Chromatography and Modification of Nucleosides, Part B: Biological Roles and Function of Modification (Journal of Chromatography Library)

JOURNAL OF CHROMATOGRAPHY LIBRARY - volume 45B

chromatography and modification of nucleosides part B: biological roles and function of modification

Chromatography and Modification of Nucleosides edited by C. W. Gehrke and K. C. T. Kuo Part A: Analytical Methods for Major and Modified Nucleosides HPLC, GC, MS, NMR, W a n d FT-IR Part 6: Biological Roles and Function of Modification Part C: Modified Nucleosides in Cancer and Normal Metabolism Methods and Applications Part D: Comprehensive Database for RNA and DNA Nucleosides Chemical, Biochemical, Physical, Spectral and Sequence

JOURNAL OF CHROMATOGRAPHY LIBRARY - volume 45B

chromatography and modification of nucleosides part B: biological roles and function of modification

edited by

Charles W. Gehrke and Kenneth C. T. Kuo Department of Biochemistry, University of Missouri-Columbia, and Cancer Research Center, P. 0. Box 1268, Columbia, MO 65205- 1268, U.S.A.

ELSEVIER Amsterdam

- Oxford - New York -Tokyo

1990

ELSEVIER SCIENCE PUBLISHERS B.V. Sara Burgerhartstraat25 P.O. Box 21 1, 1000 AE Amsterdam, The Netherlands Distributors for the United States and Canada: ELSEVIER SCIENCE PUBLISHING COMPANY INC. 655 Avenue of the Americas New York, NY 10010, U.S.A.

ISBN 0-444-88505-6

0 Elsevier Science Publishers B.V., 1990 All rights reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without the prior written permission of the publisher, Elsevier Science Publishers B.V./ Physical Sciences & EngineeringDivision, P.O. Box 330, 1000 AH Amsterdam, The Netherlands. Special regulations for readers in the U.S.A. -This publication has been registered with the Copyright Clearance Center Inc. (CCC), Salem, Massachusetts. Information can be obtained from the CCC about conditions under which photocopies of parts of this publication may be made in the USA. All other copyright questions, including photocopying outside of the USA, should be referred to the publisher. No responsibility is assumed by the Publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, the Publisher recommends that independent verification of diagnoses and drug dosages should be made. Although all advertising material is expected to conform to ethical (medical) standards, inclusion in this publication does not constitute a guarantee or endorsement of the quality or value of such product or of the claims made of it by its manufacturer. This book is printed on acid-free paper. Printed in The Netherlands

V

TABLE OF CONTENTS Preface

VII

A Dedication and Thanks

XI I

Special Acknowledgement to Dr. Robert W. Zumwalt -

XI I

Editors

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Contributors.

. XIX

Chromatography and Modification of Nucleosides: Parts A and C Introduction and Overview Chapter 1

Chapter 2

Chapter 3

Chapter 4

Chapter 5

Contents of

Dieter G. 5611.

-XLI I I B1

Synthesis and Function of Modified Nucleosides in tRNA Glenn R. Bj6rk and Jiirg Kohli

B13

Biosynthesis and Function of Queuine and Queuosine. Helga Kersten and Walter Kersten

669

Codon Usage and Q-Base Modification in Drosophila melanogaster. E. Kubli Solid Phase lmmunoassay for Determining the lnosine Content in Transfer RNA Edith F. Yamasaki, Altaf A. Wani and Ronald W. Trewyn Site Directed Replacement of Nucleotides in the Anticodon Loop of tRNA: Application to the Study of lnosine Biosynthesis in Yeast tRNA*la Keith A. Kretz, Ronald W. Trewyn, GCrard Keith and Henri Grosjean

B109

B125

B143

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Chapter 6

tRNA and tRNA-Like Molecules: Structural Peculiarities and Biological Recognition- - - - - - - - - B173 Rajiv L. Joshi and Anne-Lise Haenni

Chapter 7

Mitochondria1 tRNAs-Structure, Modified Nucleosides and Codon Reading Patterns - - - - - - - - - B197 Guy Dirheimer and Robert P. Martin

Chapter 8

The Modified Nucleotides in Ribosomal RNA from Man and Other Eukaryotes - - - - - - - - - - - - - - - - - 8265 B. E. H. Maden

Chapter 9

Modified Uridines in the First Positions of Anticodons of tRNAs and Mechanisms of ---- --Codon Recognition Shigeyuki Yokoyama and Tatsuo Miyazawa

_______ ____

Chapter 10 Subject

__

_____

Natural Occurring Modified Nucleosides in DNA Melanie Ehrlich and Xian-Yang Zhang

Index

8303

- - B327

______________-__--_____________________-------B363

Journal of Chromatography Library (volumes in the

series)

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8367

VII

Preface The central roles of modifications in nucleic acids in protein synthesis and control of biological functions have evoked intense and continued investigations on the conformation of the RNA and DNA macromolecules by scientists representing a wide spectrum of disciplines. Professors Glenn Bjork and Jurg Kohli of the Universities of Umea and Bern, respectively, in Chapter 1, present in-depth the synthesis and function of modified nucleosides in tRNAs. They describe the presence of modified nucleosides in tRNAs from different organisms, Eubacteria, Archaebacteria and Eucaryotes, and address the role of transfer RNA modifying enzymes, modified nucleosides next to the 3' side of the anticodon, at the wobble position 34, in the anticodon region and outside of the anticodon region on stabilization of tRNA conformation. Knowledge about the synthesis and function of modified nucleosides will have a strong impact on cell physiology and will contribute importantly to our understanding of the function and metabolism of tRNA modification, as well as a strong impact on applied gene technology and the production of proteins of medical importance. Professors Helga and Walter Kersten from the University of Erlangen-Niirnberg present an excellent review of their research on the biosynthesis and function of queuine and queuosine-containing tRNAs. They give a systematic analysis of prokaryotic and eukaryotic tRNAs of the Q-family from bacteria, protists, plants, fishes, mouse cells, and human lymphomas, showing causes and consequences of variations in the Q vs G34 content of respective tRNAs, and relate how the presence or absence of Q in tRNAs can cause alterations in the expression of genes encoding LDH isoenzymes and cytochromes. Eric Kubli of the Zoological Institute at the University of Zurich states that codon usage is nonrandom, genome specific, and within a species. He discusses codon usage and Q-base modification in highly and weakly expressed genes in Drosophila melanogasfer. He

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has compiled a set of codon usage tables for the unicellular organisms E. coli and yeast, and for vertebrates, and evidence is presented that queuosine seems to be one of those small molecules with many roles, possibly integrating a variety of different functions within a cell. Information is presented on Q-base modification, protein synthesis, and development. In Chapter 4, Ronald Trewyn and co-workers of Ohio State University present solid phase immunoassay techniques for measuring the inosine content in tRNAs, and in Chapter 5, Keith Kretz and colleagues discuss site-directed replacement of nucleotides in the loop of tRNA with application to the study of inosine biosynthesis in yeast tRNA. Dr. Rajiv Joshi and Professor Anne-Lise Haenni of the lnstitut Jacques Monod, Paris, follow with an excellent presentation on tRNAs in translation as molecules that fulfill an amino acid donor function in mRNA-dependent ribosomal protein synthesis; tRNA-like RNA molecules that resemble tRNAs but cannot act as a tRNA and possess a different specialized function; they also designate as tRNA-like structures domains in nucleic acids that have certain recognitory properties; and finally tRNA-like features those structural elements in nucleic acids that are related to tRNAs. Professor Guy Dirheimer and Robert Martin of the lnstitut de Biologie Moleculaire et Cellulaire of Strasbourg review in Chapter 7 the structure and codon recognition patterns of mitochondria1 tRNAs (mt tRNAs). Research in molecular biology of mitochondria during the past decade has provided a number of unexpected results about the way in which the mitochondria1 DNA (mt DNA) is expressed. These authors report that these results have contributed novel insights into gene expression mechanisms (e.g. intron splicing, codon recognition, genetic code). Nearly 300 mt tRNA structures from organisms of different phylogenetic origins have been determined either directly by RNA sequencing or indirectly by gene sequencing. However, the gene sequence alone may not be sufficient to deduce the specificity of a given tRNA because post-transcriptional base modification may influence the codon recognition pattern and because the mitochondrial genetic code differs from the standard

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code. Drs. Dirheimer and Martin have elegantly reviewed the present state of mt RNAs and mt DNAs. Information is presented on isolation of mt tRNAs from yeasts, molds, protozoa, mosquito, D r o s o p h i l a , Xenopus laevis, mammals, and plants. Structural information, modified nucleosides, and codon reading patterns are presented. Mt tRNAs, as a group, have unusual and highly variable structures. Almost any given mt tRNA has one or more odd features, including base changes. These changes affect residues implicated in secondary or tertiary interactions. This chapter provides a comprehensive wealth of information on mt tRNAs and should be considered as the first reading of investigators planning investigations in this area of molecular biology. The thrust of Professor Maden's chapter on the modified nucleotides in ribosomal RNA (rRNA) of man and other eukaryotes is based on how the findings from successive experimental approaches have contributed to the unfolding of knowledge of the modified nucleotides in rRNA from man and other eukaryotes and thus led to concluding major generalizations. He concludes that all of the methylations and perhaps all other modifications occur in the rRNA sequences of the precursor molecule, and most of them occur in regions where primary structure is highly conserved among eukaryotes. Further, the diversity of sequences which are 2',-0methylated suggests that the specificity for this type of methylations is determined by conformation. Ribosomes in man and other vertebrates contain >200 modifications. The exact locations of all the methyl groups in the primary structure of 18s rRNA from Xenopus laevis and man have been determined. The methyl groups are widely but non-uniformly distributed and occur in conserved sequence regions, some of which are in complex tertiary structures. Professors Yokoyama and Miyazawa of the University of Tokyo elegantly present the interrelationships of modified uridines in the first positions of anticodons of tRNAs and mechanisms of codon recognition. In protein biosynthesis, some tRNA species strictly recognize only one codon by Watson-Crick base pairs, while other tRNAs recognize multiple codons. They present the general concept of the regulation of the rigidity/flexibility of the first letter of an

A

anticodon by post-transcriptional modification of tRNA, and that this will be most useful for understanding the molecular mechanisms of the regulation of codon recognition and allows the efficient and correct translations of codons in protein synthesis. Completion of the mapping of the modified nucleosides in human and other eukaryote sources is the outstanding problem in the determination of the primary sequence. Attainment of this goal will be a major step toward understanding the highly specific recognition process involved in the modifications, their biological role in ribosome biosynthesis and function. An excellent review article is presented in Chapter 10 by Professor Melanie Ehrlich and Dr. Zhang of Tulane University on naturally occurring modified nucleosides in DNA. The functional significance of DNA methylation--transcription, chromatin structure, DNA replication and repair-and cancer a n d embroygenesis are thoroughly discussed. Dr. Ehrlich gives the latest information on highly modified bacteriophage DNA, the modified bases in DNA from bacteria and lower eucaryotes, methylation of DNA in mitochondria, chloroplasts, and viruses. Most types of DNA, in addition to the four bases, contain one to three additional bases as modified forms of Ade or Cyt. Usually these modified bases are found in minor amounts with exceptions for bacteriophage and dinoflagellates. The only modified bases in DNA from all other sources are m5C, m4C, and m6A. It is considered that the functions of these DNA modifications are diverse and range from protection of bacterial host DNA against a pathway for degrading foreign DNA to control of prokaryotic and eucaryotic transcriptional activity as well as DNA repair. Most DNAs contain minor amounts of m5C, m4C, and rn6A. Any one of these can be found in most bacterial DNAs, whereas in all studied vertebrates and higher plants, m5C, and only m5C, is the naturally occurring modified base. Further, the levels and patterns of m5C in vertebrate genomes are tissue-specific. Bacterial DNA methylation is often involved in restrictionmodification systems. However, there is no evidence for this function in the case of vertebrate DNA methylation. Since 1981, Professor Gehrke and Mr. Kuo have collaborated on many research

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projects with Dr. Ehrlich on DNA methylation. We have used our advanced quantitative chromatographic protocols to measure the modified bases in DNA from many sources. This collaboration has resulted in many publications cited in this chapter and in other scientific journals. (See refs. 3, 33, 50, 61, 95, 146, 150, 151, 155, 220 in Chapter 10.) Columbia, Missouri 1989

Charles W. Gehrke Kenneth C.T. Kuo

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A DEDICATION AND THANKS

This volume i s dedicated t o t h e e f f o r t s o f t h o s e r e s e a r c h e r s across the world who a r e searching f o r a better understanding of r o l e s and functions of modified nucleosides found i n a l l n u c l e i c a c i d s , the complexities and f i d e l i t y of p r o t e i n synthesis, conformational e f f e c t s , codon-anticodon i n t e r a c t i o n s , and t h e development of new research t o o l s t o probe the b i o l o g i c a l s i g n i f i c a n c e o f these important macromolecules. Charles W . Gehrke Kenneth C . Kuo

SPECIAL ACKNOWLEDGEMENT TO DR. ROBERT W. ZUMWALT

Dr. Robert W . Zumwalt, Research Associate and Analytical Biochemist i n our research group over the p a s t 20 y e a r s i n t h e Department of Biochemistry, University of Missouri-Columbia, and t h e Cancer Research Center, has been an u n t i r i n g resource and consul t a n t i n b r i n g i n g this four-volume t r e a t i s e , C h r o m a t o g r a p h y and Dr. Zumwalt has Modification o f Nucleosides, to a finality. placed his t a l e n t s o f g r e a t technical d e t a i l , knowledge of chromatographics, and patience i n achieving completion of these works. Dr. Zumwalt was a c e n t r a l a u t h o r / e d i t o r , w i t h Kenneth Kuo and Charles Gehrke, i n our f i r s t three-volume book e n t i t l e d Amino A c i d A n a l y s i s b y G a s C h r o m a t o g r a p h y , p u b l i s h e d by CRC Press i n 1987. Those volumes contain two major chapters on t h e search f o r amino a c i d s i n l u n a r s o i l and cosmo chemistry by Gehrke, Kuo, Zumwalt, and Ponnamperuma. The e d i t o r s extend t h e i r deep a p p r e c i a t i o n t o Dr. Zumwalt f o r his technical a b i l i t i e s and e d i t o r i a l e x p e r t i s e i n accomplishing t h e completion of t h i s t r e a t i s e .

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EDI TORS

The e d i t o r s , Kenneth C. Kuo and C h a r l e s W. Gehrke, a t t h e Wood1and and F1o r a l Garden, U n i v e r s i t y o f M i s s o u r i , Col umbi a, Missouri

.

CHARLES W. GEHRKE C h a r l e s W i l l i a m Gehrke was b o r n i n 1917 i n New York City. He s t u d i e d a t Ohio S t a t e U n i v e r s i t y , r e c e i v i n g a B . A . i n 1939. From 1941 t o 1945, he was p r o f e s s o r and chairman o f t h e Department o f Chemistry a t M i s s o u r i Val l e y C o l l e g e , M a r s h a l 1 , M i s s o u r i , t e a c h i n g c h e m i s t r y and p h y s i c s t o s e l e c t e d Navy midshipmen from d e s t r o y e r s , b a t t l e s h i p s and a i r c r a f t c a r r i e r s o f World War I 1 i n t h e South P a c i f i c .

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These young men r e t u r n e d t o t h e war t h e a t e r as deck and f l i g h t officers. I n 1946, he r e t u r n e d t o O h i o S t a t e U n i v e r s i t y as i n s t r u c t o r in a g r i c u l t u r a l b i ochemi s t r y and r e c e i v e d h i s Ph.D. d e g r e e i n 1947. I n 1949 he j o i n e d t h e Col 1 ege o f A g r i c u l t u r e a t t h e Uni v e r s i t y o f M i s s o u r i Columbia, r e t i r i n g i n t h e F a l l o f 1987 f r o m p o s i t i o n s as P r o f e s s o r o f B i o c h e m i s t r y , Manager o f t h e E x p e r i m e n t S t a t i o n C h e m i c a l L a b o r a t o r i e s , and D i r e c t o r o f t h e U n i v e r s i t y I n t e r d i s c i p l i n a r y Chromatography Mass-Spectrometry f a c i l i t y . H i s d u t i e s a l s o i n c l u d e d t h o s e o f S t a t e Chemist f o r M i s s o u r i D r . Gehrke i s now F e r t i l i z e r and L i m e s t o n e C o n t r o l l a w s . S c i e n t i f i c C o o r d i n a t o r a t t h e Cancer Research C e n t e r i n Col umbi a. P r o f e s s o r Gehrke i s t h e a u t h o r o f o v e r 250 s c i e n t i f i c p u b l i c a t i o n s i n a n a l y t i c a l and b i o c h e m i s t r y . H i s research i n t e r e s t s i n c l u d e t h e development o f q u a n t i t a t i v e , h i g h r e s o l u t i o n gas- and 1 i q u i d - c h r o m a t o g r a p h i c methods f o r amino a c i d s , p u r i n e s , p y r i m i d i n e s , m a j o r and m o d i f i e d n u c l e o s i d e s i n RNA, DNA, and m e t h y l a t e d " C A P " s t r u c t u r e s i n mRNA; f a t t y a c i d s ; and b i o l o g i c a l m a r k e r s i n t h e d e t e c t i o n o f c a n c e r ; c h a r a c t e r i z a t i o n and i n t e r a c t i o n o f p r o t e i n s , c h r o m a t o g r a p h y o f b i o l o g i c a l l y i m p o r t a n t molecules, s t r u c t u r a l charact e r i z a t i o n o f carcinogen-RNA/DNA a d d u c t s , and a u t o m a t i o n o f a n a l y t i c a l methods f o r n i t r o g e n , phosphorus, and p o t a s s i u m i n f e r t i l i z e r s . Automated s p e c t r o p h o t o m e t r i c methods have been d e v e l o p e d f o r l y s i n e , m e t h i o n i n e , and c y s t i n e . P r o f e s s o r Gehrke has been an i n v i t e d s c i e n t i s t t o l e c t u r e on g a s - l i q u i d chromatography o f amino a c i d s i n Japan, China, and a t many u n i v e r s i t i e s and i n s t i t u t e s i n t h e U n i t e d S t a t e s and Europe. He p a r t i c i p a t e d i n t h e a n a l y s i s o f l u n a r samples r e t u r n e d b y A p o l l o f l i g h t s 11, 12, 14, 15, 16, and 17 f o r amino a c i d s and e x t r a c t a b l e o r g a n i c compounds w i t h P r o f e s s o r Cyri 1 Ponnamperuma, U n i v e r s i t y o f Mary1 and, and w i t h a consortium o f s c i e n t i s t s a t t h e National Aeronautics and Space A d m i n i s t r a t i o n Ames Research C e n t e r , C a l i f o r n i a . I n 1971, he r e c e i v e d t h e annual A s s o c i a t i o n o f O f f i c i a l A n a l y t i c a l C h e m i s t s ' (AOAC) Harvey W. W i l e y Award i n A n a l y t i c a l C h e m i s t r y and was r e c i p i e n t o f t h e S e n i o r F a c u l t y Mem-

xv b e r Award, UMC C o l l e g e o f A g r i c u l t u r e , i n 1973. I n August, 1974, he was i n v i t e d t o t h e S o v i e t Academy o f Sciences t o make a summary p r e s e n t a t i o n on o r g a n i c substances i n l u n a r f i n e s t o t h e Oparin I n t e r n a t i o n a l Symposium on t h e " O r i g i n o f Life." I n 1975, he was s e l e c t e d as a member o f t h e American Chemi c a l S o c i e t y C h a r t e r Review Board f o r Chemical A b s t r a c t s . As an i n v i t e d teacher under t h e sponsorship o f f i v e C e n t r a l American governments, he t a u g h t chromatographic a n a l y s i s o f amino a c i d s a t t h e C e n t r a l American Research I n s t i t u t e f o r I n d u s t r y i n Guatemala, 1975. He was e l e c t e d t o Who's Who i n M i s s o u r i Education and r e c i p i e n t o f t h e Faculty-Alumni Gold Medal Award i n 1975, and was t h e r e c i p i e n t o f t h e p r e s t i g i o u s Kenneth A. Spencer Award from t h e Kansas C i t y S e c t i o n o f t h e American Chemical S o c i e t y f o r m e r i t o r i o u s achievement i n a g r i c u l t u r a l and food chemistry, 1979-80. P r o f e s s o r Gehrke r e c e i v e d t h e T s w e t t "Chromatography Memorial Medal " from t h e S c i e n t i f i c Counci 1 on Chromatography, Academy o f Sciences o f t h e USSR, Moscow, 1978, and t h e Sigma X i Senior Research Award by t h e U n i v e r s i t y o f Missouri-Columbia Chapter, 1980. I n 1986, he was t h e r e c i p i e n t o f t h e American Chemical S o c i e t y Midwest Award. He was an in v i t e d speaker on "Modi f i ed Nucl e o s i des and Cancer" i n F r e i b u r g , West Germany, 1982, and gave p r e s e n t a t i o n s as an i n v i t e d s c i e n t i s t throughout Japan, mainland China, Taiwan, P h i l i p p i n e s , and Hong Kong i n 1982 and 1987. He was s e l e c t e d f o r t h e Board o f D i r e c t o r s and E d i t o r i a l Board o f t h e AOAC, 1979-80; President-Elect o f the Association o f O f f i c i a l A n a l y t i c a l Chemists I n t e r n a t i o n a l O r g a n i z a t i o n , 1982-83; and was honored by t h e e l e c t i o n as t h e Centennial P r e s i d e n t i n 1983-84. He developed " L i b r a r i e s o f I n s t r u m e n t s " i n t e r d i s c i p l i n a r y research programs on s t r e n g t h e n i n g r e s e a r c h i n American U n i v e r s i t i e s . D r . Gehrke i s founder and chairman o f t h e Board o f D i r e c t o r s , A n a l y t i c a l B i o c h e m i s t r y L a b o r a t o r i e s , I n c . , 1968 t o p r e s e n t , a p r i v a t e c o r p o r a t i o n o f 200 s c i e n t i s t s , engineers, b i o l o g i s t s , and chemists s p e c i a l i z i n g i n chromatographic i n s t r u m e n t a t i o n , and addressing problems worldwide in t h e e n v i ronment.

XVI

Over s i x t y m a s t e r s and d o c t o r a l s t u d e n t s have r e c e i v e d t h e i r advanced degrees i n a n a l y t i c a l b i o c h e m i s t r y u n d e r t h e d i r e c t i o n o f P r o f e s s o r Gehrke. I n addition t o h i s extensive c o n t r i b u t i o n s t o amino a c i d a n a l y s i s b y gas chromatography, D r . Gehrke and c o l l e a g u e s have p i o n e e r e d i n t h e development o f s e n s i t i v e , high-resolution, q u a n t i t a t i v e high-performance l i q u i d c h r o m a t o g r a p h i c methods f o r o v e r 100 m a j o r and m o d i f i e d n u c l e o s i d e s i n RNA, DNA, mRNA, and t h e n a p p l i e d t h e i r methods i n c o l l a b o r a t i v e r e s e a r c h w i t h s c i e n t i s t s i n molecular b i o l o g y across t h e world. P r o f e s s o r E r n e s t Borek a t t h e 1982 I n t e r n a t i o n a l Symposium on Cancer M a r k e r s , Freiburg, West Germany, s t a t e d t h a t P r o f e s s o r G e h r k e ' s c h r o m a t o g r a p h i c methods a r e b e i n g used s u c c e s s f u l ly b y more than h a l f o f t h e s c i e n t i s t s i n attendance a t these meetings. P r o f e s s o r Gehrke, w i t h D r . R o b e r t Zumwalt and M r . Kenneth Kuo, i s t h e s e n i o r a u t h o r / e d i t o r o f a t h r e e - v o l ume comprehensive t r e a t i s e e n t i t l e d "Amino A c i d A n a l y s i s by Gas Chromatography, " pub1 ished by CRC P r e s s ( 1 9 8 7 ) . The v o l umes i n c l u d e 19 c h a p t e r s c o n t r i b u t e d b y l e a d i n g s c i e n t i s t s f r o m twelve nations. I n 1989, P r o f e s s o r Gehrke and P r o f e s s o r C y r i l Ponnamperuma o f t h e U n i v e r s i t y o f M a r y l a n d were named cop r i n c i p a l i n v e s t i g a t o r s on a p r o p o s a l t o p l a c e on t h e moon a c h e m i c a l l a b o r a t o r y w h i c h w i l l be automated, m i n i a t u r i z e d , computer r o b o t i c - o p e r a t e d and w i 11 s u p p o r t NASA programs i n t h e s t u d y o f f i v e a s p e c t s o f t h e e x p l o r a t i o n o f space; (a) a s t r o n a u t h e a l t h , (b) c l o s e d e n v i r o n m e n t l i f e s u p p o r t , ( c ) l u n a r r e s o u r c e s , (d) e x o b i o l o g y , and ( e ) p l a n e t o l o g y . I n 1989, P r o f e s s o r Gehrke and Kenneth Kuo a r e a u t h o r s / e d i t o r s o f t h i s f o u r - v o l ume t r e a t i s e e n t i t l e d "Chromatography and M o d i f i c a t i o n o f N u c l e o s i d e s , " p u b l i s h e d b y E l s e v i e r i n t h e J o u r n a l o f Chromatography L i b r a r y s e r i e s . These t h r e e volumes address " A n a l y t i c a l Methods f o r M a j o r and M o d i f i e d Nucleosides", " B i o c h e m i c a l R o l e s and F u n c t i o n o f M o d i f i c a t i o n " , " M o d i f i e d N u c l e o s i d e s i n Cancer and Normal Metabol i s m " , and "Comprehensive Database f o r RNA and DNA Nucl e o s i d e s " .

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KENNETH C. T. KUO Kenneth C. T. Kuo was born i n 1936 i n China. He s t u d i e d a t Chun-Yen I n s t i t u t e o f Science and Engineering, Taiwan, r e c e i v i n g a B . S . degree i n Chemical E n g i n e e r i n g i n 1960. A f t e r f u l f i l l i n g a m i l i t a r y s e r v i c e o b l i g a t i o n , he e n r o l l e d a t t h e U n i v e r s i t y o f Houston. I n 1963, he j o i n e d t h e Chevron Chemical Company i n Richmond, C a l i f o r n i a , d e v e l o p i n g p e s t i c i d e r e s i d u e a n a l y t i c a l methods and s t u d y i n g p e s t i c i d e metabolism. Recognizing t h e power o f g a s - l i q u i d chromatography (GLC) and t h e need o f h i g h r e s o l u t i o n , s e n s i t i v i t y , and speed i n t h e a n a l y s i s o f amino a c i d s , he a p p l i e d and was accepted as a member o f t h e research team under P r o f e s s o r Char1 es Gehrke a t t h e U n i v e r s i t y o f M i s s o u r i -Col umbi a in 1968. He developed mixed phase columns f o r h i s t i d i n e , a r g i n i n e , and c y s t i n e , which a l l o w t h e dual column complete q u a n t i t a t i o n o f p r o t e i n amino a c i d s i n 30 minutes by GC. He, a l o n g w i t h Drs. Gehrke, S t a l l i n g , and Zumwalt, i n v e n t e d t h e Patent No. S o l v e n t - V e n t C h r o m a t o g r a p h i c System ( U . S . 3,881,892), which e l i m i n a t e s t h e sample s o l v e n t e f f e c t i n GC a n a l y s i s . T h i s s o l v e n t - v e n t i n g d e v i c e was used i n t h e search f o r amino a c i d s i n t h e r e t u r n e d A p o l l o l u n a r samples o v e r t h e p e r i o d from 1969-1974, t h u s p r o v i d i n g a s e n s i t i v i t y f a c t o r o f 100 g r e a t e r t h a n c l a s s i c a l ion-exchange a n a l y s i s a t t h a t time. He r e c e i v e d h i s M.S. degree i n a n a l y t i c a l b i o c h e m i s t r y under P r o f e s s o r Gehrke i n 1970. D u r i n g t h e l a s t 20 years, he and D r . Gehrke have d e d i c a t e d t h e i r research e f f o r t s t o t h e developments o f q u a n t i t a t i v e h i g h r e s o l u t i o n chromatographic methods f o r biochemical and biomedical research. He p a r t i c i p a t e d i n t h e NASA Apol l o Returned Lunar Sample c o n s o r t i u m o f s c i e n t i s t s search f o r evidence o f chemical e v o l u t i o n i n t h e l u n a r samples from A p o l l o m i s s i o n s 11 through 1 7 (1969 t o 1974). He has s t u d i e d b i o m a r k e r s f o r cancer, and developed q u a n t i t a t i v e h i g h r e s o l u t i o n chromatographic methods f o r polyamines, protein-bound n e u t r a l sugars, 8 - a m i n o i s o b u t y r i c a c i d and 8 - a l a n i n e ; and m o d i f i e d r i b o n u c l e o s i d e s i n human u r i n e and serum. I n t h e l a s t f i v e years, h i s m a j o r e f f o r t s have been d i r e c t e d t o t h e development o f a package o f methods

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f o r t h e complete q u a n t i t a t i v e composition a n a l y s i s o f DNA, mRNA, and t R N A by h i g h r e s o l u t i o n HPLC. Through these methods, more than 70 m a j o r and m o d i f i e d r i b o n u c l e o s i d e s , 15 deo ynucleosides, and 9 mRNA cap s t r u c t u r e s can be i d e n t i f i e d He was an and measured i n n u c l e i c a c i d s or body f l u i d s . inv t e d s c i e n t i s t by t h e Chinese Academy o f Science i n 1982 and l e c t u r e d throughout China on t h e chromatography o f He has c o n t r i b u t e d t o over f i f t y s c i e n t i f i c nuc eosides. Pub i c a t i o n s i n a n a l y t i c a l c h e m i s t r y and b i o c h e m i s t r y .

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CONTRIBUTORS

Glenn R. B j G r k

The research grou o f Glenn Bjbrk. The scientists a r e s t a n d i n g i n deep snow i n f r o n t o f the s c u l p t u r e "Northern L i h t " a t the campus o f the U n i v e r s i t y of Umea. From l e f t : GQenn B j C w k , Yvonne Jonsson In e l a Sanstrom, Tord Hagervall, Johanna Ericson, S i v Lundmark, 6 u n i Q l a JLger, Kerstin Jacobsson, C l a e s Gustafsson, and Makael Wi kstrom. Glenn R. Bjiirk, born 1939 i n Sweden, received h i s B.Sc. i n 1963 a t the U n i v e r s i t y of Uppsala, Uppsala, Sweden, and h i s P h . D . i n 1968 i n Biochemistry a t t h e same u n i v e r s i t y . His t h e s i s work was on t h e p u r i f i c a t i o n and c h a r a c t e r i z a t i o n o f t R N A methyl t r a n s f e r a s e s from y e a s t . He s p e n t one y e a r a t the U n i v e r s i t y of Umea

xx s t u d y i n g m i c r o b i o l o g y and i s o l a t e d t h e f i r s t b a c t e r i a l m u t a n t d e f e c t i v e i n t R N A m o d i f i c a t i o n . Between 1969 t o 1971, he was a p o s t - d o c t o r a l f e l l o w i n t h e l a b o r a t o r y o f D r . F.C. N e i d h a r d t , f i r s t a t Purdue U n i v e r s i t y , L a f a y e t t e , I n d i a n a , USA, and t h e n l a t e r a t t h e U n i v e r s i t y o f M i c h i g a n , Ann A r b o r , M i c h i g a n . D u r i n g t h i s t i m e he s t u d i e d t h e g e n e t i c s and p h y s i o l o g y o f b a c t e r i a l He r e t u r n e d t o t h e mutants d e f e c t i v e i n tRNA m o d i f i c a t i o n . Department o f M i c r o b i o l ogy, Uni v e r s i t y o f Umea, Sweden, as a Research A s s o c i a t e and c o n t i n u e d h i s work on t h e s y n t h e s i s and f u n c t i o n o f m o d i f i e d n u c l e o s i d e s i n tRNA, b u t a l s o i n i t i a t e d t h e studies o f the regulation o f the synthesis o f the tRNA modifying enzymes and t o e l u c i d a t e t h e o r g a n i z a t i o n o f t h e c o r r e s p o n d i n g genes. I n 1977 he r e c e i v e d a p o s i t i o n as A s s o c i a t e P r o f e s s o r a t t h e Department o f M i c r o b i o l o g y , U n i v e r s i t y o f Umea. I n 1980, he s p e n t a s a b b a t i c a l y e a r i n t h e l a b o r a t o r y o f D r . John Roth, Department o f B i o l o g y , U n i v e r s i t y o f Utah, S a l t Lake City, Utah, USA. The same y e a r he was a p p o i n t e d f u l l P r o f e s s o r a t t h e Department o f M i c r o b i o l o g y , U n i v e r s i t y o f Umea. S i n c e 1981 he has been c h a i r m a n o f t h a t d e p a r t m e n t . Guy D i r h e i m e r , G e r a r d K e i t h , and R o b e r t M a r t i n Guy D i r h e i m e r was b o r n i n 1931. He s t u d i e d pharmacy, b i o c h e m i s t r y , and p h y s i o l o g y a t t h e U n i v e r s i t y o f S t r a s b o u r g . I n 1955 he was engaged b y t h e C e n t r e N a t i o n a l de l a Recherche S c i e n t i f i q u e (CNRS = French Research C o u n c i l ) as " S t a g i a i r e de Recherche," w o r k i n g i n t h e l a b o r a t o r y of P r o f e s s o r Ebel on t h e b i o l o g i c a l r o l e o f i n o r g a n i c polyphosphates. I n 1957 he was D u r i n g h i s m i 1 it a r y s e r v i c e nomi n a t e d " A t t a c h e de Recherche. " (1958-1960) he worked i n Lyon i n a r e s e a r c h l a b o r a t o r y o f t h e M i l i t a r y H e a l t h S e r v i c e on t h e i s o l a t i o n o f a s o l u b l e s u b s t r a t e He s t u d i e d t h e o f lysozyme f r o m m i c r o c o c c u s Jysodeikticus. composi t i o n o f t h i s g l u c o p e p t i d e and t h e enzymology o f lysozyme on t h i s s u b s t r a t e . He p r e s e n t e d t h i s s t u d y as Ph.D. i n Pharmacy i n 1961 and became "Charge de Recherche" ( S e n i o r Research F e l l ow). Back t o S t r a s b o u r g he showed w i t h i s o l a t e d enzymes t h a t p o l y p h o s p h a t e s can r e p l a c e ATP i n 2 r e a c t i o n s and he s t u d i e d t h e A l l his i n t e r a c t i o n o f n u c l e i c acids w i t h polyphosphates.

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research on polyphosphates was t h e s u b j e c t of a P h . D . he presented a t the Faculty of Sciences of Strasbourg i n 1964. The same y e a r he presented the "Agregation" (habi 1 i t a t i o n ) of the

From l e f t : Robert Martin, GCrard Keith and Guy Dirheimer a t the Institute De Biologie Moleculaire E t C e l l u l a i r e . F a c u l t i e s of Pharmacy i n Biochemistry and became " a s s i s t a n t p r o f e s s o r " a t t h e U n i v e r s i t y of Strasbourg t e a c h i n g biochemistry and t o x i col ogy . A f t e r a s h o r t post-doctoral a t t h e l a b o r a t o r y of P r o f e s s o r Holley a t Cornell U n i v e r s i t y i n I t h a c a , N . Y . , USA, he s t a r t e d t o work on tRNAs. In p a r a l l e l he a l s o began t o study the mechanism of a c t i o n of r i c i n and was the f i r s t t o show (1967) t h a t t h i s t o x i c p r o t e i n from c a s t o r beans i n h i b i t s p r o t e i n s y n t h e s i s i n e u c a r y o t e s a t t h e ribosomal level. His l a b o r a t o r y ' s work on tRNAs was devoted t o t h e i s o l a t i o n and sequencing o f t h e s e molecules. Thirty-seven tRNAs, mainly from y e a s t , b u t a l s o from

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o t h e r b i o l o g i c a l sources ( b a c t e r i a , p l a n t s , i n s e c t s , mammals), and more r e c e n t l y from y e a s t m i t o c h o n d r i a , were sequenced. Also, s e v e r a l enzymes working on tRNAs were i s o l a t e d and s t u d i e d The gene o f y e a s t (aminoacyl-tRNA synthetases, tRNA methylases) c y t o p l a s m i c a s p a r t y l - t R N A synthetase was sequenced a f t e r c l o n i n g . Several p a r t s o f t h e y e a s t and l u p i n m i t o c h o n d r i a 1 genome were a1 so sequenced. I n t o x i c o l o g y t h e mechanism o f o c h r a t o x i n A, a mycotoxin g i v i n g a human nephropathy, was e l u c i d a t e d i n c o l l a b o r a t i o n w i t h This t o x i n i n h i b i t s P r o f e s s o r Roschenthaler from Munster (FGR). p r o t e i n s y n t h e s i s by c o m p e t i t i o n w i t h p h e n y l a l a n i n e . Other t o x i n s from p l a n t o r mushrooms o r i g i n a t i n g from Madagascar and a c t i n g on p r o t e i n s y n t h e s i s were i s o l a t e d and s t u d i e d . F i n a l l y , t h e i n t e r a c t i o n o f carcinogens w i t h t h e b i o l o g i c a l m e t h y l a t i o n o f DNA i s a l s o under s t u d y i n h i s l a b o r a t o r y . G. D i r h e i m e r has p u b l i s h e d about 230 papers. He was P r e s i d e n t o f t h e S o c i e t e de Chimie B i o l o g i q u e (=French S o c i e t y o f B i o c h e m i s t r y ) i n 1980-81. I n 1980 he became FEBS ( F e d e r a t i o n o f European Biochemical S o c i e t i e s ) F e l l o w s h i p s O f f i c e r , and from 1984, General S e c r e t a r y o f FEBS. He was a l s o P r e s i d e n t o f t h e Soci e t e Francai se de Toxi c o l ogi e (=French S o c i e t y o f Toxi c o l ogy) 1980-81, and i s s t i l l a member o f t h e E x e c u t i v e Committee o f t h e European S o c i e t y o f Toxicology. Dean o f t h e F a c u l t y o f Pharmacy o f Strasbourg 1969-70, he i s now A s s i s t a n t D i r e c t o r o f t h e CNRS I n s t i t u t e o f M o l e c u l a r and C e l l u l a r B i o l o g y i n Strasbourg. He was awarded t h e L. Bonneau P r i z e o f t h e French Academy o f Sciences (1973) and t h e S i l e r Medal o f CNRS (1977).

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Ghrard Keith was born i n 1940 i n Haguenau (Alsace-France) where he grew up. He s t u d i e d a t Strasbourg U n i v e r s i t y i n 1958, and s t a r t e d research i n 1963 under t h e d i r e c t i o n o f J.P. Ebel He was engaged by t h e "Centre N a t i o n a l de l a Recherche The S c i e n t i f i q u e " (CNRS) = French Research Council i n 1964. i n i t i a l researches were on chemical m o d i f i c a t i o n and b i o l o g i c a l a c i t i v i t i e s o f y e a s t tRNAs. I n 1966 he spent h i s 16 months m i l i t a r y s e r v i c e p e r i o d a t t h e Laboratory o f V i r o l o g y o f t h e F a c u l t y o f Medicine (Strasbourg) under t h e d i r e c t i o n o f o f A . K i r n where he worked on t h e i n h i b i t i o n o f DNA s y n t h e s i s by i n t e r f e r o n i n

.

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vaccine v i r u s i n f e c t e d c e l l s . I n e a r l y 1968 he went back t o J.P. E b e l ' s l a b o r a t o r y , where he became a co-worker o f G. D i r h e i m e r , s t a r t i n g w i t h t R N A p r i m a r y s t r u c t u r e determi n a t i o n s . He m a r r i e d i n 1967 a teacher, w i f e Agathe. He f i n i s h e d h i s t h e s i s (French D o c t o r a t d ' E t a t ) i n 1971 on p u r i f i c a t i o n , b i o l o g i c a l a c t i v i t y and One p r i m a r y s t r u c t u r e o f b r e w e r ' s y e a s t t R N A Asp and tRNATrP y e a r l a t e r he j o i n e d P.T. Gilham's research team a t Purdue U n i v e r s i t y ( L a f a y e t t e , I n d i a n a ) where he worked on s p e c i f i c s t e p wise d e g r a d a t i o n o f RNAs. Son Emmanuel was born i n t h e S t a t e s d u r i n g t h i s s t a y (1973) and daughter C k c i l e was b o r n i n 1977 i n S c h i l t i g h e i m (France). Since 1974 he i s back i n S t r a s b o u r g a t t h e IBMC ( I n s t i t u t f o r M o l e c u l a r and C e l l u l a r B i o l o g y ) , w o r k i n g m a i n l y on tRNA p r i m a r y s t r u c t u r e d e t e r m i n a t i o n s . He c o n t r i b u t ed a l s o e x t e n s i v e l y on t o p i c s such as t h e a c t i o n o f plumbous i o n o r bromomethylbenzanthracene on tRNAs. Other works were devoted t o s t u d i e s o f p h e n y l a l a n i n e t R N A synthetase, p l a n t t R N A methyAs f a r as RNA p r i m a r y s t r u c t u r e i s l a s e s and DNA methylases. concerned he went s u c c e s s i v e l y through a l l s t e p s o f e v o l u t i o n o f t h i s t y p e o f research: sequencing o f c o l d t R N A i n n e a r l y gram amounts (1968-1973), more r e c e n t l y (1974-1980) sequencing p o s t l a b e l e d s p e c i f i c fragments a f t e r s p e c i f i c enzymatic h y d r o l y s i s u s i n g mg amounts and now ( s i n c e 1981) sequencing p o s t - l a b e l e d s i n g l e h i t h y d r o l y s a t e s i n pg and even ng amounts. He sequenced t h e p r i m a r y s t r u c t u r e o f o v e r t w e n t y - f i v e tRNAs from a l l k i n d s o f organisms: b a c t e r i a , y e a s t , p l a n t s , i n s e c t s , b i r d s and mammals. The p r i F i v e y e a r s ago he s t a r t e d c l o n i n g and sequencing DNA. mary s t r u c t u r e o f t h e r a b i e s v i r u s RNA i s one o f h i s l a t e s t cont r i b u t i o n s . G. K e i t h i s t h e a u t h o r o f o v e r 75 s c i e n t i f i c p u b l i c a t i o n s i n b i o c h e m i s t r y . Since 1984 he i s a D i r e c t o r of r e s e a r c h a t t h e French "Centre N a t i o n a l de l a Recherche S c i e n t i f i q u e . " He i s a member o f t h e French "Socikt;! de B i o c h i m i e . "

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Melanie E h r l i c h and Xian-Yang Zhang M e l a n i e E h r l i c h was born i n 1945 i n t h e Bronx, New York City. While a s t u d e n t a t t h e Bronx High School o f Science, she began research work i n t h e v i r a l oncology s e c t i o n o f t h e Sloan K e t t e r i n g I n s t i t u t e f o r Cancer Research. A f t e r r e c e i v i n g a B . A . degree from Barnard College, she m a r r i e d Kenneth E h r l i c h and

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j o i n e d him i n graduate s t u d y a t t h e S t a t e U n i v e r s i t y o f New York a t Stony Brook. They now have two daughters A n i l i n , age 13 and Emily Myung-Hee, age 17. A t Stony Brook i n 1970, she r e c e i v e d t h e Ph.D. i n m o l e c u l a r b i o l o g y under t h e d i r e c t i o n o f Monica R i l e y . Next, she went t o t h e A l b e r t E i n s t e i n School o f Medicine f o r a p o s t - d o c t o r a l p o s i t i o n which i n v o l v e d research on SP15 DNA, t h e most h i g h l y m o d i f i e d DNA known. I n 1972, a t t h e c o n c l u s i o n o f t h a t f e l l o w s h i p , she j o i n e d t h e f a c u l t y o f Tulane U n i v e r s i t y , School o f Medicine. She i s c u r r e n t l y a p r o f e s s o r i n t h e Department o f B i o c h e m i s t r y a t Tulane. Her r e s e a r c h i n t e r e s t s t h e r e have

front of the Drs. M e l a n i e E h r l i c h and Xian-Yang Zhan s t a n d i n i? C a p i t o l b u i l d i n g i n Washin ton, D.C. ?his was l u r i n g a break a t a conference on DNA methygation t h a t was h e l d a t t h e N a t i o n a l I n s t i t u t e s o f Health.

xxv centered around n a t u r a l l y o c c u r r i n g m o d i f i c a t i o n s i n DNA. Among t h e t o p i c s pursued i n her l a b o r a t o r y a r e t h e p h y s i o l o g i c a l s i g n i f i c a n c e o f v e r t e b r a t e DNA m e t h y l a t i o n ; r e l a t i o n s h i p s o f b a c t e r i a l DNA m e t h y l a t i o n t o thermophily; i n t e r a c t i o n s o f DNA and sequencespeci f i c DNA-bi n d i ng p r o t e i n s ; r e p a i r o f m i smatched DNA bases; and p r o t e i n s i n v o l v e d i n p l a n t DNA m e t h y l a t i o n . I n c o l l a b o r a t i o n w i t h P r o f e s s o r Charles Gehrke and Kenneth Kuo a t t h e U n i v e r s i t y o f M i s s o u r i , she r e c e n t l y discovered a new base, N4-methyl c y t o s i n e , i n b a c t e r i a l DNA. Also, h e r l a b o r a t o r y has r e c e n t l y made t h e f i r s t i d e n t i f i c a t i o n o f a p r o t e i n which b i n d s s p e c i f i c a l l y t o 5-methylcytosine-containing DNA sequences. T h i s human p l a c e n t a l p r o t e i n may p r o v i d e a v i t a l l i n k i n understanding how v e r t e b r a t e DNA m e t h y l a t i o n r e g u l a t e s chromosomal f u n c t i o n s . X i an-Yang Zhang was born and educated i n Shanghai , Chi na. I n 1964 she graduated from Shanghai U n i v e r s i t y o f Science and Technology. I n 1968, she f i n i s h e d h e r graduate t r a i n i n g under i n t h e Shanghai I n s t i t u t e o f B i o c h e m i s t r y o f P r o f e s s o r Chen-Wu Qi t h e Chinese Academy o f Sciences. She became r e s e a r c h a s s i s t a n t and then research a s s o c i a t e i n t h a t i n s t i t u t e . Her m a j o r i n t e r e s t s were p u r i f i c a t i o n and c h a r a c t e r i z a t i o n o f p r o t e i n a s e i n h i b i t o r s , serum p r o t e i ns and c h r o m a t i n p r o t e i n s , I n 1980, she r e c e i v e d a p o s t - d o c t o r a l f e l l owshi p from t h e A1 exander von Humboldt Foundation o f West Germany and became a Humboldt f e l l o w i n P r o f e s s o r s H. Zachau and W. H o r z ' s l a b i n t h e I n s t i t u t e o f P h y s i o l o g i c a l Chemistry a t t h e U n i v e r s i t y o f Munich. There she worked on c h r o m a t i n s t r u c t u r e and nucleosome phasing. Since 1984, she has been a s e n i o r research a s s o c i a t e i n M. E h r l i c h ' s l a b i n t h e Department o f B i o c h e m i s t r y o f Tulane Medical School. She has worked on t h e i n t e r a c t i o n o f m e t h y l a t e d DNA and m e t h y l a t e d DNA-binding p r o t e i n s and t h e t i s s u e s p e c i f i c i t y o f human DNA m e t h y l a t i o n . She i s m a r r i e d and has two daughters, Fu and L i . Her husband, Ren-Zhi Cai, i s a l s o a b i o c h e m i s t .

XXVI

Henri Grosj ean

Henri Grosjean was born i n 1941 i n Brussels, Belgium. He studied a t the University of Brussels in the Department of Molecular Biology of Professor Jean Brachet under the supervision of Professeur Hubert Chantrenne. He graduated i n 1963 and The thesis was received his P h . D . i n Biochemistry i n 1969. r e l a t e d t o the mechanism and the s p e c i f i c i t y of the aminoacylt R N A s y n t h e t a s e s . D u r i n g t h e academic y e a r 1973-1974 he was an i n t e r n a t i o n a l post-doctoral research fellow of the National I n s t i t u t e of Health (NIH) a t Yale University (USA) in the l a b o r a t o r i e s of Professors Dieter S o l 1 and Donald Crothers. In these l a b o r a t o r i e s , he learned about f a s t r e l a x a t i o n techniques i n r e l a t i o n t o the studies of anticodon-anticodon complexes as a model f o r codon-anti codon recognition. Back i n Europe, he spent several months i n two l a b o r a t o r i e s i n Germany, a t the Medical Highschool in Hannover w i t h Professor Gunter Maass, and a t Max Planck I n s t i t u t e f o r Biophysical Chemistry i n GBttingen w i t h Professor Manfred Eigen. Since 1980, t o g e t h e r w i t h Dr. Claude

XXVII

Houssier, he developed t h e t e c h n i q u e o f f a s t r e l a x a t i o n a t t h e U n i v e r s i t y o f L i e g e (Belgium) i n r e l a t i o n t o t h e problems o f n u c l e i c a c i d s s t r u c t u r e and dynamics. Simultaneously, a t t h e U n i v e r s i t y o f B r u s s e l s (Belgium), he developed new techniques t h a t a l l o w e d one t o r e p l a c e enzymatic a l l y t h e a n t i c o d o n i n s e v e r a l tRNAs. T h i s recombinant RNA technology was e s p e c i a l l y designed t o s t u d y t h e s p e c i f i c i t y o f t h e s e v e r a l m o d i f i c a t i o n enzymes a c t i n g i n t h e a n t i c o d o n l o o p o f a t R N A molecule d u r i n g i t s complex m a t u r a t i o n process. I n 1965 he was appointed t o t h e permanent s t a f f o f t e a c h e r s a t t h e U n i v e r s i t y o f B r u s s e l s where he p r e s e n t l y h o l d s a p r o f e s s o r s h i p i n B i o c h e m i s t r y . Together w i t h h i s c o l l e a g u e s A . Burny, G. Marbaix, G. Huez and A. Sels, he i s a c o - D i r e c t o r o f t h e L a b o r a t o r y o f B i o l o g i c a l Chemistry a t t h e Department o f M o l e c u l a r B i o l o g y , U n i v e r s i t y o f B r u s s e l s . He i s a l s o a l e a d e r o f a small research group s t u d y i n g t h e m o l e c u l a r b a s i s f o r t h e accuracy i n v a r i o u s biochemical processes, e s s e n t i a l l y i n t h e s y n t h e s i s o f RNA and o f p r o t e i n s . He i s an a c t i v e member o f s e v e r a l s c i e n t i f i c s o c i e t i e s i n Belgium. I n 1982 he became an e l e c t e d member o f t h e European Mol e c u l a r B i o l o g y O r g a n i z a t i o n (EMBO) He i s t h e a u t h o r o f about 70 papers i n c l u d i n g 12 r e v i e w a r t i c l e s o r c h a p t e r s o f m u l t i - a u t h o r s books. H i s c u r r e n t research i s i n accuracy i n t r a n s l a t i o n process, e v o l u t i o n o f t h e g e n e t i c code and t h e p r o t e i n s y n t h e s i s , and macromolecular o r g a n i z a t i o n o f RNA m a t u r a t i o n enzymes i n h i g h e r e u k a r y o t i c c e l l s . For these works, he i s c o l l a b o r a t i n g w i t h s e v e r a l l a b o r a t o r i e s i n France ( P r o f e s s o r J.P. Ebel i n S t r a s b o u r g and Professor M. Grunberg-Manago i n P a r i s ) , i n S w i t z e r l a n d ( D r . E. K u b l i i n Z u r i c h ) , i n Canada ( P r o f e s s o r R. Cedergren i n M o n t r e a l ) , and i n t h e USA ( P r o f e s s o r R.W. Trewyn, Columbus, Ohio).

.

XXVIII

Anne-Li se Haenni

Anne-Lise Haenni was born i n S w i t z e r l a n d , and h o l d s Swiss and French c i t i z e n s h i p s . She a c q u i r e d h e r u n i v e r s i t y t r a i n i n g i n Harvard Medical School, Boston), and i n S w i t z e r t h e USA (A.M., 1 and (Docteur es Sciences , U n i v e r s i t y o f Geneva). A f t e r h a v i n g accompl i shed a s e r i e s o f p o s t - d o c t o r a l t r a i n i n g p e r i o d s i n France ( D r . E. Lederer, I n s t i t u t de Chimie des Substances N a t u r e l l e s ; Gif-sur-Yvette; D r . F r a n c o i s Chapevi 1 l e , Departement de B i o l o g i e , Saclay), S w i t z e r l a n d ( D r . A. T i s s i k r e s , Dbpartement de B i o l o g i e M o l e c u l a i r e , Genive), and t h e USA ( D r . F. Lipmann, The Rockefell e r U n i v e r s i t y , New York), she became a member o f t h e Centre She N a t i o n a l de l a Recherche S c i e n t i f i q u e i n France i n 1969. h o l d s t h e p o s i t i o n o f D i r e c t e u r de Recherches a t t h e I n s t i t u t Jacques Monod i n P a r i s . From h e r o r i g i n a l i n t e r e s t s i n t h e enzymatic o x i d a t i o n o f c y c l it o l s and t h e d e t e r m i n a t i o n o f t h e s t r u c t u r e o f p h y t o t o x i ns,

XXIX

she t u r n e d t o p r o t e i n b i o s y n t h e s i s and t o t h e r e g u l a t i o n of expression o f p l a n t v i r u s e s . She has l e c t u r e d i n o v e r 15 c o u n t r i e s . She i s t h e a u t h o r o f more t h a n 90 s c i e n t i f i c a r t i c l e s , and i s co-author o f one t e x t book on p r o t e i n b i o s y n t h e s i s . She i s a member o f EMBO, and was awarded t h e " P r i x J a u b e r t " by t h e U n i v e r s i t y of Geneva i n 1984.

