve a Recombination Intermediate with Two HoUicSay Junc:tion5 How the Holliday junctions present ., a recombination inter rnOOate are cleaved has a huge impact on the structure of the prodtJct DNA I'Tldecules. Products will either have the DNA fla!1blg the site of recombination reassorted (in the splice/crossover products) or nof (in the patch/non-crossover prole d eavage sites (labeled site 1 and site 2). The sirnpk! nJe that determines whether a not resolution wi1l re;Ut in crossover versus 11Clfl-Crossover products ts as folows. 1f both j~ are deaved in the 50fTJe way, that is either both at site 1 or both at site 2, then nrn-crossover products wiR be generated. An ~ of this type of product is shovm in panel b of the figure; 1hese ete the molecu1es generated when both Holrtday junctJons are cleaved ot site 2. Nooce, the allele mati:.efS NB and a/bare stil on the same DNA mole<:ures as they were i1 the parental chromosomes. Cleavage of both junctions at site 1 also gEnffi!tes noo-aossover products.

a

• Cleavage of both junctions at site 2 wJ give a patch product {patdl + patch = patch, non-aossover prodOOs). • Oeavage crt both jlllC:OOns at site 1 also gives a patch product (~e + splice = patch because the second spice-type resolution essentialy "undoes" the rearrangement caused by the first deavage)

• deavage of one iunctioo at Site 1, but the other at site 2 therefore generates crossover products (splice + patch = splice), because the rearrangement caused by the site l cleavage is retai1ed in the final product

jmclionx (j)

junction y

'

~

A

5' ~ -===

3' 4i=:

In eotltrast, when the two Hdliday junctions are deaved using different sites, then the crossover products are generated. All example of 1his type of resolution is shovvn in panel c of Box 10-1 Fag~Xe I . Here junction x was cleaved at sate I whereas jU1Clion y was deaved at site 2. Notice that nov-1 gene A is lirlked to gene b. whereas gene a is lrnked to gene B; thus reassortment of the Hanking genes has OCOJrred. Oeavage of junclioo x at site 2 and junction y at site l also generates crossover products. Why is 1he sifl'llle rue true710 understand trcs, compare 1he junctioos shown here to lhe single Holiday JUnction shovvn en F.gure lo-2 Vou should see that, at a single jrnction, cleavage at site 1 WOtJd geve the sPice products, ~as deavage at site 2 wcUd generate patCh ~l.ds. So when you (Qf"li:;ine the restJts of cleavage at the tiM) JUrctions. ths is "~Mlat happens:

®-1£:

-

~E:

ff

a

B

t

t

(j)

b

resolution of x at

resotltion at site q) in both X and y

site (j) BOd y at site @

c

b

A

A

B !

a

B b

8

n on-crossover pr<XLds

,

5' 3'

3' 5'

-

a

B

a

B

A

A crossover products

8 0 X 10 •1 F I (; U R E 1 Two po$$1Die Wil)'$ of ~I'K an intentJed;.te from die DSB-tepait' pathway.

The parerltal 01\lA

moleaJes were like lrose in ftgtJ~e to-3. The regions of red DNA are those that were resynlhesiled rui~ recombincltion.

Models for Homologoos RecornbinaliDil

Double·Stranded DNA Breaks Arise by Numerous Means and Initiate Homologous Recombination Double-stranded breaks in DNA arise quite frequently_If these breaks are not repaired. the consequence to the f..'ell is disastrous. For example. a single OSB in the E. coli chromosome is lethal to a cell that lacl<s the ability to repair it. The m ajor mechanism used to repair DSBs in most cells is homologous recombination via the DSB-repair pathway described above.. Some cells also use a Rinlple.r ntechan.i~.m. calloo nonhomologous end joining (NHEJ} as well. This process is described in Cbapt£'.1' 9. In bac.1eria. the major biological role of homologous rec'()mbination is to ropair DSDs. These broken DNA ends arise from several causes {see C.haptoc 9). Ionizing radiation and other damaging agents sometimes directly b((l.ak both strands of the DNA backbone. M:any typ€S of DNA damage also indirectly give rise to USEs by interfering with tho progress of a replication fork For example. an unrepaired nick in one DNA strand will lead to collapse of a passing replication fork (Figure 10-4),

~--<:hrorrto --s-ornal --rep-tica_tion_fork _ _ __

~ ONA lesion in template strand

FIGURE 10-4 Damage in the DNA tetnplate cat) lead to DSB formation durif\s DNA repf.cation. This is edS.est to see when the tE!fll)laae contc1111S and (left panel). but also can oo:u when the tem-

plate cames a fork-sropptng leseoo (ngflt pancl). ln this case, the two newly synthewed strands {shown in red) can bose-pdir and rile folk CiJCl regress. This Sfruc.11..Jfe G3l1 be flll'thef processed by a l1l.lt'li>er of me~ns.. The broken end can seTVe to initidte reaxnbination.

267

Similarly. a lesion in DNA that makes a strand unable to serve as a template will stop a replication fork. This type of stalled fork can be processed by several different means (for example, fork regression or nuclease digestion; see Figure 10-4) that give rise to a DNA end witb a DSB. These broken DNA ends ttum initiate l'CCOmhination witb a homologous DNA moJecule. a pro<.'eSs which w iJl . in turn , hP.a.l the

break. In additi on to repairing DSBs in chromosoma l DNA. homologous recornhihatinn promotes gen e tic exchange in b acteria. This ext.:hange occurs between the c hromosome of one cell and DNA t"hat enters that (;ell via p h age-medi ated transduction or cell-cell conjugation (sec Chapter 21). In these cases, the Elnt..ring DN A comes into the cell as a linear molecule. and thus provjde.s the critical "broken'' DNA end n eeded to initiate rec.:om bination. ln eukaryotic ceUs, homologous recombination is critical for repairing DNA brealcs and collapsed replication forks. However, there are other times when recombination is also needed. As we will describe below, recombtnation is Q$Sent:lal to the proc:ess o f chromosome pairing during meiosis. In this case, as cells enter meiosis they produce a specific protein to introduce DSBs into the DNA and therefore initiate thi!': recombination pathway (see below). Thus, although they arisE! &om many different sources. the appearance of a DSB in DN A is a key early event in homologous recombin ation .

HOMOLOGOUS RECOMBINATION PROTEIN MACHINES Organisms from iill bianc.ues of life encode enzymes that c:atal}rze the biochemical steps of recombination. ln some cases, members of homologou s protein families provide the same function in all organisms. ln contrast, other recombination stops are catalyzed by different clasS4:1S of proteins in different organisms but 'lrvith tho same g~neral outc:osntJ. Our mo~t detailed understanding of the mechanism of' recombination comes from studies of E. coli and its phage. Thus, in the follow ing sections, we first focus on the proteins that promote recombjnation in E. coli via a major OSB-repair pathway. known ac; the RecBCD pathway. I fomologous recombhilation in eub.ryotic cells. and the proteins involved in there Hvents. are considered in later sections. Table 10-1 lists th€l proteins t hat catalyze critical recombination steps in bat.:tew:ia as well as those that serve these same funL'tions in eukaryotes (the bt.Jdding yeast S. cerevisiae is the best-rmderstood e>sult of DNA damage or failure of a repUcation fork are the major source of th~ initiating events in cht'Omosomal DNA Tha foll owing scc.tions dc.c;cribe the K coli recombination proteins and how they perform their fundions during rec.umbination Ly the

TABlE JD-1 Prokaryotic and tuluwyotic: facton that Catalyze Recombination Steps

Recombtnation Step

E. coU Protein Catalyst

- -- - - -RocA protein

Paw1ng hunok>gous ONAs and strand invastOO lr1ro00ctioo of OS8

DNA brooks to generate Sl~le strands

PrOOOS&ng

Of t5tfal1d

Rad51 Ocm1 (in meiosis)

1\iooc

Spa11 (in meiOSIS) HO (for maung-type switctuny)

RecBCD

MAX protem (also caJIP.d

hellcase,rnuctease

((;( InVasion A~

Eukaryotk: Protein Catalyst

Rad50158J60 f'luctease)

AecBCD and RccFOR

Rad52 at1d RadSY

RUvC

Pefhaps Must! 1 and

exchange pro<ens Hdliday Jt..rlction recogntlioo and br'c:IJ1ch migta taon Resduoon ot HOifaay JLif1CIIOOS

others

DSB-repair pathway. These proteins are discussed in the order in which they appear during the reaction pathway. First. we will see how the RecBCD enzyme processes DNA at the site of the OSB to generate single-stranded regions. Next. the structure and mechanism ofRocA, thP. strand-exchange protein. is d~cribed. RecA, after assombling on tlle single-stranded DNA, fiuds regions of sequence homology in the UNA molecules and generates new base-pairing partners between the.oo:e regions. The RuvA and RuvB proteins tha t drive DNA br.rnch migration are then described. Finally, 1he Holliday junctionresolving enzyme, RuvC, will be considered.

The RecBCD Helkase/Nuclease Pt"OCesses Broken DNA Molecules for Recombination DNA molecules with single-stranded DNA extensions or tails are the preferred suLstrate for initiating strand exchange between regions o1' homologous sequence. The Recl30J enzyme pl'OCeSSf>S broken DNA molecules to generate these regions of ssDNA. RecBCO also oolps load the RecA strcmd-exchange protein onto these ssDNA end<>. In addition. as we will sec, the multiple enzymatic activities of RecBCD provide a means for cells to ..choose., whether to recombine with. or destroy. DNA molecules that enter a cell RecBCD is cotnpo!'ed of three !\Ubtmit.s (1he product-; of the recB, rec.C, and reeD genes) and has both DNA heUcase and nuclease a<:tivitics. U hinds to DNA moleculCR at the site of a double-stranded break and tracks along DNA using the e11c.rgy of ATP-hy drolysis. As a result of its action, the DNA is unwowld, wi.th or without the accompanying nuclcoJytic destruction of one or both of the DNA :<>trends. The activities of RecBCD are controlled by spociflc DNA sequence elements known as ciU sites (for goS&Over hotspot ,instigator). O.i sites were discovered because they stimulate the frequency of homologous recombination. H guro 10-5 ~hows a schematic of RecBCO processing a DNA molecule containing a singJe chi site to activate this DNA for recombination. RecEGD enters the DNA ::tt the site of tht1 double-strand break

27ll

Homologous Recombinotinn ol ti1P Mn{ec:ulor LevP.l

-F I c:;------- -U R £ lo-S steps of DNA process;,g by Re broken ends. However, ch site; funclton orty in one onentation. On lhe ONA molerule shown. the c:t1! site is ooented such lhlt it will only rrodrfy a RecOCD enzyme that is rnMng hom 11ght to left The RecBCO enzyme has two [l\IA hefKaSeS: ReeD. moves rapdly on the 5' -ending Sband (bottom strand) <~nd RecB, v.tldl moves slowly a1 ltle 3 · ~ding snand (top strand)- Beca~Se these two stJbunts travel at different speeds, d-Ie DNA moleades at:OJmu late a si~S'!JaOd DNA loop on the tcp stral1d Wing ~g. A red )( IS sl1ov.in oo the ReeD solulit, after the eu:yme has encountered the ch site. to defde the if1activation or loss of thiS SIJbrnit

and moves along the DNA, unwinding the strands. The Reel~ and ReeD suhnnits are both ONA he1icascs, that is. enzymes that use ATP hydrofysjs to me1t DNA base pairs (see Chapter 8). The nudease activities of RecBCD frequently cleave each strand during unwinding and thereby tlestroy the DNA

Upon el1<..."0untering the chi sequence, the nuclease activity of the RecBCD enzyme is altered. As RecJ3CD moves into the sequence distal to the chi site (with respect to the broken DNA site at which the enzyme entered), it no longer cleaves the DNA strand with 3' --. 5' polarity. Furthermore. after the encounter with the chi site, the other DNA strand (the one w ith the 5'---+ 3 ' polaritylis dP.aved even more frequently than it was prior to the chi ~te. As a result o f this change in activity. a duplex DNA molecul~ is converted into one with a 3' singles tranded extension termina ting w ith the obi sequen ce at the 3' end. This structure i:. irl~.al for assembly of RecA and initiatio n of strand exchange (see belo w}. Tho m olecular basis of the change in RecBCD's enzyme activity after the encountt::r with a chi site is unclear, but appears

to be associated with either the inactivation or Loss of the ReeD subunit. The ssDNA tail generated by RecllCO must be coated by the RecA protein for recombination to occur. However. cells also contain singlestra.ndad DNA-binding protain (SSB} that can bind to this DNA. To

ensure that RecA, rather than SSB binds these ssDNA tails. RecBCD interacts directly with RecA and promotes its assembly. Chi sites increase the frequency of recombination about tenfold. Thi:o; stimuJalion is most pronounced directly adjcu.:.mt to tht~ chi site. Although elevate d recombination frequencies are observed for about 10 kb distal to the chi site, they drop off gradually over this distance

(Figure 10-6). The obser vation that recombination is stimulated specifically only on one "side,. of the chi site was initially puzzling. lt is now clear, however. why this pattern is observed: the DNA between the DSB (where Red:JCD enters) and thr: chi site is cut into small pieces by the enzyme and is therefore not available for recombinatinn. In oonuast, DNA sequ~nces m el by RccBCD aftm- its eoooun t~r with dli are preserved jn a .recombinagenic, single-stranded form and are specifically loaded with RecA. The ability of chi sites to controJ the nudeaS(} activity of RecBCD also hnlps bacterial cells p rote<:t thP.rosel ves from foreign DNA that may f.mter via phage infection or conjugation. Th~ eight-nucleotide chi site (GCI'GGTGC) is big hly overrepresented in the E. <."''li genom e: whereas it is predieted to occur only once every 6 5 kb, or about 80 times. the chromosomal sequence reve.als the presence of 1 ,009 chi sites! Because of this overrepresentation. E. coli DNA that enters an E_ coli cell i:; likely to bo proces.c;ed by RecBCD in a manner that gen erates thH 3' ssDNA tails, and thus activatecl for recombination. In contrast, DNA from another species (in which E. coli chi sites are not overrepresented) will lack frequent chi sites. RocBCD action on this DNA will Jead to its extensive degradation, rather than activation fo:r recmn bi nation. ln summ~ry. the DNA-degradation acti vity of Red3CD has multiple consequenU!s: this degradation is needed to process DNA at a break site for the subsequent stops of RecA assembly and strand invasion. In this manner. RecBCD promotes recombination. J-Iowever, because RecBCD degrades DNA to activate it, the overall process of homologous .recombination mu!>1 also involve DNA synth esis to regenerate thp, degraded stranrl.s- In addition, RedK'.J) sometimes functions simply lo destroy DNA-as it does when foreign DNA lacking frequent chi sites enters <Ells. In this w ay, RecBCD can protect cells from the potentially deleterious consequences of taking l•p foteign sequetJ<.'Cs, which . fen· example, may carry a bacteriophage or other harmful agent. r-0

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Homoktgou&Recom binaUrm at the lldoleculflr LP.vP-1

272

RecA Protein &-semhles on Single...Stranded DNA and Promotes Strand Invasion RccA is the central protein in homologous re.combination. It is the founding member of a family of enzymes called strand--exchange proteins. These proteins catalyze the pairing of homologous DNA molecules. Pairing involves both the sean.:h for sequence matt:hes between two molecules and tbu generation of rngi<ms of base pairing between these molecules. ThP. DNA pairing anrl strand-exchangP. activiti~ of RecA can be observed using simple DNA substrates in vitro; examples of DNA pairing and strand-exchange reactions useful for demonstrating the biochemical activitie5 of RecA are shoWn in Figure 10-7. The important features of these DNA molecules are: {1) DNA sequence complementarity between the two partner molecules; (2) a region of s jngle-strandecl DNA on at least one moieculo to a llow RocA asscmLly; and (3) the presence of a DNA end within the region of complAmr-mtarlty. enabling the DNA s lrnnds in the newly-formed duplex to intertwine.

The ac:tivc form of R•3CA is a protein-UNA filament (Figure 10~). Unlike most proteins involved in m olecular biology, that func;ti on in ~ller disc-:rete protein units, such as monomers, rlimers, or hexarners, the RecA Olamr.nt is huge and variable in size; filaments that contain approximately 100 subunits of RecA and 300 oudooti des of DNA are common. The filament can aurommodate one, two, three. or even furn strands of DNA. A s described below. filaments ·with either one or three bound strands are most common in recombination intermediates. Tbe structure of DNA within the filament is highly extended mmpared to either uncoated ssDNA or a stanrlarcl B-form helix. On average. the distance between adjacent bases is 5 A ratber than the 3.4 A spacing normally observed {Chapter 6). Thus. upon Re<:A binding. the length of a DNA molecule is extended approximately 1.5fold {Figure tO.&). lt is within this RccA-filament that the search for homologous DNA sequences is conducted and the exchange of DNA strands executed. To form a filament, subunits ofRecA bind coopHratively to DNA Ret:A binding QJ1d assembly are much more rdpid on singlc-stmnded than

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FIGURE 10-8 lMee views of the RecA fiament. (a) Electron moograph of circular DNA molecules that are fully or partially coated '-"'11h RecA. An tJncoated DNA rndewfe as~ shewn to ~rate hovv 1he DNA is elongated LpOO RecA bio<:Mg. (Source: Reprinted with permission from Stasiak A. and Egeiman EH 1988. V~llllion d Rec~ Readions. p. 265-307. lfl GMetk R€'COfT'Ibmotion R Kuchet1apc!li and G. Smith, eds., ASM Press. From flglxe ~-) (b) A higher resotution VteN of the tiJilrlent gener.lted by ~many Erlll trl'l
reso\J'Iiof'l ~w generated~ X-.ay ay~. Here one tun of the heicat tilanert is shcrM1 from a tq> down view. lrl&vi~ subt.nt.s are col ored; 1he red 'S\l;unit is doses\ to the~.

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274

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FIGURE J0...9 Polarity of RecA assembly. Note that new subui'Wt3 of RecA pn the lilamertf on the ONA 3' Stde to an ~lflt subom much faster than tf1ese sthmits JOin on the s• Side l1ecause of thts polanty of assetrbfy, CNA molecules wth 3 ' ssONA ~ wi be etfloentty coated Wid' RecA. In (l:)f'lbast, mol&ules with 5' ssONA ewtensi()(IS w¢uld r.ot serve as ~bates fOJ fb"rlent assemUy.

on double-stranded VNA, thus explaining the need fur regions of ssDNA in slmnd-exchange substrates. The filamoot grows by the addition of RccA subunits in tlm 5' to 3' direction, sut':h that a D NA suand that terminates in 3' ends is most likely to be coated by RecA {Figure 10-9). Note that in the OSB-repair model for recombination, it js DNA molecules with just this structwe that participate in strand invasion.

Newly Base-Pcrired Partnet"S Are Established within the RecA Filament RecA-catalyzed str-dJ1d exchange t:an be divided into rlistiuct reaction stages. First, the RecA 6Jament must assemble 011 one of the partic:ipating DNA molecules. Assembly oo::urs on a molecule containing a region of sjngle-st.randed DNA. such as an ss()NA tail This HecA-ssDNA complex is the active form that participates in the search for a homol· ogy. During this ~ih . RecA must "look., for base-pair w m plementa.rity between the DNA within the filament and a new ONA molecule. This homology se.arch is promoted by RecA because the filament structure has two distinct DNA-binding sites: a primary site (bound by the. first DNA molt:!eul.e). and a secondary site (Figure 10-10). This secondary DNA-bmding site can be occupied by double-stranded DNA. Binding to this ~ite is rapid. weak, transient and- importantly-independent of DNA sequence. in this w-dy, the RecA filament can bind and :rapidly "sample" huge stretches of DNA for sequence h omology. How does the RecA filament sense sequence homolQgy? Details of this m echanism are stiU not dear. The DNA in lhe secondary binding site is transiently opened and tested for complementarity with the ssDNA in the primary site. This "'testing" is presumably via base-pairing interactions~ although it occurs i.n1tially wlthout disrupting the global base-pairing between the two strands of the DNA in the secondary site. In support of this idea, e>.J>eriments suggest that the initial aligrunent may involve bas&flipping of some of the bases in the DNA duplex (see Chapter 9 for a discussion of base-flippjng during DN A rep air). (n vitro experiments indicate that a sequence match of just 15 base pRirs provides a sufficient signal to the RecA filament that a matf'.h h as been found,. and lhereby trigger strand exchange.

Homoloynm; Rf!CXm~binatinn Pratt-tin Machin~s

2 75

FIGURE 10..JO Modeloflwostepsin the seard\ for homology and DNA slfM!d exchange within the RMA filament. Here the RecA filament is represen'led tram a top OO...n \.1ew as in figure to-&:. The ll"lCCmirlg ONA~x IS shown in~ (Source: Adapted from Howard-Aanders et at. I 984. Norure 309: 2 15 - 220. Copynght @ 1984 N.3ture

Publistang Group. Used with permissiOn.)

1 DNA in secorldary sm is tested for oomplemefllarity

base--pairing between strands is switched

Once a region of base-pair complimentarity is located , RecA promotes the formation of a stabJe oomplex between these two DNA molecules. This RecA-bound thcee-str.andcd structure is called

a joint molecule and usually COIJtains several hlllldrod base p airs of hybrid DNA . It is within this joint molecule that the actual exchange of DNA strands occurs. The DNA strand in the primary binding site becomes base-paired with its complement in the ONA d uplex bound in the secondary site. Strand exchange thus requires the breaking of one set of base pairs and the formation of a n tiW set of identical base pairs. Completion of strand exd1ange also requires that the two newly-paired strand s be intertwined to form a proper double helix. RecA binds preferentially to the DNA pToduc:ts Rftar stnnd exc;hange bas occurred anrl it is this binding energy that aetually drives the tixchange reaction toward the new DNA configuration.

RecA Homologs Are Present in All Organisms Strand--exchange prote ins of thP. RecA family are present in all forms of

life. The best;.;haracterized members are RecA from Eubactoria, RadA

276

Homologous RtK:ODJbinolion allhe MaleculDr Le~/

from Archaea. Rad51 and Dmcl from Eukaryot.a. and the bacteriophage T4 UvsX prultiill. These proteins form similar filaments to that made by RocA (Figure l o-11} and likely funcHon in a11 analogous manner (although some features of the proteins are specificaHy tailored for their speci fie cellular roles and interaction partners). We will discuss the roles ofRad51 and Dmc1 recombination in eukaryotjc cells below.

RuvAB Complex Specifically Recognizes Holliday Junctions and Promotes Branch Migration After the strand invasion step of recombination is complete. the two rP.c:ombining ONA molecules are connected by a DNA branch known as a Holliday junction (see above). Movtlment of the site of this bran<:h requires exchange of ONA base pairs between the two homologous DNA duplexes. Cells encode proteins that greatly stimulate the rate of b.ranc.h migration. RuvA protein is a Holliday junction specific DNA-binding protein that recognizes the strur.ture of the DNA junction. regardless of its spocific DNA sequen~:e. RtnlA ret:ognizes and binds to Holliday junctions and recruits the RuvB protein to this site. RuvB is a hexameric ATPase, similar to the hexarneric helkases involved in DNA replica-

tion (see Chapter B). The RuvB ATPase provides lhe energy to drive the exr.hange of base pairs that move the DNA branch. Strur:tural models for RuvAB complexes at a Holliday junction show how a tetramer of RuvA. together with two hexamers of RuvB work together to power this DNA exchange process (Figure 10-12).

RuvC Cleaves Specific DNA Strands at the Holliday Junction to Finish Recombination Completion o[ recombination requires that the Holliday junction (or junctjons) betw-een the two tecombining DNA molecules be resolved. In bacteria. the major Holliday junction resolving endonuclease js RuvC. RuvC was discovered and purified based on its ability to cut

FIG lJ R E I 0·11 RecA-Iike proteins in three branches of life. NudeoprOlein filaments are shewn for (a) 11uman Rad51, (b) E. co6 RecA and (c) A fuJ9dus RadA proteins The RadS1 and RecA PI~ are also sl1own i1 figure 10-8. Notice the similar tQcaJ str\.Jctl,Jre o( the ftameflts reveated by the stnpes in these EM IIT'C~ges. (Source: West S.C et af. Nature RevJeWS in Molerolor and Cell Biology 4: 1 - 12 1m~ provided by A sity of Li!l.!Sanne. Switzeriand.)

Stasi~ Univef-

Homologous ~ombinatiofl Prott~in MocMnt!S

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fIG U R £ JQ-11 High resolution $tT\K1UJe of RuvA and sChematic model of the RuvAB complex bound to Holliday jundion DNA {a) The oy'Stal sttuctl.l'e of the RuvA taramer shows the foorfolcl ~ H.. Shioagawa H., and Morb.va K 2CXXl Proc /Votl. AaJd. Sa USA g]; 8257-6262) Image prepared ~t~1th BobSaflt MolSaipt. a1d Rester 3D. (b) Asd"lematic mcdeJ d the crystal WUCillle IS shown with tvvo RuvB hexarrErs. Notice hem a te11amef ol RuvAbinds !Mih foufok! symmetry to the jurld.lon. Tv.o hexamers of Rt.M3 hind on ~ Sides of RlNA. and functlOn as a motor to pump 0\JA through the jl.lndjon. The RlM3 hex.Yners .Ye shown io cross-sections, so 1tlat the DNAthreading tiYough these complexes can be seen (S
DNA jWlctions made by RecA in vitro. Genetic evidence indicates that it functions in ccmcert with RuvA and RuvB. Resolution by RuvC occurs when RuvC recognizes the Holliday junction (likely in a complex w ith RuvA and RuvBJ and specifically nic:ks two of the homologous DNA strands that have t·he same polarity. This deavase results in DNA tmds that term.inate with 5' phosphates and 3'0H groups that r.an be diroc:tly joined by DNA ligase. Depending on which pair of strands is cleaved by RuvC, the resulting ligated recombination products will be of either the ··splice.. (crossove£) or "pak.h'' (non-
5'

216

Homnlogous Recombrnation at the Molt>Cular LeH~/

a

b

s· F I (i U R IE

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RUVC dimer bound to Holliday juncUon DNA

(a) The aystol stnx:ture of the RlNC. protein (Ariyoshi

M.. Vassytyev O.G. lvl'i!SaiU 1-l~ Nahmurn H , Shir-.,gawa H , af'ld Mcrtkawa K 1994. CeH 78: 1063-1072.) hlage prepared wrth BobSaiPf, Mt:JISaipt, and Raster 30. (b) Model fa binding d a RuVC dimer lo 3 Holltday 1UI'lCtlor1 Notice how. tn tNs modef. a &-ner of RuvC can btfld the Holliday Jllf!COOn and 1ntroduce symmell\G!l cleavages into the two 1denbcaa DNA sttamk (Source: Rofterty H i et al. 1996. Gystal struct\Jre ot DNA rerorntr.ation protein RtNA.5cJence 274: fig. l b. p. 416. flg. 3e, p. 418. Copynght 0 l996 American Assodabon fOf ~he Advancement of Soeoce. Reprlrlted wrth permisSton.)

HOMOLOGOUS RECOMBINATION IN EUKARYOTES Homclogous Recombination Has Additional Functions

in Eukaryotes As we have just described, homologous recombination in bacteria is required to repair double-stranded breaks in ON A. to restart coJiapsed

replication forks, and to allow a cell's chromosomal DNA to recombine with DNA that enters via p hage infection or conjugation. Homolo~us Ter..ombination is also required for DNA repair and the restarting of rollapsed replication forks in eukaryotic cells. This requirement is iUustrated by the fact that Gell$ with defeds in the proteins that promote recombination are hypersensitive to DNA damaging agents, especially those that break UNA strands. Fm1honnore, animals carrying mutations that interfere with homologous recombination are predisposed to c;ertaln types ot' cancer. However, as we will d iscuss below, homologous recombination plays important additional roles in eukaryotk organisms. Most importantly. homologous t'ecombination is critical for meiosis. During meiosis, homologous recombination is required for proper chromosome pairing and, thus. for maintaining the integrity of the genome. This recombina· tion also reshuffles genes between the parental chromosomes. ensuring variation in the sets of genes passed to the next genel'ation.

Homologous R ecombination Is Required for Chromosome Segregation during Meiosis As we saw in Chapter 7, meiosis involves two rounds of nuclear division, :resulting in a reduction of lhe DNA content from lhe nonnal content of diploid cells (2N). to the content present in gametes (lN).

Figure 10-14 shows sr.hcmatically how the chromosomes are configured during these two division cydes. &fore division, the cell has two copies of each r:hromosome (the homologs), one eac.h that was inherited from its two parents. Dining S phase, these cJJromosomes are replic:ated to gjve a tota.l DNA C()otenl of 4N. The proouds of replic.ation- that is the sister chromatids- stay together. Then, in pmparntion for the first nuclear division, these d vplir:oted homologous chromosomes must poi.c and a lign at the center of the cell. It is this pairing of homologs that requirP.s homologous recombination (Figure 10-14). Th~ ev~n ts are carefully t;med. Recombination must be oo.mplete before the first nuclear division to a llow the homulogs tu prop€rly align and then sepa· rete. During thls p~. sister chromatids remain paired (see CJlapter 7, Figure 7-16). T hen. in the second nuclear division, ;1 is the sister chr-omatids that separate. The products of this division are the four gametes~ each w ith one copy of each chrom()S()me (that ;s, the lN DNA content). Without re<:omLination, \.hromosomes often fai l to align properly for the fi.rsl meiotic d ivision. and. as a result, there is a h igh incidence of chromosome loss. This improper segregation of chromosomes. called nondisjunction, leads to a large number of gametes without the correct chromo5()D1e c()mplrunent. Gametes with either too fuw or too many chromosomes cannot develop properly once fertiliLed; thus, a fa ilure in homologous recombination is often reflected in voor ft:rtility. The homologous roc()fnbination events that occur during meiosis are called meiotic recombination. Meiotic recombination also frequently gives rise to crossing over between genes on the two homologous parental chromoson1es. Thi~;; genetic exc·har.ge c;a n be observed cyrologk.ally (Fjgure 10-15, top pw1el}. An important consequence is that t he alleles prese nt on the parental DNA molecuJes are ~ssortt;~d fQt the nm..1. generation.

Programmed Generation of Double,Stranded DNA Breaks Occurs during Meiosis The developmental program needed for cells to Sllcces.sfull y complete meiosis involves turning on the expression of many genes that arc not needed during normal growth. One of these is SP01I This gene encodP.s a protein that introduces double-strand breaks jn chromosomal DNA to initiate meiotic recombination. The Spoll protein cuts the DNA at many chromosomal locations. wjth little sequence selectivity. but at a very spedfic time during meiosis. Spolt-mediated DNA cleavage occurs right around the time when the replicated homolog<ms chromosomf'l6 start to pair. Spol l r.ut-s ites, although frequent. are not randomly distributed along the DNA. Rather, the cut-sites are located ruost commonly in duomosomal regions that are not tightly packed with nudeosomcs, such as promot~rs rontroiling gene transcription (see ChapleJ'S 7 anc.l17). Regions of DNA that experience a high frequ ency of DSBs als
200

Hcmwlogous RFX'Dmbinatiun at the Molt:cular Levtd

fiGURE to-14 ONAdynamks during meiosis. Here. only one t)Pe of ciYomosome is sl1own for da11ty. The 1\Ml homologs are 5hown, tn red ond blue. aftet they have been rlupic.ated by a mufld of ONA replication. Homologous recombination is required 10 pair these horrclogoos chromosomes in preparation for the fiCSt l1lJCie;lr division. !hiS recombir.i!tion can also lead to crossing over, as is stiDwlllere ~ the A afld B genes.

Homologous Rt:combs1wtioo in Bv/wryott'S

Ml

FIGURE Jo-15 Cytologicalviewof

crossing ova Reoprocal crossing ove:J directly visualized in harm.ter reUs in tissue OJltute. Chromosomes whose ONA cool:ilins bromode~.e in place of thymidine ifl both -strands
combir.ant chromosomes are also seen Vvhen mrtotically growing c~ ile treated with a DNA-damaging .agefll (bottom). (SOOfce: Couf· tesy of Sheldon Wolff and Jody Bodycote.)

The mechanism of DNA cleavage is as follows. A specific tyrosine tside cllain in the Spoll protein attar:ks the phosphodiester backbone to cut thtJ DNA and generate a covalent complex between the protein and the severed DNA sttand (Figu re 10-16). Tw o subunits of Spo11 cleave the DNA two nudeotides apart on the two DNA strands to make a staggered double-strand break. SpoJ 1 shares this DNA cleavage mechanism with the DNA topoisomerases and the s ite-specific recombinases (see Chapter 6 and Chapter 11). In fact, Spo11 appears to be a distant cousin of these enzymes. The fact that Spo11 cleavage involves a covalent protein-DNA oomplex has two consequences. First. the 5' ends of the DNA at the site of Spoll cleavage are covalently bound to lhe enzyme. It is these Spo11linketl 5' DNA ends that are the ioitial sites of DNA processing to create the ssDNA tails required for assembly of RecA-liktl proteins and initiation of DNA strand invasion (see below). Second. the energy of the deaved DNA phosphodiester bond is stored in the bound proteinDNA linkage, and so the DNA strands can be resealed by a simple reversal of the cleavage reaction (see Chapter 11, Figure 11·7). This resealing can or:cur when r.ells receive a signal to stop pror.eeding with meiosis.

2132

Hvmolosous S f!CCJ111binolion al tl1c Mnferolar Level

FIG U R f. I D-16 Mechanism of deavage

by Spo 11. The OH group of a lyt0510e lf1 the Spell prae.n attacks the ONA to form a covcdent proten- ONA li~e r~ subunits of Spo 11 are reQUired to generate a double~ DNA bre.3k. one to attaCk eadl of the

Spo11

DNA deal/age

two DNA str.Jnds. Note. because d thi5 cleavage IT':edlantsm, the OSB car. be reseak!d by the Simple ~sal of the cleavage reacllon.

MRX Protein Processes the Cleaved DNA Ends for Assembly of the RecA~Iike Strand.-Exchange Proteins The DNA at the site of the Spoll-<:atalyzed double-strand break js processed to generate single-stranded regions needed. for assembly of the RocA-Iike strand-exchange proteins. As was observed in the RecBGD pathway from bacteria, this processing generates long segments of singlt}-stranded ON A that tetm.inate in 3' ends (Figure 10-17), During meiotic recombination. the MRX-eneyme complex is responsible for this DNA processing event. This complex. althvugh not humoiogous to Rec.BCD, is also a multi-subunit DNA nuclease. MRX ls composed of protein subunits called Mreu. Rad50, and XrsZ; the first letters of lhese suhunits give the complex its name.

Processing of the DNA at the break site oc(;Urs exdusiveJy on the DNA strand that terminates with a 5' end- that is. che strands covalently attached to the Spo11 protein (as described above). The strands terminating with 3' ends are not d(Jgiaded. This DNAprocessing readion is therefore called 5' to 3' resection. The MRXdependent 5 ' to 3' resection generates the 1ong ssDNA t~i1s with 3' ends; that are often 1 kb or longer. The MRX cvmplex is also thought to remove the DNA-linked Spol1.

Dmc l Is a RecA~Iike Protein that Specifically Functions in Meiotic Recombination Eukaryotes tmrode two well-characterized homoJogs of the bacterial RecA protein: Rad51 and Dmcl. Doth proteins function in meiotic:

HQmologous 1/et;OITII.Iinafiorr in Eukoryotes

5' 3'

================

FaG U R E

(l'lClnSNer)

recombination pathway. f ormation d the dooble stranded breaks dutiog meiosis requires

homOiogs

the presence of boUl Spo 11 and the MRX com-

5'

3'

Spo11

MRX

s

3'

===============

DSB fcrmation

5'

3'

1 o-11 Overview of meioOC

replicated

bySpo11

plex. Ths observation~ that DSSf{J(Jll(ltloo and sU>sequent strand processing are noonaly coupled by 1he IIRX protem is respor!Sitle for resection of the S·-ending str.lnds at the break !tile. The~~ p~ Q-ncl

RadSl

and then asserr~Jie en the ssOI'JA ta~ Both

proleKlS pdftiopate 11'1 fe<:cx-rhnatiol\ but hoN they work together IS not known. They are

shown forming separate filaments for clarity.

5• 5' to 3'resection

~·..~:::;::::::::::::::::::::::::: a·

byMRX

(Source: Lidlten M 2001. Breaking rhe genome to save ft. Qlrrent. Biology 11· fig 1. p. 10~5. Cop).flght © 2001. with perrn;ssion from E1se\iier.)

assen'tlly of the strand exchange protem t1laments

strand invasion

recombination. Whereas Rads1 is widely expressed in cells dividing mitotically and meiotically, Dmcl is expressed only as cells enter meiosis. Strand exchange during meiosis occurs between a particular type of hom<)logous DNA pattnef_ Recall that meiotic recombination occurs at a time when there are four complete. double-stranded DNA molecules representing each chromosome: the two homologs each of which have been copied to generate two sister chromatids {Figure lD-16). Although the two homologs likely contain small sequence differences and carry distinct aUeles for various genes. the majority of the DNA sequenr.e among these fuw copies of the chromosome will be identical. Interestingly. Dmct-dependent recombination is preferentially between the nonsistec h omologous chromatids. rather than between the sisters (Figure 10-18}. Although the mechanistic basis of this selectivity is unknown, there is a clear Nological rationale: meiotic recombination promotes interhomoJog connections to assist alignment o£ the chromosomes for division.

interhomolog fecombioation by(lmcl FIGlJR£

lo-18

Omc:l-dependent

re
Many Proteins Function T~ther t<> Promote Meiotic Recombination As we have described. proteins involved in the critical stages ofDSB for-mation. DNA processing to generate 3' ssDNA tails. and strand exchange during meiotic recomhination have been identifit>d and chamMerized. Ceoetic experiments indicate that many additional proteins also participate io this process. Furthermore, many proteins appear to interact with the kno \ 'Vll recombination enzymes and it seems lilcely that these proteins function in the context nf a Jarge multicomponent complex. 1bese laJ"ge protein-DNA complexes, known as reoombination fadories. can be vis ual i7.ed in ceU~. For example, the co-localization o f Rad51 and Dmcl to thestt factories during meiosis is shown in Figure 1(}-19. Rad52 is another essential recombination protein that interacts with Rad51. Rad 52 functions to promote assembly of Rad51 ONA filaments. the active form of Rad51. It does thjs by antagonizing the action of RPA, the major s ingle-stranded DNA-binding protein present in eukaryot\c ceUs. lo this respect, Rad52 shares an activity with the E. coli RecBLTI protein, which. as we learned, helps RecA load ontn ssDNA that would otherwise have been bound by SSB. By analogy with bacteria, we e>.- pect that euka:ryotic ceJis encode proteins that promote the branch migration and Holliday jund ion resolution steps of recornhination. In fact, en?.ymcs capable of promoting these reactio ns are being identified For example, the Mus81 protein, which js highly conserwd in eukaryotes. js required for meiosis, and may function as a Holliday junction resolvase. As we have seen, n1eiotic recombination aligns homologotlt) chromosomes and promotes genetic exchange between them. These recombination reactions oflen lead to crossing over between the parental chromosomes.. Recal I, however, that depending on how the HoUiday junctions in lhe recombination intermediates are reSolved, recombiuation via the OSB-repair patllway can also give rise to non-crossover products (see above). These events may provide the essential chromosome-pairing function ooeded for a successful meioHc division . yet leave no detectable change in the genetic makeup of the chromosomes. But, even non-crossover recombination can have genetic cmlSequences. such as ~iving rise to .a gene conversion event. G~n ~ conversion happens when an aUele of a gene is lost and replaceJ by an a lternative aJJ eJe. Examples of how gene co nversion occurs both in m itotically-growing cells and during meiosis are d escribed in tbe following sections.

FIGURE 10-19 Co-~oftheRad51 a'MSDnK1 proteinsto "recombiMtion fadories" in cells undefgoing meiosis. ProtelflS were detecled by im~a11vng \Mth flJorescently labeled anribodles to Rad5 1 (green) and Dmd (red). When 1he two protelt)o; cob:ak7e 1he merged image appears ve'ow (Source: Adapted from Shinohara M. et at. Ttd1/l)

Mating-Type S witching

MATING-TYPE SWITCHING In addition to promoting DNA pairing. DNA repaif~ and genetic exchange. l10mologous recombination can also serve to change the DNA sequence ar a specific chromosomal location. This type of recombination is sometimes used to regulate gene expression. For example. recombination controls the mating type of the budding yeast S. cerevisiue by switching which matjng-type genes ru·e present at a specific location that is beiog expressed in th~f organism's genome. S. cerevisioe is a single-cell eukaryote that can exist as any of three differen t cell types (see Chapter 21). HaploidS. ceJtwisiae cells can he either of two mating types, a or a. And. when an a and a cell come in close proximity they can fuse (that is. "mate") to form an a/o diploid cell The ala cell may then go through meiosis to fo.rm two haploid a-cells and two haploid a-cells. The mating-type genes encode transcriptional regulators. These regu]ators control expression of target genes whose products define each cell t ype. The mating-type genes expressed in a given cell are those found at the mating-type locus fMAT locus) 1n that cell (Figul'() 10-20). Thus, i n a-celJs the at gene is present at the MAT locus , whereas in a-cells, the cxl and n2 genes are present at the MAT locus. In the diploid ce ll , both sets of mating-type control genes are expressed. The regula tors encoded by the matingtype genes, tngelher with others found in all three c-ell types, ac1 in v~uious combinations to ensure that the correct patte~n of genes is expressed in each cell type (see Chapter 17). Cells can sw itch the ir mating type by recombination as we now describe. rn addition to tlle a or o: genes present at t he MAT locus i.n each cell , there is an additional copy of both the a and n genes present (but not expressed) e]sewhere in lhe genome. These additional silent copies are found at loci called HMR and H!YIL (Figure 10-20}.

HML

MAT e<pressed locus

silent locus

HMLa

a~

co.L-1_:_---~.:.-~!

'

a1

Yn

acet

HMR silenl loclls

Ya~-------------T-_Y. _a~---

.. ~ ._______ ___.1_

ll

MATa

f

HMRa

HO endOI"llldease

deavage

n1

+n2

Yn ~~----~----------------------~-4~~ar=! :H 0: ~ - - -- - ---'---

HMLc:t.

MATc:t.

« ·type silent cassette FIC V R E 1o-20 G~tic loci e~~toding mating-type info rmation. AI~ thrOI'I'I
.....Lor:...__

These flMH and HML loci are therefOre known as silent cassettes. Their function is to provide a "'storehouse., of genetic information that can be 1.1sed to switch a cell's mating lvpe. This switch requires the transfer of genetic infonnation from the liM sites to the /tvfAT locus via homoJoguus recomhination.

Mating..Type Switching ls Initiated by a Site...Spedfic Double--Strand Break Mating-type switching is initia ted by the jntr()duction of a OSB at the MAT locus. T his reaction is perfo rmed by a specia lized DNAcleaving enzyme, called t he HO endonuclease. Expression of the IlO gene is lightly regulated to en sure tha t switc hing o ccurs only when it should. The mechanisms responsible. for this regulation aa·e uiscusse
Mating,Type Switching Is a Gene Conversion Eve11t, Not Associated with Crossing Over Although the OSB-repair pathway could explain lhe mechanism of mating-type sw itch recomb ination, current evidence indicates that,

Mating-Type Switr:hins

a information

287

FICOUR£ 1&-21 Recombination MOdel for mating-type swikhing: synthesis-

t

HOdeavage )

D

dependent strand annea&mg {SOSA). 1he figure shows lhe st~ leading to gene corrversicn at the MAT loaJs. Tne HMR and MAT regions are shown in green; the region of HMR encoding the a rlOlf"lla\100 ss represented Ill dark green whereas the regioo of N.AT the a infOlfl"liltion IS shown 1n ~me green. Upon

encodirs

completion of ~ss of SDSA, ltie a regtOn on.gii"ICIUy present a MAl has been replaced by. that is. cowerted to. the a 1111formatloo present m 1he HMR regiOJ\.

b

3'0

tL (J

5'

c

-..

Su

3'0

replication fort< assembly hf:.re

3'

s·o

d

..

)

..

5'o

~:· 5' 0

""")

lR_1__ strand invasion

l

Synfuesis ol,_ new ONA strands from a iOfonnation ~en"t>late

at HMRa

·~

~--~ n

i

/

1>trand eKcised

branch migtal!on to disengage dttpte,<es ~emoval ol sevond old strand at MAT

repar synthesiS and sealtng

of DNA strands

e J-IMRa 5 0 3'0

--..

a information

..,

~

a information

after the strand i.nvasinn ~:;tep, this recombination pathway diverges from the DSB-repair mechanism. One hint that the mechanism is different is tltat the crossover class of recombination products is never observed during mating-type swUching. Recall that in the DSB-repair pathway, resolution of the Holliday junction intennediates giv~ two classes of products: the splice, or crossove!' class. and the patch. or non-cmssover, class (see Figure 10-2). According to the DSB-repair modeL these two types of products are predicted to occur at a similar frequency, yet. in mating-type switching. crossover products aro never observed . Therefore, models for recombination that do not involve Holliday jtJnction intermediates bettor explain mating-type AWitr:hing. To explain gene conversion without crossing over. a new recombination model tcnne curs. In oontrast lo normal DNA replication, however, the newly S)mthcsized strands are displaced from the template. As a result, a new double-stranded DNA segment is synthesized, joined to the DNA site that was originally cut by HO. and resected by MR.r'X. This new segment has the sequence of the DNA segtuent used as the template (HMRa in Figure 10-21). Completing recombination Tequires that the other ··otd" DNA sttand present at MAT (the 3 '-ending strand not dcttved by MRX} be removed {fhe bottom strand in l'igure 10-2ld). Titen, the newly synthesized DNA - an exact copy of' the intoonation in the partner DNA molecule - replaces the information that was originally present. Thls mechanism nicely explains how gene conversion occw-s without formation of a HolHday junction. Thus, by this model. the absence of crossover prnducts during mating-type recnmbinatinn is no longer mysterious.

GENETIC CONSEQUENCES OF THE MECHANISM OF HOMOLOGOUS RECOMBINATION As discussed in the beginning of tbis chapter. initi al models for the mechanism of homologous recombination were fonnu lated hu·gely lo explain the genet! consequences of the process. Now that the basic steps invol\led in recombination arc underStood , it is useful to review how the pTOcess of homologous recombination aJters DNA molec:ules and thereby generates specific genetic changes. A ct:Jntral feature of homologous recombination is that H can ocelli between any two regions of DNA ~ regardless of the sequence. prov•ded that these regions are sufficiently similar. We now understand why 1hi.s is true; nooe of 1he s teps io homologou.c; .rect1mbina1ion require rec:ognitirm or a bpecific DNA sequence. F'or s1eps that have some sequence preference (such as the transformation of Rec1;3CD by chi sites and DNA deavage by RuvC protein). the preferred sequence s are very common_ The committed step during recombination between two DNA molecules occurs when a strand-exchange prote in of the RecA family successfully pairs the molecules, a proc:ess dir:tatcd only by the normal capacity of DNA strands to form proper base pairs.

t.':eneric Co11S8fJ.uences of the Mechanism of Homologous Re

A corollary of the fact that recombination is generally independent of StJqUence is that the frequency of recombination between any two genes is generally proportional to the distance between those genes.

This proportionality is observed because regions of DNA are, in general, equally like1y to be used to initiate a successful recombination e vent. This fundamental aspect of homol~ous recombination is what rnakes it possible to use recombination frequencies to generate usefuJ genetic maps that display the order and spacing of genes along a clunmosome. Distortions in genetic maps compared to physical maps occur when a region of DNA does not have the ..average,. probability of partidpaling in recombination (Figure 10-22). Regions with a higherthan-average probability are .. ho t spots," whereas regions that participate less commonly than an average segment are "cold.'' Therefore. two genes that have a holspot between them appear in a genetic map to be farther apart 111All is true h1 a physical map of the srune region. In oontrast. genes separated by a ..cold"' interval appear by genetic mapping to be closer together than is true from th e ir physical distance. We have encountered two exampl~ for the anolecu)a r expletni:llion of hot and cold spots in chromosomes. Regions near chi s iles and Spoll cleavage sites have a hlgher·than-average probabilit}' of initial· ing recombination and are "hot," whereas regions having few such sites are oorrespondingly "cold."

Gene Conversion Occurs ~use DNA Is Repaired during Recombination Another gen~ti c consequence of hornol~us recombination is gene conversion. We have introduced the concept of gene conversion during the specialized recombination events responsible for mating-type switching in yeast. However, gene conversion is also commonly obseJVed during nonnal h omologous recombination events. such as those respflns ihJe for genelic exchange in bar:teria and for pairing d Hnmosomcs during meiosis. To illustrate gene conversion during. meiotic recomhinat ion. consider a cell undergoing meiosis that has the A allele on one homolog and the o allele on the other. After DNA replication. four copies of this gene are present and the genotype would be: A A a a. In the ahsence of gene

~~

~

I L.__._l "cdd &pol" "ttd spot"

I

f 1c:; URE 1G-22 Com,.tson of tf1e genetic and physicaiiiWip$ of a typic;il region of a yo5t chromosome. Markers shCMt thE" locabon of \lariOUS geres Nobce in lhe regton bEtween 5po7 and Ct;k15 thdt the genetk rnap r.; t:Ontacted due to a low fi'eQtJMC:Y of UOSSII'lg 0\1€1'. In c.otltf<)S(, "' the reg100 beM-een Cdc 15 and FW t the genebc map rs epanded dtJe to a high frequency o1 crossang 0\/t!'f (Source. ~ from Al>erts B. et ill 2001 Mdewlar biology of the cell. 4th edition p. l \38, ftg 10· 14. Cq:>vllghl. Cl 2002 Reproduced by~ of Roulledge!Taylor & Frands Books. Inc)

1

240kbp

zoo

Homologoos llecombinotioTI at the MnJP.Cvlor ~..e.iml

conversjon, two gametes carrying the A allele and twn gaauetes carrying the a allele would oo generated. If instead, the gametes with genotypes A. a, a, a (or A , A, A, a) are formed, then a gene conversion event has occurrerl, in which one copy of the A gene has been converted into o (or vice versa). How might this arise? There are two ways that gtme conversion can occur during the DSB-repair pathway. First, consi.Jer what would happen if the A gene was vocy close to the site of the double-strand break. In this case, when the 3 1 ssDNA tails invade the homologous dtlplexes, and are elongated, they may copy the a information. which could replace the A information in the product chromosome upon completion of recombination (see Figure 10-3d). The second mechanism of gene conversion involves the repair ofbaso pair m.i:9matches that occur in the recombination intennediates. For example, if either strand invasion or branch migration includes the Ala gene, a segment of heterodup1ex DNA car.-ying the A sequence on one strand and the o sequenc:e on the other strand would be fonned (Figure 10·23; see also Figure 1&1d inset). This region of DNA carrying base-pair mismatches could be recognized and acted upon by the cellular mismatch repair enzymes (which we discussed in Chapter 9). These enzymes are specialized for fixing base-pair mismatches in DNA. When they detect a mismatched base pair. these enzymes excise a short stretch of DNA from one of lhe two strands. A repair DNA polymerase then fills in the gap. now with the properly base-paired sequence. When working on recombination intermediates. the mismatch repair enzymes wiU choose randomly which strand to repair. Therefore. after their action, both strands wHl cairy the sequence encoding either the A infornlation or the a information (depending on which strand was ..fixed'' by the repair eneymes), and gene convel'Sion will be observed. fl CURE

10-D

Mi.smatdl repair of

a

het~ex DNA within recombination irdermediales can giwe me to gene

~r:=====

conve~Sion..

s

~

A

}A

new ONA syntheSized during repair c

parental DNA

I'Tl06ea.Jies

Bihliogrophy

SUMMARY Homologous recombinatjon occ:urs in aU organisms, for generic exchange. the r~omnent of genes nlvng chromosomes, and the repair of broken DNA strands and m llapsed replication forks. The recombination prooess involves the breaking and rejoining of DNA molecules. The double-strand repair p athway o( h omologous recombination well desccibes many recombination evcots. By th~ mode). initiation of exchange requires that one of the two homologom DNA molectJles have a d ot,lblestranded break The broken DNA ends are processed by DNA-desrading enzymes to generate single-stranded DNA segments. These ~ngle--stmnded regions participate in DNA pairing with the homologous partner DNA. Once pairing lJCCllrti, the two DNA mol~ule!' are joined by a branched structure in the DNA called a HolJiday jlmcliun. Cutting the DNA at the Holliday iunction l"eSQlves tlw junaion and tenninates recombination. HolJiday jllllUions can be cut in two alternative ways. One way generates crossover produr:ts, in which regions from two parental DNA molecules are now covalently joined. The alternative way of cleaving the junctkm generates a ••patch'' of recombi ned DNA b ut does not result in crossing over. Cells encode enzymes that cata.l~e all lhe sleJ.lS in homologous l'eccwbinalion. Key enzymes are lhe stJ:andexchange proteins. Of these, E. coli RecA i<> the premier example; RecA-like proteins are foW1d in all organisms. RecA..tike stmnd~change proteins promote the search for al~wjng

homologous sequences between two PNA rnolecul€8 and the exchange of DNA strands within the recombination intetmediale. RecA functions as a l arge protein-DNA complex, known as the RecA filament. Euka.ryotic cells encode two strand~exchange proteins. called Rad51 and Dmc1.. Other important recombination enzymes are the ONA-c1e~wing enzym.cs that generate doub le-stranded break.<> in DNA to initiAte recombination; these proteins appear to be found only in cu karyotes and include Spot 1 and HO. Nu.d eases that process the DNA at the break site to generate the required single-stranded regions include the RecBCD enzyme in proka.ryote.c; and the MRX enzymfl complex in eu.ka.ryotes. Arldilional enzymes promote tha movement (brand1 migration ) and clea\lage (resolution) of Holliday jtmctions. During meiosis, recombination is essential fo.r the proper homologous pairing o{ chromosomes prior to the fim nudror division. Therefore, recombination is highly regulated to ellfiurc it occurs on all chrom~omes. The Spo11 DNA-cutting enzyme and the Dmcl strand-exchange protein are both specifica.tly jnvoJved in these recom· bination reactions. Homologou!oi recombination is ruso some-

times used to control gene expression. The mating·type switdung of yeast is an e}..-cellent example in this type of regu,IAtiun: il is alc;o an example of gene convers,ion- Analysis of the mec:hanb;m of mating-type switching has a 00\1\' d ass of models to describe some homologous reoombination events caJ.Jed synthesUi-dependent strand annealing.

BIBLIOGRAPHY Books Brown T.A- 2002. Genvmes. 2nd edition.. John Wiley.

New York and BIOS Scientific Publishers Limited, ());ford. United Kingdom. Griffiths A J.F., Mi ller J.H., Suruki O.T., Lewontin RC., Gelbart W.tvl. 2000. An introduction to genetic analysis, 7th edition. W.H. Freeman. New Yock. New York.

Rec001bination in Bacteria Court D.L, Sawitzk.e }.A., and Thomason L.C. 2002. Genetic engineering using homologous recombination. Annu. Rev. Genet. 36: 361 -368 (Epub 2002 )une t 1 ). Cox M.M. 2001. Recombinational DNA repair of damaged replication forks in Escherichia ooli: Questions. Annu. Rev. Genet. 35: 53-82 . .Kowalczykowski S.C., Dixon D.A .. Eggleston A.K., Lauder S.D., and Rehrauer W.M. 1994. Diochemist.ry of homologous recombinAtion in F.schericma coli_ Micl·obio/. Rev. 58; 401-465.

Ltlsetti S .L and Cox M.M- 2002. The bacterial Rec.A p£Otein ancl1he rec:ombinatorial DNA repait of stalled tepli£:ation forks. Annu. Rev. Biochem. 71: 71-100.

SI'n.ith G..R. 2001. HomoiQgous recombination

near and

rar

from DNA breaks: Alternative roles and contrao;fing views. Armu. Rev. Cfmet. 35: 243- 274.

Recombination in Eukarvotes "Eichler £.E. and Sankoff 0. 2003. Structural dynamics of eul<aryotic chromosome evolution. Sde(l("_-e 301: 793-797.

Keeney S, 200 I . MechaniRm and control of meiotic reccmbinahon initiation. Curr:. Top . Del' Dial. 52:1 -53. Page S.L. and Hawley R.S. 2003. Chromosome cho.reogtaphy: Th e meiotic h allP.I. Science 301: 785-789. Paques F. and Haber J,E. 1999. Multiple pathways of recombination indu.r.ed by double-strand breaks in Saccharomyces cerevtsioe. Microbiol . Mol . Bioi. Re1r. 63: 349- 404 .

Pas rink A-. Eeken J_C, and Lohman P.H. 2001. Genomitintegrity and the repair of double-strand DNA breaks. Mvtat. Res- 48CJ.461; 37-50. Prado F.• Cortes~LedesltUI F., Huertas P., and Aguilera A. 2003. Mitotic rncombination in Si'!ccmaumyces cerevisiae. Cul7'. Genet. 42: l 85- 198 (Epub 2002 Nov .29).

29:l

llomologous RtK:ombmafion a/ che Molecular LsVF-1

Symington LS. 2002. Role of RAD52 epi~tasis group gP.nes in homologous recombination and double-strand break repai1. Microbial. Mol . Biol. Rev. 66:630-670 (table of contents). van den Boscll l\11., Lohman P.H., and Pastink A. 2002. DNA doubl~lranrl hreak. repair hy homologous rer.ombrnation. Bioi. c.:l!em. :i83~ 873-692. West S.C. 2003. Molecular views of reoomhinal lon proteins and their control Nalure R~t-iews: Molerulnr Cell Biology. 4 : 435-445.

Mating,Type Switching in Yeast Haber J.E. 2002. Switchmg of Saccharomyces cerevisiae mating-type genes. ln Mobi/P. DNA Tr (ed. N..L, Craig. .R. Craigie, M. Gellert, A.M. Lamlxlwitz). ASM Fross, Washington, D.C.

CHAPTER

Site.-Specific Recombination and

Transposition of DNA NA is a very stable molecule. DNA replication. repair, and homologous recombination. as we have learned in the previous chapters, aU occur with high fidelity. These processes seJVe tu ensure that the genomes of an organism are nearly identical from one generation to the next. Importantly, however. there are also genetic processes that rearrange DNA sequences and thus lead to a

D

O U T LINE

• Conservative Site-Spedf~e Recorrmation (p. 294)

mom dynamic genome structure. These processes arc the subject of

•

this chapter. Two classes of genetic recombination, conservative site-specific n~rombination (CSSR) and transpositional recombination (generaUy called transposition). are responsible for many important DNA rearr-c1ngements. CSSR is recombination between two defined sequence elements [Figure 11-1). Transposition , in contrast. is recombination between specific sequences and nonspecilic DNA sites. The biological proctJSSCS promoted by these recombination reactions include the insertion of viral genomes into the DNA of the host ce ll during infection. the inversion of DNA segments to alter gene structure. and the movement of transposable elements-often calJed .. jumpingu genes- from one chromosomal site to another.

Bdog!Cal Roles of Site-SpeatiC Recnmbinatiofl (p. 302) Transposlbon (p. 3 10)

• Examples of Transposable Elements and Their Regulation (p. 327)

• V(O)J Recombination (p. 338)

11-1 Two dasses of genetic recombination. The top pa11el shcJ\NS an ~of srte-5pecific recombtnat100. Here recombination betvJeen ·me red and blJe recombirnnion sites inverts the DNA segment carrying the Aand B genes. The bottom panel shows Clf'\ example of transposdion in wt1IC.h lhe red transpos:abte element excises from 1he gray DNA and lrlSErtS mto afl unrelated s.te in the blue DNA. FIGURE

+

transpos!lion

+

293

2.94

S ite-Spcrtfir RF.combillotiDfl and Tra11SJ7C1$ilirm (Jf PNA

The impact of t hese DNA rearrangeme nts on c hromosome struct ure and function is profound. ln. many organisms, transposition is the major source of spontaneous mutation a nd nearly half the human genome consists of sequences derived from t:ranspusable clements. Furthermore, as we will see, both viral infection and development of the vertebrate inunune system depend critically on t hese specialized DNA rearrangements. Conservative site-spedfic reoombinatiun and transposition share key mechanistic features. Proteins known as recombin.a5es recognize specific sequences where l1:!l:umbiniition will uccur within a DNA tuo locule.

The reoombinases bring these specific sites together to form a proteinUNA complex bridging the DNA sites, known as the synaptic complex. Within the synaptic. cQmpJex, the recombi.J:41Sc catalyzes the cleavage and rejoining of the DNA molecules either to invert a DNA segment or to move a segment to a new site. One recombinase protein is usually responsible for all the~>e steps. Both type; of recornl:lination are also carefully controlled such that the danger to the ceJI of introducing lx-eaks in the DNA. and rearranging DNA segments, is minimized. As we ~hall see. however. the two types of rerornbination also have key mechanis· tic differences. In the following sections the s impler- site-specific recombination rP.actions are introduced first. foUQwed by the discussion of transposition. Eoch of these sections is organized to describe general features of the mechanism first and then to provide some specific examples.

CONSERVATIVE SITE..SPECIFIC RECOMBINATION Site..Specific Recombination Occurs at Specific DNA Sequences in the Target DNA Conservative site-specific rocontbination (CSSR) is tesponsiWe for many reactions in which a defined segment of DNA is rearranb'€d. A key feature of tht>Se reactions is that the segment of DNA that will be tnoved carries specifi c short sequence clements, called l'eCOmbination sites, where DNA exchllilgc occurs. An example of this type of rocombination is the integration of the ph age X genome into the bacteria) chromosome (Figure 11-2 and Chaptllr 21). During X integration, recombination always occurs at exactly the same nucleotide sequence within two ..-ec:ombination sites. one on the phage DNA, and the other on the bacterinl DNA .Recombination sites

F I C lJ R E 11-2 Integration of tbe A genome into the chromosome of the host cell. DNA exchange octUf'5 speafica1ty between the recombtnabon Sites oo the two ONA rnolerues. The relatnie lengths of the A Mid ceJUar chromoscxnes are 1101 shcMtn to scale.

Phage recombtnatioo site bacterial --l~--;;~r

recomblnabon Site

carry two classes of sequence elements: sequences specifically bound

by the rocombinascs, and sequences where DNA cleavage and rejoining ocorr. Recombination sites are often quite shon. 20 bp or so, although they may be nsuch longer and carry additional sequences bound by proteins. Examples of t he more complex rocombination sites are discussed when we consider specific recombination reactions. G<>SR can generate three different types of DNA rearrangements (Figure 11-3): (1) insertion of a segment of DNA into a specific site (as occurs during phage ~ DNA integration): (2) d eletion of a DNA segment; or (3) inversion of a DNA segment. Whether recombination results in DNA insertion, deletion, or inversion depends on the organization of the recombination recognition sites on the DNA molecule or molecules that participate in recombination. To understand how the organi7.ation of recombination sites determines the type of DNA rearrangement. we mus t louk at the sequence e{ements within the recombination sites in more detail (Figure 11-4). Each recombination sit e is organizf!d as a pair of recombinase ret:ognition sequences, positioned symmetrically. These recognition sequences flank a centra l short asymmetric sequence. known as thfl crossover region, where DNA cleavage and rejoining occurs. Because the crosso\ler region is asymmetric, a given recombination site always has a defined polarity. The orientation of two sites present on a single DNA molecule will be related to each other either in an in\lerted repeat or a direct repeat manner. Recombination between a pair of inverted sites will invert the DNA segment between the two sites (f'igure 11-3. dght panel). fn contrnst. recombination using the identical mechanism but uccwing between sites organized a.s direcl repeats deletes the DNA segment between the two sites. Finally. insertion specifically oCCl.lrs when recombination sites on two differena molecules are brought together for DNA exchange. Examples of each of these lhree types of remrangements will be consjdered belo'~'· after a general discussion of the recombinases.

deletion

insertion

X

A

X

0 0 ::l X

+

y

e

A

X

A

e

y

X

+

y

X

e Fl CiU R £ 11-3 T1uee types of CSStUec()rnbtnatioft. lr\ each case. ittS 'tl'e red se.grnent of DNA lhat IS moved c:x -eananged c.'ulng recombinatiOn 1\ B, X.
zm>

Siti:'·S,-:w.cific EIP.combmation and Tram;position of DNA

fl G IJ R E 11-4 StnKttwes irwolwd in CSSR. Tne patr of s~ re<:orrbnase rea>gmlon sequences flank the GOSSO\Ief region where rec~tion ocrurs. The st.bunitsof the reccxnl:linase bnd these re
No1ice that the sequeoce of the aossove~" reg1011 is not paln::lr01111C. resulting in an intJinstJC: asymmetry to the recCII'Tlbnation sites. (Srure; frcm Craig N. et 2002 Mobile DNA II, p. 4, f I . Q 2002 ASM Press)

a.

l

recombinase t>if\ds recognitiofl sequences

~c·~~'~r =~l,~.n~cc;;~~~~~J

3'C

:::N l e G :&

:=>

Site,Specific Rccom'binases Cleave and Rejoin DNA Using a Covalent Protein-DNA Intermediate There are two families o f conservative site-specific recombinases: the serine recombinases and the tyrosine recombinases. Fundamental to the mechanism used by both families is that when they cleave the DNA, a covalent protein-DNA intermediate is generated. For the serine recombinases , the side chain of a serine residue within the protein's active site attacks a specific phosphodiester bond in therec ombination site (Figure 11·5). This reaction introduces a singlestranded break in the DNA and simultaneously generates a covalent .linkage between the s erine and a phosphate at this DNA cleavage si te . Likewise, for the tyrosine recomhinases. it is the side chain of the active -site tyrosine that attacks and then becomes jo ined to the DNA. Table 11· 1 classifies a number of important rccombinases by family and biological function. The covalent protein-DNA intennediate conserves the energy of the cleaved phosphodieste.r bond within the protein-DNA linkage. As a result. the DNA strands can be rejoined by reversal of the d eavage proces.o;;, For rc"eTSal, an OH group from the cleave.l DNA a ttacks the covalent bond that links the protein h• the DNA. This process covalently seals the DNA break and regenemtcs the free (non -DNA bound) recombinase (see Figure 11-5}. It is this mechanistic feature that contributes the "conservative" to the CSSR name: it is called "conservative" because every DNA bond that is broken dUJ'ing the reaction is rf'.sealed by the recumhinase. Nn extem al eneagy. such as that released hy ATP~hydrulysis, is needed f01·

<.:onSf't'Valive Sir~Sf1ecific Recvmbinotion

deaved DNA end

-- ------------

fl GU R E 11-5 eov•nt-inlet'rnediat2 anechanism used by the serine and tyfosine recombinases.. Here oo the bt:kn DNA can then reattack the proreirrCNA c:ovalent bond co t~s.e this cleavage readtoo, resea

tre ~. and release the protein. 1he recombtna-;e, ldbeled Rec. t5 shown 1n blue.

DNA cleavage and joining by these proteins. This cleavage mecl1anism. with its covalent intennediate, is not tmique to the recombinases. Both DNA topoisomerases (C..'hapter 6} and Spo11. the protein that introduces doublc-stranderl breaks into DNA h l initiate homologous recombination during meiosis (Chapter 10), use this mechanism.

T A8 l E

11-1

Re«
Serine Famil}' Salmonella Hin invertase

Transposon Tn3 and ~ resolvases

lrlverts a chromosomal region to flip a gene promoter by recogni.zlfiQ h1x silcs. Allows expressiOn of two dis(inet surface anrigenS.

Promotes a DNA deletion reaction to resolve !he DI\IA fusion event that results frcm replicative transposition. Rccombinat1rn 51188 Hre caUed fflS s1tes

J)rrosine Family Phage " iotegrase

Promotes DNA 1ntegratia1 and excision of the phage 11 9Cf10"ne .nto. and OUl d . a specific seql.lef"lCe on the C. coli chromosOfne Rocombination sites are called att sites.

fltiage P1

ere

E. coli xere and Xe1D

YeastflP

Promotes circularizauoo of the phage DNA durtng lfllecttOO by rtlC(lgNZifl9 srtes (called lox setes) on the phage DNA. Promotes several DNA deleuon reacooi'\S thar convert dlmenc cilculaf DNA molecules ir1to rno110mers. Rocognizcs both plasmid-borne sites (eel'). aod chromosomal sites (dif) sites lfWetts a region of the yeast 2~-t plasmid to allow for a DNA amplilicatioo re-dCiion called rolling

Cll'cle replecat100. Recombnatioo srtes arc called /rtSiles.

Z97

.';itn-Specific Recombination a11d Transposition of DNA

Serine Recombinases Introduce Double,Stranded Breaks i.n DNA and tlren Swap Strands to Promote Recombination CSSR always occurs between two recombination sites. As we saw above, these sites may be on the same DNA molecule (for inversion or deletion) or on two different molecules (for integuttion). Each recom-

bjnation site is made up of double-stranded DNA. Thcn:fore. dwing recombination, four single strands of ONA (two from each duplex) must be cleaved and then rejoined-now with a different partner strand-to genera te the rearranged DNA. The serine recornbinascs cleave all four strands prior to strand exchange (Figure 11-6}. One molecule of the recornbinase proteio promotes each of these cleavage reactions; therefore a minimum of rowsubunits {that is a tctramer} of the recornbinase is required. These double-stranded DNA breaks in the parental DNA mo l e~ c ulcs generate four double-stranded DNA segments (marked by the proteins bound to them as R1, .R2, R3, and R4 in Figure 11~6) . For recombin ation to o ccur, the R2 segment of the to p ONA m olecule

FIGURE

crossover region

11-6 Recombination by a

serine rec:OI11binase. Each of the four DNA stfands is cleaved within the ctOS50Ver regic:n by one Sl.lbunit of the protein. These suburlits are labeled Rl, R2, R3, and R4. Oeavage of the tvvo niivld.Jal strIex is 5ta8gered by tvvo bases. This t-J\10 base regioo fc::Jfms a hybt1d ~ tn the recor-nbinant ~The recombinatioo sites are sim~ar to those shown in FigUre 11-4.

I

I

5'

3'

recorminase recoglllfion s1tes 5'

3'

oennet

rec:otr~birnlse

eavage

3'HO J t ( R1 5 3'

R2

~ OH3'

"

3'HO 5' 3 •

R4

0 H 3•

1

swap llNA """'"" '

5'

3

, R2 (

5' 3' •

R4

-t--·

re 5'

3'

5'

~

recombinase

CorJServvtive Sjte-Specific RFcvmUnauon

299

musl recombine with the R3 segment of the bottom ONA molecule. Likewise, the Rl segment of the top molecule must recombine with the R4 segment of the bottom DNA mttlecule. Once this DNA "swap" has occurred. the 3'0H ends of each of the deaved ONA strands can attack the rocombinase-DNA bond in their new partner segment. As discussed above, this reaction liberates the recom~ binase and covalently seals the DNA strands to generate the rearTanged DNA product.

Tyrosine Recombinases Break and Rejoin One Pair of DNA Strands at a Ttme In contrast to the serine recombinases, the tyrosine rccombinases cleave and rejoin two DNA strands first, and only then cleave and rejoin the other two strands (Figure 11-7). Consider two DNA molecules with their recombination sites aligned. Here also. four molecules of the recombinase w-e neede
F I G lJ R E 11-1 ltecombination by a tyrosine recomllinase. Here ltle Rl af1d R3 sa.bur1its deave the DNA in lt1e fl'st step (a); In the ex<JI1l)le shcMn. the protcin becomes lirll<ed to the cut ONA by a '5 P-1yr051ne bond. Exdwlge of the first par cl stfMds oo::us when the tw·o

5' OH group; at tnP. W>..al<. sites each attack •he prdeln DNA bond oo the other DNA mo1eaJe (b). The secood stiand exchange OCCU1'5 by lhe S(lfl"le rnedlaisrn. us;ng lhe R2 and R4 subunits (c Md d). (SOllee: From Crag N. et ill 2f:J(Jl. Mobile DNA II, cdor pate f. d'lapter 2 ~ 'EIJ2 ASM Press.)

individual DNA strands. To start recombination. the subunits of recom~ binase bound to the left recombinasc binding sites (marked as Rl and R3 in Figure 11 ~7t4) each cleave the top strand of the DNA molecule to which they are bound. This cleavage oa:urs a t the first nucleotide of the cr<.JSsuv~r J'egion. Next the right to p strand from the top (gray) DNA molecule and the risht top strand from the bottom (red) DNA molecule "swap .. partners. These two ONA strands ore then joined, n o\o\ in the recombined configurations. This ..first strand" exchange reaction generates a branched DNA intermediate known as a Holliday junction (see Chapter 10) (Figure 11-7b}. Once the first stnmd exchange is coittplete, two more recombinase subunits (those marked R2 and R4} cleave the bottom strands of each DNA molecule (Figure 11-7c). These strands again switch partners, and then
Structures of Tvrosine Recombinases Bound to DNA

Reveal the Mechanism of DNA Exchange The mechani sm of site-specific recombination is best understood for the tyrosine rccombinases. Several structures of members of this protein class have been stJLved, and these structures reveal the recombinases .. caught in the act" of recombination. One beautiful example is the structure of the Cre recombinase bound to two different conflgurettions of the tecombining DNA. lnsights into the mechanism:- derivE:-d from tl1ese structures are explained below. r.re is an enzyme encoded by phage Pl. which functions to circularize the lineaT plutge genome during infection. The recombination sites on the DNA, where Cre acts. are caUed lox sites. Crc-lox is a simple ex~pl e of recombination by the t,yrosine rocCimbinase fami ly; only C:rc protein and the /n](. sites are ncerlcd for complete recombination. Cre is also " ride)y used as a tool in gGnetic engineering {see 13ox 11-1, Applicati on of S ite-Specific Recombination to Genet ic Engineering). The Cce-lox structures reveal that recombination requires four subunits of Crc, with each molecule bound to one binding s ite o n the substrate DNA molecules (Figure 11-8 ). Tim confuiTllation of the DNA is generally a square planner four-way junction (see the discussion or Hollid~y junctiuns in Chapter 10) ,...,lth each ..~nn" of this junction bound by one subunit of Cre. Although at first glance the structures appear to have fourfold symmetry. this is not really the case. Cre exists in two distinct conformation s with one pair of suhun\ts in confornu1rion t , shown in green, and the other pail' in ~.:on· fomtation 2, sh own in purplr. (f igure 11-Bb). Only in one of the.--e conformaUons (the green subunits in the figure) can Crc cleave and rejoin DNA. Thus, o nJy one pair of subunits is in the active conformat;on at a time. The pair of subunits in this active conformation switches as the reaction progresses. This switching is critical for controlling the progress of recombination and ensur.i ng the sequential ..one strand a t a time" exchange mechanism.

Con:~J·ervotive Site-Specific RecombilKJtion

a

lf!r

301

a's

~ ~d

sobstrate

DNA

\lYr 5' •

:r

ll·~~

,S'

cleavage

s·

3:r

;r

'}

'fYr·~ H6~

Cre-CitiiA intemlediale I

C»>

~

s·3'

s

3..

ll:=:

b

11-8 Medtanism of site-specific recombination by the Cre recombinase. (a) The left pancl shaM the series of ~ntmnediate Oe·DNA structures that r~ fleet the sequential "one strand at a time" mechanism of exchange. In eac:h of the panels only the two StJbunits colored in gteeo are in the active cCll'ltorma~. Note that after first strand cleavage. the coloo of the suburits swttdl as the second ptW of Cre subunrts ~ 'ome acuve for recomblrlatloo. (Source: From Foog Guo et aL 1997 Structure 1exedwth CNA. Noture 389: 4 1. Copyrigflt ~ 1997.) (b) The nghtpanel st,ows the ayst~ Slructure of Cre bound to the Hollit1ay II.Jnction rntermediate (corresponding to the third panel in part a). Note that the two subunits cooed in gree-n~ 10 a alfef· eot coofOfmcltion than are those robed irl purple. 1he COOlllex. ~rc.. docs not have fOI.lffdd symmetry; notiCe. for e~ilrll'k\ that tvvo of the pa.s of a~acent DNA "ilrffi5" in the structure are msch doser ~ther than are the other p.ws. (Gopali O.N~ Guo F., in:l Van Ouyne G. D. 1998. EMBO J. 17: 4175.) lrl'1dge prepared v.$ Bob.Srnpt MoiScripr. and F I Ci lJ R £

Rast« 30.

Box ll-1 Applkation of SJie..SpedtK RecumbiniJtion to Genetit En~

&cause SO"ne sie-spedfsc recombenati011 systems ate so SllllpiE; they have become WldeJy used as toots in expenmental genencs. Cre recombtnase. and Its dose relative A.P recom~se, are both used expenmentally to delete genes in eukaf)'Otic uganisms (ciso see example 10 Chapter 21 ). An exarf4)1e of me usefuness ot U115 strategy becomes dea- ~ we constder the folow!ng hypott1ettcal exarrpe. A researcher IS inlerested in the role of .a spe. cific gene tn the development of lu1g carlC(!r and sl1e wishes to study tns process the moose as a model tvgantsm (see Chapter21 ). When the gene of Interest IS disrupted ("'<nocked out"), ~. the rr«:e at die during early embryoge-test>. Appcll'a'ltly the geoe is requwed v«y early in dev€1q:>rnent Huw can its role ., luog cancer be studied in the aWit animcJI? Site-speoflc re<.:o~Tlbmtion can often prOVIde the Mswer. ~g routine metlr ods, re5€ilrd-.ers can llltroduce rerombinanon sites recognized by Ge (or RP) flankMl& the gene of IOtEll'est These Sites will have no effect on the gene's functw..--, urr less the reoombinase is .also present Therefore. the Cre protan (or FlP prdein} can be •ntroduced into the same organism, under- the cootrd d a promoter that can be carefully regt.lated (see Olapter 17) The mJCe can lherefore be alowed to develop in the absence ci the recomblnase, but then ~ bil1h, ere e~ can be "turned on" The presence d the rec.orrbnase causes delelOl d the gene d nerest In ttls case, the propen9ty of the Cre-treated mce (an v.A11Ch the gene is daeted) for k..rlg canes c.an oow be ronpared With theu- '"normal" 1«te- mates, tn which the gene of Interest ~ stJI lfllact Thus, rerornbinaticn using ere allows ~ potmtici functlons of the gmes 10 be ~ 111 different stages of developnent US!flg

BIOLOGICAL ROLES OF SITE..SPECIFIC RECOMBINATION Cells and vil'llses use conservative site-specific ret:ombination for a wide variety of biological functions. Some of these functions are discussec1 in the following sectjons. Many phage insert their DNA into the host ch:romnsome during infection using this recombination mechanism. ln other cases. site-spccilk recombination is used to aJter gene expression. for example. inversion of a DNA segment can allow two alternative genes to be eJ~..-presscd . Site-specific rocombination is also widely used to help maintain the structural inlegrity of circular DNA molecules during cycles of DNA replication, homologous recombination, and cell division. A comparison of site-specific recombination systems reveals some genoral themes. AIJ reactions d epend critically on the assembly of the recombinase protein on the DNA, and the bringing together o f the two recombination sites. For some rewmbination reactions this assembly is very s imple. requiring onl}' lhe recombinase and its DNA recogni~ tion sequences as just described fur Crc. In contrast, other react ions nr quira accessory proteins. These accessory proteins include so-called architectural proteins that bind specific DNA sequences and bend the DNA. They o rganize DNA into a specific shape and therchy stimulc.~te the recombination. Arcbitocturill proteins can also control the direction of a recombination reaction. for example. to ensure that integration of a DNA segment o t.: curs while preventing the reverse 1-eat'r lion- DNA excision. Clearly, this type o f regulation is esstmtial for a logical biological outcome. Finally. we l'Vill also see that recombinase.~ can be regulated by other pruteins to control when a partit..-ular DNA rearrangement takes place and coordinate it with other cellular events.

Biological Roles of Site-Specific Recombif!Dtion

30:1

).. lntegrase Promotes the Integration and Exdsion of a Viral Genome into the Host Cell Chromosome When bacteriophilge J\ infects a host bacterium. a seri~ of regulatory ev~n ls result either in ostablishment of the quiescent lyso~enic state or in phage multiplication, a process called Jytic growth (see Chapters 1£ and 21 ). r~tablishment of a lysogen requires the integration of the phaga DNA into the host chmmosome. Likewise, when the phage looves the lysogattic state to Tcplic:ato and make new phage particles. i t must excise its DNA from the host chromosome. The analysis of this integration/excision Te<.tction provided the first molecular .insights into sitc·specific recombination. To i.nlegratc. lhe >.. inlegrase protein (~ l nl) f:atalyzes recombination

between

tv~ro

speciflc sites. known as the att, or attachment, sites. The ottP site is on the phage DNA (P for phage) and the attB site is in the bacterial chromosome {B foc bacteria; see Figure 11-2)..Mnt is a tyrosine cecombinase. and the mechanism of stn-tnd exchiillge follows the pathway described above for the Cre prote in. Unlike Cre recombinat ion, however, J\ integration requires accessory proteins to h elp the mquired protein-DNA complex to assemble. The.sc proteins control the reaction to ensure that DNA integrntion and DNA ~xcision occuc a l the right time in the phage life cycle. We will first consider the integration pathway and then look at how excision is triggered. Important to the regulation of l\ integtation is the highly asymmelric organization of the attP and attB sites (Figure 11 -9}. Both s ites carry f I~ U R E

11· 9 Recombfnatiof) sites

inwtvecl in A. ineepation and excision sho1Mng Ute important sequence elements. C. C, B, and B' are the core Mnt binding sltes. The addioonal proten bmdif1g sites a~e oo at1P and flri the C and C srtes. These regions are <:<:led the "ilfTnS;" the sequences m the left are called the P arm and those oo the right CJ(e called ltle P" arm. The small ~ boxes labeled P1• Pz, artd P1' are theam ~fnt~ S4tes. Sites marlc.ed H 01e the IHF ~sites, and ~!ES rntll\(ed X are !he sites OOd1 'bind )(is. F is the site bound by Fts, another architectural pt'otein not tis· cussed f\JrtheJ here. lhe 'ifll'l ffg\OOS ate me CI'OSSO\Iel regions. fol dar«y, Mnt IS not st'OMl boln:l to the COR: S\tes. Note tNt not ill protein bindtng sre:s are filed dtmg either H1legratnle vr el\CI:>we feCOfllblnation. After ·~ the P Mn IS part of attL Vlhereas. ltle P' arm becomes partofattR.

304

Sfte-Specific Recombination and 1h:msposition of DNJ\

a central core segment (approximately 30 bp}. These core recombination sites each consist of two Alnt binding sites and a crossover region where strand e.xchange occurs (as described above). Wheteas attB consists only of this central core region, ClttP is much longer (240 bp) and carries numerous addiliona1 protein binding sites. Flanking each side of the core region of <JltP are DNA regions known as the "arms." These arms carry a variety of protein binding sites, including additional s ites bound by >..Jnt {labeled as Pt. Pz, and P', in Figure 11-9). Mnt is an unusual protein because it has two do-

FICURE 11·10 Modelforlt.q;~

DNA ID bring DNA-birlding sites~ The Alrt afld It-IF boding sites ftom the f'' arm of atrPare shown lt-iFbincbng to the t-i' srte bends the DNA ro a~ow one rnoleetlle oJ Alnl to bind bott1 the P,' and C' sites. lhe breat; "' the ONA wlhin the l·r site reflects a fliCk that was JX~ en the DNA ~ed fer 5tructural iYlalySIS of the IHF-DNA complex. (Souce: From Rice P. er al. 1996. Oystal strtJctlJre of M IHF-ONA complex. Ce/187· 1303. Copyright© f996, with

mains involved in sequence-specific DNA binding: one domttin binds to the arm recombinase recognition sites and the other binds to the core recognition sites. In addition, the a.rms of attP carry sites bowtd by several architectural proteins. Binding of these proteins governs the directionality and efficiency of ffiC'.Ombination. Integration requires attB~ attP, ~lnl, and an architectural protein called integration host factor (IHF). IHF is a sequence-dependant DNA-binding protein that introduces large bends (> 16lY') in DNA (Figuro 11-10). The cu-rus of ottP carry three IHF sites Oabeled H 1, H 2 • and H' in Figure 11·9). The function of IHF' is to bring together the >Jnt sites on the DNA arms [where Alnt bjnds strongly) with the sites present at the central com (where it binds only we-illy) but whero it must bi:nd to catalyze recombination. When recombination is complete. the circular phage genome is stably integrated into the host chromosome. As a result, two new, hybrid sites are generated at the jlmctions between the phage and the host DNA. These sites are ca.Hcd attL (left) and attR (right) {sec Figure 11- 9). Both of the:r;e sites contain the core region, but the two arm regions are now separated from one another (StJe the location of the P and P' regions in Figure 11-9}. Thus, neither of thP two core regions in this new arrangement is competent to assemble an active Mnt rocornbinase complex via the mechanism that was used to generate Jhe complex for integration; the DNA site:; important for assembly are simply not in the right pL'lc~.

permissioo from E.iscvief.)

Phage A Excision Requires a New DNA,Bending Protein How does ~ excise? An additional architectural protein, this otJA phage-encoded, is essential for excisive w combination . This protein, called Xis (for excise), b in ds lo specific DNA sequence s and introduces bend s in the DNA. In this ru~umer. Xis is similar in function to lHF. Xis recognizes two sequence motifs present in one a(m of attR (and also present iJt attP- markcd X, and X7 in Figure 1l-9}. Binding these sitos 1ntrodut:f.'IS a Ja.-ga bend {> 140°) and together. Xis, Unt. and IHF stimulate exch;ion by assembling an active proteinDNA complex at attR. This complex then interacts producti vely with proteins assembled at attL and recombination occurs. ln addition to stimulating excision (recombination between atiL and attR), DNA binding by Xis also in hibits integration (recombination between attP und a ttB). The DNA structure created upon Xis binding to attP is incompatible with proper assembly o f >Jnt and lHF at this site. Xis is a phage-encoded protein and is only made when the phage is triggered to enter ly1ic growtb. Xis expres~ sion is desr.ribed in detail in Chapter 16.. Its dual action as a stimulatory cofactor fur exci~ion and an inhibitor of integration ensures thai the phage genome will be free. and remain free. from the host chromosome when Xis is present.

Biologiool RolesofSite-Specific Recombination

305

The Hin Recornbinase lnvens a Segment of DNA Allowing Expression of Alternative Genes The Salmonello Hin recombinase inverts a segment of the bacterial chromosom e to a llow expression of two alternative sets of genes. H in recombination is an example of a class of recombina tion reactions. relatively common in bacteria. known as programm ed rearr.m.gements. These reactions oft en ft.1nction to .. pre-ad apt" a portion of a population to~ sud den clumge in the environment. In the c~se of Hin i nversion, recombination is used to help the bacteria evade the host imm\.lllc system as w e will now explain. The genes that a re co ntro lled by the inversion p ror:ess encode two alternati\'c fonns of tlagellin {called the H1 and H2 form s)- thc protein comp onent of the flagellar filament. Flagella are on the surface of the bacteria and an: thus a common ta.rget fur the immune systf'..m {Figure 11-11). By using Hin t o switch bet ween these a lte rnative forms, at least some ind ividuals in the bacterial popula1ion can a "Void recognition of this su rlacc ~t ruc:ture by the immunA system. The chromosomal region inverted by Bin is about 1 ,000 bp and is flanked by specific recombination sites called hixL (on the )eft) and hixR (on the right) (Figure 11-12). These sequences arc in inven ed orientation with respect to one another. Hin, a serine recombinas.e. prom otes inversion using (he basic mechanism described ahove for this enzyme family. The invertible segment carries lhe gene encoding Hin, as well as a promote r, which in one orient<1tion is p ositio ned to express the gtmes located outside the invertible segment directly adjacent to the hixR site. When the invertible segment is in the .. on •· orien· tation, these adjacent genes are expressed , w hereas when t he segment is flipped into the "off" orientation, the genes cannot be transcribed, because they Jack a funct ional promoter. The two genes undel' control of this '•fiip pi.J::J,g" promoter are fljB. which encodes the H2 flagcllin, and fljA, which encodes a trdllSCriptional rep ressor o[ the ge11e for the Hl flagellin. The Hl flage llin gene is located at a d istant site. Thus, in the " on .. orientation , H2 flageHin and the Hl repressor a.-e expressed. These cells have exclusively H2-type .flagella on their surfa(:e. ln lhe ...off' orientation. h owever,

F I CiU R £ 11-11 Micrograph of bacteria (Solmonelfo) showing flagella. lhe colol

enhanced SCdrlfliog electron micrograph shows SalmoneJJo typhimuri(Jm ('ed) invading cultl.Jied human cells. The hair-~ke pm1rusioos on the bactena are the flage«a. (Source: COOl· tesy of the Rody Mountain l abolatones, NIAfD, NAH.)

300

Site-Spec.ifir. Recombination and Trrmsposiriop of DNA

Fl C\J R E 11-12 DNA inversion by the Hin rewmbinaw of Solmonello. Jnve~'nn or me DNA sewoeot betweer. the 11nc sites ffips

hm

•P___________.

----- ·-- -I

ON

a Pf(lfllO(er (P) to give two alternative patterns of tlagelfio gene expfessiOO

lli>
hixR

fljB

fljA

hixR

fljB

ftjA

-~"1 hin

OFF

neither H2 nor the H1 repressor is synthesized. and the Hl-type flagella aft! present.

Hin Recombination Requires a DNA Enhance£ Hin recombination requires a sequence in addition to tbe hix sites.. This short ("-'60 bp) scqtJence is an enhancer that stimulates the rate of recombination rvl,OOO-fo!d. Like enhan.c;er sequences that stimulate transcription {see Chapter 17), this sequence can function even when located quite a distance from the recombination sites. Enhancer function requh:l:'.s the bacterial Fis protein (named because it was discovered as a factor fol' j_nvei'Sion ~ulation). L:ilce IHF. Fis is u sil&specific UNA bending protein. In addition. it makes protein-protein contacts with Hin thaLBie important for recombination. The enbancer-Fis complex activates the catlliytic .steps of recombination. Hin can actually a.sserublc and pair the hix recombination sites to fonn a SYJlaptic complex in the absence of the Fis-enhancer complex (Figure 11-13). This contrasts to the role of lHF in ~ integration. where the accessory protein is essential for assembly of the recombinase-DNA complex. For Fis activation of HU:1, the three DNA sites {bi.xL, hixR. and enhancer) need to come together. Formation of tl1\s three-way complex is greatly facilitated by negative DNA J' I c; U R E 11-13 Complexes formed

durint Hin-
Hin invertasome

Biologicol flolcs of Site-Specific R6COITibinalion

:«17

SUJlflrcoi ling [see Chapter 6), which stabilizes the aRsociation o( th~ distant DNA sites. Another bactflrial architectural p rotein , HU also facilitates assembly of this invertasome complex. HU is a dose structural homologue of IHF. yet in contrast to fi-lf, il binds DNA i.n a sequence-independent manner. What is the biological rationale for control of Hin in version by the Fis-enhanc.er complex? The principal function is to ensure that recombination only {)(:curs between hix sites that are present on the same DNA molecule. This selectivity e11S\1res th~t the invertible segmant is flipped frequently while intermolecular ONA rean.angernents that oould disrupt the integrity of the Lacterial chromosome are avoided. In contrast to integration and excision of phage ).., Hin-catalyzed inversion is not highly regu1aterl. Rather. inversion occurs s tochastically. such tllitt ·w ithin a population of ce11s there will always be some flf!lJs that carry the invertible segment in each orientation .

Recombinases Convert Multimeric Circular DNA Molecules into Monomers Site-specific recombination is critical to the maintenance of circu lar DNA molecules wilhin cells. The chromosomes of mo!':t bacteria ace drcular, as are m ost p )asmids in both prokaryotic and eukaryotic cells. Some viral genomes are ulso circular. An intrinsic problem w ith circular DNA m olecules is t hat they sometimes form dimers and even higher multimeric forms during the process of homologous recombination. Site-specific recombination can be user! t o convert these DNA multimeTS bar:k into monomers. Consider whal happens wh\:ln a DNA t:ro.sSOVi:l'f OCCUl'S between two identical circular molecuJes. This process is shown occuring between two copies of a bacterial chromosome (Figu.re 1 1-14) (soo Chapter 10 for a discussion o f homologous recombination ). A single homologous recombination event can generate a single Jarge circular chromosome with two copies of aU the genes- that is. a dimeric chromosome. At the time of ce1J division. this di mer poses a major problem, as there will be only one rather than two DNA molecules to be segregated into tl1e two d au,g htel' cells. Becaus~ of tltis multimerization problem. many circular DNA mo1ecules carry sequencos recogni200 by site-specific rucombinases. Proteins that function at these sequences are sometimes called rerrolvases. as they "rnsolve" dimers (and larger mullime rs) into monomers. Clearly. it is essential for their function that these proteins specifically cata1y?..e r~sol ution (a DNA dcletiou rP.action) but not the reverse reaction (convecsion of monomers to dimers), which would onJy make the muhimerizalion problem worse! As we wm see, s pecific mechanisms are in place to enforce this d irectional selt-1cti.vity on the recombination reaction. The Xer recombinase calalyzes the monom erization of bacterial chromosomt>S and of many bacterial pJasmids. Xer is a member of the tyrosine Tecornbinese family, ancl it s mechanism for promoting recombinat ion i s very siotilat to tht:1t described above fur the Cre protein. Xer is a hetcrotclramer. containing two subunits of a protein called XerC and two subunits of a protein caUed XerD. Both XerC and XerD a:re tyrosine rocombinases bu t they recognize different DNA sequence. Thert-fore. tbe recombination sites used b y the Xer recombinase must carry rP.Cognition sequences for each of these protein s. The recombination sites in bacterial chromosomes. called dif sites~

F I (j U R E 11-14 Cii'OIIar DNA

rnoleaJie5 can fonn multirners. Homcklgous ~ti011 betv.een 1ht! 'M/0 daogllter DNA~ ck.lrlng DNA rerflC.:J!:loo general\.~ a dlmeric ~ (01 plasmd).

Site-speofic recorrDinaticlt by~ XetCD f'ECOI1'bt. ~'laSe IS fheo needed to geJlelate the moc>Omef!C DNA rnolerues needed for eel dv5too

308

Site-Specific RPcomhimllion and Transposition of DNA

have a :XerC recognition seqllence on one side and an XP.rD l'ccognitio:n ~uencc on the other side of the crossover region {Figure 11-15). There is one dif site on the chromosome. It is located within the region wh ere DNA ccplication terminates (see Chapter 8). When the chromosome forms a dimer, tMs dimer will of course have two dif sites (see figure 11-14}. How do cells make sure that Xer-mediated recombination at dif sites will convert a chmrnosorne dimer into monomers without ever prom o ting the reverse reaction? This directional regulation is achieved

through the interaction between the Xcr recombinase and a cell division protein called FtsK. This regulation is shown in Figures 11·15 and 11-16, and occurs as foJJows. When FtsK is unavailable for interaction wilh lhc XelCD com plex af the dif site, the recombinase complex adopts a conformation in which only the two XerC subunits are active. As a resull. XerC \.vill promote exchange of one pair of l.JNA strands to form the HoHiday junction intermediate (see the discussion on the genera) me<:hanism of tyrosine recombinase recombination. above). Beccn.t~e XcrD is n evt!r Hclivated, recumhimtl.ion is mwt.>£ coutpteted. lnstead, l'Cvcrsal of the XerC cleavage reaction often occurs. This rever-saJ sim ply regenerates lhe original DNA arrangement {see Figure 11-15). In contmsl. when the Fts K protein is available and inter-dcts with the Xe.rCD complex, it alters the conformat ion of the complex and activates XerD protein. [n 1his case. Xed) promotes recombination of the fin;t pair of strands to generate thE{ Hollid ay junr.tion intermediate. O nce this reaction is completed. XetC p1·omotes the second pair of stnmd exchunge reactions, yielding the recombined DNA products (see Figure 11-15). XeO

XerC

FIGURE 11-15 Palbw.Jysfor

Xe.--mediated rKOJnbination at dil In the

absence of~ (FtsK-Irldependem pathvvay shown .... the left parlef), only )(etC is active ttl pc()I'TX)l:e strand exchange to form a t-blliday junction rteanediate k1 thi> ase (because XeD is not active). recombination IS not completed and tfle XaC teaction is frequernfy teversed. In lt1E: presence of R5l< (FtsK dependent pathway shown tn the light panel), XeiD, no.v active, calillyzes klrmation of the Hclliday jxx:\100 lltefTTlediate. and Xf>rC promotes serond stiand exdlonge to complete the remmboaOOil event and geoetate ch(omosome monomets. {Source: Adapted from Aus5el L. Batre F-X. Aroyo M. Stasiak ~. and SherlC:Itt D. 1002. Ce/1 10& 195-205. FtgUre 6, p. 202.)

1

1~·

_.,., _.-.,,_=-~ 3'

~--~~'

~~~~ ~

Jr~

/

ftsK

BiolDg~col Roles

of Sile-Sperific lkt:omt;irnJiioo

309

11-16 Repjation of dtromosome segregation by Ftsl<. Just before eel d.'v;sion, the ~ replicated origins, shown in FIGURE

Breen. mcNe to 1he poles of the eel, wheleas the repkat10n tefTT1R5 that includes dif. shown as a tioog1e. typicalfy remains locdlized at the rndcel When the dff site is repi.cated, the two daJghtet

dif sites om recombine to ram a Hciday juT tion, wiVch is resolved by XeTC. If the repkated

no crossovers

chromosome forms~ segregaron will txeak rhe synapoc complex and the 4fSites~ move ~ay from the rnictcal lo
PO (><) !.·;;,;,.;. -. - -...-

.. ---------.---.-..-1-.------.--

d;.;...., site do..... ------ - •. .

activation of XerObyFtsK XelC atso active

1 c::eu dtvision

"ronsposition of DNA

FtsK iR :t.n ATPase that tracks along DNA. ll functions as a "DNApumping protein" similar to the RuvB protein that p romotes DNA branch migration during homologous recombination {discussed in Chapter 10). FtsK is a lso a mcmbnme-bound protein that is l ocalized in the ceU at the site where ceJI division occurs. It functions to mov DNA away from the centc.r of the cell prior lo division so th&t the cell can divide at this sile (figure 11-16). This localiution of FtsK to the division site is ~)' to how the cells insure tlutt XerO is liCtivuted specifically when o dimeric chromosome i s present. ln this ca..~. the chromosome will be "'stuck" in the middle of the dividing cefJ as one half of the chromosome dimer is moved into each d aughter cell. The two dif sites in this dimcr, with bound XerCD prote ins, therefore inh·lract with FtsiG ln this manner. sile-spedfir: recombination is regulated to occur at the right time and place with respect 1o the cell division l.j'd e.

There Are Other Mechanisms to Direct Recombination to Speci6c Segments of DNA Although we have limited our discussion to oonservative site-specific rP.combination, there are other rccornbination events that occur at specific sequences and servo similar biological functions. Some of these l:eaOtions, for example , mating type switching in yeast, OCC'ur by a targeted gene-con version event, as we describcrl in Chapter 10. Thl:! gene rearrangemcntc; responsible for assembly uf gene segments encoding critical proteins for the vortebrate immune system - known as V(D)} recombination - also Ut:Curs at specific sites. This reaction is mechanistically similar to transposiliOil, however, und therefore is considered later in this chapter.

TRANSPOSITION Some Genetic Elet11ents Move to New Chromosomal Locations by Transposition Transposition is a specific form of genetic recombination thai moves certain genetic elements fmm one ONA site to another. These mobHe genetic elements are r.alted transposable elements or transposons. Movement occurs through recombination be1ween the DNA sequences at the very ends of the transposable element and a sequence in the DNA of the host ceU (Figure 11-1 7): movetnent can occur willt or without duplication of the t'Jement, as we will see. rn some l:ases the recornbina~ tion reaction involves a transient RNA intcrmPdiate. Wh en transposable eJeme.nts move, they often show little sequence sl{]ectivity in their t.:hoice of insertion sites. As a msult, t.cansposons can insert withan genes, often completely di~Tupting gene fundion. They can also insert within the regula tory sequences of a gene where their p.re::ienr.e may 1(>.8d to dumges in how that gene js expressed. ll was these di~ru ptions in gene function and expression that led to the discovery of transposable e lements {see Box 11-3. Maize Elements and the Discovery o f "Jfansposons later in this chapter). PcJ'baps uot surp risingly, lhercfure, transposable elemebts are the most common source of new mutations in many organisms. In fact, these clements ara an important cause of mutations leading to genetic disease in humans. The ability of tram.-posable eJem~nts to insert so promiscuously

Traflsposilion

transpoSon ·------------------------~ C?old sile CO)-- -------------f1eW--""""si,....te______,)

~~genome DNA()

exciSed from old site and inserted io new site

copies of element at oJd and new sies

Fl GU R E I 1•17 Trans'postlion of a mabie genetic element to a new site in the host DNA. Recombll"'abon, -111 s.crne cases, tnvolve:s ~ of the transposoo from the cld DNA ltlca6oo (left). In other cases. one copy of the lraflsposoo stnys at the old locatJoo Mid Clrother cq:>y IS tnSEfted Into the new DNA 5lte (~).

in DNA has also Jed to their modification and use as mutagens and DNA d elivery vet:tors in experimental biology. Tr~msposable elements are present in the genomes of a] 1 life-forms. The c.:omparati"re analysis of ~.nome sequences nweaJs two f.ascinat.ing observations. First, transposon-related sequences cnn make up huge fractions of the genome of an organism. For exampl~. more than 50% of

both the human and maize gP.nomes are composed of transposonrelated DNA sequence. This is in sharp contrasf to the smaH pe«:entage (< 2% in human) nf the sequence that actually encodes ceJJular proteins. Second. the tmnsposon content in diffurent genomes is highly variable (Figure t1 -18). For example, compan:d to humans or maize. the fly and yeast gcnomes a re very "gene~rich.. and '"'transposon-poor." There are many different lypes of transpos~ble elements, These elfmen ts ccm be divided inlo families that share common aspects of struc:rure and recombination mechanism. In the following sections. we

introduce the three major families of tnmsposable elements and the recombination mechanism associated with oach funaily. Some of the best--studied individual elemen ts. will then be described. ln the description of individual clements. we focus on how transposition is regulated to balance the maintenance and propagation of these elements \'\lith their potential to disrupt or misregulate gene!-~ within the host organism. The genetic recombination mechanisms responsible for transposition are also used for functions other t han the movement of transposons. For example, many viruses usc a recombination pathway nearly identical to transposition to integrate into the genome of the host cell during infection. These viral integration reactions will therefore be considered together with transposition- Likewise, some DNA rearr-~mgement& used by ceJls to alter gene expression patterns occur using a mechanism very similar to DNA transpositiun . V(D)J recombinntion, a reaction required fur developrnenl of a fundional immune sy.stem in vert~brates, is a well ~ llildcrstoocl example. V(D)J rocombination is discussed at the end of this ('lu:tpter.

There Are Three Principal Classes of Transposable Elements Transposons can be divided into the following three families on tho basis of their overall organiz-.-~.tion and mechanism o f transposition: 1. DNA transposons.

Sile-SPf!Ctflc Recomrot~afiun and Transposifil)n of JJNI\

312

2. Viral-like retrotransposons- this duss includes the retroviruses. T hese e lements are also called LTR retrotrnnsposons. 3 . Poly-A retrotransposon s. These elenw.nts are also caJled nonvi.-al rctrotransposons. F igure 11-19 shows a schematic of the general genetic organization of

each of these element families. DNA transposons remain as DNA throughout a cyd c of recombination. They move usiog me chanisms that involve the cleavage and rejoining of DNA strands. and in this way they are s imiJac to elements that m ove by conservati~e sitespecific recombination . Both types of rctrotran.sposons move to a new DNA location using a trd.Tl.Sient RNA intecmedicde.

DNA Transposons Carry a Transposase Gene, Flanked by Recombination Sites DNA transposons carry both DNA sequences that function as recombination sites and genes encoding proteins tltat participate in recombination {Figure 1J..19a). The recombination sites are at the two ends of the element and are organized as inverted rept>.at sequences. These terminal inverted repeats vary in length from about ~5 to a few hundred base p:Urs. ate not exact sequence Tepeats. and carry the recombimse roc:ognition sequences. The recombinases responsible for transpos;tion are usuaJly called transpos.a ses (or, sometimes, integrases). a Maize repeated elements

b Human V28

om:mmDIDJl)

V29-1

TRY4

[:J )

c Drosophila melanogaster

d Saccharomyces cerevisiiJe

e

Escherichia cOli

thrB

thrA

lhrC

J 1 -18 Tr.ul5pD!EOftS jq ~ oaurerKe iJIIIf ~n. f<Epeated eemE!Ilts, mostly crrnpose:j of trensposons or transpo~ed ~nces (sudl as truncated efernef1ts} are sMir.'O Ill greer>. CelkAar genes ace~ io blue. (a) MaizE. (b) Human. (c) Drosopfula. (d) BodOOg ~ast (e) f coTi. (S~ fr(lm Brown TA 2002. Gent:vnes 2nd edition, p. 34, ftg. 22 af1d references theeifl Fl GURE

Copyrigt'l © 2002.)

'li'cmsposition

a

DNA lranspo500S

) trar1sposase

target sne

termm inverted

duplicatloo

repeats

b wat-!Jre relrotransposonslrettovin.t9es

t

d

integrase af1d RT

c

LTR

r-

pdy-A rettotransposons

r--+

5' UTR

-_,.,..- . ,. f...1-..J

(~)

ORF1

FIGURE 11-19 <Jenetic organiution of the thtee daues of transposable eleme11tS. (a) DNAtransposons. The ek:ment •ndudes th~:~ leml!l"lal inveried repeat sequences (greer1 arrows) which ate the rec~ sttes, and a gel'l€ encoding transposase. (b) "'ral~ke retrctransposons and retrovuuses. ll•e element 1f1CI\Xjes two long termnal r~at ( LTR) sequences that 1\?d a fegKlll enco enzymes, 111~rase and reverse transa1p-

element

rt

313

ORF2

DNA transposons carry a gene encoding their own transposase. They may carry a few additional genes. sometimes encoding proteins that regulate transposition or provide a function useful to the element or its host cell. For example, many bacterial DNA ttansposons carry genes encoding proteins that promote resistance to one or more antibiotic. The presence of the transposon, therefore. cam;t::S the host cell to be resistant to that antibiotic. The DNA sequences immediately flanking the tran.sposon have a short (2 to 20 bp} segment of duplicated sequence. These segments are organized as direct repeats, are called target ~ite duplications, and are gen~ rated during the process of recombination 8$ we shaH discuss below.

Transposons Exist as Both Autonomous and Nonautonomous Elements DNA transposons that carry a pair of terminal invertt'!l repeats and a transposase gene have ewrything th~y nood to promote their own transposition. These elements are called autonomous transposons. However, genomes also contain many ewn simpler mobile DNA segmenl.s known as nonautooomotlS transposons. These elements carry only the terminal inverted repeats, that is tlte cfs-acting sequences needed for transposition. In a cell that also mrries an autonomous transposon, tJnco
ViraJ..Jike Retrotransposons and Retroviruses Carry Terminal Repeat Sequences and Two Genes Important for Recombination Vual-likc retrotransposons and retroviruses also t.arry inverted ~erminal repeat sequences that are the sites of recombinase binding and action {Figure 'l1-19b). The terminal inverted repeats are embedded within longer repeated sequences: these sequences are organized on the two ends of the eJ~ment as direct repeats and are

tase (RT). (<) Poly-A retrotranspo50ns. The element termtr10tes 111 the 5' cYld 3 • UTR seQUef1C£S and

eJJt.odes two enzyrr>eS, an

RNA-bn:ling enzyme (ORFI)and an enzyme haVIng both fe\le.-se t.raf'SCJ1ptase ill1d endonudease adlvitie.s (0Rf2),

called long terminal repeats OT LTRs. Viral-like rctrotransposons e ncode two proteins needed for their mobility: integrase (the transposase) and rt!verse transcriptase. Reverse lranscriplase (R'O is a special type of DNA polymerase that can use an RNA template to synthesize DNA. This enzyme is needed for transposition because an RNA intermediate is required for the transposition reaction. Because these elements convert RNA into ONA. the reverse of the normal pathway of biological information flow (DNA to RNA]. they W'e known as "retro" elements. The distinction between viral-like retrotmnsposons and retroviruses is that the genome of a retrovirus is packaged intu a viral particle, escapes its host cell, and infects a new cell. In contrast, the rotrotransposons can move only to new DNA sites wirhin a coli bul never leave that cell. Jjke the ONA transposons. those e lements are flanked by short taiga silt=~ uuplicatiuns that are ~n ermed during reoombinal ion.

Poly..A Retrotransposons Look Like Genes The poly-A retrotransposons do not have the terminal inverted repeats present in the other transposon dasses. Jnstead. the two e nds of the element have distinct sequences (Figure 11-19<.:). One end is called the 5' UTR (for untranslated region) w hereas the other end has a region called the 3' UTR foll owed by a stretch of A-T base pairs called the poly·A sequetu;e. These elements are also flanked by

short target site d upli cation~ Retrotransposons carry two gt!ll~. know as ORFl and ORF2. 0RF1 encodes an RNA-binding protein. ORF2 eneotltlS a pmttJin with bolh reverse transcriptase aetivity and an endonuclease activity. This pnr tein, although distinc..1 from the transposases and int~~es encoded hy the other classes of elements. plays essential roles during recombination. Like their DNA and viral-like transposon rounterpart.s, poly-A retrotr~nsposons exist u:>mmonly in both autonomous and nonauto nomous fonn s. Furtbennorc. genome sequence analysis reveals that ther£: are many t.nmc..'Btod clements that do not have a complete 5' UTR .sequence and have lost their abllity to traru;pose.

DNA Transposition by a Cut,and-Paste Me<:hanism DNA transposons. viral-like retrotransposons, and retroviruses all use a similar mechanism of recombination to insert their DNA into a new site. First, let us consider the simplest transposition reacti on: the movement of a DNA transposon by a nonreplicative mechanism . T his recombination pathway involves the excision of the transposon from U.s initial location in the host DNA followed by integration of this excised transposoo into a new DNA site. This mechanism is thetllfore cal1cd cut-and-paste transpos.iUon (Figure 11-20). To initial~ recombination. the transposase binds to the terminal inverted repeats at the end of tl1e tra.nsposon. Once the tra.nspo.sase recogn~ these ~~nces, it brings the two enlls of th~ lransposon ON.A together to gencrate a !o1able protein-DNA e.;omplex. This romplex is called the synaptic complex or transpososome. It contains a multi mer of tmnsposase-usually two or four subunits-and the two DNA ends (sec bclowj. This complex fundions to ensure that tht~ DNA cleavage and joining reaetions n eeded to move tha transposon {}(:CUr simultaneously on the two ends of the dement's DNA. ll also protects the DNA e nds from ceJiular enzymes during red>nlhination.

Transposi'tion

11-20 The cut~paste mechanism ()f transposition. The ftgl.f'e shows movernem of .'l tr.mspOSOn hom a target site 1r1 the gray host DNA to a new site in the tlue DNA NQte the stagga-ed cleavage sites on the target DNA dtuing the DNA st1a11d transfer readloo that give nse to short re~ sequences at the new target site (tre tatget 5rte dt.pcafiOnS).The ONA at the origlnai iOSertlon sire (here if'l gray) Will be le-ft With a dotJI.:.Ie. stranded DNA break as a re5IJit of transposoo excisolon This break CMI be repan-ed l7y FIGURE

DNA deavage of ooltt sCtands

1 s•

:ii 5

'

~ ef1d joining or homologous

recon'bmtioo (see Chapte5 9 and 10)

t~~ ~~~ 1 ~~:;o ';(' . ct~ ONA repaW' synthesis

to filgaps

elemem in pew DNA location

L...J

L-J

target site

largelsDe

duplication

d.4Jiicalion

The next step is the exdsion of the transposon DNA from its original location in the genome. To ochieve this, the transposase subunits \-vithin the transpososome first cleave one DNA strand at each end of the transpo.-con, exm;tly at the junction between the transposon DNA and the host sequence in which it is inserted (a region called the Oanking host DNA). The lransposase deaves the DNA such that the transposon sequence tenninates with free 3'0H groups at each end of the element's ONA. To finish the excision reac:tion, the other DNA strand a.t each end of the element must also be cleaved. Different transposons use different m.t:!Chanisms tu cleave thestl "st>eond'" DNA strands (those strands that term.inate with 5' ends at the transposon host DNA junction}. TI1ese mechanisms are described in a following section. After excision of the transposon. the 3'0H ends of tLe t.ransposon DNA-the ends first liberaterl hy the transposase-attac"k the DNA

phosphodiest.ar bonds at the site of the new insertion. This DNA seg:rnenl is called the target DNA. Recall that for most transposons, the target DNA can have essentially any sequence. As a result of this

attack, the tt·ansposon DNA is covalently joined to the DNA at the tar· get slle. During each ONA joining reaction. a nick is also inttoduced illlo the target DNA (Figure 11-20). This DNA joining reaction OL't.:urs

by a one-ste p transesteriftcation reaction that is called DNA strand transfer. A similar mechanism for joining nucleic acid strands is used for RNA s plicing (see Chaptr.r 13), Th e ttanspososome ensw es that the two en ds of the transposon DNA attack the two DNA strdnds of the same target site together. The s ites o f attack on the two strands are usuaUy separated by a few nut.:leotides (for example. 2, 5 and 9 n ucleotide spacings nre com~ m on), Tl1is distance is fixed for each type o f transposon a nd gives rise to the short targot-s ito duplications that fla nk tnmsp osed co pies of 1ht: elt~ment (as is e xp lained in Lh~ n ext StlCtion). On ce DNA str:md tran s fer is com p lete, the job of the lran spososome is also oomplet~. The remaining recombination steps are carried out by cellular DNA repait proteins.

The Intermediate in Cut..and~Paste Transposition 15 Finished by Gap Repair The structm c of the DNA intcnnediale gE".nerated after ONA s trand tran:r fer has the 3' ends of the transposon DNA attached to the target DNA. This stn1cture also carries the two nicks in the target DNA that were generated during the procE>.ss of D NA strand tlr family of

transposons.

There An: Multiplt= Mechanisms for Clt!aving the Nontransferred Strand during DNA Transposition As just d escribed, the transposase cleaves the 3 ' ends of the element DNA an d promotes DNA strand transfer to catalyze (:ut-and~pasto trans~ position. However, transpOt;ons that move by this mechanism also n etJd to cleave the 5'~termi nating s bonds a t the junctions betw(.'tln tbe transposen and the flanking host DNA. These DNA strands arc called tl1c nontransferTed strands, as their 5' ends are n ot directly linkct1 to the target DNA during the DNI\ strand tran~fet reaction. Diffenmt transposons use different mechanisms to catalyze this sewnd strand cleav-

age reaction (Figure 11-2 11. Three methods are described here. An enzyme other than tha tronsposasc wm be lJSod to cleave the nonrransfurred s trand {Figwe 11~2 1}. For example, the bacterial llans-poson Tn7 encodes a specific protein {called 'fnsA) that does this job (Figure 11-2ta), TnsA has a structure very similar to tbat of a restric· tion t:ndonudeasc. TnsA assembles with tho Tn7-uru;o
Transposition

a

s·• 3'

9

• *

HO 6 3'

+

OH

t·=_

;:::)

=:>

s--

{L_

c

+

IS911

•

3'~

OH

OH

~

1

ln1~Tn5

b

Tn7

317

~

5'

DNA ..and transfer 3'

5' • 3'

~

hairpin ope~T~g

OH 5#. 3' •

•

...... IC ___.

3'

OH

+ 5' 3'

llJ 3'

!DNA.._

DNA""'nd

transfer

S' ~

FIGURE 11-21 Three mec:hartismsfordeaving the nomansferted strand. (a} An enzyme other than lransposase is osed. (b) The lrcli15JXlSaS€ ca~ the attad of one DNA strand oo the oppOSite strand to fOfm 1he PM l1aifptn intermed4ate. The rvYO h.lifptn E:~s are subse(}uet~tly trydrotyred by the ~se. (c) The tran5poSdSe c.atafyzes the attack of the 3'0H from one end of ltte element's DNA on the same strand at ltte oppoSite end Subsequent steps (not shrntvo) then result 1n ar1 excised trMSpOSOn.

(the TnsB protein) By worldng tugether, the transposase and TnsA excise the transposon from its original target site. The other ways of cleaving the nontransfcrred strand are promoted by the transposase itself-using an unusual ONA transesterification mechanism that is similar to ONA strand transfer. For example, the transposons Tn5 and TnlO cleave the nontransferred strand by gener-ating a structure .known as a "DNA hairpin_" To form this hairpin, the transposase uses the initially cleaved 3'0H end of the transposon DNA to attack a phosph odiester bond directly across the DNA dup lox on the opposite strand {Figure 11-:Zlb}. This reaction both cleaves the attacked DNA strand and L"'valently join the 3' end of the transposon DNA to one side of the break. As a result. the two DNA strands are covalently joined by a looped end, reminiscent in sh ape to a hairpin. Thi.s hairpin DNA end is then cleaved (that is ..opened") by the trans-

posases, to generate a standard double-strand break in the DNA. This opening I'E'.at;tion occurs on both ends of the transposon DNA. Once

l .+ 1 00

s·

transfer

5'

3'6

thPse steps arc complete, the 3'0H ends of the element DNA arc ready to be juin(ld to a new targat DNA by the DNA strand transfer reaction DNA cleavage via a transcsterifica1ion rea~;tioo um also O(lllf between the two ends of the transposun. This is the third mechanism used by transposons to cleave the nontransferred strands. In this case, one cleaved 3'01-l end attacks the same DNA strand at the opposite end of the element·s DNA {Figure 11-21c). The resulting DNA antennediate is further p~ to generate the excised transposon. The IS3 family of transposum; us~ this mechani&m. \ 1\fhy might transposat>es Ut;e tran~t$telificatiun as a ci..avagA medl· anism? It is probably an economic solution. Transposascs have the Intrinsic ability to promote (1) s ite-specific hydrolysis of tho 3' ends of the transposon DNA and (2.) transest~::rification of this end into a nonspe(.:Ul<: DNA site. These same activities, with the transcsterification reaction simply applied to a new DNA site, can aflow the transposase to promole transposon excision. This mechanism, thtirefor~. avoids the need for the transposon to encode a second enzyme to cleave the nonttansferred stnmd.

DNA Transposition by a Replicative Mechani~m Some DNA transposons move using a mechani$m caJied replicative lransp05iljon. in wbit.:h the element DNA is duplicatro during each round of trnnsposition. Although the products of the transJJosition reaction are d early different, as we will now see, the mechanism of recombination is very similar to that used for cut-and-paste transposition (Figure 11-22). The first slep of replicative transposition is the assembly of tho transposasc prutei.u on the two ends of the transposon DNA to generate a transpososome. As we saw for cut-and-paste transposition. transpososome formation is essential to coordinate the DNA cleavage and joining reactions on the two ends of the t'r'"dllsposon's UNA. The next step is DNA cleavage at the ends of the transposon DNA This reaction is catalyzed by the transposase within thR trarupososome. The transposasc inlroduces a nick into the DNA at each of the two junctions between the transposon sequence and lhe flanking host DNA (see Figure 11-221. This cleavage liberates two 3'0H DNA ends on the transposon sequence. Jn contrast to cut-and-paste transposition, the transposon DNA is not exdsoo from the host sequences at this stage. This is the major difference between replicative and cut-anci-paste trcm.sposition. The 3'0H ends of the transposon UNA are then joined to the target. DNA site by the DNA s trand transfer Teactiun. The mechanism is the same as we saw above for cut-and-paste transposition. However. the inrennP.Cliate generated by DNA strand tnmsfet is in this case a doubly branched DNA molecule (s~ Figure 11-22). fu this intermediate, the 3' ends of the transposon are covalently joinetl to the new target s ite, while the 5' ends of the transpoS()Jl sequence remain joined to the old flanking DNA. The two ONA brant;hes within this intermediate bave rhe structure of a replication fork (see Chapter 8). After DNA strand tTansfer, the DNA replication proteins fmm the host cell can assemble at these forks. ln the best understood example of replicative transposition (phage Mu, which we discuss below}, this assembly specifically occurs at only one of the two forked structures (see Figure 11-22 bottom pnnels). The 3'0H end in the cleaved t.argel DNA serves as a

Tramposition

traf\sposon

FIGURE

3 19

11-22 Mechanism lor

replicative transposition. The transpososome .,troduces a single-strand nld: at each of

the ends ol ttte transposon ONA. ThiS cleavage g~ a 3'0H group ill ead'l end These OH

groups then ar..xk tile target DNA .and berorne joined to the target by DNA strand trnnsler Note that at eadl end of the t0'\sp0s0f\ ooly one strand ts transferred lflto the t.Mget at this poo11, resuUil'lg in the loo'natiof1 of a
poson seq..e;ce. The resUtirtg product. caled a comtegrate, has the two star11ng aroJar DNA molea.ks joined by two CDpies the tra~50f'l The ssOOA gaps m the brnnched intermediate gJVe r.se to d1e t3rget site d.plic:atims. These d~ are not sho~ irl the cointeglate fol darity.

or

gap

..---,

l l

replication fork

asserr-Uy (at left gap)

rep!i(;ation throUgh the transposon

•

00111tegra1e with 2 copies of traosposon

primer for DNA synthesis. Rep1ication proceeds through the transposon sequtmce and stops al the S(," CCnd fork. This re plication reaction genaates two copies of the tran.6poson DNA. Th est: copies are flanked by the shor• direct target site duplications. Replicative tran:oposilion frequently causes chromrn;omal inversions and deletions that can be highly detrimental to the host cell. This propensity to cause rearrangements may put replicative transposons at a seloctive d isadvantage. Perhaps this is w hy so many e lements have de velopod ways to exciw completely from their or iginal DNA IO<;ation prior lo io ining to a new UNA site . ny excision, transposun~ avoid generating these ma jor disruptions to tb e host gtmome.

Viral~like Retrottansposon~

and

Retroviruse~

Move Using

an RNA Intermediate Viral-like retrotra nspo.sons and retroviruses insert into new ~i tes in tbe genome of the bost cell. using the same s tep s of DNA cleavage anti DNA strand tran sfer we have described for the DNA transposons. In con trast to the DNA transposons, however, Tecombinalion for these retroelements involves an RNA intermediate. A cycle of transposition starts with transcrjption of Lbc retrotrml&pos on (or rotroviraJ) DNA sequenw into RNA by a cellular RNA poJy· merase. Transcription initiates at a promoter sequenl..' e within one of the LTRs (Figure 11-23) and continues across the element to generate a nearly full-length RNA copy of the element's DNA The RNA is then reverse ttanscribt:Jd to genorale a double-stranded DNA molecule. Thi~ DNA molecule is called the eDNA (for copied DNAJ and is fre<' from auy flanking host DNA sequences. It is the eDNA that is rocosnized by the i nt~ase protein {a protein highly relaloo to the transposases of DNA elf!mcnts. as w~ shall see below) for recombination with a new target DNA sjte. Integrase assembles on the ends of t his eDNA. and then cleaves a few nucleolides otf the 3' t.md of each stTand. This d eavage rcal:tion is identical to tl~A DNA cleavage stHp of DNA transposition. As the direct precursor DNA for integration is generated fro m the RNA template by rev ers~ transcription, it is already in the form of a n exctsed transposon. Therefore, a mechanism to cleave the scL"'nd strand is u nnecessary for these elements. lntegrasa then catalyzes the insertion of these cleaved 3' e nds into a DNA target s itt- in the hos t ~u g~n ome us ing the DNA strand transf~ rt!al...1io n. As WH d iseussed aho ve, this target site can have essential!)' any DNA sequence. Host c ell ONA repair proteins fill the gaps at the target site generated during UNA strand transfer to complete recom bination. This gap-repair reaction generates the tru-get·site duplications. Because transcription to generate the RNA intermediate initiates within on e of the LTRs. this RNA does not carry the P.ntire LTR sequence; the sequence between the transcription start site and the end o f the element is m issing. Therefore a special mechanism is needed to regenerate the full-length element sequence during reven;e transcription . The pathway of reverse transcription involves two internal priming eve uts and two strand switc;hes (see details of the process In Rox 11-2, Tt1e Pathway of Retroviral eDNA Formation). These switching events result in the d uplication of sequences at the tm ds of the eDNA. Thus, the eDNA has complete. reeonsiTud cd LTR sequences to compensate for regions of sequence Jost during traJ1SCrip-

'l'ranspcJsitiDil

e1emerrt

FIG tJ R £

DNA

1~~s3' . ~~~~~~~~~~ . . .~ - . .C ~s·

~~ed 3'0H 5

5'->---::§§~:::::;;:::::;:§§~wh S',.

5

' 3"

3'0H

~+

targetONA

11·23 Mechanism of retroWal

integration aad transposition of Wal-like retrotransposons The tq:> panel~ integrated pi'OVirus. foe a mom detailed vievv of the LTR sequences, see the figure. in Box 11-2 The promoter for trcYlSCJ'iptioo of the waS RNA is en-bedded in the left liR as shov.n dJNA synthesls from this viral RNA IS exp?aiped 1r1 BOll 1 J.2. The lntegrase-catafyzed DNA deavage af'd IJC\IA strand trar6ftJ .steps Me shown.

~~

l

32J

~=========== integase-catalyled DNA strand transfer

gaprepaw and igalion

lion. This reconstruction of the LTRs is essential for recognition of the eDNA by integrase and fOT subsequent recombination.

DNA Transposases and Retroviral lntegrases Are Members of a Protein Superfamily As we have seen, DNA cleavage of the 3' ends of the transposon DNA (or cDNAJ and DNA strand transfer are common steps used for DNA transposition and the movement of viral-like retrotransposons and rettoviruses. This conseTVed recombination mechanism is reflected in the structure of the transposasclintegrase proteins (Figure 11-24). High-resolution structures reveal that many different transposaw.s

32Z

Siltl-Specific RecombmoUon and 1ronsposilion of DNA

To understand the process of retroviral reverse transcription (or that of the viral-like ll!trotransposons), we ferst need to look in ITIO!'e deta~ at lhe s1ruc:ture of the lTR sec.uooces. Each tlR is tonsltucted of three sequence elements. These are cafled: U3 (fof' unq.Je .3' end), R (for repeat), and U5 (for unique 5' end). TICI'lSOiption from the i'ltegrated cnpy cJ the retrovilaf gErome generates the wal RNA with the R sequence at each end (Box 11-2 Figln! 1). Therefore. ruing the process of re\IE!fSe traiSCiipOOf\ 011e additionaJ U3 and US region mLJSt be sylhesU-ed. Jls explained below, d~tion Jwpens beCdi.JSe priming of DNA synthesis OCOJrs at IOlerTial siles within the RNA genome and the R sequenre allovvs tvvo "strand ~hes" to ocax during the replicalioo proces.s. l is the viral RNA that is padc.aged rnto virus particles, and ttls RNA erters the neiiV eel during i"1fedlor1 The viral RNA .s padcaged !Mlh a ceULiar tRNA molecule (see Chaptec 14) that serves as the pnrner for synthesis of lhe first eDNA strand ~ tRNA forms b.:tse pars with a specific se<J,JeOCe near the Us regtOO. ~ as the primer-binding sh (PBS) (Bole 11-2 Figure 2a). DNA synthesis by the reverse transcriptase enzyme then cnpiEs the U5 regK>O and the fwst R segment (Box 11-2 Figure 2b). Re\lefSe lranscr¥:>tase has two enzymatic acbvities that are 11'T'f'0'1ant for eDNA formation: a DNA polymerase activity ard an RNAse H ac:tMty. RNAse H enzymes degrade RNA thai 1S b.:tse-paired vdth DNA (as we di50.6Sed in Chapter 8 ). Dunng JeveiSe ~ RNAse H r~ the template RNA strards. Wleo this step occ;urs on the first RNA- OOA hybrid intermediate (see Box 11-2 Figures 2b and 2c), the U5-R OOA strand ts reteased in a ~e-stranded form

This Us-R DNA strand can then base-pal- with the R regten en the other end of the viral RNA rnoleclJe (Box 11-2 Hglxe 2d). This step is the first of the tvvo strand ~ Once ths ~ch

ing OCClJIS. reverse ~tase conti'lues DNA synthesis to copy ILe remanderofthe RNA template (&1< l 1-2 ftgt.re 2e). The restAiu1g DNA strand ends witt1 lhe PBS seqt.61Ce at its 3' tentinus. The RNA ~ strand is removed, as befcre, by RNAse H (8o{ 11-2 F1gutes 2d and 2e}RNAse H-meaated degradahon of the viral RNA also generates a11 RNA fragment that serves as the primer for synthesis of the second eDNA strand. Jhrs reg100 of RNA remains base-paired with a seQUence cofted thE! polypurine Ired (PPl) at the edge of the U3 sequence (Box lt-2 f-igures 2e and 2f). Elongation of thts primer rop1es the U3, R.

u-.s

RUS

RNA

s·•

gag

ll5, and PBS 5€QUE!f'ICes into DNA

Once the tRNA pnmer IS rerno..ed from the fvst cOM strand, the seoond strand SWitch OCCtKS. The romplefflrot.cuy sequence d the PBS on the 5' ends alo:ws base-pairing neractJons between the two DNA strands arld tarnation d a orw· lar ~nt:ermedlate. Elongatioo of each of the 3• DNA ends pre· sent in lhis irterrneaate to the el1d of the other strand generates the dwble-stranded eDNA with two complete LTR sequences. lhis 0\IA molecule 1s rhen ready to be integrated mto the ce4rs genome by the integrase protein. Reverse transcriptase is a Vlrus--enroded (cw retrotranspo-

soo-encoded) enzyme and serves no essent~ c.e41ular function. It is. ho\tvever, absolutely es5elltial for ret:rowus replication. Thus, it is a common target of antiviral drugs. 1nduding many of the drugs that have bem used to fight the AIDS epidemc.

pol

env

l -

U3

- 3'

~

pol

ei1V

U3R U5

-

HR prc:Mrus

1RU5

RNA

s•

gag

pol

env

U3 R

-

3'

BOX 11-2 f I GUR E 1 Oetalechiew of the sequence elements f1Nr the encls Of the rettoviral RNA and eDNA. ~like retrottansposons have a very sim1ar sequence aganizmion. The pol gene encodes both reverse transOl'{'ase {llldudingthe RNAse H adivity) arld il1tegfase.

~

Sox 11-1 (Continued}

a RNA

fliiiiJ.!~I.II~J:Il!ll'JIDsO~======:::IliHI!H.t.[JrDWr[I:Q3" ] =~ =!.!_

r

IP.NA primer

b lptt lllli:W3•

lRNAseH _/_

+ S ::l JMl!RI!======::n1P~nJ:liUIXW3"

r.

l

d

U~ ONA~

as primel&l U3

s• fUI

e

PPT RNA prlrnet'

f

I

~3· ltm ltf'DIUS ~

3'U88!

l

RNAse H ramoves tRNApffiler"

a

g

l

ONA fOOensicn cf both ~ ef1dS by RT

h DNA ~:Ull li!e$1

3'«)11-x- U()DI I

m:IIIIO£WllD:r] ~cONI integtaliarl

!PI Ud w:;wufJ5'

for

11•2 f' I & U R E 2 Pathway of reverse tnnsotption to generate the eDNA copy of the retrovinl 01' ret.rotRnsposon RNA.

BOX

324

b

Sitc-Specifo: Recmnbinarion CJJ1d TraiJSpat>ition of DNA

ONA bOOing

MuA

Tn5 ltansposase

RSV inteSfase

FIG U Rt

11-24 Similarities of catMytic

domMJSot ~~and integfases. (a) SlructlJ'es of the cor.served cOfe domainS of Tn5 IRriSpOSdSe (Davies DR Gay'.lwl i.Y, Remkoff W5.• and Rayment l 2000. Soence

289: n - 85), of phase Mu ~(Rice P

and Mlz.utJCht K. 1995.

w 81:209- 220), and

ot RSV ~ase (Chc><:* YM, ~ JV, Ke H.. and tp.;c.ombW.N. 1994.1 Mol. FhoJ. 240: Common secondary slrudJ.xe eleIT1E!f\1s are shovvn tn 1te same colm. The IXlf mobt adJii~ srte re9dues are stlo.Nr. 10 bell and 476- 500)

sbdc.lmages prepared Wl1h ~ MoiSapt. and Raster 30 (b) Schematic or the oomain ocganzolion ot IJ-.e three proteins shawl 111 part a. lhe N-teTTT"trill domains bod to the eleJnent DNA.

The rr..ddle> domans rontclln the catatytic rcg.ons shown .., (a) lhe C_.eoninal clomaf'1s are &nvotvt.'<1 if1 proteir.-proten contac::15 needed to as· sernhle the transpososotl'le and/or to 1rtefact with other pn:te~r.s that reglJate tansposilior~ (Soun::e· fiom Rice PA ar.d Baiter TA 2001 ~ ardlteclure d ~ md tntegrase c:onr plexes. Nature B. 302. C~ «:> 2001

and integrases cairy a catalytic domain that has a common threedimensional shape. This catalytic domain contains three evolutinnarilv invariant acidic ami no acids: two aspartates (D) and a glutamate (E). Therefore, recombinases of this class are refened to as ODE·motif transposase/\ntegrase proteins. These acidic Hmino acids form parl of the active site and coordinate divalent metal ions {such as Mg2 • or MnZ+) that are required for activity (as we described for the DNA polymerases. see Chapter 8). An unusual feature of the transposase/integrase proteins is t hat they use this same active site to cat:Uyze both ~he DNA cleavage iind DNA strclnd li'Hnst~r. rather than IYtvang two achve s ites, each specialized for one chemical reaction. . In contrast to llae highly conserved structure of the cata lyhc domains. the rel11Dining regions of pl'oteins in this family are not conserved. These regions encode s ite--specific DNA-binding domains anrl regions involved in protein-protein interactions needed to assemble the protein-DNA complex specific for each individ~al element. Thus, these unique domains ensure that transposases and 1.n tegrases catalyze Tecombination specifically only on the element that encoded them or on a very highly reJatoo element. Trnnsposases and integ.rases are only active when assembled into a synaptic complex. also called a transpososome, un DNA (see above). The co-crystal structure of Tn5 transposase bound to a pajr of transposon end DNA fragments provides insight in to why this is the case (Figuro 11-25). The transposase subunit that is bound to the ~ combinase recognition sequences on one of these DNA fragments (that is, on one transposon end) donates the catalytic domain that promotes the DNA cleavage and DNA stJ'an.d transfer reactions on the other end of the transposon. Because of this subunit organization, the transposase will be properly positioned for recombination only w hen two subunits and a pair of DNA ends are present together in the complex.

Poly, A Retrotransposons Move by a "Reverse Splicing" Mechanism The poly-A rotrotransposons, for example. human LfN~ ele.rnents. move using an RNA intermediate bm use a mechanism dtfferent than that used by the vical-like elements. This mechanism is calle d target site primed reverse lranscriplion (Figura 11-26 ). The first step is tran-

Tron.r;position

3Z5

fIGURE 11-25 Co-cry$tal of Tn5 bound to substrate OM The cOfll)lex contains a dmerof transposase. The catayticdornains are colored as 1!'1 Hgt.tle l l-24. The green balls are &valent met~ ionS bcond in the protan's active Site Note that the Sl.lbl.m .booM ~ its ONAt~cling
sciiption of the DNA ui an integrated e lement by a cellular RNA polymr.rase (Figure 11-26a). Although the promoter is embedded in the 5JI JTR. it can in this case direct RNA synthesis to begin at the first nucleotide of the element's sequence. This newly synthesized RNA is exported to the cytoplasm and translated to generate the ORF1 and ORF2 prn1eins (see above). These proteins remain associated with the RNA that encoded them (figure 1126b}. In this way, an element promotes its own transposition and does not donate proteins to competing elements. The protein-&\JA complP.x then reenters the nuclease anrl associates \.vith the cellular DNA (Figure 11·26c}. Recall that the ORF2 protein has both a DNA endonuclease activity and a reveiSe b'anscriptase activity. The endonuclease initiates the integration reaction hy introducing a nick in the chromosomal DNA (see Figure 11-26d}. T-rich sequences are preferred cleavage sites. The presence of th~ Ts at the cleavage site permits the DNA to base-pair with tbe poly-A tail sequence of the element RNA. The 3'0H DNA end generated hy the nicking reaction then serves as the primer for reverse transcription of the element RNA (Figure 11·2&). The ORF2 protein also catalyzes this DNA synthesis. The remaining steps of transpositjon, although Jl()t yet well und erstood, include synthesis of the St">COnd eDNA strand, repair of DNA gaps at the insertion site, and ligation to seal the DNA strands. Many of the poly-A retmtran sposons that have ooen detected by large-scale genomic sequencing aro truncated elements. Most uf these are missing regions from their 5'ends and do not have complete copies of element·encoded genes or an intact promoter. These truncated elements therefore have lost the ability to transpose.

3Z6

SilP.-...'\pt:>cific Rs>combinotion and Tronsposition of DNA

11-26 Transposition of a poly-A retrotransposon by tatgel siteF fG lJ R E

primed reverse transcription. The figure outlines a model toe the mcNem ent af a UNE element (a) Ace!U& RNA polymerase lfWate5

a

p ~

LINEONA

E5'UTR

0

~ d ~~~~~~~re

{b) The ~ng rr.e>senger RNA is trcmslated to rroduce the products of the two encoded ORfs that ther. btrld to the 3' end of their mRNA. {c) The protein-mRNA complex thai t.nds to a T.och Site 111 the target DNA (d) l he proteins mih·

b LINE rrRNA

ate deavage in the tat~ DNA. lea1A115 a 3'0H at the [')NA end a'ld ~an ~A:DNA ~d. (e) The 3'0H end of the target~ seNes as a pc-imer fcr- reverse transdiption of the element RNA tQ produce c.tlNA (first straod. synthesis). (f) The final steps of the ~on reaction include second S!rand synthesis, DNA JOif\ill& and repcjf to c:reate a newly n>erted llNE el€n1enl

5"

5' UTR

OOF1 and ( ORF2 proteins

ORF1

ORF2

~~~"£

1•

!" "'" " " 1-

3' UTR

3'l!TR 3'

lb~-M

llf\EmRNA

+

c

target

S'

DNA

l'

5'

lbindiog to twget

ONA

tar~t

l

site de8\fa9!!, RNA·ONA hybrid formation

d

1•>"'""""'

e

d fi<sl cONAsnand

RNA dega
ll

second strand synthesis

DNA joining and repair

LINE DNA

)

Example.'> of Th:msposable f1P.ment1

EXAMPLES OF TRANSPOSABLE ELEMENT S AND THEIR REGULATION Transposons bave successfully invaded and colonized the genomes of all life-forms. Clearly they are very robust biological entities. Some of this sua:ess can be attributed to the fact that transposition is regulated in ways that help to establish a harmonious coexistE>.nce with the host ex!ll. This coexiste nce is essential for the survival of the ele ment as ltansposons cannot exist without a host organism. On the other hand, as introiluced above, transposons can wreak havoc in a cell, causing insertion mutations, altering gene expression. and promoting large-scale DNA reammgements. These d isruptio ns are particularly noticeable in plants, a fuatWP. that lfld to the discovery of transposons in maize (Box 11-3. Maize Elements cmd the Disco vet)' ofTransposons). In the fuHowing sections we bri~fly describe some of the bestunderstood individual transposons and transposon .fumilies. (A larger list of trans posons and some of their important features is swnmarized in Table 11-2.) Each s ubsection provides a brief overview of a spoc.ific element and an example of regulation that is of particular impnrtanc.f! to that elemenl. As we will s~e. two types of reguJalion appear as recurring themes: • Transposons control the number o f their copies present in a given celL By reguJating copy number, these e lements limit their deleterious impact on the genome of the host cell • Transposons control target site choice. Two general types of target site regulation are obs~rved. ln tb~ first, some elements preferentia lly insert into regions of the chromosome that tend not to be harmful to the host cell. These regions are caHPA:l safe havens for ttansposons. In the second type of regulation . some transposons specifically avoid transposing into thejr own DNA. Thi~ phenomenon is called tr8D5position target immunity. IS4~Family

Transposons Are Compact Elements with Multiple Mechanisms for Copy Number Control The bacterial transposon T n10 is a well-characterized representative of the JS4 family. which a lso includes Tn5. Tn10 is a compact element of 9 kb and encodes a gene for its o\-vn transposase and genP.s imparting resistaoct' lo lhe antibiotic te.tracycline (Figure 11- 2 7).

gene I

I: 1510 left tS10 right

FIGURE

11-27 Genetic DtXanttatiOn of btKterial transposon TniO. The map shows the func-

llooal elemen!!; m the baaerlal trc1!15fXlSOn lh 10. rn 10, lt;e many bactEflal ~. ~ cames •m "muli ~· at Its termn. For Tn/0, these efements ate c.alled 15101.. (tef'l} and ~lOR (nght) Both types of 1510 aement. can tJMspose. and are found lf1 DNA separate1y frorn l'n 10. n,e whte tnangles show the .n verb!d repeat ~es at the ends of the IS e4emt:mts and Tn 10 Mhough these four c~ He ro exadly !he same .n st:quence, dl are ~ by tht< Tn/0 lraf\SpOsaSe am:! are used as recombanahon Sites.

328

S ite-.t:;per:ifir. Rccombinotion and 'Tiunsposition of DNA

Box IJ•J Maize Elements imd the Discovery of Tramposons Plant geflornes are very rich in transposons. FUrthermore. lhe ability of transposable clements to alter gere e<pression cao often be readily observed as dramatic variation in the colOfation of the plant (Box 11-.3 Figure 1). Thus, it is not surprisirlg that transposable elements, and many of their salient features. were first discovered in plants. Barbara Mcdintnd< discovered "controling elements" in rnaize in the late 1940.>. tt \\es ac::tuanv the cblity ot transpa;able elements to break crtOffl.DSOO'eS that fi'st came to McCiintoc:l<'s & tent:ion. She found that some strc.lins experienced brden dvomosomes vey freq.Jently. iY1d she named the genetic elemerl respaiS(lle for these chromosome breaks Ds (dssoL-iator). Surprisingly, she ct>saved that the sites of these ''holspots" tor momosome breaks were different in diti:!rent straras, and could even

BOX 11-3 FIGURE 1b &le oholorwriepoonin SNpdrapn flowers due to fam.J transposition. The Sile of v.hite patches is related to ~e frequency of trnnsposition. (Source. Chatterjee M. and Wertln C 1997. The Plant Joomof 11 : 759 - 771,

Figure 2a, page 762.)

be in differert dy0f1"10S(lllal locations in the descendents d an in
eox 11-3 fiGUIU let Exampfeohom(maize)c:obsbowing co1ot variegation due to bilmpOsitic>n. (Sourt:e: Ft-otograph ~ by Ba1b&o tv\c.Cinto&; lfl"lage courtesy Cold Spnng Harbor Labcdory ArdWes.)

sanes. Ds, in fad. is a nonautonomous ONA transposon that moves by cut and paste transposition. Ds moveiTI€nt requires the Ac (ad!VatOf) element - also discovered by McClintock- to be present in the same cell and provide the l:ransposi)se protein Ac is now recogniled to be part cJ a large family of DNA transposons called the hAT family named for the hobo elements from flies, the Ac elements from maile, and the Tom element:; from snapjragon.

T n10 transposes via the cut-and-paste m echanism (describP.rl above). using the DNA h airpin flt rategy to cleave the nontrans{(!rred sb:ands (Figtrres 11-19 and 11-21). The Tn10 sequence also has a site for lHF binding. IHF h elps in the assembJy of proper trans pososome complex n eeded for recom bination a s it does during phage)... in tegration (see above). Tn 10 is mganized jnto three functio nal m odules. This o rgani7.ation is relatively common. and eloments that have it are called composite trnnsposons. The two outen nost modules, called IS10L (left) anrl ISlOR (right). are actually mini transposons. "IS" stands for insertion sequence. lS1DR encodes the gene for the transposase that recognizes the terminal inverted rep eat se<Juences of IS10Rt IS10L. and Tn10. IStoL. although Vt.!l'Y similar in sequence to IS10R. d oes not encode a ftmctional trans· posase. Thus. both IS10R and TnlO are autonomous, whereas IS10l, is a

E:.'i:ompiP.s of ·rranspm;nhiP Elements one! 1'lzeir Regulation

TABLE-

329

11-l.Major Types of Tnnsposabf.e £\ements

Type

Structural Featvres

DNA-MEDIATED mANS~ aacterial rP.pftcative Termin~l inverted l"f'Poats transposons

Bacterial cut-and-~te

trenSpOSOns

Eukaryooc transposoos

that flank antibiOticresist
inverted repeatS ft!at flank antibtotJcres.stance and transposase genes Inverted repeatS ft\dt flank coding region with introns

Mechanism of Movement Cop~ngof~tONA

Tn.~. yli, phage Mt.t

accompaoymg each round ot irlserOOn into a nevv tclfget sitP.

E.xcis1011 ci DNA from old target st:e and illseftion into new site Excision of DNA from old target sne aod insertion

into new sita

Tn5. Tn 10. Tnl. IS9 11. Tn917

P elemenlS (frosophlta) 11A1 family Ellerrents lc1/Mardlerelements

RNA-MEDtATEOTRANSPOSfTION

V.mJ..fl<e retrotransposons

- 250 to 600 t>p direct terminal repeats (LTRs)

flanking genes for reverse

transcnptase. integrase. and reli'OvlraHtke Gag

Transcnptton 1nto RNA kOITl p rof'l10tef ln left LTR by RNA po1ymefaSe II followed by revefse uanscriplion and

Ty elements (yeast)

Copiaefemeots (Drosophila}

f'ISertion at target Stte

orotetn Puty-A retrotransposons

3' A-T-rich sequence and 5' 0 1A flar* genes e11ooding an RNA-binding proteirl and

reverse transctiptase

Transcriptlon into RNA from

tnfemal promoter; targetprimed rPverse transcription miated by endonuclease cleaVage

F and G et~ents (Drosophila ) LINE and SINE elernetlts (mammals)

Alu sequences (t'IUTl8flS)

nonautonomous transposon. Both types of fSlO elements are found, as expected. unassociated with Tn 10. Tn1 0 limits its copy number in any given cell by strategies that restrict its transposition frequency. One mechanism is the use of an antisense RNA to control the expression of the transposase gene (Figure 11-29) (see the discussion of aJ)tisense RNA regulation in Chapter 17). Near the end of lSlOR are two promoters that direct the synthesis of RNA by the host cell's RNA polymernse. The promoter that directs RNA synthesis inward (called PIN) is responsible for the expression of the transposase gene. The promoter that directs transcription o utward (P0 liT). in contrast, serves to regulate transposase expression by mald n~ an ttntisense RNA. as follow s. Thfl RNA~ synthesized from P""' and POlff overlap (by 3 6 base pairs) and therefore can pair by hydrogen bonding between these overl~pping {complementary} regions. This pairing prevents binding of ribosomes to the PIN transcript. and thus sy11tbesis of the tran sposase protein. By this m echanism, cells that carry more copies of Tn10 will trdl1scribe more of the antisense RNA. whi.ch i n turn w itt Jimil expression of the transposase gene (Figure 11·28. see legend for m ore det
TnlO Traru~position Is Coupled to Cellular DNA Replication Tn 10 a]so couples tranr;p osi tinn to celh.Jlar DNA replication. Recall lhat bacteria suclt as B. coli {a conunon host for TnlO) methylate thei1·

S:!O

Sitf'·Spedfic Rcc.cmbillCJiion and 1'mnspo.
F I G U R E 11-28 Antisense regulittion of

of DNA a

IS10right

TniO expression. (a) A map of lhe overlapping promoter reglOOS is shoWn. The leftward promoter (piN) promotes eXpl'eSSIOO of the tranposase gene; the r~~d promotef

(pOVf), wtlich lies 36 bases to the left of plN promotes expression of an antisense RNA.. 1he first 36 bases of ead1 1raclscnpt are complementary to one anoltter. Note that lfl eels the antiserlse traf'lscnpt inrtlated at pOUT is ~ ~ than is the mRNA initiate~ at piN. (b) In celts hiMftg a high ropy nt.r.'lb€1 of Tn1 0, the RNA.~ pclimg OCCUr.> frequently and blocks tTaflslatiOr'l ot the ~ rnRNA {thereby evenM'My reducing the copy rumber of the element). (c) In eels hav~ a low ropy l1.t.Jfflber of the transposon. RNA:RNA pairing IS rare; ft1e traf1slaboo ot trafll053SE' mRNA rs eftiaent and 1he a:py llllfT1be.r in the ceft is lflCreased

RNA:RNA pairing iS l"equent

c lo.N Tn10 ccpy number:

or

translation lransposase mRNA is efficient

DNA at GATC sites (see Chapter 8. Box 8-4). 1'his m ethylation occurs after DNA replication, such that GATC s ites are hemin1ethylated for the few minutes between passage of the rep(ic:ation furk and' rP.COgm: tion of these seque nces by the methylase enzyme. It is during this brief p eriod-when the Tn10 DNA is hemimethyJate d - that transposition is most likely to occ ur. This coupling of transcription to the methylation state is due to the presence of two critical GATC sites in the transposon sequence. One of these sites is in the promoter fot" the transposase gene; the second is in the binding site for the transposetse within one of the inve\1ed te rminal repeats. Both RNA polymerdSe and transposase bind more tightly to the hemimethylated sequences than to their fully methylated versions. As a result. when the DNA is hemimethylated, the transposase gene is most efficiently expressefi, anrl the transposase protein binds most effici ently to fue DNA. Therefore, transposition of Tn10 occurs at its highest frequency
E:x(Jmples of Tronsposoble Elemenrs wzd Tlmir Regulation

331

fiGU ltE 11-29 Trilmposition oftnro

after passage of .- replication toR. Tr.1nsposi!ion is ac1ivated by the llem~methy1ated DNA that e)IJSts JUst afterONA rep1+catior1 (methylallon Sites ere not sllown). OUlng lranSpO.SitiOf'. d double-stranded tl(eak tS m00e tn the d1JUrno.. sQfllCJ DNA where 1t1e ~nt euised This break can be repaired by the DS~rep&r pathway (~ Chapter 10)• ., process that regenerates a alPY of T1110 allhe site cf exdsion By lhs

medlarusm, transposition may ~r to be

"n=p' cative" 111 nature, although the actual rew~n process goes 1hrough the

cut-and-paste {nornepkcative) p81hway.

l

P'odrls after replication and repair

....

Phage Mu l s an Extremely Robust Transposon Phage Mu. like phage A, is a lysogenic bacteriophage (see Chapter 21). Mu is also a large DNA transposon. This phage uses transposition to insert its DNA info the genome of the hosl ceiJ during infection and in this way is similar to the retroviruses (ruscussed abo~e) . Mu also \15eS m\lltiple Tounds of replicative transposition to ampHfy its DNA during lytic growth. During the lytic cycle. Mu completes about 100 ro\lnds of transposition per hour, making it the most efficient transposon known. Furthermore, even when present as a quiescent lysogen, the Mu genome transposes quite frequently. compared to trdditional transposons such as Tn 10. The narne Mu is short for mutator and stems from this ability to lrdllSpose promiscuously: cells carrying an inserte.d copy of the Mu DNA fn..quently accumulate new mutations due to insertion ofthe phage DNA into cellular genes. The Mu genome is about 40 kb and carries more than 35 genes, hut only two encode proteins wi.th dedicated roles in transposition. These are the A and B genes, which encode the proteins MuA and Mti.B. MuA is the ttansposase and is a member of the ODE protein superfamily we discussed. MuB is an ATPase rhat stimulates MuA activity and con-

trols the choice of the DNA target site {Figure 1 t-30). This process is explained in the next section.