Rajiv L. Joshi

Rajiv L. Joshi was born i n I n d i a i n 1959 and a r r i v e d i n France i n 1971 f o r h i s secondary and h i g h e r e d u c a t i o n . S t u d y i n g a t t h e U n i v e r s i t y o f P a r i s V I I , he o b t a i n e d h i s B.Sc. degree i n 1980, h i s M.Sc. degree i n 1982, and h i s t i t l e of "Docteur 6 s

xxx Sciences" i n 1986 i n Fundamental B i o c h e m i s t r y and M o l e c u l a r P l a n t Virology. He c a r r i e d o u t h i s r e s e a r c h work i n t h e group o f D r . Anne-Lise Haenni, i n t h e l a b o r a t o r y o f D r . F r a n c o i s C h a p e v i l l e a t t h e Jacques Monod I n s t i t u t e i n P a r i s and has c o l l a b o r a t e d w i t h t h e groups o f Drs. Mathias S p r i n z l ( U n i v e r s i t y o f Bayreuth, FRG) and Joanne M. Ravel ( U n i v e r s i t y o f Texas a t A u s t i n , USA). He has i n v e s t i g a t e d t h e s t r u c t u r e and f u n c t i o n o f tRNA-1 ike r e g i o n s p r e s e n t a t t h e 3 ' end o f many p l a n t v i r a l RNA genomes, and has a l s o c o n t r i b u t e d t o t h e understanding o f t h e r e c o g n i t i o n o f tRNAs by tRNA-speci f i c p r o t e i ns. D u r i ng h i s d o c t o r a l work, he o b t a i n e d a 1 ong-term f e l l owshi p from t h e French M i n i s t r y o f I n d u s t r y and Research, and two s h o r t term f e l lowships from EMBO and t h e French-German Cooperative Program. He has g i v e n s e v e r a l seminars i n v a r i o u s r e s e a r c h i n s t i t u t i o n s and u n i v e r s i t i e s i n France and Germany, and has a c t i v e l y p a r t i c i p a t e d in numerous i n t e r n a t i o n a l congresses and workshops. Since 1987, he h o l d s a permanent p o s i t i o n o f "Charg;! de Recherches 2 " a t t h e Centre N a t i o n a l de l a Recherche S c i e n t i f i q u e i n France, and i s S t a f f S c i e n t i s t o f t h e Jacques Monod I n s t i t u t e i n Paris. He i s p r e s e n t l y working on p l a n t t r a n s f o r m a t i o n i n c o l l a b o r a t i o n w i t h Drs. Francine Casse-Delbart and Mark T e p f e r (Centre N a t i o n a l de l a Recherche Agronomique, Versai 1 l e s , France) Helga and W a l t e r K e r s t e n Helga Kersten was born i n 1926 i n Hannover, Germany. She s t u d i e d Chemistry a t t h e U n i v e r s i t i e s o f Wurzburg and F r e i b u r g , and r e c e i v e d t h e Ph.D. i n Chemistry i n 1955 f r o m t h e U n i v e r s i t y o f Freiburg. Her t h e s i s work was on t h e b i o c h e m i s t r y o f c o r t i c o s t e r o i d s under t h e d i r e c t i o n o f Hans-Jurgen S t a u d i n g e r . As a p o s t - d o c t o r a l f e l l o w , she e s t a b l i s h e d i n 1958 t h e Moore-Stein a n a l y s i s f o r wool research a t t h e Woll f o r s c h u n g s i n s t i t u t a t t h e U n i v e r s i t y o f Aachen under t h e d i r e c t i o n o f Helmut Zahn. She and her husband, W a l t e r Kersten, j o i n e d t h e department o f P h y s i o l o g i c a l Chemistry a t t h e U n i v e r s i t y o f Munster. There t h e y s t a r t e d t h e i r c o l l a b o r a t i o n and research c a r e e r w i t h t h e d i s c o v e r y o f complex f o r m a t i o n o f a n t i b i o t i c s w i t h DNA. Helga K e r s t e n obt a i n e d t h e degree o f h a b i l i t a t i o n i n 1964 and was a p p o i n t e d as a

XXXI

P r o f e s s o r of B i o c h e m i s t r y a t t h e Medical F a c u l t y of t h e U n i v e r s i s i t y o f Munster i n 1968. From 1964-1965 she spent a y e a r as v i s i t i n g i n v e s t i g a t o r i n t h e McArdle I n s t i t u t e o f Cancer Research, working t o g e t h e r w i t h Charles Heidel b e r g e r . I n 1979

she was a p p o i n t e d P r o f e s s o r o f B i o c h e m i s t r y a t t h e Science F a c u l t y o f t h e U n i v e r s i t y o f Erlangen, Germany, where she s t i l l teaches B i o c h e m i s t r y f o r Chemists and Pharmacists. P r o f e s s o r Helga Kersten i s a u t h o r o f 180 s c i e n t i f i c p u b l i c a t i o n s . She and h e r husband were c o - e d i t o r s o f P r o g r e s s in M o l e c u l a r a n d S u b c e l l a r B i o l o g y . Both w r o t e f o r t h e s e r i e s , M o l e c u l a r B i o l o a v . B i o c h e m i s t r v and BioDhvsics, and t h e book, I n h i b i t o r s o f Nucleic Acid Synthesis. Helga Kersten w r o t e s e v e r a l i n v i t e d a r t i c l e s ; among these, O n t h e B i o l o g i c a l S i g n i f i c a n c e o f M o d i f i e d N u c l e o s i d e s in t R N A i n Proaress i n N u c l e i c A c i d Research and M o l e c u l a r B i o l o a y , Vol. 31, 1984. Over 50 s t u d e n t s have r e c e i v e d t h e i r diploma o r d o c t o r a l degrees under h e r d i r e c t i o n .

XXXII

W a l t e r K e r s t e n was born i n 1926 i n Obersuhl, West Germany, where h i s f a t h e r was a medical d o c t o r . A f t e r Wartime m i l i t a r y s e r v i c e he s t u d i e d medicine a t t h e U n i v e r s i t i e s o f Wurzburg and Marburg, where he r e c e i v e d h i s MD i n 1954. T r a i n i n g and e d u c a t i o n i n c l i n i c a l c h e m i s t r y and biochemiss t r y f o l l o w e d under t h e d i r e c t i o n o f Hans-Jurgen S t a u d i n g e r a t t h e Z e n t r a l l a b o r a t o r i u m o f t h e S t a d t i s c h e Krankenanstal t e n Mannheim. W a l t e r Kersten was appointed P r o f e s s o r o f P h y s i o l o g i c a l Chemistry a t t h e U n i v e r s i t y o f Munster i n 1962. He spent one y e a r a t t h e McArdle Laboratory, Madison, Wisconsin, w i t h W . Szybal s k i w i t h whom he worked on t h e p h y s i cochemi c a l p r o p e r t i e s o f DNA a n t i b i o t i c complexes. I n 1968 t h e c h a i r o f B i o c h e m i s t r y a t t h e Medical F a c u l t y o f t h e U n i v e r s i t y Erlangen-Nurnberg was o f f e r e d t o him. Here he b u i l t up a new Department o f Biochemistry.

D u r i n g t h e l a s t 10 y e a r s W a l t e r K e r s t e n was t h e o r g a n i z e r o f a j o i n t p r o j e c t i n Cancer Research between C l i n i c s and T h e o r e t i cal Institutes. T h i s Sonderforschungsberei ch a t t h e U n i v e r s i t y o f Erlangen was supported by t h e DFG. From 1979-1982 he served as Dean of t h e Medical F a c u l t y . Professor Walter Kersten's s c i e n t i f i c c o n t r i b u t i o n s are documented i n o v e r 100 p u b l i c a t i o n s on t h e mode o f a c t i o n o f a n t i b i o t i c s , t h e b i o c h e m i s t r y o f polyamines, tumor markers e.g. polyamines, m o d i f i e d nucleosides and p t e r i d i n e s .

Jiirg E. K o h l i

JUrg E. K o h l i was born i n 1945, i n Chur, S w i t z e r l a n d . He s t u d i e d a t t h e U n i v e r s i t y o f Bern m i c r o b i o l o g y , c h e m i s t r y and zoology and o b t a i n e d a diploma i n 1973. Then he worked a t t h e I n s t i t u t e o f General M i c r o b i o l o g y o f t h e same U n i v e r s i t y ( P r o f . U. Leupold) on g e n e t i c chromosome mapping i n t h e y e a s t S . pombe and o b t a i n e d t h e Ph. D. degree i n 1976. He spent a s h o r t t i m e a t t h e Genetics I n s t i t u t e o f t h e U n i v e r s i t y o f Munique working on cytochromes i n y e a s t . From 1976 t o 1979 he spent h i s p o s t d o c t o r a l y e a r s i n t h e Department o f M o l e c u l a r B i ochemi s t r y and B i o p h y s i c s a t Yale U n i v e r s i t y ( P r o f . D. S a l l ) w i t h t h e i s o l a t i o n , c h a r a c t e r i z a t i o n and sequencing o f nonsense suppressor-tRNAs f r o m

XXXIII

A f t e r r e t u r n i n g t o S w i t z e r l a n d he worked w i t h h i s own group on t h e s t r u c t u r e , f u n c t i o n and e v o l u t i o n o f t R N A and t R N A genes. The h a b i l i t a t i o n f o l l o w e d i n 1984. The f o l l o w i n g y e a r he stayed s h o r t l y i n London a t t h e I m p e r i a l Cancer Research Fund I n 1986 he became p r o f e s s o r o f m i c r o b i o l o g y w i t h D r . P. Nurse. and g e n e t i c s a t t h e I n s t i t u t e o f General M i c r o b i o l o g y a t t h e U n i v e r s i t y o f Bern. H i s c u r r e n t i n t e r e s t s concern t h e mechanisms o f g e n e t i c r e c o m b i n a t i o n i n S . pombe and t h e development o f v e c t o r and g e n e t i c e n g i n e e r i n g systems f o r y e a s t s . S.

pombe.

K e i t h A. K r e t z

h i s undergraduate degree K e i t h A. K r e t z r e c e i v e d c h e m i s t r y from C a p i t o l U n i v e r s i t y i n 1982, and t h e Ph.D. f r o m

in

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Ohio S t a t e U n i v e r s i t y in 1987. His d o c t o r a l t h e s i s was e n t i t l e d "Anticodon M o d i f i c a t i o n o f T r a n s f e r RNA and C e l l D i f f e r e n t i a t i on. " His research evaluated the r o l e o f m o d i f i c a t i o n s i n t h e anticodon o f tRNA on normal and n e o p l a s t i c c e l l d i f f e r e n ti a t i on. C u r r e n t l y D r . K r e t z i s a p o s t - d o c t o r a l f e l l o w i n t h e Department o f Neurosciences, U n i v e r s i t y o f C a l i f o r n i a , San Diego, where h i s research focuses on t h e m o l e c u l a r g e n e t i c s o f lysosomal enzymes and r e l a t e d s t o r a g e diseases. E r i c Kubli E r i c Kubli was born i n 1940 i n Neuchatel , S w i t z e r l a n d . He s t u d i e d zoology a t t h e U n i v e r s i t y of Z u r i c h where he r e c e i v e d h i s Ph.D. i n zoology i n 1970 w i t h d i s t i n c t i o n . He c o n t i n u e d h i s work a t t h e same i n s t i t u t i o n as a s e n i o r r e s e a r c h f e l l o w o f t h e I n s t i t u t e o f Zoology u n t i l 1973. F o l l o w i n g t h a t he spent two y e a r s on

xxxv a f e l l o w s h i p p o s t - d o c t o r a l a s s o c i a t e w i t h P r o f e s s o r D. S(il1, Department o f M o l e c u l a r B i o p h y s i c s and B i o c h e m i s t r y , Yale U n i v e r s i t y . I n 1975 he r e t u r n e d t o h i s s e n i o r r e s e a r c h f e l l o w p o s i t i o n a t t h e Z o o l o g i c a l I n s t i t u t e . I n 1983, w h i l e on s a b b a t i c a l leave, he spent s i x months a t t h e MRC L a b o r a t o r y o f M o l e c u l a r B i o l o g y i n Cambridge, England. I n 1985 he was a p p o i n t e d Associ a t e P r o f e s s o r a t the University o f Zurich. H i s r e s e a r c h has c e n t e r e d around t h e e l u c i d a t i o n o f t h e s t r u c t u r e and f u n c t i o n o f t R N A and t R N A genes i n D r o s o p h i l a m e l a n o g a s t e r , e s p e c i a l l y t R N A dependent nonsense suppressors and nonsense mutants. A t p r e s e n t h i s i n t e r e s t i s i n t h e i n t e r a c t i o n o f genes t r a n s c r i b e d by polymerases I 1 and 111.

B.E.H.

Maden

Ted Maden i s Johnston P r o f e s s o r o f B i o c h e m i s t r y , and t h e c u r r e n t Head o f t h e B i o c h e m i s t r y Department a t t h e U n i v e r s i t y o f Liverpool. He was born i n Derby, England i n 1935. B e f o r e commencing as an undergraduate a t Cambridge U n i v e r s i t y , he h e l d an E n g l i s h Speaking Union School boy Exchange S c h o l a r s h i p f o r a y e a r

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a t Mercersberg Academy, Pennsylvania. He graduated from Camb r i d g e U n i v e r s i t y i n 1957 w i t h a B.A. i n p h y s i o l o g y , and then went t o medical school a t K i n g ' s C o l l e g e H o s p i t a l , London, where he won t h e f i r s t p r i z e i n C l i n i c a l Medicine and graduated i n 1960. D u r i n g h i s s t u d i e s as a medical s t u d e n t he became i n t e r e s ted i n molecular biology. F o l l o w i n g i n t e r n s h i p s he r e t u r n e d t o Cambridge as a research s t u d e n t and o b t a i n e d h i s Ph.D. i n 1967 from t h e MRC L a b o r a t o r y o f M o l e c u l a r B i o l o g y f o r s t u d i e s on p r o t e i n s y n t h e s i s . From 1967-69 he was a p o s t - d o c t o r a l s c i e n t i s t w i t h P r o f e s s o r s J.E. Darnel1 and J.R. Warner a t t h e A l b e r t Eins t e i n C o l l e g e o f Medicine, New York, where he began h i s research i n t o mammal ian ribosome b i osynthesi s , He was a p p o i n t e d L e c t u r e r i n B i o c h e m i s t r y a t t h e U n i v e r s i t y o f Glasgow i n 1969, S e n i o r L e c t u r e r i n 1972, and Reader i n 1976. D u r i n g t h i s p e r i o d he and h i s r e s e a r c h group made a d e t a i l e d s t u d y o f t h e m e t h y l a t e d nuc l e o t i de sequences in v e r t e b r a t e ribosomal RNA by f in g e r p r i n t i ng and r e l a t e d methods, as summarized h i s chapter. D u r i n g a sabbat i c a l y e a r i n 1977-78 a t t h e Carnegie I n s t i t u t i o n o f Washington Embryology l a b o r a t o r y a t B a l t i m o r e he i n i t i t a t e d t h e experiments which l e d t o a map o f t h e d i s t r i b u t i o n o f methyl groups i n X e n o p u s rRNA, and gained experience i n working w i t h DNA. Upon h i s r e t u r n t o Glasgow he and h i s r e s e a r c h group sequenced X e n o p u s 18s rDNA and i t s f l a n k i n g r e g i o n s . He was e l e c t e d t o t h e Royal S o c i e t y o f Edinburgh i n 1978, and was appointed t o t h e Johnston C h a i r a t L i v e r p o o l i n 1983. He i s shown here i n h i s l a b o r a t o r y a t L i v e r p o o l w i t h a f i n g e r p r i n t o f methyl l a b e l e d X e n o p u s 18s ribosomal RNA. He i s m a r r i e d w i t h two grown c h i l d r e n . His l e i s u r e a c t i v i t i e s a r e mountaineering, t r a v e l w i t h h i s w i f e , and distance running. D i e t e r G. SO1 1 As a graduate s t u d e n t i n s y n t h e t i c o r g a n i c c h e m i s t r y D i e t e r G. Sbll's research focused on t h e chemical s y n t h e s i s o f N-glycos i d e s of p t e r i d i n e s f o r i n v e s t i g a t i o n as analogs o f n a t u r a l l y o c c u r r i n g nucleosides. These s t u d i e s l e d t o h i s n a t u r a l i n t e r e s t i n p o l y n u c l e o t i d e s y n t h e s i s , and t h i s caused him t o become a There he was i n p o s t - d o c t o r a l f e l l o w i n D r . Khorana's group. v o l v e d i n d e v e l o p i n g t h e e a r l y methods f o r r i b o - o l i g o n u c l e o t i d e

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s y n t h e s i s which a1 lowed t h e s y n t h e s i s o f t h e 64 r i b o - t r i n u c l e o s i d e diphosphates, compounds e s s e n t i a l f o r t h e subsequent e l u c i d a t i o n o f t h e Genetic Code. His i n t e r e s t i n tRNA also started d u r i n g t h a t time; t h e demonstration o f t h e p o s i t i o n o f t h e anticodon i n t R N A and o f t h e f a c t t h a t one t R N A m o l e c u l e can recognize m u l t i p l e codons were t h e f i r s t r e s u l t s o f t h i s work. Towards t h e end o f h i s p o s t - d o c t o r a l t r a i n i n g he became i n t e r e s t ed i n s t u d i e s o f n u c l e i c a c i d enzymology. Upon j o i n i n g t h e Yale f a c u l t y he began work on b i o c h e m i c a l / m o l e c u l a r b i o l o g i c a l problems. Since t h a t t i m e h i s r e s e a r c h has been focused on s t u d i e s of s t r u c t u r e , b i o s y n t h e s i s , r e g u l a t i o n and f u n c t i o n o f t R N A and o f aminoacyl-tRNA synthetases. To t h i s end he used chemical, biochemical, g e n e t i c , and m o l e c u l a r b i o l o g i c a l t o o l s and systems r a n g i n g from E . c o l i , y e a s t , c y a n o b a c t e r i a and D r o s o p h i l a .

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Ronald W. Trewyn Born i n Edgerton, Wisconsin, D r . Ronald W. Trewyn r e c e i v e d a Bachelor o f Science i n b i o l o g y l c h e m i s t r y from t h e U n i v e r s i t y o f Wisconsin-Whitewater, and t h e Ph.D. i n m i c r o b i a l p h y s i o l o g y l b i o c h e m i s t r y from Oregon S t a t e U n i v e r s i t y . He t h e n j o i n e d t h e U n i v e r s i t y o f Colorado H e a l t h Sciences Center, s e r v i n g as r e search a s s o c i a t e and i n s t r u c t o r i n t h e Department o f B i ochemi st r y , B i o p h y s i c s and Genetics. D r . Trewyn then j o i n e d t h e Ohio S t a t e U n i v e r s i t y P h y s i o l o g i c a l Chemistry department, and served as a r e s e a r c h s c i e n t i s t a t t h e Comprehensive Cancer Center. C u r r e n t l y D r . Trewyn i s Professor, Department o f Physiol o g i c a l Chemistry, and a t t h e Comprehensive Cancer Center serves b o t h as D i r e c t o r , Tissue Procurement Service, and Research S c i e n t i s t . D r . Trewyn has p u b l i s h e d e x t e n s i v e l y on h i s r e s e a r c h on t R N A and cancer, and has been an i n v i t e d speaker a t numerous symposia i n t h e USA and Europe.

A l t a f A. Wani A l t a f A. Wani r e c e i v e d t h e B.S. degree i n c h e m i s t r y , botany, and zoology from S.P. College, Kashmir U n i v e r s i t y , Srinagar, I n d i a . He then o b t a i n e d an M.S. degree i n b i o c h e m i s t r y from A.M. U n i v e r s i t y , A l i g a r h , I n d i a , and t h e M. P h i l . degree from t h e Department o f Biochemistry, A.M. U n i v e r s i t y , m a j o r i n g i n b i o chemical g e n e t i c s , and m o l e c u l a r b i o l o g y . He r e c e i v e d t h e Ph.D. degree i n b i o c h e m i s t r y from A.M. U n i v e r s i t y , A l i g a r h , and h e l d a p o s t - d o c t o r a l p o s i t i o n a t Ohio S t a t e U n i v e r s i t y , Department o f Radio1 ogy, where h i s research focused on mol ecul a r c a r c i nogenesis C u r r e n t l y D r . Wani i s a s s o c i a t e p r o f e s s o r , Department o f Rad ology, a t Ohio S t a t e U n i v e r s i t y .

E d i t h Fusayo Yamasaki Born i n Auburn, Cal if o r n i a, E d i t h Yamasaki r e c e i v e d t h e Bachelor o f A r t s i n b i o c h e m i s t r y from t h e U n i v e r s i t y o f C a l i f o r n i a - B e r k e l e y , and t h e Master o f Science i n c h e m i s t r y from Oregon S t a t e U n i v e r s i t y . She served as a Peace Corps v o l u n t e e r

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i n Accra, Ghana, West A f r i c a f o r two years, t h e n j o i n e d t h e l a b o r a t o r y o f D r . Bruce N. Ames i n t h e Department o f B i o c h e m i s t r y a t B e r k e l e y i n 1972. D u r i n g t h e f o l l o w i n g t e n y e a r s Mrs. Yamasaki p u b l i s h e d numerous s t u d i e s concerning mutagens, c a r c i n o I n 1982 gens, and t h e i r t e s t i n g w i t h D r . Ames and c o l l e a g u e s . Mrs. Yamasaki j o i n e d C o l l a b o r a t i v e Research, I n c . i n Wal tham, Massachusetts, then i n 1985 she j o i n e d t h e Department o f P h y s i o l o g i c a l Chemistry a t Ohio S t a t e U n i v e r s i t y i n Columbus.

Shigeyuki Yokoyama and Tatsuo Miyazawa Shigeyuki ( ' Y u k i ' ) Yokoyama was b o r n i n 1953 i n Tokyo, Japan. He majored i n b i o p h y s i c s and b i o c h e m i s t r y , and o b t a i n e d h i s degree o f Bachelor o f Science from t h e F a c u l t y o f Science, U n i v e r s i t y o f Tokyo i n 1975. He r e c e i v e d h i s degree o f Doctor o f Science a l s o from t h e U n i v e r s i t y o f Tokyo i n 1981. He was appointed t o i n s t r u c t o r i n 1982 and t h e n t o a s s o c i a t e p r o f e s s o r i n 1986 i n t h e Department o f B i o p h y s i c s and B i o c h e m i s t r y , F a c u l t y o f Science, U n i v e r s i t y o f Tokyo. I n 1987, he r e c e i v e d t h e Award f o r Young Chemist from t h e Chemical S o c i e t y o f Japan. D r . Yokoyama has been working w i t h P r o f e s s o r Tatsuo Miyazawa o f t h e Department o f B i o p h y s i c s and B i o c h e m i s t r y . He has publ i s h e d , as t h e a u t h o r o r co-author, more t h a n 40 papers i n t h e area o f b i o p h y s i c s and b i o c h e m i s t r y . He has s t u d i e d , i n graduate school , n u c l e a r magnetic resonance spectroscopy and o t h e r p h y s i cochernical methods f o r c o n f o r m a t i o n analyses o f b i o m o l e c u l e s . He has been d i r e c t i n g a research group on t h e mechanisms o f p r o t e i n synthesis a t the l e v e l o f molecular conformation. D r . Yokoyama has developed novel methods f o r c o n f o r m a t i o n a l analyses o f nucl e o s i des and n u c l e o t i des in aqueous s o l u t i on by NMR spectroscopy. He has a p p l i e d t h e s e methods t o m o d i f i e d nuc l e o s i d e s found i n tRNAs. As f o r h y p e r m o d i f i e d u r i d i n e s i n t h e f i r s t p o s i t i o n s o f tRNAs, he has found t h a t these p o s t - t r a n s c r i p t i o n a l m o d i f i c a t i o n s remarkably enhance o r reduce t h e conformat i o n a l f l e x i b i l i t y o f t h e n u c l e o t i d e u n i t , and t h e r e b y r e g u l a t e t h e range o f codons r e c o g n i z e d by those t R N A species, which guarantees t h e g e n e t i c code. H i s group i s a l s o i n v o l v e d i n t h e s t r u c t u r e analyses o f novel m o d i f i e d n u c l e o s i d e s i n tRNAs i n

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Shigeyuki Yokoyama (left) and Tatsuo Miyazawa (right) collaboration with Dr. Susumu Nishimura of the National Cancer Center Research Institute, Tokyo. Dr. Yokoyama's group has been studying the molecular mechanisms of the strict recognition of amino acids and tRNAs by aminoacyl-tRNA synthetases, 2'/3'-isomer specificity o f aminoacyl-tRNA and peptidyl-tRNA in protein synthesis, and the roles of polypepti de-chain elongation factor Tu. He is a1 so i nvol ved i n studies on the structure and function of oncogene products, growth factors, and carcinogens i n foods.

Tatsuo Miyazawa was born i n 1927 in Kyoto, Japan. He was graduated from the Department of Chemistry, Faculty of Science,

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U n i v e r s i t y o f Tokyo i n 1950. I n t h e graduate school f o r f i v e years, he majored i n p h y s i c a l c h e m i s t r y i n t h e l a b o r a t o r y o f Professor S . Mizushima, Department o f Chemistry, F a c u l t y o f Science, and o b t a i n e d t h e degree o f D o c t o r o f Science i n 1956. I n 1955 he was appointed i n s t r u c t o r i n t h e Department o f Chemistry. For t h e p e r i o d 1956-58 he was a p o s t - d o c t o r a t e i n t h e l a b o r a t o r y o f P r o f e s s o r Kenneth S . P i t z e r , U n i v e r s i t y o f California-Berkeley, and subsequently f o r one y e a r he was a research chemist i n t h e l a b o r a t o r y o f D r . Elkan R. Bout, C h i l d r e n ' s Cancer Research Foundation, Boston, Massachusetts. He was promoted t o a s s o c i a t e p r o f e s s o r o f t h e I n s t i t u t e f o r P r o t e i n Research, Osaka U n i v e r s i t y i n 1959, and p r o f e s s o r i n 1964. From 1971 he was a l s o a p r o f e s s o r o f t h e Department o f B i o p h y s i c s and Biochemistry, F a c u l t y o f Science, t h e U n i v e r s i t y o f Tokyo u n t i l he f i n a l l y moved t o Tokyo i n 1974. He served as t h e p r e s i d e n t o f t h e S p e c t r o s c o p i c a l S o c i e t y o f Japan f o r t h e p e r i o d 1985-87. He i s now a c o u n c i l member o f t h e U n i v e r s i t y o f Tokyo, and a member of t h e Science Council o f Japan. D r . Miyazawa has p u b l i s h e d , as a u t h o r o r co-author, more than 290 papers and more than 100 a r t i c l e s i n t h e f i e l d o f p h y s i c a l c h e m i s t r y ( i n t e r n a l r o t a t i o n and m o l e c u l a r v i b r a t i o n ) , i n f r a r e d and Raman spectroscopy (molecular v i b r a t i o n s ) , n u c l e a r magnet i c resonance spectroscopy, and p h y s i c a l b i o c h e m i s t r y o f pept i d e s , p r o t e i n s , n u c l e o t i d e s and n u c l e i c a c i d s . For t h e p e r i o d 1950-55 he s t u d i e d t h e i n f r a r e d a b s o r p t i o n and i n t e r n a l r o t a t i o n o f model molecules r e l a t e d w i t h t h e p o l y p e p t i d e chain, and e l u c i dated t h e n a t u r e o f t h e c h a r a c t e r i s t i c i n f r a r e d bands o f amides. For h i s " S t u d i e s on P o l y p e p t i d e s and Model Compounds by I n f r a r e d Absorption Spectroscopy, he r e c e i v e d t h e Award f o r Young Chemists from t h e Chemical S o c i e t y of Japan. I n 1955-1974 he s t u d i e d t h e v i b r a t i o n a l s p e c t r a and c h a i n conformations o f polymers i n c l u d i n g p o l y p e p t i d e s ( p r o t e i n s ) , and p o l y p r o p y l e n e and a l s o t h e c r y s t a l v i b r a t i o n s o f polyethylene. For h i s " S t u d i e s on Polymer S t r u c t u r e s by M o l e c u l a r Spectroscopy," he r e c e i v e d an Award from Yamaji Science Foundation (Tokyo) i n 1973. A f t e r he moved back t o t h e U n i v e r s i t y o f Tokyo i n 1974, he s t a r t e d t h e p r e s e n t s e r i e s o f s t u d i e s on t h e s t r u c t u r e s and f u n c t i o n s o f b i o m o l e c u l e s by t h e a n a l y s i s o f NMR. He analyzed t h e NMR o f snake n e u r o t o x i n s , and

XLII

e s t a b l i s h e d t h e method of NMR ( l a n t h a n o i d probe) a n a l y s i s of the conformation of f l e x i b l e molecules i n s o l u t i o n . For h i s " S t u d i e s on t h e S t r u c t u r e s of Polymers and Biomolecules by Molecular Spectroscopy," he received an award from the Chemical S o c i e t y of Japan i n 1980. C u r r e n t l y , i n c o l l a b o r a t i o n with Dr. S . Yokoyama, he i s c a r r y i n g o u t a p r o j e c t r e s e a r c h "Dynamic S t r u c t u r e s and Function Regulation of P r o t e i n B i o s y n t h e s i s System," i n c l u d i n g the biochemistry, physical chemistry and molecul a r biology o f the system of t R N A , aminoacyl-tRNA s y n t h e t a s e s , polypeptide chain e l o n g a t i o n f a c t o r and ribosome. Robert P. Martin Born in 1948 in France, Robert P. Martin studied at the UniversitC Louis Pasteur, Strasbourg (France), where he graduated in 1973. His postgraduate education followed first at the Faculty of Pharmacy and then at the Institut de Biologie MolCculaire et Cellulaire du CNRS in Strasbourg, under the direction of Professor Guy Dirheimer. His initial work concerned the purification and sequence determination of yeast cytoplasmic tRNAs. In 1975 he was appointed by the French Research Council (CNRS) as an 'Attache de Recherche' and started to work on mitochondrial tRNAs and their involvement in mitochondrial protein synthesis. He received his Ph.D. degree in 1980. The thesis was devoted to yeast mitochondrial tRNAs, their isolation, the determination of their genetic origin and primary structure, and the study of their codon reading patterns. In 1981, he became a 'ChargC de Recherche' and in 1982, he was recipient of the Maurice Nicloux Award of the French Biochemical Society and of the 'Bronze Medal' of the CNRS for his contribution to the elucidation of the genetic code in mitochondria. Leading a small research team at the Institut de Biologie MolCculaire et Cellulaire, his main research interest was then focused on the study of the structure, transcription, and expression of tRNA, rRNA, and protein coding genes in mitochondria from diverse sources. Since 1988, he has been a 'Directeur de RecherchC' at the CNRS. Dr. Robert P. Martin is the author of over forty scientific publications in biochemistry and molcular biology. He is a member of the French Biochemical Society. His current research is concerned with genetic and molecular analyses of mitochondrial frameshift suppressor tRNAs; study of mitochondrial aminoacyl-tRNA synthetases and their genes; and investigation of the nature and mechanism of action of enzymes involved in the biosynthesis of mitochondrial tRNAs such as the mitochondrial RNAase P.

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CHROMATOGRAPHY AND MODIFICATION OF NUCLEOSIDES PART A:

ANALYTICAL METHODS FOR MAJOR AND MODIFIED NUCLEOSIDES AND OLIGONUCLEOTIDES HPLC, GC, MS, NMR, UV, and FT-IA TABLE OF CONTENTS

Introduction and Overview

Charles W. Gehrke and Kenneth C. Kuo

Chapter 1

Ribonucleoside Analysis by High Performance Reversed-Phase Liquid Chromatography Charles W. Gehrke and Kenneth C. Kuo

Chapter 2

HPLC of Transfer RNAs Using Ionic-Hydrophobic Mixed-Mode and Hydrophobic-Interaction Chromatography Rainer Bischoff and Larry W. McLaughlin

Chapter 3

Nucleic Acid Chromatographic Isolation and Sequence Methods Gerard Keith

Chapter 4

Affinity Chromatography Methods for Purification of tRNAs Using Immobilized Elongation Factors Mathias Sprinzl and Karl-Heinz Derwenskus

Chapter 5

Structural Elucidation of Unknown Nucleosides in RNA and DNA Charles W. Gehrke, Jean A. Desgrks, Klaus 0. Gerhardt, Gerard Keith, Paul Agris, Hanna Sierzputowska-Gracz, Michael Tempesta and Kenneth C. Kuo

Chapter 6

Three-Dimensional Dynamic Structure of tRNAs by Nuclear Magnetic Resonance Paul F. Agris and Hanna Sierzputowska-Gracz

Chapter 7

Evaluation of the Effects of Modified Bases in the Anticodon Loop of tRNAs Using the Temperature-Jump Relaxation Method Henri Grosjean and Claude Houssier

Chapter 8

High-Performance Liquid Chromatography of Cap Structures and Nucleoside Composition in mRNAs Kenneth C. Kuo, Paul F. Agris, Christine E. Smith, Zhixian Shi and Charles W. Gehrke

Chapter 9

lmmunoassays for Modified Nucleosides of Ribonucleic Acids Barbara S. Vold

Chapter 10

Chromatography of Synthetic and Natural Oligonucleotides Heiner Eckstein and Herbert Schott PART C: MODIFIED NUCLEOSIDES IN CANCER AND NORMAL METABOLISM METHODS AND APPLICATIONS TABLE OF CONTENTS

Introduction

Nucleoside Markers for Cancer T. Phillip Waalkes and Charles W. Gehrke

Chapter 1

Progress and Future Prospects of Modified Nucleosides as Biological Markers of Cancer Robert W. Zumwalt, T. Phillip Waalkes, Kenneth C. Kuo and Charles W. Gehrke

Chapter 2

Ribonucleosides in Biological Fluids by a High-Resolution Quantitative RPLC-UV Method Kenneth C. Kuo, Dat T. Phan, Nathan Williams and Charles W. Gehrke

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Chapter 3

Ribonucleosides in Body Fluids: On-Line Chromatographic Cleanup and Analysis by a Column Switching Technique Eckhard Schllmme and Karl Siegfried-Boos

Chapter 4

HPLC of Free Nucleotides, Nucleosides, and their Bases in Biological Samples Phyllis R. Brown and Yong-Nam Kim

Chapter 5

Isolation and Characterization of Modified Nucleosides from Human Urines Glrlsh B. Chheda, Helen B. Patrzyc, Henry A. Tworek and Shlb P. Dutta

Chapter 6

Modified Nucleosides in Blood Serum Edith P. Mitchell, Llsa Evans, Paul Schultz, Rlchard Madsen, John Yarbro, Kenneth C. Kuo and Charles W. Gehrke

Chapter 7

Modified Nucleosides in Blood Serum as Biochemical Signals for Neoplasias Francesco Salvatore, L u c k Sacchettl, Marcella Savola, Fabrlzlo Pane, Tornmaso Russo, Alfred0 Colonna and Flllberto Clmlno

Chapter 6

Biochemical Correlations between Pseudouridine Excretions and Neoplasias Flllberto Clmlno, France Esposlto, Tommaso Russo and Francesco Salvatore

Chapter 9

High-Performance Liquid Chromatographic Analysis of Nucleosides and Bases in Mucosa Tissues and Urine of Gastrointestinal Cancer Patients Katsyukl Nakano

Chapter 10

Modified Nucleosides as Biochemical Markers of Asbestos Exposure and AIDS Opendra K. Sharma and Alf Flschbeln

Chapter 11

RNA Catabolites in Health and Disease Irwin Clark, Wln Lln and James W. Mackenzle

Chapter 12

Classification of Lung Cancer and Controls by Chromatography of Modified Nucleosides in Serum John E. McEntlre, Kenneth C. Kuo, Mark E. Smith, David L. Stalling, Robert W. Zumwalt and Charles W. Gehrke

Chapter 13

Modified Nucleosides and Nucleobases in Urine and Serum as Selective Markers for the Whole-Body Turnover of tRNA, rRNA, and mRNA: Future Prospects and Impact Gerhard Schbch, Gernot Sander, Helnrlch Topp and Gesa Heller-SchOch

PART D: A COMPREHENSIVE DATABASE FOR TRNA AND NUCLEOSIDES HPLC, GC, MS, NMR, UV, AND FT-IR Charles W. Gehrke, Jean A. Desgres, Mathlas Sprlnzl, Klaus 0. Gerhardt, Gerard Keith, Paul F. Agrls, Susan Dltson, Dat Phan, Henna Slerputowska-Gracz, Michael S. Tempesta, John A Hayden, Kenneth C. T. Kuo

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D I E T E R SOLL D e p a r t m e n t o f M o l e c u l a r B i o p h y s i c s and B i o c h e m i s t r y , Y a l e U n i v e r s i t y , New H a v e n , C o n n e c t i c u t , U . S . A .

TABLE OF CONTENTS t R N A S p e c i f i c i t y o f Ami noacyl - t R N A Synthetases. T r a n s f e r RNA and t h e Genetic Code Role o f t R N A i n P r o t e i n Turnover , Role o f tRNA i n C h l o r o p h y l l B i o s y n t h e s i s . Other Roles o f tRNA. Role o f M o d i f i e d Nucleosides Outlook. References.

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Although t h i s t i m e l y book d e a l s m o s t l y w i t h t h e f u n c t i o n and a n a l y s i s o f m o d i f i e d n u c l e o s i d e s found i n a l l n u c l e i c a c i d s , I would l i k e t o devote t h i s i n t r o d u c t o r y c h a p t e r t o a b r i e f , s e l e c t i v e and n e c e s s a r i l y personal r e v i e w o f r e s e a r c h on t r a n s f e r RNA. O f course, I do n o t wish t o i m p l y t h a t n u c l e o t i d e m o d i f i c a t i o n i n t R N A i s more i m p o r t a n t t h a n t h a t o c c u r r i n g i n o t h e r n u c l e i c a c i d species found i n t h e c e l l . Rather, i t s i g n i f i e s t h e h i s t o r i c a l f a c t t h a t t R N A has been t h e m a j o r source o f m o d i f i e d nucleosides s t u d i e d t o date. I n p a r t t h i s i s due t o t h e u b i q u i t o u s presence o f t R N A i n t h e cytoplasm and i n t h e o r g a n e l l e s o f c e l l s , b u t p r i m a r i l y because t R N A i s t h e most abundant source o f m o d i f i e d n u c l e o t i d e s amongst n u c l e i c a c i d s . As a m a t t e r o f f a c t , t R N A has been t h e source o f o v e r 60 m o d i f i e d nucl e o s i des w i t h known s t r u c t u r e s . Given t h e improved techno1 ogy o f n u c l e i c a c i d i s o l a t i o n and t h e s t e a d i l y i n c r e a s i n g s e n s i t i v i t y o f a n a l y t i c a l methods, new m o d i f i e d n u c l e o t i d e s a r e s t i l l i s o l a t e d and c h a r a c t e r i z e d a t a r a t e o f 1-2 p e r y e a r from t R N A species, even from organisms as w e l l e x p l o r e d as E . c o l i . When I d i d my f i r s t experiments i n t h e t R N A f i e l d i n t h e m i d - s i x t i e s t h e r e was much excitement about tRNA. The g e n e t i c

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code had j u s t been e l u c i d a t e d ( r e f . l), t h e d e t a i l e d mechanism o f p r o k a r y o t i c p r o t e i n b i o s y n t h e s i s was a h o t l y c o n t e s t e d f i e l d ( r e f . 2 ) , and t h e understanding of t h i s process n e c e s s i t a t e d a In d e t a i l e d knowledge o f t h e s t r u c t u r e and f u n c t i o n o f tRNA. 1965 t h e f i r s t tRNA sequence was determined ( r e f . 3), t h e mechanism o f aminoacyl a t i o n and t h e s t r u c t u r e o f ami noacyl-tRNA synthetases were a c t i v e l y i n v e s t i g a t e d , and t h e f i r s t d e f i n i t e t h e anticodon, f u n c t i o n a l r e g i o n s i n t h e tRNA m o l e c u l e (e.g., r e f . 4 ) were e s t a b l i s h e d . The c h e m i s t s ' c u r i o s i t y was aroused by t h e many m o d i f i e d n u c l e o t i d e s found i n t h i s molecule ( r e f . 5) and I t was s t u d i e s ' o n t h e i r b i o s y n t h e s i s were s t a r t e d ( r e f . 6). recognized e a r l y t h a t t h e much g r e a t e r chemical r e a c t i v i t y o f m o d i f i e d nucleosides, compared w i t h t h e f o u r m a j o r n u c l e o s i d e s , and t h e f a c t t h a t t h e y were much l e s s abundant i n t h e m o l e c u l e allowed t h e f i r s t i n s t a n c e s o f " s i t e - s p e c i f i c ' ' m o d i f i c a t i o n w i t h f l u o r e s c e n t groups o r s p i n - l a b e l e d m o i e t i e s (e.g., r e f . 7 ) . T h i s aided b i o p h y s i c a l s t u d i e s on t h e n a t u r e and dynamics o f t R N A s t r u c t u r e ( r e f . 8 ) . I n a d d i t i o n , s p e c u l a t i o n s about t h e f u n c t i o n o f m o d i f i e d n u c l e o t i d e s abounded, as everyone wanted t o f i n d a reason f o r t h e 1arge amount o f f a s c i n a t i n g biochemical elegance d i s p l a y e d i n t h e b i o s y n t h e s i s o f t h e r a r e bases! However, although the determination o f the c r y s t a l s t r u c t u r e o f tRNA ( r e f . 9) was a landmark achievement i n t h e f i e l d , r e s e a r c h a c t i v i t y i n t h i s area waned, as i t became c l e a r t h a t t h e m a j o r q u e s t i o n s o f protein-tRNA r e c o g n i t i o n and o f t R N A f u n c t i o n r e q u i r e d more complex i n v e s t i g a t i o n s than expected. The s i t u a t i o n has now changed. D u r i n g t h e p a s t few y e a r s new experimental approaches o f g e n e t i c s and m o l e c u l a r b i o l o g y , i n a d d i t i o n t o modern b i o p h y s i c a l i n s t r u m e n t a t i o n , have opened t h e t R N A f i e l d t o i n n o v a t i v e a t t a c k and shed l i g h t on q u e s t i o n s which have vexed us f o r many years, Very l i t t l e m a t e r i a l i s now r e q u i r e d t o determine a t R N A sequence by r a p i d g e l sequencing methods ( r e f . l o ) , a l t h o u g h t h e unambiguous d e t e r m i n a t i o n o f t h e m o d i f i e d nucleosides c o n t a i n e d i n i t may t a k e much more m a t e r i a l We now know t h e sequences o f 413 tRNAs and 664 and time. sequences a r e a v a i l a b l e from t h e a n a l y s i s o f t R N A genes ( r e f . 11)

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and t h e scope o f organisms which a r e b e i n g worked on has widened significantly. I n t h e f o l l o w i n g I would l i k e t o o u t l i n e a few areas which t o me make c u r r e n t t R N A r e s e a r c h e x c i t i n g . t R N A S P E C I F I C I T Y OF AMINOACYL-tRNA SYNTHETASES The development of a f f o r d a b l e automated chemical methods f o r DNA s y n t h e s i s and o f 1arge-scal e enzymatic t e c h n i q u e s t o t r a n s c r i b e DNA w i t h T7 RNA polymerase i n t o RNA ( r e f . 12) has l e f t a s t r o n g new impact on t h e f i e l d . The chemical s y n t h e s i s o f genes f o r many amber suppressor tRNAs w i t h d i f f e r e n t amino a c i d s p e c i f i c i t i e s has p r o v i d e d a f a c i l e way o f o b t a i n i n g a f a m i l y o f a l t e r e d p r o t e i n s by suppression o f amber mutants o f t h e r e l e v a n t However, t h e gene w i t h these d i f f e r e n t suppressors ( r e f . 13). n u c l e o t i d e requirements f o r s p e c i f i c i t y o f t h e v a r i o u s aminoacyltRNA, as w e l l as c o n s i d e r a t i o n s o f t h e s t a b i l i t y o f t h e complex of such aminoacyl-tRNAs w i t h mRNA d u r i n g p r o t e i n b i o s y n t h e s i s , may make i t i m p o s s i b l e t o o b t a i n a w e l l - f u n c t i o n i n g suppressor t R N A f o r each amino a c i d . T h i s approach a l s o i s o f r e l e v a n c e t o Using t o t a l chemical the o l d question o f tRNA s p e c i f i c i t y . s y n t h e s i s a new t R N A gene was c o n s t r u c t e d which d i f f e r e d i n 12 The n u c l e o t i d e s from an E . c o l i t R N A L e u species ( r e f . 14). r e s u l t i n g t R N A c o u l d be charged by s e r i n e i n v i v a . I n a s i m i l a r v e i n , chemical ( r e f . 15) and i n v i t r o mutagenesis ( r e f . 16) has been used t o change t h e amino a c i d s p e c i f i c i t y o f an E . c o 7 i t R N A Y e t and an E . co 1 i t R N A S e r species t o g l utami ne acceptance. These r e s u l t s suggest t h a t a l i m i t e d number o f n u c l e o t i d e s i n t R N A determine t h e m o l e c u l e ' s s p e c i f i c i t y towards aminoacyl-tRNA synthetases. A t h e o r e t i c a l t r e a t m e n t ( r e f . 17) o f t h i s problem based on t h e s t a t i s t i c a l examination o f t R N A sequences has been rendered and appears t o be i n agreement w i t h some o f t h e e x p e r i mental data. Another approach t o t h e i n v e s t i g a t i o n o f t R N A s p e c i f i c i t y was taken by i n v i v a and i n v i t r o mutagenesis o f E . c o 7 i glutaminyl-tRNA s y n t h e t a s e (GlnRS). Using a s t r o n g g e n e t i c s e l e c t i o n based on t h e c h a r g i n g o f supF t R N A T y r w i t h t h e i n c o r r e c t amino a c i d glutamine, mutants o f GlnRS have been o b t a i n e d ( r e f . 18) which have r e l a x e d s p e c i f i c i t y o f t R N A

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binding. Such mutants m i s a c y l a t e a small s e t o f E . c o l i t R N A species i n v i t r o . When t h e i r sequences were examined a s m a l l number o f conserved bases a t c e r t a i n p o s i t i o n s i n t h e t R N A sequence were i d e n t i f i e d ( r e f . 19). I n v i t r o mutagenesis o f an E . c o l i t R N A S e r species i n these p o s i t i o n s may l e a d t o c h a r g i n g o f t h i s tRNA w i t h g l u t a m i n e ( r e f . 16). Thus, some o f t h e bases i n t h e s e p o s i t i o n s appear t o be i m p o r t a n t f o r s p e c i f i c r e c o g n i t i o n o f t h e t R N A by GlnRS. Recent s t u d i e s w i t h a number o f s y n t h e t i c amber suppressors have shown t h a t , i n a d d i t i o n t o GlnRS, lysyl-tRNA synthetase i s capable o f m i s c h a r g i n g ( r e f . 20). TRANSFER RNA AND THE GENETIC CODE The sequence a n a l y s i s o f tRNAs and t h e i r genes has shown t h a t a v a r i e t y o f organisms show m i n o r d e v i a t i o n s from t h e " s t a n d a r d g e n e t i c code" ( r e f . 21). T h i s most f r e q u e n t l y i n v o l v e s t h e use o f UGA i n m i t o c h o n d r i a o f c e r t a i n organisms ( r e f . 22) and o f UAA and UAG as a sense codon i n o t h e r organisms. More r e c e n t l y , two o t h e r e x c i t i n g phenomena have emerged. N a t u r a l suppression d e s c r i b e s t h e f a c t t h a t i n c e l l s normal tRNAs e x i s t which a l s o have t h e a b i l i t y t o suppress c h a i n t e r m i n a t i o n codons under c e r t a i n circumstances. T h i s was f i r s t d e t e c t e d by t h e readthrough o f t h e UAG s t o p codon d u r i n g i n v i t r o TMV RNA t r a n s l a t i o n by D r o s o p h i l a ( r e f . 23) and p l a n t ( r e f . 24) t R N A T y r species. I n t h i s case t h e s t a t e o f m o d i f i c a t i o n o f t h e a n t i c o d o n base G ( m o d i f i e d t o Q) appears t o be i m p o r t a n t . The c o n c e n t r a t i o n o f a n a t u r a l suppressor t R N A may m o d i f y t h e l e v e l o f e x p r e s s i o n o f genes w i t h nonsense codons i n t h e i r r e a d i n g frame. I n wheat s t o r a g e p r o t e i n genes a t l e a s t two i n s t a n c e s o f presumably s i l e n t genes a r e known. I n b o t h cases premature, i n frame t e r m i n a t i o n codons (UAG and UAA) i n t e r r u p t t h e r e a d i n g frame ( r e f . 25). The p o s s i b i l i t y o f i n v i v o read-through cannot be excluded, g i v e n t h e presence o f such n a t u r a l suppressors i n p l a n t tRNAs. Presumably, t h e codon c o n t e x t i s v e r y i m p o r t a n t f o r such a readthrough. I n o t h e r e u k a r y o t i c organisms t R N A G L n i s I n T e t r a h y m e n a t h e macronuclear DNA such a suppressor tRNA. codes f o r s e v e r a l t R N A G l n genes, one o f which c o n t a i n s a TTA a n t i c o d o n ( r e f . 26). The corresponding t R N A was shown t o decode

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UUA; o t h e r T e t r a h y m e n a tRNAGin species decode t h e normal T h i s suggests t h a t t h e ochre g l u t a m i n e codons CAA and CAG. t e r m i n a t i o n codon UAA i s used as a g l u t a m i n e codon i n c y t o p l a s m i c p r o t e i n s y n t h e s i s i n t h i s organism ( r e f . 26). I n y e a s t i t was i s o a c c e p t o r can decode t h e UAG found t h a t t h e normal tRNA;Y; amber codon, presumably by C - U m i s p a i r i n g ( r e f . 27). Another i n t e r e s t i n g case i s i n mouse l i v e r , where two tRNAGin species were i s o l a t e d and sequenced ( r e f . 28). While t h e m a j o r one serves t h e normal g l u t a m i n e codons t h e m i n o r one i s a b l e t o r e a d UAG w i t h i t s a n t i c o d o n UmUG. The s y n t h e s i s o f t h i s p a r t i c u l a r i s o a c c e p t o r species i s g r e a t l y i n c r e a s e d a f t e r Moloney muri ne leukemia v i r u s i n f e c t i o n . T h i s and o t h e r d a t a s u p p o r t t h e p l a u s i b l e h y p o t h e s i s t h a t t h i s t R N A suppresses t h e UAG t e r m i n a t i o n codon l o c a t e d a t t h e gag-pol gene j u n c t i o n o f Moloney leukemia v i r u s and thus f a c i l i t a t e s t h e s y n t h e s i s o f t h e v i r u s encoded protease ( r e f . 28). N a t u r a l suppressor tRNAs may r e p r e s e n t an a d d i t i o n a l r e g u l a t o r y pathway c o n t r o l l i n g t h e l e v e l s o f expression o f c e r t a i n genes. An e x c i t i n g new development has been t h e d i s c o v e r y t h a t s e l e n o c y s t e i n e i s encoded f o r by a UGA codon i n b o t h E . c o l i and I t has been found t h a t t h e gene encoding i n mammalian c e l l s . formate dehydrogenase i n E . c o l i i s i n t e r r u p t e d by a UGA nonsense codon w h i c h s p e c i f i c a l l y d i r e c t s t h e c o t r a n s l a t i o n a l i n c o r p o r a t i o n o f s e l e n o c y s t e i n e i n t o t h e p r o t e i n ( r e f . 29). Obviously, t h e r e a d i n g c o n t e x t o f t h e UGA codon i s i m p o r t a n t . C u r r e n t l y t h e search i s on f o r t h e t R N A which d i r e c t s t h e i n c o r p o r a t i o n o f t h i s amino a c i d . This finding reinforces the q u e s t i o n o f whether t h e r e a r e some i n s t a n c e s o f c o t r a n s l a t i o n a l i n s e r t i o n o f phosphoseri ne i n t o p r o t e i ns as a UGA-readi ng phosphoseri ne t R N A has been c h a r a c t e r i z e d from b o v i n e and human sources ( r e f . 3 0 ) . ROLE OF tRNA I N PROTEIN TURNOVER Degradation o f i n t r a c e l l u l a r p r o t e i n s can be achieved by t h e u b i q u i t i n and ATP-dependent p r o t e o l y s i s pathway. The in v i v o h a l f - l i n e o f a p r o t e i n w i t h an exposed amino terminus depends on i t s amino t e r m i n a l r e s i d u e . Recent experiments have shown t h a t

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aminoacyl-tRNA i s r e q u i r e d f o r t h e p o s t t r a n s l a t i o n a l a d d i t i o n o f N-terminal amino a c i d s t o a c i d i c amino t e r m i n i o f p r o t e i n s . T h i s m o d i f i c a t i o n i s e s s e n t i a l f o r p r o t e i n d e g r a d a t i o n by t h e u b i q u i t i n pathway ( r e f . 31). ROLE OF t R N A I N CHLOROPHYLL BIOSYNTHESIS The f i r s t s t e p o f c h l o r o p h y l l b i o s y n t h e s i s i n t h e c h l o r o p l a s t i s t h e r e d u c t i o n o f glutamate t o g l u t a m a t e - l semialdehyde. Recent i n v e s t i g a t i o n s ( r e f . 32) showed t h a t t h i s At first conversion i n v o l v e s t R N A i n two d i f f e r e n t r o l e s . g l utamate i s charged by c h l o r o p l a s t g l utamyl-tRNA s y n t h e t a s e t o c h l o r o p l a s t t R N A G L U . I n t h e second s t e p t h e glutamate r e s i d u e i s reduced i n an NADPH-dependent r e a c t i o n by a dehydrogenase which This reaction r e q u i r e s t h i s s p e c i f i c t R N A as c o f a c t o r . r e p r e s e n t s a novel r o l e f o r t R N A : a p a r t i c i p a t i o n i n a m e t a b o l i c conversion o f i t s cognate amino a c i d i n t o another l o w m o l e c u l a r weight m e t a b o l i t e , which i s n o t used subsequently i n p e p t i d e bond synthesis. OTHER ROLES OF t R N A Unexpected evidence o f tRNA involvement i n a v a r i e t y o f b i o c h e m i c a l pathways has been o b t a i n e d from c l o n i n g and sequencing s t u d i e s o f v a r i o u s genes. Thus i s was e s t a b l i s h e d t h a t t h e f. c o l i d n a Y gene, which i s thought t o be i n v o l v e d i n t h e p o l y m e r i z a t i o n phase o f DNA r e p l i c a t i o n , encodes a m i n o r S i m i l a r l y , t h e temperature-sensitive a r g i n i n e t R N A ( r e f . 33). d i v E m u t a t i o n , which a r r e s t s t h e growth o f E . c o l i c e l l s a t a s p e c i f i c stage, i s l o c a t e d i n t h e D-stem of a s e r i n e t R N A species ( r e f . 34). I n S t r e p t o m y c e s c o e l i c o l o r mutants ( b l d A ) e x i s t which a r e d e f e c t i v e i n a n t i b i o t i c p r o d u c t i o n and i n t h e development o f hyphae and s p o r e s . DNA sequence a n a l y s i s o f s e v e r a l i n d e p e n d e n t l y i s o l a t e d mutant b l d A genes showed t h a t t h e In m u t a t i o n s occur i n a p u t a t i v e l e u c i n e t R N A gene ( r e f . 35). each case t h e c l o n e d w i l d - t y p e t R N A gene complements t h e corresponding m u t a t i o n a l phenotype. However, t h e e x a c t biochemical s t e p i n which tRNA i s i n v o l v e d i n t h e p a r t i c u l a r process i s s t i l l unknown.