Mu Uses Target Immunity to Avoid Tt'allsposing

into Its Own DNA Mu, like many tran~posons, shows very little sequence preference at its target sites. As a result, ..good.. taJgel sites occur very frequently in DNA

332

Sit,.._<;~ific ElecombillCJtion

and 'fransposition of DNA

F1 C lJ It E 11 • 30 Owerview of the early

-lvluA transposase-

steps of Mu trartsposition. Klt6 subunits ol the MtJA transposase asseni>le on the ends of Mu DNA. MuB hlr-.ds ATP and then ~ k> DNA of allY sequence A proteit-1-protetn trtteracbon between WtlA and Mu8 brings tt1e MuA ONA-trl!lr.SpOSOSOme ~to a new DNA target Site. Mt.e IS not shCMirl 11'1 the final panel because, after DNA strand transfer, it lS no tor.ger needed and probably ~the

MI.IB

~

MuB target compCex

Examples ofTronsposnble Elements and Their Regv.lation

including the DNA of the Mu genome itself. Given this nearly ra~1dom sequence preference. how does Mu avoid transposing into its own DNA. a situation that would likely result in serious disruptions of the phage's genes'?

This problem is solved because Mu transposition is regulated by a process called transposition target immunity (Figure 11-31). DNA sites SUITounding a c<>py of the Mu e lement, including the ele ment's own DNA, are rendered very poor targets for a new transposition event. Interplay between tl1e .MuA transposase Hlld the MuB ATPase is at

the center of the mechanism of transposition target immunity. MuAMuB interactions prevent MuB from binding to the DNA near where MuA is hound The interactions responsible for this i:nterp1ay are • MuA inhibits MuB from binding to nearby DNA sites. This inhibi tion requires ATP hydrolysis. • MuB helps MuA find a target site for transposition.

To see how individual protein-protein and protein-DNA interactions function together to generate target immunity. consider transposition immune DNA

Fl G 0 R E 11-:J1 The inlerplav between MuA and MuB on DNA leads to the

de\lelopmealt of.., immune target DNA. The~ sites are lrl ti-e terminal invested

tepeats on the ends of the lranspOsOh (shcMtn in dark green). tv'tuA ;s shown bound to my ooe of the two repeat regior6 fer clarity. Ellel}' tTne MtlB hydroty:res ATP rt cllssoodc:ltes from the DNA M.i3 bound to PIP is shown 111 lhe datta green; MuAMtJ3 ((l(!tad stroufates ths hydroly'.>is reddm. IV though sl"IOINil mntacting only t\IVo mdemtty to ItS ONA-b!OOng site. tx\!A lengths of 5 to l S kb can be r~ed "immune• by a sir@e MuM:.xJund lefiTllflCll invested repeat sequence.

1

very poor target

into two candida te UNA segments: onP. is any representative segment of D NA; wh ereas, the second has a copy of Mu already inserted {see Figure 11-31). We will call the first DNA segment the naive region and the second DNA segment the immUJ'\e regio n. What happens at each of these DNA regions as Mu prepares to transpose? First we consider events at the naive region. M\JB. in complex with ATP (MuH • ATP), will biorl the DNA. using its nonspccifitDNA-binding activity. At the same time, MuA transposase will assemble a tra nsposome on the Mu DNA. 1'hUl MuA in Lhe transpososome can then make protein-protPin contacts with th(l> MuB-DNA complPx at the naive region. As a result of this interaction , MuB delivers trus DNA to MuA for use as a target site. In con trast, both MuA and MuB bind to DNA in the immune region. MuA in te r-dc.1s With its specific binding sites on the Mu ge nome that is already present; MuB·ATP again binds using its affinity for any DNA sequence. However, when both MuA and MuB are bound to this region, they will interact. As a result. MuA stimulates ATP-hyd rolysis by MuB a nd tho uisassociatiou of MuB from this DNA. Mu.B t.lmmfut'fl doos not accumulate on th;s immune DNA segment. By this mean s, the Mu transposition proteins use the energy stored in ATP to protect the Mu genome from becoming the target of ttansposition. As expected from this mechanism. e ven a single MuA-binding s ite within a DNA molecule is sufficient to impart target immunity. 1hmsposition target immunity js observed for a number o f different transposable elements and can work over very Ioog distances. For Mu, sequences within approximately 15 kb of an existing Mu insertion are imm une to new insertions. For some elemen ts-for example T n3 and Tn7- target immunity occurs over distances greater than 100 kb. Targot immunity protects an e lement fi:o m transposing into itse1£, or from having another new copy of the same type of e Jement insert into it~ gunome. Fl1J1Jlcrmore, this type of regulation of target ONA selection also provides a driving force fur elemen1s t o move to naw locations ..far .. from where they are initially inserted, a feat ure that may also be advantageous for their overall propagation and survival .

Tel/Mariner Elements Are Extremely Successful DNA Elements in Eukaryotes Reoognizdble members ol the Tel/mariner family o f e lements are widesprearl in both invertebrate and vertebrate organisms. Elements in this family are the most com mon DNA transposons present in ou karyolP.S. Although these elements are dearly related. members isolated from niffeTent organisms ha,re distinguishing footures and are named differently. For example, elements from the worm C e.Iegans are called Tc elements. whnreas the original element named Mariner was l.solatcd from a

Drosophila species. Tc iimorim~r e lements are among tho simplest autonomous trans-pooous known. l}rpically, they are 1 .5 to 2.5 kb loog and carry only a pair of tenninal inverted repeat sequences (tht? site of tTansposa..11oe binding) tiild a gene enooding a transposare protein of the DOE transposa5e superfamily (see above). hi contrast to many transposons, no accessory prote.ins are rcqu.in1d for transposition. a lthough the final steps of recombination do require ceHular DNA repair protRios.. This simplicity in shucture and mechanism 1nay he> responsibla for tha huga suocess of these elmnents in s uc.:h a wide range of host organiSJ1ls.

'Exumples of Transposable Elements and Their Regulation

Tc11mariner elements move by a cut-and-paste transposition mechanism (Figure 11-ZO). The transposon DNA is cleaved out of the old flanking host DNA using pairs of cleavages that are staggeiAd by two base pairs. These elements strongly prefer to insert into DNA sites with the (obviously, very common) sequence 5'TA. ObviouslJ'• this is a very common sequence. What happens to the •· empty" site in the host chromosome when a transpos<>n excises? In the case of Tc1/mariner elements. DNA sequaooo analysis of some sites tha t once caniad a trao sposon reveals that sometimes the broken DNA ends are tilled in (by repair DNA synthesis) and then directly joined (see the discussion on nonhomologous end joining in Chapter 9). These repair reactions result in the incorporation of a few extra base pairs of DNA at the old insertion site. Tllese srnall DNA insflrtions are known as "footprinfs ... as they are tlle traces left by a lnmsposon that has ..traveled through" a site in the genome. ln contrast to many transposons. the transposition of Tcz/rnorirer elements is not w ell -regulated Perhaps as a result of this lack of control. many elements fuund by genome sequencing are ..dead... -tlult is. unable to transpose. For example, many ~Jernents carry mutations in the transposase ~e that inactivate H. Using a 1~ number of sequences from both inactive c..nd active elements. researchers constructed an artificial hyperactive Tc1hnariner element. This element, named Sleeping Beauty. l.rai:'.sposes a.t very high frequencies compared to naturally isolated elements. Sleeping Beauty is promising as a tool for mutagenosis and DNA insertion in many eukacyotic organisms. Furthermore, this r~struction expP.riment reveals that the frequency of transposition by Tc1/moriner elements is naturally kept at bay due to the suboptimal activity of U1eir transposase proteins.

333

A

Yeast Ty Elements Transpose into Safe Havens in the Genome The Ty elements (Iransposons in Yeast). prominent transposons in yeast, are viral-like retrotransposons. In fact, their similarity to retroviruses extends beyond the ir mechanism of transposition: Ty RNA is found in cells packaged intu viral-like particles (Figure 11-32). Thus, these elements seem to he viruse$ lha t cannot escape one cell and infee( new cells. There are many lypes of well-studied 1)t elements; fur example. S. cerevisiae carries members of the 1}'1, Ty3. Ty4. and Ty5 classes (although the Ty5 elements in this yeast species all appear to be inactive). Each of these classes of Ty e lements promotes its own mohility but cloes not mobilize de.m ents of another class. Ty el~ments preferentially integrate into specific chromosomal regions (Figure 11-33). For example. Tyt elements neatly always transpose into DNA within ......200 bp upstream of a sta rt site for transcription by the host RNA polymerase Ill enzy1n~ (see Chapter 12}. RNA Pol fH specifically transcribes tRNA genes, and most 'I)rl insertions are near these genes. Ty3 integration is also tightly· linked to Pol lll promoters. ln this case. integration is precisely targeted to the s tart site of transcription (~ 2 bp}. ln coob-ast, 'Ty 5 preferentially intf!gTBtes into regions of the genome thai are in a silenced. transcriptionally quiescent state. Silenced regions targeted by Ty5 include the telomeres and th~ silent copies of the mating-type loci (see C'.hapte:r 10). In all these cases. the mechanism of regional target-site selection involves the formation of specific prote in-protein comp1exes between the elemenfs integrase- bound in a complex to the eDNA-and hostspecific protftins bound Lo thP..st=> chromosomal sites. For example. 'JY5

B

FIGUtt£ 11-~2 YeDst Ty elemenlJ padtaged into virus~ (~) AA dec· lr0f1 macgraph of 5. r:e~ celts overe>e-

pressrng Ty I wtJS-llke pattides. The pamdes are seen CIS (lVaf, ~ JectfOO mcroscopy shown"€ the thfee.dimen Stonal recor.structlonS of Ty I WIOOS. These Ty I

elern€flts CiJffY a~ Gag protetfl ~ lom1.s the sp4\y shells Wttll tl'lmellc v~ of lt1e J:loc'ftldes. {Sow::e. CJat! ~ et a!. 1002. fV/obiJe DNA I( P5M Pres._, Washngtor~. D.C. (b)Also Courtesy of H s.a~il )

. .I •

ct1romosome m

tRNA genes: sf 0

I

:

100

:

' l

I

I

$ 200

'

:: 300

400

500

kb Ead, colored box~· Tyl, Ty2. Ty3,
t 1-33 dusteted integration sites observed f01Jy elements.

sents a knc:wn site for lrclOSpOsa'l insertiOn. Note lt1at

M

integrase forms a specific c<Jmplex with the DNA silencing proteiu Sir4 (see Chapter 17).

Why do iy elements exhibit this regional target site prefere11ce? lt is proposed that this ta.""get specificity enables the transposons to persist in a host organism by focusing most of their insertions away from important regions of the genome tha~ are involved directly in coding fot proteins. The use of this type of targeted transposition may be especiaJly important in organisms with small, gene-rich genornes, such as yeast.

LINEs Prom<>te Their Own Transposition and Even Transpose Cellular RN As The autonomous poly·A retrotransposoos known as IlNEs are abundant in the genomes of vertebrate o)·ganisms. In fact, about 20 pe~:eent -of the human genome is composed ()f LINE sequenres. These elements were first recognized as a family of repeat sequences. TheiT name is derived from this initial jdentification: LINE is the aCr()nonym for long interspersed nudear ~lement. L1 is one of the best understood UNEs in humans. In addition to promoting their own mobility, LINEs also donate the pt-oteins needed to reverse tmnscribc and integrate another related class of repeat sequences, the nonautonomous poly-A retrotransposons, known as ~hort !nte~:spersed n uclear ~lements (SINEs}. Genome sequences reve<~l, once again, the presence of huge munbers of these elements, which are typically only between 100 and 400 bp in length The Alu sequence is an example of a widespread SINE in the human genome. A comparison of the structures of typical UNE and SINE e(ements is shown in Figure 11-34.

The sequences of LINEs and SINEs look like simple genes. rn fact, the cis-acting sequences important for transposition simply include a promoter. to direct transcription of the element into RNA, and a polyA sequence. Recall that these A residues pair with the DNA at the target site to help generate the primer terminus for reverse transcription (see F'lgUie 11-23).

Examples ofTrrmsposabk El11ments and 1'he,ic Res ulntion

liNE EN

5'UTR

0Rf1 SINE

o

RT-RNAseH ORF2

a.

-=~~~~:-::::-::::::::""'::'-:-r~r--"'"'""

'\.tar~ site duplicatiOn

337

FIGURE 11-34 Geneticcrganization of

a typical UNE and SINE. Note tf1e vartable-

length poly-Asequence at the ~ end oft~ deme(llS. Thts IS a defin(lg fea1ute of the Pdy·A rctrooanspCJSOI15. n 1ese eemEfltS are il.lso flanlred by target-site dtJpicationc; that are v.mable "' length (blue arrows). Sequence dements are not shewn lo .scale. Both types of elen"\eflts also Q!I'T)' promoter sequences, see figur€5 ll-19 and 11·16. (Sauce: frOfTI Bt.r;trnan F 2002. Lateral DNA transfer. p. 2~ 1. f 8.4. c 2002 Cold Spirts Harbor labOfarory Pl'ess.)

These simple sequence requirements for transposition pose a problem for LINEs: how do they avoid transposing cellular mRNA moleo.des? AU genf'IS have a promoter, and most are transr::ribed into an mRNA that will carry a poly-A sequence a t the 31 end of tile molecule (Chapter 12). Thus, any mRNA should be an a ttractive "5ubstrate" for transposition. In fact, genome sequences provide dear evide nce for transposition of cellular RNA via the targetprimed reverse transcription mechanism. For many cellular genes. there are additional COpies of a highlyrelated sequence in the genome. These copies appear to have lost their promoter and their introns (regions of sequence present within a gene but removed from the mRNA by RNA splicing; see Chapter 13), and of~ tm c-arry ttoncali.ons near thoir 5 ' ends. These sequences are known as pi'OeeSSed pseudogenes and usually are not expressed by tha cell. These pseudogtmes are often flanked by short repeats in the target DNA. This structure is exactly that expected of LlNE-pr-omQted tJdDsposition of a oollular mRNA.

Although transposition of cellular RNAs can occur. it is a rare event. The principal mechanism u sed to avoid this process is that the UNE..encoded proteins bind immediately to their own RNA during translation (see Figure 11-23). Thus, they show a strong bias to catalyzing reverse transcription and integration of the RNA that encoded them.

V(D)J RECOMBINATION We ha\le seen that transposition is involved in the movtunent of mauy

different genetic elements. Cells. however, have also harnessed this recombination mechanism for functions that directly help the organism. The best example is V(D)J recombination, which (.)CC\.!I'S in the cells of the vertebrate immune system. The immune syster:n of vertebrates has the job of recognizing and fending off invading organisms. including viruses, bacteria, and pathogenic euka.ryotes. Verteb-rates have two specialized cetl types dedicated to recognizing these invadeiS! 6 cells and T cells. B cells produce antibodies that circulate in rhe bloodstream. whereas T cells pronur..e cell surfuce-bound receptor proteins (called T cell l'eeeptors). Recognition of a ..foreign., molecule by either of thrnro classes of proteins starts a cascade of events focused on destruction of the invader. To fulfill their functions successfully. antibodies and T coli receptors must be able to recr>gnize an enormously diverse group of molecul.es. The principal mechanism cells use to generate antibodies and T cell receptors with such diversity relies on a speciali.zed set of DNA rea.rra~ement reactions known as V(D)J recombination.

3:.8

Site-Specrfic Recomhinatiorl und Transposition of DNA

Antibo\iy a nd T cell receptor genes are composed of gene segJuents tha t are assembled by a series of sequence-specific DNA reWTangements. To understand how this recombination generates the needed diversity, wo need to look at t he structure of an antibody molecule (Figure 11-35]; T cell receptors have a similar modular structure. A genomic region enc<Xling an antibody molecule is shown in Figure 11-36. Antibodies are constructed of two copies each of a light chain and a h~.avy chain. The part of the protein that interacts with fureign molecules is called the antigen-binding site. This binding region is constructed from VL and VH domains of the antibody molecule. shown in Figure 11-35. The "V' signifies that the protein sequence in this region is highly variable. The remaining domains of the antibody are called "C.,. or constant, regions and do not differ among different antibody molecules. figure 11 -363 shows the genomiJ:; region encoding an antibody light chain (from a mouse), called the kappa locus. This region carries about 300 gene segment s coding for different versions of the lightchain VL protein region. There w-e also four gene segments encoding a sh ()ft region of protein sequence called the J region, folJ owed by a single coding region for the \'L domain. By the m ech:misn1 we shall describe below. V(O}J recombination can fuse the DNA between nny pajr of V and J segments. Thus, as a resuJt o f recombination. 1.200 variants of the antibody light chain can be pcoduced from this single genomic region. These segments Me then brought together with the C1. coding region by RNA splicing (C'.haprer 13}. The situation for assembly of the gen~ segments encoding the antibody heavy chain is simila.r. In this case, however, there is an additional type of gene segment, called D (for diversity) {Figure 11-3&). Heavychain genes can be vety complex. For example, a specific heavy-chain locus in a mouse has more than 100 V regions. 12 D regions, and 4 l regions. V(D)) recombination can assemble this gene to generate more than 4,600 differeht protein sequenc£>S. Because functional antibodies can be constructed from any pair o f light and heav)f chains. the diversity

FIGURE. 11-55 structureof~

arllibody moleaAe- The tv.o fight chains are st10\M'1 if'l pili\. whereas lhe heavy chams are in

blt.Ie. The varntle and constant regions ate labeled on the left SKle of the molecute only. ~ that the antige(1 birdng region 1s forrno:l a the interface between the vl and vti ~s.. (Harris u .. Sk<Jletsky E., and Wk:Pherson A. 1998.1 Mol. Bioi. 275: 861- 672.) Image Pf€P'fed Wlt.t1 BobScllpt, MoiScnp. ard ~3D-

V(l)Jj R PCOmb ination

a germ.lioeONA V 1 \12 V3

0::: .J: 1,

FIG U R.E

Vn

J1 J2 J3 J4

C

DNA rearrangement duong

have rot experienced V(O)J recotrt:Jinat10t1 (germ-ire DNA). (b) Rea:lmbtnat10r1 bel'-Neerl two speof\c gere sewnents (V3 ald E) as ocazs ruing B<eJ development n-.s ~only one of the many types of rE!OOiTOOdbOO events that can oroJC m afferent p.-e-6~. The:: rec:ornbirled low> is thro transcri:Jed af1d the RNA spliced ( Chaptet J3) to j\DII tf)O'Se a COf1slan~ gene segment lh.s mRNA is tnen ~ to gener· ate the ight chclift prolen (c) Schemabc cJ ltle ever~ more complex heavy chain genebc f€8ion. With tts <'lddinooa11J" gere segments cYid mutt~ types of CDf'IStdf1( regiOnS segme1ts (QJ. c'Y ek:.). (Sourre From &mnao 1-.• 1002. l.ateroJ DNA trr:JrrS!er p. 345, f 1 J 3 . ~· 2002 Cokl Spr~ Hatba l.c!boratcty Press.)

B ceu afferertiatlon

b

a ce1 DNA

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.;.:;, i Jn.I.;......)-

I-~

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v

1··J

c

~

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CC

DDDD . . 1 JJ )l

J1-36 Overview of the pt'OCe$5

of \I(D)J recombination. The top po1'\els <;how~ steps invclved in producing lhe ~ c:hatn cf an antibody p-oten (a) 1he genebc Ofga.-.zalion of part ~t
Jr-------.l------.J . )

TJ:~J----;;.D.....,D~D~J

339

Cy

Cr

Cct.

ir Jl ::::1)

generated by recombination at tbe light- and heavy-loci have a multiplicative impact on protein structure.

The Early Events in V(D)j Recombination Occur by a Mechanism Similar to Transposon Excision Recombination sequences, called recombination signal sequeru:es.

flank the gene segments that are assombled by V(O)J recombination. These signals all have two highly conserved sequence motifs. one 7 bp (the 7-mer) and the second 9 bp (th e 9 -mer) in length (Figure 11-37). These motifs are bound by the recombinase (see below). The recombination signal Sfl<)Uenccs corn~ in two d(!Sscs. One d ass has the 7-rner and 9-mer motifs spaced hy 12 bp of sequence. whereas the second class has these motifs spaced Ly 23 bp (Figure 11-3 7a). Recombination always occurs between a pair of recombination signal sequences in which one pat1ncT hos the 12 bp "spacer" and the other pa rtner h as the 23 hp ·•spacer. •• These pairs of recombination signal sequtmces atP. organized as inverted repeats flanking the DNA segments that are destined to be jojned (Figure 11-37b). The recombinase responsible for recognizing and cleaving the recombination signal sequences is composed of two protein subunits called RAG1 and RAGZ (RAG for Recombina tion Activating Gene). Tl1ese proteins funct ion in a manner very similar to a tram;posase

Site-Specific RecombincUon and Trom•posi1iDll of DNA

b

(C

V

D

J

!a-J_)F~~.~3'"-,/.......Jj) lgH

Af!!f>t>:..____:: ..

(Q- z:::.·~~----:<1 ..::......--'-')) lgK

)>

c : }) IQA

~~~~~~---=~-=--.. ~;=--J~) Tffi~TC~

s: I G u It£ II- 37 Recombination signal sequences recognJJ.ed ih \I(D)J recombination. (a) ~P of thE two types of reaJI'flbif1ation siJ?11C!1 seqt.JenCeS {RSS). The 12 bp spacer is .shown in btue, lhe·23 bp ~ 11"1 gmef1 and the conserved 7-mer a11d 9-rner sequePU.' elements. 'Shored by both types of sequef1Ces, are in yellow The nuc;Jeotide sequence 1n the wacer region is neil it;1portanl The length, however. tS cntical. (b) Examples of RSS arrangements in the ger.etk reg.ons oocoding antibodies (lg genes) ar>d TeeMJ~Of proteins (Trn genes). (Source: (a} f rom BusW..an F. 2002. Lotero/ DNA transfer. p. 346. f 1J.S. () 2002 Cold Spring Hatbo!' l.abora10ry Press.)

(Figure 11-38). They recognize the recombination signal sequences and pair the lwo sites to form a protein-DNA synaptic complex. The RAGl proteins within this complex then introduce singlescranded breaks in the DNA at each of the junctions between the recombination signal se-quence and the gene segment that will be rearr.mged {Figure 11 ·38 a). The site of cleavage is such that the prote in coding segment now has a free 3'0H DNA end (figure 11-38b}. Then, as we saw previously for some ll'ansposon excision reactions (Figure 11-20), this 3'0H DNA end attacks the opposite strand of tbe DNA dolthle helix. This attack results in the coupled ONA cleavage aru:l joining reaction that generates a h airpin DNA end. lt is the p rotein coding sequence segments that have the DNA hairpin ends, whereas the recombination signal sequences now have normal double-stranded breaks at the ir ends (Figuw 11 ·3&). This same mechanism generates a DNA hairpit\ at each of the two ,.ecombining DNA

segments. Once 111e two DNA sequences in the synaptic complex have been nicked and ''hairpinnoo" by the R..A.G m combioase, cellular DNA repair

proteir1s take over to finish the recombination l'eaction (Figure 11-3Bd). The DNA hairpin ends on the two protein-coding segments must be opened, and these ends must then be joined together. Cellular nonhomologous end-joining proteins {see Cllapter 9} patticipate in this reactiou. Interestingly, DNA joining is often accompanied by the addition (or deletion) of a few n udeotides. These additions are analogous lo the ..footprints,. left in the old target DNA when transposons excise, as w e described for the Tel / mariner transJWsons. These additions add an

exlta

compon ent to the sequence diversity of the rosulting protein

molecule. The pair of clP.avAd recombination signal sequences are also joined together during recoJnbination. This event generates a c ircular DNA molecule that is usually discarded by the ceU. The similarities between the mecl1anism of DNA cleavage to initiate VfD)J l'eco rnbination and transposon e:A-cision are remarkable. ln fact, the recombination signal seqUences also look similar to the tenninal invE!rted repeats found at the ends of a transposon, and RAG1 protein

l '1D)f RP.combmution

a

reconi,)inatioo signal sequences

v

fiGURE

---.~~'==J~·~

.L

341

11-18 TbeV(D)J

recombihation pathway: deot~ ocast' by a mechanism similar to transposon exasion.. The recombtnases c.ntyze ~ strand cleavage at tre eOOi of the signal seqJenees, leaving a ftee 3'0H. Each l 'OH theP ritiate:s anad<. on the opposite $1lilnd.s to form a hairpin irlrermaiate (see FJ&urt> 11 22b).

rr.e

mpn struct\.res are subsequently hydroly~ i'lf!d lt1ef1 jOined togcthf.'f to form a coding JOint ~een the V and J regions. The to.w mi> cor· ty~ng t1le recornbNtlon ~ sequences are

c

also joined to fofm a signill joint lhe b mer stfUCIUre undergoes flll1t1er re~

whereas the latter IS discarded (Source: rrom Bustman f. 2002 toteral DNA transfer, p. 348. I t I .6, ttl 2002 Cold Spnt1g Harbor latoraklry Press.)

d hairpifl opening, fusion of coding

ONAno~

~gmen1S

v

homologous end joiniflg

J

proteins

~~ ---------~====~;;;

1

further recombination

l

appears to have some similarity to the UOE tran$posase protein family. These observations, together with many others. have provided overwht"Jming evidence for the propOsal th at V(D)J recombination. now a critical feature of the immune system of higher animals, evolved from a DNA transpo$on. This conclusion speaks to the cdtical importance of trnnsposable elements in lhe evolution of cellular ~enomes.

SUMMARY ------------------------------------------------------ ------------------------ ---Although DNA is normally thousht of as a very static molecule that archives the genetic material. it is also subject to numerous types of rearrangements. 1wo dassf!s of genetic recombinalion-conservalive sitc-spocific recombination and transposition-are responsible for many ur these

evencs. ConseiVative sile-speciJk recombination ocCUis al definerl sequence elements in the DNA. Rocombinase proteins teeogni2e these sequence elements and act to cleave and join DNA strands 1o reattange DNA segments containing tho recomLihatiuo sites. Three types of rearrange.numt.s are common~ DNA insertion, DNA deletion,

----

anrl DNA inversion. These .rearrangcmonts have many functions, including insertion of a viral gt.'llome into that of the host cell during infection, resolving DNA multimet s, and altering gene expression. Tht~ organization of the recombination sites on the DNA as we ll as the participation of DNA architcctlll".al proteins dictate the outcome of a specific recombination reaction. The architocturnl proteins function lo bend ONA sL-gments and can have u la~gc influence on the reactions occurring oo a specific region of DNA.

There al'e nvo families of conservative ~ile-svecific reoombinascs. Both fami)ies deave DNA using a protein-

342:

Site·Spcteific: Rf!lXJmbinalion and Tmnspa~>irion cf DNA

DNA covalent intenncditJie. For lhe serine recombina•:es, this linkage is via an active-site serine residue: for thi! tyrosine recombinases.. i1 is v ia a tyrosint>~ SlM.IClures of the tyrosine recornbinases yield many insights into the details of the .recombination mechani~"'ll . Transposition is a dass of recoml-lination that moves mobile genetic elements. called t.ransposons, to new RCJlOJnic sites. ThP-re are three major classes of lransposoos: DNA transposons. viral-like retrolransposons, and poly-A ~trotransposoos, 'fhe UNA tran.s~ons exist as ONA throughout a cycle of trans position. Tl1ey move either b}' a cut-and-past& recombination mechanism. which involves an cxciood transposon intermediate, or a replicative mccha· nism. The two dasses of retrolransposons move using an RNA interme-diate. These "retro"' elements reqUire the RNA~

dependent DNA polymtlrnse, called reverse transcriptase. as well as a re()Ombinascprotein for mobility. Ttansposons are pr05ent in Lhc gl'Jnomes of all orgrurisms, where they can co nstitute a h uge .fraction of the toti:ll DNA s~quem::e, Th~y are a major cause of mutHtions and genome rearrangements. Transposition is often 1'(,.-gulat.t:d to help ensure that transposons don't cause too much of a disruption ro th~ genome of the host cell. Control of tr~nspo son copy number and ~ulation of the choice of new iJ1senionl>iles are commonly observedFinally. a transJlosit ion-ltke mecha nism r:an he w;od for other types of DNA rearrangem ent reactions. The prjmc example of this is the V(O)J recombinat ion reaction, responsible for assembly of gtme fragments during developmen• of the vertebrate immune system.

BIBLIOGRAPHY Books

Transposition

Buslmmn F. 2002. Latcrol DNA ttunsfcr: Mecllonisms and cOllSequences. Cold Spring Harl:Jor Llborutory Press,

Haren L.. Ton-Hoan.g B .• and Chandler M. 1999. In tegrating DNA: Ttansposases e nd viral intcgrases. Ann. Rev. M icmbiol. 53: 245-281 .

Cold Spring Hamor. New York Craig NJ~. Craigie R. Gellert M., and Lambowitz A.M.. cds. 2002. Mobile DNA II. American Society for Microbiology. Washingt on. [)C.

Plaslerck R. 1995. The 'l'C11/mar iner tro.nsposon family. Curren t Topics in Microbial. lmmuno/. 204: 125-143.

Site#Specific Recombination

Rice P.A. and .Baker T.A. 2001 . Companitive archHectum of transposasc and integrative complexes. Not Strt1ct. Bioi. 8: 302-307.

Baker T.A. 1991 ..... _ and then there were two." Noture 353! 7 94 - 795.

Own Y and Rico P.A. 2003. f\:ew insight into sites pecific recombination from FLP recombin~ONA structures. Arlnu. Rf!ll. Biophys. BiomoJ. Stwcl. 32: 135-159.

Hallet B. and Shcrratl D.J. 1997. Transposition and sitespecific recombination: Adapting DNA cut-and-paste m<.-chanisms to a variety of genetic rcarnmgements. FEMS Microbiol. Rev. Zt: 157- 178. Smith M.C. and Thorpe IJ.M. 2002. Diversity in the setine rocombinascs. Mol Nncrobiol. 44: 299-307. Stark W_M, .Boocock M.R, and Shcrr.ltt D.J. 1992. UitaJys is hy sHe-specific recombinases, Trends Genet. 1$! 432 -~39.

Prak E.T.L, and I
Smit A. t-'.A. Hl99. JnterspeTsed repeats and o\hcr mementos of transposabl': clements in mammalitm genomes. CurrOp. Genet. Dett. 9 :657- 663.

Williams T.t. and Baker T A. 2000. Transposose team puts a hood lock on DNA. Science 289~ 73-74 .

V(O)J Recombination

1.r..

FtJgmann S.D.. Lee A.l.. Schockctl P.E.. Villey and &halz D.G. 2000. The RAC proteins and V{D)J recomhi-

nution: Complexes. ends. and tronspo.silion. Annu. Rev. Immuno/. 18: 495- 527 .

Cc:llcrt M. 2002. V(D}J recombination: RAG p roteins, repair foctors, and regulation. A.nriU. Rev. Biochetn, 71: 101 - ·132.

P A

R T

EXPRESSION OF THE GENO ME

Port 3 Exp~t>iOII

344

PART

tJ/ Lhe Genome

OlJ T l l N [

• 01<1ptef 12 Me
13 RNA Spl1oog

Chapter 14 ltan.sL31100 dlopter 15 lhe Genetic Code

art 3 is concerned with one of the great challenges in understanding the gene- how the gene is expressed. 1n other words . how is infonnation in the fonn of the linear sequence of nucleotides in a polynucleotide chain converted into the linear sequence of amino acids in a p<Jiypeptidc chain'? The flow of information from genes to proteins and the concept that information flow is unidirectional. is known as the central dogma and was enunciated by Francis Crick in 1958: .

P

The central dogma states that once ''information" ha
thetases that load ammo adds lmto the tRNA adapt01s; and tbP. protein-synthesizing factory itself. the Tibosome, wh ich is composed of RNA and protein. The remainder of the chapter describes how these four components, with help from a number of key aux.illiary factors, manage the remarkable process of converting the nucleotide code of a given mRNA into the correct orde r of amino acids in its protein product. F"mally. Chapter 15 describes the classic experiments that led to the elucid at ion of the genetic code. and lays out the rules by which the code is translated. The n ucleotide sequence information is based on a three letter code. while the protein sequence information is based on twenty differPnt amino acids. The code is degenerate with two or more codons (in most cases} specifying the same amino acid. There arc also specific codons that indicate where translation should start and where it should stop.

PHOTOS FROM THE COLD SPRING HARBOR LABORATORY ARCHIVES

------

Much of Roberts' researdl .,as focused on the rui'IC'OOn and di~I"SSty of restrietioo enzymes (Chapter 20). bul he was also a wdisrolererof "splitgenes," forv.fuctl he shared the Nobel Pnze \Mth Ptlil~p Sharp ICI 1993. Shown here with him are taft to r'l:t1t. Yasha Glaman. the tumor virdugtst Ahmad Bullhari. who WOI~~ oo phage Mu tlansposmon (Chapter ll); and James Damell, ~ v.a1.: focuses on ggnal transductKxl in gene regulation (CMpter 17). Ridwtrd Robet'ts, t977 Sylnposiuln on Chromatin.

DiMd Baltimore. ~·Jacob, and Walter c.ilbert, 1985 Symposium on the Molec&dar Biology of Development. &ftimore Co-drs0011ered. wth HoNafd Temin, the er1zyme reverse tl?.tlSCllptase, wtUch makes 1JNA lJSIIl8 RNA as a template (chapter I I). Jacob, IMth Jacques Monad. proposed the basic model tc. how gene ~ession is regt.JI~ (Chapter 16) end a&J Jlfoposed a model for OO.V ONA replication is regulated (~ 8). Gilbefl provided b1ochem1cal vaidation fa aspects of the .J
-- ---- -----------Sydney lkennef and James watson. 1975 Symposium on 'die Sym.pse. Brenner, shov.tn here with 'Natson, coooibuted to the dis· O':lVenes of ~NA and the nat\Jie of the genetiC code (Chapter 2); his share of a Nobel F'Jire, in 2002, 11owe~ was for estabkshing the v.orm, C Elegans, as a model system tor the study of rlevelopmefltal biology (Olapter 2 1).

196J Symposium on Synthe-sis and Structure of~ In addition to his role in solving ttJe struallre of IJNA.. Cnck was oo intellectual Crnllng force m rhe develr..pment of molecular t:..ology dunng the field's critical early ~s. His "adaptor 11ypothcS!S" (ptillished in the RNA T.e Oub newsletter) predicted the existence of molecules req\llfed to translate the genetic rode of RNA into ttle amioo acid seqtJence d protens. 01ly later .....ere tRNAs found to oo jUSt that (Chapter hand~~

14)

Phillip Sharp. 1970 Sfmposium on Tumor \linlses. ShiMp and Richard Roberts shared the 1993 Nobel Pr11e itl fllledione for &scovenng lhat m
Paullantecnik. 1969 Symposium on The

Medlanism of Protein SynthesG. Zamemik developed f1 vitro systems of protein synthesis that proved critic~ to

undefstanding how the gefletic code~ (W)d tnw cells manufacue prote.ns (Chapter 2). Together witfl Mahlcn Hoagland, he also disco~ tRNAs. a key ro~ in that J)focess (Chapter 14 .)

CHAPTER

Mechanisms of Transcription

P to this poinl we have been considering maintenance of the genome- that is, how the genetic material is organized, protected, and replicated. We now tum to the question of how that genetic material is e.xpressed-that is, how the series of bases in the DNA d irects the production of the RNAs and proteins that perfoTITI cellular functions and define cellular ident;ty. ln the next few chapters we will describe the basic prucesses responsible lor gene expression: transcription, RNA processing, and translation. 'D"anscription is. chemically and f.mzymaticaUy, very similar to DNA replication (Chapter 8). Both involve enzymes that synthesize i1 new strand of nucleic acid complementary to a DNA template strand. There are some important differenCP..s, of course-most notably that in the case of tra.Iu;c:ription I he new s1Iand is made from rlbonudeotides .rather than dooxyribonucleotides (see Chapter 6). Other mechanistic featm-es or trnnscription that differ from replication include the following:

U

• RNA polymerase (the enzyme that catalyzes RNA synthesis) does not need a primer; rather, it can iniriate transcripUon de novo (though in vivo initiation is pennitted only at certain sequences, as we wiU see). • The RNA pl'oduct does not remain base-paired to the template DNA strand-rather, the enzyme displaces the growing chain only a few nucleotides behind where each ribonucleotide is added (fib"llfe 12-1). This displacement is critical for the RNA to be (as is typically tbtt case) translated to pmduce its protein product. Furthermore, because this relt!aS4:l follows 80 dosely oohind the site of po1ymerization, multiple RNA polymerase molecules can transcribe tbe same (,rene at the same time, each following d oseJy along behind another. Thus. a ~lJ c.sn s ynthesize l:qrge n umberR of t.ra11script.s from ::~ singiP. gene (or other DNA sequen~ in a short time. • 1ianscription, though ve.ry accuratt-, is less accurate than replication (one mistake occurs in 10.000 nucleoticles added, compared tu one m 10,000,000 for replication). This difference reflects the lack of extensive proofreading mechanisms for transcription, although two forms nf proofreAding fnr RNA synthesis do exist. It makes sense for the cell to worry more about the accW'8cy of replication than of transc..:ription. DNA is the molecule in which the genetic material j~ stored, and DNA replication is the process by which that genetic material is passed on. Any mistake that arises dwing replication can therefore easily be catastrophic: it becomes permanent in the genome of that individual and also gets passed 011 to subsequent generations. 1'canscriptiun. in runtrast, produces only transient t..'Opies anu normally several from each transcribed regiun. Thus. a mistake dwing transcription will rarely do more harm than render one out of many transient transcripts defective.

OUT LIN E

• RNA Polyroooses and fh(:. lranscnption Cyde (p 348)

. The TranscnptJon Cyde 1n ~ia(p

353}

• Transcnptlon 1n Eukaryot~ (p. 36

3411

Mechonisms ofTronsc.riptiat1

---------------------- -12-1 lrnnsc:ription ol DNA

J= I G U R E

inlo ~ The figtJre shows, lfl the absence of the enzymes iflv~ hotll the DNA double 1\Efjl( is unwound and an RNA strand .s built oo the template strand.

Beyond these mechanistic differences between DNA replication and transcription, there is one profound difference that Teflects the different purposes served by these processes. Transcription selectively copies only certain parts of the genome and makes anything frorn one

to several hundred. or even thousand, copies of any given section. In contrast. replication must oopy the entire genome and do so once (and only once) every cell division (as we saw in Chapter 8}. The choice of which regions to transcribe is not random: each typically includes one or more genes, and there are specific DNA sequences that direct the initiation of transcr;pHon at the start of eriCh region and others a t the end that tenninate transcription. Not only are different parts of the genome transcribed to different extents, but the choice of whicb part to transcribe. and bow extensively, can be regulated. Thus. in diftare11t cells. or in the same cell at different times. different sets of genes might be transcribed. So, for example, two genetically identical cells in a human will , jn many cases. lranscrjbe different sets of genes, leading to differences in the charcteter and fum::tion of those two cells {for example. one might be a muscle c."t:lll, the other a neuron). Or. a given bacterial cell will transcribe a different set of genes, depending on the medium in which it is growing. These questions of regulation are dealt with in Part 4.

RNA POLYMERASES AND THE TRANSCRIPTION CYCLE RNA Polymera..~ Come in Different Forms1 hut Share l\tfany Features RNA polymerase performs essentially the same reac:tion in all cells,

from bacteria to humans. lt is thus not surprising that the enzymes from these organisms share many features, l3specially in those parts of the enzyme directly involved with catalyzing the synthesjs of RNA. From bac.teria to mammals. the cellular RNA polymerases are made up of multiple subunits (although some phage and organelles do encode single subunit enzymes that perform the same task). Table 12-1 shows the numbers and sizes of subunits found in each case and also shows whicb subunits arc conserved at the sequence level between different enzymes. As can be seen from the table, bacteria have only a single RNA polymerase, while in eukaryoti(,: cells thet·e are three: RNA Pol r. lJ, and liJ. Pol D is the enzyme we will focus on when dealing with eukaryotic transcription in the second haJI of this chapter. That is because it is the most studied of these enzymes, and il is alsn the polymerase .responsible fur transcribing most genes-indeed,

lA BLE 12 · 11beSuburritsofRNAPolymerases Prokaryotic

Eukaryotie

Bacterial

Archaeat

RNAPI

RNAPM

RNAPIM

Core

care

(Pot1)

(Pof I)

(Pol iti)

p

A'/A"

RPA1

RP81

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B

RPB2 Rf'63

RPC2 RPC5

a'

0

RPA2 RPC5

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(+9others}

[+ 7 others}

(+ 11 othersj

Note·The ~ 111 eactt coturm f.l'fl riStad 111 order of decreaslflg rT'IOieCUru ~M~tgN Soutr.e. Oala adaptad lrom EVigtll n.H 2CXlO J Mol. Bid. 304: 687 ·698. f ig. 1. p eat\. ~ 2000 Acac::femic Presl.

essentially aU protein-encoding genes. Po) 1 and Pol m are each involved in transcribing specialized. RNA-encoding genes. Specifically, Pot l transcribes the large ribosomal RNA precursor gene. whereas Pol mb'anscribes tRNA genes, some small nuclear RNA genes. and the 55 rRNA gene. We return to these enzymes at the end of ahe chapter. The bacterial RNA polymerase core enzyme alone is capable of synthesizing RNA and comprises two copies of the a subunit and one each of the ~. (3' . and w subunits. That enzyme is closely related to rbe eukaryotic polymerases (see Table 12-1). Specifically, the two large subunits. J3 and J3', are homologous to the two large subunits found in RNA Po.l U (RPBl and RPB2) . The a subunits are ho.molo-gous to RPB3 and RPBll and w to RPB6. The sti'lJUUJ'e o f a bacterial RNA polymerase cure em~yme is similar to that of the yP..ast Pol lJ enzyme. These are shown side-by-side in Figure 12-2. Later we will describe son1e of the structural details that shed light on how these enzymes work. For now we just highlight some of the general features. The bacterial and ye.asl enzymes share an overf'IU shape and organi· zation; indeed. they are more ali.ke than the compariS<m of tbe subunit sequences would predict. This is particularly true of the intemaJ parts. near Oto active site, and less so on the periphf,ries. The distribution or these sirnilar;tics and diffe-ren ces presumably reflects. in the former case, the fact that the enzynlP.S carry out the same fundion (synthesis of RNA on a DNA template), and in the lattP.rc.aso, that. to function in the wll. the two enzymes interact with other proteins and those are specific and d ifferent in the two cases. as we shall S $. Overall, the shape of each enzyme resembles a crab claw. This is reminiscent of the .. hand" structure of DNA polymerases described in Chapter 8 (Figure 6 -5). The two pincers of the aab claw are made up predominantly of the two largest subunits of each enzyme ( W and ~ for the bacterial case, RPBl and RPB2 fm the eukaryotic enzyme). The nctive site, which is made up of regions from both these subunits, is found at the base of the pincers w ithin a region called the "active center cleft'' (see Figure 12~2). The active site can bind two Mgt · ions, consislEmt witb the proposed two-metal ion catalytic mechanism lor nucleotide additirm proposed for all types of polymerase (see Chapter 8}.

350

FIGURE

Mechanisms

of 1h mscriplion

12-2 Comparison of the crystal

structures of prokatyotic and eukaryotic RNA polymemses. (a) Structute of RNA pdymer.;se rote enzyme from r aquatraJS. The St.Jbuncts are colored .as lollovvs: I} s

500wn in pur

pie. p· in blue. the two a s.lhlolts m yelow Clf"ld green B!ld (I) In red (Seth oarn. The Rockefe8er Unive-s.ty, personal COfT!ml.ricatioo.) (b) Structure of RNA Pol~ 11 from~ s. cereviSicJe. The sub.Jnlts are robed to shov-.1 mew relatedne~ to thol;e 10 the bacten~ encyme

(see Table l2· 1). "fuJs, RPB 1 CY1wn in yelow and green; and t
There are various ch annels that allow DNA, RNA, and ribonucleotides into and out of the enzyme's active center cleft. These we d iscuss later when t:onsidering the met:hanisms of uanscription.

Transcription by RNA Polymerase Proceeds in a Series of Steps To transcribe a gene. RNA polymerase proceeds through a series of well-defined ste ps which Are groupod into three phaSP.s: inilialion, elongation. and lennin ation. Hero, and in Figure 12-3r we s u mmari?.e the basic features of each phase. Initiation. A promoter is the DNA sequence that initialJy binds the RNA polymerase {together with initiation factors in many cases). Once formed. the promoter-polymerase complex unde.:goes structural changes required fur initiat ion to proceed. As in replication initiation. the DNA around t:M point where transcription will start unwinds. and lhe base

RNA PDlymeroses alld tile 'Tr:mscrlption Cyck

RNA

351

FIGURE 12~3 The phases of the

polymerase

transuiption cycle: initiation, elongation, and terminalioA. The hgure sl1ows the gmeral scheme for the transcrrption cycle. The

features shown hdd fa both bac1enal Clfld eukaryotii: cases. Otht.'f factors reqwed for inib
downstream ONA

p

binding

{dosed canptex)

<:~re referred to as down:>t~m of the st«' site L.ik~ S€QU61C5 preceding the start site are rcfecred to as upstTeam seq;cn:es. When refetrirlg to a specific posibcln lf1 the upstream sequence. lhcs is given a negative vak.Je. Oov.nstream sequences are aloned ~ values.

Initial trat'lscfiption

1 c: 0

~

0>

c: 0

iii

polymefase

x-------------------,.

terminates. and releases

RNA

pairs are dismpted, producing a .. hubble~ o£ single-stranded UNA. Again like DNA ~"Ppli cation, transcript;on always occurs in a 5' to 3' directirut. lllat is, the new ribonucleotide is added to the 3 ' end of the growing chain. Unlike replication. however, only one of the DNA strands acts as a template on which the RNA strand is built. As RNA polymerase binds promoters in a defined orientation. the same strand is always transcribed from a given promoter. The choice of promoter determines which stretch of DNA is tran· scribed and is the main step at which regulation is imposed. Tha t is, the decision whether or not to initiatP. transcription of a given gcoe is chiefly how a cell regulates which proteins it will make at any given

or

time. Elongation. Once the RNA poly merase has synthesized a short stretch of RNA (appTOximatoly ten bases),. it shifts into the elong-.nion ph~. This transitinn 1-equires further confomtational changes in po'Jymerase that lead it to grip the template more firmly. Dw'iflg elongation, the enzyme performs an impressh.te .range of tasks in addition to the catalysis of RNA synthesis. Jt unurinds the DNA in front and re·anneals it behind, it dissociates the growing RNA chain from the template as it moves along, and it perfonns proofreading functiuns. Recall that during replication, iD contrast, several different t!nzymes are required to catalyze a simi lar nmge of functions.

Termination. Once tho polymerase has transcribed the Length of the gene (or geoes), it mUb1 stop and release the RNA product. This step is called lP.rminalion. In some cells there are specific. w ell-characterized. sequen ~ that t:Jiru,'P.r t.f!mlinat ion; in others it is less clear what instructs the enzyme to cease transcribing and dissociate from the template.

Transcription Initiation Involves T hree Defined Steps The first phase in tl1e transcription L-ycle- initiation- can itself be broken down into a setios of defined steps (as indicated in Figure 12-3). The first step is the initial binding of polymerase to a promoter to form what is called a closed complex. In this l0rm the DNA remains doublostrandcd, and the enzyme is bound td one face of the helix. ltl the second step of initiation, the closed complex undeTgoes a transition to the open comp~ in which the DNA strands separate uver a distance of some 14 bp around the start site t.o form Lhc lranBcri.ption bubble. The opening up of the DNA frees the template strand. The firs1 two ribon ucleotides are brought into the active sire, aligm~d on the template strcmd, and ioined t~ther. The enzyme then begins to move a1nng the template strand. opening the DNA helix ahead of the site of polymerization and a llowing it to reseal behind. In this way, subsequent ribonucleotide.~ are inc.mporated into the growing RNA cha~n. Incorporation of the first tell or so ribonudeotides is a rather inefficient process, and at that sta~ the enzyme often releases bhort rnmscripts (each uf le...-.s than ten or so nucleotidcs) and then begins synthesis again. Onm an enzyme gets further U1an the 10 bp. it is said to have escaped the promoter. At this poinl it has Conned a stab~ ternary COltlplex, containing enzyme, DNA. and RNA. This is the transition to the elongation phase. hi the remainder uf this chapter, we will describe the transcription qcle in more rlet1il - first fur tbe bacterial case, and t hen for eukaryotic systems.

The Transcriptipll Cycle i n 8octeria

353

THE TRANSCRIPTION CYCLE IN BACTERIA Bacterial Promoters Vary in Strength and Sequence, hut Have Certain De6ning Features The bacterial core RNA polymerase can, in pr~ndple. initiate ttanscription at any point on a DNA molecule. In cells, polymerase initiates transcription only at promoters. It is the addition of an initiation factor called u that converts t.:ure enzyme into the form that initiates only at promoters. That fotm of the enzyme is C"dtled the RNA polymerase holoenzyme {Figure 12-41. In the case of E. coli, the predominant ('f factor is called de (we will consider other. alternative u factors. in Chapter 16). Promoters recognized by polymerase <'.ofltaining c-r70 share the following charac.teristic

structure: two conserved sequences. each of six nucleotides. are separated by a nonspecific stretch of 17-19 nucleotides {Figure 12-5). The two defined sequences are centered, respe<.:tivt!ly. at about 10 base pairs and at about 35 base pairs upstremn of the site where RNA synthesis starts. The sequences are thus called the -35 (minus 35) and -10 (minus 10) l'egions. or elements, according to the numbering scheme described in Figure 12-3, in which the DNA nudcotide encoding the beginning of the RNA chah1 is designated +1. Although the vast majority of if0 promoters contain recogni~ble - 35 and - 10 regions, the sequences are not identical. By comparing many different promoters. a consensus sequence can be derived (see Box 12-1 , Consensus Sequences~ for a discussion of how these are derived). The consensus sequence reflects prefened - 10 ancl - 35 regions, separated by the optimum spacing (17 bp). Very few promote•·s have this exact sequence, but most differ from it only by a few nucleotides. Promoters with sequences closer to the consensus are generally ..stronger" than those that match less weJI. By the strength of a promoter. w~ mean bow mauy transcripts it initiates in a given time. That measure is influenced by how well the promoter binds polymerase

FIGURE 12-4 RNA polymerase holoemyme 1:. fJIIUOiicus. The RNA polymerase ~ ffom ThermlJS ~ Shown tn gray is the core enzyme (the sarne enzyrne shown WI part (a) of figtJre 12.-2.). In pt.J!ple is the o:l:l subunit (reg10115 2. 3, and 4 see F18Ufe 12-6). On the rit'hl tS regson 2, at the tOJ) regton 3. and at the bottom region 4 As desaibed letter 1n the teet, it 15 o: regions 2 and 4 that recognile the - 10 and - 35 regioos ol the promotet" r~..fy. (Mllrakam11(5., Masuda S.. and Darst SA 2002 Soence. 296· 1280.) Image prepared \IIIIth MoiScnpl &bScript and Raster 3D.

354

Mechanisms ofTranscripti<m

f'tCiURE 12-5 features of bacterial promoters. Varioos comb~ of bcKterial promoter elernenJ.S i:lfe shown Details of how each Ele-nent contributes to polymerase bn&nE and function a~e Oesaibed tn the text

c

0

"e>
n

1:1 : J - 10

+1

.,. J

initmlly, how efficiently it supports isomeri7~tion. and how readily the polymerase can then e..-.cape. The correlation between promoter strength and sequen~..-e explajns w hy promolt-Jrs are so heterogeneous: some genes need to be expressed more highly than others and the former are likely to have sequences closer to the consensus. An additional DNA elemen t that binds RNA polymerase is fnw1d in some strong promoters, for example those direc:ting expression uf the ribosomal RNA (rRNA) genes. This is called an UP-element (see Figure 12-5b) and increases polymer-riSe bin ding by IJl'OViding an additiona] specific interaction between the enzyme and the DNA. Another class of cr 71t promoters la<.:ks a -35 region and instead has a so-called ••extended - 10" element. This comprises a standard ·-10 region with an additional short sequence element at its upstream end. Extta colllacts made between polymerase and this additional sequence element compensate for the absence of a - 35 region. As we will see in Chapter 16, the gal genes of E. coli use such a. promoter.

Tbe rr Factor Mediates Binding of Polymerase to the Promoter The
The - 10 region is also recognized by an<~ helix. But in this case, the interaction is less weU-cbaracterized and is mere complicated for the following reason; whereas the - 35 region simply provides binding energy to secure polymerase t o the promoter~ the - 10 region has a more elaborate role in 1rnnscription initiation, because 1t is V\rithjn thaJ elE>ment that DNA melting is initiated in the transition from the

Th~

Transcriptivn C}de in &lcterio

355

Box 12·1 Cmlsensus SequetKes

l1le DNA sequences of binding sites recognized by a given protein may not always be exactly the same. likewise. a stretch of amino acids that bestows upon a protein a particulal- function may be slightly different in different protetnS. A consensus sequence is, m each case, a version of the sequence having at each position the nudeotK:!e (or amino acid) most commonly found there in different examples.. Thus the consensus seqtJence for promoters in E. coli reoognized by RNA polymerase containing u 70 is sho\1\111 in the figure (Box 12- J Figure f). lhis consensus sequence was derived by aligning 300 sequences known to function as u 70 promoters and ascertaining the most common base found at eoch position in the - 35 aod in the 10 hexamers. That nodeotide is then chosen as the nucleotide of dloice at lhat positioo in the consensus; its relative frequency and 1he frequencies with vvtlich the other three nudeotides ocwr at each position is portrayed in lhe grapll. Note that there is no significant consensus amoog the 17 to 19 nudeoodes that ie in the regiorl between - 35 and - 10 .

In that example. eadl individual promcter sequence had prev.ously been tde.fitified, so afigning the sequences is IJMat But consider a rather different example. In tlis case. r:o binding site has been identified for the DNA-bindirg protein in question. Havvever, severat regions of a chrcxnosome are known to contain binding sites somevvhere within their fengths. A computer algorithm is empk)yed that scans each of the sequences of rnese chromosomal regions. seardling tor a poten~ bindilg site oomrnon to them aU. A second approach to denving the consensus sequence fur a

ONA-bindng proten vvhen the binOOg site is not already ~ takes advantage of dlembll methods for synttlesimg vast se(S of shoo ttJA fragments of random sequence (Chapter 20}. The pi«eio of interest is mixed with the poptjation of DNA mo1ecufes and those a-JAs to \1\hich it binds are retrieved and sequenced A comparison of the ~ boond reve
100 ~--------------------------------------------~

~ 75

-35

17- 19

- tO

fll..<deOOdes BOX

12-1

FlGURl

I l'romoter consensus sequence and spacinJconsensus. (Scx.lrce: Redrawn from~ B. et al2002. Molec-

ular biology ofthe celT. 4th edtion, p. 308, f.g 6 .12. Copyngflt © 2002. Reproduced by permission of Routtedge/Taylor & Frands Books, Inc:.)

-extended - 10"

-

-35

L

n

- 10

li

3 2 1 C '--...o..;.;......,_.....,........_____ ~~;;;:;"~!'~ 11 ) AII 2A?.3;.22.t ) 12 'l 1t jN LJ _ _....L._ _........,._ melting

f iGURE 12-6 Regions of 0'. Those llegioos of u factor that re
merase cere enzyme to a standctrd promoteJ.

see Figure 12-7. (Source: Redrav.fl irom Young BA. Gruber TM, and Gross CA 2002. VieiNs of tra~100 initta1lon. Ce// 1{)9: 4 17-420,

fig_ ' - Copynght e 2002, IMlh pemisslon from Elsevier.)

356

MeohcmislllS cfTronscription

dosed to open complex. Thus. the region of u that inleracls w ith the - 10 regivn is doing more than simply binding DNA. In lcooping with this expectation , the a helix involved in recognition of the - 10 region contains several essential aromatic amino acids that can interact with bases on the nontemplate strand in a manner that stabilizes the melted DNA In Chapter 8, we described a similar role for the single-strand binding protein (SSfl) during DNA replication. The extended - 10 element. where present, is recognized by an a helix in u region 3. This helix makes contact with the two specific base pairs that ('.Onstitute that clement. Un like the other elements within the promoter. the UP-element is not .recognized by u but is jnstead recognized by a carbo.>..-yJ terminal domain vfthe a subunit, called the aCID (Figure 1 2- 7J. The aCTO is connected to the oNTO by a tlex.ible linker. Thus, although the aNTO is em~ bedded in the body of the enzyme, the aCTD can reach the upstream element and can do so even when that element is not located immediately adjacent to the - 35 region, but instead is located further upstream. The u subt,mit is posjtjoned within the holoenzyme stn.,1cture in such a wny as to muke feasible the recognition of various promoter elements. Thus, the DNA~binding regions point away from the body of the enzyme rather than being embedded. Moreover, the spacing between those regions is con sistent with the distance between the DNA elements they recognize. Th us, u region s 2 and 4 are separated by about 75 A when u is bound in the holoenzyme; and this is about the same distance as that between the centers of the - 10 and -35 elements of a t:ypical v-70 promoter {see F igure 12-5). This .rather Iatge spacing of the protein domains is accommodated by the region between u regions 2 and 4 , that is. by region 3-espedaUy region 3.2 (see Figures 12-4 and 12-6}.

Transition ro the Open Complex Involves Structural Changes in RNA Polymerase and in the Promoter DNA The initial binding of RNA polymerase to the pro moter DNA in thfl closed complex leaves rhe DNA in double-stranded form. The next stage in initiation requires the enzym e to become more intimately engaged w ith the promoter. jn the open complex. The transition fr<>m d osed to open complex involves structural changes in the enzyme

jnNTD

aero--

(]

: UP-element

! - 35

I

:

!

I'~

- 1()

~

J

F I(; U R IE 12-7 cr and a SUbunits recruit RNA polyJner.He cote enzyme to ttle promotel. The C-terrmnal doma1n of the a subunit (aCID) recogniles the lJP-ekrnent (where presertt). \Mlile u regions 2 and 4 rfnlgrnze the 10 ood -35 regtons respectiVely (see r~e 11 ~) In this figure, RNA fl0¥nerase LS shown 111 a rather dif4erent sc:hematic; form ~ presenred _.. e.vlier fimJres. 1t«s form IS particularly tJSPJul for it1diC8ling st.rlaces that touch DNA and regulating proteins and we LISe it ag<Jirl tn son1e figtXes 111 Chapter l6 oMlf:!l1 WE a:Jf'IS«:ler regulation ot tJanscriptiOf'l in bacterta

")

The Transcriplicn Cycle in 8oc:terio

357

and the opening of the DNA double helix to reveal the template and nontemplate strands. This "melting" occurs between positions - 11 and + 3 , in relation to the trans<::ription start site. In the case of the bacterial enzyme bearing if", this transition, often called isomerization, does not require enexgy derived from ATI hydrolysis, and iS instead the result of a spontaneous conformational ch1lllge jn the DNA-enzyme complex to a more energetically favorable fonn. Isomerizati on is esSf)fltiaUy irreversible and, once complete. typically guarantees that transcription will .subsequently iuiliate (though regulation can still be imposed after this point in some cases). Formation of the closed complex, in contrast, js readily re\letsible; polymerase can as easily dissociate from the promoter as make the transition to the open complexTo picture the structural changes that accompany isomerization, we need to eXilmine the structure of holoenzyme in more detaiL A channel runs between the pinrers of the claw-shap ed enzyme, as we described earlier (see Figure 12-2). The active site of the enzyme. which is made up of regions from both the 13 and P' subunits, i s found at the base of the p incers within the ..active center r:left." There are five cha nnels Into the enzyme. as shown in the picture of the open corople:x in FigUJe 12-8. The NTP-upta ke channel allows ribonucleotides to enter the active center (see Figure 12-8 caption) . The RNA-exit channel a llows the growing RNA chain to leave the enzyme as it is syotl1esized during elongation - The remaining three channels allow DNA Elntry and exit from the enzyme, as follows. The downstreaiil. DNA (that is, DNA ahead of the enzyme, yet to be transcribed) enters the active centec cleft in double-stranded form tbJ'ough the downstream DNA channel (between the pincers). Within the active center clP.ft, the DNA strands separate from position + 3. The nontemplate strand exits the active center d eft through the non template-strand (N'f) channel and travels across the surface of the enzyme. The template strand, in contrast, follows a [.tath through the active center cleft and exits through the template-strand (T) channel. ihe d,ouble helix re·fonns at - 11 in the upstream ONJ\ behind the enzyme.

12-8 Channels into and out of the open~ ihiS tigure shoNs the relative positions of the ONA strands (tempate strand in £faY, nontemplate strand 1n orange); the four regrons of a, the - 10 and - 35 regw;>ns of the promoter and the start Sl!e of trdllscriptiOn ( + I). U'le channels ttvough \';fich DNA and RNA eflter or leave !he RNA potymEfase enzyme lYe also stloMl The orty chanroel not shcMf1 here as 1he nudwttde entJy channet through ~h nudeotides enter the act-.ve site dett for lf'1CO!pOrat1on ln1o the RNA chairl as it as made. As dra\Nn, thilt channel WOllld eoter the active site down into the page at dboot the posr bon stlown as "+ I" on tJ'le DNA. Where a DNA str.md passes llfldemeath a protein, il is dr<Mfl as a dotted ribbon. S1gma rcgson3.2 IS the ~nks regaon between f,T1 1 .and lT.o. fIGURE

RNA en

Two striking structural cllanges are seen in the en zyme upon isomerization fron1 tlw clo:;tJd to open com,,Jex. First. the pincers at the front of the enzyme clamp down tightly on the downstt'earn DNA. Secund, there is a major shift in the pusition of the N-tenninal region of cr (region 1.1) as we now describe. When n ot bound to DNA . a region 1 .1 lies tvithio the active center cleft of the holoenzyme, blocking tlae path that, in the open complex, is followed by the template DNA strand. In the open complex, region 1.1 shifts some 50 A and is now foun d on the outside of the enzynu~ . allo"ving the DNA access to the cleft (see Figure 12-B). Region 1.1 of (J is highly negatively charged (just like DNA). Thus, in the holoenzyme, regivn 1.1 acts as a molecular mimic of DNA. The space in the active center cleft, which may be occupied either by region 1.1 or by DNA. is highly p ositively c.hargoo.

Transcription Is Initiated by RNA Polymer-a~ without the Need for a Primer Recall from Chapter B that DNA polymerase does not synthesize new UNA strands de novo- that is. it can only extend an existing polynucleotide chwn. For this reason. replication always requ ires a primer strand. The primer is typically a shol't piece of RNA that binds to the DNA template sb'and to form a shorl hybrid double-stranded region: DNA polymerase then adds nucleotides to tho 3' end of the primer. RNA r oJym erase can initiate a new RNA chain on a DNA template and thus does nol need a prime£. This impressive feat requires ttlat the initiating .ribonudootidt: btl brought into the active site and held stably on the temp late while the next NTP is presented with correct geometry for the chemistry of polymerization to occtu. This is particularly d;ffi. cult because RNA polymerase starts most transcripts with an A. and tha t ribonucleotide binds the template nucleotide (T) with only two hydrogen bonds (as opposed to the three between C and G). Thus, the t~Dzyme has to make spedfic interdctions with t h~:~ initiating ribonucleotide, holding it rigidly in the correct orientation to allow chemical attack on the incoming NTP. The requirement for such specific interactions between the enzyme and the initiating nucleotide probably e)(ptains why most transcripts start with the same nucleotide. T he interactions are specific fur that n ucleotide (on A). and thus only chains beginning w ith A are held in a manner suitable for efficient initi~ atioo. n is beHoved that the interactions are p.rovid()d by various parts of polymerase holoenzyme, including part of sigma. Consistent with this. in experiments using an RNA polymerase containing a c?O derivative tackiog thjs part of sigma, initiation requires much highAr tha.1 norm
RNA Polymerase Synthesizes Several Short RNAs before Enterjag the Elongation Pbase Once rihonudcotides ent~:~r the active renter deft and RNA syntht~sis begins, there follows a period called abortive initiation. ln this phase. the enzyme synthesizes short RNA molecul PS of loss than ten nucleotides in length. Instead of being elongated further, these transcripts are released from the polymerase, and the enzyme, without disassociating .from the template, begins RNA s}·nthesis aga in. Oncp, a polyruftfas~ manages to make an RNA longer than 10 bp, a stable

lP.rnary r.omplex is formed-that is, a t:omplex containing the en~

zyme. the DNA templatt:, and a growing R.~A chain. This lS the start of the elongation phase, which continues until polymem:se ls instructed to tem•;nate transcription by specific sequences downstream of the gene. It is not clear why RNA poJyme.rnse undergoes t.his period of abortiwt initiation, but once again a region of the o factor appears to be involved, acting as a molecular mimic. In t his case it is region 3.2. and it mimics R.~A. This .t'f)gion uf rr lies in the middle of the RNA exit channol in the open complex (see Figure 1 2.~8) , and for an RNA chain to be made longer than about ten nucleotides, this region of u must be ejected from that location. a process that con take the enzyme several nttempts. The elec.:tion of u region 3.2 probably accounts for a being more weakly associnted with the elongating enzyme than it is with the opell complex; indeed it is often lost altogether from the elongating con1plex. In Box 12-2, The Single-Subunit RNA Polymerases, we see how these simple RNA polymerases. duspite lacking a u subunit, undergo a ,.o·udurally comparable shift in trnnsition fmm the initiatlng tu the elongating complex.

The Elongatjng Polymerase Is a Processive Machine that Synthesizes and Proofreads RNA DNA passes through lne e longating enzyme in a mrumer very s imilar to its passage through the open complex. Thus, double-stranded DNA mters the front of the enzyme betwoon the pinl:ers. At the OJJOning of the catalytic: d eft. th€> strands separate to folJow different paths through the enzyme heforo exiting via their respective channels and roforming a double helix llebind the e longating polymerase. Ribonucleotides enter the a(.tive site through their defined ch annel and are added to the growing RNA chain under the guidance of the tem{Jlate DNA strand. Only eight or nine nudeotidcs of the growing RNA chain remain bas&pa.ired to the DNA template at any given time: the re.m ainder of the RNA c ha;n is peeled otT and directed out of the t~nzyme through the RNA exit channeL ln addition to all this, RNA polyn'IE!IOSe c.arrics out two proofreading functions as well. The first of these is called pyrophospbotol}•lic editing. In this. the enzyme uses its active site, in a simple back-reaction , to catalyze thf' removal of an incon-ectly inserted ribonucleotide, by reincm~ rotion ofPPi. The enzyme am then iru;orporate dJlothe~: ribonudeotid.e in its place in the growing RNA chain. Note that the enzyme can remove either mrrert or inrorrect basP.s in this manner. but spends longer hovering over mismatches than matches, and so removes the former more frequently. In the soc.ond proofreading ruecllanism. called hydrolytic editffis, the polymerase backtracks by one or more nucleotides ax1d deaves the RNA product, removing the error-containing sequence. Hydrolytic editing is stimulated by Cre factors, which, as well as enhancing hydrolytic f!diting func;tion. a lso serve as elongation stimulating factors. That is, they ensure that pulymerase elougattlS ef.fic..:ieotly and heJps overeo me "nrresf' at sequences that are difficult to t.ronscribe. This co.rubination of functions is comparable to those imposed on the eukaryotic RNA polymerase II by the transcription factor TFllS (see belowj. Another group of proteins- the Nus proteins- joins polymerase in the elongation phase and promotes. in still rather undel1ned ways, the process~s of elongation and termination (see also Chapter 16 for examples of regulation dlU'ing elongation).

Box 12-2 The Single-SUbunit RNA Polymerases

In the 1ext

\life

discuss the rooltt-sobunit RNA polymer~

found an bacteria and etA<aryolic eels. But there are several examples of single-sul>t..Jlit RNA potvmffilSes that are cap.?ble d performing the same basic reaction as their more complex multiceltufar munterpatts. Thus, many bdcteriophage- fcr example, the E. coli phage T7 - encode polymerases of this type with which, upon infection, they transcribe most of their genes. Similarly, the majority of mitoc:horldriai and dboplast genes are lri:Vlsaibed by polymerases dosefy related to the sirr gle-subt..rlit phage enzymes. " is remac~ that €\IO{ution has produced these ~ sim~ enZ')'lreS capable of cartyi"lg out transcription a task that we, ir1 ~ text ~size as an inlpressive achievement even for the mudl larger and more complicated multt-subunit en.zyrnes. The T 7 polymerase is the most widEly studied of the single-subunit enzymes. tt has a moleo.Jtar weight of 1001<0 - compared to 40t:i0 for the bacterial COle enzyme

(Without cr factor)-and a structure shown in Box 12-2 Figure 1. OveraH it looks tike the Pot I fatrily of DNA pofymerases that we considered in Olapter B. Thus. the T7 RNA polymerase resembles a right hand, with the fingers, thumb, and palm representing d()(T)ains arranged around a central deft, within which lies the active site. Although it more dosety resei'TINes DNA polymerase, the T7 enzyme does have features in common with the a:lluJar

RNA polvmerases as wel~ features that have become (Tl()re apparent since the structure of the T7 and bacterial enzymes have been compared in mmple< wllh ttletr templates. 1\S we saw i1 the text the bacterial enzyme has various dlannels intn

and out of the active center deft (see F'tgure Jl-8). One of these, for example, allow.; the NTPs access to the active site and template, where they are polymerized, under the influence of the template, intD the grovving RNA chain Another chameJ provides the ~ RNA chatn an exit from the enzyme. Comparable ci-w3nnels are seen 11i tht= structure of the~~aswell.

The ir-Vtiation and elongation complexes of the bacterial and T7 polymeroses have been COCllJared These compa;sons highlight ooe stnking example of bovv a comparable fuoctional transition can be ad"lieved through different kinds of structurat change in the two cases We noted In the text that, in the bacterial case, the transition from initiation to

e1ongation involves a signifacant shift in the ~i0l1 Qf a domain of the a factor. This movement opens up the RNA exit channel, thereby allo\.ving production of tJansrnpiS larger than 10 nudeotides io 1ength The T7 enzyme has no a factor; but a -romparebte structural diange i1 the body of that silgle-subunit enzyme mediates the transition from tri€ inittating to elongating com~ex. and this structure! change is reqUired to form the RNA extl channeJ.

8 OX J:Z-2 FI C U R E 1 Baderiophaze T7 RHA polymeqse_ (Jeruzalrru 0 . and Steitz TA

1998. EM80 J 17: 4 Wl .) Image prepared wtth Mo!S
-- - - - - - - - -

The 1ivnscripfi<Jn Cycle in BoderiCI

Transcription Is Terminated by Signals

widtin the RNA Sequence Sequenc.es called terminators trigger the elongating polymerase to dissociate from the D NA and release the RNA chain it has made. In bacteria, terminators come in two types: Rho-independent and Rho-dependent The first kind causes polymerase to terminate without the involvement of other factors. The second kind, ns its name suggests, requires an additional protein called. RJto to induce termination. We will deal with each kind of terminator in turn. Rho-indepen dent tetm in~ttol'S. also CAlled intrinsic terminators. ronsist of t wo sequence elements: a short inverted rep eat (of about 20 n ucleotides) followe.d by a stretch of about eight A:T base pairs (Figure 12-9). These elements do not affect the poJyrnerase until after they have been transcribed - that is, they function in the RNA rather than in the DNA. Thus, when polymerase transcribes a n inverted repeat sequence, the rosulling RNA can fonn a stem-loop strudure (f;lften called a "hairpin") by base-pairing with itself (see Chapter- 6}. The hairpin is believed to cause termination by disrupting the elongation complex. This is achi~ved either by forcing open the RNA exit channe l in RNA polymerase, or. BlJcording to another model. by disrupting RNA-template interactions. The hairpin only works as an efficjeni terminator ·when it is foJ iowed by a stretch of A:U base pairs. as we have described. This is because. under those circumstances, at the time the hairpin forms. the growing RNA chain will be held on the template at t he active site by only A:U base pairs. As A:U base pairs are the weakest of e~ U base pairs (weaker even than A:T base pairs), they are more easily disrupted. by tl1e effects of the stem loop on the trdJlscribing polymerase. am] so the RNA will more readily dissociate (Figure 12-10). Rho-rlependent terminators have less well-charactetized RNA elements, as we shatl discuss below. and for them to work requires the action of the Rho factor as well. Rho, w hich is a ring-shaped protein with six identical subunits, binds to single-stranded RNA as jt exits the polymerase (Figure 12-11). The protein also has an ATPase activity: onre attached to the transcript, it uses the energy derived from ATP hydrolysis to wrest the Ri\lA from the template and from polymerase. dyad

ONA ~A

5'

,. symmefly • ~--~~-----.. .CCC .rtGC C C G i!C 'l'l'.ATO AGC OCJO C n T T'l"TT"l'GAACAAAAJ

3'

~ ~O if~ C GGGCGG A'l''ZA C 'l:C GCCCG"''AAAAAAACT'T G TTTT

FIC>URE 12-9 Sequence of • rho-independent tefmiMtor. AJ the top IS rlte sequeore. IIi me OW\ d the tEminatot. Below is stlQWn the sequence of thEe' RNA. and at the bottom the StrUcture of the t~ator ha•rpin. Thetemunator in questiOn IS from the tq> attenuator. diswssed 10 c.haprer 16, me boxes .show mUlalions i~ted In the sequence that disrupt the terminatot. (Sotwce: Adapted from Yanofsky C 198 L Nalllre 2B9: 75 1 758. ftg 1. Copyflght <e1 1981 Nature PUblishing Group. Used W1th permission.)

Mechonzsms of TronEcrzpfion

F I G U R E 12-1 0 1'hln:KJiptiot1 ~ Shovv'n.s a model for how the rho-independent terTnNlOr ~t \1\011<. (a) The hairpn forms ir1 the RNA (Figue 12-9) as sooo as that regJOO has been transoibed by fXJ¥nerase (the enzyme IS rot shown here). (b) That RNA stnrlu"e disrupts polymerase J1..!St clS the enzyme is lrarf;C~ 1he AT nd1 strE:tch d ONA dc:Mnstrecrn (c) fne commatiOfl of the haHpn structl.Jre and the weak irlaac.tions between the str~ oi Us in the RNA and As in the terr"fllate OJf"lsptre to pl..S the !Tansatpt from the t~ terrr~~ fl..lthea- elongcst;or (Source. Adapted kom Plat1 T. 1981. Cel24: 10- 23. Copyright ~ 1981, wnh ~ from Elsevief.)

a

b

c

Fl G U R IE 12-11 The p transaiption termination factor. The aystal stl\lcture of the rho tellT'IT1atlOn factor is shown "' a top dom1 \/lew. Jt consiStS of a hexamer of rt-c prorein. ead1 monomer hete lfl a dfferent ccb. The six rnoctOmers foon Clfl opm ring. The ring IS not flat- the six1h subunit es further dov-.n ., the plane of lhe pc~ge than the fvst The gap between the two Slh.nts IS 12 A. dl1d the heMcal prtd1 be1ween them is 45 A. The RNA transcript on which rho aru (not shown) 1s beleved to br.d along the bottom of each ~ and then threz,d tfvOl.gh the md:Je of the mg. (SkOI"dalal<es E- and Befger J.M 2003. CeJI 114: 135.) Image prepared With MoiSaipt, BobScfl>l,

stlO\-."

and~er30 .

!

How is Rho directed to a particular RNA molecule? First, there is some specifkity in the sites it biTld~ (the 50-CC'Illed rut sites. for Rho u tilization). Optimally these sites cunsist o f stretches of about 40 nucleotides thai do n ot foJd into a secondary structure (that is. they remain largely single-slJ'andedj; they are also rich inC residues. The second level of sp ecificity is that Rho faits to binrl any transcript that is being translated (that is. a transcript bound by ribosomes). ln bacteria. lransc.ription ancl lrnnslation are tightJy coupled-translalion initiates on growing RNA transcripts as soon fiS they start exiling polymerase, while they are still being synthesized. Thus, Rho typicoUy teiTilinales only those lt-dllscripts still being transcribed beyoJJd the end of a gene or operon.

TRANSCRIPTION IN EUKARYOTES As we have already discussed, transcription iJ1 eukaryol.e s is undertaken by polymerases closely related to RNA poJymerases found in bacteria. This is hardly surpri.c;ing: the process of tmnscription itself is identica l in the two cases. There are, however. diffCJ-ences in the machinery used in each case. One we have already seen: eukat yoles h ave three different polymerases (Pol I. fl, and ill). whereas bacteria have only one. Also, whereas bacteria require only o ne additional initiation factor (a), several initiation factors are required for efficient anrl promoter·speci.fic initiation in eukaryotes. These are called the general transcription ractors (GTFs). In vitro , tho general transcription fuclors are all that is required, togEther with Pol n, tu injliate tr.=mscription on a DNA template. ln vivu, however, the DNA templato in eukruyotic cells is incorporated into nucleosomes. ss we saw in Chapter 7. Under these circumstanres, the general transcription factors are not sufficient to promote significant e>.pression. Rather, aclditionaJ factors a_re n~quired , including the so-called Mediato.r Complex, D~A-bi.nding regulatory prote.i ns, and, often, c:hrmnatin-modifying enzymes. We w iU first oonsider the basic mechanism by which Pol n nnd the geneml transcription factors assemble a t a promotel' to initiate transcription in vitro. We thnn consider the roles of the additional components required to promote tTansc.ription in vivo.

RNA Polymerase 11 Core Promoters Are Made up of Combinations of Four Different Sequence Elements The eukaryotic core promoter refers to the minimal set o£ sequence elements re<.]uired for accurate transcription initiation by the Po) [I machinery, as measured in vitro. A core promoter is typir:ally about 40 nudeotides long. extending either upstream or downstream of the transcription start site. Figure 12-12 shows the locution, relative to the transcription start site, of four elements found in Pol TI core promoters. These arc the TFIIB recognition element (BRE), lhe TATA element (or box}. the Initiator Ond and the downstream promoter element (DPE). Typically. a prornoter includes only two ur three of these four P.lements. The consensu s sequence for each element, and the general transcription factor that binds it, are also shown, and we shaH describe these features in more cl~tail in conung sections.

Mechcmu;ms pf 'lhm scripticn

TFNO -+28

+32

F ICURE 12-12 Pol II c.ore prontoler.. The figure shows the po54ttOOS of vaMus DNAelcmer'lls relative to the transcription start Stte (!Odicated by the arrow above the DNA) These elements, described I'\ the text, are as follom: BRE (TFU!l recogr4jofi deme'lt); TAlA (lATA Box). lm (~nibator element}, and OPE (Qownstream llfomoter ~ement). Also shown (below) dl"e the (OOSei'\SUS secp.mce for ~ element (determined 111 the same way as dcsalt>ed for the bacterial promoter elements. see aox 12 I): «ld (above) ttle name of the gene~al tfilr\Scr1pllon faclor that rerognaes-each element (Soi.Kce: Butler J.E.f. et al. 2002 CMeS and Development 16; 2563-2592. Fig 1.)

Beyond-and typically upstream of- the core promoter, there are other sequence elements required for efficient transcription in vivo. Together these elements c:onslitute the regulatory sequences and can be grouped into various categories, reflecting their location, and the organism in guestion. as much as their function. These elements include! promoter proximal elements; upstream activator sequences {UASs); enhanc-ers; and a series of repressing elements called silencers, boundary elemen~s. and insulators. All these DNA elements bind regu· latory proteins (nclivatms and repressors). which help or hinder transcription from the core promoter, the sub]oot of Chapter 17. Some oJ these regulatury sequences can be located many 10s or even 100s of Kb from the core prnmoters on which they act

RNA Polyme~:ase II Fonns a Pre~lnitiation Complex with General Transcription Factors at the Promoter The generaJ transcription factors collectively perform lhe functions performed by a in bacterial transcription. despite sh owing no significant sequence l1omology to that protein. Thus, the general transcrip~i on factors help polymerase bind to the promoter and melt the DNA (£:omparable to the transition from closed to open complex in the bacterial case}. They also help polymerase escape from the promoter and embark on the elongation phase. The complete set of general transcription factors and polymerase. bound together at the promoter and poised for initiation, is caHed the pre·initiation complex. As we desr.rlhed above (and in Figure 12-12) many Pol II promoters contain a so-called TATA cleme11l (some 30 base pairs upstream from the transcription start site). This is where pre-initiation complex fol'mation begins. The TATA element is recognized by the general transcription factor called TFIID. (The nomenclature "TFII'' denotes a transcription factor for Pol!l, w i.th individual factors distinguished as A. B. and so on.) Like many of the general transcription factors, TFliD is in 1acl a multi-subunit complflx. The component of TFUD that binds to the TATA DNA sequence is called TI)P (TATA binding protein). The other subunits in this complex are calle d TAt's, for TBP assoc.iaterl factors. Some TAFs help bind the DNA at certain promot~ ers, and others control the DNA-binding activity ofTBP. Upon binding DNA, 'fBP extensively distorts the TATA sequence (we shall discuss this event in more detail presently). The resulting TBP- DNA cumplcJC provides a platform to recruit other general

7rorrscription in Eukaryvtes

transcription factors and polymerase itself to the promoter. In vitro, these proteins assemble at the promoter in the following order lFigure 12- 13): TFllA, TFJffi, TFIIF together with polymerase (in complex with yet more proteins, such as those in the Mediator Complex. which we describe below). and then TFllE and TFIJH, wh ich bind upstream of Pol n. Fotmation of the pre-initiation complex containing these components is followed by promoter melting. In contrast to the situation in baderla, promoler melting in eukaryotes requires hydrolysis of ATP end is m ediate d by TF'Jlli. It is the helicasc-like activity of that factor which stimulates unwjnding of promoter DNA. TBP-.__

fl
TFIID

0~------

~:~~-}

shovvn here. and described il deta~ in the text TFUB

Once assembled ~ the pt"omoter. Pol II leaves the pre-~~iticrtioo complex ~ addition of the nudeotide precursors reQUired for RNA synthesis, and after phosphorylation of Ser Jestdes withm the enzyme•s "'tail" The tal contains ITilllbpfe repeats of the heptapeplide ~: Tyr·Ser·Pro-lhr·Ser·Pro-Sef (see figure 12-18).

36ti

MecbaniBill$ of Transcl'ipricn

jusl as we saw in tho bacterial case, there now follows a period of abortive initiation before the polymerase esc.apes the promoter and enters the e longation phase. Recall that, duri ng abortive initiation. the polymerase synthesi zes a series of short t.ranscrlpts. [n eukaryotes~ promoter escape involves a step not seen in t.he bacterial case, t hat of phosphorylation of lhe polymerase as we nm.v describe. The large subunit of Pol U has a C-terminal domain (LID), which extends as a "tail.. (see Figure 12-13). The CTD contains a series of repeats of the heptapeptide seque nce: Tyr-Sel'-Pro-Thr-Ser-Pro-Ser. There are 27 of these repeats in the yeast Pol 11 ern and 52 in the human case. Each repeat contains sites for phosphorylation by spe· cific kinases including one thai is a subunit of TFUH. T he form of Pol U recruited to the promoter iuilial1y contains a ]atgely unphusphorylated tail, but the species found in the elongatiun compJe-.x bears multiple phosphoryl groups on its tail. Addition of these p hosphates helps polymerase shed most of the genernl transcription factors used for initialion, and which the enzyme leaves behind as it escapes the prmnot£n: As WP. will SM, regulating the phosphoryla tion state of the CID of Pol 0 controls later steps- those involving processing of the RNAas well. Indeed, jn addition to TFlrH, a number of other kinases have been idenlified that act on the ern .as well as a phosphatase that :removes the phosphates added by those kinases.

TBP Binds to and Distorts DNA Using a ln~rted into the Minor Groove

~

Sheet

TBP uses an ex'tensive region of B sheet to recognize the minor groove of the TATA element (Figu re 12-14). T his is unusual: more typically, proteins recognize DNA using a he1ices inserted into the major groove

FIGURE 12-14 1UP-DNAcomp1e._ The TAlA binding protem {TBP) IS sflo.vn hefe in purple~ INith the. DNA lATA sequenre (shown 1r1 gray) found at the stdn of many Pd II &enes- The details thts 1111eractlon are ~ scribed Jn the text ( Nikolov 0 .6., Olen H. Halay

or

P..E.. Usheva A.A., Hisatake K, Lee O.K. Roedef RG.. and Budey S.K 1995. 1\'oture 3n: 119.) Image prepal'ed wtth Mol5cnpt BobSc.npt, and Raste' 3D. Extended DI\JA on eiltler side or im-

age modcJed by l.eemor Joshua Tcr.

Transcriplicm i n Euknrytlles

367

of DNA. as we saw in Chapters 5 and 6. and a lso for u f~c.tor earlier in this chapter. The reason for TBP·s unorthodox a·e cognilion mechanjsm is linked to the need for that protein to distort the local DNA structure. But tbis mode of recognition raises a problem: how is specificity achieved? WEl have seen in Chapter 6 that. compared to the major groove. the minor groove of DNA is less rich in the chemical information that would enable bnstt pairs to i)P. clislinguishHd. Instead, to select th e TATA soquance, TBP relies on the ability of that sc;,quence t u undergo a s~ific structural distortion, as we now describe. Whan il binds DNA, TBP causes the minor groove to be w idened to a n almost tlat conformation; it also bends the DNA by <m angle of approximately 80Q. The interaction between TBP and DNA involves only a l imited number of hydrogen bonds between the protein and the edges of the base pairs in the minor groove. lnstend. much of the specificity is imp osed by two pairs of phenylalanine side chains that interr.alate between tbe base ~j rs a l either end of th~ recognition sequence and drive the strong bend in the DNA. Thus, A:T b:1se pajrs arn hworP.d because tbey are more readily distorted to allow the initial opening of the minvr groove. There are also e:xt<~nsi ve i ntt~ractions bt:~tween 1he phosph~;te backbone and basic residues in the p sheet, adding to the ove rall binding energy of the interaction.

The Other General Transcription Factors also Have Specific Roles in Initiation We do not know in detaiJ the functions of all the other general transcription fnctors. As we have noted. some of these !actors are ju fuct complexe.s made u p of two or more subunits (shown in Tablfl 12~2). Below we c:onunent on a few st-ructur--dl and functional characteristics.

TAFs. TBP is associRted w ith about ten TAFs. Two of the TAFs bind DNA elements al the promoter; for example. lhe initiator element Onrl and tbe downstream promo•er element (DPE) (see Figure 12-12). Several of the TAFs have structurnl homology to histone p roteins. And it has been proposed that they might bind ONA in a sim1lar manner. although evidence for such a fonn of DNA binding has not been obtained. For example. TAF'4 2 and TAF62 from Drosophila have bef:n shown to form a structure similar to that of the H3• H4 tetramet (see Chapter 7 ). T hese histone-like TAFs are fou nd not only in the TFIJD complex but are also associated with some histone modjficatiou enzymes. sucb as the yeast SAGA complex (see Table 7-7). Another TAF appears to regulate the binding of T lW to DNA. U does this using an inhibitory flap that binds to the DNA~binding surface of TBP-another example of molecular mimicry. This flap must be displaced for TBP to bind TATA.. TFIIB. This protein. a s ingle polypeptjrlt! r;hain, enters the preinitiation complttxafterTBP (Figure 12-13). The cl'ystal structure of the ternary cumplex of TFilB - TBP - DNA shows spe<;-jfic TFIIB-TBP and TFliB- DNA contacts (Figure 12-15). These include base-specific interactions with the major gwove upstream (to the BRE- see Figure ·12~ 1 2) and the minor groove downstream, of lhe TATA element. The asymmetric binding of TFIIB to the TBP- TATA complex accounts for the

TA 8l E

12-2 the CA!neral Ttansa'iption fadors ofRNA Pol)merase II Number of

GTF&

Stlbunits

lBP TfiiA TFIIB

TfiiE TfiiF TftiH TArs

--1 2

1

2 3 9 tI

~ 11UT1bets shown arC11or Vt!flst but are SlJI"lltal for

other eukeryo!es. ~ tunaos

Mf'Chanisms cf Transcription

FIGURE

12-JS mte-TBP-promoter

comp\ex. This strt.JClUfe shows the TBP protein bound lO the lATA seqtJen(.e, just as we ~in the pllMOUS fi~ Here, the genm.t tRinSC11P(ioo factor lftiB (shO\Ivfl in turqUOISe) has bem added lhts 1ripar1ile complex forms

the platform to 1IVhtch other~ transmpllon tactors. and Pol U·~.ac-e recTl.lited ckAing ~itiation rornptex assentJ(y. (Nikclov 0.8., Cf'en H.. Halay E.D., Usheva AA. Hisatalce K.. Lee O.K.. RQCder RG.•
leemor Joshua-Tor.

asymmetry in the rest of the assembly of the pre·initiation complex and tbe unidirectional transcription that results. TFIJB also c ontacts Pol IJ in the pre-initiation complex. Thus, this protein appears to bridge the TATA-b01md TBP and pol}'·merase. Recent struch1ra l studies suggest. that the N-terminal domain of TF11B inserts into the RNA exit r.hannel of Po) IT in a manner ana logous to lT32 in the bacterial case. TFilF. This two-subunit factor associates with PoJ D and is recruited to the promoter together wi1h that enzyme {and other wctors}. Binding of

Pol fi- TFllF stabilizes the ONA-TBP- Tflm complex and is required before TFUE and TF1JH are recruited to the pre-initiation complex {Fig· ure 12.-13).

TFIIE and TFllH. TFIIE. which. like TFIIF. consists of two subunits, binds nex1. and has roles in the recruitment and regulation of TFlJJ-1. TFrtH QOntrols the ATP-dependent transition of the pre-initiation complex to the open complex. It is also the largest and most cumpfex of the genera] transcription factors-it bas nine s ubunits and a molecular mass comparable to that of the polymerase itself! Within TFI!H are two subunits that Function as ATPases. and another 1hat is a protein kinase. with mles in promoter melting and 8SC':ape. as described above. Togethor with o ther fActors. the ATPase subunits are also involved in nucleotide mismatch repair (see Chapter 9).

In Vivo, Ttan..~riprion lnt.tiation Requires Additional Proteins, Including the Mediator Complex Thus far we have described what is net'ded for Pol TI to initiate transcription from a naked DNA template in vilro. But we have already noted that higb. regulated levels of transcription in vivo require. additionally. the Mediator Complex, transcriptional regulatory proteins, and. in many cases, nudeosorne-mooifying enzymes (which are themselves often parts of large protejn complexes) (Figu:re 12-16). The charocteristiL"S of various modifying complexes are ghtP.n in Table 7.7. One reason for these additional requirements is that the DNA ternplate in vivo is packaged into nucleosomes and chromatin. as we discussed in Chapter 'J. This condition complicates binding t o the promoter of polymerase and its associsted fuctors. Transcriptional regulatory

Trcnscriplion in Eul..aryotes

~9

12-16 Assembl)'oftbe pre-initiation complex in Pfeset\Ce of Mediatllf. nudeo$ome rnocifiet.s and rernodeien. ;and tAnscriplional ac:tivalors. In addlti:ln to the general tlilnSC'riptim facklrs stx:Mnin Figure l2cl3, ~ill activators boond to sites near the geoe recruit rodeosomes rTlCXifyi.'1g andre~ ~es. c"!nd the W..ediatct ColllJiex. wl11Ch togethe1 hefp form the pre-KIItiatioo complex. FIGURE

proteins ca1led activators help recruit polymerase to the promoter. stabilizing its binding there. This recruitment is mOO.iated through interactions between DNA-bound activators and parts of the transcription machinery. Often the interaction is with the. Mediator Complex (hence its name). Mooiator is associated with the CfD ,.tail .. of the large polyrnetase subonit through one surfar:e. while presenting other sutfaces for interaction wjth DNA-bound activators. This explains the need for Mediator to achieve significant transcription i.n vivo. Despite tl1is central role in transcriptional activation, deletion of indiv)dual subunits of Mediator often leads to loss of expression of only a smaU subset of genes. different for each subunit (it is made up of many subunits). This result likely reflects the fact that dift~nent activators are believed to interact with different Meruator submits to bring polymerase to different genes. In addition, Mediator aids initiation by regulating the ern kinase in TFilll. The need for nucleosome modifiers BIJd remodellers also differs at difft'.rent promoters or even at the same promoter under different circumstances. Wben and w here required, these complexes are also recruited b y the DNA-bound activators. \f\Te will discuss the role of Mediator and modi.fier.s in stimulating transcription i1l Cbapter 17. We now consider some of the structural and functional properties o( Mediator. yeast mediator

Mediator Consists of Many Subunits, Some Conserved from Yeast to Human As shown in Figure 12-17, the yeast and human Mediator each include more than 20 subunits. of which 7 show significant sequence homology between the two organisms. (The names of the subtmits are differP.nl in each c-.ase. reflecting the experimental approaches that led to their identification.) Very few of these subunits have any identified function. Only one. (Srb4), is e~ntial for transcription of essentially all Pol IT genes jn vivo. Low-resolution structural comparisons suggest both Mediators have a simHar shape, and bolh are very large-even bigger than RNA polymerase itself. The Mediator from both yeast and humans is organized in modules. These modules can be dissociated from one another under certain conditions in vitro. This observation, together witb the fact that human Mediator varies in its composition (and size) depending on how it is isolated, has led to the idea that there are ' 'arious fonns of Mediator (partir:ularly in metazoa1ts), each containing subsets of Mediator subunits. Furthermore, it has been argued that the different forms are involved in regulating different subsets of genes, or responding to

human mediator

fIG U R £ 12-17 eomp.ri.son oHhe Yea5t

-"'Human Methtors.

The~

~are shown in dark~ (Source Modified v.ith parniSSI(I(l from M6k s_ and Roeder R G. 2000 Tr<:llSCTJ>Iional regulation the~ mediator-ike coaaiVatofS tn yeast a"ld metazoan eels.. T;ends Btochem. Sa. 25~ 271-283. Copyngtrt @ 2000. w«h pemliSSlOfl from Else\llef.)

Mechanismt~ of'Ihm:;cripticm

370

different groups of regulators (activators and repressors). It is ec.,uatly possible, however, tbat the variations seen in subunit composition are artifacts, simply tdlecting different methods ofisolation. In some stuclies il has been shm.vn that a complex consisting of Pol U, Mediator, nnd some of the general hanscription factors can be isolated from cells as a single complex in the absence of DNA. Th is led to the speculation that ~he bt..dk of the proteins ret~uired to initiate trdnscriplioil might arrive at the promoter in a single preformed complex. rathe£ than in a stepwise manner. The putative preformed complex was named the R.'IA Pol TI holoenzyme, after the bacterial enzyme containing the u factor, and thus able to initiate. Despite this parallel in naming. there are essential factors (su<'.h as TFIID) that do not associate with the eukaryotic RNA polymerase. Jt is unclear whether the holoenzyme exists in signifirnnt amounts in vivo, compared to separate poly· merase and Mediator Complex.

A New Set of Factor's Stimulate Pol II Elongation and RNA Proofreading Once polymerase bas initiated transcription. it shifts into the elongahon phase, as we have discussed. This trnnsition involves the Pol I1 enzyme shedding most of its initiation factors-for example. the general transcription factors and Mediator. Cn their place another set of factors is recruited. Some of these (such as TFlJS and hSPTS) are elongation foctot"S-that is, fuctors that stimulate elongation. Others are required for RNA processing. The enzymes involved in all theoo processes ere. li.1<.e several of the initiation factors we have discussed. recruited to the C-terminaJ taU of the large subunit of Pol U, the CTD (Figure 12-18). In

l

1.

~Q

enzyme

POiyadenytatiOI'l and cleavage

fat::t
b N

y

1

s

2

p

3

T 4

s

5

p 6

'f- c s I 7

-

-

fI GU R E 12-18 RNA processing enzyllle5 •e recRiited by the hi1 of polymemse. The 1Dp part of d•e iigu'e shows vanous ~ itwclved 111 RNA pocessing Jem.~ted by the "tail" or polyme.ase.

Differ61l enzymes are recrukrl deprocing on lhf: pho~ stale of the tat Those enzymes are lher. lransfmed to the RNA as they are needed (see next sect100 H"' text). The bottom part of1Me flg\Jfe ilfustrates a scherncn: of the ta.l With lhe se:JU€OCf! of ooe CllJY of lt1e heptapeptide repeat c;hown. lhe positions of setine residues that get ~ated <".re tndieoled ~n of serirleat po5ibon 5 l5 assooated w.lh r~ of cawing factors, W1Ereas ~laoon of seooe at position 2 1$ aSSQOated ~ ~~ of splicing tactOIS.

this case, however. the factors favor the phosphorylated form of tho CID. 1n us phosphorylation of the CTO leads to an exchange of initiation factms fur those factors required for elongation and RNA procf!SSing. As is e vident from the crystal slntcture of yeast Pol U, the polymerase ern lies direct]y adjacent to the chaiUle l through which the newly synthesized RNA exits the enzyme. This. together with its length (it can extend some 800 Afrom the body of the enzyme) a llows the tail to bind several components of the elong;dion and procf'..ssjng machinery and to d eliver them to the emerging RNA. Various proteins are thought to stimulate elongation by Pol ll. 0 11e of these, the kinase P TEFb, Is recruited to polymerase by transcriptional activators. Once bound to Pol D. this protein phosphorylates the sel'iJie residue at position 2 of the CTD repeats as described earlier. That phosphorylation event correlates with elongation. In addition, P-TEFb phosphorylates and thereby act ivates another protein. called bSPTs. itself an elongation factor. Lastly, TAT -SF1, y t;!t anolhef elongation factor. is recruited by P-TEFb. Thus. P- TEFb stimulates ~Jongation in three separate ways. Another factor that does not affect initiation, hu t stimulates elongation, is TFW:i. This factor stim ulates the overall rate of eJongalion by limiting the length of time polymerase pauses when il enoountP.rs sequences that would otherwise Lend to slow the em~yme•s progress. It is a feature oi polymerase that it does not transcribe through all sequenC'.es at a con stant rate. Rather. it pauses perjodically, sometimes for ta lher long period.s, before resuming transcription. In the presence ofTFllS. the length of time polymerase pi\uses at any given site is reduced. TFllS has M other function: it contn'butes to proofreading by polymerase. We saw at the start of the chapter how polymernses are able, inefficiently, to remove misincorporated bases using the active site of tl1e enzyme to perforro the reverse reaction to nucleotide inc-.orporation . In addiUon . TFlJ~ stimulates a n inherent RNAse ac.livity in polymerase (not part of the act ive site), allowing nn alternative approach to remove misint.'Ofporatt.'>£1 bases through foc:al limited RNA degradation. 11tis feature is comparable to the hydrolytic editing we described io the bacterial case stimulated by the Gre factors we discussed there.

Elongating Polymerase Is Associated with a New Set of Protein Factors Requiretl for Various Types of RNA Processing Once transcribed, eukaryotic RNA has to be pl'<X:essed in various ways before bei ng exported from the n ucleus where it can be translated. These processing evenls 1ndude the following: capping of the 5' end of the RNA; splicing; and pofyadenylation of the 3' end of the RNA. The most complic-.ated of these is splicing- the process whert:by n on· coding introus are removed from RNA to generate the mature mRNA. The mechanisms and regulation of that process and others, such as RNA e djting. are the subject of Chapter 1 3. We consider the o ther two processes here. Strikin gly, there is an overlap in proteins involved in elongation, and 1hose requjred for RNA processing. In one cas~ fur example, or1e elongation factor mentioned above (hSPT5) also recruits and stimulates lhe 5' capping enzyme. (n another case. efongatjon factor TAT- Sf1 recruits components of the splidng machinery. Thus it seems lhat elongation, termination of transcription , and RNA processing are interconnectedpresumably to ensure their proper coordination.

372

Mechanisms ofTranscripli<m

The fi rsl RNA processing even' is capping. This involves the addi· tion of a modified guanine base to the 5 ' end of the RNA. SpecificaJly. it is a n1e thylated ~uanine. and it is joined to the RNA lranscri pt by an unusual 5'-5' linkage involving three phosphates (see bottom ol Fig· ure 1 2-19). The 5' cap is created in three enzymatic. steps, as detailed in lhe n gure and legend. Jn the first step. a phosphate group is removed from the 5' of the transcript. Then, the GTP is added. And in the final step, that nucleotide is modified by the adrlition of a methyl group. T he RNA is capped when it is still only some 20- 40 nucleotides long-when the transcription cycle has progressed only to the transition between the initiation and elongation phases. After capping. dephosphorylation of Ser5 within the tail repeats leads to dissociation of the capping machinery, and further phosphorylation (this time of Ser2 \'ltithin the tail repeats) causes recruitment of the tnachi nery n ooded for RNA splidng (see Figure 12-18}. The fhlill RNA processing event. polyadcnylation of the 3' end of the mRNA, is intimately linked with lhe termination of transcription (Figure 12-20}. Just as with capping and splicing. the polymerase C1U tail is jnvolved in recrWting the enzym l?.s necessary for polyadenyl.ation

FICUR£ 12-19 Thestructureand formation of the S' RNA ap. In the first ~the "'fphcrsphate at the 5' end of the RNA is remove:J by an enzyme called RNA

initlatir€ C'1\JCIElotide of tr
triphosphatase (the il

gi.Jai'\VM ll'ar\Stefase catalyzes the nudecphiltc at:lad< of lhe restAting termllial {j-pl1osptlale 011 the cx"{lttospl1ccyl gr~ of a moleo.Ae of GTP, With ~ and )'-phosphates of the GTP seNing as a pyrophosphate 1ea\ling W'OOP· Orce !his laage is made, the new1y ~ 81J1ll"'lne and the puine at the<Jng.inaiS' end of dle rnJs by meth~~ rransterase The restiting 5 ' cap structure late- reaUitS the ribosome to the mRNA for translatioo to begin (see O lapfer 14).

3'HO

1-~-3'

3' HO

1-ttan-.se 3'

-~-

-------

RNA cleavage

------

12-20 Poiyadenylidionand terrination. The vaoous steps en tNs process are desoi:led in the text FIGURE

CstF

5'

poly-A polymerase {PAP)

poly-A-bincfJng p.-ot:OO

5'

additional poly-Abinding protein

5'

-A200

(Figure 12-18). Once polymerdse has reached the end of a gene, it encounterS specific sequences that. after being tTanscriberl into RNA. trigger the transfer of the polyadenylation enzymes to that RNA, leading to throo events: cleavage of the message; addition of many adenine residues to its 5' end; and, subsequently, te.m1ination of transcription by polymt>.rase. This process works as fotluws. Two protein complexes ore carried by the ern of polymerase as H approaches the end of the gene: CPSF (cleavage and polyadcnylation specificity factor) and CstF {cleavage stimulation factor}. The sequences which. once transcribed into RNA, trigger transfer of these factors to the RNA. are called poly-A signals and are shown in Figure 12-20. Once CPSF and CstF are bound to the RNA. other proteins are recru ited as well. leading initially to .HNA cleavage and then polyadenylati<m.

Polyadenylation is ro~lia tP.d hy AA P.neymA call~d poly-A polymetHS4=!, whicl1 adds about 200 adenines to thc RNA"s 3 ' end prodi..I.COO by the cleavage. This eneyme uses ATP as a pl'PCursor aud ~dds the nucleotides using the sarna chflrnistry as RNA polymerase. But it does so without a template. Thus, the long tail of As is found in the RNA but not the DNA. lt is not clear what dctcm1inflS the length of the poly-A tail, but that process involves other protP.ins that bind specifically to the poly·A sequence. Thf'l mature mRNA is tlu•n t:tansported from the nucleus, as we shall discu:ss in Chapter 13. It is noteworthy that the long tail o f As is unique to transcripts made by Pol 11, a feature that allows experimental isolation of protein coding rnRNAs by affinity chromatography. Thus. we see h ow a mature mRNA is rei~ from polymerase once thP. gene has been transcribed. But what lennimttes transcription by polymen:tse? In fact, the enzyme doos not terminate immed iately whe11 the RNA is d cavcd an d polyadenylated. nather. it continues to move along the template. generating a second RNA moleCule that can become as lm1g as several hundred nudeotides before terminating. The poly· merase then dissociates from the template. releasing the new RNA. which is d p,graded without P.Vnr leaving the nociP.us. It is not understood what links polyadcnylation to termination, but it is clear that the polyad enylatlon signal is required for termination (interesting ly, RNA cleavage is not)_ Two basic models have been proposed to explain the link between polyadcnylation and tennination: fust. that the transfer of 3' processing enzymes from the polymerase CfD tail to the RNA triggers a conformational change in the polymerase that reduce; processivity of the tmey:me. )e
RNA Polymerases I and Ill Recognize Distinct Promoters, Using Distinct Set.~ ofTranscription Factors, hut still Require TBP We have already mentioned th at eukaryotes have two other polymerases-Pol J and Pol Ill - in addition tn Pol n. These en zymes are mlatr.d to Po) (( and P.Ven share sev~al subun its (Thble 12-2) , but they initiate transcription from distinct promoters and transcribe distinct genes. These genE'.s encode specialized RNAs. rather than proteins as we discussed earlier in the chapter. Each of these enzymes also works with its own W1ique set of general transcription factors. TBP, however. is universal , because it is involv£.-d i n h1itiating transcription by Pol ( and Pol IU, as well as Pol U. Pol I is required for the expression of only one gene. that encoding the rRNA precursor. Then: are rnany copies of that gene in each cell, dlld indeed it is ~xpressed at far higher levels than any other geneperhaps explain ing why it h as its own dedicated polymerase. The promoter for the rRNA gene oompdses two parts: the oorc elem en t and lhe UCE tupstream control etement) as shown in Figure 12-21. The former is located around the start site o f transcription, the latter betwf"fm 100 and 150 bp upstreum (in h umans). ln uJdit.ion to Pol l.

TronscctpUon In Eukaryotes

a

375

12-21 Poll promoter region. (a) St!OOure of the Poll pronlCllet. (b) Pel I tm f3CTOIS. The case shown here IS the vertebrate F f (; U R E

c=

UCE

- 150

-45

- 100

+20

S'(stem. The set ot proteins invOOied •n hdptng

Poll transolpOOO ., yeast IS rather diffm>nt

b ._,..TBP

initiation requires two other factorS, called SLl and UBF. SL1 comprises TBP and three TAFs specific for Poll tnmscri ption. This complex binds to the downstream half of UCE {called site A}. SL1 binds DNA only in the presence of UBE That factor binds to the upstream half of UCE (called site B), bringing in SLt and stimulating transcription from the oore promoter by recruiting Poll PoilU promoters come in various forms, and the vast majority have the urmsual feature of being located downstream of the tr.mscription start site. Some Pol UI promoters (tor example, those for the t.RNi\ genes} consist of two regions. called Box A and Box B, separated by a short e lement (Figure 1 2-22 )~ others contain Box A and Box C {for example, the SS rRNA gene); and still others contain a TATA element like those of Pol JL Just as with Pol II and Pol I. transcription by Pol ill requires transcription factors in addition to polymerase. ln this case, the factors are called TFJIIB and TFlJJC {for the tRNA genes}, and those plus Tf111A for the SS rRNA gene. Figure 12-22 shows the tRNA promoter. HA.re. the TFOIC compl.e x binds to the promoter region. This complex recruats TFUlB to the DNA just upstream of the start site, where it in turn recruits Pol m to the start site of t ranscription. The enzyme then initiates, prcswnably displacing TFlliC from the DNA template as it goes. As with the other two classes of polymerase. Pol Ill uses TBP. ln this case. that ubiquitous factor is found within the TF'ttlB complex.

a

0~--ir-------~--~~----~--~· aoxB

BoJ( A

b -

T13P TFIIIB

\

TFJIIC

BoxB

ffGU RE t2-22. PoHh core promoter. Shovvn here tS the promoter b a yeast tRNA

gene The ordet of events leaOng to trilf'lsc:npt'ia1 nl~ation

is desot:>ed in ~ text

SUMMARY Geoo expression is the process by which the intonnation in the DNA douL1e helix is converted into the RNAs and pn:teins wha;e activities bestow upon a C6Jl Us morpttology and functions. Transcription is the first step in g('.ne expression and involves copy ing ONA into RNA. This process, catm1)•2.fld by the enzyme RNA palymcrose, is in many ways similar to the process of DNA repliU~Uon diSt.·ussoo in Chapter 8 . b;J both cases, a new chain of oudel'Jtides is synthesized upon a DNA tt~mplate; anrl both DNA and RNA synthesis proceeds in a 5' to 3' direction (tlwt is. the emyme adds each successit.~ nucleotide to the 3' end of the growing chain). Hut there ar(l several critical differ-ences between thest.! two pi'OCeSf;es, some mechanistic. othets ~ecfingth~ different roles they ~mrve. f<'or example, in DNA replicatioo the entire genome is duplicamd ona3 and Ol').ly once each cell di\'ision. ln traa. scription. only some regions of the genome are trapscribed, and the regions cltosen \181)' in differoot cells or in lhe same c..-etl at diffew.nt times.. Differt'.llt reg~oos can be transcribed to diffef{'Jlt extents- thaf is. anything from one to severn} thousand lranscripts can be made of a given region in a single cell. Mechanistic difffll'em:es betwet>.n tr.wscription and replication include the (()))oWing: the nuclootides wrod to build a oew DNA ch ain ere dem:yribonudeotides, whereas in transcription they are ribonudeotides. Also. whereas DNA polymerase can onfJ• elongate eXisting polynucleotide r.hcios. and thus requires a primc.r. RNA polymerase can inilkl!tle RNA synthesis de novo. RNA pnlymer~sw from bat.-teria to humans are highly oonservcd. Eukazyotes have three diffurent ptJlycnera.'*ls ~ bacteria have just one. 'fhc three eukaryotic enzymes are calk:.ocl RNA Pol l, U, and m. ln this chapter we [{)(;used primarily on Pol n, as this is rhe enzyme- that transcribes the vast majority of genes in the c.:e U and a11 the protein coding genos. The bask e~yme frnm £ . coli, called the core enzyme. has one cop)' of each of three subunit:s-p, P', and wand two copies of a . AU these subunits have homologues in the eukaryotic enzymes. TlJ£ structures of the bacterial and yeast Pol II enZ)'me aa·t: also s imilar. Both resemble a crab claw in shape. rhe plncers being made up of the largest subunits, fl and W in the case of the baclf!ria l enzyme. The active site is -at th(' base of the pinarrs, and access lo and from the active site is afforded through five channels: onc allow~ do~ble--strandcd DNA to enter b6tween the pinUJrs a1 tlu1 froot of the enzyme; two orhcrs allow the two single strands- the tcroplate and non-template strands- to lca\'e the enzyme behind the ac;th·e site : a nother channel pro"ides the routo by whicll NTPs enter the active site: and the RNA pro dut.:t. which peels off the ONA template a sh ort dista11ce behind lhe site of polymer• ization. exits the OOZJ'me tluough the .fifth channel. Pol U differs from the bacterial rnzyme in nnf! important way The fon:ncr has a so-called "tail'' at the C. terminal end of the large subun it. wd this is absent from the bacterial tallZ)rme. This tail is made up of multiple .repeats of a heptapP.ptldc sequenc:e.

A round of tTanscription proceeds t.hrougb three pheses ce.Ued initia tion. t+longation, a nil termination. Though RNA polymera:Jes r:an synlbesizc RNA unaided, other p roteins- called initiation factors- are required for accurate- and efficient initin1ion. These factors ensure that the enzyme initiates trdnscriptlon only from appropriate sites on the DNA, called promoters. l n bacteria there is only one initiation factor, o , whereas in eukacynles there are se11eral. collecU11P.ly c alled t11e general transcription f01ctors, ln eul<aryotes. the DNA is wrapped within n ucleosomcs and, in vivo, effici(>.lll inllialion veTy oftfln requ i~ a d d itional proteins . including t~ Mediator Complex and nudet1some modifYing enz )'mes. Transcriptional activator p roteins are a Jso n eeded (set' Chapter 17). Durtng initiation , RNA polymerase (tOJ3cthcr with the initiation facton;) binds to the promotm- in a closed com· plcx. ln that state the DNA remains io a double-stranded form This closed complex then undP.rgoes isomerizatioo to the ope.n complflX. In that form, tbc DNA around the transt.:ription start site Ui unwound. disrupHng the base paits, and Jorming a bubble of single--stranded DNA. This transilion a llows SCt:es.s to the l(';mplate strand, which determines the order ofbasoo ia the O(!W RNA strand . This phase of initiation is followed by promoter escap~ once the enzyme has synlhesi~ed a series of short RNAs. called abortive initiatioo. it manages to mako a (ranscript that grows ooyood 10 bp. At this point the cn zsme leaves the promoter and enters the elongation phase. During this phttSC, polymerase moves along the gene while the enzyme perlnrms several functions~ it op ens the DNA downstream nnd reseals it upstream (behin.dl the active site; it adds rlbomlcleotide$ to the 3' end of the grow ing transcript: it peels the newly-formed RNA off the templafll some 6 or 9 base pairs behind the point of polymerization; and lt also proofreads the transcript checking for {and replacing} incorrectly inserted nudeotides. Transcription in both bacteria and cukaryotes follows these same step,c~. There are differences in the two cases, howe, ·ar: For example, in bac1eria. isorneri:zntioo to the ~pen complex OC(.U ts sponreneo11sly and does n vt require ATP bydrulysis. In uukaryotes this step does require ATP hydrolysis. More stlikif"lgl}'• ira ffilkatyotes, prom(lter cscapti is regt.llatcd by the phosphorylation stole ot· the CT'D tail. Thus, thr. form of Pol U that binds the promoter in the pre-in itiation complP.x has an uopbosphor}'iated CfV. This domain becomes phOsphorylated br ()OEl or more kineses, including one that is part of one of the general rraoscraption fa<.1ors, ·rFriH. Terminarion alro works difffll-entJy in bactetia and eukaryotes. Thus. in bacteria there are two kinds of terrninators- intriosic (.Rho-independent) and Rh
Bibliogrophy

Rho-dependent terminators require the ATPasc Rho, a pr'Oklin that hops oo elongating transcripts and .. pulls" them frorn thfl enzyme. lo eukuryotes, termination is. closely )inked lo an RNA pr('lcessing went called 5 ' polyadcnylstion Once phosphorylated. thfl CfD tail of the Pol H freos itself from rhe other proteins at the promoter, reloosing polymerase into the elongation phase. The CTIJ then binds fac-tors in\lolved in transcriptiona.J elongation and RNA proce~fng. Thus. there is an exchange ofinitial~n for e lnn-

377

gatioo and processing factors as the polymerase moves away from the promote~" and starts transcribing the gene. There are also illtel'actim1s between the elm1gation fuctors and those mvolved in processing. ensuring proper t:oordi-nBtion of these events In this chapter we considered capping of the 3' end of the RNA transcripts, polyadenylation of the 5' end. and the link between the lasr of thP..se lilld transcriptional terminati.<Jn, Splicing is described in the next chapter.

BIBLIOGRAPHY Books

Transcription Initiation

C,()/d Sprif18 Harbor Symposia on Qu011titative Biology. 1998. Volume 63: Mechanisms of 'franscriptioo. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, New York. Ptashne M . and Ganv A. 2002. Ctmes ontl Slgnols. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, New York.

RNA Poly.merase

Malik S. and Roeder R.G. 2000. Transcriptional regulation through mc..>diaror-like coactb:ators in yea:-1 and meta· zoao cells. Tronds Biochem. Sci. 25: 277- 283. Myers L..C. and Komberg RD. 2000 Mediator of transc.Tiptional regulation. Annu. Rev. Biochr:m. 69: 729-749. Woychik N.A. aud Hampstiy fvL 2002. 'The RNA poly· merase U madtinery: SlJUcture itfuminat~s function. Cell lOB: 4 53 -463. Young B.A., Gruber T.M.• and Gross C.A. 2002. Views of transcription in itiation. Cell 109: 417-420.

Borukhov S. and Nudler E. 2003. RNA polymemse holoen-

Elongation and RNA Proc~ing

White R.J. 2001. Gene transcription: Mechanisms ond control. Blackwell Science, Malden. Connecticut.

zyme: Structure. function and biological implications. Curr. Opin. Microbiol. 6:93-100.

Darst S.A. ZOOl . Bacrerial RNA

polymeraS~;J .

CW'I.'. Opin.

Stt'tlct. Bioi. 11:155- 162.

Ebright R.H. 2000. RNA polymerase: Stntctura1 similarities between bacterial RNA polymerase and eukaryotic RNA polyrDP..rase lL }. Mal. Bial. 304: 687- 698. Murakami K.S. and Darst S.A. 2003. Bac..tel'ial RNA poly· mecases: The whole story. Curr. Opin. Strvct. B1o/. 13: 31-39.

Paget M .S. and Hclmano J.O. 2003. The ~igma 70 f~mily of

sigma fa&:.:tors. Cenoroo Bioi. 4 : 203.

Promoters Ilutlcr J.E. an.:! l
Howe K.J. 2002. RNA polymerase n conducrs a symphony of pre-mRNA processing actiVities. mochim. Biopllys. Acto 1577: 308-324.

Maniali'!' T. and Reed R 2002. An exteosi.vEJ network of coupling among gene expression machiites. Notum ~16: 499- 506.

Termination Richardso.a J.P. 2002. Rho-depeodent termination and

ATPases in transcript termination Biocltim. Biophys. Acto 1577:251- 260. - - - 2003. Loading Rho to te.tminate transt.'tiption. Cell 114: 157-159.

C H A P T E R

RNA Splicing

he coding sequence of a gene is a series of three-nucleotide oodons that specifY the Jinear sequence of amino acids in its polypeptide producl Thus far we have tacitly assumed that the coding se<Juence is contiguous: the codon for one amino acid is imrned.iataly adjacent to the codon for the next amino acid in the polypeptide chain. This is true in the vast majority of cases in bacteria and thei.c pbage. But it is not al ways su for eukaryotic genes. In those cases, the coding sequence js periodir.aUy interrupted by stretches of noncoding

T

svquence.

Thus many eukaryotic genes are mosaics, consisting of blocks of coding sequences separated from each other by blocks of noncoding StqUences. The coding sequences are called exons and the intervening sequences are caUcd introns. & a consequence of thjs alternating pattern of exons and introns. genes bearing noncoding interruptions are often said to be .. in pieces" or "split... Figur~ 13-1 shows a typical eukaryotic gene in which the coding region is interrupted by three introns. splitting it into four exons. The munber of introns found within a gene varies .=
OUTl iNE

• The Chemstry of RNA Splaang (p. 380)

• The Spliceosome Mochinery (p 383)

•

Splic1ng Pathways {p. 385)

• Alternative Splteing (p. 394)

• Exon Shufffirlg (p. 401)

• RNA Ed•ting (p. 1104)

• mRNA 1ransport (p. 406)

that correspon d to 2 kb of mRNA . Thus, in this case~ the coding portion of the gene is lflSs than 10% of its total length.

f I Ci ll R E

1 3-1 typica et.lkaryotic IMe.

lhe d~JCted gene wntalflS four E!XOf1!>

2

separated by three lfltrons. Trnnscnption &om the promoter geoecates a pre-mRNA. ~ in the l1lKklle line. that containS al1 the exons and lntrons. Splldng r~ the mtrons ClOd fuses the eXOns to gef'lt:rate the fTli'llJ.if€ rnRNA t1at once processed further (see polyoo€f1Yiation, Chapret 12) and e>qxlf'ted frcrn the nucleus. can be translated to give a protein product

!tran~liOo 2

3

lspftc:ftlg spliced rnRNA

'

2

3

4

3'

Ub the unintnrrup t ~€1 genes of prokaryotcs, thfl s plit genes of eukaryotes Hre t.ranscriboo into a single RNA copy of the c nti1'e g~ne. Thus, the primary trdn script for a typical eukaryotic gene contains introns as well as exons. This is shown in the middle part of Figure 13-1 . Becaus e of the length and number of introns, the primary transcript (or pre-mRNA) can he very long i ndeed. In thA extreme CasA of the htJman dystrophio gene, RNA polymerase must travetsf! 2,400 kb of DNA to copy the entire gene into RNA. (Given that transaiption proceeds at a rate of 40 nucleot1des p er second. it can readily be seen that it takes a staggering 17 hou rs to make a single transcript of this gene!) Th>,spite this seemingly odd gooe aganization, the protein-synthesizing maLtainery of the cell (Chapter 14) is equipped only to translate messenger RNAs coDtaining a contiguous stretch of codons; it has DO way of identifying a nd skipping over a block ofnonr.oding sequence. And so the primary transcripts of split genes must have their introns removed before they can be translated into protein. lntrons are removed from the pre-mRNA by a process called RNA splicing. This process converts the pre-mRNA into mature niPSsenger R'.JA and m ust occur with great precision to avoid the Joss, or addi· tion . of even a single nucleotide at t he sites at w hich the exons are joined. A!; we shall see in Chapters 14 and 15. the triplet-nucleotide codon s of rnRNA are trnnslated in a fixr.d reading frame that is set by the first codo11 in the protein-coding sequence. lack of precision in splicing-if, for example, a base were lost or gained at the boundary between two exons - would throw the reading frames of exons out of register and downstream codons would be inCQnectly selected and the wrong amino acids incorporated into proteins. Some pre-mRNAs can be spliced in more than one way, generating alternative mHNAs. So, fur example. different combinations of introns might be removed. This is called alternative splicing, and , by this strategy. a gene can give rise to more than one polype ptide product. lt is estimated that 60% of the genes in the human genome are spliced in a lte rnAtive ways to genel'ate more than one protein per gem~. T he m•mber of diflel'ent variants a given gene can encode in this way w ries frorn two to hundreds or even thousands. Por example, the Slo gene from rat which encodes a pot~ssium channel t"Xpressed in neurons has thr1 potential to encode 500 alternative versions of that pood uct- And, as we shall see, there is e Drosophilo gene that <:an cncod~ as many as 36,000 possible products as a result of a1tflrnati ve sp licing! (n thi s chapter we discuss, not only the mechanisms and regulation of RNA splicing. but also ideas about why eukaryotic genes have interrupted coding regions. We a lso describe RNA editing, another \V"~Y initial tJRnscripts can be altered to change what they Ancode.

THE CHEMISTRY OF RNA SPLICING Sequences within the RNA Determine Where Splicing Occurs We now consider t he molecu 1i:lt m echanisms of the splicing reoc.-tion. How am the intr:Qns and exons distinguished from each other? How a re introns r~movad'? How idre exons joi.ned with hish precision? The bordets between introns and exons are marked hy specific Dudeotidr

sequooces w i1hin thP. pre-mRNAs. These sequences delinP.atc wh P.re splicing will occur. Thus. as shown in Figure 13-2, the exon-intron boundary- that is, the boundary at tlle 5' end of the intton - Js marked by a sequence caUed the 5' splice site. The intron-exon boundary at the 3' end of the intron is marked by the 3, sp~ site. (The 5' and 3' splice sites were sometimes referred to as the donor and aa:epiOI' sites, respectivP.ly, b ut this nomP.K~clature is rruvly used today.) T he EigW'e shows a third sequence n~ary for splicing. This is ceUed the branch point site (or br.mch point .sequ ence). It is fc.>und entirely within the intron, usually close to its 3' end, and is foHowP.d by a polypyrimidine tract (Py trdct), as sh own. The consensus sequence fo.r each of these elements is shown in Figure 13-2. The most highly con.crerved sequences are the Gll in the 5' s plice s ite, the AC in the 3' splice site. and the A at the branch sitn. These highly consecved nucleotides are all found within the Jntron itself- perhaps not surprisingly, as the sequence of the exons, in con· trast to the introns, is con str<W1ed by the need to encode the specific amino acids of the protein product.

The lntron Is Removed in a Form Called a Lariat as the Flanking E.xons Are Joined

tat us hflgin by considering the chemistry of spl icing. which is achieved by two succe~ive traosesferi6cation reactions in whic h phosphodiester linkages within the pre-mRNA are broken and new ones aJ'e fo rmed (Figu~ 13-3). The first reaction is triggered by the 2' OH of the conserved A at the branch site. This group acts as a nucleophile to attack the phosphoryl group of the conserved G in the 5' splice site. (This is an SN2 reaction that proceeds through a pen~ tavalent phosphorou ~ intermediatr.) As a consequP.nr:e, the phosphodiester bond between t he sugar and the phosphate at the junction between the intron Hnd the exon is d eaved and the freed 5' end of the intron is joine d to the A within the branch s ioo. Thus, in addition to the 5' and 3' backbone linkages, a third p hosphodiestcr extends from thP. Z'OH uf that A to creatP. a thrt::t!-way ;u nction (henL'C its description ~s a branch p oint). The structure of the three-way JU nction is shown in F.igm e 13 ~4 . Notice that the 5' exon is a leaving group in the fll"St tt'dnsesterifi· cation r eaction . (n the second reaction. the 5' exon (more precisely. th<' newly llbera t~d 3'0H of Uw 5' (>xon) reverses its role and becomes a nucJeophile that a lta cks the phosphoryl group a t tha 3' spli ce site (Figure 13-3)- This sP.Cood reaction has two consequences. First, and most importan tly~ it joins the 5' and 3' exons;

btanct• site

FlcuR l U-2 sequences .-t the inttotHU~o" boundcny. Showu10 the figtre are tl~ con!i€nSUS ~for bo1h lhe 5'

and 3' splice sites• .ni also the conserved A at the l:lrar1<:h site. As 111 dher cases of consensus sequences. whefe two aftemanve bases are .strnitat1y favored, V1ose bases are both tndic.:lfed at thal posit100. In lt11S flgtre. lh~ c:onsei1SUS sequences shown are fur llumans. Tl-is IS true lor all other ligures, IJlless ~estated-

38Z

FIGURE

RNA Splici~

Shown are the two steps of the spltcing reaclion described trl the text In the first step, the RNA fcm'ls a loop SM.JatJre. v.t1ict1 is shovJn in detdil If\ the next figure.

3" exon

introo

Il-l The spJidng reac:tion.

"----~~ ~

~lc

3'

"--- - - - -- - Ct12'

1

5' W J - OH:J

I

l

l

+

5'

s-GpG

3'

spliced el
thus, this is the step in which the two coding sequences are actually "spliced" togethnr. Second. this same reaction liberates the intron. which serves as a lea\'ing group. BecausH the 5' flnd of the intron had been joined to the branch point A in the first transesterificati.on reaction . the 11ewly liberated intron has the shape of a lariat. In the two reaction steps, there is no net gain in the number of chemical bonds- two phosphodiester bonds are broken, and two new ones made. As it is just a question of shuffling bonds, no enm:gy input is demanded by the chemistry of this process. But. as we shall see

FIG U R £ 13~4 The strudure of the

three-way junction formed during the .sptidng reaction.

slko~ ~ 0

)

OH

I O= P-o f 0

0

II

0

1 I o= r-o~ o- bale o - ~o

.

f

O= P-o-

o I

OH

1 o-

~

0

O= P- cr

I

0 ~end__. .

of irltron

0

OH

I o = P- o-

bP.low. a large amount of ATP is cun~um ed during the splicing rP.dCtion . This energy is requiced, not for the chemistry. but to properly assemb le and operate thr splicing machinery. Another point about the splicing reaction is direction: whal ensures tha t splic ing on ly goes forward-that is. toward the products shown in f igure 13- 3? Two features that could contribute to th is a re as follows. Fiest, the forward maction involves an increase in entropy - a single pre-mKNA molecule is split into two molocules, the mRNA and th~ liberated lariat. Secon d . the e xcised exon is rapidly degr-.:tded after its rnmoval a nd so is not available to p~rtake in the reverse reaction.

ANAl

s

f

RNA II

exon 1

!

~AA

THE SPLICEOSOME MACHINERY RNA Splicing Is C arried Out by a Large Complex Called the Spliceosome The transesterification reac tion s just described are mediated by a h uge molecular " machine" called the spliceosome. T his compte}( comprises about 150 proteins and 5 RNAs and is similar In size to a ribosom e (Chapter 14) . In t.--ar.ryi ng out even a single splicing re action. the spliceosome hydrolyzes several molecules of ATP. Strikingly. it is believed that many of the functions of the sp Uceosome aro c.:p liceosome.

~2

I

3

l 3' Bi

5'

Exons from Djfferent RNA Molecules Can Be Fused by Trans.. Splicing l n our description of s p licing above. we assumed that the 5 ' splice s ite of on e exon is joined to thP. 3 ' splice s ite of the exon t hat i mme di::~ fely follows it. This is not always t he case. In a lternative sp licing, exons can be skipped . a nd a given exon is joined to one fu rther downstream (as we seP later in the text). In some cases, two exons carried on d iffe rent RNA molecu les can be spJiced together in a process called trans--splicing. Although generall y rdre. bans-sp licing occu rs in a lmost all thfl mRNAs of trypanosomes. In th~ nematode worm {C. elegans), all mRNAs u ndergo tra ns-splicing (to attach a 5' leader sequence), a nd many of them nndergo cis-sp lking as well. Figwc 13-5 shows how the basic sp licing reaction just described is a dapted to carry out trans·splidng.

::AA

CGll

f

~ Ff G U R £

..--.3"

11·5 lrilns-Splicing.

tn trans-

splicing. two exons, inibally found 111 two separate RNA moleoJes, zre spliced rogethef 1n1o a ~ mRNA- fhe dlel'nlstry ot this reaaoo IS lhe same as that ot the standanf sp!ia1g reactton descd>ed pre~~~ousty. lllld the sp\red pmduct 6 i~able. The O'Ay dlflerence IS lhal lhe cthef product- che lariat W1 the Slandard 1'e«too- 1S, 1"1 trans-spliong. a Y· shaped br~dl SWCtl.lre ~c.:!- Tl"4$ IS becdt.&.: the ll'llticll readim brMlgs togethef two RNA ~les rather lhan fom1rtg a loop Vv1l.htr1 a Slrlgle molecule-

384

RNA Splicing

The suRNPs have three Ioles in splicing. They recognize the 5' splice site and lhe branch siti3; they bring those sites together as required; a nd they catalyze (o r help to catalyzej the RNA cleavage and joining r eactions. To perfonn these functions. RNA-RNA7 RNA-protein, and protein-protein interac tions ate all important. We stru-t by co nsiderin g some of the RNA-RNA interactions. These operate within individual snRNPs, between different snRNPs, and between snRNPs and the pre-mRNA. Thus, for example, Figure 13-63 shows the interaction, through complementary base-pairing, of the Ul snRNA and the 5' splice site in the pre-mRNA Later in the reaction. that splice site is recognized by the U6 snRNA. In another example, shown in Figure 1 Ub. the branch site is recognized by the U2 snRNA. A third example. in Figure 13-6c. shows ~rt interadion between U2 and U6 snRNAs. This brings the 5' splice s ite and the branch s ite together. It is these and other similar interactions, and the .reanangements they lead to, that drive the splicing reaction and contribute to its precision, as we will see a little later. Some RNA·frce proteins a.re involved in splicing as mentioned ;tbove. One example, U2AF (U2 auxiliary factor). recognizes the polypyrimirline (Py) trnct/3' splice site, and, in the initial step of the splicing reaction, helps another protein, branch-point binding protein {BBP). bind to the branch site. BHP is then displaced by the U2 snRNP, a s shown in Figure 13-6d. Other proteins involved in the splicing reaction include HNA-annealing factors, which help Joad snRNPs onto 1he mRNA. and DEAD-box helicase proteins. The latter usc their ATPase activity to dissociale given RNA-RNA inte.raclions, aJlowing alternative pairs to form and 1hereby driving the rcammgcn1enls that occur through the splidng reaction. Finally, before tuming to 1he spliceosome mediated splicing pathway itself. we looJc at one further intel-action. Figure 13-7 shows the crystal structure of a section ()f the Ul snRNA bound to one of the protein s of the U1 snRNP.

1J-6 50me RNA-RNA hybrids formed durinc the splicing reaction. In

f'I GUR E

some cases, (a) diffEfent snRNPs recognize the same (or O\l€flappng) seq\Jefl(es in the premRNA at Cifferent stages of the spidrlg reoction, as shOIM'l here for U1 and U6 recogmmg the 5' !f'llce Site. In (b) snRNP lJ2ts 5huwn retogt11Zlng the band\ Site. 111 {c) the RNA:RNA pau-eng bet'NeeJ the snRNPs U2 and lJ6 is 511owrl. Finally, in (d), the same seqvence w thwl the pre-mRNA ts re<:ogr11red by a prct€111 (not part of an snRNP) at one stage and displaced by an sr:RNP at another. of these changes accornpa.-.es the amval or departure of a:lmponents of the spri(eos0111e and a structi,Jfaf rearrangemenlltlal is requi!ed fur lhe splicing reactollo proceed.

Earn

a

b

exoo 1

s·

exon2

s O"C~~ ov

'!!

exoo1

~

~~~

'liAGJ\CA'.....-... U6

c

d ' jj

U6

:~~

....u

u : : ~ u~

•

eaP 5

~

exon2

LJ ~l:Dl A ~

'ru

mo~

3'

l =

eJC.Otl2

:r

Sp/icirtg Palhwo.ys

13-1 Sbudl8'e of 5fJiiceosomal protein-ANA complex: UIA binds hairpin n of Ul snRNA. (Oubridge C., Ito N., Evans P.R., Teo C.l-1., and Nagae 1<. 19 94. Nature 3 72: 432.) Image prepared\\""' Mo~ Bd:ISal>t. and Raster 30.

FIGURE

SPLICING PATHWAYS Assembly, Rearrangements, and Catalysis Within the Splkeosome: the Splicing Pathway The steps of the splicing pathway are shown in Figure 13-6. Initially. the 5' splice site is recognized by the Ul snRNP (using base pairing between its snRNA and the pre-mRNA. shown in Figure 1rJ.6}. One subunit of U2AF binds lO the Py tract and the other to the 3' splice sHe. The former subunit interacts with BBP and helps that protein bind to the branch sHe. This arrangement of proteins and HNA is called the Eady (E) complex. U2 snRNP then binds to the bcanch site, aiderl by U2AF and displacing BBP This arrangement is called the A complex. The base-paiTing between the U2 sn.Rt\JA and the branch site is such that the branch site A residue is extruded from the resulting stretch of double helical RNA as a single nucleotide bulge as shown in Figure 13-6h. This A Tesidue is thus unpaired and available to react with the 5'splice site. The next step is a rearrangement of the A complex t o bring together all three splice sites. This is achieved as follows: the U4 and U6 snRNPs. along with the US snRNP. join the complex. Together these three snRNPs are called the tri-snRNP particle, within which the U4 and U6 snRNPs are held togethe.- by complementary base-pairing between their RNA components, and the US snRNP is mort:! loosely associated through protein;protein intf!factions. With the entry of the tri-snRJ\.tP,. the A complex is converted into the B complex.

IWA Splicing

FIGURE 13-8 Steps of the spkecsome-mediated splicing reaction.. 1he assembly and action of lhe sPiceosome are

._1u1~·:=========~~= aA~=-j0W ~u~ ==jf .35.J...

5 ..

~ovvn. and the details of

each step ..e descrtled Components of the spliCing fl'Ja<:hnery amve Ot leave the complex ill each step. dlanges

111 the text

that are associated Wth struc1tJial r~nts necessary fer the splicing reaction to proceed. There IS evidence to suggest that some d the components shown do not amve oc leave precisely Vlklen 1ndicated 10 this f~e; they may, b' example, rem<~n p-esent but wealr.en their association wich the ~x ralheT than dissociating C<:lfT'!pletefy. It is .also oot possible to be sue of lhe crder of some dlanges showt1, par1leularly the two steps tnvdvlng changes tn U6 parmg: v.hen it takes 0\lef frcnJ Ul at the 5' splice site. ~ed to lll.t.en it takes. over from U4 in binding U2. Despite these uncert<Woties. the aiCical imdvement ot different COfTl>Oflents cllhe madlinery at afferent stages c1 the spliong react\oo, and the ~al dynarruc nalure of the

snRNP

snRNPs

spllC.eosorne, we as shoMi. U1

U4

s----3'

Splicjng Pathways

In the next step, U 1 leaves the complex, and U6 replaces it at the 5' splice site. This requires that the base-pairing betweep the lJl snRNA and the pre~mRNA be broken? allowing the U6 RNA to anneal

with the same :region (in fact. to an overlapping sequence, as shown in Figure 13-6a). Those steps complete the assembly pathway. The n ext rearrangement triggers catalysis, and occurs as follows: U4 is released from the complex, allowing lJ6 to interact with 02 (through the RNA:RNA base-pairing shown in Figure 13-6c). This arrangement, called the C complex, produces the active site. That is. the rearrangement brings together within the spliceosome those componePts-believed to be solely regions of the tJ2 apd U6 RNAs-that together :furm the active s ite. The same rearrangement also ensures the substrate RNA is properly positioned to be acted upon. It is strilcing that, not only is the acti"Ve site primarily formed of RNA, but also that it is 011ly fonned at this stage of spliceosome assembly. Presumably lhis stra tegy lessens the chance of aberrant spl icing; linkin g the formation of the active s ite to the successful completion of earlier steps in spliceosome assemhly makes it highly likely that the active site is available only at legitimate splice sites. formation of the active site juxtaposes the 5' spl.i ce site of the prernRNA and the branch site. facilitaling the first trnnsestcrification reaction. The second reaction. between the 5' and 3' splice sites. is aided by the lJ5 snRNP, which helps to bring the two exons together. The final step involves release of the mRNA product and the snRNPs. The snRNPs are initially still bouncl to the lariat. but gel recycled after rapid degradation of that piece of RNA. It might seem odd that the machinery and mechanism of splicing is so complicated. How did it evolve that way? Would it not ha ve been s impler to ftJSe the cxons in a single reaction. rather than undergo the two reactions just described? Th consider this question, we turn to a gLDup of introns that-unlike those we have considered thus far-can splice themselves out of pre-mRNA without the need for tlle spliceo-

some. They are called self~splidng introns.

Self..Splicing lntrons Reveal that RNA Can Catalyze RNA Splicing The three classes of splicing found in cells (nol including tRNA processing, which we discuss in Chapter 14} are shown in Table 13~ 1 . Thus far we have dea1t only with nuclear pre-mRNA splicin g, that mediated by the $pliceosome found in all eukaryotes. Also s hown m Table 13-1 are

Mechanism

C&ass Nuclear pre-mRNA

Group ll rl.OOS

Group I lnlroos

Very common; used for most eukaryotic genes

Rare: soroo eukaryohc genes trCf'11 organeles and prokaryotes Rare; llUdeal rRNA in S()I'T)e eukaryotes. organelle

genes. 8f\d a lew prdlaryotic genes

catafytic Machinery

-------------------------Two tvlajor spttCeOSOme t~ansesterification

reactions; brandl s stu A

Same as pre-mANA

RNA enzyme encoded by tntron (ribolyrne)

Two transe.ste-ificatiUil w..acbons. branch site G

Same as QfCt.Jp II

th.A so-called group I and group ll self-splicing in trons. By self-splicing, we mean that the intron itsell folds into a specific conformation within the precursor RNA ana cataly.teS the chemistry of its own release (recaiJ that we discussed the genm:at features of RNA enzymos in Chapter 6). In terms of a practical definition. self-splicing means that these introns can re.move themselves from RNAs in the test tube in the abseJlce of any proteins or othef' RNA molecules. The self-splicmg introns are grouped into two df'tsses on the basis of their structure and splicing mechanism. Strictly speaking. self-splicing introns are no1 enzymes [catalysts) because they mediate only one round of RNA processing (as we shall consider later in Box 13-1). ln the case of group n introns, the chemistry of splicing, and the RNA intermediates produced. are the same as for nuclear pre-mRNAs. That is, as shown in Figure 13-9, the intron uses an A residue within the branch site to auack the phosphodiester bond a t the boundary between its 5' end and the end of the 5' exon- tbat is, at the 5' splice site. This reaction produces the branched lariat, as we saw before, and is followe<1 by a set.'.flnd reaction in which the newly freed ~'OH of the exon attacks the 3' split;e site, releasing the intmn as a lariat and fusing the 3' and 5' exons.

Group 1 lntrons Release a Linear l ntton Rather than a Lariat Group r introns splice by a different pathway (Figure 13-9c).l1lstead of a branch point A resid ue, they use a free G nucleotide or nucleoside. This G species is bound by the RNA and its 3'0H group is presented to the S' splice site. The same type of tr.msesterif1r.ation :reaction that leads to the lariat fonnation in the earlier examples, here fuses the "G" to lhe 5' end of the intron. The second reaction now proceeds just as it does in the earlier examples: the freed 3' end of the exon attacks the 3' splice site. This fuses the two cxons and releases the int.ron, t.hough in this case the intron is linear rather than a lariat structut"C. introns, share a Group l introns. which are smaller than group conserved secondary s tructure (RNA folding is discussed in Chapter 6}. The structure of group J introns includes a binding p ocket that will accommodate any guatline nudeotide or nucleoside as long as it is a ribose form. lJJ addition to the nudcolide-l.Jindjng pocket, group J in1rons contain ao "internal guide sequence" that base-pairs with the 5' splice site 5et1ucnce a nd, thereby detennines the precise site at whlch nudeophllic attack by the C nuclootide takes place (see Bo" 13l . Converting Group llntroJtS into Ribo:zymes). A typical self-splicing intron is between 400 to 1,000 nucleolides long. and, in contrast to introns removed by spliceosmnes, much of the sequence of a selfwsplicing intron is critical for the spliciJJg reaction_ This sequence requirement holds because the intron must fold into a precise structure to perform the n~ction chemistry. In addition, in vivo, the intron is complexed with a n umber of protei.n s that help stabilize rhe mrrect structure-partly by shielding regions of the oockhone from each other. Thus, the folrling requires certain sections of the R.'JA backbone to be in close proximity to othP.r sections, and the negativn charges provided by the phosphates in those backbone regions would repel each other if not shielded. In vitro, high salt concentrations (and thus positive ions) compensate for the absence of these proteins. This is how we know that the proteins are not needed for the sp licing reaction.itself. The similar chemistry seen in self· and spliceosome-mediated splicing is believed to reflect an evolutionary relationship. Perhaps ancest ral

n

Splicivg Pofhways

Box 13-J C::onvetting Group I lntrons into Ribozymes

Once a gmup \ s~-~ mttm has ~n spliced 00\ the active site it contains remains intact. So what prevents this splicing reaction from reversmg ;tsett! One thing IS the htgh cellular concentration cl G nudeotides-this strongly favors the forward reaction. But in additJon, the intron undergoes a 'furthel reaction that effectively prevents it fr«n participating m the back reaction. Conveniently. at the extreme 3' end of the intron is a G, which can b111d •n the G-binding pocket. MeanwMe. the 5 • end of the intron can bt1d along the internal guide sequence. Thus, a third transesterificabon reooion can occur tu cydize lhe 1ntmn. The new bcmd formed with the terminal G is labile and hydrdyzes spontaneously. As a 0011seq.rence, the intron is relinearazed, but it is truncated and so precluded from the back spliOI'lg reaction.

As ~ined earlier in the text, %foup I (and II) introns are not enzymes

because they have a tumaver number of 01ly one.

But 1Jley can be reaOiy converted IntO en.zymes (rixuymes) in the folo\N~ wat (13ox 13- 1 figure 1): the relru~orized intron described atxNe retains its active site. If~ provide it with free G and a substrate that indlldes a sequence c~ to the intt:mal glAde sequence. it wil repeatedy catalyze cleavage of substrate molecules. We have cooverted a grot.p I intron into a rOOz.yme, smar to the 'Mf( that the self-deaving hammerhead could be runverted to a ribozyrne by separating lhe ective site frvrn the substrate (~ 6). We can go a step further by da'lgu-,g the Se.ql.J€1'lre of the 1ntemal guide seqU€flCe and thereby generate tailor-made riJonudeases that deave RNA mdecules of oor chOre.

wi•

RNA

5'

3'

+ 3'

s

~

5'G BOX

13-1

FIGURE

1 Groaplintronscanbeamvertedintotrueribozymes.

+ ___

3'

~90

RNA Splldng

apre-mRNA ~

c gr~ I self-splicing

b group II sel-splicing

5'

1 3'

!

5'·--·3' +

---3'

3'

fJ---

13·9 Croup I and BTOup II introns. This f.gue rompares thE reaction of the self-splicing I and ll lf111ons and the sPJceosorne·med".ated reactton alreirly desaibed. The chemcstry in the case of group n 10tr
g~

~GeU44: 207 -210,ftg 1.)

group Il-like self-splicing introns were the 6tarting point for the evolution of modem pre-mRNA splicing. The catalytic functions provided by the RNA were retained. but the requirement for extensive sequence specificity within the intron itself was relieved by having the snRNAs and their associated proteins provide most of those furu::tions in trons. In this way, introns had oniy to retain the minimum of sequence elements required to target splicing to the correct places. Thus, many rnore and varied sizes and sequences of introns were permitted. 11 is interesting that the structure of the c..atalytic region that perfonns rhe first transesterific.ation rear.tion is very similar in the grot1p ll intron ami the pre-mRNA/snRNP c.omplcx {Figure 1 3-10). This obseiVation fuels the broader speculution (discussed in Chapter 6) that ear]y in the evolution of modorn organisms, many catalytic functions in the cell were carried out by RNAs and that these functions have. on the whole. since been replaced by proteins. In the case of the spliceosome and the ribosome, however, these activities have not been entirely replaced by proteins. Rather, the vestigial RNA-catalyzed mechAnisms remrun at the heart of the present complex madtinery.

Splicing PathiVays

391

FiGURE 13-10 Proposedfoldingolthe RNA ~alytic ftgioos for splicing of gtoup 0 introns M1d pre-tnRNAs. The dotted reg10ns c:J the RNA in the gfOl.f) I case l'efllace an ilddltional four folded domains net shcN.n lf1 th!s

depicticn

group2

How Does the Spliceosome Flnd the Splice Site~ Reliably? We have already seen one mechanism that guards against inappropriate ~plidng-the active site of the spliceosome is only formed on RNA sequences that pass the test of heing recognized by multiple elements during splic:eosome asscmbty. Thus. for examplo, the 5' sp1ice site must be recognized initially by the U1 snRNP and then by the U6 snRNP. It is unlikely both would recogn ize an incorrect sequence. and so selection is stringent. Vel. the problem of appropriate spticesite recogn ition in the pre-mRNA remains formidable. Consider the following. The average human gene has eight or nine exons and can he spliced in th~ alternative fonns. But there is one human gene with 363 exons and one Drosophila gene that can be spliced in 38,000 alternative ways (Figure 13-11). If the snRNPs had to find the correct 5' a nd 3' splice sites on a complete RNA molecule and bring them together in the coJTCCl pairs, unaided. it seems inevitable that many errors would occur. Remember, also, that the average exon is only some 150 nudeotides long, wh€reas the average intron is approximately 3.000 nuclcotidf'.s long (as we have seen, some introns caJI be as long as f!OO.ooo nudcotides). Thus, the ex<ms must be 1dentified within a vast ocean of intronic sequences.

6)(()(19

exon 17

33 attem~ves

2 altematives

I

n

I

l

mRNA

F IGURE 13·I 1 The multiple exons of the Drosophikt DSCAM gene. fus gene was cloned as an axon gudance recept01 responsible fOt" directlng grOW1h COI'lE!S to their poper target The DSCAM gene (5ha.M"l at the top) is 6 12 kb long; once tJansalbed and spliced. It produc:Es one 01 rr.ae Vet'SIOOS of a 7.8 kb, 24 exQJ'l, rnRNA (the ftgUre shows the genenc stnJ<:ture of those mRT\IAs)_Ps shovvn, &here are se:.verill motualy exdustve ahematrves fc r exoos 4, 6. 9, and 17 Thus, each rnRNA v.111contain ooe of 12 poss1ble alten)atlves for exon 4 (tn orange), one of 48 for exon 6 (purple), ore of .53 for exon 9 (bfue). ~ one of 2 for exon 17 (red). If all possible combinations of these e
Splice-sHe recognitlon is prone to t wo kinds of erroTS (Figure 13-12). First, splice sHes can be skipped. with components bound at. for example, a given 5' splice site pairing with those at a 3 ~ site beyond the

correct one. Second, other sites, close in sequence but not legitimate splice sites. could be mistaktmly recognized. This is easy t o appreciate when nnn r-e<".alls that the splice sita consensus sequences are n:~ther loose. And so, for example. components at a given 5' splice site mighl pair with components bound incorrectly at such a "pseudo.. 3' splice site (see Figure 13-12b}. Two ways in which the accuracy of spl ice-site selection can be enhanced are as follows - First, as w e saw in Chapter 12, while transcribing a gene t o prodlJce thn RNA. RNA pulymeraStl 11 carries wit h H various protein s with roles in RNA processing (see FigurP 12-18).

aexon~

exon1

b pseudo spice-site seleCtion 2

3

pre5' --=::::===--IE:=====-- ~ nflNA

5'---3' F ICU R E J 3 - 12 &rurs produced by mistakes in splice-site selection.

exon 1

f{

2

-.:=============-~ 3'

t

bpseudo" splice sire

5' -

(a) srows 1he consequence of skiptJt~ an exon ThiS happens tf the spbceosome compooents bo..orrl at the 5' splice slte of one exon Interact IMth spftceoSorne c~s bound at the 3' splice Stte o( not lilc next ecm, but one beyond. (b) Jfiustrates the effect of spltceosome components reccgJliZ1f18 Hpsecdd' spice sites- sequences that resemtle (but are not) leglbmate ~ice Slle5. In the case sha,vn the p.;eudo Site 15 withU) an exon and lea~ to rE£ions near the 5' end cf that ~ being rnst:at\enly spliced OIA along w.th the intron.

3'

These include proleins involved in splicing. When a 5' splice site is encountered in the newJy synthesized RNA, those components are transferred from the polymerase C-terminal ••tail .. (that part of the enzyme where they hitch a ride) onto tbc RNA. Once in place. the 5' splice site componen ts are poised to interact with those that bind to the next 3' splir:t- site to be synthesized. Thus, the correct 3' splice sif'e ran be recognized before any compoting sites f••rt her downstream have been lranscriboo. This co-transcriptional loa rlin~ p.rl)(;ess greatly diminishes the likelihood o f exon skipping. It is worth noting that even though much of the splicing machinery assembles wbile the gene is being transcribed- and on individual introns in the order they are transcribed-this does not mean the introns are themselves spliced out in that orrler. Thus. in contrast to many other activities we have heard abou1- transqiption, replication, and so on - there appears to be no ''tracking" mechanism involved. whereby the machinery assembles at 011e end of the gene or message and acts as it tracks to the othet end. A second mccbar1jsm guards against the use of incorrect sit<.>S by ensuring that splice sites close to exons (and thus likely to be authentic) are recognized preferentially. So-called SR (Serine Argenine nch) proteins bind to sequences called exonic splicing enhancers (ESEs) witbin the exons. SR proteins bound to these s it es interact with components of the splicing machinery. recruiting them to the nearby splice sitrn;. ln this way, the machinery binds more efficiently to those splice sites than to incorrect sites not close to exons. Specifically, the SR proteins recruit the U2AF pr<Xeins to lhe 3' splice site and t J1 snRNP to the 5' site (Figure 13-13). As we saw earlier. these factoTS demarcate lhe splice sites for the rest of the machinery to assemble correctly. SR proteins are essential for splicing. They not only ensure the accuracy and efficiency of constitutive splicing (as we have just seen) but also regulate alternative splicing (as we wiU see pl'esently}. They come in many varieties, some controlled by physiological s ignals. others constitutively activP. Some arc expressed preferentially in certain cf:lll types a nd control splicing in cell-type specific patterns. We will discuss some specific examples of lhe roles of SR proteins in the next section.

exon

8XOI"'

F1 CuRE 13 ·13 SR proteins nuuit splkeosome c:ampor~euts to the 5" and r .spHce sites. l egitllll<'le spltee sites are recOWJtzed by the splicing machinery by wtue of bemg dose to exms Thos, SR r-oteiilS bend to SE!Quetl<es wtthin the exons ( eJ~OOIC spliong enhancers), and from there rt?crult U2AF and UlsnRNP to the downstream 5' and upstream 3' splice sttes respectively. ThiS tn.tlates &he ~emtly of the sphong madunery on the corf1:!d SiteS and spltang can proceed as out~ned ear~er In an intton 1s dra>ovn in lhe center; bounded on each S*de by an exoo Ttws tooklllg at lh1s figure, note is 1n contrast lo many of the t'.ar1ier medlanistlc ligures ln which a Single central mlron rs depc.ted ly1ng between tvvo tntrOfiS. (Sotxce. Ftom M
mat

RNA Splkhlg

ALTERNATIVE SPLICING Single Genes Can Produce Multiple Products bv AJtemative Splicing As we described in the introduction to this ch apter. many genes in higher eukaryotes encode RNAs that can be spliced in allernativa ways to genernle two or more ditfetent mRNAs and, thus, different prolpJn products. In som e cases, the number of potential alt e:rnatives that can be generated from a single gene is breathtaking-hundreds (in the rat Slo gene, for example) or even thousands (for the Drosophila DSCAM gene (Figure 13-111). For a simple case. consider the gene for the mammaJian muscle protein Troponin T. Shown in Figure 13-14 is a region of the premRNA made from this gene and containing five exons. This RNA is spiked to fonn two ahQrnative mature mRNAs, each containing foW' exons. A different exon is eliminated from each of the two mRNAs, so the two messages have three exons in common. as well as each carry· ing one unique exon. But, as shown in FigurP, 13-15, alternative splic ing caJ1 arise by a number of means. Thus, as well as alternative exons being chosen. exons can be extended, or {deliberately) skipped. Also. introns can be retained in some messages. rather than being deleted. again generaling diversity in the proteins produced. Jn the previous section , we described mechanisms that ensure variations of this sort do no t take place-that e.xons are not skipped and splice sites not ignored. So how does alternalive splicing occur .so often? T he basic answer is that some splice sites are used only some of the time. leading to the production of different versions of the RNA from different transcripts of the same gcnc. Alternative splicing can he e ither constitutive or regulated. In the form er case. more than one product is a lways made from the transcribed gene. Jn the case of regulated splicing, dift'emnl forms are generated at d iffel'ent times. under different roodition s. or in different cell or

tissue types. Another example of cons1itutive alternative splicing is seen with the

T a,ntigen nf the monkey virus SV40 [FigtrtP. 13-16}. 1l1e T antige..fl ,gene encodes two protein products- the large T antigtm (T-ag} ood the small t antigen (t-ag). The two proteins result from alternative splicing of the pre-mR.J'\JAs from the same gene. Thus, as shown in the figure, the gene has two exons and difft=...renl mature mRNAs result from th e use o f two different 5' splire sites. In the mRNA encoding Jarge T, e:xon 1 i.s spli~d directly to exon 2, dR1eting the intron that }ip,s belwnen. The mRNI\ for t-ag, on tha other hand, is fonned using the nlternativa 5 ' splice site. Thus, in this case, the mRNA includes some of the intron as

feCURE 13•14 AlternatiVe splic:it1g in tile troponin T gefle. Shown here is a regJOI) of u.s gene encoding ftve econs wuch gereri!!t5 tvvo afte~ spliced fum1s ~ lf'lOICate:f

spliced mRNA

One cortatns exons J. 2, 4, and 5; the other" contalliS e
5' RNA transcfjpt

s·

1

2

3

5

splidng

5'

ntroponinT pnrnary

SSllired mRNA ~ lroponirl T

exon1

2

5'

1

3

2

4

5

5

s·

l l

3'

splicing

Altermtive Spiking

39 5

DNA

3

2

!

f3}(()(11

lranscriptico

spiced 5' mRNA

t 1-

2

3'

1

5'

normal

3

1

3

l

s·

3'

3

1

pr-imary RNA 5' trar\SCI'ipt

1

3'

i 3'

11

1

2

3 exon extonded

exon sl
l

5'

2

l

s·

3'

3

1

inlron retained

+

3'

2

5'

;:!'

1

3

altemati\e exoos

Fl G u RE 13-1 S Five W exons. In ar.olher case. a con1pete irltron is retained rn the mature mf
Cher exon 2 or 3 .

well. (It is, therefore, an example of the ..extended exon,. shown in Figure 13-15.) The reason th.is larger message encodes the smaller protein is because there is an in-frame stop codon within the region of the i.ntron retained in this mRNA. Both forms ofT antigan are made iJ1 a cell infucted by SV40. But the ratio of the two fonns produced does differ depending on the level of the splicing protein SF2/ ASF. When present at high levels. this protein directs the machinery to favor use of the closest 5' splice s ite and stop 5'SST codon

ONA exon 1

primaJy

RNA

s

1

b'anscript

mRNA

protein

N cc:-::===..... ::: c

2

1

'

2

l

5'

'

3'

5'

2

5'

3"

2

1

+

l

! 2

3'

!

l

Nc : : : : - - '7-ag"

13-16 COnstitutive afternative splicing. Spliong of the SV40 T antigal RNA is shown. Both folms are typ1cally ptodtJced. and both proteins mac!Ef. upon infection The smal t antigen is encoded by the longer of the two rnRNAs; that message CXJntains an in-frame stop codon lflWe<'lm of exon 2 5 ' SST refels rome 5' splice~e used to generate the la~g.e T mRNA; 5' sst tf-.atfor smalf t 3 ' SST IS the 3 ' splice site used m generating both mRNAs. f t GU R E

rne

•C

3

RNA Splici.ng

thus produces more of the t-ag mRNA. SF2/ASF js an SR protein and. when abundant, presumably binds sites within exon 2 and helps the splir:e060me assemble thP.re.

Altecoative Splicing ls Regulated by Activators and Repressors Proteins that regulate splicing bind to specific sites called exoni<:

(or intronic) splicing enhancers (ESE or ISE) or silencers (ESS and ISS). The former enhance. and lhe latter repress. splicing at nearby splice sites. We have already encountered enhancers and the SR proteins that hind to them (Figure 13-13). h1deed, these elements and proteins are important in directing the splicing machinery to many exons-even when alternative spiking is not involved. Also, in the example of constitutive alternative splicing we just described, it was an SR prote in that ensured alternative splicing occurred. But this protein family- which is large and diverse-has specific roles in regulated ahf!matjve splicing as well, by directing the splicing machinery to diffP.rent splice sites onder dUierenl conditions. ThusJ tl1e presence or activity of a given SR protein can delermjne whether a patticu)ar splice site is used in a particular cell type. or at a particular stage of development. Figure 13-17 shows hypothetical cases of regulated splicing b y an activator bound to a splicing enhancer and a repFessor bound to a splicing silencer. The SR proteins bjnd RNA using one domain-for example, the well-charac.1eri1.ed RNA-recognition motif (RRM). Each SR protein

13·17 Regulated alternative spftdng. Some ~ernativcly spfiC€d exons appear In mRNAs l.ll1less ~eflted from doing FIG ll R E

so by a repessor prolan (shown 111 part a). Othefs appear only if a specific: actili
a

ceU type1

eel type2

~icC"lgsife+

primary RNA

transcriPt

repressor Si1e I 3"

5'

3'

~~;~g ~

macf1tnery primary RNA

transaipt

3'

5'

5'

Epliced mRNA

b 5'

5'

unsplicec:J 'fl

l

:I'

spliOOg splicing sJte enhancer I \ 3'

~1 l

5'

5'

3' tJnSPiice<.1

5'

3'

t~3'

!i'

3"

5'

l

3" spflced RNA

has another domain. rich in ;uginine and serine. called an RS domain. The RS domain, found at the C-terminal end of the protein, med iates interactions between the SR protein and prote ins within the splicing machinery. recruiting that machinery to a nearby splive site. An example of an activator that promotes a particular aJtemative splicing event in a specific tissue type is the Drosophila Half-pint protein. This activator regulates the alternative splicing of a sel of prem..RNAs in the fly nvary. It works by binding to sites near the 3' splice site of specific exons in those pre-mRNAs and recruiting the U2AF spJicing factor. Most silencers are I'E'..Cognized by members of the heterogeneous nuclear ribonucleoprotein ( hnRNP) family. These bind RNA but lack the RS domains and so cannot recruit the splicing machinery. Instead, by blocking specific splice sites, they repress the use of those sites. One example is hnRNPA1, w hich binds to an exonic silencer elmmmt within an exon of the HIV tat pre-RNA and represses the inclusion of that exon in the final mRNA. By binding to its site, the repressor blocks binding of the activator SC35 {an SR pNJtein) to a nearby enhancer element. Thls blocking is not direct the two binding si tes do not overlap-but hnRNPAl promotf'..s cooperative binding of add i.ti onal molecult!S of hnRNPA1 tn lldjamnl sequences, spreading over the enhancer sile_ When preSent, another SR pro~in {Sf2/ASF) can overcome this repression, because il has a higher affinity for the enhancer sequence than does SC35 and therefore displaces the repressors bound there. We will see similar themes of cooperative and competitive binding in examples of transcriptional regulation in Chapters 16 and 17. Another mammalian splicing repressor Is the hnRNPI protein. In some cases Lhis protein blocks the binding of the basic splicing machinery by binding directly to the Py tract (explajning why hnRNPI is also called the polypyrimidine tract-binding protP-in). In other cases it excludes a given exon from the mature mRNA by binding to sequences that flank that exon. This exclusion occurs either because molecules of hnRNPI at each end of the exon in teract and loop out the exon. which is then passed over by the spliceosome; or because the molecules of hnRNPI at each end bind cooperatively with oth.-:r molecules or hnRNPI. coating the RNA across the whole exon. This too would rf.lnder the exon invisible to the splicing machinery (Figure 1 3-16}. In Chapter 17 (Figure 17-28) we consider a particularly elaborate example of regulated alternative splicing-that involvi11g the doublesex gene of Drosophila. The sex of a given fly depends on which of two altemativP. splicing variants of this mRNA it produces. We have emphasized alternative splicing as a way in which multiple protein products can be produced from a single gene. These different proteins are called iso(orms. They can have similar functions, di stinct functions, or even antagonistic functions (thus, one form might act as a dominant negative of another}. But even some genes that encode only a slngln functi onal protoin show ah rtmativp splicing. In those cases, a lte rnative splicing is used simply as a way of switching expression of the gene on and off. This is achieved in two w ays. Most straightforwardly. an exon contains a stop codon, and. when i.ncmporated into mRNA, this prematurely terminates translation generating R truncRted polypeptide. Typically, such an incomplf;lle prote i11 is nonfunctional and rapidly degraded. Alternative splicing determines whether or not the exon with the stop

RNA Splicins

398

F Ui U RE

( YhnRNPI

13- 18 lnhillition of splidng b)'

hnDIPI. Two modefs are f'(esented. In one the prtJtetn coats the eotire ewn. In the odler it bnds at each end of the exon and lnnceak it

s

1

j

within a lcl:lJ.

5'

3'

or

~ 5' [

3'

l ~

3'

codon is included in a given mRNA. and thus, in effect. whether or not tho gene is expressed. The second way alternative splicing can be used as an on/off switch is by r egulating t he use of em intron. which, when retained in the mRNA. ensures that species is not transported out of the nucleus and so is never translated. Splicing was discovered in studies of gt.'lle expression in the mammalian adenovirus. where mRNAs are alternatively spliced, as desctiberl in Box 13-2, Adenovirus and the Discovery of Splicing.

- - -- - - Studies with bacteria and thetr phage led to the view that tl-1€ mRNA is ao exact repka in terms of nt.deobde seqJeOCe of lhe gene from whd1 It 1s tr~osoibed {see Chapter 15). It therefore came as a sf1od< when, irJ 1C.J77, it was d&ovefed

that certcM'l (ood. as we now know. milny) eukocyotic !"T'lRNAs are spliced together in patchWOfk fashion from mt.JC.h longer prrnary trcmscnpt5. HOIN was ths startirlg discovery made? In an effort to understand gene transoiptiot'l in eukol)'otes. scientlsts focused on the human Cf\IA virus called adencMrus. This virus was lflterx:led to SerVe as a l1)()def for understat'lding the molecutar biOlogy of the eukal)'otK g~ Just as phage T4 and l\ had c'lol1e for the prokaryooc geoe (see Chapter 21) 1he VIrion of adenowus IS composed of several
tne

trip311ite leader_The leader ts then alternatively spiced to the coding ~ for the hexon. fiber, and other vtrion proteins to generate each of the late vtral mRNAs. That these messEngefS are spliced together rrom RNAs ansing from several Jegions of the genome emerged from a variety of expenments- one of which ts knovvn as. R-loop mappmg (Box 13-2 flgllfe 2). \Nhen RNA is lf1Cubated. under the awropriate coodittons, wtth a dooble-suaoded DNA coo-

taning a stretch of sequence identical to thilt of tne RNA. the RNA anneals to its complement, di~ng a stretch of the noocomplementary strand in the form of a loop (Box 13-2 Ftgure 2a)- FoiiOWVig the staining pmcedure used to visualize nucleic aods, this R-loop can be observed irJ the electron microscope. as RNA-DNA and DNA-DNA dup&exes appear thder ~n single-standed nucleic <1C::ids. \t'vhen St,Jch an experiment was perfomed with adeno"rus messengers. the re.st.Jiting R-loops were found not to be fully contiguous with

a single region of DNA. Instead. aod depending on vvhich fragment of VJral DNA was used, one or both ends of the RNA were found to pr~ from the RNA loops as sin~ stranded tails (Box 13-2 Figure 2b). In other cases, one of the tails IS seen to anneal Wtth a DNA fragment from a different region of the viral genome (Box 13-2 Ftgure 2c). d early, these mRNAs were composite molecules that had been joll')ed together from sequences c.ompfemeorttdry to noncOf)tiguous regioos of the genome. These cYld other kinds of Df\IA.f
Box ll-2 (Ccmtinued)

tripartite leader

hexOn

- .. 10

0

30

20

40

so

60

70

80

map units

13-l

I Milp of the human adenovirus-1 genome. The map stlO'NS the IJ'anscnption pattems of the late «Rf'~As. indud~ng the ~ary tran~ (shov..'!'l as a long datk green anON at the top): the lllpartite leader sequences fouoc:l at p:lSibol'lS 16.6, 19£, and 26.6 (shown as green bars); and !he ~ pclSibOOS of tt\e DNA sequences that encode the various late rriWA.s (the late ll1RNAs e~e shaM) as short dark green arrows}. BOX

fl GUtl ~

a

b

DNA cvt with restrictioo e nzyme

~a===========~===:~========~==========================:3~ mRNA INih poly-A tail f) -

cap

R~

n

~=

cispfaced

~

~

ONA~:::·==:::::=:::::==:=:::~ ·~,

strand

c

------------

BOX

13-2 Fl GUR~ 2 R.foop mapping of the adenowirus-21ate messeager RNAs. (a) The schematlcshows the formatioo ot a n

R-loop stn/Cil.Jre. A dcd>le-5tranded CNA fragmart get~erated by &gestion with a rewiction endonuclease is ioo.bated with mRNA and heated to jt.st above1ht> Tm of the 0\IA in 8ffi'o furmerride. fhe hybrid famed between the messenger and its cOfll'lementary DNA sequence results tn disp1acernent of the second~ 51ral1d. The pcly-A tail of the rnRNA (ne1 encoded by ONA, see Chaptec 11) IS seen prqectir1g from the end cf the h).bld ooplex, (b) Electron rrnaogrelph a'ld schemobc diagram ot an R-loop obsecvEd after inaiJatlng hexon mRNA with a comple~ DJA ~nee from the late regioo of the aclerovlnJs-2 genome Note the extensions of both the ~and 3' ends a lhe mes~. Jhe DNA is re,xesented by black m : the RNA is represented by green lines in the cf.agram. (c) Electron lllCIOgfi!f'h and sctlemabe diagrclrn cf an R-loop <Jbsaved alta incubating fber mRNA with two ONAs, the cc.:rrPae ~rus ~ and c restriclion elldoru:fease frcgmeflt derived from the eaf1y region of rhe genome (~ce: EMs courte5¥ of (b) Chow I T.. Gelinas RE., Bcd<e- TR, and Robet1s RJ_ 1977. hi ~ sequeoce ar.angerrtent at ~ 5 ' ends of ader& virus 2 messenger RNA. Ce/J 12! 1- B, page 1. Cop~Jllt €:1 1977 Used With penri5sion fran Elsevier-(c) Berget S.M., Moore C., and Sharp PA 1977. 5t*:ed segments at the 5 ' terrmnus chdenowus-21ate mRNA 11-oc. Natl Aco::l. Set 7 4:3171 - 3 175.)

A Small Group of lntrons Are Spliced by an Alternative Spliceosome Composed of a Different Set of snRNPs High er e ukaryotP.S (including mammals. p lants. and so on) use the major splicing machinery we have discussed thus far to direct splicing of the majority of their pre -mRNA. But in these ocganisms (unlike in ycastj some pre-mRNAs are spliced by a low-abundance form of s pl iceosome. This .rare form conta ins some componflnts common 1o the major spliceosome but other uniqu e components as w e ll.

Thus, 011 and U12 componeniS of the alternative splicesome have the same roles in the splicing reaction as Ul and U2 of the major form, but t hey recognize dis tinct sequences. U4 and U6 have equivalenl counterparts in both spliceosome forms-altllough t hese snRNPs are distinct, they share the same n ames. Finally, t he US component is identical in both the major and in the altem ativEl spliceosome. The minor spliceosome recognizes rardy occurring introns having consensus sequenres distinct Irom the seqUtmces of most premRNA introns. This recentJ}'· discovered form is known as the ATAC spliceosome, because the termini of the originally identified rarP. int.rons contain AU at the 5' splice site ana AC at the 3' si te (in RNA or AT and AC i n DNA). Later it transpi red that many introns spliced by this pathway have CT- AG tennini (like mainstream int.rons), but otherwise their consensus sequences are distinct from those of the major palhway. Despite the djfterent spUce sile and branch site sequences recognized by the two systems. these major and minor forms of spliceosomes both remove introns using the same chemical pathway (Figure 13·19). Consistent with this conserved mechanism, the differenr;es in splice-s ite sequences recognized by these snRNPs ate mirrored by complementazy differences in the sequences of their snRNAs. Thus. it is the ability of the snRNAs and splice site sequences to base-pair that is CfJnserved, not any particulai sequence within either. II is a1so worth noting that AT-AC ln\fons might fit into the evolutionary ~chem e discussed earlier. Thus. as we mentioned. it has been proposed that the group n introns represent the oJdest form of introns. Further to this, it is suggested that the AT-AC introns evolvad from the group U introns and. eventually. give rise to the major pre-mRNA introns (Ftgure 13-20).

EXON SHUFFLING Exons Arc Shuffled bv Recombination to Produce Genes Encoding New Proteins As we h ave noted , all eukaryotes ha ve jntrons, and yet these elelllf)nts are rare-almos1 nonexistent - in bacteria. There are two likely explanations for this situation. First-in the so-called introns early model - inlmns existed in all organisms but have been lost from bacteria. lf introns originally did exist in bacteria, why might they subsequently have been lost? The argument is tl1at thesP. ..genl3 ric:h .. organisms (see ChaiJtf'. rs 7 and 11). have strtJamlined their genomes in response to Sl;!lectivtt pressure to increase tl1e rate of chromosome replication and Q:ll divisioni (Recall also that tllllOJ"Jg

EKon Shuffling

fl--

ft(i URE

13·19

401

TheAT~ACspfteeoSOme

qtalyzed spficing. This minor~ wocks on a mmoty of exons (pe:f'h.3ps ooe in a thousand 111 humans, for example), and those have &stinct spice-site sequences. Regardless. the chemtstJy 1> lhe same, and so ate some ot the spkeosome c0f'l1'C)nents, and others are dosefy r&ted.

U1t

13'

5'---·3· FIG u Jl E

pre-mRNA introns

13-20 Sequer.c:es conserved in

different l
3'

Sf::keS~re,

and brarx:h~ of nuc:tear and trans·

pc~ introns-rTii!jof, I!J-AC.

. .

.

t VA;U'C'l:JlD .

group I introns

G U G YG

_ _ _ _ _ _ _ _....,." A"-_ __

_ _ _M

splioog- and gfOliD nllitrons. Shaded regions show nudeotides that are identlc.alirl major, AT·AC, and trans-splirng introns. (Soutce: Adapted from Vu 'H .• Scharl E..C... Srnrth C.Mv and Stetz JA •999. The growing \Mllld of smatl nuclear nbot-.udeoproteins. In The RNA W~ 2nd e
bor, New Yod<.)

RNASplidng

eukaryotes, yeast- which are uniceilulai and rapidly growi~-have fewer introns than clo complex multicellular organisms.} 1n tile alternntive vjew. intxons never existed in bacteria but rather arose later in evolution. According to this sCK:alled introns late model, introns were inse.rted irHo genes •hat previously had no introns, perhaps by a transposoj1-Jike mechanism (see Chapter 11}. Irrespective of which explanation is true- ana at this stage it is impossible to rler.ide the mHtler unambiguous1y~there is the second. perhaps more interesting, question: w hy have the introns been retained in eukaryotes. and. in par ticular. in the extensive form se~n in multicellular eukaryotes? One clear advantage is that the presence of introns, and the need to remove them, allows for alternative SJllicing which can generate multiple proteiil products from a single gene. But. on an e ven grander scale. another advantage afforded these organisms is believed to be the following: having the coding sequence of genes clividAd jnto several exons allows new genes to be created by reshuffling exons. Three observations strongly suggest that this process actually occurs:

~2

exon1

• 3" ~· :=:1---====---===

1_IIiil-==

E1 5' =:J........

E2

~

1-00 02

01

domain 1 domain 2

! Fl GU RE

m

02

01

01

13-11

JdimeriZ.atlOn

J

DNA-binding

EXon$ •nc:ode ~n

domains. In lhis e011ple. the ONA-bvld~ng dorT'4n of a protem rs erxuded by ooe eJ~Dn, vJ\ile the dimenlabOrl ~m of tNL same prole!M i'S ~ncoded by a sepjll(lte l'j.Qn. Protein domains~ lf'ldepeldenlly of the rest of lf'IE' p(oteirt lf1 V'olfj(:h they are found, and often carry Ollt a SJngle fLflction (CIS we discussed .., Chapter 5). Tht.r.., ewns ~:an of!en be exdwlged

between proteitls rxoductlvely

• First, the bort1crs between e"'ons and lntrons within a given gene often coincide with the boundaries between domains (see Chapter 5) wHhin lhe protein encoded by that gene. That is. it seems lhat ead1 ~on \'ery often encodes an independently folding unit of p rotein {often corresponding to an independent function as well). For example. consider tll.B DNA-binding protein depicted in F iguit! 13-21. Like most DNA-binding proteins, this ooe has two d omains- the DNA recognition domain and the dimeri7..ation domain. l'ls shown in the figure, these domains (Dl and 02} are encoded by separate exons (El and EZJ within the gene. • Second, many genes. and the proteins they encode. have apparenlly arisen duriog evolution in part via exon duplication and divergence. Proteins made up of repeating units (such as immunoglobulins) have probably ati~n this way (see Chapter 11 Figure 11· 35}. The presence of intmns bt:!tween each exon makes the duplication more Ukely. • Third, relared exons are sometimes found in othenvise unrelalr.d genes. That is. there is cvldeuce that exons really have been reused in genes encoding differru;t proteins. l'ls an example, consider the LDL receptor gene (Figure 13~22). This gene contains some exons that are dearly evolutionarily related to exons found in the gene encoding the EGF precursor. At the same Lime, 1t has other exons that are dearly related tn exom; hom tl1f! C9 complement gene {Figure 13-22}. More extensive examples of exon accretion are apparent from the complete sequences of genomes-for example. the human genome. As shown h1 f'igu re 13·23. there are numerous examples of proteins made up ol hlghly related domains used in various combillations. encoded by genflS made up of shuffled exons. As we have seen, exons tend to be ruther short (some 150 nucJeotides or so) while i.ntrons vary in length and can be very long indeed (up to several hundred kbl. 1b.e size ratio ensures that . for the ~verage gem: in a higher euka.ryote, rP-Combination is morF. likely to ocua within the introns than within the exons. Thus, exons are more likely w be reshuffled than disrupte
Exon Shu:fflUl$ exw 1 2 3 4

LOL receptor gene 5 6 7 B

9

10 11 12 13 14 15 16 17 18

C9~nt

gene

Fa G u R 1£ 13-22 (iene:S made up of parts of other genes. The LDL receptor (the plasma low derr.itty lipq:lrotein receptor) gene ct11tains a sttetch of SU< extn. dosay rel&ed to six exons trom the C9 cnmplemeflt gene and eigt-. dosefy related to eight from the EGF (epidermal growth factor) pcec~soc gene_ Thus, ffie illl. rKepmr gene IS made Lp of ei<005 shuffled b£1\.\'een other genes; and, though n(l( snov,, he-e. these same parts appear tn yet other genes as ~Nell. The cntrons are. 111 many cases, l'iOt posrtioned in exactly the same positions witho tne ECF prerusor gene 4Yld the comparable region of the lOt. recept.or gene. When they ere in the SQme place. this IS incf!Cilted t;.y dotted ~Jines.

use of the 5' and 3' splicf! sites-guarantees that almost all recombinant genes w ill be expressed. becau~ the spHce sites in different genes are largely interchangeable. ln addition. alternative splicing can allow new exons to be tried without discarding the original gene product.

b

y

t

Y. F

y

c

{

f 1G U R E 13-23 A«umulation, Joss. and reshufflillg of domains during the evohruon of a

family of proteins. The 1\gure'shows proposed routes whereby different related protems mtghl: ha\e ~ t;.y gain and loss of specific domams. Three e<arnples .are given. 111 each ca-:;e the proteins in question are dvomatin modi~ enzymes (Chaf:te£ 7) from yeast M. worms 0N}, flies (F), and humans (H). Each protein IS depaed by a senes of
SW12

403

404

RNA SplicifW.

RNA EDITING RNA Editing ls Another Way of Altering the Sequence ofanmRNA RNA editing, Ji.ke RNA splicing, can change the sequence of an RNA after it has been transcribed. Thus the prott:rln produced upon translation is diffeient from that predicted from the gene sequence. There are twc;J mechanisms that mediate editing: site-~pocific dt!ruuinaHun Hlld guide RNA-directed uridine insertion or deletion. We consider each in tum. In one form of sit{."-specific deamination, a specifically trugeted cytosine residue within mRNA io;; cc;Jnverted into widine by deamination. Typically. for a given nlRNA species, the pi'()(:(!SS occurs only in certain tissues ot cell types and in a regulated manner~ Figure 13-24 shows the mammalian apolipoprotein-B gene. This gene ha~ several exons, within one of which is a particular CAA codon that is targeted for editing; it is tht: C within this codon that gets deaminated. That deaminalion, carried out by the enzyme cytidine deaminase, converts the C toe U (Figw:e 1325}. In this example, the doa.nrlnation occurs in a tissue-spocillc manner: messages are edJtro in intestinal cells but not in Jiver cells. These two forms of apolipoprotein B are both involved in lipid metabolism The longer form, found in the liver. is involved in the transport of endogenously synthesized cholesterol and triglycerides. The smaller ver~ sian. found in the intestines. is involved in tho transport of dietary lipids to various tissues. Thus the CAA codon. which is translated as glutamine in the unedited message in the liver, is converted in the intestine. to

-

pre-rnRNA

codon2,153 within exon 26

r1

-

.lof:IIJ

-

~==:•-•·::;=:~

1

l

~

lraosport Co intestine (site-speaflC ~~on)

CAA-UA A

1:::.1----~~...------3· glUtamine

N c===========~============~ c 4 ,563 aa protein

c.. . . . . . . . . .~. . . . . . . . . . . .~

N~:-=============' C 2,153 aa proleirl

fiGURE 11-24 RNA~"' by de~inaliun. The RNA made from the tll.l'nafl apoilpoprotc:Bl ~ IS edited m a tissue-specifiC ~by deammt~Qt~ of a specific cytodine to generate a tn:line. This evert ocoss In RNAs destired for the intestine, but not those for the liver. lhe resub. descrbed in the text is that a stop c.odoo i1froduc:ed iflto lf1e 111tes1:inat mRNA ger.erc'l1eS a shorter protetn than tNt produced tn 1he iver. The figufe is llOl dra1Nn to suje: thus the edited exon tS exon 16; and !he codon ~ ~ fiire it is 111

re
RNAEdifillg

UAA-a ~top codon. The result is that the fu ll-length protein (of flOrne 4,500 amino) acids is produCf'.d in the liver, but a truncated polypeptide of only about 2,100 amino acids is made in the intt.ostine (see Figure 13-24). Olh& examples of mRNA editing by enzymatic dcamination include adenosine doornination. This reactio11 carried out by the enzyme AOAR (adenosine deaminase acting on RNA)- of which there are three in lnunans-produces lnosine. lno..c;ine can bruo~pair with cytosine, and so this chango can readily niter the SE-quence cf the protein encoded by the mRNA. An ion channel expressed in mammalian brains is the larg~l of this type of ooiting. A single ooit in its rnRNA elicits a single amino acid change in the protoin. which in tum alters the Co.2 perm~lity of the channeL In the absence of this editing. brain devdopmmt is seri-

ously impaired. A very different form of RNA editing is found in the RNA transcripts that Qncode p roceins i n the mitochondria of trypanosomes. In this case, multiple Us nre in..c;erted into specific regions of m.RNAs after transcription (or, in other cases. Us may be d eleted). These iru;ertions can be so extensive that in an extreme case they amount to as many as half the nucl eotidss of the mahrre mRNA. The addition of Us to the messaJ1.e changes codons anrl reading frames, completel y al tering the " meaning.. of the mossagtt. As an example. consid~r &he trypanosome coxlf gene. In a specific region of the mRNA of this gene. four Us are inserted between adjacent bases at three sites (two Us at one si te and one U at each of lwo additional sites)_ Thase additions alter some codons and cause a .. -1" change in the reading frame. a sbi.ft that is required to goooratt~ lhe corrt~ct open-rtJading frame, as shown in Figure 13-26a. How are these additional bases inserted? Us are inserted into the message by so-called guide RNAs (gRNAs) , as shown in Figure 13-26. These gRNAs range from 40 to 80 nucleolides in length and are encoded by genes distinct from tb.o se that encode the mRNAs they act on. F.ach gRNA is divided into three regions. The first. at the 5' end, is called the "anchor'~ and directs the gRNA to the cegion of the mRNA it will edit; the second determines exactly where the Us will be inserted wilhitt thtt edited sequence; and the third. at the 3' And . is a poly-U stretch. We now look more closely at how the gRNAs direct editing. The anchor ~on of the gRNA contains a sequence that can base-pair with a rugion of tht-t message immediately beside (3' to) thP resinn that will be edited (figure l3-26b). This is followed by the ooiting "in~truc

tions;" a &tretch of gRNA cor:np!ementa.ty to the region in the messag~ to be edited, but containing additiQna] As. The As are at positions in the gR_I\JA opposite where Us will be inserted into the mRNA At the 3' end of lhtl gRNA is the poly-( 1region. The rolt: of thtl nuclP.Otides ill this region is unclear, though it is proposed that they tether the gRNA to purine rich sectucnces in the mRNA upstream (5' to) the adited region. As shown in Figure J3-26c, thtl gRNA and mRNA form an RNA-RNA duplex with loopoo out single-stranded regions opposite where Us will be inserted. An endonuclease recognizes and ~.:uts tht1 mRNA opposite these loops. Editing involves the transfer of Us into the gap in the message. This process is catalywd by the enzyme 3' tP.mllnal uridyJyl transfutase (TIJTase). AftflT the addition of Us, the two halves of the rnRNA arc ;oinod by ~rn RNA ligase, and the "e
13·15 The deamination of the t>.se cytosine to prod&Ke uracil

fIG U R£

RNA Splicing

a

site of u insertion

I

DNA sequence

5'1

primary

5'

RNA

~At't-C""l ~----_., 3'

~ ~

- - - DAA- tA'TIJ:C.CU - - - - - - 3

+

DA:U:I e®1g

mRNA

rnRNA 5'

~A

3'

=§

3' IIJOfr:UJ CUAACAUAUGGA I

editing anchor region (region of homology}

2UUU

~

s·

5'

I

!

3'

eAA

1pay.uJ uc AJI AUGGA

l

3'

3'

5'

~dease cuts at mismatch

b 3' guide RNA

3'

IPDf't·tH CUtAACA..UAUGGA. __ _ ....JL_ _J edit~

s

anchOr

region ~

3'

3'

tlld)>-tncul!c.AuAuoGA

!

5

ligase joins s· to 3' ends of meSSCJ9e

~

3'

-

.

f~l CUAACAUAUGGA

- 3' 5

- - -----------------

F IGURE 13-26 RNA editing by guide RNA meGated U in.settiot'l. Edimg of the tfypatlosome cox!/ gene RNA (a) Shows the posnions of the four U nudeolides InSerted i.nto the pre·mRNA of the cox// gene These genetate the cooect reading flame and coding lnfoonation in the mRNA. (b) Shows these--

qvence of the guide RNA rhat deterrntnes the U inserf100 pattern. and the sequen:e of the I.XleCfited S1retch of mRNA. (c) Shows the edibng readiOI1 ttself.

responsible for inserting seVL>rdl Us at difforunt sites (as is the case for the one shown in Figure 13-26}. Furthurmore. in some cases, several different gRNAs work on rliffertmt regions of the same mes..c.;age.

mRNA TRANSPORT Once Processed, mRNA Is Packaged and Exported from the Nucleus into the C ytopJasm for T rans)at ion

Once fully processed- capped. m tron-froo, and polyac.le11ylatt:dmRNA is transportud out of the nucleus and into the cytoplasm (Figure 1 3-27) where it is trans]atcd to give its protein product {Chapter 14). Movement from the nucleus to the cytoplasm is not a passive process. Indeed, it m ust be carefully regulated: the fully processed mRNAs represent only a small proportion of the RNA found in the nucleus,

mRNA Tm11$po l1

407

F I Ci U R E 13-27 Tran5J)Ort of mRNAs out of the nucleus. RNA export from the ooclws lS an active process, and only certain (appropriate)~ <Jre selected fortrar~spott To be selected for IJa1sport. the RNA must nave the coned cotledion of prot005 bound to it. These wdl disbngwh If from olhet RNA$. wtlich mt.J!>1 be retamed 111 the nucleus or destroyed. f'rotens that rerogni.zE exon:exon boundare;. fof ~. lrdlc.ate an m~ that has beEn

appropnat.ely spiked, ~ Jlro\elns that blfld 11'ltfoos 1nckate an RNA that si1C'Jid be retdlfled tn the nlldeuS Once 10 tht! c:y'IOplasrn. S<JtOe prolelfls are shed c:!nCl olhers ar~ taken ro ll'l

cytosol

and many of the olher RNAs would be detrimenta l to the cell lf exported. Thcst! indude, for examp)fl, damaged or misprocessed RNAs. and liberated introns h ·vbich. being, as they tend to so much larger than the ex{Jns, represent a lnrger population of RNA than do tht! mature mRNAs). How are RNA selection and transport achiuved? As we havo t:!mphasized in this and the previous c hapter. from the moment an RNA molecule starts to be transcribed, it becomes associaterl with proteins of various sorts: initially proteins involvfld in capping. then r-oplicing factors. and finally the proteins that mediate polyadrnylation. Some of these prot ems are replaced at various steps a long the proce.ss~ ing path. but others (induding some SR proteins. for example} are not; and, moreover, ad ditim1a1 proteins join. A.<:. a result. a typical mature mRNA carries a collection of prott:!ins that irlflnt.Hies it as being mRNA destined for rranspnrt Other RNAs not only l ack the parlicutar signature collection required for ttansport, bul have their own altf!fnative set of proteins thar actively blocks export. Thus, for ttxample, excised introns will often c:arry hnRNPs, and these probably mark s.uch an RNA for nuclear retHntion ~r1d destruction. Mature mRNAs carry residual Sl{ proteins. and even another group of proteins that bind spocifica lly to exon·exon junctions (which are only found in spliced species o f course). The mRNAs do also contain some hnRNP::s, but fewer than are typkally bound to introns, and in a different context as weU. This emphasizes the fact that it i~ the set of proteins, not any individual kind of protein, that marks RNAs for either export or f'etention in the nucleus. Export takes pla£.-e through a special structure in the l1UcleaT mP.ITI.brane called the nuclear pore complex. Small nlUlcculcs-tho:;e under abvut 50 Kd-can pass through these pores unaided; but larger mole<.ules and compl~xes, including mRNAs and thP.ir associated proteins, require active transport. (Ofher molocules-protcins made in the cytoplasm but with functions in the nucleus. for e.xaro.pJo- are transported in the other direction. from the cytoplasm into the nucleu..c;. through these same pores.)

oo.

readiness for translatlco (Chapter 14).

408

RNI\ Splicing

Tho mechanisms of nuclear transport are beyond the scop(j of thi!? book; suffice U lo say that some of the proteins associated with the RNA carry n odear export signals that are recognized by export rocepto.-s that guide the RNA out through the pore. Once in the cytoplasm. the proteins are discarded, a nd ate th~n recogniwd for import back into the nucleus where they associate with another mRNA and repeat the cycle (Figure 13-27). E:.>."]Jort requires energy, and this is supplied by hydrolysis of GTP by e CTPase protein called Ran. Like other G'TPasos, Ran tlxists in two

conformations dupemH ng on wh.t1t.her compJexed with GTP or GOP, and tho transition from one state to the other drives movero~nt into or out of the nucl~us.

SUMMARY Most genes encode proteins. and the s~uencc of amino adds within any given protein is determined by the sequence of "codons,. i.n its gene. Each codon is made u p of a group o r throe adjacent nudeotidcs. l n atinosl aU bac· terial and phage genes, lhe open-reading fra me is 8 single stretch of codons wtlh no break But the coding sequence of many eul<aryotic g~nes is split into stretch<.->s of codons interrupted b)' stretches of nonr.utling sequence. The codtng stretchf'.S in th~ split genes ~ called exons (for "expressed sequences") and the noncoding stretc11os are c.sllt~ introns (for " intervening sequences..). The nurnb~Jrs and sizes of the i ntrons and exons vary enormously from gene to gene. Thus. in yeast. only a relatively small prOportion of S~Ot!S ha\le mh:uns. and where lbcy (ICCUf they te nd to be short and few in number (one or occusionaJiy two par gene}. ln multiceUuJar organisms such as humans. the number:' of gent.'S containing introns is much l~rger, as is the number of introns per geoe (up to 362 in an extreme case). The sizes of cxons do vary bot are uften around 150 nuclt-!(l1ide.s; intruns, un the other hand. val"y frum 61 hp to as mur:h as a st~(ni.ng &Kl kb. Whcu a gene oontairung introns is transcrilx!d, the JU'.IA jnilially retains those introns. Th~-e arc then remowd to produce the mature rnRNA. The process of intton removal is caUc..>d splicing. Many intron-<:ofllnining g~nes give rh;e to a uni(tun mRNA spedes. That is. in each caw, all the introns are remuved from tb£ original RNA. lt:>aving an mRNA composed of ell tho exons. But in other cases. splicing can produce a number of difftftlnt mRNAs from the same gene by splicing lhc orig.ioa) RNA in different patterns. Thus, tor e xam1llc, some genElS contain alternative exons, only one of whicb ends up in a givt)n mHNA. ln other cases. a givoo cxon might Lc I'Vmovcd (aloug with the i ntrous ) from somtl co pies of the RNA-again producing a4lt:Tlllt'tlw ver.sions of roRNA from the same gone. Sequ~nces fi..1und al tho boundary bctw<.>cn introns and cxoos allow tho ceiJ to identify &ot.n:ms for removal. 'fh£$e splicing scquetu~cs arn almost m<elusivaly within the introns (where tlwre are no restrictions imposed by tlte net1d In t.'flCodc amino acids. as there are in exons). Th~o SCt)UCn~s are called the 3· and 5' splice sites, dcnot.irtg their rolijftve

locations at one or the other end o f the intrun. To splice out an intron also requitt!S a sequence t!hmmnt, callr.d the branch site. ncar lhe 3' end of the intron. Introo removal proceeds v ia two transestcrification coaction.~. Jn the first. an A in the branch site aUat:;ks a C in the 5' splice s ite. In the socood, the liberated 5' exon altocks the 3' ~plicc sitr.. These reactions havo two oonscqmmccs. First and foremost, thuy fuse the tw u exons. Se«~ond . they release thv intro n in the form oJ a branched structure called a lariat. Splicing of nudoosomat pre-mRNAs requu-es a large complt.-x of proteins and RNAs called tht! spliceosomc. This is made up of so-cnlled snRI\"Ps, of which there arc five-L'1, ll2, U4. U5 , and U6 snRNPs. f'..ach of these compriJifl& an RNA m.olecule, catted the UJ lO lJ6 snRNA. respectively. and a number of proteins. the majority of which are different in each case. The action uf abc spliceosnme is paMiculady interesting in two regards. First. the RNA cutnponnnts have a central mit~ in recognizing introns ond r:atalyzing their removal Sf£· nnd. the comple;x is very dynamic. That is, at different steps during the process of splicing. tbc sp)iC()osomc CtlflStitution alters-di{ferunt subunits of the mHChine join aud leave the complex. each performing a partic:uJar function. Thus. P.arly on. lJ 1 snRNP rncogni01.fls the s· s plice site, while tbe! Uz snR.NP ttJCogni?..lJS fht• branch site. l.J4 and U6 then join, loget~r with U5, bringing the branch site and 5' s pli c:e site togtthcr and stimulating tho first r.1action con· rJomitant with. Ul and U4 leaving. t 'inally, th~ 3' and 5' splice sUes arc brought 1ogcU1er and t:>xons are fuserl . Tht:~re are a fuw rare intrnns that can remove thcmsclH!.S from w ithin RNA moloculcs by a process known as selfsplicing.. Though not strictly an enzymatic reaction. the RNA of the intron nevertheless medjat(.os lhe ch emistry of removal. Tllt!tle 5(11£-splicing introns rome in two classes, one of whjch (group J1) splico by tbe same c ht:imical ~th way as thai mt!di~tt~d by the splic~ome. These introns probably tcprescn' lba evolutio nary origin o[ modern spliceosomaJ i.nttuns, and the two"step chemical palhw~y ured by both rdlects that evoh .rtionary reJaUonsltip (and perhaps c"plains why introns are not removed by a rnure dirt!t;t singlr.-step mccltani~m).

Bibliography

The splice s ites d~lw.d above at'f! defined by rather short soqocnCfJS with low levels of conservation. It thus tepreS()nts a s ignificant chall<mge fur the slJlicing machiotlry ty are selected bd'nre other downstream sitos arc available to compete. Second, lhere are other proleins- SR proto~ns-that b ind .n.ear ll'gitimato splice sites and help rocruil the splicing machinery to those sites. ln this way, au•hentic sit·es eftec. tively have a high~r affinity fot tha machinery than do so· cnti(Jd psocrlo :<~it(:s of similar sequence. There arEl a taq:;o variety of SR prott'?ins. Each binds R.l\JA with one surface and with another interacts with componen1s of the splicing machinecy. So m£ SR proteins regulate splicing. That is, a ~aven SR protein may be found ooly i.n oue ooll •ypc and mediate a parlicular splicing event only in that cell type. Otht:>.r SR vrotcins are only active in the presence o( specific pbysiolot:;ical signals , f;lld so a given spficmg event only occurs in response to 1ha• signaL (n this way, SR proh!ins resemLit! trnnscrip-

40ll

tional activators, as we wtll soo in latur chapters. Also waJogously with transcriptional rcgulatiun. there are tllpressors of splicing tlutt exclude splicing of S?--"Cific introns under· certain circumsta nces. Tugt!th~r w•th tltti other modilktttiou."> d~lt wilh in Chaplel' 12, splicing is required hf1fore mRNAs can btl transported out of the nudr.us through nuclear pures. This too can be regulated. lt is believed that a given eJCon typicaHy encodes an independt'ntly folding land functtonal) protvin domain. Thus, such HD exon can readily function in combination wilh dilferenl exons.. This s uggt1sh1 .it has beeu relatively easy. thruugh evo lution, lo generate new proteins by shuffling existing exons betW(.'Cn genes. RNA editing is another mechanism ahar aUows an RNA to be c hanged a fter Lranscription so as to encude a different prot{!iJl from tllot encoded by the gone. 1\vo mechanisms fnr cditit1g ere: enzymatic modifiali on of bases (generating forms that alter how lhey are read by tRNAs) and the insertion or deletion of multiple U nu· clenlides within the message.

BIBLIOGRAPHY Books Alberts B., Johnson A .• Lc\.vis J.. Raff M., Roberts K.. and Walter P. 2002. Molecular biology o.flhe cell. 4th editioil. Garland Scijcnce. New York Gestclanrl R.F•• Cech T.R.. Atkins ).F .• eds. 1999. The RNII wol'ld, Znd edilauo. Culd Spring .Harbor Lai:M,ralory Pwss, Cnld Spring f larlJOr, N~w Ynrk Lewin B. 2000. Genes Vll Oxford University Press. New York. Lodish 11•• Berk A •• ZipUl'sky S.L, Matsudalra P. , Baltimore D .• and Darnell ). 2000. Molecular cell biolop;y. 4tll ediliou. W.H. Fn:emao. New York.

Mechanisms of Splici~ and the Spliceosome Crid< F. 1979. Spht genes and RNA splicing. Science 204! 264-271. Cmvc.1ley El.R. 2000. Son\ng uut the cnmplcxHy of SR protein function. RNA 6: 1197- 1211. Hastings M.L. and Krainer A.~ . 2001 . f:lre-mRNA splicing in the new milh:mnium. Curr. Opin. Cell Bioi. 1~! 302-309. Jurica M.S. a1td MoO(& M.J. 2003. Pre-mRNA splicing; Awash in a sea of proteins. Mol. Cell 12: 5-14. Maniatis T. a nd Reed R 2U02. An oxteosive nf!tworl< of cuupling among gene P.Xpressinn ma r.hinos. Nature 416: 499-50(;. Reed ft 2(100. Mechanisms of fidelity in pre·mRNA splicing. Cun: Opin. Cell Bioi. 12: 340 -354. Sh:lley J.P. and Gothlie G 1998. Mcchsnical d~vjses of 1hc spliu.. osome: MoloTS. clocks, springs and things. CeU 92: 315-326,

Tam W.Y. and Steitz J.A. 1997. Pro-mRNA splicing: The discoverv of~ new spliceosome doubles the cballcmge. Tre11ds Biod!em. Sci. 22: l 3Z- 137.

SeU-Splidng Cech T.R. 1990. Nobcl ll>.etorc. Self-splicing and Hnzyomtac activity of an intervening sequence RNA lrum Tetrah_v~ mena. Biosci. Rep. 10: 239- 261 .

Altetnative Splicing at\d Regulation Barass J.D.. aod Beggs J.D. 2003 . Splicing gues global. Trends Genet. 19: 295-29A. Cra\;cley fJ .R. 2001 . Alter~ative splicing: locreasi OB d iversity in thl{ prutcomic world . Trends Genet. 17: 1.00- 107.

Ladd A N. and Cooper T.A. 2002. Finding signals that regulate alternative splicing in the post-genomic era. Genome lJioJ. 3: rcviews0008.1 - 00U8.16 .

fvlamatis T. and Tasic: B. 2002. Alternative pro-ml
Smith C.W. end Valr.Jtrccl f. 2000 . A1tl1rualivc pnHfl.RNA

splicing: The logic of combinatorial control. Tl'ends Biochem. Sci. 25~ 381- 38lt

mRNATransport Dreyfuss G., Kim V.N,. aod 1\at&oka N. 2002. MessengerRNA-binding prutcins and tht: messab>t!S lhcy carrv. Nat . Rev. Mol. Cell Biol. 3! 195-.W5.

410

RNA Splicing

RNA Editing Bonne R. 1996. RNA editing: How t} messal:,le is chnrtgeci Curr. Opin. Gen. nw. 6: 221 - 231 Blanc V. and Davidson N.O. 2003. c-to U RNA roiling: Mochanisms leading to 8'~m·.tic d1versity. f. 810/ Chem 278: 1395-1398.

W. A. and Fournier M.J. 2003. RN/\-gu;dcd nud (lotid•• mod lfiOHtion of ribo.or;u mal and nthr1r RNAs.

Dccahu

f. Bioi . Chem. 278: 6 9 5 -

696 .

Keegan L.P., Gallo A., and O'ConncU M.A. 2001. TI1a many roJes of an RNA ooHor . Nat. Rev. Genet. 2; 869-878.

Maas S .• Rid1 A., and Nishikura K. Zl iiJ3. A--tn-1 RNA editJog: Recent news and residual mysten(IS. /. Bioi. Chem. 278~ 1391 - 1 394. Madison-.t\ntenucd S .. f'.rams f., and Ha jduk SJ .. 2002. t-:diting machines: The comp h::xities of trypwu'ISomc RNA ooiting. CeJ/100: 435-4-36. Simpson L., Sb1cego S., and A.phaStzhvv R. 2003. Undine insmtion/dclolion RNA cililing in trypanosome mitochondria: A complex busillSS$;. RNA 9: 265- 276.

CHAPTER

Translation

he etmtral question addressed in this chapter and the next is how genetic information contained wjthin the order of nuclootidliS in messenger RNA {mRNA) is used to generate the linear se
T

or

O U T LINE

• Messenger RNA {p, 4 12) Transfer RNA (p. 415) Ahadlment of Amino Aads ID tRNA (p. 417)

. O~e

Rrosome (p

4~3)

lnttcatton d Translabon { p 432)

• rranslaaJon Eforlgaoon (p 440)

• Terrooation of TrcmsLatioo (p. 448)

• TranslatiOil-Depcndenl Regt.Jiato; of mRNA and Protein Stabaity (p. 45/)

ta~l of trc~.nf'l l ating a code writtP.n in a four-ba~e alphahet into a second code written in the language of thtt 20 amino acid'ii. The mf.WA providtiS thtl informalion that must be interpreted by the translation machinery, and is the template for translatiorL The protein-coding rt!gion of the mRNA consists of an ordered serios of lh~nudootide long units calltxi wdons that specify the order of amino acids. 'Ille tRNAs provide the physical interface between the amino acids being added to the growing polypeptide chain and the rodons in the mRNA. Em;ym~ C'.all~ O»minoacyl tRNA synthetases co~plt: amino acid& to spacific tRNAs that recognize the appl"()priate w don. The final central player io translation is the ribosome, a remarkable, mulli-megadalton machine composed of botll RNA and protein. The ribosome coonlinates thtt correct recognition of tho mRNA by e.a('.h tRNA and catalyzes peplide bond formntion between the growing polypeptide chain anrl rhe &nino acids &Uachod to tho seloc.'tttd tRNA. We will first consider the key attributes of each of these four compo-ne:rlls. W~ thtm dt~scribe how these components work to~uther to ac· oomplisb tmn.slation. Rectm.t progress in t~lucidating the st.ruL1ure of the components of the translational machinery make this an eJ the decoding of n ucleotide sequence infonnation and the addition of amino acids to the growing polypeptide chain?

MESSENGER RNA Polypeptide Chains Are Specified by Open~Reading Frames The translation machinery d ecodes only a portion of each rnRNA. A.o; we saw in Chaptor 2, and will consider in dP.tail in Chapter 15. the information for protein synthesi£ is in the form of threo-nucleotide cndons. which tiar.h specify one amino acid. The protein coding region(s) of each mRNA is cotnpoStJd of a contiguous, non-overlapping string codons called an open-reading frame (conunonly known as an ORF). Each ORF specifius a single protein and starts and ends at internal ~ ites within the mRNA. That is, the ends o( an ORF are di!>1i.nct from the ends of the rnl{NA. Translation starts at the 5' end of the open-reading framl;)l and proceed!; one rodon ctt a tim t• to the 3' end. The:: t1rst and last codoos of an ORF are known as the start and stop rodons. In bacteria, the start codon is u.crually S'·AUG-3' but 5' -GUC.3' and sometimes even 5' -Ul.IG--3' are also used. Eukaryotic cells always use 5'-AUC..3' as the start codon. This codon has two important functions. First. it spocifies the first amino ar.id to be incorporated into the growing polypt~ptide chain. Second, it dtlfincs th~ reading frame for all :subsequent codons. Because codotLc; are inunediatu1y adjacent to each otht:~r and boca~ codons are tbrtlt3 nucJootides long. any stretch of mRNA could be t.ranslated in three different rending frames (}tigure 14-1). However. o~ translation starts, each subsequent codon is always immooiately adiit· cent lo (but not overlapping) the previous three-base codon. Thus, by s~tti.ng the lol.;8t.ion of tbtJ first codon, the start codon d dennines the location of all following codons.

or

~

5

fJ~~~mi!J~I!B!l UJU~~~~~~[£1!1aJ stop

(

t<) (_a) (0 C!:J 0J

slop

0)

(V ~

®0

Fl cuR E 14-1 Three possible readif11 fralne$ of the E coli trp leader sequence. Start a,)(lons are shaded in green and stop codons are shaded m red fhe amlf'll 1 acid se.qJenc:e cllhe encoded 5e{)t.Jence IS ll'ldicated in lhe Single letter code below each codon.

Stop codons. ~f which there are three (5'-UAG-3', 5'-UGA-3'. and 5'UAA-3'), dt!fine the end of the open-reading frame anll signal termination of polypeptide synthesis. We a;m now fully <:!ppreciate the origin of the tenn open-reading frame. It is a contiguous stretch of coclons "read" in a particular frame (as set by the first codon) that is "open .. to tl'allslation because it lacks a stop codon (that is. until the last codon in the ORF). MessengP.r RNA<> contain at )east. one open-reading frame. The number of ORFs per mRNA is different hvt\veen eukaryotes and prokaryotes. Eukaryotir. mRNAs almost always contain a single ORF. In contrast, prokaryotic mRNAs frequently contain two or lllQre ORFs and bence can encode multiple polypeptidf:! chains. Messertgm- RNA" contairJing multiple ORFs are known as polyci~1ronic mRNAs, and those encoding a single ORF are known as monoch;tronic m.RNAs. As you learned in Chaptar 12. polycistronic mRNAs often encode proteins thai perfonn relate
Prokaryotic mRNAs Have a Ribosome Binding Site that Recruits the Translational Machinery For translation to ~cur, the ribosome must be recruited to the mRNA. To fucilitate binding by a ribosome, many prokaryotic open-reading frames contain a short sequence ups(rt'.am {on the 5' side) of the start codon called the ribosome binding site (RBS). This ulemenl is also referred to as a Shine-Da1gamo toequence after the scientists who discovered it on the basis of comparing the sequences of multiple mR.NAs. The ribosome binding site, 1ypicaJly located three to nino UastJ pairs on the !1' sidf:! of the s tart codon, is complomtmtary to a sequence located neaT the 3' end of one of the RNA r.omponents, thu t6S ribosomal RNA (ri~NA} (sua Figure 14~2a). The ribosome binding site base-pairs with this RNA component. thereby aligning the ribo-some with the beginning of the open-reading frame. 1be core of this region of the lGS rRNA has the sequence 5'-CCUU::U-3'. Not surprisingly, prokaryotic ribosome hinrling sites are most ofttm a suhset of the sequence 5'~AGGACG-3' . The extent of compJementarily and lhc spacing btitwoen the ribosome binding site and t hu stan codon h& a

(0 (P)

414

Translation

FtGURE 14-2 Structlaeofmessenget

RNA.. (a) A pclyc:istr0111C prokaryotic message. The nbosome binding site is mdtcated by RSS. (b) A monCJCIS!fOI"ll<: eukaryotx: message. lhe 5' cap 15 indicated by a "bar' a l the E!fld of me mRNA.

s·

"'olil":~~~

·--·s t t

o;QXn UXI(XXJO

·--·· s l 3' l

ReS start

RBS st;J{Iil l - - ---·

cx11•

!

•oaoo (Q)

-

·

~

b

S' NN~NNN AU( GNN 3' 5'

5'~

~~. . . . . . . . . . . . . . . .~~ - ~-o~AAAAAn3'

&QJ ).

strong intluence on how actively a particular open-reading frame is tran slated~ high complementarity and proper spacing pmmotcs active translation. wherea..~ limited complementarity and/or poor spacing generally supports lower levels of translation. Some prokaryotic ORFs internal to a polydstronir message lack a strong ribosonu:! binding site but are nonetheless actively translated. ln these cases the start codon often ovedaps the 3' end of the adjacent ope.Hcading frame (most often as the sequence 5'-AUGA-3'. which contains a stm1 and a stop codon). Thus. a ribvsome that has just completed translating the upstream opan-reading frame is appropriately positioned to begin translatmg fmm the start codon 'foe the downstream open-reading frmne. circunwenting the need for a ribosome binding site to recruit the rihosome. This phenomenon of linked translation between overlapping open-reading frames is known as tratlSJaliu.nal wup)ing.

Eukaryotic mRNAs Are Modified at Their 5 ' and 3' Ends to Facilitate Translation UnJike their prokaryotic counterparts. cukaryotic; mRNAs recruit ribosomes using a specific chemical modification caUed the 5' cap, which is 1ocaled
scanning. Two other features of eukaryotic mammalim1 mRNAs stimulate translation. One feature is the ptesence. in some mRNAs, of a purine

Tro115fer RNA

three hasPS upstream of tho start codnn and a gu ao.ine immediately downstream (5'-G/ANNALIGG-3' ). This sequence was Of'igi.nally identified by Marilyn Ko?..(jk and is referred to as the Kozak. sequence. Many eukaryotjc mRNAs lack these bases, but their presenr.e increases the efficiency of translation. ln contrast to the situation in proka.ryotes. these bases are thought to interact with initiator tRNA, not with the smaH rRNA. A secoud feature that contributes to effident trnnslatjon is the presence of a poly-A tail at tl!c extreme 3' end of the mRNA. As w e saw ill Chapter 12, this tail is added enzymatically by the enzyme poly-A polymerase. Pespite its location at the 3' end of the mRNA. the poly-A tail enhances the level of translation of the mRNA by promoting efficient recycling of ribosomes (as we shall discuss later).

TRANSJ.'"ER RNA tRNAs Are Adaptors between Codons and Amino Acids At the heart of protein synthesis is the ••translation .. of nucleotide sequence information (in the form of codons) into am.in n adds. This is aa:omplishcd by tRNA molecules. whkh act as adaptors between mdons and the amino adds they specify. There are many types of tRNA molecules, but each is attached to a specific amino acid and each recognizes a particular codon. or cod.o ns. in the mRNA (most tRNAs J'OCognize more than one codon). tRNA molecules are between 75 and 95 ribonucleotides in le ngth. Alt hough the exar::t sequen~;e varies, all tRNAs have certain features in r:ommon. First, all LRNAs end at the 3' terminus with the sequence 5'-CCA-3'. This is the site tl1at is atlached to the cognate amino add by the edlzyme aminoacyl

tRNA synthetase, a:-; we will c;ons~dvr below. A second striking asp ect of tRNAs is the pt·esence of severn l unusual bases in their primary structure. These unusual fcHtures are created pust-tr•.msr..riptiunaiJy by enzymatic modification of normal bases in t he polynudeotide chain. For example, pseudouridine ('I'U) is derived from uridine by atl isomerization in which the site of attachment of the uracil base to the ribose is switched ftom the nitrogen at ring position 1 to the carbon at ring position 5 (f'igure 14-3). Likewise. dihydrouridi.ne (DJ as derived from uridine by enzyma1ic reduction of the double hond between the carbons at positions 5 and 6. Other unusual hases found in tRNA include hypoxanthine, thymine. and methylguaoinc. These modified bases aJ'e not essential for tRNA funr;tion. but cells lacking these modified bases show reduced rates of growth. This suggests that the modified hases lead to improved tRNA functiou. For example. as we will see in Chapter 15. hypoxanthjne

pseodouridine

H~

)z)

0

N

~

14-3 A sllbset of modmec nudeosides found in tRNA. FIGURl

416

Translaliort

plays an important role in the process of codon tRNAs.

re~nition

by r:ertain

tRNAs Share .a Common Secondary Structure

that Resembles a Cloverleaf 1\s we saw in Chapter 6. RNA molecules typ icalJy contain regions of sf>Jf-complemcntarity that enabJo tbern to fnm1 limited stretches of double helix that 8.ff.l held together lJy base pairing. Other regions of RNA molecules have no complement and hence, are single-stranded tRNA moJecules exhibit a characteristic pattem of single-stranded and double-stranded regions (sec;ondary structure} that can be illustrated as a rloverl~f (Figure J4-4 ). ThP. prinr:ip~l f~tu res nf' the 1RNA doverleaf are an ar.ceptor stem; three stem·loops. which are referred to as the 'I!U loop. the 0 loop, and the aJJticodon loop; and a fourth variable Jnop. Descriptions o f each of these features follows:

• The acceptor stem. so-named hocause it is the site of attachment of the amino acid. is formed by pairing between the 5 ' and 3' ends of the tRNA mnJocoJe. The 5'-CC.A-3' sequenr.e at the extreme 3' e nd of the mole<~ule protrudes from this d ouble-stran d ed stem. • The 'l'U loop is so-named because of the characteristic presence of the tmusua) base 'l1 U in the loop. The modified base is often found within the sequence 5'-T\11UCG-3'. • The D loop takes its name from the characteristic presence of !!ihydrouridines in the loop. • The antkode)n Juop. as its name lmpJies, contains the anticodon, a three-nudootide-Jong decoding e1ernent that is responsible for recognizing the codon h y base-pairing with the mRNA. The anticodon is bracketed on the 3' end by a pwine and on its 5' ond by ur-acil. • The variable loop sits between the anlir.odon loop and the 'IJilJ loop , and. as its name implies. varies in sizt> from 3 to 21 bases.

fIGURE

3'

14-4 c:Jovelfeftf representation

of the secorad«y str~e of UlNA. fn ltlrs representatiOn ot a (RNA, the~ OC-'tWeen dlffererrt patts of the tRNA <~re- •trlcated by the dotted red tines.

5'

acceptor arm

oroop varH>Ie loop

1\ttochment of /lmillCJ Ac:ids to tRNA

a

3'

b

c

417

\jiU loop

I ..._acceptor

arm

F I (j U R f 14..5 Conversion between the dover1eaf ilnd die .ctu.t ttwee-dimensionat sbvcturr of a tRNA. {a) doverleaf represenlatloo. (b) l-shoped representatiOn showing the location of the basepalf'ed reglOOS of the finat folded IRNA. (c) fQbboo f€Jlfesent.atl0n ot &lC actual told~ struaure of a M-JA 1\'ot<; that although th(s diagram illustrates how the «tua1 tRNA structure IS related to fhe dovede.:l repreS€flt.1b0n,

a tRNA does not att31fl its hnatstructure b) first Wse-pal~ and then fokf111g 1nto .aol-4-.ape..

tRNAs Have an L.Shaped Three~Dimensional Structure The cloverleaf reveals regions ot' self-complomenlarity withitl tRNAs. Whot is the actual three-dimensional configuration of this adaptor molecule? X-r ay crystallography reveals an L-shAped tertiary structure in which the terminus of the acceptor stem is Rt one end of the molecule and t11e anticodon Joop is ahout 70 A 3\1\'ay at the other end. To understand the relationship of this L-shaped structun~ (depicted as an upside-down L in Figure 14-5) to the cloverleaf. consider the following: the acceptor stern and the stem of the ~U loop form an 9JI.1ended helix in the fina) tRNA structure. Similarly, the anticodon stem and the stem of the D Joop fonn a second extended helix. These two extended helices align at a right angle to each nther, with the D loop and the 'l!U loop coming together. In the final irnage, the two extended helices adopt l.beir proper helical configuration. Three kinds of interactions stabilize this L-shaped structure. The first is hydrogen bonds between bases in different h elical regions that are brought near each other in three-dimensional space by the tertiary structure. These are generally unconventional {non-Watson-Crick} bonding. The second are iuteractions between the bases and the sugar·pbosphate backhone. The third kind of stabilizing interaction ls the adrliUonal base stacking gained from formation of the two extended regions of base pltirilll$·

ATTACHMENT OF AMINO ACIDS TO tRNA tRNAs Are Charged by the Attachment of an Amino Acid to the 3' Terminal Adenosine Nucleotide via a High-Energy Acyl linkage tRNA molecules to which an amino acid is attached are said to be charged, and tRNAs that Jack an amino acid are said to be uncharged. Charging requires an acyl linkage between the carboxyl group of the

418

Tl't111skrrion

amino add and the 2'- or 3'-hydroxyl group (see below) of the adenosine nucleotide that protrudes from the acceptor stem. Trus acyl linkage is considered to be a high-energy bond in that i~ hydrolysis results in a large change in free energy. This is significant for protein synthesis: the en ergy released when the bond is broken helps drive the formation of tho peptide honds that link amino acids to each other i n pnJypeptide chains. as we will sP.C boJow.

Aminoacyl tRNA Synthetases Charge tRNAs in Two Steps All aminoacyl tRNA synthetases attach an amino acid to a tRNA in two enzymatk steps (Figure 14-6}. Step one is adenylyJation in which

the amino add reacts with ATP l o heoome adenylylated with the con-

14-6 The two steps of arinoK)'J-tRNA dta.ging. (a) Acleny¥afion fJC:OURE

of amino aad. (b} li'ansfer of the aden~ amino i!Cid to tRI\JA. The process shown IS for

a R

NH.,z-

a class II tRNA synthetase

J0

0

I II .. C - C - <J I

+

0II

0II

0I

o

o-

cr

-o - j-(l- r-o-

H

1

-o~

~

AlP

amino acid

OH 3'

l R

NHt-

0

I II c- c-

W

+

o-P-o

!-

I

H

adenylylated amino acid

0

0

o-

0

II ft -o- p-o- P-o1 I

OH '5

b adeny1ylated amino add

N-~z-J-~-o-~-o~ . I !. . H 0

R

0

OH 3'

l R

0

I I

II

~ bond

\

~- c - e-o ~~

H

+

0

~

~-t-0\:? OH 3'

J ABlE

14•1 Clases of Amino.qf tRNA synthetases*

ClassU

Quarternary Structure

Class I

euartemary Structura

Gly Ala

(o~~

Glu

Gin

(a ) (a)

Arg

(a)

Cys tvlel

(a:J

fht-

(a ..) (o.2) (o2) (n:o)

HIS

(a.-)

A~p

(Cl,.) (a t ) (a l)

l eu Tyr

(u2) (u) (u) (a ) (u)

(a~.,)

Ttp

(a)

Pru Ser

Asn Lys Phe

Val lie

SQurce. Oata fran Oelarue M. 1995. Amlnoaeyl. AltA syntnetases. CUrent 0plfli(Jnlfl SfnJCtvraf BID/cgy 5: .48 55. acapled from Table 1 "'ass J en7)1111es BIO generally mcrorroerie. wtJereas Class P enzymes ~re ciWT!enc or teti<\ITlel!c. \.'11th res~s fru n two SlAlur-11.5 contrtb\Jtirlg to ll'le t:Jndl"'g site •~ ra si'lgle lANA o and prefer to IIIAJU'ils cllhe lANA ~s and the subsl::np\S f'dicale t1lw sl.o4«;hiometfV

oomitant release of pyrophosphate. Adenylylation refers to transfer of AMP. as opposed to adenyJation. which w ould indicate the transfer of adenine. As we have seen in the case of polynucleotide synthesis {see Chapter 8), the principal driving force for the adenylylatinn reaction is the su bsequent h ydrolysis of py rophosphate by pyrophosphatase. As a result o f' adenylylation, the amino acid is attached to adeuyJic acid via a high-energy ester bond in which the onbonyl group of the amino acid is join ed to the phosphoryl group of AMP. Step two is tRNA charging in which tho adenylylated amino acid, \hthich remains tightly bound to the synthetase. reacts with tRNA. This reaction resu lts in t.he transfer o f the amino acid to the 3' end of the tRNA via the 2'· or 3'-hyd.roxyl nnd the concomitant release of AMP. There are two classes of tRNA synthetases (Table 14-1}. Class I enzym es attach the amino acid to the 2'0H of the tRNA and are generally monomerit.:. Class n en zymflS a ttach the ant.ino add to the 3'01"1 nf the tRNA and are typically dimeric or tetrameric. Although the iniliaJ coup ling between the lRNA aud the amino <1cid a.re diftereot, uuce released from the synthetase, the a mino acid rapidly equilibrates hetween attachment at the 3'0H and the 2' 0H-

Each Aminoacyl tRNA Synthetase Attaches a Single Amino Acid to One or More tRNAs Each of the 20 amino acids is attached to the appropriate U
420

1rcrru;lation

amino acirt Instead, a singiP. spP.C~i es of aminoac:yl !RNA synthetase charges tRNAGtn as well as t RNAGJu with glutamate. A second enzyme then converts (hy aminatiou} the glutamate m oiety of the charged tRNAGtn molecules to glutamine. That is, CJu-1RNACln is a minated to GJ n-tRN N-~1 " (the prefix idenlifies the amino acid anrl the superscript identifies the na1 ure of the tRNA}- The presence of lhis second e nzyme removes the need for a gJulamine t RNA syn· thetase. Nevertheless, a n amjnoacy) tRNA synthetase cau never attach rom-e than one kind of amino acid to a given tRNA.

tRNA Synthetases Recognize Unique Structural Features of Cognate tRNAs As we can see from the above considt>.rnt ions. aminoacyl tRNA synthetases face two in'lportant chaJJenges: they m ust recogui7.e the corre<:t set of tRNAs for a particular amino add, and they must charge all of these isoaccepti ng tRNAs with the correct ami.no add. Both processes must be carried o ut with high fidelity. Let us fll'st consirler the specificity of tRNA recognition: w hat features of the t RNA molocu le enable a synthetase to discriminate cognate, isoaccepting tRNAs from the tRNAs for the other 19 amino acids? Genetic. biochemical. and X-n'ly crystallographic evidence indicate tbat the spocifidty determinants are clustered at two distatlt sites on the m olocule: the ar.ceptor stem and tho antir.ndon loop (Figure 14 -7)_ The acceptor stem is an especially important determinant fr1r the specificity or tRNA synthetase recognition . In some cases changing a single base pair in the acceptor stern {a particular base pair known as the discriminator) is sufficient to c.onvcrt the recognition s pecificity of a tRNA from one synthetase to another. Nonetheless, the anticodon loop frequently contributes to discrimination as welL The synthetA.se for glutamine, for example. makes numerous contacts in both the acceptor stem and across the anticodon Joop. induding the an ticodon itseJf (Figure 14-8).

- ----- -- ----

Fl G IJ Rl 14-7 strum.e of tRNA: elements requftd fo, aminoacyl

synthetase ret:ognition.

3'acooptor end

\

llttacbmanl of Amino Adds to tRNA

4Zl

FIG U R l 14- 8 Co-crystal 5truc:t1.1re of glutaminyl aminoacyf tRNA syntMtase with tRNAe~t~. The enzyme fS sllown 111 gray and tRNAQ

1S sholllin 1n

pu!ple. The yellow,

red.

and green moleo.Jie IS glufilmlf1\A MIIP. Note the proximity of thas moleOJie to the 3 • end of the tRNA and rhe poV-lls of c<Jntact between the ffiNAand the synthetase (Roth V.L, S~llkln LF., ~ B, Sproat B.S.,llnd Stectz TA l998. 5truaure 6 : 439 - 449.) Image !"t;>aredwith &bScnpt, Mol<\a~pt,
You might expect that the anticodon would almost always be used for recogniUon hy tRNA synthetases because it is the ulUrnate defining feature of a tRNA- the anticodon dictates the amino acid that the t&"lA is responsible for inrorpomtinp, into the growing polypeptide chnin. However, because each mn.ino acid is usuaJly specified by more than une codon, recugnition of t he antic:odon cannot be used in all cases. For example. the a mino add serine is specified by sb: rodons, including 5 '-AGC-3' and 5' -UCA-3', which are completely difterent from one QJ:lother. Hence, the tRNAs for serine Jlecessarily have a variety of different antir.odons, whicb r..ould not be easily rec..ognized by a single tRNA synthetase. So. to recognize its tRNAs, the synthetase for serine tl'\U$1 rely on detenninru•ts that lie outside f,lf the anticQdou . The set of tRNA determinants that enable synthetases to discriminate among tRNAs is sometimes referred to as the "second geneUc code'· because of its centcal importance li1 information flow, As we discussed above. th is code is significantly more complex than the ..first genetic code,. and cannot be readily tabula1ed. Without such a oode, synthetases oould not distinguish one tRNA from another, and the tran slation machinery would not produce po ly peptides w ith a reproducible sequence.

Arninoacyl..-tRNA Formation ls

V~ry

Accurate

Tho chaHenge faced by aminoacy] tRNA synthetases in se]ecting the correct amino add is perhaps even more:~ daunting than the challenge the ~nzyme faces in recognizing the appropriate tRNA (Figure 14-9). The reason for this is the relatively smaJJ size of amhto adds anti , in some cases, their similarity. Despite this cballenge, the freq uency of mischarging is very low ; typically. Jess than 1 .in 1,000 tRNAs is charged with the i ncorrect amino acid. ln certain cases it is easy tn Wlde.rstand how this h igh accuracy is achieved. l-'nr example, the amino adds rysteine and tryptophatl differ suhstanllaUy in size, shape. and chemical groups. Even in the case of the similar-looking

Tronslction

422

b CH 3

'

~2. / Cfia

Ctfl

Q-i

CH

I

I

/ c"H

~N"

CH!

'\/

COati.

~

/c"'\:H H,.N

COOH

valine

~ l GU R IE 1 4-9 Oistinguishing feah..lres of similar.amino acids..

amino acids tyrosine and phenylalanine (see J<'igure 14-9a}, the opporttmity for forming a strong and energeticaUy favorable hydrogen bond with the hydroxyl moiety of the former but not the latter allows the synthetase for tyrosine (tyrosyJ tRNA synthetase) to discdm.inate effectively against phenylalanine. (t is more challenging to understand the case of isol ~udne and \ta}ine. which differ by only a s ingle rnethylene grnup (see Figure 14-9b}. Valyl lRN/\ synt hetase can sterically exclude isoleucine from its catalytic pocket because isoleur.ine is larger than vali-.e. ln con· trast, valine should slip easily into the catalytic pocket of the iso1eucyJ tRNA synthetase. Although both amino acids will fit iilto the synthetase amino acid binding site, interactions with the extra methylene group on isoleucine wiU provide an extra - 2 to - 3 kcallmol of free energy (see TalJle 3-1). As we described in Chapter 3, even this relatively small difference in free energy w il1 make bind1ng to isoleucine approximately 100-fnld more likely than binding to valine. if the two amino acid s are present at equal concentrations. Thus, valine would \Je attached to isoleucine tRNAs approximatP.ly 1% of the time. however, this is an unacceptably t.igh rate of error. As we have seen, the actual hequenr.y of misincorporation is < 0.1 %. How is such hi~h fidelity achieved?

Some Aminoacyl tRNA Synthetases Use an Editing Pocket to Charge tRN As with High Accuracy One common mechanism to inc:rease 1he fidelity of au aminoacyl tRNA synthetase is to proofread the product·s of the c.hargiug reaction as we hflve seen for DNA polym.e rases in Chapter 8. For example, in Hddition to its r.atalytic pocket (for adenylylation), the isoleucyl tRNA synthetase has a nearby editing pocket (a deep deft in the ell7.yme) that allows it to pmofread the product of the adenylylation reaction. AMP-valine (as well as adcnylylates of other sn1aJJ amino acids, sucb as alanine} can fit into this editing pocket. where it is hydrolyzed and released as free valine and AMP. In r:ontrast, AMP-isoleuciue is too large to e nter the editing pocket and h£Juce is not subject to hydrolysis. Therefore, the editing pocket is a molecular sieve that exdudes AMP-isoleucine but not AMP-,raline. As a consequence, isoleucyl tRNA synthetase is able to discriminate against va line twice; in the initial binding and adenylylation of the amino acid [discriminating by a factor of approximately 100), and then in the oditi11g of the atlonyly)atecJ amino acid (again discriminating by a factor of approximately 100), for an overall selectivity of approximately 10,000-fo}d (that is, an eJTOr rate of approximately 0.01 % ).

The Ribosome Is Unable to Discnminate between Correctly and Incorrectly Charged tRNAs The reason that so much responsibility faUs on aminoacyl tRNA synthetases to ensure that the proper amino add has heen attached to the proper tRNA is that no further discrimination takes place after· the chArged t RNA is released from that enzyme. In other words, the ribosome "btlndly'' accepts aoy charged tRNA that exhibits a proper codon-enticodnn interaction, whether OT not the tRNA caJ't'i.os its cognate amino add. This conclusion is supported by two kinds of e>.periments: one genetic and the other biochemical. The genetic experiment involves

8ox ,._, Selenocysteine

Certatn p-oteins, suCn as fue enzymes ~ooe pet~ and formate dehydrogenase, contain an unusual amino aod (ailed selerlocysteine, whi
req..Y.res ~e p~ of a ~al sequenre etanent elsewhere 1n the fi1RNA Thus, selenocystetne can be "thought of as a 21st cn1ino acid that is incorporated ttto proteinS by a modificatJOrl of the standard translation mach&necy d the cell

BOX

14•1

FI
I ThestnJCtures

of cysteine and sefenocystei~.

the iSolation of a mutant tRNA that carries a nucleotide s ubstitu tion in the amicodon. Recall that tRNA synthetases frequently do not rely on interaction with the anticodon to r ocognizc cognate tRNAs. J fence. a subset of tRNAs can be muta ted in the ir enticodon s but st ill he charged w i1h their usual cognate amino adds. As a consequence uf the anticodon mllt atinn~ however, the mut8Ill tRNA c.leJivers its amino acid to the wrong codon . ln other words, the r ibosom e and the auxiliary prote ins that work in conjunction with t·he ribosome (whkh we will discuss shortly) primarily check that the charged tRNA makes a proper codon-anticodon interaction with the mRNA. The ribosomA and these proteins dn liUie to prfw P.nl an incorrectly chargecl tRNA from adding an inappropriate amino add to the grrrwing pf)lypeptide. A dassic bitx.:h emical experiment nice ly illustrates the point tha t the r ibosome recognizes tRNA and nut the amino acid that it is carrying. Consider the charged tRN A cysleinyl-tRNACyl< (remember that the prefiX identifi es the amino acid and the superscript identifies the n ature of the tRNA). The cysteine attach~d to cysteinyl-tRNAcys can be converted to au alanine by chemirnl reduction to give alanine-tRNACys (Figure 14 -10). Whe n a dded to a cell -free proteinsynthesizing system , alanin e-tRN Ac:Y~ introduces alanines at codo ns that s pecify ins ertion of cys teine. Thus, the trnns )atiuu machiue ry relies on the high fi.dcHty of the aminoar;y) tRNA synthetases to ens ure the ar.curate d ecoding uf each mRNA (see Box 14-1. Selenocy steine).

cysteine

SH-

NH2 H

0

C-

C

I

I

C-

II

'

H

alafline NH2 H

THE RIBOSOME The .ribosome is the macromolecular machine that d trects the synthesis of pwteios. Consistent with the additional cha11enges of translating a nucleic add c:ode into an amino add code , fhe ribosome is larger and more complex than the minimal machinery required fur DNA nr RNA sy ntht.>sis. Indeed, single p olypeptides can perfurrn DNA or RNA

H-

0

f I U c - c - c -41r.llll.,

I

H

alaoine-lRNA

F I G U R E 14-I 0 C)l51eit't)1·tRNA charged w1th c 01' A.. Chemu::
424

1lvmslation

synthesis (although DNA replication and transcription are also often mediated by larger multisubunit complexes). ro contrast, the mdchinery for polymerizing amino adds is composed of at least tl1ree RNA molecules up to about three kilobases in size and more than SO dHferent proteir1s, with an overall molecular mass of greater than 2.5 mcgadaltons. Compared to the speed of DNA replication- ZOO to 1.000 n ucleotidos per second- translation takes place at a rate of only 2 to 20 amino acids per second In pro karyotes, the transcription machinery and the tronslation machinery are located in the same compartment. Thus. the ribosome can commence translation of the mRNA as it emerges from the RNA polymerase. This situation allows the ribosome to proceed in tandem with the RNA polymerase as it elongates the transcript (Figure 14-11). Recall that 'he 5' end of an RNA is synthesized first, and thus transla· lion, which also starts at the 5' end of the rnRNA. can conunence on nascent transcripts as soon as they emerge from the RNA polymerase. Interestingly. there are several instances in which the coupling of transcription and translation is exploited during the regulation of gene expression, as we shall see in ChapteT 1£:1. Although s low relative to DNA synthesjtl in prokaryotes. the ribosome is capable of keeping up with l11e transcription mat:hinery. The typical prokaryoHc rnte of translation of 20 amino acids per second corresponds to the translation of 60 nucleotides (20 codons) of mRNA per second This is similar to the rate of 50 to 100 nucleotides per second synthesized by RNA polymeracre. In contrast to the situation in prokaryotes. translation in eul<ary· otes is com pletely separate from transcription. Indeed. these events occur in separate compartments of the cell: transcrip tion OCL'UTS in the nucleus. whereas translation occurs in the cytoplasm. Perhaps due to the lack of coupling to transcription, eukaryotic tra.nslation p:roceeds at the more leisurely speed of 2 - 4 amino acids per second..

RNAP

14-11 Prokaryotic RNA polymerase and the ribosome at work JIG V R E

on the same mRNA.

Tlw Ribowme cefltrifuge

425

F I
rillosomar sut>oots

The Ribosome ls Composed of a Large and a Small Subunit The ribosome is composed of two subassemblies of RNA and protein known as thc> large and small subunits. The large subunit contains the peptidyl transferase center, which is responsible for the Formation of peptide bonds. The small subunit contains the decuding center in which charged tRNAs read or "decode" the codon units of the

mRNA By convention, the large and small subunits are named according to the velocity of their sedimenlation when subjected to a centrilugal force (Figure 14-12). The unit used to measure sedimentation velocity is the Svedbe~ (S; the larger th<~ S value the faster the sedimentation ve1ocit:y), which is named after the inventor of the ultracentrifuge, Theodor SvedbP.rg. In bacteria the large subunit has a sedimentation velocity of 50 Svedbet-g units and is aC~;ordjngly known as the 50S subunit, whereas the small subltilit is called the 30S subunit The intact pmkaryotic ribosome is refeu ed to as the 70S ribosome. Notice that 70S is le..c;s than the sum of 50S and 305! The explanation for this apparent discrepancy is that sedimentation velocity is determined by both shape and size and hence i.s not a measure of ntass. The eukaryot ic noosome is somewhat larger. composed of 60S and 405 subunits. which together fonn a n 80S ribosome. The large and small subunits are each composed of RNA known as ribosomal RNAs, and many ribosoiDill proteins (Figure 14·13). Sve
almost 1,000 kDa. The Large and Small Subunits U ndergo Association and Dissociation during each Cycle of Translation C-entral to the mechanism of translation is a cyde in which the small and large subunits of the ribosome associate w ith each othct and the mRNA, translate the target mRNA., then dissociate after each round of protein synthesis. Th is sequence of association and dissociation is

Translation ,.._ 5.8S rRNA

I' I G U ~ E 14-JJ Composition of the prokaryotic and eukaryotic ribosomes. The rRNA and prore1n caT'fXlSifion of the rltffesert subunits are •ntitc.ate<:l. The sizes of the !RNA

am the J1I.JITiber of proteir6 are lrldicated.

•tn (160 nucleotides) 60S (MW"' 2 ,800,000)

.._......., ~

5S~NA

G-(120 OUdeelides)

eukaryotic

ribosome 28SrRNA

80S

(4.700

(fvlW= 4.200,000)

nucteolides)

b

' - - - - • 49 proteins

tv

40S

18S rRNA

1 ,400.000)~ {1,000

c=:::>

(Jv!W=

nucteotides)

- 33 proteins

S

50S (MW= 1 ,600,000)

prol<aryotic r1bosome

_..r-.

70S {MW:. 2.500.000)

i

SS~NA

{120 nucleotides)

23SrRNA

(2,000 nudeotides)

'-------.• - 34 proteins

30S (MW= 900.000)

~

-L

16SrRNA (1 ,540 f1Ucleobdes)

21 proteins

known as the ribosome cycle (figure 14-14}. Briefly, translation begins with the blndinf?, of the mRNA and an initiating t RNA to a free, small subunit of the ribosome. The small s ubunjt-mRNA complex then recruits a large subunit to create an intact ribosome with the rnJU~A sandwiched between the two suhunits. Protein synthesis is initiated in the next step. conunencing at the start codon at the 5' end of the message and progressing downstream toward the 3' end of the mR.'I\IA. As the ribosome translocates from codon to codon, one charged tRNA after another js slotted into the decoding and peptidyl trans ferase

centers of the ribosome. When the elongating ribosome encounters a stop codon, the now completed polypeptide chain is released, and the ribosome disassodates from the mRNA as separate large and small subunits. The separated subunits are n ow available to bind to a fresh mRNA molecule and repeat the cycle of protein synthesis. Although a ribosome can synthesize only one polypeptide at a lime. eoch mRNA can be translated simultaneously by multiple ribosomes {for simplicity Jet us assume that the message we are considering js monocistronic). An m.RNA bearing muJH p1e ribosomes is known as a polyribosome or a poly.smnt: (Figuro 14-15). A single ribosome is in con·

The Ribosome

'FIGURE 14-14 Overview of the events of translation.

tact with approximately 30 nucleotides of mRNA but the large size of lhe ribosome only alJows a density of one ribosome for every 80 nuclootides of mRNA. Thus. a single mRNA molecule is able to direct the simultaneous synthf.'.sis of multiple pol~ypeptidcs using an array of ribosomes. The ability of multiple ribosomes to function on a single mRNA explains the relatively limited abundance of mRNA in the cell {typically 1-5% of total RNA). lt' an mRNA could be translated by only one ribosome at a time, as few as 10% of the ribosontes would be engaged in protein synthesis at any time. Instead. the association of multiple ribosomes with ear::;h mRNA indicates that the majority of the ribosomes are engaged in translation.

New Amino Acids Are Attached to the C-Terminus of the Growing Polypeptide Chain As we know, both polynucleotide and polypeptide chains have intrinsic polarities. Thus, for each of these molecules we can ask which end of the chain is synthesi:zed first. We learned in Chapters 8 and 12 that

4Z'1

TranslatiCIIl

428

polypep~

chairl

H 0

I 11 H ---N- e - e , I 1 1 N- c - R H

...

I

~ - e- R

f ~ ~- I H C=o C=O

R

I)

I

0

0

ONA and RNA affl synthesized by adding P.ach new nucleotide triphosphate to the 3' end of lhe growing polynucleotide ch~ in (olten refecred to as synthesis jn the 5' - 3' direction). What i.s the order of synthesis of a growing polypeptide chain? This was first determined in a classic experiment performed by Dint.zis that was described in Chapter 2. This experimt-nt found that each new amino acid must be added to the C-tcrminus of the growing polypeptide chain {often referred to as synthesis in the N- to C-terminal direction). As described in the next section, this directionality is a direct result of the chemistry of protein synthesis.

Peptide Bonds Are Fonned by T r.msfer of the Growing Polypeptide Chain from One tRNA to Another

H

The ribosome catalyzes a single chemiaJl reaction; the formation of a peptide bond. Trus reaction oc£Urs between rho amino acid residue at the carbo11:y-tennina l end o f the growing polypeptide and the incoming amino add to be added to the chain. Both the growing chain and the incoming amino acid are attached to tRNAs; as a rest~lt, duting peptide bond formation, the growing polypeptide is continuously attached to a

1

0

I II R 0 ~ t II I I I 'N-c-c- N-c H R I I I \

--N-C- C

H

H

H

c =o I

0

Ot'\

F I CURE

14-16 Thepeptidyt

bansfeta!e readicn.

tRNA.

The actual substrates for each round of amino acid addition are two charged species of tRNAs-an aminoacyl-tRNA and a peptidyJtRNA. As we discussed earlier in this chapter {see the sec.iion, Attachment of Amino Acids to tRNAs) the aminoacyJ-tRNA is attached at its 3 ' end to the carboxyl group of the amino acid . The pt!p&idyllRNA is attached in exactly the same manner (at its 3'end) to the tarboxyJ-terminus of the growing polypepUde chain. The bon d between the aminoacyl-tRNA and lhe amino acid is not broken during the formation of the next peptide bond. lnstead , the 3' ends of these two lRNAs are brought into close proximHy to t;Jach other on the ribosome. This positioning allows t.he amino group of the aminoacyJtRNA to attack the c.arbonyl group of the most carboxyl-terminal amino acid attached to the peptldyl-lRNA to form a new peptide bond (Figure ~4-16). There are t wo consequences of this method of polypeptide synthesis. First. this rnechanism of peptide hond fomtation requires that the N-terminus of the protejn be synthesized befon! the C-terminus. Second , the growing polypeptide chain is trans· ferred from the peptidyl-tRNA to the aminoacyl-tRNA. For this reason. the reaction to form a new peptide bond is called the peptidyl transferase reaction. fnterestingly. peptide bond formation takes place wj,hout the simultaneous hydrolysis of a nucleoside triphosphate. This is because peptide bon d funuation is driven by b reaking the hlgh-energy acyl bond that joins the growing polypeptide chain to the tRNA. You will recall that this bond was created d uring the tRNA synthetase-catalyzed reaction that .is responsible for charging 1RNA. Tbe charging reaction involvas the hydrolysis of a molecule of ATP. Thus, the energy for peptide bond formation originates from a molecule of ATP that was hydrolyzed during the tRNA charging reaction (Figure 14-6).

llibosomal RNAs Are Both Structural and Catalytic Determinants of the Ribosome Al though the ribosome and its basic functions were discovered more than 40 years ago. the recent determination of the high-resolution.

The Ribot>crne

14- 17 Two .iews of lhe ribosome. The 505 Slb.nt IS ~ the 305 ~~ lfl both fhe caYJty between the 50S and 305 S\Jbulits in the rl8f1t hand imaae rEpreserts the Site of IRNA as-soaaba'l (see rJ8Ure 14 19) l he RNA canponent of lhe 505Slhne 1s shov..n 10g~ and the protetn compooent is shown 10 pt.lfpk. The RNA~ of the 305 Sthrit IS sl1own K'1 '&f1t blJ£: and the PfO t8\ component 10 &rk bLe. (Yusupov MM., Yusupova G.Z., Baucom A.., Ueberman K . Earnest 1.111., Ca1e J.J-i , Md Nollef H.F :?001 5oence 292: 883.) Images Pfep.'!red ~h ~. Bob.Scnpl
\hree-di.mensionaJ structure of the prokaryotic ribosome has vastly increased onr understanding of the workmgs of this molP.cuJar machine (Fjgure 14-17). Perhaps the most important outcome of these studj~ is lhe definitive d ('-lffionslralion that ribosomal RNAs are not simply structural components of the ribosome. Rather, rhey are directly responsible for tbe key functions of the ribosome. The most obvious example of this is the demonst:rntion that the peptidyl tr!'lnsferase center is composed entirely of RNA. as we will discuss in deta il below. RNA also plays a central role in the function of the small subunit of the ribosome. The anticodon loops of the cha:rged tRNAs and the codons of tl1e mRNA contact the lGS rRNA, n ot thE> ribosomal proteins of the small subunit. A further indication of the importance of RNA in t he structure and function of the ribosome is that most ribosomal protejns are on the periphery of the ribosome, not in its interior {see Figure 14-19). The core functional domains of the ribosome (the ~tidyl transferase center} are composed either entirely or mostly from RNA Portions of some ribosomal proteins do reach into the core of Ihe subunits, where their function seems to be to stabilize the tightly packed rRNAs by shielding the negatiw ch arges of their sugar-phosphate backbones. Indeed. it js likely that the con lempomry ribosome evolvod from a primitive protP.in·synthesizing machine that was composed entirely nf R.l\IA.

The Ribosome Has Three Binding Sites for tRNA To carry out the pcptidyl transferase reaction, the ribosome must be able to bind at least two tRNAs simultaneously. In fact. the ribosome contains

429

1hmslaticm

14-18 1'he ribosome has three tRNA binding sites. The schematic ittustratioo of the nbosome shows the three ~ng sites ((, P. and A) that span the two submts. f I G U It E

throe tRNA bind ing sites, caJied the A, P. and Esites (Figures 14-18 and 14-19). The A site is the binding site ior the am.inoacylated-t.RNA, the P site is the binding site for the peptidyl-tRNA, and the E site is the binding site for the tRNA that is released after the growing polypeptide chain has been tm.nsferred to the aminoacyl-tRNA (E is for exit). Each tRNA binding site is formed at the interface between the large and the small subunits of lhe ribosome (Figure 14-lga and b). In this way. the bound tRNAs can span the distance between the peptidyl transferase center in tl1c large subu.rUt {Figure 14·19cJ and the decoding center in the small subunit (Figure 14-19d}. The 3' ends of the tRNAs that ate coupled to the amino acid or to the growing peptide chain are adjacent to the large subunit. The anticodon loops of the bound tRNAs are located adjacent to the small subunit.

Channels through the Ribosome Allow the mRNA and Growing Polweptide to Enter and/or Exit the Ribosome Both the decoding center and the peptidyl transferase centt:r are buried within the intact ribosome. Yet, mRi'JA must be threaded through the decoding center during translation , and the nascp,nt polypeptide chain must escape from the peptidyl transferase renter. How do these polymers enter (in the case ofmRNA) and exit the ribosome? The answer is provided by the structure of the ribosomE>,. which reveals "tunnels"' in and out of the ribosome. The mRNA enters and exits the decoding cenhrr through two narrow channels in the smaH subunit The entry channel is only wide enough for unpaired RNA to pass through. This feature ensures that the nlRNA is in an extended form as it enterS the decoding center by removing any intramolecular base-pairing intm·actions that may have formed in the mRNA. In between the two channels is a region that is accessible to tRNA$ and where adjacent codons can bind to the arninoacyl-tRNA anti peptidyl-tRNA in the A and P sites, respoctively. fnterestingly. there is a pronounced ~ink in the mRNA between the two codons that facilitates maintenance of the correct reading frame (Figure 14-20). This kink p lace; the va<;ant A site codon created after a cycle of ribosome translocation in a distinctive position that prevents the incanting aminoacyltRNA from aC(:f'.ssing bases immediately adjacent to the codon. A sec.:OJid channel through the large subunit provides an exit path for the newly synthesized po1ypeptide chain {Figure 14·21). As with the mRNA c hannels. the size of the channel limits the folding of the growing pOlypeptide chain. ln this c:ase, the polypeptide ca.n form Cln a heUx within the channel but other secondary structures {such as ~ sheets) and tertiary interactions a:tn only farm after the polypeptide exits the 1ar:ge ribosomal subunit. For this t'OOson. the llnal threedimensional structul'e of a newly synthes~d pmtein is not attained until after it is re)f'.<'tSed frorn the ribosome. Now that we have described the four primacy cmupommts of the llanslation process. the remainder of the chapter wiU focus on the individual stages of translation in more detail. Our description will proceed in order through the throe stages of translation: initiation of the synthesis of a new polypeptidP. chain. elongation the growing polypeptide. and termination of polypeptide synthesis. As we wnl see. lhere arc important similaritlt~ W1d djfff~rcnces between probuyotes and eukaryotes in the strategies they employ to carry out protein syntllesis. We shall cons,ider the nature of the translation mru;hinery from both kinds of cells in each of the following sections. As we have seen for DNA and RNA

or

The RihoSCJrne

a

b

c

d

.FIGURE 14-19 \liews of the three-dimensional structure of the ribosome indudingthree

baund tRNAs. The E, P. and A site tRNAs are shown II'\ yellow, red. and Efeef'l respe<:tively. The colors

representing the RNA ilOd protein ~ of lhe smaft artd large Slh.rits i'lfe the same as those in f1gute 14-17 (a) and (b) Two vievvs CJf the ribosome bound to the three tRf\JA!, 111 the E. P, and A Sites. Note tr.at the left (a) Clf1d right (b) \liews shown here correspond lo lho!"of! viev..rs or the rlbosorne shown in f 'SL'e 14-17. (c) The ISolated 505Slb.Jnrt bound to tRNAs. The peptJdvt transferase center I!> orded {d) lhe ISOlated 305 subunit bound to lRNAs The decoding cer1ter IS circled. (VLJS4JOV M.M., Yusupova G2, Baucom A.. lteberman K., Earnest lN., Cate lH~ and Noller f-lf. 2001 Soence 292: S83.) Images prepared IMth MoiScnpt. l3obScnpl, and Raster 3D

431

432

Translation

14--20 Theinteraction between lhe A site and P site tRNAs and tbe mRNA within the ribosome. Two views FICURE

of lhe structute of tt1e mRNA and tRNAs are stx:.v.Kl as 1hey are W1d &n the tibosome. R)r d
in the lower lrl1age. (YUSl.pOII MM., Y~.J5t4>0Va Gl.. Bau::an A, lieberrnoo K. E.amest Ut, CateJ.H, and Noller H.F. 2001. Soen<:e 292: 883.} Image pepared W!th MoiScript. Bobsoipl, and Raster 30.

synthesis. although the ribosome is the center of activity. a uxiliary factors play critical functions in each of the steps of translation and are required for protein synthesis to occur in a rapid and acr;urate fashion.

INITIATION OF TRANSLATION For trans1ation to be successfully initiated, three events must occur {Figure 14-22). First. the ribosome m ust be recruited to the rnRNA. Second. a charged tRNA must be placed into the P s ite of the ribosome. T hird, the ribosome must he precisely positioned over the start F I CUll E

14-21 The polypeptide exit

tu1nel In this image the 50S subunit tS cut in

hal ro reveal the polypeptide exit tunnel. The .mAtS stlO'Mllll wh1te and the ril>osomal prolelnS Ne shown in yellow The three bou'ld 1RNAs are colored as follows: E-sltc (brOIMl}, P-5ite (pwple} and A-site (greeo).The red cYid gold p
lnitjafion cf TronsJqtinn

433

OO'Ion. The correct p(1Sitiolling of th(' ribosome over the s1art codon is critical, because this establishes the reading frame for the transl~tt.ion of the rnRNA. Even a one-base shift in the location of tlte ribosome would resul1 in the synthesis of a completely unrelated po lypeptide (see the discussion of messenger RNA above and in Chapter 15 ). T he dissimilar strudures of prokaryotic and eukaryotic mRNAs resu lt in c\istjnctly different means of accomplishing these events. We will start by addressing the initiation events in prokaryotes and then discuss the differenc~ observed 1n eukaryotic cells.

Prokaryotic mRNAs Are Initially Rect"Uited to the Small Subunit by Base..Pairing to rRNA The assembly of the ribosome on an rnRNA occurs om~ subunjt at ll time. Tne small subunit associates ·with the mRNA first. As we discu.-.sed eaJ'. lier, for prokaryotes the association of the small subunH with the mRNA is mediated by base-pairing interactions between the ribosome binding site and the 16S rRNA (Figure 14-23). For ideally positioned ribosome binding sites. the small subunit is positioned on the mRNA such that the start codon will be in the P site wllen the large subunit joins the com~ plex. The large subunit joins its partner only at the very end of the initiation process, just prior to the formation of the first peptide Land. Thus, many of the key events of translation initiation occur in the absenCP. of the full ribosome.

!

fl . . .r'-~~~---

~-...3'

A Specialized tRNA Charged with a Modified Methionin~ Binds Direcdy to the Prokaryotic Small Subunit Typically chargad tRNAs enter the ribosome .in the A site and only reach the P site after a round of peptide bond synthesis. During initiation, however, a charged tRNA enters the P site directly. This evenl r~ quires a special tRNA known as the initiator tRNA. which base-pairs with the start codon - usually AUG or GUG. AUG and GllG have a different meaning when they occur within an open-reading frame. where they arc read by tRNAs for methionine (tRNAMf11} and valine (tRNAVa'). respectively (see Chapter 15). Neither methionine nor valine is attached to the initiator tRNA. lnstead, it is charged with a modified form of methionine (N-fonnyl methionine) that bas a formyl group attached to its amino group (Figure 14-24). ·rhe charged iniliator tRNA is referred to as fMet-tRNA 11Md. Because N-fonnyl methionine is the first amino acid to be incorporated into a polypeptide chain, you might think that all prok;uyotic proteins have a forn,yl grou p at their amino tenninus. This is not the case. however. as an P.n.zyme known as a defonnylase removes the formyl group from the antino terminus during or after the synthesis of the polypeptide chain. ln fact. many prokaryotic proteins rlo not even start with a methionine; aminopeptidases often remove the amino terminal methion.lne as well RS one or two additional amino acids.

Three Initiation Factors Direct the Assembly of an Initiation Complex that Contains mRNA and the lnitiatOI' tRNA The initiation o f prokaryotic translation conunences with the ~ma H subunit and is catalyzed by three translatmn initiation factors called

FUilJRl 14-22 AnoveniewoftJt~ events of translmon initiaUon.

434

Translation

rs

Q-i3

30S

H

I s I CH2 I H c~ I I ~ N - C -COOH I

I

r

H-

11 I fz C-

I

H

H

methionine FIGURE

14-23 The 16SrRNAirlteracts with the ribosome binding site to position the AUG in tbe P ~ lhs dkJStrabon sf10ws an mRNA With the ideal ~tJoo between ~

N - C-COOH

N-formyl methionine (fMet)

14-24 Methionine and N-formyl methionine.

fiGURE

ritx:>5oMe bll'lding Site and the iniboting AUCL 'llis spacing ~es the AUG m the regioo of 1tle P. Many mRNA have ro11 !deal spil
FIGURE 14-15 A model of initiation faciDI binding to the lOS •ibosomal subunit. The esumated kx:a1Joo of IJJ, tf2. and IF3 btoding im.> shown ak:Jng with ttlt feg1Clr1.; of the .S05 fb:lSOmal subunlf that wij became part of the A, P. <md E sites. (Source Adapted fran Ramato-ishniln, V. 1002. !Ubo.some stn.JC1Ure cY1d the fTleChatl;sm ct translattoo Cell 108: 560, fig 2 Cqryn111t ~ 2002 'Mth permission from Bsev.a)

IF1, IF2~ and IF3. Each factor facilitates a key step in the initiation process (Figure 14-.25):

• lFl prevents tRNAs from binding to the portion of the small subunit that will become part of the A site. • IF2 is a GTPase (a protein that binds and hydrolyzes GTP) that interacts with three key components of the initiation machinery: the small subunit,. 1Ft, and charged initiator tRNA {ftvJet-tRNAtiMet). By interacting with lhese components. IF2 facilitates the ~ub5equent association of fMet·t&.~~fMe1 with the small subunit al1d proven~ other charged tRNAs from associating with the small suburuL • U:t3 binds to the small subtmit and blocks it from reassociating with a 1ruge subunit, or from binding charged tRNAs. Because initiation requires a free small subunit, the binding of lf"3 is critical for a n~w cycle of translation . IP3 becomes associated w ith the small subunil at t he eod of a p.revi.ous round of translation when it helps to disassociate the 70S ribosome into its large and small subunits. Each of the init.ation factors binds at. or near. one of the three tRNA binding sites on the small subunit (Figure 14-25). C-onsistent with its role in blocking the bi~:tding of charged tRNAs to the A site, IF1 binds directly to the portion of the small subunit lbat will become the A site. 1P2 binds to IF1 and reaches over the A site into the P site to contact the flvleHR.li.JA/'Met. F-Inally~ IF3 occupies the part of the small subunit that will become the E site. Thus, of the three potential tRNA binding sites on the small subunit, only the P site is capable of bi nding a tRNA in the presence of the initiation factors. With all three initiation factors bound. the sm all subunit is prepared to bind to the mRNA and the initiator tRNA (F'igur(' 14-2£). These two R...'\IAs can bind in either order and independent Iy of each o1he•·. As rliscussed above. binding to the mRNA typically in volves hase-pairing between the ribosome b inding site and the 16S rRNA in the smaiJ subo.nit. Meanwhile. binding fMet-tRNA.EMet to tho small subunit is facilitated by its interactions with IF2 bound to GfP anrl (once the mRNA is bound) base-pairing between the anticodon anrl the start codon of the mRNA. The last step of initiation involves the association of the large subunit to create the 70S initiation c;ornplex. When the start codon and fM.et4RNAlMt't h-.se-pair. the small wbunit undergoes a change in c:onfnrmation. This altered confonnatinn results in the reJease of IF3.

fniliation of n<2nslarion

fn the absence of 1F3, the large subunit is free to bind to the small sub· unit w ith its cargo of IF1 , IF2, mRNA, and fMet-tRNA,fM1" . The binding of the large subunit stimulates the G"flla.se activity of1F2•GTP, musing it to hydrolyze GTP. The resulting IF2·GDP has reduced affinity for the ribosome and the initiator tRNA leading to the release of [f2·GDP as well as TF1 from the ribosome. Thus, the net result of initiation is the formation of an intact (70S) ribosome assembled at the start site of the mRNA with fMet·tRNA;'Me' in the P site and an empty A site. The ribosome-mRNA comp18')( is now poised to aooept a charged tRNA into the A site and comment;e polypeptide synthesis.

b

Eukaryotic Ribosome~ Are Recruited to the mRNA by the 5' Cap Initiation of translation in eukaryotes is simiwr to prokaryotic initiation in many ways. Both use a start codon and a derlicated joitiator tRNA, and both use initiation factors to form a complex with lha small ribosomal subunit lhat assembl es on the mRNA prior to addition of tht:l large subunit. NevertheJp,ss, eukaryotes use a fundamentall}l distinct melhod to ~uize the mRNA ar'd the st
In eukaryoles, the small subunit is already associated with aninitiator IRNA when it is recrui ted to the capped 5' end of lhe mRNA. 11 Iben '"scans" along the mRNA in a 5'-+ 3' direction unt il it reachP.s the first 5'-AUG-3' in the cocrect context [see the discussion of the Kozak sequem;e in tha preceding secrion on mRNA), which H recognizes as the start coclon. Thus, in mosl in ~tances [see Box 14,2. uORFs and IRESs: Exceptions that Prove the Rule), only the first ALIG can be used as the stert site of translation in eukaryotic cells. Note that thjs method of initiation is consistent with the fact that the vast majority of eukaryotic RNAs are monocistmnic and e[lcocle a sin~le polypeptide; recognition of an jnternal start codon is gcne1-ally not possible or required. As we have seen for other molecular processes (such as promoter recognition during transcription}, eukaryotic cetls require many more auxiliary proteins to drive the ini.liation process than do prokacyotes (although eukaryntes have initiation fa(;toa·s that correspond to the p rokaryotic IF1. lF2. and lF3). Remarkably. more than 30 diffP.renl pulypeplides are involved in initiation of ttansla·

lf2

. +1Ft +Gm c

_J1

3 ~

..__

d

tion in eukaryoles.

In contrast to the prokaryotic situation, in eukaryotic cells bind ing of tho initiator tRNA to the small subunit always precedes asso. dation wilh the mRNA {Figure 14-27a). As the euLuyotic ribosome completes a cycle o f translation, it dissociates iuto free large and small subunits t hrough the action of factors (caJled elf'3 and eiFtA. respectivel y) analogous to the prokaryotic initiation factors lF3 and JF'l. Two GTP-binding proteins, eW2 and elF5B, mediate the recruit · ment of the charged initiator tRNA. For eukaryotes thi s tRNA is charged w i1h methionine, not N-formyl methionine, and is referred to as Met-tRNA1Mc'. Jn a case of unfortunate n omenclature, the eukaryotic analog of IF2~GTP is e1F5B-CTP. This fac:tor associates with the small subunit in an elf'1A-depende.n t manner. [n turn. e lF5BCTP helps to recruit a complex of eiF2~GTP and Met-tRNA/"''e' to the small subunit. 'lhgether these two GTP~binding proteins position the Mel·tRN~MJ.t in tb o future P site of the sma1l subunit. resulting in the formation of the 43S pre-initiation cvmplex.

fi~URE

14-26 A summary of

translation initiation in prokaryotes.

436

fiGVRE

Translation

14·27 JWembfyofthe

a

b

,

eubryotic SAN~ ribosomal subunit and initiator tRNA onto the mRNA. Note that elf4F is romposed of three prote~rn.: elr-4A. eiF4E. and eiF4G. eiF4E diredy btnds me5' Cilp, tethemg the other two p-oteirr. to !he end

, --1~-

5'cap

st;lrt codon

eiF4F

(AUG)

ofthemRNA. eJF1A eiF3

(:~) ~

Recognition of eukaryoti.c mRNAs by the 43S pre-initiation comple:x begins with the .recognition of the 5 .. cap found at the end of most eukaryotic mRNAs. Recognition is mediated by a three-subunit protein called elF4F (see Figure 14-27b). One of the three subunits binds directly to the 5' cap and the other two stAbunits bind non· specifically to the associated RNA. This romplex is joined by ciP4B which activates an RNA helicase in one of the cll'4.F subunits. The helicase unwinds any secondary structures (such as hairpins) that may have formed at the end of the rnRNA. Removal of sscondary structures is critical as the 5' enc.l of mRNA must ho unstructured to bind to the small subunit. The c1F4F/B bound unstructured mRNA recruit:<> the 435 pre-initiation complex to the rnR"'A through inter--actions between eiF41c' and elF3.

lniliaricn of Translaricm

The Start Codon Is f·ound by Scanning Downstream from the 5r End of the mRNA Once assembled at the 5' end of the lll.Ri'\JA, the small subunit an'1 its associated factors movo along the mRNA in a 5' - 3' llireclioo in an ATP-dependent process that is driven by the elF4F-associated RNA helicase (Figure 34-28). During this movement, the small subunit .. scans" the mRNA for lhe first start codon. The start codon is l 4..28 lderttification of the initiating AlJC by the eukaryotic SJMit ribosomal subunit.

Ft G U R f

elf4F

start codon (AUG)

~""""""" (~+0,

438

1hmslotion

recognized through base-pairing between the anticodon of the initiator tRNA and t he shtrt codon (this is why it is c ritkal that the initiator tRNA bind to the small subunit before it binds to the mRNA). Correct base~pairing triggers the release o f eJF2 and elFS_ Loss of elF3 (which h ad preven ted binding o f the large subunit} and elF2 [wh ich was bound to the initiator tRNA) allows the large subunit to bind to the small s ubunit. As in the prokaryotic situ11tiou . binding of the large subunit toads to the release of the remaining initiation factors by stimulating GTP h ydrolysis by the lF2 analog, elF5B. As a result o f these events. the Met-tRNA;Met is placed in the P site of the resulting 80S initiation complex. With the start codon and Met-tRNA""1t:t placed in the P site, the eukaryolic rihosorne is now poised to accept a charged tRNA i nto its A site at1d carry out the formation of the first peptide bond.

Translation Initiation Factors Hold Eukaryotic

mRNAs in Circles In addition binding to the 5' e nd of eukaryotic mRNAs, the initiation factors are closely ac;sociated with the 3' e nd of the mRNA through its poly-A tail (Figure 1 4-29}. This is mediated by an interaction between e£F4F and the poly-A binding protein that coats the p oly~A t ail. A consistent interaction between the two ends occurs because both e1F4F and tho poly~A binding protein are bound to tl1e mRNA through multiple rormds of translation. The interaction between these prot eins results in the mRNA being held in a circular configuration via a protein bridge between the 5' and 3' ends of the molecul e. It has long been known that the poly-A tai l contributes to efficient translation of mRNA. The finding that translation initiation factors "circularize"' mRNA in a poly-A-dependent manner provides a ~im ple catiouale for this observation~ once a rihosome finishes translating an mRNA that is circularized vja its poly-A tail. the newly released ribosome is ideally positioned to rc-iuitiate translation on the same mRNA.. FIGURE

14-.29 A modf:l bthe

ucularization of eukaryotic mRNA. GrcularizatJon IS pr~osed to be mediated by an •nleJactic n betwec:n the elf4G 5lburit of eiF4f 4lnd the pdy-.A bndit1g protem

- - stop {AGU)

Initiation of TranslCJtion

439

Box 1~2 uORFs and IRESs: Exceptiom that Prove the Rule Not all eukafyotic polypeptedes are encoded by an openreading frame that starts with the AUG that is most proximal to the 5' tenninus. In some cases, the first AUG is not in a prDPef sequence context resulting in its bypass. In other cases, short. upstfeom. open-reading f.-ames ( uORFs, encod-

ing pepMes less than ten <1mino aads long) are found upstream the principal open-reading frame, lhat encodes a large polypepbde (Box 14-2 F'8lJre Ia). In these ~ses, the uORFs act to regl.Aate the extent of translation of a larger, downstream. open-readtng freme. At least some of these uORFs are fullowed by RNA sequences thclt CGuse a proportion (30- 50%) of the small subunits that translate l:hem to

or

be retained on the rnRNA after termillclllon. The retatned small subunits continue scanning for the next AUG but can only locate it after a newly char-ged initiator tRNA is placed in the P site by eiF2. As you will seE' in Chapter 16, this charac· teristic can be explvited to regulate translation of downstream, open-reading frames. In other cases, these uORFs are Simply bypassed at some frequency, allowing initiation at

a doiNnstream AUG- ab:it at a greatly reduced rate. A more extreme e><.ample of ir.lti61ing translation at sites downstream of lhe AUG tha is dosest to the 5' terminus is represented by 1ntemol ribosome entry sites (lRESs). tRESs are RNA sequences that fooction like the prokaryotic rtlosorne banding sites. They recruit the small subunit to bind aod inittate at an intemat site tn the mRNA (Eiox 14-2 Frgure l b). These a.e relatively rate in et.!aryotic transcripts and are most often enroded in virel n~ that often lad a 5 ' cap end and helve a need to exrJoit d1e sequences of their genome maximol y. By using an IRES. a Wdl mRNA can encode more than one protein, reducing the need for extended transcriptional regulatory sequerces for each proteiJH:oding ~ Different IRES Seq.Jences work by dfferent mecharlisms. At least one viral IRES oredy binds to elf4F, mimicking the normal recruitrnetlt of this complex throogh interoctions with the 5' cap. Others are thought to interact directly with the srnal SLbunit rRNA in a mrmner analogous to the prdaryuic riJosome blnd~sile.

uORF

~ .,. . . . .'WUG. . . . . . . . . . . .ar~~lU.....................~................~,~~....~IGDA~. . . .~

a

rare S

rarE~

s·

I

I

IG4W

3'