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ROLE OF MODIFIED NUCLEOSIDES Is t h e r e a r o l e of m o d i f i e d n u c l e o s i d e s i n t R N A f u n c t i o n ? C u r r e n t l y we have t o o few f a c t s t o g u i d e us t o a c o n c l u s i v e I do n o t b e l i e v e t h a t t h e r e w i l l be a answer t o t h i s q u e s t i o n . u n i f o r m answer. Presumably m o d i f i e d n u c l e o s i d e s a r e needed f o r f i n e - t u n i n g of t R N A f u n c t i o n , as i t i s c l e a r t h a t t h e y a r e n o t c r u c i a l f o r t h e b a s i c s t e p s o f a m i n o a c y l a t i o n ( r e f . 12) and p r o t e i n s y n t h e s i s ( r e f . 36) as judged by i n v i t r o experiments. However, some s t u d i e s have shown t h a t t h e presence o f m o d i f i e d bases leads t o o p t i m a l i n t e r a c t i o n s o f t R N A w i t h t h e components o f t h e p r o t e i n s y n t h e s i z i n g system (see e.g., r e f . 6). Genetic and biochemical s t u d i e s w i t h mutants d e f e c t i v e i n t R N A m o d i f y i n g enzymes have d e m o n s t r a t e d i n a few cases t h a t l a c k o f m o d i f i c a t i o n r e s u l t s i n decreased o r a b o l i s h e d suppression o f It i s puzzling, nonsense codons ( f o r r e v i e w see r e f . 3 7 ) . however, t h a t t h i s e f f e c t m a n i f e s t s i t s e l f o n l y w i t h suppressor tRNAs and n o t w i t h o t h e r t R N A species c o n t a i n i n g t h e same m o d i f i e d bases. i t remains t o be seen whether t h i s i s t h e r e s u l t o f t h e r e l a t i v e c o n c e n t r a t i o n o f t h e p a r t i c u l a r t R N A species i n the c e l l . An e x c i t i n g r e c e n t i n v i t r o s t u d y shows t h a t m o d i f i c a t i o n o f a s i n g l e n u c l e o t i d e n o t o n l y changes codon r e c o g n i t i o n o f t h e tRNA, b u t a l s o i t s i d e n t i t i y towards aminoacyl-tRNA synthetases. The m i n o r t R N A 1 t e species i n E . c o l i r e c o g n i z i n g t h e codon AUA c o n t a i n s t h e novel m o d i f i e d n u c l e o s i d e lysylcarbamoyl-cytidine i n the f i r s t p o s i t i o n o f the anticodon ( r e f . 38). When f u l l y m o d i f i e d , t h e tRNA recognizes t h e codon AUA and i s aminoacylated w i t h i s o l e u c i n e . B u t i f an unmodified C occupies t h e f i r s t anticodon p o s i t i o n , t h e n t h e t R N A w i l l be charged w i t h m e t h i o n i n e and recognizes t h e codon AUG. OUTLOOK What excitement w i 11 f u t u r e research b r i n g ? With developments o c c u r r i n g as r a p i d l y as i n t h e p a s t few y e a r s i t i s d i f f i c u l t t o p r e d i c t . However, I t h i n k t h a t two areas w i l l r e c e i v e more a t t e n t i o n . The e v e r i n c r e a s i n g power o f b i o p h y s i c a l methods w i l l p r o v i d e a d e t a i l e d knowledge o f t h e c r y s t a l s t r u c t u r e o f t R N A

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complexed w i t h t h e cognate aminoacyl-tRNA s y n t h e t a s e ( r e f s . 39, 40) and p r o b a b l y a l s o w i t h o t h e r p r o t e i n s . These s t u d i e s w i l l be complemented by f u r t h e r i n s i g h t i n t o t h e dynamics o f t R N A molecules gained by NMR experiments (e.g., r e f . 41). With t h e s t r u c t u r a l i n f o r m a t i o n a t hand i t w i l l be p o s s i b l e t o do d i r e c t e d i n v i t r o mutagenesis t o engineer b o t h t R N A and t h e i n t e r a c t i n g p r o t e i n s t o pose v e r y s o p h i s t i c a t e d questions, as has been demons t r a t e d b e a u t i f u l l y i n t h e p r o t e i n e n g i n e e r i n g work w i t h aminoacyl-tRNA synthetases alone ( r e f s . 42, 43). I n t h i s way t h e question o f the underlying principles f o r the high s p e c i f i c i t y o f aminoacyl-tRNA synthetase: t R N A i n t e r a c t i o n should f i n a l l y emerge. Another re1 a t i v e l y uncharted area d e s e r v i n g more a t t e n t i o n i s t h e f i e l d o f t R N A and r e l a t e d enzymes i n o r g a n e l l e s . Sequence a n a l y s i s o f t R N A species may uncover a d d i t i o n a l tRNAs w i t h unusual f e a t u r e s as have been found i n m i t o c h o n d r i a l ones ( r e f . 11). T h i s may h e l p o u r understanding o f what f e a t u r e s a r e r e q u i r e d f o r t R N A f u n c t i o n (see a l s o t h e a r t i c l e on v i r a l RNAs and t R N A i n t h i s volume, r e f . 44). I n addition, organellar metabolisms may r e q u i r e t R N A f o r y e t unknown f u n c t i o n s as was seen i n t h e involvement o f t R N A i n c h l o r o p h y l l s y n t h e s i s ( r e f . 32). Other s u r p r i s e s may come from e n c o u n t e r i n g pathways (e.g. , Gln-tRNACLn f o r m a t i o n i n v o l v e s m i s a m i n o a c y l a t i o n and t r a n s a m i d a t i o n i n o r g a n e l l e s , r e f . 45) d i f f e r e n t from t h e accustomed ones, o r f i n d i n g macromolecules w i t h dual f u n c t i o n s (e.g., m i t o c h o n d r i a l aminoacyl-tRNA synthetases i n v o l v e d i n RNA s p l i c i n g [ r e f . 461, o r t h e involvement o f t R N A G L u i n b o t h c h l o r o p h y l l and p r o t e i n b i o s y n t h e s i s [ r e f . 451). 1.

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CHAPTER 1 SYNTHESIS AND FUNCTION OF MODIFIED NUCLEOSIDES I N TRNA Glenn R. B j 6 r k 1 and J u r g Kohliz 'Department o f Microbiology, Umea University, S-901 87 UHEA, Sweden ZZnstitute o f General Microbiology, University o f Bern, Bal tzer-Strasse 4 , CH-3012 Bern, Switzerland

TABLE OF CONTENTS 1.1 I n t r o d u c t i o n 1.2 Presence o f M o d i f i e d Nucleosides i n T r a n s f e r RNA From D i f f e r e n t Organisms 1.2.1 M o d i f i e d Nucleosides i n C y t o s o l i c tRNAs. . . . 1.2.2 M o d i f i e d Nucleosides i n O r g a n e l l e tRNAs. . . . 1.3 S y n t h e s i s o f M o d i f i e d Nucleosides i n Transfer RNA . . 1 . 3 . 1 T r a n s f e r RNA M o d i f y i n g Enzymes . . . . . . . . 1.3.2 Genetics and R e g u l a t i o n o f t R N A M o d i f y i n g Enzymes i n B a c t e r i a and Yeast . . . . . . . . 1.3.3 M e t a b o l i c and Developmental Aspects o f t h e Synthesis o f M o d i f i e d Nucleosides. . . . . . . 1 . 4 F u n c t i o n o f M o d i f i e d Nucleosides i n T r a n s f e r RNA. . . 1 . 4 . 1 M o d i f i e d Nucleosides Next t o t h e 3 ' - S i d e o f t h e Anticodon ( P o s i t i o n 37) Influence Translational Efficiency, Translation F i d e l i t y , S e n s i t i v i t o f the tRNA t o the Reading Context and geading Frame Maintenance 1.4.2 M o d i f i e d Nucleosides a t t h e Wobble P o s i t i o n ( P o s i t i o n 34) I n f l u e n c e T r a n s l a t i o n a l F i d e l i t y and Codon Choice . . . . . . . . . . . . . . .

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B14 814 B18 B20 B20

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825

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B32 B37

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838

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1.4.3

M o d i f i e d Nucleosides i n t h e Anticodon Region Other Than i n P o s i t i o n 34 and 37 I n f l u e n c e T r a n s l a t i o n a l E f f i c i e n c y and F i d e l i t y 1.4.4 M o d i f i e d Nucleosides O u t s i d e t h e Anticodon Region May S t a b i l i z e t R N A Conformation . . . . . R e g u l a t o r y Role o f t h e Synthesis o f M o d i f i e d Nucleosides i n tRNA . . . . . . . . . . . . . . . . . . . . . F u t u r e Prospects and Impact . . . . . . . . . . . . . .

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1.5 1.6

845 B46 B48 B50

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1.7 1.8

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Acknowledgements References

INTRODUCTION T r a n s f e r RNA p a r t i c i p a t e s i n t h e decoding process i n a l l I t i n t e r a c t s w i t h many d i f f e r e n t p r o t e i n s , l i v i n g organisms. i n c l u d i n g e l o n g a t i o n f a c t o r s and aminoacyl-tRNA l i g a s e s , and w i t h ribosomal RNA. T h i s d i v e r s i t y o f i n t e r a c t i o n s f o r t h e t R N A may be one reason f o r i t s complex c o n t e n t o f m o d i f i e d n u c l e o s i d e s which a r e d e r i v a t i v e s o f t h e f o u r c a n o n i c a l n u c l e o s i d e s adenosine(A), guanosine(G), u r i d i n e ( U ) and c y t o s i n e ( C ) . A t p r e s e n t more than 50 d i f f e r e n t m o d i f i e d n u c l e o s i d e s have been c h a r a c t e r i z e d ( r e f . 1). Some a r e m o d i f i e d by t h e a d d i t i o n o f a s i n g l e m e t h y l group t o t h e base o r t o t h e 2 ' - h y d r o x y l group o f t h e r i b o s e w h i l e o t h e r s a r e formed by a very complex sequence o f r e a c t i o n s r e s u l t i n g i n h y p e r m o d i f i e d n u c l e o s i d e s . The s y n t h e s i s o f most m o d i f i e d n u c l e o s i d e s occurs on t h e p o l y n u c l e o t i d e l e v e l f o l l o w i n g t h e t r a n s c r i pt i o n o f t h e t R N A genes. T h i s r e v i e w w i l l c o n c e n t r a t e on t h e s y n t h e s i s and f u n c t i o n o f m o d i f i e d nucleosides i n t R N A . Other aspects o f m o d i f i e d nucleos i d e s i n t R N A n o t covered i n t h i s review, such as t h e i r r o l e i n tumor f o r m a t i o n and as d i a g n o s t i c t o o l s , a r e d i s c u s s e d elsewhere ( r e f . 2). Furthermore, r e s u l t s n o t covered i n d e t a i l h e r e can be found i n e a r l i e r r e v i e w s on t h i s t o p i c ( r e f s . 1, 3-14). 1.1

1.2

PRESENCE OF MODIFIED NUCLEOSIDES I N TRANSFER RNA FROM DIFFERENT ORGANISMS 1 . 2 . 1 Modi f i e d Nucl e o s i des in Cvtosol ic tRNAs F i g u r e s 1.1-1.3 show t h a t c y t o s o l i c t R N A f r o m a l l t h r e e k i ngdoms (Eucaryotes, Archaebacteri a and E u b a c t e r i a) c o n t a i ns modified nucleosides. A few ($13, Cm32, mlG37, t6A37, $38, $39, $40, $55, mlA58) a r e p r e s e n t i n tRNAs f r o m a l l kingdoms, i n d i c a t i n g a common e v o l u t i o n a r y o r i g i n o r convergent e v o l u t i o n ( r e f . 15). E u c a r y o t i c t R N A c o n t a i n s t h e l a r g e s t v a r i e t y and abundance o f m o d i f i e d nucleosides, and consequently i t c o n t a i n s s e v e r a l m o d i f i e d nucleosides s p e c i f i c f o r t h a t kingdom. However, t h e r e a r e a l s o s p e c i f i c m o d i f i c a t i o n s f o r t h e o t h e r two kingdoms, e.g. mnm5s2U f o r e u b a c t e r i a and ml$ f o r t R N A f r o m a r c h a e b a c t e r i a ( r e f .

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Eubacterial t R N A s

mcrnosu mnm’s b cmnm’s2u cmnm’u mnm’SezU ac4C Cm Gm

a

m6 A 1‘ A rns2i6A ms2io 6A tk mtsA ms2t ‘A m ‘G

I

Figure 1.1 Modified nucleosides resent in tRNA from Eubacteria, (Data compi 1 ed from reference $1) Structures of different modified nucleosides as well as the abbreviations can be found in reference 239. An index and an exponent indicate the number and the position of the substitution respectively, e.g. 6-dimethyladenosine is abbreviated m$A. m-, c-, ,:n 0 - , t-, i - and s- are abbreviations of methyl-, carbon-, amino-, oxy-, threonine-, isopentenyl- and thio groups. Other abbreviations: pseudouridine; I , inosine; W, nucleoside of Y base; oyW, nuc koside of peroxy Y bas.e. An enzyme catalyzin the formation o f m5U at position 54 in the tRNA is denoted tR\A (m5 U54)methyl transferase and likewise for other tRNA modifying enzymes.

t

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Archaebacterial

tRNAs

I

ac4C Cm

I

m'G tsA

M o d i f i e d nucleosides p r e s e n t i n t R N A f r o m Archaebact e r i a t Data compiled from r e f e r e n c e 21). S t r u c t u r e s o f d i f f e r e v t Figure m o d i f i e nucleosides as w e l l as t h e a b b r e v i a t i o n s can be found i n r e f e r e n c e 239. An i n d e x and an exponent i n d i c a t e t h e number and t h e p o s i t i o n o f t h e s u b s t i t u t i o n r e s p e c t i v e l y , e.g. 6-dimethyladenosine i s a b b r e v i a t e d mqA. m-, c-, n,: 0 - , t-, i- and s- a r e a b b r e v i a t i o n s o f methyl-, carbon-, amino-,. oxy-, threonine-, i s o p e n t e n y l - and t h i o groups. Other a b b r e v i a t i o n s : side o f u r i d i n e ; I , i n o s i n e ; W , n u c l e o s i d e o f Y base; oyW,. nuc k o pseudoAn enzyme c a t a l y z i n t h e f o r m a t i o n o f m5U a t peroxy Y bas.e. p o s i t i o n 54 in t h e t R N A i s denoted t R \ A (m5 U54)methyl t r a n s f e r a s e and l i k e w i s e f o r o t h e r t R N A m o d i f y i n g enzymes.

a'"

Y

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Figure 1.3 Modified nucleosides resent in tRNA from Eucaryotes, Structures of different (Data compiled from reference 51) modified nucleosides. as well as the abbrevjatjons can be found in reference 239. An index and an exponent indicate the number and the position of the substitution respectively, e.g. 6-dimethyladenosine is abbreviated mqA. m-, c-, nr, o-, t-, i - and s- are abbreviations of methyl-, carbon-, amino- oxy-, threonine-, isopentenyl- and thio groups. Other abbrev;ations: pseudoof uridine; I , inosine; W , nucleoside o f Y base; oyW,. nuc eoside peroxy Y base. An enzyme catalyzin the formation of m5U at position 54 in the tRNA is denoted tR\A (m5 U54)methyl transferase and likewise for other tRNA modifying enzymes.

tr

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15). Most tRNAs from b o t h eucaryotes and e u b a c t e r i a c o n t a i n m5U54, w h i l e tRNAs from a r c h a e b a c t e r i a u s u a l l y c o n t a i n ml$ i n t h i s position. Since t h e p o s i t i o n o f t h e methyl group i n mV54 and m5U54 i s t h e same i n r e l a t i o n t o t h e r i b o s e m o i e t y and t h e phosp h a t e - r i bose backbone, an e v o l u t i o n a r y convergence f o r t h e methyl group i n t h a t p o s i t i o n o f t h e tRNA has been suggested ( r e f . 16). Although n o t shown i n F i g u r e s 1.1-1.3, t h e r e a l s o e x i s t s species s p e c i f i c i t y o f t R N A m o d i f i c a t i o n w i t h i n a kingdom; e.g. mo5U34 i s p r e s e n t i n tRNAs s p e c i f i c f o r Val, a l a , t h r , and s e r from Gram p o s i t i v e organisms, w h i l e t h e corresponding tRNAs from Gram n e g a t i v e o r g a n i sins have mcmo5U/cmo5U34. Recently, selenium-containing m o d i f i e d n u c l e o s i d e s have a l s o been i d e n t i f i e d i n t R N A from b o t h b a c t e r i a and eucaryotes ( r e f s . 17-19). One, mnm5SeW, has been i d e n t i f i e d i n p o s i t i o n 34 o f tRNAGLu from Clostridiom stricklandii ( r e f . 20). Modi f ied Nucl e o s i des in O r a a n e l l e tRNAs A compilation o f the modified nucleosides present i n chlorop l a s t (23 sequences) and m i t o c h o n d r i a l tRNAs (63 sequences) of t h e S p r i n z l c o l l e c t i o n ( r e f . 21) leads t o t h e f o l l o w i n g general conclusions. Fewer s i t e s a r e m o d i f i e d ( c h l o r o p l a s t 24, mitochondr i a 30) i n t h e o r g a n e l l e t R N A molecules i n comparison w i t h c y t o The s o l i c tRNAs (55 s i t e s o f m o d i f i c a t i o n i n tRNA o v e r a l l ) . chemi c a l s t r u c t u r e s o f modi f i c a t i ons a r e 1ess v a r i e d i n organel 1e tRNAs. Special f e a t u r e s o f m i t o c h o n d r i a l tRNAs a r e t h a t t h e m5U54 and $ m o d i f i c a t i o n s i n t h e T$C-loop occur l e s s f r e q u e n t l y than i n nonorganel 1e tRNAs. Pseudouridi ne i s o f t e n found in o t h e r p a r t s o f m i t o c h o n d r i a l tRNAs, e s p e c i a l l y i n t h e h i n g e r e g i o n between t h e T$C-loop and t h e acceptor stem which r a r e l y i s m o d i f i e d i n c y t o s o l i c tRNAs. I t may be t h a t these s p e c i a l t r a i t s o f m i t o c h o n d r i a l t R N A m o d i f i c a t i o n a r e r e l a t e d t o t h e unusual s t r u c t u r e o f some m i t o c h o n d r i a l tRNAs (e.g. m i s s i n g D loop, bulges i n stems). Special f e a t u r e s i n c h l o r o p l a s t tRNAs a r e t h e o b l i g a t o r y m o d i f i e d n u c l e o s i d e s Gm a t p o s i t i o n 18, D a t p o s i t i o n 20, m5U a t p o s i t i o n 54 and $ a t p o s i t i o n 55. A t p o s i t i o n 46, m7G i s o f t e n found i n c o n t r a s t t o m i t o c h o n d r i a l tRNAs where t h i s n u c l e o s i d e i s m i s s i n g . Another s p e c i a l i t y o f c h l o r o p l a s t tRNAs i n c o n t r a s t t o p l a n t c y t o s o l i c tRNAs i s t h e abundance o f acp3U a t p o s i t i o n 47. 1.2.2

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More d e t a i l s are noteworthy concerning the modification pattern in the anticodon region of organelle tRNAs. A t position 34 (the wobble position) of mitochondria1 tRNAs an unmodified U i s very common which renders the t R N A the potential t o decode the whole family of four codons ( N N U , N N C , N N A , N N G ) . I f necessary, the wobble position i s modified t o cmnm5U t o avoid misreading in those families of four codons t h a t specify two d i f f e r e n t amino acids ( r e f . 2 2 ) . In mitochondria of Sacharomyces cerevisiae there also e x i s t s a t R N A A r g with an unmodified A a t the wobble p o s i t i o n and t h i s t R N A i s also able t o read a l l four CGN codons f o r arginine ( r e f . 2 2 ) . An unmodified A a t position 34 has not been observed in cytosolic tRNAs where the A i s always converted t o inosine. The adaptation of the t R N A sequence and modification pattern t o the altered genetic code in mitochondria has recently been reviewed ( r e f . 23). Transfer RNAs with an unmodified U or Um i n the wobble position have a l s o been found in chloroplasts ( r e f . 2 1 ) , b u t l e s s i s known on the actual decoding rules in t h i s organel 1 e . There i s no $ a t position 35 in organelle t R N A T y r which correlates with the absence of introns of the nuclear type i n organelle t R N A genes. Pseudouridine a t position 35 can only be introduced a t the precursor level and the enzyme seems t o require an intron (see 1.3.1). A t position 36 of chloroplast tRNALeU the modified nucleoside m7G has been found ( r e f . 2 1 ) . Nucleosides a t t h i s s i t e are rarely modified a n d the role of m7G i n the anticodon i s u n k n o w n . 7methylguanosine i s o n l y present a t position 46 i n the case of cytosol i c t R N A (Figures 1.1-1.3). The nucleoside adjacent t o the anticodon a t position 37 i s frequently modified in organelle tRNAs as in other tRNAs. The base ms2i6A c h a r a c t e r i s t i c f o r eubacterial tRNAs a t this s i t e has been found in organelle tRNAs, although i t i s usually not present in cytosol i c tRNAs of eucaryotes. Several observations mentioned above on modification in organel 1 e tRNAs pose i nteresti ng problems concerning the b i osynthesi s of mi nor nucl eosides in organel 1 es. Based on biochemical experiments Smolar and Svensson ( r e f . 24) suggested t h a t the same

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nuclear gene encodes both the enzyme c a t a l y z i n g the formation of mZG26 i n cytoplasmic and mitochondrial t R N A and t h i s was l a t e r supported by g e n e t i c evidences ( r e f . 25). This i s a l s o t r u e f o r the formation of m5U54 and i6A37 ( r e f . 25, 2 6 ) . T h u s , these r e s u l t s suggest t h a t the enzymes c a t a l y z i n g the formation of mgG26, i6A37 and m5U54 a r e expressed from nuclear genes, and synthesized i n t h e cytoplasm and can be t r a n s p o r t e d i n t o the mitochondria a s well a s i n t o the nucleus where the formation of mgG26 and m5U54 i s l i k e l y t o occur ( r e f . 27, 28). C e r t a i n modific a t i o n s may be missing i n mitochondrial and c h l o r o p l a s t tRNAs, because the corresponding enzymes a r e not imported from the cytoplasm. I t i s u n l i k e l y t h a t genes coding f o r tRNA-modifying enzymes w i l l be discovered in the mitochondrial genome s i n c e i t has been thoroughly c h a r a c t e r i z e d ( p o s s i b l e exceptions a r e p l a n t mitochondria) . Chloroplast genomes a r e 1 a r g e r and 1 ess i s known about t h e i r genes. Therefore, the p o s s i b i l i t y e x i s t s t h a t c e r t a i n c h l o r o p l a s t - s p e c i f i c t R N A m o d i f i c a t i o n s (m7G a t p o s i t i o n 36, ms2i6A a t 37 and acp3U a t 47) are introduced by enzymes t h a t a r e expressed from t h e c h l o r o p l a s t genome. 1.3

1.3.1

SYNTHESIS

OF M O D I F I E D NUCLEOSIDES I N TRANSFER RNA

T r a n s f e r RNA-Modi f v i na Enzvmes T r a n s f e r RNA-modifying enzymes t h a t c a t a l y z e the s y n t h e s i s of m5U54, mlG37, mnm5s2U34, mcmo5U, 434 and $38,39,40 have been p u r i f i e d t o near homogeneity from E s c h e r i c h i a c o l i / S a l m o n e l 7 a t y p h i m u r i u m ( r e f s . 29-36) a s we1 1 a s the tRNA(GmI8)methyl-transf e r a s e from Thermus thermophilus ( r e f . 3 7 ) . The tRNA(m1A) methylt r a n s f e r a s e from r a t 1 i v e r and tRNA(mgG26)methyl t r a n s f e r a s e from Tetrahymena have been p u r i f i e d t o homogeneity ( r e f . 38, 39). The l a t t e r enzyme has both mono- and dimethylating a c t i v i t y . Most tRNA-modi f y i ng enzymes a r e small , a c i d i c p r o t e i n s composed of one polypeptide i n t h e a c t i v e s t a t e . However, t R N A (m5U54)methylt r a n s f e r a s e from S t r e p t o c o c c u s f a e c a l i s i s composed of two equal s u b u n i t s i n the n a t i v e s t a t e ( r e f . 40, 41) and the tRNA(s4U8)synt h e t a s e , which seems t o have a complex s t r u c t u r e composed of a t l e a s t two s u b u n i t s , i s encoded by two d i f f e r e n t genes ( r e f . 42, 43). The tRNA(mnm5s*U34)methyltransferase from E. c o l i , which i s

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coded f o r b y t h e trmc gene, possesses t w o e n z y m a t i c a c t i v i t i e s ; one d e m o d i f i e s cmnm5szU t o nm5s2U w h i l e t h e o t h e r a c t i v i t y t r a n s f e r s a m e t h y l g r o u p t o nmss*U and mnm5szU i s f o r m e d ( r e f . 36). The tRNA-modi f y i n g enzymes have been d i f f i c u l t t o p u r i f y s i n c e t h e y a r e p r e s e n t i n t h e c e l l i n s m a l l amounts, e.g. t h e t R N A (m'G37)methyl t r a n s f e r a s e has been e s t i m a t e d t o be p r e s e n t i n t h e amounts o f o n l y 8 0 m o l e c u l e s as compared t o more t h a n 800 m o l e c u l e s aminoacyl-tRNA l i g a s e s p e r genome e q u i v a l e n t i n €. coli ( r e f . 3 5 ) . I n Gram n e g a t i v e b a c t e r i a and i n e u c a r y o t e s t h e m e t h y l g r o u p donor i n t h e f o r m a t i o n o f m5U54 i s S-adenosyl-L-methionine, whereas i n Gram p o s i t i v e b a c t e r i a t h e m e t h y l d o n o r i s 5 - 1 0 - t e t r a h y d r o f o l a t e ( r e f . 44, 45). The enzymes a r e u s u a l l y s t i m u l a t e d b y Mg2+ i o n s o r p o l y a m i n e s b u t t h e tRNA(mlG37)methyl t r a n s f e r a s e f r o m E . c o l i i s n o t and tRNA(mnm5s2U34)methy1t r a n s f e r a s e f r o m t h e same o r g a n i s m i s i n f a c t i n h i b i t e d b y Mg2+ i o n s ( r e f s . 29, 35, 3 6 ) . Most o f t h e enzymes a r e dependent on SH-groups f o r e n z y m a t i c activity. O n l y t w o m o d i f i e d n u c l e o s i d e s , q u e u o s i n e (Q) and i n o s i n e (I) i n p o s i t i o n 34, a r e s y n t h e s i z e d p r i o r t o i n s e r t i o n i n t o t h e p r e existing polynucleotide. Queuosine i s t h e most complicated m o d i f i e d n u c l e o s i d e so f a r d i s c o v e r e d and i t s s y n t h e s i s has r e c e n t l y been r e v i e w e d ( r e f . 4 6 ) . The enzyme, t R N A g u a n i n e t r a n s g l y c o s y l a s e , exchanges t h e p r e e x i s t i n g G34 w i t h t h e Q-base ( q u e u i n e ) l e a v i n g t h e tRNA backbone i n t a c t ( r e f . 4 7 ) . I n o s i n e ( I ) p r e s e n t i n t h e wobble p o s i t i o n i s a d e a m i n a t e d d e r i v a t i v e o f a d e n o s i n e w h i c h i s why i t was t h o u g h t t o be s y n t h e s i z e d b y a t R N A deaminase. However, i n o s i n e i s f i r s t s y n t h e s i z e d f r o m a d e n o s i n e and l a t e r r e p l a c e s an a d e n o s i n e i n p o s i t i o n 34 o f t h e p r e e x i s t i n g tRNA ( r e f . 48). G e n e t i c a n a l y s e s have shown t h a t d i f f e r e n t enzymes c a t a l y z e t h e s y n t h e s i s o f t h e same m o d i f i e d n u c l e o s i d e s i n r i b o s o m a l RNA and i n t R N A ( r e f s . 49, 50). However, t h e tRNA(mSC)methyltransf e r a s e f r o m mammalian t i s s u e s shows some a c t i v i t y t o w a r d s r R N A f r o m E. c a l f as w e l l as t o w a r d s s y n t h e t i c p o l y m e r s ( r e f . 5 1 ) . F u r t h e r m o r e , t h e r e a r e d i f f e r e n t enzymes p r o d u c i n g @ i n t h e a n t i c o d o n stem ( p o s i t i o n s 38, 39, and 4 0 ) , $ i n p o s i t i o n 55 and i n p o s i t i o n 32 ( r e f s . 52, 5 3 ) . T h e r e a r e a l s o d i f f e r e n t enzymes i n

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yeast, which catalyze the synthesis of mlG in position 9 and 37 in the tRNA (ref. 54). Two distinct tRNA(m*G)methyl transferases from rat liver with different site specificities have been characterized (ref. 55). Thus, there are different enzymes catalyzing the synthesis of the same modified nucleoside not only in different nucleic acids but also at different positions of the tRNA. Some modifications are correlated to the decoding capacities of the tRNA (see Figure 1.4). Therefore, it was suggested that at least a part of the recognition signal for the corresponding enzymes must reside in the sequence surrounding the nucleotide to be modified, I n fact, for 12 modified nucleosides a correlation between the modified nucl eosi de and the surrounding nucl eoti de sequence was pointed out (ref. 56). However, the tertiary structure o f the tRNA is also part o f the recognition signal for tRNAmodifying enzymes (refs. 57-61). Transfer RNA reading codons that start with U usually have an i6A derivative in position 37. The recognition signal for the corresponding enzyme is the sequence A36-A37-A38 as well as a five base-paired anticodon stem (ref. 56). Seryl tRNAs species I and V do not have an i6A37 derivative, Codon a (nucleotide)

I

I

1st

2nd

3rd

Modifications in position 37 in t R N A from:

I Eucaryotes

I Eubacteria I

I I

Archaebacteria

N= any o f the four nucleotides

F i g u r e 1.4 Presence o f m o d i f i e d n u c l e o s i d e s .at. p o s i t i o n 37 1 3 ' s i d e o f . t h e a n t i c o d o n ) and t h e coding. c a p a c i t i e s o f tRNAs rom e u b a c t e r i a , e u c a r y o t e s and a r c h a e b a c t e r i a .

B23

a1 though t h e y have t h e sequence A36-A37-A38 ( r e f . 62). However, these tRNAs do n o t have a f i v e base p a i r e d stem s u p p o r t i n g t h e n o t i o n t h a t t h e tRNA-modifying enzymes c a t a l y z i n g t h e f o r m a t i o n o f i6A37 a l s o r e q u i r e o t h e r i m p o r t a n t r e c o g n i t i o n s i g n a l s than t h e p r i m a r y sequence s u r r o u n d i n g t h e t a r g e t n u c l e o t i d e ( r e f . 62, 63). F o l l o w i n g i n j e c t i o n o f c h i m e r i c tRNAs i n t o Xenopos l a e v i s oocytes, t h e 634 t o 434 c o n v e r s i o n i n y e a s t tRNAAsp was measured. The formation o f Q34 was s t r o n g l y dependent on t h e presence o f t h e U33-G34-U35 sequence ( r e f . 64). Furthermore, t h e r e a c t i o n o f A34 t o I 3 4 r e q u i r e s a Purine-35 and does n o t a l l o w a U36 u s i n g c h i m e r i c y e a s t t R N A A s p as s u b s t r a t e ( r e f . 65). Formation o f I 3 4 i n c h i m e r i c y e a s t t R N A A r g was a l s o dependent on sequence o f t h e a n t i c o d o n r e g i o n b u t n o t as s t r i c t as t h e one observed f o r tRNAASp (H. Grosjean, p e r s . comm.). Therefore, t h e dependence on t h e a n t i codon sequence f o r t h e tRNA(I34)synthetase d i f f e r s from one t R N A t o t h e o t h e r suggesting t h a t r e c o g n i t i o n s i g n a l s beyond t h e anticodon a r e i n v o l v e d . This i s c e r t a i n l y t r u e f o r the tRNA (Gm34)methyl t r a n s f e r a s e , which i s n o t dependent on t h e n e i g h b o r i n g n u c l e o t i d e s ( r e f . 66). The tRNA($55)synthetase seems t o have a s t r i c t requirement o f a U54 ( r e f . 67). Thus t h e h i g h l y s p e c i f i c tRNA-modifying enzymes have d i f f e r e n t requirements t o r e c o g n i z e the target nucleotide. For some t h e sequence s u r r o u n d i n g t h e n u c l e o t i d e t o be m o d i f i e d i s c r u c i a l , b u t f o r o t h e r s t h e t h r e e dimensional s t r u c t u r e may be a more d e t e r m i n i n g f a c t o r . Enzymes c a t a l y z i n g t h e f o r m a t i o n o f 434, PA37 and yW37 belong t o t h e f i r s t group w h i l e enzymes c a t a l y z i n g t h e f o r m a t i o n of Gm34 and I 3 4 belong t o t h e second group (H. Grosjean, personal communication). Both i n e u b a c t e r i a and i n eucaryotes, tRNA i s t r a n s c r i b e d i n u n i t s longer than t h e i r functional s i z e whereafter they are e n z y m a t i c a l l y trimmed ( r e f . 68). I n e u b a c t e r i a , p o l y c i s t r o n i c as w e l l as m o n o c i s t r o n i c p r e c u r s o r s a r e p r e s e n t . The m a j o r i t y of t h e e u c a r y o t i c p r i m a r y t r a n s c r i p t s a r e m o n o c i s t r o n i c a1 though d i c i st r o n i c p r e c u r s o r s have been i d e n t i f i e d ( r e f . 69, 70). I n both types o f organisms t h e m o d i f i c a t i o n s occur a t d i f f e r e n t stages during the processing of the precursor. I n bacteria, several m o d i f i e d n u c l e o s i d e s have been i d e n t i f i e d i n p o l y c i s t r o n i c p r e c u r sors. However, r i b o s e - m e t h y l a t e d n u c l e o s i d e s have n o t been found,

B24

s u g g e s t i n g t h a t t h i s m e t h y l a t i o n r e a c t i o n r e q u i r e s an a l m o s t The m o d i f i c a t i o n m i g h t o c c u r s t e p w i s e s i n c e t h e mature tRNA. m u l t i m e r i c p r e c u r s o r f o r tRNA:=" c o n t a i n s m5U, $ and D b u t n o t mlG37 w h i c h i s p r e s e n t i n t h e monomeric p r e c u r s o r , w h i c h i n t u r n l a c k s Gm18 ( r e f . 71). Modified nucleosides i n t h e precursors have been i d e n t i f i e d i n c e l l s w i t h d e f e c t s i n t h e p r o c e s s i n g reactions while the actual kinetics o f the modification reactions i n l o g a r i t h m i c a l l y g r o w i n g w i l d t y p e c e l l s a r e more d i f f i c u l t t o assess. I n f a c t , t h e r e i s e v i d e n c e t h a t t h e m e t h y l a t i o n r e a c t i o n s a r e p r i m a r i l y o c c u r r i n g on m o l e c u l e s o f t h e same s i z e as m a t u r e tRNA i n c e l l s a t balance growth ( r e f . 72). This i s consistent w i t h e x p e r i m e n t s i n v i t r o w h i c h showed t h a t t h e RNAase P-cleaved precursor i s a b e t t e r s u b s t r a t e than t h e uncleaved p r e c u r s o r f o r t h e tRNA(m5U54)methyl t r a n s f e r a s e ( r e f . 7 3 ) . Despite t h i s , the f o r m a t i o n o f m5U54 and #55 precedes t h e f o r m a t i o n o f $40 and i6A37 Thus, t h e r e s u g g e s t i n g s t e p w i s e m o d i f i c a t i o n as f o u n d i n v i v o . seems t o be a b a l a n c e between t h e t r a n s c r i p t i o n and p r o c e s s i n g o f t h e tRNA and i t s m o d i f i c a t i o n . To be a b l e t o f u l l y assess t h e dynamic a s p e c t o f t R N A m a t u r a t i o n we m u s t have more knowledge about t h e r e g u l a t i o n o f n o t o n l y t h e t r a n s c r i p t i o n o f t h e i n d i v i d u a l t R N A genes b u t a l s o o f t h e r e g u l a t i o n o f t h e s y n t h e s i s and a c t i v i t y o f t h e enzymes w h i c h p a r t i c i p a t e i n t h e m a t u r a t i o n process. By i n j e c t i n g t h e y e a s t tRNATyr gene i n t o X.7aevis o o c y t e s i t was e s t a b l i s h e d t h a t a l l m o d i f i c a t i o n s e x c e p t a t p o s i t i o n s 34 and 37 t a k e p l a c e i n t h e n u c l e u s b e f o r e t h e s p l i c i n g o c c u r s ( r e f . 27, 28). M o d i f i c a t i o n s i n t h e T$U-loop p r e c e d e d t h e p r o c e s s i n g o f t h e 5 ' - l e a d e r sequence w h i l e t h e f o r m a t i o n o f $ i n t h e a n t i c o d o n stem as w e l l as $35 p r e c e d e s t h e s p l i c i n g r e a c t i o n . However, t h e f o r m a t i o n o f 434 i s one o f t h e l a s t s t e p s i n t h e m a t u r a t i o n p r o c e s s and t h e r e s p o n s i b l e enzyme m i g h t be a c y t o p l a s m i c enzyme ( r e f . 74, 7 5 ) . I n f a c t , enzymes c a t a l y z i n g t h e f o r m a t i o n o f Gm34, 134, 434, P A 3 7 and yW37 a r e a l l c y t o p l a s m i c enzymes s u g g e s t i n g t h a t m o s t enzymes i n v o l v e d i n t h e m o d i f i c a t i o n o f a n t i c o d o n r e g i o n ( e x c e p t p o s i t i o n 35 see below) a r e p r e s e n t i n t h e c y t o p l a s m (H. Grosjean, p e r s o n a l c o m m u n i c a t i o n ) . So f a r , no m o d i f i c a t i o n has been shown t o be a p r e r e q u i s i t e f o r t h e s p l i c i n g e v e n t ( r e f . 25,

B25

76). Yeast t R N A T y r from the cytosol c a r r i e s II, a t p o s i t i o n 35. This modified nucleoside can only be introduced i n t o precursor t R N A s t i l l c a r r y i n g the i n t r o n ( r e f . 7 7 ) . This suggests t h a t t h i s anticodon modification occurs a t the nuclear membrane. Also, i n t h e case f o r Drosophila t R N A T y r , i t has been shown t h a t the i n t r o n i s needed f o r modification, s i n c e i n j e c t i o n of i n t r o n - l e s s D r o s o p h i l a t R N A T y r genes i n t o Xenopus oocytes r e s u l t s i n a l o s s of II, a t the anticodon i n the r e s u l t i n g mature t R N A T y r (E. Kubli, Univers i t y of Zurich, personal communication). T h u s , during the maturat i o n process s i z e reduction and modification a r e i n t i m a t e l y r e l a t e d processes occurring i n c o n c e r t .

Genetics and Reaulation of t R N A Modifvina Enzvmes i n Bacteria and Yeast I t can be estimated t h a t a t - l e a s t 30 d i f f e r e n t modified nucleosides e x i s t i n b a c t e r i a l t R N A . As s t a t e d above, there a r e d i f f e r e n t enzymes c a t a l y z i n g the formation of the same modified Some modified nucleoside a t d i f f e r e n t p o s i t i o n s i n t h e t R N A . nucl eosi des, 1 i ke ms*i 06A and m n m 5 s 2 U f have a compl i c a t e d s t r u c ture, and more than one enzyme i s involved i n t h e i r s y n t h e s i s . From such c o n s i d e r a t i o n s i t can be i n f e r r e d t h a t a t l e a s t 45 d i f f e r e n t tRNA-modifying enzymes e x i s t i n a e u b a c t e r i a l c e l l , Assuming an average gene s i z e of one kilobase (kb) f o r a modifying enzyme, a t l e a s t 45 kb of DNA would be required t o encode f o r tRNA-modifying enzymes. T h u s , a s u b s t a n t i a l p a r t of the g e n e t i c information (about 1%of t h e t o t a l DNA c o n t e n t ) i s devoted t o t R N A modification. However, each gene seems t o be expressed a t a low l e v e l , and thus the e n e r g e t i c load f o r the c e l l i s not t o o extensive. Mutant E . c o l i s t r a i n s exist t h a t show a l o s s of several d i f f e r e n t modified nucleosides i n t h e i r tRNAs. T h i s i s the case f o r relaxed ( r e T A ) E . c o l i s t r a i n s grown under amino a c i d s t a r v a t i o n conditions ( r e f . 78). More interesting a r e mutations t h a t lead t o l o s s of a s p e c i f i c modification i n tRNAs, without a f f e c t i n g o t h e r minor nucleosides. Table 1 l i s t s the known mutants w i t h s p e c i f i c d e f e c t s from the two b a c t e r i a ( E . c o l i and S. t y p h i m u r i u m ) and the two y e a s t s ( S a c h a r o m y c e s c e r e v i s i a e and S c h i z o s a c c h a r o m y c e s p o m b e ) t h a t have been i n v e s t i g a t e d . These mutants have 1.3.2

B26

been i d e n t i f i e d e i t h e r s e r e n d i p i t o u s l y ( r e f . 25, 79, 80) o r by d i r e c t s c r e e n i n g o f mutagenized s t r a i n s f o r u n d e r m o d i f i e d tRNAs ( r e f . 49, 50, 81-83). A l t e r n a t i v e l y , t h e y were o b t a i n e d by t h e s t u d y o f mutants i s o l a t e d t o have a s p e c i f i c phenotype: informat i o n a l suppression ( r e f . 84), r e s t r i c t i o n o f i n f o r m a t i o n a l supp r e s s i o n ( r e f . 85-90) Grossenbacher e t a1 ., ( i n p r e p a r a t i o n ) , r e d u c t i o n o f ribosomal m i s r e a d i ng ( r e f . 91), operon d e r e g u l a t i o n ( r e f . 92, 93), and loss o f r a d i a t i o n - i n d u c e d growth d e l a y ( r e f . 94, 95). The m o d i f i c a t i o n - d e f i c i e n t mutants have been used t o s t u d y t h e pathways i n m o d i f i c a t i o n b i o s y n t h e s i s and t h e c h a r a c t e r i z a t i o n o f m o d i f i c a t i o n enzymes. They a r e a l s o h e l p f u l f o r t h e m o l e c u l a r c l o n i n g o f genes t h a t code f o r enzymes i n v o l v e d i n m o d i f i c a t i o n . Many, b u t n o t a l l , mutants a l s o y i e l d i n f o r m a t i o n on t h e b i o l o g i c a l f u n c t i o n o f modified nucleosides. The absence o f a p a r t i c u l a r modified nucleoside i n tRNA i n c o r r e l a t i o n t o a specif i c phenotype o f t h e mutant s t r a i n has o f t e n produced t h e c r u c i a l evidence f o r t h e e l u c i d a t i o n o f t h e b i o l o g i c a l f u n c t i o n o f a m o d i f i e d n u c l e o s i d e (see Table 1.1). I n some cases mutants have y i e l d e d i n f o r m a t i o n on unexpected connections between m o d i f i c a t i o n pathways and i n t e r m e d i a r y metabolism ( r e f . 83, 95). The f i n d i n g t h a t m o d i f i c a t i o n s i n m i t o c h o n d r i a 1 and c y t o p l a s m i c t R N A s a r e i n t r o d u c e d by t h e same enzymes was r e v e a l e d by t h e a n a l y s i s o f mutants ( r e f . 25). Table 1.1 summarizes t h e g e n e t i c s o f t R N A m o d i f i c a t i o n . The genes a r e n o t c l u s t e r e d i n e u b a c t e r i a . I n most cases, one gene governs t h e s y n t h e s i s o f one m o d i f i e d n u c l e o s i d e . However, t h e f o r m a t i o n o f s4U and mnm5s2U r e q u i r e s more than one gene p r o d u c t . The s y n t h e s i s o f mnm5s2U34 i s governed by t h e asuE (25 min.), t h e t r m E (83 m i n . ) , trmF (83 min.) and t h e t r m C (50 min.) genes. The p o l y p e p t i d e synthesized from t h e t r m C gene has two enzymatic a c t i v i t i e s s i n c e two d i f f e r e n t mutations, t r m C l and t r m C P , accumul a t e two d i f f e r e n t d e r i v a t i v e s , cmnm5s2U and nm5s2U, r e s p e c t i v e l y , The s e q u e n t i a l f o r m a t i o n o f mnm5szU34 i n t h e t R N A ( r e f . 36, 96). i s suggested t o be: U34asUE > s5U34t r m E t cmnm5s2ut rmC 7 mn5 s2 ~ t r m C Z > mnm5s2U34. Thus, one i n t e r m e d i a t e (cmnm5s2U) i s more complex than t h e end p r o d u c t i n t h e b i o s y n t h e t i c pathway o f

B21

mnm5s2U.

The formation of s4U8 requires a t l e a s t two genes, n u v A which code f o r two polypeptides constituting the tRNA(s4U8)synthetase ( r e f . 43). Since only 11 of the 45 potential structural genes for t R N A modifying enzymes in bacteria have been i d e n t i f i e d , i t is obvious t h a t more mutants defective i n t R N A modification b o t h i n bacteria and i n yeast must be isolated before a complete picture of the complex biosynthesis as well as the function of the modified nucleosides i n t R N A will emerge. The genetic organization of a few genes has been established. The trmA gene, which codes f o r the tRNA(m5U)methyltransferase, i s monocistronic and i s transcribed counterclockwise on the E . c o l i chromosomal map ( r e f . 9 7 ) . The trmA promoter i s homologous t o the corresponding P 1 promoter of r R N A genes (P.H.R. Lindstrom, manus c r i p t i n preparation). These sequence homo1 ogi es may expl ai n the similar regulatory behavior of the trmA gene and the rRNA genes. The trmD gene, which codes f o r the tRNA(mlG37)methyltransferase, i s part of a t e t r a c i s t r o n i c operon. Unexpectedly, the f i r s t gene and the l a s t gene i n the trmD operon code f o r ribosomal proteins S16(rpsP) and L 1 9 ( r p 7 S ) , respectively ( r e f . 98). These two ribosomal proteins are made i n 100 fold larger amounts than the tRNA(m'G37)methyltransferase. Furthermore, the regulation of trmD gene expression i s n o t the same as t h a t of the surrounding ribosomal protein genes. (P.M. Wikstrom, unpublished r e s u l t s ) . Measurements of transcription under several physiological conditions revealed o n l y one large t r a n s c r i p t constituting the whole trmD operon. (A.S. BystrBm, unpublished r e s u l t s ) . Thus, the mechanism(s) behind the d i f f e r e n t i a l expression as well as the noncoordi nate regulation of expression of the genes w i t h i n the operon must operate a t the post- transcriptional l e v e l . The h i s r gene, which codes f o r the tRNA($38,39,40)synthetase I , i s p a r t of a d i f f e r e n t i a l l y expressed multicistronic operon ( r e f . 99, 100). The h i s r gene i s expressed 10-14 folds lower t h a n the upstream p d x gene, which i s involved i n the synthesis of vitamin B,. T h u s a genetic l i n k between pyridoxal phosphate biosynthesis and t R N A modification e x i s t s . Eucaryotic tRNAs contain more modified nucleosides than tRNAs from eubacteria, and consequently more genes f o r t R N A modifying enzymes must be present i n , for instance, yeast than i n E . c 0 7 i . and

nuvC,

B28

In S. c e r e v i s i a e the mutants trml and t r m 2 , lack m;G26 and m5U54, respectively, i n their tRNA (ref. 25, 79). The s i n 1 gene in the fission yeast S. pombe and the m o d 5 - l gene in S . c e r e v i c i a e govern the synthesis of i6A37 (ref. 86, 87). The m i a mutant of s. c e r e v i s i a e is deficient i n dihydrouridine (ref. 80). Two antisuppressors, s i n 3 and s i n 4 , of S. pombe reduce suppressor efficiencies of ochre and opal suppressor tRNASer, The genes were shown to be unlinked, but they influence the synthesis of the same In a single mutant of nucleoside, mcm5s2U34 (ref. 88, 102). either s i n 3 or s i n 4 the level of mcm5s2U34 is reduced 6-13 fold but in a double mutant the level of mcm5s2U34 was almost undetectable. However, another unidentified modified nucleoside is also absent in the s i n 3 and s i n 4 mutants. Thus, it is possible that these genes are required for the synthesis of another modified nucleoside. Since no intermediate in the synthesis of mcm5s2U34 was observed in these two mutants the biosynthetic pathway of this hypermodified nucleoside is still unknown. This is i n contrast to the pathway for the synthesis of mnm5s2U34 which has been unravel 1 ed by analyzing the derivatives of mnm5s2U present i n different mutants of E . c o l i (see above). Another antisuppressor mutant, s i n l 5 , reduces the efficiency of an opal suppressor tRNALeU (ref. 103). Recently, the tRNA from the mutant was shown to be deficient in ncm5U, a nucleoside present at the wobble position of tRNAs (A.-M. Grossenbacher, University of Bern, and C. Gehrke, University of Missouri, personal communication). The yeast loci, TRMl and MOD5, which are required for the synthesis of mZG26 and i6A37, respectively, have been cloned and sequenced (ref. 104, 105). Results suggest that both loci are structural genes for the corresponding enzymes and as expected they each consist of a single cistron (ref. 106). Until recently, little was known about the regulation of the synthesis of the tRNA modifying enzymes and even less about the different processing enzymes. However, the level of tRNA(m5U54)methyl transferase increases with increasing growth rate, while the activity of the tRNA(m'G37)-, tRNA(mnm5~2U34)methyl transferases, and the tRNA($38,39,40)synthestase I is invariant with the growth

TABLE 1.1 Known M u t a t i o n s That A f f e c t tRNA M o d i f i c a t i o n Nucleoside a f f e c t e d 4 - T h i o u r i d i ne,

54

U

D i h y d r o u r i d i ne, D

Primary d e f e c t

B i ol o g i c a l functions affected

E. co 7 i, nuvA

F a c t o r A o f s4U modification enzyme

Ami noacyl a t i o n o f tRNA, Growth delay, Photoprotection

43, 94, 226

E. c o l i, nuvC

Factor C o f s4U m o d i f i c a t i o n enzyme

L i k e nuvA b u t i n addi t i on t h i azol e b i osyn t hes is

95

S. cerevisiae,

Not i d e n t i f i e d

Not id e n t i f ied

80

Not id e n t if ied

79

Position w i t h i n tRNA

Organism and gene

8, L i n k acceptor-[) stems

D loop

References

mia

N* , N2 ;Dimethyl guanosi ne, m$ G

26, L i n k Danti-codon stems

S.cerevisiae, tr m l

m$G Methyl t r a n s ferase

7-(4,5-Cis-dih droxyl - c y c l openten- - y l aami no-methyl ) -7-deazaguanosi ne, Q

34, Wobble Base

E.coli

Accumulation o f Q precursor i n t RNA

E. coli,

tRNA-guanine t r a n s g l y c o s y l ase

N i t r a t e reductase expression

82, 210

Y

tgt

240

U r i d i ne-5-oxyaceti c a c i d , cmo5U o r V

34, Wobb e Base

E.co7i

Chorismic a c i d b i osyn t hes is

Not i d e n t i i e d

83

Uridine-5-oxyacetic a c i d methyl e s t e r , mcmo5U o r mV

34, Wobb e Base

S. typhimur ium, supK

mcmo5 U Methyl transferase

Decoding: UGA and f r a m e s h i f t suppression

31, 84

5-Methyl ami nomethyl 2 - t h i o u r i d i ne, mnrn5 s2 U

34, Wobble Base

E.coli, t rmE and t rmF

s2U i n s t e a d o f mnm5s2U i n tRNAs

Decoding: Reduction o f mis-reading and r e s t r i c t i o n o f nonsense suppressors

91

E . co 1 i , t rmC

Accumul a t i o n o f rnnm5 s2 U p r e c u r s o r i n tRNA

Decoding: R e s t r i ct i o n o f nonsense suppressors

96

E. col i , asuE

mnm5 s2 U-Methyltransferase

Decoding: R e s t r i ct i on o f nonsense suppressors

89

Unknown U d e r i v a t i v e i n T4 tRNAs, mnm5s2U i n E . c o 7 i tRNAs

34, Wobble Base

E.co7i

Not id e n t i f ied

Decoding: R e s t r i c t i o n o f T4 nonsense suppressors

85

5-(Methoxycaybonylmethyl ) - Z - t h i o u r i dine, mcm5s2U

34, Wobble Base

S . pombe, s i n 3 , and sin4

Not id e n t i f ied

Decoding: R e s t r i ct i o n o f nonsense suppressors

88, 102

5-Carbamoylm t h y 1 u r i d i n e , ncrn U

34, Wobble Base

S. pombe, sin15

Not id e n t i f ied

a

m1 G

l-Methylguanosine,

37, 3 ' - s i d e o f anticodon

E.coli t rmD

m1 G-Methyl t r a n s -

Decoding: R e s t r i ct i o n o f nonsense suppressors Not id e n t i f i ed

m1 G

l-Methyl guanosi ne,

37, 3': s i d e o f a n t i codon

S . t y p h moriom t rmD

m1

G-Methyl t r a n s ferase

Decodi ng : Frames h i f t i n g i n runs of c

b

2-Methylthio-N6isopentenyl-adenosine, m s 2 i 6 A

37, 3 ' - s i d e o f a n t i codon

E . co7 i , miaA,

Not id e n t i f ied

92, 171 Decoding: Attenu-174, 176 a t i o n a t t r p operon, r e s t r i c t i o n o f nonsense, suppressor codon c o n t e x t sens i t i v i t y , 1ower e r r o r l e v e l , reduced C rp and reduced growth r a t e .