440

Translation

Box 1.C.2 (Continued) b

S

AUG

~~~t:J~&·--·NIG --rt~~

--,~~

B0 X

14-2

Fl GU R E 1

Two methods for euJc.yutk tnsnslaticJn to

initiate at internal AUC5.. (a) ~ can alow the smal subunit cornpex difeoly to the m~

10 CDll1nue scanrllr1g after CClrtlf'*e~g trc1nslaticn. {b) IRESs can recrut the 43S pre-lflitiatJon

TRANSLATION ELONGATION Once the ribosome is assembled with the charged initiator tRNA in the P she, polypeptide syn thesis can begin. There are three key events that must occur for the correct addition of each amino add (F'igure 14-30). First, the correct aminoacyl-tRNA is loaded into the A site of the ribosome as dictated by the A-site codon. Second . a peptide bond is formed between the aminoac:yl-tRNA in the A site and th.e peptide chain that is a ttached to the peptidyHRNA in the P site. This peptidyl transferase reactioll, as we have soon. results in the transfer of the growing polyptiptidc from the tRNA in the P site to the a mino add moiety of the charged tRNA in the A site. Third, the resulting peptidyl -tRNA in the A s ite and its associated codon must translocated to the P site so that the r ibosome is poised for another cycle of codon recognition and peptide bond fon na.tion. As with the original positioning of the m.RNA. this shift must occur _prodsely to maintain the correct reading frame of the message. Two auxiliary protains known as elongation factOI'S control these events_Both of

oo

Ttan!>laticm Elongarion

these factors use the energy of

(,.JP

441

binding and hydrolysis tn enhance

the rcite and accuracy of ribosome function.

Unlike the initiation of translation, the mechanism of elongation is highly conserved between prokaryotic and eukaryotic cells. We will limit our discussion to translation elongation in prokaryotest which is understO<:l
Aminoacyl.-tRNAs Are Delivered to the A Site by Elongation Factor EF-Tu

aminoac)lf-tRNA

binding to A site

Aminoacyl-tRNAs do not bind to the ribosome on their own. bJstea.U, they are ··escorted 11 to the ribosome by the elongation factor EF-Tu (Figure 14-31). Once a tRNA is aminocylated, EF-Tu binds to the tRNA's 3' end, masking the coupled amino add. This interaction prevents the bound aminoacyl~tRNA fTom participating in peptide bond fonnation until lt is r&leased from EF-Tu. Like the initiation factor lF2, the elongation factor EF-Th binds and hydrolyzes GTP and the type of guanine nudeot i(Je bound governs its function. EF-Tu can only bind to ah aminoacyl-tRNA when it is associated with GTP. EF·Th bound to GOP~ or lacking any bound nucleotide. shows little affinity for aminoacyl-tRNAs. Thus, when EF-Tu hydrolyzes its bound GTP. any associated aminoacyl-tRNA is released. EF-Tu bound to an aminoacyl-tfu'\JA cannot hydrolyze GTP at a significant rate. The trigger that activates the EF-Tu GTPase is the same domain on the large subunit of the ribosome that activates the IF2 CTPase when the large subunit joins lhe initiation complex. This domain is known as the factor binding center. EF-Tu only interacts with the factor binding center after the tRNA is loaded into the A site and a correct codon-anticodon match is made. At this point. EF-Tu hyd~ Jyzes its bound GTP cmd is released from the ribosome (Figure 14-3 1). As we discuss below. control of GTP hydrolysis by EF-Tu is critical to the specificity of translation.

The Rihosome Uses Multiple Mechanisms to Select Against Incorrect Aminoacyl..tRNAs The error rate of translation is between 10 -s to 10 -4. That is, no more

than 1 in every 1,000 amino acids incorporated into protein is incorrect. The ultimnte bac;is foe the selection of the r.ocrect aminoacyl-tRNA is tha bac;e pairing between the charged tRNA and the codon displayed in the A sile of the ribosome. Despite this, the energy difference between a correctly fonned codon-anticodon pair and that of a near match cannot account for this level of accuracy. [n many instances only one of the tbree possible base pairs in the anticodon-codon intct'al:tion is mismatched, yet the ribosome rarely allows such mismatched arninoacyl-t.RNA.s to continue in the translation process. At least three rlifferent mechanisms contribute to this specificity (Figure 14-34}. In each case. these mechanisms select against incorrect r.odon-anticodon pairings. One mechanism that contributes to the fidelity of codon recognition involves two adjacent adenine residues in the 1fiS rRNA component of the small subunit. These bases form a tight interaction with the minor groove of each corred base pair fOI'rnad betwoart the anticodon anrl the

3' FIGURE 14-30 Sununary of the steps of translation.

442

Tromlation

first two bases of the codon (f igure 1.4-323}. As you will recall (see t 1gure 6-tO), the edges of a G:C and 8Jl A:U base pair are very similar in the minor groove. The adjaoont A residues in the 16S rRNA do not discriminate between G:C or A:ll base pairs and recognize either as correct. 1n contrast, non~WatsonOk.k base pairs fonn a minor groove that cannot be recognized by these bases, resulting in significru1tly reduced affinity for incorrect tRJ\JAs. The net result of these interactions is that corre.ctly paired tRNAs roc:hibit a much lower rate of dissociation &om the ribosome than Ct base-pairing interactions dramatically enhances the rate of a critical biochemical step. For the

EF-Tu--GDP

'

DNA polymerase. this ,step was the fonnation of the phosphorHester bond. In this case, it is the hydrolysis ofGTP by EF-Tu. A third mechanism tha1 ensures pairing accuracy is a form of proofreading that occurs after EF-Th is ~leased. When the charged lRNA is first in troduced into the A site In a complex with EF-Tu -GTP, its 3' end is distant fro10 the site of peptide bond formation. To participate successfully in the peptidyl transferase reaction, the tRNA must rotate into the pepridyl transferase centRr of the lacge s ubtm.i t in a process caJlcd accommodation (Figure 14-3.2c). Incorrectly paired tRNAs frequently dissociate from the ribosome during acoomrnodation. It is hypothesized that the rotation of the tR..~A places a strain on the codon anticodon interaction and that only a cOJTectly paired anticodon can sustain this strain. Thus. ¥nispaired tRNAs are more likely to dissociate from the ribosome prior to participating in the paptidy1 transferase reaction. In summary, in addition to the codon-anticodon interactions. the ribosome exp loits minor groove interactions at1d two phases of proofreading to enstJre that a correct aminoacy1-tRNA binds in the A !;ite. Each of these three additional selectivity mechanisms enhances the rate of peptide bond formation with correct codon-antil:odon interac· 4

tions and selects against incortect inte ractions .

The Ribosome Is a Ribozyme 14-31 Ef-Tuescorts 41mi~A co ate A 5ite of me ri005ome. Charged tRNAs are bound to EFTu-GTP as they first IOteract With the A ~te of the ribosOOIE. ~ the ((){reCI c:ock::>rt-anticodon lf1 tetacnon occurs, Ef-Tu mtsads \l\llttl lhe facta blr.ding c.er.ta-; h¥lrolyzes rts bound GlP and is rdelsed from the tRNA and the lilOSCKne. FtCURf

Once the correctly charged tRNA has been placed in the A sire and has rotated into the peptitlyl transferase center. peptide bond fonnation takes place. This reaction is catalyze d by RNA. specificaUy the 23S rRNA component of the large s ubunit. Early evidence for this carne from experiments in which it was shown that a large s ubunit that had been large ly stripped of its proteins was still able to cany out peptide bond formation . Proof that the peptidyl trdnsferase is entirely composed of RNA has come from the high-resolution. three..dimensional structure of the ribosome. which reveals tha t no amino acid is located closer than 18 A from the active site {Figure 14-33}. Because catalysis requires dista nces in the 1 - 3 A range, it is dear that the peptidyl transferase center is a ribozyme. 7hat is an enzyme composed of RNA [see Chapter 5).

a

b

correct pairin g

correct pairing

5'

EF-Tu-Ga:> \

O +o.

ina>n"ed pairing

s·

tncORect pairing

factor Wl
eerier rot

c

no GTP hy<)Oiysis EF-Tu-lRNA r9eased

I 4-32 llwee mechanisms to ensd'e correct pairing between the tRNA and the mRNA (a) Ad&timal hydrogen bonds are fotmed between two~ re9dl.lf!S of the 165 .-RNA and~ minor groove of the antic:oOOrrcodon pail only \1\f;en they are c.Qf'l'eC11y base-pa'led. (b) Cooect base-pM~ atiCMIS Ef Tu boood to the ar<~II1WCy!-IRNA to ~ad V\M the fuctor ~ o:nte .-dicing GTP hydrcfysis tYXi EHu rdease {c) Ontv cooooly ~ ~tRNAs rema10 assooated with the rixlsome as f1ey rotate into ~ croect position fa ~tide bond loorwltioo. Ills rrnatioo $s referred 1o as tRNA accommodation F 1cuRE

443

Translouon

fiGURE 14-31 RNA surrounds tfle pep~ tidyt transt1!rase £eAter ~f the large ribosomal s.Jbunil The three-dimerlSlooal stiU:.tUI't:> of the bacterial 50S Sllhunit is shown. The M\As e~re

sho<M1 in gray tn1 the nbosof'r.al protens are

sho1M1 111 purple The 3' ends of the A and P sile

1RNAs that are lfomediately adjacent to the peptidy! traflSfernse CEflter are shown in green and red, respediveJy. ('(USl.lpOV MM, YUSUJXYV
How does the 23S rRNA catalyze p eptide bonrl formatjon? The exact m echanism remains to be determined, Lut somt! answers to this question are beginning to f3merge. First, base-pairing between the 23S rRNA and the CC.A ends of the tRNAs in the A and the P sites help to position 1he alpha-amino group of the ominoacyl-tRNA to attack the carbonyl group of the growing polypeptide attached to the peptidyl-tRNA. These interactions are al so likely to stabilize the am i noacyt-tRNA after accommodation. Because dose proximity of substrates is rarely ,;ufficienl to generate high level., of catalysis. it is hypothesized that other elements of the ribosomal RNA change the chemical environment of the peptidyl transfurase active sitA. For example, i t has beAn proposed that nu· cleotides in the peptidyl transferase a3nter accept a hydrogen from the alpha amino group of the am}noacy.!·tRNA. making the assodatcd nitrogen a stronger nucleophile. This is a common mechanism used by many proteins to stimulate nucleophilic attack of catbonyl groups.

Peptide Bond Fonnation and d1e Elongation Factor EF..G Drive Translocation of the tRNAs and the mRNA 011ce the peptidyl transferase reaction has occurred. the mNA in the P site is deacetylated (no longer attacherl to an amino acid ) and the growing polypeptide chain is linked to the tRNA in the A site. For a new round of peptide cha io elongat.ion to occur, the P· site tRNA must move to theE site an d thtl A·siw tRNA mu~t movtl to the P site. At the same time, the mRNA must move by three nucleotides to expose the next codon. These movements are coordinated within the ribosome ~ nd are collectively referred to as translocation. Tlre initial steps of translocation are coupled to the pepti~yl transfurase reaction (Fig1,1re 14-34)_ Once the grm.ving peptide chain has boon transferred to the A-site tRNA, the 3' end of tbis tRNA moves into the P-site portion of the large subunit (Figure 14-34 panel 2). In conttast, the anticodon en d of the A-site tRNA remains in the A site. Similarly. the now deacetylnted P-site urnAis located in the E site of the large subunit and the P s ite of the small subunit. Thus, translocation in the large subunit preet3dcs transJoc"tion In the small subunit

TTUnslafiOn Elongation

and the tRNAs are said to he in " hyhrid stat~ ... Their 3' ends c;hift into a new location but their anticodon enrls are still in their pre-pep· tidyl transferase position. The completion of translocation requires the action of a second elongation factor called EF-G. EF-G can only bind to the ribosome when associated witb GTP. After the peptidyl transferase reaction. the shift in the location of the A-site tRNA uncovers a binding site for EF-G in the large subunit portion of rheA site. When EF-G-GTP binds. it Cf:m1ac ts the factor-binding center uf the Jarge s ubunit. which Btimulatcs GTP hydrolysis. GTP hydrolysis changes the conformation of EF-G-GDP. allowing it to reach into the small subunit and trigger translocation of the A-site tRNA (Figure 14-34 yane1 3}. When translo-cation is complete, the resulti ng ribosome b'tructure has dramatically reducecl affinity for EF-G-GDP. allowing the elongation factor to release from the ribosome. Together these events result in t he translocation of the A-site tRNA into the P site, t.he P-site t.RNA into theE site . Wld the movement of the mRNA by exactly lhree base pairs {Figure 14 -34 panel 4).

EF-G Drives Translocation by Displacing the tRNA Bound to the A Site The exact means by which EF-G induces trans location is not clear, but part of the mechanism involves l.he ability of EF-G-GOP to occupy the A-site portion of the decoding center. By interacting with the decoding center, EF·G--COP displaces the A·site tRNA into the P site. Lik~ dominoes, the displacement of the A-site tRNA into the P site means that the P-site tRNA mlliit move into the E site. During the movement of the tRNAs, the mRNA is shifted by t hree base pai[s. Movement of the mRNA is mediated by base-pairing between the moving A-site tRNA and the mRNA, which is maintained during trans location. Essenlially. the mRNA is p u lled along with tlle moving A-site tRNA. Indeed, rare •·frame-shifting .. tRNAs that have four-nucleotide-long anticodons (and am therefore compensate for Ctlrtaio frame-shift mutations) move the mRNA by four nucleotides instead of three. In contrast to A-site tRNA movement. movement of the P-site tRNA into the E site disrupts base-pairing of the tRNA with the mRNA. Henm , the nnw unclmrgerl tRNA in the E site is free to dissociate from the ribosome and to become recharged with a fresh amino acid by ami.noacyl tRNA synthetase. Changes in the small subun it of the ribosome also contribute to translocation. For example. change!; in the structure of the small subunit must occur to allow the release of EF~OP after translocation is complete. In addition, prior to translocation. portions of the t:~mall s uhamU separate the A. P. and E sites. Thus, for t he tRNAs to ttanslocate to their new positions. these region!) m ust move out of the way. The irreversible nature o f GTP hydrolysis and the occupancy of the A-site decoding center by EF-G-GDP ensures the forward m ovemen t of the ttanslation process. How does EF-G-GDP interact wi1h the A site of the decodi ng center so effectively? Crystal structures ofEF-Th bound to tRNA and EF-G rnveal a d ear answer to this question. EF-G-GDP and EF-Tu -GTP -tRNA have a very similar structure (Figure 14-35}. Recall that EF-Tu -GTPtRNA also binds to the A~s ite decoding center. What is most remarkable about this similarity is that. although EF-G is composed of a single polyp eptide. its structure mimics that of a tRNA hound to a

F I c:J U R £ 14-34 Ef-G stimut.tiOI\ of

translocaticn reqtMtes QP hydrof)'sis.

4-45

446

TcqnslaNcn

FICU Rl: 14-35 Structural COII!Iparison

of e1ongation factors. EFiu-GDPNP-Phetf
M. ~ S.• ~G., Reshetnlkova L, Clar\ B.FT and Nyborg J. 1995. 5c;ief!ce 270: 1464-1472. Right struct\Jre: ~ ~ AevarsSOfl A, Garbef M-. lhelle410sava 1 , and ltljas A 1996. 5mlctvre 4: 555-565.) Images prepared with Mo&1Jpt, 69t:>So.pt, and Raster 30.

protein. This is an example of "molecular mimicry" in which a protein takes on the appearance of a tRNA to facilitate as:;odation with the same binding site.

EF-Tu..GDP and EF-G~GDP Must Exchange GDP for GTP Prior to Participating in a New Round of Elongation EF-Tu and EF-G are catalytic proteins that are used once for each round of tRNA loading onto the ribosome, peptide bond formation. anrl translocation. After (;'J'p hydrolysis. both proteins must release their hound GDP oncl bind a new molecule of GTP. For EF-G this is a sjmple process, as GOP has a )ower affinity for EF-G thoo does GrP and is rapidly released after hydrolysis of CTP. The unoound EF-G rapidly binds n n aw GTP molecuJp.. ln the case of EF-Tu. a second protein is required to exchange GOP for GTP. Tht~ e long
A Cycle of Peptide Bond Formation Consumes Two Molecules of GTP and One Molecule of ATP Let us conclude our discus-c;ion of elongation with a simple cost accounting. How many mo1ccules of nucleoside triphosphate does it cost per round of peptide bond form ation (leaving aside the energetics of amino acirl biosynthesis and the energetics of initiation and lennination)? As you will recall. one molecule of nucleoside triphosphate (ATP) is consumed by the aminoacyl-tRNA S}'nthetase in creating the high-energy acyJ bond lhat links the amino acid to the tRNA. The breakage of this high-energy bond drives the peptidyl transferase reac-

Tf:TITiination ofTronslati<m

447

tion that creat.P,S the peptide bond. A second molecule of nucleosirle triphosphate (GTP) is consumed in the delivery of a charged tRNA to the A site of the ribosome by EF-Tu and in ensuring that correct codon-anticodon recognition had taken place. Finally, a third n uclecr side triphosphate is consumed in the EF-t;-mediated process of translocation. Thus, making a peptide bond costs the cell two molecules of GTP and one of ATP, with one n ucleoside triphosphate being const.tmed for eacb step in the translation elongation process. Interestingly, of the three n1olt:Jcules, o nly one (.ATP) U; energetically connected to peptide bond form ation. The energy of l.he other two molecules (LlP] is spent to ensure the acCUI"acy and order of events during translation (see Box 14-3, CTP-Binrling Proteins, Confonnational Switchi ng, and the FideJ i ty and Ordering of the Events of Translation). Tim:mghoul the discussion of transwtion elongation we have nol oic;tinguished between prokaryote.c; and eukaryotes. Although the eukaryotic factors analogous to EF-Tu (eF..Fl) and EF'-C (eEF2) are named different1y. their functions are remarkably similar to their prokaryotic counterparts.

Bo• 14-J CTP-Binding Proteins. Confonnatiooal Switching. and the Fidelity and Ordering

GTP is used throu~h~Jl transfation to control key events. lhe el1€fgy of GTP hy<:\'olys.s is not cot()led to chemical rTl()G. 1H:ation as ATP is tn the coupling of amno acids to tRNAs. Jnstead. the energy of GTP hydrolysis is used to control the order and Mclity of events during translation How IS this ac~

teased fran the

mthe Events of Translation

~tRNA

alowing peptide hood forma-

llOn lo ensue. The mechalliSfll tha1 acwates GTP hydrolysis by each d the GTPiegtlated at.JXMy proteins is the same. In each case. GTPase adMty is stimUated 1hi'Ot€h an Vltffilction with a specific

region of 1he large st.brit caled tre factor binding centa lhts

A key featae of the GTP·binding proteins invdved in transla-

intetaction is nd of sufficient affinity to ocet.Jt in lsclation. Instead,

6on ~s that thew confoJTT\ation changes depenang m the guanne nucleotide (such as GOP vs. GYP) to v-hch they Clfe bound Thts ctr~ be seen for EF-TU in Box 14-3 ftgue l, vvhicn shows the wee-dtnensia.at structure of Ef-Tu bou1d to GTP or GOP. Ef-Tu l.fldergoes a major COflformational change ~ it binds to GTP that resuhs 10 1he ~ oi its tRNA bnding site. In palba.lla.; me d()I'T'la.n cl Ef.Tu {shovvn 111 fl1i'g€flta In Box 14-3 f.gue 1) shifts its rotioo ~e to the aher doma\ns of the protell dependng en the nucleotide that is bound This chal1ge in domain kx:ation as well as Changes in the confoonalion of the other two domains (sho.Nn in tutQUOise aid d.n blue) results in the fOtTTiB101 of a rew surface oo EFru that binds ughtly to
each GTP;dng site able to interact prcxi.Jdive4y with the iactor birding center. leadng to GTP hydrdysis and the associated dm~ in pniew1 COI'lbmatkn The use of GTP dui:ng translatim is analogous to the use of AlP by che siidng darrp loaders (see Chapts- 8, Box &2). Reccil 1hat in that case, ATP bi'ldir1g was r~ed to assemble an 1ni1Jal ~ Wth the siding danv. but ATP hyadysis and release of the ~ c~ could ady occu when the canp looder en&ded the p\nl6~te jlntion_ In ~nsl~ GTP IS ~ fcr the llliual assooatloo With the ribosome {a-d 1n some i1stn:e5 0~ RNAs ~nd proteinS), and G1P hydrc*f-;15 only ocaxs Q'lce 1he fador has correc;1Iy rtteracted ~ the ribosome. As in the case of 1t1e sf.OOg darrp, GTP h~ genaally rest.Jts in the retease of the factor from the fibasane.

f{)( exafTllle. Ef·Tu-GTP can bind to an anirl0a<:y1-1RNA but ErTu-GOP camot. By cc;.uping GTP hydroly% to the ronplebon of key events 10 translation, the otrler d trese ~ can be tightry cootroled. For EF·Tu. the GTP-deper.dffit assodation of Ef-Tu \"Ah amnoacyltRNAs €n5Ures 1hat peptide bond formaoon does not oc:cti prior to oorrect codon-anticodon pairing. Formation of the mrrect base pairs triggers GTP hyao!ysis. Once b
*

Translation

Box 14-3 (Continued)

BOX 14-J FIGURE 1 Comparison of EF-Tubounclto GDP and GTP- (a) Ef-lU bound to(l)P_(b) EF-Tu boundtoGTP. The GTPbrd· ing ~ is shown in red. The rotation of the magenta domain and the changes in the structure of the greefl and blue clomatns lea
TERMINATION OF TRANSLATION Release Factors Terminate Translation in Response to Stop Codons The ribosome's cycle o f aminoacyl-tRNA bjnding, peptide bond form at ion , and translocation continues until one of the three stop cocions enters the A site. It was initiaJly postulated that there would be one or more chain-terminating tRNAs that would recogni:t.e these codons. However. this is not the case. Instead, stop codons are recognlzed by proteins callerl release factors (RFs) that activate the hydrolysis of the polypeptide from the peptidyl-tRNA

There ere two classes of release fact ors. Class I release factors rec· ognize the stop codons and trigger hydrolysis of the peptide chain from the tRNA in the P site. Prokaryotes have two class I release factors called RF1 and RF2. RF1 recognizes the stop codon UAG. and Rf'2 rer.ognize.CJ the stop codon UGA. The third stop codon. UAA, is recognized by both RF1 and RF2. In eukaryotic cells there is a single class 1 release factor catled eRFl that recognizes a l1 three stop codons. Class TI release factors stimulate the dissociation of the class 1 factors from the ribosome after release of the polypeptide chain.

Te.rminat;<Jn <Jj Trqnslation

449

Prokaryotes and eukaryotes have only one class n factor called RF3 antl eRF3, respectively. Like EF-G, EF·Tu, and other translation factors, class II release factors are regulated by GTP.

Short Regions of Class I Release Factors Recognize Stop Codons and Trigger Release of the Peptidyl Chain How do release factors recognize stop codons? Because release factors

are entitely composed of protein, recognition of stop codons must be mediated by a protein~RNA interaction. Experiments in which short coding regions were genetically swapped bet ween RFl and RF2 (which have different stop-coclon specificity) pinpointed the region of this ~gnition to a stretch of lhnx~ omin o acids. Exchange of these three amino acids between RFl and RF2 re.stllts in hybrid release factors that acquire the stop codon recognition specificity of their counterpart but are otherwise identical In fundion. Evidently. jur:;t three amino acids are re..finecl by these three amino acids represents a peptide anticod<m that interacts with and recognizes stop codons_ In keeping with this view, the tJnee.dimensional ~tructure of RF2 bound to a ribosome reveals that the peptide anticodon i:; lm:ated d ose to the stop codon in the decoding center {Figure 14-36). A region of class I release factOTs that conlribotes to polypeptide release has also been irlentified. All class I factors 6hare a conserved, throe-amino acid sequence (glycine glycine glutamine, GGQJ that is essentia l for polypeptide release. Moreover, the structure of RF2 bound to the ribosome oonfirms that the GGQ motif is located in close proximity to the p eptidyl tr'dl1sfurase cente r (Figure 1 4~36-) . It remains unclear whether the GGQ motjf js directly involved in the hydrolysis of the polypeptide from the peptidyl-tRNA or if it induces a change in

FI C. URl 14-36 Modelofa~tn lease f~ bollnd to 1M A • of the riboJome. 1h!s model iklstrat.es the location of a dass 1 r~ faa.or bound lO the ribos:ome.. The P site and E srte tRNAs are shown as L--shaped surfaces. lhe GGO CJmiOO aod mom that IS irwolved lf1 ~-pept\de hydrolysis IS tocated adiaceo1 to the 3' end of the P site tRNA. The SPf peptide anticodor1 1s l~~ceflt to the anticodon loop of the P site tRNA in ~ position 1M woUd allow easy access to the stop c.odon (Source: Adapted from Brodesen, D. f_ ard RamaJcnshoan. V 2005. stli=!pe CN1 be sedur.nve.. Not. Struct. Bfo/. ro. 79, fig 2, part a.)

450

nanslatiQf'!

the peptidyl transferasP. center that allows the center itself to catalyze hydrolysis. Together, these studies have Led to the hypothesis that class I release fuctors functionally, but not structurally. mimic a tRN.A~ having a peptide anticodon that interact1l with the stop codon and a GGQ motif that reaches into the peptidyl transferase center. Comparing the structure of the release factor that is bound to the ribosome with that of a free release factor providP.s an additional insight into the role of slop codon terognition in polypeptide relE;:ase. As we have seen. the pelJtide anticodon and the GGQ of a release factor extends from the decoding center to the peptirlyl transfer center of the ribosome. In the ahsence of a ribosome, however. tl1e peptide anticodon and the CGQ motif are quite dose to each other (approximately 20 A), roo rlose to reach both the decoding center and the peptidyJ transferase center. (For comparison, the amino acid-accepting stem at the 3' end of a t.Ri\lA molecule is about 70 A from the anticodon loop at the other end of the molecule.} Thus, release factors must undergo a change in conformation upon binding to the ribosome. This finding has led to a model in which release factors can only assume the extended, chain-terminating conformation (in which tbey can reach into the pepaidyl tranl)ferase center) when a stop codon is present in the decoding center.

GDP/GTP Exchange and GTP Hydrolysis Control the Function of the Class II Release Factor Once the class ( release factor has triggered the hydrolysis of the (JHptidyl-tRNA linkage , it must be rHrnoved from the ribosome (Figure 14-37). This is accomplishe d by the class II release faetor, RF3 (or eRF3). RF3 is a CTP-binding ptotein but, unlike the other GTP-hinding ptoteins involved in translation, this factor bas a higher affinity for COP than GTP. Thus. free RF3 is prHdominantly in the GDP-bouod form. RF3-GDP binds to the ribosome in a manner that depend s on the presence of a da:;s I release factor. After the class I RF stimulates polypeptide release, a change in the conformation of the Yibosome and the dass I factor stimulates RF3 to exchange its bound GDP for a GTP. The hinding of GTP to RF3 leads to the formation of a highraffinity interaction witb the ribosome that displaces the d~ l factor from the ribosome. This change also allows RF·3 to associate with the fuctor binding center of the bnge subunit. As with other GTP-binding proteins involved jn translation. this interaction stimulates the hydrolysis of GTP. In the absence of a bound class l factor, RF3-GDP has a low affinity for the ribosome and is rel.:>.ased.

The Ribosome Recycling Factor Mimics a tRNA

F rG I) R E

14-]7 Polypeptide release is catatyzed by two refuse Uctors. The class ' release facta (shcMir1 here as RF 1) recognizes the stop codon ar~d Sflmclates p~e release~ a GGQ rrw:AA that is locabred 10 the peptidyf transferase aner. The dass Y release f.x.1or (RF3) binds only after polweplide release and dfNes the oi550Ciatioo of tne ctass ' re~ease tactOt

After the release of the polypeptide chain and the rell*!se factors, the ribosome is stiU bound to the mRNA and is lefl with two deacylated tRNAs (in the P andEsites). To participate in a new round of polypeptide synthesis, the tRNAs and the mRNA must be removed from the ribosome and the ribo:-ome n1ust dissociate into its large and small subunits. Collectively. these evenl<; are referred to as ribosome recycling. In prokaryotic cells a factor knoVv'n as the ribosome recycling factor (RRF) cooperates with EF-C and lF3 to recycle ribosomes after polypeptide release {Hgure 14-38). RRF binds to the empty A s;tc of the ribosome, where it mimic.s a tRNA. RRF also recruits EF-G to the ribosome and, i n events that mimic EF..C funct ion dllring elongation. the EF-G stimulat~s the release of the uncha.ged tRNAs bound in the P and E

Termination of Translation

451

14-38 RRf and Ef-G combine to stimulate lhe release of tRNA and mRNA hom a tlmninated ribo5ome. fIGURE

RRF- -

em _J EF-G-GTP

l

Ef-G-GOP

sites. Although exactly how this l'elease occurs is unclear, it is thought that RRF is displaced from the A site by EF-G io a manner s imilar lo the d.isplarement of a tRN A from lhe A site during eJongation. Oru::e the tRNAs are removed, Ef'-G and RRF are relea!ied from the ribosome along with the mRNA. IF3 (the initiation factor) may afro participate in the relP.ase of the mRNA and is required to gpparate the two ribosomal subunits from each othcc. The fina.J outcome of these events is a smaU subunif bound to IF'3 (bur nol tRNA or mRNA) and a free latge subunit The released ribooomc can now participate in a new round of translation. Rei nforci ng the view that the RRF is a mimic of IRNA. it resembles a tRNA in i•s tlm::e.ilimensional structure. Ne\iertheless. it interacts with the ribosom~ in o very different manner than does a LRNA. RRF is closely associated only with the large subunit portion of the A site. We can rationalize thls difference between the rocycling factor and tRNAs in the following way. If the ribosome recycling factor precisely m imickeel an A-s ite tRNA, then the P-site tRNA would be moved into theE site by EF-C. Instead , EF-G and the recycling factor lead to the release of the P-site tRNA from the ribosome directly from the P site. It is likely that EF...C and the ribosome recycliug factor cause a more dramatiL change io Lhe structure of the ribosome than normally occur:r-o during tran slocation. allowing both the mRNA and the tRNAs to be released T..ike initiation and elongation. the tennination of translation is medi· ated by an ordered series of interdependent factor binding ancl release events. This ordered nature of tran.r;lation ensures that no one step occurs before the previoll'~ step is complete. For example. EF-Th cannot escort a new tRNA into the A s ite unlil EF-G complP.tes translocation. Similarly. RF3 cannot bind to the riblsome unless a da.><S J relea~ fador has already recognized a stop codon. There is a wectkness to this orderly approach to translation: if any step cannot be completed, then the entire process stops. It i ~ just this Achilles heel that antibiotics P.xploit when they target the translation process (see Box 14-4, Antibiotics Arrest Cell Division by Blocking Specific Steps in Translation).

TRANSLATION ..DEPEl\T))ENT REGULATION OF mRNA AND PROTEIN STABILITY At some frequency, mRNAs will be made that are mutant or damaged. Such defective mRNAs can arige from mistakes in transcription or from damage that occun; after they are synthesized . For example, because they are siogle-srran ded. mRNAs are more susceptible to breakage. Such damaged mRNAs have the possibility of making incomplete or incon-ect proteins that coul d have negative effects on the cell. In some cases. such as point m\!talions that change only a single amino acid. there is little that can be done to eliminate the mutant mRNA o r its protein vroduct. However, in other cases desc.rihed below. the process of translation is used to detect defective m.RNAs and eliminate either them or their protei n produ<:ts.

The SsrA RNA Rescues Ribosomes that Translate Broken mRNAs Normally a st·o p codon is required to release the :ribosome from an rnRNA. What happen~ to a ribosome that initiates translation of an ml{NA fragment that lacks a termination codon in the appropriate

Trcm5lation-Dependent Regulation of mRNII end Prote;n SttJbility

453

Box 11-• Antibiotics Arrest Ceft Division by Bloddng Spedfk Sleps in Translation

Antifiotics represent a powetful tool to fight disease. Many of the most widety used antibiotics in medicine kill boctena but IleNe rrt1fe or no effect on eukaryotic reUs, arld hence are not tooc to the patient Since thelr discovesy in the first half of the last centuJy, antbotlCS have helped make previously untreatabte iflfecoons such as tubera.lk.lsiS, bactertal pnei.XT\Oflia, syphilis, and goocxrhea largely rurOOte (although the emergence d antt>iotic-resistant bacteria is becomir1g an lrlO"easing obstade to effective treabneflt). Antib!otKs have marly different kinds of targets In the bacterial cett but approxwnately 40% of the known antibiotics are inhibtors of the translation machirr ery (Bo>l 14-4 Table 1). In gener;( these anttiotics btnd a COfl"l>OO< of the translabro apparatus and irhbit its function. Because different antibiotics arrest translus medical bene-fits, antibotics have cane to ~ an "I"JlX)rtant rde in helplf"lg us understi.Jnd the IN()j"king of the lranslaboo machinery.

Puromycin is one anriootic ~ used in studies of translation. It binds to the large slf>unit region of the A site.

--BOX

---- --Targets and TABLE 1

14-4

~cs:

Once bound, pt.romyon can substi1ute for an aminoacyltRNA 111 111e peptidyt transferase reaction (Box 14-4 fig-

ure l). Because purornycill is very small compared to a tRNA. its binding to the A site is rd suffident ro reta1n the pdypeptide chain m the ribosome Thus, peptidyl chains that are transferred to puromyon dissodate from the rbosome as an IOC~ete, puromycin-bound pclypeptide. In other words, puromyan causes f.dypep(tde synthesis to terminate premcrt\Jrely. Ottter antb.ottcs target other features cl the riboscrne, such as the peptide exu tunnel the peptidyl transferase center. the factor binding center, 1he decoding center. and regaons c.riticaf for translocatioo. Yet other cmtibiotics are inNbitors of translation factas. For example, kirromycin and fusldic acid 01e inhibitors of the elongation factOJs EF-Tu and Ef-G, respectively (Box 14-4 Table 1). In both cases, the antibiotic interacts with the GTPbound form of the tran!',ilal\01 factor and prevents changes 1n conformation rhat woukl normally occur after GTP hydrolysis. Thus, kirromycin arrests ribosomes with bound Ef-lu·GDP aminoacyHRNA. Sirn1tarly, fusklic acid arrests ribosomes \Mth bound EF-G·GDP. rn both cases, the next step in translatim is prevented by the fa~ure to release tl1e ekmgalion faaQf.

Co~

Antibiotic/Toxin

Ta,get Celts

Molecular Target

Tetracycline

Prol®yoticcells

Hygrcmycin B

Prokaryotic aro

A site of 30S SUbu'\il Near the A site of 30S

Paromyoo

eukaryotic celts Prd<aryotic cells

Chlorarnphencd

ProkaryotiC cells

Puomyon

Pr~af)/Olic 6nd

eukaryobc cells

SUbUnit Adjacenllo lhe A site codon-anticoda1 interaction sate in 30S Slbulit Peptidyt bansferase cenlf.r of 50S subulit

PeptidYI trnr'JsfP.rase r..enter of large ribosunal subulil

Pra<aryottc celts

Peptide e>
Fu$idiC acid

Prokaryotic c;ells

Ef..G

Thiosb"P.Ptorl

ProkaryotiC cells

Factcr binding C€fltef of the 50S subunit

Erythrcmycin

50S Sltlt.tM

EF-Tu

PrP.vents thP confc:lrma6onal changes associated INith GTP hydrolysis and. therefore. EF-Tu rele.ase

Prokaryotlc and W<aryotie cells

Chemically fl'!oditiCS lhe RNA In the factor bind;ng cooter d large ribosomal sublllit

Prevents activatiOn of translaoon factor

EukalyOlic cells Eukaryotic cells

Cllernieatly modifies ff·TU Peptidyt transferase center d the 60S st.IJUnil

Inhibits EF-Tu function tnhiblrs pPptldyllnmsferase activity

KifTOO\y-cirl

Ricin and a-Sarcin (protein toxins)

Oiptheria Toxin Cyctohex1mide

lnhibts aminoacyi-lRNA btndtig 10 the A site Prevents translocation of A-sem !RNA to P .site lncreaSP.S fJiror rate dt.Jrlf"YJ translal:ioo by decreasing selectivity of codon-anlicodoo paimg Slacks correct positioning of the A site aminoacyt-tRNA for pepbdyl transfer reaction Cham wrn.inat()(; mimics the 3' end of ami110acyJ-tf!NA in A site end acts as acoept(l{ for 1t1e nascP-nt poiypf>pljde chain Blocks exit of the growing pOlypeptide chain from the riboscroe; arrests translation Prevents reft>..ase otEF-G-GOP from me ribOSome Interferes wirh the assOcia.l ion of IF2 and EF-G witn factor btndirg CCI""ller

GiPa.ses

454

nanslaticm

Box 14-4 (Cofltinued)

1 4- 4 FIG U R t 1 Pulcmydn tenninates ttanslation by mirnking a tRNA irl the A site. Pllromycirl binds. in the A site and participates io peptide bond fQf'mat;i()n. Once completed. puromyon and any ~sodated po})peptide diffuses out of the ribosome

B0 X

reading frame? Such an rnRNA can bP. generated by incomplete transcription or nuclease action. Translation of this type of mRNA can initiate normally <1nd continue until the 3' end of the mRNA is reachc>..d. At this point. the rilX>somc cannot procef!d. Thero is no codon either to bind an aminoacyl-tRNA or to bind a release factor. Withou t some mechanism to retease them from these defective mRNAs. many ribosomt".s would be pennannntly trapped . removing them from polypnptide synthesis. ln prokaryotic cells, such stalled ribosomes are 1'f'.scucd by the action of a chimeric RNA molecule that is part tRNA and part mRNA, called a bnRNA. Ssl'A is a 457-nudrotidc tmRNA that includes a region at jts 3' end that strongly resembiP.S tRNA"w (figurn 14-39). This similarity allows the SsrA RNA to be ('.barged with alanine and to bind EF-Tu--GTP. When a ribosome is stalled a t the 3' end of an mRNA. the SsrA" 18-EF-Tu-GTP complex binds to the A site of the ribosome and participates in the peptidy I transferase reaction, as would any other tRNA. Translocation of the peptidy-SsrA RNA results in the release of the broken mRNA i{emarkably, translncation of the SsrA RNA also results in a portion of this RNA entering the mR'\JA-binding channel of the ribosome. This portiOil of SsrA RNA ex1.ends the open-reading frame of the im;omplete mRNA by ten codons followed by a stop codon. The net result of SsrA binding is that when the defective mRNA is released from the ribosome. the incomplete polypeptide is fused to a ten-amino-acid ..tag" at its carboxyl terminus and the ribosome is rel:yded. Interestingly, the ten-amino-add tag is recogn ized by t:ellular proteac;cs that rapidly dflgrade the tag and the truncated polypeptide to which it is atta<::lw.d.

too

Ttanslntion-Dependent Regulation of mRNil and Prol~in Stal>i/it.v

455

14-39 The tmftNA and 5sr"A rescue ribosomes stalled on prematurely tenninated mRNAs.. 1he SsrA RNA mrnics a tRNA but can c:rly bind a ribosome that tS stalk!d at the 3' end of an mRNA.. Once ba.Jnd. the SsrA mRNA substitutes part of its sequence FIGURE

to act as a new"~

recognition by

SsrARNA

wi>l EF-Tu-GDP

transpeptidatiOn

continued translocation of mRNA reading frame

Thus, trallblation pn Jducts ansmg from broken mRNAs are rapidly cleared to prevent these defective proteins from harming th e ool\. How does the SsrA RNA bind to on ly stalled ribosomes? Because of the large size of SsrA lit is morP than fow times bigger than a standacd tRNAl. it cannot bin d to the A site during normal e longation. In contrast. w hen the 3 ' end of the mRNA is missing. additi<mal room is c.meted in the A s ite to Jtccommodate the larger RNA. Thus, only ribosomes stalled at the 3' e nd of an mRNA represent a pote ntia) bincling site for the SsrA RNA.

Eukaryotic Cells Degrade mRNAs that Are Incomplete or that Have P remature Stop Codons Translation is tightly linke d to the process of mRNA decay in eukaryotic cells (Figm ·e 14-4Ga). This linkage is exploited by two mechanisms that rncJnit()r thn integrity of rnRNAs that are being trans· latcd. For example , when an mRNA contains a premature stop ,codon (known as a n onstmse codon; see Chapter 15}, the mRNA is rapidly degraded by a pro01ss called nonsense mediated mRNA decay (Figuro 14-40b). In mammals , rP.cognition of m RNAs with premature stop codons relies on the a ssembly of pro1ein complexes w ithin the o pttnreading fra me of the mRNA. These exon·junction comp lexes arc as.-;emblod on tho m RNA as a consaquence of splicing and are located just upstream of <'ach exon-exon boundary (sre Chapter 13}. Ordinarily. when the fi rst .ribosome tr.:ms,ates an mRNA, these complexes aro displaced as tl1t1 mRNA enters the decoding center of tlw ribosomf'~ H owever, if a premat ure stup t:odon is present in the mRNA ldue to mutation of the gene or mistakes in transcriptio n or splicing). then the rioosome i s re leased prior to the displacement of the complexes. Under these conditions. the comp lexes interact with the prelnaturely torminating ribosomR, which activates an enzyme that removes the cap at 1he 5' end of the m.HNA. Because the mRNA is ordinarily protected from degrada tion by the 5' cap. removal of the cap cau.ses rapid degradation of tho m RNA by a 5' - 3' exonuclease. A d ifferent process called nonstop mediated decay .rescues riboS
'lhmslation-Dependent Regl.llation of mRNA ond Prot~in Stability

a

451

b nonsense mediated mRNA decay

norma~

exon jurlction complexes

decapping enzyme

~

f 5' translation of

1 CQ_-o--~

cap

mRNA stop codon

5'-3' endorll.ldease

5' cap

"

s- 3' endonuclease degades ~..nCaJJs>ed RNA

c non-stop mediated decay

14-40 Eubryotic mftNAs wittl premature or no stop c.odons are targeted lor degradation. (a) li'ansiation of a riQlmal ~~all of the exoo junctic:ln rompexes. (b) Nooserlse med4at€d decay. TranslaIIC¥1 of .an mRNA with a premature stop codon does not displ;tce one Of more of tne exon JOI"'C· tion rorr.pbes. This results in tt-e recruitment of the Upf 1, Upf2, and Upf3 prdens to the ritJo. some. Once OOund to the OOoSOf'lle. these protE:'IflS act•vale a decapptng enzyme that rernolleS the 5' cap of the mRNA. The uncapped mRNA ts ltlen rapidly degraded by 5' to 3' exonudeases that are ~ tJflable to degrade the mRNA dtJe to the ~e of the s· GlP· f JGU R £

5'

cap

I

exosome

{c) Nonstop medi
a stq:~ codon, the pnly-A til~ of the rrRNA 15 translated A complex that includes the Ski7 protein a11d a 3' to 5' exonudease called the exosome bods aoy fi>osome staled at the 3' end of the pdy·A tail This resUts in the re4ease of the ribosome from the l'llRNA and its degtadat!on. Simlar to SsrA mediated nonstop decav. the poiy-4ysl!fle found at the end of proteinS derived from such mRNAs targets the p!O{etn fer degradation.

.-----------\'~ . 4A.4

exosmne

s

~

~~

•

4

~

protease

•

TrollSlation

SUMMARY Proteins are synthesized on RNA templates known as mes· ~l'lger HNAs (tnRNAs) in e pmcess known as translation. Translation iovol\les the decoding of nuclootide seqoc"nce information into lhe linear St!lluence of amino acids of the polypeptide chain. The machinery for protein synthesis consisls vf four principa.l components: the messenger RNA: adaptor RNAs known as tRNAs; aminoacyl tRNA s ynthetases that attach amino acids to the tRNAs: and the ribosome, wluch is a moltisubunil complex of protein and RNA that catal yzes peptide bond formation. The rnRNA contains the coding sequence for protein 8Jld recognition elements for the initiation and termination of tmnslation. 'I'he ooding sequence js l.-nown as an ope:n-re.ading frame (ORF). and coosi~ts of a series of three-nudeotide-long onits known as cotlons that are in register with eacb other. An ORF s pecifins a singlo polypeptide chain. Each OR.F begins with a start oodon and ends \"lith a slo p r.odon. The s tart croon is us ually AUG or GlJG in prokaryotes and always AUG in eukaryotes. ln prokaryotes, the start codon is p receded by a region of roguenc~ complementarity to tlle 16S rRNA component of the ri~me. which is responsible for alir,ning the ribosome over the start codon. In euka:ryott;!s, the rnKNA c-ontains a special structure at its 5' termi11us known as Lhe cap. which is responsible [or recruiting the ribosome. Eukaryotic mRNAs tcJlllinatc in a string of A residues known as the poly-A tail, whicb enhances the efficiency of translation. Prokaryotic mRNAs often contain two or more opell·Teading munE'J>; tbey art' refe-rn!'C] to 8S being polycistronic. Eukaryotic rnRNA§ usually c<mtain only a single open-reading frame. tRNAs are a physical interrace between rndi:>ns in the rnRNA and the atnino acids that are added to the growing polypeptide d:laio. tRNAs are L-shaped molecules with a loop at one end that displays the anticodon and a 3' pro· truding 5'-CCA-3' sequence at the other end 1he anticodon is oornprementary to the codon. which it reoognizes by base-pairing. Tite 5'-CCA-3 1 terminus is the s ite of attachment of an amino add to which it is )ns of the -ribosome. Tha intact ribosome contains three

t.RNA binding sites that reach between the two subunits: an A site where the charged tRNA enters the ribosome, e P site that contains the peptidyl~tRNA, and an E site, where deacylatt)d tRNAs exits the ribosome. Translation o( one protein tnvolves a cycle o[ association and dissociati.vn of the small and Jarge subumts. 1n thil:l ribosome cycle. (he small and large subunits assemhlo at the beginning of an open -reading frame and then di5sociat€' into free subunits when o-anslation of the ORr is complete. The mRNA is translated starting at tht! 5' entl of the ORf and tht! f-IOlypeptide chain is syntlw.siZP.d in an amino-terminal to oartJoxyl-termina( direction. Translation takes place in three principal steps: initia tion. elongation. and termination. Initiation in prokaryotes lnvQives the recruitmeflt of the small ribosomal soburut to thu mRNA through the interaction of tbe ribosome binding sire with tho 165 rRNA. l11is interaction is 18c;jlitated by three auxiliary proteins (called initiation fac:tors IFl , 11"2, and JF3). that help to keep the two subunits apart and recmU a special iniliator tRNA to the start ccdon. Pairing between the anticodon of the charged initiator IRNA and the start codon triggers the recru.ilroent of the large subunit, lhe release of the initiation factors, and the plac~um1 of the t:harged inili:jltor tRNA in the P site. This is the prokaryotic initiation complex, and it is poised to acupt a charged tRNA into the A site and carry out tbe (ormation of the first peptide bond. Eukaryotic mRNAs recruit the small subunit through recogniti.on of the 5' cap and the actiou of nwnerous auxiliary initiation factors. The small subunit then scans downstream until il eocounters an AlJC. which it recognizes as the start codon. As in pmkaryotes. only when the starting AUC is recog11ized does the large riboso~l subunit associ~ eta with the rnRNA. The first slcp of the elongation phase of transJation is the introdudion of a charged tf{NA into the A site.. This is catalyzed by the GTP·binding protein EF-Tu if1 prokaryotes a nd its equivalent in eukaryotes, Multiple mechanisms ensure tbat proper base-pairing has taken place between the C{)don .and. t.h e anticodon befnre tht> arninoaqtl g10up is allowed to enter the peptidyl tranfr ferase center. Next. p eptide bond form~llon takes pfaoe by the transft:r of the peptidyJ chain from the tRNA in the P sire to the aminoacyl-tRNA In the A site. Peptide bond formation is catulyzed by RNA in the peptidyl transfera.c;e c.entet of tbe large subunit This ribozyme stimulates the nucleophilic attat:k of the amino group of the aminoacyl·t~NA in the A site on the carbonyl group that attaches the growing polypeptide chain to the tRNA in the P site. Finally, the ribo$ome trans)ocates to the next vacant codon in a process thl:lt is driven both by the peptidyl transferase rf:action and the action of the e lon galion r.actor EF--G (or its eukaryotic equivalent}_ As aresu lt of translocation. the deacylated fRNA in the P sib: is shifted into th~ E site where it e)(itS the rlb0$orne ano the peptidyl-tRNA in the A site is shifted into the now vac.anf P t>ite. The ad jacent codon in the mRNA is shihP.d into the now vac.ant A sitP., which is poi$9(} to accept the delivery ofacharg(ld tRNA by EP-iu.

'ftanslat.ion terminates when the ribosome eorouo~rs a stop codon. which is recogui.zed by o ne of two class J teleao;e facton; in prokaryotes and a single class 1 re.k>.ase fac~ tor in eukaryotes. The release factor triggers the hydrolysis of the polypepllde frOfn the peptidyl-tRN/\ a,nd henc:e tha re~se of lhe oomplctl!d polypeptide. Finally. a class It relf>ase factor, a ribosome recycling factor. and ail initiation

factor (IF3 in prokaryotes} complete termination by causing the release of the rnRNA and the deacylated t.RNAs and the dissoc1atioo of the ribosome into its large and small sulr units. The ribosome cycl,.. is now complettl and the small tmbuoit is ready to commence a new cycle of polypeptidP. synthesis.

BIBLIOGRAPHY Books Alberts B., Johnson A •• Lewis J, RafT M., Robert$ K., and Waller P. 2002. Molecular biology of the cell. 4th edition. Garland ScienCE, New York. Brown T.A. zooz. Cen ome.s. 2nd '&lition. niOS Scientific Publishers Ltd ., Oxfurd, United Kingdom. Sonenberg N.• Hershey j .W.B.• and Mathews M.S.. eds. WOO. lranslational conttol of gene expression. Cold Spring H"atbor Labomtory Press, Cold Spring J larbor~ New Yo rlr..

tRNA Arnez J.C. and Moras D. 1997. Structural and functional considerations of the aminoacyJat.ion reactinn. Trends BiochP.m. Sci. 2: 189-2~2 . Cusack S. 1997_ Aminoacyl-tRNA sy nthetases. Ol.rr. Op. StrrJcl. Bioi. 7: 8B1 - 869.

OeJarue M. 1995. Aminoacyl -tRNA synthetast'~. Curr. Op. Struct. Biof. 5: 4B-55.

The Ribosome Dahlberg A..E. 2001. The ribosome io action Science 292: 866-669.

Fnmk J. 2000. The ribosome- A macromolecular machine pal' excellence. Chern Biol 7: Rl33-Rl41 . Moore P.B. and Steitz T.A. 2002. T be involvement of RNA in ribosome function. Natura 418~ 229-235. Ramakrish nan V. 2002. Ribosome structure and the mechanism o f translation. G!JJ 1.08: 557-572. Roli-Mecak A., Shin A., Dever T.E.• and Burley S.K 2001 . Engaging the ribosome: Universal lfs of translation. Trends in Bw. Sciences 26; 7U5-709.

Translation Broderson D.E. and Ramakrislman V. 2003. Shapes can be seductive. Not. Slm ct. Bivl. 10:78-80. Gl'een R. 2000. Ribosomal translocatiol'l; £F~C turn~ the crank. Curr Bioi. Ht: R369- Ra73. Nissen P.. Hansen J_, D&J N .. Moore P.ll. and Steitz T.A. 2()(}{).a. The structural basis of ribosome activity in peptide l;lond synthesis. Scie.nce 289: 920- 930. Nis~n P., ~dgaard M .. and Nyborg f. 200ob. Macromoleoular m imicry. EMBO }. 19: 489- 495.

C HAPTER

The Genetic Code

t the very heart of the Centml Dogma is the concept of infbrma· lion transfer from the ijnear sequence of the four lett<Jr alphabet of the polynucleotide chain into the 20·amino acid language of the polypeptide chain. As we have seen . the translation of g~neti c information in to amh1o acid ftcquences takes plan~ on ribosomes and is mediated by speciaJ adaptoT moloct.JlP.S known as transfer RNA.-. (tRNAs). These tRNAs recognize groups or three consecutive nudeotides known as codons. With four possible nucleotides at (l.acb position, the total humber of permutations of these triplets is 64 l4 x 4 X 4), a value well in excess of the n umber of amino acids. Which of these triplet codons crre responsibte for specifying which amino acids. and what are the rules that SO''tlm their use!' In (his chapter, we dis· cuss the nature and undet1ying loglc of the genetic code, how the code was ··cracked.'' and the effect of mutations on the coding capacity of messenger RNA.

A

OU T LlNE

• The Code Is Degenerate (p. 461)

• Three Rules Govern thE' Gene& Code (p. 'l69)

SUppressor MutD6ons Can Restde m the Same or a Different Gene (p. 471)

• The Code lsi'Jealiy universal (p. 475)

THECODEffiDEGENERATE Table 15-1 lists all 64 permutations, with -he lflft·hand column indi· eating the base at the 5' end of the trip1ot. the row across the top spec· ifying the middle base and the rl ~ht~hand co)umn identifying the b8se in the 3· position. One of the most stril
ing three triplets being chain-terminating signals (see below). This means that many amino acids are specified by more than one codon, a phenomenon called degeneracy. Codons specifying the same amino ad d arc synonyms. For example, UUU and UUC are synonyms for pl1enylalanine, whereas serine is em;oded by the synonyms UCU, UCC, UCA. UCC, ACU, and AGC, In fact, when the fi rst t wo nudcotides arc identical, the tltird nuclw tide can ba e ither cytosine or urncil and the cod on will still code for the same amino acid . O ften, adenine and guanine arc similarly interchangeable. However, not a ll degenAracy is ba.c;ed on equivnlence of the first two oudeotidcs. Leucine, t'or example, is coded by UUA and UUC. as Wf=fll as by CUU, CUC, CUA , and CUG (Figure 15-1). Codon degeneraey, especially the frequent third·place equivalence of cytosine and uracil or guanine and adenine. expJains how there can be great variation in the AT fCC ratios in the DNA of various organisms without correspondingly large changes in U1e relative proportion of amino adds in their proteins. (For example, thft g•momcs of certain bacteria display vastly differe nt AT/GC ratios, and yel are closely related fl.nough to encode proteins of highly similar amino acid sequences.} 461

The GenP.tic Cede

462

TABLE

15-1 The Genetic Code

uuu uuc UUA UUG 3' ,

0

H

H

II

I

I

I

ctl

S' JC c

,

o' .. c

r

e

'

~

C

leucme

srem

~

CtJ'IIic .sl...., I -. "cccccLGA .

lt!U

UAlJ Ser

UCG

1m 1m

ccu

CI>U

CCC

CAC Pre

Cl>

CUA

~

CIJG

CCG

eN;

~Q.

AUU

ACU

M.J

1li u:

f:.JJC

ACC

AAC

CCA

CM

J

A

~

loop

..

lie

Thr

~~'G c " G~ ·. D'~

PCA

AUGt Met

KJG

AN3

o-1

GW

GCU

GAU

lJ . ,

muc

Tyr

UGU

Cys

UGC

UAC

NJA

1. - 1J

3' -_

cue

ucu ucc UCA

stop

11m

stop

UGG

His

Trp

CGU CGC CGA

Gr1

stop

Arg

0c

~

t

MA

Asn lys

N;:.C

N3A

AOO

:0

'8

CGG

N3U

7

Q. ill

;:; ()"

~

~

Sef

~

•:J

~

Arg

A

~

~l~~~~ f'rRNA

'ti" c: c:

G G GGG;..,c l];·

stem

01:!

u

~

D llllem

NH~ H

Leu

cw

3

ro-c-y--,-~

Phc

] S'

L......-J

GUA

GtJG

GAC

GCC

GUC

Val

GCA

JlJa

GCG

(3GU

Asp

GAA

GAG

GGC GAA

Glu

Gly

GGG

codon

• Chain-terminating or Anonsense· oocjons t I>Jso used in baefena to speaty the ll'litialor f~Met-tRNAJMet

Perceiving Order in the Makeup of the Code

F I GU R £ 1 5-1 Coclon-antkodOfl pairing of two tRNA leU molecutes. Cnncal stem and loop regions of the tRNA strudllre <"re WJeled (see Chaprer 14) 1'he ra1 hexagons firlked to the C (3' to the ant1codon) denotE meth~tloo at the N I positions of the base Note thai 1he codon ~ shovvn rn a 3' to 5 oriertalloo.

fnspection of the distribution of codons in the genetic code suggests that the code evolved in such a way as to minimize the deleterious effects of mutations. For instance. mutations in the first position of a codun w ill oft en give a s imilar (if nut the same) amino acicl.. furthermore, codons with pyrimidines in the second position specify mostly hydrophobic amino acids, whereas those with pur;nes in the sa.-ond position correspond mostly to polar amino acids {sec Ta ble 15-1 and Chaptflr 5, Figure 5-4). Hf:nc:e, bocsn..1sc transitions {A:T to G:C or C:C to A:T substit utions) are the most common type of poin t mutations. a change in the second position of a codon will usually replace one amino acid with a very similar one. Finally. if a codon suffers a transition mutation in the third position, rarely will a different amino acid be specified. Even a transversion mutation itt this position will have no consequonce about half the rime. Another consistency noticeabtc in the code is that whenever the first two positions of a codon are both occupied by G or C, each ufthe four nucleotidcs in the third position specifies the samc-t amino acid (such as proline, alanine. arginine, or ~lyc:ine). On the other nand. whenever the first two positions o f the codon are both occupied by A ur U, the identity of the third nucleotide does make a rliffnr~nCt!. Since G:C base pairs arc stronger than A;lJ base pairs. mismatches in pairing the third codon base are often toleratoo if the fi rst l wo posjtions m ake strong G:C base pairs. Thus, having all four nudeotides in

the third position specify the same amino acid may have evolved as a Sflfety mcr.hanism to minimize ennrs in tho reading nf such c'Odon s.

Wobble in the Anticodon It w as first proposed that a

s~cific

tRNA anticodon would exist for every codon. (f that were the case, At least 61 diffcmnt tR.l\IAs. possibly with an additionr~l 3 for the chain-terminating codons. would be pn~scnt.

Evidence began to appeat, howENf'>.r, that highly purified tR"'JA SpPt.:ics of

known sequence could rocognjze smJeral
Three Codons Direct Chain Termination As we ha ve seen . throe oodons do not correspond to any amino acid. Instead , the.y signify chain termination. A5 we discussed in Chapter 14. thtlse chaiu-terrninating cod ons. UAA. tJAG. and UGA, are read not by

TAtiLE 15-2 Pairing Combinations with the Wobble Concept

Base in AnticOOon

Base in CodOfl

G

UorC

C

G

A

U

U

AorG

A U. or C

a

b

3'

r:.1 L:

5'

- -anticodon arm oftRNA anticodon

~

5' r==A Gtr=]3•

rnRNA chain

5'C:~~d3'

~codon

U in the first (5')anticodt:n positiOn can pair With A a G

c

3'

s

3'

5'

3'

5'

I ill the first {5') anticodon positiOrl can pairwilh U, C, or A

codon or anlicodon

H QUarine-LI'acil

f l Ci u R IE 15-2 Wobble base pairing. Note that the ribose-ribose diStances fOf al the wobble pa~rs are dC6e to those of the standard A:U or G:C base pairs.

specie) tRNAs but by sped.fk prot<~ins known as release factors (RFl and RF2 in badcria and eRFl in eukaryotcs). Release factors enter the A site of lhe ribosome and trigger hyd rolysis of the peptidyl-tRNA occ;upying the P site. resulting in the release of the newly synthesized protein.

How the Code Was Cracked The assignment of amino acids to specific oodons is one or the great achievements in the history of molecular biology (see Chapter 2 for an historic account). How were these assignments made? By 19f30, the general OLltline of huw messenger RNA (rnRNA) partidpates in protein

11Je Code Is Degene.rate

b

a Tstem

T loop

"

54

3'accepto( end

\

\

58

20

69

FIGURE 15-3 Stcuctwe <1f yeast tRNAIIM. (a) The left panel shows a view of rhe l ~ moleaAe based~ X-f
synthesis bed been estab1ish€Cl. Nevertheless, there was little optimism that we would soon have a detailed understanding of the genetic code itseJf. It was believed that identification of the codons fur a given amino acid would reqillre exact knowledge of both the nucleotide sequences of a gene and the corresponding amino acid order in its protein product. At that time. the elucidation of the amino acid sequence of a protein, although a laborious process. was already a very practical one. On the other hand, the then-current methods for deter· mining DNA sequences were very primitive. Fortunately, this apparent road block did not hold up progress. In 1961. just one ye
Stimulation of Amino Acid Incorporation by Synthetic mRNAs Biochemists found that f':Xtracts prepared from cells of E. coli that were actively engaged in protein synthesis, were capable of incorporating radioat-1ive)y-labeled amino acids into proteins. Protein synthesis .in these extracts proceeded rapidly for several minutes and then gradually came to a stop. During this interval. there was a corresponding Joss of mRNA owing to the action of degradative enzymes presen1 in the extract. However. the addition of fresh mRNA to extracts that had stopped making protein caused an immediate resumption of bynthesis. Tho dependence of cell extracts on extemaUy added mRNA provided an opportWl;ty to eluddate the nature of the code llSing synthetic

0-~

+

+

HOOH

ritxmvdEoslde diphosphate (ppA)

poty-An-+ 1

- - - - - - -·- - - - - - - - - -

1 S-4 PDI)'nudeotide phosphotylase rea
polyrihonucleotides. These synthetic templates were created using the enzyme polynucleotide phosphorylase. which catalyzes the reaction;

{XMPJn + XDP c= 1Xl'v1P)11 + 1 +

0

(&Juation 15-1)

where X represents the base and IXMPI, represents RNA of length 11 nudeotides. Polynucleotide phosphorylase i.s normally responsible for breaking down RNA and under physiological conditions favors the d~dation of RNA into nucleoside diphosphates. By use of high nucleoside diphosphate concentrations. however, this enzyme can be made to catalyze the formation of intemu.deotide 3 '--. 5' phosphodiester bonds and thus make RNA molecules (Figure 15-4). No template DNA OT RNA is required for RNA synthesis with this enzyme; the base composition of the synthetic product depends entirely on the .ratio of the various ribonucleoside diphosphates added to the reaction mixture. for e~ample, when only adenosine diphosphate is used, the rer>ulting RNA contains only adenylic acid and js thus called polyadenylic acid or poJy~A. ft is likewise possible to make poJy-U. poly-C. and poly-G. Addition of two or more different diphosphates produces mixed copolymers such as poJy-AU. poly-AC, poly-CU. and poly~AGCU. In al1 these mixed polymers, the base sequences are approximately random. with the nearest-neighbor frequencies deter· mined solely by the relative concentrations of the reactants. For exomple. poly-AU molecules with two times as much A as U have sequences like UAAUAUAAAUAAUAAAAUAUU. . . .

Po)y..U Codes for Polyphenylalanine Under the right conditions in vitro, almost all synthetic polymers will attach to ribosomes and function as templates. Luckily. high cxmcentrstions of magnesium were used in the early experiments. A high magnesium concen1ration circumvents the need for iniliation factors and the special i nitiator tMet-1RNA. allowing chain initiation to talce place without the proper signals in the rnRNA. Poly-U was the first syn1hetic p
The Code Is Degenerute

467

from genetic experimeflts, as indicated in Chapters 2 and 21, and beJow.) On the basis of am,,ogous experiments with poly-C and poly-A. CCC was as5igned as a proline codon and AAA as a lysine codon. Unfortunately. this type of experiment did not tell us what amino acid CGG specifies. The guanine residues in poly-G firmly .hydrogen bond to each other and form multistranded triple helicas that do not binrl to ribosomes.

Mixed Copolymers Allowed Additional Codon Assignments Poly-AC molecules can contain eight different codons. CCC. CCA. CAC. ACC, CAA. ACA, AAC, and AAA. whose proportions vary with the copolymer A/C ~1io. When AC copolymers attach to ribosomes. they cause the incorporation of asp~ragine. glutamine. histidine, and threonine-in addition to the proline previously assigned to CCC codons and the lysine previously assigned to AAA oodons. The proportions of these amino acids incorporated into polypeptide products depend on the A/C ratio. Tnus, since an AC copolymer containing much more A than C promotes the incorporation of m any more asparagine than histidine residues, we c(mclude that asparagine is coded by two As and one C and that lli.stidine is coded by two Cs and one A (Thble 15·3}. Similar experiments with other copolymers a11owed several additional a-ssignments. Such experiments. however. did not reveal the order of the different nudeoUrJes w ithin a codon. Ther-e is no way of knowing from random copolymers whether the histidine cQclon containing two Cs and one A is orderecl CCA, CAC. or ACC.

TA 8 L E

J 5·3 Amino Acid Incorporation into ProteiM•

Amino Acid Poly-AC (5 : 1) Asparagine

Gk.ttemire

Histidine Lysine

Obsefved Amino Add

Tentatl\'e Codc>n

tncorpor~

Assignments

- - - -24 24

6 100

1A2C 3A 1A2C, 3C 2A1C, 1A2C

7 26

Poiy-AG (1 5) Asparagine Glutamine

5 5

2A1C

Histidine

23

\A2C

1

Proline

100

Threooifle

21

3A

2A1C 2A1C

Proline Threonine

Lys1nc

calcui8tedTriplet Frequency

fA1C

3C

20 20 4.0 4 .0

4 100

OB

48

24

40 3 .3 3 .3

3.3 3,3

167

07 3 .3

SUm of Calculated Triplet Frequencies

20 20

tOO

2A1C

3A tA2C.3C 2A1C. 1A2C

2A1C

16 7 16.7

83.3

16.7 0 .7 100 20

'The BmiOO aod ..-.corporat•on no fJI(l(eins was dlserved after adding randcm cq>e\ymefs cl A 8Jld C to a ceR·Iree f?ldlact The incolpa'a!Jofl is given as a pacentage of the rn8Jtlmal incctporetiOn d a~ ruT'inc> aod The oopol)omer r.ltio was then \lSW 10 calculate the frequer,cy with wt1lctt a given (;odon YICIUid appear in the polynudeollde poduct. 1lle retaiJVf: freQtePeS ()f lhe cOdOrlG are a lulclloq otlha proba,biU~ thai a part~CUw tltJCieC:4i(le W1U occur if1 a g~ven

Fa example , v.t.en ltle A/C letio IS 5: I Ire ratio d AAAJAJ>..C = 5 X 5 X 5 :5 X 5 >.. 1 = 126:25. If we lhos BSSign to the 3A codOn a freQVet1CY ot 100, then the 2A arld 1C CCIV! isassqted a Ire~ ct 20 By cooelalill.Q II"E rellllive ~es ct aminO ac.id inGOfPOfat.iOn with the~ lreQuenciBS with wt-iCh given codons eppe8f, tentallve codon aSfi'lgnrl'efits can be m.acJe

pasiVon d a c.otloO

TA 8liE

15-4 Bindlns of Aminoacyl tRNA Molec:ules to lrinudeo~e Complex~

Trinucleotide 5'-liUU~'

uuc

UUA AAU

Ul.JG

AUC

Phenyfalantne

cuu AUA

cue GUG UCG CCG

CUA

CUG

Leucine Isoleucine

AUG

MeU1iooine Valine

GUU UClJ

GUC

ucc

GUA UCA

ccu

CCC

CCA

AAA UGU

AAG

UGC

lysine Cvste1r1e

GAA

GAG

GlutamiC add

UCU'

Serine

Proline

'Nore that lhls codOn was fl'ISaSSigned by U--.s me4hOO

Transfer RNA Binding to Defined Trinucleotide Codons A direct way of ordering the nucleotides within some of the codons was developed in 1964. Th\s method utilized the fact that even in the absence of all the factors required for protein synthesis, specific aminoacyi-tRNA molecu les can bind to ribosome-mRNA comp(('.xes. For example. when poly·U is mixed with ribosomes, only phenylalanyJ tRNA wil1 attach. Correspondingly. poly-C promotes the bincling of prulyl-tRNA. Most importantly. lh.is specific binding does not demand the presence of long mRNA molecules. In fact. tbe binding of a trinucleotide to a ribosome is sufficient. The addition of the trinucleotido UUl) results in pbenylalanyl·tRNA attaclunent. whereas if AAA is added. Jysyl-tRNA specifically binds to ribosomes. The discovery of this trinucleotide effect provided a relatively easy way of determining the order of nucleotides within many codons. For example, the ttinuclrlotide 5'-GUU-3' promotes valyl-tRNA bindmg, 5'-UGU-3' stimulates cysteinyl~tRNA binding, and 5'-UUG-3 ' causes leucyl-tRNA binding (Table 15-4). Although a ll 64 possible bi.nuclootides were synthesized With tbe hope of definitely assigl'lil lg the order of every codon. not all codons were determined in this way. Some trinudeoHdes bind lo ribosomes mucb less efficiently than UUU or GUU, making it jmpossible to know whether they code for specific amino acids.

Codon Assignments from Repeating Copolymen At the same time that the trinucleotide binding technique became available, organic chemical and enzymatic techniques were being used to prepare synthetic polyribonucleotides with known repeating sequences [Figure 15-5). Ribosomes start protein synthesis at random pcints along these regular copolymers; yet they incorpordle specific amjno acids into poJypcptides. For example. the repeating s~uence cucucuru ... is the messenger for a regular polypeptide in which 1eudne and serine a1temate. Similarly. UGUGUG .. . promotes the synthesis of a polypep1ide containing two amino acids, c.ysteinc and valine. And ACACAC ... directs the synthesis of a polypeptide alter-nating threonine and histidine. The copolymer built up from re~t ition of the three-nucleotide sequence AAG (AAGAACAAG} directs the synthesis of tbree types of polypeptides: polylysine. polyarginine. and polyglutamic acid. Poly-AUC behaves in the same way, acting as a

DNA template

f I ti U R E I 5-5 Pfeparing digo-ribonudeotides Using a canblnation of orgCiflic synthes1s C!l1d cq>ying by DNA~ meJase 1. dou~anded DNA With~ repealing sequences can be generated. RNA

"'itj; · n~·In~ ;nc '~ SIU~ IQ: Iwta ~~

pclymerese vvill then S}'Jlthesize long potyribonuc:leotides COTTesponding to one or the other ONAstf<Jnd. depmding on the choice ot ri1»

JS

RNA polymerase

nucleoside ~te added to the reaction CTP UTP ~I

3'

TA 8 L £ 15·5 Assignment of Codon.s Us"'g Repeating Copoaymers Built from Two or Three Nudeotides

Amino Acids

Codons

Incorporated or

Copolymer

Recognized

Polypeptide Made

Codon Assignment

(C U).

CUCjUCUICUC

leudne

5 '-CLJC.-3'

UGUIGUG/UGU

Serine Cysteifle

UGU

Valine

GUG

(UG). {AC)..

ACA!CACIACA

Threonine HIStidine

(AG)..

AGAIGAG\AGA

ArgiflK\e Glutamire

(AUC}N

AUCIAUClAUC

Po&yisdeucine

UCAIUCAIUCA

Polysenre

CAUICAUlCAU .

Potyhistidne

ucu

ACA CAC AGA GAG 5'·AUC-3' UCA CAU

template for polyisoleucine, polyserine. and polybistidine (Table 15-5}. Further codun assignments we.-.e obtained from repeating tetranucleotide sequences. The sum of all th~e observations permitted the assignments of specific amino acids to 61 out of the possible 64 codons (see Table 15-1), with the remaining three chain-terminating corlons. UAG, UAA, and tiGA. not specifying any am ino acid. (Note, as discussed in the previous chapter, that in the special oontext of tmnslation initiation in E. coli. AUG is used as a start codon to specify N-forrnyl methionine

.rather than its usual codon assignment of methionine.)

THREE RULES GOVERN THE GENETIC CODE The genetic code is subject to three rules that govern the arrangement and use of codons in messenger RNA. The first rule holds that codons are road in a 5' to 3' direction. Thus. in principle and as an exarnpJe, the coding sequence for the dipeptide NH[Thr-Arg-COOH could be written as 5'-ACGCGA~ 3' (where 5'·Af'.G-3' is a threonine cndon and 5'-ffiA-3' an arginine codon) or as 3'-GCAACC-5' wherein the codons are written in the same order as before but oppositely to their original

mooure..

orientations. Because messenger RNA is translated in a 5' to 3' direction, however, only the former is the correct coding sequence; if the latter were translated in a 5' to 3' direction. then the resulting peptide would be NH2 -AJ'g-Thr..(X)()H, rather than NH2'Thr-Arg..COOH. The second rule is that codons are nonoverlapping and the message ("A>ntains no gaps. 'J1ais :rnRans that successive codons are represented by adjacent trinucleotides in register. Thus, the coding sequence for the tripeptide NH 2-Tbr-Arg-&r-ax>H is represented by three contiguous and nonoverlapping ttip1ets in the sequence 5'-AC.GCGAUCU-3'. The final rule 1s that the message is translated in a fixed reading frame. which is set by the initiation codon. As you w iU recall from Chapter 14. trAnslation starts at an initiation codon. whicb is located at the 5' ood of the protein-coding sequence. Because codons are nonoverlapping and consist of three consecutive nucleotides. a stretch of nucleotides could be translated in principle in any of three roadlng framos. 1t is tbe initiation codon that dictates which of the tbree possibltl Teadiog frames is used. Thus, fur example. the sequence 5' ... ACC.ACGACGArr.ACCACGACG . . • 3' could be translated as a series of threonine codons (5'-ACG'-3'), a series of arginine codons (5'-(..:GA-3'), or a ser~es of asparate codons (5'-GAC-3J) depending on the frame of the upstream start codoo.

Three Kind~ of Point Mutations Alter the Genetic Code Now that we have considered the nalure of the genetic code, it. is insfmcti\le to re\lisit the issue llf how the coding sequence of a gene is altered by point mutatioru; (see Chapter 9-}. An alteration thai changes a codon specific for one amino acid to a codon specific for another amino acid is called a missettse mutation. As a consequence, a gene bearing a misflense mutation produces e protein product in wlljch a single amino add has been substituled for another, as in the classk example of th~ human genetic disease sidde cell anemia. in which glutamate 6 in the (3-globin subunit or hemoglobin lu:ts boon replaced with a valine. A more drastic effect results from an aheration causing a change to a chain· tennination c.'Odon, which is known as a nonsense or stop mutation. When a nonsense mutation arises in the middle of a genetic message. an incomplete polypeptide is released from the ribosome owing to prem ature chain termination. The size of the incomplete

polypeptide chain depends on th., location of the nonsense mutation. Mutations occurring near the beginning of a gene result i n \lcry short polypeptides. whereas mutations near the ond produce polypeptide chains of almost nonnallength. As we saw in Chapter 14 , mRNAs that contain a premature stop codon arc rapidly degraded in eul<.eryotic cells by a process known as nonsense-mediated mRNA decay. The third kind of point mutation is a frameshift mutation. Frameshift mutations are insertions or deletions of one or a small number of base pairs that altBT the reading framt". Consider a tandem repe-dt of the SE!<.]Uence GCU in a frame that wuuJd be read as a series of alanine codons (the codons are artificially set apart from each other by a gap for clarity but are. of course, contiguous in a real messenger RNA): Ala

Ala

Ala A la

Ala

Ala A1a

Ala

5'-GCU GCU CCU GCU C.CU GCU GCU GCU-3'

StJppressor MtJtafions Con Res

Now imagine the insertioo of an A in the message. thereby generating a serine corlon (AGC) at the site of the insertion. The resulting frameshift causes triplets downstream
Ala

Ser Cys Cys Cys Cys Cys

5'-GCU GCU AGC UGC UGC UGC lJGC UGC-3' Thus, th(l insertion (or for that matter the d eletion) of a single base drd5ti cally alters th~ coding capacity of the message not o nly est the site of the insertion but for the remainder of the messenger as well. Likewise. the insert ion (or deletion) pf two bases Warting frame. Finally. consider the instructive case of an insertion of" three extra bases at nearby posm oos in a message. It is obvious that the stretch of message, at and between the three insertions. will be drastically altered. But because the code is read in units of three, messenger RNA dow n.-.tream of the three inserted bases will be in its proper reading framn and hence, completely unaltered;

Ala

Ala

Ser

Cys Met Leu

His

Ala

Ala

Ala

5'-GCU GCU AC.C UC.C AUG CUG CA U GCU f'.f..1J GCU-3'

Genetic Proof that the Code Is Read in Units of Three The preceding example is the logic of a dassic experiment by Frdncis Crick, Sydney Brenner. and their coworkers, involving bacterio phage T4 that established thol the code is read in units of three and did so purely on the basis of a genetic argument (that is. without any biochemical or molecu lar evidence). Genetic crosses were carried out t<, create a mutant phage harboring three inferred single base pair insertion mutations ot nearby positions in a single g~ne. Of course, the three insertions would hav~ scra.mbJecl a short stretch of cooons but the protein encoded hy the gene in question {called rll) was able to tolt-:>Jate the local alteration to jts amino acid sequence. nus finding indicated that the overall coding capacity of the gene had been chiefly left unaltered despite the presence of three mutations, each o( which alone, or any two of which alone, would have drastically altered the reading frame of the gene's message (and rendered its protein product inactive). Because the gene couJd toteratt3 three inseJ'tions but not one or two (or, for that mattc-:r, four). the genetic code must be road in units of three. See Chapters 2 and 21 for a discussion of the historic figures who showed that the code is read in units of three, and fa a description of the role of bacteriophage T 4 as a model system for elur.idating the nat ure of1hc code.

SUPPRESSOR MUTATIONS CAN RESIDE IN THE SAME OR A DIFFERENT GENE Often. the effects of harmful mutations can be reversed by a seoond genetic change. Some of these subsequent m utations are easy to understand . being simple reverse (back) mutations. which change an altered nuclcmtide sequence back to its original arrangement. More difficult to understand ate the mutations occurring at different locations on the chromosome that suppress the change due to a mutation at

472

The Genetic Code

site A by producing an additional genetic change at site B. Such suppressor mutations fall into two main cates ories: those OC:Ctlrring within the same gene as the original mutation. but at a different site in this gene (intragenic suppression} and those occuning in another gene {intergenic suppression). Genes that cause suppression of mutations in other genes are called suppressor genes. Both of the rypes of suppression thai we are considcri.nv; here work by causing the production of good (or parHaHy good) r.:opies of the protein made inactive by the or\ginal hal'Tilful mutation. For exam.pJe, if the first mutation c;aused the prodt~ction of inactive copies of one of the enzymes involved in making arginine, then the suppressor mutation allows arginine to be made by restoring the synthesis of some good copies of this same enzyme. However, the mechanisms by which intergenic and intragenic suppressor mlltations cause the lftlumption of the synthesis of good proteins are cmnpletely different As an e)(ample of intragenic supression, consider the case of a missense mutation. its effect can sometimes be reversed through an additional missense mutation in the :.arne gene. In sucb cases, the original loss of em:ymatic activity is d ue to an altered threedimensional configuration resltlting from the presence of an im::orrect amino add in the encoded protein sequence. A second misse nse mutation in the same gene can bring back bidogica] activity if jt somehow restores the original configuration around the functional part of the molecule. Figure 15..() shows another example of int.ragenic suppression, this time for the case uf a framcshift mutation.

lntetgenic Suppression Involves Mutant tRNAs Suppressor genes do not act by changing the nudeotide sequence of a tnutant gene. Instead. they change the way the mRNA template is read. One of the best known examples of suppressor mutations are mutant tR.'l\JA genes that suppress the effects of nonsense mutations in proteincoOing genes (but mutant tRNAs that suppress missense mutations and

fi GURE 15·6 Suppressionof mmeshift mutatians. {a) A deletlcllll'l the ntldeotide coding sequE'.nce ccm resUt. in an lf!C()mplete. inactive fXJlweptlde-dla~n (b) 1he effecl of the deletion, st1own 111 panel a, cdfl be overcome by a second ITit.JtatiOf\ c.'lll l15ertioo 1n the codiflg seqoer.ce This ti'"L'\el1i0f1 results 111 the production of ., complete polypeptide d-an having 1\vo amino acid replacements.. Depending on the change in seQUetltt:. the pcore111 may have partial or full activity.

a

b

11, 5•

-:1 "

ONA

:~ I

l {UUIIIIIUIUUIIttill(: deletion of a fiUCleotide during gene replication

insertioo of a nuyleotJde during

1

gene replicalion

;rt

s sense codons

= cmrect

missense nonsense codons codons

l t

I

\

incocrect aborlive amino aoos amino adds cham ending

""'""'mR....

!

~rT.~~~

1

Suppres.-;or Mutt~ticms 0111 Hesidli? in lhe Spme or o J]j{fe~nl Gene

473

even frameshift mutations are also known). In E. coli. suppressor genes are known for ()acb of the three stop codonfi. They act by reading a stop oodon as if it were a signal fa- e specific amino acid. The£e are. for example. three weU-characterized genes that suppress the UAG codon. One suppressor gene inserts serine. another glutamine, and a third tyrosine at the nonsense position. ln each of the three UAG suppressor mutants, the anticodon of a tRNA ~pecies specific for one of these amino acids has been altered. For example, the tyrosine suppf€550f arises by a mutation within a tRNA1Yr gene that changes the anticodon from GUA (3'-AUG-5') to CUA (3' -AUC-5'}. thereby enabling it to recogni~e UAC oodons (Figure 15-7). The serine and glutamine suppressor tRNAs a}s() arise by single base changes in their anticorlons. The discovery thai cells with nonsense suppressors contain mutationally-altered tRNAs raised the question of how their C'.odons correspond1ng to these tRNAs could continue to be :read normally. In the case of the tyrosine UAG suppressor. the answer comes from the discvvecy tha1 three separate genes code for tRNA1}-T. One codes fur the major IRNA"l}'r a

gene coding for a minot tyrosine tRNA

mutated gene containing nonsense codon

5·'1JcJeiiOuo)J'

DNAs·•ouu,~eooo)3' OOOI~aOOO ~

H

~

0~ 1~1 1000 ~ I

tmnscfiption

tyrosine tRNA rerognaes the lYf'OSIIle (X)dons 5'..UAC-3' and 5 -UAlJ.-3'

mutafll mRNA containir.g nonsense codon read

' factor by a release to form a nonfundiooal inCXlfflllete ptotein ptodud

~ ...

!

J;:l ~

s b

mutated gene fOf

mutated gene contaifrirg nooseose codOO

Sl

l'fl&fl()r tyrosirle

ooot8eooo OODIIIOOO}~ ~·

DDOIIIUOU 3'.OOOIIIDDO L

nanscription 3.

5'

tR NA

5'1

)~

s

..

recogniZeS the nOflSeflSe codon 5"..tJAG-3"

J

3'

the nonsense codon is St4Jpressed; tyrosire is inserted al the posibon

of lhe nonsense codon to alow the formatiOn of a complete po'lypeptide chain.

}3'

)5'

Fl (; U ~ £

15-7

NonsenM $Uppt'essiol\.

The ~re s11o.Ns ha.~~~ a mmor tyrosine tRNA species acts to sq:>ptess the nonsense codon lflmRNA.

species. whereas the other two are dupJicate genes ooding for a species present in smeUer acHOU11Lq, One or tbc other of tho two duplicate genes is always the site of the suppressor mutr1tion. No such dil<>mma exists for UGA suppression, which is mediated by a mutant form of tRNA1'P; the suppressing tRNA nv retains its capacity to read UGG (tryptophan) codons while also recognizing UCA stop codons. This is possible because the anticodon was changed from CCA (3'-ACG-5' ) in the wildtype to UCA (3'-ACU-5' ) in the mutant lRNA"lrp' and wobble rules, as we have tmen, allow t ecognition of A or G in the 3 ' p osition of the codon by U in the 5' position of an anticodon.

Nonsense Suppressors also Read Normal Termination Signals The act of nonsense suppression can be viewed as e competition between the suppressor tRNA and the release factor. When a stop codon comes into tl1e ribosomal A site, either read-thm ugh or polypeptide chain termination will occur, depending on which arrives first. Suppression of UAC codons is efficient. In the presence of the e;uppressor tRNA. more than half of the chain-terminating signals are read as specific amino add codons. E. coli can tolerate this misreading of tbe UAG stop codon because UAG is used infrequently as a chain-terminating coclon at the end of open-reading frames. In contrast, suppression of the UAA oodon usuaiJy averages between 1% and 5Cf'o and mutant c::clls producing UAA-suppressing tRNAs grow poorly, This is expected from the fact that UAA is frequently used as a chain-terminating codon and its recC'Ig.Jl it ion by a suppressor tRNA would be expected to result in the production of many more aherrnntly long ]Xlly peptides.

Proving rhe Validity of the Generic Code The code wns cracked, as we have seen, by m~ans of biochemical methods involving the use of cell-free systems for carrying out protein synth~is. But molecular biologists are generaUy suspicious of a m el hod thut relies on in vitro analysis alone. So how do we know definitively that the c...ode as depi~ted in Thblc 15-1 is true in living cells? Of CO\..lf'6e, in the modem era of large-scale DNA sequencing. in whlch the entire nucleotide sequences of the geno.rnes of diverse organisms ranging from microbes to man have been determined. the genetic code has not <1nly been va lidated but shown to be universal or ne.arly so (see below]. Nonetheless, a classic and instructive experimtm.t in 1966 h elped tc vaH· date the genetic oode well before DNA sequencing wa.s possible. The e>.-perimenl was baserl nn the COllStruction by genetic recombination of a mutant gene of phage T4 that harbored a mu tually suppressing pair of insertion and deletion mutations (similar to the example given in Figure 15-6). The gene in question enroded a cell-wall degrading enzyme called lysozyme. ch osen because it is small. easy to purify, and its complete amino ac1d sequence was known. The experimental strntegy was to compare the amino acid sequence of the doubly mutant protein with that of wild -type lysozyme. When the amino acid sequences of the mutmt (. • . NH2 - Th.r Lys VaJ His Tlis Leu Met Ala Ala Lys-CQOH . . .) and wild-type (.. . NH2- Thr Lys Ser Pro Ser Leu Asn Ala Ala Lys-(;OOH . .•) were compared. they were fmmd fo differ by a stretch of live amino acids (highlighted in bold). This observation suggested that the insertion and deletion mutations had scrambled a shorl stretch of r.odons in the message o f the mutant. Knowing the consequent effect of the scrambled codons on the amino acid sequence c f the protein imposed important

constraints on the nature of the gtmetic rnde. Specifically~ if the genetic code as elucidated in biochemical experiments is valid, then it should be possible to identify a set of codons for the wild-type sequence Ser Pro Ser Leu Asn that, when properly aligned and bracketed with an insertion at one end and a deletion at the other. would specify the mutant amino acid sequence. Indeed. such a solution exists. which requires a deletion of a nucleotide at the 5' end of the coding sequence and the insertion of a n\l,c.:leotide at the 3' end:

Nl-I2 - Lys

Ser

Pl'o

Set

Leu

Asn

Ala ~ COOH

5'-AAA ACU CCA UCA CUtl AAU CC-3' 5'- AAA GUC CAl l CAC UUA AUG GC-3'

NHz-Lys

Val

His

His

Leu

Me1

Ala- COOH

As you can see, the solution verifies several codon assignments and demoPEt.rates that more than one synonymous codon is used to specify the same amiJ1o acid in \'ivo (fm·e:xample. 5'-cAU-3' andS'-CAC-3· for histidine). Lastly, a11d importantly. you should he able to convince yourself from the solution that translation proceeds in a 5' to 3' direction. (Hint: see if you can accoUJlt for the two amino add sequences in their proper NHa to COOH order when you align each of the codons in your solution in a 3' to 5' orientation.)

THE CODE IS NEARLY UNIVERSAL - - - - - - - - -- - - - - - - - - - - - - -- - - The results of large-scale sequencing of genomes have lrugely confirmed the expected universality of the genetic code. The universality of the code has hod n huge impact on our understanding of evolution as it made it possible to directly compare protein coding sequoncet> among all organisms for whkh a genome sequence is available. As we shall see in Chapter 20, powcrfn.l computer programs are available that can search for and iderttify ~imilaritics among predicted coding sequences from a wjde range of organisms. The unjversality of the code also helped to create the field of genetic engineering by making it possible to express cloned copies of gene.s encoding useful protean products in surrogate host organisms, such as the production of hrunan insulin in bacteria (see Chapter 20). To 'm~rstand t"he conooJVative nature of the code, consider what might happe n if a mutation changed the genetic codf!. Such a m utation migltt, for example, alter the sequeJ1Ce of the serine tRNA moJ ecuJ~ of the class that oottesponds to UCU. causing them to recognize UUU sequences instead. This would be a lethal m utation in haploid cells ccntaining only one gene directing the production of tRNASew. for serine wouJd not be jnserted into many of its normaJ positions in proteins. Even if there were more than one gene for tRNASec- (as in a diploid cell}. this type of mutation would stiJI be Jethal since it would muse the simultaneous repJacement of many phenylalanine residues by serine in cell proteins. In view of what we have just sajd, it was completely Ullexpected to liud that in C'.eJtain subcellular organelles. the genetic code is in fact slightly diffetent from tltc stfmdard code. This realization came during the elucidation of the entire DNA sequence of the 16,569-base pair human mitochondrial genome but is obsenred for milrochondria in yeast. the fruit tly, and higher plants. Sequences of the regions known

to specify proteins have revealed the following differences between the standard and mitochondrial genetic codes {Table 15w6): • UGA is not a stop signal but codes for tryptophan. Hence, the anticodon of mitoch ondria) tRNA'OJ! recognizes both UGG and UGA. as if obeying the trailltional wobble rules. • Intarnal methin.rdn~ is encoded by hath AUG and AUA. • ln mammalian m itochondria, ACA and ACG are not argunne codons (o f which there are six in the nuniver.sal" code) but specity

chain termination. Thus, there are four stop codons (UAA, UAG, AGA. and AGG] in the mammalian milochondrial code. • In fruit fly mitochondria, AGA and AGC are also not arginine codons but specify serine. Perhaps not

surprisingly~

mitochon drial tRNAs arc likewise

unusual with respect to the rules by which they decode mitochondnal messages. Only 2 2 tRNAs are present in mammalian mitochondria. whereas a minim um of 32 tRNA molecules are required to decode the "universal" code according to the wobble rules. Consequently, when

TABU 15-6 Genetic Code of Mammalian Mitochondria• second po&iticn

:cc:: Cll

uuu uuc

Ph4l

ucu

(GAA)t

ucc

UJA

Leu

UUG

(UAA)

cuu cue

Leu

CUA

(UAG)

in _. s:

CUG

'in 0

AUU

Wi

...

AUC

~

m1

UAU

SEW

UCA (UGA)

UAC

1m

Tyr (GUA) stql

UGU (GCA)

UGC

l!m

Trp

UGG

(UCA)

CGU

LCG

m:a

stop

ccu

C.AIJ

His

CCC

CJIC (GUG)

CGC

CAA

CGA (UCG)

Pro CCA (UGG)

CCG

CPG

Gtn (\A.IG)

N;t)

AftU

A:$1)

Arg

AUG

lie (GAU)

Met (~)t

GUU GUC GUA

GUG

Val (UAC)

AJlC

(GUU)

N:A (UGU)

AAA

lys

M;G

AAG (UUU}

GCU

GAU

HX

TlT

GCC Ala GCA (UGC) GCG

Asp GfC, (GUC}

GAA

GltJ

GAG (UUC)

a

'8Ill

CGG

;:r

~

c.

!,;

N3U

SE!1'

AGC

(GCU)

aD

step

8

..

~ :ll

rm

.s

stop

GGU

GGC

Gty

GAA

(UCC}

GOO

" OOetenees between U1e mik>Ch'Ofldial and "unlvernal'" ger.ebc code (Table 1~1) are ~ by greef1 shading.

t Eacfl QtOt.P of oodons is sl1aded if1 gray and is read by a sifVe tRNA wf10Se antiCOdOn, written 5 ' - ~. io pc.enl heses. Each fotx-codoo gr~ is read by a tRNA having a U in the fif"sl (5'} positioo of ltle anlioodon. Two-c;odon groups with oodoos ending rn either' UfC ()( NG are read with GU ~by tRNAs. with G or U, respectively, in lhe first positioo of lt1e anticodon. The anlicodons oftoo oontaifl mc:>dified bases.

t Note that lhe C in lhe fm M\loodoo J>oSitioo engages in Lf'lUStJal pairing.

Bibliogt'(Jphy

477

an amino acid is specified by four codons (with the same .first and second positjons), only a single •nitochondrial tRNA is uwolved. {Ret.:all that a minimum of two tRNAs would be required by nonmitochondrial systems.) Such mitochondrial tRNAs all have in the 5' (wobble) position of their anticodons aU resid ue. wbich is able to engage in pairing with any of the four nucleotides in the thlrd codon position . 1n cases where purines in the third position of the codon con-espon d to different amino acids fmm pyrimidines in that position, a m odified U in the first position of the anticodon of the mitochondrial tRNA restricts wobble to pairing wHJl the two purines only. Exceptions to the "universal" code are not limited to mitrochondria but are also found in several prokaryotic genomes and in the nuclear genomes of certain cub.ryotes. 1he bacterium Mycoplosmu CGpricoJum uses UGA as a tryptophan codon rather than a chai~termination codon. Likewi::;e. some unjcctlulm· protozoa use UAA UAG, which are s top codorn; in the "universal"' code. as g1utamine codons. Finally. a rodon (CUG) for one amino acid UeucineJ in the .. universal'' code has become a codon for another amino acid (serine) in the yeast Gmdida.

anu

SUMMARY In the '"'universal" genetic code u sed by every organis m from bacteria to huutans, 61 codons signify specific amino acids; the remaining three are d 1ain-termination cot.km.s. Th6 t'.()(!tl is llighly degenerate, w ith sevewl codons {~-yn .onyms) usuaUy corresponding to a singJc amino acid . A given tRNA can sometimes s pecifically recognize several codons. This tthility arises {rom ~nibble in the base at tlw. 5' end of the anticodon. The stop codons UAA. UAG. and UCA are read by spec-:ific proteins, not s pecialized tRNA mola·ules. The genetic cod£1 is subject to three principal rules. Codons are read in a 5' to 3' d irection. codons are nonoverlapping and the message contains no gflpS, and the message is translated in a fi"'ed reading frame, which is set by the initiation t.'Od on . The genetic t1llde was cracked through the f'tudy of prob:in synthesis in cell-free extracts. Addition of new mRNA to an eJ-1.ract depleted of its original messenger componPnt results in the production of new proteins whesl Bmino ocid sequ~nces are dewm1ined by the externally add ed mRNA The first (a1ld pmbahly mosl iropqrtant) st1Pp in c.:cad<.ing llle genet i•~ code occurred when the synthetic polyri-

bonuclootide poly-U was found to code specifically for pO)yphenylelanine. Use of other synthetic polyribonudeotidas, both homogoneous (poly-C, and so on) and mixed (poly-AU, and so on), then ;:~)lowed assignm~nl of <."'(lons {or tho various amino acids. Dctennination of the exac1 order of nucleolide.s in codons subsequently caJTit> from A sturly of specific trinudeotide-tRJ\IA-Iibosoott> interar:tions and the use o f regular copolymers as messens,rers. Point mutations that alter the end() are nusseru;e m uta· lions, whir:h change the codon for one amino acid inlo I he codon lor anothllr amino acid; nonsense m utations, which cau S6 protein synthesi5 to tenninate prematurely; and frameshift mutations, which alter lhe re2ding frame of the mossage. In some cases the effects of missense, nonsense. and fta:rneshiCt mutations can bt! partially suppressed by extragenic suppres.<;ors. For example, mutant tRNAs read s top codons generated by nonsense m utations as if they were codons for a spet:ific amino acid. A slightly differnnl genetic code is utili?..ed in mitochondria and in the pcindpa) gen omes of certain prokaryote~ and p•·oto:;r,oa. such as the use of UGA, a stop codon in the ''universal code," as a tryptophan codon

BffiliOGRAPHY Books Celis J.E. and Smitb f.D.~ erls. 1979. Nonsense mutations

und tRNA suppressors. Academic Pross, New York. CliiTk B. and PPtEJN.l:!O l·t, e<)s. 1984. Cena expression: The translational step and its r.ontro.. Alfred Btmzon SJ7T7posium. vol. 19. Copenh agen . Mtanksgaatd. Cold Spong Harbor Symposia on Quantitative Biology. 1006. Volume 31: The genetic rode. CQid Spring 1-lamor Laboratory, Cold Spring Harbor. New York.

SoU D.C., Abelson J.N .• and Schimmel P.R., ed.nce). New York

Features of the Gt."'\etic. Codt: Crick F.H.C. 1006. (;odon-anticodon pairing: The wobbte hypothesis. f. Mol Biol. 19:548- 555.

4-n

T he Gen(!tic Code

J. and Grosjean H. 1YHI. Usage of the three termination codons: Co.m pilation and analysis of the known

KohH

e ukatyotit: and prokaryotic transJation terminiltion sequ ence.f\. Mol. Om . Gtmel. 182: 430- 4 39 .

Lagerkvisl U. 191'11. Unorthodox codon .reading and tht> evo1ution of the genetic code. Cell 23: 305-300.

How the Code Was Cracked Crick FH.C,. 1003. T h e recent excit~ment i.n the c oding problem. Prop,. NtJcleic Acid Res. 1:164.

Khorana H.C. 1 968. Polynucleotide synthe.'ilS and the geneti t~ code. Harvey UJCh.JTf.' Series 1966- 67. VoL 62. Academic Press, New Yruk

Nirenberg M. and Leder P. Hl64. The effect of trinucleotide.<; upon the lJinc:ling of sRNA to ribosomes. Sc1tmcc 145: 1399- 1407.

Speyer J.F.• Lengyel P.. Basilio C., Wahba A.J•• Cardner R.S.• and Ochoa S. 1Y63. Synthetic polynucleotides and lhe amino acid code. C'.cld Spring Harbor Symp. Quant. Bioi. 28: 559- 566.

Three Rules of the Genetic Code Brenner S .• Stretton A..O.W., and Kaplan S. l H65. Genetic code: The nonsen se triplets fur chain terminat M'ln and thP.ir ~~ 1ppression. No tore 200: 994 - 998.

Oick F.H.C., Barnett L., Bw.nner S .• and Watt&-Tobin R J. 19tH . CeneraJ nature of the genetic r:orle for pro(eins. Nature1S2: 1227 - 1232.

Geren A. 1!168. Sense a nd nonsense in the gene tic code. Scie11ce ltiO: 149 - 159 .

1el7.aghi E. , Okada Y., Slteisinger G .. Emrkh 1.. Inouye M.. and Tsugita A. 19ti6. Change of a sequence of amino acids in phage T4 lysozyme by acridine-induced mutation G. Proc. Notl. Acod Sci. USA 56: 50()- 5 07

SuppTession Duddngbam R.H. and Klll'lanrl C.G. tYOO. lntenu::lion.<> between UCA-su pp ressor tRNA'P and the ribosome: Mechanisms of lRNA selection. rn Tronsfer RNA~ BinltJgicaJ aspects (ed. 0 . Si.ll et al.). pp. 421 - 426. Cold Spring Harbvr Laboratory. CI:>Jd Spring Harbor. New York-

Ozekl H., l.ookuchi H., Yamao F. . Kodaira M.. Sakano H.. fkemura T., e nd Shimura Y. 1960. Genetics of nonsense suppressor of IRNAs in Escheric/lia cofi. ln 'Tronsfer RNA.- l:liol ogica/ aspP.Cts (ed. D. et al.), pp. 341-349. Cold Spring f1arbor Laboratory, Cold Spring Horbor, New York,

scm

Steege D.A. and SOli V.G. 1979. Suppression. In BioJogir.:a l reglllalion and development I (ed. RF. Goldberger),

pp. 433-486. Plenum. New Vork

480

Parr 4 Regulation

PA RT

OU T l i NE

• dlaptes 16 Gerle Regulation m Prokaryotes

Chapter 17 Gene RegulatiOfl tn Eokaryotes

Chapter l 8 Gene Reguiaton dur•ng Development Chapter 19

Compa~awe Geoorntcs artd

the Evclutlon of Anrnal Dtvefsity

n the preceding part. we considerfl.d lmw tl1e genetic information encoded in the DNA is expressed. This involves the transcription of DNA sequences into an RNA form '\vh ich is then used as a template for translation into protein. But not all genes are expressP.d in all cells aU the time. lndood, much of lite depends on the ability of cells to m:press their genes in different combinations at different times and in different places. Even a lowly bac.'tetium expretises only some of its genes al any given time -ensuring it can. for example, make the enzymes needed to metabolize the nutrients it e ncounters \i'v'hile n ot makjng enzymes fOf' other n utTients at the same titne. Development of multicellular organisms offers a striking exam ple of this so-called ~'differential gene expression... Essentially all the cells in a h uman contain the same genes. but the set of genes expressed in fanning one cell type is different from that expressed in forming another. Thus, a muscle cell expresses a set of genes different (at least ill part) from tha t a-pressed by a neuron, a skin cell, and so on. By and large these differences m:cur at the level of tf'dnscriplion-mo~t commonly, the jnjtie~tion of

I

lr.mscription. In the following chapters~ we look at how genes are regulated starting in Chapter 16 with how this is done in bacteria. It is here that the basic mechanisms can most readily be appreciated First. we deal with simple cases that illustrate different mechanisms of transcriptional regulation. These include the case of the lac operon. These genes encode proteins needed for metabolizing the sugar lactose. and are expressed only when that sugar is available in the growth medium. Then we look at examples of gene regulation that operate at later steps in gene expression-RNA elongation and tran~l atio11, for example. Finally. in this chapter we describe how phage ~. chooses between alternative developmental pathways by expre:;~ing different sets of genes upon infection of a bacterial cell

In Chapter 1 7 . we consider basic mechanisms of gene

expr~'Sion

in eukaryotes 1 frorn yeast to some of the simpler cases found in higher eukaryotes. Mechanisms of transcriptional ~ctivation and rcpre~sion are compared to those in bacteria, a nd we see where mechanisms ace conserved. and where there are additional ft'.<Jtwes- most notably the effects of chromatin modifications of the type cliscllssed in Chapter 7. We also see how smaJI RNA molecules can regulate gene expression in various ways. As we saw in Chapter 13, eukatyotes very often have to splice RNA before they can be translated. This offers another step at which expression of a given gene can be regulated. In this case. regulation can determine not only when a given gene is exptesserl. but also which of several alternative protei,ns is made. In Chapters 18 and 19. we cons ider gene regulation in the context of developmental biology. In Chapter 18, we look at exan1ples of how genes are regolated to bestow ceU type specificity (differentiation) and pattern fonnation (morphogp,nesis) in a group of genetically identical cells- for example, those found in a developing embryo. Chapter 19 looks at diversity among closely-related organisms and sees how. in many of these., the differences in morphology or behavior result not hom changes in the genes, but from differences in where and when those genes are expressed within each organism during development.

Port 4 Regul (lliOfl

481

PHOTOS FROM THE COLI) SPRING HARBOR LABORATORY ARCHIVES Edward Lewis. CMilindegren. Alfred Hetshe~ and Joshua l..ededJerg. 1951 S)mpGsium on Cenes and Mutatiotls. Levv1s instigated the genetic analysis of devclopment uSing the fruit fly a~ fns model (Olapter 18). He shared the 1995 Nobel Prize io Medtcine fof hs work. L•ndegren was a pioneer of yeast ger1etics (Chapter 21 ) . Hershey WM. togethef with MdX

Delbrud and Salvador Luria. the leader of the gtoup that used phage as their model system in the early days d molecular blok>gy (Chapter 21 ); the ttYee of them shared the 1969 Nobel Prize fof Medicine_ L€detberg discova-ed rhat DNA coud pass betweEn bactena by a rna~ process cairo conjugation (Ol~er 2 1). for which he shared in the 1958 Nobel Prize for Me
Jeff Rclllerts atd Ann Burgess. 1910 Symposium on Transcription of Ciet'tetK: Mat2rial. Roberts' fesearch has focused on regulators of 8€fle e;<presSIOO lf1 bactena and phage. portJctJarly anlltenninators u'l phage 1.3mbda (Chapter 16). Burgess bocaf1le a bfoJogy edt.JCator and 15 i1volved in national efforls to improve 5Cieoce t:dUCDtioo. Rd:>erts was an authot of lhe previ>t.6 ediiJon of this book, while Bu~s has a cousin among the cunenl authors (TB).

Christiane Nlisslein-\foJhard,. 1996 CSHL Meeting on Zebrafish Development and Genetia. Mutant sa~f1S caroed out in fruil Qies by NUss~oltwd arJd 11ef colle~ Enc: WlesdtatJS identified many genes oib· cal to the early embryonic development of that organtSm, and probably all animals (~er 18). For this the two of

lt1em sllared 111 lhe 1995 Nobel Pfire with Fdv.'
Part 4 Regulction

Math ptasltfte ald Joseph Coldstein. 1988 Symposium on Molecular Biology of Signal Transduction. Ptashne Wc)S lnstrument.:li in t~!ll8 the eady ideas of Jacob and Monod about how gene expression is regulated, and desaibillg flow these wo"'- at a moleaJiar (a.oef (Cilaprers 16 and 17}. Goldstein. \IIIith tis longtime ccllclborator l'v1idlael Br""m, v.uked oul lhe stgnal transdudlon pathways (Chapter 17) that control expression of genes irwclved in cholesterol metaOOiism. for which they won the 1985 Nobel Prize in Meacine.

JacqUes Monod and leo Szilard., 1961 CSH Laboratory. fvlonod. together with Ff~

Jacob. foonulated the operon model for the reguJation of gene expression (Chapter 16). The two of them. together With their colleague Andre lwoft shared the I963 Nobel Prize in Me&cine for this achievement leo Szilard was a wartime nuclear physicist \hOO turned to molecular biology ~er talong the phage course at Ccld Spring Harbor in 1947. He ran a lab with AiYoo No!.1d in Chicag:>. (Source: Courtesy of fsthef Subley.)

Mrs.. LH. Herskowitz with sons. ka and

Joel, 1947 Symposium on Nucleic Adds and Nudeoproteins. Ita Herskowitz pioneered the use ()f the yeast 5. cetellisiae as a model organ1sm for rroleoJiar ~ (Chapter 2 1), and made I11Cljor cootribc.Jtio1s t() ideas about gene regulation M'llhis organism as tte had. eader, in bacteriophage lamtxla (Chapters 16 and 17). tis father; lrvM. later the authoJ d a geneti<s texlbool<. was attending lhe symposaum that year.

CHAPTER

Gene Regulation in Prokaryotes

n Chapter 12 we saw how DNA is transcribed into RNA by the enzyme RNA polymerase. We also described the sequence elements that constitute a promoter-the region at the start of a gene where the enzyme binds and initiates t-ranscription. In bacteria the most common form of RNA polymerase (that bearing o 70) recognizes promoters formed from three clements- the .. _ 10"... - 35••. and ..l JP" elementsand we saw that the strength of an}· given promoter is determinoo by which of these elements it pos."esses and how well they match optimum "consensus•· sequences. In the absence nf regulatory proteins. these clements deter:.mirle the efficiency with which polymerase binds to the promoter and. once bound, how readily it initiates transcription. Now we rum to mechanisms that regu)ate expression-that is, mechanisms that h1crease cw decrease expression of a given gene as the requirement for its product varies. There are various stages at which expression of a gene can be regulated. The most common is transcription inHiation, and the hulk of tbis chapter focuses on thfl regulation of that step in bacteria. We start with an overview of general mechanisms and principles and proceed to some well·studierl examples that demonstrate how the basic mechanisms are used in various combinations to control genes in specific biological contexts. We also consider mechanisms of gene regulation that operate at steps after transcription initiation, including transcriptional entitennination and the regulation of translation.

I

OUTL I NE

• Pnnaples of Transcriptmal RegulatiOn (p. 483)

Regulation of lfansaipt1orllnitiatinn:

ExaJillles frnm Bactena (p. 488) • Examptes of Gene Regulation at Steps after Transaipbonlnitiation (p. 504)

• The Case of Phage A. toyers of RegulatiOn (p. 5 12)

PRINCIPLES OF TRANSCRIPTIONAL REGULATION Gene Expression Is Controlled by Regulatory Proteins As we described in tho introduction to tills section. genes are very oftEn controlled by extracellular signals-in the case of bacteria, this typically means molecules present in the growth medium. These signals are communicated to genes by regulatory proteins, which come in two types: positive ~ulators, or activators; and ~E}Jl8tive regulators, or repressors. l)picaUy these regulators are DNA-binding pro~ teins that recognize specific sites at or near the genes they controL An activator increases transcription of the regulated gene; repressors decrease or eliminate that transcription. How do these regulators work'? RecaH the steps in transcription initi~ ation described in Chapter 12 (see Figure 12-3). First. RNA polymerase binds to the promoter in a dosed complex (in which the DNA strands remain together]. The polymerase-promoter complex then undergoes a transition to an open complex in whlch the DNA at the start site of 483

484

Gene Regulation 111 ProkcryQtes

transcdption i~ U!'IWound and the polymerase is positioned to initiate transcription. This is followed by promoter escape the step in which polymerase leaves the promoter and starts transcribing. Whic.h steps are stimulated by activators and inhibited by repressors? That depends on the promoter and regulators in question. We consider two general cases. outlined lUlder the next two headings.

Many Promoters Are Regulated hv Activilt()('S that Help RNA Polymerase Bind DNA and by Repressors that Block that Binding At many promoters, in the absence of regulatory proteins, RNA polymerase binds only weakly. This is because one or more of the promoter elements discussed above is absent or imperfect. When pol}'IDecase does occasionally bind, however~ it spontaneously undergoes a transition to the open com]_Jlex and initiate& transcription. This gives a low level of constitutive ro:pression called the basal level. Binding of RNA polymerase is thfl rate f.imiting step in this care (f'igure 16-l a). To contm l expression from such a promoter, a repressor need only bind to a site overlapping the region bound by polymerase. In that way. the repressor blocks polymerase bmding to the promoter. thereby preventing transcription (Figure 1&1b), although it is important to note that repression can work in other ways as well. The s ite on DNA whP..re a repressor binds is called an operator. To activate transcription from this promoter, an activator just helps polymerase bind the promoter. Typically this is achieved as follows: the I' I G U R f

16-1 Activation by re<:ruitment

of RNA ~ase. (a) In the absence of bott1 ~IVator «ld repressor, RNA polymerase occaslOI'lally binds the promoter 5p01'1tarleously and n llates a low level (basal level) ()f ~ ( b) &'!ding of the reprESSOf to lhe operntor sequerce l:*x:ks birrlrtg of RNA polyrnefase and so tnhlbtts b
me

a

basal level

activator ~site

-

of~on

q>e~~or

promoter

no transc:ription

spontaneous isoo'leriz3tion

leading to activated level of transcriplion

l'rinQples of 1i'anscriptioool Regulatwn

485

ar.tivator uses one surface to bu1d to a s ite on the DNA near the promott::r; with anotlu>.r surface. the activator simultaneously interacts with RNA polymerase, bringing the enzyme to the promoter (Figure 16-lc). This mechanism, often called recruibnmt. is an example of cc:NJperativc binding of proteins to DNA (sec Chapter 5). The inter~tions between the activator and polymerase. n.nd bctv.reen activator and DNA, serve merely "adhesive" roles: tbe enzyme is active and the activator simply brings it to the nP.arby promoter. Once there. it spoptaneously isomerizes to the open complex and Initiates tTanscription. The lac genes of E. coli are transcribed from a promoter that is regul ated by an activator and a repTessor working in the simple ways just outlined. We will describe this case in detail later in the chapter.

Some Activators Work by Allostery and Regulate Steps

after RNA Pol}merase Binding Not all promoters are limited in the same way. Thus. consider a promoter at the other extreme from that described above. In tbis case, RNA polymerase binds ef-ficiently unaided anrl forms a stable dosed complox. But that closed complex does no1 spontaneously undergo transition to the open complex {Figure l&2a)- At this promoter, an activator must stimulate tho transition from closed to open complex, since that transition is the rate-limiting step. Activators that stimulate this kind of promoter work by triggering a conformational change in either RNA polynierase or DNA. Thal is, they interact with the stable closed complox nnd induce a conformational d1ange that causes transition to the open complex (Figure 16-2b). This mechanism is an example of allostery. In Chapter 5 we enco untered allostery as a general mechanism for ronttolling the activities of pl'oteins. One of the examples we considered thel'e was a protein (a cydin) binding to. and activating, a kinase {Cdk) involved in ceU cycle regulation. The cyclin does this by inducing a confonnational change in the kinase. switching it from an inactive to an active state {l-1gurc 5-27). In this c.hapter, we will ~two examples of transcriptionaJ activators working by allostery. In one caSe (at the ginA promoter), the activator lNtrCJ interacts with the RNA polymerase bound in a closed complex at the p romoter, stimulating a

16-2 Ahosteric adiwllion of RNA polymerase. (a) BiC'Iding of RNA polymerase to the promoter tn a stalk dosed

fIGURE

no spontaneous isomerization and thus

no transaipti
CDmplex. (b) The activator Interacts Uth polymerase to lr~ tfarr.>11ion k> the open

activator

complex and hrgh levels of tJansenption. 1he rep~esentatlons of the dosed and oper1 complexes are shown only dicgrammaocclly; fcr a more~ desolpcion of those states

binding Site

see Chapter 12

b

a~:«tvated levef of transoiption

4Hti

CeJle Regfllaficm in PrcJ<,aryott·s

trcmsition to the open complex. In the other ex8mple {at the merT promoter}. the ru.:tivator (MerR) achieves the same cffed but does so by inducing a conformational change in the promoter DNA. There are variaCions on t hese themes: some promoters are inefficient at more th an one step and can be activated by more than one mechanism. Also. ~pressors can work in ways other than just blocking the binding of RNA polymerase. For example, some repressors inhibit transition to thn open complex, or p{Omoter escapt~. We will consider examp1es of lh""se later in the chapter.

Action at a Distance and DNA Looping 1'hus far we have tacitly assum~ that DNA-binding proteins that interact with each olher bind to adja.:;ent sites (for example. RNA polymerase and activator in Figures 1&1 and 16-2). Often this is the case. But some proteins interact with each other even when bound to sites well separated on the DNA. To accommodate this interaction, the DNA between thP. sites loops out. bringing the sites into proximity with one ano1her (Figure 16-:1). We wiH encounter examples of this ldnct of interaction in bacteria Tndeerl. onf! of the acti vators we have already mentioned (NtrC) activates "from a distance"; its binding sites are normally located about 150 bp upstream of the promotel', and the activator works ev~n when those site..'\ are placed furthet away (a "kb or more). We will a lso .:;onsidcr repressors that interact to form loops of up to 3 kb. ln the next chapter-on eukaryotic gene regulation-we wi ll be fuccd with more numerous and more dramatic examples of thi$ "action at a distance.·· One way to help bring distant DNA sites closer together (and so help looping} is tho binding of other proteins to sequences betweAn those sites. In bacttlria there are cases in wh ich a protein binds between an activator binding site ~d the promoter and helps the activator interact with polymerase by bending the DNA (Figure 16-4). Such "architectural" proteins facilitate interactions between proteins in other processes as wel.l (for P.>..'Blllple, sit&-spp,cifir. t'R<;Ombinatinn; see Ch~pter 11).

fl G U RE

16-3 InteractionS between

a A

proteim bound to ONA. (a) Coopruative bindifl,g of proteins to ~acent SiteS. {b) Cooperattve blndtng of proteos to separated Sltes.

)

b

site

site

A

8

Plilicipl r>$ of Tt·Qnscriptioncl Rf'gt.tlation

41'7

FIGURE 16-4 DNA-bending protein can facilitate intefaction between DNA-binding pt'oteins. A protetn that bends

DNA brnds. "=> a

51te be4:v.:een

the .activator

bn:lu;g site and the promoter. 11-.is brings the two srt:es doser together tn space and thereby t1Elps the interaction bet\I\/Eet1 the ONA-Lound adNetor and polymerase.

CoopeJative Binding and A llostery Have Many Roles in Gene Regulation We have already pointed out that gene .a ctivation can be mediated by simple cooperative binding: the activator interacts s imultaneously with DNA and with po)ymera.se and so l'f';Cruit.s the cn?.ymP. to thP. promotP.r. And we have described how activation can. in other cases. be mediated by aUosteric e vents: an Hctivator interacts with polymerase already bound to the promoter and, by inducing a conformational cl1ange in the enzyme or the promoter. stimulates transcription initiation. Both cooperative binding and allostery have additional roles in gcme regulation as well For example, groups of regulators often bjnd DNA cooperatively. TIJat is, two or moro activators and/or oopressms intcl'ac:l with ear.h other and with DNA, and thereby help each other bind near a gene they all regulate. As we will see, this kin d of interaction can produce sensitive switches that allow a gene to go from completely off to fully on in tesponse to only small changes in conditions. Cooperative binding of acfivatol'S can also serve to integrate signal~; that is, some genes am activated o nly when multiple signals (and thus multiple regulators) are simultaneously present. A particularly striking and wellunderstood example of cooperativity in gene regulation is provided by bacteriophage~. We consider the basic mechanism and consequences of cooperative bintling in more d etail when we discuss t hai P.Xamplc later in the chapter. and also in Box 16-5. AUostery, tor its part, is not only a mechanism o f gene ar.tivation, it is also often the way regulators are controlled by their specific signals. Thus, a typir,nl bacterial regulator can adopt two conformations-in one it can bind DNA; in the other it cannot. Binc\ing of a signal molcculn loc.ks U1e Togulatory profP.in in one or another conformation. thereby determining whether or not it can act.. We saw an example of tl1is in Chapter 5 (Figure 5-25), where we also considered the basic mechanism of allostery in some detail: in this and the next c.hapter we will see several examples of allostctir. contro l of regulators by their signals.

Antitermination and Beyooo: Not All of Gene Regulation Targets Transcription Initiation As stated at the beginning of this chapter, the bulk of gene regulation takes place at the initiation of transcription. This is true in eukaryotes ;ust as it is in bacteria. But regulation is certainly not teStrictccl to that step in either class of organism. 1n this chapter we will sco examples, in baclflria, of gen~ rogu1atioo lhat involve transcriptional elongation. R.f\IA processing. and translation of the mRNA into ptotcin.

4U8

Gent~ Regularic.n

i11 Proknryotes

REGULATION OF TRANSCRIPTION lNITIATION: EXAMPLES FROM BACTERIA Having outlined basic p rin ciples of ttanscTiptional regt1lation. we tum to some examples that show how these principles work in real cases. first, we consider the genes involved in lactose metabolism in E. coli- those of the lac operon. Here we will see how an activator and a tepressm.· regulate expression in response to two signals. We also describe sorne of the experimental approad1es that reveal how these regulators work.

An Activator and a Repressor Together Control the lac Genes The Lhroo Jacgenes- IacZ. lacY, anrllacA-are arranged adjacently on the E. coli genome and are called the lac operon (Figure 16-5). 'lbe lac promoter, located at t he 5' e nd of JacZ. d irects transcription of all thrOe genes as a single mRNA (called a polyci..o;;tronic message because it includes more than one gene); this mRNA is translated to give tbe three protein products. The lucZ gene encodes the enzyme J3-galactosidase. which cleaves the sugar lactose into galactose and glucose, both of which are used by the cell as energy sources. The lacY gene encodes the lactose permease. a protein tl1at inserts into tho cell membrane and transports ladose into the cell. The /acA gene encodes tbiogalactosirle transacetylase. which rids tho cell of toxic tbiogalactosides that also g<:,'l transported in by lacY These genes are expressed at h igh leve)s only when lactose is available. ami glucose-the preferred energy source-is not. 1\vo l'~ula tory proteins are involved: one is a n activator called CAP. the other a ropressol' called U1e Lac: repres.410r. Lac repressor is encoded by the lctcl gene, which is located ncar the other lac genes, but transcribed from its own (constitutively expressed) p romoter. T he name CAP

stands for Catabolite Activat()r Protein, but this activator is atso known as CRP (for cAMP Receptor Protein, for reasons we will explain later). The gene encoding CAP is located elsewhere on the bacteria) chromosome, no t linker\ tn the lac genes. Both CAP anrl Lac repressor are DNA-binding proteins and each binds to a specific site on DNA at or near the ]{)c promoter (see Figure 16-5). .Each of tbnsc regulatory proteins responds to one environmental signal and communicates it to the lac genes. Thus, CAP mediates the cffflct of glucose, whereas Lac repressor mediates the lactose signaJ. This regulatory system works in the following way. Lac repressor can bind DNA

CAP skle

16-5 the /D.C. operoo. rhe three genes (~ Y. and A) are transabed as a Sltlg1e mRNA from the promotes' {as indicated by the ClfTOIN)- lhe CN' Site and the operator are e£ICh about 20 bp. n'le operata ltes Wllhen the regwon bound by RNA polymerase at the promoter, and 1he CAP sfte ~es jUSt l4JSUeam of ti-.e pJomoter (sec figure 1&8 for more details d the rclati\.-e arrangements ot ther-e binding sites and the te>-1 for a desaiwlt•oo of the proteins that bind to throl). lhe pidure is ~fled '" lha thefe are two addtional, weaka; kx operators located nearby (see Bgure 1& 13), but we do not need co c.t;flSI
Regulation of 'lfansaiptioll l llitiatioo : Examples from Bar.:teria

.

+

lacZ

+

489

basal level of transcriptioo

CAP site

no transcription

+

FJ GUR£ l6-6 E.xpressiott of the lac genes. The presence or- absence of the sugCYS 1.x:tcse and glucose control the level of ~ession of the loc genes. •;tgh levels of apJession feqllire the presence of tactose (and hence the absence of fundlonal l d(. repressor) and absence or the pwened t!rlel'gy S«Jrc.e, glurose (and l1eoce p~esence of the actiVator CAP). wtJefl bound ro the operatcr. Lac repressor exc.Jude:c; polymerase whether or not actiVe OJ' ts present. CAP and lac repressor are shown as Single IXIItS, btJt CAP act\j(!Uy binds DNA as a d.-r1er, and lac repres50f binds as a tetTamer (see Ffg~.re 16-13}. CN> recruits polymerase to the lac promoter whefe it ~pontooeoosfy undergoes isomes~on to the ~ complex (the state~ 1n the bottom line).

and repress transcription only in the absence of lactose. In the presence of that sugar. tho repressor is inactive and the genes de-repressed (expressed}. CAP can bind DNA and activate the lac genr>.s only in lhe absence of glucose. Thus, the combined effect of these two regulators ensures that the genes are expressed at significant levels only when lactose is present and glucose absent (Figure 16·6].

CAP and Lac Repressor Have Op~ing Effects on RNA Polymerase Binding to the lac Promoter

g

lee ~ ~~~~~~~~ opendor. This 21 bp sequence is twofold symmetric and is recognized "half-site'' by n vo subunits of Lac repressor. one binding to ca~h half-site (sen tac operator Figure t£-7]. We will look at that binding in more detail later in this chapter. in the section "CAP and Lac Repressor Bind DNA Using a Common Structural Motif." How dues repressm, when bmmd to the FICURE 16-7 the symmetric half·5ite5 of theloc operator. operdtor, repress transcription? A s we have seen, the site bound by Lac repressor is called the

CAP~

DNA covered by RNA polymerase

site

·~------------------·· mRNA

5f~CN:A~~NlOCACCCCA~;:: I ~~OTG'rollM~l.-rl\ACU~OC'ij

3"\ ~~~~~~,..n~GTG-rec"l'" ~

' -10

' ·35

..'1

1he control region of the loc operon. The 11ud€otide sequence and organizabon of ~ Joe operon ronlrolreg100 are sl'lovm. The colored bars above and befow the DNA show regiOnS covemi by RNA po!'yl'\'IE!faSE' and the regularOfY prote.ns . 1\'ote that Lac repressor covers t'l"'IO! DNA than that sequence defll'led as the mirum.al ~~ btndifig Site. and RNA polymerase lllCfe lhao that defined by lhe seQ\J«lCes that rncll<.e up lhe promoter. f' 1cUR IE 16-8

The lac operator overlaps the promoter. anrt so repressor bound to the op erator ph ysically preventc; RNA polymerase from binding fo thr; prontQter and thus initiHting RNA synth~is (see f\gure 16-8). Protein binding sites in DNA can be identified, anrl their loc..ation mapped. using DNA footprinting and gel mobility assays described in Box 16·1. Detecting DNA-Binding Sites.

DNA Footprinting How can a protein bindif'lg site en JJN.A. such as an operatol, be tdentlied? A series of powerfU approtKhes aUOINS identificatJon

of the sites Vvhere prot'eil\s act and the cherrical groups .in ONA (methyl arrino, or ~e) a prote111 contacts. The basic principle that underttes these methods, is as follows. It a IJNA fragmef1t .is labeled with a radioactive ataTI only at ooe end of one strand, the location of any break ., this strand can be deduced from the s~re of 1he labeled fragment lhat results. The size. tn turn, can be determined by high-resolutioo ekctrophaesis in a po1yacrylamide gel In the nuclease proIKtion footprinting method. the birdng site is marMd by intemudeotide bonds that are shielded rmm the cutting ~ of a nudease by the binding protUI (Box 16-1 ftgure 1). lhe resubing ''footprint" is reve~ by the absence of bands of par-ticular SJzes. The relatod ~cal ptotection footprinting method refies on the ab*ity of a botrld protein to protect bases j, the binding site from base-spedOC chernkaf reagents tt"at (after a fufther reaction) give rise to ba<:kbooe ruts. By c:h.anging the order of the first two steps, a tJiJd method, chemical inteffl!ft!nce footprinting, determines which featllt'es of the DNA struc1Ute are necessary tor the pt'otein to f:Wld. AA cwerage of one chemical change per DNA ss 111dde, and lhe.l the modified ONA is mixed \Mlh the b1nding protein. Protein-DNA complexes are isolated. tf a modification at a particular site does not p-event binding of the protein. DNA isolated fr001 the complex will cootal'l that modifiCation and the harmless modification alows the DNA to be brden at lhts stte by further chemical treatment If, oo the other hand, a modification blocks DNA binding. then no DNA mOlexed with bending prot6n and the

ISOlated fragments \lllill not be bml<en at this site by subsequent chnmicaltreatment. By using aft lhree methods. we can learn where a prctein makes specific cortt.octs both v.tith bases and v.1th rhe phosphates in the 5l.Jgar-phosPf1ate backbone of DNA

Gel Mobility Shift Assay Ps JUst noted, h(M' far a DNA mderue migrates during gel electrophoresis vanes \1\iith saze: the smaller the molerue the more e~ it moves frtough the gel and so the further it gels 1n a given time In addOOn. if a gwen DNA molecule has a protein bound to it. JYtigraticl'l of 1hat DNA protein OJft1plex through lhe gel l5 mtarded ~red lo migratim of the oobound DNA ITderulc. lhis forms the basis vf an assay to detect specific DNA bi-lding actMties. The general approach is as folb.vs. A short DNA fragment containing the binding site of interest is radioactivdy IClbeled so it can be detected in smal q.J?I1tities by polyaoy1amide gel electrophoresis and autoradiography. lhss DNA "probeH is then mixed \Mth the proteJn of tntcresl and the mixture IS run on a get. If the protem binds lo the probe. a band appears higher 1-J> the gel than bands formed from free DNA (see Box 16-1 Figure 2). 1his ~od can be used ro idefltify muftjpte proteins in a crude extract lhus. iJ lhat probe has sites for a runber of proteins found ., a given eel typ::, and that probe is mixed With an extract of that ceU type. mulrip~ bands can often be resdve
lf~ulalicn of TraliSCriptivn

l11iticmon: £/
491

Box 16-1 (Continued)

J:....:::::=::::~

*-=·

BOJ(

J

*'===__::•====:l

]DNA fragment

1 Foo1plinting

labels at the ends of the DNA fragmetlts, irdcate sites ~e DNAse ruts. and red circles represer~t lac represSOJ bound to op:or<nor. On the feft. DNA molecvJes CtJt at random by DNAse are separated by S\l.e by gel e!ectfophoresis. On tl'le right, ONA molerules are first bound to repressor thm stbjected to ONAse treetment The "footpflnt• is in&:ated on the ~t. This c.orresponds to the colection of fragments geraated by DNAse rutlrlg at sites 111 free DNA. btA not 1n DNA with repressor bound lo it. In the fatter case. those sites are inaccesstie because they are within the cperator sec:pence and hence covefed by repr~-

~fragment+

ONA-bil'1dir'G protein

*'

1£ _ _ _-_ -=....JJ

FIGURE

MOWS

-

*..

16•1

methocl 1'he stars rept"esent the radioactive

•

BOJC

----------

16•1 FICiURE l Gel mobility

sMt assay. The prinople of the mob..lty I

*'=- - --.,-' free DNA

sl-ift assay iS shown 5Chematically. A protein is mxed with radio labeled probe ONA OOf"lt.aanir'lg a binding site foe .t1in rxoteln. The mixture is

-

bound ONA

- - : freeDNA .____~

..J

resolved by ~amde gel electrqlloresis and w;uafized using autora~. DNA not mixed with protein FU'lS as a single band corresponding lo the size of lhe DNA ffagment (left lane} In ttle mixture with tt1e protein, a proportion of the DNA molecules (but not aW of them at the conc.entrations used) binds the DNA molectJe. lhu>, tn the right-hand lane, there iS a baOO cooespondng to free DNA. <:~nd another corresponding to the DNA fragment 1n ccrnplex With the prote!fl.

Cene Regulation i'll Prokaryotes

As we:~ have s~en, RNA polymerasP. bi.nrls the toe promotf!t poorly in the absence of CAP. even when there is no active repressor present. This is becuuse the sequence of the - 35 region of the lac promoter is not optimal for its hinding, and the proro(1tor Jacks an UP-element (Sf:le Chapter 12 and Figure 16-8). This is typical of promoters that are con· trolled by activators. CAP binds as a dimer to a site similar in length to the Joe operator, but different in sequence. This site is located some 60 bp upstream of the s tart site of transcription (see Figure 16 -tJ). When CAP bincls to that site, too activator helps polymerase bind to the promoter by interacting with the enzyme and recruiting it to the promoter (see Figure 16-6). Thls coo)Jflrative binding stabilizes the binding of polymeraso to the promoter. We now look at CAP-mediated activation in more cletail.

CAP Has Separate Activating and DNA.-Binding Surfaces Various experiments support the view that CAP activates the lac genes by simple recruitment of RNA polymerase. Mutant versions of CAP have been isolated that bind ONA hut no not activate transcription. The existence of these so-called positive c:ontrol mutants demonstrates that, to activale ttanscriplion, the activator must do more than simply b ind DNA near the promoter. Thus. activation is not caused by, for exarnple. the activator changing local DNA structure. The amino acid subs(itutions in the positive controt mutants idP.ntify the rogion of C.AP that touches polymerase, caUed the activating region. Where does the activating region of CAP touch RNA polymerase when activating the Joe genes? This site is revealed by mutant fom1s of po1ymerase that can transcribe most genes norma1ly. but cannot be activated by CAP at the Joe ~nes. Those mutants have amino acid sllbstitutions in the Cterminal domain (CTD) of tl1e a subunit of RNA pol}rmerase. As we saw in Chapter 12. this domain is attached to the N-terminal rloma in (NTD) of a by a flexible linker. The aNTD is embedded in the body of the enzyme, but the nerD e.x tends out from it and binds the UP-element of the promoter [when that e lement is present) (soo Figure 12-7). At th(: lac promoter. where therr! is no UP-elemm1t, uCTO binds to CAP and adjacent DNA instead (Figure 16-9). This picture is supported by a crystal structure of a complex containi~ CAP. aCfDr and a DNA oligonucleotide duplex containing a CAP site and an adjacent U P-element

(Figur~

16-10). ln Box 16-2, Activator Bypass Exptlri-

ments, we describe ~n experiment showing that activati.on of the lac p romoter requires no more U1an polymerase recruitment. Having seen how CAP activates lransoiption at the Joe operon and l1ow Lac repressor counters that effect -we now lool motEl closely at bow these regulators recognize their UNA-binding sites. 16-9 Acflva(iOtl of the IGc promotet- by CAP. RNA polymerase b..nding .:It the lrx proolOfa With the heJp ut CAP CAP is recogrvzed by the c:TlJ!, of the o. subunits. lhe aCIDs abo C
1'1 CUR E

R~ulati(}n

of1lum;cripticm lt1itiation; E~mp/es from

Bcu:r~Pfa

493

FIGURE J6-10 StructUfeof CAP--oCTD-DNA complex. CAP is shown boiXld as a dirner to its site iust as we s;rw in figure 5-113. In addition. In thi!i case, the nero of RNA polymefaSe IS shoiMl boond to
The site of inter~ on each protein invo~s the ~dues identified genetically. In thiS f1gure. UP as shoWJ1 in tuquotse and the aCTD of po¥nerase tn putple. One rnoleaJe of ATP is shown bound to eilch monomer of CAP. (Benotf B. et al 2002 Science 297: 1562.) !n,.-,ge prepared ""lttl MoiSCTtpt Clf1d Raster 30.

CAP and Lac Repressor Bind DNA U sing a Conunon Structural Motif X-ray aystaUography has been used to determine the struclural basis of DNA binding for a number of bacterial activators anrl repressors, including CAP and the Lac reprossor. Although the details differ, the basic mechanism of DNA recognition is similar for most bacterial regulators, as we now describe. Jn the typical case. the protein binds as a homodimcr to a site that is an inverted repeat (or ncar repeat). One monomer binrls each half-site, with the axis of symmetry of the dimer lying over that of the binding site (as we S8W for Lac repressor, Figure 16-7). Recognition of specific DNA scquencos is aC'.hievcd using a conserved. region of secon dary stmctu re called a helix-turn-heJix {fjgure 16-11). This domain .is composed of two a helices, one of which-the recognition helix - fits into the major groove of the DNA. As we discussed in Chaptf1r 5, an o- helix is just the right size to fit into the major groove, allowing ~ino add

If an activator has only to recruit polymerase to the g€ne. then other Wq:Jeriment, another protein:protein iflteradion is used in p\ace d 1hat between CAP af1d polvmetase. nli:s ts 00ne by taking t\<\0 proteins known to interact with eadl tther. atrad'Jjng a-.e to a DNA-binding doma•n, and, with the other. replacirlg the C-temunal domain of the potymerase ~ subunit {oCTO). The modified potyrneJose can be i!Ctivaled by 1he mal<eshift "activiltol' as long as the appropriate DNA-binding SJte is Introduced near the prorooter. In another experiment, the nOD of pdymerase lS replaced with a DNA-bndilg dc:x"nak1 (for example; that of CAP). Thts modified polymerase effioently initiates transcriptioo from the lac promoter •n the

absence of any activator, as roog as the appropriate DNAbin&ng 5ite 1S pJaced nearby. A thnd experiment is even simJ*t: pdymerase can transcribe the loc genes at hi&flleve!s in vitro in the absence of any adlvator if the enzyme as presef1t at high COlCentration. So we see that either reatJiti1g polyrnerctse artificiaUy a s~ng it at a ligh concentratim is St.Jffecient to produce act\vated levels of express&oo ot the loc genes. These experi"nents are consistent with the activator h<Mng onty to help polymeras€ bind to the p10moter. For an explanation of why simply 1110'easing the concentration of a proten (tor p_xample, RNA pclymerase) helps it bind to a Site on DNA (lfl tflis case the promoter'), see Box 16-5. The restlts discussed in the box would r10t be expected if the activator had to induce a specifiC ~tlosteric in polymerase to

mange

acwate lrarlsaiption.

Box J&-2 (Continued)

a

y

1 DNA-

- 10

binding site

b

- - - ·• actiVated lacZ transcription

CAP site

--35

- 10

BOX 1fi.2 FIGURE 1 TwoadivaiDfllypassexperimePts.. (a} rheoCTD is repLred by a prcrein )\ Wich Wllefacts wi1h pl'otein Y. Protein Yis fused to a ONAbnding domain, «~d the site r~ by 1hclt domain is shovvn placed near the lac genes (b) The o.CTD ts rerJaced by the DNA-birdng portion of CAP.

residues on its ouh~r fuce to interact with chemical groups on tho cdgtJs of base pairs. Recall that in ChaptP.r 6 we saw how each baso pair p~ scnts a characteristic pattern of hydrogen bonding acceptors and donors (Figure 13·10). Thus. a protein Q}n distinguish different DNA sequences in this way \'\Tithout unwinding the DNA duplex (sec Figure 16-11}. Thu contacts made hetw•~n tho amino acid side chains protruding from the l'eCOgnition helix and the edges of the bases can be mediated by diroct H-bonds, indirect H-bonds (bridged by water molecules), or Van der Waals forces. The nature of these bonds is discussed in Chapter 3, and their roles in DNA recognition in Chapters 5 anrl 6. Figure 16-12 illustrates an example of the interactions made by a given recognition helix and its DNA-binding site.

Fl G U R E 16-Jl Binding of a protein with a ltetix-turn-t\elix domain to DNA. The protem. as is typically the case, binds as a elmer. and the two subunits are Wldtc.ated by the shaded ardes. The ~-tum-helix motif on ~ monomer l5 indicated; the "recognition heilc" is labeled R

RegtJiatiort o/Trrm $Ccipticn lrrifiati OJ'I: &lf!f> f rom Bact~trio

FIGURE

495

16-11 Hydrogen bonds

betweefl A repressot and bese pain in the major~ of its operator. Oiagrarn of the repressor-operator c~ showing ~ydlo

gen bonds (in dctted lines) between i'liTlln() ac\d side chains and bases in the consensus NlkttE'. Onty the felevant ~mmo acid Stele d-tains are shown. In ~n to Gif144 and SeJ45 m the ~gnitioll helix. Asrl55 in the loop folbMng the recoanital helill also rncl\es contact \Mth a specific base Futhennofe (iJld llfll~Sl.Jal 1tl this case. see later .., the text) LysA in the N-termm arm of the p;olm make. a aJOI:ad 111 the fruJOf gtoove on the opposite face of the DNA hfix. G1n33 contilCfs the backbone. (Source: R~ from Jordoo. S. and Pabo, C 5cJEnao 242· 896, F".g. 38)

The second h t:Jlix uf the helix turn-helix doma in sits across the major groove and makes contact with the DNA backbone, ensuring proper p.-es~ntation of the recognition helix. and at the same time adding binding en ergy to the overall p rotein-DNA inte.ractlon. This desr.ription is essentially true for not only CAP (Figures 5·18 and 16-10) and Lac repressor. but for many other bacterial regulators as well. including the phage A rcprussor and Cro prott~ins we will encounter in a Jater section : there are differences in detail, as the following axamples illustratl!. 4

• Lac repressor hinds as a totrnmer, not a dimor. Nflverthelcss. each upl".ralor is (:Ontacted by only two of these subunits. Thus, the~ different oligomeric form does not allt~r the me~;han.i sm of DNA recognition. The other two monomers within the tetramer can bind one of t wo other lac operators, located 400 bp downstream and 90 bp upstream of the p rimary operator. In su ch cases. the intervening DNA loops out to accommodatA th e reaction (Figure 16-13). • ln some cases. other regions of th e prote in. ouls ide lhe helixturn-helix domain. also interact with the DNA. A mpressor. for example, makv.s additiona l contacts using N-tcrminal arms. These

lac operator

laC lac promoter operator

16-13 lac repressor binds as a tetomer to two operators. 10e loop shown is between rhe Ldc repressor bouid at the pmn~ operatcr Clf1d tfle upstream l. The pnrTialy ClpffiliDr-thar one 5hown ogamst the promoter-IS the operate. referred to W1 disctlS:SlOO of regu~tion of lac gene eltpression. 1n thiS ftgure. each repressor d!mer h shown as two ordes. tather t1wl as a ~owl (as used rn ~ller fi~es)

F 1cuRE

r~ch

around lht.l DNA and inteN\c.l with the minor groove on thE1 b01ck face of the helix {see Figure 1tH.2}. • ln many easElS, binding uf tht1 protein does not a]ter the structure of the DNA ln some cases. howewr. various distortions arc seen in the protein-DNA complex. For example, CAP ind~ a dramatic bend in the ONA, partiaUy wrapping it armmd the protein. This is c-aused by other regions of the protein, outside the helix·turn·helix domain, inleracting with SE!fiU Elnc~ outside the operator. In other cases, binding results in twi5ting of the operator DNA. Not a ll prokaryotic repressors bind using a h elix-tum -hcli>-. A f fl\\ have been described that emploJ' quite different a pproach~. A striking example is the Arc repressor from phage P22 (a p hage related to~ but one which infects Salmonella). The Arc a:eplt'..ssor binds as a dimer to an inverted repeat opctator, but instead of an a-helix, it recognizes its binding site us ing two Antiparallel J)-strnnd$ inst:rted into 1he major groove.

The Activities of Lac Repressor and CAP Are Controlled AUosterically by their Signals When lactose enters \he 0111, it is convertod to 3 Uolactose. Jt is aJiolactose (rather than lactose itsoiO that controls Lac ruprnssor. Paradoxically, the conversion of lactose to allolactose is cataJ}'zed by ~alactosidast:, itself encodBd by one of tha lac genes. How is this possiblE!? The answer is that expression of the lac genes is leaky: even when they are rnpressod. an occasional transcript gets made. That happens ~cause every so uften RNA polymerase will manage to bind the pwmoter in p lace of Lac repressor. This leakiness ensures there is a low ltwel of 13-gal ~ctos idasf> in the r:ell even in the absence of lactose, and so I hero is enzyme poist:~d to catalyzu the conversion of lactosa to f'ltO· Jactose. Allolactose binds to Lac repressor and triggers a change in the shape (conformation) of lhal protein. In the absunce of a!Jolactose , repressor is present in a form that binds its s ite on DNA (and so keeps lhP. Joe gtl.Of'B switched off). Once allolactose has altered the shape of repressor. th{t prot~in can no lm.g~r bim1 DNA, and so tht~ lac ge~ ar~ no longer repressed. In Chapter 5 we described the structural basis of this allost.P.ric chAn ge in Lac repressor (Figure 5-25). An important point to t!mphasize is that allolactose binds to a part nf lac rP.pressor <list inct from its DNA- binding domain. CAP activity is rugulatt!d in a similar manner. Glucose low~rs the intractillular con~ntration of a small molecule . cAMP. This moltf· cule is the allosteric effector for CAP: only when CAP is complexed with cAMP does the protein adopt a conformation that bin ds DNA. Thus, on ly when glucose leve ls are low (and cAMP levds high) d oes CAP bind DNA and activate 1he lac gl1nes. Thfl pnrt o FCAP thut binds the effector. cAMP, is s eparate from 1he part of the prohrin that binds DNA. The lac operon of E. coli is one of the two systems used by French biologists Fran~ois Jacob and Jacques Monod in formulating the early ideas about gene regulation. In Box 16-3. Jacob, Monorl. and the ldeas Behind Gnn~ R~ul ation, we give a brief d~scription uf lhnSt! Hady t:~IHdi t~s ahd why the idt)as they gent.!J'att:~d h ~ v(• proved so influentiaL

Rt'fJUllllitJn ()/ 1ronsaiption JnitiaUon : BxampiP-S from lkrcteria

--The idea that the expression of a gene can be contrclled by the pn:xh:t of anothef gene-that there exiSt regulatoty genes the sole functioo of which is regulating the expcesstOO cA other genes-was ( lf1e of the great insights frcm the earty years of molecular oology. It was proposed by a group of scientists woci<Jng in Paris in the 1950s and earfy 1960s, in particular Fra~ Jacob and Jacqt.Jes Monad They sougtlt to explain tvvo apparently unrel(rted ~: the appearance of ~-galactosidase Ill E. coli g«JMl in lactose, and the behavior of the bacterial virus (bacter~e) A upon infection of E. coli lhetr "II'JOfk romnated in publicaOOn of thelr operon modet in 1961 (and the 1965 Nobel Prize fcr meclidne. wtnch they shared \Mth th~ coleague. Andre Lwoff). lt is Gffia.At to appredate the magritude of their achievement now that we are so familiilr with thetr ideas and have suet. drect vmys of teshng their models. To put it in perspective. consider IM'lat was known at the time they began 1heir dasSlc experiments: ~lactosidase actiVIty apPeared in E. coli cells only \\/hen lactose was prrNidecl in the growth mec:fiUITJ It was not dear that the appearClllCe of this enzyme invdved switching on expression of a gene. Indeed, ooe eany ~ was that the cell contolled a gener~ (genetic) enzyme. and that enzyme tool< on whatever properhes were required by the ciro..mstances. Thus, Vvhen lactose was present. the generic enzyme ~ on the appropnate s~ to metabolize loc1ose, using the sugar itself as a tailJiate! Jacob. 1\110100. ar~d I.OOr coworkers ~ected 1he ~ genet~Ull~y. We \'\Iii not go thrrugh their experiments in any detoi~ but a brief sunmary gives a taste of their ingenuity. First. they tsolated mutants of £ coli that made ~-gal~ dase irrespective of whether lactose was pesent- that is,

----------------------------------

mutants in which the enzyme was produced constitutively. These mutants came 1r1 two dasses: in one, the gene enc.cx:btg the lac repressor was inactivated; rn the other, the opetator site was defective. lhese t\f./0 classes eotAd be cistilguishecl using a ds-rrans test. as VJe row desaibe. Jacob and Monod coostructed partidly ~id eels (see Chapter 21) in v.hich a sectioo of the dvomosorne from a ~ type eel tArTying the foe genes (that 15, the Lac repressor gene, lacl. the genes of the lac q:>e100, and their regUa!ay elements) was Introduced (on a pascrid caled on f') into a eel c.arryilg a mutant vers~on of lhe lac genes on tts chromosome. l11is trillsfef restAted in the cl t\N{) ~s d lhe /oc genes in the eel, rnaj(jng it possible to test whether the wid-type copy roJd romplement ;r,y grven fl1\Jtant cq>y. When the chromosomal genes were ~ WlStilutively because of a mutation in the loci gene (enroding repressor). the w4d-type ropy oo the plac;mid resrored repression (arrl indudbity)-for example, I)-galactosidase was once agan 00y made vvtlen lact.ale was present (Bo.x 16-3 Fl81Jfe l ). lhis IS becaJse the repressoc made from lhe wid-type loci gene on the tJ'asmid could dffUse to the chromosane- that is, it could act ., trans. When the mutation causing C01Stitutive expression of the chn:Kmsomal genes was in the foe q>erator. it coold nct be COfllllemenled in trans by the wild-type genes (Box 16-3 Rgure 2). The operattif fmcOOns onfy In CIS (that is, it only acts on the genes directly linked to lt on tl1e same DNA molea.Jie)lnese and ott1er results led }(!cob and Moood to propa;e that genes were expressed from specific sites ~led prorncAefs found at lhe start of the gene, and that this expressO'l was regtjated by repressors that act through q:>€fala' sites located on the DNA beside the promoter.

rxesence

A

active repressors

llU.an dvomosome

(L

r

X !

--

no transcription

16-3 FIG tJ R E 1 Partial diploid cells $how that fundional repreuor$ work in trans. In Che i!bsence of lactose. the foe galeS are not ~. and \hus no 5igllihcant level

B0 X

of ~sjd;,se is fl'liK1e in these ceb.

497

498

Gene Regulatic;n in Prokaryotes

Box 16-3 (Continued)

16-J F Ui U R ~ 2 Partial diploid eels show thai Gp4ntors wed< Cllllv in cis. {a) Haploid cell contaning l1lUta1t opetaror

80 X

{Oc)· (b) Pai1ially cipk>id eel containiflg a normal opetator (0) and a mutant ope~atol (Oc). The lex: genes (Z, Y, and A) attached to the nuant operator continue to be eJ€fator oo another dlromosome in the same eel. ThtJs, the opeiClteronly 'IA'CJI\s .-. cis.

a mutant dYomosome

A

y

A

y

A

1

l

b wild-type chromosome

z

operan sles. The similanly of these tvvo regtlatory systems (despite the V€fY different biology) CDI'M1Ced Jac.OO and Monod \hat they had identified a fundamental rnedlanism d gene regulat:ffl and that their model would apply throughout natu'e. ft6 we wil see. aUhough thei' description was not complete- most noticeclbly. they did not include activators (such as CAP} in their scheme-1he basic model they proposed d cis regtjatay sites rea>gnized by trans regulatory factofs has dommaled the map-

But these e>
BOX 16-3 FIGUR E 3 11lis drawing, showing the loc operolt and Its regufatiol\ was rendered by FranpJis Jacob, 1001.. {Sou'ce: Courtesy of Jan IMtJ..owskl.)

v

ity of subseqJeflt thtnJ
_,L R~

"t' S4, l

'

S4~

i ~

fit

l ~

4

I

t

~ ~

?71

~ RNA.

~t.

Elegvlorior1 of 'fuutscriptlon

Combinatorial Control: CAP Controls Other Genes As Well The lac genes provide an example of signal integration: their expres~ sion is rontrolled by two signals. each of which is commllllicated lo the gen es via a s ingle regulator - the Lac .repressor and CAP, rnspoctively. Considt~r another Stit of E. coli genes, the gal gont!S. These eneooo ~nzymes involved in galactose metabolism. As with thf' lac genes. the gal genes aro only expressed wlmn their substrate sugar- in this case galactooo- is present. and the preferred energy source, glucose, is absent. Again, analogous to lac~ the two signals are communir.ated to I he genes via two regulators- an activator and a n:pressoc. 'The «'pressor, encudtld by the gen e galR. mt:diates the effects of the inducer galHctose, but the activator of the gal genes is again CAP. Tims, a regulator (CAP) works together with different repressors at different genes. This is an example of combinatorial t:ontrol. In fact. CAP acts at more than 100 genes in F. coli, working with an array of partners. Combinatorial control is a characteristic reatm'tl of gene ~ul a t ion. Thus, wh~o the same s ignal controls multiple gen ~. it is typically communicated to each of th ost~ gt:nt~s by the same regulatory protein. That regulator will be comro Wlicating just one of perhaps several signals involved in regulating each gene; tlw other signals, different in most cases. will eacb be mediated by a scpamte regulator. More a:>ln· p lex organisms-higher eukaryotes in partiC\llar-tend tn have more signaJ intt.'giOtion, and theru we will see greater and more elaborate • examples of combinatorial control (Chapter 17}.

Alternative a Factors Direct RNA Polymerase to Alternative Sets of Promoters RP.calJ from Chapter 12 that it is the o subunit of RNA polymerase thot recogni?:es the p romoter sequences (.Figure 12-6). The lac promoter we ha ve been discussing, a long with the bul k uf othor E. cob promoters, is rt.>cognized by RNA polymijrase bearing the a-7° subun:it. E. coli encodes several otht:~J a subunits that can replace u 10 under ccrtai n circumstances and din:ct tht:! po1ym€:1rase to alternative p romoters. One of the$6 a lternativ~>.s is the heat shock (T fac:tor, a 32• ThtLS, when E. coli is subject to heal shock, the amount of t·his new cr fact(Jr increases in the ct:~U, it displaces a7t1 from a proportion of RNA polymerases, and directtl those enzymes to transcribe ~nes whosA {lroducls proted thtl cell from the effects of btm.l shock. The l~veJ of cr12 is increased by two mechanisms: first, its transla1ion is stimulatoo- that is, its mRNA is lmnslated with grnater efficiency aftclr he::tt shor:k than it was ba fort~; and second. the protein is transiently stabilized. Another c.xamplt! of an allt!roalive a factor, d", is considell!d in the next section. ~ is associated w ith a small fraction of the polymerase molecules in the cell ar,d directs that enzyme to genes involved in nitrogen metabolism. Sometimes a series of a lternative sigmas directs a particular program of gene expression. Two examples are found in the bar:teriu m B. sub1ilis. We consider the most elaborate of these, which controls sporulation in that organism. in Cbaptm- 18. The other we describe briefly here. Bacteriophage SPOt infects 8 . subtilis. where it grows JyticaUy to produce progany phage. This process requires that the phage express its genes in a carefuUy controlled order. That control is imposed on polymerase by a series of a lternative cr factors. Thus, upon jnff!ction, tllf' bactflria1 RNA polymerase (bearing the R. suhtili.~ Vt1rsion of· cf0l

Cene JiegulaUon in ProkaryotPS

500

rP-cogn izes so-c-alled "early" phage promotP.rS, which direct transcript ion of genes that enrode proteins n eeded E!arly in infection. One of these genes {called gene 28) encodffi an a)tem alivfl o. This displaces the bacterial cr factor and dirt.>cts the polymeras~ to a second set of promoters in the phage genome, those associated with the so-called ..middle" genes. One of these genes, in turn, encodes the a factor for the phage "l ate'' genes (Figure 16-14).

NtrC and MerR: TranscriptiOilal Activators that Work by Allostery Rather than by Recruitment Although the majority of activators work by recruitment. there are exceptions. 1\-vo exampl~ of activators that work not by recruibneot but by allosteric mechanisms are NtrC and MerR- RtscalJ what we mean by an allosteric mochanism.. Activators that work by recruitm.e nt simply bring an active form of RNA polymerase to the pJomoter. ln the case of activators that work by allosteric m echanisms, polymerase initially binds the promoter in an inactive complex. To activate tran· scription, the activator triggers an allosteric r;hangti in that compltsx. NtrC controls expression of gtnes involw.d in nitrogen metabolism, such as the ginA gune. At lhu ginA g~nu, RNA polymerase is prebottnd to the promoter in a st able closed complex. The ac.tivalor NtrC indual8 a conformationaJ change in the enzyme, triggering transition to the open complex. Thus the activating event is an allosteric change in RNA polymerase (soo Figure 1&-2). MerR controls a gem: called mt'.rT, which encodes an tmz.yme that makes cells resistant to the toxic effects of mercury. MerR also acts on HO inactive RNA polymerase- promoter complex. Like NtrC, MerR induces a oonfonnationaJ ch ange that t riggers open c..omplex formation. 1n this case. however, the allosteric effect of the activator is on the DNA rather than the pn lyrnerase.

NtrC Has ATPa"e Activity and Works from DNA Sites Far from the Gene As with CAP, NtrC ha~ S£:parate activating and ONA-bi.oding domains and binds DNA onl)! in the presf;lnce of a specific signal. In the case of NtrC, that signal is low .oitrogtm levels. Undtt.r those conditions. NtrC is p hosphorylated by a kinase, NtrB, and as a result undergoes a confonnational change that reveals the activator's DNA-binding domain. One~

28

34

l

1

~ -

promoter eariy genes

F tG uR E

middle genes

16-14 .AJtemative a factors axrtrol the ordered expression d genes in a bacterial wrus. The bacteru~f phage SPO 1 uses thlee a faaors 111 succession to regulate exJXess.on cl1ts genome Thts ernures Chat vwal genes are expressed 1n the order 10 ~lich they i:lfe needed. (Source: Ad.'lpted from Ai:lerts B et at. 2002. Molecular bofDgy ofthe CPS, 4th edibon, p 415, fig 7-63. Cq>yr\ght © 2002. Reproduced by pe~ ot Routledge~ & frnoos Bool\s, Inc,)

tategeoes

RegulatiQil tJ/ TtanSCTipliort lnitiatiQn: EJ!amples from E

ar.tive, NtrC binds four sites located some 150 base pails upstream of the promoter. NtrC binds to each of its sites as a dimer, and. through protein:protein interactions between the dime.rs, binds to the four sites in a highly cooperative manner. The form of RNA polymerase that transcribes the ginA gene contains the oM subunit. This enzyme binds to the ginA promoter in a stable, closed complex in the absence of NlrC. Once active, NtrC (bound to its sites upstream) interacts direcll_y with rr4 • This requires 1hat tlw DNA betw(H;:n the activator b inding sites ond the p comoh:Jc

form a loop to accommodate thEl intoraction (Figure 16-15}. lf the NtrC binding sites are moved further upstream (as much as 1 to 2 kb} the activator can still work. NtrC itself has an enzymatic activity- it is an ATPase; this a ctivity provides the energy needed to induce a conformational change in polymerase. That conformational Change triggers polymerase to i nitiate transcription . Spt::cifically, it stimulates conversion of the stablu. inacth ra, dosed complex to an active. open complex. 1\t some genes controlled by NtrC. there is a binding site for another protein. ca11Ho UIF. located betwoon the NtrC bind ing sites ancl the promoter. Upon binding, IHF bonds DNA; when Lh t IHF binding site-and hence the DNA bund-are in the conect register, this event increases activation by NhC 'Ine explanation is that. by bending the DNA. (Hf. brings the DNA-bound activator closer to the promoter, helping the acti~tor interact with the polymerase bound tharu (see Figure 16w4 and, for a closer look. at bow DiF bends DNA, sre Figure 11-10).

MerR Activates Transcription by Twisting Promoter DNA When bound to a s ingle DNA-binding site, in the presence of mercury. MerR ac:\ivates the merT gene. As shown in Figure 16-16, MerR binds to a sequence located between the - 10 and - 35 regions of the merT promoter (this gtme is transcribed by a ro-contain lng polymerase). MerR binds on the opposite face of the DNA holix from that bound by RNA polymerase . and so polymerase can (and does} bind to the promoter at th~ same time as Mel'R.. T he merT promoter is tmusual. The d istance between the - 10 and -35 elements is 19 bp instead or the 15 to 17 bp typically found in a a 7c promoter (see Chapter 12, Box 12-1). As a result, these two sequence elftmen ls recognized by a ara neither optimaUy separated no r- aligned: t hey

an~

som(:jwhal ro ta ted around the face of the h(!lix

in relat.ion to nach other. Furthennore, lhe bind ing of MerR (in the

- - -- - - - - -

16·15 Acfivation by Ntre. The promoter sequence rc:cogrnzed by cr•·contalnt~ holo€fl~ zyme ~ different from triM rerogntzed by a 70--ron~re holoenzyme. Alttloogh •'lOt speofted in ttre ligule, r-.ltC contacts \he oMsubt.ril of polyrnercJse. NttC IS ~wn as a dcmer, but lfl fact forrns a h•glier-or<:ler oornp&ex Ofl DNA. Ff G U R f

Gene RegtJJation i tr Prokaryores

FIGURE 16-16 ~byMef'R.. The - 10 c:~nd - 35 eements of the merT p.-omoter ~e 011 ,-,eady opposite s~ of the helix. (a) In the absem:e of merru~y. MetR b•nds al1d stabitizes the 11"\adJVe form of the promota: (b) 'lrl the presence ct rnercu~y. f.AerR twlStS the ONA so as to propetl} a' gn the p~omol€r elements.

-

a

- 35

merT

MerR

-10

t~

rnerT

''--

- 35

MerR

absence of Hg2 +) locks the promoter in this unpropitious conformation: polymeFase can bind, but not in a manner that allows it to initiate transcription . There fore. there is no basal transcription. Whon MerR binds Hg2 ' , howP.Wr, thfl protein undergoes a confor· mational change that causes the DNA in the c~nter of the promoter to t wist. This structural distortion restores the disposition of the - 10 and 35 regions to something dose lo lh~t found at a strong u 70 promoter. In this new configuration, RNA polymerase can efficitmtly irutiate transcription. The s tructures of promoter DNA in the ..active" and .. inactive., stales have benn determined (for another promoter regulated in this manner) and are shown in Figure 16·17. It is important to not~ that in this example the activator does nol interact vvith RNA polymerase to activate transcription, but instead alters the conformation of the DNA in the vicinity of the prcbound enzyme. Thus. un like tho earlier cases, there is no sepal'ation of DNA binding and activating regions: for MerR, DNA bi nding is intimatel:y !inked to the activation process.

Some Repressors Hold RNA Polymerase at the Promoter Rather than Excluding It Lac repn.>ssor works in the simplest possible way: by binding to a Site overlapping the promoter, it blocks RNA polymerase binding. Many repressors work in that same way. In tlu~ MerR case. wn saw a rliffert.mt fonn of repression; in thal cas~ the protein holds the promoter in a confonnalion im::ompatlbl~ with transcription initiation. There are other ways repressors can work. one of which we now consider. FIGURE 1 6--17 Structure of a mei'T~fike promoter. (~) P10moterwith a 19 bp space~. (b) Promoter with a t9 bp spacer v.hen lf1 romplex v..'llh active activator. (c) Prorr.oter with a 17 ~~e. The promoler si10Wfl in parts (a) a11d (b) tS trom the bmr gene of &c.ilkl5 svbcilis, ll'hltch I.S controlled by rhe segulatof SmrR BmrR works. as an activator "vhefi complexed 11\ith the dttJg rewpl1i.'ll)'lpho5phooiUTI (TPP). "The 35 (nc.Ad) and - 10 {TACAGI) elements ot one are .si-l
- 35

a 1 9bpspace~

b

straoo

w~en.

respedJVely (Source: Adapted, With permi~l(}tl. fr001 Zheieloova ~~n E.£ and

c

Breman R.G. 2001 Natllfe 409: 378; Flg\.lre

3 b, c. d Copyright {0 2001 Nature Pttblishtng

Gr01.4>. Used with p€fmSSion.)

17 bp spat;ef"

- to

RegaJiution of TroCJscripfion l T!itialiott. Emmplf¥.> }rom lfaelf't'lo

Sorne repressors work from binding sites th::tt d o not overlap thn promoter. Those repressors do not block polymerase binding-rathlfr they bind to sites beside a promoter, interact with polymerase buund at that promoter; and inhibit initiation. One is the E. coli Cal repressor, which we mentioned earlier. The Gal repressor controls ~nes that encode enzymes involved in galactose meta bolism; in the absence of galactose thf;f repressor keeps the genes «.JfL l11 this case, the repressor int~ct.s with the polymerase in a manner lhat inhibits transition from the clust~d t o open complex. Another example is provided by tlw P 4 protein from a bacteriophage (
AraC and Control of the araBAD Operon by Antiactivc1tion The promoter of the amBAD operon from E. coli is activated in the presertce of arabinose and the absence of glucose and directs expression of genes cnc.ocling enzymes {'(.}(}Uired for arabinose metabolism. Two activators wort... togoth& here: AraC and CAP. When arabinose is prosent. AraC binds that sugar and adopts a configuration that allows it to bind DNA aHa dimer to lhe adjacent baH-sites, aro/1 and oro/:t (Figure 16-18a).

a + arabinose

araC

b -arabinose

- -----

FIGURE 16-18 CCntrol of the .aBAO operotl. (a) ~e binds toAiaC, chang1ng tne shapecf that Clctlvalorro itbnds as a dimertoorol, a1d oral1 This places ooe trooornerof haC dosett1 ihep-ornot:er from whtdl•t can activate trMsenptlOf\ {b) tn lhe absroce d arabinose. 1he AraC cirner M:lptt a different confOill1CIIOI'l and l:xnds toaro~
!\s prcxmter 1:> illso cmrolled by CAP. but that «S net ~ 111 thiS f@Jte

Str"-'

504

Gene RegulatiOll in Prokaryotes

Just upstream of these (but not shown in the figure) is a ('..AP site: in the absence of glucosti, CAP binds hnrc and helps ar.tivnrion. ln the absence uf arabinos~ the aro.BAD genes are not mcpressed. This is because, when not buuncl tu arabinose, AraC adopts a different conformation and binds DNA in a different way: one monomer stiU binds the orof1 site, but the other monomer binds a distant half-site caUed araq. as sbowo in Figure 16-18b. A!S these two half-sites are 194 bp apart. when AraC binds in this fRshion lhfl DNA ben.veen the two sites forms a loop. Also, when bound in this way. Lht..-re is no monomer of AraC at arol'!, and a~ that i& lhe p osition from which activation of araBAD promoter is mediated, there is no activation in this configuration. The magnitude of induction of the nroBAD promoter by arabinose is ver y large, and for this reason the promoter is often used in expression 'Vectors. Expression vectors are ONA constructs in w hich efficient synthesis of any protein can be ensured by fusing its gena to a strong promoter (see Chapte.r 20). In 1his cas~. fusing a gene to the mnBAD promoter a1lows expression of the gene to be rontrolloo by arabinose: the gene can be kept off until m.-prossiun is desirable, and then ••induced" when its product is wanted simply by addition or arabina;e. This allows expression even of gtmes with products that are loxic to the bacterial cells.

EXAMPLES OF GENE REGULATION AT STEPS AFTER TRANSCRlPTION lNITlATION Amino Acid Bio~)'nthetic Operons Are ControUed by Premature Transcription Termination Jn .E. coli the five contiguous trp genes encode enzymes that synthesiztJ thf> amino add tryptophan. These genes are expressed efficiently only when tryptophan is limiting (Figure 16-19). The genes are controlled by a repres.•:;or. just as the lac genes are, but in this case lhe ligand that controls the activity of that repressor (tryptoph~n) ac\s not as an inducer bul as a corepressor. That is, whM tryptophan is present, it binds the Trp repressor and induc.es a conformational change in that protein. enabling it lo bind the trp operator And prevent transcription. When the tryptophan concentration 's low. the rrp repressor is free of its corepressor and vacates its opt..~lot. aflmving the synthesis of lrp rnRNA to commence from the adjacent promoter. leader

Pfomoler

trpE

trpD

trpC

trpB

!

F l GtJ RE 16-19 The trp operon.. The WIPtq)han q-,..eroo of E. cob. si10Wif'l8 lhe relatiOn of the leader {see text) to the structu~ genes tl1i'lt code for the T!p enzymes. The gene products are anthranilsase indole glycad fAl~dte synthetase (tip('), ttyplqlfwl synthetase ~ (rrpB), and tryplqlhan synlhetase
frpA

-

Exflmple:; of Ge1w Ht:g~~lflriOtl ot Sleps oft« Transcript iot1 Initiation

Surprisingly. however, once polymerase has initiated a t.rp mRNA molecule it does not always complete the full transcript. Indeed. most messages are terminated prematurely before they include even the first trp gene (tTpE), unJess a second and novel device confirms that little tryptophan is available to the cell. This second mechanism overcomes the premature transcription termination, called attenuation. When tryptophan levels are high, RNA polymerase that has initiated transcription pauses at a specific sita. and then temlinates before getting to trpE, as we just described. Bt.tt when tryptophan is limiting, polymerase does not terminate. end instead reads through the trp genes. Attenuation . and the way it is overcome, rely on the close link between transcription and translation in bacteria. and on the ability of RNA to form alternative structures through intramolec.ular base pah·ing. as we now describe. The key to understanmng attenuation came from examinjng the sequonce of the 5' end of trp operon mRNA. This analysis teveaJed that 161 nudeotides of RNA are made from the tryptophan promoter before RNA poiymerase encounters the first codon of trpE (Figures 16-19 and 16-20). Near the end of this leader sequence, ;md before trpE, is a transcription terminator, composed of a charact~ristic h.ctirpin loop in the RNA (made from sequences in regions 3 and 4 of Figure 16-20), followed by eight u~:idine resid ues (see Figure 12-9). At this so-called attenuator9 transcription usually stops (and, we rnigh1 have thought, shoulrl always stoplr yielding a learler RNA 139 n uclootides long (Figure 16~20). This is the RNA product seen in the presence of high levels of tryptophan. How, then. can mRNA for the whole operon ever be made, as is seen in the absence of tryptophan? Three features of the )P.ader sequence allow the attenuator to be passed by RNA polymerase when the cellular concentration of tryptophan is low.

r- GUUGGUGGCOCACUU cc·

1

139

I ~~~~~~u~u~u~u~u 4

I Met-Gin-n.- •.

UGAACAAAAlJUAG~GAAUAACAAUG

t

end of leader (slte of attef'lllation)

Fl CuRE 16-20 Trp operator leader RNA. Features of the rudeobde ~of the trp operon leader ~

161 trpE polypeptide CAAACA . . •

• f jrst. them i.s a second hairpin (besides the terminator hairpin) that can form between regions 1 and 2 o f the leader (see Figure 16-20), • Second, region 2 also is complementary tu regioo 3; thus, yet another hairpin r.onsisting uf regions 2 and 3 can form. and when it does it prevents the terminator hairpin (3. 4) from forming. • Tirird. the leader RNA contains an open-reading frame enooding a short leadet" peptide of 14 amino acids, and this open-reading frame is preceded by e. strong l"jbosome binding site (see Figure 16-20).

The sequence encoding the leader peptide he.s a strjking feature: two tryptophan codons in a row. Their importance is underscored by corresponding sequences found in si milar leader peptides of other oper'Ons encorling enzymES tbAt make amino ar.irls (~ Table 16-1). Thus. the leudne operon lead er peptide has lour adiacent leucine oodons, and the histidine overon teader peptide has seven histidine codonl:i in a row. In each case these operons aoo controlled by attenuation. The function of these c:odons is lo stop a ribosome attempting to translate the leader peptide: thus. when tryptophan is scarce, there is very little charged tryptophan tRNA available. and the ribosome staJJs when it reaches the tryptophan codons. Unrler those circumsta.t'lf'.es. RNA around the tryptophan codons is withln the ribosome and cannot be part of a hairpin loop. (Recall that transcription and translation proceed simultaneously in bacteria.) The consequence of this is shown in Figure 16-21 and d escribed below. A ribosome caught at the tryptophan codons (part b) masks region 1, leaving region 2 free to pair with ~ion 3; thus the terminator hairpin (fOfm~ by regions 3 and 4) cannot be m ade~ and RNA polymerase passes the attenuator and moves on .into the operon. aJlowing Trp enzyme expression. If, on the other hand, there is enough tryptophan (and thctefore enough charged Trp tRNA) for the ribosome to proceed t hrough the tryptophan corJons, the rihosome hlor.ks sequenr.e 2 by the time RNA containing regions 3 and 4 has been made. H.ibo..c;om~ block ing region 2 allows formation of fue terminator hairpin (from l't1gions 3 and 4), aborting transcription at the end of the leader RNA. The leader peptide itself has no function and is in fact imrnediateJv destroyed hy cellular proteases. The use of hoth repression and attenuation to control expression allows a liner t"U ning of the level uf intrat.:elh.J.Iar ti"yptopban. Jt prov ides a two-stage response to progressively more stringent tryptophan starvation- the .initial response being the cessation of repressor binding. with greater starvation leading to relaxation of atten uation. But attenuation a1one can provide robust regulation: other amino acid op erons like his and /ell have no represso rs; instead. t hey rely enUre ly on attenuation for their control. This exampJe of attenuation shows that transcription of a gene can be regulated without the use of a regulatory protejn. In Box 16-4, Riboswitchcs. we see other examples of reguJation without regulatOI)' prote ins.

Ribosomal Proteins Are Translational Repressors of their Own Synthesis Regulation of translation often works in a manner analogous to transc.:tiptionat repres. and blCJcks initiatiCJn of that prot..'ess. In .some cases, this bintli ng

TABLE. 16·1 Leader Peptldes of Attenuator·Controlled Operons Containing Cenes for Amino Add Biosynthesis' Operon

Amino Acid Sequence of Leader Peptldes

Tryptophan Threonine 1-itsttdtne lsolaucrne-va/fne G£DA

Met Met Met Met

Lys Lys

Thr Thr

Ala Arg Arg Ala

lie

Phe

Val

lie

Thr Phe Atg

Ser

Ser Gin Leu Pro Val Gly Pro Met

Val

Arg

Val

Val Leu Pro

Loucme Phenylalamne lsoleucine-valme B

Met Met Met

Ser Lys Thr

He

His

His Thr

Val lie

Cys Arg Gly Phe

Leu

Leu Thl' Lys Val Gly

Phe

Lys

us HIS

lie Ala

Gly Thr His Ser Ala Gly

lie

Thr Gin

Phe Asn Val

Phe Ala Ala Lys Val Val

Trp Thr HiS Leu Leu Leu

Trp Thr HIS Val Gly Leu

Arg

lie His Val Arg

Thr Thr His lie Gly

Ser lie His Ser Lys

Leu Leu Asn

Thr Pro Val Ala Ala

Gly

Asn

Gly

Ala

Val

Vat

lie

lie

lie

Phe

lie

Val

Arg

G•y

Arg

Ser

Ala

Ala

Val

Val

Val

Thr

Gly

Asp Pro

HIS

Phe Phe Phe Thr

Leu Leu val

Gly

Pro Asn

Thr Ala

Phe Ala Pro

Pro Pro

'The b•osynthes•s of ISOieuc;ne end ~aline Is comolell:. the genes are eneooed In several oporons and tne pathway 10 leucine synthesl&Is a branch of lhe valine pathway Tl'lus. •soleuclne, valine, and klucine ~re (Sourc~· Adapted from Bauer C et al 1983 Gene fUf!CtiC/1 m proJt.aryotrJs. Copyngl'\t e 1983Cold Spring Harbor LabOratory Pres& usea will'\ perm•s·

aH•nvo1vad In altenualloo of the isoleucine-vaMeop&rons slor> l

~OB

Gene Regulol ion i n Prokaryotf!S

a high tryptophan

3

\

leader peptide coding regtOO

b low tryptophan

4

trp opefOfl rri
tryptophan c:od
---

c no protein synthesis

f I(; U RE 16-11 Transcription t~ at 1M ttp attenuator. 1-lowtraflSCrl'tion termnation at the flp qJeron atterua10r is cortmlled by the avatl.:bily of tryptq:*wm. ~ (a) (coodrtions ot hgh ~),

sequence 3 CAO parWlh ~ 4 to form the transcf~ tamination fl
b'ypto(Jharl), the dJosome stalls at~ ti)'P\opharl cocbns.leavifl& ~ 2 free lO pair v.ith ~ 3, therebyprevenllng fonnct!OO of tl-e 3, 4, termination hclirplr_\1 (c) (no prote111 syrJhestf>), if no rl>osome begins transfatlon of the~ peptide AUG. the~ forms by pillriog ot ~~ 1 and 2. prevel"ti1'1g foona6oo of the 2. 3, hatfpin, aod allowing forma!ion of the hailpin at sequences 3, 4.1he 1rp enzymes are 001 expressed_

involves recognition of specific secondary structmes in the mRNA. We consider here the regulation of the genes that enc:ode ribosomal prot~jns. Cor·roct expression of ribosomal protein gt>.nes poses au intercsling reguJatory problem for the cell. Each ribosome contains some 50 dUr tinct proteins that must be made at the same rate. Furthermore. the rate at which a cell makes protein, and thus the number of ribosomes it needs, is tied closely to the cell's grbwth rate: a change in growth conditions quickJy leads to ;m lnCIT'.ase or decrease in the rate of synthesis of all ribosomal components. How is aJJ this coordinated reguJation accomplished? Control of ribosomal protein genes is simplified by their organ.i?..ation into sevcml operons. each containing genes for up to 11 ribosom al proteins (fjgure 1&22). 1'l1e genes for some nonribosomal proteins whose synthesis is also linked to growth rate are contained in these operons. including those for RNA polymerase subunits a, J3, ami fl'. As with o th er operons. t~ operons are sometimes regulated at the level of RNA synthesis. But, the primary control of ribosoma l

Examples cj Gene Regulation at Steps after1i'anscriplion I11iUaCion

509

protein synthesis is at tho level of tronslntinn of the mRNA, not transcription. The following simple experiment shows the distinction When e>.1ra copies of a ribosomal protein operon are introduced into the cell, the amount of mRNA increases correspondingly. but synthesis of the proteins stays nearly the same. Thus. the cell compensates for extra mRNA by curtailing its activity as a template. This happens hecau~ ribosomal proteins are .-epressors of their own translation. For each operon. one (ol' a complex oftwu) ribosomal proteins ltinds the messt=mger near the translation initiation SEquence of one of the first genes in the o~ron, preventing ribosomes from binding and initiating translation. Repr.:>.ssing translati01J of the first gene also prevents expression of some or all of the rest. This strategy is very sensitive. A few unused molecuJes of protPin L4, for example, will sh ut down synthP-Sis of that protein. as we1I as synthesis of lhe orher ten ribosomal proteins in its operon. In this way. these proteins are made just at the rate they are needed for assembly jnto ribosomes. How one protein can function both as a ribosomal component and as a. regulator of its own translation js shown by comparing the sites where that protein binds to ribosomal RNA and to its messenger RNA. ThCSP. sites are similar hoth in sequence and in secon dary structure

Gene regi.Ja6on typically invclves regulatay proteins that ron·

trol the expression of genes at the levef of transcription or translation. NOt al gene expression is governed by regUatOf}' proteins, however_The (fyptephan QDeron c.J E coli, as we have seen, responds to the celular levef of ns end product (tryptophan) by an atteruation rnechaflism invoLvJlg a leader RNA but 110 dedicated regt.llatay protein Anorher example of gene regtr latm that &les nc.t irn.dve a regUatoly protein is the ribosanal RNA (f'RNA} genes of E. coli, IM-.ose rate d tfan~n is ~ ~nfluenced by the gll:Mith rate ollhe c:d. It tums oot that RNA polyme.-ase foons unstable ccxnpk!xes at the promoters tor ~A genes, and these complexes are htgHy sensitive to the c:oncentJatic:J of the nud~ tha\ lfitiates trari~oon (usually ATP). lienee. under c()fl(itions of rClpid growth when the celJlar levels of ATP are high, the RNA po¥rJerasepromoter cCJI'T1Plexes are productJve. and the rRNA genes ~e transcribed at a htgh tate Converselyl under~ of rument Imitation when the grtM!th rate and cellular A1P levels are ~ matUI by RNA pclymerase is refficient and fRNA genes are tTaf'&ribed at a Jow rate. lhis nucJeotide.sensing system IS pethaps the S4fllplest cl all transcriptional rontrol mechanisms as d rfVdves 00 regulatory proteW\5 and IS sclely determined by the special prq:aties d IRNA gene pmmoteiSYet another example of ge£~e regulation without regulatory protetnS is the riboswitch Riboswitches are regulatory RNA elements that act as direct ser\SOIS of small molecule metabcr lites to c«ttrol gene transcription ot tranSiatirn. FOI' oomplt;

many genes whose fi..w1ction is related

to the amUlo acid methionine in the bacterium Bodlfus subtllis are c:orlTOIIed by

a 200-nUcleotide-klng, untranslated leader RNA that can ad~t attema6ve strUctlA'es: one involving a stem-loop transcription

terminator and the other an ilntitroninator- s..a~ methio.me. but not methiorlifle itself (a other metf1iorwle-related srnaW moJecules), binds to these leader RNAs to stabilize the transcription termll'iilbon structure. These leadef RNAs a-re therefae 5\Mtches (!iboswitches) that sense cettlAar levels of S-adenosyl methlooine iYld therefJv control I.Talsaiptional read-through into the downs11eam gene. Many examples of riboswitches are now known, each responding to a diffetfflt metabolite, such as vitamin B12, ttlamine pyrophosphate, flavin mononudeotide, lysine, gua~. and adeflirle (l3ox 16-4 figure l ). Some riboswitdles opernte at the level of transcfip-

00.1 temination but others operate at the level rl translation, controlling the foonaoon of an RNA structure that bkrls binding of the ribosome to the mRNA fa the downstream gene. Riboswttches are found not only in bacteria, but evidendy also in ardlaea, tung~ a nd pfants. Another kind of tiboswitch deserves special mention Rather thdl"' resr..a1ding to a metitdite, these leader RNAs respond to UCY::haqsed tRNA Thus, certairl genes. notably genes fOf aminoacyl tRNA s;nthetases (see Chapter 14), are controlled by a transcnpoon termination mechanesm that 1f11101ves a 200· to 300-nucleobde tong. untranslated, leader RNA that directly and specificaly lflteracts Wth the cognate, uncharged tRNA for the synthetase This interaction stabibzes the leader RNA in its antitetmination stn.KltJre so 1hat trclnscrtption IIllO the adjacent synthetase gene can proceed. Sperifiaty IS achieved 111 part by a ·~anticodttl'' interoctim betwcefl the tRNA and the lea<;!er RNA. Because onJy uncharged !RNA can bind to the leader, transcriptionat read-thro\.Jf;fl is only stim~ated ~!~.hen the cognate ameno acid is in short stlpply and the tevet of unchafged tRNA in the cell rises.

510

Q.JW

Box IH

Rey,uloljon in Prokaryott?S

(Continued)

B12 nboswitch

FMN riboswitch

lPP ribosWitch

flavlnmonon.ldeotide

r

~o-fo I 6tt_

·o-P=o I

0

C1

1

cc~o

~x=o

..-lJ

guanine riboswitch

~ l

5'

0

•"'\..,s

QL.Jaf'ioe 0

~ H

-- - - - -

adenine riboswitctl

adenine

~

0

1

I

H

~-

-

- -

BOX 164. F l G U R E 1 Riboswikhes pactiejpate in f\mdamenta1 genetic control. The secoodal)' structures of the seven kf'lOWI"' ribosY.itches
(Figlm3 16-23). The comparison suggests a pr€Cise mecha.nism of regulation. Since the binding site in the m l!ssenger indudes the initiating AUG, mRNA bound by excess protein S8 (in this examplf:) cannot attacb to ribosomes to initiate translation. {This is analogous to Lac repressor binding to the lac promoter and thereby blocking access to RNA polymerase.) Binding is stronger to ribosomal RNA than to mRNA. so translation is repressed only when all nood for the protein in ribosome as~mhly is satisfiad.

Exnwple~ of Gene Regulation at

Steps after T'rollS(:rijAn

L11

L1

L10

l7112

f3

S12

S7

EF-G

EF-Tu

S 10

l3

L4

l23

L2

(L22. S19)

S3

l16

l29

l14

L24

l5

S14

S8

l6

l18

S5

l 30

L15

S 13

S11

S4

a

l 17

l 11 operon

poperoo

stroperoo

S10operon

spcoperon

o operon

16-ll E.. coli ribosomal protein operons.. RiboSOf11
a

S8 binding site in 16S ribosomal RNA

b banslation u"Wation re9on in lt1e messenger RNA for S8

U•

f 1c tJ R E. 16-23 Riboscmal protein 58 binds 16S rRNA. A companson of lhe region vJla"e rbo SOO'Ial ~ S8 (encoded by the spc operon; Figure 16-22) bind!. 165 rRNA m 1he ribosome. with the tlaristaoon «111latioo Site fl'l llS mRNA 5rnilar sequences are shaded io dark green. The dashed fines box oft thi!t r€g10n d the 165 rRNA protected by the 58 protein (Source: CerrettJ D.P., Matthealas LC., Kearney KR, Vu 1 • ard Nornura M 1988. J. Mot Biol. 204; 309 - 329.)

512

Gtme Regulation in ProkrJ,·y otes

THE CASE OF PHAGE A: LAYERS OF REGULATION Bacteriophage A is a virus that infects E. coli. Upon infection. the

phage can propagate in either of two ways: lylically or lysogcnically, as illustrated in Figure 16-24. J..ytir: growth requires replication of the phage DNA and synthesis of new coal pwteins. The.se compone.nts combine to form new phage particles that are released by lysis of the host cell. Lysogeny - the alternative propagation pathway-involves integration of the phage DNA into the bacterial chromo..c;ome where it is passively replicated at each cell division- just as though it were a legitimate part of the bacterial genome. A lysogtm is extremely s table unclur normal d rr:ums tant..-es. bu t the phage domtant wilhin it -the prophage-can efficiently switch to lytic growth if the cell is exposed to agents that damage DNA (and thus threatnn the host ceU•s continued existence). This switch from lysogenic to lytic growth is called l}rsogenic induction. The choice of devf:l1opmental pathway depends on which of two alternative programs of gene expression is adopted in that cell. The

I bactecial

>.. repressor

' induction

I lysogeric growttl

1

• F I GU R E t 6-24 Growth and induc'tion af A lysogen. Upon Infection 11. can grow ather lyticaliy or lysogenicoly. A lysogen U3t1 be propogared st
The Case IJ/ Phage "'. Lcyers IJ{ Kegvlot ion

phage

!ill

Q

recombirlatioo

proteini'

exdsiooase integase

xis int

head genes

- -------

fIGURE 16·25 Map of phage A in the circular fonn. ~ genome is linear lfl lhe ~ge head, but, upon infection, orcularizes at the cos site. vvt\en .-.IEgrated 111lo the bacterial chromosome i\ is in a inear foiTI\ With ends at the on site (see Chapter 11 fOf a desaiptlen cf lfltegration).

program responsible for the lysogenic state can be maintained stably for many generotions, but then. upon induction. switch over to the lytic program with great efficiency.

Alternative Patterns of Gene Expression Control Lytic and Lysogenic Growth A has a 5o-kb genome and some 50 genes. Most of these encode coat proteins, proteins involved in DNA replication, recombination and lysis (Figure 16-25). The products of these genes are important in making new phage particles during the lytic cycle, but our concern hem is resbicled to the regulatory proteins. and where they act. We can. therefore, concentrate on just a few of them, and start by considering a very smaU area of the genome, shown in Figure 16-26. The depicted region contains two gtmes (d and cro) and three promoters (FR. P._, and PRM). All the other phage genes (except one minor

cl

DNA

cro

FIGURE

16-26 Promoten in the right

and left corttrol reP,ns of pbage A.

514

Qme Reguiotirm i fl Pl'Qkpryotes

F I G U R E 16-27 Ttansoiption in the A. control regions in lytic and l)sogenic powth. N[fYIIIIS indicate V>hch !=fOmDI€IS Df€ adJve at ~ ~oe penod durlflg lylic and lysogenc grow1h. respectil.€ty. Tne arrrJ.NS also show the drecton ot ~~oo lrom eadl promoter

lysogeric

one) are outside this region and are transcribed directly from PR and PL (which stmtd for !'igh tward and leftward promoter. respretivcly). or from other ptomoters w hose activities are controlled by products of genes transcribed from PI! and Pv PRM (yromoter for repressor maintenance) transcribes onJy the cl gene. PR and I\ are strong. constitutive ptomoters- that is, they bind RNA polymernse effidentJy and direct transcriptjon without help from an activator. P11M, in contrast. is a weak promoter and only directs efficient tranSLTiption when an activator is bound just upstmam. P RM resembles the foe· promoter in this regaro. There are two atrarlgements of gene expre55ion depicted in Figure 16-27: one renders growth lytic. the othe.r lysogenic. Lytic groWlh proceeds when PL and Pn remain switched on , while Pro..~ is kept off. Lysogenic growth, in contrast, is a consequence of PL and PR being switched off. and Pru.1 switched on. How are these promoters controlled?

Regulatory Proteins and Their Binding Sites

FIGURE 16-18 ). ~ llle f~ stoNs a fT10f10ll1el of " repressor, l"ldicating vaflOI,lS surfaces involved m afferent activttie:; c-aned out by lhe prolein N indicates the
T he ci gene encodes }\ repressor. a protein of two domains joined by a flexible Jjnker region (Figure 16-28). The N-terminaJ domain contains the DNA-binding reg)on (a heli x· turn~heHx domain. as we saw earlier). As wit h the majority of DNA-binding proteins. >.. repressor bin ds DNA as a dimer; the ruah1 dimP.rization con tacts rue made betw een the C-tenninal domains. A single dimer recogn izes a 17 bp DNA sequence. each monomer recognizing one half-site. again just as we saw in the lac system. (We have a lready looked at t he details of DNA recognition hy A repressor in F igure 16-12.} Despite its name, X rep ressor can both activate and rep ress transcription. When functioning as a represso r. it works in the same way as does Lac repr<>.ssor-at binds to sites that overlap the p rcr rooter a nd exdudcs RNA polyme rase. As an activator, X t:ept:essor wor'ks like CAP, by recruitment. X repressor's activating region is jn the N-tcnnina l domain of the protei n. Its target on polymerase is a region of the a subunit adjacent to the part of (T that recognizes t he - 35 region of the promoter (region 4. see Chapter 12. Figure 12· 6 ). Cro (wh ich stands for _QOntro1 of repressor and gther things) only represses tran.c;cription. Hke Lac repressor. It is a sint;le domain pto~ tein and again binds as R dimer to 1 7 bp DNA sequences. ~ repressot and Cro can each bind to any one of six operators. These sites are rer:ognizoo with different affinities by each of the proteins. Three of those sites are found in the l~ft-control region, an d Uuee in the right We will focus on the binding of~ repressor and Cro to the sites in the right-hand region, and these are shown in Figure 16-29. Binding to sites in the left·band c::o ntrol region follows a similar pattern. The three binding sites in the right operator are called (~ 1 • OR2, and O.R.1 ; these sites are similar in sequence. but not identical, and each one- if isolated from the others and examined separately- can bind

The Case of Phage 1\: Loyer." of Regulation

51 S

a

b

Fl cuRl 1~29 Relative positions of promoter and aperator sites in o.,. Note that ~J
wrth pesmss1011.)

either a dimer of repressor or a dimcr of Ceo. The affinities of these various interactions. however. are not aJI the same. Thus, repressor binds Oru tenfold better than it binds OR;· In other words, ten limes more repressor- a tenfold higher concentration-is n eedt}d to bind Dtu than ~1 • OR3 binds repressnr with about the same affinity as docs ~. Cro. on t he other hand, binds OR:! with highest affinity. and only binds ORL and OR, when present at tenfold h igher concentration. The significance of these differences wiU become apparP.nt presently.

A Repressor Binds to Operator Sites Cooperatively ~ repressor binds DNA cooperatively. This is critical to its function and occurs as foJlows. C.onsider mpressor binding ro sjtes in ~. In addition to providing the d imerization c-.ontacts. the C-temrlnal domain of ~ repressor mediates intemctions between dimers (the point of contact is thE patch marked ..tetramerization '~ in Figure 16-28}. In fhis way, hNo dimcrs of repressor can bind cooperatively to adjacent sites on ONA. For example, repressor at ~ helps repressor bind to the tower affinil.y site O R'J by coopf'.rative binding. Repressor thus binds both sites simultaneously and does so a.t a concentration that would be sufficient to bind only ~t were the two sites tested se::parotely (Figure 16-30 ). (Recall thal. withottt coopE~rativity. a t enfold higheT concentration of repressor would be needed to bind OJQ.} ~3 is not bound; repressor bound cooperatively at ~1 ~d ORZ cannot simultaneously make contact with a third d imer a.t that adjacen t site. We have already discussed the idea of cooperative binding and seen an examplt>..: activation of the lac genes by CAP. As in that case. cooperative binding of repressors is a simple consequence of their touching each other while simultaneously binding to sites on the same DNA molecule. A more detailed diSCl1'iSion of the causes and effects of cooperative binding is given in Box 16-5, Concentrntion , Affinity. and Cooperative .Binding. Cooperative binding of regulatory proteins is used to ensure that changes in the level of expression of a given gene can be dramatic even in response 1o smaJI changes in the level of a signal that controls that gene. The lysogenic induction of A. discussed below. provides an exceUent example of this sensitive aspect of controL In some systems, cooperative binding between activaton; is also the basis of signal intcgratit>n (see the discus.c:;ion on 13-interfcron in Chapter 17).

FICURE

16-JO

Coopefa~bindingof

~ r~ w DNA.

l nc "repress«

and thos~ duners interact to form tetramers. lhese inteorac rT\OO(.)r'!1()fS

IJ"lteract to form dimers.

1100s £f\Sllfe tha bindlrJE of ff!J:%e5Sor to DNA rs cooperatiVe. That ~live bin&ng ts helped

further by inter;)CtlOOS between Jepres.sor t~ at~ intErading IMt~ others at Ot. (see latt!t m teXt and F1gure 16-31).

516

Gene Regulati011 in Proknryotes

Box 16-5 CDncentration, A~ and Cooperative Binding

What do we l'l"'eaf1 when we talk about •str'or€wand "vveak" binding sites? \rVhen we say two rrolecules rerognize each other, or irteract with each other- ~ as a protein and its stte on OOA-we mean they have some afftntty for each other. \Nhethef they are actualy found bol.fld together at any giVen 1ime depends on two things~ 1) how high that affinity is- i.e., how tightly they int€f0Ct,. and 2) the concentJation of the

sor at OR2 periotically lets go of OOA. it is hold'"€ on to repressor at OR, and so remains in the proximity of ~ This effectively roeases the local ~on of repressa in the vidlity of mat site and ensures rep-essor rebinds frequently.

If you d15pense with cooperativity and jt;st increase the corr certration of repressor in the cell, when repressor faRs off OR2 rt vviNnot be hetd nearby by repressor at 01!1 and ~ t..JSUally drift away before it c43n rebind CJm. But at the higher conce~ ot repressor. another moteaJe of repressor wll ~ety be dose to OIU and bind there. Thus, even if ead1 repressor dimer crly ::;its on 0 112 for a short time, by either holding rt nearby or

molecules. As we emphastzed an Chapters 3 and 5, tile molea.Jlar interactions that underpm regtjabon 111 biological systems are reversible: \o\tlen interactJng molecules find eadl other, they stk:k together for a periOO of time and then separate. The 1-ighel 1he affinity, the t!!t'ter 1he tV\Q ooecules stick together, and in general the longer they remain together befcre parting. The higher the concentration, 1he more often they wil find earn other In the fi-st place. Thus, higher affinity or higher roncentrntion have similar effec1s: they both resl.At in the two J1"ld.. erules, in general, spendi~ more time bound to each aher.

irueasing the runber d possa:Ae replacements, )'OU InCrease the Ukelihood of repressor being bolJ"Id at ooy giVen time. Yet anothef way of thi~ about cooperative bin
Cooperativity Visualized CooperatMty can be expressed 10 teiTTIS of increased afi!"1ly. Repressor has a higher affrity for ~1 than fcr 0~. But once rep-essor is bound to ~~ , repressor can bind ~ more tightly because it illtefaOS v,;th rliX only ~ but with repressor bound ~ ~~ as well. f\.'either of these interactioos is very strong alone, but when combined they stbstantialy increase me affinity d bOOing of that secoo:J represscr. As we saw in Chapter 4, the raationsh1p between bindng energy aM equilibrium is an exponential one (see Table 4-1). Thus, increas111g the bYlclif€ energy as •ttte as twofold increases affinity by an order of magritude. Another way to picture hoJ.I cooperati\iity works is to think of It as iocreas;ng the local concent7a1ilxl of repress
, .,

100

~

,

,,

'1:1

c;

::;)

, , .,

,

,' ,,

I

.8

z

0

0

----- - - -

-

I Cooperative lbincfing reaction. The ~ ine shcM<s the c;urve lhdt descroes binding of a prolt:in to ~ Single site. The steeper SlgmOirl curve shows cooperative bindng ot for 80 X 16-5 FIGURE

example, t. tep"essof to ns operator sites. (Sc>u"re: Adapted from Ptasfme M. 1992 A genetic SWitch: Phage ond higher otgOfliSITJS. 2nd edfnon Copyngtt @ 1992 Bladwell 5oe1'lc.e ltd. Used with peimiSSmn.)

The Cose of Pltoge ~: l.ayf:rs ofR~«ilotion

517

Sox 16-5 (Continued)

fraction of free repressor (1.e., that not bolnd to ONA) is found as monomer., the eel; thus ~ &S in essence a cooperntive b\nding of ftu rncnomers rather than tvvo stable dimm, addV"lg to the coocerted nature of cofllllex fOflT\Cition on OIIA, and so adding to the steepness of the curve. But~ does cooperativity make the binding rurve steeper? We have already seen how the site is filed at a lower concentrntloo of represSOf than its affinity wcUd suggest; but haN IS it that, as repressor concentration decreases, birdng fats i!1WiJ1f so qJCk!y? Cons'der in\eractions between components of any system 35 the concentrcltion of the c.vrJllOO€nt5 is reduced, i!!TIY given irteraction between two of them wt OCOJr less frequefltly. If the system reqt.ft-s mUtiple interactions between several dEferent components, this \'\All bee~ very rare at lcmer concentrations. Th.Js, binding of feu- moncrners of a t=rotein to tvvo Sites reqwes sever-at (111 ~ seven) anteroctJons; the chance of the ~ c~ts coning together is drastically re
Cooperativity and DNA Binding Specificity A final ImpOrtant aspect of cooperati\te tinding is that it imposes spedfdy on DNA binding. CAP activation of loc promoter shows this CAP brings RNA pdymerase to prorrders that bear Cfl.Psites ~(as opposed to other promoters of COI'Jl)(r rable affioity that lack OP sites). l J<ewise, ~ represscr at ~~ directs arother mdeaJe ot repressor to bind to the ~ site ac:\3cern to tt not some other site of equal affinity else\M1ere in the eel. k1 fact. cooperativity is ~ to ensuring that proteins can bind with sufficient specificity for lfe to vvor'K as we krcw it. To llustrate this, tonsader a protem bmding 10 a sit.e oo DNA. This protein has a ~ affinity for its cooect srte. But the DNA wtthin the cell rept"esen1s a huge rumber of potent\al (but incorrect) bind;ng sites fer that J:rotein. What is importart. therefore. is not simply the absolute affinity of the protetn for its rcrrect site, but its affnty for that site rompared to its affinity fOf al the other, lllCOO'ect sites. And remember, those incorrect sites are at a much higher concentration than the rorrect site (represenbng. as they do, aU me DNA in the eel except the cooect site). So even if the affinity for the incDrrect sites is kmer than fOr the correct site, the higher concentratiOn of the fooner

ensures the protein wil often sample them whie attempting to reach its cooect site. Vvtlat is needed is a strategy that increases affinity for the COO'ect site without adng interactions with the incorrect s.ites. tncreasing the num~ of contacts between the protein and rts DNA site (for exarrpe by rnal\lng the protem larger) does not necessarily ~ because it also tends to tnctease binang to the incorrect sites. Once affinity for the II1CClrfeCt sites gets too hifjl, the protein essenb
spends too long san-Ping incorrect sites. 1'ht.6 a kinebC. prol:r Jem re,paces the specificity one and it can be jUSt as disn.ptive. Cot:l)erativiry solves the problem. By binding to two adjacent sites cooperatively, a protein increases aamaticaly Its affinity for those srtes, without inaeas;ng affinity for other sites. The reason It does not increase affinfty for the incorrect sites is simply because the chance of two molerues of protein bindiog tncorrect sites dose together at the same time (aiiOWI~ cooperatMty to stabitize that binding) is extremely remote. Only when 1hey flhd the rorrect sites do they remain bornd long enough to give a second protein a chance to tum ~Coopet'ativity and Alklstety

AlttoJgf1 in ttis chapter we use 1he term cooperotMly to refer to a partx:Ular mechanism of cooperative blnding. 1he term tS also used in other rontexts \Nhere different rnec:harisms ~ly. In general we might 5iJY that <:clq)erativity describes any situation in wa-.c:h two igands bind to a tharcl molerule in such a way that the binding of ooe of those ligands ~ the bnding of the cther. ThtJs. fct the DNA-binding proteins we ~ here, coopeiOb\lity 1S ~ated by Slmple acl1estVe rnteractJons, but In cth€r s1tUaticns ~vily cnn be mediated by allosteric events. Pee-haps the best &le of that as the binding d wt gen ll10iea.ks to hemoglobtrt Hemoglobin is a homotetramer. and each subunit binds one molecule of oxygert That binding l5 CDVperative: v.tlen the first oxygen bmds, 11: aruses a ronfoona~ change wtll(;h fu<es the binding s1te for the next oxygen in a conformation of higher affinity. lhus, in this case there is no direct interaa10n between the ligands, but by triggering an alosleric transition one ligand increases affirity fer a seoond

Repressor and Cro Bind in Different Patterns to Control Lytic and Lysogenic Growth How do repressor and Cro control the different patterns of gene expression associated ~ith t he different ways A can grow? For lytic growth. a s ingle C'.ro dimer is bound to OR3; this site overlaps PRM and

so Cro represses that promoter (which would onl y work at a low leveJ anyway in the absence of activator because the promoter is weak) (Figure lf>.31). As n~ither repressor nor r:ro is bound to 0~, and OR2• PR binds RNA polymerase and directs h:anscription of lytic genes; PL does likewise. Recall that both PR and PL are strong promoters that need no activat or.

518

Gene ResviCJiion in Prokvry ales

lysogen

cl

cro

lybcgrowth

cl

F 1Gl1RE 16-11 The actim of A repressor and Cro. R€pressor bound to ~1 and OIQ tums off lranscrlptlon from P... ReprE'SSOf boond at ~ conlartS RNA po1vrrl6ase at f\w. activating e>q:.JreSSIOO of the cl (repesoof) gere. Ore lies ltolllhln PP....,; Cro bour!d there rep~eSSes ~ of cl. (Source: ~ed from

Plc:dlne M. and Gann A 2002. Gelles &signols. p. 30, Fig 1-13. ~ Cdd 5pnng Harbor I aboratcry Press.)

During lysogeny, Pru-.1 is on. while PR (and PJ] are off. Repres>sor bound cooperatively at ~ ~ and Ostz blocks RNA polymerase binding at PI<• repressing transcription ftom that promoter. But repressor bound at ~ activates transcription from P RM• We return to the question of how the phage chooses between these alternath•c pathways shortly. But first we consider induction-how the Jysogenic state outlined above switches to the alternative lytic one when the cell is threatened.

Lysogenic Induction Requires Proteolytic Cleavage of A. Represscr E. coli senses and responds to DNA damage. It does this by activating the function of a protein called RecA. Thls enzyme is involved in recombination (which accounts for its name; see Chapter 10) but it hBs another function. That is. it stimulates the proteolytic autocleavage of certain proteins. The primary substrate for this activity is a bacteria) reprt...oc;sor protein called LexA that represses genes encoding DNA repair enzymes. Activated RecA stimulates autudeavage of LexA. releasing repression of those genes. This is called the SOS response (sec Chapter 9). If the cell is a lysogen. it is jn the best intflrests of the prophage to escape under these threatening circumstances. T o this end, ). repressor has evolved to resemble Lex A. ensuring that A repressor too undergoes autocleavage in response to activated RecA. The cleavage reaction removes the C-terminal domnin of .repressor, and so dimerization and cooperativity are immediat~ly loRt. As these functions are critical for repressor binding to OR1 and OR:J (at concentrations of repressor found

1'ht? Cose pf f'ho8f! .,... l.ayecs oJ Jlegulution

519

in a l ysogen). loss of cooperativity ensures that repressor dissociates from those sites (as well as from 01.1 and <Ja.a). Loss of repression triggeiS transcription from Pfl.. nnd PL leading to lytic growth. For induction to work efficiently. the le,·el of repressor in a lysogen must be tightly regulated. If levels were to drop too l ow. under normal conditions. the lysogen mjght spontaneously induce: if levels rose too high. appropriate induction would be ineffident. The reason for the latter is that more repressor vmuld have to he inactivated (by RecA) for the concentrat ion to drop enough to vacate 0 1u ancl ORz· We havP. already sp,en how repressor ensures that its level never drops too low: it e~.ctivat es its ow n expression. an example of posilive autoregulation. But how does it ensure Jevels never get too high 7 Repressor also regulates itself negatively. This negative autoregulation works as foJiows. As drawn. Figure 16-31 shows P RM being activated by repressor (at O~tt) to make more repressor. But if the concentration gets too high, repre!':sor will bind to ~ as well, and repress PIW {in a manner analogous to Cro binding Ol!!f ond repressing PRM during lytic growth). This prevents synthesis of new repressor until its concentration fulls to a level at which it vacates ~a· ~an aside, i1 is interesting to note that the term .. induction•· is used to describe both the switch frorn lysogenir; to lytic growth in ~.and the swit~hing (Jfl of the lac genes in response to loctose. This common usage stems from the fact that both phem>mena were studied in parolleJ by Jacob and Monod (see Box 16-3). It is a lso worth noting that, just o.s lactose induces a conformational change in Lac repressor to relieve repression of the lac genes, so too the inducing signals of"- work by causing a structural t:hange (in this case proteolytic cleavage) in). repressor.

Negative Autoregulation of Repressor Requires Long,Distance I nteractions and a Large DNA Loop We have discussed eooperati\'e binding of repressor dimef't' to adjacent operators such as OR1 and Ottz· There is yet illlothcr level of cooperative binding seen in the prophage of a lysogen, one critical to proper negative autoregulation. Repressor dimcrs at OR, and OR2 interact with reprf'. ssor dimers bound coopP.rat ively at Oa.1 and 01 ~. These interactions prorluce an o(:tomcr of repTessor. each climer wilhin the !JCtamer is OOUDO to 8 separate Dperator. To accommodate the long-distoncc interactio n between repressors at Ott and Q . the DNA bP.tween thos~ operator rcgions - so(ne 3.5 kb, including the cJ gene itself- must form a loop [Figure 16-32). When the loop is formed, OR3 is held c lose to Dt..~- This allows another 1wo dimers of rcpref\sor to bind coopeTativeJy to these two sites. This coopemti\!ity means O~tt binds repressor at a lower con(;entrat ion than

FIG tJ R E 16-32 lntenc:tion of Ol. Repress
cl

ae~ represSIOn of P~;

aod Pl. and allow repressor to b1nd O,u at a lower coocentrabon than It othe!WlSe could. (Source· Adapted f1001 Ptasllne M. end Gclnn A 2002 _Genes & signals. p. 35, ftg 1 I E. Cl Cold Spong ~lbor Laboratot)l Press.)

520

Gene Ffegulation in Prokaryotes

ll otherwise wuuld-indeoo. at a concentration only just a little higher than that required to bind O.o and ORz- Thus, repressor concentration js very 1ightly controlled indeed- small decreases are compensated for by increased expression of its gene, and increases by switching the gene off. This explains why lysogeny can be so stable while also ensuring that induction is very efficient. The struchtrf.l of the Crterminal domain of.>.. repressor. interpreted in the light of earlier genetic studif'..s, reveals the basis of d imer formation. But it also shows how two dimers interact to form the tetrnmcrlc fOrm (as occurs when mp.ressor is bound cooperatively to ~1 and~)_ Moreover, the structure reveals the basis for the octomer form-and shows that this is the highest order oligomer repressor can form (Fig ure 16-33).

Another Activator, "ell, Controls the Decision between Lytic and Lysogenic Growth upon Infection of a New Host We have seen how }1. repressor and Cro control lysogenic and lytic growth. and the switch from one to the other upon induction. Now we tum to the early events of infection, those thnt determine which pathway the phage cboos€8 in the first place. Critical to this choice are the products of two other }1. genes. en and clll. We need only C>.'}Jal1d slightly our rnap of the regulatory region of }\ to see where ell and em lie: en is on the right of cf and is transcribed from PR: dll. ort the 1eft of d. is transcribed from PL (Figu re 16-34). Th~e and other genes were isolated in elegw1t genetic screens outlined in Box 16-6. Genetic Approaches that ldcntifi~d Genes Involved in the Lytic/Lysogenic Choice. Like ')1. repressor, Cll is a tronscriptional activator. It binds to a site upstream of a promoter Galled PR.C (for repressor gstablishrnent) and

• potential interfaces

d itner---dimer

dirrla-dirnF!f

Interface

Interlace

"'

intertace FIG U R £ 16-Jl Interactions between the C--tenninal dGmiJin of ). ~B- The ftgt.te shows. at the top. a schematic representatiOn of two dmers of the C-term~ domain of A represSOf lr.diCclted are the tvo10 patc;he ~re C3Wed Band Ron lf1e s~ of that domain that~ interocbcf"IS

between tvvo dirners to gl\le a tettorner. in tM! first I"IStcln<:e. and then between two tettane~ to gl\le
1'he Cose ofPhage 1\: Wy4!)rs of Regvlalion clll

cl

cro

elf

FIGURE

16-34 Genesartd promoters

involved in the lytic)lysogerJic: chotc:e. Not shown hefe is the gene N which lies between P. ClOd em (see Figure 1& 25)

stimuJatcs transcription of the d (repressor} gene from that promoter. Thus. the repressor gene can be transcribed from two different promoters (PRE anrl PRM). PJ<E is a weak promoter because il has a very poor - 3 5 sequence. Cll pootein binds to a site that overlaps the -35 region but is Located on the opposite face of the DNA helix; by directly interacting with pol)'· mP.rose, err belps polymerase binrl to the promoter. Only once sufficient repressor ha<; been macle from P RE r.an that repressor bind to OR1 and ~ and direct its own synthesis from PRM. Thus we see that repressor synthesis i" established by transcription from one p romoteT (stimulated by one activator) and then maintained by tcansaiption from anolhflr (undet its own control- positive autoregulation). We can now sec in summary h ow CIJ orchestrates the choice between lytic and lysogenic dP.velopment. Upon infe<:tion, tronscription is immediately initiated from the two constitutive promoters PR

Sox 16-6 Geraetk Approaches that Identified Genes Involved in the

lytic/lysogetlic Choke Genes involved in lytic/lysogenic dltice vvere identified by screen11:1g for ~ mutants that efficierrt.fy grow only either lytically Of' lys~. To ...-.derstand how these mLtants were found, we need to OJI1sider hCMt phage are grown in the laboratory (see Choptff 2 1). Bactenal cells CcJl be gf'l:Min as a confluent opaque lawn aooss an agar plate. A lylic phage, grown on that lawn. prcxllces dear pf~ or holes (Figure 21-3). Each plaque is typically initiated by a single phage infecting a bacterial eel. The progeny ptlage from that infection then infect sum:>l.lliding eels, and so on. killing off (lysing) the bactelial eels in the vicinity of the ongml infected cell ilfld causing o clear eel-free rone 10 the otheriNise opaq..Je lawn of bactenal eels. Phag€ A forms plaques too, but they are turbid (or doudy)-that is, the region Wlthn lhe plaque is dearer than the undected &awn, but only margu'\Cilly so. The reas<J"l fOf this is lhat A, unlit.e a purely lytic phage, kills only o prqxJ(tion of the cells it snfecls, the others suvivwlg ~ lysogens. Ly;ogens are resistant to subseqJeflt infection oncl so can FJOW within the plaque unlwmed by the mass o1 phage partiCles found rhere. The reason for this ...lfl"ll1ll.lnity is qt.rite simple: in a ¥ogen, tlle Integrated phage DNA (the prophage) continues making repressOf from Pr«.,. Any ne.N ~ genome entering that cell will at once be bound by repressor. giving no chance of lytic growth. In one dassk study, rrutants of A that funned dear plaqL.JeS were isclated These mt..Jtant phage ore ~.Nble to form lysogens but stib groo lyncaly. The >. degens. By analogy to the lac system, the ct mutants are c;orr.parabre to the Lac repressor {fad) muta~ts, vir mutants are the equiValent d the lac operator (locO) mutants (see Box 16-3). Another revealing mtJtation was identified W1 a different experiment this one a rnutatkn in a host gere_ The mutant is called hfl fur high frequency of lysogeny. When infected with wild-type A. this strain almost always forms tysogens. very rarely allow1ng the ptJage to gro.v lyticaly. This bacterial strnin lad<s the protease that degrades lhe A dT protein (see text}.

s.;

522

Gene El~lation in Prokary«es

and PL. PR directs synthesis of both Cro and CU. Cro expresf.lion favors lytic development: once t:ro .reaches a certain level it w iiJ Lind ~ and block P RM· en expression, on the other hand. fa vors lysogen ic growth by directing transcription of the repressor gene (Figure 16-35). For successful lysogeny, repressor must then bind to ~. and OR:l and activate PRM befme t.:ro can inhjbit that promoter.

Growth Conditions of E. coli Control the Stability of CII Protein and rhus the Lytic/Lysogenic Choice The efficiency with which OT directs transcription of the cl geneand h ence the rate ot which repressor is made -is the critir..al step in deciding how >.. will develop. What determines b ow efficiently en wotk.s in any given infection? When the phage infects a p opulation of bacterial cells that are healthy and growing vigorously, it tenrls to propagate lytically, releasing progeny into an environ ment rich in fresh host cells. \Vhen conditions are poor for bacterial growth. however, the phage is more likely to fnrm ly~ogens a nd sit tight; !here will likely be few host celJs in the vicinity for any progeny phage to infect. 7ltese diffenmt wowtl1 conilitions impinge on 01 as foJlows. en is a very unstable protein in E. coli; ll is degraded by a specific protease called FlsH (HflB). encoded by the hfl gene. The speed with which CU can di.rect synthesis of repressor is thus detennined by h ow quickly it is being degraded by FtsH. Cells lacking the hfl gen e (and thus FtsH) almost always form lysngens u pon infectinn by >..: in tho absence of the protease. OT is stable and direct5 synthesis of ample repressor. FtsH activity is itself regulated by the growth conditions of the bact£rial .cell. and. although it is not understood exactly how that is achieved, w e can say the following. If growth is good, FtsH is very active. 01 is destroyed efficiently, repressor is not made. an d the phage tend to grow lytically. In poor growth conditions the opposite happens: low FtsH activity. stow degradation of en. repressor accumuJation. and a tendency toward lysogenic development. Levels of Cil are also modulatvd by the phage p rottiin ern. un stabilizes en. p robably because it ads as an alternative (and thus competing) substrate for FtsH.

FIGURE

16-35 Estabtishmentof

fysozeny. The d gere es transoibed from J1.T Vvhert estL'Iblishlng lysogeny allCl from P,u., v..41en ~ng tha1

PAM

statE>_ Reprt:-sscr bound at OR,

controls the clll ~ne Vltudl hefps establish tysogeny (see telrt}. (Srurce; Adapted from Plastme M . cYid Gatln A. 2002. Genes & s:gna/s, p. 31, rtB 1-14. Cl Cold Spring Harber labooltory Press.)

•

PR

~

and Qu not ooly acwates \he mamteMilC of expresSKlO. Ncte that f'F. controls not ooly lytic genes but ~so ~ession of cil, and as thus important in lysogeny ,as wei as lybc dev€1-

opmenl ~ilafly. th~ not sllown irJ the figure, Pt , which CXlr'lf.l'cls IT1ally lytle genes, also

~

cro

cf

~p· c l.

PRE

~p· o~~.

PRE ell

OR

•

ell binding site

btnding site

A second di protein-dependent promoter, Pt. h as a sequence similar to that of PRB and is located in front of the pbage gene int (see Figure 16-25); this gen e encodes the integrase enzyme that cataJyzes site-specific recombinAIivll of ~ DNA i nto thfl bacterial chromosome to form the prophage (see Chapter 11 ), A third dl-depcndent promoter. PAQ, located in thr middle of gene Q. acts to retard lytic d evelopment and thus to promote lysogenic development. This is OOt:ause the PAQ RNA acts as an antisense mf'n<;sage, b inding to the Q message and promoting its degradation. Q is another regulator, one that promotes the late stages of lytic growth, as we will see in the next section. Transcriptional Antitermination in A Devel{)prnent We earlier saw examples of gene regulation that operated at stages after transcription initiation. 1\vo more examples are found in A development. as we now describe, starting with a type of positive transcriptional regulation callfx.l an1itermination. The transcripts controlled by A N and Q proteins are initiated perfectly well in the ~bsence of those regulators. But the transcripts tcrminale a few hundred to a thousand nudeotidm:: downstream of the promoter u nless RNA polymerase has been modified by t he regulator: A N nnd Q proteins are therefore called antiterminators. N protein regulates early gene expression by acting a l three terminators: one to the left of the N gene itself. one to the right of cro, nnd one between g~nes P and Q (Figures 16-25 and 16-36}. Q protein has one target. a teTminatm 200 nucleotid es downst ream of the late gene promote!', Pp,-, located between the Q Wld S genes (see Figure 16-36). '111e late gene operon of >., transcribed from PR·· is remnrkohly large for a prokaryotir: transcription unit: about 26 kb, a rli:r:t1anat that takes about 10 minutes for RNA polymerase to traverse. Our u nderstanding of how antitenninators worl: is incomplete. J.ike other regulatory proteins, N and Q only work on genP.s that carry particular sequen ces. Thus, N protein prevflnls termination 1n the early operons of ~. but nol in other bacterial or phage operons. Tite specifk recognition sequences for antiterminators are nflt found in the terminators where they act, bllt instead occur in the operons well he(orc th~

f1CiUR£ 16-36 Recocnffion she$ and sit8 of action ofttle AN" Q tnnsaiption antitemainators. The-uRJet hne shows the early ngh(w.arrl promoter P., Cll'ld rts ritJal terminclt.or,t,11• TI1e nvt Site IS cWtdecl mto two regK>OS, called 8oxA (7 bp) and BoxB, ~epaated by a spac.er reg.D'I of 8 bp lhe seqttenee ol BoXB f,as dyad symme(ly e~nd loons. a stern-loop SII\.IC1ufe ol'lCE ~ransmbt.'CJ lflto ~The Seq.JeO<:e Of the r
terminators. N protein .requires s ites named nut (for N utilization) thot me 60 and 200 nucleotides downstream of .PL and PR (see Figui'P. 16-36). .But N does not bind to these sequena;s w ithin ONA. Rather, .it binds to RNA transcribed from DNA containing a nut sequence. Thus. one£ RNA polymerase has passed 8 nut site, N binds lD the RNA and from thue is loaded on to the polymerase itseJ£ In this state. the polymerase is resistant to the terminators found just beyond the N and em genes. ). N worl;s along with the products of the bacterial ~nes .nusA, nusB, n usE, and .nusC. The N\\SA protein is an important cellular transcription factor. NusE i~ the small ribosomal subunit protein Sto, but its role in N protein function is unknown. No cellular function of NusB protein is known. These proteins form a complex with N at the nut !-lite, but N c-.an work in their absence if present at h igh concentration, suggesting that it is N itBelf that pJ'Omotes antitennination. Unlike N p rotein. thfl 11. Q protein recognizes DNA sequences (QBE) betWeen the - 10 and - 3 5 regions of the late gene promot~r (Pd (sec Figure 16-36). Jn the absence of Q . polymerase binds Pw and initiates transcripUm1, only to pause after a mere 1 6 or 1 7 nucleolides; it thtJn continues but term inates when it ~ches the tP.rrnin<~to r (tR.) !;Ome 200 bp downstream. If Q is present it binds to QEE once the polymerase has left the promoter, and tra..nsfers from there to the nenroy paused polymerase. With Q on Loard . the polvmerase is then able to transcribe through tR··

Retroregulation: An Interplay of Controls on RNA Synthesis and Stability Determines int Gene Expression The CII protein octivatp,s the promoter P1 that directs expression of the int gene, as wcJl as the promoter P~ responsjbJe for repressor synthesis (see Figure 16-25). The Int protein is the enzym e that i ntegrotns the phage genome into that of the host cell during formation of a lyS
Summmy

52.5

.,

site of termination in absence of N protein

5'

jTGATGACAAAAAATTAGCGCAAGAAGACAA.AAA'TCACC'l''l'GCGC'l'AA'l'GC'l'C'lGT

3'

&.C'l'AC'rG'l''l'"ETT'l'AATCGCG'l''l'C'l'TCTGTT"l'T'l'AG'l'GGJ\ACGCOA~'.fACGA· GACA

It'll

· gene

I

direction of transcription

16-37 DNA $ite and transctibeclllNA structures active in rettoreguta&ion of int exprKSion. At the top IS shown the DNA SE<)U€f'lC.e and. bekm. the smaM cylrndas shew the symmetriC setJlleflCC"S that fofm hauptllS 111 RNA. The stn.dure on the Jeft shows the tenmlator klfrr,ecJ II! RNA ttanfIGURE

soibed from P,. witJlout antit€1TTlination by N protetn. v.tuch is tesiStant to degradation by nudeases_The str\.Jctl.n> on the rigtlt shows an extended loop formed In RNA 1Janscnbed from PL. tK'lder the inflt.'erJC.e of N pr~ antltermm~ta, v.fltett tS ., target fa clE-avage by RNAse Ul af1d degraclabon by nudeases.

There is yet a further subtlety in this regulatory device. When a prophage is h1duced, it needs to make integr-.tse (together with another enzyme. called excisionase; see Olapt« 11) to callilyze r'f'J onnation <>f free phage DNA by recombination out of the bacterial DNA; and it must do thls whether or not Cll activity is high. Thus, under these circumstances. the ph
SUMM:ARY A typical gene is switcbOO on anrl off in response lo the need for its product. This regulation is predominantly at the level of transcription ini tii!lion.. Thus, for example. in E. rol1, a gene oncorling the enzyme that metaboliz.~ lactose is tran·

s r:ribed at high k>Vels only when tact~ is available in the growth medium. Furthetrnore, when ghl<.'Ose (a belle£ energy

somce) is also available, the gene js not expressed eve,n w hen lactose is present.

Gene RegulatiOI' in Prokal}'otes

Signals, such as the ~ence of a. sprific sugar. are communicated to gt=)nes by regulatmy protajm. These are of two types: oclivntors, positive N:golators that swilch genes on: and repressors. negative regulators tbat switch genes ntl'. Typically these regulato\'s e.re DNJ\-binding proteins that f('(;()gnit.e specific sites at or oear-thc genes thoy control. Activators. in the simpl~:.st (and rl10b1 t·ommon) cases, work 011 promoters that are inherendy weal<. That is, RM polymerase :b inds to the promoter (and lhos irutiates transr;riprlon) poor·Jy in the absence of any n ..o.gulatof. An aclivator binds to DNA wi1h one :r;urfac.e and with another surface binds polymerase and re<:n1its jt to the promoter. This process is an example of cooperative binding. and is sufficient to stimulate lra:nseriplion. Repressoa-g can inhibit tr-..nscription by binding to <1 ~:~itt~ thut overlaps the promoter. thereby blocking I{NA polymerase binding. Repressor$ can work in other ways as well, for example by binding to a site beside the promtter and, by inler9(:ttng with pol ymerase bound at tl'l€! promott•r, i..nhlbiting inihi1l1on The Joe genes of E. r.oli are rontroller! by a1.1 a•:tivator and a reprossor that worl in the simpl~t way just outlined. CAP. in the absenl:e of glucose. binds DNA nero the lac promoter and, by recruiting polyme1-ase to U1at promoter activalas expreQ!lon of lhOSf' genes. Tile Lac rcpressm binds a Sile tbal overlaps tlv> promoter and $butt> off expl'ass:ion in lhe absence of lactose. Another way in which RNA potycnerasc is rooruitccl to different genes is by the use of alternative IJ f~tors. Thus, dHferent cr factors can rcplaoo thll most prevalent one err~•• in E, w li) and direct the enzyme 1o promoters of di.ffel'ellt t>equt1nces. Exa•nples include tr'2, whid• dirct..:ts tianscription of gene~ m l'esponse to lle.at shOt:k. and tr"'. which direcls lranscrlptlon of genes involved in nil£08f!n mctabo· tism. Phagt> SPOl uses a series of ahemalive cr to ~ o ntrol lh.Q ordered e>.-pression of its genes during i.nfechon There are. in bacteria, examples of olht;C kinds of tran scriptional at.1ivaliou es well. Thus. et some promoters. RNA polymernse binds efficiently unaided. and forms a stable. but inactive. dosed compl~. That d osed complex does not sponlaDOOusly undergo transition to tht! open complex and initiate transcription AI such a promoter, an activator must stimulate lhe transition from dosed to open romplex. Activators that stimulate lhis kinc1 or promoter work by allostery: they i nter~t:l with the stable dosed complex and

induce a conformational change that causes transition to the opt=)D cou1ple;x. ln lhis chapter we saw two examples of transcriptional activatVtS working by a llo.~tery. In Ot'lE! c.;ase, the activator (NbC) interacts with \he RNA polymerase (bearing u·~) bound in a stable dosed ·oo~nplcx at the ginA promoter. stimulating transition to the open cornpl€?<. ln th~ other example, the a(lh\lalor (M&·R) induces a conlormational change in the merT promote.- DNA. ln all the cases we have consl.nes a 11.d antitermination by the N and Q prorejns of phage>... The trp genes cncocle en~ymes ruquirOO for the synthesis of the amin.o dcid tryptophan. These genes are only transcribed whoo the t.:clt !acks lryptopban. One way that amino acid con· trols e>--pression of these gem.lt; is attenuation: a transcript initiated J' with a detailed dis..:ussion of how baclcnophage >. dlf)()t:CS ~-tween two ahomaJive ~cs

of propagation. Several of Ihe slral~ uf getre .rt.-gu·

lation cncounternd in this system tum out to operate in other systems as well. including, as we will see in tate· cbaptf'li'S, those that govern the development of animalsfor example. the use of -.:oopcrative binding to give stringent on/off swilches: and the use of separate pathways for estab· Jishing and maintaining expression of genes.

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6857- 6864.

.1\omura M.• CourseR, and Baughman C. 1984. Regulation of the s ynthesis of ribosomes and ribosomal mmponcnts. Ann. Rev. BiochP-m. 53 ; 75-117.

Roberts j.\1\T., Ysrooll W.• Brut1eu E.. Guo J., Marr M .• Ko O.C.. Sun B .• a nd Roberts C.W. 19AA. Antite rru.ination by bocleriopbage A. Q protein. Cold Sp ritl8 flllrbor Symp. Quonl . Bioi. 63: 319-325. Wet~:>berg

R.A. and Gottesmun ME. 1999. Processive

antder mination. J. BoctP.riol . 181:359 - 367.

Yanofsky C. 2000. Transcription attenuation: Ont:e vi~we
CHAPTE-R

Gene Regulation in Eukaryotes

n eukaryotic cells. expression of a gene can be regulated at all those steps wa saw in bacteria (Chapter 16}, and a few additional ones besides. Mosl striking among the additional steps is splicing. As we saw in C.hapter 13. many eukaTyolic genes come in pioces cmd, ~fter tronscription, Che c-oding region in tho RNA is spliced together to generate the mature message. ln many cases, a given tr;mscript can he spliced in alternative ways to generate differen1 products, and this too can be regulated. But just as in bactt!ria, it is the initiation of transcripHon that is lhe most pervasively regulated step. lndecd. many of the p rinciples we encountered when considering how transcription is regulated in Lhose organisms apply to regulation of tnmscription in cukaryntes as welL Those principles are laid out in the first fe:1w pages of the chapter on prokaryotic gene regulation {Chapter 16) and in the summ&y at thP. end of that chaptor. We urge reeders who have not previously (or recently) read that chapter to lonk at thotw passage5 before continuing with this chapter. We have also already seen that the cuk
I

OUT L I NE

• ConseJVed Mec:hanl5fl1S of TrcmSCilptJC>rli!l RegulatiOn from Yeast to Mammals (p. 531)

• Recruitrneflt of Protem Complexes to Gerles by Eukaryolic AdMttots {p 537)

• Signal tntegrabcn and CufnbJnatonat Control (p. 544)

• Transcripttonal Repressors (p. 549)

Signal Transduction and ltle Control of Transatption
• Gene ~s,lenong" by Modifaca•on uf Histones and DNA (p. 556)

• Eukaryooc Gene Regulation at Steps after Trafl501Ption h·utiation (p. 562)

• RNAs .n Gene Reg\J"'t10n (p. ~7)

529

530

Gene Rewklticn in Eukaryot~

associated with a typical gane. As tn hacterl.a, individual regulatcrs bind short sequences, but in eukaryotes these binding sites are often more numcllJUS and positioned furtlwr from the start site of transcription than they are in bacteria. We call the region at the gene where the transcriptional machinery binds, the promoter; the individual binding sites, regulator binding sites; and the stretch of DNA encompassing the complete roltection of regulator binding sites fo.r a given gAAP. the regulatory sequences. The expansion of regulatory sequences- that is, the increase in the number of binding sites for regulators at a typical gene- is most striking in multicellular organisms such as Drosophila and mammals. This situation reflects the more extensive signal integration found in those

organisms: that is. the tenrlency for more signals to be required to slvitch a given gene on at the right time and place. We saw e.:xamples of signal integmlion in baderia (Chapter 16), but those examples typically involved just two di.fft..rent regulators integrating two signals to contrd a ,gene (glucose and lactose at the lac genes. for example). Yeast have less signal integration than multiceJlula.r organisms- indPecl they are not so rlifferP.Jlt from bacteria in this regard-and their genes have lP.ss extensive regulatory sequences than those of multicellular eukaryotes (Figure 17-t). In multicellular organisms, regulatory sequences can spread thousan ds of nucleolides from the promoter -both upstream and downstr~..am-and c-.an ~ made up of tens or regulator binding sites. Often these hinding sites are grouped in units called enhancers, and a p,iven enhancer binds regulators responsible for activating the geno at a given time and placR Alternative enhancers bind different groups of regulators and control expression of the same gene at different times and p laces in rosponse to d ifferent signals. Having more extensive regulatory sequences means that some regulators hind sitc:>s far from the genes th ey control, in some cases 50 kh OJ more. How can regulators act frnm such a distance? ln bacteria we e.ncountered DNA-binding proteins that communicate over a range of a few kb: }.. repres.<>ors at Of< interacting with those at O..: and NtrC, whid1 can activate tl1e ginA gene from sites pli!ced 1 Kh or more u,pstrearn. In those P.xamples of "'actjon at a distance," the intervening DNA l11ops ou t to accommodate the inte,r-dctiun between the proteins. The same mechan ism explai ns «!Ction at a distance in man y. if not all.

FIG U Rl. 17~ 1 The regWalol'y i!tetnents

ol a bacterial yeast, and human ~ene.. •~ustrated here f5 the increasng complexity of regul.atDf}' sequences from il si'nple bilCtmal gc-11e mntrolled by a l'epn:SSOf to a human geJ'l(t controled by ~ dC.tivators and '~sors:. tl eadl case. a promoter 'is shov\n at the site where transcription a& iniliated. While this is aca.are toT the boctemll c..ase. tn the et.A
regulatOry promoter sequence

...--

-

~ukaryotic cases as well, though in some case...; the distances uver which proteins work is very large and it is not dear how the looping

occurs. Activation at a distance raises another problem. When bound a1 an enhana-.r, there may be several genos within range of an activator, yet a given onhancer typically regulates only one gene. Other regulalory sequences-called insulators or boundary elements-are founrl bet ween enham:ers and some promoters. lllSulators h)ock activation of the promoter by adivators bound ilt the enhancer. These elements, although s till poorJy understood , ensure activators do not work indisaiminately.

CONSERVED MECHANISMS OF TRANSCRIPTIONAL REGULATION FROM YEAST TO MAMMALS

--------------------------------------------------------In this chapter we consider sene regulation in orgcmisms r-dnging from singl(H:elled yeast to mammals. AU these organisms have both tlu• more el~borate transcriptional machinery and the nudeosomes and their modi fiers typical of eukaryotes. So it is not surprising that many of the basic features of gene regulation are the same in all cu.karyotes. A s yeast are the most ~menable to a combin ati on of genetic and hi ochemica~ dissection. much of thA information tiboul how activators and repressors work comes from that organism. lmpoJ1ont for the conclusions drawn from thit' work, when expressed in a mammalian cell. a typicnl yeast activator can stimulate transcription. This is tested using a repo[ter g~ne. The reporter gene consists of binding sites fur tho yeast activator inserted upstream of the promoter of a gene whoso expression Javel i:; readily measured (as we discuss below). We will see that tho typical eukaryotic activat or works in a mann er similar tn the simplf'flt bacterial case: it has separate DNA binding and adivating .rcgimJs, and activates transcription by re<;ruiting protein complexes to specific genes. In conuast, repressors work in o4 variety of ways. some dlfforent from anything we encounter<~ in baclcl'ia. These inr:lurle exumpl£1s of what is called gene silencing, in which modification to regions of chromatin keep gen6S in sometimes large stretches of DNA switched off. Despite having so much in common, not all details of gene regulation are the same in aJl Aukaryotes. Most imporhmtty. os we have mentioned, a typical yeast gene has less extensive regulatory sequences tl1an its multicellular oounterpa.rt. So we m ust look to higher organisms to see h ow the basic mechWlisms of gone resulatio n are exten ded to acoommroate more oomplimted cases of signal integration and comhina1oria) cootrol Regulation at later stages of genH e.xpres· sion-transcript elongation. RNA splicing 8Jld trans lation - arc dealt with later in the chapter.

Activators Have Separate- DNA Binding and Activating Functions In bacteria we saw that a typical activator, such as CAl~ has separate DNA binding and activating functions. We described the genetic demon· stration of this: positive control (ur pc) mutants bind DNA normally, but

532

('.ene Regularion in Eokary(Jles

-ac:Walion d001i:11Fl

FI(;URE

17-2

Gal4bowtdtpitssiteon

The yeast activator Gal4 brlds as a dimer to <~17 bp Site Ofl ONA. The s::NA-binding DNA.

domain ot the protein IS ~e from 1he region of the prote.tn c~irlg lhe aclfvabng regia! (lhe ac1Jvation domam}.

:ire defective in activation. Eukaryotk activators have separate DNA binding and activating regions as well . [ndHed, in (hat case, the two s urfaces are very often on sepa.rale domains of the protein. We take as an example the most studied eukaryotic activator, Gal4 {Figure 17-2). This protein activates transcription of the gc5lactose genes in the yoo.st S. cerevisiue. Those genes, like their bacterial coun· terparts, encode enzymes Tequircd for galactose metabolism. One such gene is called GALl. Caf4 binrls lo four sites located 275 bp upslceam of GAL1 {Figure 17- 3)- When boUild there, in tbe presence of gulacto..c;e, Gal4 activate!' transcription ofthe GALl gene 1,000-fold. The separate DNA binding ltlld activating regions of Gn14 were revealed in two complement ary experiments. In one experiment. expression of a fragment of the GAIA gene-enooding the N·terminal third of the activator- produced a protein that bound ONA normully but did not activate ll'onscriplion. This protein contained the DNAbinding domain bu t lacked the activating region and Wi:!S, therefore, formally comparable to the pc mutants of bacterial activ&:ltors (Figure 17-4a). ln a second expP.rbnent, a hy'h rid gene was constructed that encoded tlu: C-terminal three~qut:Utets of GaJ4 fuserl to the DNA-binding domain of '' bacteJiall'eprc..c;sor protein, LexA. The fusion protem was expressed in yeast loge1hcr with a reporter plasmid benring LexA binding sit{~ upstream of the GAL1 promoter. The fusion protein activatod tran$Criplion of this reporter (Figure 17-4b). This experiment shows that activation is not mediated by DNA binding alone, as it was in one of the alternative mechanisms we encountered in bacteria- activation by MerR lnstead, the DNA-binding domain serves merely to tether the activdting region to the promoter just as in the most common mechanism we saw in bacteria (Chapter 16}. Many other euk.aryotic activators have been examined in similar experiments and whether from yeast. flies, or tnaiJUlUlls, the same st(lry typically holds; DNA-binding dumains and activating regions are separable. ln some cases thoy are evon carried on separate polypeptides: one has a DNA-binding domain. the other an activating region. and they form a complex on DNA An example of this is the herpes virus ac\iwtor VP16. which interacts \.vilh the Octt DNA-bindiog protein found in cells infected by that virus. Another m:ample is the Drosophilo activatm Notch, desoibed in the next chapter. The separable nature of DNA binding and activating regions of eukaryotic activators is the basis for a widely used assay ln deted protein-protein interaction."' (soo (30J( 17-1, The Two Hybrid Assay).

275bp

17-l The rezulcltmy sequencu of the yeast GAU gene. 1k UASc (Upsttei!m Mlvating Sequence for GAL) cOI'Itoos 4 binding sites, each ofvhd'l bnds a Oimt:.,. ofGal4 as shcMn 1n figure 17-2 Though rot shewn ~ !her{? 15 anolhff sstc bew1ea1 these and the CAL 1 gene that btnds a repressor Cillled Mig1, ~ y,oe ,...,11 heal' alxu later 1t1 the cl~ (see ftgt~e 17-20). FIGURE

Con~ Mechanisms o{1h mscriptional fle8tJiolioo from

a Gat4

l 005t to Mnmmols

533

FtGt#RE 11~4 Docnainswap

uperiment Part (a) shows that the DNAbnd.ngdomaln ofeak, Without that protein's aclrva11on dotnalo. can sti11bmd DNA. bU annat activate ~- In aoolt1er ~

DNA-binding domain '\.

Gal4stte

lacZ

...__, oFF Gcll4sile

b

(not shown) 1t1e adivation doiT'.airl. IM!hru the DNA-binding domain, <Jso does not activate tcansc.nption. Part (b) sho.-..ts that &tachtng the ac.tnta1l01i domaifl of Gal4 lO h DNA-btr"dng domain of the bacietial protecn LE~ aeates a hybrid protein that activates transcription of a gene in ye:ast as long as !hat geoe beafs a bird11'18 s te for le!A. f.J
Gal4

activating --...... region

Le>
c!omain- - - -

l exAsite

BoX 17-1 ~Two Hybrid Assay

This assay is used to iderrtity p10teins that interact with each other. lhus, in the case shown in Box 17- 1 Figure 1, activa-tion vt a feporter gene depends on the fact that protein A interacts with pro1ein B (even though those proteins need not themselves nDffn<311y have a role in IIanscnptional octivaoon). The assay is predicated on the finding, discussed in the tel
that the DNA-binding domain and activating region can be on as long as those protetns .nteraa, and the actNating region is thereby tetllered to dle DNA near the gene to be activated. Practkalty, the assay is canied out as follows. The gene encoding protein A is fused to a DNA fragITI€flt encoding the DNA-binding domain of Gal4. The gene fof a second protern (B) tS fused to il fragment encoding an activating region. Neither pmt~ alone, when expressed in a ye3St: cell, activates the reporter gene Glfrying Gal4 binding sites (as shown in the firSI two lines of the figure). \Mlen both n~rid genes are expressed together in a yeDst eel, however, the interaction betvveen proteins A and B generates a COOlplete CKtivator, and the reporter is expressed. as shown in the sep01ate protei~

bottom line of the figure.. In a widely used elaboration of this Simple assay, the two hybnd assay 13 erl'f)Joyed to screen a library of candidates to find any protein that wifl interact With a known starting protein. So flOW, protein A in the figure would be the starting protein (called the "bait"), whiCe protem 8 (the '"prey'') represents one of many aiWrna lives encoded by the library (see Chapl€f 20 for a description of how fibranes ~re made). Yeast eels. are transfected with the construct encoding protem A fused to the DNA-binding domain, together ~th the library encoding many unmown proteins fused to the «tivating region ~us. each tJ'ansfected yeast ceU contains ptotan A tethered to DNA and cne or another oiternative protejn B fused to a n activating reg1011. Any cell cootaintng a combtnation of A and B &hat 1nterects wal activate the reporter gene. SUCh a cetl will form a cdony that can be identified by plating on suitable indicator medium. Typtcaly the reporter gene would be locZ. and posttive cokmies (those comprising eels e>
534

r-ene Regukltion in Eukaryotes

Bok 11-1 (Continued) - - activating regivn gene

-----"r _

_ _,_J_ _ ___..I.... . _-:1_

NO rranscr~tion

DNA-bindWlg sile

A

A

DNA-!Jin
gene ........_:):~~~~-..... -_.... _·~==--=--

domam-...__

_

(..,;......_........,_ _)_!_

A

t

NO transcnpbon

A

Y'

lOOM '

(~}----~~ ~ ~)-----~[

~~n

ONA-tlitldirlg si1e

lO X

17-1 F I& URE 1

Eukaryotic Regulators Use a Range of DNA~Binding Dornajns, but DNA Recognition Involves the Same Principles as Found in Bacteria The experiments described above show that a bacterial DNA -binding domain can function mplar..e of the DNA-binding domain of a eukruyotic activator. That result sugg~ts tht:re is no fundamental difforonce in the ways DNA-binding proteins from these organisms recognize their sites. Recall from lhe previous chapter that most bacterial regulators bind as dimers to DNA target sequences whicb are twofold rotationally symmetric ; each monomor i nserts an «X helix into the major groove of the DNA ove r one-half o f the site and detects lhe edges of base paiJS

found there. Binding typically requires no significant alteration in the structure of either the protein or the DNA. The vast majority of bacter~ ial regulaloTy proteins use the so-called helix-tum-helix motif. This motif, as we saw, consists of two a helices separated by a short turn. One helix {tho recognition helix) fits in the major groove of the DNA and recognizes specific base p ain. The other helil< makes contacts with the DNA backbone. positioning the recognition helix properly and increasing the strength uf binding (see Figure 5-20). The same basic principles of DNA recogn ition are used i11 mosl eukaryolic cases. dP.spite variations in detaiL Thus. proteins ofhm bind as dimers and recogniZE! specific DNA sequences using an u helix inserted into the major groove. One class of eukaryotic regulatory protein presents the recognition helix as pan of a stwcture very like the helix-turn-helix dornain; others present the recognition helix within quite different domain struct'ures. In a \l'ariation we did

C.QilUli'VM f'AE>d!ofiisms of 1hmscnptionol Regulation from Y6'Q.~ to MammaLs

535

not soo in prok.aryot cs, several of lh P. regu la tory protei ns we encou n ter in eukaryotes hind DNA as heterodimerst tm d in som e cases even as m onomers (tho ugh often only when binding cooperativel y with other protei ns). Heterodimers ex tend the range o f ONAh inding specific it ies available : when each monomer hi:is a d ifferent DNA-b inding s pecificity. the site recognized by the hcterodimer is differen t from that recognized hy either homodimer. HerP is a hrim sw'Vey of some eukaryotic DNA-binding doma ins. llomeodomain Proteins. The homoodomain is a class of helix-tumhelix DNA-binding domain and recognizes DNA in essen tially the same way as those hacteri1:1l p roteins (Figure 17-5). Hom eodomains from different proteins are strucluralty very similar: not only is the recognition helix sim ilar, the s urrounding protein structure rhat presents that ~l ix to the DNA is similar too. l n contrast, as we suw 1n the previous chapter, the detailed structures of helix-tum-helix domains vary to a weater exlent. Homeodomain proteins arc found in aU eukaryotes. They were d iscovered in Drosophila wher:e they control many basic dflve topffiflntaJ pT\llfclllJS, jus\. as they d o in higher cukaryote:r; as welf, and we wiJl cons1de::r their func tions in that w.gard in Chapters 16 and 19. Homeodornl!in prolr.ins are also found in yeast- some of the malingtypc control genes we discuss below encode homeodomt:Si n p roteins. Indeed, it is the structure of one of those that is shown in Figure 1 7-5. Many horueodoma..in proteins bind DNA as hetf!rodirners. Zinc Conta ining DNA-Binding Domains. Thare are various different forms of DNA-bin ding domain that incorpomte a zinc atom(s). Those include the classically defined zinc fif18et' p roteins (such as the general transcription factor TFlliA (Chap ter 1 2) that is in volved in the expression of a ribosomal RNA gene) and the related zinc cluster d omain found in the yeast actil>·ator Gal4. In these cases, the Zn atom interacts with Cys and His residues and sttrves a structurdl mle essenlial for integrity of the DNA-binding domain (Figure 1 7..£}. The DNA is again recognized by an u helix inserted into the maier groove. Some proteins oontain two or more 7Jnc finger d omains linked end to and. Each finger insorts an u holi ~ into tho major groove, exte ndin~- wit h each

c

recognttion h<>b

/

FIG U RIE 17-5 ONA n!Cognition by a

homeodomilin. The horneodort"an conslStS of thee a helcces, d IMldl two (helices 'J and 3 fl lhf. fltu'e) focm lhe slruc:ttJe r~ the hel&x~ll.m-hckx moM (<."OO"l>ft tru fi&~.~e\Niltl F'lg(JI"E- 16-11, for ex~). Thus, heb j IS the l'eCogl'lioon heik and as stoMi, tt 6 i1sertcd in1n the mara grooye ot ONA. Amino ac~ ~ a1~ 1ts OlJ\er edg!? mcr.e specif1c contacts With basf. ~ tn the case shcMotl. 1hc yeast a2 tr~ repressa: i!fl arm EtrleOOng from he!« 1 ~r..akes addttonat contacts \oWh basf>. pcm 111 ~ mi(I(J" giOOie. (Sot.Jt;e· AdapCed from ~gt.>J C et ~ 1991. Ce-11 67· 5 17 - 528. CcpyriEht ~I 1991. \Jsed wfl"1 ~from Elsei.;B'.)

FIGURE ) 7-6 Zinc; fingK ~lit. The a helle a~ the left of the stn.Jct\Jre iS the re<egrvtion ~Oc.. and 1t lS pteSE>nted 10 the- DNA by the ~ 4-.eet on thE: rittt The zjrc 15 ccx:lftinated by the two His re:5ldues 11 lhe " heb>. and two Cys resn.es W1 me p sheer as ~- l'ftiS ~ stabbes the stru:tu"e and IS esset1tral tor DNA bt~. (SOli"CC;. ~ed from LeeM. S. etal. 1989. Sdenc:.e

254: 635 - 6l7.)

Qmf! Regulation in Eul
additional finger-the length of the DNA sequence recognized, and thus the affinity of binding. There are other DNA-binding domain~ that use zinc. ln thol'e cases, the Zn is coordiruited by four Cys residues, and stabilizes a rather different DNA recognition motif- one Iesembling a helix-tum-helix. An example of this is found in the mammalian regulatory protein, the glucorticoid receptor, which regulates genes in response to certain hoTmones. Leucin~

~ aca1 R E

17-7 Leurine zipper bound to OffA. 1Wo farge u tteices. ooe frorTl eadl ~ form both the t:llrnerizattrn CJnd DNAbanding dCIIilM at different .sediclrls along theu lergfi1. lh.Js, as shoiNn. toward the top the two heteeS 1n1er<1ct to f oon a t:.ailed-mJ that holds ltle IT1Cn:JITiefS toget:he!'; futhe£ down, the helices separate enou~ to ernbraa= the DNA, irlsening into the map groove on ~te ~ of the 0\lA·~IX. 000: agan, specit\cJty as p o111ded by contacts mtJde llefvlleerl .amno side chains on the u flehces and the edge of base pans in the major groO\Ie. An e~ cl this is fcund in the yeeS1Ircli\SCI1;>!D'Ial ildr.l&a. GCN4 (f'ifpe 5- 15) (Soorre: Adapted tfOfT1 ~ T,G. et al. 1992. Cel/71 1713. Ccpyright ~ 1992. Used 1Mth pmrissioo from E~.)

aoo

Zipper Motif. This motif combines dimerization and DNAbinding surfaces within a single structural unit. As shown in Figure 17-7, two long a helices form a pincer-like structure that grips the DNA, with each a heJix insetting into the major groove h alf a turn apart. Dimerization is mediated by another region within those same n helices: in this region they form a short stretch of coiled coil, wherein the two helices are he ld together by hydrophobic interactions between appropriately-spaced leucim~ (or othet hydrophobic) residues. We discussed this protein-protein interaction in more detail in Chapter 5 {:Figure 5·15). Leucine-zipper-containing proteins often form betemdimers as welJ as homodimers. That is aJso true of our final category. the so-called helix-loop-helix prot&ins (HJ..H proteins).

Helix-Loop-Helix Proteins. As in the example of the leucine zipper, an extended a helical region from each of two monomers inserts into the major groove of the DNA. As shown in Figure 17-8. the dimer.i.zation surface is formoo from two helical regions: the first is pari of the same helix involved in ONA recognition; the other is a shorter o helix. These two helices are separated by a flexible loop that aJiows them to pack together (and gives the motif its name). Leucine zipper and HLH proteins are often c~llcd basH: zipper and basic lfLH proteins: thls is because the yegion of the a he lix that binds DNA contains basic amino acid residues.

Activating Regions Are Not Well#Defined Stru~tures ln contrast to DNA-binding domains, activating regions do nnt always

have weJI-defined structures. They have been shown to form helical structures when interacting with their targets within the lr'dnscriptionaJ machinery, but it is believed those s tructures am "induced" by that bind-

17--8 Helix· loop-helix motif. f111t115 case. w~ again see a long u ~ wwolved en balh DNA rerngllllDl and m CO!Tihnation with fiGURE

a second. shorter; a helix am~ {Sol.c'c.e: ~d f10m MaP C., Rotld M A, \Nentiaub H.,

Cl'ld PitJo C. 1994 Cryst<j structllle of MyoO bHl H dcm~ (.()t1"'fJex. PeJspectives en t>NA retognmoo and m'fllteabons for tJimsalpt:tM octivabon Celf77· 451. f-J801e 2A. ~L - \994. Used with pemassion from c:lsevter.)

ing. As we shall see, the lack of defined structure is consistent with the idea th~t activating regions are adhesive surface.s capable uf interac1ing with sevP-ral other protein surfaC$. Instead of being characterized by structure, therefore, activating regions are grouped on the basis of amino acid content The activating region of Gal4, for example, i..r; called an "acidic" activating region, reflecting a preponderance of acidic amino acids. The importance of fhp.se acidic residtJes is highlighted by mutations that mcrease the activator's potency: such mutations invariably increase the overall acidity (negative charge} of the activating region. But despite this. the activating region contains equally critical hydrophobic residues. Many other activators have acidic activating regions like Ga14. Although these show little sequence similarity, they retain the characteristir. pattern of acidic and hydrophobic residues. It is believed that activating regions consist of reiterated scnall uni te;, eac·h of which bas a weak activating capacity on its own. Each unit is a short sequence of amino acids. The g«-'.ater the number of units, and

&Cltlitment of Protein Ccmp/exes

the mnre acidir. r.ach unit, the stronger the resulting actjvating region. This is consistent \'\ri.lh the idea that activating regions lack an overall structure and act simply as rather indiscriminate "sticky'' surfaces. (To understand this reasoning, imagine instead that an activating region folded into a precise, stable three-dimensional stnu:: ture-comparabJe to, for example, a UNA-binding domaia Under those circwnstances, fragments of that domain would not he expected to ~tain a fraction of the DNA-bittding activit)' of the intact domain-rather, the entire domain i~ neE'
RECRUJTMENT OF PROTEIN COMPLEXES TO GENES BY EUKARYOTIC ACfiVATORS Activators Recruit the Transcriptional Machinery to the Gene We saw in bacteria that, in the most common c.ase, an activator stimulates tmn!;cription of a gene by binding to DNA with one surface. and with another, interacting with RNA polymerase and recruiting t~ enzymP to that gene {see Chapter 16, Figure 16-1). Eukaryotic oct ivators also work this way, but rarely, if ever, through a dir~d interaction between the activator an d RNA polymerase. Instead, the activator recruits polymerase indirectly in two ways. First. the activator can interact with parts of the transcription machinery other than polymerase, m1d, by recruiting them. recruit polymerase as well. Second, activators can recruit nucleosome modifiers that alter chromatin in the vicinity of a gene and thereby help polymerase bind. ln many cases, a given activator can work in both ways. We first consider recruitnum t of the transcriptional machinery. The eukasyotic transcriptiona l machinery contains n ummuli.S proteins in addition to RNA pofymern.w. as we saw in Chapter 12. Many of these proteins come in preformed complexes such as I he Mediator and the TFITD comple-x (see Table 12-2 and Figure 12- t 6 in Olapter 12}. Adivators interact with one or more of these complexes and recruit them to the gene {Figure 17-9). Other oornponents that are not directly recruited by the activator, bind cooperatively with those that a.ra recruited. This means t hat. despite the large number of components needed to transcribe a gene, activators may have to Tecruit only a relatively few entities. Indeed. according to one view. most of the machinery comes to the gene in a single, very large complex called the holoenzyme, which contains the mediator, RNA polymerase. and some of the general transc:ription factors (as we described in Chapter 12). This leaves just a couple of other <.:ompl~xes to arrive separately, such as TFJJD and TFDE. These IaUer components may be recruited themselves by activators or bind cooperatively with holoenzyme.

TATA

---

--·

Fl c; u R £ 17-9 ~atiq, of transcriptiol\ initiation in NafYOles by reauitment of the b'aMoiption mac:hinefy. A 5t11Sie adivatof is shi:Jv.in reauting twO possible rarget romplexes: the Mediator; and through that, RNA polymernse II; and also the general trar\S01(:lbon factor lfliO. Other gerter.JI tra11501ptlon factors are reauited $pat of the Mc-diatcr/Pol U comptex (holoenzyme); separately, (through direct reautlment by th€ octtvalor); or btnd spontaneously in the pest".rlCe of the recruited cwpcoeots. ltlese Me not ShCM'fl here. In reatity. th1s. reotitrnent would usualty be mediated by fTlOfe thM one activator bound upst~earn of the gene.

- - - -- -

17-10 Activatk)n of transcription through dired tethering ftGUU

of mediatQJ to DNA. ThiS is <Ji exarnpe of ar activillOf bypass experiment. as desatbed ~ ~· 16. 8())( t&2. 1t11t'603Se. the GAL 1 gene IS activated. in the absence of its lJSllal ael)v"ottlf Gal4, by the fUSion ot tht DNA-bnang dornan ci lexA to a component of the Medil'ltc.- Complex (Gall1-see Chapter 12, Rgure 12· 16) . ACilYatlon depends on L.e..A DNA-btndlog sites being lllSef1ed 4JS(re
Whatever the precise details. an actiVHtor promotes formation of the entire pre-initiation l.:omplex by l'ecrui ti.ng one or more of the constituents to the promoter. Many proteins in the tr.mscr.iptional machinery have been shown to bind to activating regions in vitro. For example. a typical acidic activating region can interact with components nf the mediator and w ith subunits ofTFIID. Recruitment can be visualized using the tedmiqup, caJied chromatin im.mWloprecipitalion (ChiP), described in Box 17-2, Chromatin Immunop.recipitation. This technique reveals when a given protein binds to a defined region of DNA within a cell. At most genes. the transcriptional machlnery appears at the promotei only upon activation of the gene. That is, the machinery is not prebound. and so activation is not typically mediated by an alternative mechanism we encountered in rare cases in bacteria: the aUosteric modification of prebound polymerase. In bactAria we saw that genes activated by recruitment (such as the lac genes) can be activated in so-called actjvator bypass experiments (Box 16-2). In such a.n experiment, activation is observed when RNA polymerase is recruited to the promoter withotJt using the na tural adivator-polymer-ase interaction. Similar experiments work in yeast Thus, the GALl gene {normally activated by CaJ4) can be activated equally well by a fusion protein containing the DNA-binding domain of the bacterial protein LexA fused directly to a component of the Mediator Complex (Figure 1 7-10}. lt is important to note that these experiments do Tlot exclude t.1-1P possibility that at least some activators not only recruit parts of the transcriptional machinery, they also induce allosteric changes in them, Such changes might stimulate the efficiency of transcription initiatinn. Nevertheless. the recruitment of the machinery to one or another gene is the basis of specificity; that is, which gene is activated depeuds on which gene has the machinery recruited to it. Also, the sur.cess of the activator bypass experiments suggests that any

Rti'Cll.litment of Protein Compl exes to Ce~ by EJJkoryotic A c:livotors

-- ----

539

--------------------------------------------------------------

BGll 17-2 Cbrornatin lmmllrloprecipitatioo

Nthough this teWef1tJ and rootinely used. it does have ~ of ~ the investigator needs to be aware. F'arst, the reso\Jtion d the method is irnded. Lt is not possible 1D shotv that a protein is OOund to a spe<:ifte site. merely that it is lxuld to a srte witlin a gwen 200-300bp fragment llus, it is ade<;ulf:e to show rhat a regulatory protein is bound upstream of one rather- than another gene. but it does not show you exactly VIA1ere upstream of the gene the protetn IS bound Second,
This technique, often just called ChiP. enables an investigator to identify vvhere a given protein is bound in the genome of a living cell ~s. for ~. it is possllle to determine ~ther componeniS of the trans~tional madrinery are bound to a given prorooter ill a gwen time It is atso possible to determine \'Vhether a specifte regwtory protein is boond at a given gene. and so on. In outtine, the ~ ts perfooned as folows: fomaldehyde is added to eels, cross-linb"lg to the DNA f.Yly proteins that are bound to DNA at that momert. lhe eels are then lysed and the DNA is broken 1r1to smal fragmerts (200- 300 bp each). Using an antlbody specific for the protein of interest. the fragrr.ents of OOA attadled to that protein can be sep«ated from the majority of the DNA ill the cell lhe ooss-inmg is then reversed and the protein removed. To detetmine ~ a particu~ region of DNA is bound by the prol€11\ PCR is perforrred (Chapts 20) using primers designed to amplify that P<J1)ru1ar region (a pronoter. for example). If the prottYI had indeed been bound there. DNAwl be presert and get amplified. As a crntrol, PCR ~ targeting another region of OOA {O(Ie to lllthich the protein is mown not to bind) are used; in that case, no DNA should be am~ified (Box 17-2 Figure l). proteins cross-linl\ed to ONA fr'agmeflts

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fiGURE

flVf!llts that might exif'l are nnt essential fnr s ucr.ns!OOfHJ genP. expression il1 these cases.

allnstP.ric

Activators also Recruit Nucleosome Modifiers that Help the Transcription Machinery Bind at the Promoter In addition to direct recruitment of the transcriptional machinery, recruibnent of nucleosome modifiers can help activate- a gene inacoos.c;ibly packaged within duomalin. As discussed in Chapter 7 fTable 7·7), nudeosome modifier~ come in two types: those that add chemical groups to the tails of histones. surh as histone acetyl lransfet:ases (HA1SJ, which add acetyl groups; and those that remodel the nuclRosomes. such as the ATP-dependent activity of SWIISNF. How do these 1nodifications help activate a gene'? ThP.re are two basic models to explain how changes itt nudeosmnes can help the transcriptional machinery bind at the promoter (Figure 17-11). First, remodeling, and certain modifications, can uncover DNAbinding sites that would otherwise remain inaccessible within the nucle.nc;nme. For example, by increasing the mobility of nuclaoso mes, remoclelern free up bindinf.?, s ites for regulator~ and for the transcription machinery. Similarly, the addition of acetyl groups to hlstone tails alters the interactions betwoon those tails and ad;acent nucleosomes. This modification is also ool ievcd to ·• loose.n" c-hromatin structure, fmeing up sites (sec Cru~pter 7 fnr a more r.omplete description). But adoing acetyl groups also helps binding of the transcriptionaJ machinery (aud other proteins) in another way: it creates specific binding sites on nucleosomes for proteins bearing so-called bromodomains (Figure 7-39). One component of the TFIID complex bears bromodornains, and so binds to acetyJated nucleosomes better than to UJlacety· Jatcrl ones. Thus, a gene rearing acetylated n ucleosomes at its promo ter wiJJ likely hRve a higher affinity for the transcriptional machinery than one witl1 unaretylated nucleosomes. Whic.h parts of the transcription machinm:y, and which nucleosome m odifit'rs, are required tu transcribe a given gene? And which components are directly recruited by a given acti\l·ator working at a given gene? The a nswers to these questions are nol known in most cases, but some components of the trc1nscriptional machinery are more stringently required at some genes than at othars, and the same applies to nucleosome modifiers as well. These differences are in m.nny ('.8ses not absolute. Thus, whiJe all genes absnJutely Teqllire RNA polymerase itself, a given gene may depend on another particular component of the transcription machirlery. or a nucleosome modifier. or it may not. ln some cases, a component of the transcription machinery might be required partially (lbat is, in the ab!:lence of that component. activation is reduced but not eliminated). ln addition , what is needed 1o adivate n given gene am vary d epending on circumstances. such as the stage of the cell cycle. f or example. Gal4 usua lly activates the GAL1 gene efficiently in the absena:? o f a histone acetylase. During mitosis, however-when chromatin is mofi' C()ndeosed {Chapter 7) -activation is eliminated unless that acetylase is rocroitcd to the gene. Action at a Distance: Loops and Insulators Many eul<aryotic activators-particularly in higher eukaryotes - work from a distance. Thus, in a manunaJian cell, for example, enhancers r.an be found several tens or even hundreds ofkb upstream (or downstream)

R(ICnlitmenl of Protein C..omplexes to Genes by /ir,Jknty(Jlic 1\ctivofor$

ctromatin mnodeling c omplex

histooe acetylase

mstone acetytallon

+

transcription mad'linery binds promoter

f 1G u R E 17-1 f Local alterations in chromatin structure mre\tlid'l alters the structure of nudeosomes artUJd the pmmoter, renderu-tg it accessible a00 capable of binding lhe tla'lScr1ption machinery. Clri the left-hand Side, the acbvator IS sho.m fe<:rlliling a htstme acetylase. That eruvme adds acetyl groups w reslt:l.E Witt.'~ the htstor1e latls (~wn as blue flags)_This alters the pock1ng of the nudeosomes somewhat, and also creates btrlding stes ra profetns cai'J)'Iig the apprq:xiate recogf1itia1 domains (bromodomains; fig\Kes 7-39 and 7-40). Together these effects again ~•ow hindiflg of the transa~on ml!Chmay to the promoter.

of the genes they control. We saw in bacteria that proteins hound to sepal'8ted sites o n DNA can nevertheless interact - a reaction acconlmc>dated by DNA looping. But in those cases. we were considering proteins binding only a few hundred base pairs apart. Under that condition. lhe proteins are bound sufficiently close to each other that their chance of interacting is much higher on DNA than off it. Once the s ites lo which they bind are separated by mnre than a few kb, this advanta~ ic; lost. Mechanisms exist to h elp corrununication between distanUy-bound proteins. Recall, from bacteria, one way this can be done. The "architectural " protein 1HF binds to sites on UNA and ben ds it. At some genes controlled by NtrC, IHF sites are found between the activatorbinding sites and the promoter. By bellding the DNA, n lF helps the DNA-bound activator reach RNA p olymerase at the promoter (see Chapter 16, Figure 16-4). Various models have been proposed to expfain how proteins binding in bPtween enhancers and promoters might help activation in the cell~ of higher eukaryotes. Jn Drosnphile), the cvt gem~ i.
542

(if!ne Regulpfillfl in Euknryotes

iliA strer1gth of activation . How Chip works is still not clem. but one modd is that it bind'S to muttiple DNA sites between the et•hancer <:m d the promoter. and, by intnracting with itsct£. forms multiple miniloops in tbe intervening DNA, the cumulative effect of which is to bring the promoter and enhancer into closer proximity. There are other models. In eukaryotes, the DNA is wrapped in nucleosomes as we have seen. and the histones within those nucleosomes are subject to various modifications that affoct their di')position and compactness. Thus, s ite::; separated by many base pairs may not, in effect, be as far apart in the cell as might have been thought~ Also, chromatin may in some places form special structures that actively bring enhancers and promoters closer together. If an enhancer activates a specific gene 50 kb away. what stops it from activating other genes whose promoters are within that range? Specific elements a:tlled insulators etmtrol the actions of activators. When placed between an enhancer and a promoter, an insulator inhibits activation of the gene Ly that enhancer. As sho"l'ffi in Figure 17·12, the insulator does not inhibit activation of that same gene by a cliffercnt enhancer. one placed downstream of thP. pnxnoter; nor rlnP.S it inhibit the original activator f.rnm working on a different gene. Thus. the proteins that bind insulators do not actively repress the promoter. nor do they inhibit the activities of the activators. R~ther. they block C'..ommunir.ation between the two. ln other a,;say:;, immlators a lso seem able to inhibit the spread of c hromatin modifications. As we have soon, the modification state of JocaJ chromatin influences whether gem•,.s are expressed or not. We w ill see belo w that pro pagation of certain reprt>.ssing histont> modifications over stretches of chromatin lies at the heart o f a phenomenon c a lled transcriptional silencing. Silencing is a specialized form of repression that can spread along c hromatin. switching off multiple genes without the need for each to bear bl nding sites for sper.ific .

--

17-12

tnsulators block ac1Wation by el1hancen.. tn part (a) IS

fiGUR£

shovm a promo1e ~ivated by activators bOLfld to (lfl In w (b), an tnst;la~ IS placed betweef1 the enhancer and the pro meter. When bolJlld by ~opi•ate ios\Aator· btndlng proteins, ad.lvatlOn ot the PfOmotet by tile enhanc.et IS blocl<ed. despte activators tJind. lfl& to the enhancer. Parts (c) and (d) show tllat nether the acti\.'Ctors at the el'lhoocer. fl(J' me promoter, are lllactl\fated by the action ot the insulatoc 1hus 111 (c), the ac.trvatOf can activate .aoother P'~ l'le
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fttJCniifment of ~t'in Complexes to Genes by Eukaryotic: J\ctiYOtcrs

543

wpressors. Ins ul ator eiP.Illflnts c
Appropriate Regulation of Some Groups of Genes Requires Locus Control Regions The human globin g£lnes are expressed in red blood CE-lls of adults and in various cells in the lineage that forms red blood celts during development. There are five different globin genes in humans {Figure t 7J3a}. Although clustered. these genes are not all expressed at the same time. Rather. the different gem:s are expressed al different stages of development starting with £. then thP y gene~. followfld by J3 and ~ How is their express·i on regulated? Each gene has its own Collection of reguJatoty sites needed to switch that gene on at the right time during development and in the proper tissues. Thuos the f3-globin gene (which is expressed in adult bone marrow} has two enhancers: one upstroam of the p romoter, the othe.r downstream. Only in adult bone marrow are the correct reguJators all active and present in appropriate concentrations to hind these enhancers. But more than this is required to switch on these genes in the correct order. A group of rcguJatory elements colJer.tiveJy called the locus control region, or LCR. is found 30-50 kb upstream of the whole duster of globin genes. How the LCR works i.-s still undear. hut it binds regulatory proteins that cause the chromatin :.;tructure around the whole globin gene cluster to ''open up,·· allowing access to the array of n Jgulators that control expression of the individual genes in a dcfinoo order.

a

lOcus control region {LCR} £

FICtJRE 17-13 Regulation bylCRs.

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ClUster of gfObln genes

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The hl.xt'-iln gfobrl g61e5, and the lCR that ensures their crdered expreSS
ThP. LCR is made \IP of multiplc-sequenC€ P.lements. Some of these have the properties of enhancers: that is. if those seq uences are attachfld experimentaiJy upsl«'.am of a reporter gene, they can activate that gene. Othe r pacts of the LCR act more like insulator elements and still othe.rs seem to have properties of promoters. This diversity of elements has led to numerous models for how LCRs might work The simplest is that regulatory proteins bind to the LCR and recruit chrnmatin modifying complexes to the region. Reoent experiments h
SIGNAL INTEGRATION Af'D COMBINATORIAL CON TROL Activators Work Togethe.- Syt"~ergistkally to Integrate Signals In bacteria we saw examples of signal integration in gene regulation. Recall. for examp le, that the lac genes of E. coli are effici ently expressed only when both Jactose is present and gJucose absent. The two signal!ti are r:oan cnunicated to the gn:n~; thmugh ~eparflte regulators-one an activator and tbe other a rP.pressor. In muJticellular organisms s ignal integration is u sed extensively. ln some cases nume rous s ignals are required t o switch a gene on. But just as in bacteria , each s igna l is tmns mittod to the ge ne by R ,;eparate regul ator, so at many genes ml1ltipl e activators must work together to switc.:h the gane nn. When mu.ltiple activators work t<1gether, they rlo so synergistically. That is. the effect of, say. two activators working together is greater (usually much greater) than the sum of each of them worki ng alone. Synergy can result from multiple activators recruiting a single component of the transcriptionaJ machinery; multiple actlvatofS each recruiting a different oomponent; or multiple acti vatnrs helping each other bind to their tiites upstream of the gene tbey control. We briefly cnn~ider a ll three s trategies before givi.ug exru:npJes. Two activators can recruit a single complex- for exampla, the Mediator- by touchlng ruffcrent parts of it. The combinf'ld bindjng energy w ill have an e>:ponential effect on recruitment (see Chapte r 3. Table 3-1 1. In cases where the activators recruit different complexes (neither of which would bind efficiently without help), synergy i:r5 even easiP.r to picture.

Signollntegrotion ond Ccmbinatoriol Control

545

Synergy can a lso result from activators helping each other bind u nder conditions where the binding o f one de pends on binding of the othe r. This cooperativily can be of the type we encountered in bacteria, whereby the two activators touch each other when they bind their site.s on DNA. But it can work. in other ways as weJl: one activator can recruit something that helps the second activator bind. Figure 1 7-1 4 illustrates the different ways activators help each other bind DNA. 111~ indude ..classical" cooperative binding; rccn1itme nt of a morli· fier by one adivotnr to he lp a secon d bind; a nd binding o! one activa-

tor to nucJeosomaJ ONA uncovering the binding site for another. Synetgy is critical for signal integration by activators. Consider a gene whose product is only needed when two signals are received. Each signal is communicated to lhe gene by a separate activator. The gene must be efficiently expressed when both activators are present but be relatively impervious to t he action of either activator alone. a A

•

Jo

b

r

X A io --A

X

-1

d

I site A

6feeB

i)

f i GURE 17-14 CooperaWe binding of adivators. Hereare 5howo four W~ys that the tlinljng oJ one pro«..ofl to a site co Df\JA can he~ the binding c1 <1nother to a nearoy ~ In part (a) lS shown co~ binding through direct tnteractsoo betwea1 the two poteins, as we saw for ). represscr tn Olapter 16, and IMII see betweEn mooy tegulators in etJkaryotes as well. In (b) a .simile. effect is achieved by both proteins 1nt6Ctcting 1Mth i' CCifTW'I1CI1 thrd protein Parts (c) oo6 (d) show meet dfE.'Ct$ 111 v..hKh binding of one prolan to ItS site oo DNA within nudeosomes t $ blncf~ng of a second proten. In (c) the ftTSt protein recuts a nuc::Jeosome remodeller v.ho5e actioo reveals a bmding Site for a second pr~. In part (d) the bending of the ftrst protein to its site OCCU"s because that Site is on the DNA j\Jst where t exits the ntJdeosome. Ey birlding there. it unwinds the DNA from the 11udeosorre a little, reveaing the ooding site for the second potein. Each of these mechCII"'tsms can ~ tnv one regulator UWl tlelp others bind. or,ll16eed, hew an actJvatc.r can hap the trallsc:rfJtioo mac'lltnely bend lo d promoter:

Gene Regulot.ion in Eukaryoles

Signal Integration: the HO Gene Is Controlled bv Two Regulators; One Recruits NudeosO(ne Modifiers and the Other Recruits Mediator The yeast S. cerevisioe divide::; by budding. That is, ins1ead of di\>i ding to produce two identical daughter ceJJs, the so-called mother cell buds to prod uc.e a daughter cell. We will focus here on the expres.c;iun of a gene called HO. (We need not concern ourselves with lhe function uf this geno, w hich is described in Chaper 11.) 1'he HO g()ne is expre~sed only ifl mother r;ells and only at a certain point in the cell cycle. These two conditions are communicated to the gene through two activators: S WI5 and SBF. SWI5 binds lo multiple sites some distance from the gene, the nearest being more than 1 kb from the promoter (Figure 17-15). SBF also binds multiple sites, but these are located closer to the promoter. Why does expression of the gene depend on both activators? SBF (which is active only at the correct stage of the cell cycle) cannu1 Lind its s ites unaided; their d isposition within chromatin prohibits it. SW15 (which acts only 1n the mother cell) can bi nd t o its sites unaided but cannot, from that distance~ activate th~ HO gene (remember that in yeast. activator.:: do not work over long d istances). SWI5 can. how{lver. recn1it nucleosome modifiers (a histone acetyl trans ferase followed by the remodelling enzyme S\-\.'1/SNF). These act on nucleosomes over the SBF sites. Thut~. if both acti vators arc prosen1 and al:tive, the action of SWI5 en ables SBF to bind, and that activator. in tum. r-ecruits the trahscriptional machinery {by directly binc.H ng Mediator} and activates expression of the gene.

Signal lntegrntion: Cooperative Binding of Activators at the Human ll-lnterferon Gene The human 13-intcrferon gene is activated in cells upon viral infoction . Infection triggers three activators: NFKB, lRF, and Jun/ATF. These prot~ins bind cooperatively to sites adjaoont to one another wH'hin an tmhancoc located about 1 kb upstream of the promoter. The structuw. formed "by these regulators bound to the enh;mc:er is called an enha.ncemitlme (Figure 17-16). The binding of the activators is cooperative for two reasons. First, the activators interact with each other. Second. an additional protein,

FIG U R l 17.. 15 Control of the HO ge11e.. SWI5 can bind ats 9tes within chromatin unaided, but SBF cannot. Remodeltes-s and

histone acet}43ses ~d by SWI~ alter rn.Jdeosanes over the Sl3F Sites, allow4r.g that activator to bind near the promoter ~nd activate the ger~e 1r1 the figure, for sifl'Pit:il't 1he oudeosomes ate not drawn. (Source: Adapted from PtashneM and Gann A 2002. Genes & Slgnds, p. 95, Ftg 2-18. © Cold Spring Harbor l abofatory Press.)

SWI5 \

HO

SBF

SElF site

---~ ON

Signo/ Integration and Combinutorial Control

F I Ct U R E

547

17-16 The humi111 ~-interferon

enhanceosome. Cooperatrve bin
enhancer

protein Hv\G-1, activates the j3-fnterfezon gene.

( INF-f3 gene

caUed HMG-1. binds within the enhancer n.nd aids binding of the activators by bending the DNA in a way that facilitates the interactions among them . HMC-1, which is consititutiVAiy active in t he ceU, 1hus has an architectural roltj in the process. Thestj layers uf cuuperativity ensure tight integration of signals: for the gene to be activated. an three activators and HMG-1 must be present. Once formed, activators ~"'ithin the anhanceosome contact 1he transcriptional machinery and act ivate the gene.

Combinatorial Control Lies at the Heart of the Complexity and Diversity of Eukaryotes We encountered simple cases of combinatorial control itt bar:teria. For example, CAP is involved in regulating many genes. in collaboration with other regulaton;. At the Joe genes it works with the Lac repressor; at the gal genes w ith the Cal repressor. There is extensive combinatorial control it1 euka:ryotes. We first consider a generic case (Figure 17-17). Gtme A is controUed by four signals U. 2, 3, and 4), each working through a separate a(:tivator {activators 1, 2, 3 , and 4). Gene B is controlled by three signaJs {3. 5, and 6). workjng through activawrs 3, 5, and 6. Note that there is one signal in common between these two cases. and the activator 1hrough which that signal works is the same at both genes. ln compJe.x multiceUular organism.<>. such as Drosophila and h umans. combiniitOriRl control involves many more regulators and genes than shown in this k ind of example; ancl, of coUJSe, repres:;ors as well as activators can be involved . How i:; it that the regulators can intermix so promiscuously?

Ccne Regul ation in EuJ.:t;llJ'otes

FIGU RE 17-17 Combinatorial conbal TWo genes are shown. each controled by mtJhiple ~gnals-fotx in the case of gene Jl.;, three m the case of gMe B. Each S~S:nal is COOil1il.J"'ICed to a gene by one regulatory proten Rt.gclatory prott:it1 3 acts at t>oth- genes, in ~on with different addCtiortaf regt.Jators 111 ~ tvvo ca~es.

Sile 1

site 2

As we- discussed above. muhiplP. activators wOJk synergisticaJly. ln fnd. even multtpJe copi~s of a singla a clivntor work synergistically. suggesting that a given activator can interact with multiple targets. This provides an explanation for why diirerent regulators can work together in so many combinations: because each can use any of an array of targets , the comhinations that work together are unrestrir.tE!lL Both the e.xampJe~ of Rignill integrntion we considered above - the HO gene in yeast and the h uman (3-interferon gene- involve activalors that also regulate other genes in examples o[ combinatorial con trol. Thus, from the yeast example , SWl5 is involved in regulating several other genes. And in the manm1alian case. NFKH regulates not only the {:)-interferon gene bu t numerous other genes including the immunoglobulin K light chain gene in B cell:o;. Jun/ ATF, Jikewise. works with other regulators to control other genes. We described earlier that some DNA-binding proteins hind as heterodirners with alternative partners. 11ris offers another level of combinatorial controL

Combinatorial Control of the Mating..Type Genes from Sacchat·omyces c~evisia.e The yeast S. cerevisioe exists in three forms: two hflploid ccHs of dif. fcront mating types- a and a-and the diploid formed when an

a and an a cell mate an d fuse. Cells of the two mating ty pe,._ differ because they express d ifferent sets of genes: a specific genes and a specific genes. These genes aro controlled by activators and repre.ssors in various combinations, as we now briefly describe. T he a cell and 1he a cell each encode cell type specific regulators: a cells m;<~ke the regulatory protein at; a r:ell ~ make the protein~ al and o2. A fourth regulatory prorein, called Mcml, is also involved in regulatin g tho mating-type specific genes {and many other genes) and is p1'1J11;ent in both Ct:!ll typP.S. Hnw do these various regulators work together to ensure that in a r.ells. a specific genes are switc:hed on nnd a spoctfic genes are off: vice versa in a celJs: and in diploid cells, both sets are kept off? The arrangement of regulators at the promoters of a specific genes and

ex specific genes is shown in FigurE" 17-18. • ln a cells. the a specific genes are (JJf because no ac:ti\'ators are bound there, while the a specific genes are on because Mcm 1 is bound and activates those genes. • Jn u cells, the a specific genes are on because Mcmt is bou.nd upstream and activates them. At these genes, Mcm1. binds to a weak site and rloes so on ly when it binds cooperatively with

'fulnscriptional Repressors eel type;

a cell (h
a eel (ha(Jioid)

a

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l

l l

l

549

a/o. cel {diploid)

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MAT locus:

l

gene regulatory proteirls:

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a1

Mcm1

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target genes:

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OFF

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gertes

F I cuR~ 17-18 Control of eel-type sp«ifk genes in yeast. As~ in deta~ lfl the text, the ihree eel types of the yeastS. Cel€ii1Sioe (the ~loid a and a: ~s.. and 11-!e a/o. d4pCad} are detined by the sets of genes they expfess. One ublqllltotJS regu!atOf (Mcml) and three cell-ftpe speOfic regl.Jiators (al. a 1. and o2) together reglAatc ttYee dasses of talgel genes. The MAT lociJS Ci the region of the genome which encodes the moti~ typE regulators (Chapter 1 I).

n monomer of the protein u1 . This en!'ure..'i lh<1t Mcml ar:tivates these genes on1y in a cells. The a s pecific genes are kept o rf in a cells by tl1e repressor u2. Tlris repressor binds. as a dimer, coop· eratively with Mcml at these genes. Two properties of cc2 en-;ure a-specific genes are n ot e>..-pressed here: i t covers the activating region of Mc ml, preventing that protein from activa ting; and it also actively represses the genes. The mechanism by which «X2 acts as a repressor is described in the next section. • In d iploid cells. both a and a specific genes are o tt. Tllis is done as follo ws: the a specific genes bind Mcm1 and a2. just as they d o in a cell~. This keeps those genes off. The a specific genes art! off because. as in a cells, no activators hind there. • Both the h aploid cell types (a and (l() ex-press another cla..'>s of gen es called haploid-specific genes. These are switched off in the diploid cell by n2 which binds upstream of them as a heterodirner with the a1 protein. Only in diploid cells are both these regulators present.

TRANSCRIPTIONAL REPRESSORS ln bacteria we saw that many repressors work by binding to sites that overlap the promoter and thus bloc.k binning of RNA polymerase. But we also saw other ways 1hey can work: they can hinrl to sites ndjar...'Onl to promoters and. by interacting with polymerase bound th oro,

550

Gene Regvlotion in Eukaryotes

inhibit the enzyme from initiati.,g t.ranca:ription. They can also interfere with t he action of activators. ln eukaryotes we see all tht1s~ except the first {ironically the most common jn bacteria). We also see another form of .repression, perhaps the most common in eukaryotcs, which works as follows: as with activato~. repres.-:;ors can recruit nudeosome modifiers, but in this case the eneymes have the opposite effects to those recruited by activatorsthey compact the chromatin or remove groups recognized by the transcriptional machinery. So. for example, histone deacetylases repress tran.'lCI'iption by removing actetyl groups from the tails of histones~ as

we have already set>..n, the presence of acetyl groups helps transcription. Other enzymes add methyl groups to histone tails. and lhis frequently represses transcription. These kinds of mooification also fonn the basis of a type of repression ca)lod ..silencing," which we consider in some dct.1i1 lat€1' in this ch apter. These various examples uf repression are s hown schematically in Figure 17-19. Here we conside.- jusl one specific example, the repressor called Migl which. 1ike G:\14, i~ involved in r.nntmlling the GAL genes of the yeast S. cere-.•i siae.

FU>UR£

17-19 ways inwhkb

eukaryotk repressors work. h1soipllon be repressed 111 various ways. These Uxiude the fOIX mechanisms shown in the figure. Pm (a) shows that, by bending tD a site O'l DNA that~ the btr.ding s11e of an .adivator; a repressor can rni'Jibll btndlng of the activator to a gene. and thus blod< ~dlvalion of that gene. In a vanation on this theme. a repressor can be a derivattve of tt'e same pn~ein as the activator. but lad the ac1ivating region. In another vanaoon, an ac:ttvator that binds 1o DNA as a dirnef cat1 be jntlibtted from doing so by o derivative that retains the region of the protem requcred ra dimera.ation, but lad.s the DNA-binding dcmain Such a derivati\ie foons lniKtlve heterodlmers wiltl the actvator In part (b}, a repff!SSOf bends to a site on DNA lieStde an actJvata NJd inter· acts IMth that adivdlor, oa:ludmg 1ts ad!vatmg tegion. In part (c). a repcessor binds to a site tJpstream d a gene and, by ~ with the

a

of ~ genes (an

acnvator tiflding sile

7~

promoter

repressor binding site

b

transcfl>llo!l
some speofic way. mhib1ts transcription inlba--

RNA polymerase II

tiort Paf1 (d) shows repressioo by re0\Jltlr1g histone moc!itier.. that alter lll.ldeosomes m Wil}IS that mhiblt transrnpllOn (fOf example, ~1100. as Ylowt1 here. bur also melhylatlcft r~ some cases. or even remodeling

at some promoters).

indirect repression

Si1PJal7rCU1sduclion 011d the Control

u~

F J
Figure 17-20 shows the GAL genes as we saw them earlier {Figure 17-3). but with the addition of a site, between the Gal4 binding sites and the promoter: this is where, in the presence of glucose, Migt binds and switches off the GAL genes. Thus, just as in E. coli, tbe cell only makes the enzymes needed to metabolize gaJactose if the preferred energy source, glucose, is not pf$ent. How doos Migt repress the GAL genes? Migl recruits a ''repr~sing £'Omplex.. containing the Tupl protnin. This complex is recruited by many yeast DNA-bincling proteins lh ~l repress transcription. including the n2 protein involved in controlling mating-type specific genes we described above. Tupl also has counterparts in mammalian cells. 1\vo mechanisms have been proposed to explain the repressin g effect of Thpl . Fim, Tupl recru its histone deaC«3tylases. which deacetylate nearby nucleosomes. Second, Thp1 interacts directly with the transcription machinery at the promoter and inhibits initiation.

SIGNAL T RANSDUCTION AND THE CONTROL OF TRANSCRIPTIONAL REGULATORS Sibrnals Are Often Communicated to Transcriptional Regulators through Signal Transduction Pathways As we have seen. whether or not a given gene is expressed very often depends on enviromental signals. Signals come in many fonns- they am, as we saw was typically the case in bacteria, be small molecules such as sugars. But they can also be proteins released by one cell and received by another. This is particularly common during the development of muJticellular organisms (C..hapter 18). There are various ways th;lt ~ ignals arc detected by a cell and commun icated to a gene. In bacteria we saw that signals controJ the activities of regulators by inducing allosteric changes in thost> regulators. Often that e ffect is direct: a smal1 molecular signal , s uch as a sugar, e nters the cell and bin ds the transcriptional regulator directly. Hut we saw one example where the effect of the signal is indirect {control of t hP activator NtrC). In that cnse. the signal (low ammonia levels) induces a kinase that phospborylates NlrC. This type of indirect sig11aling is an QXample of a s ignal transduction pathway.

The term .. signal" refers to the initiating ligand itself- that is, the sugar or protflin fur example. This is how we have defined it previously. It can aJso refur to the .. information .. as it pr~sscs from de tection of1hat ligand to the regulators that rlirectJy control the gcn010- that )R, as it passes along a signal transduction pathway. ln the simplest of bacterial cases there was no dis1inction of course, but once a signal transduction pathway is involved. there is. And in eukaryotes we will see- particu larly in Chapter 18- that most signals are communicated to gen es through signal transduction pathways. som etimes very elaoorate ones. In this section we first look at a couple of cases of signals being passed along signal transduction pathways in eukaryotes. We then consider more generally how signals, emerging from such pathways, control the transcription al regulators thems.elves. In a s ignal trnnsduction pathway, the initiating ligand is lypir:ally clotected by a specific cell s urface receptor: the ligand binds to an extracel lular domnin of the receptor and this binding is communicated to the intracellular domain. F'rorn there the s igna! is relayed to the relavant tr.mscriplional regulator. often through a ca~>Cade of kinases. How is the binding of ligand lo the extracellular dom::~i n oommu nicaterl to the jntracellnlar domain? Thls can be through an allosteric change in tlle .receptor, whereby binding of ligand alters t.he shape (and thus activity) o r the intraceUular domain. Alternatively. the ligand can act simply to bring together two or more receptor chruns, a llowing i ntemclions between the intr<Jccllular domains of those receptors to activate each other. figure 17-21 sho"vs two examples of s ignal transduction pathways. The first is a relatively simple case, the STAT pathway (Figure 17-2Ia). ln this example. a kinase is bound to the intracellular domain of a receptor. When the receptor is activated by its ligand (a cyto.k.ine), it brings together two receptor chains and triggers the kinase l o phosphorylate a particular sequence in the intracellular domain of tha opposing receptor. This phosphorylated site is then recognized by a particular STAT protein which. onr:e bound. gets pbosphorylatad itself. Once phosphorylated, tho STAT dimerizes, moves lO the nucleu s, and binds DNA. The other example i::: more elaborate (Figul'e 17-2lb): the MAP kinase pathwoy that controls activators such as Jun. In this case. the nctivated receptor induces a cascade of signaling events. ending in activation of a MAP kinase that phosphorylates )un (and otl1er transcriptional .regulat01·s). The most common way in which information is passE!d through signal transduction pathways is via phosphory~tion, but proteol}'Nis, dephosphorylation, and other modifications a rc also usoo.

Signals Control the Activities of Eukaryotic Transcriptional Regulators in a Variety of Ways Once a signal has heen communicated, direc:.1ly or indirf'ctly. to a tra n~>Cription a l regulator, h ow does it control tha activity of that regulator? In bacteria we saw that the allosteric changas tha i control transcriptional regulato{S very often affect the ability of the regulator to bind DNA, This is true in cases where the signalling liga nd itself adll' oirccl 'y on the transcriptional regulator and in cases w here tho pre:::ence of the signallmg ligand is communicated to the regu lator through a signal trans duction pathway. Thus, Lac repressor binds DNA only when free of alloJactose. and phospohorylation

a outside cell

b outside eel

.inactive Ras I

MAP KKK

.

Ftm

~

...

MAPKK

Ftm

(ADP]

enters nucleus and bends DNA and

..,.•t---

actiVates lran$Cription

<

.... • 7 am (AOPJ

F1G u R f. 17·21 Two signaUransduction pathways from nJCVI1n14llian eels. StlONn are she STAT and Ras pathways. (i!) A cytoi<Jne IS sOOv.rl bn:ling its rea:ptot bnnglflg together two rec€ptor chr:Wls. E.;x;h chain has a kinase called a J.AJ< attached to its in1Iac.ellular dom<wt. Blinging the chains together (probably accompanied by a c.onformational char)ge tritlgeted by cy1okine bin&ng) leads to phosphaytation cJ the rec:eptrr chains by the IAK kinases (wtlic:h also ~e each cther. stimtlating !heir kinase adivity) The sites phosph:).rylated in the receptor chain are lherl recogmed by cylq:llasmic prctens Cilled STilTs. Eilch STAT has a so-called SH2 dorn.1n These recogrne phosphorylated lyr resdt.Jes W1 c£ftan sequence contex\5, ard lhiJl is the basis of specificity in thts pathway. That is, tl-e portct.h SlAT recruited to a given receptor determines ~Alhtch genes will ~ly be adlvated Once reauated to the rerepta, that STAr rtsdf gas phosp~ by the JAA 'ana!ie.lhis illbws two SlAT proteins to fonn a dimef (the SH2 domain oo each STAT rec.ogniziflg the pl~ed site on the other). The dimer moves 10 1Jl€ nucleus -where 1t birlds specific sires on DNA (different for diffo-ern STATs) and acbvate; ~iption of ~gcfleS. (b) Shows &he

Ras pathway leading into the do.tvnstream r.J.N> M1ase pattnNCIY. A growth factor (such as EG) binds its

receptor, bringing together the d>,a~ns v..bch, as in the STAT case, therl p~ each other: Ths reauas an s senne ard threon.ne residues io the next kinase (<~ rvw> llinase kinase, called tvlek). "This dct!Vates IV'el<, which'" tum phosphoryiates a1d activates the MAP lunase (Eft:). Ths MAP ~Jmse lheo ~otytafes a nurter of SlJbstra(es, tnekJdlng ~ activatofs (for ~ Jun) which r~te a nurtler of spe6fic genes.

MAPK

~rn

554

Gene Regulation in Ellkaryotes

or NtrC 1riggers :Jn :lllostorjc change controlling DNA binding by that activator. In eukaryotes. transcriptional regulators are not typically controlled at the leveJ of DNA binding (1hougb there aro exceptions}. Regulators are instead usually controll~d in one of two basic ways: Unmasking an Activating Region. This is done either by a conformational change in. the DNA bound activator, revealing a previoul'ly buried activating region; or by release of a masking prote in that previously interacted with, and eclipsed, an activating region. The confmmational changes required in each e2se can be triggered either by binding ligand directly or through a ligand-tiependtmt phosphorylation. Ca14 is controlled by a masking protein. In the absence of galactose. Gal4 is bound to its sites upstream of the GAL1 gene, but it docs nol activate that gene because another protein. Ga180, b;nd~ to Cal4 and oc..cludes its activating region. Calactuse triggers the release of Gal60 and activation of the gene (Figure 17-22). h1 many cases the masking protein not only blocks the activating region but is itself {or recn1its) i:l deacetyla.c;e. <md "o :~ctive1y represses the gene. An ex
OFF (basal)

1 ON

FIG U R£ 17-22 JlJe V~ itdiv.tor Gitl4 is reptated by the GalBO protein. Gal4 IS aciJVe Gflfy ;., the presence of galactose. Even in the absence of galactose, Cal4 is found bound to 11s sites upstream of the GPl l gene. But it does not under these rirrurnstcJnres ~te that gme because the actNeling region 1!; t:wnd by a protEin ti.Yied GaiSO. In the presence of galactose, GalBO unOef80eS a <enbmational ch~ the activating regions are revealed. ar~d the GPJ..1 gene is ac;IJiiated In the figure, Gal8o 1!. ~ OISSOC.IiltJng from Qll4 ln the presence (lf ~. lrl f'eitty it IS thought to dJange its posilbon and weafcerl il!o I:J4ndin& but not c.OI'l'lfAelely taa off As shOM'I, Mig 1 Is not OOu1d at tts srte bearuse there is no glucose present (see Figure 17-20).

proliferation in these cells. Mutations affecting this pathway are often assodated with cancer.

Transpoctlnto and Out of the Nudoos. V\'hen not activt>, many activators and repressors are held in the cytoplasm. The signalling ligand causes them to move to the nucleus where they act. There are many variations on thiR theme. Thus. the regulator can be held in the cytoplnsm through inter::Jction with an inhibitory protein or with the cell membrane , or it can be in A conformation in which a s ignru sequen et? required for its nuclear import is concealed. Release and transport into the nucleus in response to a signal can be mediated UtrOugh proteolysis of an inhibitor or tethering region, or by allosteric changes. We will see an example of this in thP. next ch;1pter when we consider the formation of the dorsal-ventra] axis of tl1e Drosophila embJyo. There, Cactus is an inhibitory protein that binds the transcriptional regulator Dorsal in the cytoplasm. In response to a specific signal, Cactus is phosphorylated and destroyed, allowing Dor· ::w1l to enter the nudeus and act (Fi gure 18-13).

Activatoi"S and Repressors Sometimes Come in Pieces We h ave, on the whoJe, considered activators and repressors in their simplest foJTOs. though we have alluded 1o some additional complexities. For example. the activator can come in pieces~ the DNA-binding domain and activating rogjon am be on separate polypeptides, which come together on ONA to fonn the activator. In addition, in (10hRidt~ring the regulation of regulators by their signals. we again see examples of protein com plexes forming on DNA. and the nalure of the complex can determine whether tho DNA-binding protein activates or represses nearby genes. For example. we just saw a case {E2F/Rb) where an activator can bind a protein and b€come a repressor. There a.re even more elaborate cases, such as the glUCO(;Orticoid receptor (GR). This mammalian protein can either activate or mpress transcription depending on the nature and arrangeme nt of its DNA-binding sites at a give11 gena. ln the absence of its ligand, CR is held in the cytoplasm through jnteracliun with " protein called hsp90. Upoh ligand binding, the recepto r is ralcased and moves to the nucleus. (Thus GR is another example of a regulator whose activity is controlled by nuclear localization.) Once in the nucleus, the GR binds sites called GREs. These sites come in two types. When bound to one, it activates lTanscription; when hound to the other, it repres~es. as we now rlescrihe. When bound to the second of these sites, the receptor adopts a conformati on that aLLows it to bind a histone deacetylast>. When bound lu the firsl site. the C<:lnfonnation of the receptor is such that it does not bind the histone deacetylase but xather binds another molecule called CBP. Binding of CBP leeds to activation of the nearby gene, partly because CBP is itself a hi:;tone acetylase but also because it can recruit components of the •ranscri ptional machinery. (CBP is recrui1ed by m m1y activators in mammalian cells~ nnd often severnl activators can interacl with it al once. Indeed, 1:he activators bound at the human p-inlerfcron enhancer- Figure 17-16-are an example.} The lenns ··co-repressor" and "'co-octivator" are ofttm npplicd to any auxiliary protein whkh is neither par1 of the transcriptional machinery nor itself a DNA-binding regulator, but which is n t=~venheJ ess involved in transcriptional regulation. CBP is an example. Th11 term is also often appJied to other nucleosome modifying complexes.

GENE "SilENCING" BY MODIFICATION OF HISTONES AND DNA We have thus far considered regulation by activators and .repressors that bind near a gene and switch it on or off. The effects are local. and the actions of the regulators are often controlled by specific extrac.ellular signals. We now turn to mechanisms of gene silencing. Silencing is a pasitioll effect-a gene is silenced because of where it js located, not in responSfl to a specific envirorunental signal. A lso. silencing can ..spread'' over Ia~ stretches of DNA. switching off m ultiple gent!S, ewo ont'!S quite distant from the initiating eveut. Despite these differences, unrlerstanditJg silencing does not require entirely new priuciples, just extension s of those we have already encountered in thls chapter-. The most common fomt of silencing is associated with a den~>P forru of chromatic callod h.eteroclu-omadn. HeterochrOmatin was named for its a ppearance under the light microsco pe: it appears dense compared to other chromatin , the euchromatin. Heterochromatin is frequently associated with particular regions of the ch romosome, notably th~ relom<>.res-the structures found at the ends of chromosomP.s-amJ rhe ce n lt'Clm~res. As you learm~d in Chapter 7~ telomeres and ctmtromeres are typically ~;omposed ur repctjtive sequences and oontain few. if any. p rotein cociing genes. If a gene is experimentally moved into these regions, that gene is typically s witched off. In fact, there are other regions of the chromosome that are also in a heterochromatic state, ann jn which genes are found, such as in the s ilent mating-type locus in yeast. And i n mammalian cells, about 50% oftho genome is estimated to be in some form of heterochromatin. We have already seeo that the density of chromatin can be a ltered by enzymes that chemically mod.if)r the tails of histones. Such pac.kaging affects accessibility of the DNA and therefore alTec:ts processes such as replkatio n, recombination, as well as transcription. As we have describf'td, both activatiOJl and repression of transcription oft4:ln involve modification of nuchrosomes to alter the accessibi1~ ity of a gene to the trensc.ripUonal machinery and ot.hel" regulatory pr-oteins. We have also encountered proteins that t'eCogu ize modified nucleo...~mes and bind specifically to them. JIeterochromatic silencing can be unrlerstoorl as an 61\tension of these same principles. as Wft describe momentarily. Transcription a m a lso be sileuced by methylation of DNA b y enzymes called DNA methylases. This kind of sile ncing is not found in yeast bul is common in mammalian cells. Methylation of DNA sequences can inhibit binding of proteins. including the transcrip· tional machinery, and thereby block gene expression . But methylation can inhibit exprAssion in a nother way as well: some sequen ces are

ra:ognized only when mMhylated by specific repressors that then switch off nearby genes, often b}• recruiting histone dcacetylase.

Silencing in Yeast Is Mediated by Deacetylation and Methylation of Histones The te lomeres. the si lent mating-type locus (Ompter 10). and the rDNA genes are all "silent" regions in S. cerevisiae. We consider the telomere as an exam pJe. The final 1- 5 kh of each chromosome is founr\ in a fo lcied. dense structure, as shown in Figure 17-23. Genes taken from other chromoso-

Gene -silencing .. by Modification of Histvnes and DNA

--, ~ Rap1 protem

- -Sir 2 protein

F1G lJ R £ 11-2J SileN:int at 1he yeast telomHe. Rap 1 re
bind SIR3 and SIR4, recruting more SIR COC!"f'lel!, alowing 1he SIR2 wrtt.l It to act on nudeosotnes h1nher away, Md so oo. This explains the spreading cf the Sllerlc.ing effect produced by declcetylation (Source: Adapted from Grunste~n M. et aL 1998. Yeast lle\e.-odlromatin· RegulafJOtl of 1ts ~errt>ly and~ tty ootones. Celf 93' 325 - 328 ~· ~) 1998 Used INith perrmSSIOO from esevier.)

rna) locations and moved to this region are often sileDced, particulnrly

if they are only weakly expressed in their usual location. The r.hromatin at the telomere is less acetylated than that found in most of the rest of the genome, wher·e genes are more readily expressed. Mutations have been isolaterl in which silencing is relieved-t hat is, in which a gene placed at the telomere is expressed at higher levels, These studies implicate three genes encoding regulators of silencing. SIR2, 3, and 4. (SIR stands for ~ilent i nformation regulator). The thre.e proteins encoded by these genes form a complex that associates with silent chromatin, and Sir2 is a histone ciea<;:etylase. The silencing complex is recruited to tbe telomere by a DNA-binding protein that cecogni7.f'.S tht:: telomere's 1-ep::atcd sequences. This recruitment initiates local deacetylation of histone tails. The rlt>.acetylated hlstones are, in turn. recognized directly by the silencing complex. and so the local deacetylation readily spreads along the chromatin u1 a selfperpetuating manner. producing an extended region of dense b eterochromatin. How is this spreading ljmited to the telomere (and other silenced regions)? Other kinds of histone modification bJock binding of the Sir2 proteins, and thAreby stop sprf>.ading. Methylation of the tail of h istone H 3 is believed to do this. Histont! m ethyl transferases attach methyl groups to h.iston~; tails. As we saw in Chapter 7, these enzymes acid methyl groups to specific lysine w.sirlues in the tails oJ' histonvs H3 anri H4. Histone methyl ~ferases have ro.cently been described in S. cerevisioe. where they are believed to help repl'cssion of some genes, and. as just noted. block spreading of Sir2 mediated silencing in others. But histone methylases have been better c::haracterized in higher eukruyotes and in the ywsl Schizosaccharomyces pomhe. b1 those organisms. silencing is typically associated with chromatin containing histones that are not only deacety lated . but methylate
557

just as acerylatf'd residues within histones are recognized by prot.r:ins bearing bromodomains. methylated residues bind proteins wlth chromodomains (see Figure 7~39). One such protein is t.h e Drosophila protein HPl , a component of silent h eterochromatin in that organism.

Histone Modifications and the Histone Code Hypothesis It has been proposed that a histone code exists. According to this idea, different patterns of mollifications on histone tails can be "read .. to mean different things {Figure 7-3 9). The -meaning" w ould, in part, b•~ the .result of the direct effects of these modifications on chromatin de nsity and form. But in addition, the particular pattern of modificatio ns nl any given location would .cecruit specific proteins, the particu-

lar set depending on the number. typa, anrl dis position of recognition domains those proteins can:y. We bave already seen that a component of rhe TFIJD compJex recogni~s acetylaled iysines (it has two bromorlomains and f'f!COgnizes, specifically. H4 N-tenn inal tails modified on two particular lysine groups). Anc1 we have just seen that HPl recognizes H3 tails rnudified by methyl groups on a particular lysine residue. There are also proteins that p hosphorylate serine resirlues in H3 and H4 tails and proteins that binrl those modifications. Thus, multiple modificati ons af several positions in the histone trois are possible; the examples of H3 and H4. together with H2A and B, are shown in Figure 7-40. Add to this the observation that many of the proteins that carry modificationrecognizing domains aro themselves enzymes that modify bistonos further, and we start to see how a process of recognizing anrl maintaini ng patterns of modification could be achieved. Consider one simple case-lysine 9 on the tail of hi~on e H 3 (Sf~ Figure 7-39). Different modification states of this rosirlu~ have different meanings. Thus. acetylation of this residue •s associated with acrively transcrib€-.d genes. 1l1at msidue is recognized by various histone acetyIases bP.aring bromodomains, and these stimulate additional acetylation of other n earby nucleooomes. When lysine 9 is unmodified, it Is associatnrl with silenced regions (as we saw in S. cerevisioe above). Uuacetylaterl histones often recruit rleacetylating enzymes betrer than acetylated hl~tones, reinforcing and maintaining the deacetylated state (as we saw in the s preading of s ilenced region s inS. cerevisioe). Finally, that same lysine can in some organisms he methylated: in that case, lhe modified resich te then binds proteins that establish and maintain a heterochromatic state, stronger than that associawd with ele.aretylate.rl histones.

DNA Methylation Is Associated with Silenced Genes in Mammalian Cells Some mammaJian genes are kept silent by methylation of neruby DNA sequences. Jn fac~ la1-ge region s of the mammalian genome are marked in this way, and ofhm DNA methylation is seen in r~ious that are also heterochromatic. This is because methylated sequences are often rec<)gnized by ONA·binding proteins (s uch as MeCP2) that recruit h istone deacety)ases and histone metbyiases, which the n modifY nearby chromatin. Thus. methylation of DNA can mark sites where h.eterochromati.n subsequently fonns (Figure 17-24). DNA methylation lies at the heart of a phenomenon callP.d imprinting, as we now describP. ln a diploid ceU. there are two copies of most genes,

Gene MSilem:ing·· by ModifiroiiOII of fii.stones and DNA

559

0 proteins that bind

methylaled DNA

(Off)

ctuomalin remodeling complex

gene COfl'llllelely