E

former1 y trpX

ferase

50

msz i 0 6 A

37, 3’-side of anti codon

S. typh. miaA

Not i denti f i ed

Reduced growth rate, 90, 174 derepressi on, restriction of nonsense suppression, increased codon context sensensitivit , lower err01 leve , reduced reduced cell iield

Y

“,Lc!

N6-i sopentenyladenosine, i 6 A

37, 3‘-side of a n t i codon

S. cerevis i a e , mod5

Isopentenyl transferase

Decoding: Restricti on of nonsense suppressors

86, 87, 238

S . pombe, sin1

Isopentenyl transferase

Decoding: Restri ction of nonsense suppressors

88

Pseudouri di ne

Pseudouri d y l ate S . t y- p. h i m 38. 39, 40, anti codon u i r i u m , h i sT synthetasel o o p and stem

7-Methyl guanosi ne, m7 G

46, Variable Loop

€. c o l i trmB

,

m7G Methyltransferase

Not i denti f i ed

81

Ribothymidine, T , m5 U

54, T loop

E . col i

,

t rmA

m 5 U Methyltransferase

Small reduction in growth rate

49, 227, 245

s. c e r e v i -

m5U

Methyltransferase

Not i denti f i ed

25

s i a e , trm2

* c- r : p o l y p e p t i d e c h a i n growth r a t e . a: Grossenbacher, J . K o h l i , K.C. Kuo, and C.W. Gehrke, i n p r e p a r a t i o n . b : P.M. Wikstrom, A.S. Bystrom, and G . R . B j o r k , unpublished r e s u l t s .

’3-M.

53 212, Reduced growth rate! derepressi on, restri c- 2 4 i t i on of nonsense suppression, reduced c r i * , lower error ]&el

B32

r a t e ( r e f . 30, 107). Furthermore, t h e e x p r e s s i o n o’f t h e t R N A (m5U54)methyl t r a n s f e r a s e i s s t r i n g e n t l y r e g u l a t e d , w h i l e t h e o t h e r two t R N A m e t h y l t r a n s f e r a s e s a r e n o t ( r e f . 108). I n fact, the expression o f t h e tRNA(m5U54)methyl t r a n s f e r a s e i s r e g u l a t e d 1 ike s t a b l e RNA under a l l p h y s i o l o g i c a l c o n d i t i o n s so f a r analyzed. Thus, a l t h o u g h t h e d i f f e r e n t enzymes a r e a l l i n v o l v e d i n t h e Only m o d i f i c a t i o n o f tRNA they are n o t coordinately regulated. t h e tRNA(m5U54)methyl t r a n s f e r a s e i s r e g u l a t e d 1ike b u l k t R N A ( r e f . 107). Temperature as w e l l as composition o f t h e growth medium can s p e c i f i c a l l y i n f l u e n c e t h e expression o f genes ( r e f . 109). The f o r m a t i o n o f Gm i n B a c i l l u s s t e a r o t h e r m o p h i l u s as w e l l as t h i o l a t i o n o f m5s2U54 i n T . t h e r m o p h i l u s a r e s t i m u l a t e d by h i g h temperature ( r e f . 110, 111). However, i n t h e case o f t h e forma t i o n o f Gm18, mlA, and m5s*U54, t h e a c t i v i t y b u t n o t t h e amount o f t h e enzymes i n c r e a s e s upon s h i f t i n g t o h i g h e r temperature ( r e f . 112). M e t a b o l i c and Developmental Aspects o f t h e S v n t h e s i s o f M o d i f i e d Nucleosides I t i s w e l l e s t a b l i s h e d t h a t many m o d i f i e d n u c l e o s i d e s have amino a c i d s as d i r e c t p r e c u r s o r s . The n u c l e o s i d e s t o be m e t h y l a t e d r e c e i v e t h e methyl groups from m e t h i o n i n e v i a S-adenosyl-Lm e t h i o n i n e except i n Gram p o s i t i v e organisms i n which t h e methyl group o f m5U54, o r i g i n a t e s from t h e t e t r a h y d r o f o l a t e pathway ( r e f . 3, 44, 45). The s u l p h u r atom i n t h e t h i o l a t e d n u c l e o s i d e s o r i g i n a t e s from L - c y s t e i n e ( r e f . 113), w h i l e t h r e o n i n e i s i n c o r p o r a t e d Furthermore, t h e t 6 A r e c e i v e s a carbon i n t o tRNA forming t6A. atom from b i c a r b o n a t e ( r e f . 114-117). I n the biosynthesis o f the h y p e r m o d i f i e d n u c l e o s i d e yW, m e t h i o n i n e and l y s i n e a r e used as p r e c u r s o r s ( r e f . 118). Thus, methionine, c y s t e i n e , t h r e o n i n e , l y s i n e and carbonate a r e d i r e c t p r e c u r s o r s t o some o f t h e m o d i f i e d nucleosides. D e f i c i e n c y i n anyone o f these amino a c i d s leads t h e r e f o r e t o t h e f o r m a t i o n o f undermodified t R N A . Changes i n t h e metabolism o f t h e c e l l , which do n o t a l t e r t h e c o n c e n t r a t i o n o f t h e d i r e c t p r e c u r s o r s t o m o d i f i e d nucleosides, can s t i l l i n f l u e n c e the synthesis o f modified nucleosides leading t o formation o f undermodified t R N A . S t a r v a t i o n f o r l e u c i n e o r a r g i n i n e r e s u l t s i n a severe unbalanced growth t h a t induces s p e c i f i c u n d e r m o d i f i c a t i o n 1.3.3

B33

o f tRNAPhe and t R N A L e " ( r e f . 78). I m p o s i n g u n b a l a n c e d g r o w t h b y o t h e r means, l i k e a d d i t i o n o f a n t i b i o t i c s , a l s o r e s u l t s i n t h e s y n t h e s i s o f u n d e r m o d i f i e d tRNA ( r e f . 119, 120). However, unb a l a n c e d s y n t h e s i s as such i s p r o b a b l y n o t t h e r e a s o n f o r t h e appearance o f u n d e r m o d i f i e d tRNA; amino a c i d l i m i t a t i o n u n d e r b a l a n c e d g r o w t h a l s o r e s u l t s i n u n d e r m o d i f i c a t i o n o f tRNA ( r e f . A m u t a t i o n i n i l v U changes t h e c o n c e n t r a t i o n s o f t w o 121). i s o a c c e p t i ng tRNAVaL and t R N A L L e , and a1 so i n f l u e n c e s t h e r e g u l at i o n o f t h e i s o l e u c y l - t R N A l i g a s e ( r e f . 122). The i l v u ' p r o d u c t i s suggested t o a1 1ow d e r e p r e s s i o n o f is o l e u c y l - t R N A 1 igase, b u t a l s o t o i n h i b i t t h e m o d i f i c a t i o n o f tRNAs s p e c i f i c f o r v a l i n e and isoleucine. Thus, changes i n t h e i n t e r m e d i a r y m e t a b o l i s m o f t h e c e l l may impose s e v e r e a l t e r a t i o n s i n t h e m o d i f i c a t i o n o f t h e t R N A w h i c h i n t u r n may change t h e l e v e l o f e x p r e s s i o n o f s e v e r a l operons (see below, s e c t i o n 1 . 5 ) . Unexpectedly, t h r e e d i f f e r e n t k i n d s o f a u x o t r o p h i c mutants ( T h i - , Thy-, Aro-) a r e a l s o d e f e c t i v e i n t h e m o d i f i c a t i o n o f tRNA. M u t a n t s o f E . c o l i t h a t r e q u i r e t h i a m i n e f o r g r o w t h a l s o l a c k s4U8 i n i t s t R N A ( r e f . 9 5 ) . The m u t a t i o n maps i n t h e n o v c l o c u s and i t was h y p o t h e s i z e d t h a t f a c t o r C o f t h e tRNA(s4U8)synthetase i s a s u b u n i t o f an unknown enzyme i n t h e b i o s y n t h e s i s o f t h i a m i n e . However, n u v c may be p a r t o f an operon w h i c h a l s o encodes an enzyme i n v o l v e d i n t h e b i o s y n t h e s i s o f t h i a m i n e and t h e nuvC m u t a t i o n can a f f e c t a n o t h e r gene i n t h e o p e r o n ( c f h i s T , p d x operon, and t h e t h y A l o c u s ) . A t h y m i n e - r e q u i r i n g m u t a n t (Thy-) suppresses nonsense as w e l l as f r a m e s h i f t m u t a t i o n s , and i t was suggested t h a t some tetrahydrofolate-dependent m e t h y l a t i o n o f t R N A i s i n f l u e n c e d ( r e f . 123). However, r e c e n t l y t h e g e n e t i c o r g a n i z a t i o n o f t h e t h y A r e g i o n has been e s t a b l i s h e d . An u n i d e n t i f i e d gene upstream o f t h e t h y A gene o v e r l a p s t h e l a t t e r gene. Conseq u e n t l y , t h e s u p p r e s s o r p h e n o t y p e o f some Thy- m u t a t i o n s may be due t o a m u t a t i o n i n t h e o v e r l a p p i n g sequence f o r t h e s e t w o genes I f so, t h e c o u p l i n g o f t R N A m o d i f i c a t i o n and syn( r e f . 124). t h e s i s o f thymidine i s n o t metabolic, b u t g e n e t i c ( c f hisT, p d x above). The t h r e e a r o m a t i c amino a c i d s as we1 1 as f o u r v i t a m i n s a r e s y n t h e s i z e d b y a common pathway i n E . c o l i . The i n i t i a l s t e p , t h e c o n d e n s a t i o n o f e r y t h r o s e - 4 - p h o s p h a t e and p h o s p h o e n o l p y r u v a t e ,

B34

i s c a t a l y z e d by t h r e e isoenzymes ( F i g u r e 1.5). The f o l l o w i n g t h r e e s t e p s a r e c a t a l y z e d by p r o d u c t s o f t h e a r o B , a r o D and a r o E genes i n t h a t o r d e r and s h i k i m a t e i s produced. The a d d i t i o n a l two enzymatic s t e p s c a t a l y z e d by t h e p r o d u c t o f t h e a r o A and a r o c genes y i e l d c h o r i s m i c a c i d , which i s t h e p r e c u r s o r f o r t h e synt h e s i s o f n o t o n l y t h e t h r e e a r o m a t i c amino a c i d s b u t a l s o f o r t h e four v i t a m i n s ubiquinone, f o l a t e , menaquinone and e n t e r o c h e l i n . M u t a t i o n s i n any of these f i v e a r o genes r e s u l t i n a r e q u i r e m e n t f o r a r o m a t i c amino a c i d s f o r growth (Aro-), and g i v e r i s e t o a d e f i c i e n c y o f cmo5U34/mcmo5U34 i n t h e t R N A ( r e f . 83). The presence o f s h i k i m i c a c i d i n t h e growth medium f o r an aroD mutant r e s t o r e s t h e a b i 1it y t o s y n t h e s i z e cmo5U34/mcmo5U34 i n t h e t R N A , w h i l e t h i s c a p a c i t y i s l o s t i n an a r o c mutant. M u t a t i o n s i n any o f t h e genes s p e c i f i c f o r t h e s y n t h e s i s o f t y r o s i n e , phenyla l a n i n e , t r y p t o p h a n , ubiquinone, f o l a t e , menaquinone o r e n t e r o c h e l i n do n o t i n f l u e n c e t h e s y n t h e s i s o f t h e m o d i f i e d n u c l e o s i d e s i n tRNA ( r e f . 83, T.H. H a g e r v a l l , Y . H . Jonsson and G.R. B j d r k , Therefore, c h o r i s m i c a c i d it s e l f o r some unpubl i s h e d r e s u l t s ) unknown r e l a t e d m e t a b o l i t e t h e r e o f must p l a y a key r o l e i n t h e f o r m a t i o n o f cmo5U34/mcmo5U34 i n t h e tRNA. One i r o n t r a n s p o r t system i n b a c t e r i a l c e l l s i n v o l v e s e n t e r o c h e l i n , an i r o n c h e l a t o r t h a t o r i g i n a t e s from c h o r i s m i c a c i d ( F i g u r e 1.5). B a c t e r i a growing under ir o n 1 i m i t a t i o n produce an o u t e r membrane r e c e p t o r f o r t h e Fe3+ e n t e r o c h e l i n complex. Such c e l l s c o n t a i n i6A37 i n s t e a d o f ms2i6A37 ( r e f . 125, 126). An i d e n t i c a l e f f e c t on t h e t R N A m o d i f i c a t i o n occurs when c e l l s o f € . c o I i grow i n body f 1 u i ds where ir o n - b i n d i ng p r o t e i ns a r e p r e s e n t ( r e f . 127). I t has been suggested t h a t t h i s a l t e r a t i o n o f t R N A m o d i f i c a t i o n i s connected t o t h e a d a p t a t i o n o f E . c o l i t o grow i n A body f l u i d s and w i t h b a c t e r i a l p a t h o g e n i c i t y ( r e f . 127). d e f i c i e n c y o f t h e m e t h y l t h i o group o f ms2i6A s t i m u l a t e s t h e t r a n s p o r t o f t h e a r o m a t i c amino a c i d s ( r e f . 128). The m i a A mutant, which has an A37 i n s t e a d o f mszi6A37, has an i n c r e a s e d s y n t h e s i s o f e n t e r o c h e l i n ( r e f . 129). T h i s may i n d i c a t e t h e presence o f a r e g u l a t o r y c i r c u i t i n which t h e t r a n s p o r t o f arom a t i c amino a c i d s i s s t i m u l a t e d t o save more e n t e r o c h e l i n t o t r a n s p o r t t h e l i m i t i n g i r o n i o n s and a t t h e same t i m e s t i m u l a t e

.

B35

the synthesis o f the enterochelin. Thus, t h e s y n t h e s i s and t r a n s p o r t o f t h e aromatic amino a c i d s and e n t e r o c h e l i n a r e i n t i m a t e l y coupled t o t h e f o r m a t i o n o f msZi6A37 i n €. c o l i and msZio6A i n S. t y p h i m u r i u m . F a c u l t a t i v e a e r o b i c b a c t e r i a , l i k e S. t y p h i m u r i u m , which a r e a b l e t o grow under b o t h anaerobic as w e l l as a e r o b i c c o n d i t i o n s , change t h e i r i n t e r m e d i a r y metabolism when s h i f t e d between t h e s e two c o n d i t i o n s . The f o r m a t i o n o f one m o d i f i e d nucleoside, m s 2 i 0 6 A37 i n S. t y p h i m u r i u m r e q u i r e s m o l e c u l a r oxygen i n t h e process o f t h e h y d r o x y l a t i o n o f t h e i s o p e n t e n y l group t o form msZio6A37 from msZibA37 ( r e f . 129). This r e a c t i o n was suggested t o be i n v o l v e d i n t h e r e g u l a t i o n o f a e r o b i o s i s i n S. t y p h i m u i r i u m .

A

N5:) N‘ rnlaA+

I

IR

IPP

R

F i g u r e 1.5 L i n k s between t h e a r o m a t i c pathway and t R N A m o d i f i c a t i o n i n E . coli/S. t y p h i m u r i u m . Dashed arrows t o Phe, T y r and T r from i6A37 i n s t e a d o f msZi6A37 ( r e f . 128). The arrow between Fey+ and e n t e r o c h e l i n i n d i c a t e d t h e involvement o f t h e l a t t e r i n t h e t r a n s p o r t o f Fe3+.

B36

B a c i l l u s s u b t i l i s i s a d i f f e r e n t i a t i n g eubacterium i n which entry into the stationary growth phase i s the f i r s t stage of the sporulation process. Therefore, B . s u b t i l i s has been used as a model system for studying the developmental process. Changes in the r e l a t i v e amount of t R N A isoacceptors have been observed during development and the structural differences have been established for tRNAPhe, t R N A T v r , t R N A T r p , a n d t R N A L y S . In vegetative c e l l s the formation of ms2i6A37 i s n o t complete ( r e f . 130-133). During conditions which repress s p o r u l a t i o n the thiomethylation reaction i s also inhibited suggesting t h a t t h i s t R N A modification reaction may be coupled t o the sporulation process ( r e f . 1 3 4 ) . The pattern of isoacceptors of t R N A L v S changes during d i f f e r e n t growth phases a n d a t R N A L v S species, deficient in ms2t6A37, i s predominant in spores ( r e f . 135, 136). T h u s , developmental changes a s well as changes in the growth conditions strongly influence the modificat i o n of t R N A . During the development of the slime mold D i c t y o s t e l i u m d i s c o i d e u m , the level of m5U, 3 , and m5C decreases in the t R N A . However, t R N A containing m5U i s preferentially used in protein synthesis ( r e f . 137, 138). A decrease i n the level of modified nucleosides in t R N A during the development of the b u l l f r o g ( R a n a c a t e s b e i a n a ) has also been observed ( r e f . 139). Since the presence o f 434 has been implicated i n the codon preference ( r e f . 140) as well as an a b i l i t y t o read amber s t o p codons ( r e f . 141), i t s 1 eve1 during development i s o f p a r t i cul a r i n t e r e s t . Changes i n the level of 434 occur during the d i f f e r e n t i a t i o n of murine erythroleukemia c e l l s ( r e f . 142-145), development of D r o s o p h i l a m e l a n o g a s t e r ( r e f . 146, 147) a n d D . d i s c o i d e u m ( r e f . 148-150). Furthermore, tumor tissues completely lack 434 in t R N A ( r e f . 151). When D . d i s c o i d e u m i s grown in a defined medium, the presence of 434 i n t R N A i s dependent on queuine i n the growth medium ( r e f . 149). A1 t h o u g h the presence or absence of queuine in the medium does n o t influence the growth r a t e i n the vegetative growth phase, the presence of queuine-and thus the presence o f 434 i n tRNA-is a prerequisite f o r normal development of the slime mold ( r e f . 150). The cell cycle of mammalian c e l l s comprises f o u r successive phases. F o l l o w i n g the M phase, the c e l l s divide a n d enter the G,

B37

phase. In the G, phase the cells prepare themselves for the S phase, in which the cells synthesize DNA. One undermodified tRNALvs i soaccepting species has been correl ated to cell di vi si on (ref. 152-154). Purified growth factors stimulate specific tRNA modifying enzymes involved in the maturation of this tRNALvS species (ref. 155, 156). A temperature-sensitive hamster cellline which is blocked in the G, phase, showed an increase in the level of the undermodified tRNALvS species at nonpermissive temperature. The temperature-sensitive molecule in this mutant is suggested to be a tRNA-modifying enzyme (ref. 157). These results suggest that specific tRNA modifications may be necessary for the commitment of cell division. Less conclusive data for a relation between cell cycle and tRNA modifications exist for the yeast S . p o m b e . The antisuppressor mutation s i n 3 (see above) leads to an increase in cell length (ref. 102). Furthermore, allosuppressor. mutations that also modulate the efficiency of nonsense suppressor tRNAs, have been shown to be allelic with the cell cycle control gene c d c 2 5 (ref. 158). Prel iminary data on a1 tered tRNA modification patterns in a1 losuppressor strains have been obtained (C. Gehrke, University of Missouri, personal communication). Finally, also the c d r mutants that have a changed division response to nitrogen depletion have been shown to be allosuppressors (ref. 159). FUNCTION OF MODIFIED NUCLEOSIDES IN TRANSFER RNA None of the modified nucleosides present i n yeast tRNAPhe appear to be essential to maintain the three-dimensional structure (ref. 160). However, the presence of the modified nucleosides increases the surface area of the tRNA molecule by 20%, suggesting that the modified nucleosides are present to be recognized by various protei ns/nucl ei c acids. A1 so, many parti ci pate in unusual hydrogen bonds, e. g. mlA58-m5U54 of yeast tRNAPhe (ref. 160) and the role of certain modified nucleosides is probably to bring about these interactions and thereby contribute to the final tRNA structure, especially where loops I, 111, and IV interact. Since some modified nucleosides, like m7G and mlA, have a positive charge, this and not the methyl group p e r s e , is important for maintenance of the structure of tRNA as well as recognition sites 1.4

B38

f o r p r o t e i n s ( r e f . 161). A l l mutants so f a r c h a r a c t e r i z e d , except ( r e f . 84) and a mutant d e f i c i e n t i n mnm5s2U34 ( r e f . 85), appear t o be v i a b l e . Furthermore, some species o f M y c o p l a s m a have t R N A w i t h a v e r y low c o n t e n t o f m o d i f i e d n u c l e o s i d e s ( r e f . 162). T h i s suggests t h a t most o f t h e m o d i f i e d n u c l e o s i d e s i n t R N A a r e n o n e s s e n t i a l f o r c e l l growth b u t m i g h t have an i m p o r t a n t r o l e i n The presence o f a t h e f i n e t u n i n g o f t h e f u n c t i o n o f t h e tRNA. m o d i f i e d n u c l e o s i d e may i n c r e a s e o r d e c r e a s e v a r i o u s i n t e r a c t i o n s Therefore, each m o d i f i c a t i o n may have i t s unique o f the tRNA. f u n c t i o n and may have evolved as a consequence o f changes i n t h e t R N A molecule. The m o d i f y i n g enzymes themselves may a l s o have a r e g u l a t o r y f u n c t i o n besides c a t a l y z i n g t h e f o r m a t i o n o f m o d i f i e d n u c l e o s i d e s ( r e f . 163, 164). supK

1.4.1

M o d i f i e d Nucleosides Next t o t h e 3 ' - S i d e o f t h e Anticodon ( P o s i t i o n 37) I n f l u e n c e T r a n s l a t i o n a l E f f i c i e n c y . Transl a t i o n F i d e l i t y . S e n s i t i v i t v o f t h e t R N A t o t h e Readina Context and Readina Frame Maintenance. P o s i t i o n 37 i s h i g h l y prone t o be m o d i f i e d , and a c o r r e l a t i o n e x i s t s between t h e k i n d o f m o d i f i c a t i o n p r e s e n t i n t h i s p o s i t i o n tRNAs from and t h e c o d i n g c a p a c i t y o f t h e t R N A ( F i g u r e 1.4). eucaryotes and e u b a c t e r i a r e a d i n g UXX codons u s u a l l y have a b u l k y hydrophobic m o d i f i c a t i o n a t t h i s p o s i t i o n . However, such tRNAs from a r c h a e b a c t e r i a l and M y c o p l a s m a c o n t a i n mlG37, why t h e e a r l i e r suggestion t h a t these b u l k y hydrophobic m o d i f i c a t i o n s a r e necess a r y t o s t r e n g t h e n t h e weak A36-U i n t e r a c t i o n must be questioned. I n f a c t , tRNAs from e u b a c t e r i a w i t h an unmodified A37 a r e a b l e t o However, read codons s t a r t i n g w i t h U ( r e f . 62, 63, 165, 166). when p r e s e n t i n t R N A b o t h t h e ms2- and mszi6A- m o d i f i c a t i o n s have been shown t o s t a b i l i z e anticodon-anticodon i n t e r a c t i o n ( r e f . 167, 168) and t h e msz-group t o s t a b i l i z e t h e secondary s t r u c t u r e o f a p o l y n u c l e o t i d e ( r e f . 169). T r a n s f e r RNAPhe from i r o n s t a r v e d E . c o l i c o n t a i n s i6A37 i n s t e a d o f msZi6A37 and i s much l e s s e f f i c i e n t i n p o l y U - d i r e c t e d However, no e f f e c t o f t h e polyphenylalanine synthesis i n v i t r o . m o d i f i c a t i o n d e f i c i e n c y was observed u s i n g n a t u r a l mRNA as temp l a t e ( r e f . 170). C e l l s grown i n i r o n - l i m i t i n g media c o n t a i n ms2d e f i c i e n t t R N A and have a derepressed t r y p t o p h a n operon, most

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l i k e l y due t o a l o w e r a b i l i t y t o r e a d t h e two contiguous t r y p tophan codons i n t h e t r p l e a d e r mRNA ( r e f . 171). The ms2-defi c i e n t t R N A may t h e r e f o r e read contiguous codons such as t h o s e p r e s e n t i n polyU and i n l e a d e r mRNA l e s s e f f i c i e n t l y t h a n codons I f so, t h e ms2n o t r e i t e r a t e d as i n genes coding f o r p r o t e i n s . m o d i f i c a t i o n i s more i m p o r t a n t i n some codon c o n t e x t s t h a n i n o t h e r s (see below). The m i a A mutants o f E . coli/S. t y p h i m u r i u m l a c k ms2i6A37/ms2i06A37 and have an unmodified A37 ( r e f . 90, 171). The m i a A l mutant o f S. t y p h i m u r i u m shows a l a r g e (up t o 50%) r e d u c t i o n i n growth r a t e and a reduced p o l y p e p t i d e c h a i n e l o n g a t i o n r a t e i n v i v o . Furthermore, t h e r e g u l a t i o n o f s e v e r a l b i o s y n t h e t i c operons i s a f f e c t e d ( r e f . 90). Lack o f ms2i6A37/ms2i06A37 reduces t h e e f f i c i e n c y o f nonsense suppression i n v i v o , o f t R N A b i n d i n g t o t h e ribosome, and UGA-suppression i n v i t r o ( r e f . 168, 172-174). However, t h i s m o d i f i c a t i o n i s n o t e s s e n t i a l f o r t h e a c t i v i t y o f t h e tRNA, because t h e m i a A mutant i s v i a b l e , ( r e f . 90, 92) and s e v e r a l f u n c t i o n a l tRNAs e x i s t t h a t have an unmodified A37 ( r e f . 62, 63). Thus, t h e e x t e n t o f t h e e f f e c t o f ms2i6A37 v a r i e s w i t h T h i s has a l s o been observed ext h e t R N A o f which i t i s p a r t . p e r i m e n t a l l y ( r e f . 174). Suppressor tRNAs l a c k i n g ms2i6A37/ms2io6A37 a r e more s e n s i t i v e t o t h e sequence s u r r o u n d i n g t h e codon than t h e w i l d t y p e tRNAs which suggests t h a t t h e ms2i ( o ) 6 - m o d i f i c a t i o n i s d i r e c t l y i n v o l v e d i n d e t e r m i n i n g t h e i n t r i n s i c codon c o n t e x t s e n s i t i v i t y o f t h e t R N A ( r e f . 174). T h i s m o d i f i e d n u c l e o s i d e seems n o t t o be i n v o l v e d i n t h e amino a c i d c h a r g i n g r e a c t i o n ( r e f . 92, 175). The t r a n s l a t i o n a l e r r o r l e v e l Since i s reduced b o t h i n v i v o and i n v i t r o ( r e f . 172-174, 176). an improved accuracy r e q u i r e s energy ( f o r r e v i e w see r e f e r e n c e 177) t h e decreased c e l l u l a r y i e l d ( r e f . 90) and p e p t i d y l r e l e a s e Combination o f t h e m i a A m u t a t i o n ( r e f . 178) may be reasonable. and ribosomal mutations, which a l s o i n c r e a s e s t h e f i d e l i t y , reduces t h e growth r a t e , and i n some cases t h e c e l l becomes s t r e p t o m y c i n dependent, s i n c e t h i s a n t i b i o t i c i s known t o suppress t h e p r o o f r e a d i n g f l o w s ( r e f . 90, 173, 176). Thus, t h e ms2i(o)6m o d i f i c a t i o n p l a y s an i m p o r t a n t p a r t i n t h e e f f i c i e n c y and f i d e l i t y o f t r a n s l a t i o n as w e l l as i n t h e s e n s i t i v i t y o f t h e t R N A t o t h e sequence s u r r o u n d i n g t h e codon.

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T r a n s f e r RNA from y e a s t which reads codons s t a r t i n g w i t h U c o n t a i n i6A37 ( c f F i g u r e 1 . 4 ) . A n t i s u p p r e s s o r m u t a t i o n s o f y e a s t , s i n l and m o d 5 - 1 , b o t h c o n t a i n an unmodified A37 i n s t e a d o f i6A37 ( r e f . 86, 87). Lack o f t h e i 6 - m o d i f i c a t i o n reduces t h e a c t i v i t y of t h e s e r i n e i n s e r t i n g UGA and UAA suppressors and t h e t y r o s i n e However, b o t h mutants grow r e l a t i v e l y i n s e r t i n g UAA suppressors. w e l l , showing o n l y a 10% r e d u c t i o n i n growth r a t e i n t h e case o f Thus, l i k e t h e msZi( o ) 6 - m o d i f i c a t i o n t h e s i n l mutant ( r e f . 8 7 ) . i n e u b a c t e r i a l tRNA, t h e i 6 - m o d i f i c a t i o n i n y e a s t t R N A i s i n v o l v e d i n t h e anticodon-codon i n t e r a c t i o n . T h i s suggests t h a t a p a r t o f t h e e f f e c t observed i n m s 2 i ( o ) 6 - d e f i c i e n t t R N A i s due t o t h e l a c k o f t h e i6-modi f i c a t i o n . The removal o f yW37 o f y e a s t tRNAPhe lowers i t s a b i l i t y t o b i n d t o ribosomes and t o s y n t h e s i z e p o l y p h e n y l a l a n i n e i n a polyUprogrammed t r a n s l a t i o n system ( r e f . 179). Furthermore, t R N A P h e d e f i c i e n t i n yW37 shows a v a r i a b l e e f f i c i e n c y o f decoding, r e l a t i v e t o t h e f u l l y m o d i f i e d tRNA, a t d i f f e r e n t s i t e s i n g l o b i n mRNA, and e s p e c i a l l y a t tandem Phe-codons ( r e f . 180). Furthermore, a t such tandem codons t h e f i r s t s e l e c t e d t R N A i n f l u e n c e s t h e selection o f the next. T h i s m o d i f i e d n u c l e o s i d e has been suggested t o i n t e r c a l a t e between t h e two codon-anti codon t r i p 1 e t duplexes p r e s e n t i n t h e A- and P - s i t e on t h e ribosome ( r e f . 181). The model suggests t h a t b o t h i n t h e A and P - s i t e t h e yW-base i s i n c o n t a c t w i t h t h e mRNA and s t a b i l i z e s t h e codon-anticodon i n t e r a c t i o n by s t a c k i n g . t R N A from a l l t h r e e kingdoms r e a d i n g AXX codons u s u a l l y c o n t a i n s a t 6 A d e r i v a t i v e i n p o s i t i o n 37 ( F i g u r e 1.4). T h i s conserved f e a t u r e i m p l i e s a common f u n c t i o n i n these groups o f t R N A , such as t h e suggested s t r e n g t h e n i n g o f t h e weak b i n d i n g o f U36 t o Accordingly, E . c o l i t h e A i n t h e f i r s t p o s i t i o n o f t h e codon. t R N A * L e , d e f i c i e n t i n t6A37, has a reduced a b i l i t y t o b i n d t o poly(A,U,C) o r poly(A,U,U)-programmed ribosomes b u t behaves normal l y i n t h e c h a r g i n g r e a c t i o n ( r e f . 182). E u c a r y o t i c tRNALvS d e f i c i e n t i n t6A37 i s l e s s e f f i c i e n t compared t o f u l l y m o d i f i e d tRNALvS i n t r a n s l a t i n g AAG codons i n v i t r o , and s i t e s p e c i f i c e f f e c t s r e l a t i v e t o f u l l y m o d i f i e d t R N A was observed ( r e f . 180). A t tandem AAG codons t h e f u l l y m o d i f i e d tRNALvS i s p r e f e r r e d as t h e f i r s t s e l e c t e d t R N A w h i l e t h e t637 d e f i c i e n t tRNALvS i s

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p r e f e r r e d as t h e second s e l e c t e d t R N A ( r e f . 180). From measurements o f a n t i c o d o n - a n t i c o d o n a s s o c i a t i o n as w e l l as b i n d i n g t o p o l y (A,G)-programmed ribosomes o r f r e e AGA t r i p l e t s , i t has been c o n c l u d e d t h a t t 6 A s t a b i l i z e s t h e base p a i r between t h e n u c l e o t i d e (U36) a d j a c e n t t o t h e 5 ' - s i d e o f t h e m o d i f i e d n u c l e o s i d e and t h e f i r s t n u c l e o t i d e o f t h e codon most p r o b a b l y b y an i n c r e a s e d s t a c k i n g i n t e r a c t i o n ( r e f . 183). tRNAs r e a d i n g AXX codons c o n t a i n an U36-PA37 sequence. E x p e r i m e n t s u s i n g Upt6A showed t h a t t 6 A s t a b i l i z e s t h e s t a c k i n g i n t e r a c t i o n compared w i t h UpA, and p r e v e n t s wobble a t t h e 3 ' - s i d e o f t h e a n t i c o d o n ( r e f . 184). F i g u r e 1.3 shows t h a t tRNAs t h a t r e a d codons s t a r t i n g w i t h C o r G u s u a l l y have an u n m o d i f i e d p u r i n e n u c l e o s i d e o r a s i m p l e m o d i f i c a t i o n l i k e mlG, m2A, e t c . , p r e s e n t i n p o s i t i o n 37 ( F i g u r e 1.3). The c o n s e r v e d p r e s e n c e o f m1G37 i n tRNAs t h a t r e a d codons s t a r t i n g w i t h C i s noteworthy. The e n e r g e t i c a l l y more s t a b l e GC p a i r s s h o u l d have a l o w e r r e q u i r e m e n t o f s t a b i l i z a t i o n o f t h e anticodon-codon i n t e r a c t i o n . However, some o f them, l i k e m'G and m6A, d e s t a b i l i z e t h e Watson-Crick d o u b l e - h e l i c a l s t r u c t u r e b y 1.01.8 K c a l h o l o f m e t h y l s u b s t i t u e n t ( r e f . 185). They may a l s o p r e v e n t t h e f o r m a t i o n o f hydrogen bonds t o n u c l e o t i d e s on t h e 5 ' s i d e o f t h e codon as has been s u g g e s t e d f r o m t h e o r e t i c a l cons i d e r a t i o n s ( r e f . 186). The p r e s e n c e o f m1G37 i s n o t e s s e n t i a l f o r t h e f u n c t i o n o f a t R N A s i n c e a few tRNAs e x i s t , w h i c h have an u n m o d i f i e d G37, and c h i m e r i c tRNAs w i t h an u n m o d i f i e d G37 a r e f u n c t i o n a l in v i t r o ( r e f s . 21, 1 6 5 ) . However, a m u t a n t ( t r m D 3 ) o f 3. t y p h i m u r i u m , i n w h i c h mlG37 i s a b s e n t a t 41°C b u t n o t a t 30"C, has a d r a m a t i c r e d u c t i o n i n g r o w t h r a t e a t 41°C. Simultaneously, t h e m u t a n t has a c q u i r e d a c a p a c i t y t o f r a m e s h i f t a t r u n s o f C a t 41°C (Wikstrom, P.M., A.S. B y s t r o m and G.R. B j o r k , u n p u b l i s h e d results). One p o s s i b i l i t y i s t h a t t h e p r e s e n c e o f a m e t h y l g r o u p i n p o s i t i o n 1 o f G i n f l u e n c e s t h e p r e c i s i o n o f t h e anticodon-codon i n t e r a c t i o n b y p r e v e n t i n g an i n t e r a c t i o n w i t h t h e 5 ' - n e i g h b o r i n g n u c l e o t i d e i n t h e mRNA. I n summary, t h e m o d i f i c a t i o n a t t h e 3 ' - s i d e o f t h e a n t i c o d o n ( p o s i t i o n 37) i n f l u e n c e s c e l l p h y s i o l o g y i n a p r o f o u n d way. More precisely, these m o d i f i c a t i o n s i n f l u e n c e n o t o n l y t h e e f f i c i e n c y and t h e a c c u r a c y o f t h e t R N A i n i t s d e c o d i n g f u n c t i o n b u t a l s o

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c o n t r i b u t e i n t h e sensing of t h e n u c l e o t i d e n e x t t o t h e codon, and t h e y may be i n v o l v e d i n r e a d i n g frame maintenance. 1.4.2

M o d i f i e d Nucleosides a t t h e Wobble P o s i t i o n ( P o s i t i o n 34) I n f l u e n c e T r a n s l a t i o n a l F i d e l i t v and Codon Choice The wobble n u c l e o s i d e a t p o s i t i o n 34 i s , l i k e t h e n u c l e o s i d e a t p o s i t i o n 37, o f t e n m o d i f i e d . A c o r r e l a t i o n e x i s t s between t h e k i n d of m o d i f i e d n u c l e o s i d e p r e s e n t i n p o s i t i o n 34 and t h e decod The s2U d e r i v a t i v e s w i t h i n g c a p a c i t y of t h e tRNA ( r e f . 1). d i f f e r e n t 5 s u b s t i t u t i o n s ( e u b a c t e r i a and eucaryotes) a r e p r e s e n t i n tRNAs t h a t r e a d codons o f t h e t y p e NAAfG where N can be C, A, o r G. I n t r i p l e t - d e p e n d e n t b i n d i n g t o t h e ribosome and i n p r o t e i n s y n t h e s i s i n v i t r o , s 2 U - d e r i v a t i v e s r e c o g n i z e p r i m a r i l y A more e f f i c i e n t l y t h a n G as t h e t h i r d l e t t e r o f t h e codon ( r e f s . 187, 188). P r o t o n NMR analyses have shown t h a t t h e s e d e r i v a t i v e s almost e x c l u s i v e l y have a conformation t h a t a l l o w s t h e r e c o g n i t i o n o f A b u t n o t o f U and G ( r e f . 189). Such a n a l y s i s have shown t h a t b o t h t h e 5 s u b s t i t u t i o n and t h e t h i o c a r b o n y l group s t a b i l i z e t h e c o n f o r m a t i o n necessary f o r p r e v e n t i n g m i s r e a d i n g o f codons which Howend w i t h U o r C as w e l l as r e s t r i c t t h e wobble towards G. ever, t h e presence o f t h e t h i o c a r b o n y l group has been shown n o t t o be i n v o l v e d i n t h e p r e f e r e n t i a l r e c o g n i t i o n o f A b u t has been suggested t o i n c r e a s e t h e s t a c k i n g i n t e r a c t i o n w i t h i n t h e ant i c o d o n ( r e f s , 190-192). Two mutants, t r m c l and trmC.2, o f E . c o 7 i a r e b o t h d e f e c t i v e i n t h e s y n t h e s i s o f mnm5sW34, which i s n o r m a l l y p r e s e n t i n tRNA:X: sop^). The e f f i c i e n c y of tRNA:j;; i n r e a d i n g b o t h UAA and UAG codons i s reduced i n t h e trmC mutants, and t h e u n d e r m o d i f i e d tRNA:j;; i s a l s o more s e n s i t i v e t o t h e codon c o n t e x t than t h e normal tRNAbj;; ( r e f . 96). The d e r i v a t i v e s o f mnm5s2U34 p r e s e n t i n t h e trmCl and trmC.2 mutants a r e cmnm5s2U and nm5s2U, r e s p e c t i v e l y ( r e f . 36). Mutants ( t r m E , t r m ~ ) t h a t have s2U34 i n s t e a d o f mnm5s2U34 i n t h e i r t R N A a r e more a b l e t o r e a d UAA t h a n UAG codons. Such u n d e r m o d i f i e d t R N A a l s o b i n d s AAA b e t t e r t h a n AAG i n v i t r o ( r e f . 91). A mutant o f E . c o l i , a s u ~ , p r o b a b l y d e f i c i e n t i n s2U34, has been i s 0 1 a t e d as an a n t i suppressor t o s u p L , (tRNAh1:) ( r e f . 89). Thus, a l t e r a t i o n s o f t h e s i d e c h a i n a t p o s i t i o n 5 as w e l l as t h e t h i o group seem t o be i m p o r t a n t i n t h e decoding

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capacity of mnm5s2U34. These mutants a r e a l l viable, even though they a l l lack mnmsszU34. However, they a l l possess a derivative of m n m 5 s 2 U t h a t e i t h e r has the t h i o group or some k i n d of a s i d e chain. A temperature-sensitive mutant of E . c o 7 i has been isolated t h a t i s deficient in mnm5s2U34 ( r e f . 85). The chemical s t r u c t u r e of the undermodified derivative present i n t h i s mutant has not been determined. I f the temperature s e n s i t i v i t y of t h i s mutant i s due t o the lack of mnm5s2U34 and the presence of a completely unmodified U, the reason might be t h a t t h i s mutant misreads codons, which end w i t h U or C , a t h i g h temperature because the mnm5s2-modification was suggested t o r e s t r i c t the a b i l i t y t o bind t o U ( r e f . 189). The other mutants (trmCI, c2, f , F , and a s u f ) which contain e i t h e r a t h i o group or a s i d e chain a r e s t i l l r e s t r i c t e d i n the misreading of codons t h a t end with U or C , and thus these c e l l s are viable. However, they read codons ending w i t h e i t h e r A o r G w i t h reduced efficiency. Yeast tRNAs t h a t read s p l i t codon families have mcm5s2U34 or nm5U34, and i t has been suggested t h a t the presence of t h i s nucleoside explains why yeast ochre suppressors d o not read UAG and why the ncm5s2U34 containing tRNAz;; can not read UCG ( r e f s . 193-195). The antisuppressors s i n 3 and s i n 4 of 5. pombe inact i v a t e the s e r i n e inserting ochre suppressor and the double mutant has almost no mcm5s2U34 b u t an increased level of s2U34 ( r e f . 102). Independent of t h e i r e f f e c t on t R N A suppressors, the two mutations reduce the growth r a t e . I n v i v o decoding of the s e r i n e codon UCG by the UCA reading s e r i n e t R N A i s not promoted in the presence of the mutations s i n 3 and s i n 4 ( r e f . 102). These r e s u l t s support the r e s t r i c t i v e function of the s2U-derivatives as suggested from e a r l i e r experiments i n v i t r o as well as from model building, NMR analysis and crystal s t r u c t u r e s of such modified nucl eosides. Another group of modified uridines (moSU, cmo5U, mcmo5U) has been found i n position 34 of tRNAs s p e c i f i c f o r valine (GUN), s e r i ne (UCN) , pro1 ine (CCN) , threoni ne (ACN) and a1 ani ne (GCN) . In t r i p l e t dependent binding ( r e f s . 196-199) and i n v i t r o synt h e s i s of MS2 coat protein ( r e f s . 200, 201), these modified uridines can read n o t o n l y codons ending w i t h A o r G b u t a l s o

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codons ending w i t h U. From NMR a n a l y s i s i t has been concluded o f t h e 5 - s u b s t i t u e n t i n t e r a c t s w i t h t h e 5'-phost h a t t h e -OCH,phate t o b r i n g about a f l e x i b i l i t y o f t h e wobble n u c l e o s i d e so as t o r e c o g n i z e U, A, and G ( r e f . 189). These m o d i f i e d n u c l e o s i d e s a r e p r e s e n t i n tRNAs r e a d i n g f a m i l i e s o f f o u r codons s p e c i f y i n g t h e same amino a c i d . A l l these codon f a m i l i e s a r e a l s o r e a d by a t l e a s t one o t h e r t R N A species, which reads codons ending w i t h U o r C a c c o r d i n g t o t h e wobble h y p o t h e s i s . Furthermore, t R N A from m i t o c h o n d r i a w i t h an unmodified U i n t h e wobble p o s i t i o n , and t h e o n l y e x i s t i n g tRNAGLy, t R N A A L a , tRNAPro and tRNAvaL f r o m M y c o p l a s m a m y c o i d e s a r e a b l e t o read a l l f o u r codons i n such a f a m i l y ( r e f s . 202, 203, 204; Samuelson, T., Y.S. Guindy, F. L u s t i g , T. Boren and U. L a g e r k v i s t , personal communciation). Thus, i t i s n o t obvious why these m o d i f i c a t i o n s a r e p r e s e n t . However, an Aromutant o f E . c o l i t h a t l a c k s cmo5U34/mcmo5U34 i n t R N A grows 20% slower than Aro' c e l l s i n r i c h medium, i n d i c a t i n g t h a t t h e presence o f cmo5U under some p h y s i o l o g i c a l condi t i ons is i m p o r t a n t . (BjCirk, G.R., unpublished r e s u l t s ) . T h i s wobble base i s w i t h i n 4 i o f a p y r i m i d i n e i n 16s r R N A when t h e t R N A i s i n t h e P - s i t e on t h e ribosome ( r e f . 205). T h i s may extend t h e a n t i c o d o n s t a c k i n t o t h e 16s r R N A and s t a b i 1 ize t h e t R N A - r i bosome i n t e r a c t i o n . The importance o f t h e m o d i f i c a t i o n as such i n t h i s i n t e r a c t i o n has n o t been e l u c i dated. A r e c e s s i v e UGA suppressor ( s u p K ) , which a l s o suppresses some f r a m e s h i f t m u t a t i o n s , has been i s o l a t e d ( r e f s . 84, 206). The supK mutants a r e d e f i c i e n t i n a t R N A methyl t r a n s f e r a s e , which most l i k e l y c a t a l y z e s t h e f o r m a t i o n o f mcmo5U i n some t R N A species ( r e f . 207). (See F i g u r e 1 . 4 ) . These r e s u l t s suggest t h a t a t R N A which has cmo5U34 i n s t e a d o f mcmo5U34 i s a b l e t o r e a d t h e UGA codon o r t o f r a m e s h i f t . However, t h e suppressing agent has so f a r n o t been i d e n t i f i e d as a t R N A species, and t h e mechanism behind t h i s suppression i s s t i l l unknown. E l o n g a t o r t R N A Y t o f E . c o l i c o n t a i n s ac4C i n p o s i t i o n 34, w h i l e i n i t i a t o r tRNA:et c o n t a i n s a C. By c h e m i c a l l y removing t h e ac4-modification, i t was shown t h a t t h e t R N A r t which l a c k s ac4C34 b i n d s t o AUG-programmed ribosomes almost t w i c e as w e l l as t h e t R N A r t c o n t a i n i n g ac4C ( r e f . 208). However, t h e presence o f ac4C

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decreases t h e misreading in v i t r o o f AUA ( I l e ) . Thus, t h e funct i o n of a c 4 - m o d i f i c a t i o n appears p r i m a r i l y t o reduce t h e misreadT h i s i s achieved by a somewhat i n g o f t h e AUA ( I l e ) codon. reduced e f f i c i e n c y t o read t h e AUG (Met) codon. I n 5. c e r e v i s i a e a l e u c i n e - i n s e r t i n g amber suppressor t R N A n o r m a l l y c o n t a i n s m5C i n t h e wobble p o s i t i o n . By d e l e t i n g t h e i n t r o n , t h e tRNA(m5C34)methyl t r a n s f e r a s e does n o t r e c o g n i z e in v i t r o t h e almost mature tRNA as s u b s t r a t e which r e s u l t s i n a tRNA&," t h a t has an unmodified C i n t h e wobble p o s i t i o n . Such tRNA&,", produced from an i n t r o n l e s s gene i s l e s s e f f i c i e n t i n suppression ( r e f . 209). I f t h i s tRNA a l s o l a c k s m5C34 in v i v o t h i s r e s u l t suggests t h a t t h e presence o f m5C34 improves t r a n s 1 a t ion e f f ic i ency The hypermodified n u c l e o s i d e Q i s p r e s e n t i n p o s i t i o n 34 o f tRNAs s p e c i f i c f o r t y r o s i n e , h i s t i d i n e , asparagine, and a s p a r t i c a c i d t h a t reads codons ending w i t h U o r C. A mutant, ( t g t ) , o f E . c o 7 i t h a t l a c k s 434 d i e s i n s t a t i o n a r y phase and i s unable t o s y n t h e s i z e n i t r a t e reductase under anaerobic c o n d i t i o n s ( r e f s . 82, 210). Q - m o d i f i c a t i o n was suggested t o p l a y a r o l e i n t h e express i o n o f n i t r a t e reductase ( r e f . 210). However, i t was n o t r u l e d o u t t h a t t h e mutant harbors a d d i t i o n a l m u t a t i o n s , which may e x p l a i n t h e observed p l e i o t r o p i c e f f e c t s . The presence o f Q a f f e c t s t h e codon choice, because tRNAHiS c o n t a i n i n g G34 i n s t e a d o f 434 p r e f e r s CAC t o CAU, w h i l e low p r e f e r e n c e was observed w i t h However, presence o f 434 i n f u l l y m o d i f i e d t R N A H i s ( r e f . 140). t R N A H i s t r a n s l a t i n g g l o b i n mRNA in v i t r o d i d n o t i n f l u e n c e t h e t R N A T y r from D r o s o p h i l a m e l a n o g a s t e r o r codon c h o i c e ( r e f . 180). tobacco p l a n t s and c o n t a i n i n g G34 i n s t e a d o f 434 a r e a b l e t o read UAG s t o p codons ( r e f . 141, 211), and i t has been suggested t h a t p a r t i a1 suppression o f t e r m i n a t i o n codons m i g h t be a r e g u l a t o r y d e v i c e ( r e f s . 212, 213). Thus, t h e m o d i f i c a t i o n o f G t o Q may be i n v o l v e d i n t h e r e g u l a t i o n o f gene expression (see S e c t i o n 1.3.3).

.