~~~~4-~------------------~--~' 0FF fl G U R E 17-24. Switdling i1 gene off tbrough DNA methylation and histone mocifk:ation. rn its l.f'll11()difted state, the mammalian gene shovvn Cdfl readity ~h between beitlg ex~ed or not

expressed itl the p!'esence of adlvatOt'S and the Ira~ macllnefy, as sklwn ifl d1e top fine rn tHs situation ~ IS never filmfy shu! off- it IS leaky Often that is not good eoougf> -sometimes a sene must be romplet<>ly shut off, on occasion pctrndnendy. 1hs is acheved througll meltlylabon of the DNA and modification of the locat nudeosomes. n-tis, when the gene IS not being e:pressed, a CfiiA me~dse (a meltlvJase) can gain ¥.Cess llnd ~ cytosines IMtlin the prcxnoter ~. the gene ttself, and the upsteNn acwatot bindilg Sites. Them~ &J0l4' IS added to the s· pos.tim tn the cytosir£ ring. geliefatlng 5methylcytosme {see chtpter 6).. This mod'lftcabon alone c.c.1 disrupt ~ of 1he ttansaiptlon mach~ry and adlvators tn some cases. But tt also binds otha poteins (fOf' e~.ample. MeCP2) that recognsre DNA ~ences c.ontaiotng rnethy1cytostlle. ~ proteins, in tum. recruit complexes that remodeJ and modify local nodeosomes, S\llllt1:hing off exptessiOO of the gEne completely.

one ropy on a chromosome inherited from the fathPC, the orher on the equivalent chromosome from the mother In most cases, the two alleles are expressed at comparable Jcvels. This is hardly surprising: they mrry 1he same regulatory sequences anrl are in the presence of the same regu· lators; t hey a re also located in an equivalent region of two very similar chromosomes. But there are a few cases where one copy of a gene is expressen while the other is silent. Two weJI·studied examples are tlle human H 19 and lgf2 genes (Figure 17-25). These are located close to each other on l1wnan chromosome 11. In a given cell. one cop y of ll19 (that on the maternal chromosome) is expressed, while the other copy {on the paternal chnr mosome) is switched off; for Jgf2 the reverse is true-the patemaJ copy is on and the maternal copy off. Two regulatory sequences are critical for the differential expression of these genes; an enhauc~r {downstream of the H19 gene) and an insulator (located between the H19 anCI Igf2 genes). The enhancer

Gene Regu/Qfivn in EukClfJ'Oles

fiGURE 17~25 ln'4Niadi-ng. ShO\IVnare

a malemal chmmosome

CTCF'

two~ of genes contro~ by imprint-

JOg-the mamrnolian f£12 cpressed from only the maternal chm· ~ Jgl2 from ·the patenal chromosome. The methylalion slate of the insutator element determ~t~eS whether or not the •nsulator binding protWI (GO) can bind and block activatiof1 of the Hf9 gene from the downstream enhancer.

X·

OFF

,....--ON

~

H19

enhancer

b paternal ctlto6ome

''

wiSlAator

H19

(when bolUld by activators} can, in principle, activate either of the two genes. So why does it activate only H19 on the maternal chromosome and Tgf2 on the patemal chromosome? The answer lif'.s in ~he rolo of the insulator and its methylation state. Thus. the enhancer cannot adivate the lg/2 gene on the mat.e rnal chromosome because oo that chromosome, the insulator binds a pmtein, CTt."F. that blocks activators at the miliancer from activating the lgfZ gene. On the paternal chromosome. i n contrast, the insulator element and the H19 promoter are methylated . In that state, the transcription machinery cannot bind the H19 promoter, ancl CTCF cannot bhtd the insulator. As a result, the enhancer now activates the Igf2 gene. The H19 geilB is further repressed on the paternal cluomosome by the binding of MeCP2 to the ffi(~thylated insulator. Tbis, as we have seen. recruits deacetylases , and these repress the H19 promoter.

Some States of Gene Expression Are Inherited through Cell Division even when the Initiating Signal Is No Longer Present Patterns of gene expression must sometimes be inherited. A signal released by one cell during developJJJent causes n eighboring cells to switch on specific genes. Those genes may have to remain switched on in those r:eUs for many cell generations. even if the signaJ that inducOO them is present on ly fleetit!sly. The inheritance of gene expression p nltP,ms, in the absen~ of either mutation or the initiating signal, is called epigenetic regulation. The imprinting eXample we discussed above revr.als one way the expression of a gem~ can be regulaten ep igem~ticalJy. Contrast this with some of the examples of gene regulation we have discussed. lf a gene is controlled by an activator, and that activator is only active in the presence of a given signal. then the gene wiD remain on only as long as the signal is present. lndeecl, under nom1aJ conditious, the lac genes of E. coli will only be expressed while lactose is present and giucose absent. Likewise the GA L geoes of yeast are expressed only as long as glucose is absent and galactose present. and h'Uillan P.,iJUcderon is made on ly while cells are stimulat~d by viral infection. But we have also already enoountered an example of gene regulation which can be inhe rired epigeneticnlly. The reason that case. maintenauce of a phage A. lysogen {Chapter 1£). can be described as epigenetic is discussetl in Box 1'7·3, A LysogeJ:J~ and the Epigenetic Switch.

Gene -Silell cing " byJ1,1odific:ation of' Hilltones cmd n NII

561

Nuclt'losome and DNA m odifications can provide th~ basis for epigenetic inheritance. Consider a gene switched off by methylatiou of local bistones. When that region of the c hromosome is replicated d uring cell division, the methylated histones from the parental DNA molecule end up distribu ted equ ally between the two daughter duplexes (see Figure 7-42). Thus. each of the daughter molecules carries some methylated and some unrnethylatHd n•Jdeosomes. The methylated nudeosomes recruit proteins heating chromodomains . includiug the histone methylase itself which then methylales the adjacent uumodifierl nucleosornes. A daughter strand that lacked methylated histones altoget..ht?..r (that is, one from an unmethylated parent) would not recruit the methylase. ln this way. the state of chromatin modification can be maintained through generations. DN A methylation is even more reliably inherited . as shown in Figure 17·26. Thus, certain DNA methylases can methylate, at low frequency, previously unmodified DNA; but far more efficiently. so-called maintenance methylases modify hemimethylated DNA-the very substrate provided by replication of fully m Rthylaterl DNA. ln mammalian cells, DNA methy lation may be the p rimary marker of regions of the genome that are silenced. After DNA replication.

13' methylation

3'

DNA replication

I

not feoognized by maintenance ~

3'

f.

G .. 5'

3'

\

•

5'

maintenance

methylase

5'

5'

3'

s·

3'

3'

'3

mechytation

3'

F I CURE

s

17-26 Patterns of DNA methrlatiotl un be untamed througtl cell diwision.

1>5 we saw in figure 17·24. DNA involved in ~sion of a vertebrate gene can get methylated. and

e,.;pressioo of that gene s\!Vitched off. l hts Initial methyldtion is perlOI'med by a dt novo methylase. fof the shutdown state to keep a gEne off petmanendy, the methyla1lon state mtiSt be tnhetited througtl cell divls&on. l hls figure shows how that IS actueved. A 0NA s~nce is snown 111 wtndl two c.ytosmes are present on each strand. one methytated. ttl£' other not This pattern is mainti'lincd through eel division,

because, upon OOA repbc.allon, a mamtenance mcthy2sc rec.ogrnzes lhe helmcnethylated DNA. and adds a mettlyl groop to the unmelhylated cyklstne within it. The completely unmethyla1ed sequCflce 1S not recognized by this en:zyrne. and so l"efl''ains unmeth~ted. U1us. both ddugtlter DNA duplelces end up with the same p.mem of methylation as the parent (Source· Adapted from Alberts B. et aL 2002. Molewtar biology of the cefl, 4th edillaf\ p. 4e T, fig 7·81 . eopynght ~· 2002. Reproduced by pemus s.10n of Roudedge/Tayb & Francis Books, Inc)

562

Gene Iregul afion ill E11l<.ory(Jles

Box 17·3 A lysogens and the Epigenetic SWitd1 Heritable PQttems of gene e>I{JI'essiQ) can be estabftshed ~ DNA. moditlcation. Consider a bacterial example we disCussed 11 Chapter 16, a ~ lysogen. In a lysogen, the phage iS 111 a dormant stae Vv1thin the bacterial host cell. lhs state is associatecl with a specific pattem of gene expre:sstoo, and in particular with sustained expression of the ~ out the use of 1'\ldeosome. or

repressor protein (see Figure t 6-27}. lysoger~~c gene expressiol is established il an infected cell irl respoose to poa growth condilioos. Once estabished, haJvever. the lysogenic state is mailt:ailed !.tabfy desr;ire ffll:lrOJements in gfCINth cooditions: I"'Cl
received.

Maintenance ofthe lysogerjc state thf"CM.€h eel division is thus an example d epigenetic regutarioo Instead of any rom d I'TlOdfficalioo, this eptgenetic control resdts from a t.w-step strategy for repfessof synthesis. In the ftrSt. sysihesis is initlcfy estal:ished bough acb\lalioo of 1he repressa (d) ~ by the adJVOiof 0 1 (which is.sensitive to gr<JJVth conditions). In the se:ood step, represscr synthesis IS mairltalned by auforegt.Aation: repressor activates expression of its OJv'n gene (see Olapter 16. figure 16-35). In thts vvay. Vvflen the lysogEric cell divides; each daughter celnherits a copy of the dormant phage genome and some repressor prdein. That repressa is suffiaent to stmulate funher repressor symhe.sis from the phage genome in both cells. t.Auch of gale r~latioo dJring the devdq.ment d rruri:elu&ar Otganisms v.-al<s in just ttfs Wifo/. We vvil see examples in the next~-

hemimet.hyJated sites are remethylated. 1hese can then be recognized by the r-ept-cssor MeCP2, which in turn recruits histone deacety lases and methyl ases. reestablishing silencing {Figure 17-2 4).

EUKARYOTIC GENE REGULATION AT STEPS AFTER TRANSCRIPTION INITIATION Some Activators Control Transcriptional Elongation rather than Initiation h1 the previous chapter we encountered the N aud Q proteins of phage ~ ; these .regulatOI's control the elongation of a transcript after initiation (Figure 16-36). Specifically. t hey act as .. antiterrninators." h l eukaryotes we se•J I'E'glllation at this step as well The elaborate transcriptional machinery of a eukaryotic cell contains nurnerous proteins required fo r initiation. It also contains some that aid in elongat,oh {~e Chapter 12). Al some genes there are sequences downstream of the pmmoter that cause pausing or stalling of the polymerase soon after initiation. At those genes. the presenoo or abscnc.:e o f certain elongation factors greatly inJJuancas the leve l at whic h the gene is expressed. One example is the HSP70 gent! fl'om Drosophila. This genr.. activated by heat shock, is controlled by two activators working together. The GAGA binding factor is believed to recruit enough of the ttanscrlption machinery to the gene fo.r initiation of transcription. But. in the absence of a second activator. HSF. the initiated polymerase stalls some 100 bp downstream of the promoter. In CP.sponse to heat shock. HSF binds to specific sites at the promoter and recruits a kinru;e. P-TEF, to the stallt3c:l initiated machinery. The kinase phosphotylates lhr.' C-terminal domain of the largest subunit of RNA polymerase (the so-called polymerase "tail ,.) freeing the enzyme from the stall and allowing transcription to proceed through the gene. We saw in Chapter 12 that phosphorylation of the polymerase tail is an important step in the early stages of transcription at all genes, and the ~inase TFilli can pe.rform tha t phosphorylation. Whethe.r P-TEF is also needed at most gen~s is not cleC!r. A strong acidic activator like

Gal4 is able fo recruit P-TEF along with tbfl rest of the machinery. It may be tluH only at certain genes is the recruitment of the machinery partitioned between regulators in the way w e see at 1fSP70 gene, allowing an extra layer of control. The HJV virus, that which causes AIDS. transcribes its genes fro m a promoter coutro1led by P-TEF. Again. polymerase initiates transcription at t hat promoter, under th e cootrol of the activator SP1. but s tRfls soon afterward. In that case, P-TEF is brought to the stalled polymerase by an R.'IJA-binding protein, not~ DNA bound one. The protein •-espnnsible is called TAT. TAT recogn izes a specific seque nce near the start of the HlV RNA and present in the t.re.nscript made by the stalled polymerase. Another domain of TAT interacts with P-TEF and recruits it to the stalled polymerase.

The Regulation of Alternative mRNA Splicing Can Produce Different Protein Products in Different Cell Types As we saw in Chapte1· 13. the coding region of many individual eukaryotic genf:'S is split. with stretches of coding sequence (e:xons) interrupted by (sometimes much Jarget) regions of noncodi11g sequenCE! (caHHd introns). The whoJc gene is transcribed before the ooding regions are spliced togetht'.r. discarding the n oncoding regions. The number of genes with introJ:ls, and the number of introns per gene, increases with the complexity of the organism. In some cases a given precursor mR.t'IA can be splicerl in alternative ways to produ~ different mRNAs that encode different protein products. The choice of splicing variant produced at a given time or in a given cell type can be regulated. The regulation of alternative splicing works in a manner reminiscent of tra.nsc:riptional reg11lation and was ruscusserl in Chapter 13. To recap, the splicing machinery binds to splice sites anti carries out the spli cin~ reaction . Binding of the machinery to a givtill splice sita deptmds on tha affinity of that s ite for the machinery and the actions o f proteins that regulate splicing. For example, a strong splice site can direct efficient constitutive splicing. But that can be blocked by a splicing repressor that binds to sites overlapping the s trong splice site and exclurles the splic· iug machinery (Figure 'l3-~7a). This n1echanism of splicing repression is analogous to mechanitims of both ll'anscriptional and translational repression we encountered in E. coli. ln other cases. SE:quences called s plicing enhancers are found near splice sites. These sequences are recognized by regula tory proteins that recruit the splicing mar.him~ry to thP. splica sil o. T.ikc tran~rip

tio naJ activators. these regulatory proteins have separate domains, on t-J that binds the nucleic acid (in this case RNA) and one that binds th ~ splicing machinery (Figure 13-17b). The regula tion of a splicing cascade by repressors and activators lies at the heart of sex detennination in Drosophila. as we now briefly desc.t ibe. The sex of a fly is determined by the ratio of X chro mosomes to autosumes. A female results from a ratio of 1 (two Xs and two :rots ot autosomes), and a maJe from a Talio of 0.5. This ratio is initially measured at the level of transcription using two activators. caUed Sis A and SisB. The gt:nes encodillg these regulators are both on the X chrornosom•~. and so, in the earty embryo. the prospective female makes twire as much of their Jlroducts as does the male (Figure 17-27). These activator::; bind to sites in the regulatory sequence upstream uf the gene Sex-lethal (Sxl). Another regulator that b inds to and controls

Gene R.egtJiatloll in Eut..:aryottlS

fEMALES

.-

MALES ,~ _1X ....;,_:_2A ___,.

--2X:2A

sis-a sis-b

SIS-a

d[Jn

sis-b

dpn

1X

1X

1X

1 DNA

DNA no trans~

!spiKing spliced mRNA

eatty Sxl protein

c = J - - - -:::J3'

~

N ::::::==:::::>c

s»

fl G u R E 17-27 ~ Transaiptional regulation of in male and female flies. The Sis A and Sis Bgffies are fOtJnd On the X chrocnosorne and encode ttar~salJtional acWcJto(S thai control elep(e551()(1 of 1t!e Sxl gene Dpfl. a ~ of SX/, ts encoded by a gene Of'l chornoSClOlt"! 2- \l\lt)iCe both males and femak>s ~ the same ar1omt of the autosomally enc.oded Opi'l. females rnake tv-..ce as mud1 of the actlvators ~mates (because females have two Xchromosome; and males ooly one}. l hc differ€fla.! in ratio of actiVators to repressor ensures the Sx11s e>ed .., lhe teXt and the next flgl.Jre (Source: ~pted fran Es1es P A. et al 1995. MuftJp4e response elements in !he sex--le'lh<'ll earl)' ptomoter ensure ifs fanafe...s.pecific expre<;ste.m pattern. Mol CeO Bioi. 15- 904-9 17 Cq:>yright ~~ 1995 Natu'e PWiishing Companies.)

men

the Sxl gene is a repressor called Dpn (Deadpan); this is encoded by a gene found on one of the autosomes (chromosome 2)_ Thus, the ratio of activators to repressor dffffll's in the two sexes, and this makes the. i11ffetence between the Sxl gene being activated fin females} and repressed (in males). The Sxl gP.ne is P.xprt'lS..~il from two ptmnoters, Pe and P m- The former (pronlotp,r for ast.ablishment} is lhe one controlled by SisA and SisB (and hence expressed ill females only). Later in development, this promoter is switd1ed off permanently. In female embryos, expression of Sxl is maintainect by expression from Pm (promoter for mnintvnance). Transc.ription from Pm is constitutive in both females and ma1cs, but the RNA produced from this promoter contains one exon more than the transcript produced from P .,. If that exon remains in the mature message~ it fails to produce an active protein. That is what happens in the male. Eut. in the female spliciog removes that exon and functional Sxl protein continues to be produced_ As shown in Figure 17-26, it is Sxl protein itself, present in the female but n ot the male (thanks to earlier expression from P.,), that directs splicirlg of the RNA made from P,11 aml ensures the inhibitory cxon is spliced o ut. Sxl does this by working as a splicing repressor. Thus, functional Sxl protein continues to be made in females. That protein regulates the splicing of other RNAs in the female as wen as its own. One of these is the RNA made constitutively [in males and fema les) from the tra gene (Figure 17-28), Again , in the absence of Sxldirected spl.icillg, this RNA fails to give p.rotein (in males), but in the pre$t>..nce of Sxl it is spliced to give furtctionaJ Tra ptotein (in females).

Et!kDI}Vtic Cerre ltto'gvltlticm at Steps after Transcription lr!itialifm ma\e primary RNA

female priinary RNA

transcript

bn$Ctipt

regtJlaled 3' splice site

I

s·

3"

FI G t.IRf

..(

s·

3'

3'

3'

l

l

no functional

Sxl protein

PfOlein regulated 3' splice sile

/

5"

:r

5'

J 3'

tra gaoe 5'

5'

!

!

no func6onal proleYl

Tra-2

regulated 3' spice site

\

5'

response to Tra pJOfEir1. a \lel31011 of ~ protem IS ;produred (in females) with a stretch

of 30 ai'Tlino acids at cts C term~ end that

Traprotem

l

3'

3'

dsx g ene

3"

!)

S'

N•

--

' ~

-

-.c

Osx protms

N,

'

r

c

~

represses female genes

represses ma)e gei1Eis and

!

activates female genes

male development

A cascade of alternative

spliOng events detetmmes the sex of .a fly. As desolbed in detail ", the texl. lhe SeJ< lettlal p101:cin t5 produced 1n fltes that INifl develop 1100 females (shown on the right of the figure) but rd those lilat wil de\leJop IntO males (shown en the left) The preset'lC.e of that prurelfl JS maintamed by autoregulatiOn of the~ of its own m~ In the absence or11'-.at regu!i'ltion no fLJI'Cll()nal proten IS Pfoduced (in males). SeJ<-Iethal also wntrols spliong of lile tro g61e, producing fur1Clionat T~ protoo tn females (but not male:.). Tra ~ •Jself a ~10ng regulatOf. lt acts on pre-mRNA frtlm the doubfe.. sex gene. When the dsx mRNA IS spliCed .,

Sxl gene 5'

17-26

565

~

female development

Tra protein is also a splicing regulator. Whereas Sxl is a splicing repressor. Tra is an activator {Figure 17-28). One of its targets is RNA made from the gene encoding Double sex {Dsx). This RNA is spliced in two altemative forms. both encoding regulatory pi oleins but with different activities. Thus, in the presence of tro, dsx RNA is spliced in a way that gives rise to a prote in that •·e presS('.s e>-'J'ression of malesp~dfic g~nes. In the absence ofTra protein. the fonn of Dsx produ03d represses female~specitk genes.

Expression of the Yeast Transcriptional Activator Gcn4 Is Controlled at the Level of Translation Ccn4 is a yeast transcriptional activator that regulates the expression of genes encoding enzymes that direct amino acirl biosynthesis. Although it is a transcriptional activator, Ccn4 is itself regulated at the level of translation. In the presence of low levels of amino acids. the Gco4 mRNA is translated (and so the biosynthetic enzymes are e>.'J)ressed}. In the presence of high levels of amino acids, the Ccn4 mRNA is not h an.slated. How is this regulation i!cltieved?

mtlflglJish tl from the fofrn of the protein prodLJced 1n the abserce of the Tro regWtcr {In ma'es) lhe female fOITil of Osx act1Vate; genes reqwred for f~ rlevelopmer!t af1d represses tlose for JN!Ie developt;IMt Tne male form. wtnch lias a stretch of ISO amino aods i!t the C·temnnal end. represses genes tha\ difoo bnalo development Sxl protan acts as a splic· tng represSOf by bin
17-29 Translationat control of Gcn4 in response 10 amino add stMVation. As desaibed m detail•n the rex1, the open-re.adFI GU RE

nonstarvation conditions

.~.f~

tRNAMcll~

il18 ftame encoding the yeast adivatorGrn4 ts preceeded by four other ORFs. The first of these upstream ORk IS translcltee ini1ially, \.Nhen amino acids are scatte (starvation c.onclibons), it takes longer for the ttanslatlonalrnadllf)£f}' ro re-m1b&e translahoo, and so 11: tend~ to reach the Gcn4-eocoolf'lg opErl-reaCing frame before re-lnitiatlng aod translates 1hat to give Gcn4 Pfotein. When arneno aads are plertifiJ (l'lOOStaiVo1ion conditions) re-il"liti$on talles p lace at •ntef-

'

40S Slh.>nil

-eos- l 5' I

1

vening open reaang frames. and the tlans!abon mactlioery then di.ssooates from the RNA temp!G1e and Gcn4 IS never translated (Sou-ce: Hinnebusc.h A G. t 997../oumo/ oiBiokJgy of the CeJ1272 2 1661-21664, fig. 1 COpyrigtlt CJ 1997 The Amencan Society for Biochemistty & Mole<:ular Biology,)