1.4.3

M o d i f i e d Nucleosides i n t h e Anticodon Reaion O t h e r Than i n P o s i t i o n 34 and 37 I n f l u e n c e T r a n s l a t i o n a l E f f i c i e n c y and F i d e l it v A m u t a t i o n i n t h e h i s r gene o f s. t y p h i m u r i u m r e s u l t s i n a $ d e f i c i e n c y i n p o s i t i o n s 38, 39, and 40 i n many t R N A chains, and

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among them tRNAHiS ( r e f s . 52, 214, 215). The growth r a t e , t h e p o l y p e p t i d e c h a i n e l o n g a t i o n r a t e , and t h e e r r o r l e v e l a r e reduced i n a h i s T mutant ( r e f s . 216, 217). Such mutants have been i s o l a t e d by t h e i r a b i l i t y t o derepress t h e h i s t i d i n e operon ( r e f . 93). The h i s t i d i n e l e a d e r mRNA c o n t a i n s seven h i s t i d i n e codons i n a row, which a r e r e a d i n e f f i c i e n t l y by t h e u n d e r m o d i f i e d t R N A H i S . T h i s l e a d s t o d e r e p r e s s i o n o f t h e h i s t i d i n e operon ( r e f . 218). Since t h e a n t i c o d o n stem o f s e v e r a l tRNAs n o r m a l l y c o n t a i n s $, a h i s T m u t a t i o n has a p l e i o t r o p i c e f f e c t and a c c o r d i n g l y i n f l u e n c e s t h e r e g u l a t i on o f s e v e r a l amino a c i d b i o s y n t h e t i c operons, probabl y through an a t t e n u a t i o n mechanism ( r e f . 215). Lack o f $ i n t h e a n t i c o d o n stem a l s o reduces t h e e f f i c i e n c y o f suppression by s u p € (tRNAiA2) and s u p F (tRNAA1;) b u t has no o r o n l y a m i n o r e f f e c t i n sensing t h e sequence s u r r o u n d i n g t h e codon ( r e f . 219; B j o r k , G.R., unpubl.ished r e s u l t s ) . D e l e t i o n o f t h e i n t e r v e n i n g sequence o f a y e a s t tRNAL1; ochre suppressor gene r e s u l t s i n inabi 1it y o f t h e modi f y i ng enzyme normally catalyzing the formation o f $ i n the middle o f the a n t i c o d o n ( p o s i t i o n 35) t o p e r f o r m i t s f u n c t i o n . The r e s u l t i n g ochre suppressor tRNAL1; has an unmodified U35 i n s t e a d o f $35, and such t R N A has a much l o w e r e f f i c i e n c y o f suppression ( r e f . 7 7 ) . These r e s u l t s suggest t h a t $35 i s i m p o r t a n t i n anticodon-codon in t e r a c t i o n . By chemical m o d i f i c a t i o n o f s T 3 2 o f t R N A A r g , i t has been shown t h a t t h e s t r u c t u r e o f t h e a n t i c o d o n l o o p i s a l t e r e d . Such t R N A A r g a l s o suppresses t h e n o r m a l l y o c c u r r i n g f r a m e - s h i f t i n g in t h e t r a n s l a t i o n o f MS2 RNA i n v i t r o ( r e f . 220). 1.4.4

M o d i f i e d Nucleosides O u t s i d e t h e Anticodon Reaion Mav S t a b i l i z e t R N A Conformation Lack o f m:G26 i n y e a s t tRNASer reduces t h e i n v i t r o c h a r g i n g a b i l i t y by 20% s u g g e s t i n g t h a t some tRNASer s p e c i e s a r e n o t chargeable ( r e f . 221). T r a n s f e r RNA, d e f i c i e n t i n d i h y d r o u r i d i n e s u p p o r t s p o l y p h e n y l a l a n i n e s y n t h e s i s i n v i t r o w i t h t h e same Chemical d e r i v a t i z a t i o n o f e f f i c i e n c y as normal t R N A ( r e f . 80). acp3U47 o f t R N A P h e does n o t i n f l u e n c e t h e a c t i v i t y o f t h e tRNAPhe i n t h e aminoacylation o r polyphenylalanine synthesis i n v i t r o ( r e f . 222). Chemical r e d u c t i o n o f m7G47 d i s r u p t s t h e C13-622-

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m7G47 base triple, which leads to a slightly less ordered tRNA structure (ref. 223). A mutant of E . c o l i , trmi?, defective in the formation of 1117647, grows more slowly than a t r m P cell, supporting the idea that m7G47 stabilizes the structure of the tRNA (ref. 224). Methylation i n v i t r o producing m7G47 or m2G10 results in altered kinetics of aminoacylation (refs. 224, 225). An E . c o l i mutant, lacking s4U8 in its tRNA, shows identical growth characteristics as the wild type cells. However, the s4U8 is involved in the photoprotection phenomenon (refs. 94, 226). Thus, modification in parts other than the anticodon region may be involved in the stabilization of the tRNA structure. Therefore, more specific assays are necessary to reveal the function(s) of these modified nucl eosi des. Ribothymidine (m5U54, rT54) is one of the most abundant modified nucleosides, and because it is present in tRNA from both eucaryotes and eubacteria, its presence was thought to be essential for growth. However, a mutant ( t r m A 5 ) of E . c o l i , completely lacking m5U54, is viable but is outgrown by a trmAt cell in a mixed population experiment (ref. 227). The difference in the growth rates of the wild type and the trmA5 mutant is 4% (Bjark, G.R., unpublished results). A yeast mutant ( t r m 2 ) lacking m5U54 grows normally (ref. 25). Furthermore, some bacterial species Lack of normally lack m5U54 in their tRNAs (refs. 228, 229). m5U54 facilitates initiation of protein synthesis i n S t r e p t o c o c c u s f a e c a l i s with unformylated tRNAYet, and a similar mechanism may operate in E . c o l i (refs. 12, 230-232). All these results show that m5U54 is. not essential for cell growth but may slightly enhance the function of tRNA. Some eucaryotic tRNAs normally have U54 and are able to accept methyl groups i n v i t r o , which results in an m5U54 containing tRNA. Using pairs of such tRNAs, only differing in the absence or presence of m5U54, it was shown that the activity of tRNAPhe and tRNACLy was increased and decreased, respectively, when m5U54 was present, suggesting that the degree of m5U54 in some eukaryotic tRNAs may regulate protein synthesis (refs. 233, 234). The content of m5U54 seems to influence the elongation factor directed A-si te binding and the misincorporation of leucine in a polyU directed polyphenylalanine synthesizing

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system ( r e f . 235). Lack o f m5U54 augmented t h e i n t r i n s i c misreadi n g c a p a c i t y o f tRNAZeu, and d i d n o t induce o t h e r tRNALeU species t o m i s i n c o r p o r a t e l e u c i n e ( r e f . 12). The s t a b i l i t y o f t e r t i a r y s t r u c t u r e o f t R N A M e t i s i n c r e a s e d by t h e presence o f m5U54 and i t i s f u r t h e r increased, i f t h e t R N A c o n t a i n s s W U 5 4 ( r e f . 236). Therefore, t h e presence o f m5U54 and s2m5U54 s t a b i 1i z e s t h e t R N A structure. The f u n c t i o n a l d i f f e r e n c e s observed i n v i t r o may e x p l a i n t h e small b u t s i g n i f i c a n t growth r a t e d i f f e r e n c e s o f t h e E . c o l i trmA mutant observed i n v i v o . 1.5

REGULATORY ROLE OF THE SYNTHESIS OF MODIFIED NUCLEOSIDES I N

t RNA .

I t has been suggested t h a t t h e degree o f m o d i f i c a t i o n may be a r e g u l a t o r y d e v i c e ( r e f s . 12, 96, 215). The r a t i o o f m o d i f i e d t o unmodified t R N A may r e g u l a t e t h e expression o f s p e c i f i c operon(s) o r gene(s) by t h e d i f f e r e n t i a l e f f i c i e n c y o f decoding e i t h e r a l e a d e r mRNA i n an a t t e n u a t o r c o n t r o l l e d operon o r a s t r u c t u r a l gene o r b o t h . Furthermore, small changes i n t h e p o l y p e p t i d e s t e p t i m e i n t r a n s l a t i n g a s t r u c t u r a l gene may be i m p o r t a n t t o assure a I f so, t h e degree o f m o d i f i c a t i o n proper f o l d i n g o f a protein. may be i m p o r t a n t , as w e l l as t h e codon choice, t o a d j u s t t h e p o l y p e p t i d e s t e p t i m e i n r e l a t i o n t o t h e process o f p r o t e i n f o l d i n g . Since t h e t R N A i s a l s o l i k e l y t o decode genes o t h e r than those t o be r e g u l a t e d , an element o f s p e c i f i c i t y must e x i s t . E i t h e r t h e impact o f t h e m o d i f i e d n u c l e o s i d e i n q u e s t i o n i s more i m p o r t a n t f o r t h e f u n c t i o n o f a c e r t a i n s p e c i f i c t R N A molecules, o r t h e m o d i f i e d n u c l e o s i d e i s i n v o l v e d i n sensing t h e n u c l e o s i d e s c l o s e t o t h e codon. Both these requirements have been shown t o be t r u e ( r e f s . 62, 63, 174). Thus, c e r t a i n s p e c i f i c codon c o n t e x t sequences may have evolved, e.g. i n a l e a d e r mRNA sequence, making t h e f u n c t i o n o f t h e decoding t R N A e x t r e m e l y s e n s i t i v e t o t h e degree o f m o d i f i c a t i o n . Since t h e f o r m a t i o n o f m o d i f i e d nucleos i d e s so f a r analyzed i s an i r r e v e r s i b l e r e a c t i o n , such a r e g u l a t o r y d e v i c e i s slow, and i t i s l i k e l y t o s e t a c e r t a i n degree o f expression r a t h e r than o p e r a t i n g q u i c k l y . Although l a c k o f $ i n t h e a n t i c o d o n r e g i o n o f t R N A H i S l e a d s t o d e r e p r e s s i o n of t h e h i s t i d i n e operon, i t i s d i f f i c u l t t o see a m e t a b o l i c c o n n e c t i o n between t h e s y n t h e s i s o f $ and h i s t i d i n e . The c o n n e c t i o n between

B49

ms2i6A37 and t r y p t o p h a n s y n t h e s i s i s 1ikewi s e d i f f ic u l t t o r e c o n c i l e (ref. 92). However, l a c k o f i r o n l e a d s t o i6A37 i n s t e a d o f ms2i6A, w h i c h a l s o l e a d s t o i n c r e a s e d t r a n s p o r t o f t h e a r o m a t i c amino a c i d s ( r e f . 1 2 8 ) . T h i s may save more o f c h o r i s m i c a c i d f o r t h e s y n t h e s i s o f e n t e r o c h e l i n ( c f F i g u r e 1 . 5 ) , w h i c h e x p l a i n s why t h e degree o f m s 2 - m o d i f i c a t i o n may i n f a c t be a r e g u l a t o r y d e v i c e f o r i r o n metabolism. Ames and c o l l a b o r a t o r s ( r e f . 163) have p o i n t e d o u t t h a t t h e They h i s t i d i n e l e a d e r mRNA i s i n p a r t homologous t o t R N A H i S . s u g g e s t e d t h a t some p r o t e i n , e.g. t h e tRNA m o d i f y i n g enzymes, b e s i d e s b e i n g i n v o l v e d i n t h e b i o s y n t h e s i s o f tRNAs, may a l s o i n f l u e n c e t h e e q u i l i b r i u m o f t h e d i f f e r e n t stem and l o o p s t h a t can I f so, some t R N A m o d i f y i n g enzymes may f o r m i n t h e l e a d e r mRNA. have t w o f u n c t i o n s - - o n e c a t a l y t i c and a n o t h e r r e g u l a t o r y one. However, no d i r e c t e v i d e n c e f o r such a d u a l r o l e has so f a r been p r e s e n t e d . I n t h i s c o n t e x t one can a l s o ask why some t R N A m o d i f y i n g enzymes, e.g. t h e trmD enzyme and t h e h i s T enzyme, a r e p a r t o f m u l t i c i s t r o n i c operons ( r e f . 237). The i n t r o n s o f r a t b r a i n c o n t a i n i d e n t i f i e r sequences ( r e f . 238), some o f w h i c h c l o s e l y r e s e m b l e v a r i o u s s p e c i f i c tRNAs ( r e f . 164). The i n v i t r o t r a n s c r i p t f r o m an ID-sequence c o u l d be m o d i f i e d t o c o n t a i n m5C i n a sequence c o r r e s p o n d i n g t o p o s i t i o n 49 o f y e a s t tRNAPhe, s u p p o r t i n g t h e s u g g e s t i o n t h a t t h e t r a n s c r i p t has a s t r u c t u r e s i m i l a r t o tRNA. The a u t h o r s a l s o s p e c u l a t e t h a t t h e f o r m a t i o n o f m5C m i g h t be p a r t o f a r e g u l a t o r y p r o c e s s i n w h i c h t h e t r a n s c r i p t s f r o m t h e I D sequences a r e i n v o l v e d . The h y p o t h e s i s t h a t t R N A m o d i f i c a t i o n o r tRNA m o d i f y i n g enzymes i s p a r t o f a r e g u l a t o r y d e v i c e i s a t t r a c t i v e . Although p a r t o f t h e r e q u i r e m e n t s o f t h e h y p o t h e s i s has been f u l f i l l e d , so f a r no b i o s y n t h e t i c pathway, d e v e l o p m e n t a l changes, o r c e l l c y c l e e v e n t s have been d i r e c t l y shown t o be r e g u l a t e d a c c o r d i n g t o t h i s h y p o t h e s i s . T h e r e f o r e , i t s g e n e r a l i t y a w a i t s more know1 edge a b o u t t h e f u n c t i o n o f t h e m o d i f i e d nucleosides, about t h e g e n e t i c o r g a n i z a t i o n o f a r e g u l a t e d o p e r o n and how i n t e r m e d i a r y m e t a b o l i s m o r d e v e l o p m e n t / c e l l c y c l e e v e n t s i n e u c a r y o t e s a r e l i n k e d t o tRNA m o d i f i c a t i o n . Knowledge i n t h e s e a r e a s w o u l d a l l o w us t o e v a l u a t e t h e r o l e of tRNA m o d i f i c a t i o n and t R N A m o d i f y i n g enzymes i n t h e metabol ism o f t h e c e l l .

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FUTURE PROSPECTS AND IMPACT Our knowledge about t h e s y n t h e s i s and f u n c t i o n o f m o d i f i e d n u c l e o s i d e s i n t R N A has widened c o n s i d e r a b l y i n r e c e n t y e a r s . Not o n l y does t h e presence o f a m o d i f i e d n u c l e o s i d e improve t h e e f f i c i e n c y o f t h e tRNA i n t h e decoding step, b u t i t may a l s o i n f l u e n c e t h e f i d e l i t y o f p r o t e i n s y n t h e s i s as w e l l as sense t h e r e a d i n g c o n t e x t , i.e. t h e n u c l e o t i d e s s u r r o u n d i n g t h e codon. Furthermore, some m o d i f i e d n u c l e o s i d e s may a l s o be i n v o l v e d i n m a i n t a i n i n g t h e r e a d i n g frame. Therefore, t h e presence o f d i f f e r e n t m o d i f i e d n u c l e o s i d e s i s l i k e l y t o have d i f f e r e n t impacts on Due t o i t s importance i n t h e decoding the a c t i v i t y o f tRNA. steps, t h e l e v e l o f t R N A m o d i f i c a t i o n a l s o i n f l u e n c e s t h e express i o n o f s e v e r a l operons through an i n e f f i c i e n t r e a d i n g o f a t t e n u a t o r r e g i o n o f some operon t r a n s c r i p t s . Thus, l a c k o f a m o d i f i e d n u c l e o s i d e w i l l have a s t r o n g impact on c e l l p h y s i o l o g y . M e t a b o l i c as w e l l as g e n e t i c l i n k s / c o r r e l a t i o n s e x i s t between t h e s y n t h e s i s o f m o d i f i e d n u c l e o s i d e s i n t R N A and i n t e r m e d i a r y metabol i s m , development and c e l l c y c l e . Therefore, t h e l e v e l o f t R N A m o d i f i c a t i o n has been suggested t o be a r e g u l a t o r y d e v i c e a l t h o u g h no d i r e c t evidence e x i s t s a t p r e s e n t f o r such a d e v i c e . F u r t h e r more, o n l y a few genes i n v o l v e d i n t h e s y n t h e s i s o f m o d i f i e d nucleosides have been i d e n t i f i e d . Therefore, f u t u r e s t u d i e s must t r y t o widen o u r knowledge i n these areas as w e l l as i n t R N A m o d i f i c a t i o n i n h i g h e r organisms. New and improved methods i n g e n e t i c m a n i p u l a t i o n , u t i l i z a t i o n o f transposons, DNA sequencing methods, and improvement o f i n v i t r o t r a n s l a t i o n system and i n development o f methods such as t h e advanced HPLC methods f o r n u c l e o s i d e s as developed by Gehrke and Kuo ( r e f s . 246-249) and des c r i b e d i n these t h r e e volumes w i l l c o n t r i b u t e t o o u r unders t a n d i n g o f t h e f u n c t i o n and metabolism o f tRNA m o d i f i c a t i o n . Such r e s e a r c h w i l l have impact on o u r knowledge o f t h e mechanism o f t r a n s l a t i o n i n such aspects as f i d e l i t y , codon c h o i c e and i n f l u e n c e o f codon c o n t e x t . Furthermore, such knowledge w i l l have a s t r o n g impact on t h e a p p l i e d gene-technology and t h e p r o d u c t i o n o f p r o t e i n s o f medical importance.

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ACKNOWLEDGEMENTS T h i s work was supported by t h e Swedish Cancer S o c i e t y ( P r o j . No. 680), Swedish N a t i o n a l Science Foundation (BU-2930) and t h e Swedish Board f o r Technical Development ( g r a n t No. 4206). The c r i t i c a l r e a d i n g o f t h e manuscript by J. U. Ericson, C. E. D. Gustafsson, T. G. H a g e r v a l l , Y. Jbnsson, K. K j e l l i n - S t r a b y , S. Normark, a l l Umea, and L. Isaksson, Uppsala a r e g r a t e f u l l y acknow1 edged. 1.8 REFERENCES 1. S . Nishimura, M o d i f i e d nucleosides i n tRNA, in: P. R. Schimmel, D. S611 and J. N. Abelson (Eds.] T r a n s f e r RNA: S t r u c t u r e , Pro e r t i e s and Recognition, Co S p r i n g Harbor Laboratory, 1979, pp. 59-79 2. G. Nass, (Ed.), M o d i f i e d Nbcleosides and Cancer, Volume 84 o f Recent R e s u l t s i n Cancer Research, S p r i nger-Verl ag, B e r l in, 1983. 3. E. Borek and P. R. S r i n i v a s a n , The meth l a t i o n o f n u c l e i c acids, Annu. Rev. Biochem, 35 (1976) 275-288 4. J. L. S t a r r and B. H. S e l l s , M e t h y l a t e d r i b o n u c l e i c a c i d s , P h y s i o l Rev., 49 (1969) 623-669. 5. R. H. H a l l , The M o d i f i e d Nucleosides i n N u c l e i c Acid, Col umbi a U n i v e r s i t y Press, New York, 1971. 6. D. S b l l , Enzymatic m o d i f i c a t i o n o f t r a n s f e r RNA: M o d i f i e d nucleosides form a t t h e p o l y n u c l e o t i d e levee, b u t t h e i r f u n c t i o n i s n o t e s t a b l i s h e d , Science, 173 (1973 293-299. 7. F. Nau, The m e t h y l a t i o n o f tRNA, Biochemie, 5 (1976) 629645. 8. P.F. A g r i s and D. S o l l , The m o d i f i e d n u c l e o s i d e s i n t r a n s f e r RNA, i n : H. Vogel , (Ed.), N u c l e i c A c i d - P r o t e i n Recognition, Academic Press, Inc., New York, 1977, pp. 321-344. The 9. M. Ya. Feldman, Minor components i n t r a n s f e r RNA: l o c a t i o n - f u n c t i o n r e 1 a t i o n s h i p s , Prog. Biophys. Mol ec. B i o l , 32 (1977) 83-102. 10. G. Dirheimer, Chemical nature, p r o p e r t i e s , l o c a t i o n , and ph s i o l o g i c a l variations of modified nucleosides i n t R g A , Recent R e s u l t s i n Cancer Research, 84 (1983) 15-46 11. G. R. B j o r k , M o d i f i e d nucleosides i n RNA - T h e i r f o r m a t i o n and f u n c t i o n , i n : 0. A p i r i o n 6Ed.) Processing o f RNA, CRC Press, Inc., Boca Raton, F l o r i a, 1984, pp. 291-330. 12. H. Kersten, On t h e b i o l o g i c a l s i g n i f i c a n c e o f m o d i f i e d nucleosides i n t R N A , Progr. Nucl. A c i d Res. Mol. B i o l . , 3 1 1984) 59-114. R. B j b r k , M o d i f i c a t i o n o f s t a b l e RNA, i n : J . L. Ingraham, 13. B. Magasanik, M. Schaechter, K. B. Low, F.C. N e i d a r d t and H. E. Umbarger (Eds.), E s c h e r i c h i a c o l i and S a l m o n e l l a t y p h i m o r i o m c e l l u l a r and m o l e c u l a r b i o l o g y , American S o c i e t y 1986. f o r M i c r o b i o l o y, i n 14. G. R. B ' o r k , U. !Ff",;on, C. E. .D. Gustafsson, T. G. H a g e r v a l i Y. H. Jbnsson and P . M. Wikstrbm T r a n s f e r RNA modificat;on, Ann. Rev. Biochem., 56 (1987) 263-287.

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!

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\

A

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t rmA gene c o d i n g f o r t r a n s y e r r i b o n u c l e i c a c i d (5-methyl u r i d i n e ) m e t h y l t r a n s f e r a s e i n E s c h e r i c h i a c o l i K-12, J. B a c t e r i o l , 142 (1980) 371-379. C. W . Gehrke, R. W . Zumwalt, R. A. McCune and K. C . Kuo, Q u a n t i t a t i v e high-performance 1i q u i d chromatography a n a l y s i s o f m o d i f i e d n u c l e o s i d e s i n p h y s i o l o i c a l f l u i d s , t R N A , and DNA, Recent R e s u l t s i n Cancer Researc! 84 1983) 344-359. C. W . Gehrke, K. C . Kuo, R.A. McCune, k.0. e r h a r d t and P.F. Agris, Quantitative enzymatic hydrolysis of tRNAs. Reversed-Phase h i h-performance l i q u i d chromato r a p h y o f tRNA n u c l e o s i d e s , Chrom. Biomed. A p p l i c . , 230 (Q982) 297-

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!(

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CHAPTER 2 BIOSYNTHESIS AND FUNCTION OF QUEUINE AND QUEUOSINE TRNAs HELGA KERSTEN and WALTER KERSTEN I n s t i t u t f u r P h y s i o l o g i s c h e Chemie d e r U n i v e r s i t a t E r l a n g e n - N u r n b e r g , F a h r s t r a s s e 1 7 , 0 - 8520 E r l a n g e n , F . R . G .

TABLE OF CONTENTS 2.1 Introduction , . . . 2.2 Occurrence, Chemistry and B i o s y n t h e s i s o f Queuosine intRNA 2.3 A n a l y s i s o f Queuosine and o f Q-tRNAs . . 2.3.1 The Epoxide D e r i v a t i v e OQ and V i t a m i n e B,, i n Escherichia coli . . . . . . . . 2.3.2 O r i g i n of Q i n tRNAs o f D i c t y o s t e l i u m d i s c o i d e u m 2.3.3 Q-tRNAs i n E r y t h r o l eukemi c C e l l s . . . 2.4 tRNAs from Pro- and Eukaryotes w i t h G34 i n Place o f Q . 2.4.1 Occurrence i n i r o n - l i m i t e d E s c h e r i c h i a c o l i . 2.4.2 F l u c t u a t i o n s i n Developing D i c t y o s t e l i o m d i s c o ideum . . . . . . . , . 2.4.3 T i s s u e - S p e c i f i c Occurrence i n P l a n t s . . 2.4.4 Occurrence i n t h e S k i n and Melanoma o f X i p h o p h o r i n e Fishes 2.4.5 S p e c i f i c P a t t e r n s i n Human Leukemias and Lympho m a s . . . . . . . . . . . . . . . . . . . . . . . 2.4.6 Q u a n t i t a t i v e Analyses and Synopsis 2.5 Causes o f Q - D e f i c i e n c y i n E u k a r y o t i c tRNAs 2 . 5 . 1 Enzymatic Assay f o r Free Queuine 2.5.2 I n h i b i t i o n o f t R N A Guanine Transglycosyl a s e s by P t e r i d i n e s 2 . 6 R e g u l a t i o n o f Gene Expression by A l t e r e d Q-Modifica. . . . . t i o n i n tRNAs 2 . 6 . 1 F u n c t i o n a l P r o p e r t i e s o f tRNAs C o n t a i n i n g G34 o r Q

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2.6.2

Respiration

2.6.3 2.6.4

and

Fermentation Pathways i n E .

............. .. .... . .. Redox-Systems i n D. d i s c o i d e u m . . . . . . . . . LDH-Isoenzyrnes i n E r y t h r o l e u k e m i c C e l l s . . . .

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2.7 2.8

Summary and Perspectives References . . . . . . .

2.1

INTRODUCTION

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The most important achievements in research on t R N A modificat i o n were the discoveries of a b o u t 60 modified nucleosides and the elucidation of t h e i r s t r u c t u r e s and positions i n s p e c i f i c tRNAs. In several instances the precise steps in t h e i r biosynthesis have yet t o be c l a r i f i e d . W i t h respect t o t h e i r functions, specialized roles in transcription and t r a n s l a t i o n have been described, (1) In t R N A H i s of S a l m o n e l l a t y p h i m u r i u m two pseudouridines in the anticodon region are essential f o r the attenuation of the transcription of the h i s operon ( r e f s . 1 - 4 ) . ( 2 ) Ribosylthymine ( T ) i n position 54 of elongator tRNAs of pro- and eukaryotes i nfl uences, i n cell -free protein synthesizing systems, the r a t e of elongation ( r e f s . 5, 6 ) . The absence of T increases the misreading of U U U by Leu-tRNA;"" (NAA) in the poly(U)-directed synthesis o f polyphenylalanine ( r e f s . 7 , 8 and summarizing references in r e f s . 9-11). A preference f o r T54 versus U54-containi ng el o n g a t o r tRNAs on polysomes was found i n lower eukaryotes, suggesting t h a t the methylation of U54 t o T serves t o modulate the a c t i v i t y of the respective tRNAs i n protein synthesis in v i v o ( r e f . 1 2 ) . (3) The presence of T54 i n prokaryotic i n i t i a t o r t R N A r e s t r i c t s i n i t i a t i o n of MS2-phage RNA t r a n s l a t i o n in v i t r o t o the formylation of Met-tRNAfH;t ( r e f , 10 and r e f s . t h e r e i n ) . When T i s absent, the unformylated Met-tRNAfM;f U54 s t a r t s t r a n s l a t i o n a t the i n i t i a t i o n codon AUG w i t h o u t prior formylation. In a d d i t i o n t o the gene metX, coding f o r Met-tRNAfMTf,a second gene metY f o r t R N A Met-tRNAfMst has been found in E s c h e r i c h i a c o l i . This gene i s p a r t of a multi-gene operon and i s co-transcribed w i t h genes encoding proteins involved in transcription termination control mechanisms. The Met-tRNAf 5 t has one nucl eosi de exchange; m7G a t position 47 i s replaced by an unmodified A-residue. This t R N A i s synthesized in several amino acid auxotrophic s t r a i n s o f E . c o l i . Since Met-tRNA'Mp' i n i t i a t e s mRNA t r a n s l a t i o n in v i t r o w i t h o u t prior formylation ( r e f s . 13, 14 a n d Y. Kuchino a n d H. Kersten,

B71

unpublished results) the expression of the metY gene might relieve the metabolic control of protein synthesis, exerted by tetrahydrofolate-dependent formylation of the initiator tRNA, i n special environmental conditions. (4) In E . c o l i , starved for iron, N6-(2-isopenteny1)adenosine (i6A) at position 37 cannot be methylthiolated to ms2i6A in tRNAs specific for aromatic amino acids, for serine and for cysteine. The incompletely modified tRNAs are thought to relieve the attenuation at corresponding amino acid operons, thereby providing sufficient amounts of serine and aromatic ring systems that are- utilized as precursors for the synthesis of the highaffinity i ron chel ator enterochel in. A hydroxyl ated derivative of ms2i6A, e . g . msziobA, has been found in tRNAs of aerobically grown Salmonella typhimurium. The hydroxylation step is dependent on molecular oxygen and is suggested to play a role in the regulation of aerobiosis (ref. 15). The molecular mechanism by which a tRNA with an alternate A37 modification exhibits a specific function probably involves a1 terations in codon context sensitivity: tRNAs having A or i6A in position 37 in place of ms2i6A or ms2i06A respectively, become more sensitive to the codon context (ref. 16). Modified nucleosides in tRNA might also influence their ribosome-independent functions, e . g . cell wall and lipid biosynthesis or the terminal addition of amino acids to proteins (ref. 17). The chloroplast tRNAGLU in plant leaves provides glutamic acid for the biosynthesis of chlorophyll (ref. 18). It might be of importance that the hypermodified nucleoside 5-methylaminomethyl-2-thiouridine (mam5s~U), found otherwise in microorganisms, is present in the chloroplast tRNAGLu. Several modified nucleosides in tRNA developed in facultative anaerobic microorganisms and were conserved during the evolution of organisms. The modifications are not essential for the fundamental reactions in the living cell; they probably serve as a mean for a1 ternative biosynthetic pathways and adaptation mechanisms when cells are exposed to environmental changes, e . g . a switch from anaerobiosis to aerobiosis and vice versa. A possible regulatory role of tRNAs and of modified nucleosides in differentiation was postulated by Borek (ref. 19).

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The a i m o f t h i s a r t i c l e i s t o d i s c u s s t h e b i o l o g i c a l i m p o r t a n c e o f q u e u o s i n e (Q), i d e n t i f i e d as 7-( ( ( 4 . 5 - c i s - d i h y d r o x y - 2 c y c l openten-1-yl ) -ami n o ) m e t h y l ) -7-deazaguanosi ne ( r e f . 20). Q i s p r e s e n t i n p o s i t i o n 34 o f s p e c i f i c tRNAs o f f a c u l t a t i v e a n a e r o b i c b a c t e r i a and i n t h e c o r r e s p o n d i ng c y t o p l asmi c and m i t o c h o n d r i a1 tRNAs o f l o w e r and h i g h e r e u k a r y o t e s . Significant alterations i n t h e e x t e n t o f Q - m o d i f i c a t i o n were f o u n d i n tRNAs f r o m v a r i o u s b i o l o g i c a l sources. Here we p r e s e n t a s y s t e m a t i c a n a l y s i s o f p r o k a r y o t i c and e u k a r y o t i c tRNAs o f t h e Q - f a m i l y from b a c t e r i a , p r o t i s t s , p l a n t s , f i s h e s , m o u s e - c e l l s and human lymphomas, showing causes and consequences o f v a r i a t i o n s i n t h e Q - v e r s u s G34 c o n t e n t o f r e s p e c t i v e tRNAs. We d e s c r i b e p r o k a r y o t i c and e u k a r y o t i c b i o l o g i c a l model systems t h a t p r o v e d t o be v a l u a b l e f o r o b t a i n i n g i n s i g h t i n t o t h e b i o s y n t h e s i s and f u n c t i o n s o f Q. The r e s u l t s o f t h i s r e s e a r c h a r e o v e r v i e w e d and show t h a t i r o n and v i t a m i n B,, p l a y a r o l e i n t h e b i o s y n t h e s i s o f Q i n e u b a c t e r i a . I n l o w e r and h i g h e r e u k a r y o t e s , t h e p r e s e n c e o r absence o f Q i n r e s p e c t i v e tRNAs causes s p e c i f i c a l t e r a t i o n s i n t h e e x p r e s s i o n o f genes e n c o d i n g LDH-isoenzymes and cytochromes. OCCURRENCE, CHEMISTRY AND BIOSYNTHESIS OF QUEUOSINE I N tRNA Q u e u o s i n e and Q - d e r i v a t i v e s o c c u r i n p o s i t i o n 3 4 o f t h e a n t i c o d o n tRNAs s p e c i f i c f o r Asn, Asp, H i s and T y r . On t h e e v o l u t i o n a r y s c a l e o f o r g a n i s m s , Q appears f i r s t i n r e s p e c t i v e tRNAs o f f a c u l t a t i v e anaerobes, i t i s h i g h l y c o n s e r v e d and f o u n d i n p l a n t s , i n f i s h e s , i n i n s e c t s and i n mammals. However, Q i s Eubacabsent i n y e a s t t R N A (summarizing references i n r e f . 21). t e r i a synthesize Q d e n o v o , t h e p r o t i s t Dictyoste7ium discoideum and h i g h e r e u k a r y o t e s o b t a i n t h e f r e e base, q u e u i n e , b y n u t r i t i o n o r t h e i n t e s t i n a l f l o r a ( r e f s . 22-24). High contents o f f r e e q u e u i n e were f o u n d i n wheat germ, and i n f r u i t s ( c o c o n u t m i l k and t o m a t o ) and i t s o c c u r r e n c e i n p l a n t f r u i t s has l e d t o t h e suggest i o n t h a t p l a n t s may s y n t h e s i z e q u e u i n e d e n o v o ( r e f . 25). The c h e m i c a l s t r u c t u r e o f Q was e l u c i d a t e d ( r e f s . 26, 27) (see F i g u r e 2 . 1 ) . I n e u b a c t e r i a o n l y Q was found, whereas i n e u k a r y o t i c t R N A A S p and tRNATyr g l y c o s y l a t e d d e r i v a t i v e s o f queuo-

2.2

B73

s i n e have been i d e n t i f i e d . Q u e u i n e was s y n t h e s i z e d c h e m i c a l l y ( r e f . 28). R e c e n t l y an e p o x i d e o f Q has been d i s c o v e r e d i n tRNAs o f E . c o l i MRE 600 and has been d e s i g n a t e d as OQ ( r e f . 29). I n bacteria, t h e deazaguanosine-derivative q u e u o s i n e i s s y n t h e s i z e d f r o m guanine. The carbon-8 i s e x c l u d e d t o g e t h e r w i t h n i t r o g e n N-7 ( r e f . 3 0 ) . The m o d i f i e d bases 7-cyano-7-deazaguanine and 7-ami nomethyl-7-deazaguani ne were f o u n d in E . c o I i tRNAs ( r e f . 31) ( F i g u r e 2.1). The deazaguanine-derivatives (a, b i n F i g u r e 2.1) a r e t h o u g h t t o be p r e c u r s o r s o f queuine, t h e y can be i n s e r t e d i n t o t h e r e s p e c The enzyme t i v e tRNAs b y t h e t R N A guanine-transglycosylase. exchanges t h e g u a n i n e r e s i d u e a t p o s i t i o n 34 f o r t h e p r e c u r s o r s ( r e f . 32). The c y c l o p e n t e n d i o l m o i e t y i s s y n t h e s i z e d a t t h e l e v e l I n contrast o f t R N A f r o m as y e t unknown p r e c u r s o r s and enzymes. t o b a c t e r i a , t h e b i o s y n t h e s i s o f Q-tRNAs o f a n i m a l c e l l s i n v o l v e s tRNA guanine t r a n s g l y c o s y l a s e s t h a t i n s e r t f r e e queuine i n t o t h e r e s p e c t i v e tRNAs ( r e f s . 33, 3 4 ) .

ANALYSIS OF QUEUOSINE AND OF Q-tRNAs The c o n v e n t i o n a l t e c h n i q u e o f RPC-5 ( r e f . 35) i s s t i l l u s e f u l t o a n a l y z e t h e e x t e n t o f Q - m o d i f i c a t i o n i n r e s p e c t i v e tRNAs. U n f r a c t i o n a t e d t R N A p r e p a r a t i o n s a r e used and t h e s p e c i f i c tRNAs a r e a m i n o a c y l a t e d w i t h t h e c o g n a t e r a d i o a c t i v e l y l a b e l e d amino a c i d . The tRNAs a r e a p p l i e d t o RPC-5 columns and e l u t e d a t l o w e r s a l t c o n c e n t r a t i o n s f r o m t h e columns t h a n t h e i r u n d e r m o d i f i e d The Q-tRNAs can be f u r t h e r c o u n t e r p a r t s w i t h G a t p o s i t i o n 34. a n a l y z e d b y p e r i o d a t e (10;) t r e a t m e n t o f t h e p r e c h a r g e d t R N A ( r e f .

2.3

F i g u r e 2.1 queuine (c)

.

S t r u c t u r e s o f queuine p r e c u r s o r s

(a),

(b)

and o f

B74

36). The v i c i n a l OH-groups of the cyclopentendiol moiety a r e o x i d i z e d , and rearrangement a t t h e cyclopentene r i n g i n c r e a s e s t h e hydrophobicity of the r e s p e c t i v e t R N A ( r e f . 26 and r e f s . t h e r e i n ) . Therefore, t h e oxidized Q-tRNAs a r e e l u t e d from the column a t s i g n i f i c a n t l y higher s a l t c o n c e n t r a t i o n s than Q-tRNAs. 2.3.1 COTi

The EDoxide D e r i v a t i v e 00 and Vitamine B , ,

i n Escherichia

In the course of our i n v e s t i g a t i o n s on the p o s s i b l e r o l e of Q-modification i n t R N A on r e s p i r a t i o n and f e r m e n t a t i o n pathways i n E . c o l i ( s e e r e f . 10 and s e c t i o n 2.6.2, t h i s c h a p t e r ) tRNAs of the Q-family from two E . c o 7 i hemA mutants have been analyzed. The hemA mutants SHSP-19 and SASX-77 were obtained from t h e c o l l e c t i o n of B. Bachmann, (Yale U n i v e r s i t y , New Haven, CT, U S A ) . The mutants a r e unable t o form 5-aminolevulinate s y n t h a s e , t h e key enzyme i n the b i o s y n t h e s i s of heme. Both hemA mutants u t i l i z e f e r m e n t a t i v e pathways when grown a e r o b i c a l l y without added 5aminolevulinate and i r o n . The n u c l e o s i d e s from p u r i f i e d t R N A A S p and from t R N A T y r of t h e mutants were analyzed, with the r e s u l t being observance of a modified n u c l e o s i d e with a s i g n i f i c a n t lowered r e t e n t i o n time a s compared t o queuosine ( F i g u r e 2 . 2 ) . Combined HPLC and mass spectrometry of p u r i f i e d t R N A A s p and t R N A T y P (performed by J.A. McCloskey and coworkers, U n i v e r s i t y of Utah, S a l t Lake C i t y , UT, USA) revealed t h a t t h e s e tRNAs c o n t a i n a Q-derivative i d e n t i c a l t o oQ. The epoxide was o r i g i n a l l y found i n commercial t R N A from E . c a l f M R E 600, the s t r u c t u r e being e l u c i dated a s d e s c r i b e d i n r e f . 29. Applying HPLC-combi ned mass-spectrometry, HPLC and RPC-5 f o r t h e a n a l y s i s of Q-nucleosides we have r e c e n t l y observed t h a t : (1) The cyclopentane epoxide r i n g system i s formed i n tRNAs of the Q family o f E. co7i s t r a i n MRE 600 o r s t r a i n MC 4100 when the b a c t e r i a a r e grown i n g l u c o s e / s a l t medium under a e r o b i c o r s t r i c t a n a e r o b i c c o n d i t i o n s ; t h e formation of oQ i s t h e r e f o r e independent of mol ecul a r oxygen. (2) Q i s p r e s e n t i n r e s p e c t i v e tRNAs of E. co7i MRE 600 and MC 4100 when the b a c t e r i a a r e grown i n L-broth; oQ-tRNAs a r e converted t o Q-tRNAs a f t e r a s h i f t from g l u c o s e - s a l t medium t o Lbroth a l s o i n the presence of r i f a m p i c i n .

B75

cu

G

E

Gm

11 1119

0

20

40

60

minutes

F i u r e 2.2 HPLC-analysis of nuc!eosides from t R N A T y r o f E . c o l i SHZP-19 (hemA) Enzymatic d i g e s t i o n o f t h e t R N A and HPLC a n a l y s i s were performed according t o r e f . 37. (Unpublished r e s u l t s , B. Frey and H. Kersten.)

(3) The a d d i t i o n o f v i t a m i n e B,2, cobalamine, t o t h e g l u c o s e / s a l t medium prevents oQ f o r m a t i o n and Q-tRNAs a r e synthesized. None o f t h e o t h e r n u t r i e n t s from L - b r o t h a r e r e q u i r e d i n t h i s case. (4) I n tRNAs o f t h e Q - f a m i l y f r o m t h e hemA mutants auxotroph I n these f o r m e t h i o n i n e (mete) OQ i s p r e s e n t i n p l a c e o f Q. mutants t h e a d d i t i o n o f cobalamine does n o t accomplish t h e convers i o n o f OQ i n r e s p e c t i v e tRNAs. The metB gene, coding f o r c y s t a t i o n 7-synthase i s l o c a l i z e d a t about 90 min i n t h e same r e g i o n as

B76

t h e btuA gene. This gene codes f o r a membrane r e c e p t o r i n v o l v e d i n cobalamine t r a n s p o r t . We suggest t h a t i n these m u t a n t s the u p t a k e o f cobalamine i s i m p a i r e d . The present d a t a ( s e e T a b l e 2 . 1 ) i n d i c a t e t h a t OQ o c c u r s a s an i n t e r m e d i a t e i n the b i o s y n t h e s i s of the c y c l o p e n t e n d i o l - m o i e t y and t h a t Q i s formed from oQ by an adenosylcobalamine-dependent r e d u c t a s e i d e n t i c a l w i t h , or simi 1 a r t o , ri bonucl e o t i d e - r e d u c t a s e t h a t c o n v e r t s r i b o s e t o d e o x y r i b o s e . The p r e c u r s o r o f the c y c l o p e n t e n d i o l r i n g s y s t e m a p p e a r s t o be d e r i v e d from a r i b o s y l - o r r i b i t y l - d o n a t i n g m o l e c u l e (B. Frey, J . McCloskey, W . Kersten and H. Kersten, 1 2 t h I n t e r n a t i o n a l Workshop on t R N A Umea, Sweden, 1987). TABLE 2 . 1

Queuosine i n t R N A i s formed from the Epoxide i s p r e s e n t i n t h e Growth Medium Strain

Growth c o n d i t i o n s med i urn

E. c o l i MC 4100

E. coli MRE 600 E. c o l i SHSP-19

glucose s a l t L-broth g l ucose s a l t + O . 5% casami no a c i d s glucose salt+0,5% y e a s t e x t r a c t g l u c o s e s a l t + v i tamines g l ucose s a l t + v i tamine B glucose s a l t anaerob2i c g l y c e r i n/NO,

OQ

w h e n Vitamin B,,

moles 100 moles o f $ Q oQ <

<

0.30 3.26 0.80 0.50 2.93 3.24 0.30

< <

3.81 0.30 2.36 2.42 0.30 3.72 2.56

g l ucose-sal t L-bro t h

<

g l u c o s e s a l t +5-A1 a g l ucose s a l t -5-A1 a L-broth +5-A1 a

<

0.30

3.24 3.18 3.04

<

0.30 3.26

3.43 0.90

S . typhimurium GT 316 glucose s a l t L-broth

0.30 4.71

<

4.27 0.30

I f n o t s t a t e d o t h e r w i s e , E. coli s t r a i n s were grown a e r o b i c a l l y up t o the s t a t i o n a r y phase.

tRNAs c o n t a i n i n g OQ i n p l a c e o f Q do n o t show t h e h y d r o p h o b i c I n F i g u r e 2.3 ( l e f t s h i f t on RPC-5 columns upon 10; o x i d a t i o n . p a n e l ) t h e e l u t i o n p r o f i l e o f a Q - t R N A A S p f r o m E . c o l i MC 4100 i s shown as a c o n t r o l . The e l u t i o n o f Q - t R N A A S P i s s h i f t e d a l m o s t Howt o t a l l y t o h i g h e r s a l t - c o n c e n t r a t i o n s upon 10; t r e a t m e n t . e v e r , OQ tRNAASp f r o m €. c o l i SHSP-19 and SASX-77 and f r o m €. c o l i MRE 600 do n o t show t h e h y d r o p h o b i c s h i f t when t r e a t e d w i t h IO;, (see r i g h t p a n e l i n F i g u r e 2.3). The r e s u l t shows t R N A A s p f r o m s t r a i n SHSP-19 as a r e p r e s e n t a t i v e .

E.coli (hemA)SHSP 19

(RPC-5)

F i g u r e 2.3 L e f t : RPC-5 column p r o f i l e o f Q - t R N A A s p f r o m MC 4100 ( c o n t r o l ) and r i g h t : o f oQ-tRNAASP f r o m E . c o l i (hemA) 0-0-0 u n t r e a t e d , 1 4 C As - t R N A A s p ; 0 - 0 - 0 IO- t r e a t e d tRNAASp. U n f r a c t i o n a t e d tRNd7s were i s o l a t e d tram t h e

€.

coli

SHSP-19 3 H Asp-

€. c o l i s t r a i n s , grown a e r o b i c a l l y i n g l u c o s e ( 0 . 4 % ) s a l t medium. tRNAs w e r e a m i n o a c y l a t e d w i t h 3 H Asp o r 1 4 C Asp r e s p e c t i v e 1 and I O -

tram

t r e a t e d as d e s c r i b e d i n r e f . 36. The tRNAs were e l u t e d RPC-b w i t h an i n c r e a s i n g . N a C 1 g r a d i e n t f r o m 0.4M 1.5M ( a l l € . c o 7 i s t r a i n s were o b t a i n e d f r o m t h e c o l l e c t i o n o f B. Bachmann). ( U n p u b l i s h e d r e s u l t s , B . F r e y and H. K e r s t e n .

-

B78

O r i a i n of 0 i n tRNAs of D i c t v o s t e l i u m d i s c o i d e u m The p r o t i s t D . d i s c o i d e u m i s the s i m p l e s t m u l t i c e l l u a r eukaryote. Wild-type s t r a i n s grow n a t u r a l l y on b a c t e r i a , e . g . on E . c o l i , and a x e n i c s t r a i n s grow v e g e t a t i v e l y a s s i n g l e c e l l s o a p a r t i a l l y o r f u l l y defined medium ( r e f s . 38, 39). RPC-5 has been s u c c e s s f u l l y a p p l i e d t o show t h a t D . d i s c o i d e u m o b t a i n s queuine from b a c t e r i a , the n a t u r a l food s o u r c e , and t h a t v e g e t a t i v e c e l l s can be grown i n a well d e f i n e d medium with o r without queuine ( r e f . 2 2 ) . S u r p r i s i n g l y , the c e l l s c o n t a i n twofold higher l e v e l s of t R N A A S p and t R N A T y r when grown i n a defined medium i n the presence of queuine, compared with t h o s e grown i n the absence of queuine. The l e v e l s of t R N A A S p and t R N A H i s a r e not a f f e c t e d by the presence o r absence of the modified base. The r e l a t i v e i n c r e a s e i n t h e amount of t R N A A S p and of t R N A T y r i s thought t o be caused, a t l e a s t i n p a r t , by a s t a b i l i z a t i o n of t h o s e tRNAs by g l y c o s y l a t i o n of the cyclopentendiol moiety. Glycosyl a t e d d e r i v a t i v e s of Q were found i n e u k a r y o t i c t R N A A S p and t R N A T y r , b u t not i n t R N A A S n and t R N A H i s . The t u r n o v e r r a t e s of Q - t R N A T y r and of Q - t R N A A S p a r e lower than t h o s e of Q - t R N A A and Q-tRNAH i s ( r e f . 4 0 ) . During t h e s e s t u d i e s , some c h a r a c t e r i s t i c s of t h e t R N A guanine t r a n s g l y c o s y l a s e of D . d i s c o i d e u m have been e l u c i d a t e d . ( i ) The enzyme i s being synthesized independently of the presence o r absence of queuine. ( i i ) The enzyme d i f f e r s from o t h e r eukaryo t i c t R N A t r a n s g l y c o s y l a s e s i n t h a t i t exchanges e x c l u s i v e l y , and i n an almost i r r e v e r s i b l e r e a c t i o n , queuine f o r guanine 34. However, the enzyme cannot u t i l i z e guanine a s a s u b s t r a t e a s can other t R N A transglycosylases. The enzyme has been a p p l i e d f o r q u a n t i t a t i v e determination of queuine i n t i s s u e e x t r a c t s ( s e e section 2.5.1). 2.3.2

2.3.3

0-tRNAs i n Ervthroleukemic C e l l s f r i e n d - v i r u s transformed erythroleukemic c e l l s of mice a r e well e s t a b l i s h e d a s a model system t o s t u d y biochemical a l t e r a t i o n s i n growing o r d i f f e r e n t i a t i n g c e l l s ( r e f s . 41, 42). The transformed c e l l s can be s t i m u l a t e d t o d i f f e r e n t i a t e by various a g e n t s , e . g . d i m e t h y l s u l f o x i d e (DMSO) o r b u t y r a t e . Differentia t i n g c e l l s a r e analogous t o e r y t h r o b l a s t s t h a t a r e a l r e a d y

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s p e c i a l i z e d f o r t h e s y n t h e s i s of hemoglobin. Hypomodification with r e s p e c t t o Q was found i n tRNAs of growing c e l l s , however, the Q-content i n r e s p e c t i v e tRNAs i n c r e a s e s when t h e c e l l s a r e induced t o d i f f e r e n t i a t e ( r e f s . 43, 4 4 ) . In t h e RPC-5 e l u t i o n p r o f i l e Q - t R N A T y ' and G34-tRNATyr a r e found when the c e l l s ( l i n e age F,6) a r e grown i n a medium supplemented with f e t a l c a l f serum, however, only one s p e c i e s having G34 i n p l a c e of Q i s found when grown i n a medium supplemented with horse serum. Queuine a n a l y s i s of t h e horse serum (see s e c t i o n 2 . 5 . 1 ) shows t h a t the serum c o n t a i n s only t r a c e s of queuine. Growth of F,6 c e l l s i n media supplemented with horse serum e i t h e r with o r without queuine, i s t h e r e f o r e used t o study biochemical a1 t e r a t i o n s i n mouse c e l l s caused i n response t o queuine. This b i o l o g i c a l model system has been applied t o o b t a i n i n s i g h t i n t o the f u n c t i o n of queuine, of QtRNAs, and t h e i r r e s p e c t i v e G34 c o u n t e r p a r t s ( r e s u l t s a r e p r e s e n t ed i n s e c t i o n 2 . 6 . 2 ) . tRNAS FROM PRO- AND EUKARYOTES WITH 634 I N PLACE OF Q The t R N A guanine t r a n s g l y c o s y l a s e from E . c o l i c a t a l y z e s t h e exchange of G34 i n r e s p e c t i v e tRNAs of the Q family from both p r o k a r y o t i c and eukaryoti c sources (cytoplasm, mitochondria and c h l o r o p l a s t s ) . With t h e E . c o 7 i t R N A guanine t r a n s g l y c o s y l a s e and 3 H guanine the amount of G34-tRNAs was determined i n u n f r a c t i o n a t e d tRNAs ( r e f . 4 5 ) . The 3 H G34 l a b e l e d tRNAs were recovered, subsequently s e p a r a t e d by one-dimensional gel e l e c t r o p h o r e s i s and d e t e c t e d by fluorography ( r e f . 4 6 ) . In the following s e c t i o n s q u a l i t a t i v e and q u a n t i t a t i v e analyses of Q - d e f i c i e n t tRNAs from v a r i o u s sources a r e presented.

2.4

2.4.1

Occurrence i n Iron-Limited E s c h e r i c h i a c o 7 i I t has been reported t h a t b a c t e r i a accumulate G34 tRNAs i n t h e s t a t i o n a r y phase when grown i n glucose s a l t medium ( r e f . 4 7 ) . tRNAs, i s o l a t e d from s t a t i o n a r y phase o r a n a e r o b i c a l l y grown c u l t u r e s of E . c o 7 i MRE 600, o r from E . c o 7 i hemA mutants (SHSP-19 and SASX-77) can be e l e c t r o p h o r e t i c a l l y s e p a r a t e d i n t o f o u r G34t R N A s p e c i e s (Figure 2 . 4 ) . The slow-running tRNAs r e p r e s e n t two t R N A T y r i s o a c c e p t o r s by comparison with p u r i f i e d t R N A T y r . E . co7i t R N A s T y r c o n t a i n a long extra-arm and thus have higher molecular

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weights than the o t h e r tRNAs o f the Q-family ( s e e a l s o s e c t i o n 2 . 4 . 3 ) . The major Q - d e f i c i e n t t R N A band probably r e p r e s e n t s t R N A A S p , because t h i s t R N A s p e c i e s i s h i g h l y abundant i n p u r i f i e d bulk t R N A from E . c o l i .

Figure 2.4 E l e c t r o p h o r e t i c p a t t e r n s o f G34-tRNAsI p o s i t i o n 34 with 3 H Gua from: a)

c)

l a b e l e d a-

E . c o l i MRE 600; anaerobic growth E . c o l i SHSP-19, hemA; anaerobic growth E . c o l i SASX-77, hemA; anaerobic rowth p u r i f i e d t R N A T v r from E . c o l i SHS -19.