uORF4

UORF1 (translated)

~~~ 3'

I

L____J

UORF1

GCN4

uORF4 ribosome re-tnitiates

$tarvation conditions

eiF2e y

G pMs phoryJation

siQW binding

-r~

tRNAMet~

(

40S StJblnit

s·

3'

I

GCN4

I

uORF4

UORF1 (tr.mslated)

amifloacid

£~

biosynthetic enzymes

~

~

5.

3'

I

I

oORF1

I

uORF4

GCN4

The mRNA encoding the Gcn4 protein contains four small open reading frames (called uORfs) upstream of the coding sequence for Gcn4. The most upstream of these short open·reading frames (uORF't) is effidently recognized by ribosomes that scan along the mes~age from the s · end (see Chapter 14). Onre they have translated uORFt. a uni(jlle property of this ORF allows 50% of the small subunits of the ribosome to remain bound to the RNA and resume scanning for downstream initiation (AUG) codon s (Figure 17-29). Before intiating translation of any downstream open-reading fram e , scanning 40s ribosome subunits must bind the trcmslation factor elF2 complexed with the initiating tRNA molecule fMET-tRNA. (Recall from Chapter 14, in the absence of the initiator tRNA, the 40s subWlit r.annot recognize the AUG sequence in the mRNA.) Under conditions of amino acid starvation. eiF2 is phosphorylated, a modification that reduces the efficiency with which it binds fhe ribosome~ also, under those conditions, there is less charged initiating Me t·tRNA available. Thus, when amino acids are scarce, ribosomes that resume scaru1ing after lransla· tion of uORFl pass through u0RF2-4 before robindillg eiF2- tR:'\JAMet. They therefore faH to initiate t:rans.Jation from any of these intervening AUGs. But the AUG of the ITue Gcn4 OJ
RNAs IN GENE REGULATION We saw in Chapter 16 a few examples in which RNA molecules m-e central to regulating expression of a gene or set of genes. Recall. for example, attention of the trp genes of E. coli. In that case. the secondary structure of short R.l'\IA transcript detennined whether RNA polymerase transcribed thP trp genes, or terminated transcription before rraching them (Figure 16 -2 1). We a lso saw (Box t,fi.-4 ) h ow s o -<:alled riboswitches work in a similar way. Once again, alternative secondary structures of leader RNAs detennine whether polymerase continues transcribing a set of genes, or lArlllinatcs instead . In this chaprer we have seen hoY< regulatory elements in RNA can bind proteins involved in transcriptional regulation. The HN TAT protein was an example. Recently, however. it h as become apparent that RNAs have a more general and mechanistically distinct role in gene reg· ulati on. Short &'\lAs, gP.nernted by the action of enzymes we will discuss in this section, can direct repression of genes with homolog)' to Lhose short RNAs. 1his repression, called RNA interfenmce (RNAi), oan mani· fest as translational inhibition of the ITI.RJ'I.JA, destruction of the mRNA or tra.nsaiptional silencing of the promoter that directs expression of that mRNA How widespread the action of RNAs will turn out to be is still Wlclear, and the details of the mechanism u<>ed to silencfl the target genes in any given case is also typically unresolved. But as we will see, the role nfthesc RNAs mnges from developmental regtslntion (in. for ex-

ample, the worm C. elegon$1) to the protecti011 ~jn$1t infect inn by CP.rtain viruses (in plantsj. RNAi hns also been adapted fur use as a powerful experimental technique aJJowing specjfic genes to be switched off in anJ' of rnany organisms.

Double-Str.:mdt:d RNA Inhibits Expressio n of Gene~ Homo logous to that RNA The discovery that simply introduClilg double-stranded RNA (dsRNA) into a ceH can repress genes containing sequenCflS identical to (or very similar to) that dsRNA was remarkable in 1900 when it was reported. In t hat case. the experiment was done in the wonn C. elegans (see Chapter 2 1). A similar effect is seen in many other organisms in which it has subsequently been lTied. Earlier than this report, ho\1\"ever. it had been kuown that in plants genes could be silenced by copies of homologous genes in the same cell, Those additional transgenes were often found in m ultiple copies, some integrated in direct repeat orientation. Also, in plant~. it was known that infection hy viruses was combated by a mechanisnl that involved destruction of viral RNA. These two cases were brought together in the following observation: infection of a plant with an RNA virus that carried a copy of an endogenous plant genr led to silencing of that endogenous gene. All these phenomena are now known to be mechanistically linked. In this section we consider how dsfU-JA can switch off expression of a gene.

Short Interfering RNAs (siRNAs) Are Produced from dsRNA and Direct Machinery that Switcht!s Off Gene.s in Various Way~ Diccr is an RNAseDI-Iike enzyme that recogni1.es and digests Long dsRNA. The products of this are short double-stranded frabrments about 23 nucleotides long. This is shown in the first step of Figure 17-30. These short fu'JAs (often called short interfering RNAs, or siRNAs) inhibit expression of a homologous gene in three ways: tbey trigger d eslruc.:tion of it~ rnRNA; they inhjbil translation o f its mRNA; or tbfly induce chromatin modifications within the promoter thttt silence t he gene. Remarkably. whichever route is u sed in any given case. much of the same machinery is required. That machinery jncJ udes a complex caJled RISC (RNA-in duced silencing complex). A RJSC complex contwns, in addition to the siRNAs themselves, various pmteins ioclucU.ng members of the Argonal.JI family, which ai(> believed to interact with the RNA component. As shown in Figure 17-30, once a given siRNA bas been produced and assembled within RISC, it is denatured 1n an .ATP-
RN~ in

dsRNA

JJU!!ammommmmnl:mmn:::mnm: cytoplasm

Gene Regul(Jfitm

F1GURE t1·30 RNAisilencil!g. RNAi swikhes off the expressiOn of a given geoe \'\hen ~ RNA mo1eaJes v.ith her 1'110\ogy to Jhat gene are tntroduced, or made, in that cell Ths effect invol\les processmg of the OsRNA to make shoft 1nterferlf1g RNAs by the enzyme JXer. These siRNAs then direct a complex c:alled RISC (RNA-inc:iJced silencing complcM) to repress genes in three ways. It 2Jttad<s and digests mRNA with homology to the siRNA; at interferes With translauan of those mRNAs; or it c:twects chtomatin modifyt~ enzytrleS to the prornotm that direct expressl0f1 of lhose mRNAs. Although in the figure RI5Cperfonr6 S. Used with pernisslon)

complex recruits other proteins that modify the chromatin around the promoter of the gene. This modification leads to silencing of transcription . We have already d escribed silencing med iated by chromatin modification. Establishing silencing in the centromeric regions of the ye.ast S . pombe has recently been shown to require the RNAi machinery. In that case, it is believed that regions of the centromere (see Chapter 7) are transcribed to produce RNAs that either £old to form stem Loops or hybridize with other RNAs from the same region. The resulting dsRNAs are recognized by Dicer and cleaved to produce the siRNAs responsible for directing the RNAi machinery to the centromc.res. It is still unclear the extent to which RNAi might t urn out to be involved in other cases of chromatin modification and silencing in other organisms. There is another featwe of RNAi silencing worth noting-its ex1reme efficiency. Thus. very small amounts of dsRNA are enough to induce complete shutdm.vn of tatget gem•.s. While it remains unclear why the effect is so strong. it might involve an RNA-dependent RNA polymerase which is required in many cases of RNAi. The involvement of this enzyme s uggests some aspect of the inhibitory ''signal"

might be amplified as part of the process. One way this might bo achje, •ed is revealed by the following obscrvaO.on. When a given siRNA targets a region of a specific mRNA. additional siRNAs a re often generated that target adjacE'.nt regions of that same rnRNA. The RNA-dependent RNA polymerase might have a role in generating these additional siRNAs after recl'uitment to the tnRNA by the original siRNA (see Figure 17-30, on the right).

MicroRNAs Control the Expression of some Genes during Development We have alluded to tbe long dsRNA precursors of the siRNAs as e ithAr heing provided experiment
protect cells from any infectious, or otherwise disl'uptive, e lement that employs a dsRNA intermediate in its replicative cycle. This woulu include certain viruses and many transposons that replicate via a dsRNA intermediate (see Chapter 11}. RNAi turns off genPS expressed by those agents. as well as destroying the dslU\IA intennecliatcs lhcmselves. The importance of this function for RNA.i remains evident in plaots. Many plant viruses have evoJved mechanisms to counteract the host mounted RNAi defe nse response. These viral functions, called viral suppressors of gene silencing (VSGSs) are nonnally essential virulen<:e deteTT11i11ants, but can be dispensed with when infecting plants defective in RNAi patbwa}'S. Jt bas also been reported that some mutants of C. elegan s that -.ffect RNAj have increased endogenous transposon activity. As an experimental method. RNAi has had swift and wide ranging impact lt enables on experimenter to silence any given gene in almost any organism simply by introducing into that organism short dsRNA molecules with sequence complementary to that gene. 'I1le effectiveness with which RNAi eliminates expression of target genes is critical, as is the relative ease of the procedure. Thus, when it comes to inactivating the gene, it is much easier than disrupting the coding sequence within the genome, an operation which, even where possible, is laborious in all but the most amenable of model organisms.

&mmary

571

SUMMARY As in bacteria. transcription initiation i$ the lilOSL fJ'equently regulated step jn gene expression in eukaryotes. despite the additional s teps that can be rE>gUlated iD tb.ere organismt;. Also as in the bacteria. transcription initii300il is typical!)' regulated by proteins that bind to specific st1qUenc.es on DNA near a gene and eithet Gwitch thai gene on (activators} or s witch it off (represscm;). This mo.smvatinn of regu1atDI'y mechanism holds in the face of several complexities in the otEanizati011 and transcription of eukaryotic genes not found in bacteria. as we now sumrnruize. )\.\K:)eosomes and their modi&atioo. The PNl\ in a eu.karyotic oeU .Ls wrapped in histones to for m nudeosomes. Thus, th~ DNA sequellQ15 to wlrich the transcriptional Jl1a(jhinery lllld t11e regulatory proteins bind are in many cases occluded. Eo~ymes thai modify hi.st01l€S. by adding {or removing) small chemical groups, alter the hi,stones in rwo ways. they change how tightly the nuclrosomes are packed (and thus how aa;e:ssibtc the DNA within them is); aou they forro (or remove) binding sites for other proteins iJwolved in transcribing the gene. 0th£.rr eozymas •remodel.. the nucloosomes: they use the L'llergy from ATP hydrolysis to move the oucleosomes around, influencing which sequences are available. Many regulatOl'S and target" distft.llal5. Genes of multicellular eukaryote.s are typically controlled by more regu~ latory proteins •han their baclenal counterparts, some bound far from the gene. •f11is retlacts the larger number o[ physiotogicaJ s ignals that control a typical gen e in m ulti-

oollular organisms. 1lle elaborate IJ"anscriptkmal machinery. The enzyme RNA polymerase is (t~rgoly conserved between bacteria Md eukclryofes (Chapter 12). But tbe eukaryctic l:'.nzyme con· tains mote subunits. antl there att> some 50 or so additional proteins that bind at the typical eukaryotic promoter along with polymerase. While we do not Jcnow what many of theSE~ pro~ns do. the m~jority are essential for affident transaipt'Ofl of many genes. Many of these pl'Oteins come to the promo•er as large prowhi complexes. In eul<aryotes. jusf as we saw in bacteria. f!ctivators predominan tly work by rect'l.lilmenl. Jn lbese organisms. howevet". the activators do not recruit polymeraoo directly. or alone. Thus. they recruit the other protein romplexes required to initiate ltanSCTiption of a gtvoo geoe. RNA poly· merase itself is brought in along with thf!SB othor complexes. The actil•ator £;all recruit histone-modifying enzymes as vv~ll. and the effem or th~ modifications may help the transcription machinery h ind the promote£. The ac.tivatcli"S can interact with one or more of many different comporlimts of the transcriptional mach.i.rlel)' or the nudeosome modifiers. This explains how they can so readily work together i.n large nwnllers and variou s comlli· nations and accvunls for rhe widespread use of signal integrati.on and combinatorial control we see. particularly in multiceUuJer organisms. Some activators work £rom sites rar from the gene, requiring lhaf. the DNA between their binding sites nod the promoter loops oui. Jiow loops (;aft form over the very large distances caliE>d for jn some cases is not clear, but lt very likely involves chanBf1s in the chromatin strur.-

ture betw~en the activator binding s:ite and the promote r, bringing those two elements closer 1oget.her. DNA sequences catled i.11$ulators bind proteins that inle,rfere with the interaction between activarors bound et distanl enhanoors and their promoters. The~*! could work by inhibiting mechahisms tha1 far:ilitate looping (s uch as chang.e s in ch romatin structure). lnsuL'\tors help ensure that activators work only on the rorrecl genes. Eul..-aryotic reJW"SSon> work iv various way~. just as they do in bacteria However, the simplest anrl toORt commun mechanism S(':f$a in bacteria is for the repTessot" to bind to a site overlapping the promoter. thus blocking binc.ling of RNA poJymen~se. That mer::hanism is not lypidllly seen in e1-1karyotes. Most commonly, eulatyolic reprossors work by recruiting his1ooe mrnlili~rs that reduce transcription.. FOI' example, whereas a histone acet_y lase is typically associal&t witl1 activation, a histon£> doocetyla:>e-thaf is, an enzyme that removes acetyl groups-acts to repress a gene. In some casas.. hmg stmtches of nudeosornal DN.A can be kepl i.n a relatively inert state by appropriate nucloosome roodificalion, most notabl.f deacetylation and owthybtlion. In this way. groups of genes can be .kept in a "silf.)nt" state without the need for specific repressors bound at eacll itldividual gene. Once established. Ibis condition can maintained because the modification enzyn~ themsehres are often preferentially recruited l o nudeosomes thol are in thar Slate. Thus, the .modification slate recruits the enzymes thai produ~ that parli<:ular patle.rn of modllicariollS. Thi,; mean&" that oooo initiated, the silent s tate can be exten~ and inherited ralhereasUy. Jn some eukaryoti£ organisms, such as mammals. silent germs are also associated with methylatf:ld DN.A. Methylated sequences can either block lhe binding of the transcription machinery and acti\lators. or those sequences can specifi· cally bind a class of repressors that rectuit histone-modifying anzymp.s that repress nearby gooes. We ~lso saw how various steps in gE~ne .regulalion after transcription initiation can be regulalud_ These include transcriptionAl elongation and translation, just as we eaw in bacteria. But most stri king (and something we cHd not see in bacteria) ' s tho regulation of splicing. ln multi.c()Uular eu..karyotes the majOI:i.ty of RNAs requjre s plicing. In some ca.se6, atternativP patwros of splicing lead to different protain products. Thai proc:ess r:;an be regulated. We considered the example of sex d etermination in Droscphila, whem a r.ascade o f regulated, aUemalive splicing events deJ·eTmi.nes whether a tly develops as a male or fema le. Another Conn of gen e reguJalinn we rle~Scribed in this chaptm itwoh '6s s ma1J RNA moJeculf'.s that inhibit exvress ion of homologous genes. These RNJ\s include regulatory RNJ\.s used en animal development and others generated in plants upon viral infoction. The .DlBChanisnts by which these RNI\s inhibit expression of genes can involve destruction ot' mRNA, inhibition of tt
oo

572

Gene Regulolion in EuJ..:f)ryvtes

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Cold Spring Harbor Symposia on Quanlito.h"ve Biology. 1998. Volume 63~ Mechanisms of transcription. Cold Spring Harbor Laboratory Prcn.s. Cold Spring Harbor. New York.

Harux)Jl G.J. 2003. RNAi: A guidR to gene silencing. Cold Sprin~:; Haxboc Laboratory Press. Cold Spring Harbor. New York. Ptasho.e M. and (;ann A. 2002. Ce.nes und signcz.ls. Cold Spring Hllrbor Laboratory Press. Cold Spring Harbor,

NP.w Yorl.. White R.J. 2001. GenE> transcription: Mech anisms ond control Blackwell Science, Malden. Massachu setts.

DNA Recognition Carvie CW. and Wolberger C. 2001. Recognition o( s pecitlc DNA sequena>s. Mol. CelL 8:937- 946. Hanbon S.C. 1991. A structural taxoDumy of DNAbirlding domaifUf. Noturo 953: 715- 719.

Activation Bulger M. and Groucline M. 2002. TRAPping enhancer fund ion. Nature (.,enetics 32: 555-556.

Fry C.}. and Poterson C.L 2001. L"hromatin remooellng enzymes. Who's on first? UJIT. Bioi. 11: Rl85-Rl97. Jones K.A an d Kadonaga J.T. 2000. Explori rtg the transcripHoo-ch romatin interface. Gen es Dev. 14: l992- 19tm. Lefshn J.A. and Yamamoto K.R. 1996. Allo:st~ric effects of DNA on lr<~nsc.Tiplior•a l reguJaiOTS. Nature 392: 685-888. Malik S . and RoooRr R.G. 2000. TJ1lJ1Scriptional regulation through mediator-lib: coaclivatocs in yeasl and metazoan cells. Trends Biochem. Sci. 2&: 277- 283. Myers LC. and Kornberg RD. 2000. Mediator of ttanscrlfr tiona! regulation. Annu. Rev. Biochem. 69: 729- 749. Naar AM, Lemon B.D., and T)ian it. 2001. Transcriptiooa) ooadivalor rnmplex.es, 1\nnu. Rev. Bwchem. 70: 475-501 . Ptashne M. and Gann A. 1~97. 1ranscriptional activation by rocruitmcnt. Nature 386: 569-577.

Slnl.hl K. 199~. fundamentall.} diOerent logic of gene r~ ultttion in euk.iryotes and prokaryotes.. Cell 98: 1- 4.

Repression MaldoDado E., Ham psey M., and Rflinbef-g D. 1999. Repres-

Targeting lh~ heart or the maller. Ce1199: 455-458. Smith R.L. ~:tnd John~n A.D. 2000. Turnifls senes off by & nfi.Tup1 : A COI)S6("Ved system of transcriptional ropres-sio.n in eukaryotes. 1l'ends Bioc/lem. Sci. 25: 325-330. ~i on:

Nlldcosome Modifiers and Transcriptional R egulation Borger S.L. 2002 llistone modifications in transcriptional regula1ion. CmT. Opln. Genet. Del'. 12:142- 148. flaus A. and Owen·Hughes T. 2001. Mcchanh;ms for ATPdependent chromatin remodeling. Curr. Opin. C.renet.

Dm·. 11: 148- 1 54. Jenuwein T and Allis C.O. 2001. 1k'ansJa ting the histone code. Srrence 293: 1074-1080. Marmoi'Sltlin R. dnd Roth S. Y. 2001. Histooo a(;elyltraDsferases: PunC1ion, 1;tructure, and catalysis. Gurr. Opin. Genet. D811. tt: 155 - 161. Narlikar G.J.• Fan H.Y., anci Kingston R.E. 2002. Cooperation between complexes that regulate chromatin struv lui'£' and transcription . Ce/J 108: 475-487. Ptlleroon r:.L. and Workman f.!. 2000. Promot.er- targeting and r.hmmalin remodel ing by thP. SWJISNF rnmplex. Cun: Opin. Genet. Dett. 10: 187- 192.

Sile ncing and Imprinting Bird A.P. and Wolffe A.P. 1999. Methylation-induced re pression - Belts, braces, and chromalin. Cell 99: 4 5 1- 454 .

Gartenberg M.R. 2000. The Sir proteins of Socrhoromy,'fls CPreviSloe: Mediators of transcriptional silenciog snd much more. CutT. Opin. Micrubiol. 3: 1 32-137. Gonschling O.E. 2000. Gene si lencing; 1Wo faces of SlR2. CutT. fJiol. 10: R708- R711. Grunstein M. 1fJ98. Y6a$l heterochromatin: Regulation of its assembly and i nheritance by h i,;tonP.s. Cell 93: 325-328. Mrutienssen RA. and Colot V. 2001. DNA methylation and aptgE>neti.c inlwritance i.Jl plants and filamentous fuoRt Sciem:e 293: 1070 . Rich ards E.J. and Elgin S.C. 2002. Epigenetic £.:1Jdes for h etM t'tro()hr·omalin fonnarion aod silencing: Rounding up tho usual suspects. Cell 108: '189-500. Tilghman S.M. 1Y99. 1be sAns of ttJe laaheo; and mot h~ Genomic unprinting in mamma lian development. Cell 96: 165-1us. Wolffe A.P. 2000. Trnnscriptional control : lmpnn ting illfiulation . Curr. Bioi. 10: R463 - R465.

C ombin atorial C ontrnl and Synergy Carey M. 1996. The enhanceosom e and transcriptional synergy. Cel192: 5- 8. l ohnson A.D. 1995. Molecular mcch ani:>ms of celt type determination in budding yeast. Cun : Opi.n. C.enet. Dev. 5: !i52- 558. F~ l vo J.V., Kim T.H., Kin1 T.I<., Lin CH., Parekh B.S•• and Wathelel M.G. Hl98. Strur.lurc and function of the interferon-beta en hanceosome. Cold Spring Hal'b. Symp. Quam. Bioi. 63: 60Y- 620.

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Pawson T. and Nash P. 2000. Protein-protein interacrions define spedficily in signal transduction. Genes De''· 14:

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Dorsett D. 1999. Distant liaisons: Long-range cnhcmceTpromoter interactionti in Drosophila. C..urr. Opin. Cenel.

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West A.G., Gaszner M., and FelsenfeJd C. 2002. Insulators~ Many functions, m any me{:hanisms. C.enes Dev.. 16: 271-288.

Signals and Signal Transduction Bromberg J.f. 2001. Activation of STAT proteins and growth controL Bioessoys 23: 161-169 . Brown M.S., Ye L Rawson R.B., and Co)dslcin }.L. 2000. Regulated intramembrane proteolysis; A control mecheniJ;m conserve
IJBmell J.E. Jr. 1997. SI"ATs ancl gone regulation. Scien ce 277: 1630- 163 5.

Hm C.S. and Traisman R. 1SJ95. Transcri ptional regulation by exlracellular signal s: Mechanism and s peclticity. Ce/100: 199 - 2.11.

Bailis J.M. and Forsburg S L . 200Z. RNAi huslws heterochromatin. Genome Bioi. 3:12 . Der!li A.M. antl Haooon G.J. 2003. RNAk An ever-growing pw..zle. 1Tends Bioc.hrun Sci. 4: 100- 201 . Grewal S.l. and Moaud D. 2003. Heterochromatin anrl epigenetic control of gene e xp ression. Scitmce 301: 796-802 .

Hannon G.}. Z002. RNA interference. Nature 418; 244- 251.

l
CHAPTER

Gene Regulation during Development

here are more than 200 different cell types in a human. all of which arise from a single cell, the fertilized egg. These genetically identical cells come to differ from one another by expressing distinct sets of genes during development. For example, developing musc)e cells express specialized fonns of ac:tin, myosin, and tropomyosin that are absent in other organs s uch as the liver or kidney. To appreciate the extent of differential gene expression, consider the following. A typical invertebrate. such as a fruit fly or worm. contains approximately 15,000-20 .000 genes. whereas vertebrates con tain perhaps double this nwnber, betwoon 3U,OOU and 40,000 genes. Whole-genome microarray methods make it possible to identify which genes are expressed in a given tissue. As an example. approximately 7% or 8% (- 1500 genes} of all genes in the gen ome of the n ematode worm C. e.Iegms are expressed in the muscles (Figure 18-1). Different cell types-say. a muscle cell and a neuron-express somewhat different. but overlapping, subsets of genes. Typically. less than half of the genes expressed in one cell type are also expressed in another given cell type. and a specific cell may be df'.fined by the expression of about 100 to 200 ..signa1ure" genes that are responsible for its unique characteristics. (See Box 18-t. Microarray

T

OUTL I NE

1hree Strelegies by IM'lich Cells Are Instructed to Express Speciflc Sets of Genes during Development (p ~76)

• Examples of tt-e Three Sli~te~ tor Establishing Differertbal Gene ExpresSion (p_580)

• lhe Molecular Biology of Dm.sophilo l~s(~ 590)

Assays: Th eory and Practice.) How do rnlls that are derived from the same fertilized egg establish

different programs of gene expression? Most diifcrential gene expression is regulated at the level o f transcription initiation, and we described the basic: mechanisms of this regulation in the preceding two chapters. In the

FIG U R E 18-1 Mic:roarray grid$ COIT!fJarint eJfpre5sicn pattuns in two tissues (muscles and l'leutonS) in C elegar7s. Each arle in the grid contams a short ONA segment from the r:odir1g regt011 of a single gene irl the C elegans genome.. RNA was e
respectively). Thus. the red crdes irldicdte genes ~essed in ITliJSc:le. v.hereas the gJeen reflect genes ~essed in neurons.. The yeftow ordes lfldicate genes eJ
575

57fl

G~-tlll! R(:'Sul®on

ottrii18 /J(?11elopm(:(Jl

first half of this chapter, we dt>.scribe how cells communicate with each other during development to ensure tbat each e>..-presses the particular set of genes required for their proper development. Simple examples of each of these strategies are then described. In the second half of the chapter. we describe how these strategies are used in combination with the tran~ scriplional regulatory mechanisms described in Chapter 17 to control the development of an entire organism- in this case, the fruit fly.

THREE STRATEGIES BY WHICH CELLS ARE INSTRUCTED TO EXPRESS SPECIFIC SETS OF GENES DURING DE\'"'ELOPMENT

a fertilization

Ul1ferUized egg with lrifonn

fertilized egg wi1fl focalized

distrihorioo of RNA

RNA

b

celiA _ _ _ _ _.,.. cell B signal

FIGUJlE

18-2 The three strategies fer

initialing ctiffwe~ gene dvity durirlg (a) In SOfTIE animals, certain "matemal" RNA$ present in the egg become ~ed either l::efore or af!er F erotaz
ner«t wrth c.cll B 'D stirrllAate the rt:eeptor pesent on 1he Sllrioce d celt B. Thts ts beatlJse the iigarld'' produeed by cell Ais tethere
We have already seen how gem~ ff>.'Jlression ~an be controlled by "signals .. received by a cell from its environment. For example, the sugar lactose activates the transcription of tlte lac operon in E. coli, while viral infection activates the expression of the f)-interferon gene in mammals. In this r.hapter we focus on the strategies that are used to instruct genetically-identical coils to express distinct sets of genes and thereby differentiate into diverse c ell types. The three major strategies are mRNA localization. cell-to-cell contact, and signaling through the diffusion of a secreted signaling molet::ule (Figure 18·2). Each of these strategies is introduced briefly in the following sections.

Some mRNAs Become Locali2ed within Eggs and Embryos due to an Intrinsic Polarity in the Cyt~keleton One strategy to establish differences between two geneticaHy-identical cells is to distribute a critical regulatory molecule asymmetricnlly during cell division, thereby ensuring that the daughter cells inherit different amoW1ts of that regulator and thus fo llow different pathways of development. TypicalLy. the asymmetrically dllilrlbuted m olecule is an m.RNA. These m.RNAs can encode RNA-binding proteins or cell signaling molecules, but most often they encode transcriptional ac:tivatm:s or repressors. Despite this diversity in the hmction or their protein products, there is a common mechanism for localizing mRNAs. Typically, they are transported along elements of the cytoskeleton. ac.tin £laments. or microtubules. The asymmetry in this process is provided by the intrinsic asymmetry of these elements. Actin £laments and microtubules possess an intrinsic polarity. with directed growth at the + ends (figure 18-3). An mRNA molecuJe can be transported from one end of a cell to the otlwr by means of an ..adapter'' protein, which binds to a specific sequence within the noncoding 3'untransiated trailer (3' llTR) region of an mRNA. Adapter proteins cOntain two domains. One recognizes the 3• UTR of the mRNA~ while the other associates with a specific component of the cytoskeleton, such a... myosin. Depending on the specific adapter that is used, the mRNA· adapter complex either ··crawls'' along an actin filament, or directly moves with the + end of a growing microtubule. We will see how this basic process is used to localize mRNA determinants within the egg or to restrict a determinant lo a single daughter cell after mitosis.

CelJ,to--Cell Contact and Secreted Cell Signaling Molecules both Elicit Changes in Gene Expression in Neighboring Cell~ A cell c.-an influence which genes are expressed in neighboring cells by producing extracellular signaling proteins. These proteins are

Three Slrot~ by which C-ells Are lnStroc;ted to £xpre5S Specific Sets of Geney during Development

Microanay assays permit the genome-wide analysis of gene expression proRes. The rricroanoy, typicaly encom~g thousands to tens of thousands of kncMA1 sequences lfl"lO')Ob;ized on a !TlCfa5C(lpe slide. CCir1 be subjected to a series of ~zanoo e
codr,g

capc!!city of tre tn. rnan genome on a single slide. To •nvestigate whole-genome patterns of gene expression. the skle is hybridized with differefltialy labeled fluorescent

577

RNA probes. Conslder the case shown ;n Figure 18-1, whK:h compares gene activity in the rnusdes and neuron5 of 1he

nematode worm, c elegans. Total rnRNA was ISdated from each tissue Mid labeled With different dyes. It is possible to tabet tile musde ;nRNAs red and the neurona' mRNAs green. These two samples of labeled mRNAs are then simultaneously hybridized on the same gtass slide containing PCR fragments representing each of the nearly 20,000 genes in the C. (!/egcns genome Wlen both samples hybridize to a partia.Jar spot, or gene fratrnen~ a yellw cola 1s emitted. This hybridization result indicates that the partic:tJor gene is significantly expJessed ~ both tissues. Spots that suo~ stain red correspond to genes that are mariy expressed in 1he muscles, but not neurons. Conversely, those spots. that stain green represent genes that are e>cpressed tn neurons but not musdes.

The ~ mefhod carl be used to compare the gene expression profiles of any tv.o samples. for exarrPe, there have been extensive .studies that compare mRNA profites in normal tissues and tumors.. It is also posstie to isolate RNA from normal y~ celts, or Drosophik;J embryos, and COJ"rl)are these wi1h mutant 'y'east eels. or mutant fly ernbfya;.

synthesi2ed in the first cell and then either deposited in the plasma membrane of that cell or secreted in to the extracellular matrix. These two approaches have featu res in common, so we consider them together here . We will the n see b ow secretetl signals can be used in other ways. A given signal (of ~ither s ort) is generally recogniZf>d by a specific receptor on the surface of recipient cells. Whoo that receptor binds to the sign aling molecuiP., it triggers changes in gf!fle expw..ssion in the recipient cell. This t!OJT\mtmication from the cell surface :receptor to the nucleus often involves signal transduction pathways of the sort we considered in Olapter 17. Here we summarize a few basic features Q{ these pathways. Sometimes llganil-receptor interactions induce an enzymatic cascade that ultimat~Jy modifies regulatory pmteins almady present in the nucleus (Figure 1&4a). In other cases, activated m ceptors cause the release of DNA-binding proteins from the et11l surface or cytop lasm into the nucleus (Figure 18-4b). These regulatory proteins bind to specific DNA recognition sequences and either activate or repress gene expression. Ligand binding can also cause proteolytic cleavage of the receptor. Up on cleavage, the intracytoplasmic domain of tlm receptor is released fro m the cell surface and enters the nucle us, where it associates with DNA-hinding p roteins and influences how those proteins regulate transcription of the associated genes (Figure 18-4c}. For example, the transported protein might co11vert what was a transcriptional repressor into an activator. In this case. target gen es that were formerly repressed prior to sign aling a re n ow induced. W e will consider examples of each of these variations in cell signaling in this chapter.

5'

RNA

0 f I Ci U RE 18-l An adapter prottirl binds to specific sequences within the 3' UTR of tile ~NA.. The adapter also btnt!s to fTlYOSll\ Vllfich "~s.· along the actin fil.arnent 10 a directed fashion, from the • • end to the grov.;ng "+e end of the fiamenl

5'18

Gell(' Regulal ivn durifl8 DtNelopment

Signaling rnolHC:ules that remain on the s urface control gene expression on1y in those cells that are in d irect. physiCC'll contact with the signaling cell. We refer to this process as cell-to-cell contact. In contrast. signaling molecules that are secreted in to the extracellular matrix can work over greater distances. Some travel over a distance of just 1 or 2 c:ell diameters, whereas others can act over a Tange of 50 cells or more. l ong-range signaling molecules are somPtimes responsible fo r positional infom1ation, whic.:b is discussed in the next section.

Ceil

membrane

' f

t

kifla~ ~scade causes~

ptlorytatioo d

~DNA-bwdng ~

prQtein in 0\JdeUs

~':;;;t'. .

b

-

~

J release of DNA-

, binding protein

intonudeus

intracytoplasmte d~

A recurring theme in development is the importance of a cell's position within a deVt:tloping embryo or o.rgan in cletennining what it will become. Cells located at the front of a fr uit Oy embryo (that is. in anterior regions) will form portions of the e:1dult head such as the antenna or h.rain but will no t develop into posterior structures such as thE> abdomen or genitalia. Cells located o n the top, or dorsal, s urface of a frog embryo can develop in to portions of the backbone in the tadpole or adult but do not form ventral~ or ..belly... tissues such as the gut. These exampl es illustrate the fact that the fata of a cellwhat it will become in the .adult-is constrained by its location in the developing embryo. Th e infiut1nce of tocation on d evelopment is called positional information. The most common way of establishing positional information involves a simple extension of one of the strategies we have a lready encountered in (..napte r 17 - the use of secreted signaling molecules {Figure 18-5) . A small group of celts synthesize and secrete a signaling mo lecule that becomes distributed in an extraceUul~ gradient (Figure 18-Sa). Cells located near the ..source" recei ve high concentrations of the secreted p rotein and develop into 8 particular cell typp. Those cells located at progressivoly farth er distances follow different pathways of deve lo pment as a result of receiv1ng lowP.r c.oot:entrations of th~ signaling molecule. Signaling molecules that control position information are somctimcs called morphogens. Cells located near the source of the morphogen receive high concentration s of the signaling

c

deave-~

Gradients of Secreted Signaling Molecules Can Instruct Cells to Follow Differe nt Pathways of Development based on Their Location

l 1..-

1

mole~

and, the refore, experie nce peak

activation of the specific cell surface receptors that bind it. In contrast. cells locatP.cl far from the S()ttrce receive low leVft)s nf the signal, and consequently, only a small fraction of their cell surface receptors are a ctivated. Consider a IOW of three ceJls adjacent to 8 source of a secreted morph ogen. Something like 1 ,000 receptors are activated in the firSt cell. while only 500 receptors are activated in the next cell. transport into nucleus lo aSSOdate with ONA-l>flding protein

1S.4 Different mecNnml$ of signal tnnsdudion. A ligand (or~51gm4ing molecule") bends to a cell SU'fac.e f«Ep!Of (a) The actJvated receptonnduces lat€nt cellular k111a5eS tticlt !Jitllil(j~ Cilll5e the phosphofyfobon of ON/\-btnding proteins v.ithal the ru:leus. This phos~ c.auses the regulatory protein (0 activate (Of repress) the~ of spedfic genes. (b) The ac1rviltf!d recepklf releases a dormant DNA~ prctEV~ from d-e cytoplasm so that It ca1 t1CMt enrer the flUdeus Once 111 the nodeus, tile regulatory proten activates (or represses) tf1e transaPfun d specifte genes. (c) 1he actNated receptor IS d eaved by cdular proteases that cause a C·tamm portlon of the rec.ep10r lo enler the flt.Jdeus and •nteract With speotK ON~g J.lro(etnS. The resulting protein ~ adivate:.lhe tr~n of speofic genes. FIGURE

Tbree Strategies by whith Cells Are Instruc:led to Express Specific Sl!ts of Gen es d uring Development

a

18-5 Adusterofcelb produces B signaling Rdecufe. cw morphogen. that diffuses through

FIGURE

ED==o···a·· o source

b

number of

TTT

~pa 1 1 l

levelS of acti\!ated transcripllon facklf in nuclei

d

579

~ 00

J~.c

---"~--

gene A

gene B

gene C

OFF

gene A I OFF

and just 200 in the n ext (Figure 18-Sb). These different leveJs of receptor occupancy are directly responsible for di:ffen:mtiaJ ge ne expression in the responding cells. As we have rean, binding of signaling molecules to cell surface receptors leads (in on e way or another) to an increase in the concentration of specific transcriptional regulators, in an active form, in the nucleus of the cell. Each receptor r.ontrols a specific t~rlptional regulator {or regula tors), and this controls expression of particular genes. The number of cell s urface recepto.rs that are activated by the binding of a morphogen determines how many molecules of the particular regulatory protein appear in the nucleus. The cell closest to the morphogen source-oontaining 1,000 activated receptors- will possess high ooncentrations of the transcriptional activa tor in its nucleus (Figure 18-5c). In contrast. the cells located farther from the source contain intennediate, and low levels, of the activator, respectively. Thus, there is a correlation between the number of activated receptors on the cell surface, and the amount of transcriptiona l regt.,Jlator present in the nucleus. n ow are these rli fferent levels of the same t ranscriptional regulator able to trigger diffP.rent patterns of gene ID..'p ression in these difforent cells? In Chapter 16 we learned fuat a small L:hange in the levels of the 11. rep ressor determines whether an infected bacterial ceJl is lysed or lysogenized~ Similarly, small changes in the amount of morphogen. and hence small differences in the levels of a transcriptional regulator within

the extract!llul• matrix. (a) Cels 1, 2, and 3 receiVe progres~ IOINE!f amou-ots of the signaling mclecule since they are located prosreswely E.Yther frorn !he S()U(Ce. (b) c~ t, ~. anrl $ con~n ~ ~ fltJ~ of activated surfclce receptors. (c) The three eels contain different levels of one or more regulaloJY proteins. In the simplest 5Ca'lario. th:re IS a inear correlation beMeen the numbef ot actiVated cell surfuce r~~ and the
A, because it contains 111teanediate 1€\/els of the reguLatory factor These levels are not Sllfftcier.t to ae11vate gene A. Fioalfy. cell 3 contaions the lowest levels of lte regulatory fadOl and expresses Ofiy gene c sne expression of geres Ae~oo 8 recp.lites ~er leYels.

the nucleus, dP.terrnine ceU iclentity. Cells that contain high concentra-tions of a given transcriptional regulator express a variety of target genes that are inactive in cells containing intnrmerliate or low levels of the re~ ulalor (Figure 18-Sd). 'The differential regulation of gene expl'ession by d ifferent concentrations of a regulatory protein is one of the most impor-tant and pervasive mechanisms encountered in developmental biology. We ·will consider several examples in the course of this chapter.

EXAMPLES OF THE THREE STRATEGIES FOR ESTABLISHING DIFFERENTIAL GENE EXPRESSION The Localized Ashl Repressor Controls Mating Type in Yea~t by Silencing the HO Gene Before describing mRNA Localization in animal embryos. we first consider a case from a relatively simple singlEH:~ll eukaryote. the yeast S.. cere~isioe. This yeast can grow as haploid cells that divide hy burlding (Figure 1 B-6). Replicated chromosomes are distributed between

- - - - - - - -- --

FIGUR£ 18--6 A haploidyeast.:el cl mati11g type • undergoes budding to produce a mother c:ell artd stnalla daughter eeL (a) Initially, both eels are mating type a. but sometimes the ITlOC:her eel C<Jn tJJ1dergo s'l\litc::hngto then type (b) The

a

a daugtlter

daughter eel comet vndergo sv..itching since it 's t~nabfe to express the HO gene due to the loo!lited Ash 1 t~l repres501: In contrast. t1le mother cell can S\Mtd"l because it J.rls Ash 1 and is able to ecpress HO.

a

a

a mother

t~

a b HOOFF

e

a daughter

HO ON

a mother

(l

Examples of the Three Strate8ie5 for Estahlis/ling f)jfferential Gene Expression

two asymmetric CP.lls-the larger progenitor cell, or mother cell, and a smaller bud. or daughter rell (Figure 18-68). These cells can exist as either of two mating types. called a and a, as discussed in Chapters 10 and 17_ A mother cell and its daughter cell can exhibit different mating types. This difference arises by a process called mating-type switching. After budding to prodl.lce a daughter. a mother cell can ..switch., mating type, with, for example. an a cell giving rise to an a daughter, but subsequently switching to the « mating type (Fig\Jre 18-6b). Switching is COlltrollE>d by the product of the HO gene. We saw in Chapter 10 that the HO protein is a sequence-specific endonuclease. HO triggers gene conversion within the mating-type locus by creating a double-strand bw.ak at one of the two silent mating-type cassettes. We also saw in Chapter 17 how HO is activated in the mother cell. lt is kept s ilent in the daughter cell due to the selective expression of a repressor called Ashl (Figure 16-7), and this is why the daughter cell does n ot switch mating type. The ash1 gene is transcribed in the mother cell prior to budding, but the encoded RNA becomes localized within the daughter cell through the following process. During

a

b

myosin-driven

.. myosinV

~

_ _ _...,.. 5' - -

-=- 3'

0 polarized actin filament mother eel

18-7 L~lbatioo oloshl mRNAduriag budding. (a)The os/JI gene is tJclnso1bed 1n the 1'1'10ther ceUdunng budding The encoded mRNA moves from 1he mother ceO into lhe bud by sltdlng along polarued octn filaments. Movement ts dJrated
561

582

Geflf! lfP.l{ulotion during Devel opmetlt

budding, the ash! mRNA attaches to the growing ends of microtubules. Sevt>.rnl proteins fu nction as ..adApters'" that bind the a· trrR of the ash1 mRNA and also to the microtubules. The microtubules extend from tht~ nucleus of the mother cell to the site of budding, and in thjs way the ashl mRNA is transported to the daughter eel). Once localized within tht~ daughter cell, thl:l ash 1 m RNA i~ translated into a rapressor protein that binds to, and inhibits the transcription of. the HO gene. This silencing of HO expres.~ion in th e daughter cell prevents that cell from undergoing mating-type switching. 1n the second half of th is chapter, we will see the localization of mRNAs userl in the development of the Drosophila embryo. Once again this localization is mediated by adapter proteins that bind to tht>. mRNAs, specifically. to sequences found in their 3' UTRs. (See Box 18-2. Revir.w of Cytoskeleton: AsymmetJ:y and Growth.) A second general principle that emerges from studies on yeast matingtype switching is seen again whP.n we consirler Drosophila development: the interplay between broadly d istributed activators anti localized repressors to establish precise patterns of gene fl.Xpression within

Box 11J..:l Review of Cytoskeleton: Asymmetry and Growth

The cytoskeleton is CIJil'l'05ed of thee types of filaments: intermedate filaments. actin filaments, and mlCioo.hks. Actin filomerns and microll.Vules are used to localize specifk mRNAs in a vanety of different eel types, indl..King buddng yeast and Dtosophlo ~ Actin filaments ore rnmposed of polymers of act1n The ac:1ln pol}rners are organtzed as two paralld

helices that form a corrplete Mist every 37 nm Each actin f1"l()()()mer tS located •n the same orientation v.ithin the polymer, and as a result. actin filaments contaW-1 a dear polarity. lhe ph..rs ( + ) end 8f0Vv'S more rapidly than 1he mif1us (- ) end. and consequen11y, mRNAs stated for localization move along with the ~8 '-+"end (Box 18-2 figure 1).

0

·- - - - - - - -- - - -

- - -----

1 5tnlctUfes of the actirl monomeu ncl filament. Uystat ~ure ot the actin monomer. (a) lhe four domains of the monomer are 5ha.ovn. in different colors. with AlP (in red and yelow) i41 the center. The "'-!' end of the I'T'DOOI1'lef is at the tcp; the "+" end is at the bottom (OttEibein LR., Graceffa P... and DocTinguez R 2001. Science 293- 708 - / 11.) Image ~ed With rv1o!Scopt. BobSCJl)l. afl
18-2

F1C\.1 u :

Bxompl<~ of the Three Stratf:gies for £..<;~(lb/islling DiffenmtirJI CellfJ Expre:;;sion

Box 18-2 (Continued}

MicrottJbules are composed of pdyrras of a protm ccMed tubulirl, vJnch is a heterodimer c0flll05€d of related o i30d p chalf'ls. Tubt.llin heterodimro form extended, il5yfl1metriC protofilaments. Each 1:\..h.jn heterocirner i.s located in the same ooentanon v..thin the protofdament Thirteen different protcftaments assooate to form a cylrdical microtubule. and al of the p-otofilaments are aligned in paralel. Thus, as seen fur actin fi& ments, thee is afl irtroslc polarity in micr<>tJ.JblJes. vvith a ~ growing"+" end ctnd more stai:Ae "-.,end (Box JB-2 f~re 2) Both actin and tth.M funcoon as enzymes. Acfu catalyzes the hydrolysis of All' to ADP. while tubulin hydrolyzes GTP to GIY. These enzymatic ilCtivi6es are responsi'Jie for the dynamic growth. or "treadmJing," seen for actin filaments and micro-

tublAes. Typkaly, it is the actin or tl.tlulin sutunits at the .._ .. end of the fit.ament that mediate the hydro¥ls of P\rP or GTP, i30d as a result. these stb xnts are somewhat unstable and lost from the "- n eod In contrast.. nevvly added subt.rits at the "+" end have not hydrolyzed AlP Cf GTP, and this causes them to be more stable components of the filament Directed growth of actin fkments a rniaotubllles at the "+" ends depeflds on a vanety of proteins that associate with the cytoskeleton. One suulin protoftlaments at the "+" ends of miaotubules.

b tuhofin ~erodimer (== rnierotul:lule Sltlontt)

50nm

0

2 Structures of the tu~Mtn lftOI'Iomer and filament. (a) 1he crystal structute ot the tubtlm monomer shows the a subunit m turQJOise and the ~ sul:uVt in purple. TI-e GTP molecUes in each subunit are shown in red. (lowe 1~ l 1H, Downing KH.. and NogaJes E 200 I. J. fWol. Biol 3 13: 1045- 105J.} Image prepared wm MolScript , BobSoipt and ~
BOX

Sists

1&-2

F 1C u R E

of adjacent mooomers assembled in the same orientation.

individual cells. In y~ . the SWIS protein is responsible for activating of the HOgene (sec Chapter 17). This activator is present hoth in the mother C()ll and the daughter reH during budding, but its ability to turn on HO is restrid€d to the mother cell because of the presence of the Ashl repre.ssor in the daughter celt In other words, Ashl keeps the HO gene off in the daughter rnll despite the presence of SW15. expr~ion

A Localized mRNA Initiates Muscle Differentiation in the Sea Squirt Embryo Localized mRNAs can establish differential gene e:)..-pression among the genetically-identical cells of a developing embryo. Just as the fate of the daughter cell is constrained by its inheritance of the ash1 mRJ\JA in yeast, the cells in a d evelopiug embryo can be instructed to follow specific pathways of development through th~ inheritance of localized mRNA& (See Box 18-3, Overview of Cinna Development.) In the case of muscle differentiation in sea squir ts, a major determinant for programming cells to form muscle is a regulatory protein called Macho-1. Macho·t mRNA is initially distributed throughout the cytoplasm of unfertilized eggs lJut becomes restricted to the v~ taJ (bottom) cytoplasm shortly after fertilization (Figure 1R-8). It is ultimately inherited by just two of the cells in eight-cell embryos, and as a result thm;e two cells go on to form the tail muscles. The Macho-1 mRNA encodes a zinc finger DNA-binding protein that is believed to activate the tran&:-Tiption of muscle-specific genes. such as actin and myosin. Thus. these genes arP. P.xp.ressed only in muscles because Macho-1 is made only in those cells. In the second part of this chapter, we will see how regulatory proteins synthesized from localized m RNAs in the Drosophila embryo activate and repress gene expression and control the form~tion of diff~rent ~u types. Cell~to--Cell Contact

Elicits Differential Gene Expression in the Sporulating Bacterium, B. subtilis

The second ma jor strategy for establishing differential gene expression is cell-to-cell contact. Again, we begin our discussion with a relatively simple case, this one from the bacterium Bacillm; subtilis. Under adverse conditions. B. subtilis t:an form spores. The first step in this process is the formation of .a septum at an asymmetric Jocation within the sporangium. the progenitor of the spore. The septum prorluc~s two

18-8 the PMcho-1 mRHA becomes locali'red in~ fertilized egg. (a) rhe mRNAis Ill!bally distrbJted throughout tile <:y1oplasm oJ urfertiltZed eggs. #. fenilizatm 1he egg rs Induced t o l.ll1dergo a highly asymmet· ric division to produce a smal polar OOdy (top). AJ this~. the Macr.o-l mRNA becomEs Joc.at.. aed to bottom (vegetal) regions. ShortJy thereafter, and well belore the first dvistOn of the I eel embryo. the Macho"I mRNA undecgoes a second wave of localtzabon This oro.ss &.n1g the secood l'jghly asynmetric rreK:k di\IISiorl of the egg The Macho- I mf
a

polar bodies

A l

seCOnd phase of segegatioo

unrertiized egg

- - - . ..-.musde

Exampks of the Three Strategies for Establishirlg Differenlial C.ene Expression

565

reHs of differing size that remain attached through abutting membranes. The smaller cell is called the forespore; it ultimately forms the spore. The larger cell is called the mother cell; it aids the development of the spore (Figure 18·9). The forespore influences t he expression of genes in the neighboring mother ccll, as follows.

AcUt sea squirts are irrfrdlile filter-feeders that ive ll"l shallow ocean watefs (Box 18-3 Figl.xe 1). They ore ~es and possess both sperm and eggs. They can self-fenize tut prefer not to do so. lnstedd, sperm from one arilld t)1X<11fy feraizes eggs from another; The resUtl'lg embryos are ~ent and c:ornposed of ~ few eels {tlt;OO'eds. rather tl-a1 the tens of thousands seen i1 vertebrate embryos). These embryos develop rapidly into 5\Mrrrning tadpdes just 18 - 24 txus after fertiza. tion Complete eel lineages are kiDM1 for each of the rnajCir tissues. This m~es it possi:Jie fD VISI.JC!IIze me sequence of eel divisiofls from ferti\izatioo to the foonatioo of spedaized liSSJeS in the tildpole. for example, the tadpole tal rontaiiS 36-40 musde eels (depend'"r)g oo the species), and the lineage that fOOT6 these cells c.YI be tram:! bad< to the feniled egg

The tail muscles represent the first cell lineage that was visualized in any onlrnal embryo, about lOO years ago. This visualiziltioo was made possible by a yellow pigment that is present in the unfffiilized eggs of certain dSCK.Iioos.. The pigment is initially distribllted throughout the egg but

becomes localized to vegetal (bottom) regions shortly after fertilization ( Box 18-3 Fig~Xe 2 ) . The localized pigment is inherited by just two of the cetfs, or blastorneres, in eight-cell embryos. These two eels give rise ro most of the tail rnusdes in the tadpdc. The yellow pigment Is not the actual musde "determinant"- that is, it ls. not responsible for programm~ng

the cells to form muscle. Rather, the

pigment is merely a visible markeJ that is associated with the determinant

18-3 F I<; U R£ 1 Cioncr life cyde. The adult sea squirt is sOOIM"' in the t.wer left panel. 1he orange material corresponds to develop!ng eggs and the v.-hite is the sperm Progressivt.'ly older errtJryos are shown in the remaining panels. The embryos in the third row are t.lfldergOtng g~. A~ l~ can be seen in the lowe' right panel This stage is reached 12- 14 rours cltef fertilization (see the 1-c.eiJ ernl:lf}'O 11 top center panel). (Sotxce: Repfo&xed from Oehal et al 2002. The draft ge-t0111e of Ciona intestinolis: Insights into dodate and vertebrate OOgins. !C enre 298: 2 157-2167, fig 2, p.215S.) 80 X

C..ene Regulotion

diJI'itW [Jeve/opmetJf

Box 18-3 (Contifwed)

a ectoderm

vegetal pole

b

anterior

posleriof vegetal pole

c

developing tail muscles

view from vegetal pole

18 -l F' G U R E 1 laity deavages in Jbddicms. The fertiiz.ed. 1-ce!l asadian embryo contawls a number of localized "deterrrinants" thclt con1rol the development of different tissues. fOf example. the yellow ooerminant is irt.ec\ted by eels tf-0t form the t:il muscles. The red detetTT11r\aflt is irtrented by cells that form the ern1oderm. or gtn. (Sot.ree; Redrawn from Gilbert 5.E 1997. Developmentc:JI ~ 5th editioo, p. 179.1lg 5.17. Copynghl ~ 19S7 Sin.1oef Associares.. Used with pemi>Sion.)

80 X"

The forespore contains an active form of a specific a factor, uf, which is inactive in the mother celL ln Chapter 16 we saw how a factors associate with RNA polymerase and select specific target promoters for expression. crF activates the spoiiR gene which encodes a secreted signaling protein. SpoHR is secreted into the space between the abutting membranes of the mother celJ and the forespore where it triggers the protP.olytic proc~ssing of pro-a£ in the mother cell. Pro-uE is a n inadive precursor of the uE factor. The pro-a£ protein contains an N-terminal inhibitory domain that blocks
Gen(1 Regulation during DevE/ppment

Box 18-~ (Continued)

a

neural ectoderm

endoderm

vegetal pole

b

vegetal pde

c

dewloping ~mt.Jscles

view frofl'l vegetal pole

I 8-3 FIGURE 2 Early deavages in Asddlons. The fertiized, 1-cell asodian errbyo conta.ns a l'lUrT'ber of localtzed "determtnants" that control the deve!Clplrent of different tissues. For exarrple. the ~ determinant is inllerited by cells that fonn the tail muscles The red determinant LS irtlerited by celts that foon che endoderm. Of gut {Sotxce: Redrawn from Gilbert S.E. 1997. Developmental bi~ 5tl'1 edttion, p.. 179. fig S.l1 Copynght C 1997 Sinauer Associates. Used with pennissim.) BOX

The forespore contains an active form of a specific a factor. aF, whi(;h is inadive in the mother cell. ln Chapter 16 we saw how

a fa<.:tors assodate with RNA p olymerase and select specific targe t promoters for expression. aF adivates the spollR gene which encodes a secreted signaling protein. SpoiiR is secreted in to the space between the abutting membranes of the mother cell and the forespore where it triggers the proteolytic processing of pro-ul' in the mother (;e)). PTo-uE is an inactive precursor of the aE factor. The pro-aE protein contains anN-terminal inhibitory domai n t hat bl od~s uE activity and tethers the protein to the membrane of the mother ooll (Figure 1 8-9). SpoUR induces the proteolytic cleavage of the N-terminal pep tide and the release of the mature and active form of a£ from the memb rane. u £ acOvates a set of genes in the mother cell that is distinct from those exp resserl in the forespore. In this example. SpoHR functions as a signaling molecule that acts at

Examples of the 1'hree Slrotegir:s for Es1abli8hii1g Differen

~~{1JTI SpciiR

F I G U R IE 18-9 Asymmetric geM activity in the mothef cetl •nd fCKeSpOre of B. subtilis depends on the activation of diffet-ent classes of 6 facton.. r~ spo!IR gene is ac!Mlted by lher cd (Source: Losick R and Stralger P. 1996. Spc:ndatJon
the interface between the forespore a nd the mother cell and elicits difftrren tial gene e xpression in the abutting mother t:ell through t he processing of c E. Induction r~uires cell-to-cell contact be..;ause tl\c foresporc produces sma ll quantities of SpollR that can interact with the abutting mothe r cell but which ar-e insufficient to elicit the processing of erE iJ1 the other cells of the population.

A Skin-Nel've Regulatory Switch Is Controlled by Notch Signaling in the Insect CNS We now tum to an example of cell-to-cell contact in a n animal embryo that is surprisingly similar to the one just des<.:ribed in B. subtilis. In that earlier e.xarople , SpollR causes the proteolytic activation of ali, which, in its active state, dirt1(;ts RNA p olymerase to the promoter scquenc(,)S of specific genes. In the foHowing example. a ceU surface receptor is cleaved and the intracytoplasmic d omain moves to the n ucleus w here it binds a sequence-specific DNA-binding protein that activates the transcription of selected genes. For this example, w e m u.st first briefly describe the developu1.ent of the ventral nerve cord in inset:t embryos (Figure 1& 10). This nerve cord functions in a manner that is roughly com parable to the spinal cord of humans. It arises from a sheet of cells rnlled the neurogenic ectodcna This tissue is subdivided into two cell populations~ one group remains on the surface of the embryo and fonns ventral skin (or epidermis); the other p opulation moves inside the embryo to form the neurons of the ventral nerve cord (Figure 18-108). This decision about whether to become skin or neuron is reinforced by signaling between the two populations. The developing neurons contain a signaling molecule on lheir surface called Delta, which binds to a Teceptor on the skin cells called Notch (Figure 18- lOb). Th e activation of the N ot<~h receptor on sl
Gene Re8 ulGiiCIJ1 dtJtinljl Development

! b lateral irhtJjtjOO of surroUlding eels by ne\I'Qblasl

of Notch {Notc.h1f.) to be released from the cell membrane and enter nuclei, w here it associeites with a DNA~binding protein called Su(H}. The resulting Su(H)-Notch1c complex activates ge nes that encode transcriptional repressors whicl1 bloc.k the development of neurons. Notch signaling does not cause a simple indu(:fion of th e Su(H) activator protein but instead triggers an on/off regulatory swit(;h. lo the absence of signaling. Su(H) is associated with several proteins. induding Hairless. CtBP. ancl Gmu cho {Figur-e 18-11). S u(H) com" plexed with any of these proteins adively represses Notch target genes. When NotchJt ' enters the nucleus. it displaces the repressor proteins in compJex with Su(H), twning that protein into an adivator instead. Thus . Su{H) now activates the very same gent:JS that it fonnerly repressed. Delta-Notch signaling depends on cell-to"cell contact. The cells that present the Delta ligand (neuronaJ precursors) must be in direct physical contar;t with the cells that contain the Notch receptor {epidermis) in order to activate Notch ~igna Hng and inhibit neuronal dufe rentiation. ln the next SE)(:tim1 we will see an example of a secn::~ted signaling molecule that influences gene expression in cells located far from those that send I he signal.

A Gradient of the S011ic Hedgehog Morphogen Controls the Fonnation of Different Neurons in the Vertebrate Neural Tube

Fl GU R E

18-10

The neuwogeric ecto-

derm fonns two major cell types: neurons and skin cells (or epidermis). (a) Cetls en 1he early oetAOgenic ooodem can tonn either type of cell ~ ooce one ct the ceWs begiffi to foon a neurm q "neuroblast" (dati< cell m the a.>n1er of the grid of cells), it iflhibcts all of the negttxmg eels that it directly touches. (b) This •rhbltion c.auses most of tte eels to rerna~n on the surface of the embryo and foon skir~ ceiCs. In contrast. tte developing neuCJ1 moves rnto1he erriJrJ(l ~and brns neurons. (Source· llt1oto from Skeath !.B. and Cclrroll S.B. 1992. Regl.llalion of P'~ ge11e e<pr~ and cell fclte dunng narcblast segreg&JOn 1r1 theDra>ophio ~ ~elapmer1t

114: 939 46.)

We now tum to an example of a long-nmge signMling molet;ule, a morphogen. that imposes positional information on a developing organ. For this example. we continue our discussion of neuronal differentiation, but this time we consider the neural tube of vertebrates. (n all vertebral~ embryos. there is a stage when reUs located along the future back- the dorsal ectoderm-move in a coordinated fashion towa.rrl internal t"egions of the embryo and fonn the neural tllbe, the forerunner of the adult spinal cord. Cells located in the ventralmost region of the neural tube form a specialized structure called the floorplate {Figure 'lR-12). The floorplate is the site of expression of a secreted cell signaHng mt'.llecule called Sorric hedgehog (Shh). which functions as a gradient morphogen. Shh is secreted from the floorplate and furrns an extrat;ellular gmdicnt in the ventral half of the neural tube (figwe 18-12a). Neurons develop within the neural tube into different cell types based on the amount of Shh protein they receive. This is dclermincd by their loca· tion relative to the floorplate; <.:ells located near the floorp late reuJive lhe highest <.:oncentrations of Shb. while those located farther away receive lower levels. The extracellular Shh gradient leads to different degrees of activation of Shh receptors in different ce lls in th~ neural tube. The Shh gradient specifies at leas• four different types of neurons (Figure 1B-12b), Cells Jacated nt:ar the floorplat~-1hose that receive the highest concentrations of Shh- have a high number of Shh rt:lL'Hptors activated on their surface. This instructs those cells to form a neuronal cell type called V3, whkh is distinct from the other neurons that arise from the Shh gradient. Cells located in more laterol regions of the neural tube (farther from the floo rpJate) receive progressively l ower levels of the ShlJ protein. This results in fewer Shh receptors being activated in those c~Us. whkh th~refo.re become mutbrneurons. Yet

Exampl es of flle Three Strategies far Establishing Differential Gf'.rte Expres.sion

589

lower levels of Shh direct the formation of the V2 and Vt inte.rnewons, respfX.:tively (Figure 1B-12b). How d oos this differential activation of Shh receptors produce different cell types? The activation of the Shh receplO£ causes a transcriptional activator called Gli to activate the expression of spocific .. target" genes. The induction of the Cli activator is controlled, in part. by its rllgulated transport into the nucleus. Binding of Shh to its receptor on the cell surface allows a prev iously inodive form of CH to enter the nucleus of that cell in an active form. The e.J\.1.racellulac Shh gradient present in the neural tube thus leads to the formation of a corresponding Gli activator gradient. That is. lhe amount of active Cli in the nudeu s of any given cell dep ends on how far that cell is hom the floorplate- the closer it is, the higher the concentration ofGii. Once in the nucleus. Cli activates gene expressio n in a concentration-dep end ent fashion. Peak concentrations of Gli, present in cells immediately adjacent to the floorplate, activate target genes needed fur the differentiation of the V3 neu rons. Slightly lower levels of Gli activate target genes that spe<..ify the formation of motorneurons.

while intermediate and low levels of Gli induce the formation of the V2 and Vt interneurons, respectively. We will see, in the next section, that the different binding affinities of Cli recognition sequences within the .regulatory DNAs of the various target genes

likely play an important role in this diffen mtia) regulation of Shh-Cli target genes. Thus, Vt genes can be a<..tivated by low levels of Cli because they have h igh-affinity recognition sequences for that activator in the ir nearby regulatory DNA. In contrast, V3 target genes might contain regulatory DNA with low-affinity Gli recogni-

tion sequences that can be activated only by peak levels of Shh signaling and the Cli activator. This prindple of a regulatory gradient p roducing multiple ••thresho lds'" of gene expression and cell differentiation is again illush-att:Jd particularly well in the early Drosophila embryo. a

b

FIGURE 18-12 Formalion of diffefent neurons fn the vettebmte neural tube. {a) The secreted S@1aling molecule Sonic hcdgetlog (Shh) is expressed 1r1 the f~ of the developire neural ttbe (see the bfown Orde at the bottom of the diagram). The 'Shh proten dif. fuses through tile extraceAAar matnx of the ~ ttbe. The highest levels are Pfesetlt in ventral {botlom) regJOOS Clod progressively lower in mote krterci ~ (anOM) (b) The graded distn bution of the Shh protein &eMs ro the formation of dlst\nct neuooal eel types tn the ~ntral h.?Jf or the neural tube. H.gt1 and interrrle&ate levels lead fO the devclcpmert o1 tre V3 tleJrOOS and mctomarrons, ~. LoW and lowest levels lead to ttle cleveJopmf!Jlt of the V2 an
I

I

neurotlal repressor gene {Notch target genes)

FIG 0 R E 18~ 11 Notd\~Su(H) regulatory switd\. "fu! developing neuron (neuronal prewrsor wK) dces not express !'lellronat repressor

genes (~} l'hese gefleS are kept off by a ONA-binding protein called SU(H) at1d associ· a1ed repcesscr prot£>111S (H~. CtlW. Groocho)- The net!fCf'\al precur.;or cell e.presses a Slglialing mdectlle. called IJelta, thar ts tethered to the cell surface. Delta bhl to the Notch re<eptor '" neighl:xnlg. celts that are tn direct physical rontart IMth the neuroo. Delta-Notch M1tera<:tinr1g eels. ll'llhdl dffferenti· ate i.-.to ep4dermis. The activoted Notch recep10r is d~ by celltJar prDteases (scissots) ar.d the tntlocytoplasmic regKJn of the recepfCf ts released into ltle nudeus. This piece of the Notch protan c.auses the SU(H) regulanty protein to fw<;l,oo as an ac:tiv.ltor rather than a reprESSOr. As o resuft, 1he nettrooal repressor genes are adiViltl:d tr1 the epidefl'l'laf eels so that they cannot develop il'l(o neurons

THE MOLECULAR BIOWGY OF DROSOPHILA EMBRYOGENESIS rn the remainjng sections oi this chapter we focus on the early embryonic development of the fwit fly, Drosophila m elanogaster. The molecular detai Is of h ow developmem is regulated are better understood in this system than in any other animal embryo. The various mechanisms of cell communication discussed in the first half of this chapter, and those oi gene regula tion d iscussed in the previous c hapters, are brought together in this ~Xil mplc. Localized determinants and cell signaling pathways are both used to establish positional information lhnt result in gradients of regul a~

tory proteins thnl pattern the anterior-p osterior (head-tail) and dorsaJ~ ventral (bad.. ~belly) body axes. These regulatory proteins-activators and repressors- c.;ontrol the expression of gt>nes whose products defme different regions of the embryo. A recurring theme is the use of complex regu latory DNAs- particularly complex enhancers- to bring transcriptional activators and repressors to genes where they fum:lion in a combinatorial manner to produce sharp on/off patterns orgene expression.

An Overview of l>Tosophila Embryogenesis Life begins fo r the fruit fly as it does for humans: adult males inseminate females. A single sperm ceJI enters a mature egg, and the haploid sperm and egg nudci fuse to form a diploid. "zygotic .. nucleus. This nucleus u ndergoes a series of nearly synchronous divisions within the ' :entral regions of 1he egg. Because there are no plasma membran~s s~parating the nuclei, the embryo now becomes what is call ed a syncitium - t hat is, a single cell with multiple nudei. With the next series of divisions. the nuclei begin to migrate toward the <:ortex or pt:.riphery of the egg. Once located in the cortElx. the n\td ei undergo another three divisions leadi ng to the formatinn of a monolayer of approximately 6,000 nuclei surrounding the centra) yolk. During a 1-hottr period, from 2 to 3 hours after fertiH2ation, t."P. tl membrane5 form between adjacen t nuclei. Before the formation of c~ll membranes. the nuclei are totipotent or uncommitted; they have not yet taken on an identity and can still give rise 10 any cell type. j ust after celluJarization, however. n uclei have becomP. irreversibly "determined.. to differentiate into specific tissues in th ~ adult fly. This process is descriLed in Box 18-4, Overvi~w of Drosophila Devclopmf.!nt. The molecular mechanisms res pon!1ible for this dramatic process of dete.nnination are described in the remaining set;lions of thiN chapter.

A Morphogen Gradient Controls Dorsal, Ventral Patterning of the Drosc(>hila Embryo The dorsal-ventral patlflcning of the early Drosophila embryo is controlled by a regulatory protein caJJed Dorsal. which is initially distributed throughout the cytoplasm of the nnfortiHzed egg. Afte r fertilization , and aftor the nuclei reach the cortex of t he embry-o. t he Dontfll protein e nters nuclei in ventra l and latct1:ll regions bur remFJins in the t:ytoplasrn in dorsal regions (Fi~u re 18~13). Tbe form ation of this Dorsal gradient in nuclei across the embryo is very similar, in

The Molecular Biology o{Urosophila Embry ogenesis

a

591

FIGURE 18•13 Spih:le-Toll and Dorsal

Dorsal

gradient. (a) The aedes represent aossseaions throt.Jgh early Drosophila embryos. The

~ Tel rec;eptoi'S ~---7' nudei at

Toll reccpta IS un1formly distributed lt.rough<JUI the plasma mernbfane of the precelluar embryo. The Spatzle ~tiflg mdeoJe is dsrixlted in a wadiem with peak levels io the

.\,;"'"" peripl1ery

vent!almost ~- As a result more Toll receptoTs are acnvated in ventral r€g!OOS than if1

lateral and dorsal regions. 'This gradient mloll signaling aeates a broad Dooal nucle<Jr gradiem. (b) Details the Toll signalirlfl cascade Activation of the T
venttal

b

p

A

Dorsal

v '.'\., '

'.

.

Pelle

'

'

''

. \

cytoplasm

Cactus

f6 t

TLA:Ie

t:==i

principle. to the fonnation of the (;li activator gradient within ventral t:eUs of the vertebrate neural t ube. Regulated nuclear transport of the Dorsal protein is controlled hy a cell signaling molecule cal led Spatzle. This signal is distributed in a ventral-to-dorsal gradient within the extracellular matrix present between the plasma mexnbrd.lle of the unfertilized egg and the outer egg sh ell. After fertili?~tion, Spat?Jt:! hinds to the cell ~urface Toll :re<:eptor. Depending on the t.'Oncentration of Spatzle, and thus the degree of receptor occupancy in a given region of the syncitial ~mbryo, Toll is activated to a greater or lesser extent. There is peak activation of Toll receptms in ventral regions-where the Spatzle c.oncentration is h i~h est -and progressively lower activation in mote latr:ral regions. Toll signaling causes the degradation of a q 1oplasmir. inhibitor, Cactus. and the release of Dorsal from the cytoplasm into n uclei. This leads to the fonnation of a corresponding Dorsa] nuclear gradient in the ventral half of the early embryo. Nudei located in the ventral regions of the embryo conl;~in peak levels of the Dorsal protein. while those nuclei locate.d in lateral regions contain lower levels of the protein. The activation of some Dorsal target genes requires peak levels of the Dor8al protein. whereas others can be activated by intermediate and low leve ls. respectively. In this way, the Dorsal gradient specifies three major thresholds of gene expression across the dorsal-ventra1

5Y2

Cfme Regulaticm d uring Develop ment

After the sperm and egg haploid nuclei fuse, the diploict zygotic nucleus undergoes a series of ten rapid and nearly ~s cleavages withtn 1he centJill yo~ regiOOS of the egg. Large microtutx.Jie arrays emanating from the centrioles of the
replication corJlOt slnultaneous!y sustain

transal>tion These

and olhef observations have led to the suggestion that there is

.-.ocate

1 IJrosophlltJ embryogenesis. Drosophifio errbyos c:re orierted Wth the fullJre he
18-4

COO"petilion betvv'een the large maaomdecular ~ promoting replication and tran501ptffi Because transcriptional COOlJetence ts only achieved INhen the nudet reach the artex, tt has been suggested that ~ regions cootain localiZed determinants. HcN.tever, recent gene expression stucSes have Slripped rruh of the mystef)' from the cortex. Fa exarrple, the segmentation gene, hunchback, is U1iforrnly transaibed in all of the nuclei present in the anterior hatf of the early embtyo. This eJCPreSSion enrompasses both the peri~l nudei lhat have entered cortical regions, as weD as the vitclophages thcn remain in lhe yolk. Mer the nuclei reoch the cortex, they undergo another tl-ree rounds of cleavage (for a total of l3 dMsions after ferUizatioo), leading II> the dense packi1g of about 6,000 colliTT!Or'-shaped nuda encklsing 1he central yolk (Box 18-4 F.gure l, Nuclear cleavage cyde 14). Ted-ricaly, the errbyo is stl a syndtUm, illthough listochemical staining of earty embryos Wth artibodies against cytoskeletal ~ a highly structured mesh\Nork ~ each nudeus. Du1ng a 1-tn.r perod. from 2 to 3 hours after fertilization, lhe errbyo undefgoes a dramatic ceiLJarization process, ~eby eel membranes are fcrmed between aqacenl rulei (Bol< 18-4 F®Ke 1, Nudeac deavage C)'Cfe 14)_ By 3 hcus after fertililanon, the embryo has been trans-

FI GURE

The Molecular Biology (){Drosophila Embryogene6is

593

Box 18-4 (CDntinuedJ

formed into a ceiJiar blastoderm. ~to the 1ldlaN bal of eels" that characterize the Hasttiae of most other errbyos. One of the most COf11Jelng aspects of dassical embryology is the ~beauty of the mateilal. The early embryos d most f'Tidrine organisms, such as ascidians, are visualy st.unning. l.lnfortu1atdy. the Drosophila errbyo is rather ugly; rts salvation tl3S been the llf'P'ccedeoted visualization of gene el
are large nucleoprotein mmplexes, caled polar grarules. The nude• bud off from the main body of the embryo along with the polar granules, ilJld the resulting pole eels Qfferenliate into either sperm Of eggs, depenchng 01 the sex of the embryo. The microin,ection of polar plasm into abl"lOfmal locations, such as central and anterior regions, results in the posteror

diff~ of supernumerary pole

ceKs.

Corocal nude that do not enter the pdar ~ are destiled to form the sanatic tissues. "'sail. these nudei are l:otlloteot

and can form any adult cell type. However, wilhin a very brief period, perhaps as little as 30 minutes, each nucleus is r~ programmed (a specified) to fob.v a pat1ia.Jiar pathway of diflerefltialion_This specification process occurs ruing the peOOd of celularization. although there is no reasm to believe that the depositioo of eel membrdnes between neightxrng nuoo is critical for determining cell fate Different nudei extlbit dstina patterns of gene transcriptioo prior lo lhe COI'l'lptetion of eel fcr~ rr'l3ticn By 3 hwrs aftel fertiizatioo, ec1ch cell pa;sesses a foced positiooal identity, so that tl-ose located 11 Clf"ltenor regions d the embryo w~l fcxm head structures i1 the adult fly, v.tlefeas c.clls located in postenoJ regions wl fam abdominal strt.Jdl.lres fertilized egg

l manyN.Jde4iJ1 a syncytium

l

nuctet migrate to~hery,

•

eel boLwldaries starl to form

pole eels

8 0 X 18-4 ~I CURE 2 Dftelopmetlt of 'enn cells. Polar gl
594

Gene RegJllalic n during lJel-elopmenf

Box 18-4 (Corrtinued) A variety of gef"letic and eJCperimental stuaes have shaM11Nt eel fae specificat1m is contrcted by locaized maternal determi"lants that are deposited no the egg during oogeneses. lhe &-st evidence for such det~ came from ligabOn e.xperunents_ sn wlich a hair ~ lied aroond the midcle of Dn:ropl-11/a embryos. If tt-Js separa~Jfl betvveen the anterior and posterior halves ocrurred early, dumg S)tlciti<>l blastoderm stages, then central regions of the embi)Q faied 10 fmn thoradc structures SlKh as wi1gs and hatteres (Box 18-4 Ftgure 3a). Hcmever, if the tigat0-1 was done later, tJfter c.elkkriz.aticn, then 1hese sbUCtures were properiy formed (Box 18-4 Figure 3b). These and related expe1Vnents suggested that one O" more critical determinants diffused i11o posterio' regions from the anterior pole and that this deter1T11"Klf1t(s) COl.lk1 be trapped in anterior regi(JflS by separating lhe halves d earfy embryos with a hM. Systematic genetic SOeerlS by Eric Wleschaus and 0rntJane N~a identified appro>r}Q. This irwlved the examination of ttnJsands of dead

errbyos. H.. the midpoint of embryogenesis, the ventral 5100, or epidermis, secretes a GJtide that artains many fine ~Ws. or drotides. Each lx>dy segment of the embryo cmaW"Is a chmxteristic pattern d dentides. Ttree different classes of segmentati( lrl genes were tdentified oo the basis of caus.ng specific dsrtlptia1s in the dentide patt~ of dead ~- Mutations 111 the so-called "sap" genes cause the deletion of severnl adjacem segments(&:»< 18-4 Figure 4). For example, mutationS in the gap gene knirps cause the bs of the second It-rough severth atxhnna1 segments (~ crrt1t)os possess etght such SE:1iffients). Mutirtims in the "pair-n.Je" genes CaJSe the loss of alternating segments. fa example. rootations i1 the evenskipped (e~~e) gene cause the toss of the E.'lfEfl-fUTlbeJed abdonTlal segments. finaly, mutations 1n segment polarity genes do not ilker the noona1runbef of segments, but instead, couse patterning defects within every segmertt For exafrllle. fiCimal segmert.s cootain dentides tn one reglOO\ but are n.*ed in the other. ~ certain segment pclarity mutants, such as hedgehog, both regions of every segment oonta~ denlides.

l

no thorax formation

BOX 18-4 FtGURIE

tmax forms

- - - --

J Ligation experiment. \M"len a hair is used to S€pafate the antenof and postenof IlaNes of eafy embryos. then

determinants emanating from the antenor pole fail to enrer posteria regions. As a result. the embryos develop toto abnormal flies that lad tlloracic stl'Ul.1Lfes. k1 cootrast. when tile hair separa~es older embryos (series oo the rigtlt), then the determinant already entered pcsterior regioos and a normal thorax forms.

Bo~

111-4 (Continued)

a

b

BOX 1 8 -4 FIGURE 4 Oadcfield imazes of normal and rnua.1t cirdes.. (a) The paltem of deot1de t"lalrs lfl ns ncxmal efTtx)o are slightly dtlmn ~the ditferert body segrt"enlS ~ Ttlhrougf'l Aa in the tmage). (b) The ler and polarity in Drosophla. Notvre 2frl 795- 80\ Images courtesy d &ic ~ ~n t.Jnwerslty.)

axis of embr}•os tmrlergoiflg cellularization about two hours atler fertilization. These thresholds initiate the diffcrenti~tion of three distinct tissues: mesoderm, ventral neu~enic ectoderm, and dorsaJ neurogenic ectoderm (Figure 18·14). Each of these tissues goes on to tom1 distinctive cell types in the adult fly. The mesoderm forms flight nlUscles and internal organs, such as the fat body. whkh is analogous to our liver. The ventrw and dorSal neurogenic eclodenn form distinct neurons in the ventral nerve cord. We now consid er the regulation of three different target'genes that are activoted by high. intermediate, and low levels of the Dorsal pmtein - twist, rhomhoid, and sog. The highest levels of the Dorsal gradient- that is, in nuclei with the highest levels of Dorsal proteinactivate the expression of the t wist gene in the ventralmost 18 cells that form the mesoderm {f'igure 16-14). The tnist gene is not ar:tivated in lateral regions. the neurogeni~ ectoderm, where there are intermediate and low levels of the Dorsal protein. The reason for this is tlmt the twist 5' regulatory UNA r.ontains two low-affinity Dorsal binding sites {f'igure 18-14 ). ·r herefore, peak levels of the Dorsal gradient are re::quired for· the ttflicient occupancy of these sites: the lower levels of Dorsal protein present in lateral regions are insufficient to bind and activate the transcription of the twist gene.

sog

L....-1

1 high-affinity Dorsal binding sne

2 low-affifli\y Dorsal birlding sites (fined at high fOorsaiJ)

------------------------------------------FIGURE

18-14 Tbfee thresholds and three l)
DNA contctns two tovv-afhtuty Dotsar btrld!rlg sites that are oa:uped only by peak levcls cl the DotsalgradJeJJt As a result. twist eqJressioo rs re>lficted lo ventral noclei. The thomboid !">' amaocer rontams a duster of Dozsal t»nding sites. Only one of 1he5E' srtes represents an optJmaf. htgh affinity Dotsdt recognition sequence. This m!Xlllll' of high and lew affinity Sites aNows both high and ~ntmnedidtt- Jevas of the Dorsal gr~ to activate rhomboid~ in venlritJ.-lctterat regPlS. f~nally. the 509 intronic enhancer contains frur everlty-spac:ed optimal Dot!>al btndiog .sites. Th5e aiow hi&t\ mtennedJate, and b.'l! levels of the Dofsal grad!rnt 10 aCTAICITe sog a!press!On 1hrooghout tdreral regions.

The rhomboid gene is activated by intern1ediata levals of the Our~ sa] protein in the ventral n eurogenic ectoderm. The rhomboid 5 1

flanking region contains a 300 bp enhance•· located about 1.5 kb 5 ' of the transc.ription start site (}'igure 18-15a). This enhancer contains a duste r of Dorsal binding sites, mostly low-affinity s ites as seen in the lwisl 5 ' regulatory region. At least one of the sitas,

dorsa

NE

NE

b sog

n G u R E 18- IS Reglllatory DNAs. (a) The ihomboJd enhancer cont.-ms binding siteS for bo1h [)a$0) and the Sl1ilil repressor. Slslce the Snal pmtetn is only p-esent 10 \lff!l!aJ regions (tl1e mesodelm), thomboKJ 1S kept off in the mesodetm and restricted to \l€rllJal regions of the neurogenic ectOderm (Ve ntt<Jt Nf) (b) The monic scg eohanrer also contains Snail repressor sites. ~e ~
--------

The Molt'.cvlo.r BioiOi,Y oflirosophila Embzyagenesis

597

however, is an optimal, high-affinity site that pernilts the binding of intermediate levels of Dorsal protein- the amount present in lateral regions. In principle. the rhomboid enhancer can be activated by both the high levels of Dorsal protein present in the mesoderm and the intermediate levels p~nt in the venh·al neurogenic ectoderm. but it is kept off in the mesoderm by a transcriptional repressor calle
Perhaps as little as a twofold olfterence in the levels of the Dorsal ~otan detennirle whethet a naive embryontc <:el foons a musde eel or neuron. Thts regulato!y s'Mtch in cell identity depends on the sharp lateral irnits of the Snail expresston pattern, which demarcate the b
18~5 tlgllre 1). Cels that express Snail invag.nate to fotm ffieSOOerm, while cds located ., more latetal regiOilS (and lack Snail expressial) form del1\latives of the neorogenic ectoderm The formation of the sharp Snail borders depends, in par1. on the multiplialtion d the Dorsal and Twist gradients. The tdea is that the broad Dorsal gradient tnggers a slghtty steeper

598

Gene Regulation dvring /Jf.t"elopment

Box 18-5 (ContUwed)

Twist pattern. and t:hef1 the Dorsal and TVVISt proteins function synergistically ~in the limits of the snail 5' reguSatoty DNA to activate expression {Box 18-5 Figure 1). lhere is a duster of lcJIN.affinity Dorsal sites located about

of the snail gene snail promoter. BecatJse of

1 kb upstream of the trar&npt)on start site and~ Twist binding sites near the

the d.stance separaUr!g these sites, it is unJtety that Dorsal and Twist physically m ract to facitate rooperative bindng to GJA. lrlst.ead, they might make separate contacts with different rate-limiting 1Jarl5Crl>tion complexes ("promiscuous syrteTgy." see Chapter 17). Fa~ Dorsci migl-t render the snail 5' regulatoty region in ar~ "open" oonfoonatioo by recruiti11g an enzymatic complel< that modifies chromatin. stJch as SWI/SNF or HAT. llis opening of the snail 5' regulatory region night facii-tate the ~ of Twist. li'IA-lich subsequently remits the TAJOPolll complex to 1he core promoter (see Chapter 17). We see later in thiS chapter that Bicoid and Hunchback function 1n a synergistic fashion to activate ell€ ~ 2 A simib principle is used to specify the dorsal mesoderm in a vertebrate embryo, as

The 5' regulatory DNA of the goosecold gene cootains binding sites fof Siamois as wei as for "'Sm&f' J:Xotarls. Smads are ITanscrl>tion factors that are induced by the activation of TGF-13 eel surface receptors (Box 5 Frgure 2b). In the absence of signaling. Smads are inactive due to their assooation t.vith the Jnkacytq:>lasmK: domains of the TGf-13 receptofs at 1he eel surface. Upon Stgrldling. hovveve.; the Smads are released into nudei. This results in the binding of Srnads to the goosea»d 5' regtJiatory DNA. Smads and Si.amois now function ~ caly to activate goosecoJd expression within the dorsal mesoderm. The site of expression correspoods to 1he one reg10n of the errt>ryo where there are h@llevels of tdh activators_

we now discuss. lhe dorsal mesoderm of the Xenopus embryo is the soucte of important signaling molecules that control the development of the central neMJUS system (CNS) ruing gastrulation The formation of the dorsal mesoderm depends on locahzal

!

rnRNAs in the unfertiized egg. tnd.uding VegT. The 1/egT gene encodes a sequenc.e-specific transcription factor that leads to the activaQon ci the Xnr gene t!Tooghrut the presumptive mesoderm. Xnr encodes a TGf-f3 signaling mole<:ule that is necessary but not suffident to activate gene expression wit.tun the dorsal mesodetm tnstead, activanon depends on Xnr and Wnt signaling. After fertijzation, a process caled corttcal rotation occurs, duri"lg whtch the ~rttemal cytoplasm of the egg rotates relative to the plasma membrane (Box 18-5 FtgtJre 2a). Cortical rotation leads to the stabilization d ~-catenin aloog one side of the early embryo, v..hich corresponds to the future dorsal surface. A ceM surface r:rotem, ~-caten1n, as IIOilTICIIIy released into nuclei upon activation of Frfzzled receptors by secreted, extracelular signaling prtteins called Wnts. However, cortical rotation may cira..mvent the need for Wnts and directly induce FrizlJed receptas to release ~ caternn. Once in the

nucleus, ~-catenin int€fa<;ts Vvith a seqoence-spedfic transcription factor, called Td or Pangolin. lhe Tcf/13-n is distributed thooghout dorsal regions, vvhere there are ~ levcls of txatenn This Siamcis ecptession profile intersects with the Xnr s~tng mcle<:ule.s di5tTbJted throughout the mesoderm (Box 18-5 FigUre 2b). The pool of 11tersection corresponds to the dorsal mesoderm; SJamois func~ boris synergisticaly WJth Xnr to activate target genes tn 1he dorsal mesoderm One of the fiJst genes lO be activated is c.ated

goosecoid, ~ encodes a hofneodomain regulatory protein.

1

r

Dors~sifes

80X

18-5

FI GURE

snaff

1 roodeifoUJCBai-TwUt~JlL

111f:. broad Dot sal ru:leaJ gradient activ;,tes the twist sene in venlr.ll 1tg100$ [)QfSC!I 4'1nd TWist pfoteifl5 wort. syneqsistic;ally to ~Cite tt variety of ger-.es W1 \.IEf"ltrnl aocl ventrai-Jateml regions. It has been suggested mar Dorsal recruits chlom~g complexes Vti* TWISt stimulates transaiption by~ with Medator u lfiiO complf'Kes. (Sot.Kee: StathopoUos A. and l.evine lvt 2002 Dorsal gmdient netV~ lrl the Drosoptufa embfyo. Dev. Biology 246: 57-67, fig ]., p. 59. Copyright © 2002 Yl-ith pennisston from Elsevier.)

rne

The MoJecvlar BioJagy of Drosophila Embryogenesis

!i99

Box 18-5 (Continued)

... llcatenn (stabtlized) ~r

\lg1 rnRtJA

Xnr(TGF-f)) proteirl

b Tcf-.3 proleins

siamois

gene

siamois

gene

BOX 18-5 FIGURE l Specification of the dorsahnesodertn in the Xenopus embryo. (a) The Xenopus egg contains a number of local· ized mRNAs including VegT i:1f1d Vgl ~T encodes l} T-box DNA-binding pmleio vh1e 1.!11 encodes an activinJTGr 13 signaling rnoleOJle. They lead to the expr€SS100 of Xnr in vegetll regions. ~ rotation OCOJtS aft~ f~ df'dleads to the stabiJrzat1on of fj-catenin along t!-1t' future dorsal suface. The JXW11 of 1ntersec:.t10n betwee1 the XnT and fkatenin doma.ns defines the dorsal mesoderm and leads to the ac:tTvation of a ntJrTtler cl genes such as goosecOid. (b) p-caterm., dorsal l'egiOf\Sieads to the actNaoon ot the siamOis gene. 'o'\'hch encodes a hcmeobox rf!gU!amsy prott:in The Xnr 51gr'a!ing molecule leads 10 the ac1ivanon of <mOtt1t'r dass of regtAa!ory proteW"Is, Smacls.. Bolh regl.Aato~y pwreins, Smads and Siamds, .ve located Oliy 111 thedorsi31 mesoderm. rn this regron theyVIIOfi. syrerg1Sticalytoac1Nate thegoosecoidgene. ( Source: (a) Adapted from Alberts B. et al. 2002. MoleclbbtOiogyofthecell, 41.hed'l1l011. p. 1211, f2 t-66. eopynght © 2002. Reproduced by~ofR~& Ffflncis Bod-s, k (b) .AdapiL>d from Gibert S.E. 2000. Developmentol btofogy, 6th OOtion. p. 322. f~ )(}25. Cqlyr\ght 0 1000 Sinauer ~ Used v..1th perrrisgon. And kcm Moon R Clnd Krne..-nan D. 1998 rm-n cortK:al rotaton to orgcnzer Bene ~icn BioEssays 20: 542, fig. 3. ~ C• 1998. Used by p enTISSlOn ci John \Miey & Sons. Inc.)

Segmentation Is Initiated by Localized RNAs at the Anterior and Posterior Poles of the Unfertilized Egg At the time of fertilization, the Drosophila egg contains two localized mRNAs. One , the bicoid mRNA, is 1ocatoo at the a nterior pole, whHe the other, the oskCJr mRNA, is located at the posterior pole {Figure 'l8-16a). The oskor mfu'\lA encodes an RNA-binding protein that is rfl'SPOnsihle for the assembly of polar granules. These are large macromolecular complexes composed of a variety of differenl proteins and RNA.~. T he polar granules control the development of tissues that arise from posterior reg.im1s of the early embryo, including the abdomen and the pole cells, wh ich are the precursors of the geJm cells (Figure 18-16h). The oskar mRNA is synthesized within the ovary of the mother fly. It is first dcposilcd at the anterior end of the immature E'lgg, or oocyte, by "helper" ceJls called nurse c:;eUs. But. as the oocyte e nlarges to form the mature Rgg, the oskor mRNA is lranspo.rted from anterior to posterior regions. This locali7.atio n process depends o n specific sequem:P.S within lhe 3' UTR of th~ oskar mRNA (Figure 18-17}. We have already

Gene Regulaticm dvrif18 Dc~lopmenr

oskarmRNA

(polar gramAes)

oskarRNA ] if1 pole ceJis

c

early oocyte nucleus.. anlerleJr

soen how the 3' UTR of the oshl mRNA merhates itc; localization lo the daughtel' cell of budding yeast by interacting with the growing ends of microtubules. A remaikably similar process contmls the localization of the oskar mRNA in the Drosophila oocyte. The Dmsophila oocyte is highly polarized. The nucleus is located in anterior regions; growing microtubules extend from the nucleus into the posterior cytoplasm. The os.kar mRNA interacts with adapter prote ins that are a...c;sot:;ated with the growing + ends of the microtubules and are therehy transporte d away from anterior regions of the egg. where the nucleus resides, into the posterior plasm. After foctilization. tl1e cells that inherit the localized oskar mRNA (and polar granules) form the pole cells. The localization of the bicoid mRNA jn anterior regions of the unfertilized egg also depends on sequences contained within its 3' UTR. The nudeotide sequences of the oskar and bicoid mRNAs are distinct. As a result, they interact with rliffere11t adapter proteins and become localized to different Tcgions of the egg. The importance of the 3' UTRs in determin ing where eacb mRNA becomes localized is revealed by the foJJowing experiment. )f the 3' UTR from the oskar mRNA is replaced with that from bicoid. the hybrid oskoc mRNA is located to anterior regions (just as bicoid normally js). This mislocalization is sufficient to jnduce the forma tion of pole cells at abnorma1 loc.a.tions in the early embryo (see Figure 18-171. In addition , the mislocalized polar granules suppress the expression of genes required for the differentiation of head tissues. As a result, emb1yonic t:ells that normally form bead tissues are transformed into germ cells.

mature egg

s·--~-

FIGURE 18-16 Localilali~ of maternal mRWb in the Drosophila egg and embryo. (a) The tklfertlllzed Drosophtlo egg

t:Drltains two locai'red rnRNAs, birold rn anterior regiOf'IS and oskx in posterior regions. (b) The Os1\ar protein helps (OOI'cinate the assembly of

the po\ar gtinJies 1n 1he poster101 cytoplasm. N4Jdei 1tw e()ter thrs re&on bt.x}Qff-ttle postt· f!Of end of th: embrto and form th£' pole ceWs. (c:) Dt.lrlng the forrnation of the Otosophifo egg, polanzed mrcrotubules ~ trolled that els.

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f I CURE 18-17 lh• bicoid and oslu:lr mRNAs contam different UTR sefi'U!IICeS. 1he btrod UTR ca~s tt to be locaued to the ar~tellOf pole while the disbnd os.J
The Molecular BiOI08J' vf Drosophila Embryogenesis

The Bicoid Gradient Regulates the E~'Pression of Segmentation Genes in a Concentration-Depende nt Fashion The Bicuid regulatory protein is .synthesized prior to the r-.ompletion of ooUulari7.ation. As a result , it ditfuses away from its source of synthesis at the anterior pole and becomes dh.;t:ributed in a. broad concentration gradient along the length of the early e mbryo. TI1e Bicoid grad ient is formed in a way that is differenl from the Cli and Dorsal gradients. We have a lrp.ady sean that th P.Se latter gradients depend on the dilTefential activation uf ce ll surface receptors. By simply diffusing across the syncitial embtyo, Biruid bypasses the need for cell signaling. Once formed, however, it produces multiple thresholds of gene expression, just like the Gli and Dorsal gradients. There are pt-.ak levols of the Bic:oid protein in anterior regions. intermediate levels in centraJ ~ions. and low levels in posterior wgions (Figure 16·18]. Differe nt conamtrf'!tioJlS of tl1e Bicoid protein are required for the regulation of different target genes, just as we have seen for DorsaL Peak levels of Bicoid are required for the activation of ~nes in anterior regions that will form head structures; intermediate Jevel s are sufficient for the activation of those genes required for the differenliation of lhe thorax. We consider the differential regulation of two Bicoid target genes, orthodenticle and hundlbocA. Only high concentrations of Bicoid activate the exp~sion of orthoLlcnticle. which is essential for tbe differentiation of head structures (Figure 18-16a). In contras1. both high a.ncl intermediate concentrab

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lB-18 The Bicoid gra&feent activates gene expi'I!SSion in a concenltalion--~,_.,.nt fashion. {a) The broad ilf1teoor·postefior BICOid protein grddent prodoces Mferent ttvestlo&ds of orlhcr dentK/c rlld huf'IChbadc. gene ~SIOr•. Orthodenbde c; adivated only by t'llflllevds nf the S.coid gri'di· frt irl lhe head; hunchtxxlr. rs acfJ\ett
c:entr"'

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lions of flkoid are sufficient to activate hunchback, which is required lor the developn1ent of the thorax. Thls tlifl"e-renUaJ regulation of' orthodentic/e and hunchwck depends on the binding affinities of Bicoid recogniiion !'equences. We have already seen that Dorsal binding affinities are important for ensuring different thresholds of gtme. expression across the do:rsal~venlral axis. The restricted expression of the ortlwdenticle gene is regulated by a 186 bp euhancer locatRd 5' of the transc:ription stal1 s ite (Figure l8-16a). This E".JJbance:r contains a series of low-affinity Bic.;oid binding sites, which can be occupied only when Bicoid is present at high concentrations-that is, in nuclei at the high end of the Bicoid gradient. As a. result. orthodenticle is transaribcd only in anterior regions and not in posteiior regions where there are lower levels of the Eicoid activator (Figure 18-lBb). In contrast, the hunchback gene is regulated by a 5' enharn.:e r that contains htghaffinity Bicoid binding sites. These are bound by both high and intermediate levels of the 13icoid protein. and consequent1y, hunchbad.. is transr.ribed in both anterior and cenual regions of the embryo. The Bit:oid protein biuds to DNA as a monomer. This is different from many other regulatory proteins, such as the A repressor and Dor.;al. which bind to DNA as dimers. 13iooid monomers interact with one another to fosser tbe C()Ope:rative ocr.upam:y of adjacent sites. This cooperative binding pro(iuoos sharp on/off bordffl's m the hunchback expression pattern. Perhaps as little as a twofold def~line in the levels of the Bicoid graditmt determines whether the Bicoid binding sites in the hunchback enhancers are occupied or not. and heOl:e, a sharp border of hunchback expression is established in the middle of the embryo. We have already em:ountered this principle with regard to the >.. repressor (Chapter 16) and the regulation of the J3-interferon gene in mammals (Olapter 17).

Hunchback Expression Is also Regulated at the Level of Translation The localized eJq)tessi.o n of the hunchback gene in the antexior half of the early embryo is a major event in the subdivision of the embryo into a series of segments. We will see that the encoded Hunchback regulatory protein controls the expression of sevet·al genes that are essential :for segmentation. Before describing this process we fU"St consider the

FIGURE 18·19 Hunchbadlprotein gtadient and translation inNbilion by Nanos. lhe Nonas mRNA is assooated with pdar granules.. Aft6' its l.raf'!Siatioo, the prott;in diffuses fJOfll posteoor n>gtonS to form a grddi ent The maternal hi.Jt7Cht:x:rl mRNA ~ dlstnbut£'0 throughout the etlfly embtyo, btJt its trMSIatol is arrested by lhe N;n)S Pfol£'ir1, which bir-ds ro 'Sp(d.c sequences in the hutrchbod: 3' lJTR The Nanos gradiert thereby leads to the tarnation of a rroprocal Hunchba<:k gr~1 in M~teria regioos..

regulation of HU11chback expression in. a bit more detail The hunrhl>ack gene is actual ly transcribed from two promote.rs: one activated by the Bicoid gradient a" discussed above; the other controls expression in the developing oocyte. The latter, "maternal" promoter leads to the synthesis of a hunchback mRNA thai is evenly distributed throughout the cytoplasm of unfcttilized eggs. The translation of this matemal transcript is blocked in posterior regions by an RNA-binding protein called Nanos (Figure 18-19t. Nanos is found only in posterior regions because its mRNA is, in turn, selcctiw~ly localized there through interactions between its 3' UTR and the polar granules we encountered earli(Jr. Nanos protein binds specific RNA sequences, NREs (~anos response ~lements). located in the 3' l JTR of the maternal hullchbock mRNAs, and this binding causes a reduction in the hunchboc k poly·A tail, which in turn destabilizes the RNA and inhibits its transla1·ion (see Chapter 14). Thus. we see that the Bicoid gradient

The tviolecular Biology of Drosophila Emblyogenesis

activates the zygotic hunc.:h/Jar:l promoter in lhe anterior ha) f of the embryo. while Nanos il1hihits the translation of the maternal hunchhack mRNA in posterior regions (see Figure 18-19). Thi~; dual regulation of hu nchback expression produces a steep Hunchback protein gradient with the highest concentrations locaterl in the anter ior hall of •he embryo, and sharply diminishing levels in the posterior half.

The Gradient of Hunchback Repre..~sor Establishes Different Limits of Gap Gene Expression Hunchback fun ctions as a transcriptional repressor to establish different limits of exp ression of lhe so-called "gap" genes , Knippel, knirps, and giant (discussed in Box 16-4). We wiJl see that Hunchback also works in concert with the proteins encoded by these gap genes to produce segmentation stripes of gene expression, the first step in subdividing the embryo into a repeating series of body segments. The Hunchback protein is distributed in a steep gradient that extends through the pre5UIIlptive thorax and into the abdomen. High levels of the Hunchback proteiD repress the transcription of KriJppel. whereas intermediate and low levels of the protein repress the exp~ sion of knirps and giant. respectivP.ly (Figure 18-20a}. We have seen that the binding affinities of the 1:3icoid and Dorsal activators are responsible for producing different thresholds of gene expression. The llunchback repressor gradient mi~ht not wor.lc in the same way. Instead, the n umber of Hunchback repressor sites may be a more critical determinant for distinct patterns of Kri.ippel. knirps. and giant expression (Figure 16-20b). The KrtlpPfll enhancer contains only three Hunchback binding sitP-s and is repressoo by high levels of the Hunchback gradient. In contrast, the giant enhancer contains seven Hunch back sites and is repressed by low le vels of the Hunchback gradient. The underlying mocban;sm hero is unknown. Perhaps different thresholds of repression are produced by the additive effects of the individual Hunr:hback repression domains.

a

b

18·20 fflmchbadcfonns sequential gap upression p.ttterns. ~) lhe antt.ffOfpostenor Hl.lllChbad< l't'pfeSSOf' gradent tstablishes different lirr4t!: of Kri:ippe/, Ju11rps, ill1d grant exprE;SS&On. High levels of Hunchbad< are require(} fC»' the repre5sion of KtDppe{. but low fevels are sllffldent to fepress gi(Jf)L (b) The KrOppel and glont 5' regulatory ONAs c0111:aifl dllfurert nLJr'l1bets of Hunchback repres.$0r .sites. l'hef'e are three sil£-s in KrOppel, but 5e\lefl Sites 111 giant The inaeased rY.X7'lbef of HI.JI1dlback srtes tn the gtonf enhancer n"lay be responsible for ll5 represSlOO by low levels of rhe litlf1chback gradient {Source (a) Re,E 199"/ Developmentol biOlogy, 5th eation. p. 565. fig 14 23. U:f>Ynght it! 1997 Sinauer Asscriates.. Used With permissioo) flc:iURf

SO:

Hunchback and Gap Proteins Produce Segmentation Stripes of Gene Expres~ion A culminating event in the regulatory cascade that begins with the localized bicoid and cmkar mRNAs is the expressjon of a .. pair-rule" gene c:alled even-skipped, or simply e~~a. The eve gene is expressed in a series of seven alternating. or •·pair-rule," stripes that extend along the length of the embryo (Figure 18-21). Each ere stripe encompasses four c-ells. and neigbboring stripes are separated by '•interstripe" regions - also four r.ell.s wide-that express little or no m ~. These stripes foreshadow the subdivision of the embryo into a repeating series ofbody segments. The eve protein coding sequt;>.nce is rather small, less than 2 kb in length. In contrast, the flanking regulatory DNAs that control el'e expression encompass more than 12 kb of genomic DNA~ about 4 kh located 5' of the eve transcription start site. and about 8 kb in the 3 ' flanking region (see Figure 18-21). The S' regulatory region is Tesponsible for initiating stripes 2 . 3, and 7, while the 3' region regulates stripes 1~ 4. 5, and 6. The 12 kb of regulatory DNA contains five separate enhancers that together prodU(.'e the seven different stripes of eve expression seen in the early embryo. Each enhancer initiates the expression of just one or iwo stripes. We wiJl now consider the regu1ation of the enhancer that controls the expression of eve sttipe 2. The stripe 2 enbance.r is 500 bp in length and located 1 kb upstream of the e ve transcription s tart site. lt contains binding sites for four different regulatOI)' proteins: Bicoid, Hunchback, Giant, and Ktiippe) (Figure 18-22). We h ave seen how Hunchback functions as a repressor when controlling the expression of the gap genes; in thP. context of the eve stripe 2 enhancer. it works as an activator. We will return to this issue-how Hunchback can fun ction as both an activator. and repressor- a bit lat eJ'. ln principle, Bicoid and Hunchback can activate the stripe 2 enhancer in the entire anterior haJf of th e embryo because both proteins a rf! presen t there, but Giant and Kriippel function as ropressol's that m;tablish the edges of the stripe 2 pattern-the anterior ond p osterior borders, respectively (see Figu re 1 8-2:.!). (See Box 18-6. Bioinformatics Method s for Iden tification of Complex Enhance rs.}

F IGURt;

18-21

f~ of the

a

e..e gene in the devetopingembryo. (;,) Eve exprESSIOn pattem irJ the eatfy errbyo. (b) The eve locus contains CNeT 12 kb of regularory DNA The 5 • regUaf.oty regton contains two enhaocets. These cont.rol the expression of stripes 2, 3, and 7. Each~ is 5oO bp in length. reg~t01y l'egion (OntainS three enhalcers. These confrd the expression of stnpes

The 3'

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The Molec;ular Biology ofDrosopbila Embryogenesis

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18-n Regulation of W. stripe 2. (a) The 500 bp enhancer~ a total of tv\'elve lmdirJ8 site5 for the 8K:01d, Hunch~ ~ and Giant potefis. The clistribtJ· FIGURE

lions d rhese r~ proteins in the early DroSCJphila ernblyo is~ in the Qagr.lm shown in (b). There~ t1!gh levels of the Btccid

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eve S'1l1pe 2 The borders of the strspes am famed by the Giant and ~pel repressors. (Giant IS ep~~sed 1n antE~ and posterior regials.. Cnly the anteliof pattern ~s st1011vn; lhe posteoor patrem. wh;ch l$ regulated by t-turtehback, 15 •IDf stlCMin) (SoLrc.e: Adapted from Alberts B. et a 2002. Mdeo.Jor bology of the celt .llth «itoo

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Kriippel mediates transcriptional repression through two d istinct met:h anisms. One is competitio n . which is simj}a,. to the strategy employed by many prokaryotic repressors {discussed in Chaptf!r 16). There are three Kruppel binding sites in the stripe 2 enhancer (Figure 18-23). Two of these sites directly overlap Bicoid activator silc.s, and so it appears that the binding or Kri.ippel to these sites precludes the binding of the activator. The tltird Kriippel 1'f1prossor

Box 18-6 Bioinformatics Methods for the Identification of Complex Enhancers

A variety of computer programs have been developed lo identify regulatory Dl\lAs within genomes that have been completely sequenced, l<novvn as "whooe-genome assemblies:' l hese programs take advantage of the fact that regulatory DNAs contain dense dusters of Df'JA..binding .srtes. For example, the eve stripe 2 enhar~cer is 500 bp and contalls 12 separate binding sites for four different reglAatory proteios: Bimd, Ht.mchbock, Kr\q>el, <1nd Knitps (see Flgt.re 18-22). 1hus, there is more than one binding site per 50 bp ove1 the length of the eriwm<;er. This denSity of b4nding sites ts typccc-4 of enhancers that direct localized patterns of gene expression in the early Dlosophi/a errbyo. As we have discussed in this chapu. a number of regulafofy proteins have been irr\pk.ated in the regulation of pair-rule strapes of gene elpet Giant, and Knups. Urlortunately, an insuftident nurrber of Giant binding sites have been eden1ified to determine the ra~ of seqrena;s that this pfOtein is lik€fy to recognize. In contrast there is extensive ()'JA bincing tnformation for the othe- four regulatory Pf()teins, as well as fa- a homedomain protEjn called Caudal wf-Vch is expressed m a broad gradient m the postenor half of the errt>ryo where it functions as a lrcmsoiptlonal acuvator.

Bicoid, Caudal, Hunchback, Kruppel, and Kntrps each bn-.d DNA as a monomer and recognize relati\fcly simple sequences that are present 10 extremely high copy llliTibef in the Drosophila genome. eicoid, for example, remgnizes a sequence that contains an ATIA region e11compassing the eve locus (Box I 8-6 Figure I). There are thousands of Bicold, Cat.KW. Huncti:>ad, KrCJ>pel, and KnlfPS binding sites in this lfltefVat

sin'*

(Box 18-6 Figure 1a). There are, however, only three clustes that contain at least 13 binding sites in a \Nindow of 700 bp or less (a density of neafly one binding site per 50 bp; (Box l B-6 Figure lb) Re~bJy. these three dustes 1118p in the s· and 3' regulatory region of the eve gene.. One duster cmesponds to the eve stripe erflancer, another duster wnades with the eve stripe 2 ertlancer, and the thttd

3n

Ge11e Regulotio n dvriliE D(!V(J]opm ent

clustetS correspood to actual enhancers. tt 1S conceivable that a higher hit rate will be obtaJ-Jed by pradng spatial constraints on binding srtes rather than relying sotety on sample dusterirlg of SiteS. We saw 1n Chapter 17, for exampte, that the interferon enhanceosome contains bindng sites wilh ilxed spaong, indud~ helical phastng between netghboring stte..

duster is located in the 3 ' regulatory region and coincides

with the eve stripe 4/6 enhancer (Box 18-6 Figure 1). CLstemg cl DNA-hnding sites has proven to be a valuable tool for identifying enhancers in the Drosophoo genome. However, the current corq:lllta programs are not 100% accurate. In the best cases, only approx.nately one--third of 1he cdentified

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8 oX IS.fi Ftc; u R E 1 Cluslets of bindirt~ sites identify eve stripe enhancers. (a) kldMOOal Bicoid , Caodaf, 1-toochbad, t::rOppel,

and Knifps tAnding sites in a \ Mb region that rontains the even4.ipped kxus ( in renter elong with other lrotron-exon structures of netghtloMg genes). (b) High density dustermg of bindirg ~ IS llfJiq\Jely detected near eue and not efsev..hese 1n the 1Mb region (c) There are three hitt1 density d.lstas of b~ sites assoaated v.1th eve These ccindde with the ~ 3/7. stripe 2, and stnpe 4/6 e r,hancas. (Sou ICC . Redrawn from Berman P. et al. 2002. &ploltitlg ~n iactoJ binding site clustetitlg to Identify os-regutatory modules ~d ~ patem fotmatiotl in the Drosophila genome. Proe Natl. Arod. So. .99: 757- 762. fig 1. p. 759.)

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607

s it ~

m aps about 5U bp from thP. n earest Bicoid activator s iiP. wit hjn the s tripe 2 enhancer. In this cast> KriippcJ and Bicoid can co-occupy lhP nP.ighbo ring sitos. Once bound to DNA. however, Kruppl!l is able to inhibit the action of the Bicoid activator bound ncarhy. QuP.nching dopends on the recruit mP.nt o f a transcriptional repressor called CtBP (sec Figure 18-23), which we considered earJicr in the conto.xt of tho Notch s ignaling pathway. Recent studies sugg(~ that Ct.BP possessffl an flnzymatic activity, wh ich somRhow impairs the fun ction of neighboring activators. It is Likely that Giant employs a s imilar combination of competition and inhibition to establish tho a ntorjor border of U1e stripe. This basic mechanism of stripe formation-broad ly dislributnd activators and localizfXl repressors-is a rocwrin g the me in devolopm ent T hr. samr. pri ndpal governs HO expression in yflast, and we also s«~w how tht> l oU~ Iizod Snail repressor rostricts tlw action of the bwad Dorsal nucl('.ar gradient 8J1d limits the expression of the rhomboid and sog genes to lateml regions that fOrm the neurogenic cctodt!rm. It is not known how Hunchbark is able to function as an activator in thP. cont('.xt of thP. evP. str ipe 2 cnhanr:P.r, but it is indj!'>fl"!OSable in this role. The l'omoval of tho single Hunchback binding site within thft stripe 2 onlwncer essentially abuBshcs stripe 2 expression. Morf!OVer. replacing this sito with an optimal Bicoid recognition sequence causes only a partial restoration in cnhancnr function. We have seen other examples of this type of transcription synergy jn Chapter 17, including thf' activation of HO cxpmssjon hy SWIS and SBF in yeast. and the activati(Jn of the intP.rleron gene by Nf-t
Gap Repressor Gradients Produce many Stripes of Gene Expression is formed by the interplay of hroadi.Y distributed ac:tlvalors {Bicoiri ~nd Hunc hback) a nd localized repressors (Gia nt a ncl K..UppP.I). Thr. same basic mechanism a pplies to the rngulation of the other eve enhancers as welJ. f'or example , the enhance r that directs the expression of eve stripe 3 can be activated through out the early embryo by uhlquitous transcriptional activators. The stripe borders arc defined by localizod gap mpresso.rs: Hunchback establishes the anteri or border. while Knirps spP.CifiP.S the posterior borde>r (Figure 1A-24). Ttm onhanetjr that controls the expression of eve stripe 4 is a tso roprossed by Hunchback and Knirps. Howevor, difforcnt c:oncent rations of these repressors are required in P.ach case. Low levels of the Hunchback gradient that arc insufficient to rnprcss thf! eve stripe 3 enhancer aw sufficiont to repress thn el'e stripe 4 enhancer {Figure 18-24). This dtrfnmotiAl mgulation of the two oohance.rs by the Hunchback reprc~ssor grad innt produces distinct ante rior borrters for the st ripe 3 and ~1ripo 4 e.xp~ ion patterns. The Knirps pr'Otoin is also distributfld in a gradient in the pre-cr.Uutar embryo. J-Ugher levels Eve stripe 2

FIG U RE

18-23 Twodislinctmodes of

tnlnsaiptional repression_ (a) 11 te brdfl: of KnJppel re,xessor to the t
Gerw. Regulalion dt1ring IJewlopment

t-k.snctlback

Kflirps

.....-.. . . l -I ..

K-

l

bi~sites

• •

stripe 3 enhancer

stnpe 4 enhancer

O[LJ])

fIGURE

r: : ~ :D l []

I 8-24 Differential tegulation of the stripe 3 and stripe 4 enbanc:ers by opposing

gradients of the tfuiKhbadl and Knirps ft!Pfe550r5. The two stf{les ack biMir.g Sites, and as a resul(

high leve4s of tile Hun:tbad gredient are requ.red for .ts repres51on The ~ 3 enhancer mrtams many Kn11ps binding sites, and C~, low le\lefs Of l<sYps
of this gradient are required to .repress the stripe 4 enhancer than are needed to repress the stripe 3 e nhancer. This distinction produces discrete posterior horders of the strip(! 3 and stripe 4 expression patterns. We have seen that the Hunchback repressor grrtdicnt produces different patterns ofKrilppo). Knirps. and Ciant exprc~ion. This diffurcntial regulation might be due to the increasing number of Hunchback binding si t~ jn the Kruppel, I
Short#Range Transcriptional Repressors Permit Different Enhancers to Work Independently of one Another within the Complex ~ Regulatory Region We have seen that eve expression is regulated in the earJy ombryo by five separato enhancers. In f(K;t, tllere are additional cnl1ancers that control eve expression in the heart and CNS of older embryos.. This type of

SummQI}'

complex regulation is not a peculiarity of eve. There arc genetic loci that contain even more r.ohanccrs distributed over even larger distances. For example. in the next chapter we VviH discuss the regolation of homoeotic genes, whk..h are responsible for making the body segments of thfl adult fly morphologically distinct from one another. Several of these genes are regulated by as many as teo different enhancrtrs, perhaps more. that are scattored ovor eli stances approaching 100 k'ilobaoos. Thus, gr.nes cn~ed in important developmental processes are often regulated by multiplo enhancors. How do these enhancers work indRpendAntly of ono another to produce additive patterns of gene expression? In the case of eve. fl ve separate enhancnrs produce seven different stripes. Short-range transcriptional repression is one mechanism for e nst uing P.Ohancer autonomy- the independent. action of m ult iple enhancers 1o gtmcratc additive patterns of gone expression. This means that repressors bound to one cnhan.ccr do not iotnrff'.re with 1he activators bound to another enhancer within the regulatory reg;on of tho same gcno. For example, we ha ve seen that the Kriippel repressor binds to the eve stripe 2 e nhancer and estab1ishf'.s the posterior border of thP. stripe 2 pattern. The J
{lflllancer 3

stripe 3 acrtvators

F I CURE 18-25 §tlortof'Cinge repNSSion and enhaMet" autonomy. Different enhaflcers won:. indeper1dently of cne ;rother en ~he eve ~atoi'Y regKJO due to sholt-fat18e trarr ~ ~SSIOn. Repre;so•s boiJ('ld to one ~ do rnt~ntffiere 'With actiVators 1r1 the n~~ Forexarrrk. the ~pel represror bnds to the sttpe 2 elilafl(:el' ~ keq>S stripe 2 express.on off m central regioos of the embtyo. lhe"'"" stnpe 3 e:nhancef is e,~~J.Xessed 111 these regt()f1S. It IS flOlrepr~ by t<Ji,ppel becaJse it laOO tte speclic Cf\JA se~ tf1al. are rerog111.Zed by the I<J\4lpel ~oteln. In addition. l
enhancer.; are sepaated by a J.5 kb spacer ~

SUMMARY The cells of a developing embryo follow divergent pathways of development by expressing different sets ttf genes. Most differentia) gene e-,._ptession is regulated Ell lhe l~el of transcJipfioo initiation. TI1ere ate three majOI' [email protected]: mRNA localiY.ulioo, cell~tn-cell contact, and the di ffu~iuu of secrelecl signaling m o lecules. mRNA localization is achieved by the attachment of specific 3t UTR Sequt'.Il()e5 to the growing ends of microtubulEs. Tlus mechanism is used to localiZtJ the osh1 mRNA to the daughter cells of budding yeast. It is also used to localize the oskor mRNA to tLe posterior plasm of the unfertiltted egg in Drosophila. l n ce.ll~to-cell contact, a membn1Ue-bouud signaling rno.lecvle a lters gene expression itt n£ighbcdng m ils b y activating a cell signaling pathway. 1n some cases. a dol'mruu transroptional activator"" or co-activator protein. is released frttm the cell surfiK.:e into th~ mtcleus. ln other

cases, a quiescent transcription factor (or transcrlptional repressor) already present in th~ nucleus is modffied so that i I can activate gene expression. Cell-to-cell contact is USP.d by B . sl.lbtilis to establish different programs of gene expression in the rmothel.' cel1 am1 forespore. A remarkably similar me<'banism is used io prevent skin cells from becoming neurons dw'ing the development of the inse
610

Gelle 8egulafion during Development

neuronal cell types. Similarly. thP. Dorsal gracliPilt m Ole early Drosophilu embryo elicits different patterns of gane expression across the dot'Sal-ventral axis. This rlifferential regulatioo dopends on the binding affinilins of Dorsal binding s ites in t~ targe• eathiinc>erS. The segmentaH(lJl of the Drosophila embryo c1epenc:\s on a combination of localized mRNAs nnd gradienls of regulatory factors. Localized I:Jicoid and oskor mRNAs, at the anterior and poslerior poles. respectively. lead to the formatton <>f a steep Hunchback repressor gradumt across the a uterior-pos,erior axis. Tl1is gtad1eut e stablishes sequential patterns of KrUppel. Knirps. and Ciaut in the presumptive rhora:x and abdomen. "these four proteins are collectively called gap proteins; they function as triDJscriptional repl'essors that establish

locE\lizecl stripes of pair-rule gP.ne expression. {ndividual stripes are regulated by separate enhancers located in the regulatory regions of pair-ntJe genes s uch as eve. Each enhaf!Cer contams multiple binding sites for both adivatQrs ar1d gap repres~ors. lt is d1e i nterplay of broadly distributed activators. such ~:~s Bicoi.d. and localized gap repressors that establish fhe anleriot• and posterior borders of individual pair-rule stripe.. &::parate stripe enhancers work i ndependently of one another to produce composite, 7-stripe pauerns of pairrule expression. This tmhuucer ~-utouomy .is duo, in part , to short-range lranscdptional t~pt'ession. A gap repressor bound to one E>Jllumcer does not i.Jlterfere v.rith lhe activilie,s; of a neighboring stripe enhancer locaterl in the same gene.

BIBLIOGRAPHY Books Alb«>rts B., Johnson A., Lewis J., ·Raff M.• Roberts K. aod Walter r. 2002. Molec ular biology of the Cf'Jl, 4rh edition. Gailand Science. New Vorl<. Davidson E.H 2UOt. Genomic ll'.gulat~ry system $. ,A.r;ademic Press. San Diego. Gilbert S.E. ZOOO. Developmental biology, lith edition. Sin· auer Asoociates, Sunderland. Massach usP.tts. Lawrew:e RA. 1992. The making of o fly: 1'he geneUcs of an.imol design. Blackwell Sci6nce. Oxford. Wolpert t .. Beddington R.. Lawrence P-. Meyerowitz E.. Smith ).• and Jessell T.M. Z0Q2. Principles of development, 2nd edition. Oxford University Press, England.

mRNA Localization Doe C.Q. and Bowennan B. 2001 . .Asymmetric cel1 division: Fly neuroblast meets worm zygote. Ct!rr. Opin. Cell Bioi. 13:66-75. Grunert S . .and St. Johnslon D. 1996. RN/\ localization and the dc,.,elopmenl of asyrometq during Drosophila ooge-n~iJCL Cun: Opin Gtmel Dev G: 395- 402 . Kwon S. and Schnapp B.}. 2001. RNA l~:alizahon: Shedcling light on the RNA-motor linkage. Curr. Bioi. 11: Rt66- 16B.

Mowry KL. and Cole C.A. 1999. RNA sorting in Xenopus oocyt.es and embryos. PASEB f. 13: 435- 445. Riechmann V. aud Epl:1russi A. 2001. Axis formation dUI'ing Drosophila oogenesis. wrr. Opin. Genet. Dev. U: 374-383.

Strolll€ S. 19H9. GeneraUon of roll diversity during early embryoget1esis in tlJe nematode Caenorhobdilis elegans.lnl. Rev. Cytol. 114! 81 - 123.

Cell..r~U Contact Artavanis-Tsakonas S .. Rand M.D., and Lake R.J. 1999, Notc.h signaling" Cell fate control and signal int.egrntion in development. Scienre 284: 770- 776.

Doe C.Q. and S~lh J.B. 1996. Neurogenesis in the insect central nervous system.. Curr. Opin. Neurobiol. 6: 18-24.

Goodman C.S., Bastiani M.J., [)(Je C.Q. and Dulac S. 1986. Growth cone gujdance and cell recognition iu i nsect embryos·. Dev. Bioi. 3: 2 83 - JOO. Greenwald 1. 1998. UN-12/Notch signaling: Losson.c; from worms and llies. Cenes Dev.12: 1751-1762. Losick R. and Dworkin J- 1999. Linltiug. asymmetric division to cell fate: Tear:hlng an old microbe new tricks. Genes Dev. 13: 377- 381 .

Morphogen Gradients Belvin M .P. and Anderson k.V. 1996. A conserved signalinA pathway; The Drorophilo Toll~Dorsal palltway. Ann1.1. Rw. 0:111 Dell. Bioi. 12.: 393- 416. Orittr E.A. and Sleward R. 1997. The dorsoventral signal transduction pathway and the Rel-like transcription factors in Drosophila . Semin. Cancer Bioi. 8: 8 3-92. Ericson f., Briscoe J., Rashbass P.• van Heyningen V., and JeS$ell T.M. 1997. Graded sonic hedgehog signaling and the specification of cell fate in the ventral neural luhe. Cold Spring Harbor Symp. Quant. Biol, fiZ: 451-400. Freeman M. and Gurdon J.B. 2002. Regulatory principles of dcvdopmen1al signaling. Aniltl- Rev. Cell. De•r. BioL tH; 515- 5 39.

GurdoJJ J.B. and BourillOI P.Y. 200L Morpho,gen gradient h1terpretntion. Noture 413: 797 - 803. Rusch J. and Levine M. 1996. Threshold responses to the dorsal regulatory gTadieDI and the subdivision or pri· mary lissue res-rilories in tho DmsophiJa embryo. Cucr. Opin. Genet. Dev. 6: 416- 423. Smilh J.C. 1995. MP..soderm-indudng factors and m esodermal pallerning. Om: Opin. Cell Biol. 7:856-861. St. ftlhoston 0 . aod Nilsslein-VolhaTd C. 1992. The origi11 of palfem and po1arity i n the Drosophila enilityo. Cell 68: 201 -21Y.

Bi bliography

Slrigjni M. Utld C..ohen S.M. 1999. Formation of morphogoo gradients in the Drosophilu wing. S emin . Cell Dev. Bioi. 10: 335- 344. Wolperll" 1W6. One hundn~d years of positional infurmaUon. Trends C,enet. 12~ 359- ::t64.

till

Pankratz M.J. ··md Jackie H. 1900. Making stripes in the Drosophila emhryo. Trends Genet. 6:287-292. Mannervik M.• Nibu Y.• Zhang H.. rmd Ltwine M 1999. Transcriptional coregulatO£s in development. Science 264: 606- 609.

Segmentation

Pick L 1998. Segmentation: Painting s lripes hom flies to vertebrates. Dm~ Genet. Z3: 1 - 10.

Hulskarnp M. and Tautz D. 1991 . Gap gones and gradients- The logic behinrl the gap~:. Rio~_vs 13:

Rongo C.. Broihier H.T.• Moore L., Van Doren M.• ForlJes

26 1 - 268.

Johnstone 0 . and Lasko P. 20C.ll . Transltiliorusl regula~ion cmd RNA localit.ation in Drosophila oocytes and embryos. Annu. Rev. Genet. 35: 365-406. Lawrence P.A. and Struhl G. HlOO. Morphogens. compartments, and pattern: Lessons from Dro.mphilo'! Cell 85: 951-961.

Malmwald A.P. 2001. Assembly of the DrosopiJJla germ plasm. Int. Rev.. Cytol. 203: 187-2'13. Nusslein-Vnlhard C. 1996. Gradients that organize embt-yo development. Sci. Am. 275; 54- 55; 56- 61.

A .• itHd Lt!hmunn R 1997. Ctlrm plm;m assembly and

germ cell migration in Droscphila. Cold Spring Harbor Symp. Quant Bioi. 62: 1 - 11. Scolt M.P. and O'Farrell P. H. 1986. Spat1al programming of gene expression in early Drosophila embryq,>enesis. A rmu. Rev. Cell Bioi. 2 :49-80 . Struhl C. 1989. Morphogen gradients and the control ot body pattern in insect embryos. Ciba Found. Symp. 144= 65-86: discussion 86- 91, 92- 98.

Wilson J.E. and MacdonaJrl P.M. Ul93. Formation of genn cells in Drosophila. Curr. Opin. Genet. Dev. 3: 562- 565.

C HAPTER

Comparative Genomics and the Evolution of Animal Diversity t the end of his book. On the Origin of Species, Charles Oarwin speculates that all animals arose from a common ancestor. It has been suggested that at a remote time in the past. perhaps 600 million years ago. a flat worm lived in burrows beneath the ancient oceans. Over the course of many millions of years of evolutio n, this creaturn spawnod the rr.markC~hJe diversity we now see ~mong modem animals. There are 25 different animal phyla; each phy1wn represents a basic type of animal {Figure 19-1). For example. anneljds are composed of simple repeating body sogments, whereas many mollusks are twjsted or coiled (consider snails. for example). In terms of sheer numbers and diversity, the arthropods are the most successful anima] phylum. They include sea creatures such as horsPShoo crabs, lobsters and shrimp, as well as land animals including insects, centipedes. and spjders. Many m nmbciS of this phylum can fly. \!Vhero did all this evolutionary diversity c:omc from? We am just starting to get some ans wers. Most animal phyla fal1 into three major groups: the lophotrochozoans, ocdysozoans, and doutnrostomcs {see Figum 19-1). (In oorl icr times~ the Joph otrochozoans and ecdyso .....oans wer~ caUed. collectively.

A

OU TL I NE

• M ost AnlmaJs Have Esserlrially 1he Same Genes {p. 6 14)

lhre€ Way.. C.ene ExpresSIOn I~ Changed dunng Evolutioo (p. 619)

• Expenffiefltal MafllptAatJons that Alter Aninal Morphology (p. 620)

• Maphological O!anges in Crustaceans and Insects (p. 630)

• Geoome E\/Oiuticfl artd Human 0rigil"6 (p. 635}

~--------------~

r---------- ~~

r.--- - - - gqtrt*k:t>« ,....---

.....loltlyndl& ~h·

-SP<~·• any~

M1bfopoJt.

"""' "",.,..... t-
..---- ~---

Fl CUR£ 19-1 5urnmlvy of phJta. Each phylum represents a basic type of animal. The bilaren~s are divided iflto three map Bfcql§: the deuterosomes (puple}, the ~roans (~). and the ecdyso· zoans (blue). (Source: ~ted from Oa\lldsoo EH 2001. GenomiC tegu/ou;ry systems, p. 22, f 1.6. Copynght © 2001 wth pecrruSSIOn from Elsevier.)

~;)nat~

'---- t'~·L-~------- n~~~

.

, . - - - .-tu,..,.

~·

lldllt.....,..

tno!ltt:K• ~ ~

ecfllnodllnns ~

613

514

Comparalivt: Gf'nomics one! tht~ ~ttolt~lion of ArlimaJUi~~et"Sity nematode

fIGURE 19-2 Phyloseny of iiSSembled

worm

geNNneS. The fJgt!fe shows 1he relationstups am<Jr'8 ltlose acun-Ws ~~~ genomes have been seqwel'l<:ed to date. the genomes of the orgi'!nisms shown in the figure represent three phyla: tlefl')atode 11\''0111\ arthropods (fruit fly af'ld mosqtnto), and dlord
fruit fly. mosquito

common ancestor

5e8SQlltrt (C;ona)

pufferlish

--protostomes.J Chordates such as vertebrates are deuterostomes. T.be ccdysozoans include the two m~jor model organisms for stuaios in genetics and dcvcJopmcntaJ biology: the fruit fly, DroS()p/1ila melanogaster. and tho nematode worm, CaenoThabditis elegans (Chaptrr 2l). Whole-genome sequence information is now available for both ecdysozoans and d~ut-P.I'()stomes. Unfortunatoly. there i~ very litt1P. molecular information available for any of the loph otrochozoans, whk.h include two fascinating phyla, moUusks and annelids. Tho systematic comparison of different animal gcnomes offers the promise of identifying the genetic basis for diversity. As of ttJs writing, thP.. genomes of scvf-~ll different <mim~ls from thrue phyla (ue::matodes, arthropods. and chordates) have boon sequenced and assembled [Figure 19-2). It is likdy that genome assemblies will be available for species representing most of the remaining animal phyla in the ne>-1 few years.

MOST ANIMALS HAVE ESSENTIALLY THE SAME GENES Comparison of th~ c\lm'lntly available gnnomflS rovf'.als one particularly striking fcat\Ire! different animals share essentially the same genes. Thus, the three known vertebrate gr.nomes- pufferlish , mice. and humans - each contain about 30.000 genes. With very few exceptions. just about every human gene has a dear count11rpart in the mouse gcnornR In other words, no new genes were "inVfmtecl'' during the 50 million years of evolutionary divergence that separate mice and humans from their last shared ancestor. Similarly. humans and pufferfish last s hared a common ancP.stor ovr.r 400 million years ago. Yp.t, the two gcnomc-,s conta in the same number of g.mes, and most of these genes-(J}ore than tltlre quarters-can bo unambigot>usly t~lignoo.

MO!it Animals Have E$$enliclly Ute Some Gen~

f:l G U R £

1>15

19-3 Phytogettet:ic: tree

sho\-Mg ge~~e duplkation Df the fibtobfast growth factOf genes (FGf)Ciona FGFs are shovm tn orange, whereas

vertebrate FGfs are m black lettemg. &am:hless 1S an FCf klt.Jnd in Drosophila. EGL· 17 and let-756 are found 111 C elegans. (Source: Adapted rrom S&ou Y. et at. 2002. fGF geoes rn the basal chordate Ciooo mtestlnolis. DEll. Cienes &ol. 212: 437, fig 3 Cow nght Q 2002 Springe.- VeJ!ag.)

The genetic conservation seen among vertebrates extends to the humble sea squirt, Ciona inte.stinalis. wh ich is an invertebmte chordate {sec Chapter 18). It contains half the numLer of g enes present in vertebrates and last shared a common ancestor wjth that group more than 500 million yP.ars ago. Nonotholnss, nearly twothirds of the protein coding genes in sea squirts contain a clear. recognjzabiP. counterpart in vertebrates. MoreovP.r. the increase in gene number seen in vertobratos is primarily due to the duplication of genes already present in the sea squirt. For exa mple, the sea squirt genome contains six different F'GF (fibroblast growth factor) genes (Figure 19-3). There am at lnast 22 FGF genes in the mouse and human gcnomes - each gene in tl1e 5P.a squirt duplicated into an average of four copies in vertebrates. The genetic conservation seen among chordat~ appcru·s to extend to other phyla. The genomes of three different ecdysozoans (nematode worm. fruit fly. and mosquito) have been sequ enced and assembled. They contain an average of 15,0UU genes- similar to the number in SP.a squirts. As seen for the Sfla squirt. increase in gene number in vertebrates is primarily due to the duplicatjon of genes

tl16

Compar<Jtive Qmomics and lht• Evolulioo of Animal Viversitv

already pwsent in the ecdysozoans rathor than the invPntion of entir-ely new genes.

How D oes Gerte Duplication Give Rise to Biological D iversity! The increase in gene number seen in vertebrates is largely duo to gene duplication. But how doos increasing tl1e numbP.r of copjes of certain genP.S lead to increased morphological diversity? Thnre are two ways this can happen. First, the conventional view is that an ancestral gene produces mul· tiple genes via duplication, and thP. coding regions of the new ~encs undergo mutation. This duplication process does not typically produce new genes that encode proteins of enti rely new fu nction. Rather, it creates genes encoding related proteins with slightly ditfHrent activities. Thn s~ond way that duplicated genes can generate diversity has bP.P.n rather neglectfld until ve:ry recently. According to this model. the duplicated genes do nor necessarily take on now functions. but instead acquire new regulatory DNA sequen~. This allows diffcl'lml copies of the gene to be nxpressed in different patterns within the developing organism. Consider th e specifir. example of thn FGF genes. The 22 FCF genes of vertebrates are P..xprt'lss~d in a far broader spectrum of cell typos than is t ho singln genn present jn Drosophila. Thus, wh ile PGF is expressed in the developing respi ratory ot'gans of fruit OiP.S and those of h igher vertebrates as well. s ove ral of tho " new" FCF genes are additionally expressed in the developing limbs of vertebrates where flies do not exhibit a comparable patlorn of e-xpression. Another example is described in Box 19-1, Gone Duplication a nd the Importance of Regulatory Evolution. Thus, we ha.ve two models for how dup licated gnncs can creatf' diversity. According to on e scenario. tho function of the gene is moditied, through mutation of the coding sequence. According to the other scenario. lhe two genes are expr~ed in djfferent pattern ~ witllin

Box 19-1 Gene Duplkation and the lmportana! of Rezulatory Evolution The regulatory prot~ns Gooseberry and Paired probabty arose

Earn

from an ancient gene. d.Jpl1cation event contains two disb'\Ct DNA-binding domams: a h<Jmeodomain and a paired dorna111 (Box 19-T rlgUre 1). The l'MJ proteins possess similar overct structures, but !.hare on1y 25% ~mino acid sequence tdenllty In additioo to substanbal sequence &vergence, the tvvo ge.nes e>t.ibit totally distinct patterns of expressaon tn the developing embryo. The paired gene ts expressed in a series of seven str~ across 1he anteriof-postenor axis of cellulanzing embryos. In c.ontrnst, gooseberry Is expressed in every segment and exhibits 14 strPe5 of ex~n in somewhat older embfyos. Mutant erntxyos exhibit distinct phenQtypeS: fXllred mutants lack alternating segment~ while gooseberry rnt.Jtants cmtan patterning defects in every segment What is ITlOI'e important in the evolution d these a stinct activi1ies: changes in protein sequenc.e or ch.mges in gene expression? The fd-

fowirlg experiment pmiides a definitive answer It is the ~ lfl ~es51CI'I that produce 1he d~nctive a<:tivrties d Paired and Gooseberry. The Paired protein coding regioo was placed under the mnwl of the gooseberry reg\Jatocy ONA. The restlting gooseberry-paired fusion gene was expressed In transgenic Drosophila errtnyos that lad the endogenous gooseberry gene (Box 19- 1 FlgU'e 1}. Nmnally, gooseberry mutant ernbryos die and exhibit pattemf1g defects in every body segment However, 1he gooseberry-pa~red fusion gene completely rescues gooseberry 11lll~ Normal embryos are fCfrT'Ied, and these go a-. ID hatch and produc~ normal (but stefi~) ad.Jit flies. Thts expen.ment demonstrates that the Pait'ed protein. although qurte distinct fran Gooseberry, ca-. fuliil most of the regulatcry adMtes d Goosebeny v.A1en gillen the chance- that is, Vvtlen expressed W1 every segment usmggooseberry regu1at:ory seq.;ences.

Most Animols Have ~11tinlly tile

Sanw Genes

617

------------------------------------------------------------------------------19-1 FIGUR£ 1 Comparisonol the Goosebeny and Paired proteins. The tl.vo ciagrams SI.Jl)marize and c~re 1he S1ruerures of 1he sene> encodi1g the Goosebeny (Gsb) and PaJred (Prd) PfOtens. 8oth protens cx>ntain FN< Md hooleObox ONA-bndng domai'IS, a"ld lhe regions WI the [NA eno:xl1g these are in&cated The nJe IXlder these strucrures in6cates me apprc»Umate b:alions of these domains widlin the proteins BOX

o t m: :J 0

100

t 200

a

)

) 300

400

500

600

- - --M.•TAAA 3'

B R I l

G€E

Gl E gooseberry

I

R I

~

paired

L_.__J

1 kb

b

c

BOX 19-1 FIGURE 2 lhePtdproteincanreswegsb m.utants. (a)Thegooseberty-pozred fi.Jso'l gene. The fusion gere contains about 6 kb of S' ~ seqJenee from the gsb gene attached to the Prd prc>t9l COryo displays a comPeteJy normal pattern of denbdes. (c) The gsb rnuta'lt lhat lades the gslrprd tJanSgene t'l the gsb mAant (withod the ttcmgene) the pattern of det'ltide t0rs is abnormal and thet-e tS very little~ t~separating n6ghbomg segments. (Sotwce: COlltesyof Menus Nol; Li X and Nol M. 1994. Nature 367: 83-87, f~re 3.)

the organism. 1n some cases both mechanisms operate. In Box 19-2. Duplication of Globin Genes Produces New Expression Pattems and Diverse Protein Functions, we describe the cluster of human globin genes. These arose by gene duplication, and, while the different protein products all bind oxygen as part of hemoglobin, they show subtly different affinities for lheir ligand. The different genes are expressed at different times during development as well. The h igh degree of L"'nservation of the genes found in differenl anima ls has recently focused atten1ion on the role of changes in gene expression as a general mechanism in generating evolutionary diversity. The importance of this mechanism is highlighted by the striking changes in morphology caused by misexpressing genes in new places during the development of the fruit fly. ln this chapter. we emphasize how evolutionary diversity can be generated by expressing a fixed set of genes in different patterns.

6 UJ

C!Jmpwative Genomk'$ and lhe Ev11JtJU11n of l\s1imal Diwrsity

Box 19-2 Duplkation of Globin Genes Produces New Expression Patterns and Diverse Protein Ftmdions Gene~ evercs offer the opporturVty to expc!lld the repertoire of potein functions and ~ressioo profiles. Both fOffTlS of evolutl0r1 are seen fot the f3-globin genes tn mammals (see dlapter 17). Four related globins have arisen from gene duplication events tn humans: E, -v. 6, and 13 (Box 19-2 figu"e 1). All folJf genes ate linked within a common "complex:' lhe four genes exhibit subtle changes in lhe• expression profiles and protein strudtires. The ~ an:!

y-globins bind oxygen more ~ than cb ~ and p. They are used by the fetus, which lacks funct.ioring l~s and must ol:lain oxygen by exchange from itS mother's blood. 1he &- and p-globins bend oxygen Wlth lower affinity, and are used by ne\\boms and adults. which contain higher levets of oxygen. In this e)\c}l"')ple, the evolution of both the prcteln codng genes and associated regulatory OOAs lead to the speaalizabon of globin function

replieS and birds ~

monotreme& higher fish and vertebrates

£gene "'/gene I

I

mammals

ygene

artiod~s

\

I l

placeL

~ gene

primates

l-1 B 0 X 19-l F I CURl 1 l:klplication of~ gene family in the evolutiofl of wrtebrates. (Source: Adapted fTom Criffiths et al 200J. An troodvctlon to genetk onalysis, 7th ecitlon, p. 787, fl@ 26-15. Cclpyright C 2000 W H. Freeman Used with pemrission)

Box 19-3 Creation of New Genes Drives Bacterial Evolution Simple bacteria appeared mOfe than three bellion years ago,

while an1mals have been around for just over half a billion years. The rapid evolution of bacteria. along With their extended evolutionary history, have created different forms of metabolism so that they can live in hishly diverse e~nd extreme environments. Some live withCn thermal vents beneath the sea, while others ltve 1n SlAfur hOI sptings on

land. There is tJemendous varidtion in both the niXrber and types d genes present in differerll bacterial getlOfT\eS. The sM"nplest bacteria such as mycoplasma contain as few as 500 genes. IA.tl~e the most S<Jptjsticated bacteria strlt as Streptomyces encode 0\ler 7,000 genes. 111s huge range in g€f'le nunber sharply contrasts \Mth the modast, tvvofold variation

seen among ddferent animals. lhe genetJC content is also highly
In contrast. all animals irtubt .similar. and td more temper~ ate, enllifonmef'lts. lhey employ similcr tTleUboK pathways, but exhibit dstinctive morphologies. Ps we will see m the course of th•s chapter, these averse mOfphologles depend 00 changing the acti\.1ties of a fixed set of genes rather th8fl inventing n€W ooes.

Three Ways Gene Expc<

Before beginning that discussion. however, it is worth noting that evol ution need not work -by redeploying the same genes to generate diversity as sE~en for an imals. For example, bacteria possess the most highly diverse genomes among all living organisms. They conlain more than a tenfold range in the number of genes, and live in remarkably diverse environments (Box 19-3. Creation of New Gt!nes Drives Bacterial Evolution).

THREE WAYS GENE EXPRESSION

lS CHANGED DURING EVOLUTION How do genes acquire new patterns of expression during e volution? Regulatory genes enc.ode proteins that control the expressio11 of other genes (see f'.ha plers 16 and 17). Most often these proteins are transcription factors, but some influence other steps of gene expt-ession instead. Of pa rticular inrercst from the p€rspective of the cuiTent discussion is a class of regulatory genes called p attern determining genes. Changes in lhe ac:livities and expression patterns of these during evolution seem to t:ause significant changes in animal morphology. Tbe distinguishing characteristic of pauern determining genes is that tb~y cause the correct struC'tures to develop, but in the wrong place, when they are misexpressed during development. Foe example, we will see that the misexpression of the pattern determining gene. Pax6, causes ff}'es to develop on the legs of fruit flies. We will consider severaJ additional examples in this chapter. The average animal genome encodes approximately 1.000 different regulatory genes. We do not ha"e an accurate estimate of the number of regula tory genes that function 8!9 pattern detennining genes. but it is just a subset of them. To accurately assess the number. it would be necessary to misexpress every regulatory gene in the wrong tissues during dav1::3lopmenl to soo which cause transformations in morphology. Our best guess is that something like 10% of all regulatory genes would fulfill the operational definition of a pattern-detennining gene. So, the typical animal genome m ight contain about 100 such genes. The major focus of this chapter is to describe how changes in t.h ~ deployment or activities of these pattern determining genes produce di~ versity during evolution. There are three major strategies for altering the activities of pattern determining genes {Figure 19-4). 1. A given pattern determining gene can itself be expressed in a new

pattern. This. in turn. w ill cause those genes whose expression it controls (so-called target genes) to acquire new patterns of expression (Figure 19-4a). 2. The regulatory protein encoded by a pattern determining gene can acquire new functions. for example, a transcriptional activation domain can be t.:onverted into a repression domain. Thus. a regulatory protein that was an activator of a set of genes might now repress them (Figure 19-4b). Note that, although this strategy involves a change in protein function. the evolutionary consequence is a result of changes in expressioo pattern of target genes. 3. Tru:gel genes of a given pattern df'tenniniog gene can ncquirc new regulatory DNA St;!Quences, and thus come unuer the control of a different regulatory gene. In this way, their p.attern of expression is altered (Figure 19-4c}.

6ZO

fIGURE

Comparotivc Cer:10rmcs and tile Evolution of Animal Diwrsity

19..-4 Summary Qf the three

a

gene X

exp~essed

in T1- T7

sttategies for altering the roles of pattern detennining gene5- (a) Hypothetical mecha-

gene X expanded ~essed in

T1- T8

rvsm fa- E!YOit.monacy chdnge 111 two extinct tn-

bolites. In Zoccnthoides, repressor X IS expressed tn 1hora6c segments Tl- T7. In 0/enoicks, rep-essoc Xis eopressed in 111001Cic: se~ Tl - T8. This ~presses the devetopmenf of the anal spine. v.tndl anses from the T8 segment {b) Pro~s encoded by pattern determrniflg genes acqure new functior5 th1ough I'TlUtation (c) Different target genes i2re regulated due to changes in enhance!' sequences

0/enoides

b repres&oo domain

N

ONA-biflding

DNA-binding

domain

domain

c

en

EXPERIMENTAL MANIPULATIONS THAT ALTER ANIMAL MORPHOLOGY The first pattern determining gene was identified in Drosophila in the Morgan fly lab {see Chapter 1, Box 1- 2 and Chapter 21}. A mutation called bxd causes a partial transformation of halteres into wings. (As we shall see, normal fruit flies h ave a pair of wings and a pair of vestigial hindwings called haheres.) During the past 20 years. a variety of manipulations in Drosophila embryos and lruvae have documented the importanc.e of several pattern determining genes in development. Abnormal morphologies are obtained through eac.b of the three mechanisms described above: altering t.he expression, function. and targets of pattern determining genes. We first desc.rihe how the morphology of the fruit fly can be altered by manipulating the activities of specific p attern determining genes. We then apply these strategies to the interpretation of the evolutionary diversification soon in different groups of arthropods.

Expf'rimental McmipulaUans lhot Alter Anitool MOl'pholCJID•

621

Changes in Pa.x6 Expression Create Ectopic Eves The most notorious pattern detcnnining gene is Pax6, which controls eye development in m ost or all animals. Changes in the expression pattern of the Pax$ gene are probably responsible for some of the morphological diversity seen among the eyes of different animals . Pa.x6 is normally expressed with.in developing eyes: but~ when mis~ expressed in the wrong tissues. Pax6 causes &he dcvnlopn)ent of extra eyes in those tissues (Figure 19-5). ln particular, extra eyes form in the wings and legs of adult flies. Changes in the Pax6 expression pattern dming evolution probably account fm· differences in the positioning of eyElS in different animals. Most animals contain bilateral eyes that reside within the ht>.ad caps ule. But, altered expression of Pax6 has been correlated with the formation of eye spots on the stalks of snails. Evolutionary changes in the regulation of Pox6 expression have been more important for the creation of morphologically diverse eyes than have changes in Pa>.-6 protein Function. Thus, Pax6 genes from other ani·· mals also produce ectopic eyes w hen misexpressed in Drosophila. For

a

19-5 Misexpre$5ion of PclJr6 (also called ey) .00 eye fonnation in DrostJphikl. MsexpresSIOn of the Pax6 gene results in lhe forrnabOO of eyes ., irl
Drosophila 'larva

l

l

Drosophila adUJt

imagirlal disks in the 1aNae_ (SooK-e: (a) Adapted from Alberts B. et al2002. Molerutor bology ollhe ~ 4th edibon, p. 426. r 7-74. parts a & b. Copynght @ 2002. Rep!'o
Georg Hatdef.) normal fly

-

mat gve nse to: adult eye • adult leg

eels that rni5express Pax6 gene

b

fly with ey get'16 artificially expressed in leg precursor C8W

'O/utioJJ of Animal Divetsity eJq~mp le, fruit flies wrn:e engineered to misexpress the squid Pox6 gene. Extra eyes were obtained t.n the wings and legs, similar to those obtained when the Drosophila Pox6 was misexpressed {see Figwe 19-5). The fly and squid P.ciX6 proteins share only 30% overall amino acjd sequena> identity, yet they mediate similar activities in transgenic flies.

Changes in Antp Expression Transfonn Antennae into Legs A second Drosophila pattern determining gene, Antp (Ant e1tnapedi a), controls the development of the middle segment of the thorax, the mesothotax. The mesothorax produces a pair of legs that are m o~ phologically distinct from the forelegs and hindlegs. Antp encodes a hom.eodomain regulatory protein that is normally expressed in the mesothorax of the developing embryo (Figure 19-6). The gene is not expressed, for example, in the developing head tissues. But, a domi· nant Antp mutation. caused by a chromosome inversion , brings the Antp protein coding sequence un d~r the control of a "foreign" regulatory DNA that mediates gene expression in head tissues. including the antennae {see Figure 19-6). When rnisexpressed in the head. Antp r.auses a striking change in morphology: legs develop instead of antennae.

Importance of Protein Function: lnte.rconversion of ftz. and Antp Pattern d eter mining genes need not be expressed in different places to produce changes in morphology. A second mechanism for evolutionary diveTSity is changes in the sequence and function ohhe regula tory proteins encoded by pattern determining genes that is. the second sttatero· shown in Figure 19 -4. Consider two related pattern determining genes in Drosophila, the scgmentac·ion gene ftz {fushi Tarazu) and the homeotic gene Antp (Figure 19-7). These genes are linked .and arose from an ancient duplication event that predated the divergen(..-e of crustaceans and insects more than 4.0 0 miUioo )'ears ago. The two encorled proteins are related and

FIGURE. 19--6 A dom"*'l mutation in thellnfp cetae results in the homeOtic transformation of~ into lep. The fly on the right is l'lOfTTial Note the t1Jdm>ert")' set of antemae
Expr-...rimwtUJI MnniPlJlolions that Aller Animo/ M01phol08Y

contain very similar DNA-binding domains (homeodotnains}. The Antp and Ftz proteins recognize clistinct DNA-binding sites because they form heterodimers with different "partner" proteins. These p.-oteinprotcin interactions are mediated by short peptide rnotifs that map out· side tho DNA-binding doma.\n [see Chapter 17). Antp contains a tetrapeptide sequence motif, YPWM, which media tes intemclions with a ubiquitous regulatory protein aallerl Exd (Extradenticle). In contrast. Ftz contains a pent.apeplide sequcn(:e, LRALL, which mediates intera(;tious with a ilifferenl ubiquitous regulatoty pmtei.n, Ft,zFt (see Figure 19· 7). Ptz-Ftt.F1 ilimers recognize DNA sequeru.:es that aro distinct front those bound by Antp-Exd dimers. As a result, Antp and Ftz regulate different target genes. In this example, after the gene duplication event that produced Antp and ftz. the two encoded proteins acquired distinct regulatory activities through sequence divergence. Interestingly, the ftz protein in mom primitive \nsects, such as the flour bwlle Tribolium cab1oneum, contains both the LRALL and YPWM motifs. Thus, it would appear that the TI·ibob'um f'tz protein has hybrid plnpcrties and can fu nction a s both a segmentation gene and homeotic gene. Indeed. when misexpiessed in Drosophila embryos. the Tribolium Ftz protein causes both segmentation Jefects and homeotic transformations.

62.1

c

j~d-:

F12

~N

l

N

VPWM ~

FtC U R E

19-7

Duplication of ancestral

aene ~ding to Antp and ftz.

Subtle Changes in an Enhancer Sequence Can Produce New Patterns of Gene Expression The third mechanism for evolutionary diversity (Fjgure 19-4) is changes in the targe1 enhancers that are regulated by pattern determining genes. 1n this case neither lhc expression pattern n or the function of the encoded regula tory protein is altered. This mechanism is nicely ill ustrated by lhe Dorsal regulatory gradient i n t he early fly embryo. In Chapter 18, we saw how the binning affinities of Dorsal rec.ognition sequences produce distinct patterns of gene expression. Target ~nha.ocers that contain low-affinity DorsaJ binding sites are expressed in the mesoderm, where there are hjgh lev~ls of the Dorsal grad ient. In contrast, enhancers wit h high-affinity sites are expressec1 in the neurogenic ectoderm, whure there are intermediate and low levels of the gradient. The principle that clumges in enh.a nccrs can rapidly evolve new patterns of gene express ion stems from the experimental mant pulation of a 200 bp tissue spedfk t>.nhancer that is activated only in the mesoderm. The enhancer contains two low-affinity Dorsal binding sites and is acti~ vated by high levels of the Dorsal gradient in ventral regions {the future mesoderm). Single nucleotide substitutions that convert each site into an optimal Doisal binding site cause the modified enhancer to be activated in a broader pattern (Figu re 19-& aJJd b}. Dorsal functions ~--ynetgistically with another transcription factor Twist to activate gene expr~-sion in the neurogenic ectoderm. Thfm' are no Twist binwng sites in the native enhancer. 1Towever. a total of eight nucleotide substitutions are sufficient to create two Twist binding sites {CACATG). When combined with the two nucleotide substitutions that produce h igh-affinity Dmscd binding sites, the modified enlnmner now directs a broad pattern of gene expression in both the mesoderm and nt~u rugcnk tltloderm (Figure 19-&). A few a dditions) nucleotide

An ancestral Hax ~~ undawent ~ dupkatioo event to produce lfle modem ftz a1d Antp genes. The en coded protans contain similar homeodcmaifls, but have aoPted distitd protein-protein inter~~ rnottts. Ft1 {left pathway) cootains LAAlL whd1 permits It to l(](erad with fuf \ whle Antp (11ghtpolhwoy) contalr.s \'PINM aod 11'\tBadS Vvfti, Exd Ftz· RzF 1 and Anrp-fxd dime~ recogntze distinct btn&ng sites and thefefore regtkte dfterent tdrget senes.

Comparolnl(' Ce~J~J~T~ics atld the £yo/utioll of Animal Diversity

19-8 Reg\Htion cl tnnsgene upression in 11\e earty Drosophila embryo. 1he figure shOlNS a series of aosssections ot early Drosophila embfyos that

Fl GU rt E

a lew affinity Dorsal binding sites

r-0-,

el
high-aflvuty Dorsal bmding sites. (c) Elcpr~ of lacl c.ootrolled by a rno~1ed Erllanca coo-

l:lacZZ::= +1

b

tairnng two htgfrafftnity OOT5al binding Sites and t'Ml "M1st sites. {d) B
c

Twist ll

-

changes create binding sites for a zinc finger repressor, Snail The Snail repressor is expre~!d on)y in the mesoderm. A modified enhancer. <:ontainmg optimal Dorsal sites. Twist activator si tes, and Snail repres· sor sites, is expressed only in the neurogenic ectoderm where there are low levels of the Dorsal gradient (S€e Figure 19--8o). Altogether, a Sl:".ries of 2, 10 . and 14 nucleotide substitutions produce a spearum of Dorsal target enhanrers which direct expression in the mesoderm, the mesoderm and neurogenic ectoderm. or just in the neurogenic eclorlerm. These observations suggest thai enhancers can evolve quickly to create new patterns of gene expression.

The Misexpression of Ubx Changes the Morphology of the Fruit Ry The analysis of a Drosophila pattern determining gene called llbx illustrates all three princip les of evol utionary change: new patterns of gene expression are produced by changing the Vbx expression pattern, the en<::Qdcd regulatory protein, or its target enhaJlcecs_ lfbx

Experimcmlltl Mollipu/aFiGII$ thor Alter 1\nimal Mcoephclogy

a

b

FIGURE 19-9 Ubx mutants cause the transformation of the metalbocax into a ctuplicated mesothorax. (a) A normal fty ts shown tlat c:onl
jUSt behtnd the \NihgS. {b) A mutant that IS oornozygous for a weak mu~n m lhe Ubx ge11e ts 5hown The metathorax as transfomred ir~ a dLplicated mesothofax. As a restAL the fly has two pilUS of w\ngs rather thir1 one set of Wings and one set of t)alte.res. (Source: Coo..r.esy d EB. L&.'IS.)

encodes a homeodomain regulatory protein that controls the development of the third tlloracic segment, the metailiora.x. Ubx specifically represses the expression of gen~ that are required for the development of the socond thomdc segment, or mesothorax. indeed. Antp is one of the genes that it regulates: Ubx represses Antp expression in the metathorax aod restricts its expression to the rnesotborax of developing embryo~. Mutants that lack lhe Ubx repressor exhibit an abnomtal pattern of Antp expression. The gene is not only expressed within its normal site o f action in the developing mesothorax, but it is also misexpressed in the developing metathorax. This misexpression of Antp causes a transforrnfltion of the .metatborax i nto a duplicated mesothorax (Figure 19·9). In adult llies, the mesothorax rontains a pair of legs and wings. whit~ the metathorax contains a ~ir of legs aml halteres {see Figure 19-9). The haJteres are considerably smallef' than the wings and function as ba)ancing structures dwing flight. Clbx mulants exhibit a spectacular phenotype: they have four fully developed wings, due to the trans formation of the halteres into wings. This mutant phenotype stems, in part, fro m the misexpression ol Antp. Later, we wiU look more closely at how Ubx specifies halteres through the repression of several ta.rget genes required for the development of wings. The expression of Ubx in the different lissues of the metathorax depends on regulatory sequences lhat encompass more than 80 kb of genomic DNA. A mutation called Cbx {Conlrabithorax) disrupts this Ubx regulatory DNA without changing the lfbx protein coding region. The Cbx m utation causes Ubx to be misexpressed in the mcs
FtCU RE 19-10 Mise:a~ion of t.lbx in the mesothofax results in 1M loss

of wings. The Cbx l1ltJtation disrupts T2

Wing

~\

T3(Ubx)

haltera

I

the regtktocy reg«lfl of Ubx. causq Its mise>:press!Of'\ in the mesott1ofax and 1esults in its transfoonation into the met.athorex.

addition to its n ormal site of expression in the metathorax (Figure 19-10). Ubx now 1-epresses the expression of Antp, as well as the other genes needed for 1he normal development of the mesothorax. As a result, the mesothorn..x is transformed into a duplicated copy of the normal metathorax. This is a striking phenotype: the wings are uansfonned into halteres. and the resulting Cbx mutant flies look like wingless allls. This example clearly illu~trates the consequences of misexpressing a pattern detennining gene: a dramatic change in morphology results. We will see how this mechanism is used to convert swimming limbs into feeding appendages in certain shrimp.

Changes in Ubx Function Modifv the MorpholO(.,~ of Fruit Hv Embryos We h ave seen that lhe Ubx protein can function as a tomsr.riptional repressor tha i p.redudes the expression of Antp and other "mesothorax•• gene; in U1e developing mctathorax. The conversion of lJb.x into a transcriptional 8(;tivfttor causes it to fund ion Uke .1\ntp and promote the development of the mesothorax. This example il lustrates how changes in the function of a pattern determining regulatory protein r.an alter morphology. It .is not currentJ y known how Ubx funt..--tions as a repressor. However~ the Ubx protein contains specific peptide sequences that recru it repression complexes. One such peptide is composed of a stretr.h of alanine residues. Alanine-rich repression domains are seen in other pattern determining rogulatory proteins. such as Eve. which we di&cussed in Chapter 18. Tran sgenic fly embryos have been created that contain either the Antp or Ubx protein coding sequence under the <;Ontrol of the hsp70 heat shock cis-reguJatory DNA. When these embryos are placed at elevated temperatures , the re is ubiquitous expression of either Antp or U bx in most, or all, tissues. The misexpression o f Anlp causes all of the h ead and thoracic segments of the embryo to develop as duplica ted meso thoracic s~gments . These embryos are dead. but different segments can be .identified by tlle pattern of fine hairs, or denticles, on the surface of the embryo. In the case of m isexpress ing A ntp, a ll of the thorncic segments c ontain d enticle patterns thot look like the one norii181ly present only on the mesothorax. In con1rast, the misexpression of Ubx causes aU thme thorac..ic segments to develop dentide patterns typical of the normal m eta thorax (Figure 19~11 ). Ubx normally functions as a repressor. It can be converted into an activator by fusing the Ubx DNA-binding domain (homeodom.ain) to the potent activation domain from the viral VP16 prolein, which we encountered in Chapter 17 . The protein sequences that mediate transcriptiollill repression map outside the Ubx homeodomain and are not present in the Ubx-VP16 fusion protein. Thfl misexpression of the Ubx-VP16 fusion protein causes all of the segments to develo}J as mesothoracic segments, not metathoracic segments as seen when lhe normal Ubx p ro tAin is mjsexpressed in engineered embryos. Thus. rather than behaving like the normal Ubx protein. the Ubx-W16 fusion protein produces thtJ same phenotype as that obtn.ined with Antp (see Figw-e 1~ 11).

f:xpcrimenwl Munipulmiom thm Alter Anjmo/ Morphology

a

c

d

-.'.., 12

...,

••

~'"

..~11•• 1\2. Wt

19-11

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I

.. ... _...

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sr .

..c.·~... N. UAS-Ubx

•

~

UA5-U>x-VPl6

Kl t'l&Atltp

~the

rep&atoty activities of the Ub:x ptotein. The panels shoW the anterior segments of adv<mced-slilge embryos. (a) NoorlclP e1nbryo- Note how the dentkle hairs become fldiTO'JI.<er from A1 (the fiJst
rnec:h amsm for altenng fu!"diooal specificity 1n development and evoluoon_Proc Nat}_ Acad Sa. 96: 6802 6807. tig 1, parts a. b, and c. p. 6804. Image rout1esy of Wlwn McGinnrs.)

Changes in Ubx Target Enhancers Can Alter Patterns of Gene Expression The Ubx protein contains a homeo
--- ----

-----------------------

19-t 1he HoiiM!CJtk Geftes of Drosphilo Are Organized In Speaal Chromosome dustefs

AAtp and Ubx represent only two of 1he ei~ hvmeotic genes in the Drosophlo genome 1he eight homeotic ~nes of Drosophila are located in t\NO dJsters, Of gene complexes. rn~e of the e.ght genes are located \\~thl"\ the Antemapedia complex. whtle the remairnng three geoos are located witJin tfle Bfthorax <Xlf1l>lex (Box 19-4 Figt.re 1)- Do not oonfuse the names of the cOI"T"Iplex with 1tle indiVIdual genes wittwl the complex. Fa- example. the Antemapedia comJkx is named ., honor of the Antennapedia gene (Antp), Wich was the first homeotic gene identified Wtlin the complex_ There are four other homeotic genes in the An~a ~: lobd (lob), proboscipedio {pb). Defonned (Dfd), and Sex combs reduced (Scr)- Smarty, the Btthorax rofT'P&ex is named 1'1 hooor of lt1e UltlCJbithorox

gene (Ubx), but there are w:> others in this ~ex.: ofx:k:,minoi-A (olxf..A) and AJ:xiJminoi-B (t'.lxi-/3)_ Another insect, rhe flou- beetle. contains a singk cQll)lex of homeotic gt:neS that irldudes homologs of all eght homeo6c genes mntai'led in the Drosophia Antemape
628

QJmparatiw Cenomic;s und the Evolution of Animal Diwrsity

Box 19-4 (Continued)

t t

t

mouse embryo

19-4 FIGURE. 1 OrganiutiOIUMiti elqJfession of Hox genes in Dl'o.sophiiiD liM in the mouse. The figtxe COfl'1P
embryo. In corrtrast, the Jlbd..B gene, vAich is located in the 5 '~ IX>sition of the &ttuax complex, is expr~ in the posteriormost regms (see Box 19-4 Figure l). lhe significance of this colinearity has not been established, 00t it most be rnportant because it is ~ in each of the major grolf)S of arthropods (l'ldt.dng flour beetles), as wei a.s al vertebrates that have been studed, including mice and tun.:lns.

Mammalian Hox Gene Complexe; Control Anterior-Posterior Patterning Mice
lab gene in Drosophilo, \'llhile Hoxa-9 and Hoxb-9- located

at the other end of -their respective mmple>:es- are similar to the Abd-8 gene.. In addition to this "serial" homology between mouse and fly Hox genes, each moose Hox CClll'p1ex emibilS the same

type of colinearity as that seen in Drosophila. For example, Hox genes kx:ated at the 3 ' efld of each complex. .such as the Hoxo. 1 and Hoxh-1, are expressed in the anteriormost regions of developing mouse embryos (future hinc:bain). In cmtrast, Hox genes locatet1 near the 5' end of each cornp&ex, such as Hoxo-9 and ~9, are expressed in posteriof regions of the embryo (thoracic and IL.rnbar regions of the develqjng spinal cord). The Hoxd compla exhibits sequential expression across the anterior-posterior axis of the developing limbs. A comparable pattem is not obsetVed in insect limbs, suggesting that the Hoxd genes have acquired "nover

Experimerttal Manipulations that Alter Aroma! Morphologt•

6Z9

Box 19-4 (CCHJfinued)

leb

Drosophila Hom-e 0

DfcJ

pb

0 0 I

Sa Antp r UIJx ebd-A AbcJ.B

r) 0

I ,..s., I

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,-1., ~ .1, ,-1., .------ -~ --... -- ... r't' - -- - - - - - . b1 b2 ll3 b4 b5 b6 b7 b8 b9 Mouse Hoxb O C 0 Q : l 0 0 C::O e -:rY-;~-------, sf

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19-4

FIGURE

(:5

r1. o

Hoxc(l

BOX

e5

L.-1

2

86

B7 D c8

~

d8

d4

n

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L.-1

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3

4

5

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L.-1

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dl

c10

d9 d 10

:z=: r;

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= r-: ,., ! ~ :r::

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7 9 6 6 paralogous su~

~ L.-J

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en

.~

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: j 12

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2
pc1e complexes in Df'OsophiiD and in the JnOUSa. (Soulce: Adap~ from Gilbe1 S.. E. 200£l

Developmental biology. 6th edibon. fig 11.36, part a. Ulpyrigflt C 2000 SinatJel Associates. Used with pemussion.)

regulatory DNAs dl.lflOg vertebrate evolution. Indeed, we have already seen 1n Chapter 17 that a speoalized "global control reg10n" (GCR) coordinates the el<press~oo of the indMdlli:ll

Hoxd genes in develop1ng limbs. Altered Patterns of Hox Expression Create Morphclogical Diversity in Vertebt'ates Mutations in mammaian Hox genes cause dtsruptnls in the axial skeleton, ~Mlich consists of the spml COld and the different vertebrae of the badbooe These aheri!bons are evocative of some of the changes in motphology we have seen for the Antp a1d Ubx mutants 1n Drosophila Consider the Hoxc-8 gene in mice, which IS most closely related to the obd-A gene of the DrosophJo &thorax com-

---~--- -- ~

- --

3 Partial transfannation of the first tumbar vertebr.t in a mutant mouse embryo. The~ shows a dose-up litf::w of lhe tlloracic.-lumbar regioo of a mutant moose embryo that lacks Hoxc-8 gene at'tivity. The mutant (sholrvn on the rilttt) contains a vestigia p<*of ribs oo the l1 vertebra. NOfl"nCll mice c:ontaWl ribs ooly on thaacic: vertebrae. (Source: Adapled from Gilbert S. [. 2000. Developmentd bJology; 6th edmon. ftg t 1.38, p 368. Cop~ @ 2000 SinaJET Ass<:x:ia(es Used \Mth permission.) 80 X

19-4

p1ex. tt IS normally expressed near the boundary between the developing rib cage and iumbar region of the backbone, the antenor ~il" (Box 19-4 Figure 3). (The obd-A gene is expressed in the anterior abdomen of the Drosophoo embryo.) The f1rst lumbar vertebra l'l()(Tnally lacks ribs. However, ITilitant embryos that are homozygoiJS ror a knockout mutation in the Hoxc-8 gene exhibit a dramatic mutant phe11otype. lhe first lumbar vertebra develops an extra pair of vestigial ribs (see Box 19-4 F~gure 3). This type of devel-opmental abnoonafity •s sometimes called a 1-lomeotic" ttansfonnation, one 1n wflidl the proper structure develops in the wrong J)Llce. In this case a vertebra that is lyJXal of the posteoor thoracic region develops within the anterior lumbar region.

F I<; U R IE

ComptJrative GtWJmics and the BfiOIUtion of Animal OitrtJrsity

fi
19-11

tnWcon~of

Labial and ubll bintlnz sites.. tll(lSt Hox prott!li1S contain a ~ d1e 1GPl c04e half-site. v.flie the t-lox suburi\ binds the AJi
sequence of~ res;dues strongly lnftt.eoces ~oly. for example. ExcJ.Ubx dimers plclerenbally bind to CtJillXlSde sites vvith a Tf core. while Exd-lab bods Sites v.4th a GC c::nre.. (tab IS a Hox protern that concrds !he dEvefopmet!t ol arteoor head struct\Jres )

A-T-T/G-AIG {Figure 19-12). The two half-sites are often separated by two nucleotides that are important for determining w hic:b Exd-Hox dimer can bind. For example. Exd-Ubx dimers prefer recognition sequences that contain T-T in the central position. In conb-ast, ExrlLab ia) d imers prefer G-G central residues. {Labial is encoded by the 3'-m ost Hox gene in the :Antennapedi.a complex.} This observat.iotl rais~s the possibility that t.c:trget enhancers regulated by one I lox protein can rapidly evolve into a target enhancer for a d iffertJnt Hox protein. We w iU see how this principle m ight explain the different wing morphologies seen in fruit flies and butterflies. These results suggest that altering the function or expression of the Ubx protein or its target enhancers profoundly changes patterning in the Drosophila embryo. It is easy to imagine that similar changes in protein functi on and expression have occurred d uring evolution and are respon~ sible for. making related animals morphologically distinct (see Box 194, The Homeotic Ge11es of DI'060phila Are Organized in Special Chromosome Clusters).

MORPHOLOGICAL CHANGES IN CRUSTACEANS AND INSECTS Thus far we have discussed h ow changes in pattern determining genes alter morphology in fruit fli es. We now cHscuss how the three strategies fo.- altering the activjtif.JS of pattern detennining genes can ax-plain examples of natura) morphological diversity fotmd among dilferetlt arthrovods. The first two mechanisms, changes in the expression and function of pattern dett>.rmining genes. can account for changes in limh morphoJogy seen in certain crustaceans and insects. The third mechanism, changes in regulatory seqllmces, n1ight provide an explana· tion for the different patterns of wing deve lopment in fruit flies and butterfnes.

Arthropods Are Remarkably Diverse Arthropods embrac-e fi ve groups: trilobites [sadly extinct}. hexapods {such as insects), crustace<ms (shrimp, lobsters, crabs, and so on}, myriapods (centipedes and millipedes). and che licerates (horsesh oe crabs, spiders, and ~corpi ons) . The success of the arthropods derives, in part. from their modular arch itecture. These organisms are composed of a serie.<> of rept>..ating b ody segments that can be modifled in seemingly limitless ways. Some s~ments carry wings, whereas others have antennae, legs, jaws, or specialized mating devices. We .know Dl Ote about the evolutionary processes responsible for the diversification of arthropods t han for any other group of animals.

Changes in Vbx Expression Explain MOO.ifications in Limbs among the Crustaceans Cxustaceans includ t! IDOf>i, hut not ali. of the artlu:opods that swim. Some live in the ocean, whlJe others prefar fresh water. They include some of our favorite culinary dishes. such as shrimp. crab, and lobster. One of the most popular groups of crustaceans for study is Artemin, also known as nsea m onkeys.·· Their embryos arrest as tough spores that can be pUTchased at toy stores. The spores quickly resume developlnt".nt upon addition of saJt water. The heads of these shrimp c:ontain feeding appendages. The thorarjc segment nearest lhe head, T1 , oontains swimming appendages that look

Morphol ogical Cllall[N!s in CwsttW«tn.'> nnd Insects

like those further back on the thorax (the second through elnvfmth thoracic Sflgffit::nts. T2-T11 }. Ar1em ia belongs to an order of crustaceans known as br.mchiopods. Consider a different order of crustaceans. called isopods. lsopods contain swimming limbs on the second through eighth th oracic segments, just Jike the hranchiopods. But, the limbs on the first thoracic segment of isopods have been modified. They are smaller than th e others and function as feeding limbs (Figure Jg..13). TI1ese modi£ed limbs ate l'.alloo maxiJiipcds (otherw1f;e known as jaw feet), and look like appen d ages founcl on the head (though tbPSe. are not shown in the figure}. Slightly different p atterns of Ubx expression are observed in branchiopods and isopods. These different expression patterns are correlated with the m odification of the swimming limbs on the first thoracic segment of isopods. Perhaps the last shared an.c.:estor. of the present branch iopods and isopods contain the arrangement of thoracic limbs seen in Artemia {which is itself a branchiopod}~ ail thoracic segments contain swimming Jimbs. During the di vergence of branchiopods and isopods, the Ubx regulatory sequences changed in isopods. As a resuh of t.his change, Ubx expression was elimioateo in the first thoracic segment, ancl restricted to segmen ts TZ- T8. IL is easy to imtlgine that Ubx represses one or more ''head " patterning genes in the thorax. In A rtemia, these head genes are kept cff in a ll 11 thoracic segments , but in isopods the head genes can be expressed in the Tl segment due to the loss of the U/lx repressor. lndeod, exp ression of the Scr gene is restricted to head regions of branchiopods, but is expressed in T1 cf isopods. The e.xpre~ sion of Sor in Tl causes maxilJipeds to develop in place of n onnal swi mming lim bs (s ee Figure 1 9-1 3 ). Wh at is the basis for the different patterns of Vbx expression in isopod.s and branchiopods? There are several pos~ible e>..-planations. bt1t the most likely one is that t he Ubx regulatory DNA of isopods acquired mutations. By this model, the Ubx enhanoor no longer mediates expres-sion in the firnt thor4tlcic segment . In fuct, tht>re is a tight correlation between the absence of Ubx expression in the tho.t'dX and the development of feeding appendages in different crust;3ceans. For example, lobster embryos lack Ubx expression in the first two thoracic segmenLo; and contain tw o pairs of maxillipeds. Cleaner shrimp lack Ubx expression in the first three tho.-dcit:; segments and contain three pairs c f maxiUipeds.

tiZn

head segments

T1 T2 T3 fT4 1 T5

[ Scr \Vhy Insects Lack Abdominal Umbs All insects have six l~s. two on each of the three thoracic segments; this applies to every one of the more than one million specifls of insects. In contrast, other arthropods. such as crustaceans. have a vmi able numbe r of limbs. Some crustaceans have limbs on every segment in both the thorax and abrlomen . This evolutionary change in m orphoJog_v, the loss of lim bs on the abdomen of insects, is not due to altered expression of pc:lltoro determining genes, as seen in the case of maxilliped formation io isopods. Ra ther. t he l oss of abdominal limbs in insects is due to funct ional changes in the Ubx tegulatocy protein. In insects, Ubx and abd ·A repress the expression of a criticdl gene that is required for the develop ment of limbs, called Distalless (Dl/}. ln developing Drosophilo embryos, llbx is expressed <'It high levels in the metathorax and anterior abdominal segments: abd~A (.'Xpression extends into more posterior abdominal segments. Together, Llbx and alJC!-A keep Dll ofl in the first seven abdominal segments. Although Vbx is expressHd in the metathorax. it d oes not interfere with the expression of JJJJ in that segment, becau se Vbx is n ot expressed in the d-r

,

.--

FtGURE

19-JJ
in two different groups of CJUSta«:eans. In brand' 10pOds Scr ~ IS resnlcted to head regtoros ~ it tlEfps promote the devd oprnent o1 teeding ~endoges, white Ubx ts expressed tn the tho®: IM'Iere 1t corrtrols the d('Vf;iopment of swtmmmg limbs. In isor<Jds, Su el!J'ression IS detederllfl both the head and the firSt thorcx:ic 5egmc:t1t ('ft ), cnl as a result.

tt-ae WJTf'TTI1lnE limb m r J IS uanstormed itlto a feeding appendage (the rna~. ThiS pmteJior e)(J)3f!Sion of 5cr was rnatle possible by tt1E k>ss of ut»c elq)reSsion in Tl sioce Ubx norma~ rep~esse.s Sa er.press.on. (Scufc('· Adaptf'd from levtne M. 2001. Natvre 4 1 ~. 848-649, 1i~ 2. p. 848 Ccpyngt"t © 1J.XJ2 l\'a~ PubWr.g GrOUp. Used oMtt1 perms9al )

632

QJmpwc~ti~ Ccnnmic:s

end tJw Evolution of Animal Divt>nrty

veloping Tllegs until aftf'.r the time when DH is activated. As a result. Ubx does not interfere with limb development in T3. Iu u-ustaceans. such as the branchiopod Artemia already men"tioned, there are high levels of both Vbx and Dll in all 11 thoracic segments (Figure 19-14). The expression of Dll promotes the development of swimming limbs. Why does Clbx repress Dll expression 1n the abdominal segments of insects, but not crustaceans? The answer is that the Ubx protein has diverged between insects and crustaceans. This was demonstrated in the following, experiment. The mise>..rpression of Vbx throughout all of the tissues of the presumptive thorax in transgenic Drosophila embryos suppresses limb development due to Lhe repression of Dll. In contrast, the misexpression of the crustacean ( Jbx protein in transgenic flies does not interfere with DIJ gtme expression and the formation of thoracic limbs. These observations indicate that tile Dro60phila Ubx protein is functiormHy distinct from Ubx in crustaceans. The fly protein repre;ses Dll gene expression. whereas the crustacean Ubx protein does not. What is the basis for this functional difference between the two Ubx proteins? (They share only 32% overaJl amino acid identity. but their hom~ omai.ns are "\irtually identical-59/flO matches.) It turns out tbat the Ct\.l$tacoon protein has a short motif containing 29 amino acicl 1·esidues that block repression activity. Wh£>..n this sequence is deletoo. the crustacean Ubx protein is ;ust as effertive as the fly protein at repressing Dll gme expression (Figure 19~15). J3otb the crusta~an and fly Ubx proteins contain multiple repn•ssion domains. As discussed in Otapter- 17. it l8 likely that these domains internet with one or more trdllscriptional mpl't'..ssion complexes. Tiw "antirepressjon" peptide present in the crustacean Ubx protein might interfure with the ability of thf) repression domains to recruit these complexes. When this peptide is attached to the fly protein, the hybrid pro~ t ein behaves like the crustacean Ubx protein and no longer represses Dll (see Box 19-5, Co·option of Gene Networks for Evolutionary lnnovation}.

Modification of Flight Limbs Might Arise ft·om the Evolution of Regularory DNA Sequences Ubx has dominated our discussion of morphological change in arthropods. Ch anges in th e Ubx expression pattern a ppear to be responsible for the transformation of swimming limbs into

a

FtGUftE

b

19p14

Evolutioaaty chimps in Ubx prot~ function.

c

-- --------------------

(a) TheDReflhancer (011504} lS ncrrnaiJy ooiv
in DtosopllikJ embryos. These spots go on to form the (hree paifs of legs io the adult f.y. (b) The misexptesSIOII of the Drosophila lb ptotein (Orrt.btHA) strongty suppresses explE!SSKJn from the Dll enhance... (c) In contrast the mse><pressioo of the Ubx protein from the bune;hnmp Artemia

(Afl.JI:»(HA) causes orty a sight suppresSIOn of the DIJ erhlocer. (Sou1c:e: Adapted from Ronst•augen M. et aL 2002. HQx piOtelll mutction and macroevolution of the tnSect body plan Nor.ute 415: 914-917. fig 2, part <:. p. 9 J5 Copyright «:;; 2002 Natuce Pt.Hshing Croup. Used vvith perrriSSIOO ImageS c~.,. of 'w\ldlwn McC!nniS iY1d Matt Rc:mtlaugeo.)

M(JJ'phologicoJ Changre in Crustacwns cmd Insects

maxillipeds in crustaceans. Moreover, the loss of the antirepression m otif in tiJ.e Uhx protein likely accounts fo.r the suppression of abd ominal limbs in insects. In this final section on that theme, wereview evidence thal changes in the regulatory sequences in Ubx tar~ get genes might explain the different wing morphologies found in fruit flies and buttGrflies. Jn Drosophila, l}bx is expressed in the develt;>ping halteres where it functions as a repressor of wing ili:,velopment Avproximately five to ten tmget genes are repressed by Ubx. These genes encode p:oteins that are crucial for the growth and patterning of the wings {Figure 19-16) and all are expressed in tho deve!opjng wing. In Ubx mutants, these genes are no longer repressed in the halteres. and as a result. the halteres develop into a second set of wings. Fruit flif!S are dipterans. ctnd aU of tl1e members of this order contain a single pair of wings and a set of haJteres. 1t is likely that Ubx functions as a repressor of Wing development in aU dipterans. Butterflies belong to a different order of insects, the 1epidopterans. All of the members of this order (which also includes •noths} contain 1wo pairs of wings rather than a single pair of w ings anrl a set of halteres. What is the bH:;is for these different wing morphologies in dipterans and l epidopterans? The two orders divetged from a common ancestor more than 250 million years ago. This is about the time of divergence that separates humans and nonrnamalian vertebrates such as frogs. It would seem to be a sufficient period of time to aJter Vbx gene function through any or all of the three strategies that we have discussed. The simplest mechanism would be to change the Ubx expression pattern so that it is lost in the progenitors of the hindwings in lepidoptfll'a. Such a loss would permit the developing hindwings to express all of the genes that are normally repressed by Ubx. The transformation of swinuning limbs into maxitljpeds in isoporls provides a clear precedent for such a mechanism. However, there is no obvious change in the Vbx expl'ession pattern in flies and butterllies; Ubx is expressed at hlgh levels throughout ·the developing hindwings of butterflies. That leaves us with two possibilities. First, the Ubx protein is functionally distinct in flies and hutterflies. The second is that each of the approximate ly five to ten target genes that are repressed by Ubx in Drosophila have evolved changes in their regulatory DNAs so that

The regUatOJY gene DistaJ-Iess (DII) has been implicated in the development of most or all iri'nallinbs. indudlng the antefliae and legs of Dmsr:.phila. the swrnming lirrt>s and maxiliped; of a'l.lstaeeans, the finS of fistl. and the lini:Js cl.-ric£! (Box 19-5 Frgure 1)_In ail of these cases. D/1 is requwed for the extensm of lifrb.; i!oNaY flun the body.1he exterlsiVe C00S€1Vatlon cl Distol· less expression i'l virtually cAl arwnals has led to the proposa that the cncestral anima!s, perhaps the pre-Caniman flattish round wam, contaned S!111M protuberances a lbs in the higher a1imals. DH is not de&ated to the eJClflg(ltioo of anlmallir"rhi since it is also ElllpfeSSed io other types of tjssues. One interes1lng

crustacean N

633 lnsE!Ct

N

-- repress~oo

domatn

c c fl GUll E 1 ~15 Comparison af flbx in uustaceans and in inftds. (a) tb rn ~The C-temunal.atltrretxesslon peptide bb:l;s the ac:1Mty of fhe N·femllll.S represston d~n (b) Ubx in irls€as. The C-ten'nlna1 NlttrepressKln pElltl
example is seen i'1 the wJ1gs of butterflies. Many 0Jf1sider

the evespots of butterlly wings to be among the most beautiful patterns encc>U1tered it) nattJre. ft is tflought that these eye-spots ate used as decoys ttlat abv butt€rllies to evade predators. DR as expressed W'l the progenitors of the eyespas, called fOO (Box 19-5 ftgUre 2)- It is alffiCUit to argue that the eyespot l5 a deger1erate ~mb. Rather, it woUd awe<Jr that 011 regulates a distinct set of trlrget genes in the foci to heJp control the ptgmentation pattern of the eyespot PresurM:>Iy, DIJ regulates a different set of IDrget genes in the developW"lg limbs of butterflies. just as 1t does in other cYJimals. The distinct tJse of DIJ in the eyespot.s represents. an exomple of •co-op6oo." A pre-existing regulatory gene es re.dE?ployed for a ,-,:;w pt.Jrpbse.

6:14

t:..omparali~

Genomic$ and the Evolution of Animal DivP-TSity

Box 19-5 (Continued)

Box 19-5 Fl
BOX 19-5 Fl
Carrct. Brunetti et a1. 2001. wrreot Biology n · 1S78. f~g 2. parts b and d.)

they are no longer repressed by Ubx in butterflies (see Figure 19-16). fill individual predisp osed to gambling would lay odds on the fonner mechanism: a change in Ubx protein function . It seems easier to modify rep ression activity than to change the regu latory seqUE:nces of fi ve t(} ten different Ubx target genes. \1\f'e have seen that this type of mech-

a

FIGURE Jg..J6 Cbangesinthe

regutatory DNA of Ubx target genes.

dipferafl$

b

-

lepldopterans

1

1

WI !Epdopterans.

wg

,r ::--COFF +1

(a) The U::x repressoc \5 elOJlressed in thehalteres of terans (orange}. (b) Oiffereflt target genes cootain Ubx repr5sor sites in ~terans. These have been lost

+1

anism accounts for the repression of abdominal limbs in insects as compared with crustacean s. Surprisingly, it appears that lhe less likely explanation- changes in the regulatory sequences of several Ubx target genes-accounts for the different wing morphologies. The Ubx p rotein appears to function in the same way in fruit flies and butterflies. Fm example, in bu tterflies, the loss of Ubx in patches of cells in the bind wing causes them to be transformed into forewing structtues. (See Figure 19-16 for the difference between forewings and bimlwings.) This obseiVation suggests that the butterfly Ubx protein functions as a repressor that suppresses the devdopment of forewings. While not proven, it is possible that the regulatory ONAs of the wing patterning genes have lost the Ubx binding sites (Figure 19-16b). As a result. they are no longer .fflpressed by Ubx in the developing hindwing. An implication of the preceding argtmlents is thHl evolutionary changes readUy occur in regulatory DNAs. Tb is is consistent with various experimental manipulations in Drosophila. We have seen how changing just 7% of the nucJeotidcs in a m esoderm-specific E:'.nhancer converts it into a neurogenic enhancer in the fruit fly embryo.

GENOME EVOLUTION AND HUMAN ORIGINS We have desr.ribed how changes in gene expression cause morpholog-

ical diversity among different groups of arthropods. We now consider fum::Horutl diversity among different manunals. The genomes of mice and ht~mans have been sequenced and assembled, and their comparis on should shed tight on oru ov.rn b l Jman origins.

Humans Contain Surprisingly Few Genes A variety of gene prediction programs are used to identifY protein coding genes in whole-genome assemblivs (see Chapter 20). These programs identify distinctive DNA sequence features associated with protein coding genes. including putative op en-reading frames. spliceosome recognition signals. and core promoter elements. Predicted gen es are sometimes confirmed by independent tests-most frequ ently, the isolation of cDNA.s corresponding to thu encoded mRNAs. But the gene prediction programs are not oomplet~ly accurate

'Oiulion of AnimoJ lJjlfecsjty

(Chapter 20). Short, fortnitoats open -readiug frames can be ~l sely iclnntil\cd as protein cod ing genes. Conversely, authentic genes composed of many small exons can be missed because they lack obvious extended open-reading frames..Finally. there are nume£ous inaccucacies in the intron-exon structure of predicted genes due to the degeneracy and simplicity of the sequence signals required for splicing (as we saw in Chapter 1 3}. Despite these many caveats, the hnman genome contains (lnly

nus

number came as quite 25,000- 30,000 protein coding genes. a shock to many scientists working in the area of human genetics. There was a general sense that the remarkable sophistication .in

human morphology and behavior required many more genes. Before the human genome was sequenced. there were p opular estimates for 100,000 prolein coding gf:!Iles. Bast!d on the logic that w e have introduced in this chapter, we anticipate that higher vertebrates, such as hwnans. contain sophisticated mechanisms for gene regulation in order to produre many patterns of gene expression. (n oth4.'!1' wol'ds, organismal oomp1oxity is not oorrelatoo with gene mamher, but instead depends on the nuntber of gene expres· sion patterns. Consider the foJiowing argwnent. The nematode worm, C. elegam. contains nearly 20.000 genes (see C:hapte:r 21 ). while the fruit fly. Drawphilo melanogaster, contains significantly fewer genes. less than 14,000. Nonethe1 es:~. fruit flies exhibit a far more sophisticated range of morphologies and behaviors than those seen in worms. This increased complexity might result from an increase in the number of gene expression patterns. For example, the average fly gene might be regulated by three or four separate enhancers that together produce about 50.000 total patterns of gene expre88ion. In contrnst, each worm gene is probably regulated by only one or two cnhancBS. As a result. the worm might be built from about 30,000 total patterns of gene expression-significantly fewer than the number of patterns produced in flies avnn though the worm possesses more genes.

The Human Genome Is very Similar to that of the Mouse and Virtually Identical to the Chimp Mice and humans contain roughly the same number of gen es-about 28,00U protein ceding genes. Approximately 80% of thest> genes possess a cl ear a nrl unique one-to-one sequence alignment with one another between the two species. Th e proteins encoded by these g1mes

are highly conserved and share an average of 80% am ino acid sequence idenlity. Most of lhe remaining 20% of the genes in mice and humans differ by virtue of lineage-specific gene d11plication events. For example, mire conta in more copies of a gene calkd cytochrome P4.'i0 than do humans. Of course. there aro also examples of gene families that are more extensively expanded in humans than mice. The main point here is tha t there are fl}w, if any, ..new" genes in humans that are completely absent jn m iC(l. The chimp 0:1nd hu man genomes are even more highly conserved . They vary by nn average of just 2'% sequence di v~rgence-in an a verl:lge str~tch of 100 bp there are only two nucleotide substitutions hetwt.'en tl rand om chirnp and huruan. This represents a remarkable level of conservation. By r:omparison. two random sea squirts in the same population d U'fur by tuore than 1'Yo sequence d ivergence, while Individuals from d ifferent populations (but the same species) exhibit

GenolTlfl Evolution cmd Hf.IIOOil Origins

637

as much as 2.5% sequence variation. There is also extensive synteny betwee11 chimps and humans (and for that matter, miu:). The order and distances separating neighboring genes are highly conserved. We ha,~ soon that regulatory DNA e"olve more rapidly than protein s. Perhaps the limitf'd sequence divergence between chimps and humans is sufficient to alter the activities of several key regulatory DNA.;.

The Evo1utionar)' Origins of Human Speech Given the similar genetic compositions of mice, chimps, and humans, it is interesting to consider how new evolutionary innovatiOJ~s suddenly appear in humans. We speculate on the origin of one such trait, speer;:h. as it i :'l one uf the defining fP.atw:es of heing human. We alone possess the capacity for precise conununication in the form of speech and written language. Our closest cousins, the chimpanzees, display a simple form of language that is quite crude in comparison to our own. How did our distinctive form of language arise in human evolution? Speech depends on the precise coordination of the small muscles in om larynx a nd mouth. Reduced ]evels of a regulatory protein called FOXP2 cause sevP.re defects in s peech. Afflicted individuals exhibit a variety of difficulties in articulation. The FOXP2 gene was isolated in a variety of mammals, including mice. chimps. and orangutans (Figure 19-17). The human fonn of the protein is slightly different from those present in mice and primates. In particular. there are two amino acid residues nt positions 3 03 and 325 that are unique to h umans: thr to asn (T to N) at position 303 and asn to ser (N to S) at position 325 fFigure 19-181. Perhaps these changes have altered the function of the human FOXP2 protein. For example, there is evidence that these changes occur ""ithin a repression domain of the protein, thereby ra ising the possibility that human FOXP2 fails to regulate target genes that are repressed 1n mice and <:him ps. This would be comparable to the antirepression peptide that evolved in the Ubx protein of crustaceans. Alternative1y, changes in the expression pattern or r.hanges in FOXP2 target genes might be respon sible for the ability of FOJ...'P2 to promote speech in humans, as we now discuss.

How FOXP2 Postel'S Speech in Humans In this chapter we have discussed three mechanisms for changing

the function of regulatory genes such as Ubx. T he same principles apply to f 'OXP2. Perhnps a combination of ftll three mechanisms, changes in the FOXP2 expJession pattern. changes iii its amin o acid sequence. and changes in f0XP2 tcuget genes might explain its emergence as an important mediator of humw speech. For example,

F I CtJ R 1: 19-17 Summary of amino add changes in the JOXP1 proteins of mice .and priJJM'tes. The nurrt>ers llldKate

1100C0t1seNaWe amino i!ICIC.1 substlt:uions.

(So&Xc.e·, Adaplal from Zhang J. et .al 2002. evalui!tron tJrld oogtns of humdll'"SpE'Cilic leatures. Genetx::s 162: 1829. fig 4.)

An:ele~ proten

Comparotive Cenomi':s cmd thtJ b'volulion of Animal Dnr~rsity

8p human

Nij

chlrJ1Xft1Zee N

303

'];)

c

m

c c

N

F r" u RE 19-18 Comparison of the FOXP2 gene sequences in human. ~and mouse. The fig..Jte sl--.ows the atignrneflt of ttJe FOXP2 sequences for hl.lfl'1an. chimp. and mouse wilh anino aod changes. Thefe are two difterences betweer1 h.Jman and chimp {N to T at position 303 artd S to N at posi11on 325 11'1 the human ~enre) and tnree differences be~ hll11afl and mouse {ttle third c.hanee is 0 toE at positron 80) . (Soura:!: Data from Enatd W et aL 2002. Molecular evolutiof'l of fOXP2, ., gme invclved m ~hand lat'lgt.~<~ge. Nar~ 418: 869-sn.)

changes in the FOXP2 regulatory DNA might cause the gene to ar.quire a new pattern of gene expression jn the human brain. Jn chimps the grmr. might not he cxpresSAd in thA appropriate region of the brain at the right time during development. In contrast. in humans FOXP2 might be expressed a1 the right levels in the corrert time and place to foster the development of languauge in the brains of infants. In the next soction we discuss the possibility that f'OX.P2 might regulate different :sets of target genes in ch\mps and humans. This discussion is speculative. but serves to provide a framewor.k for how suhtle changes in just a few regulatory genes and their targets might lead lo the innovation of a critir..al trait such as the use of language. Consider potential target genes of the FOXP2 regulatory pcotein. Some might encode neurotransmitters or other critical signals that are ~xpresscd within the developing larynx. Perhaps these chrulgEls have augmented the levels or timing of gene expression. so that critical s\g· nals are ar.tive in the larynx during the time when we are most susceptible tu acquiring language as infants. The c;urresponding gunes might be expressed at lowe.- levels. at later stages. ot in fue wrong regions, of the developing chimp laryn.x (Figure 19-19). FOXP2 is just one e"ample of a regulatory gen£ that undoiHes human speech. It is difficult to estimate the number of "speech mgulatory genes" that have evolved after the divergenre of chimps and humans. However. we have soon that fewer tl1an 100 pattern detennining genes are sufficient to account for the morphological diversification of different arthropod groups. Perhaps a signilicantly smaller set con account for the acquisition of language.

The Future Q{ Comparative G enome Analysis Given the extensive body of information that has been compiled for a variety of different proteins, it is possible to infer the function of roughly half of all predicted protein coding gen~ based solely on primary DNA sequenre information. ln rontrast. there is a glaring limitation in our ability to intC.r the function of regulatory DNA from siOlple sequence inspection- Fewer than 100 regulatory ONAs have lx->en carefully characterized in aU animals combined. This is not a sufficient data set to determine whether regulatory DNAs that mediate similar patterns of gene expression share a common "corle"~that is. whether conserved clusters of binding sites for particular combinations of regulatory proteins can be identilloo by simple sequence inspc>ction. If such a ode exists, then it might he possible to infur both the timing and sites of gene expression by

Su~

primate

neocortex

N

actNation domain

FOXP2

c

1 human

gene A

() +1

l

;} OFF

639

F JGtHU 19-19 A scemsrio for the evok.ttion of speech in humans. A hypolhetical regL4atay protetr\IS expressed in the neoc.os'iec of both chimp!> and t-.Jman!.. However. it possesses sf!~ different actMtJes 111 thesegroups. 1he human gene IS~ expres5ed at the Cl1bC<111ime 111 the dev~nt ct the speech cent~ cnl CM:tivales al tt.ee hypothetical wget genes 11 the neocortex These target genes l11lght encode MMOtransmittefS tmpatar\1 kif the f0fl11abon of fht> speed; ceni.E'!'.In contl'aSI, the~ f0f111 of the gene might not be expr~ ~ tn'le in the development cJ the speech ren1er. Altetre tivel}: it might be ~essed at the ri~ IJnie, but amno ocXl diiferences cause it to be a v.'eik.r acJVatOt than its human counteyart. As a rest.41. the chrnp regtiatory proton IS unal::Ae ID actJV.Jte the WI spectrum of target genes in 1he fleQCO!te)c. Consequef1dy, the. chimp possesses a JT'lOfe primitive toon d lilnguage.

+1

~B

geneB

()

I

) OFF

+1

r:=;.+ \~'"""'

{geneC • ON

ON

simply s<:a.11ning the DNA sequences associated with any given gene (jn 5', intronic, and 3' positions relative to the transcription unit). This would permit a far more robust brand of comparative genome analysis than is currently availabJe. For now. we must be content with compartsons of protein coding genes as discussed for FOXP2. In the future it might also be possible to identify changes in the expression profiles of homologous gen~. The continuing dP.velopment of nP.W t::Omputational methods and the availability of new genome assemblies offer exciting prospects fm- the use of mmpara~ tive methods to reveal the mechanisms of evolutionary diversity.

SUMMARY In Chapter 18. we saw how diffcrentinl gene expression is responsible for lhe establishment uf different ceU types in the drwf!loping embryo. ln this chapter we argued that the samo concept of differential gene expression can explain tbc evolution of animal divp.rsity. Tl i~ bec..oming dear that the evolution of d iversity among organisms is not due to ti'K- pw..8{lnce of different specialized genes. Rathr,r,

animal evolution depends on deploying the same set of genes in diffr.w.nt ways. Evolutionary change can lherofore be viewed as a problem in gene regulation. and comparolive genome analysis offers the promise of identifying the regulatory mechanisms responsible for this diversity. AI the time of this writing. seven difftwent animal genornes have been sequenced and a~rrnbled. lncreasing1y

Compc:sr~Jive GencmiQ' end 1he EvalaJtton ofAniT{Ial Oiver:;ifj'

sophisticaJed methods of genome analysi~ are revcalirlg e Humber of unexpected fivdingg. First. thnre arr1 fP-wcr protein coding genes in a typical genome thnn f'.xpec:tcd. ln h umans, for example, this number dropped from an expected value of around 100,000 gP.ncs-bcrore tbft SflqUtmcing of the ~nome fu the year 2000 -to just unSCnlially lhe slime set of ~encs. Thus. between human an d chimp. we ftnd 96% conscrvation in the protein coding g(lnes, but more surprisingly. the conservation between humen and mouse is over BO%. Furthermore. the inC'ftlase in gene l11.1mber seen fot vertebrates (as compared with invertebrates) is primarily due to the duplication of "nld'A genes rathflr than the invr.ntion of new ones. Changes in ge.nc p.xpressi.on during evolution depend on altering lhe activities of a special class of r t"gulatory genes. caUcd pattern rleterminitlg genes. Whereas a typic.'11amimaJ genome might encode approximatnly 1.000 different regulatory gc•m!S, roughJy lO«t'o, or 100. of thf".Sf' oorrespontl to pattc.m dstc.nnining genes. These genes &.re characterized by the ability, whflfl mirexp11:1~ during development, to cause the • right" structures to appear in the "wrong" place. For example., the misexpression or the pattcm d etennining gnne Pox6 uauscs the formation of extre eyes in the wings and legs of adult flies. There m:e throe major strategies for altering the activities o f rlattern determining gcn£S: thanges in their expression profilns. change~> in thn function of th~ encoded regulator}' proteins. and changes in the ~n· hancers that are recogni?.fld and reguJa t(ld by pattern detennining proteins. Th~ pattern determining gene l Tltx {V/lrobitlwmx) in Orosopbila provides examples o f a ll t hree sttatogies. Thr. rnisoxpmsf.ion of Vbx i n thr: clnvcl opang wings couses the d evelopment of wingless flies. l o an extreme cbange of function. the conversion of Ubx from a repressor i n to an activator, the mod ified Vbx

gene behaves like anothttr pattern determining gene. Antp. and c.ontrols lhf~ dP.velopment nf 1'2 (rather than 1'3) liegtnetJis in clevnloping embryos In princip le. it might be possible lo c;:onvcl't a l!bx target enhancer into ~ Antp enhanceJ" by si mply dmn~r1g the spat:ing bc twoen Exd and l]bx h a lf-sites. fn terms of sheer numbnrs a11d diversity, thP. 8rt hro~ pods can be considered the most successful of all animal phyla. More is known about che molecular basis of arthropod clrom·sity than any othCI' group of animals. U is clear that the three strnt~os for alte..ring the octivitir.s of pattern dt!te rmining genes h nve been ccllical in generating wide m orphological dhrflNtity among arthropods . O ust~M::c~n~; itnd insects ~present two of the five major groups of arth.roJmds. and ch anges in thci.- morphologies have been correlated with altered activities of pattern determini~ genes, particularly Vbx. Cbanges in the rocprossiHn profile of th~ ubx gtme arc cotrelated with the convers ion of swimming lin1l>s into maxiJH)l('ds. Functional changes io the Ubx protein might account for the represston of nbdominal limhs in i~cts. finally. changes in Ubx target enhant:f'..rs might oxplain the diffc-:wnt mmphologies of the haltru-es in diptemns and the hind\vings o£1apidoptemns. We are fflst entering a golden ora of comparative genome analysis. The amount of information thJlt is becoming nvoilflblc is s taggering. At tlm cun:nnt rate of DNA sequence prodt,.~clinn , the cqu ivolent of 20 human gfmomes w HI be sequenced every ,vcar. Thf* human genomP. cnotl"'ns *'urprisingJy Jew genes. and theso are .highly consetvod i n othct ptirnatos. mammals. and vertcbratos. It is likely, tllcrofora. that tbc acquisition of many of the remarkable characte:risli.cs unique to humans. 6UCli as language. Trsults from changeS tn r<1gulatory DNA, rather than in protein codfng Sf!quences. There is the hope that the compul3tion~l analy1iis of rEgUlatory DNA will illuminate the mechanisms of evolutionary innovatior1 ond diversity that we have briefly swnmarimd in this chaptf!r; howevor. new lf!f:hnologics mu st be developed to ensure the success of this entE'!I'prise.

BIBLIOGRAPHY Books Brown T.A. 2002. CenDmes. lnd ~dition. BIOS Scientific Publishers Ltd., Oxford, Linitf't'J Kingdom. O!n'OJJ S.B., Grenier J.l(. , a11cl W~thcrboo S.D. 2001. Ft·orn DNA fo di versity : Molecular genetics and the evolution of animal design. Elackwoll Science. Maldc.n, Massachusetts. Darwin C. 1859. On lhe ongin of specie.<> by means of 11oh.Jrol selection. John Mun-ay, London. Davidson E.H. 2001 . Cenom1c regulat01y B.yslemsllJf.w clopmcnt and e m lntion. Acad r:rnic Press. An Elsevier Scieru::e Imprint, San Diego, California.

Gerhart J. and Kimchner M. 1997. Cells. embryos. and evoltJtfon/Tow.acd a cellular ond developmental undctsloncli.ng of phenotypic vtJriolion and etrolutionnry odaptabiliry. ElaekwclJ Sciona>~ Malden. Massachusetts. GilbP.rt S.b. 2000. DcveJopmtmtol biology, 6th edition. Sinaucr Associates, SWlderlWld, Massachusetts. Griffiths A.J.F., M iJier J.H., Suzuki D.T., Lewontin RC.. Gclbart W.M. 2000. An inlrodtJctivn to geMtic analysiS", 7th edition. W.H. FretlO)."'ll, NP.w York.

Lovi-Setti R. 1995. Trilobites. 2nd edition. University o f Chicago Press, Chicago. Ulinois.

Bibliography ~ff

R.A. 1006. Th e shape of life: Genes, c}welopment, and lhe evolution of animal form. University of Chicago Press, Chicago, Illinois

Evoluticn of Dive~ity Akam M. 1998. H ox genes in cs.rthropod devruopment and evolution. Biol Bu/1. 195: 3 73- 374. - -1998. E-Jox genes: from mastrr w:n~ to microman· agocs. Curr. Bioi. 6 : R676- R678. - - 2n00. Arthropod.-.: developmli!TltaJ diversity within a (super} phylum. Proc. NoU. Ar.ad. Sci. 9?~ 4438- 4441 . lltowne W.E. and Patel N.H. 2000. Molecular genctic~o; of crustacean feeding appendage development and diversification. S emin. Cell Dev. Bioi. 11:427- 435. Carroll S.l3. 2000. Endless form~: The evolution of gene regulation and morphologic.,} divursity. Cell 101 ~ 577-580.

Chance smd noce8liity: The evolution Cl( morpho logical complexity and diver:c;ily. Nat ure 400:

- - 2U01 .

1102- 1109.

Doeble) ). and Lukens L. 1998. Transcription>tl regu1atfln> and the evolution of plant fotn\. Plant Ce/J 10: 1015- 1082.

ti41

JJuboul.e D. and Wilkins A. 1998. The evoh.ttion of '"brtcolagc." Trends Genet. 14: 54-59.

Gelloo G. and McGinnis W. 1998. Shaping animal horly plans in development and evolution by modulation of H ox. expression patterns. Bioessays 20: 116-125. Lali S. and Patol .N.l-1.. 2001. Conservation and divergence in molecular mechanisms of axis fonnation. An.nu. Rcv. Genet. 35: 407-437. Pat el N .H. and Prlnre V.E. 2000. Beyond the Ho:x oomple1C, Genome Biol. 1: RtU27. Taul2 D. ZllOU. Evolution of transcr)ptional ~lation. Curr. Opin. Genet. Dev. 10:575 - 579.

Genome Evolution and Human Origins Bish op D. V.M. l002. Putting language genes in perspective. Trends Genet. 16: 57 - 59.

Human Ct>nome. 2001. Nowre 409: 813- 96H. HumRn Gnoome. 2tXn. Science29l: U45 - 1434. Mouse Genome. 2002. Nature 420: 509 - 590. Paabo S. 1999. Human evolution. Trends Genet. 15: M l 3-M16 .

P A

R T

• METHODS

Part 5 A4F.thods PART

OUTL I NE

• Chapter 20 Techniques of Molecular Btology Olapter 2 J Model 0rgafl151i1"

n Parts 2 and 3. we outlined OW' understauding of the molecular mechanisms underlying the central dogma; Part 4 focused on the mechanisms of gene expression and how differential gene expression C',(lntrols tbe uevelo pmenl and evoJutio11 of diverstl animals. Most of what we know in these areas stems From the study of a few model organisms using techniques of genetics. molecular biology anrl biochemistry, and more recently from genome analysis. The last part of this book is devoted to summar~ing some of these methods and organisms. Chapter 20 outlines basic techniques of molecular biology and b;ochemistry. These allow molec;ulcs (DNA, RNA, and proteins) to be isolated from ceUs (isolated, that is. from complex mixttJJ"es of such molecules) and sturlied in pure form in vitro. Chapter 21 ou tlines key features of a few model organisms whose study underpins moclem bioJogicaJ thinking. These are: phage and bacterin; yeasti tbe\vorm C. eleg<m8; the Drosophila fruit fly; and the mouse. Genetic analysi~; of these organisms has eJ:tabled t he study of biological processes in vivo. The power of rnolecular biology-and the revolution in our understanding of biology gained from it over the last 50 years- stems from U.")ing in vivo genP-tic anrl in vitro biochemical approaches in combination. A golden era of molecular biology was launched once it became possible to isolate specific DNA segments representing individual genes. In eat"lier times. it was possible to obtain bulk DNA from an organism. but only during the mid-1970s 1vere methods developed that pP-m1itted tbe isolation of specific genes. The use of restriction en~ 2ymes and gd e1ectropho.resis to isolate specific DNA fragments is descr1bed early in Cltaptc.r 20, and this is foUowed by a consideratlon of how such fragments can be amplifiecl and expressed in vivo. Next, we turn to techniques associated w ith in vitro amplification by polyrneJase chain reaction, and DNA sequencing. Both or these require the chemical synthesis of DNA fragments for use as primers. This t t-'Chnique is bJ:iefly described. PCR permits the pu.l'ification of virtually unlimited quantities of any given DNA segment-even whe11 starting with just a single DNA molecule. PCR amplification has revolutionized many scientific disciplines, including forensjcs, med1dne, eco1ogy. anrl of course, molecular biology. In the mid-1970s and 1980s. methods for DNA sequencing wete still manual and somewh at laborious, During the 1990s, stimulated by the ambitions of the human genome project. DNA sequencing became highly mechanized and has now developed to the point where it is possibl tl to determine the exac:t nuc:leotirlc sequtmce or entire gp,n omes in just days or weeks. Chapter 20 also includes a desctiplion of the computational methods from the emerging discipline of bioinfoiT!llltics that are used to as~rnble complex genomes and identify both protein coding genes and associated regulatory DNAs. Considerable efforts focus on comparing the genetic content of different genomes~ and the~y determ.lne the basis for organismal diversity. ln the second half of f'.hapter 20. We deal with methods of protein purification and analysis. This doses with an out line of the new field of proteomics. Cht.tptel' 21. in which we describe a handfuJ of modP.I organisms. stresses the principle that researchers employ the simplest organism in wllich the proolem of interest can be studied. The simplest o,rgan~

I

Part 5 Methods

isms of ail-in terms of genome complexity and rapidity ot· the 1ife cycle-are bcn;terial viruses, or bacteriophage. The study of bacteria and bacteriophage determined mBny of the basic features of DNA function, including the induction of gene expression, DNA replication, recombination, and repair. E. coli was the key organisn1 of study in elucidating the genetic code during the ear1y 1960s. ln the 1970s, molecular biologists were getting restless. Many felt that pTokaryotes such as bactena and their viruses Jlad been conquered and to answer the next round of biological questions dema11ded experiments on eukaryotes. Most accessible of these is the yeast. Saccharomyces cel'V.isiae. It has a very .rapid life cycle. like bacteria, but nonetheless exhibits many of the properties of more elaborate eukaryotic cells. Yeast has been used for a variety of studies, including DNA replication. the cell cycle, and transcription regulation; these studies proved most valuable because in each cas~ it was found that yea£1 contain many of the molecular machines used in higher eul aryotes as well. Chapter 21 ends with the three most popula r animal models, the nematode worm. Ctlenorhobditis elegons. the fruit fly. Drosophila melanogoster and the bouse mouse. Mus m usculus. One of the big surprises in the past 20 years is the realization that many genetic processes are high Jy conserved a mong a broad spectrum of animals, from nematode worms to humans. Exhaustive genetic screens in the fru i1 fly, for example, have identified many of the signaling pathways an d regulatory genes that co:ntrol basic devo)opmenta] processes common to highP.r animalg as welL The development of highly sophisticated gene manipulation methods in transgenic mice have pennitted researchers to determine wbat processes are conuolled by the genetic pathways found in fruit flies. Genetic'.ally altered mice also provide models for testing ideas about . (illd treatments for. many human disorders, including Alzheimers, Parkinson's disease. and rheumatoid arthritis.

PHOTOS FROM THE COLD SPRING HARBOR LABORATORY ARCHIVES Seymow Benzec;. 1915 Symposium on ttle Synapse- Using phage genettcs, Benzer defined the smatest lll1it of rrutalion. wnch turned out later lo be a Slllgle nucleotide (Chaptel 21). llissane worftalso provicled an experimental definltJon of the gene- wt1ich he called a ostron- usif1g fUl'ldional complementatioo tests. later his studies forused on bet1avior, using lhe fnlit fly as a model

Techniques of Molecular BivlCJ8y

-- ----

Wernet Alber and Daniel ~than$. l918 Symposium on DNA: Replic.ation and Recombination. These two sflared. IMth Hamilton Smith. !he 1978 Nobel J\iZe in Medicir.e for the discovery of reslnc'oon emymes and 1tlelr appicaticn to the ~lar analysis of IJNA. rhis was one of the key discoveries ., the devetopment of reCCJJT't>irwn DNA tccMology 111 the early 19705.

Paul 8efg. 1961 SJmposillm on Synthesis and Sbudurt> of MacromoiE!aJies.. Berg \1\aS a piorleer in the constructKJn of recombtnant DNA molecules .., ~ wOO< reflected in t&s shar£ of the 1980 NOOel Pnze for Chemistry.

Dale kai~ 1985 sym.,osium on Molecut.r Biology of Development. K<'JISef contllbured to the eOOv sti.Jd6es of pNg€ lambda pmpagation. One aspect of this 1/'KJfk red hem to recognire that DNA mclerules IM1h complementary siflg1e-stranded ends can readily be jcined togett>er, a fl!ldog m6cal to the devetopment ol recornllir?alt DNA tfi:hlolog.es.

mum

Walter Qlbett and David 8otstein,. 1986 Symposium «In Molecular Biology of Homo sapiens. Gibert, who ~need a dlemiccl rr~ett10d for seqJencing DNA, is shown here with Elotstew1 during the histone debate .:boot \llklell'ler 6t 11105 feasible and sensible to att~ kl sequence the human getlOITle BotslfJin, after wOOcing wtth ~e for ITldrlY years, contributEd rnt..JCh to the development ot the yeast 5. cerevislae ~ a model eukaryote fol molecular biologists; tle was also an early figu-e 11 tre 6'1ergiog f1eid of gEOOmics {Q)apters 19 «ld 20)

Albert Keston. Sidney Udenfriend. ud Frederidt sanger, 1949 Symposium on Amil1o Acids and Proteins. Keston-inventor of the test tape for detectir€ glucose-and Udenfnerld-who developed soeens for. and te5ts a. antlfnalarlal dtl.Jgs- a.e here shcmn wilh Safigef. the only persoo to vvin two Nobel Prizes 10 Chemistry. The first, m 1958, was lor developrlg a method to decermil1e the amino aad seq.Jence of a pr01em; the seamd. 21 years. later. was for devOOping the method for sequenmg DNA that is now used atmost exdus!vcly. inci.Jding Ill the automcJted rnac:hnes used to seqJence W'lole genorrtes (Q>~rs 2 at1d 20) Beyond lhe obvio~ technolog~eal achievement, detennirl1f18 that a protem had a defuled seqt.Jence revealed for the ftrSt tune that it bke!y had a dclirled structure as \.'\'ell

CHAP TE R

Techniques of Molecular Biology

INTRODUCTION The living ceU, as we have seen, is au extraordinarily complicatecl entity, producing thousands of different macromolocules and harboring a genome that ranges in size From millions to billions of base pairs. Understanding ho-w lhc genetic processes of the ceU work requires powerful, and complementary experimental approaches including the use of suitable model OTgRnisms in which the tools of genetic analysis are available, as discussed in Chapt.:-.r 21. They a lso include, as discussed here, methods for separating individual rnacromolocules from the myriad mixtures found in the cell. and for dissecting the genome into manageably-sized segments {(ll' manipulation and analysis uf specific DNA sequences. The roccessful development of such methods has Leen one of the major driving forces in the field of moJocula.r biology over tbe Jast several deeades, as well as one of its greatest t riumphs. RecentJy, it has become possible to apply molocular approaches lD the l arg~scale analysis of the fu ll complement of RNAB and proteins in 1hA ceU and to determine the nucleotide sequence of entire genomes. With a rapidly increasing number of gertome sequences becoming B\lailable, it is possible, using eomputational OJ' biointl>rmatics approaches. to undertake large-scaJe genomic comparisons of both the coding ancl noncoding regions of various organisms. ln this chapte.-, we provide a brief introduction to these molecular and computational mPthods and to the principles upon which they are base<"\. As we s hall see, the methods of molecular biolugy depAml upon, and were developed from, an understanding of the properties of biolugical macromolecules lhemsclves. For example. an un
OU TLI NE

• tntroductloo (p. 647)

• Nucleic kids (p 648)

• Proteuls (p.. 672)

648

Techniques of Molecvlar Biology

manipulating nucleic acids and proteins are, as we shall see, based on r.ommon underlying pTiociples_ Finally. a note; it is important to appreciate that when we talk about

isolating and purifying a given macromolecule m the ensuing discussion we .rar~ly {if ever) mean that a sing1c moJecule is isolated. Rather. the goal of these procedures is to isolate u large population of identical n1olecules from all of Lhe other kind.-; of molecules in the ceJL

NUCLEIC ACIDS Electrophoresis through a Gel Separates DNA and RNA Molecules According to Size We begin by discussing the separation of DNA and RNA moleculCf> by

the teclmique of gel elecll'opboresis. Linear DNA molecules separ'dte according to size when subjoct to an electric: field through a gel matrix, an inert. jello-Jike porous material. Because DNA is negatively charged. when subject to an electrical field in this w a y. it migrates through the gel toward the positive p ole {Figure 20-1). DNA molecules are flexible and occupy an effective volume. Pores in tho gel matrix sieve the DNA molecules according to thi,-; volume; large moh~ules migrate more slowly 1hrough the gel because they have a larger effeCtive volume than do smaller DNAs. and thus have mote d ifficulty passing through the interstices of the gel. This means that once the gels have been ··run" for a given time. rnoleculre of different sizes are separated because th.:.ry h ave moved different d istances through the gel. After electrophoresis i~ complete, the DNA molecules can be visualized by st.nin.i:ng tlle gel v.rith fluorescent dyes, such as ethidium, which binds to DNA and intercalates between the s tacked bases (see Figure 6- 2-U). Each band reveals the presence of a population of DNA mo]ecules of a specific size. Two alternative ki nds of gel matrices at-e used: polyac.:rylamidc and agaro5e. Polyacrylamide has high resolving capability but can

FIGURE 20-1 DNA~ by Jet electnJphoresis.. The figlwe slnvs a gel from the s1Cie in ooss-sedloo. Thus the "INelr into whi<:t1 the DNA mixture is loaded onto the gel rs indicc1led at the left. at the head cf the gel. That is also the end at 'IM-JCh the c.athode cJ the ell:< Inc fteld as locaed. the anode being at the foot of the gel As a result lhe DNA fragments, whld1 are negalively charged. move ttvoogh the.gel from the head lO lhe fool The distapce they ITa\lelrs thverscly rerated to the size of the DI\IA fragment. as cJlown. (Sol.fce. Adi:lJ)ted from Mk;klos D.A. and Freya G.A. WQ3. DNA soenre: A fust course, 2nd edition. p. 114. Cold Sprirlg }iarbor L.aboratOJy Press. Cold Sf:mg

Hadxx. NY.)

Nucleic Acids

separate DNAs only over a nanow size range. Thus. electrophoresis through po1yacrylamidc can resolve DNAs that differ from each other in size by as little as a singla base pair but only with molecules of up to several h undred base pairs. Agarose bas less resolving power than polyacrylamide but can separate from one another DNA molecules of up to tens. and evt)n hundteds, of kilobases. Very long DNAs are unable to penetrate the pores even in agarose. Instead. they snaketheirway through the matrix with one end h~ading the way and the other eod trailing from bol:Und. As a consequence, DNA molecules above a certain size (30 to 50 kb} migr-dte to a similar extent nn.d so cannot .readily be resolved. These veJ'y long DNAs can, however, be resolved from one anolhe:r it' the electric field is applied in pulses 1hat ate oriented orthogonally to each other. This technique is known as pulsed-field gel electrophoresis (Figure 20-2). Each time the orienta tion of the electric field changes. the DNA molecule, which is snaking its way 1hrough tlm gel, must nrorienl to the direction of the nel~ -Geld. The larger the DNA. tbe longer- it takes lo reorient. Putsedfield gel electrophoresis can be used to determine the size of entire bacterial chromosomes and chromosomes of lower eukaryotes, such as fungi. That is. rnolecules of up to several Mb in length. Electrophoresis separates DNA molecules. nof only according to their molL'Cular weight. but also according to their shape and topologi-cal properties. A circular DNA molecule that is relaxed or nicked migra1es more slowly than does a linear molecule of equal mass. Also, as we hijve seen. superc:oiled DNAs. wll icb are compact and have a small effective vol.ume, migrate more rapidly during eloctl'opboresis than do tess supercoiled or relaxed circular DNAs of equal mass (Chapter 6, Figure 6-26). Electrophoresis is used to separate RNAs as well. Linear doublestranded DNAs have a uniform secondary structu.re, a nd rheir rate of migration during electrophoresis is pmport ional to their molecular weight. Like DNAs, RNAs have a uniform n~ative charge. But RNA mo]~;~.cules are us ually si nRie-stran d~d ami ha\'El, as \1\re have seen (Chapter 6), extensive secondary anCJ tertiary structure. wbich influences their electrophoretic mobility. To deal with th is. RNAs can be treated w ith reagents. such as glyoxal. that react with the RNA in such a way as to prevent the formation of base pairs (glyoxal forms adducts with amino groups in the bases. thereby preventing basepairing). GlyoxyJated RNAs are unable to form secondary or tertiary structures and hence migrate with a mobility lhat is approximately proportional to molecular weight. As we w ill see in a later section , electrophoresis i.s used in a similar way to separate proteins on the basis of thf3ir .size.

Restriction Endonucleases Oeave DNA Molecules at Particular Sites Mo,.;t natura)}y occurring DNA molecullffl .are much larger than can readily be manage
-- electrodes --~ ,_ 8

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"'

< /

20-2 Pulsed..fietd eel ~-· fr1 this figure. the agcwose

F IC U~ f

Ed 6 shown fran CJbove IMth the l'l6':ld cl the gf;l and a series of~ wells, at lhe top. Aand l3 represent two sets of electJodes. TIM:Se &e switched 011 and off .:sltemalely, as deso'lbed ,., t11e WMe

tre anode cl that parr IS situated. ~ A tS sv.;tdled off, and B IS swi!chai on, the DNA moves km.Yd the bottom left CDf11€l.. The .arrOOtS thus show the path fullowed by the DNA as e1ectrqilcre5l5 procee~ (Soufce: Adc'lfJ(ed fiOOl Sarrbook J. and RusseJI D.W 1001 Mo/roJ/a clon;f1SF A lobotorory mantral. 3rd e®oo, p. 555, f\g 5·7. Cdd Spong Hartcr LilbcratOfY Press, Cdd Spring Hdlba, NY.)

650

Techniques of Molecular Biology

Restriction enzymes used in molecular biology typicaUy recognize short (4 - 6 bp) target sequences, usuaJly palindromic. and cut at a defined position within those sequences. Thus, consider one widely used restriction enzyme, EcoRI, so named because it was found in certain strains of ~scherichia coli. and was the first (I) such enzyme found in that species. This enzyme recognizes and cleaves the sequence 5'-GAATIC-3'. (Because tbe two strands of DNA are complementary, we need specify only one strand and its polarity to describe a recognition sequence unambiguously.) This bexameric seque.nce (like any Qther) would be expected to occur once in every 4 ldlobases on ave1•age. (This is because there are four possible bases that can occur at any given position within a DNA sequrmce. and so the chances of finding any given specific (3 bp sequenc~e is 1 in 4tl.) So, consider a Jinea.r DNA molecule with six copies of the GAATIC sequence: EcoRI wouJd cut il ioto seven fragments in a range of sizes reflecting the distribution of those sites in the molecule. Suppose we then subject the EcoRl-cut DNA to electrophoresis through a gel: the seven fragments would separate from each other on the basis of their different sizes {Figure 20-3). Thus, in the exporim~nt shown, EcoRJ has dissected the DNA into specific fragments, each corresponding to a particular region of tht~ molecuJe. lf the same DNA molecule had been deaverl w ith a different restriction enzyme~ fot example. Hindm. which also recognizes a 6 bp target, but of a different sequence {5'-AAGCIT-3')- the molecule would ha\le ])een cut at ditTecent positions and generated fragments of different sizes. Thus, the use of multiple enzymes allows different regions of a DNA moJocule to be isolated. It also allows a given molecule to be idcnti.tiert. Thus. a given molecule will generate a characteristic series of patterns when digested with a set of different enzymes. Other restriction enzymt:l5 such as .SUu3Al (wbich is found in the bacterium ~tophylococcus Q.Yreus) recognize tetrameric sequtmces (5'·GATC-3') and so cut D~A more frequently, app.roximately once every 250 bp. At the other extreme is Na tl. which recognizes an octamcric sequence (5'-GCGGO:Xxr3') and cuts. on average, only once every 65 kilub.sses {Tahle 20-1 ).

EcoRI sites (

fIG (J ft E 2()..3 Dige$1i0fl of a DNA

fragment with endonudeese Ec:oRt. N. the top lS st'OM1 a DNA 111ClleaJie afld the posifu:lns witl1111 11 at \Mitch fc()RJ deaves. When thE: moleoJle. ~~ed with tl'lOt enzyn"le, is rU(l on an <18Se sef, the pal tErn cJ bands .sflown are

' A

B

0

~.

E -

aAdecreasing size

oc-

FG -

+

+ c

h

0

E

F G

Nucleic Acids

ti5J

TAB L £ 2o-1 Some Rest.rictioll fl\don\ldeases and Their Recognition SequenceS

Cut frequency" Sau3A1

5 '-GATG-3'

fecAl Noll

5'-GAATIC-3'

5'-GCGGCCGC-3'

025ktl 4kb

65kb

Cllt

Restriction enzymes diffe.r not only in the specificity and length of their recognition sequences. but also in the nature of the DNA ends they generate. Thus, some enzymes, like Hpal, generate flush ends: others. such as Ec·.oRI. Hindm and Pstl. generate ~taggered ttnds {Figum 2o-4j. For example, EcoRT cleaves covalent (phosphodiester) bonds between G and A at staggered positions on each strand. The hydrogen bonds between the 4 base pairs between these cut sites are easily broken to generate 5' protruding ends of 4 nucleotides in length [Figure 2Q-5). Notice that these ends are con1plementary to each other. They are said to be .. sticky" 1->ecBuse they readily amteal througb base-pairing to each other or to other DNA molecules cut with the same enzyme. This is a useful property that we consider when we discuss DNA cloning.

CUI

FIG ll R l

20-4 Rt!(()grutil)n ~eJ\CeS

and cut sites of various eadoi'Kideases.

As shown, not only do different efldonuc:leas5 recogme Offefent l«get sstes. lhey olso cut

at difterenl ~·l.JCnS ~ those sites_ Thus molecules with l:blt ef1ds Of Wltb 5• CY 3 • overhanging ends can be generated

DNA Hybridization Can Be Used to Identify Specific DNA Molecules As we saw in Chapte.r 6, the capacity of denatured DNA to reanneal (that is. to re-form bven by hybridizing ,;bort random hexameric oligonucleotides

f I Ci URI 2Q-5 Cleavage ohr1 EcoRI site. EcoRI a lls the tvvo sttilflds Wlhin its rec~tlOn Site to gtve 5' Oll'el'hang.ng E'lid5. These are

called ·stidY wds beccuse !hey readil)l adhere to other mdro.Aes cut \r.'lth the samf:! mzyme

bec.c~U5e they provide complementaty singl~ed eJ1ds tr.at come aogeltler

througl'l bdse-pa••n~

to DNA and a llowing a DNA polymerase to extend them. The labeled prP.CU.l'Sors are most commonly nucleotides modified with either a tluorescent moiety o.r radioactive atoms. Typically the fluorescent moiety need only be attached to the base of one of the four nudt>Otides used as precursors for DNA synthes~ (about 25% of labeling is generaJJy sufficient for most purposes). DNA labeled with fluorescent precursors can be detectecl by irradiating the DNA sample with appropriate wavelength UV light and monitoring the longer wavelength tight that is emitted in response. Radioactively labeled precursors typically have radioactive 9Zp or 35$ incorpro-eted into the alpha phosphate of one of the four nucleotides. As you wiJJ recall, this phosphate is retained in the product DNA (see Chapter 8}. Radioactive DNA can be detected by £XPOSing thu sample of interest to X-ray fUm or by photnmulhpliers that emil light in response to excitation by the beta particles emitted from a~p nnd $ S. Tltu.re are many ways that hybridization is used in the identification of specific DNA or RNA fragments. The two most common are described below,

Hybridization Probes Can ldentify Electrophoretically.. Separated DNAs and RNAs U is often desirable to monitor the abundance or size of a particular UNA or RNA molecule in a population of many other similar molecules. FOT

example, this can be useful when determining the amount of a specific mRNA that .is expressed in lwo different cell types; or the length of a restriction fragment that oontains the gene you are studying. This type of information can be obtained using blotting methods that localize specific nucleic acids after they have been separated by electrophoresis. Suppose that you have cleaved the yeast genome with th_, •-es1riction fragment llcoRJ and want to kno~ the size of the fragment that contains your gene of interest. When stained with ethidium b.rontide, the thousands of DNA fragments generated by cutting the yeast genome are too numerous to resolve into discretely visible bands, and instead look 1ike a smear centered around 4 kb. The technique of Southocn blot hybridization (named after its inVFmt or Edward South· ern) allows you to identify within the smeat the s ize of the particular fragment containing your gene of inturest. In this procedure, tbe cut DNA that has been separatad by gel e lectrophoresis is soaked in alkaH to denature the Uouhle-st.ranrled DNA fragments. Those fragments are then transferred to a positive!ychargecl membra ne to w hich they adhere, L'feating a u imprint, or blot The DNA fragmet1 ts are bound to th e membrane in positions oomparable to where they migrated in the gel during electropho~cds. The DNA bouod to the membrane is then incubated with probe DNA containing a sequence complementary to a sequence within the gene of interest. This probiog is done u nder conditions of .salt concentration and temperature close to those at which n ucleic acids denature and rooature. Under these conditioru;, the probe DNA will ouJy hybridize tightly to its exact complement. Often the probe is in high molar excess compared to its i nunobilized target on the filter, thereby favoring hybridization rather than the reannealing of the denatured DNA. Also, the immobilization of the denatured DNA on the filter tends to interfere with renaturation anyway. Where on the blot the probe hybridizes can be detected by a variety of films or other media

Nuclmc Acids

that are sensitive to the light or electrons emitted by the labeled DNA . When, for example, an X-ray film is exposed to the filter and then develope d, an autoradiogram is produced in which the pattern of exposure.on the film corresponds to the position or the hybrids on the filter (Figure 2(1-6). A similar procedure called northern blot hybTidization (to di...tinguish it from Southern blot hybridization) can be used to identify a. particular mRNA in a population of RNAs. Because mRNAs are relatively shOI't (typically less than 5 kb) there is no need for them to be digested with any enzymes (there are only a lim ited num ber of spe~ d fic RNA cleaving enzymes anyway). Otherwise. the pro1ocol is fairly similar to that described for Southern blotting. T he separated rnRNAs ar e trallsferred 1o a positively-cl1arged membra11e and pmbed with a radioactive DNA of choice. (ln this case. hybrids are formed by basepairing between complementary strands of RNA and O)';A. ) An experimenter might carry out northern blot hybridization to ascertain the amount o £ a particular tnRNA present in a sample rather than its size. This measure is a reflection of the level of expression of the gene that encodes that mRNA. Thus, for example. one might use northe rn blot l1ybridization to ask how mucl1 more tllRNA of a spedfic type is present in a cell treated with an inducer or the gene in question compared to an uninduced celL As anolhei example, northern blot hybridization might be carried out to compal'e the relative levels of a particular transcript {and hence the expression level of the gene in question) between different tissues of an organism. Because an excess of DNA probe is used in these assays, the amount of hybridization is related to the amount of mRNA present in the original sample. allowing the re lative a mounts of mRNA to be dete.nnined. The principles of Sout hern and northern blot hybridization also underlie gene microarray analysis, which we consider in 01apter 16. In microarray analysis, tbe hybridization probe C(1mprises amplified eDNA generated from total RNA from a cell m· Hssue. These probes are hybridized to an array of DNAs. each corresponding to a different gene in the orgamsm under st udy. The intensity of t.he hybridization signal to each of the DNAs in the array is a measure of the level of ex· prt>..ssion of the gene in <JUestion.

Isolation of Specific Segments of DNA Much of the molecular analysis of genes and their function requires the separation of specific s~ents of DNA from mur.h larger DNA molec:ules, and their selective amplification. This aJ lows the information encoded i n that particular DNA molecule to be a nalyzed. Thus. the DNA can be sequenced. or it can be e"Xpresscd and its product studied. The ability to purify specific DNA molecules in significant quantitics allows them to be manipuJated in various other ways as well. Thus, recombinant DNA molecules <".an be created. These <".an be used to alter the expression of a particular gene (by fusing its coding sequena! to a promoter, for example) or even to generate DNAs that encode so-called fusion proteins - that is, hybrid proteins made up of parts derived from different proteins. The techniques of DNA cloning and amplification by PCR have hocome essential tools in asking ques-

653

gel

0

--

-===

blot

-

-f I~ U R E 2o-6 A Southern b&ot. DNA fragments,. ger1efCited by dtgeSOOn ot a ONA molecLle by a restlldton enzyme. ~ run out on an agarose gel. Once statned, a panem or fr.!gments .s seen When rransfared co a fMief ar.d probed wilh a DNA fmgrnent homologcm to just one sequence in the digested rnoleaAe. a smgle band ts seen, correspondweg to the p<:Jst· lion on the gel ot the fragment
lions ahout the conu-ol of gene expression and mai ntenance of the genome.

DNA Cloning The ability to construct recombinant DNA molecules and maintain them in cells is called DNA cloning. This process typically involves a vector that p rovides the information necessary to propagate the cloned UNA in the cell and an insert DNA that is inserted within the vector and includes the DNA of interest. Key to creating recombinant DNA molecules are the restriction enzymes that cut DNA at specific sequences. and other enzymes that join the cut DNAs to one another. By croating recombinant DNA molEJculos that can he propagated in ~~ host organism, a particular. insert DNA can be both purified from othar DNAs and ampHfied tu produce large quat~tities. In the remainder of this section. we describe how DNA molecules are cut, recombined, and propagated. We then discuss how large collections of such hybrid molecules, called libraries. can be created. In a library, a common vector carries many alternative inserts. We describe how libraries are made and how specific DNA segments can be identified and isolated from them.

Ooning DNA in Plasmid Vectors Once t he DNA is cleaved .into fragments by a restriction enryme, it typically needs to oo inserted into a vector (or propagation. That is. the DNA ft'agment mut."t be inserted within 1hat second DNA molecule (the vector) to be replicated in a host organism as we described above. By far the most conunon host used to propagate DNA is the bacterium E. coli. Vector DNAs typically have three characteristics. J. T hey contain an origin of replication that allows them to re plicate

independendy of the duomosome of the host . 2. They contain a selectable marker that allows colJs tllat contain the vector (and any attached DNA) to be read ily identified. 3. They have single sites for one or rnore restriction enzymes. This allows DNA fragments tube inserted at a defined point within an otherwise intact vector.

'f he most common vectors are small (approximately 3 kb) ci:rc-.ular DNA molecules that are called plasmids. These molecules were originally derived from circu)ar ONA molecules that are found naturally in many bacteria and single-cell eukaryotes (Chapter 21). In many cases. these DNAs carry genes encoding resistance to antibiotics. Thus. naturally occurring plasmids already have two of lbe characteristics desirable for a vector: they can propagate independently in the hnst and they carry a selectable marker. A further benefit is that these J>lasmids ~re sometimt!S present in multiple co{lies per celL This increases the uru.ount of DNA that can be isolated from a populetion. In s<>me cases these plasmids also have useful unique resbiction sites. However. since their discovery t he plasmids have been simplified and modified such that a typical plasmid vector now has greater than 20 unique restriction sites within a smaU region. This allows a much more diverse array of restriction enzymes to be used to cut the target DNA. Bac.:terial viruses- phagt:! - have been modified to allow their use as cloning vectors as well (see Chapter 21).

Nucleic Acids

To insert a fragment of DNA into a vector is a relatively simple process {Figure 20-7). Su ppose that a plasmid vector has a unique recognition site for EcoRI. Treatment with that res1riction enzyme would linearize the plasm id. Because EcoRJ generates protruding 5' ends that are complementary to each other (FigW'E! 20.5 ). the sticky ends are capabh~ of reannealing to Te-foTID a circle with two nicks. Thus, treatment of the circle with the enzyme DNA ligase and ATP would seal the nick.c; to re-form a covalently closed circle. A target DNA is cleaved with a restriction enzymft to gemeratc potential in&-erl ONAs. Vector DNA that h as lx>cn cut w ith the same enzyme is mixed w ith those insert DNAs and DNA ligase is used to link the compatible ends of the two ONAs, By adding an excess of the in~ sert DNA relative to the plasmid DNA, the majority of vectors will r& seat with insert DNA incorporated (Figure 20~7). Some vecto[s noi only allow the isolation and pwificatl on of a par~ ticular DNA, but also drive the expression of genes within the insert DN A. These pl~smids arc called expression vector'S and have transcriptional promoters im mediate ly adjacent to the site of insertion . 1f the coding region of a gene (w ithout jts promoter) is placed at the site of insertion in the proper orientation, then the inserted gene will he tr.mscribed into mRNA and translate d into protei n b y tt'le host cell. Expression vectors are frequently used to express heterologous or mutant genes to assess their function. They can also be used to produce large amounts of a protein for pul'ification. In addition , the promoter in the expression vector can be chosen such that expression of the insert is regulated by thP a ddition uf a simple compound to the growth med ia (for exam ple. a s ugar or an amino acid). This control of w hen the gene wilt be expressed is particularly useful if the gene product is toxic.

Vector DNA Can Be Introduced into Host Organisms by Transformation Propagation of the vector with itc; insert DNA requires this recombinant molecule be introduced into a host ceJI by transformation. Tran!lfonnation is the process by which a host organism can take up DNA from its environment Some bacteria. but not E ooli. can do this naturally and are srud to have genetic competence. E. coli can be rendered competent to take up DNA , h owever. by treat ment with calcium ions. Although the exact mochanism for DNA uptake is nut 1-nown. it is likely that the Ca2 .. ions shi£!ld the negative charge on the DNA. allow ing it to pass throu~h the cell membrane. CaJcium-treated cells are thus said to be oom petenf to be transformed. An antibiotic to which the plasmid imparts resistance is then uSL-d to select trano;formants that have acquired the plasmid: cells harboring the plasmid w HJ be able to grow in the presence of the antibiotic w hereas those lacking it will not. Transformation generally is a relatively ineffici ent process. Only a small percentage of the DNA-treated cells tale up the plasmid . It is this low efficiency of transformation that makes necessary selection with t he antibiotic. After DNA treatment. the cells are transferred onto medium containing the relevant antibiotic and only those ceJls that have taken up the plasmid and maintain it stnbly are able to grow. The inefficiency of transformation also ensures that , in m ost cases, each celll'eceives only a single molecule of DNA. This property makes

only cds contaning recoo-A:linar'l'l plasmid SU'Viw to~ resiStal'1t COlony

fl G U R E 2D-7 lloning in a plasmid vector. A lla~ d DNA, grne~ated by cleavage With EwRI. IS tnser1ed 111to tfle plasmd vector lineallled by that Silrl"\e en~ ~

ligated (see ta1). the recooi>inant plasmd ~ ~ trocllJc:E'd ll'liO ~13. by tratlsforrnabon (see text). Ceh contall'lirg the plasmid can be st."" b!Pd by gJUIIII1h on the c.ntbobc lov.htch the plasrr!ld confas r~nce. ~e: Adaptf!d from Mtcl
Techntgues of MDJecular Biology

656

each transformed cell and its progeny a carrier of a unlque DNA molecule and effectively allows the purification of that mole.cule away from all other DNAs in t he transforming rnixhrre.

Libraries of DNA Molei::ules Can Be Created by Cloning ligate fragments

into vector

l

transformation

!

pfafe cells Of'llo filter on agar plate

l

remove flitel

and prepare for hybridization

-

l

~ hytmdized

filter to X-ray film

(_ F IGURE

20.8

Construdion ofa DNA

libtary. ro construct the htlf<JY. genooic DNA and vector DNA. digE-sted with the same restnc: bOn enzyme. 13re rnabated cogether With hgase The reS!Jtq pool 01 bart ot hybrid veoors (e<>ch vectol carl)illlg a diffecert 111sen of ~mJC. 01\lA. represented 1r1 a different cob) is then m o&Jced 111to E. coli, !lf'ld f.he eel$ ~ pcted oolo a filter placed 0\t-er ilgi!r nle!hum Onr:e colonies hav~ ETOINJ1. the filter is removed from the plate and prepared fa h}'bndzanon: eels are lysed. the DNA ts deoatlifed. cn1 the filer rs ri'ICI.Jbated \1\ith a labeled piobe The done of rnleresl ts idecitified by autorodiographv

h is trivial to generdte a specific clone if the starting donor DNA is simple. Thus, if the starting DNA is small {derived ti:om a small virus, for example, with a genome of p erhaps only 10 kb), then this can be accom plish ed sirnpl.Y by separating the DNA fragm ents aflcr digestion with restriction enzymes and gel elettrophoresis. On~ separated. ONAs of ditleront sizes r.an be excised from the gel and purified prior to insertion into a vector. This is h arder to do if the ~1arting DNA is T!lore c.omplex (for example. the h uman genome). In this casep simple e lectrophoretic separation of DNA treated w;tb a restriction enzyme will resul1 in very many fragments distributed in a broad range of sizes around the average distance between cut sites. Thus. it is easioc under these circumstances to clone the w hole population of fragments and separate the individual dones afterwards. A DNA lib.-ary is a population of identical vectors that each con· tains a different DNA inserl (Figure 2o-8). To construct a ONA library. the target DNA (for example, human genomic DNA) is digested with a restriction enzyme t hat gives a desired average insert size. The insert size can be of any size ranging frum less than 100 base pairs to more than a megabase (for very large insert sizes the DNA is typically incompletely cut with a restriction enzyme). The cleaved DNA is then mixed with the appropriate vector cut with th e same restriction enzyme in the presence of ligase. This creates a large r.ollcction used e fficien tly only wh en the organism in question has relatively little noncoding DNA. For an organism with a more complex genome. t his type of library is not suitable for this task because many of the ONA inserts will not contain coiling DNA sequences. To enrich for coding sequences in the library, a eDNA library is used. This is made as follows (Figure 20·9] . In stead of starti ng w ith genomic DNA. mRNA is converte d into DNA sequence. Th e process that allows this i.s called re\lexse transcription and is performed by a s peda1 DNA pol ymerase (reve rse transcriptase) that can make DNA from an RNA tem plate (see Chapter 11). When treated with reverse tr.mscriptase, mRNA sequences can be converted into doublestranded DNA copies that are calle-d cDNAs (for copy DNAs). Thes~ fragmen ts are than ligate d into the vector. To isolate in dividual inserts from a library, E. coli cells are transformed with tho entire library. Each transformed cell typi cally contains on ly a s ingle vector with its assodated insert DNA. Thus . each cell that propagates a fte r trans forma tion will contain multiple copies of jus t une of the possible clones from t he library. The colony produced from ceUs carrying any donecl sequence of interest can be

Nudek: Acids

identified and the DNA retrieved. There are various ways to identify the done. For t!Xample. as we will describe be low, hybridization with a unique DNA or ~'JA probe can ic..lenti fy a population of cells that include a particular insert DNA.

Hybridization Can Be Used to Identify a Specific Clone in a DNA Library When attempting to d one a gene. a common step is to identify fragments of that gene among clones in a library. This can be achieved using a ONA p robe whose s equence matches part of the gene of interest. Such a pro~ f'.an be used to identify colonies of cells harboring clones containing t hat region of the gene. as we now describe. The process by which a labeled DNA probe is used to screen a library is called t:olony hybridization. A typical eDNA library will have thousands of different inserts. each contained within a common vector (see above). After t.l'ansforrnation of a suitable bactcriru host slt'ain with the Ubrary, the cells are plated out on p€tri dishes containing solid growth medium (usually agar-see Chapter 21). Each cell grows into an isolated colony of cells. and each cell within a given colony contai ns the sam~ vector and .ins~rt from the library (there are typically a few hund red colonies per dish). The same type of positively-charged membrane filter used in the Southern and northern bJotting techniques is again used to secure smRll amounts of ONA for probing. ln this case. pieces of the membrane are pressed on top of the dish of colonies, and imprint s of ~lis (including some DNA) fron1 each colony are left on the filter. Thus. the filter re tains a sample of each DNA clone posjtioned on •he filter in a pattern that matches the pattern of colonies on the plate. This ensures that once the desired clone has been identified by probing the lUter, the colony ol coils carrying that clone can be readily identified and the pla:smid containing the appropriate insert ONA can be purified Probing of the fi lters is carried oul as follows. They are treated under conditions that cause the cells on the membrane to break open and the DNA to leak out and hind to the filter at the same loca· Uon ao; the cells the DNA was derived from. The filters can then be incubated wiCh th e labe led probe under tht! sa me conditions that were used in the northern and Southem blotting experiments. As we menti oned earlier and discuss in Chapter 21. bacteriophage (particu larly >.) have a lso been modified for use as vectors. When libraries are ma de using a phage vector, they can be screened in much the same w ay as just described for the screening of plasm id libraries. The d ifference is that the p laques formed by growth l•f the phage on bac ter ial lcw.IJls are screened rather than colonies (see Chapter 21).

Chemically Synthesized Oligonucleotides Short~

cuslorrrdesigned segments of DNA known as oligooudeotides are critical for several techniques we desc!'i.be in this chapter. Although DNA polymerases are the most effident m achines for synthesizing DNA molecules, DNA can also be synthesized chemically. The most. common methods of chemical synthesis a re performed on solid supports using machines that automate the process. The precursors used for n ucleotide

5'

:r

~~

S'

657

l t-

s

s

~

dNTPs

3' 5'

~ $'

1--3

~

1~-~

and DNA potymemse anddNTPs

5'

~

3'

!t

;r

s

3'

s· 3' !>'

3'

:r

'J'

1-;.oo-ONA

5'

e-DNA libJary f I c:; U R E

20-9 Comtnktioft of a eDNA

fibraty. The RNA dependent DNA po~ase revase rransaiptase (RT) trdf1soibes RNA lt'tO DNA (copy or eDNA). In the first step (fitst strand syn1hesis). ~s of poly·T sequence sc.-rve as primers by hybrtd,zirlg to !he pcly-A ~ of the mRNAs.. Reverse lra11SCI1Ptase- extends the dT Pfl'tlt:f" to wmplete a DNA copy of the mRNA terrplare The ~ 1s a doplelt con~ d ore S1rclnd uf mRNA 6T1d ds c:.tlfllJiemalta.y sttand of DNA. The RNA strand rs remoYed by trearnE!f'l ¥.1lh base (NaOH), and the ,~ Slr'f5le-stronded DNA fY::N.I seJVeS as template for the second step (serond strand syrtheSis) Short random ~es ol DNA uswtly apprO»· mately 6 bp long (c.;lled ram.1crn hel.amers)

serve as primers by hybndillng to \lclnous seqoeras along lhe COD'/ DNA teMplclte. These prlrTlef!> are thEn extended by DNA po~ to oeate do~ DNA~ that Cdn be doned trllO a pl<snid vedcl (see F~ 20-8) to ~tea eDNA ~bmry.

658

Techniques of Moleculor Biology

5'-hydrox)i blocked by

~\·: DMT-0-Q rr 0

N=C-Cf-b-CH:z-O~a

CH3

~'~

~/ 'Oi3"%

FIGURE 20-10 Protonated phospho-

rtanlidite. As fum the 5 ·-hydrox1i group IS blodced by !.he add.tion of a dimethoxyintyl pcotecung f!IOL'P·

addi1ion are chemically protected molecules called phosphoa.rnidines (Figure 20-10). Growth of the DNA chain is by addition to the 5' end of the molecule, in contrast to the direction o( chain growth used by ONA polymerases. Chemical synthesis of DNA molecules up to 30 bases long is efficient and accurate. and takes only a few hours. It is a rouHne procedure: a researcher can simply program a DNA synthes izer to make any desired sequence by typing the base sequence intQ a computer controlling the machine. But as the synthetic mulecules get longer. th e final product is less uniform due to the inherent failures that occur during any cycle of the process. Thus. molecules over 100 nucleotides or so are difficult to synthesize in the quantity and with the areuracy desirable for most molecular analysis. The r-dlher short DNA molecules that can readilv be made. however, are well suited for many purpuses. For example: a c.ustom-designed oligonucleotide harboring a mismatch to a segment of cloned DNA can be used to create a directed mutation in that cloned DNA. This method, called site-directed mutagenesis is performed as follows. The oligonucleotide is hybridi~,ed to the cloned fragment, and used to prime DNA synthesis with the cloned DNA as template. In this way, a doublestranded molecule \1\rith one mismatch is made. The two strands are then separated and that with the desired mismatch amplified further. Custom-designed oligonucleotides can be used in this manner to introduce restriction sites into cloned DNAs which are then used to cnr ate fusions between a coding sequence and another coding sequence or a promoter or .ribosome binding site. As another example, synthetic oligonucleotides that have been labeled fluorescend y or radioactively can be used as pmbes in hybridization experiments. Moreover. customdesigned oligonucleotides are critical in the polymerase chain reaction, which we describe n ext, and are an indispensable feature of the DNA sequencing strategies that we describe below. Therefore, a common feature in designing experiments to construct new molecular clones of genes to detect specific ONAs, to amplifY ONAs. and to sequence DNAs is to design and have synthesized a short synthetic DNA oligonucleotide of desired sequence.

The Polymerase Chain Reaction (l>cR} Ampli6es DNAs by Repeated Rounds of DNA Replication in Vitro

ro.r

1\ powerful method amplifying particular segments of DNA. distinct from d on.ll1g and propagation within a host cell. is the polymerase

chain reaction (PCR}. This procedure is carried out entirely biochemically, that is, in vitro. PCR uses the enzyme DNA polymerase that directs the: synthesis of DNA from deoxynudeotide substrates on a singl~ stranded DNA t.cmplate. As we saw in Chapter 8. DNA polymerase synthesizes DNA in a 5' to 3' directiun and can ~dd nuclcotides to the 3' end of a custom-designed oligonucleotide. Thus. if a s}'nthetic oligonucleotide is annealed to a single-stranded 1emplate that contains a region complementary to the oligonucleotide. DNA polymerase can uoo the oligonucleotide as a primer and elongate it in a 5' to 3' direction to genernte an extended region of double-stranded DNA. How is this enzyme and reaction exploited to amplify specific DNA sequences? Two synthetic. single-stranded oligonucleotides are synthesized. One is complementary in sequence to the 5' end of one stmnd of the DNA to be amplified. the other complementary to the 5 ' end of the other strand (Figure 2o-11 ). The DNA to be amplified is then denatured and the oligonucleotides mmealed to their target sequences. At this

Nucleic Acids

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the end of the lemplates). Thus. an lhlS second cyde., DNA will have been syrlth€sized that precisely spans the- DNA sequence lo be amp~ed. lhereafter. further munds cl deni!turat10n. prifrq; and DNA synthesis (not shown) ~Ail generate ONAs that cooespond to the sequeoce interval set by the two prrners. This ONA wiU ancrease in ct>undance geometracaly with each subseQuent cyde of the chain readio~

point. DNA polymerase anrl rleoxynucleotide substrates are added to the reaction and the enzyme extends the two primers. This reaction generates double-stranded DNA over the region of inter~1 on botlt of the strands of DNA. Thus two double--stranded copies of the starting fragment of DNA are produced in this, the first, cycle of the PCR reaction. Next. the DNA is subject to ano1her ronnd of denaturation and DNA syntht>Sis using the same primers. Tiris generates four oopies of tlu3 fragment of interest. ln this way, additional repeated cycles of denaturation and primer-directed DNA synthesis amplify 1he region between the two primers in a geometric manner (2 , 4. 8. 16. 32, 64, and so forth) . So a fragment of DNA that was originally preSt!nl in vanishingly small amounts is amplified into a relatively large quantity of a double-stranded DNA (see Figure 20-11). ln a sense, DNA cloning and the polymerase c hain reaction (PCRJ roly on the same concept: repeated rounds of DNA duplicationwhether carried out by cycles of r.oH division or cycles of DNA synthe5is in vitro-amplify t iny samples of DNA into large quantities. In cloning. however. we oft en rely on a selective reagent or other device to locate the amplified sequence in an already existing library of clones, whereas jn PCR, the selective l'f'.agent, the pair of oligonucleotides, limits the amplification proc-ess to the particular DNA sequence ofin teresl from the beginning (see Box 20..1, Forensics and the Polymerase Chain Reaction}.

Nested Sets of DNA Fragments Revea] Nucleotide Sequences We next conside r how nucleotide sequences are dettffmined In a sense. nucleotide sequencing represents the ultimate in probing a genom e with high selectivity. We determine the entire sequence of nucleotides for a genome, as has now been done for organisms ronging in complexity from bacteria to Homo sapiens. and thls permits us to find aoy specific sequence with great rapidity and accuracy through thE! usc of a com puter and appropriate algorithms. In other words. our "selective reagent .. when dealing with nucleotide sequences is a string of bases that we feed into a computer. 111e increasing availability of large rtumbots o£ genome sequences mal-es h possible to sewch with hig h precision for copies of l'e lated sequences both w ithin and between organisms in silico. Obviously. nucleot ide sequencing generates extraordinarily powerful databases as we shall describe below. The underlying principle of DNA sequencing is based on the separation, by size, of nested sets of DNA molecules. Each of the DNA molecules starts at a common 5' end, and terminates at one of several alternnUve 3' endpoints. Members of any given set have a particular type of base at their 3'
Imagine that you are in a lorensic li!boortory af1(1 have a DNA sample from a suspeefs of the populatm Of dlffefeoces in the length of a sjmple ntJcleotkle repeat sequence sudl as CA (see Chapter 9). What we want to do is ~ify ONA Sllrrcxm
mromosome,

svquence. Onnparing the posilions of the rungs in these four larldeTs reveals the entire sequence ~f the starting DNA molecule. Alternatively. the fout nested sets can be differentially labeled with distinct nuorophores. allowing them to be subjected to electrophoresis as a single mixture and distinguished later using fluorometry. How are nesied sets of DNA molecules created'? Two methods were invented fot doing this. In one, DNA molect.Ues are radioactively labeled at tbeir s ~ termini and are then subjected to four different regimens of chemical treatment thai cause them to l:.rteak preferentially at Gs. Cs, Ts, or As. This chemical procedure is no l onger in wide use, and we will not consider it further. The other procedure. which employs chain-terminating nuclootides. continues to be used to this day and js the tech nology upon which modern, automatic seque11cing machines called Sequenatoi'S are based. ln the chain termination melhod. DNA is copied by DNA poly~ merase from a DNA template starting from a Uxed point specified by the use of an oligonucleotide primer. As we saw in Chapter 8. ONA polymerase use.s 2'-deoxyn ucleosirle triphosphates as substrates for DNA synthesis, and DNA synthesis occills in a 5' to 3' direction. Phosphodiester bonds are formed b y the nudeopbili.c attack of the 3'-hydroxyl a1 tho 3' end of the growing polynucleotide chain on the a-phosphate of an incoming substrate molecule. (The chain termination method relies on lhe principles of enzymatic synthesis of ONA. which we discussed in {;hapter 8.) The chain terminRtion method employs special. modified substrates called 2'-,3'-dideoxynucleotid ns (ddNTPs), which lack the 3' -hydroxyl group on their sugat moiety as weU as the 2'-hydroxyl (Figure 2{)-12). DNA polymerase wiU 2'· .3'·dideoxy ATP

H H FIGURE 20-12 Dideoxynucleotides used in DNA sequencing. On the left IS 2'-..deoxy ATP, This can be iocorpofated 1nto a growing DNA tha10 and allow arotl>ef ru.ieotlde kl be •nc.orporaled d reclly after it On the right is 2' ·,3 •-dideoxy JrrP. This <.an be incOfPOOlted 111to a growng DNA chall'\ but ono'~ in pla£e It triocks Mthei nudeolldes be.1g added to the 5ame tt.alf1.

ti62

Techniques of MoJeculor Biology

FIGURE 20.13 0\ain tenninalionin tile presence of dideo.ynudeotides. In the tcp line is. a ONA cham being extended at the 3• end With addition of an adenine nucleolide onto ll.1e previously lflCOlpOfated c.yt05il1e. The presence of dideox}
primer

OH

incorporate a 2'-,3'-dideoxynucleotide at the 3' end of a growing polynucleotide chain but once incorporated, the presence of the modified nucl{.'Otide causes elongation to terminate. The reason for this is the absence at the 3' end of the growing chain of a 3'-hydroxyl, which is needed fo.r nucleophilic attack on the next incoming substrate molecule (Figure 20-13). Now suppose that we "spike" a cocktail of the nucleotide substrates with the modified substrate 2'-,3'-dideoxyguanosine triphosphate (ddGTP) at a ratio of one ddGTP molecule to 100 2' -deoxy-GTP molecules (dGTP). This will cause DNA synthesis to abort at a frequency of one in one hundred eve.ry time the DNA polymerase encounters a C on the template strand (figure 20-14a). Because aH of the DNA chains rommence growth from the same point, the chain-terminating

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nucleoticles will generate a nested set of polynucleotide fragments. all sharing thu same 5' ~Jld bur oiffeling in their lengths an d hence their 3' ends. The length of the fragments, thocefore, specilles the position uf Cs in the template strand. If the fragments are labeloo at their 5' end through the use of~ radioactively labeled primer, a primer that had been tagged with a fluorescent adduct, or at their 3' end with fluorescently labeled derivatives of cldGTP. then upon electrophoresis through a po!yocrylarnide gel the nested set or fragments would yield a ladder of fragments, each rung of the laddtir representing a C on the template stmntl (Figure 20-14b). U we similarly spike DNA synthesi~ reactions with ddCTP, ddATP. and dd'f1Tl, then in toto we wi.U generate four nested sets of fragments, which together provide the fun nudooHde sequence of the DNA. To read tbat sequence. lhe fragments generated in each of the fm.lf reactions were n.>solvetl 011 a TJOiyacrylam ide gel (Figure 20-15). As we shall see below. this conceptually simple approach, developed initially to sequence short, defined DNA fragm ents, he~s tmder· gone a series of technical adaptations and improvements that a1low the analysis of wbolc genomes (see Box 2o-2. Sequenators Are Used for High Throughput Sequencing}.

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Shotgun Sequencing a Bacterial Genome The bacterium Hemophilus influenzne was the first free-living organism to haVf' a complete ge nome sequence and assembly. It was a logi~ cal choice since it has a small. compact genome that is composed of just 1.8 megabase pairs {Mb) of DNA. The H. influenzae gen ome was randomly shEtarcil into many random fragments with an avemge size of 1 kb. These pieces of genom ic DNA were clon ed into a plasmid recombinant DNA vector. DNA was prepared from individuaJ recombinant DNA colonies and separately sequenced on Sequenators using the clideoxy method that was discussed earlier in th is chapter. Thjs method i:s called ..shotgun" sequencing. Random reco mbinant DNA colonies are (Jicked. processed, and sequence(l. In order to make certain that every single nucleotide in the genome was captured jn the final genome assembly, som ething like 30,000- 40,000 separate r ecombin<mt clones were sequenced. A Lotal of about 20 Mb of raw ge nome sequence was produced (60fl bp of sequen ce is produced in an av~rage react;on. and 600 bp x 33,000 different colonies = .20Mb of total DNA sequence}. This is called tux sequence covP.rage. In principltl. every nucleotirlP. in the gen ome was sequenced ten times. This method migl1t seem tedious. but it is considerably faster and less expensive than the techniques that were originally envisioned. One early strategy called for systematically sequoncing every defined restriction DNA fragment on the physical map of the bacterial chromosome. A drawback of this procedul'e is that most of the known r~tl'iction fragments are larger than the amount Q( UNA sequence information that can be generated in a single reaction. C..onsequcntly, additional rounds of digestion. mapping. and sequencing would be required to obtain a complete seque.nce for any given defined region of the genome. These additional steps of cloning and restriction mapping are considerably more time consuming than the repetitive automated sequencing of random JJNA fragments. In other words, the comp11ter is much faster at assembling random DNA sequences than the fime required to perform fine-scale restriction mapping of the bacterial chromosomA. The approximately 30,000 random sequencing reads derived from random genomic DNA fragments are di!ectly loaded into the computer.

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The ~ of DI\I,A chalfl.o, taminaled v.Ttll the dtdeoxynudt>Ollde mdicated at the top of each lane, at'e detefiT\If'led by fesoMng on a polyaa)lafr4de gd. as sho1Nn ~eading the gel from top to bottom gM-~ lhe 5 • to ! · sequern.

Thdmiques of Molt'!Culclr Bio108Y

ti64

and different programs are used to assemble ove rlapping DNA sequences. This process is conceptually similar to the assembly of a jigsaw puzzle. Random DNA fragments are "assembled" based on containing matching sequences. The sequential assembly or such short DNA sequences ultimately leads to a single continuous ass~mbly. also called a contig (~Figure 20-t 7 later in this chapter).

The Shotgun Strategy Permits a Partial Assembly of Large Genome Sequences From our preceding discussion it is obvious that sequencing short 000 bp DNA fragments is incredibly fast and efficient. ln fact, the automated sequencing machines are so efficient that they far surpass our ability to assemble and annotate the raw DNA sequence information_ In otheJ words, the rate-limiting step in detemtining the complete DNA sequence of complex genomes. such as the human genome. is the analysis of the data. rather than the production of the data ~r se. We now consider how the shotgun sequencjng method used to determine the complete sequence of the H. influenzoe genome was adapted for much latger and complicated animal genomes. The average human chromosome is composed of 150 Mb. Thus. the 6()0 bp of DNA sequence provided by a typtcal sequencing reaction represents only .0004% of a typical cruoroosome. Consequently, lo determine the complete sequence of the chromosome it is necessary to generate a large number of sequencing reads from many short DNA fragments (Figure 20·16). DNA was prepared from each of the 23 chromosomes that conslitute the human genome. and then reduced into pools or libraries of smaJl n-agments using small-gauge pressurized needles. Typically. two or three libraries are constructed for fragments of diltering (increasing) sizes-for example. fragments of 1, 5, or 100 kb in length. These fragments were randomly cloned into bacterial plasmids as described earlier. Recombinant DNA, containing a random portion of a human chromosome, can be rnpidJy isolated from bacterial plasmids and then httman genome

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--------------------------------------------When the sequencing of the human genane was f1rst envtSD'led, it seemed like a daunting. virtuaJiy ~ss enterprise After all, tf1e romptete human genome consists of a staggering 3 btlion (3 x 109) base pairs, and lhe early methods for determining the nudeotide sequence of even short DNA fragments were quite tedious. 1n the 19SOs and eariy

19905, an indi111dual resear-cher could produce only a few hundred ba5e J>a'rs, pemaps 500 bp, of DNA sequence in a day or two d conceotrated effort Severallechrncal •nnuva-tions have greatly accelerated the speed and reliab~ity of DNA sequencing. As we described _, the preceOOg se
depends on the identlecation of tile last nucleotide. RJr exafll'le, ONAs ending \Nith a T residue at position 50 1n the template DNA mitfu be labeled red, while those nested ONAs ending with a G residue at position 51 mrresponding to posefun 5 1 fright be labeted black Thus, ead1 nested DNA has a unique size aod Q)b; As they are fTac:t01ated on the sequencing columns based on size, fluorescent sensofS detect the color of eadt nested OOA (&»< 20-2 Figure 1). tn this way, a single column produces 600 to 800 bp of DNA sequence after less than ttuee ho!Js of size separatm Automated sequendng machines- SequeNton-have been developed that have 96, <~nd roost recently, even 384 separate fractionation columns_ In pnne~ple, the 384-mlumn machines can generate over 200,000 nudeotides (200 kb) of raw DNA sequence tn just a few 1-oors. In a 9-hour day, each machine can produre three sequencing "runs• and more tnan ~f a megabase (500 kb) of sequence information. A duster of 100 such machines could generate the equivalent of one human genome. 3 x 1C1' bp, ., JUst two months. There are currently five majcr sequencing centa-s in the

Unit.ed States and the United Kingdom. Each contai1s large

clusters of automated DNA sequencing machlnes. Together. these five centefs proch::e a staggertng 60 x I fP bp of raw DNA sequence infoonaoon per year. (This correspoods 10 the equivalerrt ol 20 hLrnan genomes per year!)

20

50

TCAC T GCCC GCTTTCC AGTCGGGAAACC TGTC GTGCC AGCT GCATTAA T GAA TC GGGCAAC GC GCGG

BOX 20-i FIG U R.E 1 DNA sequence read out. In this re<~ction, as desaibed in the text, fluofescent ~ rl~ are used and the chains me separated by colunYI chromatogfaphy. The proRe of poslticfls of As is represented In green; Ts in red; Gs in black; and Cs m blue.

quickly sequen ced using the automated sequencing machines. To ensure tha t every sequence is sampled in the complete chromosome, an average of two million random DNA fragments are p.ror.:essecl. With an average of 600 bp of DNA SefJuence per fragment. this procedure produces over one billion bp of sequence data. or nearly ten times the amount of DNA in a typical chromosome. As discussed earlier for the S€Quencing of the bacterial chromosome, by sampling about ten times thC' amount of sequence in a c h romosome we can be confident thai every portion of the duomosome is captured. The process of producing ..shotgun " racombinant libraries and huge excesses of random DNA sequencing reads seems very wasteful. Hnwever. a duster of one humlred 384-column automated sequencing machines can generate tenfold coverage of a human cluomosome in just

Techniques of MalecukJr Biology

three weeks. This is considAI"ably fastf!r than the methods involving the isolation of knmvn regions within the chromosome and sequentially sequencing a known set of staggered DNA fragments. Thus, the key technological insight that facilitated the sequencing of the human genome was the reliance on automated shotgun sequencing and then subsequent use of the computer to assemble the different pieces like a jigsaw puzzle. The combination of automated sequencing machines and computers proved to be a potent one-two punch that led to the completion of the human genome sequence years earlier than originally planned. Sophisticated computer programs have been developed that assemble the short sequences from random shotgun DNAs into larger contiguous sequences called contigs. Reads containing identical sequences are assumed to overJa.p and are joined to form larg~r contigs (Figure 2()..17). The sizes nf these contigs dept:nd on the ammmt of sequenoe obtainoothe more St:lquence, the larger lhe contigs and the fewer gaps in t·he

sequence. lndividual contigs are typically composed of 50,000 to 200,000 bp. This is slill far short of a typical human chromosome. However. such contigs are useful for analyzing compact genomcs. For example, the Drosophila genome contains an average of one gene every 10 kb. so a typical co11lig has several linked genes. Unfortunately, more compJex genomes often contain considerably lower gene densities. The human genome conta ins an avernge of one gene every 100 kb. so a typical conlig is ofte11 insufficient to capture an entire gene, let alone a series of lint..cd genes. We now consider how relatively short contigs are assembled into larger scaffolds that are typically 1-2 Mb in ltmgth.

The Paired..End Strategy Permits the Assemblv of Large Genome Scaffolds A major )i mitation to producing larger contigs is the occurrence of repetiti"e DNAs. Such sequences complicate the assembly process since random DNA fragments from unlinked regions of a chromosome or genome might appear to overlap due to the presence of the same

j

51

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repetitive DNA sequence. One method that is used to overcome this difficulty is called paired--end sequencing. This is a simple technique that has produced powerfu l results. In addition to producing shotgun D NA libraries composed of short DNA fragments, the samo genomic DNA is also used to produce recombinant libraries composed of larger fiagments, typically between 3- 100 kb in length. C..onsider a DNA sample from a single human chromosome_ Some of the DNA is used to produce 1 kb fragments. while another aliquot of the same sample is used to produce 5 kb fragments. The end result is the construction of two libraries, one with small inserts and a second with larger inserts (see Figure 20-16). Universal primers are made that anneal at the junction between the plasmid and both sides of the large inserted DNA fragment. Individual runs v.rill produce ahm1t 600 bp of sequence information at each end of the random insert A record is kept of what end-sequences are derived from the mtme inserted fragment. One end might align with sequences con tained within contig A , while the other end aligns with a different contig, contig B. Contigs A and B are now assumed to derive from the same region of the chromosome since they share sequences with a common 5 kb fmgmenl. Most repetithre DNA sequenc.-.s are less than 2 or 3 kb in length, so the ... pRired-end'" sequences fw m the 5 kb insert are sufficient to span contigs interrupted by repeti tive DNAs. The preceding results usually produce contigs that are less than 500 kb in length. In order to obtain long-range sequence data, on the order of several megabases or more, it is necessary to obtain pa iredend sequence data from large DNA fragments that are at least 100 kb in length. These can be obtained using a special cloning vector ca lled a BAC (bacterial a rtificial chromosome). The principle of how these are used to produce lnng-range sequence infomlAtion is the SArno as that described for the 5 kh inserts. Primers are used to obtain 600 bp sequencing reads from botls ends of the BAC insert. These sequences are then aligned to different conligs. which can then be assigned to the same scaffnld by virtue of sharing sequences froTH a cnrnmon BAC insert. Thfl use of BAGs often permits the assignment of m ultiple contigs into 3 single scaffold of severn\ mcgabases (Se€ Figure 20-17}. The quaJity CJf the gr1nomP. assembly is a measure o( the 3\'crngc scartold size. Those tltflt exceed an a verage of 1 Mb or more are consirlei'E!d to be high quality assemblies. For examplE>.. the pufferfish genome is 800 Mb in length and the compJete assembled sequence is positioned on about 500 different scaffolds, eoch with an average size of 1.6Mb. This assernllly is sufficie nt for most analyses, such as the identi.fi.cahon of all protein earling genes. When Bill Clinton and Tony Blaii announced the completion of the human genome sequPnce in 2{)()(), the average scaffold sire was 2 Mb. This was sufficient to produce an occurate estimate of the genetic composition of the human genome in terms of protein coding genes (approximately 30,000 genes). However, there is the stated goal of producing a "finished" sequence. This means a single scaffold for each of tha 23 chrnmosomes. As of this writing severaJ chromosomes have been finished. and the rest are slated for completion by lh1:1 end of !:!004.

Genome.-W ide Analyses F'or the genomes of bacteria a nd simpte eukaryotes, th e process of finding protein coding genP.s is relative ly straightforward, essentially amounting to the iclenlifitA3tion of open-reading fr·Rmeti. Although not

aU ope n-reading frames- especially s mall ones- are real protein coding genes. tllis process is fairly effective. and the key challenge is in iden tifying the fu n C1 ions of th<>.se genes. For ani.mal genomes with complex exon-intmn structures. the challenge is far greater. In this case, a variety of bioinfonnatics tools aw .te<]Uired to identify genes and determine the genetic composition of complex genomes. Computer programs have been developed that identify potential protein coding genes through a variety of sequenr.e criteria. including the occurre.net> of ~x1ended open-reading fra mes thclt are flanl:cd by appropriate 5' and 3' splice sitEs (Figure 20-1 8). However, these methods ha,,e not yet been refined to the point of 100% accuracy. Perhaps someth ing like three-fourths of aU genes can be identified in this way. but many dfC missed, and oven among the predicted genes that are identified . small exons -particularly noncoding exons- are missed. A notable limitation ol' current ~"ene finder programs is the failure to identify promote rs. A typical meta zoan core promoter is about 60 bp in length and contains sequence motifs, such as TATA, INR, and DPE. which are sufficient for the binding of the TFIID initiation complex and r<-<;rui tment of the Pol Jl transcription complex fsee Chapters 12 and 17). Unfortunately, core promoter elements arP. high ly d egen erc3te, and although the transcription complex is smi'\rt enough to identify these elements within the cell, we are n ot ye t s mart enough to write programs that irlen lify them in silico even when other s(l(_)uence constraints are invoked (for example, associated exons, e tc.). Jt is conc.eivable that computer programs will he created thf't exploit all of the aforementioned properties of a gene: core promoter elements, open-reading frames, splice sites, and so on, to identify protein coding genes in a consistent and efficient manner. The most important method for validating predicted protein codin.g genes and identifying those missed by current gene lln rler programs is tb A use nf eDNA sp,quence data (see Figure 20-18). cDNAs are generated by reverse transcription {see Figure 2o-9) &om n1ature mRNAs and hence represent bona fid e exon sequences. The cDNAs are used to generate EST data. An EST, or expressed sequence tag, is simply a short sequence read from a larger eDNA. Th~ read s are typically obtained

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26-18 Gene finckt methodJ: _,aft'sis of PfOCHH:ocling regions in Ciona. A 20 lc.b region of ale of me Ciona scaffdds is shown.. Ths sequmce contains an eodcsfucanase gellt'. ~ 6'\COdes an enzyme that is reqt.ro:l for the degrad;ltcon and synthe5ls ol cdutose, a major COO'p(lnent of plarlt eel walts The ~ findef program ide1tiied 15 puta!Ne exons, !Ideated as green redNlgJes tn reahty. ~heR.- 6 a 5' eron present •n the cONA (black fcctangles below) that was missed by the COfllXJ1ef progTam. Smlarly, a flanlt,ing gene, whch encodes an RNA spf4clf'lg fclctOf. IS predicted to ronti31n a srnal!rtron 10 a large coding regKX\ \Mlt:roas 1he c1J'\lA ~equence StJggests that there 6 no intra\. Tnere ~ aso a descrepancy If\ the SIZe of the 5 '-most exon. lhe llartking gelll'S ~ c~ in worms. Illes and humans, \Nhel'('iiS ttw endogfltc.a~ ~e 6 tJfliCIUC:' to Oono, which contB~nS a celttJiose shealli Note dltferenres \1'1 the detaifed entron exoo strUCltJres of 1~ flanlung geres among trE ditfefer.t arurnaSgenomes.. (SOUfel'>: Deh.:J~ et al. 2002. Thf thh gCf'IOC'ne orOono 1()/estJnoiJS- Jnsights Into chordate cmd ~e Cf®I1S FIG U R £

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using shot;gun sequencing methods and then aligned onto ~omic scaffulrls. Regions of alignment CorteSpond tn exons. while genome sequenc.-e loCAted between regjons of aligrunent often correspond to introns (a,though. alternative splicing might utilize an exon n ot cont ained in the particular eDNA or EST that was sequenced). Shotgun eDNA sequence infocmation can help link different contigs or scaffoJds. Consider the ~ of a eDNA that is lnmsctibed from a very large gene with introns of 100 kh or more in length. 'IWo different SCquences from this common eDNA are likely to arise from linked regions of the genome aud rP.present a single large gene.

Comparative Genome Analysis The comparison of different animal genome.~ permits a direct assessment of changes in gene structure and sequence that have ru:isen dwing evolution (FigW'e 2D-18). Such c1.1mparisQns also refine the idemifia~ tion of protein coding genes within a given genome. For example, the exons of ortholognus genes are highly conserved relative to noncoding DNA sequences such as introns. Simple comparisons of the mouse and human genomes have identified a large number of highly conserved exons. Given the conservation of prntein-<Xlding sequences, there is no ambiguity in distinguishing conserverl exons from othP.r conserve<\ sequences. su h as eohanc(>rs (see below). Comparative ana1ysis helps identify short exons, some located near the 5' end of the gene and lhe core promoter. thnt are often missed by gene prediction programs. One of the striking findings of comparative genome analys is is the high degree of synteny, C(')nscrvation in genetic linkage. between distantly related animals. There is extensive synteny between mice and humans (Figure 20~19). l11 many casi:'.s, this linkage cveJJ extends to the puffurfish, which last shared a common a ncestor with mammAls more tllan 400 million years ago. The extensive synteny seen for vertebrate genomes, a long with the coordinate expression of linked genes in Drosophila, raises the possibility that n eighboring genes share common regulatory sequtlnces. A recent bioinfurmatics survey in Drosophila suggests that 10-20 linked genes within a c hromosome domain spanning 100-ZOO kb exhibit similar patterns of gene expression. Each of the flStirnated 500- 1,000 cluomosome dometins in Drosophila migbl ret::\in fixed synteny due to a reliance on commnn regulatory sequences. Protein-coding sequvnces are not the only regions o1 the genome that are under functionat constraints_ Regulatory BeQnences- transcription factor binding sites and larger elements of gene regulation, such as enhancers-tend to be selectively conserved These regulator} elements can often be recognized as short but conserved non-protein-coding sequences. For example, a computer program called VISTA aligns thf' sequences contained in different genomes over short windows, on the order of 10- 20 bp. Conservation in the range of 70% identified over distam)eS of 50- 75 bp is seen for certain regulatory DNAs (Figure 20-20). Pufferfish and mice share something like 10.000 short noncoding sequences. It is conceivable that many of these correspond to tissuespecific enhancers. However, it is likely that bnth animals, particuJarly mice. have many more enhancers that were missed by simple sequence conservation. The humble sea squin. Ciono inl~stino/is, is estimated to contain on the order of 20.000 difffl.rcnt tissue-specific enhancers and it

670

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would not be surprising for mice and humans to contain more like 50,000- 100,000 such enhancers. Other methods have been used to identify enhancers. based on the clustering of binding sites for sequence-specific transcriptional activators and repressors (see Chapter 18. Box 18-6}. The recognition of regulatory sequences in DNA poses a much greate r challenge than the idenaific.ation of pmtein-coding sequences as regulatory sequences are not subjocl to constraints as stringent as that of the genelic code . Hence. it is likely that a combination of bioinformatics methods will be required to identify regulatory DNAs in whole-genome sequE!nces. The most commonly used genome tool is BLAST (basic local alignment sean:b tool}. There are varialinns in BLAST programs. but they all sl1are lhe common feature of finding regions of similarity between diffe.rent pro tein coding genes (Figure 20-21). There are many ways in which a BLAST search c::an be done. One involve:; searching a genome, ru- many genorncs, for all of the pre
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resides bt:Jtween amino acid residues 71 - 130. When tMs 60 amino acid long polypeptide is used as a query, it identifies about 75 homeoboxcontaining genes in the Dro,.oph1la genome. Thus, BLAST quickly identifies a variety of genes with similar functions. Jn this casc. gp,nes tha t enoocle regulatory proteins containing a specialized form of the helix~tum-helix DNA binding motif (see Chapters 16 and 17). There are other ways that this typ e of BLAST search could be done. In the preceding example, we used a 60 amino acid polypeptide sequenCE!. It is also possibiP, to use the corresponding 180 hp DNA sequence that encodes the homeobox. A search v.ith the longer sequence yields simi lar results. Stat istical methods are used by BLAST progrnrns tu assess the likelihood thAt the ..hits" - the genes or encoded proteins identified by the query SC<]llene.;e- po..~ess a similar funclion. ln the case of evf'. there is less than a one in a million probability that any of the 75 related genes were identified by chance alone. In summary, the availability of whole genome sequences for an increasing number of animals is providing a rapidJy expanding database for comparativt: gtm.omics. At the same lime, the exon·intron nature of eukaryot.ic genes and th~ lack of strict sequence constraints in noncoding elements create formidable challenges to the identification of protein-coding sequences and regulatory elements by computational approaches. New and rno1-e effective tools of bioinformatics will be required to fully exploit tl1e treasure trove of i nfoTmation that is being generated by i1Utomatcd DNA sequencing.

6 72

Techniques of Molecular BiologJ'

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fIGURE 20-21 Example of a BLAST search. A sequence cl S7 arrtno acid resd!es from the horneodorna!n of the Eve protein was used to ~~ the Drasopfulo genome TI"\15 sequet'1te was entered in tre publidy available P.y 8lAST web site (ltiMWJruitfly.org/blasV). There are 3 steps in this precess. f"lfSt, you are asked ~ program yw wiSh to use In this case, theM progrcwn was selected as the Eve polypeptide IS M amino aod sequence. A fliJdeo\lde BLAST ~ c,ould be d by sefect~ng the "NT" databaseThe serond step IS to sc-!ect a dataset In this el(dfl'l*, the predicted protE'!Tl'!> dataset was seleded because we are ~Clfl'1JWlng proten sequences. For a DNA seardt, one of 5€\Jt.'fill nucleotide datasets could be used. tnduding the total ge110mic OOA or jtJst the prediCted getleS. The resutts of !he seard'. pre :.ent ccnstfValive arntro aod su~). A score of ' 52 was obtaioed for the ltomeodomain proten, Unplugged (Unpg), whid'l is essential for the deve\q:lment ot lt'e centra nervous system. ., lhs case thee ilfe 30 cf 57 exact mat:ches wittl the Eve horneodoma.n. aJld 39 of 57 total Slrr.~arities. The third ~score. I 49. was Cibta1ned Wl1h the Lt>x hcmeodo.'Tla~n. Ubx .s a oorneotu: gene fhat was extensrvefy c.1~SCUS..-.ed tn Oirlpter •9.

PROTEINS Specific Proteins Can Be Purified from Cell Extracts The purificatinn of individual proteins is critical to understanding tlleir function . Allhough in some instances the function of a protein can be studied in a cnmplex mixture, these studies can often lead tn ambiguities. For examp le, ii' you are studying the activity of one spa·

Proteins

cific DNA polymerase in a crude mixture of proteins (such as a cell lysate) other DNA polymerases and accvssory proteins may be partly or e<>mpletely responsible for any DNA synthesis activity that you observe. For this reason, the purification of proteins is a major part of understanding their function. Each protein h as unique properties that make its purification somewhat different- This is in contrast to different DNAs, which all share the same helical structure ~nd are on]y distinguished by their prE;!cise sequence. The purification of a protein is d esigned to exploit its unique characteristics, includ ing si.7...e, charge , shape, anrl in many instances, function.

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Purification of a Protein Requires a Specific Assay To purity a protein requires that you have an assay that is unique to that protein. For the purification of a DNA. the same assay is almost always used. hybridization to its complement. As you wHl learn in the discussion of immunob)otting, an antibody can be used lo detect specific proteins in the same way. In many instances. it is more convenient to use a more direct measure for the function of the protein. For example, a specific DNA-binding protein can be assayed by determining its interaction with the appropriate DNA (for example using a gel shift assay, see Chapter 16). Similarly, a DNA or RNA polymerase <;an be assayed by adding the appropriate template and radioactive nucleotide precursor to a crude extract in a manner similar to the n1ethods used to label DNA described above. This type of assay is called an incorporation assay. lncorpom tion assays are useful for monitoring the puril:l~tion and function of m."llly different enzym es catalyzing the synthesis {)f polymers like DNA, RNA. or proteins.

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Preparation of Cell Extracts Containing Active Proteins The starting material for almost aJJ protein purifications are extracts derived from cells. Unlike DNA, whicb is very resilient to temperature, even moderate temperatures readily denature protein s once they are released frotn a cell For t])is rf>.Bson , most extract preparation nnd prcr tein purification is ~..rformed at 4 <JC. CeU extracts are prepared in a number uf different ways. Cells can be lysed by detergent. shearing forces, treatment with low ionic salt {which causes cells to osmotically absolb water anrl pop easily). O£ rapid changes in pressure. ln each case. the goal is to weaken and break the membrane surrounding the cell to allow proteins to escape. In some instances this is performed at very low temperatures by freezing t he cells prior to applying shearing forces (typically, using a blender similar to the one in many kitchens).

Proteins Can Be Separated from One Another Using Column Chromatography The most common method for protein purification is oolUITin chromatography. In this approach to protein purificat ion, protein fractions are passed through glass coh.unns filled with appropriately modified small acrylam ide or agarose beads. There are various ways columns can be used to separate pf(lteins. Each separation technique varies on the basis of different properties of the proteins. Tbree basic approaches are described here. The first two, in this se...--tion. separate proteins on the basis of their charge or size• .respective ly. These methods are summarized In Figure 20-22.

F IGURE 20-12 lon exchange B1Mf gel fiJtnttioQ c:hrom•ography. P6 desafled in

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Ion exchange chromatography In this technique, the proteins are separated by their surface ionic charge using beads that are modifierl with either posithrely-d.twged or negatively-c.harged chemical groups. Proteins that interact weakly with the beads (such as a wP.ak positivelycharged protein passed over beads modified with a negatively-chruged group) are released from the beads (or eluted) in a low salt buffer. Proteins that interact more strongly require more salt to be eluted (the salt masks the charged regions allowing the protein to be relP.ased from thA beads). By gradually ina-easing the coocentration of salt in the eluting buffer, even proteins with rather similar charge char-dCteristics can be separated into d ifferent fractions as they elute from the c<>lumn.

Gel filtratHJn chromatography This technique separates proteins on the basis of s ize and shape. The beads used for this type of chromatography do not have charged moieties attached. but instead have a vnriety of diffenmt sized pores throughout. Small prott~ins can tmter All thR pores and, therefore, can access more of the column and take longer to elute (in other words, they have tnore space to explore). Large prQleins can areess less of the colunm and elute more rapidly. For each type of column. chromatography fractions are collected at d ifferent saJt concentrations or elution times and assayed Cor t he protf:in of interest The fractions with the most activity are pooled and subjected to additional purification. By passing proteins through a number of different columns, they are increasingly puri£ed. Although it is rare that an individual column will purify a protein to homt>gcneity by rf'..peatedly separating fra ctions thai contain the protein of interest (as detennined by the assay for the protein). a series of cht'Omatographic steps c.an result in a fraction that contains many molecules of a specific protein. For example, although tbere are many prcteins that elute in high saJt from a posirivciy-char~ld column (indjcating a high negative charge) or slowly from a gel fi ltration colwnn (indicating a relatively smaJJ size), thf!re will ba fur fewRr that satisfy both of these criteria.

Affinity Chromatography Can Facilitate More Rapid Protein Purification Specific knowledge of a protein can fruquenlly be exploited to purify a protein more rapidly. for example, if you know that a protein binds ATP during its fundi on, the protein can be applied to a column of beads that Bff} couplud to .ATI~ On1y pmteins that bind to ATI' will bind fo the col umn. allowing the large majority of proteins that do n ot bind ATP to pass tluouglJ the column. This approach to purification is called affinity chromatography. Other reagents can be attached to coh.lJTlllS to allow the rapid purification of proteins; thmic inc:lude specific DNA sequenres (to purify DNA-binding proteins) or even specific proteins that a:re suspected to interact with the protein to be purified. Tbus, before beginning a purification, it is important to think about what information is known about the target protein and to t.rJ to exploit this knowledge. One ' 'ery common form of protein affinity chromatography is immunoaffinity chromatography. ln this approach , an antibody that is specific for the target protein is attached to beads. ldcall y, lhls anti· body will interact only with the intended target protein and allow all other proteins to pass through t he beads. The bound protein can then be eluted from the colunm using salt or, in some cases, mild detergent. The primary difficulty with this approoch is that frequently the anJi· body binds the target protein so tightly that the protein must be completely denatured before it can be eluted. Because protein denatw'8-

tion is often irreversible, the target protein obtained in this mannt:r may be inactive and therefore l ess useful. Prote ins can be modified to facilitate their p urification. This modification usually involves adding short additional amino acid sequences to the beginning (N-tenninus) or the e nd (C-terminus) o f a truget protein. These add itions, or .. tags'" can be generaled using molecular cloning meth<x:hi. The peptide tags a dd known properties to the modified proteins that assist in lheir purificat ion. For example , adding s ix histidine residues in a row to the beginning or end of a protein will mnke the modified protein bind tightly to a coluum with immobilizerl NiH ions attached to beads-a property that is uncommon among proteins in generaL In addition. specific epitopes (a sequence o f 7-10 amino acids recognized by an antibody) have been defined that can be attached to any protein. This procedure aJlows the modified prote in to be pu rified using immunoaffmity purifi~tion and a heterologous antibody that is specific for the arlded epit{)pe. lmportantly. such ani ibodies tmd epitopes can be c hosen s uch that they bind w ith high affini1y under one oondition (for example, in the absence of Ca+Z) Lut readily el u te under a second condition (such as the addi1ion of low amounts of Ca.-.2) . This ovoirls the need to use denaturlug conditioos for elution.

lmmunoafli..nity chromatography canal~ be u~ to rapiclly precipitate a specific protein (ami any proteins tightly associated with it) from a crude extract. In this case, precipitation is achieved by attaching the antibody to the same type of bead used in colWlUl chromatography. Because these beads are relatively large, they rapidly sinl to the bottom of a test tube along with the antibody and any p roteins bo11nd to the antibody. This process, called immWlOprecipitation, is used to rapidly purify proteins or protain complexes from crude e xtracts. Although the protein is rarely completely pure at this point. this is often a useful method to determine what proteins or other molecules (for example. DN~ see the section on Olrornatin lrnmunoprecipitation in Chapter 17) are associated with the target protein.

Separation of Proteins on Polyacl"ylamide Gels Proteins have neither a uniform negative charge nor a uniform secondary structure. Rather, they are constructed ft'01ll 20 distinct amino acids. some of which are uncharged, some positively charged, and stiJI ochers are negatively charged (Figure !i-4). Also, as we rliscussed in Otapter 5, proteins have extensive seoondary and tertiary structures and are often in multimeric complexes (quarternary structure). Jf. however, a protein is treated with the strong ionic detergent sodium dodecyJ sulphate (SDS) Md a reducing agent, such as mel'Qlptoethanol , the secondary, tertiary, and quarternery structure is usua1ly eliminated. Once coaled with SDS, the protein behaves as -an unstructured polymer. SDS ions coat the polypeptide chain and thereby impart on it a unifonn negative charge. Mercaptoethanol reduces disulphide bonds and thereby d isrupts intramolecular and intennolecular rlisulphide bridge..s funned betwflen cystfline residues. Thus. as i'> the case with mixtures DNA anr1 R.NA. P.lect...-rophoresis in the presence of SDS can be u sed to resolve mixtures of proteins according to the length of individuaJ polypeptide chains. After electrop horesis. the proteins am be visualized with a stain, such as Coomassie brilliant blue, that binds to protein. When the SDS is omitted, electrophoresis can be used to separate proteins according to proptrrties other than molecular weight, such as net cha:rgfl and isOE>lecUic point (see btilow).

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VL~ualize Electrophoretically~Separated Proteins

Proteins are, of course. quite different from DNA and RNA, but the procedure known as inunun()blotting, by which an individual protein is visualized amidst thousands of other prote ins. is analugous in concept to Southern and northern blot hybridization. rn immunoblotting, e1ectrophoretical1y S<~paratoo proteins are transferr~ and bound to a fiJt er. The filtE-r is then incubated in a solution of an ant ibody that had been raised against an individual purified protein of interest. The antibody finds the corresponding prote in on the filter to which it avidly binds. finally. a chromogenic enzyme is used to visualize the filterbound antibody. Southern, northern. and immunoblutting have in common the use of selective reagents to visualize particular molecules in complex mixturt)S.

Protein Molecules Can Be Directly Sequenced Although more complex than the sequencing of nucle ic acids. protein mo)P.CUJp.s can also be sequenced: that is, the linear order of am ino acids in a protein chain can be directly detemli:noo. There are two widely used methods for determining pro1ein sequence: Edman degradation using an automated protein sequencer and tandem mass spectrometry. The ability to determine a protein's sequenre is very valuable for protein identification. Furthermore, because of the vast resource of oornp)ete or nearly compJete genome sequences. the determinatjon of even a small stretcb of protein 8et.Jllence is often sufficient to identify the gene which encoded that protein by finding a matching open-reading frame. Edman degradation is a chemical reaction in which the amino acid's residues are sequentially released for the N-terminus of a polype ptide chain (Figure 20~ 23). One key feature of this method is that the N~term inal-most amino acid in a chain can be specifically modified by a chemical reagent Ui!Jed phenylisothiocyanale (PITC), which modifies the free (X-amino group. This derivatized amino acid is then cleaved off the polypeptide by treatment with acid under oonditions that do not destroy the remaining protein. ThP identity of the released amino acid derivative can be easily determined by its elution profile using a column clu:omatography method called High Performance Liquid Chromatography (HPLC) (each of the amino acids has a characteristic retention time). Each round of peptide cleavage regenerates a normal N-te.nninus with a free a-amino group. Thus, Edman degradation can be repeated for numerous cycles, and thereby reveal thP. sequP.nce of the N.ferminal segment ofthe protein. In prac:tice. 8 to 15 cycles of degradation are commonJy performed for protein identification. This number of cycles is nearly always sufficient to uniquely identify an individual protein. N-terminal sequencing by automated Edman degradation is a widespread and robust technique. Problems arise, however. when lhe N-terminus of a protein is chemically modified (for example, by fonnyl or a~tyl groups). Such blockage may occur in vivo, or during the process of protein isolation- When a protein is N-tenninally blocked. it can usually be sequenced after digestion with a proteat>e 1o reveal an internal region for sequencing. Tandem mass spectrometry (MSIMS) can also be used to detennine regions of protein sequence. Mass spectrometry is a method in which the mass of very small sampleti of a material can be determined \\rith great accuracy. Very briefly, the principle is that material travels through the instrument (in a vacuum) in a manner that is sensitive to

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its mass/charge ratio. For small biological macromolecules such as peptides and small proteins, the mass of a molecule can be determined with the accuracy of a sing1ADalton. To use MS/MS to determine protein seqoP.nCH, the protein of interest is usually digested into short peptidcs {often l t!S5 than 20 amino acids) by digestion with a specific protP.ase such as trypsin. This mixture of pt' ptidAS is subjected to mass spectrometry and each individual peptirle ";II be separated from the others in the mixlure by its mass/charge ratio. The individual pepUdes are t hen captured and fragmented into all thf! component peptides. and the mass of each of tbese component fragments is then df'termined (Figure 20-24). Deconvolution of these data reveals an unambiguous sequence of the initial peptide. As with Edman degradation, sequence of a single approximately 15 amino acid p{!ptide -from a protein Is nearly always sufficient to ide ntify the protein by comparison of the ooqu..,,nce of that predictf!d from DNA seq\tences. MS/MS bas revolution&led protein sequencing ond identilication. Only very small amounts of w.atP.rial are needed. and complex mixtures of proteins can be s imultaneously analyzed.

Proteomics T he availabili ty of whole genome sequencE>.s in combination with ana)yt ic methods for protein separation and identification has usbered in the field of proteomi cs. Protoomics is concerned with the identification of the full set of proteins prod uced by a cell or tissue under a particuJar set Qf t.:onditions, their relative abundance, and their illter-

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20-24 Analysis of lhe p«Jteome by 20 elec1rophcxesis and mass spectrutnefry.. (a) of proteins f!Om a eel extra:t separeted by 20 gel4:ctrophoresis. No~ M ,n thiS example, only pi'O teirls with a small renge of tSOelectric points (between 5 and 5 5) Clfe ~ed (here separated teft to fight). Iff- stiinds for tsod edllc forusing. The vertiaj rlifectivn sep<Wates pmtems by theJf 505- a and h Cold .Spn~ HMbor lit>or.ltory Press, CPid SptJng Haft>or NV) FIGURE E.~e

acting partner proteins. Whereas microarray analysis (see Chapter 18 ) ma!es it possible to visualize gene transcription on a genome--wide basis, the tools o f proteomk.s provide a snapshot o f the cell's fulJ Tepertoire of proteins. Proteomics is based on three principal methods: two-dimensional gel electrophoresis for protein separation , mass spcctt'oruetry for the precise determination of the molecular weight and identity of a protein (or peptides generated from the protein). anrl bioinformatics for a~igning proteins and peptides to the predicted products of proteincoding sequences in the genome. A single cell often produces thousands of different proteins, far tOQ many to separate and identify by SDS gel electrophoresis alone. As its name implies. twCKlimensional gel electrophoresis separates p rote ins in two dimensions and does so in successive steps. In the first step, the proteins are frar:tionated according to their isoelectric point by isoeleetric fucusing. During isoelectric focusing, a gradient of pH is generated in a gel. The isoelectric point is the pH at which a protein exhibits n o net charge and hence becomes stationary {focuses) in the pH gradient. In the second step, the proteins are separated according to size by SDS gel e lectrophoresis as described above. Because proteins are separaterl on the basis of two properties (isoelectric point and molecular weight), thousands of different proteins can be resolved from each other in a single expttriment. After fractionation

Bibliogaphy

671J

by two-dimensional gel P,}ectrophoresi~ each protein is separately subjected to mass spectrometry in order to determine its exact molecular weight. As discussed above, it is generally more effective to first treat the protein with ~ protease and then determine the molecular w eight of the resu lting proteolytic fragment~ rather than the intact pmtein itself. MS/MS analysis also allows the preeise sequence of the polypeptide fragments of each protein to b e identi fied. FioaHy, given a r:omp)ett! g~nome sequem;e for the organism undf'..r study ~d these peptide sequences from the proteins of interest. the tools of bioinformatics make it possible to assign P.ach prote in (that 1s. its proteo)ytic fragments} to a partic ular protein-coding sequence (gene) in tlle genome.

BIBLIOGRAPHY Books Browo T.A. 2002. GenomC!i. 2nd edition. BIOS Scientitk. Oxford, United Kingdom .

Griffiths A.J.F. , Gelbart W.M. , Lewontin R.C., and MiHer J.H 2002. Modem genetic nnalysis. 2nd edition. W-H. Freeman. New Ymk. Hartwell L., Hood L.. Culdbl:>.rg M.l-, Re y11olds A.E.. Silver L.M. , and Ve1·es R.C. Z003. Genetics~ From genes to genomes. 2ttd edmon. McCraw-1-iill. New

York.

Sambrool< ). and Russel O.W. 2001. Molecular cloning: A laboratory manual. Cold S pring llarixlr Laboratocy Pre,.ss, Cold Spring Harbor. New York. Snusta.l D.P. anti Simmons M .J. 2002. PrinclpiP.s of genetics. 3ro edition. Wiley. 1\iew York.

Genomic Analysis Human genomtt. 2001 . Nutnre. 41l9: 813-000. H umRn genome. 2001 . Science. 291: 1145 - 1434.

Mouse genome, 2002. Natv re. 4 20: 509- 590.

CHAPTER

Model Organisms

well-known adage in molecular f:,ioJogy is that fundamental problems are most easily solved in the simplest and most accessible system in which the prohlem can be. addressed For this reason. over the years molecular biologists have focused their attention on a relativ{lly small numbex of so-called model organisms.. Among the most important of tbese in order of increasing complexity are: Eschel'ichia coli and its phage, the T phage and phage ).; baker's yeast Saccharomyres cerevisioe; the nematode Co.enorhabditis elegans; the fruit fly Drosophila melanogaster: and the house mouse Mus

A

musculus. What is it that model systems have in common? An important feature of aU model systems ifi the availability of powerful tools of traditional and molecular gE'.netics, making it possible to manipulate and study the organism geneticaUy. Sec<Jnd is that the study of each model system attracted a critical mass of investigatot'S- This meant that ideas. methods, tools, and strains could be shared among scientists investigating the same organism, facilitating rapid progress. For example. beginning in the 1940s a circle of scientists gathered around Max DelrUck, Sahtadore Lurla. and Alfred D. ft{lrshey, spend ing the summers at the Cold Spring Harbor Laboratories in New York studying the rnultiplicalion of the T phage of E. coli. This grm~p. called the Phage Group, were among tho~ who were i mportant in establishing the field of molecular biology. Many of the members of the Phage Group were physicists attracted to pho.ge. not only because of their relative simplicity, but because the large numbers of phage that could be studied in each experiment generated results that were quantitative and statistically significant. By the late 1950s Cold Spring I Tarbur offered nn annual phage course, where ever-growing numb~rs of investig(ltors came to learn the new system. T his was a case where focusing o.n the same model organism. guaranteed faster progress than would have beeiJ made if these individuals had studied many different organisms. The cboice of a model organism depends on what question is being asked. When studying fundamental issues of molecular biology, if Is often conveni~-;nt to study simpler tm:irellular organisms or viruses. These organisms can be grown rapidly and in large quantities and typically allow genetic and biochemical approaches to be combined Other questions. for example those ooncerning development, can often only be addressed using more complicated model organisms. Thus. the T phage (and its best-known member. T4. in particular) proved to be an ideal system for tackling fundamental aspects of the nature of the gene and information transfer. Meanwhile. yeast. with its powerful ma1ing system for genetic anal ysis, became the premier systPm for elucidating fundamen,al aspects of ehe eukaryotic cell.

OUT l tNE

• Bactenophage (p. 682)

• &dena (p. 687)

• Bakffs Yeast, Saccharomyces C(](("'llt (p. 693)

The Nematode Worm, Caenomabdit.is elegans (p. 696)

• The fruit Fly, Drosopfulo melonogas (p. 699)

The House Mouse. Mus musrulw (p. 705)

Evolutionary conservation from fungi to higher cells has meant that discoveries made in yeast frequently hold true COI humans. The nematode and the fruit fly also offer well-developed genetic systems for tackling problems that cannot be effectively addressed in )ower organisms, such as deve lopment aild behavior. Finally. the mouse. tbough less facile to study than nematodes and fruit fiies. is a mammal and hence the best model system for gaining insights into human biology and human disease. Jn this chapter we will describe some of the most commonly s tudied experimental organisms and present the principal features and advantages of each as a model system. We shall also consider the kind of experimental tools that are available for s1udying each or-ganism and some of the biological problems that have been studied in each casa. This chapter is not intended as a comprehensive prt~se ntatton of aH the mode) organisms that have had an important impact in molecular biology. F01 example, not induded here is the mustard Arabidopsis thaliana. which h as emerged as a powerful model organism for nnd erstanding the molecular biology of plants.

BACTERIOPHAGE Bacteriophage (and viruses in general) offer the simplest system to examine the basic processes of life. Their genomes. typically small. are replicatod- and the genes they encode expressed-only after being injcctr.d into a h ost ceJ1 (in the case of phage, a bacterial cell). The genome can also undergo r ecombination dming these infections. Because of the re lative simplicity of the system, phage were used extens ively in the early days of molecular biology- indeed, they were vital to the development of that field. ~ven today they remain a system of choice whtm studying the basic mechanisms of DNA replication, gene expression, and recombination. Jn addition, they have been important as vectors in recombinant ONA technology (Chapter 20) and are used in assays for assessing the mutagenic activity of various compounds. Phage typically coiJsist of a genome (DNA or- RNA, most commonly the fooner) packaged in a coat of protein subunits. some of which form a head structure {in which the genome is stored} and some a tail structure. The tai I attaches the phage particle to the ()utside of a bacterial host cell, al lowing the genome of the phage to be passed into that cell. There is specificity here: each phage attaches to a specific cell surface molecule (usually a protein) and so only cells bearing that ..reCP.ptor.. can be infected by a given phage. Phage come in two basic types ~lytic and temperate. The former, exampJes of which include the T phage. grow only lyrically. T hat is. as shown in Figure 21-1, when the phage infects a bacterial cell. its DNA is replicated to produce multiple copies of its genome (anything up to setteral hundred copies) and expresses genes that encode new roat proteins. These e\rents are highly coorrlinated to ensure nf!w phage particles m-e constructed before the host cell is lysed to release them. The progeny phage are then free to infect further host cells. Temperate phage (such as phage A) can also replicate lytically. But they can adopt an alternative developmental pathway called lyscgeny {Figure 21 -2). Jn lysogeny, instead of being replicated, the phagp, genome is integrated into the bacterial genome, and tbe coat protein genes arc not expressed. In this int.egrated. repressed state the phage is cal led a propha~. ri'he prophage is replicated passively 8S part of the

fl G U R £ ::U - 1 The fytk growth qde of a baaeriophage. 1he phage p
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bactt-.rial chromos<Jme at cell division. and so borh daughter ceHs are lysogcns. The lysogenic statt:l can be m;tintained in this way for many genP.ratlons but is also poised to switch to lytic growth at any time. This switch from the lysogenic to lytic pathway, ca11ed induction, involves excision of thP. prophage DNA from the bacteria( genome,

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Assays of Phage Growth For bacteriophage to be useful as an experimental system, methods are needed to propagate nnd quantify phage. Propagation is needoo to generate mate rial-high titer phage stocks foc use in experiments, or for DNA ex.tniclion. Phage are typically propagated by growth on a suitable bacterial host in liquid culture. Thus, for example, a vigorously growing flask of bacterial cells can be infected with pbnge. Afte.c a suitable time. the cells lyse, leaving a dear liquid suspension of phage particles. To quantify the numbers of phage particles in a solution, a plaque assay is used (Figure 2 1-3). This is done as follows: phage are mixed with, and adsorb to. bacterial cells into which they inject tl1eir DNA_ The mix is then diluted. and those dilutions are added to "soft agar," which contains many m ore (and uninfected) bacterial rells. These mixtures are poured onto a hard agar base in a petri dish. where the soft agar sets to form a jelly-like top layer in which the bacterial cells are suspended; some are infected, but most are not. The plates are then incubated for several hours to allow bacterial growth and pbag.e infection to take their course. Each infected cell (froru the original mix) will lyse during s ubsequent i ncubation in the soft agar. The consistency of the agar allows the proge ny phage to diffuse. but not far. so they infect only bacterial cell s growing 1n the immcdiat~ vicinity. Those ce11s, in turn, lyse releasing more p rogeny. w hich again infect local cells, a nd so on. The result of multiple rounds of infection is formation of a plaque, a circular dearing in the otherwise opaque lawn of densely grown uninfected bacterial cells. This is because the uninfccted bacterial cells grow into a dense population within. thA soft aga£, whilo those b acterinJ cells located in al'eas around each initial int£.-ction are killed offr leaving a clear patch. Knowing tbc number of plaques on a given plate, and the extent to which the original stock w as diluted before plating, makes it trivial to calculate the number of phage in that original stock.

21·3 Aaques formed by pbage infection of. lawn of bac:terial «!!ls. fn the case shov.11. the plaques are prcduced by a lytic T-phage. (Sourr,e. Stent G5. Molecular biology of bacterial ~ruses, p. 41 ) fl G U R E

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The Single..St.ep Growth Curve This classic experiment revealed the life cycla of a typical lytic p hage and paved the way for many subsequent experiments that examined that life cycle in detail. The esse ntial feature of this procedure is the synchronous infection of a population of bacteria and the eli mination of any re-infection by the progeny. This allows the progress of a single round of infection to be followed (Figure 21 -4). Phage w ere mi>..ed with LcK:1eri C~l cells for 10 minutes. This is long e nough for phage to adsorb to bacterial cells. but it is too short for infection to progress much furth er. This mixture is then diluted (with fresh growth media) by a factor of 10.000. This dilution ensures that only those cells that bclllnd phage in the initietl incuLation will contribute to the infected population: aJso. it ensures that progeny phage produced from those infections will not find host coils to infect. The diluted popu lation of infected cells is then incu bated to a llow infection to proceed. A t intervals~ a sample can be removed fro m the mixtu re and the n umber of free phage oounted using a plaque assay. Initially that number is very low (comp rising just the phage from the initial infection that did nut infect a call before being d iluted). Once sufficient time has elapsed for infected cells to lyse and release their progeny, a big increase in the number of free phage is detected. (This takes about 30 minutes for the lytic phage T 4.) The time lapse between infection and release o f' progen y is called the latent pc.-iod. and tht! n umber of phage rdeased is called the burst size.

Phage Crosses and Complementation Tests Being able to count the number of phage v.rithin a population allows researchers to meru:;ure whether a give n phage derivative can grow on a given bacterial host cell (and the efficiency with which it does sofor Hxample, the burst size). A1so. the plate assay allows certain types of phage derivatives to be distinguished because ot the di fferent plaque morphologies they produce. Differences in host range and plaque n\orp hologies were very often the .result of genetic differences between otherwise identical phage. In the early days ot· rooJecular biology. this provided genetic markers in a system in which they couJd be anaJyzed, enabling researchers to ask how gen~tic information is encoded and funt:tions. The ability to perform mixed infections-in w hich a singJe cell i~ infected with two phage particles at once-makes genetic a nalysis possible in two ways. First. it allows one to perform phage crosses. Thus, it two different mutants of the same phage {and thus harboring homologous chromosomes) oo-infed a cell, recombination- and thus genetic exchange-can occur between the genomes. The frequency of this genetic exchange can be used to order genes on the genome. A high recombination frequency indicates that the mutations are relatively far apart, wh<>reas a low frequen cy indicates that the mutations am located close to eech other. The large number-s of phage pArticles ahat can be used in such experiments ensures t11at even very rare events will occur (recombination between two very rloseJy p ositioned mutatiohs) as long as there is a \'lfBY to scrt.-en for-or better still, select for- the rare event. Second, co-infection also allows one to assign mutations to complementation groups; that is, one can identify when two or more mutations are in the same or in different genes. Thus, if

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two different mutant phage are used to co-infect the same cell and as a result each provides the function that the other was lackjng. the two mutations m\Jst be in diffftl"ent genes (complementation groups). If, on the other hand. the two m utants f.ril to complement each other. then that can be taken as evidence that the two mutations are likely located in the same gene.

Transduction and Recombinant DNA Phag crosses and complementation tests allow the genetics of the phage themselves to be anal yzed These same vehicles and tedmiques can, h owever, also be used to investigate the genetics of other systems. Initially these observations were restricted to bacterial genes inadvcr~ tently picked up during an infection (as we describ e below). With the advent of recombinant DNA techniques in the 1970 s, however. th~e studies wore extended to DNA from any organism. During jnfeclion, a phage might oC(;asionaJiy (and accidentall_y) pick up a piece of b acterial DNA. The most common way in which a p hage picks up a section of dle host ONA is when a prophage excises from the bacterial chromosome during induc tion of a lysogen . That process involves a site-specific recombination event (see Chapter 1 t), and if that event occurs at slightly th e wrong position. phage DNA i~ lost and bacterial DNA included. As long as tha t exchange does not eliminate part of the phage> genome required for propagation. the resulting recombinant phage can still grow an d can be used to transftir the bar ~t~rial DNA from one bacterial host to another. This process is known as specialized transduction. The bacterial DNA included in the specialized transducing phage is amenable to the same kind of genetic analysis as is possible for the p hage itself. Because nf its ability to promote specialized transduction, it was natural that phage '-. was chosen as one of the original cloning vectors (Chapter 20). Thus, by eliminating many of the sites for a particular Testriction enzyme, and leaving only one (insertion vector) or two (re· placement vector) in a region of the phage not essential for lytic growth, }, can be made to accept the insertion (in vitro) of DNA from any source. That UNA can be propagated and analyzed much more easily than it could in its organism of origin. The restriction e ndonuclease sites in '-. were eliminated by repeatedly selecting pllage that plated with higher and higher efficiencies on strains expressing the restriction system in question. By enriching for resistance to endonucl~ ase in this way. a nd then , in vitro, mapping which sites were lost and which retained, the desired derivative was identified. Many different 11. vectors were developed. all differing in the .restriution sites used and in h ow recombinant phage could be identified. One selection system worked as follows: a X derivative was derived in which a solitary restriction site was retainoo within the cT gunc. the gene that encodes the repressor (see Chapter 16). In the parent vector, therefore. this gene is \ntcu;t and lhe phage can. if it chooses, form a lysogen; the phage. tberE'fore. forms turbid plaques. When a piece of DNA is inserted ar this site, however, the resulting recombinant phage has a disrupted cl gene. cannot form lysogens, and so it forms only dear plaques. This change in plaque morphology provides an easy way of distingtt ishing recombinant from nonrecombinant phage. Moreover, this approach ca.n be made into a selection (rather than a screen ) if

the bacterial stra in used is an hfl strain (see Box 16·5 in Chaptm 16). On that strain, a n y phage that can form a l ysogen invariably does so Thus, only recombimmt phag~ produce plaques on the hfl strain.

BACTERIA The attractjon of bacteria such as E. coli or B. subWis as experimental systems is that tht:Jy Rre relatively simple cells and can be grown and rnanipulatoo with comparative ease. Bacteria are single-celled organi~ms mwhich all of the machinery fof' DNA, RNA. and protein synthesis is contained in 1he same cellular compartment (bacteria have no nudeust. Bacteria usually have a si ngle chromosome- typically much smaller than the genome of n igher organisms. Also. Lacteria have a short generation time (the ooJl cycle can be as short as 20 minutes) and a genetically homogenous population of cells (a done) can ea~ ily be generated from a single cell. Finally, bacteria ar e convenient to stud_v genetically because. on the one hand. they are haploid (which means that th e phenotypes of mutations, even recessive mutations. manifest readily ). and, on the other hand, because genetic material can be cOTIVenic ntly exchanged between bacteria. Molecular biology owes its origin to experi ments with bacterial and phage model systems. Up untH the famous- rluctuation a na1ysis experiments ot' Salvsdore Luria and Max DeJruck in 1943, the study of bacteria (bacterioJogyJ had remained largely outside the realm of tra· dit1onaJ genetics. Taking a statistical approach. Luria and Delruck demonstrated that bacteria can undergo a change i n which they become resistant to infection by a particular phage. Critically. they showed that this change 81'ises spontaneously, rctther than as a response (adaptation ) to the phage. Thus. like other organisms, bacteria can inherit traits (for example. sensitivity or resistance to a phage). and occasionally this inheritance can undergo a spontaneous change (mutation) to an a lternative inheritable state. Th e experiments of Luria and Deh:uck showed that, like other Ol'ganis ms. bacteria exhibit ·genetically determined c.baracterjstics. But because of their simplicity. bacteria w ould be ideal experimental systems in which to eluc idate the n ature of the genetic material and the trait-determining factors (genes) of Cregor MendeL

sralklrlary phase

I

exponer~tieJ

phase

lag phase

Assays of Bacterial Growth Bacteria can be grown in Jiquid or on sol id (agar) medjum. Bacterial ceHs are large enough (about 2 J.Lffi in length ) to scatter light. allowing the growth of a bacteria) culture to b e monitored conveniently in liquid t;:ulture by the increase in optical density. Actively growing bacteria that are dividing with a constant generation time increase in numbers exponentia lly. They are said to be in the exponential phase of grm...rth. As t he p opu lation increases to high numbers of et--!lls. the growth rate s lows and bacteria e nter the stationary phase (Figure 21~5 ) , The nun1ber of bac teria can be determined by diluting the cu lture and plating the cells on solid (agar) medium in a petri rush . Single cells grow into macroscopic colonies consisting of millions of cells within a rt11ative1y brief period of time. Knowing how many colonies are on the plate and how m uch the culture was diluted makes it possible to calculate the concentration of cells in the original culture.

21 -5 Baderial growth cwve. As desaibed in d1e rexl. bacterial cells. such as £. colt~ can grow vel)' rilp\dly when 1101 over·

F I c; U R IE

crOIMled i'lrld wheo prOpagated 1n wt:'U r»:ygenated nr..h medium. Thcs ~ of growlh ts called the ~ponentiill phase becauSt: the cells are r~pkallng e!lpOner\tialy. Once the rurtJer of eels gets too h~Bh. and the rultt.re bernmes very dense. growth tails off tnlD the so-called statialary phase. ulls ~ from staticlnary phase and diluted to loN densrty 11"1 Jresh medium w.n ~n enter exponfflllal. phase but Oflly aflef a lag phase. The rate of rei number~ if\ ec?.Cl) of these phases IS ~owtr,

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688

Model Organisms

Bacteria Exchange DNA by Sexual Conjugation. Phage...Mediated Transduction, and DNA,Mediated Transfonnation A principal advantage of bacteria as a model S}''Stern in molecular biology is the availability of facile s ystems for genetic change. Genetic

Hlr

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as an ·~t mn•- cdl types cao be traflsfared to a JeOpient f celt. ff the dooor cdl is an F~ st.ain, it copies and trans1ers tust the F pasmid, ff an f', •l copes and transiers the F plasmid alof"€ v.-M the ~ hast lJNA;

it an Hfr, It copies Cll1d rr.afl!fus varying arnoools and pans of ·tt;.e llost dvornOSClfTle. depending 01 the site of lnt~ation cmO the oorat!oo of ~g. Once 1n the reoperlt, ~ DNA 1rom the host IS available fof remmbtna· lion. and hence genenc exchallge With the genome ol lhe ~<Elf.

exchange makes it possible to map mutations, to construct strains. with multiple mutations. and to build partiaJly diploid strains for distinguishing ~ ive from dominant mutations a nd for carrying out cis-trans analyses. Bacteria often harbor autonomously replicating DNA elements known~ plasmids (Figure 21 -6). Some of these plasrnids, such as the fe.rti)ity plasmid of E. coli (known as the F-fa.ctc:Jt) are capable oftransfe.rring themselves from one cell tu another. Thus, a ceJl harboring an Ffactor (which is said to be p +) can transfer the plasmid to an F - cell. Ffactor-mediated conjugation is a replicative process. Thus. the F .. cell transfers a copy of the F-factor, whUe still ret-dining a copy. such that the products of coniugation are two F .. oolls. Sometimes the F-factor integrates into the chromosome and as a consequence mobili7.t$ conjugative ll'Bnsfer of the host chw mosome to an F t.'t}ll. A strain hatboring such an integrated F-factor is said to he an llfT (for high f~uency recombinant) !t1rain and is enormous}}' useful for carrying out genetic exchange. Precisely w hich parts of the host chromosome are transferred during any given example of this exchange varies for two reasons. First. different fUr strains have the F-plasmid integrated at different locations withjn the host chromosome. Transfer of the host chromosome into the recipient «>Jl takes place linearly, starting with that region of the chromosome closest to one end of the integrated F·plasmid. Thus, where the plasmid is int{.,wated de•erm)nes which part of the chromosom e Js transferrod first. Also, it is rare that the entire chromosome gets transfelTed before mating is l>token oft Thus. genes far from the transfer start point are transfel'I'ed with low frequency. anrt distant genes may never get t.ransferred in a given mating. Note that a complete copy of the integrated f -factor is transferred last, if at all. A third and extremely important form of the F-factor is the F' plasmid. The F' is a fertility plasmid that contains a small segme"t of chromosomal DNA, which is transferred alon g with the plasmid from cell to cell with high frequency For example. one such F' of historic jmportance is F ' -Joe. an F factor that contains the lactose operon. F' -factors can be used to create pattia11y diploid strajns that biJve two copies of a particular region of the chromosome. This was preci~cly how Jacob and Monod created partiaJly diploid strains for carrying out their cis-trans analyses of mutations in the lactose operOJ1 repressor gene and the operator site a.t which the repressor binds (see Box 16-3 in Chapter 16) . The F-factor can undergo conjugation only with other E. coli strains~ however, certain other conjugative ptasmids are promiscuous and can transfnr UNA to a Wide variety of unrelated strdins- even lo yeast. Such promiscuous conjugatjve pJasmids provide a convenient means for introducing DNA. including DNA that has been modified by recombinunt DNA technology, into bacterial str ains that are otherwise Jacking in their own systems of genetic exchange. Yet another powerful tool for genetic exchange is phage-mediated transduction (Figure 21-7). Generalized transduction is mediated by phage that occasionally package a fragment of chromosomal DNA during maturation of the virus rather than viral DNA. When such a phage particle infects a cell. it introduces the segment of chromosoma l DNA

Bacteria

from its previous host ill place of infectious \liral DNA. The injected chromosomal DNA can recombine with the chromosome of the infected host cell, effecting the permanenl transfer o f genetic infonna· tion from one cell to another. This kind of transduction is called generalized transduction because any segment or host chromosomal DNA can be transferred from one cell to another. Dttpending on the size of the virion. some generalized transducing phages transduce only a few kilobases of chromosomal DNA. whereas others transduce well over 100 kb of DNA. Another kind of phage-mediated transductioo is called specialized transduction, as already mentioned. This process involves a lysogenic phage such as X that has incorporated a segment of chromosomal DNA in place of a segment of phage DNA. Suc h a specialized transrlucing phage can. upon infection, t.ransfer this bacterial DNA to a new bacterial host ceil. Finally. we come to the case of DNA-mediated transfQrmation . which we described in Chapter 20. Certain experimentaUy important bacterial species (for exwnp1e, B. subtilis but not E. «.:·oltl possess a natural system of genetic exchange that enables them to take up and incorporate linear, naked DNA (released or obtained frorn their siblings) into their own chromosome hy recombination. Often the cells must be in a specialized state known as "genetic oompetence~ to take up and incorporatA DNA from their environment. Genetic competence is especially useful as it is possible to use reoombinant DNA technology to modify a cloned segment of chromosomal ONA and thE:lll have it taken up and incorporated into the chromosomes of competent recipient cells.

Bacterial Plasmids Can Be Used as Cloning Vccton; As we have seen, bacteria frequently h arbor circular DNA elements known as plasmids that can replicate autonomously. Such plasmids can sel'\le as convenient '\lectors fot bacterial DNA as well as foa·eign DNA. Indeed. the initial (a11d s ucce.srsfuJ) attempts to clone recombinant DNA involved a plasmid (pSC101) of E. coli that con· tains a unique restriction site for EcoRl into which DNA could be inserted without impairing the capacity of the plasmid to replicata {Chapter 20).

Transposons Can Be Used to Generate Insertional Mutations and Gene and Operon Fusions Ac; we discussed in Chapter 11, transposons are not only fascinating genetic elements .in their own right but are enormously useful tools for carrying out molecular genetic manipulations in bacteria. For example. transposons that integrate into the chromosome with low-sequem:.;e specificity (that is, with a high degrP.e of randomness). Sllch ac; Tn5 and Mu, can be used to generate a \ibrary of insertional mutations on a genome-wide basis (Figure 21-8). Such mutations have two important advantages over traditional mutations induced by chemical mutagenesi-s. One advantage is that the insertion of a transposon into a gene is more likely to result in compl£-te inactivation (a null mut.ation) of the gene (when such is desired) than a simple nudeotide switch created hy a mutagon. The second advantagt. is that, having inactivated the gene, the presence of the inserted DNA makes it easy to isolate and clone that gene. Even more simply. with the appropriate DNA primers, the identity of the inactivated geJle can be

689

Fl CUR E 21-7 flhage-med"&at.ed generalized transdudion. As described r~ the text dtlmg SOO'le ~ infectJC(IS. the host chromosome Is fragmented. <Mld segrnern at that DNA ca11 be pac:kdged m the phage pani· des 111Slead nt the repicaled phage ONJ:l lhts hcs1 DNA lS thereby delivered to another cel111

the same way as the phage genome adlnanl'y \.\Wid Qna, 1r1 the new host. the [t.1A can be rernmbtoed Wtf, the chrcrnc.sorre IOU'ld there. promotif'€ genetic Eli.Change.

Model ~anisms

690

FIGURE 21-8 ~rated insertional mut.agenesis. The transposon. avried toto a eel on a plasmid, CAn then trans· pose from that vet\ide into the host geoome. Because of the hlgll densrty ot coding regtOOS (geni:S) on a lypic:al bacterial chrolllOS<Jme, the transposoo will I.e}' ofteolf1Seft tnto a gene. A mcrkerC locaoon straightlorward

determined by DNA sequem.'e analysis from chromosomal DNA harboring the transposon insertion. Transposons can also be use d. to create gene and operon fusions on a genome-wide basis. Moditled transposons have been created that harbor a reporter gene such as a. promoter-l ess lacZ {for examplet Tn5/o c}. When this transposon inserts into the chromosome (in the appropriate orientation). transcription of the rep01·ter is brought under the control of the disrupte d target gene. Such a fusion is known as an operon or transcriptional fusion (Figure 21-9). Other fusion-generating transposons have been created thnt harbor a reporter gene lacking both a promote r and sequences for the initiation of trc:mslation. In these cases. e>:pression of the reporter requires both that it is brought under the transcriptional contro] of th e target gene and that it is introduced into the reading frame of the target gene so that it can b e translated properly. A fusion in which the reporter is joined both transcriptionally and translationally to the target gene is known as a gene fusion .

Studies on the Molecular Biology of Bacteria Have Been Enhanced by Recombinant DNA Teclmology9 Whole..Cenome Sequencing, and Transcriptional Profiling With the a dvent of recombinant DNA technologies. such as DNA cloning, the availaLility of whole-genome sequences, and methods for

--

------------·

FICiURIE

21•9

Tr~necated

locZ fusions. The method of tranSpOSOn mutagenesis outliled in the previOI..lS ~ure
pr001oter

Tn

lacZ

Tn

studying gene transcription on a genome-wide basis have, of course, revolutionized molecular biological studies of higher cells . .But these sa me technologies have had an impact on the study of bacterial model systems as well. especially when used in conjunction with the traditional tools of bacterial genetics. For example, the de velopment of tailor-made derivatives of transposons for creating gene fusi ons is facilitated by recombinant DNA methodologies. As another example, the use of genetic oompetence in combination with recombinant methods for creating precise mutations and gene fusions has expanded the kinds and number of molecular genetic manipulations. The availability of microarrays representing an of the genes in a bacterium has made it possible to study gene expression on a genome-wide basis. In combination with the tools described above, the function of genes identified as being expressed under a particular set of conditions can be rapidly and conveniently elucidated. Methodr. for rapidly identifying proteins that interact with each other (such as two-hybrid analys is; sec Chapter 17, Box 1 7-1). which have h ad a greAt im ~Jact in yeast and other eukaryoUc systems, are aJso powerful tools for elucidating networks of inte ractions among bacterial p roteins. The a vailability of wboJe-genome sequences and promiscuow; conjugative plasm ids has created opportunities for carrying out molecu1ar genetic manipula· tions in bacterial species that ntherwise lack sophisticated. tradi1ional tools of genetics.

Biochemical Analysis Is Especially Powerful in Simple Celt~ with Well,Developed Tools cf Traditional and Molecular Genetics Since the earliest days of molecular biology, bacteria have occupied center stage for biochemical studies of the machinery for DNA replication, information transfer. and gooe wgulat1on, among many other topics. There are several reasoJ\fi for this. F~t. large quantities of bacterial cells can be grown in a dc:-.ftned and homogenous physiological state. Second, the tools ofuaditi.onnl and mo1c:!Cular genetics make it possible to purify protein complexes harboring precisely engineered alterations or to overproduce and thereby obtnin individual proteins in large quantities. Third, and of great importance, the machinery fur carrying out DNA replication , gene transcription. protein synthesis, and so forth is much simpler {having far fmver romponents) :iri bac.1€ria than in higher t;C il~ as we have seen repeatedly in this t ext. Thw;, elucidating fundamental mechanisms proceeds more rapidly in bacteria in w hich fewer proteins need to be isolated and in which mechanisms ate gent:rnlly more streamlined than in h igher celL
Bacteria Are Accessible to Cytological Analysis Despite their app arent simplid ty and the absence of membrane-bound cellular compartments {for example, a nucleus and a mitochondrion}. bacteria are not simply bags of enzymes. ;:~ s bad been thought for many decades. Instead, as we now know, proteins and protein complexes h a ve characteristic locAtions within the cell. EveJl the chromosome is highly organized inside bacteria. Despite their s maU size, baderia are accessible to the toohi of cytology. such as irrununofluoresence microscopy for localizing proteins in fixed cells wit h specific

antibodies, fluorescence microscopy with the Green Fluorescent Protein for localizing proteins in living cells, and fluorescence in situ hybridization (FISH) foe localizing chrom osomal regions and p la$mids within ce1Js. Tht: a pplications of such methods have provided invaluable insights into several of the molecular processes considered in this text For example. we n ow know tha1 the re plication machinery of the bacterial celi is relatively stationary and is localized to the cell center [Chapter 8) . This finding tells us that the DNA tem plate is threaded through a relatively stationary replication "factory'' d uring its duplicaUon as opposed to the traditional view in which the DNA polymerase traveled along the template like a train on a track. As another examp1e, the application of cytological methods have taught us (again contrary to the traditional view) that during replication the two newly duplicated origin regions of the chromo· some migrate toward opposite poles of the ooll. Cytological methods are an important part of the arsenaJ for molecular studies on the bacterial cell

Phage and Bacteria Told Us Most of the Fundamental Things about the Gene Molecular biology owes its origin to experiments with bacteria l and phage model systems. Jndeed. as w e saw in Chapter 2. groundbreaking wOl'k with a pneumococt-"US bacteriu m 1ed to the discovery that the genetic mate rial is DNA. Since then . experiments with £.coli and its phage have Jed the way. as we have seen throughout this book. For example, the experiment of Hershey and Chase convinced p eoplE> that the genetic material of phage is DNA: the expcrimP.nt of M~-selson and Sta hl proved that DNA replicates semiconservatively in E. cdi; the phage crosses of Crick end Brenner (Chapter 1 5) revealed th at the genetic code is built of triplet codons; while the elegent genetit; studies carried out Ly Ya11ofsky in E. coli demon~ stratw genetic colinearity; and not forgetting the work of Jacob and Monod (see Chapter 16, Box 16·3), which uncovered the fund amental strategies of gane reguJation. There are countless other examples where, by chofJsing these s implest of systems, fundamental processes of life w ere t•nderstood. An important example comes from the classic work of Seymor Benzer, who examined intensaly a single genetic locus in pbage T4, called rll. Wild-type T4 is capable of growing in either of two strains of E. coli known as B and K, but rll mutants grow only in strain B. This makes it possible to detect wild-type phage (arising. for example, frorn recombination between two different rll mutants) at frequenciPS of less than 0.01%. That is, a single wild-type phage can be detected among 10 ,000 rlJ mutant phage when p lated on a lawn of strain K bacteria where only the rare :recombinant "vill form a plaque. Taking advantage of this seemingly arcane property of rll mutations, Seymour Benzet' carried out recombination experiments between pairs of dl mutants and was the~y able to map the order of such mutations at a high level of resolution (approaching or reaching that of the nucleotide base pair). He also devised a "complementation .. test (discussed above) for showing that the rii locus comprises two adjarent genes. Benzer introdu ced the term cistron to describe the gene (based on tbe words cis and trans). As an aside, it is interesting to note that it was this wor'k that enabletl this samP. locus to be exploited by Crick and Brenner in their genetic studies on the genetic code.

Baker's Yeast, Saccharomyces cerevisiae

tl9

BAKER'S YEAST, Saccharomyces cerevisiae Unicellular eukaryotes offer many advantages as experime:ntal model systems. They have relatively small genomes compared to o ther eukaryotes (see Chapter 7) and a similar1y smaller number uf gem:1S. Like E. coli. t hey can be grown rapidly irt the laboratory {approximately 90 minutes per C€ll division under ideal conditions ) ~ allowing cloned populations to be propagated from a single precursor celL Despite this simplicity. yeasl cells have the central chnrac:teristics of all eukaryotic cells. They contain a discrete n ucleus with multiple linear chromosomes packaged into chromatin, and their cytoplasm includes a full spectrum of intracellular organelles (for example, mito-chondria) and cytoskeletal structures (such as actin filaments). The best studied unicellular eukaryote is the budding yeast S. cereldsiae. Often re fp,JTed to as brewer's or baker's yeast because of its use as a fermenting agent. S. cenwisiae bas been intensel y studied for more than 100 years.ln experiments in the Ul60s. Loui s Pasteur identified this yeast as the catalyst for fermentation (sugar was believed to break down spontaneously into alcohol and carbon dioxide). These studies evcntuaJly led to the identification of the first enzymes and the development of biochemistry as a experimental approach. The genetics of S _Cl'll'et'isiao has been studied sinCe thu 1930s, resulting in the characterization of many of its gen es. Thus. like E. coli. S. cerevisioE:~ allows investigators to attack fundamental problems of biolC>g)• using both genetic and biochemical approaches.

The Existence of Haploid and Diploid Cells Facilitate Genetic Analysis of S. cererui«e S. cerevisiae cells can grow in either a haploid state (one copy of each chromosome) or diploid state (two copies of eacb chromosome) {Figure 21-10). Com,ersion between the haploid and diploid states is

altJ.

a/a.

0 r a

FtCURE 21-10 Tbelif~ofthe ~ing yeastS. cerellisiae. As described in the text here and elsewhere, S. c:erevisroe eJCists ~n ltlre~ forms_ Two ~id cefll')1je5, ~ and ex. at~d the dipldd poouct of 1'11allng between tbese two. Replication of these difla"enl cell types. mating and spcrufatiol\ aeshown.

694

Model Organisms

i gene of interest

transfurm with linear DNA~

ends homologoos

to the ctvomosome

mediated by mating (haploid to diploid) and sporulation (diploid to haploid). There are two haploid cAll types called a- and o-cells. When grown together, these cells mnte to form a/a diploid cells. Under conditions of reduced nutrients, a/o. diploids undergo meiotic division to generate a structure known as the ascus that contains four haploid spores (two a-spores and two o-spores). When growth conditions improve, these spores can germinate and grow as haploid cells or mate to ff'r-foiTll a/a diploids. In the laboratory, these cell types can he manipulated to perform a variety of genetic assays. Genetic complementation can be perfomted by simply mating two haploid strains. each of which contains one of the two mutations whose complementation is being te::;ted. Jf the mutations complement each othru-, the diploid will be a wild type for tl1e mutant phenotype. To test the function of an individual gene, mutations am be made in haploid cel ls in which there is only a single copy nf that gene. For example. to O.
Generating Precise Mutations in Yeast Is Easy

homologOus ends recomt:line with chromosome IJNA

1

DNA between homologous regials replaces the geoe of interest FIGURE 21-11 R«ornbinational tran$fonnation in yeast. fls desaibed lfi the text, any region of the y&l.St gEflM'e Ci1ll reedily bf> rt?oced by seqllf!flCe5 d c:hotc.e. Ihe DNA to be in!;erted i~ fli:lnkEd wilt! shof1 seQL~ homdogws to thc6e flcrling the regtro tn the d'~ to be reploced ~the donor fragmef1ts are mtrodur.ed to the cSI. tngh levels of f1omolq;ous rec:ombNtion 111 thrS 0/Ea,JISfll Ef1.5Ue a high frequency d recombnattr,fl\NIIh the chromosome, te:.Uting IIi the geneoc exchcJ1ge 9'lcMfl. 1he tnserted DNA may dflfer frcm the resi.dec!t ~enc.e by as fiUie as o stngle hase P-'~~'· or at the other emetn£, it can be very different in lenglh i!fld sequence lhus. very el
or<~te ~ mOOfiCallenS C
The genetic analysis of S. cerevisiae is further enhanced by the availability of techniques used to precisely and rapid ly modify individual genes. When linear DNA with ends homologous to any given region of the genome is introduced into S. cere.visine cells, very high rates of homologous recombination are observed resulting in the Teplacernent of chrornosotnal sequences with DNA used in the tr-dnsformation (Pigure 21-11). This property c an bC! exploited to make precise changes within the genome. This approach can Le u sed to precisely delete the coding region of an entire gene, change a specific codon in an open-reading frarne. nr even change a specifie base pair in a promoter. The abilit y to make such precise changes in the genome allows very detailed quE'.stions concerning the function o f particular genes or their regulatory sequences to be pursu ed with relative ease.

S. cerevi.siae Has a Small, Well-Characterized Genome Because of its rich history of genetic studies and its relatively small genome. S. ctmwisiae was chosen as the first eukaryotic (nonviral) organism to have its genome entirely sequenced. This landmark was accnmplished in 1996. Analysis of the sequence (1.3 X Hf base pairs) identified approximately 6,000 genes and provided the first view of the genetic c:omplexity required to direct the formation of a eukaryotic organism. The availability of the complete genome sequence of S. ce.retrisiae ha~ aJJowed "genome-wide" approaches to studies of this organism. For example. ONA microarroys that include sequences from each of the appro>:imately 6.000 S . cenwisiae genes have bePn used exten· s ively to c haracterize patterns of gene expression under different physiological conditions. Indeed, the levels of gene expression in S. cerevisiae cells have now been tested in more than 200 different conditions, inuluding different carbon ooun:es (such as glucose vs. galactose), cell types. and growth temperatures. 'These findings are not only usaful to detennine the expression of individuul genes llut have

Bokec s Yeast. SacchCIJ'omyces ce.revisise

695

also led to the group ing of genes into coordinately regulated sets~ which all responrl similarly to changes in conditions. Other gP.nome-wicle resources include a library of 6,000 strains, eli(;h deleted for only one gene. Greater than 5,000 f)f these str-ains are viable as haploids. indicating that the majority of yeast genes are nonessentiaL This collection of strains has allowed the development of new genetic screens in which every gene jn the S. cerevisioe genome can be tested individ ually for- its role in a particular process. The use of microarrays has also a UowAd the genome-wide m apping of binding sites for transcriptjonal regulators using ch ~ matin immunoprecipitation techniques (see Chapter 17, Box 17-2).

S. cerevisiae Cell~ Change Shape as They Grow As S. cerevisioe cells progress through the cell cyd e. they unde~go chara t:teristic changes in shape (Figure 21-12). Immediately aftP.r a new cell is rclc:asecl from its mother, the daughter cell appears slightly elliptical in shape. As the ceJJ progresSE'.S through the cell cycle, if forms a small ..bu
21-12 the mit01icall qde in yeast. S. cetevJStae dMdes by buddng. The dPvelopma'lt a c daughter bud througtJ lhe I'Tl!totJc cycle IS shown. and desalbed tn the text FIGURE

large bUdded eel

basic molecular and cell biological questions. Studies of S . c.'t:uevisiae have made funrlamental contributions tn our under!>tanding nf eukary· otic trans<..Tiption and gene regulatio n, DNA repli(;ation. recombination. translation, and splicing. Genetic studies in baler's yeast have identified proteins involved in aH o f fuese events.

THE NEMATODE WORM, CaenorluWditis elegans Sydney Brenner. after making seminal oontributions in molecular genet· ics. identified a srnaU metazoan in whit;h to study the important questions of development ond the m olecular basis of behavior. Learning from the suc:x:ess of molecular genetic studies in phage and bacteri~. he wanted the simplest possible organism that had differentiated celt types, but that was also amenable to microbiological-li.ke brenetics. In 1965 he settled on the smaU nematode wonn Caenorhnbclitis elegMs (C. elesans) because it contained a variety of suitable cha.ro.cteristics. These mclude a rapid gc11eration time to enable genetic screens: hermaphrodite repro-duction producing hundreds of "self-progeny" so that large numbers of animals could be generated; sexual reproduction so that genetic stocks could be constructed by mating; and a small number of trdllSparent cells so that development could be followed directly. Brenner set two ambitio us initial goals that would be essentiaJ for the long-term success of this endeavor. One \rvas a complete mapping of an (lells by reconstructing serial se<.1ion electron mic..:rograph..c; (completed by John White in 1986). and the other was the mapping of the cell lineage (completed l>y John Sulston in 1983 ). Seven years later Bronner established the genetic:s of the new model organism w ith the isolation of over 300 rnorphological and behavioral mutants. These defined over 100 cnmplementation groups mapping to six linkage groups. Nearly 30 yeats later there are 400 laboratories worldwide that study C. e.legons. Due to its simplicity and experimental aa:;e.ssihility. it is now o ne of the most completely understood metazoan.

C. elegans Has a Very Rapid Life Cycle

c. elewms as cultured on petri dishes ancl fed a sirnple diet or bacteria. They grow well at & range of temperatures. growing twice as fast at 25 oc than at 1SOC:. At 25"C fertili zed embryos c.omploto rlevelopment in 12 bows nnd hatch into free.l iving animals capable of complex behnviors. The first stage juvenile (Ll) passes through four juvenile stagt>S (L1 -lA) over the course of 40 hours to become a sexuaU\' mature adult (Figure 21-13). The adult hermaphrodite c.:on produce up to 300 self-progeny over the comse of about 4 days, or c:an be mated w ith rare males to produce up to 1 ,000 hybrid progeny. The adult Jives for about 15 day~. Under stressful conditions (low food, increased temperatures. high popula. tiou density). the Ll stAge an imal t.:M enter an alternative <1evelopmental stage in whic~ it forms what is called a dauer. Oauers are resistant to environmental stresses and can live many months while waiting for environmental conditions to improve. The study of mutants that fail to enter the dauer stage, o r that enter it inappropriately. have identified genes expressed in specific neurons that function to SP.nse environmental conditions. genes expressed throughout the animal that co.ntroJ body growth, and genes that control life-span.

The Nematode Wocm, Caenorbabdilis elegans fertilzed egg

FlGURE 11- 13 lhe[lfecydeofthe

•

C. elegtJM. Shown as the file cyde in hours of dellelopment. from first stage ji.JVel'liie to adtJi. as described in the text The altemative developmertal stage that an l l juvenile enters-to become a &uer-is ~ shov.n WOfrq.

2 5"C

' 25 Oeuer larva

Activation of these latter genes in the adult can dramatically extend the lifespan of the animal and homologs of these genes have lx.-en implicated in life extension in mammals.

C. elegans ls Composed of Relatively Few, Well Studied Cell Lineages C. elegans has a simple body plan (f'igure 21-14). The p rominent organ in the adult hen naphrndite is the gonad . wh.il;h contains the proliferating and differentiating germ t:ells (sperm and oocyl(~). fertjlization chamber (spermatheca). and uterus for temporary stomge of young embryos. The embryos pass from the uterus to the outside through the vulva, a strut:ture fonned from 22 epidermal cells. Mutations that disrupt the formation of the vul va do no t interfere "vith pro~ a

b

postencw oocyte

6 97

uterus

w lva

hyopOOermiS

muscle

bodywal

1.2mm fl G U R E 11-14 The body plan of the worm. ~ (in part a) is sho'lvn a sectJon through Ml adult henn~odite worm The vanous organs are idenlirted in the sketch below (in part b) and zre de-scribed tn the text. (SC>tXc.e: (a) Suls'ton J E and Hol\r.UHR 1977. Dev. Bioi. 56: 110- 156)

anus

duction of embryos. but do prevent the eggs from being laid. Consequently. the embryos develop and hatch inside the uterus. The hatched womts then devour their mother anrl become trapped inside her skin (cuticle layer) forming a ''bag of worms." This readily identified phenotype has allowed tbe isolation of hundreds of vulva-less mutants identifying scores of genes that function to control the generation, specification . and differentiation of the vulva cells. Among these genes are components of a highly co nserved receptor tyrosine kinase signaling pathway that controls cell proliferation. Many of the mammalian homologs of these genes are uncogenes and tumor-supressor genes that when altered can lead to cancer. In C elegcms. mutations that inactivate this pathway eliminate vulva rlevelopmenl becau..~ the vulval cells are never generated, whereas mutations that activate this pathway cause ov&pmliferation ofthe vulva precursor reUs. rf'.sulting in a n rultipJe vulva phenotype. Because the animal is transparent and the vulva is generated from only 22 cells. it is possible to describe lhe mutant defect with cellular resolution such that the lyJ.Je of mu tation can be associated with a specific cellular transfotmation .

The Cell Death Pathway \Vas Discovered in C. elegans The mast notable achievement to date in C. e.legnn s n::search has been the elucidation of the molecular pathway that regulates apoptosis or cell death. Earl)' anaJysis of ceU lineages noted that the same set of cells died in every animal, suggesting that cell d eath was under genetic wntrol. The fu'St cell rleath defective (ced) mutants i.c;:o)ated were d nfective for the consumption of the cell cocpse by neighboring c:~Hs, thus in the mutants ce ll corpses persisted for many hours.. Using these ced mutants, H. Robert Horvitz and his coUeagues isolated many additional ced mutants that failed to produce persistent cell corpses. Tlmse m utants proved to he d efective at initjating the cell death program. Analysis of the ced m utants showed that, in all but oue case, devf'llopmr.ntally programmed cell death is cell autonomous, that is, the cell commits suicide. In males, a CEill .known as the linker celt is killed by its neighbor. The m ole<..u lar identifk:ation o f the c.:oo genes provided the means to identify proteins in manunals that car.ry out essentia lly the identical biochemical reaction~ to <;ontml cell cteath in all animals, in fact expressing human homologs in C. tdegons can substitute for a mutated ced gene. Cell death is as important tiS cell proliferation in d evelopment and disease and is the fncus of intense research to dPVelop therapeutics for thA contm l of cam.er and neurodegent"rative ruseases.

RNAi Was

Di~overed

in C. degans

In 1998 a remarkable cfjscovery was announced. Tl1e introduction of dou ble-stranded RNA (dsRNAJ into C. elegans silenced the gene ho.molflJ!OUS to the dsRNA. This unexpe<.:ted d iscovery and subsequent analysis of RNA interference (RNAil is significant in two respects. One is thnt RNAi appears to he universal sin~Y- introdudion of dsRNA into nearly all animal, fungal, or plant cells leads to howology-cl irec:tcd mRNA d egradation. rndeed, much of what we know about RNAi comes from studies in plants (Chapter 17), The second was the rapidity with which experimental investigation of this mysteTious process revealed the molecular mechanisms (see Chapter 17, Figure 17-30). These investig_ations intersected with the analysis of another RNA-mediated gene regulatory process that involves tiny endogenous microRN.As that have been

The Fruit 1-'/y, Drosophila melanogaster

shov.rn to regulate gene expression in plants and animals, coordinate genome rearrangements in ciliates, and regulate chromatin stmcture in yeasf. The first two miuoRNAs were discollered in genetic sctrens in C. elegans. A fraction of these worm micm.RNAs is conserved in flies and mammals whP.re their func:tions are just beginning to be revealed. It is likely that more examples of RNA-directed gene reguJation will be dis..:overed in the coming years.

THE r"'RU IT FLY, Drosophila melanogaster We are approaching the 10uth anniversary of the fruit fly as a model organism for studies jn genetics and developmental biology. In 1906 Thomas Hunt Morgan and his research associates at f'..olumbia University placed rotting fruit on the window ((~dge of their laboratory in Sche1merhorn Hall. Their goa1 was to isolate a small, quickly re producing animal that could be cultmed in the lab and u sed to study the inheritance of quantitative traits, such as eye color. Among the menagerie of creatures that were capturedr the frui t fly emerged as the anima! of choice. Adults produced large numbers of progeny in just two weeks. Culturing was done in recycled milk bottles using an inexpensive ef,ncoction of yeast and agar.

Dtosophila Has a Rapid Life Cycle The salient features of the Drosophila Hfe cycle are a vety rapid pel'iod of embryogenesis, followed by three periods of larval growth prior to metamorphosis {figure 21-15), Embryogenesis is completed within 24 hours after fertilization and culminates in the hatching uf a first-instar larva. As we diS<;ussed in Chapter 18. the early periods of Drosophila embryonic development exhibit the most rapid oucleru cleavages known for any animal. A first-instar larva grows for 24 hours and then molts into a larger, second-instar larva The process is repeated to yield a thirdinstar larva tJud feeds and grows for two to th.rre days. One of the key processes that occurs during iarvaJ development is the growth of the ill:lHginal disks. which arise from invaginations of the

F I G u R E. 11 -15 Tl'le Drosophilu fife cyde. The vanws stages of <1eve1opmenLd the fty. shown here. &e desaibed 111 the text.

pupa

o

adut

2

\embryo J1 day

Drosophila fife cycle

21h-\ 3days\

first-inStar

aa.v-a

th11d·1nstar tai'Va

700

Mock/ Qrsonisms

epidernlis in mid-stage embryos (Figure 21-16). There is a pair of disks for fwery set of appendages (for example. a set of foreleg imaginal disks and a set of wing imaginal disks). There are also imaginal disks for eyes. iilllennae. the mouthparts, and genitalia. Disks are initially small and composed of fewer than 100 cells m the embryu but contaiJ1 ten s of thousands of cells in mature larvae. The development nf the wiflg imaginal disk has become an important model system for understanding how gradients of secreted signaling molecules such as lledgehog and Dpp (TGF-f3) control complex patterning processes. hnaginal disks differentiate into their appropriate adult structures during metamorphosis {or pupation).

The First Genome Maps Were Produced in DrosQphila In 1910 the Morgan Lab identified a spontaneous mutant male fly that h ad white eyes rather than the brilliant red seen for normal strains. This s ingle tly launched an incisive series of genetic studies that led tu two major discoveries; genes are lot.:ated on chromosomes. and each gene is c.omposed of two alleles that assort independently during meiosis (see Mendel's first law; Chapter 1). The identification of additional mutations led to the demonstration that genes located on separatp, clrromosomes segregate independently (Me11det·s seeunrl law). wl1ereas those li11ked on the same chromosome du not. An undergraduate at Columbia University, Alfred H. Sturtevant (a member of the Morgan lab), developed a simple mathemalical alger rithm for mapping the distances bet\Neen linked genes bascrl on recombination frequencies. By the 1930s. extensive genetic maps were produced that iden tified the relative positions of numerous genes contrnlling a variety of physical characteristics uf the adttlt. s uch as wing si:r..e and shape and eye color and shape. Hermann J. Muller, another scientis t trai1Je.d in the Morgan fly Jab~ provided the first evidence that envirorunentaJ factors. such as ionizing radiation. can cause chromosome rearrangements and genetic mutations. Large-scale ''genetic screens.. are routinely perfonned by feedjng adult males a mut11gen. such as EMS (ethylmetha:nesulfonate). and then mating them with normal females. The F, progeny are heterozygous and

FIGURE 11-16 Imaginal disks in Dl'oStJpltila. 1he posit1011 cf variOUS wnagml

disks m the laM ere shown on the nght. On the feft .s shown ltle ~mbs ~nd organs they bm '" the adt.Jially. These disks are initially fonr•ed as sman groups of eels .n lhe embt') gTOII\, to tens of thousands of cells tn the mature larva These <W;s develop •nto their" respective adult Slrue'lures dt.Jnng popollon

gelitalia~

• adult

The Fruit Fty. lliosophila me

contain one normal chromosome anrl one random mutation. A variety of methods are used to study these mutations. as described below. In addition to its remarkable fecunclity (a single fernaJe can produce thousands of eggs) and rapid life cycle, the fruit fly was found to possess several very useful features that guaranteed it a sustained and prominent role in experimental research. It contains only four chromosomes: two large autosomes, du'Omosomes 2 ami 3 , a smaller X chromosome (which detArmines sex}. and a very small fourth chrnmo.some. Calvin B. Brjdgp,s-yet another of Muller's colleagues-discovered Ot.at certain tissues in Drosophila larvae undergo extensive endoreplication without mitosis. In the salivary gland, this process produces remarkable giant chromosomes composed of approximately 1,000 copies of each r:hmmatid . Bridges used these polytene chromosomes to detennine a physical map of the Drosophila genome (the first produced for any 01ganism) (Figure 21·17J. Bridges identified a total of approximately 5,000 ·~bands" on the four chromosomes and established a correlation between many of these bands and the locations of genetic loci identified in the classical recombination maps. For example, female fruit flies that are hete·m zygous for the recessive white mutation exhibit normal red eyes. However. similar females that contain tl1e white m utation and a small deletion in the other X chromosome. which removes polytene bands 3C2-3C3, exhibit white eyes, This is because there is no longer a normal. dominant copy of the gene. This type of analysis led to the conclusion that the white gene is l ocat~rl somewhere bf'.tween polytene bands 3C2 and 3C3 on the X chromosume. A variety of additional genetic methods were created to establish the fruit fly as the premiere model organism for studies in animal inheritance. For example, balancer chromosomes were created that oontain a oories of inversions relative to the organization of the n ative r:hmmoson1e {Figure 21-18). Critkally, such balancers fail to undmgo rel:nanhinatiun with the native chromosome eluting me iosis. As a result. it is pnssihJe to maintain permanent «.~ultures of fruit flies that conta in recessjve, lethal mutations. Considm a null mutation in the

38

1

2

3 4

3C

5

6 1 2 3 4

5 6

1

B

9 1011

w- rst• fa• w- rst• ta• w .. rst- la-

Of 258-45 Df 67c23

Vf264-32

w• r s t+ few rst- fa -

0('258-33 Df N8

I

I

phenot}'peof deletion/mutation t-..eterozygole

21-11 Genetit maps.. polytene dtromosomes. and deli~~ mappff\1. Endorephar tion in the abserlce ot m tosis generates en!atged chomosomes 10 5CliT1e ttssoes or the fly. most OOfably the salivaty glands wtlere the giant chomosames atE' COOlJO$ed of a thoos.atld chr(l(l'lalids It was possible, lex the flfSt bme. to c.ooel&e the OCCi..ll'll:nC.e of ~ for cer\illn ~t'> with givro phys.c.j se.grnffl(S of dvomo· somes. Sfleol•cally, pl1enotypes of flies (white eyes) were correlated "'lth delt>tions 1n lhe ~ (Source: Hartwel l et al2003 . Cienehcs. From genes to get?Ofl'le. 2nd editloo, p. 8 l 6. f1g 0-4} Fl Go R "E

102

Model Organisms

origina1 chromosome x

a bc

'1 -

~f

1

Jr

c

b

rd e il

!

a 2

balancer chromosome

::n-18 BaCancer chromosome. BalcnaJr chtomoscr;-oes (bcttarn panel) co~oo a series of tnversions when c-ompared With the onginal. ~ft'fltdl chromosome (rop pane!). In this diagram. a hypotheocal chromosome has tvvo Nms. The left arm of the b?lancer chromoS(Jme has an tntfffl<:ll invefSion that reverses ti-e order of gere; a, b. <Tid c: in the <Jrigni)J c.tvomosome. Sunilarly, the arm on the l1ght of the baf.. anc.er chrornctsoo1e has on inversaort that rever5e.S th€> ordE!f of genes c\ e. and f tn additiOn, ttiere mtght be an lfl\l€f'SI(ltl cetltet'ed around the C€ntramere. an thJs case revet'Sing the Ofder of genes 1 and 2. The bel.!!ncer chromosome U1us. has a slgruf~CM~tly c:Wfereot ooief of genes when compared ~nth the orig•nal As a reslit, there ~S a supp<esS!On of remnbtnation beF u; u R t

tween the chranosames In hetetuzygotes coolalntng Cll'lf." ropy of each.

even-skipped (e.ve) gene, which we di.;cussed in Chapter 16. Embryos that are homozygous for thl:s mutation die and fail to produce viable larvile and adults. The eve locus maps on chromosome 2 (at polytene band 46C). The null mutation can be maintained in a population that is heterozygous for a .. nonna l" chromosome containing the nuU allele of eve and a balancer second chromosome. which contain$ a normal copy of the gene. Since the eve null aUele is strictly mcessive, these flios are c.;ompl eteJy viable. However, only hetero:t:ygotes are obSE!rved among adult progeny in successive generations. Emhryos thai contain two copiem of the balancer chromosome die because some of tl1e inversions p rodU(:e recessive disruptions in critica l genes. In addition, embryos that contain two ropies of the normal chromosome die bacause they are homozgyous foT the twe n ull mutation.

Genetic Mosaics Permit the Analysis of Lethal Genes in Adult Flies Mosaics are animals that contain small patcht>.s of mutant tissue in a generally "normaJ" genetic background. Such small patches do not kill the individual since most of the tissues in the organism are nomml. For example, small }k-ttch es nf engmi/ed lengmiled homueygous mutant tissue can be produced by inducing n1itotic recombination jn developing larvae using X-rays. When such patches are c reated in pnsterinr region.<) of the developing wings, llien the f('.SUlting fli es exhibit abnormal wings that huve duplicatfld anterior structures in place uf the nonnul posterior structures. The analysis of genetic mosaics provided thr. (irnt evidence that Engrailed is required for subdividing the appendages and segments of flies into anterior and posterior compartments. The most f' pectaeular genetic mosaics are gynanclromorphs (Figure 21-19). These are flies that are literally ha lf male and ha lf fe m ale. Sexual identity in mes is determined by the number of X chromosomes. Individuals with two X chromosomes nre females, while those With just one X are males {theY chromosome does not d efine sexual identity iJ'l flies as it does in mice and humans : in flies, Y is only naeded for the production of sperm). Rarely. one of the two X chro· ntosomes is Jost at the first mitotic rlivision following lhe fus ion of the sperm and egg pronuclei in a newly fertilized XX embryo. This X instability occurs only at the first division. In all subsequent divjsions. nudei oonlaining two X chromosomes give rise to daughter nudei with two X chromnsomes. while nuclei with just one X chromosome give rise to daughters containing a single X. As we discussed in Chapter 18. these nuclei undergo rapid cleavages without cell membranes and I hen migrate to the periphery of the egg. This nugrntion is coherent a nd there is little or no intennixing of nudei containing one X chmmosome with nuclei containing two X chromosomes. Thus. half the embryo is male and half is female, although the .. line" separating thn male and female tissues is random lts exact position depends on the orientation of the two daughter nuclei aft.er the first de.avage. ThP. line sometimes bisects the adult into a left half that is female and a right half that is male. Suppose that one of the X chromosomes contains the recessive white allele. If the wild-type X chromosnme is lost at the first division, then the right balf of the fly, the ma}p, hf!lf. has white eyes (the male haJf has only the mutant X chromosome} while the left half {the female side) has roo eyes. (Remember that the female half has two X chromosomes and that one OOJitains lhe dominant. wiJd-typc aUele.)

The Fruit f1y. Orosophila melanogaster

The Yeast FLP Recombinase Permits the Efficient Production of Genetic Mosaics What was not anticipaterl during tb e classical era of genetic analysis is the fact that Drosophila pos.c;esse:> several fdvorahle attribut ....s for molecular studies and whole-gen ome analysis. Most notably. the genome is relatively small. It is composed of (mly approximately 150 Mb a nd conlains fewer 1han 14,000 protein coding genes. This represents jus1 5% of the amount of DNA that m akes up t he mouse and h uman genomcs. As the fruit flv entered the modern era, sevmaJ methods were esfablishAd rhaf impnwed some of the older techniques uf genetic manipulation and a lso Jed to completely oew t-.xperimental methods, sudl as the production of stable transgenic strains carrying recombinant ONAs. AE we discussed earlier. genetic moRaics are produced hy mitofit: l'OCOrnbination in som atic tissues- Initia lly. X-rays were used ttJ induce recombination, although fhis method i:t~ inefficient and produces small patches of mutant tissue. More recently, the frequency of mitotic rocombjnation was greatty enhilnced by the use of the FLP reuoml>inase from yeast (Figure 21 -20). FLP recognizes a simple sequence motif, FRT, and then catalyzes DNA rearrangement (see Chaptflr 11)- FRT sequences were inserted ncar the centromere of each of the four chromosomes using P~element transfOTmati(ln (see below). Heterozygous flies are then produced thai contain a null allele in gene Z on one chromosome and a. wild-type c:opy of that gene on lhe homologous chromosome. Both chromosomes contain the FRT sequences. These flies are stable and viable as tltere is no endogenous FLP recombinase in Drosophila. It as, however, possible to introduce the rucombinase in transgenir. strains that contain the yeast FLP protein coding sequence under the L'Ontrol uf the heat~inducible hsp70 promoter. Upon heat shock. FLP is synth& sized in a.Jl cells. FLfJ binds to the FRT tnotifs in the two homologs containing gene Z and catalyze mitotic recombination (.Figure 21-20)_ This method is quite efficient. 1n fact, short pulses of heat shoc"k are often suffi cient to produce enough FLP reco mbinase to produce large patches of z- Jz- t.issue in different regions uf a n adu lt fly.

a

' normal
b

XX

2 It Is Easy to Create Transgenic Fruit Flies that Carry Foreign DNA P~lements

are transposable DNA segments that are the causal agent of a genetic phenomenon called hybrid dysgenesis (Figure 21-21, see nlso Box 19-3). Consider the consequences of mating females from the "M" strain of Drosophila melonogaster with males from tbe ''P" strain (same species, but different populations). The F', progeny are often sterile. The reason is that the P strain contains nume rous copies of the P-.element transposon that are mnbilizcd in embryos derived from M eggs. These eggs lack a repressor protein that inhibits P-aiement mohilization. P-element excision and insertion is limited to the pole cells, the progenitors of the gametes (spenn in males and eggs in fem ales). Sometimes th e P-elemenls insert into genes that are essential fnr thf' development of the:st! germ cells, ami. as a resu lt. rhe adult flies dflrivecl from these matinp,s are sterile. P-elements aro used as transfonnation vectors to introduce ret.:tJmbinant DNAs into otherwise nonnal strains of flies (Figure 21-~2). A full-

703

xo

0

fIG ll Rl 21-19 Gyendromurphs. Gyandromorph mutants are a ~ st.•king l'orm or genellc rnosatc_ (a) lhe bUe X clvcn-Dsome cames the reces..we (white} ITllAat.wn ....+Jereas the red Xc.f1romosome has a fl(lfT11Cll danlflill1t cq1y a the gene The IT'ItJf«Jt IS the ~ of X dY~ los5 Cit the flfSt miotic.. Q\.1slon 111 iYI XX (female) lly ~ descnbEd 1r1 ~ Lext. (b) In {f te resUllng JnJt:.Til. one half of the

fty 1$ female. tl-.e cAher IS male.

Model Organisms

704

P-e~t

temlini

:

:. x

21- 20 FLP~FRT. lhe use ot this ~fiC recorr.btnation system from yeast (desaibed Chapter 11) Jlfomotes high leve1s of m~otic: rerombinatiOO m flies. The rec.nmbinatlon is contmled by ~the remrnooase an lhes Cflly when required.

F I CURE 10

a X

PO

P9

><

X

MC

X

M~

YTY c

~

nQrmaJ

progeny

progeny

normal progeny

c&sgeric troSSe5

PO

><

M~

~ F1 Pf09en}' ~lly

sterile

J

F2 progMy with rnar1y mutations

21·21

Hybrid dysgenesis. P-e!efrtenr transposons res.tde passtvely in P S(rains because they e
germ ceU loonatJOn Trn!. e114>latns the high frequency ot steolity 1111heoffspfing from sud!

crosses..

length P-f!lement transposon is 3 kb in length. If contaJns inverted mpeals at the termini that are essential for excision and insertion. The interveninR DNA encodes both a Tepl'f>~«Sor of transposition and a transposasc that promotes mobilization. The repressor is cxprossed in the developing eggs of P strains. As a result, t here is no movement of P-elements in embryos derived from females of th~ P-slrnin £these contain P-clements). Move-ment is seen onJy in embryos derivoo from eggs produced by M strain females. which lack P-elements. Recombinant DNA is inserted into defect ive P·elem ents that lack the interna] genes encoding repressor and ttansposasa. This DNA is injP.Cted inlo posterior mgions of early, procellularembryos (as we saw in Chapter 18. this is the region that contain~ the polar granules). The transposase is injected along with lhe recombinant P-elP.ment vector. As the cleavage n uclei enter posterior regions, they acquire both the polar granules and rec::ombinant P· element DNA loge•her w ith tra n ~ posase. Tite pole cells bud off from the polar p lasm and the mcombinant l1 ~)cruents insert into random positions in the pole cells. DifferP.nt pole cells cont::~i n diftP.nmt P-element insertion events. The amount of recombinant P-element DNA and transposase is calibrated so tllat, on average, a given pole cell receives just a sing le integrated P-e lernent. The embryos are allowed to develop into adults aucl then mated with appropt·iate lester strains. The recombinant P-eJemP.nt contains a "markP.r" gene such as wh ite ' and the strain used for the jnjoctions is a white mutant. The tester strains are also white · . so rhat any F 2 fly that has red eyAS must contain a copy of the recombinant P-eletnrmt. This method of P~Jement transformation is routinely used to identify regulatory s~ que nces such as those govem ing et'e stripe 2 expression (which we discussed in Chapter 18 ). In addition , this strategy is used to examine protein coding genes in various gtmetic backgrounds. ln summary. Drosophila offers many of the sophistic.ated tools of da~ sical and molecular genetics that. as we h~ve soon. are available in microbial model systems. One conspk.uous excepHon l1as beeu the absence of methods for precise manipulation of the genome by homologous recon1hination with reeombinant DNA. such as in the creation of gene deletions. However. such methods were recently dP.veloped. and are now being stw.amlined for routine use. ironically. such manipuJations are rt".adily avaiJable~ as we shall see. in the more comp!i· cated modc1 system, the moose. Nevertheless, because of the wealth of genetic tools available in Drosophila and the extensive grmmrl work of knowledge about this organism resulting from dP.Cades of investigation,

The Hovre Mouse, Mus musculus

FIGU RE 21-22 P·element traf1§fonnation.. P-elernents can be used as~ in the hc!nstamatioo Df fly embfyos. TllJs, as dsru,'i>ed In the tt'X\ ~ cf d1
the fruit fly remains one of the premier tnodP,l systems for studies of dcvelopnw.nt and behavior.

THE HOUSE MOUSE, Mus musculus By the standarcls of the C. e]egnns and Drosophila. the life cycle of

the mouse is slow and cumbersome. Embryonic dP.Velopment, or gestation, occurs over a Jleliod of three weeks and the newborn mouse does not reach puberty for a nother 5 - 6 weeks. Thus, the effective life cycle is roughly 8-9 weeks, more than fi ve times longer than that of Drosophila. The mouse. however, enjoys a specia] status due to its exalted position on the evo1utionary tree: i1 is a mammal and, therefore, related to humans- Of course. chimps and other higher primates are closer to humans than mic~. but they are not arnenabiP. to the various experimental manipulations available in rnice. Thus, the mouse provides the ]ink between the basic principles. discovered in simpler creatures ]jkc worms and flies, and human disP.ase. For example. the potclwd gene of Drosophilo cncodf-:s a critical component of tbP. Hedgehog receptor (Chapter 18). Mutant fly embryos that lack Lhe wild-type potched gene activity exhibit a variety of pattP.rning defects. Tbe orthologous genes in mice are also jmportant in development. Unexpectedly. however, certain patched mutants cause various cancers, such as skin cancer. in both mice and humans. No amow1t of analysjs in the fly would reveal such a function. fn addition, methods have been develn1'JP.d that pem1it the efficient removal of spocific genf!S in otherwise normal mice. 11lis .. lo1ockout" technolog_v continues to have an enonnous impact on QUI' understanding of the basic mechanisms underlying human development. behavior. and disease. We shall briefly review the salient featW'es of the mouse as an experimental system.

The chromosome complement of the mouse is similar to that &een in humans: there are 19 autosomomP.s in mice (22 in humans). as well as X and Y sex chromosomes. There is extensive synteny between rrUce and humans: extended regions of a given moose chromosome contain the same set of genes (in the same order) as the ..homologous•' regions of the corresponding human chromosomP.s. The mouse genome h as beRn sequenced and assembled. As discussed in Chapter l9'" the mouse hBs virtually the santP. complement of genes as those present in lhP- human

705

'706

Model Organisms

l

\

sperm head expands

Mouse Embryonic Development Depends on Stem Cells

palemal pronuCleus

2-cell embryo

S-cel embryo

F I'" U R E 21-23 embryogenesis.

genome: each contains approximately 30,000 genes and there is a one~toone conllSpondence for mort-~ than 85o/o of these gtmes. Most, if not all. of the differences between the mouse and human genomes is the selective duplication of certain gene families in one lineage or the other. Comparative genome analysis confinns what we have known for some time: the moure is an excellen1 model for human development and disease.

Overview of mouse

Mouse eggs are small and difficult to manipulate. Likf! human eggs. they are just IUU microns in diamdcr. Their small siw prohibits grnfHng experiments of the sort done in zebrafish and frogs. hut microinjection methods have bP.tm developed for in troducing recombinant DNA into mouse cell lines so as to create transgenic strains, as discussed below. In addition. it is possible to harvest e nough mouse etnbryos, even at the earliest stages. for in situ hybridization assays and the visu~lization of specific gene expression patterns. Such visualization methods can be applied to hoth normal embryos and mu$ants carrying disruptions in defined genetit.: loci. Figure 21-23 shows an overview of mouse embryogenesis. The initial divisions of the oorly mouse embryo are very slow and occur with an average fu::!quP.ncy of just once evf'J'Y 12-24 hours. The .first obvious diversification of ce U t}1Jes is seen at the l&ct>Jl stage, caUed the mo.-ula (Figure 21-23, panel 6}. The cells located in outer regions form tissues that do not contribute to the embryo, but instead develop into the placenta. Ce1ls locatf:d in inlPlnal regions generate the inner cell m ass (lCM). At the 64-cell stage, there are only 13 lCM cells, but these form all of the tissues of the adult mouse. The lCM is the prime som ce of embryonic stem ce11s, which can be cu)h.n-ed and induced to fonn any adult cell type upon addition of the appropriate growth factors. Human stem cells have become the subject of considerable social contr~ versy, but offer the promise of providing a rene wable source of tissues that can be used to replace defective cells in a variety of degenerative di~ses s uch as diabetes and Alzheimer's. At the 64-cell stage (about 3- 4 days after fertilization) the m ouse ~mhryo, now called a blastocyst, is finally ready for implantation. lntflractions between the blastocyst and uterim-! wall lead to the formation of the placenta, a characteristic of aU mammals except the primitive egg-laying platypus. After formation of the placP..nta. the embryo enters gastrulation. whcreby the lCM fom1s all three germ layen;: endodem1. mesoderm. and ectodcnn. Shortly thereafter, a fetus emerges that contains a brain. a spinal cord. and i.ntemru organs such as the heart and liver. The first stage in mouse gastrulation is rha subdivision of the JCM into two ~ll layers: an inner hypoblast and an outet P.piblast, which form the endoderm and octodenn. respectively. A groovF. called the primitive streak forms along dlE~ length or the epiblast and the cells that m igrate into the groove form the internal mesodenn. The anter.i ot end of the primitive streak is called the node; it is the source of a variety of signaling molecules that an~ U.C~ed to pattern the anterior-posterior axis of the embryo, including two secreted inhihjt:ors o f T{;F-f3 signaling, Chordin and Noggin. Double muhmt mouse embryos that lack

The House Mouse, Mus musculus

707

both genes develop into fetuses that lc"lck head structures such as the forebrain and nose.

It Is Easy to Introduce Foreign DNA into the Mouse Embryo Microinjcction methods have been develoJl€d for the efficient expression of recQmbinant DNA .in transgenic $trains of .-nice. DNA is injected jnto the egg pronucleus, and the embryos are placed jnto the oviduct of a female mouse and allowed to implant and develop. The injected DNA integrates at random positions in the genome (Figure 21-24). The efficiency of integration is quite high and usuaJJy occurs during early stages of deve lopment. often in one-CPJI embryos. As a resu lt ~ the fusion gene inserts into most or all of the cells in the embryo. including the lUvJ. cells that form the somatic tissues and germJine of the adult mouse. Approximatt::ly 50% of the transgenic mke that are produced using this simple mcthorl of microinjeclion e xhibit germline transformation; that is. their· offspring also contain U1e foreign recombinant DNA. Consider as an example a fusion gene containing the enhancer from the Hox.b-2 genP. attached to a lacZ reporter gene- Embryos and fetuses can be harvested from transgenic strains carrying this Tc}lort.er and stained to revaal thf! patlern of lncZ expression. In this case, staining is observed in the hindbrain (Figure 21-25). Transgenic mice have been used to characterize several regulatory sequences , including those that regulate the (}-globin genes and HoxD genes. Both com· plex loci contain long-range regulatory elements (the LCR and GCR, respectively) that coordinate du~ expression nf the different gP..nP.s over distances of sever.aJ hwu\red kilobases (see Chapter 17).

embryos from sactiftced female

l

Homologous Recombination Permits the Selective

Ablation of Individual Genes The single most powerful method of mouse transgenesis is the ability to disrul)t. or ..knock out,'' single genetic loci. Thls permits the creation of mouse models for h uman disease. For example. the p53 gt=>..n.e encOlies a regulatory protein that activates the expression of genes required for DNA repair. h has been implicated in a variety of human cancers. When p53 function is lost, cancer cells become highly invasive due to rapid accuroulaUon of DNA mutations. A strain of mice h as been established that is completely normal except for the removal of the p 53 gene. These mice, which are highly susceptible to canr.er, die young. There is rhe hope that these tnk:e can he used to test potential drugs and anticancer agents for usc in humans. Although Drosophila contains a p53 gene, and mutants have been isolated, it does not provide the same opportunity fO£ drug discovery as does the mouse modP.l Gene disruption experiments are clone with embryonic stem (ES} cells (_Figure 21-26). These cells are obtained by culturing mouse hlastocysts so that ICM cells proliferate without differentiating. A recombinant DNA is created that contains a mutant form of the gene of .interest. .For example , the protein coding region of a given target gene is modified by deleting a smaH tegion near t he beginnin g of the gene that removes codons for essential amino acids from the encoded protein and causes a framcshift iu the remaining coding seq uence. The modified form of the target sene is linked to a drug

pronucleUS

DNAio be injected

embryos are placed In ovidiJcl of rec-eptive female

21-24 Creation of transgenic mice by miuoinjedion of DNA into the egg pronudeus.. One-cell embryos are obtained hom a neotviy mated female mouse. Recornbtnant DNA IS 111jEded into the nudeus, and trot: embryo is 1hen implanted tnto the oviduct or a 5ll'r08<1te. Mer sevt:'fal d!ys, the embcyo impianl$ and ulomately forms a fellJs lhal c:ortairts 111tegrated c:cpies of the r~nar\t Df\11\ Fl C t,l R £

Model Organisms

FIGURE

21-25

In situ expression pat·

lems of embryos obtaiited from~

mice- A l!ansgenic stlatn ot mice was aWed that contains a por.ion d the Hoxb-2 regul
bryos were obtained trorn ttansgeoic temales and stained to re~eal sites of 13-galactOSidase (lacZ} activity. There are two prominent bands of staining deteaed ., the 11in
9339-9345, Fl c.)

resistance g~ne, such as NEO that confers resistaa1ce to neomycin. Only those ES cells that contain the transgcnc are able to grow in medium containing the antibiotic. The NEOgene is placed downs tream of the modified target gene. but upstream of a flanking region of homology with the chrombsome socb that double recombination with the chromosome will result jn the replacement of the targel gene with the mutant gene and the drug resistance gen ~. {Aitematively. the NEOgene can be inserted into the tatget gene. ) There is, however, a high incidP.nce of nonhomologous recombina~ tion in which recombination occurs illicitly at sites other than the endogenollS gene. To enrich for homologOlls recombination events, the :recombinant vector also contains a marker-the gene for the enzyme thymidine kinase (TK)-that can be subjected to counter selection by use of the drug gancyduvir. which is converted into a toxic compound by the kinase. The thymidine kinase gene is curried outside the region of homology with the chromosome in the vector. Hence. ttansfonnants in which the mutant gene has beP.n incorporated into the chromosome by homologous recombination wm shed the thymidine kinase gene bu.t transformants jn which incorporation into the chromosome occurred by illicit recombination will frequently contain the entire vector with the thymidine kinase gene and h ence can be selected against. As a result of this procedure. recombinant ES ce1ls am obtained iu which one copy of the target gene corresponds to the mutant allele. These recombinant ES cells are harvested and injected into the ICM of normal blastocyst.s. The hybrid embryos are inserted into the oviduct of a host mouse and allowed to develop to term. Some of the adults that arise from the hybrid embryos possess a transformed germHne and therefore prodoce haploid gametes containing the mutant form of tbe target gene. The ES cells that were used for the original transformation and homologoos recombination assays give

The House Mouse, Mm musculus

. .,

',. (

(f ! \ _ ----- ------- / \ )

!

hOmOlogous reoombi~tlon

1f.s)

I

~l--------~~------~J add cloned DNA to ES c:eM wlture

--

.!

select for NEO (positiVe selection)

neoi'Tl)'Cit1

-

------ - , -

r

-

!

select against Tt< GANC (counter selection)

t

create clonal tine

d one ''Mockout• tTlOf,JSt;!

rise to both somatic tissues and the germline . Once mice am produced that conta in trans formed germ cells, matlngs among siblings are pp.rformed to obtain homo zygous mutants. Sometimes these mutants must be analyzed as embryos due to le thality. With other gem~s. the mutant embryos d evelop into full-grown m ice, which are then examined using a variety of techniques.

Mice Exhibit Epigenetic lnheritance Studies on m a nipulated m ouse embryos led to the djscovery of a very peculiar mechanism of non-Mendelian , or epigenetic . inherita nce. This phenonw.non is known as parental imprinting (Figure 21-27). The basic idea is that only one of the two alleles for certain genes is active. This is because the other L'Opy is selective ly inactivated either in the developing sperm cell or thp. developing egg. Consider the case of t he lgf-2 gene. n encodes an insulin-like growth factor that is expres:t'led jn the gut and liver of developing fetuses. Only tlw. Igf-2 a llele inherited from the father is actively expressed in the embryo. The other copy, although perfectly normal in sttquen ce , is inactive. The

fiGURE

709

-----

21-26 Cenel<nock<*lvia

hofftologous recombination. The figure eutfirle; the medlod used to aeate a cellifte

lacking any gM!n gene. Homologous recorl'lbnalion that CXCI..tS IJVilt"WI a target gene (showr 1n green) resUts 111 the ll"lCCfPPJCltlon ol NEO and disn.¢on of that ga1e. Nonhomologous, or rar.dcm. recombinatiOtl can result in the incorporation of the disrupted gene rontalfling NEO, and the gene Ef'IC.oding thymidine kinase (TK). dones CarJY1118 bolh (.()llst.r\Jcts SlJI'Vive e<posore to neomyan. btit the oones also canying ll< are st.bseq.Jently coonterseleded by growth 111 gancy<:biir (GANQ. Clooes containing the> constn.ICt CiiOyl'1g the tdlge.t ger1e With the N£0 II1S€f110n ~E:' thus the only Sl...lfVf\iQIS. Once produced. these C£ts can be doned and used to ~erote a complete moose lackJng that same gene (see Figure 21·24).

710

Model Orsanisms

21 -27 1mprillting in the mouse. The pennanent slfenang of one allele

Fl CiURE

ot a g1ven gene 10 a mouse. As outlined lf1 the text. and described in de1ail W'l Chaptet 17, ifnpmt~ng ensures that orly one o:py of the mouse lgf2 gene tS expressed in each cell It ts cJways the copy earned on the paternal chmmosome that is el
somalte c~l

somstlc c.ell

'~--------~~--~'

I

meiosis removes impriiU1ng

I female imprint established

l

mate ii'Jl)rint established

@

sperm )

eggs

l

l

!?~\ ::.

.,.;>)~,.~ offspring expresses different alleles somatic eel

somatic cell

or

differential activities thP. maternal and paternal copies of the fgf·2 gene arise from the meth ylt=ttion of an associated silencer DNA that repressP.s lgf-2 expression. During spcm1ioganesis, the DNA is methylated . and as a result, thP. lgf-2 gP.nP. can be antivated in the developing fetus. T he methylation inactivatt.os the sihmcer. In contrast. the silencer DNA is not methylated in the developing oocyte. Hence, the Igf-2 a]Jele inherited from the fP.male is silent. In othe r words, the paternal t:opy of the gtme is "im printed" - in thjs case, methy lated - for future expression in the embryo. This specific exrunplA is discussed in grf'..ater detail in Chapter "t7. There are approximately 30 j mprinted genes in mice and humans. Many of the genes, including the pre<:eding example of fgf-2, oontrol the growth of the developing fe tus. It has been suggested that imprinting has evolved to protect the mother from her own fetus. The lgf-2 protein promotes t he growth of the fetus. The mot her attempts to limit this gro wth by inactivating thP. maternal copy of the gene. We have con~idered how every organism must maintain and duplicate its DNA to survive. adapt. and propagate. The overall str..ttegies for achieving these basic biological goals are similar in the vast majority of organisms and. therefore. may be examined rather successfully using simple organisms. Jt is, however. clear that the more intrirnte processes found in higher organisms, sudl as differentiation and development. .require more complicated t>ystems for- regulating gene expression and

Bibljogroplty

7ll

that these can be studied only in more complex orgauisms. We have soon that a wjoe range of powerful experimental techniques can be used \1\fith success to manipulate the mouse and to explo.rP. vari('ttts comph~x biological problems. As a result, the mouse has served as an excellent rnodP.l system for studying devfllopmE>..ntal , gtJnetie, aud bjochemicaJ processes that are likely to occur in more highly evolved mammals. The recent publication and annota tion of the mouse genome has Uild crscored the importance of the m~ as a mode) lor furl her exploring and UJlde.rstanding problems in human dev"lopment and diSP.ase.

BIBLIOGRAPHY Bnoks Burke 0 .. IJawsoo 0 .• and Stearns T. 20 00. Methods in yaost genetics. Cold Spring Harbor lclborotory Pre...--s, Cold Spring Harbor, New York. Hartwt)JI L.H.. Hood L., Goldberg M.L. Reynolds A.E.• Silver L.S., antl Veres R.C. 2004. Genetics~ From senes to gpnnmP.S. 2nd ooition, McGraw Hill. New York. New York,

MiUcr f.H. 1972. EJ>tpcriments ill molecular genetics. C'..old Spring Harbor Laboratory Pre,ss, Cold Spring Harbo.-. New York. N~ A.. Certsenstein M .. Vintersten K. ond Sehrin~r R. 2 003. Mrmzpulotmg the. mouse embi}'O. 3rd ediHun. Cvtd Spring Harbor L<•boratory Press. Cold Spring Harbor. New York.

Sambrook

J.

and Russell O.W. 2001 . Moloculor cloning: edition. Cold Sp ring Harbor Laboratory Press. Cvld Spting Harbor, N~w York Snustad D.P. and Simmons M.J. 2002. Principles 11{genelict>. 3rd edition. John WilP-y Rn~ St:>ns, New YOlk Stent G.S. and Q.lendal' R. 1978. Molecular genetics: An inlroductmy normtive W.l·f. Freeman and 0:>•• San Froncisco, CaHioi'nia. J\ loborotory momml. 3rd

Sullivan W., Ashburner M .• ond Hawley R.S . 2000. Or(lsophila prolor:ols. Cotd Spring Harbor t..aborotory Press. Colrl Spring Harbor, New York. Wolpert l.., Bedrlington R., Ltlwrence P. , Meyerowitz E..

J.. and JCt>SE>ll T.M. 2002. Princip/P.s of development, 2nd edition. Oxford Universjly Press, England.

Smith

Index Pa,ge rererences in italics rofer to intorroation found in fi.gllrel> and tablt)l;.

A A complex. 3 8 5 A ONA. 106, lOB A sites, 430, 432, -450. 454 obdmru·noJ-Il (abd-1\) gene, 627 . 631-32 A bdomJ'noi-B (Abel-B) gene, 627. 62b abortive initiation, 356- 5~ absorbali(:.c, DNA, 110. 110

acc::epror sites, 361. acoommodatioP. 442 acetate. ~6 acridine. 246 actin filaments. 582. 582- B3 activated molecules. 62 activated states. 56

acti1•alin8 regions, 492 activation energ)ea, 56. 5 7 activator hypa55 experiments. 492, ~9:\-94 activators assembly. 555 chromatin alterations, 54l eukaryotes, 531 - 33, 534-36

signal integration. 544- 45 synel'g)t, 597-99

translatiou. 4B3

transport. 555 adaptor hypotheSis-. 31- 32 adaptors. binding, 577 A OAR (adenosine dea.nin~~ actin~ on RNA), 405 adenine. 99, 100, 100, 10], 102 adenosin e monophosphatc (AMP), 63 adenosine ttiJ!hOSphate. see ATP (adenosin e triphosphate) adenovil'U&'-2 genome, 399 adenoviruses, 398. 399 ecten ylattun. tRNA. 418. 416-'19 affinity. cooperntive bindiflg and. 516-1 7 affiflity chromatography. 6 74 -75 agarose gel ~lect.rophOI'f!!flis, 2J4-1S. 216.648-49

a helices characlBistics, 7
dipole moments, 85 polypeplid~ chain folded into 78 protein structure ancl, 72 alternative splicing, 394-400 COI\8fituli\'€. 394, 395 desc:ri()tion, 380 mRNA. 563-85 regulated. sg4, 3E6 A.meti test. 243

wmnoedds ecliViltiou. 63-64. attachment totRNA .417-23 incvrporntiOo by mRNA. 465- 6 6 nropolar side groups. 52 protein incorporation of. 46'1

in proteins. 73 sl:atl•atioo. 566- 67 structures. 72 . 422 synonyiill!, 461 triple ts. 35- 36 IRNA and , 415 turns in protein structure. 76 aminO groups. 4R atninoacy\ aynthetases, 63 aminoatyHRNA amino amct attar.hmenl. 428 binning. 468 ribosome selec::tion. 441-42. 4 4.'1

SJl'..inoacyl IRNA syn!hetases, 415 classes of, 4:1 9 editing pockllts, 422 formation, 421-22 func.tioo. ~18- 19 tmnslation and. 411

Ag3ssn. )eao L . 5

lRNA recogmtion, 420 anaphase. 146. 147, 1 48. Hfl

alftntne {Ala. 1\). 51), '13 aJanjne-tRNAc"', 423, i23

atlletior, definition, 578 anletioc"-pusteriOT pall~ing. ~6- 29

c::mtk.Onformallcms. DNA. t07-,.,

alkytation. 244-45 aJiel~. 7, 260

anlibioti('.S, 453, 453 antibOdies, 338, 339

a Uo13Qose, 89. 496 allost~ric ~ffllClors,

au

allosteric l'(>gulation. 88 allostery acti\ltltors, 485 CAP control. 496- 98 cooper..uive binding and, 5 J 1 gene regulation, 487

loo repressor control. ~96-9S prole.in, 86- 91 tnmscriptional activator fut1clion. !iOO

antibody~antigtln comple~. 47. 911 anlicodc.ms, 463, 463, 46..'i a ntiparaUei{J shec.1s, 76, '17, 79 1\ntirrhinum (snapdragons), 328 anris8rlse RNA. 329 anlilermination, 48 7, 523-24, 562

1\ntp (An(ennapcdia) gfl:nes, 622, 622, 6. aptJlipoprotein-13 gene, 404, 404

apoplosiS, 69H amBI'ID operon. -503, 503-4

714

Index

arabinose. 503-4

AraC. 503-4

bacteriophage P22. 496 Cre. 29'7 bacten ophage SPOl. 499, !iOO bacteriophage T, 681 l.lactt~riophagc T2, 21.22 bacteriophage T4, 33, 275, 6~2 bacteriop~ Pl

Me reprnl!$01, 49(5

A.Jclmoo., 275 architectural proteins, 302 ~i nim~ (Arg. R), 73 Afgcmaul pwtein family. 5\18 Arremia (sea mook eyG). 630 -31 arlluopods. 630 Ascsdions, 586 (1Sh1. SlJl

Asht repressvr. 580 -83, 597 nsparegllle (Asn, N). 73 aspattate tran scalbamoylase, 89 aspat1ic tM:id {.Asp. Ol, 73 .Astbury, WilHam, 21 A.1' bare patr, 1 03 AT- AC S()hceo90me. 400. 401 atoms. 4 2 -43 ATP (adenosine triphosphale). 206-7 dtnucleotide fold8 and bimling of. 82 cme'Ey rla'nation. 61 coorgy transfet:. 58 enzyme bindi ng of. R3

lJtOup-transfer reaclioru and . 62 - 63 hyd roLysis. 6 1- 6 7 aUenuation . 305. 5D7 attenuators, 50S, 507 o tfP attacluntmt site. 3{)4 AUG trip ler. 38 auloDtJmcug t ran spa>OOs.. 313

~eriop~seT7.J60,360 b~~rioph~.68l- ~7 C:f OSSflS, fiBS- 86 ck>scriptioo. 21 growth ll!lllay&. 684 f:ll"(lWib curve, 685, 685 ly~nic gwwth. 683 lytic rsrow,h. 683 baker's yeast . .!;ce Socchnromycvs cerettlsiat baJancor chromosomes, 7D2 baMllevel cxpressioo. 484 base analogs. 2 4 5- 46. 248 base mccision repair. 24'1. '248-50, 249 base fli pping. 102 , 102. 248- 50 btlre pairing ch anges. 236

DNA, 100

DNA polymerase anrl, 185 Jlipping. 102 l1yt.lrogen bondil18 apd , 102 per t um. 104 propeller tv.t1st amJngement. 107, 10'i' recombining DNA. 2(lQ U!ulOmers. 101 wobble, 464 basflf;llacking, 70 . 71. 102

auton(JmtJusly roplicating sequencm (ARS6J. 214 l!Utoradiograpbs. 36. 653

basic z.ipfXJl'S. 536

aut~lat ion.

&adlf!.eeorw

5HI

basic HLH proteins. 53{i

w.. ·t 7.19

Benzvr.SeJ~ur.692

Avety. Oswald T.. 20-21

pshee(s

B B complex. 365 B ONA, 106, 107.108

&ci/lussuhtiti6. 564-81,587.687 BAOI (bHc.:tftrial ~rt ificial chromosomes) 667 bactruia. 5ee a}sc specitlc bacteria cytological analysis. 691-9'2

gene •""Bulalory tJtemenfli, 530 genome sequencing. 663- 64 growth cun~es. 687 as models_ 6a7- 93 phage in fectiun. S2l. 684 ribosonU!. 425 R."lA l'lyn t11esis, 34 tranr.crlpUon cycle. 353~63 transcription initiation. 4b6-SG4

lraruJfonnations in \•trulonr.e. 2U bad.u•iopha,ge \1'29. 500 bacfeliopbage A, 111 cloning veclQrs, 6&; 1llliP of f.ircu lar rorm. 5 13 as model. 681 promctem. 513 rat:omhination sileti, 294, 294 - 95 regulation, 512-25 transcription. 5 14 bacteriop~e A it)tegrase, 2!17, 303, 303-4 badorlop~ Mu. 331 - 34• .J32

ch at'ACleristics. 74 - 78 DNA distortion and. 366- 67 fonn:s, 76, 77 hydroget~ bonds. 71 pJ"()(ein structure anti. n rwisl, 713 bicoid m HNA. 5~ ~602, 600. 004. 607 ~cvcJu~nnynana. BS~

bioinfmmatic.s. 6 05-6, 008 biologicaf d ivt!l'Slt}'. 616-19 biomulccoles, free e netg}'• 58-fiY

bivaleot ettachment. 146

Blait, Tony. 667 BLAS7 (b~ic focal altgnrneot eean:h tool) J 670 -71, 672

blastocyst.s, 706 bleomycin. 245 bond angle&, 42 boundary elements. 53 • Bracht'l. Jean. 30

brachiopods. 631 btan<:h migration. 260, 262. :t76 branch poin l site, J tn Brenner, Sy
5-bromouracil. Z45-46 bubble ~res. ZtS bufflt lli7.e, 685 b"d gooe.s, Drosophila. 6 20

c C complex. 387 C ·tenoinal d omains (CfDJ, 492 CA repealS, 237 Cm:tus pwfflin~ S91 , 597

<.:.aenor/robditis t!leWJn..<: body plaa,, 697 to.xpl\1SSion p.a11crns, 575 gcnurne size. 636 life eye~. 696- 97, 697 1il700g€!S. 6 97-9 8

as model. 681,696 - 99 phy la, 614 RNAi discovmy, 69 8-99 Tel ell!ments, 334- 35 CAG repe&ts. 237

CAt... oo t:alories, 4S 5'cap, 414

c.AMP (cydic adenosine monophospbate), 83, 8 3-84 CAP-aCTD-ONA complox. -193 CAP (catabolite gem~ tt<:Livatur protein) a Uo&«t;rlc contrQI of. 496-98 combinatorial control . 4 99 DNA binding lllOt.if, 493-!:lfi dumair~s, 83 intJr.Iction with cAMP. 83-84 Joe promoter activation, 4 92 RNA 1JOiymcrase binding, 4.89-9~ surfaces, 492 lramcr·iption and , 488

capping.S7t ,372,J72 ca~nyJ bonds,56

r::mtlonyl groups. 74. 74 carboxyl groups. 18 Casper66Cin. iurbjorn. 21. 30 catabolite gene ~hvator pJ'olein. see CAP (caluboJitfl gcnb 'll«.1iva· tor protein) catalysts. activatitm energjes and, 56 catenaticn. 117 fk.atcni n . 59B

CBP.S55 Cbx (contrablthorax) mutation 625- 26 ccr:ONA (covalently dc:n>ed. circular DNA), 112- 14, t13 CCC repeats. 237 Cdc!6 recruitment. 223 Cdk& (cyclin-dependent kioases} Olctjwoon. 90. 9 1

p:ttt-RC nt:tivatit)f], 223

promoter regulatioo, 485 regulation uf pre-RCe~. 226. 226-27. 227 cONJ\S {t;CIJly ONAs}, 3 24, 656~657

Cdn reavilrnent, 223 ced (cell &:oth deft>ctive-j mtJtants. 6gs rell q~ch: clle.dpoints, l«i- 48 cluvmosome replication, 223 chromosomes during. 141-43 gap phases. 146- 4-8 pre-RCregulalion during, 2.26-2'7 cell death pathway, 698 cell diVision enlbotks and. 453

thmmCJSOmes during. 138-41 DNA methylation patterns and, 561 i~ited gene expression, 5Ci0-~tl2 origins or repJir::ation and. 219

f.:ell extra~s. 6 72 -7S cell surface n:JCi'Jptors, 552, 579. 579 ceiHu-ceU ctmt.act, 576-7S, 584- 67 CENP-A, 165 t it:ntrnl dogma. 31 - 36 ctmtro!lk!tflS, 139, 139, l•Jt.J centrosomes,. 1-43 ~urn cbluridc, 2Ci, 27

cha.in-termi.nati ng nucleotides. 001, 662, ~ chaperones. 173. J 78- 79 Chargaff. Edwin . 23-24 ChaSE>~ Martha, 21 , 691 cheliQ:!Tat es. 630

chemic:al bonds characterif;;tiu; of . 4l - 4l definiliun, 41

dura riun. 4~ e~g_\r

changes. 43

flquitibda. 43 ~dum of rotation. 42 hir,h..ertergy. 55-6? scabHity, 43 weak. 45 chemical interfen:mc~ l'uotprinfing. 490 chemical protretion footprinting fl)f!lhod, 4!lU dwmical reactions, entrgy of acnvatiun. 56 chemotherapy. 245 dti site:> (cross··over hl!lspot instigator), 26Q-71 chitnpa.nzees. 6S6-37, GaB Cbip protein, &41

Chordin. 706 chromatld6. 142 chmmatin aoces,c;ibil!ty. 169-14. 1'12 altnntion by histone variants, 165 during tJe.ll diviman. 144 DNA within, 129

electron microscopy, 150

gene acx:essibihty and. 540 bigher-orderstrocturo. 160-65, l64 h istone inhP.Titance and. 1 77 regulation of structure. 165-7& structuro.l alterations, 541 clnomatin a..'lSelllbly facil\tetors, 178 chturna.t in immutlupft:(;ipitation (ChiP ), 538, 5:\ chromatin remodeling comple:Mes. 175 cl\romatography. w lumn, 673 -75 chromodomains. 172-74 chromosome break.>tge, 221 chromosome ~de.-J satiuo. 143, 144- 46 chrumosorne mapping, JS cflf(.lmtJt;Ome ~~~on, 309 chromosomes balcuo..cer, 702 dtrringcelJ div\.!;ioo, 13B--4t . 14-3-44 chamcteristic n umber of. l3~ -32

c:roosovers, 11. 12 DNA Witb)n, 129 form vf. 130- 31 gtllle density, t34

gene linkage. 10 herediry and, 8- !J mapping. 12- H microscopy. 150 replication. 223- 24 $egreg8tion during meiOAis, 278-84 varialioos in m akeup. l 31

716

Index

dgene. 514 C U protein stability, 522-23 Cjuno mtestmalis (:;eu sqwrt), 584, 585, 615, 668 Ciuno life cycle. 585 cisti'Oilll. 692

clastogenic agents, 245 Clinton, Bill, 667 clones, identtficarion, 65:7 deming. DNA. 654 cloning vectors, 686 ctO!ll!d comple><es, 352 Cochran, William, 22 wdolt antkl.ldon JlillriTJS. 462 wdons,4t2 amino acid synonyms, 4tit assignmEnts, 468-69 defi.mhon. 36 dtrect chain tennination. 463 - b4 mixed c:vpolymers, o%7

corepresSOI'S, 504 corn (Zea mays}, 11, U , 328 Conens. Karl. 6 covalent bonds, 41, 42, 44 covalently closed, circular DNA (ca:ONAJ, 112- 14, 1 13 coxll gene. 405 CPSF {cleavage and pdyadenyl:~hon specificity factor), S:74 Cre ~.CrJmbinases. 297, 300. 301 CtP.ighton, Harnet E., ll Crick. Francis H.., 22, 31-32, 692 Cro (control of repressor and other tllings) prvlcin, 495, 514 517-18.518 t.:rti$S~ bactrouph~. 685-

cytologic:al view, 28CJ description. 259 gene l inlvage and. l l during mei
nonsense. 311

theory of,

tr.msl001tion, 440

tlrn~~:tur, 13

tRNA and, 415 cohf'SirlS. 142. 144. 145 coiled~s,79-80,80

oolon cnm~r. 241 w lony hybidnation, 657 ccJumn chromatography, 673, 073-i'S c;ombinatCJriaJ oontrol, 499,547-49. 548-49 comparative genom<.• analysis, 638-39 romparaUve ('\C!nomics, 613-41 complementwy slructures, 4 8-49 ettmplernmtation tests, 685-86, 692 composite transJ>C150ns, 328 cornputeri7..ati(.lll. btoinlonn;~t)Q;, 005-6 conc.;entration. coopen!tive bind ing and, 516-1:7 CODclensms, 144. 145 confurmatjonaJ t~·witching. 447 COilStmSUS sequences. 353, 355, 355 conservative p~ses. 2'7. 28 conservative sitl'"'sp«;ific: reoumbination (aiSRj Cru rec:;ombinases .and. 30l DNA transptJsitions and, 293 geneUc; engmetlring .and, 302 protein-DNA intennediattl5, 296- 97 structures involved in. 29F ~tsequences.294-95

types of, 295

oonatttutive expression, 484, 497 ronti~. 666. 686 Coomassit: brdliant blue. 6:75 c:oopcrnlive binding activator, 545, 545 tl·'nterferon gt'f'le. 546-47 definition, 485 genA regulsHon. 487 A rt!pl'essur lo DNA, 5 l5 pr OCtlin regulalion by, 91 reactJun, 516, 516-17 SSBs, lOO c;wperaHvity. definition. 545 copolymers.. 467,466- 69

core DNA. 151 col'fl enzyrn~. 3 49, 356 rore llistones. 153-54. 154 corJS\>f'Va tic.m of. 1 H:~ DNA interactions, 156-59 N-termmal w ils, 156 structural fo)d, 154

86

crossing o"'et dmJmosorrw mapping and.l2

11

two-factor, 12 products, 2b 2, 266 CRP (cAMP receptor protein), 488 crustaceans. 630-35, 6Jr, 633 CstF (cleavage stimulation ractor). 374 vCTD !carboxy Lennin.al domain). 356 CI'F1 , 537 CI'P. 89 cut-anrl-paste mechanh>ms, 314- 16, 3l5 crt:l6Sm'm

culgei"IP., 5 41

c:utides, 595 cyclin-dependent kioases [Cdls). 00 cydins. 90, 465 cyclobutane rings thymine dimers. Z45 cysteine (Cys. C}, 73, 423 cysteinyt-tRNAcy., 423, 423 cytidine dcaminase, 404 cytochrome. P450, 636-37 cytokillf!SlS, 146, 147

cytological analysis. b~~elerinl. 691-92 cytosine

i\:C i.ru."'mpatibilit)'. 102 deamination, 244

pairirlg. roz structure. 100 tautomeric states. 100 cytoslrelt>ton. 576. 582- B:l

D Oam methyl t ransferase, 217

DRm methylase, 240 m~ylation. 24 r

Oam

~Tvrin.~.5.61 3

dauers. 696 De Vries, Hugo. 6 dt!ocetylation, 556-58 DEi\D-00>1 heJicase prote ins., 384 deaminatioo, 244,404, 404-6 decatenation , 1 J :7

def:oding centers. 425 deficiency mapping. 701 Defonned (Dfdl gen~. 627

dafonnylases, 433 d~neracy, gel teli\: cud~:,

461-69

Index degradative pathways, 58 df..hydrogenase enzymes. 82-83 Delbruck. Max. 681. 687 Delta molecule. 587. 588 denaturelion, 71,106-11 . 109, 111 densi ty gr-ddk!nt 1'$\trifugatiun. 26, 27 denticle hairs. 595 2' -dooxyadenositle.. 99 2'-d ooxyaden05ine 5 ' phospbaht, 99
D. mP.Jnri()({OBtFf', 592-94 gcnrt ocaivity. 576 gene regulation during. 575- 611 mouse, 700-7 2'- ,3 '-dideoxyguanosin~< triphosphate (dd<.."TP), 662 2 '·.3'-dic.Joooxynuclooti.des (ddNTPs), 661.661.662 d•fsites. 307- B, 308 1,8-dihydro-8-i>xoguanine (ro.CC.), 244 dfhydrourid.inf! (D}. 415. -415 dinuc.lootide ro1ds.. 8 1. 81

di.nucleotide repeats. J 37 diploid Cfllls. 132, 146, 497 , 6{)3- 94 dtpvle moments, 45

dipterans, 6.i5 diroct nlpP...'lt. 295 discriminators. 420 dispersive p~. 27. 2B Dll (Distal-less) gene, 6 31 -32, 6.13. 634

Dmct protein . 283. 283 ONA A fonn. 106, 100- 7 HCt:essibility, 175

amplHlcntion. 658-60. 659 B form . 106. 106- 7

bent. f68 bw.aking t~inal pairs. 70 c:irculm, 111, 117. 307, 307-10 . i273 d ooVagtl. 276- 77 compaction, tb'2-63

core. 151 denaturntion, 109 duui:IIP. helix. 2 1 -:la gel elEctruphorcsis. 648- 4tl gtmomic. 212. 57? lw!lical slructure. 98 htllix fom1ation.69·-7l . 70 tS{l histone stabilizntion hybridization 651-52

or.

~ling.65t

linker. 15 1. 153 major groove of, 84 --85 methylation, 558-60. 559. 561, 710 nucleasorue-associatfld. 16/i oucleotides, 25 packagitag. J51 polvnocleotid E> chains, 98- 100 polyJ>tlptide chain, 23 precursors, 64 probr.s. 652 - 53 protein aftinHy fur. 85, ~t• recognilion by proteins, 84

regulatOI)'. 596 replication, 23. 181- 233 RNA compared with, 31 segment isolation . 653 SI!«Jl.Umre read out. 665 sequencing, 662 si n,gle-Btmn~.

195 - 98

start signals. 38

stop signals. 38 strand separa l.illn duri ng rephcatmn. 26-27 slrudure, 00- 111

supercviled. 1l3 Sytllilt!sis_ 11J2, 182 - 64, 183. 184- 85

topoisomerase cleavage, 119 tupoisome:rs, 120 topology. 111 - 22

weak bonds, 70

X-rny photograph . 22 Zform. 106 ONA, rnismatchro. 191 -9'l DNA-binding prote ins, 84-137, 487 l•i.stom.•-fretl DNA aru.l. 166 leucine zipps family. 80 methylated sequmce rtaJgnitiota, 558 nudeosome positioning -IP.pen«lent on. 168. llill-69 ret:eptor binding. 577 DNA-binding s ite detecliun, 490 IJNA cloJJing. 654 DNA dam¥o 242 46,246-57, 247- 48 IJNA ex~hang6. 300- 302 DNA footprinling, 490 DNA fragmtmts, 660- 63 DNA g lyccsyfases. 248, 2SO DNA gyrase. 116 DNA helicases. 194 - 95 biochemical assav for acltvity. 196. 197 DNA Pol m heli<-.c:~Se binding 1o. 2 11 E. coli model fur initiclhon, 220 function, 1 ~5. 222 timction a t r11plication forks.1fl9, 199-200 inactive slllte, 219 i:nteraclivn w ith primas
ftmclic•ns. 201

gap filling by. 25 J m
718

lntfli'X

DNA polymerase (Conlinued} palm domain,188 proc:essivily. 190 sliding clamps and processi.vity, 2UJ - 4, 204 ~;pedslization of. 200-205 ~;tucture, 186. 186-88 switching. 201, 2.02 telomere~oo, 140 template binding. 189 tems1la re palh through. 190 thumb don1ajn. 188, l!H Y family, 254 · 57 Y family phylogenetic tree. 256 DNA primase. 220 DNA recognillon. 53.5 DNA repair. 247 DNA repair polymcrdSe. 316 DNA replic::ahon E. coli model for initiation. 220 finishing, 228-32 hi~o.ne inheritance and. t 76, 176. 1'78 incumplete. 221 inifiiilkm in eukaryotes. ZZ3- 26 . 227-28 initiation in pmkaryotes, 22'7-28 initiation of. 2 12-14 meehylation patterns. 561 DNA sl rallf) transf er. 316 DNA trdllSposition cut-and-paste mt!Chanisms. 315 ulft:banism. 314- 16 nonlransferred strand cleavage. 3 16-18. 317 replicative mechanism fOf. 316-20 ONA lransposons. 312 - 13 OnaA protein. 83. 214. 217 OnaC lu>!iCW>B loader. :.!19, 220 DNA- histone intemdions. 156-59. 157. 158. 165-66 DNAse 1. 104, 114 Unct protein. 2.'75 Dctbzflansky. Tlwodosius. 16 domains. definition. 81 domioanl hails. 7 donor sites. 381 Dorsal protein, 590-91.596, 596-00.623 Dorsal-Tw ist syrwgy. 598 dorsal-ventral paHerr~ing, 500- !i!:l dc•uhlr. helix base pairing, 100 base pairs per tum. 104 compl•••u•mtary sequences. t 01-2 DNA . 21 -2.8 grooves. 103 - 6 helicaiJlflrioditity, 121

multiple ('()r')fom~tiOI'IS, 1Clfi - 7 prupeUer twie>t, 107, 107 right-handed. 103 RNA, 123 unwinding. 194 - 9 5. 195 double-sex ge11e. 397 duuble-strand b·eak repair. 247.253 - 54.264-66.265 doubln-stmnderl breaks (DSBsl. 264 , 266. 266-6:7. 280- 82 doublc-stt".mdcd RNA (dsRNAs). 568 downsln>.am promoter eiP.mcnt (OPE), 363- 64 Opp conrrot romp lex, 700 Drosophila mefanogaster(fntil fly) J\rtfpgenes. 622, 622 Car.tus protRin. 591 chromosome 2, tS

ctlt gene. 541

Oorsal protein. 590-91 DouhiP- SPX (DSX), 565

doublP-sex gene, 39 7 DSCAM gen~. .'ffi2. ~i94 embryo~nesis.

500- 609, 592 A'ISTOill'diPtiJ51Ylile.d mutants, 702 genome maps. 700, 7()1 genome sizt!. 636 half-pint pmtein. 397 homeutic gene complexes. 629 h omeotic gEne organi.zatioc, 627-29 lfox genes. fi28 HSP70 gena. 562- 63 hunchbarl:. gene, 592 imaginal disks, 700 Krfi)lpel mutants. 59.'5 lifu cyde. 699. fl99 - 700 morinl!r e lemPnts. 334-35 ali mod~.m determining genes. 62{l Pa.'o:6 gene. 6:11-22

phvla. 614 segmentation , 599~determination .

t5UU

563-65 . •'>6·1. 565

sex-linked genes. 10 Spii.tzle protein. 591 study of mul<mts. lV Toll receptors, 591 transgene expw.ssion 624 whitt:f.ye mutant gena. 1.0. 10 wild-lypt~ gene, 10 D~A1~e.392. 394

dyad axes. 156 d\·slrophin gene. 380

E E1 protein. 76

E sites. 430 Early {EJ oomple". 385 ecdysozoans. 614- 15 EcoHl ~ricU()n enzyme. 650. 650. 651 . 651 Edman degradation. 6:76,677 CP~ . 444-45. 445.4 46 . 450.451

ElF releasE!. 554 EF-Ts.446 EF-Tu . 441 . 442. 4~. 44fi

eggs. 576 .600,706- 7 fiiF2, 435

en-·sa. 435 e lF4F. 436. 437 elf:ctrcm pairs. 45 electronegative atoms. 45 electroph[JJ'Bsjs, 120, 120, Z14 - 15,648 49. o· e lectropositive atoms. 45 t.-l«Jrostatic: forces. 42-43, 197 - !:18 elongating pol~'mernse. 359 elongation Pol U, 370- 71 RNA synthesis befure. 358-59 transcription qrde. 350.351.352.562 63 t ranslation. 440- 4fl

elongatioo fac.tors. 440

Ef-G, 444-46,445, 4 46.450 EF-C and RRF combination, 45'1 EF-Ts. 416 EF-1\J, 441. 442. 446. 448 CTP exchange, 446

homoJClgjtiS Ifi'Combination, 27'1- 78 initiation of DNA repliCation, 223- 28 mRNA, 414- 15,438, 4 4{}

protein synthesis. 28, 30 Rt!CA homolog. 275 riboso~nes,

structural comparison, 446 embryogenesi~>. 500-609. 592. 7.(]6

ernhtyos, S'76. 578. 587- 88. 6.'1-J empty target sites. 33Q EMS (etbylmu4hanesulfonat~}. 700 En/lm· expressillfl, 634 end replicatioo prof)lem. 228-30, 22!!, 232

426. 4:Hj -3f>

slBJt cudoru;, 41 2 traJ15Q'iphon. 363 -16,530. 538, 5!19- 5 1 transla tion, 424 (11111?/ .:[email protected] g~1nc, 604. 004 - 6.. 605 . 61)6. 672. f' \ltJlUI joo

cvnserved intron seqllE!flces. 1101

endunucleases. ti4-9- 51

C(Jnscrved ~isms. 531 -37

onc\onucJoolyttc. denvage, 248

of 1liversity, tH3- 41 cxou shuffiing. 4lJ3 gene conservalioTJ. 6 t 4 - 19 gene duplf.cation. 616 - 17

ei'J8T(u/ed/engroilcd homoz~us m uiRf\18, 70Z

enhancP.OSOmElS, ~. ~nhar\C'.f!rs.

~7

530. tiOS-6, tiOS- P

ummpy. 44 t!nvironmentat factors . 242- 44 ~~ymes.. see also spectfic oozymes ;l(:tivat.ion enm-J!)es and, 57 allosttlftr. tronsfo,mations ami. uB a tta dJmtlnl to substmtes. 53 react;un rates .and. 57

Ephrusst, Boris. 1 7 epsgeDelic i.nheTitance. 709- J 1 epigenmic fl'!SUiation , 560-132 epigenetic switches. 562 ep.i.lopes. 675 equilibrium cortSta nts (I("') biosvnlh et.ic reBctions. 00 che~k'.BI b on(\s . 43 energioS of anivation and . ft?

free enetgy and. 44 &tle energy chau~ and. 57 ER\C1- SPF. 251

EschericluD r.Dli aroBADoperon. 503- 4 CD protein stability. 522-23 diCtJ Jar chromosome 111 OnaA protein, Zt 4 MDI gP-006. 499 galR J!!erte.'i , 499 genome. 134 BSmod c l. 681. £iS7 model for initiatj(Jn of IJNA repliQ'JtiOfl. 220 MutS pmlein. 238 noc.leotide excitliun repair enzymes. 251 oriC, 2 t .3 phage A regulation. 512 - 25 regul .. tiun or mplieITl('l, 210 --12 ri~maJ ~otflin up~ 5 1 t

trp loader sequcnce,413 XP..rf: function, 297 Xr.rO function, 297 EST (fiXlU'es~ 6eqllent:e tagsi. 668 t~thtdi\lm joos, 120- 22. 122, 24ti. 648- 4£1 Eullllctcria. 2?5 eukaryotE'S '-'hromosomal ONA. 135- :t(J

chromosome makeup, 131.

1~2

c:umbinatorial t.:tmrrol, 547 - 49 vff'ec:t uf antibiollc.s. 453 gene dtmSit)• in genome, 13.1 gene mgulaticm in . 529- 73 genes. 379

gElnf.l expressicm t :h <mgf'S. 619 -

zo

genome. 6JS- J9 human otigins 635 · 59 insect wings. 632- 35 Mediatru: CompJP.x r-.onserval ion. 369 369-70 mmphological changes. fi30-35 rw.w genes. 618

RNA in. 12fi repair sy!ill-!ms, 247 exon sh1.1ffii11g. 400 - 403 exonic splicing r~nlJmt<:P.I'$ (E'SfsJ.l9:J. 39R exons. 379. 402 ex•10\.ICteases DNA Pol J. 200 proofreading by. 191- 92. t92 removal of mismnh:hed DNA. 24.2 expoor.ntia l phase or growth. 687 expression \lectors. 504. 6!iS extracellular matrix . .579 (!yes_ c t:topic. 621. 621 - 22 P.xr.isir~n

F F-fatlor 6fre P' p lasrrudG. 688 F-plasmids, 688 frdor binding r:enten;. 441. -447 falty acid biosyn~is. 59 fibrcblast growth factor genes {FCF}. 6 15. 6 J 5 Fis {factor- for inversion :slil'nullrlion) protein. 306-7 fisher. Rl:lna!d A.., 15 ~llins, 305

flanking host DNA. :ns

PiA J!tornoler. 305 Pi8 promoter. 30 5 fluurplates. 588 flf.lur W.tlf'! (Triholium cP~·wneurn l. 623

FLP-FRT. 10 4 FLP l'C(.OJUbintiStl$, 297, 302.703

Ou.;n-escent l1lain-terminating fl\tc;lootidcs, ti(l5 fluores<:ent in situ llybridi7..-'ltion (flSli), 691 92 fMf.t-RNA,~~>,... 43~

folding, 79, 1<-!4 - l.S. 3fH {ootprinting. 400. 491 rorenstcs, PCR and, 661 rorespores. 585 fork rt!gressron, 268 N-fo rmyl mctlticnin&. 493. 4:M F(}JCP2gP.ne,83~637- 38 . 638

fraJJlE'.shi.ft mutatioo.s , 470 , 472

720

Index

Franklin, Rosalind . 22 free energy in bi()rntJle<.:ui('S, 56- 59 concept of, 44 COI'lSel'Vation, S8 mo)ec\llar stability and, 55-57 secondary bonds , 52 fn.'t! energy change (~C) ATP hydrulysis, 61-67 c:oncept of. 44 tllluilibriurn cxmstants l!lld, 57 lligh-energy bonds and, 58-59 in mett,bolic pathway. 60 frf!f"dom of rotation, 42 Frizzled receptors, 598 fru it fly. Sft> Drorophilo mP.JonugostP.r (fruit fly) AsH p rotein, 522 F'tsK protein, 308- 10. 309 ftz (l:UShi Tomzu)gene. 622-23. 623

G y-resolvase. ~7 GAGA binding fm::tur, 562 Calao. ss4 GAU gene, 532, S33. 551, 551

gal gem>~. 499 Cnl4. protf!in. :i3Z, 5.'1.2. 534 - 37. 554 CaJ mpressor, 503 .(J-galactosidnse, 488 gafR genes. 499 gametes. alleles in . 7 gancyd ovir. 708 gap genes. 603 gap p~. 146-.08

Cap repei.I. 3 l 6 Garrod. An :hloold E.. 16- 17 GATC sequflnce distribution 240 C:Ccontent, 110 Gcn4. 565-6?. 500 GlJP/CTP exchange. -450

gf'l e lE'Gtrophoresis ONA topoisomer separntion, 120, 12f1, 12 1 nucleic acids, 64fJ, 648- 49 polyacr3'larnidf', 675 proteomics and. 67'1-78 St:queudng. 663 gel filtration chromatognrpby, 673, 674 f:tt!l matrix, nucleic: acids, 648-49 gt!l mobility shift assays. 400, 49l gene wnversion events. 264. 284 mating-type switching and. 286-88 meiotic recombination, 289 mismatc.h repair and, 290 gtme density, r.hromPSOn~l. f ,?4 gene d\lplicatiun, 616- 19 ge~ findl!l' methods.. 668 gent finder programs, ll68 genE! linknge, 9-11 gene Q. 523 gene silencing description, 531

HOgenes. 580-B:J mechanisms.. 556-62 telomeres, 5 56. 557 gene-enzyme rela tiom;h ips; l7

genera l tnmsaiption factors (GTFs), 353, 367-f generalized transduction. 688- 8 9, 689 gflTMl!l

com(Xl5ition. 4m conservutiCJfl . 6 14- 19

definition, 1 d tmsity in S"'lomes, linkage. 10 mutant, 10

J3..1

proteins and . l ti- 17 regulRt ion ~fu!r lr.mscriplion initiation, 504 regulation d uring development , 575 - ti1 1 regulation in eukaryutes. 529-73 rP.gUlation in prokaryotcs. 483-527 s uppFessor m utations. 4n-75 viral, 2 1 wild-t}'fle, tO genetic cocle. 36. 461 - 76, 462 amino acid tripl~s. 35- 3ti cracking 464- 65 degeneracy. 401-69 mammalian mitochondria, 4 78 order and makeup. 462-63 point mutations. 470- 71 readi ng. 471 rules governing, 469-71 suppn>~S
or.

genomes

bar.terml, 663- 64 compcll'lilive analysis, 6 36 -.39 E. ooli. 134 evolutiun. 63S-39 gene density in, 133 hurrum, 13'1 intron s in, 136 large scaffold . 666- 5 7 mitochondrial. 475-77

partial assembly. 664- 66 phylogeny, 614 repe.ated sequem:es in. 136 S . cerevi:;iae. f>94-9S g.onomic DNA. 212.577 ~enomK: libnuie;, 6fi6 genQmk:s. 38-39 genotypes, 7 ~.rm coil devt!lvpmt.'llt. 593 germ line transf(JI'Itlalion, 707 Riant gene. 603- 5 Gibbs. Josiah, 44 Gli er.tivator. 589 ginA gene, SOO glttbin chains, 37-38

globin genes, 543-44, 517. 618

histidine (His. II). 73

glucooortoid receptors (GR), 555

histolli'! acetyl transferasA (HAT)

gluc::ose, 42 glucose-6-phosphatll. 59 glutamic add (Ciu. E), 73 glutamine (Cln, QJ, 73 glufamjny l amillClooetyl tRNA synlhasa, 421 glycine (Ciy. C). 46. 47, 73 glycolyBf>e, 248 Goosebmry protein,616- 19.. 6J7 ~id gene,S98

ere factors, 359 green fluoresceut prccein (GFP), 79. 691- 92 Criffi.tb, Frederick, 20 group activati()(l, 62 group-transfer readionS, 61 - 67, 63 growlh curves, bacterial, 687 CTP. 446-47,450

GTP-hm!ing proteins. 435. 447 G:U base J1airs, 123-24. 124 guanine (G)

modificaliun. 244 pairing, lOl structure, 100 tautomeric states. 100 van der Waals forces, 46 guide RNAs (gRNAs). 405 gynanmomorphs. 70 2, 703

cumplexes. 174, 540 histone <;h:ipm-ones. 173. 178-79 histone code hypothesis. 551\ histone dcocetylase. 557 hi8tone riL>OCErtylase cumplexes, 174, 174 histone-DNA interactions, 158 his!one-fold durnaim;, 153, 154 histone H t, 160. 161 hislone methyl lr8nsfeTases, 557 histon e methylases, 1 74, l74 histone£ chromosomes and, 129 gene s'lencing. 556-58 inheritance, 1 75, 176, 177, 1 78 modification. 1 74-75. 558. 559

N-tumiilal tails. 159. t62. 169-74.172. 17. nudi:!OSOli'IP. funt:Hon and. 163-65 propert~.J53. 153-54 structure, 154 -56 HJV virus. 563. 567

hix recombinat ion sites. 305. 306 hlfstr.ain, 687 HUI pmteins. basic, 5~ HMG·I. 547

hnRNPAl , 397 hnR..NPI. 397, 398 HO genes. 546. 546, 680-83

H19gene, 559- liO Haeckel, Ernst. 6 hairpin ONA. 318 hairpin loops. 79 hairpin RNA. 3tH Haldane, John Burden Sanderson.1S. 17 h;llf-pint prolein . 397 HammBislen, Ola, 21 hammt:f"head ribozyme . 125. J26 haploid cells. 132. 148.580.693- 94 hap loid -specific genes, 549 H2A.z, 165 heal denaturation. 109 h E.oa1 shock u fuctor. 4!19 Hedgehog. 700

helices. handedness, 103 hfllix-breaki ng resjdues, 74 helix·loop-hel i~ motifs. 536,536

helix-turn-nebx motifs. 85, 354. 493, -JlH bemt~globin, 29. 29. 37 Hemophilus i11Puffi20e., 663, 664-6(, ht!t'edity, 8-9

Hershey, Alfred D., 21, 631, 692 heterochromatin, 556-62 heterodimers. 535 heten,duplex DNA. 262 heterologous recombination, intermediates. 266 het~rozygous genes. 7 hexapods. 630 hfl gene, 522 Hfr (hiW1 frequency recombinant) strains, 683 h.igh~n«gy bonds. 53. 59. 60-61 Hin invertase. 297 Hin re.cunbinase, 305-7, 306 HindiH restriction enzyme, 5 50 hinge hcUces, 89

HoUiday junctions cleavage. 263 dcfinitiun, 260 (j'.j Adeav~.

2 76- 77

fun nation , 300 generation. 2t\2 recombination intennediates. 200 RuvAB complex recogmbon o f, 276, 277 Ru\:C dimer binding. 278 Holliday model. 260-64, 261 hvluenzymes. 537 humeodoroain proteins. 53S homeorlomains. 535 homeotic; genes, 62'7-29 homologous recombinaliun catalysis or. 269

chromosome segregati()(l during meiosis. 2i ciTClll.ar IJNA multii'TIEli'S, 307. 307- tO des<:rip1ion. 259 cluuble-sm.nd bn>.ak repair rnmlt!l. 264- 66 il1 eukaryotes. 2n-?8 genetic con~uenct.'S. 288- 89

Holliday m odel, 260-64 .knodc.oot tochnology. 707-9, 709 models of. 259-6 8 molecular level, 259- 61 protein machines, 263-78 homologs. 131, 148 homozygous genes. 7 Horvitz, H. Rvbert. 698 house mouse. see mouse; Mlls musculus Hox genes. 628, 629 Hoxb-2 genes, 707 Hoxb genes, 628 Hoxd genes, 628 Hpal restriction enzyme, 651 HSF,562 hsp70 vontrol, 626

722

Index

HSP70 gene, 562- 63 HU protein function, 300 humans evoluUon, 635- 39 FCF genes. 615 FOXP2 gene, 638 gsme count, 6..15- 36 gen~ regulatory ~lements, 530 genome contool, 137, 671 spooeh, 637-.39 synt~my,

670

hunchbockgene, 592. 601. 802,602-5,803 Hunch bal;k ff.!fJ"$surs. 608 Huxley, Julian. 15-16 Huxlev, Thomas H., 5 hybnd DNA. 2tl2 hybrid dysgenesis, 703- 5, 704 hybridization. 108- 9.652-53,657 h ydrogen atoms. 42 hydrogen bonds 13 s heet s tnx:ture and. 77 ba~ pairing n n d. 102 lll biolvgical molect.Jies. 48 bond lengths, 4 7 -.hara-.tt!fistics, 47-48 directional prop&hes. 49 DNA helix fmmation. 69-71. 70 pr-otein structure and. 78-84 wate£ molecules. 4 9 hydrolysis ATP, 61 - 67

e.lr:finiliun, 58 in DNA synthesis. 183-84

initiation factors lJFs), 433-35. •U4 initiBtOI' (lnt), 363 - 64 initN!tor proteins. 2 12 ,214. 214-26 iniliator lRN/\, 433

insects. 630. - :-iS. 633. set' also Drosophila mekmogaster im>el1ivn ~~uence (1~). 328- 29 i.nsertional mutagenesis. 600 insulators, 531, 542, 542 mt gme, 5 23, 524-25 lnt proteins. 524 integrases, 297, 303-4. 321 -22, 322. see al. hmlsposase5

integration h ost factor (IHF). 304, 304 intercalc:ating agents. 245- 40

J3-inteoc.>n.Jn gene. 5 46- 47, 547 inteJgenK: seqoon£28, 135, 137-38 mtermediatc filaments, 582-83 interphase. 147

interwound writhes, 112 intragenic suppression. 472-74 intrun-e:xon boundary. 381 intronic splicing enhancers (ESEs). 396 introns conserved sequ~nces, 401 contribution to genomes. 136 descrtption, 379 early model. 400-403 group I, 288-91 . 389.389-90 lariat form, 381-83

late model. 402 reactions, 390 removal.1Z5 self-spicing. 387-00 inverted repeats, 295 ion eJU;Mnge chrmuatugrnphy. 673. 674 IRESs (intemnl Fibosome entry sites). 435, 4 IRF.546 IS4 family transposons. 32'7-29 asoclectrofocusing, 675

peptide bondc., 60- 61 typtl:> of dama~ by. 244

hydrolytic editing, 359 hydrophobic bonrls, 51 - 52. 78 hy~oxylgroups,48

hypelcllromidty, 110 h)'poxanthine. 4'1 5

iwftxms• .39'7

1 Wt I initiation factor 1). 4 34-35 IF2 (initiation lactor 2), 434-35 IF3 (initiation factor 3). 434- 35 lgf2ge~. 559-60, 709-tO JHt' binding. 328. SOl . 541 imaginal disks. '100 imino groops. 74. '14 1mmun~ systems, 338- 41

&soleuctne (Ueu, l), 73, 422 isomerization. 357 ttlOpods. 631

J Jacob. f'rant".ois. 49t>, 497, 688. 692 Janssens, F. A.. 11 ioinl muleculcs. 275 Jun/ATF. 546

immunoaffinity chromatography. 674-75 i.mmunoblolting, El76 1mmuoofluorescencc microscopy, ll91 - 92 imprinting. 558- 59,$60, 7(l9 - 1 1 , 7 10 inct>tporation assays. 673 inducers, d E!St:t'iptiou. 504 inducticm, 683 Ingram. Vernon M.. 29 jnheotance, 10, 562 iniliruioo RNA primers and, 193-94 steps in, 352 transcription, 350. 35 1. 538 trlmslntioo, 432-40,433 initiation compSt>xes, 434 80S,438

I(

l:.t!ratin. t:ttiled-cuils. 8fJ Khorana, Hsr Cobind . 36 ki ~tochores.

143

knirps Ef!lle. 003-5 Knirps rnlltants. 595 Knirps protein, 607-8. 6118 knockout technology, 705. 70 9 knotti ng, DNA, 118 Korn~ Arthur. 24 Kozak. Marilyn. 415 K02ak sequeflCe, 41 5,435 Kruppel gp11e, 603- 5, 607

Kuhn,A.,t7

lndex

L lalleling. 37. 651 pub;e-hibeling, 37 labial (lobJ gene, 627 Labial prutein. 630 lac genes, 468- 89, 489, 496 lac: operate.-. 4 89 lac operons. 488, 490, 498 /oc promoters. 489- 92, .J92 /oc repressors. 488 allosteric cc.mtml of. 496- 00 IJNAlrindingmotif. 493- 96 domains. 83 functi.on. 495,495,502- 3 regulation. 89. 89 RNA polymerase binding, 489-92 lactcse permease. 488 lactose repressors, 85 IacZ genes. 6g(), 707 lagging strands. 192. 228-30 A integrm;e (Am&). 297. 303, 303-4

bar:tt!rit•phctgef;, 68.'i c,floice of, 521 C(Jiltl'ol tJf, 5 17- 18 epigenetic switcbes and. 562 establishment. 522 gene expression in , 51 3-14 Acn and. 520-23 lysogenic induction. 512 lysogenic propagation, 5 12 lysogenic state. 303 lysogens. epigenetic switt:htl!i, 562 lysc~y. 682

lytic growth, 303 bacteriophages, 683 choice of. 521 OOCllrol of. 5 1 7 - 18 gene expression in. 513- .14 Acll and, 520-23

lytic: phages. 6 82 lytic p ropagat ion. 5 12

A.!ysogen. 5l2 .\ repressm, 514

C-termimd dc•lllllin inl~:tions. 520 DNA interaction. 8 5 folding, 79 hlnction, 495,517- 18,518 geometry of ropressllt'-operAtor (:offipJex. 85 i.n majm groove. 495 negative a utoregulation , 5 19- 2() operator site binding, S I S. 515- 17 i\cU lunction. 520-22 langusge. evolution CJf, 637-39 latent pnriods, 685 law of mass act.ion, 65 LDL (low-dtmsity lipoprotein) receptor. 403 leader sequences. 505 leatlingstnmds, 192 )c,pidopterans. 635 Jeocine (Leu, I.), 73 leucine zipper DNA-binding JOOtifs, 65,536,536 leucme zipper fdlnily proteins. 80 Lax/\ fllJ>reS60t. 257. 518- 19, 532

lihr-<1ries eDNA . 657

DNA. 654, 656, 656 - 57' whole genome, 664 Hg-1tittn e xperiment. 5{14 limb!;, insect. 630·-32 LINEs (looginte:rsp!m>Ed nuclear elements), 337,337-38 Ji.nked genes, 9 - 11 linker cells. 698 linke.- DNA, 15t histone Hl binding. lfiO. 160-61, 16l ltlnglhs. 153 linker histones. 153 linking d ifterE'neeS, 114 - 15 linking numbers liJ.J, 38, 73, 11 2 - 1 4, l16, 433.434 locus control regions (I..C.Rs), 543- 44 long tennirwl I'E!pf>.ats (I.:fRs). 3 14 lophotrorhozoans, 614- 15 lox sites. 300 LTRs (Jof1E lerminal repeats). :n~ Luria. Salvarlore. 6fll, 687 lysine (~s. K). 7.1

I\'S(J8enic growth

M M pflase (mitosisj, 142, 143 Mocho-1. 584, 584

Macl...ood, Colin M.• zo mauurnolecules, 51-52. 59- 92 rn.acromutations. 15 main tenance methylnses, ~ 1 m ajUI' grooves. 103- 6. 10!;, 495 major late pt'Omoter, 398 mammals. Sf'e also chimpanzees; hm1-..1ns; mouse conserved moch<misms. 531-37 FGF genes, 615 I lox genes. 028 signal lmnsdvcticn pathways. 553 MAP kinmw. pathways, 552 map units. 13 murinerelemeots, 335- 53 masking proteins, 554 -55 mass spectrometry (MS/tv!S). 676-77, 678 MA1'1ocus. 284. 286 mating-type loa, 285, 285- 86. SB0- 83 mating-type switching. 286-8 6, 287, 581 M.attbaei, Heinrich. 36 maxaUipeds. 6al Mayr. Emst, JG McCarty, Maclyn, 20 !VkClintock. Barbara, lt Mcm complflx, 226 MPChonism of Mt>l1clf!llon Heredity. 13 Medio-"lh WQJnJplex. 368- 70,369, 537,538,544, 546 rneiosis

chromosome nu~ and. 148-4!:1 chromosome segregation during. 278-84 DNA dynamics. 279 gene crossing over. 11 phBSflS, 149 meiotic call cycle. 148 meiotic recombinaHcn, 280, 282. 283-84, 289 melting point, ONA. 110 Mendel,

eres«.6 -8, 687

Mendcl's first law. &eP. Priociple of h ld P.poorlenl Sf>.gregat Mende l's second law. see Principle of Independent Assv mercaptoethanol. 675 mercuf}' ions , 501 - 2. ~erR.466.500.501 -Z.502

724

Index

m.esOOenn differentiation. 595

morphology, 620-30 morula, 706 mosak:s. 702 mouse virus polyoma. 21

messenger RNA (mRNA), 123

Mre11 pn>tein. 283

merT gene, 500, 501 merT promoter. 48(), 502 Meselsoo, Matthew, 26, 2W, 692

alternative splicing. 583-85 assemLly, 4311 broken, 452- 56 circularization, 438, 438 degradation, 457 discover,}'. 33 locati2ation. 576 monodstrcmic, 413 polycistronic, 413

MRX pi'Ote)n, 282-83 l\4SH pt'otein, 242 Mu genome, 331 Mu transposons, 689

MuA proteins. 331, 332-33, 333 MuB proteins, a:n, 332-33, 333 Muller, Hermann J.• 10 . 13, 1t5, 700 Mus muscul us (mouse). SP.e nlsu mice as model, 681, 705- 11

premature stop <:odons. 456, 457 recruitment in probryotes, ~3 s tructure, 41.4 syntbe(ic. 465-fi6 ll'Hoslatioo and. 4"11.412- 15

tnmslalion-dependent regulation. 452-57 trnnslocation, 444 - 46 bansport.406-8.407 Me.t-RNA,Mel. 435

metabolism. "inborn errors~ in, 16 metal ions, 187, 349 mttaphase, 147, 148. 149 lllftllyl groups. removal. 248 N-mf'thy1-N' -nitro-N-nit rosoguanidine m uhlgerresis by. 244 methylatkm. 2!8. 556-56 mtdJy)guanine, 4 15

Mic:a experiment. 104 mice cbromosome count. 705

patched mutants, 705 Mm8l protein, 284

mutability. repair and. 235 - 58 mutagenesis. inserti<mal. ~o mutagens. 242-4.4,243 mutant genes. 10. 14. sse also specific gencs mutations. .st1e also specific mutants definition, 15, 235 genetic variability and. 15-16 hydrolytic damage, 244 induction. 16

rumue or. 236-37 permanent incorporation of. 23B of. 236-37 Multi protein. 238,240-41 Mutl. protein. 238. 2 40-41 MutS-DNA complex, 240 MutS prot<.'in. 238. 240-41. 242 myoglobin folding, 79 myriapuds. 630 rat~

P.mbryogefle5is, 7(}8 f'GF genes. 615

N

ft)A1P.lgene.63~638

genome. 636-37 geTlCJflle content, 671 bomeotic gene complexes. 629 lumbar mutant, 629 synteny, 670 microarray grids, 575. 577 micrococcal nuclease (Mnaso). 151. 152. 152 microinjection, 707 microRN.As {miRNAs}. 570

roicrasateUite DNA. '137 microsatelhtes. 237 m.U:roscopy. <".hromosumes, 150 microtubule ta:ganiziug <:enters. 142- 43 microtubules. 142.582- 83 Mig1,550 minor grooves, 103, 105, 366-67

mismatch repaii system, 238-4~. 239. 242, 24'7, 290 missunse mutations. 470 mitQ{:houdria, 478 mituciJoodrial genortlfl, 4 75-77 Dtito~, l 42,l43.146,

147

mitotic cell divisicm. 141-43. 142. 6!/t; mitotic spind~ . 142 modP.l orgsnisms. 681-711 molecular biology techniqlH!S, 643-79 monodstronic mRNA&. 413 ~onod.~ues,496.497.6B8.692

ma1ovalent attachment. 146, 148 Morgan, Thomas Hunt, 10, 12. 699 nuxphogens, 578,579,588-89,590-99

N pt:oteins, 523-24 Nanos protein, 602. 002-3 negative autoregulation. 519 NEO gene. 708 nt:omycin resistanre. 708 neurogenic ectoderm, 587, 586. 595, 500. 5941 neurons. 588 NF-KB, 546 nicking. 1 14 Nirenoorg, MarShall, 3f,

nHrogen

isott:~pe

tagging. 26

nitrosamin~.

244 nodes. primiti vP. stl-t:ak, 700 ~ggin. 706

non-histtJI~

proteins, 129

oonauton<JmotJS transpusons. 313 oondisjuncti<m. 280 nunbumoi<Jguus end jt1ining tNHEJ), 254 llOflpolar molecules, 45 n<Jnsense r.od04'Js, 36 nonsenSE-mediated mRNA decay, 450 nons"nsu rout::ttions. 470, 4'13. 4 74 nonf;lop-medi:Ited decay. f56 nontransferrod str.~nds. 316- 18. 317 north ern blot hybridization, 653 Notch signaling. 587-88 Notl n!Str.K.:tion enzyme. 650 NREs (Nanos msponse element), 602

oNTO (amino termmal domain).. 356

r.NTPs, t84,

185

NttC. 485 - 86, 500- 501. 501,541

0

nodea~ m~n'lfi<: I'()SOf'laJlC(! Leclmlques. 75

nudeAt por~ c;ornplf>x. 407 nuclPaJ l!Caffofd!;, 163 nuciE.
0~ mt~thvd

comparali"'e genome anaJy,..ls . f169 DNA cloning, G54- S.'i DNA hyb.ridir
400

n

DNA ~men I isolarion, 653 clectrophOI'et>is. 6411- 40 gcn~·hc m(orrnalion c;on v<>ycd by, tD-40 gcnome--w rdc a oolvses. 667- 69 oligonudeoli~ synthesis. 6!\7 - Sf.! paited-P.nt! ~ctt tntogy. 666 - 6 1

PCR. o.sa-so prE',t.Ut'S()f S,

64 649- 51

utigolluch'Otidns. 657 , 56 (ln the Or18tn of S11f'CiP~ ((},nwml. 6 1 :S oncogenos. 6'.)8 (){)(:}"f8S, 5\19

o pen compl flJ(NJ, 952. :i56-58. 351 open·.tcading frames (O R.Fs), 41 2 t l , 6fi7 ""' operntors. mpn1ssor bindint- 464

opcrons, .'50'1. 5 11 optical density. 110 ORf gtJOflS, ~114

ORf proteiru;, 3:.1.3 orgatl'H.: molcculas snlubility. .'i1

oriC.. 2 13 , 214 origin mcognition ctmtp1exes (ORCs), 217 origins mplicah o n. 138 during atll divi=>1on . 136- 39. ·u g tdflnl ificalioo of. 214- 26 mulfl(;u)
ur

sequenr.es, 660- 64 synlhtsi:r>. 64- 65 vector DNA, 655-56 virol ~. 2 r

nucle01ds. 13 1 nocleqlluhc attock. 126 nudeos1oo·.S' -triphoophates. 64 nucJ~dc

fr.lgm,mrs

r..omple li<m. 208-9 length. 2 11 prime rs. 104 replialtion rortts. 193, 193 separali(.lll, 24Z Oftmoide!>. 620 ol rgn-ribont~eleohc:lEtS. 4b~

DNA lib mrie.-;, 056-57

~rictiun endonur~.

Qll-melhylguaninr• 2 4-4, 247

phosphates. 6 4

nudeosidcs, 00 nudca;omal DNA. 16? - 63 nlle'.leosom es ern-tyr;, 161 -62 i!..'i:sflmbly. 155. 17.5 70 a tomic structure. 154- 56 8.X1S Of S)'rllmt'!ll')'. 157

pnor 10 r..ell

drvtt\100. 2 19

orthodt'1t'lt.icl~ gent;.

601. 001 - 2

oskormRNA. 599, 600. 6U4 Os.knr protein 593 o.10dahon. DNA damas;e. 244 - 45 oxoC (7 .B· dihydro-8-oxogu~nine) . 244

OKygen. 42,45

p P-c:lt9f1lflnl tmnt;fo rrn!llions. '700- 5, 705

chrom()OOI1lC slructurc and, 150- Sl

p5J~1~.7V1

tore part ie~. t sz

P siiC'!>, 430, 432, 43 -1 P-1·tf. $62 , S6:J p t~mu nfll ion f~.M.:Ior, 36 2 PliP proroofer. !)03 pair-rule, 6V4 paircd-t'nd sequencmg. 667 Pa ired protein , 616 - 19, 61 'J

DNA bindin.&. Ui8 DNA-binrung protein depende11t pusitioning, J68. 'f68- 6fl nNA pack-.,gh19., 15 1 fonni\liOI1 o(, 130

hmciiCID m eukaryotcs. 529 hl!itooe v:ml\11ts 11nd. 163 65

lackmg H2A and H2.B. 158 mu:rococcal ra~. 152 movamcnl , 166- 67. 167 negative supen:oiling and, 11 5 positioning in cells. \ 10 . 111 pq'(lfP.in ctltll, 153 J'efllOdcling, J67 retn~Jdcli.ng compl exes. 166- 67 nuc.leutide ~ion repan , 247, 2 48. Z-50 - 53, 2 52 nucleotidcs 3'-5' pl1osphurllestcrnse chain-
li oJ.;.a~. 23

COnveya ncf' of 8 A.-,ellr: mrOffOUhOf\ , 28 31 CTeRlion. !Ill formf'lfion, 98

Pangoh n.598 parallElI f3 s tJef)(s, 76, 77 pm:nlnl imprinllng, 709 · 11 Pasteur. Louis. 6!l3 patch products. 262. 266 patched mutants. 705 pallem·d~>~01Tll • rung

genes, 619-20, 620

Pauling. Linu.o;, 22 Pox6~n~ . 619.62 1 621-22 PCNA, 242 Pfi prOtlltltcr, !l64 Pcllu .kinafl<' activation, S!IJ peptrdt? bonds characteris tlc:s. 72 evolution, 126 l•ydroly.s1s. 00- 61

triplots1 36 nurse cell!>, 500 Nus proteins. 359 N11sA protein. 524

plenat shape. 42 peptide groups, 48

Nusskdn-Vol1hard. Christine. 594

peptides, staining, 675

illustnnion. 74

726

Index

peptidyl transfura.w. 126.440 center. 425, 442- 44. 444 reaction. 428. 428 LRNA binding :~nd. 430 peptir1yl-tRNA. 428 PGNA,204 Pltage Group, 881

phages. &ef> bacteriophages phenolypes, 7, 8 piJOnyl, 65 ph~phoramidite. pmtonatf:d, 6511 phosphorylation, 88, 90, 553 ph otoreactivation. 247,247,248 phyla, stmlmary. 6 13 plaques, 684, 684 plasmid vectors. 654. 654- 55 plasmids, 1 11. 13 1, 688 p lectooemic 'l.mthe. 112 Ptvt promoter. 564 po int mutafio llS, 2 36,470-71

Pol II core promolers, 3 6 3-64, 304 pvllsr gran ules, 59iJ polar moll'cules. 45 polarity. IJNA hel iC
polymet"Ase 5\o\'.ikhing. 201. ,l()2 polynuc:leolidc k inase. 651 polynucleotide pho:;phorylase, 400. 466 polynudeotides. 36. 99. 122 polypeptide backbone. 74-78 polypt=!ptidtl chains, 37, 76, 79 polypeptidos. 432 polyplw.nylaJanine. 4()6-67 polyploid cell!i, 132 polypuri® trrK:Is (PPTs), 324 polyribosomes, 33, 34. 426, 427 polysomc1S, 42.6

pol)•tene chromosomt>,s, 701. 701 poslfional infonnalion, 578 posirive aut()fegulation. 5 1!l positi ve controlrn ulants. 4g4 posterior. dofinilion. 57B Pnl prvtein . 617 prv-initialion complexi!S, 364- 00,369.435 pre-mRNA. 380

Pllf: promoter. 520 pm- replit:<~li ve complex (pre-RC), 222-27, 225, 2 26, 227 primary slructum, prolcin. 72, 75

primaseo. 193-94. 199, 199-200. 210 primates. 637. sre olso humans primer-binding s ites (PBSs), 324 prhnnn:, f!l4 RN.I\.193 - 94

primer:lemplaee junctions, U12, l86-88 p•imilive streak. 706 Princip le of lnrlependoot Assortment. H, 9. 9 Priudple ol h1dnpendoot Scgrogalion. 6 -8. 7 pro-~:' Stl6 p i"t:JQ2 coiiHgen gem l, 37g probes. 651 - 53 pmlxsscipwfia fpb} gcnP., 627 processed psm.ulQRenes, 3~7 pruc:cs.~ivily.

1119. WO. 201 4

profilin. 58 3 prufla vin. 24ti programmed rearrangements. 305 prokaryotes c.hromosomn ml'll
RNA Rl3Ss. 413-14

lopoisonw.rases. 116 lrdnslaliOil. 424. 424 translalion initiation, 435 proline (Pm, Pl. 73, 74 pro111otcrs ~:Ioria!. 354

bacteriophage A. 513 COI1Sa!SU!> set{llf!fleffi,

355

core enzyme reuuitmet1L 356 description. 530 regula tion. 484-8 5 RNA Pol f. 275, 374 -76 RNA Pol Ul. 275. 374-76

lrauscription cycle. 350 proolreading. 359. 370-n proofroou1iug vxonuclea~As, 191 - 92, J92. 237 propeller twisl a rrangvment, \ 07. 107 prophages. 512 , 61:!2--8.'• prophase. 146. 147 protf'..n di:lfnains. 402 p.rotr.in !.1ahi lil y, 4:j2 - 57 protein s uhunil:;, 72 protein synt hesls.l8. 29, 30, 37 - 31:1. Sf!fl n l 60 !ret

protein-DNA intm-actions DNA binding, 84-87 init~tiun or ONA replication and. 217-21

nonspecific, 86 weak bc•nds and. 53 protHiu - protC'in i.nteraclivm;, 53 aiJOSCP.ri(. lf<~ n:;formi:ltions

and . 88. 90

inilialion of DNA n•plic:ation and, al che lt,plication fork. 2 10 - l Z protoin - RNA comp lmc, 385 p mlcin - RNA intr.rnclion.c;. ll6- 87 proteins affinity for DNA, 41W aiii)Siory, 88- 91 amino acid incorporation, -467 amino acids in, 73 bullrling blocks, 7 I - 72

2 1 7-~1

in chromosomes. 129 clefermination of s tructure. 75 folding. 76. 78. 79 Jl(,"'lCS and. 16-17

hydrogen bonus and ~ln.tcture of. 78- 84 immunoblottiop,. 676 large. 82 level<: ol'struflu m. 72-711 purificalion, 672-73 separ
two hvlxit11l5Say. 533-34 unfoltting. 74 pr~eomics. 6 77-78

prmutlllh'd phosphur<mtid itr:, 658 procesS(l{'\. 137 J>Sf11tdoknots. RNA. 123. 124 pseudouridine (I}A.I) , 415. 415 PSTA IRE helic:es. 00 pseudf-~nes,

P-TF.l-b. 371 pulse. see pulse-labeling

purines. 100. 700 puromvc.in. 454 p yrimidi nes, 100. WO pyrvphosphalases. 184 pyropJtosphale group15, 64-65, ()5 pyrqJflosphorolylic fldi ting. 359

kinasll. 81

R R groups. <:ategories, 7'1.

R-Joop ma pping. 3!18. 399 Radso protein. UIJ ROOSl protein, 275. 2fl3. 283 Rad52 protetn, 264 radi;:~fion. DNA clam;.ge. 242.- 4q . 244 - 45 RAG rocombinase. 341

activating gene} subunit. 340

RAC2 (mcombin~hnu ar.lwah ng gL'flof subunit, 340 RALL motif. 623 Ran protein, 408 Rap1 .557 rats. Slo gene, 394

Rb (retinoblastoma) proteins, 554 reactive oxygen Spi'ClPS (ROS), 244 rmding frames. types. 413 RecA· Ii ke strand-ex£".11ange p roteins. 282- 83 Rr!CA protein assembly, 272-7<0. 274 ATP bindi~. 83 ~paired partnen; within, 274-75 function. 257. 518-19 hc-nolUJ!,S. 275-76. 276

ssDNA coating. l?O

RecOCO helirnsa/nud £'.1lS6, 269-71. 2m R(x:J3CD pathw
reducins a~s. 675 regulator binding s ites, 530 regulntory dcmoots. 5.10 regulelorv Se<ju eru::es. 135. 365. 530. 616 - 1 r€laxalioo . 1 113-20 relaxt>rl. definition. 114 release factors lRFs) clnss I. 449. 449- so function. 448- 49 peptide~ release. 450

rarnodeling. nudeosomal. 100

retJalurolion. DNA. 110 repttic. mulabilily and. 235 58 repaalr!d st:qllf!n(.es. 136 replication DNA. J!o)t -233 errors in. 235

finis hing, 228-32

Q ptolnin•.;. 523- 24 q uantum flffiCbani<:s, 42-43 quarternary Stn1Cillro5, 7Z, 75. 82 qnery sequences. 670

(recombin~lion

recruitmen t. 4115. 537-44

or tclomeres. 231 Q

RACl

recc-uLi n~Js. 294. ~7

recombination . 12. 262 n!('.ombina tion factories. 28 3-84 recombiMtioo signal sequences, 34() recombinaliun s ites. 294 recombillitliooal rep
d ass 11.450

pul&l-lah{-ling. radiOildive. :!7 pulsed-field gel eJectruphoro:;is. 649 . li-19

pyrttvflle

rK:~;sive traits. 7 roc.ognition OOlix. 85, 493 recombinants, phenotypes, 8 rocombinase rocombinalion seqllf.'nces, ~~

repltrntion bubbltJS, 216 replication [ad oiJ• hypothesis. 221-22 replication forks. 1!12-200 assembly, 225 Dam methylaticm at. 241 rlesctiption. 192 DNA synthesis at, 2£5- 9 ONA t.tllwinding oofore. 194-95. 195 enz~·llJe.oo; functioning at, 199 example. 19.1 prctteirt inler.K:Iiom. 210 - 12 stallerl. 268 TnJIJ t ransposition. 3..11 topoisomernse ftmctio11. 198, l f~H-00

repliOJflve lran:spo:;ition , JUJ- 20. 319 r('pli(a lors, 213. 213- 14, 214-16. 222 repiK:.on mcdBI, 2 12 repliums. 2 12-14 replisomes. 210- 12,237 repaler genes. 5 31

rp.prcssor-operdtor comploxes_ 85 11lpw.ssor X . 620 ffrprns50Th

assamb1y. 555 mtflf'Belion at 0 , or(>__, 519-;m trnnso:iplional. 549-S 1 ttans!ali.on. 483 t ronspm1. 555 resolution, 260 ~olvasl'S. function. 307 restridion enrlonucl..-dses, 649- !i 1. 6.H restriction f!nzymes, 654 refit:ulocyros. 37 retrOJ'f®lliilioo. 524 -25. 525

728

Index

retrotransposons LTR. 31 2

poly-A, ~•12 , 322-26 poly-A s«Juencm, 314 RNA.325 viml-like, 3 t 2 , l13- 14, 320, 321 MI'O\/Ua' int~ 32 1-22 Nl4• oviruses

eDNA fonn11tion. '324

wmposilion . :na

l4

DNA oorl~. 324 inlef.lralion, 321 mOHtment , 320

RNA ends • .32~t m utations, 471 reverse trans(.ri plaso. 136. 324 mvc>.rse tr.msaiption. 325 326 656 Rho-dependent tennlnetors, !tbl Rho-lndRpcnflent terminators. :;t6l. 36l. 362 tfWfn'f} (bacl.)

rhomhoid gene, 595. 596, 597 ribooud 08f proteins ('RNPs), 62, 87 ribonudeases, 2 1 74 nbosP. 30. 122-23 ribosam!l proteins, 506- 11 ribosomal RNA (rRNA). 413.428 - 29, 43./ ribosomf! binrllng ~tit~ (RB!'ls) , 4 13 ribosome rccyclins !acton. fRRfsl . 450-52. a!i t ribosomes. 41 1. 423- 32 wniflOOCy i-IRNA selection. 44 t - 42. 443 c.haJlnefs, 430-:32. 132 wrnpo.o;ilecn. ~ 25 aystal strtKJ ure, 42!1 cycl~.

426 mRNA trans port, <430- 32

h!cydme. 450 rnscue, 545 as ribozyrnfl. 442 44 rRNA in, 428- 29 fidlrming. 414 Sl.'parruion, 425 structure. 497 subuflils. 429 s uburuts durinR transltllion. 425 - 2.7 tRNA b111ding s ites, 429- 30 IRNA da::;crirntnatlon, 422- 23 riboswilchcs 509, 510 ri~. 125,380- 90, 442- 44 RlSC (RNA-induced silencing complex), 568 RNA-armcalmg t~oo;. 384 RNA-depmdcnl R'JA polymerase. 56!1-70 RNA hairpinll, 361 RNA hcllca.'iefi, 436 RNA mterferente (RNA1), 567, 568 70,569. 69t\-99 RNA poly~ hol()enzymes, 353. 353 RNA polymerase 1 promotws, 374-76.375 RNA polymorm>e 11. 363 64, 364- 66. 365. 367 RNA polymeH.ISf' II holoenzyme, 370 RNA polyrner-400 Ill p omoters. 374-76, ;l75 RNA polymflr
P'imastl, 10~ 94 recruitment. 484 singte-subuuil , 360 s ubunitR, 31'19 lrnn::.cnplion cycle and. 348-57 tran-;cnptJOn tntJial ion b)•, 247 lraru.c.nption pt"ocess. 350-52 RNA prif1U31'S, 193-94, J!N

RNA-recognition motifs (RRMs). :ma Rl'cA r;plidnp, 1 35. 135. 379-410

r.hern.istry of :iEIO- 83 classes, 387. 387- 88 c\cscrlplion, 360 r1is..ovnry, J!lfl evol ution. 300 00 mtror. mmoval and, 125 pathways. 3BS-D3 reaction. 3112 l hree·WI'IY lunuion. Jll2

RN/\ - RNA l1ybc1d,;. 384 ftNA- RNI\ mterac:lions. J84 RN/\s. SC
base aomposilion. 35 cnaalytic region roldmg, :J9J chain folding. 123- 24 uc:wnpos1lion. 3(). 30-:U DNA compiircd with. !1 1 rlou b!P. hcl•col characteristics.. 123 editinr"' 404- 6 electrophoreSls. 649 foltiing. 124-25 roollit ion bind Ulg to . 71 p l (l('UfMII'S, 6-4 probes, 65Z -S:i p rocussing enzym~ 310 (>fotcin nrc:ognition of. 00- 117 retroviml, 325 strudure, 33. Tl . 122.12.2- .26 syfllhesis. 35, 36 lralltiCriptlon. ;J 'l in lr,m:Jr.riplionttl regul<~ tirnt, 567 -70 tran!;latioo. 9 t IYPfl=i· 395 RNAse H. 194, 194 RNAr;e P, 125 RPA. 251. 284 RS doml'llfl.!:, :i!l7 rot sates, 3~ Ru"/\U complex, 276 Ruvt:. 276- 77

Ruvr: resol, ose. 278

s S8, 16S rRNA binth ng. 5/1 S p~ (synthesJs), 142, 142 SA profClln , !ll O Sord1nromycEtJ CO I'P\III>If/r gene sikmdng. 557 g~mtrilling mul~tions . 6!1.0 senom~. 6!)4 - 05 growth, 60!i- ll6

HO gor£ silcnt.t"H. 500-83 intron:. in gflrtOmo of. 136 life cycle . 69:-J

mating-4ype genes. 54B- 49 m.itolic ceU d ivision. 695 ac; model. 681,693-96 rep li~ tor fitructure. 2 13-14 Ty elements, 335-36, 336

Salmonella Hht invertose, 2 97 So/mom:lkt typhimurium. 243, 305 sail conceutralion. 110. 111 Sou31\1 remi<:t10n enzyme, 65() SCJ!i, 397 scaffold s. 666 scanning, process of, 4 1 4 Scluzcsrteeharomyces pombe, 557 scissile phosphates, 126

SctlU-MoncrieH, Rose. l7

skin·neiVe regulatory switch. 587-88 Sleeping Beauryelements, 335 slid il'lB· nudooscme. 166 sliding clamp l()aclers, 204- S. 206 - 7,220 l"liding clllmps, 242 s lid ing ONA c lumps, 201- 4

ATP anrt load •ns uf, 200 UNA pro~i"ity and. 204 opening, 204- 5 polymerase processivi iV and. 201- 4 s tructure, 20 3, 205 Slo gene. 394 Sm~:~ds, fur.clioo , 598 small nudear ribonuclear proteins (snRNPs)., !OmBII nuclelr R\IAs (snR.'\ IAsl, 87, 383-84

Scr gene, 631

SWZ rEactionfl. 1tl3

,;ea monkey s (Ar1emial. 630-:t 1 sea squirt (Ciono intP.tilino/i .r;}. 584, 585, 615. 668 se<Xlf'ldary bonds, 4!1 BtlCClndary s truc tun!S, 72, 75 segll'Mmtl1 tiou. 599-600, 001 -2 602 selenocysJeine , 423 selr-splicif'lg tntluns, :i1!7-BII semiconservative processes, 27, 28 SeqA protein. 217. 2 18.219.223 £pquem-ltors. 661. 665 sequf.'nce coverage, 10X. f\63

Snail repressor, 597

sequencing DNA, 062

DNA fragments. 660- 63 Edman degrudalion. 677 gel elecb'Ophoresis , 663 high throughp ut, 665 rf!ad out. 665 s.b()(gun, 663-64 seter~ci1 pitous microhomologies. 254 serina recombinases, 296-97. 2117, 200, 298-00

s erine (Ser. S). 73 Sex combs recfuet>d (Si:rt gene. 627 Smt-ffllhal i Sxl} goo.es, 5&4, 5 64

sex-linkege, 10. 702 SF2/ASF protein, 397 Shme-Oalgarno 1>equ~nces. 413 shoti inrerlerir1g RNAs (siRNAs). 568-70 shotgun sequenclnB. 663- 6<1, 666 siomoi... gene. 598 sickle a U anemia, 29. 29 u r.acturs. :154. 355 . 356. 499 - 500, Stl6 sign.!l integmtioo. 400. 544-45 signal transduction pelhways. 551 - 55, 553, 577. 578 silen cing. 531, 542 Simpsoo, Ge~nge GayiUI"d, 16 SINEs (lli1orl inlerspai'Sf!d n uc.loor elements), 337,337-38 singie-r;tranded ONA-bi.nd ing proteins (SSE!s), fl 4. 84. 195- 98 singlc-strandec1 DNA (ssDNA), 264,272- 74 SIR rot'TlJJh»c re.:ruitmenl, 557 S IR genes. 557 Sir~ protein. 336 SISA, 563

SisB, !l63, 564 sistflr chromatid cohesion. 142. 144- 46 sistl'r chromatid separation. 14:i sister chromatids, 142 Sitfl-(\irected rnuw.genesis, 658 site-specific recombin ation, 30:Z- 10, 10-J SlU7 p rotelo, 456 s lir1 ~s. ,')Bfl

38~

sm!pdragoo.'> {Antirrhin um). 8. 8. 328

sodium dodecyl s ulfate (SDS). 675 software BLAST (basic local <\lignrnent search tool) p~ . 670- 71.672 VlSTA,669 St.l8 genl', 595, 597 solenoid model. 161. 162 solubility, organic m()lecule:s, 51 Sonic hedgehos (Shh). 588-89, 589 SOS response, 256, 518-19 Southern. Edward, 652 SootlJern blot hybridization. 652. 653

SPt . 537 Spatzle protein, 59 1 s pociali7.11!d lnmsduction. r>86, 68tl speech. e\lolutiun ur. 637-39, 639 spenn. content of. 6 spindle pole bodies. 143 spiral, 1 13 splice recombination products, 262 3' s pbc~> sitfl, 381 spliro-site rerognitioo, 392. 392- 93 s plicrosomRI proteins. 67 383-87, 3 t16, 391-S'J, 400, 401 splicing, i.n aukaryotes, 529 splicing enhancers. 563 SP011 protein, 282 - 83. 283. 297 SPOliR gone. 586 SR (serine argenine rich) pi'OC~ins. 3!l3, .193, 396 SSBs (singlo-strand(>(l DNA-binding proteins), 84, 84, 195- 98, 1!17, 1!19 Ssr A RNA. 4 52-56, 545 stable ternary com plexes., 352 Stadler. l.. J., 1 6 StatJ I. Frank W., 26, 2Ei0, 692 s tart codons, 412, 413,437-38, 440 STAT pathways. 552, 5~ s pli.Ceo£0ffiC6,

stt!liona.J)' phases, 687

stem-loop structures. 123 stereoisomers, so ..stid y ends." 111 srop cedens, 38, 4 12, 413, 448-49, 456 stop m uttltio ns, 4 70 strand invasion, 260 Slreptococcus pneunJoruce. 20. 20-21 structu111l main temm<.:e ur chromosome (SMC) prntains, 144- 46, 1 65

Sturtevant, Alfred H., 10, 13, 700 Su(HJ-NotchiC complex. 5811, 589

Index

730

Sulston, John. 696 s~rcoiled DNA. 113 n~tivt=:ly c:oiiOO, 11 4-15

mpliCC~tion,

positivll!ly coiled, 115 rdaxation o[ 11 5 - 16 supercoiling, 80, ll4- 15, SUpt!rholical d en sity, 11 5

H~-9<J

supp~org~ncs,472

suppressor mlJiations definit ion. 472 framsshirt mutaliun s. 472

function, 471 - 75 OOtJ!.l'rh !.C

mulation o;. 473, •J74

ViiUS.

lenninalors, :161. 36l

tertiary s tructures. p1ofein. 72, 75 tetraloops. RNA, 123 TFIIEI recognition e lemtml. 363-64, 367-68 Tfllfl-'l'nP-promoter romplex, 368 TFunD, 364, 537,540

Sulton. WalterS., 8 - 9 SV40

231

slructure of. l4 t te lo phase. 146, 147 temp€rate phages. 682 temperature. 45 termination chain, 463-64 nonsense slJppressors. 474 poly
111 . 3!14

Sved~~·fbeodo~425

Svedberg units. 425 SWJ prctcins. 583 ~'WIISBF. 546 SWUSNF.540

TFUI:-:, 368

synapsis. It synaplic complext..>S, 2.!14, 31 4-16 s:yn<:itia. 5 00 synErgistica lly, 544 syn-confocrnations. DNA. 107 -B synonyms. am.ino ad d . 461 synteny. 669. 670 synthesi:>·dt!p+;ndcnt strand aunl-l
TFiif, 368 TPUH. 251.366. 562 TFilS, 371

TGF-p receptors, 598 TGF-p (traosronning growth Jactor-/3}. 700 TGF-p(tnmd orming growth Iactol' /3) receptors, tht>rmod}1nam ics, fil-st l11w o f. 43 thermod)'llamics, second law of. 44 lhl!mmph iles. 115 Tllcrmus oqtKIIicuB, 353

287

SYJllhesis (S phasu), 14:2. 142

T T antigen. SV40 virus, 3!14 T n :U r~tors. 33H T loops . 90

T p hages. see uncfflroocleriophages T7 polym en~se. 360• .i60 TAF ('l'UP·85sociatro factors). 364,367 t11ndern mass speclrometry (MS/MS}. 676-77 hu·gei DNA. 315 target genes. 6 19 target immunity. 331-34

target signal d uplication. 313 target site choices. 327 target site-primed rovcrnc tiCIIl.Scriplion. 322- 26, :126 TAT prot1:in. 563, 567 f t\TA-btnd ing proteins (TllPs). 85

th iogalactoside tr-~usacetylase, 488 30-nm fibf:rs. 16 1 - 6 2.. 162. 1.63 thornx. 594 . 620. 625. 625-26 lhmonine (Thr. 'f). 73 thymidine kinas.: (TK} gene. 708 thymirw.. t OO, 100. 101.415 thymine dimers, 2 45. 24 5 1iton gene, 379 lmRNA. 5 45. 545 Tn5,600

Tn10 antisense regulation of, 33() characterization , 327, 327- 20 DNA replicatio n and, 329-:m l r'~nspositioo, 33l

Tn3 resolvase , 297 Tn7 fl1!no;poson, 316 Tns.l\ p.r(ltein. 316 Tm;B prol~ill. 318 Tndd . Ale.Ktlnd er. 22

'I'A'fA-boxes. 85, 86 I'ATA c lenwJtiS, 363-64

Toll recflplolli, 591

TA'f-~Ft .

t(•pcisornerases

371

Tatum. l:Dward, l U tautomerizalion, 235 ta utomers, 101.

TBP-DNA oomp lex, 366 TEIP lTATA-binding protein), 364. 300-67 Tc1/m ariner elements, 335- 35 Td transcription factor, 598 telom ernses characteristics, 230-32 end replication problem and . 232 recruitlnent of. 140 in tP.IomCil! replication, 231

tdomeres

(:flanging linking numbers, t 16 DNA cleavage, l19 LlNA relaxation. 118- 20 function. 117-18 function at replicatioo. fod<s, 199. 199-200 relaxation ofsuperooiled DNA, 115 - 16 at replication forks, 198, 198- 99 Topon, 163 Topo U hindi ng, 228 type L 116. 1 16 type 11. 116, 118 topoisomers. 120. 120, 121 to pology. DNA. 111-22

during ceil d i vi.c;ion. 139. ug-4()

toroid, 113

composition. 230 gene sileocing. 556. 557

l o hpotf>flf Ollis, 500

tro repressor. 565

tnms-splidng. 383. 383 transcriptic:m

a bortive initiation,

:lsa- sa

occumcy, 347 bacterial, 353-6.1

baclariophage ..\, 514 c ontro l of H,cgenBS, 4tlR-89 in eukal'}•otes. 363- 76 initiation in bar.teria, 488-- 504 mech~nisrn of. 347-77 modes of repression. 607 nucleotide sequences a nd, 34

phases, 351 regulation, 48:'! - 87 repressus. 549 - 5t RNAs ir! regulation. 567- 70 lt>rmination , 361 - 63, 362 transfer of informauon via 31 tr.mscription-c()(Jpled repair, 251 . 253 transcriptioo in.itiatioo, 538. 567-67 traru>Criptional elongation. 3 50 , 351, 352,562-63 transcriptional silencing, 244. 54 2 lranscriptional lermiimtion. 5 07 transduclion generalized. tx!8-B!l, 689 spf1Ciali7.ed . 686 I.Iansestnritlcation. :na, 381 transfer RNA (!RNA), 123. 415- 16 amino acids altacbmcnl. 417-23 dtargf!tl. 41 7, 4lll. 418 - 19. 422- 23 cod011-anticod on pairing, 462 intt-agenfc suppression, 477- 74 isoaa.cphng. 41!)-2:0 modified nud eosirles. 415 ribosomal d iscrimination, 422-2:! ribosome bindings sites for. 420-30, 430. 431 t 6S, 511 seoondary s tructures, 416, 4 16-17, 417 strudure, 417. 420 tnmslation and. 4 11 translocation. 444 -46 ttinudeoticle codon binding, 468 uncharged, 417, 4 22-23 lronsferascs. 62 tramformataons bacl(>tial virulence and. 20. 20. 21 DN.A-mel\iatoo. 689 P-el00li'n1, 703. 705 \lflcloc DNA inlrorluction, 655-56 lrau~ic models. 70 3-5. 707, 707- 9, 7(J8 trnnsiti(lfll>, 236, 236 tr.ms lation. 41 1 - 59

antibiotics and. 45-J descriptiOD, 411 P.longation. 440- 48

CTP-binding prote ins in , 447 i nitN!fion. 4 32-40. 433 ro.RNA Qability and, 4 52-57 nucleotide seque~ and. 34 overview, 427 p.rocein st:-~bilil}' and. 452 -57 puromycin and, 454 start a nd s top signals, 38 f>\eps. 441 lennimltion, 448-52 transfer of informa tion via, 31 transhltion initiatior• factors (IFs}. 433-35, 438

translatiunul wupling. 414 translesion ONA synthesis, :147, 254- 57,255 tcans lP.SiOJl polymera!lt4. 247, 255 b:enslcx:atioos, 440,444 - 46 trnnspusable elemanls. 1:-rB. :i10, 311 - 12. 329 tnmsposase genes. 312- 13 transposases, 321-22, 322, 3 30 . see nl~ inlegtaNI tra nsposition target im munity, 327, :i33 --34 ltansposilions. 1:ul, 293, 310-- 2 f; transposon Tn3 reoolvsse, 297 transposons description, 259 fundion, 310- 11 in gertomtc occurrence, :n 2

ot:

in5erlion 236 TS4 famHy. 327 - 29 lor.Z fosim\S mediated by. regulation by. 327 uses, 600- 00 transpososomes. 314- 16 l.rausvcrsions, 236. 236

rmu

lri-snRNP partid es. 3e5 trilobites. 630 1ri11udeofide-ribosomf! coruplexes. 468 trip81·htc lel:lders, 3m! IJ'iple repeats, 237 tritium (lHt. 36 tRNAI'tle, 465

trombone morlnl. 200-9 lroponin 1'. 394 lrp gene. 413. 505. 507

trp Op. W), '13

Tscbcnnak. Erich, 6 tubulin, 583 lurnor-suppressa- genes, 691) Tupt protein. 551 turns, 76 ! Wist. 112 h~iSI gene, 595 twist numbers, 114

1\visl protei n . 597 1\vo hybrid assay, 533-34 'I)• d ements, 335-36. 336

tyrosine rocombinases. 2~- 97. 297.299. 299-Jf lJTOsine {1'yr, Y), 48, 73 . 422 rvrosyl tRNA synther35f',. 422

u U i11sertion , 406 U2.AF (U2 auxiUary factc•r), 384 U:A:U bast' trip~. 124

Ubxgenes iu uustacellns. 630-31 embryo m 01phology arld, 626 {ruil fly morpbology and. 624-26, 625 'l.Jbx J•rotein binding sites, 63() l'mbryo morphology and . 627 evolutionary cllanget,;, 632, 632- 35 target mhancers. 627-30 Ubx rnpressor. 635 UltrabitlJOIWC (l'bx} gene. 627 ul~cenlrifugation, 425 , 425

ultreviolellight, 109- 10,245

fncfex

UmuCptotein, 254, 2.'i5 UmuD protein . 254 unfoldir•g. 79. sa> also door~turet ioo 3'unfrc~nsl~ted regioo (:S'UTR). 576

vORFs (upstr~lll open-rl.oadmg l'rcm"K$). 435. 4:!9, 568. 567 ur-etfl11lf!tlts, ~54 uracil, 20, 1 Z2- 2J until gi}ICOSylase IWI(:tion, 249

uridine, 415 UvrA protf'.ins, 251 UvrB proteins, 2S1

White, John. 696

wi ld-type J;QI'IP.S. 10 Wilkins, Moun t.A. '2.2 wings, illsecl, 6l2-J5, 635 Wnts proteins. 598 wol;ble concept. 463, 463 work. mJ~ tlllt'fgy ;~~nd. 44 Wrif,hl, 5(:waU, 1!). 1 7 writhe. 112, 115 writh ing numbers. t 13

X

l JVrC lJJ'(J(ei llll, 25 I

lfVTO heJicase, 238 UvrD prot ems, 25 l UvsX proh1f11, 27!'

v V3 Clells, 588

X-ray crystallography. 75 X-mys. 16. 245 Xer I'BCombinases, 307 XflrC, 297, l07 - 8 XE'ffl. 297. 307-B xerodfll-n\C-t pi8)1lentosu tn :Z:5 1 Xis bimling. 304

Xnr gene, 500

V1 interneuronfl, 58U V2 int" roourons, 589 valr.nct>. detination. 42 vali ne (Vat V). 73, 42Z van der Waals hooding. 46 van der Waals ~ 42 acetate. 46 tJjslan03 and. 4 6 glycine, 46 guanine, 46 weel.. lwmts. 45 van der WMis rnr1ii, 46. 4 7

XP (xf:foderma plnmentosum a genfiB. 2 XP<..:. f unction , 251 Xrs2 protein. 283

y Y~di'C 1\he~le.

215

\' famtly DNA polymerd:ies, 256 Yan or~>ky. OlBJ'IAs. 36, 692

VBnd, \nad1ntir.22 V{OIJ reoombi nati.oo. 3 11 , 338- 41 . 339, 340 \ltldon>, deflnilioo, 654 VegT ~e. 59R. 5.99 ventrcJ, defirullon. !'>76 V81 gene. 599 viral 6t1ppresSOI'S of se"" silt"ncinR(VSG&). 57U Vlrofds. t25 vil:Ust!S. genes, 21 VISTA softw are. 66S

yeasts chromosome maps. 289 con.">t~cd nlP.Chanisms. 531-37 fLP functton, 297

t :cn4 activator. 565-67. 566 gene regulalacy clements, 530 gene silenctns. 556-58 YPWM motiF. 623, 6.''10

z Z DNA. 106. 10 7, 107 - B, 108 ZacantholdCB, 62Q

w Wt~llace, A lfi·ed R.• 5 water tnOiecules, 4!j,

Walson. ' anletl n, 22 Wmd a us. Erit . 594

49. 49

Zea 17l0J'S. soe «:on1 I ZOO rnoys} 7jg~ mod el , 162 7Jnc. clustur domnins, 535 zinc-containing DNA-binding dom<1ins t.inc f'inAor DNA-binrling moh fe, tiS, sa zi..n\. fingers. 535. 535

a

1\l:breviatioos for hni.no Acids

Alanine

pJa

A

.ArQk'ine

/J.tg Asrt

R

Asx

N 0 B

Cysteine Glutamine

cvs

c

Gin

Gluurnic acid

GkJ

Q E

Gu!:amlne OJ glutamic acid Glycine

Glx

z

Gfy

G

H stidine

His lte

H I l

Asparagine Aspartic acid Asparagine or aspartic acid

2C A(2mt)

b

12A

(1 .2nm,

lsdetscine

22A (2.2nm)

l eucil'le Lysine Mettlionine Phenylalanine Proline Sefine ThrE!Of1ft1€ TryptOphan Tyrosine

Valine

(a}

SdlernaLic mx1el ot the ib.Jble heliX.

(bl

~fillift;J

:JK

rro1el of the cblbl e he-

h.p

leu lys Met

Phe

K M f

Pro

p

Sef Thr Trp

s

Tyr

y

Val

v

T

w

t

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