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All s t r a i n s were grown i n glucose s a l t medium w i t h o u t added 5aminolevulinate o r iron respectively. (B. Frey, S . Isepp and H. Kersten, unpublished r esu l t s. ) The accumulation of G34-tRNAs i n E . c o l i MRE 600 can be prevented by t h e a d d i t i o n of iron t o t h e growth medium a t a c o n c e n t r a t i o n of 100 pM. Several o t h e r s t r a i n s o f E . c o l i , t h a t have been t e s t e d s o f a r , do not accumulate G34-fRNAs when t h e b a c t e r i a a r e grown i n glucose s a l t medium t o the s t a t i o n a r y phase. The s t r a i n - s p e c i f i c d i f f e r e n c e s a r e explained best by the g r e a t v a r i a b i l i t y i n the s y n t h e s i s and t u r n o v e r of i r o n c h e l a t o r s and i r o n t r a n s p o r t systems ( r e f . 48). In f a c t , i r o n s t a r v a t i o n d u r i n g

l o g phase i s e v i d e n c e d i n E . c o l i MRE 600 b y an e a r l y e x p r e s s i o n o f a l l i r o n t r a n s p o r t p r o t e i n s i n t h e o u t e r membrane. G34-tRNAs a r e a l s o f o u n d i n E . c o l i SHSP-19 and SASX-77 grown a n a e r o b i c a l l y i n m i n i m a l media. Addition o f 5-aminolevulinate + i r o n prevents t h i s u n d e r m o d i f i c a t i o n o f tRNAs o f t h e Q - f a m i l y , s u g g e s t i n g t h a t a heme i r o n p r o t e i n p l a y s a r o l e i n t h e b i o s y n t h e s i s o f Q. 2.4.2

F l u c t u a t i o n s i n Develooina Dictvostelium discoideum I n D . d i s c o i d e u m an a l m o s t synchronous d e v e l o p m e n t a l t r a n s i t i o n from u n i c e l l u l a r t o m u l t i c e l l u l a r stages i s induced by s t a r v a t i o n and proceeds when a c e r t a i n amount o f c e l l s i s p l a c e d on a g a r o r f i l t e r s u p p o r t s i n phosphate b u f f e r . A f t e r an e a r l y d e v e l o p m e n t a l p r e - a g g r e g a t i o n s t a g e , d u r i n g w h i c h t h e c e l l s become r e s p o n s i v e t o p u l s e s o f cAMP and f o r m cAMP b i n d i n g s i t e s on t h e c e l l s u r f a c e ( r e f . 4 9 ) , t h e c e l l s a g g r e g a t e i n t o mounds c o n t a i n i n g v a r i a b l e amounts o f c e l l s . The mounds f o r m s l u g s t h a t subsequentl y d i f f e r e n t i a t e i n t o mature f r u i t i n g bodies, comprising spores a t t h e t o p o f a vacuolized s t a l k . A n a l y s e s o f tRNAs f r o m v e g e t a t i v e c e l l s grown i n a x e n i c medium w h i c h was supplemented w i t h s u f f i c i e n t amounts o f q u e u i n e and f r o m c e l l s i n d u c e d t o d i f f e r e n t i a t e b y s t a r v a t i o n r e v e a l a t l e a s t f i v e e l e c t r o p h o r e t i c a l l y s e p a r a b l e s p e c i e s w i t h G34, t h r e e o f them have been i d e n t i f i e d as t R N A A s n , t R N A A S p and t R N A T v r ( F i g u r e 2.5) (B. L o f f l e r and H. K e r s t e n , u n p u b l i s h e d r e s u l t s ) . The p a t t e r n s show c h a r a c t e r i s t i c a1 t e r a t i o n s : I n v e g e t a t i v e c e l I s , s m a l l amounts o f Q - d e f i c i e n t tRNAs a l w a y s o c c u r even when t h e c e l l s a r e s u f f i c i e n t l y s u p p l i e d w i t h q u e u i n e ( r e f s . 22, 40). The amount o f G34-tRNAs decreases d u r i n g p r e a g g r e g a t i on and i n c r e a s e s d u r i n g l a t e r s t a g e s o f development, b e i n g h i g h e s t i n s p o r e s . T h i s m i g h t i n d i c a t e t u r n o v e r o f t h e tRNAs and r e l e a s e o f q u e u i n e . 2.4.3

T i s s u e - S o e c i f i c Occurrence i n P l a n t s I n t e r e s t i n g l y , i n t o b a c c o and wheat l e a v e s t w o i s o a c c e p t i n g tRNAsTvr w i t h a G$A a n t i c o d o n have been f o u n d ( r e f . 50). Wheat germ, however, p r e d o m i n a n t l y c o n t a i n s a Q$A a n t i c o d o n i n t h e s e t w o tRNAsTvr. Tobacco l e a f tRNAsTvr w i t h t h e G$A a n t i c o d o n p r o m o t e s u p p r e s s i o n o f t h e UAG s t o p codon t h a t t e r m i n a t e s t h e gene f o r t h e m a j o r , n o n - s t r u c t u r a l 126 K p r o t e i n o f t o b a c c o m o s a i c v i r u s (TMV), t h u s g i v i n g r i s e t o a 183 K r e a d t h r o u g h p r o d u c t . The tRNAsTvr

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Figure 2.5 E l e c t r o p h o r e t i c p a t t e r n s of G34-tRNAs, l a b e l e d a t tRNA-G34 p o s i t i o n 34 with 3 H Gua from D i c t y o s t e l i u m d i s c o i d e u m . from ( a ) v e g a t i v e l y growing c e l l s , ( b pre-aggregation, aggregation., ( d ) culmination s t a g e s , and e ) s p o r e s . S t r a i n AX-( 9 was grown i n peptone medium, supplemented with 1 x .10-7M queujne (85% of the tRNAs a r e modified with r e s p e c t t o Q d u r i n g v e g e t a t i v e growth). The c e l l s were induced t o d i f f e r e n t i a t e by s t a r v a t i o n i n phosphate b u f f e r ( f u r t h e r d e t a i l s a r e d e s c r i b e d i n r e f s . 22, 39, 40).

1

with t h e QdA anticodon, however, do not suppress t h i s s t o p codon ( r e f . 5 1 ) . The observation t h a t t h e two major cytoplasmic t R N A s T y r from tobacco l e a v e s a r e lacking Q r a i s e s t h e q u e s t i o n s whether a l l t h e t R N A s p e c i e s of t h e Q-family a r e Q - d e f i c i e n t and what the causes a r e f o r t h e absence o f Q i n tRNAs from l e a v e s . Unfractionated tRNAs from f u l l y expanded tobacco l e a v e s , from tobacco c e l l suspensions (grown i n c u l t u r e ) , from wheat l e a v e s and wheat germ were t h e r e f o r e t e s t e d a s s u b s t r a t e s f o r t h e t R N A guanine t r a n s g l y c o s y l a s e of E . c o l i a n d t h e following r e s u l t s were obtained ( r e f . 5 2 ) . The p a t t e r n s of guanine-accepting tRNAs from wheat and tobacco l e a v e s show seven Q - d e f i c i e n t tRNAs, d e s i g n a t e d t R N A , - t R N A , (Figure 2 . 6 a , b ) . The number of guanine-accepting tRNAs from both s o u r c e s a r e i d e n t i c a l , however, t h e e l e c t r o p h o r e t i c mobi 1 i t i e s of t R N A , and t R N A , a r e s l i g h t l y d i f f e r e n t and the r e l a t i v e i n t e n s i t i e s d i f f e r c o n s i d e r a b l y , e . g . t R N A , i s predominant i n

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Figure 2 . 6 E l e c t r o p h o r e t i c p a t t e r n s of G34-tRNAsr l a b e l e d a t p o s i t i o n 34 with )H Gua from ( a ) wheat l e a v e s , ( b ) tobacco l e a v e s , ( c ) wheat germ, (d) wheat l e a v e s . wheat l e a v e s . The r e s u l t s c l e a r l y show t h a t s e v e r a l t R N A s p e c i e s of the Q-fami l y ( i ncl udi ng i s o a c c e p t o r s ) a r e Q-def i c i ent i n wheat and tobacco l e a v e s . The p a t t e r n s of guanine-accepting tRNAs from wheat l e a v e s and from wheat germ show s t r i k i n g d i f f e r e n c e s ( F i g u r e 2.6 c , d ) . t R N A , , tRNA, and tRNA, a r e a b s e n t i n wheat germ and a new t R N A , designated 5a, appears. Compared with tRNAs from wheat 1 eaves , t h e tRNAs from wheat germ i n c o r p o r a t e only 1 / 5 of 3 H guanine ( f o r q u a n t i t a t i v e a n a l y s i s s e e Table 2.1 i n s e c t i o n 2 . 4 . 6 ) . The absence of t h r e e guanine-accepting tRNAs (designated 1, 2 , and 6) only p a r t i a l l y accounts f o r t h i s lowered value; the o t h e r t R N A s p e c i e s of t h e Q-family a r e a l s o p a r t i a l l y undermodified a s evidenced by t h e weak bands ( F i g u r e 2 . 6 ~ ) . This a g r e e s well with the o b s e r v a t i o n t h a t wheat germ c o n t a i n s 85% t R N A T v r i s o a c c e p t o r with Q$A anticodon and 15% i s o a c c e p t o r with G$A anticodon ( r e f . 51). Unfractionated tRNAs from wheat and tobacco l e a v e s and from wheat germ were l a b e l e d with 3 H guanine and s e p a r a t e d on onedimensional g e l s . (tRNAs i n a , b o r c , d r e s p e c t i v e l y a r e run on

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t h e same g e l . ) ( Q u a n t i t a t i v e d a t a a r e presented i n s e c t i o n 2 . 4 . 6 ) . ( D e t a i l s a r e described i n r e f . 4 9 ) . The tRNAsTvr from t h e mitochondria and c h l o r o p l a s t s of bean and o t h e r p l a n t s have been d e s c r i b e d t o c o n t a i n a l a r g e v a r i a b l e loop and thus have a c o n s i d e r a b l y higher molecular weight than c y t o s o l i c t R N A s T v r ( r e f s . 53, 5 4 ) . Indeed, t h e t R N A , and t R N A , a r e i d e n t i f i e d a s tRNAsTvr, t h e y might d i f f e r i n t h e i r modified bases a s d e s c r i bed f o r bean m i tochondri a1 tRNAsTy ( r e f . 55), o r they have the same sequences b u t d i f f e r i n t h e i r conformations a s shown f o r the y e a s t mi tochondri a1 tRNAsPh e ( r e f . 5 6 ) . With p a r t i a l l y p u r i f i e d c y t o s o l i c t R N A T v r from tobacco l e a v e s , t R N A , has been i d e n t i f i e d a s tobacco c y t o s o l i c t R N A T v r . t R N A , has been i d e n t i f i e d a s t R N A A s P by e l u t i o n of t h i s band and s u c c e s s f u l aminoacylation with 3 H Asp. From t h e s e r e s u l t s , we conclude t h a t Q-modification of t R N A i s t i s s u e - s p e c i f i c i n p l a n t s and suggest t h a t t h i s phenomenon i s r e l a t e d t o t h e h i g h l y a e r o b i c metabolism i n l e a v e s v e r s u s a more anaerobic metabolism i n germ c e l l s . Occurrence i n the Skin and Melanoma o f X i p h o p h o r i n e F i s h e s t R N A degradation and a1 t e r a t i o n s of t R N A m o d i f i c a t i o n were observed i n tumor t i s s u e s and were suggested t o r e l i e v e r e g u l a t o r y mechanisms during n e o p l a s t i c t r a n s f o r m a t i o n ( r e f . 1 9 ) . An incomp l e t e m o d i f i c a t i o n of G34 t o Q i n r e s p e c t i v e tRNAs of tumor t i s s u e s from v a r i o u s s o u r c e s was found ( r e f s . 10, 45). To d e t e r mine t h e extent of Q-modification i n t R N A i n r e l a t i o n t o d i f f e r e n t i a t i o n and n e o p l a s t i c t r a n s f o r m a t i o n , X i p h o p h o r u s , a genus of small v i v i p a r o u s f r e s h w a t e r f i s h was used a s a model. I f i n t e r populational and i n t e r s p e c i f i c c r o s s i n g s a r e performed between two genotypes, X i p h o p h o r o s m a c u l a t u s and X i p h o p h o r u s he7 7 e r i , the progeny spontaneously develops me1 anoma. A s p e c i f i c oncogene, ' T u ' , i s responsible f o r the neoplastic transformation ( r e f . 57). The oncogene ' T u ' i s probably i d e n t i c a l with the h i g h l y conserved c - s r c gene ( r e f s . 58, 5 9 ) , discovered f i r s t i n the R o u s s a r c o m a v i r u s . Genetic experiments with t h e f i s h e s showed t h a t ' T u ' i s normally r e p r e s s e d by c e l l t y p e - s p e c i f i c systems o f r e g u l a t i n g genes ' R ' , which suppress the development of the d i f f e r e n t neoplasms i n the f i s h . In a l l genotypes so f a r s t u d i e d , most of the R-genes a r e l i n k e d t o ' T u ' . Crosses of s p o t t e d X . m a c u l a t u s with 2.4.4

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a nonspotted X. h e l l e r i result in F,-hybrids that develop 'benign melanoma' instead of spots. Backcrosses of the F, -hybrids using X . he1 leri as the recurrent parent produce offsprings (BC, ) , 50% of which exhibit neither spots nor melanoma while 25% develop ' benign me1 anoma' (1 i ke F, ) , and 25% develop 'ma1i gnant me1 anoma ' . tRNAs from normal skin of X. m a c u l a t u s and X . h e l l e r i and from the skin and melanomas of their offsprings have been analyzed and specific patterns of Q-deficient tRNAs are obtained. Also, in normal skin of X i p h o p h o r i n e f i s h e s , relatively high amounts of tRNAs with G in place of Q occur. In contrast, tRNAs from the livers are fully modified with respect to Q (refs. 60, 61). These results show that the extent of Q-modification in respective tRNAs of the fishes is tissue-specific. 2.4.5 SDecific Patterns in Human Leukemias and LvmDhomas Human ma1 ignant lymphomas and 1 eukemi as are heterogenous in their prognosis as well as in their morphological features, membrane phenotypes and functional capacities. Recent advances in immunology and molecular biology have led to important insights into differentiation and cellular origin of leukemias and lymphomas. The clonal origin of B-cell neoplasias and other hemopoietic cancers is demonstrated by the predominance of cells showing a restriction in regard to the expression of distinct enzymes or immunoglobulin molecules. Comparative morphological , immunological and biochemical studies have further indicated that the neoplastic hemopoietic cells closely resemble normal cel Is, both phenotypically and functionally, and can be located at a specific point i n the differentiation sequences (refs. summarized in ref. 62). Human lymphomas and leukemias of various stages and grades of ma1 ignancy and differentiation have been analyzed for the presence of G34-tRNAs (ref. 62). A number o f human lymphomas and leukemias contain only small amounts of tRNAs with guanosine in place of queuosine. With the exception of the well-differentiated immunocytomas and plasmocytomas, the relative amount o f G34-tRNA is generally greater in high-grade than in low-grade lymphomas, which suggests that the degree of undermodification is dependent on the proliferative activity of the tumors. This is well documented by

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t h e i n d i v i d u a l c l i n i c a l course of t h e p a t i e n t s , i n which r a p i d lymphonode enlargement o r h i g h b l a s t counts a r e r e g u l a r l y accompanied by h i g h c o n t e n t o f G34-tRNAs i n t h e t R N A p r e p a r a t i o n , and by s e v e r a l s t u d i e s which have demonstrated t h a t a s i g n i f i c a n t l y h i g h e r percentage of c e l l s i n t h e S-phase can be found i n h i g h grade than i n low-grade lymphomas. The r e l a t i o n o f i n c o m p l e t e queuosine m o d i f i c a t i o n i n t R N A t o c e l l p r o l i f e r a t i o n i s f u r t h e r supported by t h e r e s u l t s i n lymphocytes from p a t i e n t s w i t h c h r o n i c l y m p h a t i c leukemia (CLL) , where t h e degree o f u n d e r m o d i f i c a t i o n i n c r e a s e s f r o m s t a g e A t o stage C o f t h e B i n e t c l a s s i f i c a t i o n . Lymphocytes o f a l l these p a t i e n t s r e p r e s e n t t h e same s t a g e o f Bc e l l d i f f e r e n t i a t i o n , b u t t h e i r p r o l i f e r a t i v e a c t i v i t y increases from f a v o u r a b l e t o u n f a v o u r a b l e p r o g n o s t i c s t a g e s . The h i g h amount o f G34-tRNA found i n r e g e n e r a t i n g r a t l i v e r suggests t h a t t h e l a c k o f queuosine i n t R N A i s c o r r e l a t e d w i t h c e l l p r o l i f e r a t i o n i n non-malignant c e l l s as w e l l as i n m a l i g n a n t c e l l s (see Table 2.4.2 i n s e c t i o n 2.4.6). C h a r a c t e r i s t i c p a t t e r n s o f 9 species o f G34-tRNAs a r e observa b l e i n t h e t R N A p a t t e r n s o f CLL c e l l s , (upper panel i n F i g u r e 2.7). The number and p o s i t i o n s o f t h e m a j o r Q - d e f i c i e n t tRNAs were almost i d e n t i c a l i n p r e p a r a t i o n s from p a t i e n t s w i t h CLL, i r r e s p e c t i v e o f t h e c l i n i c a l stage. The t R N A f r o m t h e spleen o f p a t i e n t s w i t h h a i r y c e l l leukemia (HCL) showed t h r e e tRNAs; a c c o r d i n g t o e l e c t r o p h o r e t i c m o b i l i t y , species 2, 3, and 4. P a t i e n t s w i t h AML showed i n 4 o f 5 cases, one predominant t R N A species , a c c o r d i n g t o e l e c t r o p h o r e t i c mobi 1ity; s p e c i e s 2 i n F i g u r e 2.7 tRNAs from t h e spleen, from t h e plasmocytoma and from immunocytoma r e v e a l two t R N A species ( n o t shown) and tRNAs from the four patients with chronic myelocytic leukemia/blast c r i s i s (CML) e x h i b i t o n l y one m a j o r band. I t i s important t h a t the s p e c i f i c p a t t e r n s a r e independent o f t h e e x t e n t o f undermodi f i cation. Another i m p o r t a n t aspect i s t h a t t h e t R N A p r e p a r a t i o n s from p a t i e n t s w i t h CLL and c h r o n i c m y e l o c y t i c leukemia (CML), d u r i n g b l a s t c r i s i s , were o b t a i n e d from p e r i p h e r a l mononuclear c e l l s r e p r e s e n t i n g a homogenous c e l l p o p u l a t i o n . P a t i e n t s w i t h these diseases were i n d i f f e r e n t p r o g n o s t i c stages o r had b l a s t s w i t h d i f f e r e n t c e l l u l a r c h a r a c t e r i s t i c s . However, t h e t R N A p a t t e r n s

Figure 2 . 7 E l e c t r o p h o r e t i c p a t t e r n s of G34-tRNAsr 1 abel ed a t o s i t i o n 34 with 3 H Gua from p a t i e n t s with human lymphomas and Yeukemj a s C L L = Chronic lymphatic leukemia; AML = Acute myeloic leukemia.

.

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seem t o be c h a r a c t e r i s t i c f o r t h e d i s e a s e i r r e s p e c t i v e of i t s c l i n i c a l a c t i v i t y o r i t s cytochemical c l a s s i f i c a t i o n . The o t h e r tumors, studied so f a r , have a l s o shown very c h a r a c t e r i s t i c , but d i f f e r e n t , p a t t e r n s o f G34-tRNAs ( r e f . 62). 2.4.6

O u a n t i t a t i v e Analyses and Synonsis The r e s u l t s of q u a n t i t a t i v e a n a l y s e s of t h e amount of G34tRNAs i n u n f r a c t i o n a t e d t R N A from E . c o l i and from a l l eukaryo t i c c e l l s and t i s s u e s a r e summarized i n Tables 2 . 1 and 2 2 r e s p e c t i v e l y . The t R N A t r a n s g l y c o s y l a s e from E . c o l i , used n t h e s e experiments, was p u r i f i e d a s described i n r e f . 30, however TABLE 2.2 Q u a n t i t a t i v e Analyses o f t R N A s w i t h 634 i n P l a c e o f Q i n Unfract i o n a t e d t R N A s from E . c 0 7 i s t r a i n s MRE 600, SHSP-19 hemA and SASX-77 hemA: Influence o f I r o n , Anaerobiosis and 5-Aminolevulinate

Source o f tRNA

Growth c o n d i t i o n s

FeSO,

aerob, s t a t . phase

0-lOpM 100pM

50 >5

0-100pM

50

10pM

102

I,

E . coli

MRE 600

E . coli

hemA mu t a n ts

anaerob, s t a t . phase closed f l a s k s anaerob, s t a t . phase fermenter (N, ) aerob, s t a t . phase-5-ALA aerob, " " +5-ALA anaerob, " '' -5-ALA an ae r o b , " " +5-ALA

pmo13H Gua i n c o r p . per A,,, t R N A

10pM !I

I,

It

0 37 0

25

T h e b a c t e r i a w e r e g r o w n in ' W e r k m a n m i n i m a l medium', a e r o b i c a l l y w i t h 0.4% g l u c o s e , a n a e r o b i c a l l y w i t h 0.8% g l u c o s e . 5-ALA (5a m i n o l e v u l i n a t e w a s a d d e d at a c o n c e n t r a t i o n o f 20 mg/l). (a) V a l u e s r e f e r t o s t r a i n S H S P - 1 9 a n d ( b ) t o s t r a i n S A S X - 7 7 (6. Bachmann, Y a l e University, strain collection). (6. F r e y , S. I s e p p a n d H . K e r s t e n , u n p u b l i s h e d results).

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TABLE 2.3 Q u a n t i t a t i v e A n a l y s i s o f G34-tRNAs Euk a r y o t e s Source o f t RNA

N u t r i e n t source

D. d i s c o i d e o m

E . coli

t

i n U n f r a c t i o n a t e d tRNAs f r o m

pmol

3 H Gua i n c o r p o r a t e d p e r A, tRNAl2h

axeni c medi um amino a c i d + queuine medium - queuine

3

11

tobacco 1eaves cells wheat 1eaves wheat e r m bean c l 1 o r o p l a s t

10

20

5 55

queuine-free medium

Xiphophorine fishes 1 iv e r normal s k i n benign me1 anoma ma1 ignant me1 anoma

murine e r y t h r o 1eukemi c cel, 1s, growi ng growing " d i fferentiating (butyrate induced)

c u l t u r e ) horse serum - queuine 116 medium )

"

"

calf]

Human tumors* immunocytoma p l asmocytoma CLL lymph node c c l lymph node HZL s l e e n norma spleen

7

*Number of cases, detailed analyses d e s c r i b e d in r e f . 62.

+ queuine

2 38 18

4 28 35 25 15 5' and clinical details are

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the last step was omitted. With this enzyme preparation, plateauvalues are obtained after two hours of incubation. In unfractionated yeast tRNA the enzyme exchanges 120 pmol 3H-guanine/A,6, unit within two hours. From the result that the addition of iron to aerobically grown E . c o 7 i MRE 600 prevents the accumulation of tRNAs having G34 in place of Q, it follows that either iron is essential for the synthesis of queuine precursors, or that iron serves as a cofactor to the tRNA transglycosylase. It is therefore assumed that Q-tRNAs are synthesized as long as iron is available, however, that queuine is released during turnover of tRNA and cannot be incorporated into the newly synthesized tRNAs when iron becomes limiting. As to whether queuine can be reutilized in prokaryotes is presently unknown. In higher eukaryotes a turnover of tRNAs and a salvage pathway for queuine has been observed (ref. 64). Under strict anaerobic fermentation conditions Q is not being synthesized in E . C O l i MRE 600. The accumulation of Q-deficient tRNAs in the anaerobically grown hemA mutants SHSP-19 and SASX-77 can be prevented by iron t 5-aminolevulinic acid, however, not by iron itself. This suggests that the biosynthesis of queuine involves a heme-iron protein and that the biosynthesis of Q-tRNAs and the biosynthesis of heme are somehow related. From the results of the systematic qua1 i tative and quanti tative analyses of Q-tRNAs and o f G34-tRNAs in lower and higher eukaryotes it is evident that the extent of Q-modification in tRNAs is different in various cells and tissues and that it is related to the metabolic state. In the following sections causes of variations in the levels of queuine and the extent of Q-modification in corresponding tRNAs as well as emerging regulatory functions are presented. 2.5 CAUSES OF Q-DEFICIENCY IN EUKARYOTIC tRNAS In eukaryotes, unable to synthesize queuine d e n o v o , limitation of free queuine causes hypomodification in respective tRNAs. In addition, other parameters influence the extent of Q-modification. These are (i) inhibitors of the tRNA guanine transglycosylases e . g . endogenous pteridines with Ki values ranging from 5 ~ 1 0 - ~ (pterin) to 2x10-6M (biopterin) (ref. 65), (ii) differences in the

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t u r n o v e r o f Q-tRNAs and r e s p e c t i v e G34 tRNAs ( r e f . 40), (iii) v a r i a t i o n s i n t R N A - t r a n s g l y c o s y l ase 1e v e l s a n d / o r t h e 1 e v e l s o f enzymes t h a t a r e i n v o l v e d i n t h e s a l v a g e pathway o f q u e u o s i n e ( r e f . 64). 2.5.1

E n z y m a t i c Assay f o r F r e e Oueuine To c l a r i f y w h e t h e r Q - d e f i c i e n c y i n tRNAs o f e u k a r y o t e s i s caused b y t h e absence o f queuine, a c o n v e n i e n t e n z y m a t i c a s s a y f o r t h e d e t e r m i n a t i o n o f f r e e q u e u i n e has been e s t a b l i s h e d . The assay (i)t h e tRNA g u a n i n e - t r a n s i s based on t h e f o l l o w i n g f a c t s : g l y c o s y l a s e o f D . d i s c o i d e u m exchanges i n an a l m o s t i r r e v e r s i b l e r e a c t i o n e x c l u s i v e l y g u a n i n e 34 i n t h e homologous tRNA f o r queui n e , however, n o t f o r guanine; (ii) Q - d e f i c i e n t t R N A can be i s o l a t e d f r o m D . D i s c o i d e u m , grown i n q u e u i n e d e f i c i e n t medium and l a b e l e d a t p o s i t i o n 34 w i t h 3 H g u a n i n e b y t h e t R N A g u a n i n e - t r a n s glycosylase f r o m E . c o l i ; (iii) an amount o f 3 H g u a n i n e i s released from t h e l a b e l e d tRNA by t h e tRNA t r a n s g l y c o s y l a s e o f D . d i s c o i d e u m i n t h e presence o f queuine which i s p r o p o r t i o n a l t o t h e c o n c e n t r a t i o n o f f r e e q u e u i n e i n t h e assay. Crude t R N A - t r a n s g l y c o s y l ase p r e s e n t i n tRNA-free S-100 f r o m D. d i s c o i d e u m i s used i n t h i s t e s t system. The d e t a i l s and assay c o n d i t i o n s a r e d e s c r i b e d i n r e f . 62. The k i n e t i c s o f 3 H g u a n i n e r e l e a s e were d e t e r m i n e d w i t h r e s p e c t t o t i m e and q u e u i n e concenH g u a n i n e r e l e a s e was measured w i t h o u t t r a t i o n . As a c o n t r o l , a d d i t i o n o f queuine t o c o r r e c t f o r t h e r e l e a s e o f 3 H guanine by tRNA degradation. P1 ateau-Val ues f o r H g u a n i n e r e 1 ease a t d i f f e r e n t enzyme c o n c e n t r a t i o n s were a c h i e v e d a f t e r t h r e e h o u r s o f incubation. The l i m i t o f q u e u i n e d e t e c t i o n i s 3 ~ 1 0 - ~ i Mn t h i s assay. The Km v a l u e f o r q u e u i n e o f t h e t R N A t r a n s g l y c o s y l a s e o f D . d i s c o i d e u m was f o u n d t o be ~ x ~ O - ~ MT .h i s v a l u e a g r e e s w e l l w i t h t h a t o f t R N A - t r a n s g l y c o s y l a s e o f wheat germ, 9 . 5 ~ 1 0 - ~ M (, r e f . 66) and o f t h e mammalian enzymes, 4 . 5 ~ 1 0 - ~ M ( r e f s . 34, 6 7 ) . The a n a l y s i s o f f r e e q u e u i n e has been p e r f o r m e d u n t i l now by t w o assays, t h e b i o l o g i c a l L-M c e l l t e s t ( r e f . 68) and b y combined gas chromatography/mass s p e c t r o m e t r y w i t h s e l e c t e d i o n m o n i t o r i n g ( K a t z e and McCl oskey, p e r s o n a l c o m m u n i c a t i o n ) . The b i o l o g i c a l t e s t i s t e d i o u s , and t h e b i o p h y s i c a l method r e q u i r e s s p e c i a l e x p e n s i v e equipment and e x p e r i e n c e . The e n z y m a t i c a n a l y s i s o f

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queuine ( r e f . 62) p e r m i t s d e t e r m i n a t i o n o f queuine i n t i s s u e e x t r a c t s , t h e l i m i t i n g c o n c e n t r a t i o n s b e i n g 5-10 ng p e r m l serum o r wet w e i g h t o f c e l l s . A p p l y i n g t h i s method, an amount o f f r e e queuine o f 200 ng/g wet w e i g h t was found i n wheat germ. In c o n t r a s t , i n p l a n t leaves queuine i s p r e s e n t , i f a t a l l , i n amounts l o w e r than 10 ng/g wet w e i g h t . P l a n t c e l l s , when grown i n c u l t u r e c o n t a i n Q - d e f i c i e n t tRNAs (Table 2.2) s u g g e s t i n g t h a t p l a n t s cannot s y n t h e s i z e queuine de novo. The source o f queuine i n wheat germ i s p r e s e n t l y n o t known. TABLE 2.4 Queuine A n a l y s i s i n Sera, Sources Source

f e t a l c a l f serum horse serum wheat germ wheat 1eaves tobacco 1eaves Xenopus oocytes mouse t e s t e s human tumors immun o cy toma p l asmocytoma CLL lymph node CCL lymph node HZL spleen normal spleen

T i s s u e and C e l l E x t r a c t s f r o m Various

ng queuine p e r g wet w e i g h t c e l l s o r t i s s u e (serum)

40 (30-50) 0-10 (14) 200 ' 10 ' 5 260 56 (58) 128 47 107 156 57 138

C L L : c h r o n i c l y m p h a t i c l e u k e m i a ; CCL: c e n t r o c y t i c l y m p h o m a ; H Z L : h a i r y c e l l leukemia. The values i n b r a c k e t s a r e f o r comparison. They a r e o b t a i n e d i n t h e L-M a s s a y d e s c r i b e d i n r e f . 6 8 .

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S t r i k i n g l y , high c o n t e n t s of queuine occur i n mouse t e s t e s and i n The t i s s u e - s p e c i f i c d i s t r i b u t i o n of queuine and of Q-versus G34 tRNAs might be a m a t t e r of queuine t r a n s p o r t i n t o different tissues. Application of r a d i o a c t i v e l y l a b e l e d queuine t o animals and determination of t h e d i s t r i b u t i o n of queuine i n d i f f e r e n t t i s s u e s have shown a 100-fold accumulation of t h e modified base i n e r y t h r o c y t e s , compared with o t h e r t i s s u e s . Apparently queuine i s t i g h t l y bound t o hemoglobin (Nishimura 12th I n t e r n a t i o n a l Workshop, Umea, Sweden 1987). 2.5.2 Inhi bi t i on of t R N A Guanine Transal vcosvl a s e s bv P t e r i di nes The t R N A guanine t r a n s g l y c o s y l a s e from E . c o l i exchanges i n the corresponding tRNAs t h e guanine r e s i d u e w i t h a l l p r e c u r s o r s of queuine ( r e f . 3 1 ) . Since guanine i t s e l f s e r v e s a s a p r e c u r s o r i n the b i o s y n t h e s i s of queuine, a guanine a t p o s i t i o n 34 of c o r r e s ponding tRNAs can be exchanged a l s o by guanine. The b i o s y n t h e s i s of queuine i s thought t o have s e v e r a l b i o s y n t h e t i c s t e p s i n common with t h a t of p t e r i d i n e s . Also, p t e r i d i n e s a r e s t r o n g c o m p e t i t i v e i n h i b i t o r s of p r o k a r y o t i c ( r e f . 60) a s well a s of e u k a r y o t i c t R N A transglycosylases ( r e f . 65). P t e r i n , s e p i a p t e r i n and b i o p t e r i n i n h i b i t t h e t R N A guanine t r a n s g l y c o s y l a s e of r a b b i t r e t i c u l o c y t e s a t c o n c e n t r a t i o n s of 5x10-8, 1 . 3 ~ 1 0 - and ~ 2 ~ 1 0 -r ~e s p e c t i v e l y . The e f f e c t of p t e r i d i n e s was t e s t e d i n i n t a c t c e l l s of murine f i b r o b l a s t s / l i n e L-M. P t e r i n was found t o be a b e t t e r i n h i b i t o r than b i o p t e r i n i n v i t r o , however, b i o p t e r i n i s a more e f f i c i e n t i n h i b i t o r in i n t a c t c e l l s . Biopterin occurs i n t r a c e l l u l a r l y 1 a r g e l y i n i t s reduced forms , di hydrobi o p t e r i n and t e t r a h y d r o b i op t e r i n , with the l a t t e r predominating ( r e f s . 69, 70). This may e x p l a i n why b i o p t e r i n i s a b e t t e r i n h i b i t o r than p t e r i n i n i n t a c t c e l l s ( r e f . 68). These r e s u l t s have l e d us t o the s u g g e s t i o n t h a t v a r i a t i o n s i n t h e amount and/or r e d o x - s t a t e of b i o p t e r i n may account f o r a l t e r a t i o n s i n the extent of Q-modification i n tRNAs ( r e f . 71). Although no d i r e c t evidence can be presented t h a t t h i s i s of b i o l o g i c a l importance, s e v e r a l 1 i n e s of i n d i r e c t experimental d a t a support t h i s view e.g. : (i) In D . d i s c o i d e u m , s u f f i c i e n t l y s u p p l i e d with queuine, f l u c t u a t i o n s occur i n the Q-content of t R N A ( s e e s e c t i o n 2 . 4 . 2 ) . During the e a r l y developmental s t a g e , p t e r i d i n e s a r e metabolized X e n o p u s oocytes.

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and a r e e x c r e t e d as lumazine ( r e f . 72). E x a c t l y a t t h a t developmental stage t h e e x t e n t o f Q - m o d i f i c a t i o n i n t R N A i s increased (see 2.4.2, F i g u r e 2.5b). (ii) I n X i p h o p h o r i n e f i s h e s h i g h c o n c e n t r a t i o n s o f p t e r i d i n e s occur i n t h e i r s k i n , and t h e amount and p a t t e r n s o f p t e r i d i n e s a r e s p e c i f i c f o r a c e r t a i n genotype ( r e f . 73). The a n a l y s i s o f tRNAs i s o l a t e d from o f f s p r i n g s o f d i f f e r e n t genotypes o f X . h e l l e r i and X . m a c u l a t u s showed t h a t t h e l e v e l s o f G34-tRNAs v a r y with the pattern o f pteridines i n t h e i r skin. Offsprings that have h i g h amounts o f d r o s o p t e r i n c o n t a i n about t w o - f o l d h i g h e r l e v e l s o f G34-tRNAs i n t h e i r s k i n t h a n t h o s e t h a t have predomin a n t l y sepi a p t e r i n, is o x a n t h o p t e r i n and b i o p t e r i n ( r e f . 60). (iii) I n v a r i o u s c e l l s and t i s s u e s and i n t h e u r i n e o f cancer p a t i e n t s , e l e v a t e d 1eve1 s o f p t e r i d i nes were found ( r e f . 74). Possibly pteridines i n h i b i t the tRNA transglycosylase i n tumor t i s s u e s i n c e q u e u i n e - l i m i t a t i o n does n o t seem t o be respons i b l e f o r t h e h i g h c o n t e n t o f G34-tRNAs i n lymphomas e . g . CLL (lymph nodes) and CCL lymph nodes (see Table 2.2 and 2.3 i n t h e p r e v ous s e c t i o n ) . REGULATION OF GENE EXPRESSION BY ALTERED Q-MODIFICATION I N tRNAS The r e s u l t s o f t h e d e t a i l e d analyses o f t h e e x t e n t o f Qm o d i f i c a t i o n i n s p e c i f i c tRNAs from E . c o l i , from D . d i s c o i d e u m , from p l a n t 1eaves ( c y t o p l asm and c h l o r o p l a s t s ) and wheat germ, from t h e s k i n and melanomas o f X i p h o p h o r i n e f i s h e s , from e r y t h r o leukemic c e l l s o f mice, and from human lymphomas and leukemias queuine and s u p p o r t t h e view p r e v i o u s l y r e p o r t e d ( r e f . 10): a l t e r a t i o n s i n t h e Q - m o d i f i c a t i o n o f tRNAs o f t h e Q - f a m i l y a r e p o s s i b l y i n v o l v e d i n c o n t r o l mechanisms t h a t adapt c e l l s and t i s s u e s t o environmental changes.

2.6

2.6.1

F u n c t i o n a l P r o D e r t i e s o f tRNAs C o n t a i n i n a G34 o r Q I n an a t t e m p t t o g a i n i n s i g h t i n t o t h e m o l e c u l a r mechanism by which tRNAs w i t h an a l t e r e d m o d i f i c a t i o n e x e r t a r e g u l a t o r y r o l e on c e l l metabolism, t h e f u n c t i o n a l p r o p e r t i e s o f Q - c o n t a i n i n g and Q - d e f i c i e n t tRNAs have been s t u d i e d i n c e l l - f r e e systems. I t has been r e p o r t e d t h a t t h e a m i n o a c y l a t i o n k i n e t i c s i n v i t r o a r e

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d i f f e r e n t f o r Q - c o n t a i n i n g o r Q - d e f i c i e n t e u k a r y o t i c t R N A A S p . The Q - c o n t a i n i n g t R N A i s a b e t t e r s u b s t r a t e than t h e G - c o n t a i n i n g c o u n t e r p a r t ( r e f . 67). I n prokaryotes, a t r a n s i e n t occurrence o f u n a c y l a t e d G34-tRNAASp, tRNAAsn, t R N A T v r and tRNAHiS m i g h t r e l i e v e t h e a t t e n u a t i o n a t corresponding amino a c i d operons. E u k a r y o t i c tRNAsTvr from tobacco o r wheat leaves t h a t a r e t o t a l l y l a c k i n g queuine were t e s t e d i n t r a n s l a t i o n o f TMV RNA i n tobacco p r o t o p l a s t s and found t o y i e l d a 17.5 K c o a t p r o t e i n , a 125 K p r o t e i n and a 183 K p r o t e i n which i s generated by an e f f i c i e n t readthrough o v e r t h e UAG t e r m i n a t i o n codons by t R N A T v r w i t h G$A i n p l a c e o f Q$A anticodon ( r e f s . 50, 51). Therefore, suppress i o n o f s p e c i f i c codons o f mRNA by Q - d e f i c i e n t t R N A T y r m i g h t p l a y In a r o l e i n t h e c o n t r o l o f gene expression i n eukaryotes. e u k a r y o t i c t R N A H i S t h e absence o r presence o f Q i n f l u e n c e s decoding p r o p e r t i e s d u r i n g t h e e l o n g a t i o n s t e p o f mRNA t r a n s l a t i o n ( r e f . 75). I f o n l y codons i n a s p e c i f i c c o n t e x t a r e a f f e c t e d , t h e G34 , versus Q-tRNAHi S , m i g h t d i s t i n g u i s h between d i f f e r e n t mRNAs f o r t r a n s l a t i o n . To f i n a l l y s o l v e t h e m o l e c u l a r mechanism o f t h e f u n c t i o n o f Q and G34 tRNAs i t i s e s s e n t i a l t o know t h e s p e c i f i c genes t h a t a r e r e g u l a t e d a t t h e t r a n s c r i p t i o n a l o r p o s t t r a n s c r i p t i o n a l l e v e l by queuine and v a r i a t i o n s i n Q - m o d i f i c a t i o n o f tRNA. As a working hypothesis we have assumed t h a t Q-tRNAs and t h e corresponding G34 tRNAs m i g h t be i n v o l v e d i n m e t a b o l i c c o n t r o l of r e s p i r a t i o n and t h e metabolism o f l a c t a t e i n p r o k a r y o t e s as w e l l as i n eukaryotes. T h i s assumption i s based on t h e f o l l o w i n g (i)On t h e e v o l u t i o n a r y s c a l e o f organisms, Q appears facts: f i r s t i n tRNAs o f f a c u l t a t i v e anaerobes, e . g . E s c h e r i c h i a c o 7 i t h a t u t i l i z e oxygen as t h e f i n a l e l e c t r o n a c c e p t o r under a e r o b i c c o n d i t i o n s and can produce l a c t a t e under f e r m e n t a t i o n c o n d i t i o n s . The m o d i f i c a t i o n i s h i g h l y conserved and i s p r e s e n t i n c y t o p l a s m i c and m i t o c h o n d r i a 1 tRNAs o f l o w e r and h i g h e r eukaryotes; (ii) s u r p r i s i n g l y , y e a s t was found t o be t h e o n l y e u k a r y o t e i n which Q does n o t occur i n tRNAs; i n y e a s t , l a c t a t e i s n o t t h e end p r o d u c t o f g l y c o l y s i s ; (iii)tRNAs from several n e o p l a s t i c a l l y t r a n s f o r m e d c e l l s have a h i g h c o n t e n t o f G i n p l a c e o f Q. According t o 0. Warburg, these c e l l s a r e c h a r a c t e r i z e d by changes i n t h e metabol i s m o f l a c t a t e ; ( i v ) i n mammals, s p e c i f i c a l t e r a t i o n s i n t h e

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content of Q-tRNAs versus G34 a r e observed d u r i n g embryogenesi s , d i f f e r e n t i a t i o n , and ageing; s p e c i f i c changes were a l s o observed i n the metabolism o f l a c t a t e and i n t h e p a t t e r n of LDH-isoenzymes t h a t appeared t o be c o r r e l a t e d t o changes i n the e x t e n t of Qmodification i n t R N A ( r e f s . 10, 13, 7 6 ) . 2.6.2

ResDiration and Fermentation Pathways i n f. c o l i Two p a i r s of f . c o l i s t r a i n s have been c o n s t r u c t e d which contain o r lack the t R N A guanine t r a n s g l y c o s y l a s e . The t R N A t r a n s g l y c o s y l a s e mutants ( t g t ) a r e lacking queuosine i n r e s p e c t i v e tRNAs and the mutants a r e described t o be otherwise i s o g e n i c ( r e f . 77). I t i s p r e s e n t l y not known whether t h e t g t - l o c u s a t 9 m i n . on the g e n e t i c map of f. c o 7 i comprises the s t r u c t u r a l gene f o r the tRNA-transglycosyl a s e o r whether i t encodes a r e g u l a t o r y p r o t e i n Previously we have reported t h a t involved i n Q-biosynthesis. t h e s e mutants a r e d e f e c t i v e in t h e molybdopterin cofactor-dependent n i t r a t e reductase system and a r e d e f i c i e n t i n a, and d cytochromes ( r e f . 7 8 ) . In f u r t h e r experiments, however, we found a second mutation. T h i s mutation concerns a gene, the product of which i s r e s p o n s i b l e f o r t h e phenomenon of molybdate reduction i n anaerobi cal l y grown f. c o l i . Molybdate reduction has been re1 a t e d t o some of t h e p r o p e r t i e s of t h e molybdopterin system ( r e f . 79). We have designated t h e phenotype as 'Mor', the r e s p o n s i b l e gene i s h i g h l y c o t r a n s d u c i b l e w i t h t h e gene f n r coding f o r the anaerobic r e g u l a t o r p r o t e i n Fnr. The mutation i n this region and not t h e mutation i n t g t i s c o r r e l a t e d w i t h t h e absence of t h e molybdopterin-dependent n i t r a t e - r e d u c t a s e and of a, and d cytochromes. The s t r i k i n g coincidence o f two mutations r e l a t e d t o p t e r i d i n e s remains a t present unexpl a i ned. W i t h r e s p e c t t o t h e fermentation pathways, s e v e r a l anaerobic a l l y grown s t r a i n s of f. C O T ; containing Q-tRNAs have higher l e v e l s of l a c t a t e compared t o the o t h e r fermentation products, e . g . e t h a n o l , H, and CO, than those s t r a i n s t h a t a r e Q-deficient. (U. Michelsen and H . Kersten, u n p u b l i s h e d r e s u l t s ) . 2.6.3

Redox-Systems i n

D. discoideum

has been applied as a model t o study the l e v e l s of 1 a c t a t e , LDH-isoenzyme p a t t e r n s i n a 1 aboratory s t r a i n (AX 2) t h a t was grown and induced t o d i f f e r e n t i a t e i n the Dictyostelium discoideum

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absence o r presence of s u f f i c i e n t amounts of queuine. Low tempera t u r e d i f f e r e n c e s p e c t r a of cytochromes were monitored because cytochrome b i s a c o n s t i t u e n t of t h e NAD-independent ( L + ) - l a c t a t e cytochrome c oxidoreductase. In D . d i s c o i d e u m : --Very l i t t l e a l t e r a t i o n i n t h e o v e r a l l p a t t e r n s of p r o t e i n s y n t h e s i s i s observed i n response t o queuine a s j u d g e d from 2D O ' F a r r e l l g e l s ( r e f . 80). --D(-)- and t r a c e s of L ( + ) - l a c t a t e a r e p r e s e n t , both forms occur a t considerably reduced l e v e l s i n v e g e t a t i v e growing and d i f f e r e n t i a t i n g c e l l s when queuine i s l a c k i n g . --NAD-dependent D ( - ) - L D H i soenzyme a c t i v i t i e s and NAD-depend e n t L(+)-LDH isoenzyme a c t i v i t i e s e x h i b i t c h a r a c t e r i s t i c changes i n response t o queuine. --As judged from low temperature d i f f e r e n c e s p e c t r a of cytochromes, cytochrome b, accumul a t e s i n queui ne-1 acki ng c e l l s compared t o queui ne-containing c e l l s . The cytochrome b i s suggested t o be a component of an NAD-independent l a c t i c a c i d oxidoreductase found t o be a s s o c i a t e d w i t h t h e mitochondria of D . d i s c o i d e u m ( r e f . 76, and E . Schachner and H . Kersten, unpublished r e s u l t s ) . This enzyme might be s i m i l a r t o t h e y e a s t L ( + ) - l a c t a t e cytochrome c oxidoreduct a s e (cytochrome b, ) , o x i d i z i n g 1 a c t a t e t o pyruvate. LDH-Isoenzvmes i n Ervthroleukemic C e l l s The F r i e n d v i r u s transformed mouse e r y t h r o 7 e u k e m i c c e 1 7 s , c l o n e F,6, can be grown i n a medium supplemented w i t h h o r s e serum t h a t c o n t a i n s only t r a c e s of queuine. The a d d i t i o n of queuine, 3x10-'Mr t o t h e c u l t u r e medium causes an almost t o t a l s h i f t of Qd e f i c i e n t t o Q-containing tRNAs. The o v e r a l l p a t t e r n s of p r o t e i n s from c e l l e x t r a c t s obtained from queuine-free and queuine-treated c e l l s show very l i t t l e a l t e r a t i o n . Most pronounced i s a s h i f t i n e l e c t r o p h o r e t i c m o b i l i t y of a 36 K p r o t e i n a s s o c i a t e d t o the membrane p r e p a r a t i o n s . The p r o t e i n i s not y e t i d e n t i f i e d ( r e f . 13). However, s p e c i f i c changes i n response t o queui ne concern NADHdependent LDH-isoenzyme a c t i v i t y p a t t e r n s ( r e f s . 13, 71), assumed f i r s t of a l l t o r e p r e s e n t t h e f i v e t e t r a m e r i c forms A , , A,B, , A,B,, A, B, and B , . The homotetrameric forms A, and B, a r e commonly 2.6.4

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found in s kel eta1 muscl e (M, =A, ) or heart muscle (H, =B, ) , respectively. The protein subunits are coded for by two separate genes, Ldh a and Ldh b. Another LDH isoenzyme, LDH C,, is encoded by a third gene, Ldh c, (ref. 81) Ldha and Ldhc are highly conserved genes. In mammals Ldh c, formerly designated as LDHX, is expressed tissue specific, namely in primary spermatocytes. LDH C, is a cytosol i c enzyme, however i t a1 so occurs associated to mi tochondria (refs. 82, 83). In growing, undifferentiated and queuine-deficient F,6 cells the total activity of the isoenzymes is higher than in queuinecontaining cells. When the transformed erythroleukemic cells are induced by butyrate to differentiate and to synthesize hemoglobin, the activity patterns o f the LDH-isoenzymes are changed. The activity suggested to be M, increases significantly in response to queuine (ref. 71) (Figure 2.8). Protein extracts have been separated from queuine-treated and queuine-1 acki ng F, 6 cell s on two-dimensional 0'Farrell gels and Immunoblots proteins have been transferred to ni trocel 1 ulose. have been performed by using a polyvalent antibody of rabbit anti(kindly supplied by Dr. E. Goldberg, Northwestern mouse LDH c, University, Evanston, IL, USA) and rabbit anti-pig LDH A,. The anti-mouse LDH C, antibody cross-reacts to a certain extent with the A-type, but not with the B-type subunit of LDH isoenzymes. The results of these experiments show an increase in amount and number (from 1 to 4) of LDH-subunits in growing, queuine-deficient cells as compared to queuine-containing cells as judged from the interaction with both anti-mouse LDH C, and anti-pig LDH A,. From the electrophoretic mobility it is evident that in queuine-deficient cells multiple forms of immunoreactive LDH (A) subunits appear at acid pH. In differentiating cells only one LDH A subunit responds to both antibodies, the amount being higher in queuine-treated cells (Figure 2.9). These results support the view that the observed changes in the activity patterns of LDH-isoenzymes are caused by qua1 itative or quantitative changes of the expression of the LDH A subunits in response to queuine.

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Fi ure 2 . 8

LDH-isoenzyme . a c t i v i t i e s from F46 cel!s separated by Cells were grown i n media supplemented w i t h horse serum w i t h o u t or w i t h queui ne (3x10- 7 M ) . Differentiation was induced by butyrate. M4=A4 i s s u g ested t o represent the tetrameric form of the muscle type LDH, t t e f o l l o w i n g four LDH activity bands are s t i l l t o be identified. PAEE.

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Figure 2.9 mouse LDH c, re11 gels (c). Q-, (d) anti body LDH

Immunoblottin of proteins from F,6 cells with antiantibody. T\e proteins werf separated on 2D-O’FarGrowin cells (a) Q- (b) Q * differentiating cells Q+ ?The same result is obiained with the rabbit

c, j.

2.7 SUMMARY AND PERSPECTIVES The biosynthesis of queuosine and the occurrence of Q or G at Position 34 i n tRNAASn,tRNAAsp, tRNAHiS and tRNATvr from eubacteria and from eukaryotes have been investigated to elucidate the biological significance of queuine and the Q-family of tRNAs. From the reported experimental data the following conclusions, suggestions and future aspects are derived. Queuosine appears t o be synthsized d e n o v o only in eubacteria. GTP serves as a precursor from which finally 7-aminomethyl-7-deazaguanine is formed. This part o f the biosynthetic pathway requires iron, possibly a heme iron protein. In the next steps the E . c o l i tRNA transglycosylase exchanges guanine 34 by 7aminomethyl-7-deazaguanine and the cyclopentendiol moiety is attached to the precursor at the level of tRNA. Evidence is

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presented showing t h a t t h e e p o x i d e - d e r i v a t i v e OQ i s an i n t e r mediate i n t h e b i o s y n t h e s i s of Q . The epoxide, being formed when E . C a l i o r S a l m o n e l l a t y p h i m u r i u m a r e grown i n g l u c o s e s a l t medium, i s converted t o Q a f t e r a s h i f t t o enriched media. From t h e n u t r i e n t s t e s t e d , only cobalamine, vitamine Biz, proved t o be e s s e n t i a l f o r the formation of Q. I t i s t h e r e f o r e assumed: ( i ) t h a t oQ i s converted t o Q by an enzyme analogous t o the adenosylcobalamine-dependent r i b o n u c l e o t i d e r e d u c t a s e and ( i i ) t h a t the cyclopentendiol-ring i s derived from a r i b o s y l - o r r i b o t y l donat i n g molecule. Lower and higher eukaryotes, except y e a s t , a r e s u p p l i e d with queuine by b a c t e r i a l sources. The presence of G34 i n p l a c e of Q i n c y t o s o l i c and c h l o r o p l a s t tRNAs of tobacco and wheat l e a v e s i s caused by the absence of queuine. In c o n t r a s t t o l e a v e s , wheat germ c o n t a i n s high amounts of queuine. I t has been shown t h e t R N A s T v r G34 from p l a n t l e a v e s suppress the nonsense codon UAG i n Tobacco Mosaic Virus (TMV) RNA (50, 51). Therefore, we suggest t h a t t h e d r a s t i c d i f f e r e n c e s of Q-modificat i o n i n mature l e a v e s and ebryonic t i s s u e s e r v e a s a molecular b a s i s f o r n a t u r a l UAG suppression and permit t h e formation of a1 t e r n a t e p r o t e i n s . A1 t e r a t i o n s i n codon usage by a Q-containing o r r e s p e c t i v e Q-deficient t R N A i s another p o s s i b l e mechanism of t r a n s l a t i o n a l control ( s e e t h i s s e r i e s E. Kubli Codon Usage and Qbase Modification i n D r o s o p h i l i a m e 7 a n o g a s t e r ) . In c o n t r a s t t o p l a n t leaves the low e x t e n t of Q-modification - found i n tRNAs of t h e s k i n of X i p h o p h o r i n e f i s h e s - can be t r a c e d back t o the high l e v e l of p t e r i d i n e s i n the s k i n . P t e r i d i n e s a r e potent i n h i b i t o r s of t h e t R N A t r a n s g l y c o s y l a s e r e a c t i o n . The i n h i b i t o r y e f f e c t of b i o p t e r i n on the t R N A t r a n s g l y c o s y l a s e s and i t s f u n c t i o n a s coenzyme of monoxigenases i n hydroxylation r e a c t i o n s o f t r y o s i n e metabolism might be r e l a t e d . In h u m a n lymphomas. t h e observed accumulation of G34-tRNAs i s not caused by a d e f i c i e n c y of queuine. S u p r i s i n g l y , t h e e x t e n t of undermodification i s s p e c i f i c f o r a d i s t i n c t t R N A s p e c i e s and shows c h a r a c t e r i s t i c v a r i a t i o n s i n r e l a t i o n t o the c l o n a l o r i g i n of the lymphoid t i s s u e . A l t e r a t i o n s i n p t e r i d i n e metabolism a r e observed i n tumor p a t i e n t s . Therefore h i g h l e v e l s o r changes i n

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the redox s t a t e of biopterin m i g h t be responsible f o r t h e occurrence of G34-tRNAs i n human lymphomas. D i c t y o s t e l i u m d i c o i d e u m proved t o be a s u i t a b l e model system t o analyse consequences of Q-nucleoside deficiency. The c e l l s can be grown i n c u l t u r e w i t h or without addition of queuine. Recently a fusion gene of the D i c t y o s t e l i u m gene d i s c o i d i n and o f the E . c o l i lacZ gene w i t h an UAG stop codon has been constructed and integrated i n t o the D. discoideum genome. When the transformed s t r a i n i s grown i n the absence of exogenous queuine the UAG stop codon i s suppressed and a functional B-galacactosidase expressed. However when the s t r a i n i s grown i n the presence of queuine, where members of the Q-family of tRNAs a r e modified a t position 34, a functional p-gal actosi dase i s n o t expressed ( T h . Di ngermann, unpubl ished relsul t s ) . Biochemical changes observed i n Q-deficient D . d i s c o i d e u m are: an increase i n the amount of cytochromes, especially of cytocrome b,,, w i t h an identical absorption maxium as cytochrome b, from yeast; a decrease i n the levels of D (-) - l a c t a t e and L (+) - l a c t a t e and CAMP receptors. Cytochrome b2 of yeast i s a flavo hem-protein; i t provides a short, antimycin A r e s i s t a n t , respiratory chain i n which L(+)l a c t a t e can be oxidized v i a cytochrome c a n d cytochrome oxidase t o yield one mole of ATP. In Dictyostelium discoideum an antimycin A i n s e n s i t i v e respiratory chain i s present in growing c e l l s . This might be switched on when queuine i s absent and/or the respective tRNAs contain G i n place of Q. Fri end-vi rus transformed erythrol eukemi c c e l l s of mi ce, clone F46, can be grown i n media, supplemented w i t h horse serum, t h a t i s t o t a l l y lacking queuine. The deficiency of queuine causes a four t o five-fold increase i n the amount o f cytochromes per c e l l and a s i g n i f i c a n t increase in the a c t i v i t i e s of f i v e NAD+-dependent L ( + ) - L D H isoenzymes. Analysis of LDH protein subunits reveal the presence of two d i f f e r e n t LDH A subunits. In e x t r a c t s from c e l l s grown i n the absence of queuine one o f the LDH A subunits shows a s i g n i f i c a n t s h i f t t o a c i d i c pH on two-dimensional g e l s and h i g h c r o s s r e a c t i v i t y w i t h purified anti-LDH C . No LDH-protein s u b u n i t occurs i n F46 c e l l s t h a t immunoprecipitates w i t h anti-LDH B . In previous reports ( r e f s . 10, 7 6 ) we have discussed t h a t the accumulation of Q-defi ci ent tRNAs i n embryonic t i s s u e and f a s t pro1 i f e r a t i n g tumor c e l l s and concomitantly observed a1 t e r a t i o n s i n redox-systems and LDH-enzymes may be casually r e l a t e d . New r e s u l t s included i n this report f u r t h e r support this hypothesis.

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52. H. B e i e r ,

7

1

1

.

6

b

197B 61-95. 58. !I. Sc a r t l , A. Barnekow, H. .Bauer and F. Anders, C o r r e l a t ons 59.

o f i n h e r i t a n c e and e x o r e s s i o n between a tumor gene and t h e c e l l u l a r homolog o f t h e ~ o u ssarcoma v i r u s - t r a n s f o r m i n g ene i n X i p h o p h o r u s , Cancer Res., 42 (1982) 4222-4227. F. Anders, The b i o l o g y o f an oncogene, based upon s t u d i e s on R. J a e n i c k e . ( E d . ) , 33. neo l a s i a i n Xiphophorus, in: Col oquium-Mosbach, Biochemistr o f Differenti ation and Morphogenesi s , S p r i nger-Verl ag e r l in-Hei d e l berg, 1982, pp.

7

91-115.

60. H.

61.

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62. 63.

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83.

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t i o n techniques:

1986, p p . 301-326.

methods and a p p l i c a t i o n s ,

Academic

Press

ADDENDUM UDdated references and information

P. Blihm, E. Zubrod, U . Mahr and H. Kersten, Changes of t h e express i o n of lactate-dehydrogenase isoenz mes o f LDH A subunits and of phosphoprotei ns i n muri ne erythrofeukemi c c e l l s 1 acki ng the deaza uanine-derivative gueuine a n d queuosine i n s p e c i f i c tRNAs, Mol. t e l l Biol. (Life Sci. Adv.) 7 (1988) 117-122. H. Kersten, The n u t r i e n t factor.queuine:

bios n t h e s i s

i n t r a n s f e r RNA and function, BioFactors 1 (19g8) 27-24.

occurrence

B: Frey, J . McCloskey, W. Kersten a n d H . Kersten, New function o f v i tam1 n B,.z : cobamide-dependent reduction o f epoxyqueuosine t o ueuosine i n tRNAs of E s c h e r i c h i a c o l i and S a l m o n e l l a t y p h i m u r i u m , . Bacteriol. 170 (1988) 2078-2082.

!

L . Szabo, S. Nishimura and W . R . Farkas, Possible involvement of queuine i n oxidative metabolism, BioFactors 1 (1988) 241-244. B . Frey, G. J l n e l , U. Michelsen and H . Kersten, Mutations i n E s c h e r i c h i a C o l i fnr a n d t g t genes: Control of molybdate reduct a s e a c t i v i t and t h e cytochrome d complex by fnr, J . Bacteriol. 171 (1989) 1!!24-1530.

Metabolic Control i n HeLA-Cells Mediated by Queuine W . Langgut and H . Kersten

13th International t R N A Workshop Vancouver, Canada, 1989 The modified base ueuosine occurs i n t h e anticodon a t position 34 i n tRNAs,,, i n exctange f o r guanine. t o .yield queuosine Although the occurence of queuine and queuosine i s ubiquitous, i t s function i s poorly understood. HeLa-cells, grown i n the presence o r absence of queuine, show s p e c i f i c a l t e r a t i o n s of their metabolic s t a t e : 1. Queuine causes t h e synthesis o f new proteins as well a s s h i f t s i n t h e i soel e c t r i c points of preexi s t i ng proteins. 2. Queuine influences t h e level of two d i s t i n c t LDH-activities d i f f e r e n t from t h e normal isoforms o f NAD-dependent l a c t a t e deh drogenase. One of these i s t h e anoxic s t r e s s protein asp 34dDH, described recently ( 1 ) .

Queuine-containing c e l l s grow t 0 . a higher density than queuine-lacking c e l l s , however only i n t h e presence of Ca*+. 3.

We propose t h a t queuine functions i n a d a t i n g c e l l .metabolism t o hypoxic conditions and thereby helps cel s t o survive periods of low oxygen tension.

P

(1) Anderson e t a l . (1988), Biochemistry 21, 2187-2193.

B109

CHAPTER 3 CODON USAGE AND MELANOGASTER

Q-BASE MODIFICATION I N

DROSOPHILA

E. KUBLI Zoological

Institute,

Univ.

Zurich-Xrchel,

Winterthurerstrasse

190, CH-8057 Zurich, Switzerland

TABLE OF CONTENTS Introduction 3.1 Codon Usage i n H i g h l y and Weakly Expressed Genes i n 3.2 Drosophila melanogaster 3.3 Q-Base M o d i f i c a t i o n and Decoding P r o p e r t i e s Q-Base M o d i f i c a t i o n , P r o t e i n Synthesis, and Develop3.4 ment Codon Usage and Gene D u p l i c a t i o n s 3.5

..................... 6109 . . . . . . . . . . . . . . . 8110 . . . . . 8115 ......................... 8116 . . . . . . . . . . 8117

3.6 3.7 3.8 3.9 3.10

Codon Usa e i n Genes Composed o f Conserved and NonConserved egions . . . . . . . . . . . . . . . . . . Codon Usage o f Homologous, H i h l y Expressed Genes i n N a t u r a l P o p u l a t i o n s and i n S i b q i n g Species Concluding Remarks Summary References

i

8118

. . . . . . 8118 . . . . . . . . . . . . . . . . . . 8119 . . . . . . . . . . . . . . . . . . . . . . . 8121 . . . . . . . . . . . . . . . . . . . . . . 8121

INTRODUCTION Codon usage i s nonrandom, i t i s genome s p e c i f i c and, w i t h i n a species, i t shows a s p e c i a l b i a s i n genes c o d i n g f o r p r o t e i n s s y n t h e s i z e d i n v e r y l a r g e q u a n t i t i e s ( r e f s . 1-5). The codons most p r e f e r r e d a r e t h o u g h t t o be those t h a t a r e o p t i m a l f o r t h e t r a n s l a t i o n a l system o f t h e organism. I n f a c t , codons c o r r e s p o n d i n g t o t h e m a j o r t R N A i s o a c c e p t o r s a r e as a r u l e used w i t h t h e h i g h e s t frequency i n genes coding f o r abundant p r o t e i n s i n E . c o l i and y e a s t ( r e f . 6 ) . A1 though t h e s e f i n d i n g s a r e g e n e r a l l y accepted, t h e i r i n t e r p r e t a t i o n has been c o n t r o v e r s i a l i n d e t a i l ( e . g . r e f s . 4, 7, 8). The most comprehensive codon usage t a b l e s have been compiled f o r t h e u n i c e l l u l a r organisms f . c o l i and y e a s t , and f o r v e r t e b r a t e s ( r e f s . 1, 2, 4-6). The aim o f t h i s a r t i c l e i s , f i r s t , t o 3.1

BllO

p r e s e n t codon usage t a b l e s f o r h i g h l y and w e a k l y expressed genes o f a model organism o f h i g h e r eukaryotes, D r o s o p h i l a m e l a n o g a s t e r , and, second, t o d i s c u s s t h e v a r y i n g e x t e n t o f wobble base m o d i f i c a t i o n o c c u r r i n g w i t h i n a f a m i l y o f D r o s o p h i l a tRNAs d u r i n g ontogeny i n terms o f codon usage i n t h i s organism.

3.2

CODON USAGE I N HIGHLY AND WEAKLY EXPRESSED GENES I N DROSOPHILA MELANOGASTER A v e r y s e l e c t i v e codon usage i s observed i n h i g h l y expressed genes i n E . c o l i and y e a s t i n c o n t r a s t t o t h e more random p a t t e r n i n weakly expressed genes. I n D r o s o p h i l a m e l a n o g a s t e r codon usage i n h i g h l y expressed genes i s a l s o c l e a r l y biased. The d a t a presented i n Tables 3 . 1 and 3.2 c o n f i r m and extend e a r l i e r compilat i o n s by Ashburner et a l . ( r e f . 9) and O'Connell and Rosbash ( r e f . 10). Only genes known t o be expressed a t h i g h o r l o w l e v e l s have been considered. Two r u l e s f o r s t r o n g l y expressed genes can be ext r a c t e d from Tables 3.1 and 3.2. F i r s t , codons o f t h e t y p e NNA and NNU a r e avoided, a l t h o u g h t o d i f f e r e n t degrees (N = U, C, A, G ) . Second, NNC, NUG, and NAG codons a r e p r e f e r e n t i a l l y used. For weakly expressed genes codon usage i s much l e s s biased, a c c o r d i n g t o comparable c o m p i l a t i o n s f o r E.coli and y e a s t . T h i s l a t t e r f a c t should be considered when codon usage i s employed t o a s s i g n Especi a1 l y p r o t e i n c o d i n g genes t o open r e a d i n g frames (ORF's) f o r weakly expressed genes i t seems a d v i s a b l e t o use methods which a r e independent o f s p e c i f i c codon usage. A u s e f u l measure f o r t h e e x p r e s s i v i t y o f a gene i s t h e NAC/NAU r a t i o . The sum o f a l l NAC codons d i v i d e d by t h e sum o f a l l NAU codons i s about 3 f o r h i g h l y expressed genes and about 1 f o r weakly expressed genes. A comp a r i s o n o f codon usage o f h i g h l y expressed genes a c t i v e i n v a r i o u s t i s s u e s has n o t r e v e a l e d any t i s s u e s p e c i f i c i t y ( w i t h t h e p o s s i b l e e x c e p t i o n o f t h e genes coding f o r s a l i v a r y g l a n d p r o t e i n s ; r e s u l t s n o t shown). The avoidance o f t h e codons CUA, UUA, and UCA (complementary t o t h e s t o p codons) i n h i g h l y expressed genes l e a d s o f l o n g ORF's i n t h e complementary s t r a n d . Codon usage i n D r o s o p h i l a i s s i m i l a r t o t h a t i n v e r t e b r a t e s , b u t c l e a r l y d i f f e r e n t from t h e one i n E . c o l i and y e a s t (Table 3.2; r e f s . 5, 6 ) . The r e l a t i v e synonymous codon usage (RSCU, r e f . 5) values p r e s e n t e d i n Table 3.2 a l l o w a d i r e c t comparison o f t h e d i f f e r e n t organisms, s i n c e t h e y a r e independent o f t h e amino a c i d

.

TABLE 3.1

Ccdon Usage* ~

h

1

UUU UUC UUA UUG

23 125 3 36

72 106 18 119

Ser

CUU CUC CUA CUG

13 51 11 212

56 93 37 295

Pro

Ile

AUU

60 212 5

70 122 31

Thr

Met

AUC AUA AUG

Val

GUU

46 121

GUA GUG

18 139

54 54 18 208

Ala

GUC

Phe Leu Leu

-

h

ucu ucc

25 151 10 50

30 70 29 66

Tyr

34 93 67 99

His

CCA CCG

26 140 34 27

ACU ACC ACA AC G

22 193 11 33

39 98 39 68

Asn

GCU GCC GCA GCG

83 270 26 29

97 207 66 78

Asp

UCA UCG

-

h

1

ccu ccc

OC AM

Gln

Lys

Glu

1

UAU UAC UAA UAG

29 131

CAU CAC CAA CAG

21 54 84 69 19 67 185 184

AAU ?lAC AAA AAG

40 71 190 86 17 49 220 143

GAU GAC GAA GAG

85 124 161 85 27 91 227 240

-

60 75

Cys

-

OP Trp

-

h

1

9 54

26 68

UGU UGC UGA UGG

-

Arg

CGU CGC CGA CGG

46 107 12 4

52 80 50 67

Ser

AGU AGC AGA AGG

8 104 5 17

60 98 33 38

GGU GGC GGA GGG

89 175 100 4

81 146 135 33

Arg Gly

-

*Codon usage in high ( h ) and low (1) expressed genes in Drosophila melanogaster. The absolute numbers of codons are given. Data for highly expressed genes are from refs. 10,31,33,44,45,46, and 47; for low expressed genes from refs. 48,49,50,51, and W.Bender, personal communication.

TABLE 3.2 Relative Synonymous Codon Usage Values* D-1

D-h

Y-h

D-1

E-h

D-h

Y-h

D-1

E-h -

D-h

Y-h

D-1

E-h

D-h

Y-h

E-h

~~

3.17 2.57

UAU

0.89 0.36 0.26 0.39

UGU 0.55 0.29

UUC 1.19 1.69

UCC 1.19 2.60 2.17 1.91

UAC

1.11 1.64 1.74 1.61

UGC 1.45 1.71 0.22 1.33

UUA

UCA 0.49 0.17 0 . 2 3

0.20

UAA

0.09 0.04

UAG

CCU 0.46 0.46 0.50 0.23

CAU

U U U 0.81 0.31 0.42 0.46

UCU 0.51 0.43

1.58 1.54 0.17 0.06 0.49 0.18

UUG 1.16 0.66

4.50 0.11

UCG 1.12 0.86

CUU 0.54 0.24 0.13 0.22 CUC 0.90 0.94 0.02 0.20 CUA

0.57 2.18 2.24

GUA

0.22 0.22 0.04 1.11

GUG 2.50

JL

*RSCU..

13

(ref.51 =

CGU 0.98 1.45

0.64 4.39

CGG 1.26 0.12

0.73 0.34 1.94 1.80

AAU

0.90 0.35 0.28 0.10

AGU 1.02 0.14 0.17 0.22

2.98 1.78 1.87

AAC

1.10 1.65 1.72 1.90

AGC 1.67

1.79 0.16 1.05

ACA 0.50 0.17 0.22 0.14

AAA

0.51 0.14 0.38 1.60

AGA 0.66

0.16 5.20 0.02

0.51 0 . 0 6 0.18

AAG

1.49 1.86 1.62 0.40

AGG

GCU 0.87 0.81 2.72 1.88

GAU

1.19 0.69 0.84 0.61

GGU 0.82 0.97 3.80 2.28

GCC 1.85

1.13 0.25

GAC

0.81 1.31 1.16 1.39

GGC 1.48 1.90 0.15 1.65

GCA 0.59 0.25 0.12 0.18

GAA

0.55 0.21 1.83 1.59

GGA

1.45 1.79 0.17 0.41

GGG 0.33

, n. -n. z1 j=l 1

0.88 0.40 0.52 0.45

1.47 1.81 0.11 1.78

2.65

GCG 0.70 0.28 0.04

1.72 0.13 0.50

UGG 1.00 1.00 1.00 1.00

CAG

ACG 1.18

GUC 0.65 1.49 1.65 0.15

-

0.48 0.03 3.29

ACC 1.59

GUU 0.65

-

CGC 1.50 3.36 0.01 1.56

AUC 1.64 2.3

1.00 1 . 0 0

-

CGA

ACU

1.00 1.0

-

1.12 1.60 1.48 1.55

A U U 0.94 0.65 1.36 0.47

AUG

UGA

0.53 0.19 1.89 0.22

CCG 1.35

0.42 0.05 0.06 0.01

-

CAC

CUG 2.86 3.90 0.13 5.33

AUA

-

CAA

0.03 0.04

CCA 0.91 0.60 3.44

1.58 2.53

-

0.44

CCC 1.27 2.47

0.36 0.20 0.42 0.04

-

1.78 0.69

ij

0.80

GAG

0.94 0.38 0.00 0.02 0.00 0.02

0.71 0.53 0.14 0 . 0 0

1.37 1.09 0.02 0.02 0.04 0.03 0.04

x ij = number of occurrences of the jth codon for the ith a’mino

Xij

acid, which is encoded by n

synonymous codons. i D = Drosophila melanogaster; Y = yeast; E = E.coli; 1 = low expressed, h = high expressed genes. Data for Y an E from ref.5.

B113

composition o f t h e p r o t e i n s . The most pronounced RSCU v a l u e differences a r e t h e preference of NNU o v e r NNC f o r N = G and C ( w i t h t h e e x c e p t i o n o f a r g i n i n e ) and t h e almost t o t a l avoidance o f t h e p r o l i n e CCC codon i n E . coli. Yeast p r e f e r s t h e codons UUG, UCU, UGU, CAA, AGA, GGU and a v o i d s UGC, CCC, CAG, CGC, GAG, and GGA. A comparison o f r e l a t i v e synonymous codon usage (RSCU) i n h i g h l y and weakly expressed genes o f D r o s o p h i l a , y e a s t , and f. c o 7 i r e v e a l s a s t r o n g e r b i a s i n t h e two u n i c e l l u l a r organisms. Codon usages i n genes o f m u l t i c e l l u l a r organisms m i g h t be l e s s c o n s t r a i n e d by tRNA c o n t e n t t h a n t h o s e i n u n i c e l l u l a r organisms. The e f f e c t , t h r o u g h change i n t r a n s l a t i o n a e f f i c i e n c y and accuracy, o f each synonymous m u t a t i o n on t h e o v e r a l l f i t n e s s o f a m u l t i c e l l u l a r organism m i g h t be s m a l l e r han t h e c o r r e s p o n d i n g e f f e c t i n an u n i c e l l u l a r organism ( r e f . 6). I t i s g e n e r a l l y assumed t h a t t h e codon d i a l e c t o f a species i s an a d a p t a t i o n t o t h e more o r l e s s f i x e d decoding p r o p e r t i e s and concentrations o f the tRNA isoacceptor population o f a c e l l (ref. 6). I n D r o s o p h i l a m e l a n o g a s t e r t R N A i s o a c c e p t o r p a t t e r n s f o r a l l 20 amino a c i d s have been s t u d i e d f o r t h e 1 s t and 3 r d l a r v a l i n s t a r s and i n a d u l t s ( r e f . 11). No m a j o r changes were observed w i t h t h e e x c e p t i o n o f tRNAs c o n t a i n i n g e i t h e r a G o r t h e hyperdihydroxy-l-cyclom o d i f i e d n u c l e o s i d e queuosine (Q = 7-[4,5-cis, pentene-3-ami no-methyl] -7-deazaguanosi ne; o r Q* i n tRNA-Asp: a mannose d e r i v a t i v e o f Q) i n t h e i r wobble base. These show d r a m a t i c a1 t e r a t i ons in t h e i r r e 1a t i ve abundance d u r i n g D r o s o p h i 7 a development ( F i g u r e 3.1). U n f o r t u n a t e l y , codon assignments have been made o n l y f o r a few i s o a c c e p t o r s . The ribosome b i n d i n g t e c h n i q u e has been used f o r codon assignments f o r i n d i v i d u a l tRNA-Ser and t R N A Val i s o a c c e p t o r s ( r e f s . 12, 13). A c l e a r p r e f e r e n c e f o r UCU o v e r UCA/C has been demonstrated f o r tRNA-Ser-7. T r a n s f e r RNA-Ser-2 and 5 p r e f e r t h e codon AGC t o t h e codon AGU, whereas tRNA-Ser-4 responds o n l y t o UCG. Thus, t h e abundance o f t h e tRNA-Ser i s o a c c e p t o r s w i t h known codon b i n d i n g p r o p e r t i e s corresponds t o t h e codon usage p a t t e r n o f D r o s o p h i 7 a . C u r i o u s l y , a c c o r d i n g t o t h e s e r e s u l t s , no m a j o r tRNA-Ser i s o a c c e p t o r responds t o t h e most o f t e n used codon UCC. T r a n s f e r RNA-Val-3 b i n d s m a i n l y t o GUA and weakly t o GUU and GUG, whereas tRNA-Val-3b b i n d s almost e x c l u s i v e l y t o t h e t r i p l e t GUG. An e x c e p t i o n t o t h e p r e d i c t i o n o f t h e wobble hypothesis ( r e f . 14) i s found f o r tRNA-Val-4. T h i s isoacceptor

B114

binds t o a1 1 4 Val i ne t r i p l e t s . Furthermore, codon assignements for tRNA-His with the anticodon GUG and QUG have been made by an in v i v o experiment using the X e n o p u s oocyte as an assay system (ref. 1 5 ) . Thus, for most sets of synonymous codons the observed codon usage bias cannot be discussed i n terms of cognate t R N A concentrations. In the following, I shall discuss the changes i n Q-base modification of tRNA-Tyr, tRNA-His, and tRNA-Asp w i t h

An 3 r d instar larva

1st instar larva

~~

early pupa

Figure 3.1

5 d adult (males)

35d adult (males

Changes in Q-base modification d u r i n g D r o s o p h i 7 a T h e figure shows the trends as observed f o r the tRNA-AspgHislTyr isoacceptors (refs. 26-28). The t R N A Asn isoacceptor p a t t e r n i s more complicated. Q = isoacceptor w i t h queuoslne .or Q* i n the wobble position, G = isoacceptor w i t h guanosine i n t h e wobble position.

m e 7 a n o g a s t e r onto eny.

B115

r e s p e c t t o decoding p r o p e r t i e s and usage o f t h e cognate codons i n h i g h l y and weakly expressed genes. Some t e n t a t i v e ideas, which a w a i t f u r t h e r c o r r o b o r a t i o n by a d d i t i o n a l experiments and more data, s h a l l be presented.

3.3

Q-BASE MODIFICATION AND DECODING PROPERTIES

Accurate r e a d i n g o f t h e g e n e t i c code i s i n t i m a t e l y a s s o c i a t e d w i t h t h e presence o f a s e t o f s o p h i s t i c a t e d m o d i f i e d n u c l e o s i d e s i n t h e a n t i c o d o n l o o p o f tRNAs ( r e f . 16). M o d i f i c a t i o n s i n t h e wobble p o s i t i o n o f t h e a n t i c o d o n ( p o s i t i o n 34) and o f t h e nucleot i d e 3 ' t o i t ( p o s i t i o n 37) can c o n s i d e r a b l y a f f e c t t h e decoding p r o p e r t i e s o f a t R N A and t h e s t a b i l i t y o f t h e codon/anticodon i n t e r a c t i o n s ( r e f s . 17-19). The h y p e r m o d i f i e d n u c l e o s i d e queuosine ( o r Q* i n tRNA-Asp), o r an u n m o d i f i e d guanosine, i s found i n t h e wobble p o s i t i o n o f tRNAs s p e c i f i c f o r Asp, Asn, H i s , and T y r ( r e f s . 20, 21). These tRNAs r e a d t h e codons NAU and NAC, where N i s A,U,C, o r G, r e s p e c t i v e l y . I s h a l l d e s i g n a t e t h e s e tRNAs as Qbase tRNAs, and t h e i r cognate codons as Q-base codons. The p r e s ence o f 4-34 can i n f l u e n c e t h e decoding p r o p e r t i e s o f D r o s o p h i l a t R N A - T y r and tRNA-His ( r e f s . 15, 22, b u t see a l s o r e f . 2 3 ) . Using t h e X e n o p u s oocyte as an ' i n v i v o ' t r a n s l a t i o n system we have shown t h a t t h e D r o s o p h i l a tRNA-His w i t h t h e a n t i c o d o n GUG c l e a r l y p r e f e r s t h e codon CAC t o t h e codon CAU ( r e f . 15), whereas l i t t l e p r e f e r e n c e i s shown by t h e tRNA-His w i t h t h e a n t i c o d o n QUG f o r t h e codon CAU. These r e s u l t s a r e supported by o b s e r v a t i o n s c o n c e r n i n g t h e s t a b i l i t y o f complexes formed between two t R N A anticodons ( r e f . 24). From t h e l a t t e r experiments i t can be concluded t h a t t h e h y p e r m o d i f i e d base Q i n t h e a n t i c o d o n a b o l i s h e s t h e d i f f e r e n c e i n t h e b i n d i n g e n e r g i e s t o t h e two complementary sequences NAU and NAC as i t i s observed f o r an i s o a c c e p t o r c o n t a i n i n g a G i n t h e wobble base. I n D r o s o p h i l a , w i t h t h e e x c e p t i o n o f tRNA-Asn, a l l Q-base tRNAs have o n l y two m a j o r i s o a c c e p t o r s d i f f e r i n g i n t h e wobble base (G/Q; F i g . 3.1); each decodes b o t h cognate codons a l b e i t w i t h unequal p r e f e r e n c e of c h o i c e . Thus, an adjustment o f t h e tRNAs t o t h e needs o f p r o t e i n s y n t h e s i s ( r e f l e c t e d i n t h e v a r y i n g p r o p o r t i o n s o f mRNAs u s i n g d i f f e r e n t NAC/U codon usage p a t t e r n s ) c o u l d be achieved v i a t h e degree o f wobble base m o d i f i c a t i o n . T h i s r e p r e s e n t s a unique s i t u a t i o n o f a p o t e n t i a l o n t o g e n e t i c adapta-

B116

t i o n o f t h e degree o f m o d i f i c a t i o n o f a f a m i l y o f tRNAs t o t h e abundance o f t h e i r cognate codons i n t h e t r a n s c r i p t s o f a c e l l , For a l l o t h e r t R N A species i t i s assumed t h a t an o n t o g e n e t i c a l l y i n v a r i a b l e s e t o f isoacceptors w i t h f i x e d decoding p r o p e r t i e s imposes i t s s e l e c t i v e c o n s t r a i n t s on t h e codon usage p a t t e r n o f t h e genes depending on t h e i r expression l e v e l s .

Q-BASE MODIFICATION, PROTEIN SYNTHESIS, AND DEVELOPMENT As mentioned above, t h e D r o s o p h i l a tRNA-His i s o a c c e p t o r w i t h t h e a n t i c o d o n GUG p r e f e r s t h e codon CAC t o t h e codon CAU, whereas t h e tRNA-His i s o a c c e p t o r w i t h t h e a n t i c o d o n QUG shows l i t t l e p r e f e r e n c e f o r CAU. Thus t h e tRNA-His w i t h t h e GUG a n t i c o d o n seems t o be p r e d e s t i n e d t o read CAC codons which a r e e s p e c i a l l y promi n e n t i n s t r o n g l y expressed genes. I n t h e f o l l o w i n g , I s h a l l assume t h a t t h i s a p p l i e s a l s o t o t h e o t h e r Q-base tRNAs i n r e s p e c t t o t h e i r cognate codons. T h i s seems reasonable, s i n c e t h e presence o f G o r Q i n t h e wobble base i s t h e o n l y v a r i a b l e i n t h e ant i c o d o n s o f t h e two m a j o r tRNA-His ( r e f . 15), t R N A - T y r ( r e f . 25), and p r o b a b l y a l s o o f t h e tRNA-Asp and tRNA-Asn i s o a c c e p t o r s . As a l s o s t a t e d above, t h e NAC/U codon usage o f D r o s o p h i l a genes r e f l e c t s t h e i r expression l e v e l . T h i s i s c l e a r l y t h e case f o r t h e o v e r a l l codon usage o f h i g h l y and weakly expressed genes (Tables 3.1 and 3.2). Thus, low queuosine m o d i f i c a t i o n i s expected i n developmental stages w i t h h i g h p r o t e i n s y n t h e s i s ( i . e . l a r v a l growth), and h i g h degree o f m o d i f i c a t i o n i n o l d f l i e s composed m a i n l y o f p o s t m i t o t i c t i s s u e s w i t h l i t t l e s y n t h e t i c a c t i v i t y and t h e r e f o r e l e s s demands on p r o t e i n s y n t h e s i s . The a n a l y s i s o f Qbase isoacceptor p a t t e r n s in D r o s o p h i 7 a devel opmen t ( F i g u r e 3.1) c o n f i r m s t h i s p r e d i c t i o n ( r e f s . 26-28). Hence, t h e e x t e n t o f Qbase m o d i f i c a t i o n can be i n t e r p r e t e d as an a d a p t a t i o n t o t h e needs o f p r o t e i n s y n t h e s i s i n s p e c i f i c o n t o g e n e t i c stages. D r o s o p h i l a l a r v a e i n c r e a s e i n w e i g h t (mainly i n c r e a s e i n c e l l s i z e ) about 2 0 0 - f o l d i n f o u r days, i . e . t h e y produce an enormous amount o f p r o t e i n s . The p r e v a l e n t i s o a c c e p t o r s i n t h i s o n t o g e n e t i c stage ( w i t h a G i n t h e wobble base) f i t w e l l t h e codon usage p a t t e r n o f h i g h l y expressed genes w i t h a h i g h NAC/U r a t i o . I n o l d f l i e s , however, d i v i d i n g c e l l s a r e r e s t r i c t e d t o t h e germ l i n e and no c e l l growth occurs anymore. Hence, a d u l t s produce much l e s s p r o t e i n . The p r o p o r t i o n o f messengers t o be t r a n s 1 a t e d c o n t a i n i n g 3.4

B117

a low NAC/U r a t i o m i g h t t h e r e f o r e i n c r e a s e . The i n t r o d u c t i o n o f t h e Q-base i n t h e wobble p o s i t i o n and t h e concomitant r e d u c t i o n i n d i f f e r e n c e between t h e energy i n t h e b i n d i n g t o NAC/U codons c o u l d t h u s p r o v i d e more adequate decoding p r o p e r t i e s f o r t h i s f a m i l y o f tRNAs i n o l d f l i e s . I n t h e f o l l o w i n g , I s h a l l d i s c u s s examples o f NAC/U codon usage i n gene d u p l i c a t i o n s , genes composed o f conserved and nonconserved r e g i o n s , and homo1ogous genes i n n a t u r a l popul a t i ons and s i b l i n g species. They a l l c o n f i r m t h e s e l e c t i v e c o n s t r a i n t s i m posed on these t y p e s o f codons depending on t h e e x p r e s s i o n l e v e l s o f t h e corresponding genes. CODON USAGE AND GENE DUPLICATIONS The frequency o f NAC/NAU codons i n members o f a gene f a m i l y o r i g i n a t i n g from an a n c e s t r a l gene by gene d u p l i c a t i o n ( s ) s h o u l d r e f l e c t t h e expression l e v e l o f t h e i n d i v i d u a l genes, independent o f t h e i r common o r i g i n . Two genes, one weakly, one h i g h l y expressed, code f o r c y t o chrome-like p r o t e i n s i n D r o s o p h i l a ( r e f . 29). They o r i g i n a t e from an a n c i e n t s i n g l e copy gene. The h i g h expressed gene shows a NAC/NAU r a t i o t y p i c a l f o r h i g h expression, whereas t h e l o w expressed gene has about t h e same amount o f NAC and NAU codons (12/4, 10/7). Thus, t h e t h i r d base o f t h e Q-base codons i n t h e f i r s t gene has been s e l e c t e d f o r h i g h e f f i c i e n c y o f t r a n s l a t i o n , whereas t h e t h i r d base o f t h e o t h e r gene seems t o be random U o r C. The y o l k p o l y p e p t i d e (Yp) genes i n D r o s o p h i l a m e l a n o g a s t e r a r e a s e t o f r e l a t e d genes which a r e expressed i n t h e same t i s s u e s and developmental p e r i o d s ( r e f s . 30, 31). They p r o b a b l y arose by two d u p l i c a t i o n s , t h e YpllYpP d u p l i c a t i o n b e i n g t h e l a t e s t . A l l t h r e e a r e h i g h l y expressed and show t h e expected h i g h NAC/NAU r a t i o s (Ypl-Yp3: 60/22, 49/17, 45/23). A comparison o f t h e t h i r d base i n corresponding NAC/U codons o f Y p l and Yp2 shows however t h a t , a l t h o u g h t h e o v e r a l l NAC/U r a t i o i s i s about t h e same i n b o t h genes, t h e i n d i v i d u a l p o s i t i o n s a r e n o t ( r e f . 30). Thus, i n c o n t r a s t t o t h e a l c o h o l dehydrogenase genes i n s i b 1 i n g species (see below), t h e r e seems t o be no s e l e c t i v e p r e s s u r e on t h e t h i r d base o f s p e c i f i c NAC/U codons i n t h e y o l k p r o t e i n genes. 3.5

B118

3.6

CODON USAGE I N GENES COMPOSED OF CONSERVED AND NON-CONSERVED REGIONS Each o f a f a m i l y o f small h e a t shock genes i n D r o s o p h i l a i s composed o f a conserved r e g i o n i n t h e m i d d l e and o f non-conserved r e g i o n s a t t h e l e f t and r i g h t ends o f t h e genes ( F i g . 3.2, r e f . 32). A u n i f o r m NAC/NAU codon r a t i o i s expected t h r o u g h o u t t h e i n d i v i d u a l genes (adapted t o t h e i r e x p r e s s i o n l e v e l s ) i f codon usage i s r e l e v a n t f o r t h e t r a n s l a t i o n o f t h e i r mRNAs. Lack o f s e l e c t i o n pressure should r e s u l t i n e i t h e r random usage o r conserv a t i o n o f t h e r a t i o s i n t h e conserved p a r t s o f t h e genes. D e s p i t e t h e s h o r t s i z e o f t h e coding sequences, i t can be seen t h a t t h e r e i s an unambiguous t r e n d towards a homogenous r a t i o t h r o u g h o u t each gene.

3.7

CODON USAGE OF HOMOLOGOUS, HIGHLY EXPRESSED GENES I N NATURAL POPULATIONS AND I N SIBLING SPECIES The d i m e r i c enzyme a1 coho1 dehydrogenase (ADH d e s i g n a t e s t h e enzyme, Adh t h e gene) i s coded f o r by a s i n g l e , h i g h l y expressed, r e l a t i v e l y simple gene i n D r o s o p h i l a m e l a n o g a s t e r and some o t h e r D r o s o p h i l a species ( r e f s . 33, 34). N u c l e o t i d e polymorphism has been determined a t t h e Adh l o c u s f o r D . m e 7 a n o g a s t e r ( r e f . 35) and changes i n t h e sequences coding f o r ADH have been s t u d i e d i n several s i b l i n g species o f D r o s o p h i l a ( r e f . 34). Several i n t e r e s t i n g o b s e r v a t i o n s r e l e v a n t t o o u r d i s c u s s i o n can be made. Kreitman ( r e f . 35) has sequenced e l e v e n Adh genes from f i v e natural populations o f Drosophila melanogaster revealing a large number o f hidden polymorphisms. Only one o f t h e 14 polymorphisms i n t h e coding r e g i o n r e s u l t e d i n an amino a c i d change. A l l t h e o t h e r s were s i l e n t replacements, b u t none a f f e c t e d t h e wobble base o f e i t h e r one o f t h e f o u r NAC/U codons (38 o u t o f a t o t a l o f 255 codons). T h i s i s t o be expected, i f s e l e c t i o n f a v o r s a h i g h NAC/NAU r a t i o i n a h i g h l y expressed gene. The DNA sequence o f Adh i n t h e f o l l o w i n g f o u r s i b l i n g species has been determined: D.melanogaster, D.simulans, D.mauritiana, and D . o r e n a ( r e f . 3 4 ) . A l l f o u r genes have a h i g h NAC/NAU r a t i o , w i t h t h e l o w e s t i n D . o r e n a (29/9, 30/8, 30/8, 24/13). I n t e r e s t i n g l y , most o f t h e p o s i t i o n s ending w i t h a C o r a U a r e conserved i n a l l f o u r species. Furthermore, w i t h one e x c e p t i o n , C t o U changes a r e found i n one species o n l y , D . o r e n a . The f i r s t o b s e r v a t i o n

B119 overall NAC/NAU ratio

3.2

(16/5)

1 hsp-27

1

(10/10)

2.2

(9/4)

(0/2)

F i g u r e 3.2 NACINAU r a t i o s i n t h e conserved and non-conserved r e g i o n s o f t h e f o u r small h e a t shock (hsp) genes o f D r o s o h i l a m e l a n o g a s t e r ( r e f . 32). The e f f e c t i v e numbers o f NAC a n 8 NAU codons a r e g i v e n i n p a r e n t h e s i s . i n d i c a t e s t h a t s e l e c t i o n does n o t o n l y f a v o r a h i g h NAC/NAU r a t i o , b u t a l s o r e s t r i c t s t h e p o s i t i o n o f t h e two codon t y p e s i n t h e coding sequence. Codon c o n t e x t e f f e c t s m i g h t be r e s p o n s i b l e f o r t h i s f a c t i n e x t r e m e l y h i g h l y expressed genes (1-3% o f t h e polyA+ RNA i s Adh messenger!). The second o b s e r v a t i o n suggests, t h a t l e s s s e l e c t i o n p r e s s u r e i s a p p l i e d t o t h e ADH gene o f D . o r e n a , i . e . t h e mRNA abundance i s expected t o be l e s s than i n t h e o t h e r species. A developmental p r o f i l e o f Adh t r a n s c r i p t i o n i n D . o r e n a shows t h a t t h e gene i s t r a n s c r i b e d a t v e r y d i f f e r e n t l e v e l s i n l a r v a e as opposed t o a d u l t s ( r e f . 36). I n D . m e 1 a n o g a s t e r t h e r a t i o o f l a r v a l t o a d u l t e x p r e s s i o n i s about 1-2, i n D . o r e n a i t i s l e s s t h a n 0.1. Furthermore, t h e abundance i n a d u l t s seems t o be a t most one h a l f o f t h e amount i n D . m e l a n o g a s t e r . D i f f e r e n c e s i n t h e e c o l o g i c a l niches i n h a b i t a t e d by t h e s i b l i n g s p e c i e s c o u l d be t h e e x p l a n a t i o n f o r these d e v i a t i o n s i n t h e NAC/NAU r a t i o s . I n f a c t , t h e l a r v a l h a b i t a t s f o r D . m e l a n o g a s t e r , s i m u l a n s , and m a u r i t i a n a a r e fermenti n g f r u i t s , wine c e l l a r s and beer f a c t o r i e s . U n f o r t u n a t e l y t h e h a b i t a t o f D . o r e n a i s n o t known. However, a d u l t e t h a n o l t o l e r a n c e has been measured f o r a l l t h e s e species ( r e f . 37). I t i s l o w e s t (by a f a c t o r o f t h r e e ) f o r D . o r e n a !

3.8.

CONCLUDING REMARKS Arguments concerning t h e meaning o f codon usage and t h e funct i o n s o f m o d i f i e d nucleosides i n tRNAs a r e plagued by t h e same

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problem: i t i s very d i f f i c u l t t o design and perform experiments which w i l l provide unambiguous answers t o many open questions. One of the i m p o r t a n t questions i s , whether codon usage has a modulati n g influence on the r a t e of t r a n s l a t i o n . Although evidence e x i s t s i n favor of i t , measurable e f f e c t s a r e observable only a t very h i g h transcription r a t e s ( r e f . 38). One of the m a i n f a c t o r s determ i n i n g the yield of a protein seems t o be the i n i t i a t i o n r a t e ( r e f . 39). However, a modulating r o l e of elongation upon i n i t i a t i o n has been suggested ( r e f . 39). Furthermore, every c e l l probabl y transl a t e s messengers from highly and weakly expressed genes. How does a c e l l manage t o t r a n s l a t e these mRNAs appropriately in the same cytoplasm? A possible answer i s suggested by the recent discovery t h a t messengers f o r cytoskeletal proteins a r e t r a n s l ated i n d i f f e r e n t parts of the cytoplasm ( r e f . 40). Compartmentalizat i o n of mRNAs transcribed from h i g h l y and weakly expressed genes combined with an adjusted t R N A i soacceptor popul a t i on coul d improve the efficiency and accuracy of the t r a n s l a t i o n a l process. The changes in the Q-base content i n D r o s o p h i l a development are q u i t e impressive (Figure 3 . 1 ) . Queuosi ne i s inserted postt r a n s c r i p t i o n a l l y i n t o the wobble base of the Q-base tRNAs by the enzyme tRNA-guanine transglycosylase ( r e f . 18). Farkas and Jacobson ( r e f . 41) have measured the ontogenetic changes i n the act i v i t y of t h i s enzyme i n D r o s o p h i l a . Surprisingly, a c t i v i t y i s found in 3rd i n s t a r larvae (Q-base modification very low!), is absent i n l a t e pupae, highest in 0-1 day old f l i e s , and drops again in adults of 2-3 days. The obvious lack of correlation between enzyme a c t i v i t y and extent of Q-base modification in the 3rd i n s t a r suggests t h a t t h i s enzyme might have additional roles. Other functions, besides involvement in the decoding process, have been described f o r queuosine i t s e l f (Kersten and Kersten, this volume). As of today, i t i s n o t known whether the changes observed i n the Q-base content of the t R N A s i s an enzymatically a c t i v e or passive process. Diet and temperature a r e additional factors influencing the extent of modification ( r e f s . 42, 43) , indicating t h a t the transglycosyl ase a c t i v i t y m i g h t be 1 inked t o metabol i c processes. Thus, queuosine seems t o be one of those small molecules with many roles, possibly integrating a variety of d i f f e r e n t functions w i t h i n a c e l l .

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3.9

SUMMARY Codon usage tables f o r h i g h l y a n d weakly expressed genes of

D r o s o p h i l a m e l a n o g a s t e r are presented. As found f o r E.coli

(ref. 1,) yeast ( r e f . 2), and vertebrates ( r e f . 6), codon usage i n D r o s o p h i l a i s s t r o n g l y biased in h i g h l y expressed genes. However, there are marked differences i n codon usage i n comparison w i t h the unicellular organisms E.coli and yeast. The varying extent of wobble base modification occuring w i t h i n a family of D r o s o p h i l a tRNAs (Q-base tRNAs) d u r i ng ontogeny i s discussed. 3.10 1.

2. 3.

4.

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Grosjean, S. deHenau and D. M. C r o t h e r s , On t h e physi c a l basis f o r ambiguity i n e n e t i c coding i n t e r a c t i o n s , Proc. N a t l . Acad. S c i . USA, 75 71978) 610-614. B. S u t e r , M. A l t w e g, Y . C h o f f a t and E. K u b l i , The n u c l e o t i d e sequence o f t w o omogeneic D r o s o p h i l a m e l a n o g a s t e r tRNA-Tyr i s o a c c e p t o r s : a p p l i c a t i o n o f a r a p i d t R N A a n t i c o d o n sequencmethod u s i n S-1 n u c l e a s e , A r c h . Biochem. B i o p h y s . ,

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i t y o f a t r a n s f e r RNA m o d i f y i n g enzyme d u r i n g t h e development

27.

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R. K. Owenby, M. B. S t u l b e r g and K. B. Jacobson, A l t e r a t i o n o f t h e Q f a m i l y o f t r a n s f e r RNAs i n a d u l t D r o s o p h i l a m e l a n o g a s t e r as a f u n c t i o n o f age, n u t r i t i o n and g e n o t y p e , Mech. A g e i n g Dev., 11 (1979) 91-103. 29. K. J. Limbach and R. Wu, C h a r a c t e r i z a t i o n o f t w o D r o s o p h i l a m e l a n o a s t e r c y t o c h r o m e c genes and t h e i r t r a n s c r i p t s , N u c l . A c i d s i e s . , 13 (1985) 631-644. 30. M. -C. Hung and P. C. Wensink, The sequence o f t h e D r o s o p h i l a f o r y o 1 k p r o t e i n 1, N u c l A c i d s Res. , 9 melanogaster

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K. O'Hare. C. MurPhv. R. Levis and G. M. Rubin, DNA sequence of the white locus o f D r o s o p h i l a m e l a n o g a s t e r , J. Mol. Biol., 180 (1984) 437-455. 49. S. Henikoff. J. S. Sloan and J. D. Kellv. A D r o s o p h i l a metabol i c ene. transcript is a1 ternati vely' processed, Cell, 34 11983) !05-414. 50. t,-Ha?enT-K.-Basler, J. -E. Edstrom and G. M. Rubin, S e v e n l e s s , a cell-specific homoeotic gene of D r o s o p h i l a encodes a putative transmembrane receptor with a tyrosi ne ki nase domai n, Science, 236 (1987) 55-63. 51. T. P . Keith, M. A. Riley, M. Kreitman R. C. Lewontin, 0. Curtis, and G. Chambers, Sequence of the structural gene for xanthi ne dehydrogenase r o s y 1 ocus) in D r o s o p h i l a m e l a n o g a s t e r , Genetics, 116 (19 7) 67-73. 48.

8

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CHAPTER 4 SOLID PHASE IMMUNOASSAY FOR DETERMINING THE INOSINE CONTENT I N TRANSFER RNA E D I T H F. YAMASAKI' , ALTAF A. WAN12q3, and RONALD W. TREWYN'

r 3

'Department o f P h y s i o l o g i c a l Chemistry *Department o f Radiology 3 C o m p r e h e n s i v e C a n c e r C e n t e r , T h e O h i o S t a t e U n i v e r s i t y , Columbus, O h i o , USA 43210

TABLE OF CONTENTS 4.1 I n t r o d u c t i o n . . , . . . . 4.2 M a t e r i a l s and Methods . . . . . . . . , . . . . . . 4.2.1 M a t e r i a l s . 4.2.2 P r e p a r a t i o n o f Hapten-Protein Conjugates 4.2.3 Immunization o f Animals and P r e p a r a t i o n o f A n t i . . . . . . serum 4.2.4 C h a r a c t e r i z a t i o n o f A n t i b o d y P r e p a r a t i o n 4.2.5 C o m p e t i t i v e ELISA , 4.2.6 Q u a n t i t a t i o n o f I n o s i n e . 4.2.7 HPLC . . . .. . . 4.3 R e s u l t s and D i s c u s s i o n . 4 . 3 . 1 T i t e r D e t e r m i n a t i o n and A n t i b o d y S p e c i f i c i t y . 4.3.2 C o m p e t i t i v e ELISA 4.3.3 D e t e c t i o n o f I n o s i n e i n H y d r o l y z e d t R N A . . 4.4 F u t u r e P e r s p e c t i v e s 4.5 Summary 4.6 Acknowledgments . 4.7 References

.. .

. . . . . . . . . . . 8125 . .. . . . . . . . . 8126 . . . . .. . . . . 8126 . . 8126 ... .. . . . .. . .... 8128 . . . . 8128 . . . . . . . . . . . . . . 8129 . . . . . . . . . . . 8129 . . . . . . . . . . . . . . . . . . . 8130 . .. . . . . . . . . 8131 . . 8131 . . . . . . . . . . . . . . 8133 . . . . 8135 . . . . . . . . . . . . . . . . 8139 ........................ 8140 . . . . . . . . . . . . . . . . . . . . 8141 . . . . . . . . . . . . . . . . . . . . . 8141

4.1

INTRODUCTION The u t i l i t y o f a n t i b o d i e s as probes f o r r a r e and/or h i g h l y m o d i f i e d n u c l e o s i d e s i n t R N A has been demonstrated by a number o f i n v e s t i g a t o r s ( r e f s . 1-11). Inouye e t a 7 . ( r e f . 2) f i r s t demons t r a t e d t h e use o f an a n t i - i n o s i n e a n t i b o d y t o d e t e c t i n o s i n e c o n t a i n i n g s p e c i e s i n b u l k t R N A by a p p l i c a t i o n o f an a f f i n i t y chromatography column. T h i s a n t i b o d y p r e p a r a t i o n was used t o de-

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t e c t , b u t n o t t o q u a n t i t a t e , i n o s i n e - c o n t a i n i n g tRNA. Previous work from t h i s l a b o r a t o r y has suggested a r o l e f o r i n o s i n e m o d i f i e d tRNAs i n immune r e g u l a t i o n ( r e f . 12) and c e l l u l a r d i f f e r e n t i a t i o n ( r e f . 13); thus, we embarked on t h e development o f a s p e c i f i c assay t o q u a n t i t a t e t h e l e v e l o f i n o s i n e m o d i f i c a t i o n Many s t u d i e s f o r t h e d e t e c t i o n o f m o d i f i e d n u c l e o s i d e s i n tRNA. i n t R N A have used radioimmunoassay ( R I A ) procedures w i t h r a d i o l a b e l e d a n t i g e n s ( r e f s . 4-9). We saw an advantage i n p e r f o r m i n g s o l i d phase n o n - r a d i o a c t i v e enzyme immunoassays and s e t o u t t o develop a q u a n t i t a t i v e method u s i n g i n o s i n e as o u r model nucleoside. Our r e s u l t s i n d i c a t e t h a t t h e c o m p e t i t i v e i n h i b i t i o n o f s p e c i f i c a n t i b o d y b i n d i n g i n ELISA i n c o n j u n c t i o n w i t h high-performance 1i q u i d chromatography (HPLC) would a1 l o w q u a n t i t a t i o n o f v a r i o u s tRNA nucl eosi des w i t h o u t t h e need f o r r a d i o l abel ed materials. 4.2 MATERIALS AND METHODS 4.2.1 M a t e r i a l s The f o l 1owi ng m a t e r i a1 s were o b t a i n e d from Sigma: p o l y r i bon u c l e o t i d e s [poly(A), poly(G), p o l y ( I ) , p o l y ( X ) ] , n u c l e o s i d e s (adenosine, c y t i d i n e , guanosine) , n u c l e o s i d e 5'-monophosphates (AMP, CMP, GMP, I M P ) , nuclease P1, b a c t e r i a l a1 k a l i n e phosphatase, bovine serum albumin (BSA), R I A grade, g o a t a n t i - r a b b i t IgG conj u g a t e o f a1 k a l i n e phosphatase ( a f f i n i t y p u r i f i e d ) , d i e t h a n o l a mine, e t h y l e n e g l y c o l , sodium meta-periodate, p o l y o x y e t h y l e n e s o r b i t a n monolaurate (Tween 20), and t h i m e r o s a l Sodium b o r o h y d r i d e was purchased from F i s h e r S c i e n t i f i c , i n o s i n e f r o m Chemalog, and an ammonium s u l f a t e suspension o f keyhole l i m p e t hemocyanin (KLH) from Calbiochem. Complete Freund's a d j u v a n t was f r o m D i f c o , 96we1 1 p l a s t i c m i c r o t i t e r p l a t e s (Nunc-Immuno P1a t e I)f r o m G i bco, and Vaccu-Pette/96 from Research Products I n t e r n a t i o n a l . New Zealand White r a b b i t s were purchased from Charles R i v e r . Subriden RNA s u p p l i e d t h e p u r i f i e d E s c h e r i c h i a c o l i t R N A A r ' J , a n t i c o d o n I C G , and t R N A P h e , anticodon GAA.

.

P r e P a r a t i o n o f HaPten-Protein Conjuaates I n o s i n e was conjugated t o BSA and KLH, f o l l o w i n g t h e procedu r e of E r l a n g e r and B e i s e r ( r e f . 14) as m o d i f i e d by M u l l e r and Rajewsky ( r e f . 1 5 ) . Two hundred mg o f i n o s i n e were added t o 20 m l

4.2.2

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0.1 M NaIO, and s t i r r e d f o r 15 minutes a t room temperature; t h e o x i d a t i o n r e a c t i o n was stopped by t h e a d d i t i o n o f e t h y l e n e g l y c o l . Ten m l o f t h i s i n o s i n e epoxy d e r i v a t i v e were added dropwise t o 5 m l each o f BSA and KLH s o l u t i o n (20 mg/ml, prepared i n 0 . 1 M sodium bicarbonate/carbonate b u f f e r , pH 9; t h e KLH p r e p a r a t i o n o b t a i n e d as an ammonium s u l f a t e suspension was p r e v i o u s l y d i a l y z e d against the buffer). The s o l u t i o n s were g e n t l y s t i r r e d d u r i n g t h i s a d d i t i o n , and c o n t i n u a l pH adjustment t o 9.5 was done by These m i x t u r e s were s t i r r e d a t room a d d i t i o n o f 2 M Na,CO,. temperature f o r an a d d i t i o n a l 45 minutes w i t h pH a d j u s t e d t o 9.5 when necessary. Reduction o f t h e S c h i f f ' s base was accomplished by a d d i t i o n o f a f r e s h l y prepared, 2.5 m l aqueous s o l u t i o n o f sodium borohydride, 30 mg/ml. A f t e r t h e p r e p a r a t i o n s were l e f t on The f i n a l i c e f o r 3 hours, t h e pH was a d j u s t e d t o 7 w i t h HC1. p r e p a r a t i o n s o f a p p r o x i m a t e l y 20 m l each were d i a l y z e d e x t e n s i v e l y a g a i n s t phosphate b u f f e r e d s a l i n e (PBS) , 10 mM sodium phosphate (pH 7), 140 mM NaC1. The e x t e n t o f l i g a n d c o n j u g a t i o n t o p r o t e i n was determined by d i f f e r e n c e s p e c t r a u s i n g t h e equation: E280

p r o t e i n (A260 nm/A280 nm) - E260protein

E2 6 0

n u c l e o s i de -

n = E280

nucl e o s i de (A260 nm/A280 nm)

where n equals t h e number o f n u c l e o s i d e r e s i d u e s p e r mole o f p r o t e i n ; A260 nm/A280 nm equals t h e r a t i o o f absorbance values a t 260 nm and 280 nm o f t h e conjugated hapten-protein; and E equals t h e m o l a r e x t i n c t i o n o f b o t h p r o t e i n and hapten a t t h e two wavel e n g t h s ( r e f . 1). I n o u r p r e p a r a t i o n o f inosine-BSA, we found n t o be 10.9, w h i l e a comparable c a l c u l a t i o n f o r t h e inosine-KLH conjugate y i e l d e d an e s t i m a t e f o r n o f 100. P r e p a r a t i o n o f a guanosi ne-BSA c o n j u g a t e f o l 1owed t h e above procedure, u s i n g a l o w e r c o n c e n t r a t i o n o f sodium p e r i o d a t e (50 mM) and a more d i l u t e s o l u t i o n o f guanosine. These m o d i f i c a t i o n s were made due t o t h e l o w e r s o l u b i l i t y o f guanosine compared t o i n o s i n e and t h e tendency f o r t h e r e a c t i o n product, o x i d i z e d guanosine, t o form a g e l ( r e f . 14). The e x t e n t o f guanosine c o n j u g a t i o n t o BSA was found t o be 23.6 moles guanosine bound p e r mole of BSA.

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4.2.3

Immunization o f Animals and P r e u a r a t i o n o f Antiserum One month o l d New Zealand White r a b b i t s were each immunized w i t h 5 m l o f an emulsion o f 5 mg inosine-KLH c o n j u g a t e i n 2.5 m l PBS w i t h 2.5 m l complete Freund's a d j u v a n t , i n j e c t e d subcutaneously a t m u l t i p l e s i t e s i n t h e neck r e g i o n and i n t r a m u s c u l a r l y i n each h i n d l e g . Booster i n j e c t i o n s , a t t h r e e week i n t e r v a l s , were a d m i n i s t e r e d i n t r a v e n o u s l y i n t o e a r veins, g i v i n g each animal 0.5 mg inosine-KLH prepared i n 1 m l PBS. Three weeks a f t e r p r i m a r y immunization, t h e b l o o d was c o l l e c t e d from e a r v e i n s and t h e a n t i b o d y t i t e r o f serum samples was determined by ELISA, u s i n g inosine-BSA as t h e a n t i g e n . I n t h e 8 t h week o f t h e immunization p r o t o c o l , a b o o s t e r o f 2.5 mg inosine-KLH i n 1 m l PBS e m u l s i f i e d w i t h 1 m l incomplete Freund's a d j u v a n t was a d m i n i s t e r e d i n t r a m u s c u l a r l y w i t h one-half o f t h e dose i n t o each h i n d l e g . A f t e r 16 weeks, t h e animals were s a c r i f i c e d and t h e serum f r o m each r a b b i t was b r o u g h t t o 40 % s a t u r a t i o n w i t h ammonium s u l f a t e and s t o r e d as To prepare t h e a n t i b o d y f o r use i n v a r i o u s a suspension a t 4°C. assays, an a l i q u o t o f t h e u n i f o r m l y mixed ammonium s u l f a t e suspens i o n was c e n t r i f u g e d a t 15,000 x g f o r 5 minutes. The p e l l e t was d i s s o l v e d i n PBS equal t o 0.6 volume o f t h e o r i g i n a l suspension and was then d i a l y z e d e x t e n s i v e l y a g a i n s t PBS. Subsequent s e r i a l d i l u t i o n s o f t h i s a n t i b o d y p r e p a r a t i o n were done w i t h PBS-Tween b u f f e r ; 150 mM NaC1, 20 mM sodium phosphate b u f f e r (pH 7.4), 0.05 % (v/v) Tween 20.

4.2.4

Characterization o f antibody Dreuaration Antibody t i t e r s were determined by an ELISA method. The procedure as d e t a i l e d i n Wani e t a l . ( r e f . 16) was f o l l o w e d . Using an a p p r o p r i a t e d i l u t i o n o f a n t i g e n , e i t h e r homopolynucleot i d e o r nucleoside-BSA c o n j u g a t e i n PBS, 100 p1 were added t o w e l l s o f t h e 96 w e l l p l a s t i c m i c r o t i t e r p l a t e . Attachment o f t h e a n t i g e n was done o v e r n i g h t i f p o l y n u c l e o t i d e s were used o r f o r one A1 1 subsequent hour i f nucleoside-BSA conjugates were used. i n c u b a t i o n s were done a t 37°C f o r one hour u n l e s s o t h e r w i s e i n d i cated. A f t e r t h e a n t i g e n i m m o b i l i z a t i o n , t h e w e l l s were washed w i t h PBS Tween b u f f e r , employing e i t h e r a T i t e r t e k m i c r o p l a t e washer o r a Vaccu-Pette/96 device. The unoccupied b i n d i n g s i t e s o f t h e p l a s t i c w e l l s were blocked by f i l l i n g each w e l l w i t h 1 % BSA prepared i n 50 mM Tris-HC1 (pH 8.5), 150 mM NaCl , 0.01%

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t h i m e r o s a l and i n c u b a t i n g f o r one hour. Subsequent washing o f t h e w e l l s w i t h PBS-Tween (3 times) was f o l l o w e d by i n c u b a t i o n w i t h 100 p1 o f v a r i o u s d i l u t i o n s o f t h e a n t i b o d y p r e p a r a t i o n s i n PBS-Tween. The w e l l s were then washed w i t h PBS-Tween before t h e a d d i t i o n t o each w e l l o f 100 p l o f a 1:1,000 d i l u t i o n o f goat a n t i - r a b b i t IgGa1 k a l i n e phosphatase conjugate. A f t e r i n c u b a t i o n w i t h t h e second a r y antibody, t h e w e l l s were washed w i t h PBS-Tween (3 t i m e s ) and d i s t i l l e d water (2 t i m e s ) . The a l k a l i n e phosphatase s u b s t r a t e , p n i t r o p h e n y l phosphate [l mg/ml prepared i n 1 M d i e t h a n o l a m i n e b u f f e r (pH 9.6), 1 mM MgCl,] was added a t 100 p1 p e r w e l l . A f t e r r e a c t i o n f o r one hour, o r more i f necessary, t h e absorbance o f t h e y e l l o w c o l o r e d p r o d u c t was determined a t 405 nm w i t h a Bio-Tek m i c r o p l a t e reader t o p r o v i d e a measure o f t h e amount o f bound p r i m a r y a n t i body. ComDetitive ELISA I n c o m p e t i t i v e ELISA, equal volumes o f t w o - f o l d c o n c e n t r a t e d i n h i b i t o r s o l u t i o n s , r a n g i n g from 0.2 pM t o 400 pM, and t w o - f o l d c o n c e n t r a t e d a n t i b o d y , u s u a l l y a 2 x 10-6 d i l u t i o n o f s t o c k i n PBS-Tween, were pre-incubated f o r 30 t o 60 minutes. Then a 100 p l a l i q u o t was removed and added t o designated w e l l s o f a 96 w e l l m i c r o t i t e r p l a t e c o n t a i n i n g t h e i m m o b i l i z e d a n t i g e n . A f t e r a one hour i n c u b a t i o n w i t h t h e plate-bound a n t i g e n , u n r e a c t e d a n t i b o d y and i n h i b i t o r were removed and t h e w e l l s were washed w i t h PBSTween. The subsequent steps u s i n g secondary a n t i b o d y and s u b s t r a t e were t h e same as d e s c r i b e d above. D e t e r m i n a t i o n o f t h e percentage i n h i b i t i o n o f a n t i b o d y b i n d i n g was done a c c o r d i n g t o M u l l e r and Rajewsky ( r e f . 15), u s i n g t h e formula:

4.2.5

(E2

- E,)/(E,

- E,)

x 100

=

% inhibition

where E, equals t h e absorbance a t 405 nm o f t h e w e l l s c o n t a i n i n g i n h i b i t o r , E, t h e absorbance o f t h e w e l l s w i t h o u t i n h i b i t o r , and E, t h e absorbance o f t h e w e l l s w i t h o u t bound a n t i g e n . Ouantitation o f inosine Pure i s o a c c e p t i n g species o f E . c o l i t R N A [ t R N A A r g o r tRNAP , o f known nucl e o t i de sequence and h a v i ng a n t i codons o f I C G and GAA, r e s p e c t i v e l y ( r e f . 17)] were t r e a t e d w i t h nuclease P 1 and b a c t e r i a l a1 k a l i n e phosphatase, a c c o r d i n g t o t h e method o f Gehrke

4.2.6

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( r e f . 18), y i e l d i n g a m i x t u r e o f r i bonucleosides. Chromacolumns ( d e s c r i b e d tography o f these m i x t u r e s on 3 pm C,,/C, below) was done t o i s o l a t e f r a c t i o n s o f i n t e r e s t . Each t R N A species, 10 A,, u n i t s i n 100 p l water, was t r e a t e d f o r 2 minutes i n a b o i l i n g water bath, cooled r a p i d l y on i c e , and t h e n i n c u b a t e d a t 37°C f o r one hour w i t h 8 p1 o f 20 mM ZnSO,, 40 p1 nuclease P1, 1 mg/ml i n 30 mM sodium a c e t a t e b u f f e r (pH 5.3), and 40 p l bact e r i a l a l k a l i n e phosphatase, 100 u n i t s / m l . A f t e r t h i s i n c u b a t i o n , t h e pH o f t h e r e a c t i o n m i x t u r e was a d j u s t e d by t h e a d d i t i o n o f 60 p l 0.5 M Tris-HC1 b u f f e r (pH 7. 9) . The r e a c t i o n was c o n t i n u e d a t 37°C f o r another hour t o a l l o w t h e c o n v e r s i o n o f n u c l e o t i d e s t o nucleosides by t h e a c t i o n o f t h e a1 k a l i n e phosphatase. et a l .

4.2.7

HPLC

The n u c l e o s i d e composition o f t h e t R N A was analyzed by a m o d i f i c a t i o n o f t h e reversed-phase HPLC procedure d e s c r i b e d by Gehrke e t a l . ( r e f . 18). The HPLC s o l v e n t d e l i v e r y system was an A l t e x Model 322 MP programmable chromatograph w i t h a Model 153 fixed-wavelength UV d e t e c t o r (Beckman I n s t r u m e n t s , I r v i n e , CA). Data c o l l e c t i o n and processing was accomplished u s i n g an I B M CS9001 w i t h Chromatography A p p l i c a t i o n Program ( I B M I n s t r u m e n t s , The reversed-phase HPLC columns ( l i n k e d i n s e r i e s ) Danbury, C T ) . were a1 1 Phenomenex-Techsphere w i t h 3 pm s t a t i o n a r y phases, cons i s t i n g o f one C,, guard column (50 x 4.6 mm), one C,, a n a l y t i c a l column (100 x 4.6 mm), and one C, a n a l y t i c a l column (50 x 4.6 mm) (Phenomenex, Rancho Palos Verdes, CA). The m o b i l e phase c o n s i s t e d o f 94% 10 mM ammonium formate (pH 5.0) and 6% methanol w i t h a f l o w r a t e o f 1 m l p e r minute. F r a c t i o n s were c o l l e c t e d i n t h e r e g i o n o f t h e chromatogram where i n o s i n e e l u t e d as a r e f e r e n c e s t a n d a r d ( c a . 10 m i n u t e s ) , and This were pooled from each t R N A h y d r o l y s a t e chromatographed. f r a c t i o n from each t R N A sample was l y o p h i l i z e d and subsequently s o l u b i l i z e d i n 400 p1 PBS-Tween b u f f e r . A l i q u o t s o f t h e s e samples were then pre-incubated w i t h a n t i - i n o s i n e a n t i b o d y as d e s c r i b e d i n C o m p e t i t i v e ELISA above and q u a n t i t a t e d by comparison w i t h a standard i n h i b i t i o n c u r v e generated u s i n g t h e r e f e r e n c e standard inosi ne.

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RESULTS AND DISCUSSION

4.3 4.3.1

T i t e r D e t e r m i n a t i o n and A n t i b o d v S o e c i f i c i t v The i n i t i a l immunization o f t h e r a b b i t s w i t h inosine-KLH was f o l l o w e d by a s e r i e s o f b o o s t e r i n j e c t i o n s , b o t h i . v . and i . m . The s e r a were sampled b i w e e k l y and t h e t i t e r s , d e f i n e d as t h e d i l u t i o n o f t h e anti-serum g i v i n g 50 % o f maximum absorbance i n ELISA, were found t o i n c r e a s e r a p i d l y . E a r l y i n t h e immunization p r o t o c o l , i t was observed t h a t t h e s e r a from r a b b i t s immunized w i t h t h e inosine-KLH c o n j u g a t e were s p e c i f i c f o r t h e antigen, inosine-BSA, as determined w i t h t h e ELISA ( F i g u r e 4.1). With t h e l a r g e amount o f a n t i g e n used (200 ng), l i t t l e r e a c t i v i t y was observed w i t h BSA. The r e a c t i v i t y o f t h e s e r a a g a i n s t inosine-KLH was comparable t o t h a t observed w i t h in o s i ne-BSA ( F i gure 4.2) where e q u i v a l e n t amounts o f p r o t e i n c o n j u g a t e (10 ng) were used. The s e r a was much more r e a c t i v e toward t h e unconjugated p r o t e i n T h i s r e s u l t was as expected, s i n c e t h e KLH than toward BSA. I

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esL

pre-immune and a n t i - i n o s i n e s e r a a g a i n s t Pre-immune ( t r i a n l e s and a n t i - i n o s i n e i n t h e t h i r d weefl immunization w i t h s e r i a l l y w i t h PBS-Tween and 100 p l were

02

B132 B132

F i g u r e 4.1 ( c o n t i n u e d ) assayed a g a i n s t t h e a n t i g e n s , 200 ng inosine-BSA ( c l o s e d symbols) and 200 ng BSA (open symbols), as d e s c r i b e d i n METHODS. The absorbance a t 405 nm was m o n i t o r e d a f t e r a 60 m i n u t e i n c u b a t i o n w i t h s u b s t r a t e . c o n j u g a t i o n o f i n o s i n e t o t h e h i g h l y immunogenic s p e c i e s KLH was done p u r p o s e l y t o generate an immunogen w i t h a h i g h a n t i g e n i c r e sponse ( r e f . 15). Pre-immune sera e x h i b i t e d a negl i g i b l e response t o e i t h e r inosine-KLH o r KLH ( F i g u r e 4.2). A t t h e end o f t h e 1 6 t h week o f t h e immunization p r o t o c o l , t h e sera had an average t i t e r corresponding t o 1:1,000,000, u s i n g 0.2 ng inosine-BSA as t h e p l ate-bound a n t i g e n . Therefore, subsequent ELISA d e t e r m i n a t i o n s were done w i t h a n t i - i n o s i n e s e r a d i l u t e d a t A t t h i s a n t i b o d y d i l u t i o n no n o n s p e c i f i c l e a s t one m i l l i o n - f o l d . r e a c t i v i t y a g a i n s t t h e c a r r i e r p r o t e i n was observed. Several homopolyri bonucl e o t i d e s were examined as a n t i gens i n t h e ELISA w i t h a h i g h c o n c e n t r a t i o n (1:10,000 d i l u t i o n ) o f t h e i n o s i n e - s p e c i f i c serum p r e p a r a t i o n . The use o f t h i s h i g h a n t i b o d y c o n c e n t r a t i o n was done t o d e t e c t any c r o s s r e a c t i v i t y w i t h p o l y (A), poly(G), or p o l y ( X ) . The r e s u l t s d e p i c t e d i n F i g u r e 4.3 demonstrate t h a t p o l y ( 1 ) was t h e most r e s p o n s i v e a n t i g e n , w i t h a I

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and a n t i - i n o s i n e s e r a a a i n s t A1 1 a n t i gens, in o s i ne-BS

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F i g u r e 4.2 ( c o n t i n u e d ) ( c i r c l e s ) , inosine-KLH (squares), and KLH ( t r i a n g l e s ) , were used a t 10 ng p e r w e l l . Pre-immune serum open symbols) and immune .serum (c!osed symbols) were s e r i a l l y d i u t e d and assayed as d e s c r i b e d i n F i g u r e 4.1.

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F i g u r e 4.3 S p e c i f i c i t y o f binding o f antibody against various homopolynucleotides as t h e i m m o b i l i z e d a n t i ens. Increasin c o n c e n t r a t i o n s , r a n g i n g from 0.1 ng t o 10,0il0 n o f poly(1Q (open t r i a n g l e s ) , PO!;(;) (closed (closed c i r c l e s ) , poly X) triangles) and p o l y(A) open c i r c l es) were immobi 1i z e d t o m i c r o t i t e r we f s by o v e r n i g h t d r y i n g a t .37"C. A n t i r i n o s i n e serum o b t a i n e d a t t h e 1 6 t h week o f t h e immunization was d i l u t e d 1:10,000 w i t h PBS-Tween and used i n t h e ELISA.

t

dramatic i n c r e a s e i n absorbance a t 405 nm b e i n g e x h i b i t e d when p o l y ( 1 ) was i n c r e a s e d from 0 . 1 ng t o 5 ng. The o t h e r p o l y n u c l e o t i d e s , poly(A) and poly(X), showed no b i n d i n g even w i t h g r e a t e r than a 1,000-fold excess o f a n t i g e n . S u r p r i s i n g l y , t h e response w i t h e q u i v a l e n t amounts o f t h e s t r u c t u r a l l y analogous poly(G) was also negligible. As another i n d i c a t o r o f a n t i b o d y s p e c i f i c i t y , BSA-nucleoside conjugates were t e s t e d as a n t i g e n s . The r e s u l t s shown i n F i g u r e 4.4 demonstate t h a t guanosine-BSA was r e q u i r e d a t a p p r o x i m a t e l y a 5 - f o l d g r e a t e r amount than inosine-BSA t o y i e l d a comparable response. Since a n t i b o d i e s f a i 1ed t o r e c o g n i ze guanosine i n the polynucleotide, i t i s possible t h a t the recognition o f guanosine-BSA by a n t i b o d y i s due t o t h e a f f i n i t y f o r more s u i t a b l e determinant s i t e s on t h e h a p t e n - c a r r i e r p r o t e i n c o n j u g a t e . 4.3.2

C o m o e t i t i v e ELISA Further c h a r a c t e r i z a t i o n o f antibody s p e c i f i c i t y u t i l i z e d the ELISA i n c o m p e t i t i v e i n h i b i t i o n assays. The r e l a t i v e a f f i n i t y o f the a n t i - i n o s i n e sera preparations f o r various nucleosides,

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n u c l e o s i d e monophosphate d e r i v a t i v e s , and homopolyri b o n u c l e o t i d e s was a s c e r t a i n e d w i t h t h e c o m p e t i t i v e ELISA u s i n g e i t h e r in o s i neBSA o r p o l y ( 1 ) as t h e plate-bound a n t i g e n . I n o s i n e was found t o be 1.0

-E

0.8

c

m 0.6 v

a, 0

C 0

+

0.4

0

cn

a

a

0.2

Antigen /We1 I (nanogram) Figure 4.4 Comparative b i n d i n g o f a n t i b o d y w i t h i n o s i n e and i t s s t r u c t u r a l an a1 og )e, guanosi ne. Nucl e o s i de-BSA conjugates, inosi ne-BSA ( c i r c l es and guanosi ne-BSA ( t r i a n g l e s ) , were prepared as d e s c r i b e d i n MET ODs. I n c r e a s i n g c o n c e n t r a t i o n s o f conjugates i n PBS (0.05 ng t o 1 p p e r w e l l ) were t i t r a t e d w i t h a n t i - i n o s i n e serum d i l u t e d 1:1,000,~00 ( c l o s e d symbols) o r 1:10,000,000 (open symbols)

.

t h e most e f f e c t i v e i n h i b i t o r , f o l l o w e d by t h e s t r u c t u r a l analogue guanosi ne ( F i g u r e 4.5). The r e s p e c t i v e n u c l e o t i de s p e c i e s ( I M P and GMP) gave i n h i b i t i o n o f a n t i b o d y b i n d i n g comparable t o t h e corresponding n u c l e o s i d e ( d a t a n o t presented). Much h i g h e r concent r a t i o n s o f o t h e r n u c l e o s i d e s (adenosine and c y t i d i n e ) and nucleot i d e s (AMP and CMP) used as i n h i b i t o r s were found t o e x h i b i t l i t t l e i n t e r a c t i o n w i t h t h e a n t i - i n o s i n e serum, r e s u l t i n g i n i n h i b i t i o n values r a n g i n g from 0 t o 5 %. The n a t u r e o f t h e p l a t e bound antigen, e i t h e r inosine-BSA o r p o l y ( I ) , d i d n o t a f f e c t t h e c o m p e t i t i v e ELISA response by these n u c l eosides and n u c l e o t i d e s t o an a p p r e c i a b l e e x t e n t ( d a t a n o t shown). The i n h i b i t o r s hypoxanthine, i n o s i n e , and I M P were examined i n t h e c o m p e t i t i v e ELISA t o determine whether t h e a n t i b o d y s p e c i f ic i t y was d i r e c t e d e x c l u s i v e l y t o d e t e r m i n a n t s o f t h e p u r i n e base,

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hypoxanthine, o r t o t h e r i b o s e m o i e t y as w e l l , s i n c e t h e hapten p r o t e i n c o n j u g a t e used f o r immunization i n v o l v e d a l i n k a g e t o KLH v i a t h e h y d r o x y l groups o f r i b o s e . The amounts o f each compound r e q u i r e d f o r 50 % i n h i b i t i o n o f b i n d i n g o f t h e a n t i - i n o s i n e serum t o t h e a n t i g e n p o l y ( 1 ) were determined t o be 300 pmol I M P , 850 pmol i n o s i n e , and 7 nmol hypoxanthine ( F i g u r e 4.6). These r e s u l t s a r e s i m i l a r t o those of Bonavida e t a l . ( r e f . 3), whereby i n o s i n e was r e q u i r e d a t a 3 - f o l d g r e a t e r c o n c e n t r a t i o n t h a n I M P t o produce 50 % i n h i b i t i o n i n t h e i r assay, i n a c t i v a t i o n o f i n o s i n e - b a c t e r i o phage 14 caused by an a n t i - i n o s i n e serum. Several homopolyri bonucl e o t i des were assayed i n t h e competi t i v e ELISA. As expected, p o l y ( 1 ) used as an i n h i b i t o r a t a c o n c e n t r a t i o n from 1 t o 5 ng showed t h e h i g h e s t l e v e l o f i n h i b i t i o n o f a n t i b o d y b i n d i n g ( d a t a n o t p r e s e n t e d ) . However, poly(G) i n amounts up t o 5 fig f a i l e d t o show any i n h i b i t i o n o f b i n d i n g o f a n t i - i n o s i n e serum t o t h e plate-bound a n t i g e n s , h a p t e n - p r o t e i n o r POlY(1) * 4.3.3

D e t e c t i o n o f I n o s i n e i n Hvdrolvzed t R N A The c o m p e t i t i v e ELISA method f o r q u a n t i t a t i n g i n o s i n e was applied t o the determination o f t h i s minor nucleoside species i n tRNA. Inouye e t a l . ( r e f . 2) f i r s t u t i l i z e d a n t i - i n o s i n e a n t i bodies t o d i s t i n g u i s h i n o s i n e - c o n t a i n i n g t R N A f r o m o t h e r t R N A species by a f f i n i t y chromatography. A1 though t h e chromatographic procedure o f Inouye e t a l . ( r e f . 2) a l l o w e d t h e r e t a r d a t i o n and subsequent i d e n t i f i c a t i o n o f i n o s i n e - c o n t a i n i n g s p e c i e s i n b u l k tRNA, o t h e r methods would have been r e q u i r e d f o r t h e q u a n t i t a t i o n o f i n o s i n e i n tRNA p r e p a r a t i o n s . Therefore, an a l t e r n a t e p r o cedure u t i 1 i z i n g HPLC-separated n u c l e o s i d e s o b t a i n e d from t R N A h y d r o l y s a t e s and assaying f r a c t i o n s o f i n t e r e s t w i t h t h e competit i v e ELISA was i n v e s t i g a t e d . A s t u d y u t i l i z i n g an HPLC s e p a r a t i o n o f v a r i o u s c a r c i nogen-deoxynucl e o s i de adducts and subsequent R I A f o r q u a n t i t a t i o n has been d e s c r i b e d p r e v i o u s l y ( r e f . 19). The a p p l i c a b i l i t y o f i n o s i n e q u a n t i t a t i o n was demonstrated w i t h t R N A samples o f known n u c l e o t i de sequence u s i n g procedures d e s c r i b e d i n t h e METHODS s e c t i o n . Pure is o a c c e p t i ng tRNA species from E . c o l i , t R N A A r g having t h e a n t i c o d o n sequence I C G and t R N A P h e h a v i n g t h e a n t i c o d o n sequence GAA ( r e f . 17), were used f o r t h e subsequent a n a l y s i s . The presence o f a s i n g l e i n o s i n e r e s i d u e

B136

i n the

macromolecule and t h e absence o f t h i s r e s i d u e i n a l l o w e d us t o compare i n o s i n e q u a n t i t a t i o n w i t h t h e two tRNA species. With these p u r e t R N A i s o a c c e p t i n g species, q u a n t i t a t i o n of i n o s i n e c o u l d be accomplished w i t h HPLC w i t h o u t u s i n g t h e immunoassay, b u t peak i d e n t i t y i s n o t always c l e a r i n more complex cases. Therefore, t o determine t h e v a l u e o f t h e immunotRNAArg

tRNAPhe

100

80

h

8

60

C

0 .t .-

e L

H

40

20

0

Inhibitor (picomole)

F i g u r e 4.5 I n h i b i t i o n o f antibody. binding t o hapten-protein con'ugate by v a r i o u s nucleosides i n t h e c o m p e t i t i v e ELISA. Nuc eosides were d i 1-uted . t o a p p r o p r i a t e c o n c e n t r a t j o n s w i t h PBSTween b e f o r e i n c u b a t i o n w i t h an equal volume o f a n t i - i n o s i n e serum The m i x t u r e s were t o g i v e a f i n a l serum d i l u t i o n o f 1:1,000,000. added t o t h e w e l l s c o n t a i n i n g inosine-BSA (0.2 n g / w e l l ) as t h e immobilized antigen. The absorbance due t o t h e a n t i b o d y b i n d i n g i n t h e ELISA was determined and c o n v e r t e d t o p e r c e n t i n h i b i t i o n (see METHODS)

1

.

assay method w i t h b i o l o g i c a l specimens, t h e known samples were processed f o r q u a n t i t a t i o n i n t h e c o m p e t i t i v e ELISA. Use o f t h e a n t i - i n o s i n e serum i n t h e ELISA f o r t h e purpose o f d i r e c t l y q u a n t i t a t i n g i n o s i n e i n t h e t R N A h y d r o l y s a t e was complicated by t h e abundance o f t h e i n o s i n e analogue, guanosine, i n

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these h y d r o l y s a t e s ( F i g u r e 4.7). As a l r e a d y shown i n F i g u r e 4.5, guanosine was a r e l a t i v e l y e f f e c t i v e c o m p e t i t i v e i n h i b i t o r as we1 1. A1 though p r e v i o u s r e p o r t s ( r e f s . 1, 20) had i n d i c a t e d a r e d u c t i o n in c r o s s r e a c t i v i t y towards guanosi ne b y p r e - a b s o r p t i on o f a n t i - i n o s i n e s e r a w i t h guanosine-BSA, we c o u l d n o t d u p l i c a t e those r e s u l t s . Therefore, p r e - s e p a r a t i o n o f t h e n u c l e o s i d e s from t R N A t r e a t e d w i t h n u c l ease P 1 and b a c t e r i a1 a1 k a l ine phosphatase I n o s i n e and guanosine were r e s o l v e d by ( r e f . 18) was r e q u i r e d . in reversed-phase HPLC ( r e f . 18), and f r a c t i o n s ( i n d i c a t e d as "I" each case) were c o l l e c t e d f r o m t h e r e g i o n o f t h e chromatogram where i n o s i n e ( t h e f i r s t o f t h e two peaks p r e c e d i n g guanosine i n I

I

I

10'

102

103

1

100

80

h

8

60

C

0 ._ 4.._ a ._

$ 40 H

20

0

lo4

Inhibitor (picomole 1

F i g u r e 4.6 C o m p e t i t i v e i n h i b i t i o n o f a n t i b o d b i n d i n g t o p o l y ( 1 ) The irnmogilized anti.gen .used by h oxanthine, i n o s i n e , and IMP. i n E l f S A was 10 ng p o l (I). The ELISA was performed as i n Fi u r e 4.5 u s i n g a p p r o p r i a t e c f i l u t i o n s o f hy oxanthine, i n o s i n e , and QMP, w i t h a n t i - i n o s i n e serum a t a f i n a l d i y u t i o n o f 1:1,000,000.

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t h e tRNAAr g h y d r o l y s a t e ) e l u t e d a t a p p r o x i m a t e l y 10 m i n u t e s ( F i g u r e 4.7). Using v a r y i n g c o n c e n t r a t i o n s o f i n o s i n e , competit i v e ELISA was r u n i n p a r a l l e l w i t h each assay o f t h e t e s t samples. The r e s u l t i n g i n h i b i t i o n v a l u e s o b t a i n e d w i t h t h e HPLC f r a c t i o n s f r o m h y d r o l y z e d t R N A A r g and tRNAPhe were c o n v e r t e d t o picomoles o f i n o s i n e u s i n g t h e standard i n h i b i t i o n c u r v e ( F i g u r e 4.8). The y i e l d o f i n o s i n e f r o m h y d r o l y z e d t R N A A r g (10 A,, u n i t s ) was d e t e r m i n e d f r o m F i g u r e 4.8 t o be 10.8 nanomoles. The samples r e c o v e r e d f r o m t h e p r e - s e p a r a t i o n had been r e c o n s t i t u t e d i n 0.4 m l . A l i n e a r dose r e l a t i o n s h i p between t h e amounts o f t e s t sample employed i n t h e assay and p i c o m o l e s o f i n o s i n e was o b s e r v e d w i t h inosine-containing t R N A A r g . On t h e o t h e r hand, t h e r e was v e r y l i t t l e , i f any, i n h i b i t i n i w i t h t.he c o m p a r a b l e f r a c t i o n T h e r e f o r e , an in o s i n e conceno b t a i n e d f r o m h y d r o l y z e d tRNAP h e . t r a t i o n o f 2.7 x 10-5 M was o b t a i n e d f o r t R N A A r g , whereas, l e s s t R N AArg

tRNAPhe

Ti me F i g u r e 4.7 HPLC p r o f i l e o f f. c o l i tRNAArg and tRNAPhe h y d r o l y sates. The n u c l e a s e P l / a l k a l i n e p h o s p h a t a s e t r e a t e d tRNAArg and t R N A P h e h y d r o l y s a t e s were chromatographed as d e s c r i b e d i n METHODS. From t h e r e g i o n o f each HPLC p r o f i l e shown, f r a c t i o n " I " .was collected f o r the i n h i b i t i o n o f inosine s p e c i f i c antibody binding.

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than M c o u l d be e s t i m a t e d f o r t R N A P h e , having G i n s t e a d o f I i n i t s anticodon. FUTURE PERSPECTIVES The methodology i n v o l v i n g p r e - s e p a r a t i o n o f n u c l e o s i d e s b e f o r e c o m p e t i t i v e ELISA f o r t h e d e t e c t i o n o f i n o s i n e i n t R N A c o u l d be a p p l i e d t o b u l k , u n f r a c t i o n a t e d t R N A f o r q u a n t i t a t i n g changes i n t h i s i m p o r t a n t n u c l e o s i d e , f o r example, d u r i n g c e l l differentiation. S i m i l a r methodology should a l s o be u s e f u l f o r I n cases where t h e q u a n t i t a t i n g o t h e r n u c l e o s i d e s from t R N A . m o d i f i c a t i o n s i n q u e s t i o n a r e n o t s u b s t a n t i a l l y b u l k y (such as t h e case w i t h i n o s i n e ) , c r o s s r e a c t i v i t y o f t h e serum sample w i t h unmodi f ied n u c l eosi des c o u l d be c i rcumvented by t h e reversed-phase HPLC p r e - s e p a r a t i o n o f t h e n u c l e o s i d e s as d e s c r i b e d here. In cases w i t h h i g h l y m o d i f i e d n u c l e o s i d e s u b s t i t u e n t s w i t h d r a s t i c s t r u c t u r a l changes, crude h y d r o l y s a t e s o r even i n t a c t t R N A m i g h t be employed. Such a s i t u a t i o n c o u l d a l s o be a p p l i c a b l e t o q u a n t i t a t i n g m o d i f i e d n u c l e o s i d e s i n t h e u r i n e o r serum o f cancer p a t i e n t s where t h e l e v e l s o f c e r t a i n o f these components appear t o 4.4

1400

K8

100

1200 -

;

; 1000 -

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a, 0

5

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0 .+ 60 .-

e

U

800

-

I

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102

103

Inosine (picomoles)

10

20

104

/

30

40

50

HPLC Peak I Sample Volume (PI)

F i g u r e 4.8 Q u a n t i t a t i o n o f i n o s i n e i n t h e tRNA h y d r o l y s a t e s . Peak "I" c o l l e c t e d from HPLC of E . c o l i t R N A A r g and t R N A P h e

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A

F i g u r e 4.8 ( c o n t i n u e d ) hydro1 sates ( F i g u r e 4.7 was processed and u t i l i z e d i n t h e c o m p e t i t i v e {LISA Inosine-BS (0.2 ng) was used as t h e i m m o b i l i z e d antigen, and t i e a n t i - j n o s i n e serum was d i l u t e d A standard i n h i b i t i o n c u r v e w i t h - p u r i f i e d one m i l l i o n - f o l d . The amount o f i n o s i n e i n i n o s i n e (see i n s e t ) was r u n i n para1 l e l f r a c t i o n s was determined by comparison o f . t h e p e r c e n t t h e . eak "I" i n h i E i t i o n values o b t a i n e d f o r t h e t e s t samples w i t h t h a t o f standard i n o s i n e .

.

be u s e f u l i n d i c a t o r s o f disease s t a t u s ( r e f . 21). A competitive ELISA f o r q u a n t i t a t i n g these cancer markers would a l l o w automation o f analyses, and as a r e s u l t , t h e i r c l i n i c a l u s e f u l n e s s would 1i k e l y be enhanced. 4.5

SUMMARY D e t e r m i n a t i o n o f t h e i n o s i n e c o n t e n t i n t R N A i s an i m p o r t a n t cons d e r a t i o n due t o i t s p o t e n t i a l r o l e i n a l t e r i n g gene expression I n o s i n e i s found i n t R N A i n t h e f i r s t (wobble) p o s i t i o n o f t h e anticodon, and i n t h a t p o s i t i o n , i t can i n f l u e n c e codon r e c o n i t i o n and, thereby, p r o t e i n s y n t h e s i s . As a r e s u l t , q u a n t i t a t i o n o f changes i n t h e i n o s i n e c o n t e n t o f t R N A a r e o f v a l u e f o r examining t h e r o l e o f t h i s t R N A m o d i f i c a t i o n i n t r a n s l a t i o n a l r e g u l a t i o n . Reversed-phase high-performance l i q u i d chromatography (HPLC) o f f e r s one means f o r d e t e r m i n i n g t h e i n o s i n e c o n t e n t i n h y d r o l y z e d t R N A . Nucleosides i n t h e t R N A h y d r o l y s a t e a r e r e s o l v e d by t h e HPLC method, and under many circumstances, t h e i n o s i n e c o n t e n t can be e s t a b l i s h e d by simple o n - l i n e d e t e c t i o n based on UV absorbance. However, i t i s d i f f i c u l t t o separate i n o s i n e from some o f t h e o t h e r m o d i f i e d nucleosides found i n u n f r a c t i o n a t e d tRNAs, so a1 t e r n a t i v e d e t e c t i o n methods a r e needed. Inosines p e c i f i c , h i g h t i t e r and h i g h a f f i n i t y a n t i b o d i e s were r a i s e d i n r a b b i t s a g a i n s t i n o s i n e - k e y h o l e 1 impet hemocyanin c o n j u g a t e . An enzyme-1 i n k e d immunosorbant assay (ELISA) was developed and i n o s i n e was q u a n t i t a t e d by c o m p e t i t i v e i n h i b i t i o n w i t h i m m o b i l i z e d a n t i g e n . To date, t h i s immunological approach f o r t h e q u a n t i t a t i o n o f i n o s i n e has proven most e f f e c t i v e when t h e assay i s coupled t o t h e reversed-phase HPLC s e p a r a t i o n o f n u c l e o s i d e s i n t R N A h y d r o l y s a t e s , b u t o t h e r uses a r e a l s o 1 ik e l y .

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ACKNOWLEDGMENTS The a u t h o r s would l i k e t o thank E r i c D. U t z f o r e x p e r t technical assistance. Supported by Grant AFOSR-85-0003 (R.W.T.) from t h e A i r Force O f f i c e o f S c i e n t i f i c Research, Department o f Defense, N I E H S g r a n t 02388 (A.A.W.) , and Grant P-30-CA-16058-13 (OSUCCC) from t h e N a t i o n a l Cancer I n s t i t u t e , Department o f H e a l t h and Human Services. 4.6

REFERENCES H. Inouye, S. Fuchs, M. Sela and U. Z. L i t t a u e r , A n t i - i n o s i n e a n t i b o d i e s , Biochim. Bioph s . Acta, 240 (1971) 594-603. 2. H. Inouye, S . Fuchs, M. Sera and U. Z. L i t t a u e r , D e t e c t i o n of inosine-containing t r a n s f e r r i b o n u c l e i c a c i d species b y a f f i n i t y chromato raphy on columns o f a n t i - i n o s i n e a n t i - b o d i e s , J . B i o l C\em. , 248 (1973) 8125-8129. 3. H. Inouye and M. Sela, I n o s i n e B. Bonavida, S . Fuchs, coated bacteriophage T4, Biochim. Biophys. Acta, 240 1971) 604-610. 4. Fuchs, A. Aharonov, M. Sela, F. von d e r Haar and F. Cramer, A n t i bodies t o y e a s t p h e n y l a l a n i n e t r a n s f e r r i bon u c l e i c a c i d a r e s p e c i f i c f o r t h e odd n u c l e o s i d e Y i n t h e 7 1 (1974) a n t i c o d o n loop, Proc. N a t l . Acad. S c i . U.S.A., 2800-2802. 5. R. Salomon, S . Fuchs, A. Aharonov, D. Giveon and U. Z. L i t t a u e r , D e t e c t i o n and p u r i f i c a t i o n o f i s o a c c e p t i n g t R N A P h e species c o n t a i n i n g Y base by a f f i n i t y chromato r a hy on columns o f a n t i - Y a n t i b o d i e s , B i o c h e m i s t r y , 14 (1875p 40464050. 6. S. A. Khan, M. Z. Humayun and T. M. Jacob, A s e n s i t i v e r a d i o immunoassay f o r isopentenyladenosine, Anal. Biochem., 83 (1977) 632-635. 7. D. S. M i l s t o n e , B. S . Vold, D. G. G l i t z and N. S h u t t , A n t i bodies t o N6-( A2-isopenten 1)adenosine and i t s n u c l e o t i d e : i n t e r a c t i o n w i t h p u r i f i e d tiNAs and w i t h bases n u c l e o s i d e s and n u c l e o t i d e s o f t h e isopentenyladenosine f a m i l y , N u c l e i c Acids Res., 5 1978) 3439-3455. 8. B . S . Vold, Ra ioimmunoassays f o r t h e m o d i f i e d n u c l e o s i d e s N[9-(8-D-ri b o f u r a n o s y l p u r i n-6-yl carbamoyl - L - t h r e o n i ne and 2(1979) 193-204. methyl thioadenosine, u c l e i c Acids Res., 9. B. S . Vold and H. W. Nolen, A unique method u t i l i z i n g a n t i n u c l e o t i d e a n t i b o d i e s f o r e v a l u a t i n g changes i n t h e l e v e l s o f m o d i f i e d n u c l e o s i d e s o f tRNAs from crude e x t r a c t s o f whole c e l l s , N u c l e i c Acids Res., 7 (1979) 971-980. 10. B . S . Vold, P r e p a r a t i o n and s p e c i f i c i t o f . a n t i b o d i e s d i r e c t e d toward t h e r i b o s e m e t h y l a t e d n u c f e o t i d e 2'-0-methyl uanosine 5'-monophosphate, Biochim. B i o p h i s . Acta, 655 1981) 265-267. 11. Jayabaskaran, A n t i b o d i e s s p e c i f i c f o r 1-methylguanosine as a probe o f t R N A conformation, Biochem. I n t . , 8 (1984) 5614.7 1.

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R. W. Trewyn, I n o s i n e b i o s y n t h e s i s i n t r a n s f e r RNA: a p o s t u l a t e d r o l e i n immune r e g u l a t i o n , Med. Hypotheses, 13 (1984) 369-380.

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R. W. Trewyn, K. A. K r e t z , E. D. Utz, D. E. P a t r i c k and G. M u r a l i d h a r , Hematopoiesis and t h e i n o s i n e m o d i f i c a t i o n i n t r a n s f e r RNA, Proc. SOC. E x p t l . B i o l . Med., 179 (1985) 497503. B. F. E r l a n g e r and S . M. B e i s e r , A n t i b o d i e s s p e c i f i c f o r r i bonucl eosi des and r i bonucl e o t i d e s and t h e i r r e a c t i o n w i t h DNA, Proc. N a t l . Acad. S c i . U.S.A., 52 (1964) 68-74. R. M u l l e r and M.F. Rajewsky, Immunololgical q u a n t i f i c a t i o n b high-affinity a n t i b o d i e s o f 06-ethyldeoxyguanosine i n D 6 A exposed t o N-ethyl-N-ni t r o s o u r e a , Cancer Res. , 40 (1980) 887-896. A. A. Wan!, R. E. Gibson-D'Ambrosio and S. M. D'Ambrosio, Q u a n t i t a t 1 on o f O6 - e t h y l deox guanosi ne i n ENU a1 k y l a t e d DNA by p o l y c l o n a l and monoclonay a n t i b o d i e s , Carcinogenesis, 5 1984) 1145-1150. S r i n z l , J . M o l l , F. Meissner and T. Hartmann, C o m p i l a t i o n 13 (1985 r l - r 4 9 . o f t!NA sequences, N u c l e i c Acids Res C.W. Gehrke, K.C. Kuo, R.A. McCune a d K.O. Ger a r d t , Q u a n t i t a t i v e enzymatic h y d r o l y s i s o f tRNAs. Reversed-phase h i g h performance l i q u i d chromato raphy o f t R N A n u c l e o s i d e s , J . Chromatogr. , 230 (1982 297-388 D y r o f f ; J . A. Boucheron and J . A. F. C. Richardson, M. Swenberg, D i f f e r e n t i a l . r e p a i r o f 0 4 - a l k y l t h y m i d i n e f o l l o w i n g exposure t o m e t h y l a t i n g and e t h y l a t i n g hepatocarcinogens, C a r c i nogenesi s , 6 (1985) 625-629. H. Inouye, S. Fuchs, M. Sela and U. Z. L i t t a u e r , A n t i - i n o s i n e a n t i b o d i e s , I s r a e l J . Med. S c i . , 6 (1970) 442. R. W. Trew n and M. R. Grever, U r i n a r y n u c l e o s i d e s i n l e u kemia: l a g o r a t o r y and c l i n i c a l a p p l i c a t i o n s , CRC C r i t i c a l Rev. C l i n . Lab. Sci., 24 (1986) 71-93.

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CHAPTER 5 S I T E DIRECTED REPLACEMENT OF NUCLEOTIDES I N THE ANTIAPPLICATION TO THE STUDY OF CODON LOOP OF TRNA: INOSINE BIOSYNTHESIS I N YEAST TRNAALA KEITH A. KRETZ', GROSJ EAN3

RONALD W . TREWYN', GERARD KEITH* and HENRI

'Department o f Physiological Chemistry, The Ohio S t a t e U n i v e r s i t y , Columbus, O h i o 4 3 2 1 0 , U . S . A . * I n s t i t u t e o f M o l e c u l a r and C e l l u l a r B i o l o g y o f CNRS, U n i v e r s i t y o f Strasbourg, 67084 Strasbourg, France 3Laboratory o f Biological Chemistry, 1640 Rhode-Saint-Genese, Belgium

University

o f B r u s s e l s , B-

TABLE OF CONTENTS 5 . 1 Introduction . . . . . . . . 5.2 Materials and Methods . . . . . . . . . . . . 5 . 2 . 1 Materials 5.2.2 Enzymatic Cleavage o f tRNA . Ribonuclease T, 5.2.2.1 5.2.2.2 Mung Bean Nuclease . . . Polyacrylamide Gel Electrophoresis . . 5.2.3 End-Labelling RNA 5.2.4 5.2.4.1 5 ' End-Labelling with r-[''P] ATP. 3 ' End-Labelling with 5'-[32P] pCp 5.2.4.2 Hydrolysis o f tRNA . . 5.2.5 Nuclease P, 5.2.5.1 5.2.5.2 Ri bonuclease T, . . . . 5.2.6 Two-Dimensional Thin Layer Chromatography 5.2.7 Periodate Oxidation . 5.2.8 5 ' Phosphorylation . . . 5.2.9 Dephosphorylation 5.2.10 Renaturation and Reannealing o f tRNA . . . . 5 . 2 . 1 1 Radiolabelling Oligonucleotides . 5.2.12 Ligation I n V i t r o Modification of tRNA . . 5.2.13 5.3 Results and Discussion .

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5.4 5.5 5.6 5.7

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Future Perspectives Summary Acknowledgements References

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INTRODUCTION M o d i f i e d bases i n t h e a n t i c o d o n o f tRNAs have been shown t o a f f e c t codon-anticodon i n t e r a c t i o n s and, thereby, t o i n f l u e n c e t h e e f f i c i e n c y and r e g u l a t i o n o f p r o t e i n s y n t h e s i s (reviewed i n r e f s . 1-3). I n a d d i t i o n , changes i n s p e c i f i c m o d i f i c a t i o n s have been c o r r e l a t e d t o changes i n v a r i o u s aspects o f normal c e l l u l a r development, e x c l u s i v e o f d i r e c t e f f e c t s on p r o t e i n s y n t h e s i s Among t h e p o s t - t r a n s c r i p t i onal modi f ica(reviewed i n r e f s . 4-6) t i o n s made t o tRNA, t h o s e i n p o s i t i o n 34 ( t h e a n t i c o d o n wobble p o s i t i o n ) and p o s i t i o n 37 ( a d j a c e n t t o t h e 3 ' end o f t h e ant i c o d o n ) a r e o f t e n hypermodified, and i n most cases, v e r y l i t t l e i s known about t h e m u l t i - e n z y m a t i c n a t u r e o f t h e i r s y n t h e s i s (see o t h e r c h a p t e r s o f t h i s volume). I d e n t i f y i n g and c h a r a c t e r i z i n g t h e enzymes r e s p o n s i b l e f o r g e n e r a t i n g t h e s e complex m o d i f i c a t i o n s pose a number o f s i g n i f i c a n t d i f f i c u l t i e s , n o t t h e l e a s t o f which i s o b t a i n i n g a s u i t a b l e , unmodified t R N A s u b s t r a t e f o r a n a l y z i n g t h e r e a c t i o n i n v i t r o o r i n v i v o . I f an u n m o d i f i e d t R N A s u b s t r a t e can be o b t a i n e d , an a p p r o p r i a t e method f o r m o n i t o r i n g m o d i f i c a t i o n o f the nucleotide o f i n t e r e s t i s s t i l l required. DNA-di r e c t e d t e c h n i q u e s o f f e r one approach f o r o b t a i n i n g unmodi f i ed t R N A s u b s t r a t e s f o r a n a l y z i n g p o s t - t r a n s c r i p t i o n a l modifications. These t e c h n i q u e s in c l ude t h e s e l e c t i o n o f mutants d e f i c i e n t i n c a r r y i n g o u t t h e m o d i f i c a t i o n o f i n t e r e s t (see B j b r k and K o h l i , Chapter 1, t h i s volume) and t h e t r a n s c r i p t i o n i n v i t r o o f t R N A genes from SP6 o r T7 promoters ( r e f . 7 ) . I n the l a t t e r case, t R N A genes i s o l a t e d from v a r i o u s p r o k a r y o t i c o r e u k a r y o t i c c e l l s o r t R N A genes d e r i v e d by t o t a l chemical s y n t h e s i s , coupled w i t h t h e techniques o f s i t e - d i r e c t e d mutagenesis w i t h i n t R N A genes, should be u s e f u l f o r s t u d i e s o f t R N A m o d i f i c a t i o n . However, t o d a t e such methods have been concerned more w i t h t h e p r o c e s s i n g and f u n c t i o n o f tRNAs t h a n w i t h t h e i r m o d i f i c a t i o n ( r e f s . 8-10). W h i l e each o f these t e c h n i q u e s o f f e r s d i s t i n c t advantages f o r c e r t a i n aspects o f t R N A m o d i f i c a t i o n research, o t h e r RNA-directed methods a r e sometimes more u s e f u l f o r examining 5.1

.

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the biosynthesis and function of specific tRNA anticodon modifications. One such RNA-directed method makes use of recombinant RNA technology to reconstruct the anticodon of purified tRNAs; technology based on the pioneering work of Ikehara and coworkers in Japan (ref. 11) and Ulhenbeck and coworkers in the U.S.A. (ref. 12) (reviewed in ref. 13). This methodology has been used to study the specificity of aminoacylation and decoding, to evaluate the effects of modifications on three-dimensional structure, and more recently, to examine and characterize the biosynthesis of hypermodified nucleosides in the anticodon loop (reviewed in ref. 14).

The basic approach to anticodon reconstruction is the specific removal of a portion of the anticodon loop and the replacement of that section with an oligonucleotide of interest. Enzymes which cleave single stranded RNA may be used under carefully controlled conditions to cleave the anticodon loop specifically. Using one or two different enzymes, the anticodon loop is opened and the appropriate tRNA "ha1f" molecules for reconstruction are recovered after separation by polyacryl amide gel electrophoresis (PAGE). The oligonucleotide to be inserted is labelled with [32P] adjacent to the nucleotide of interest and is then ligated into the reannealed ha1 f molecules usi ng T, -RNA 1 i gase. This procedure serves to generate an unmodified tRNA substrate with a ["PI label adjacent to a specific nucleoside. The [3*P] label allows the nucleotide of interest to be monitored easily for modification i n v i t r o or i n v i v o . Many people have made use of recombinant RNA technology to replace specific nucleotides in purified tRNAs, and while the detai 1s of each reconstruction vary, the overall pri nci pl es remain the same. Therefore, for purposes of illustration, the reconstruction of yeast tRNAA l a wi 1 1 be demonstrated, and pub1 ished studies employing recombinant RNA techniques will be summarized at the end of the chapter. The details whereby tRNAAL* (normal anticodon IGC) was reconstructed to contain the unmodified anticodon AGC will be given to illustrate how this chimeric tRNA is being used to study inosine biosynthesis in the first (wobble) position of the tRNA anticodon (position 34). Earlier work by Ohtsuka e t a l . (ref. 15) had demonstrated that the anticodon of alanine tRNA from yeast could be cleaved between G,, and C, using

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T, n u c l e a s e , and Wang e t a 7 . ( r e f . 16) s u b s e q u e n t l y showed t h a t a C3'P] l a b e l c o u l d be l i g a t e d between t h e s e p o s i t i o n s . W h i l e t h i s approach w o u l d n o t a l l o w a n a l y s i s o f t h e i n o s i n e m o d i f i c a t i o n , i t o f f e r e d u s e f u l background i n f o r m a t i o n f o r o u r work.

5.2 MATERIALS AND METHODS 5.2.1 M a t e r i a l s Pure y e a s t t R N A A a was p r e p a r e d b y c o u n t e r - c u r r e n t d i s t r i but i o n ( r e f . 17) f o l l o w e d b y e i t h e r p a r t i a l l y d e n a t u r i n g PAGE o r HPLC. The enzymes, r i b o n u c l e a s e T, , Mung bean n u c l e a s e , T,-RNA l i g a s e f r o m i n f e c t e d F s c h e r i c h i a c o 7 i c e l l s , n u c l e a s e P,, and r i b o n u c l e a s e T, , were purchased f r o m PL B i o c h e m i c a l s , and n u c l e a s e - f r e e b o v i n e serum a1 bumin (BSA) was f r o m Boehringer-Mannheim. Acrylamide, bis-acrylamide, N,N,N' ,N'-tetramethylethylenediamine (TEMED) , and ammoni um p e r s u l f a t e were f r o m I n t e r n a t i o n a l B i o t e c h nologies Incorporated, w h i l e T,-polynucleotide kinase from i n Phenol was f e c t e d €. c o 7 i c e l l s was purchased f r o m Amersham. o b t a i n e d f r o m F i s h e r S c i e n t i f i c and N-2-hydroxyethylpiperazine-N'2 - e t h a n e s u l f o n i c a c i d (HEPES) b u f f e r f r o m Research O r g a n i c s . The s h e e t s f o r t w o - d i m e n s i o n a l t h i n 1a y e r c h r o m a t o g r a p h y (2D-TLC) were purchased f r o m EM S c i e n c e . The f o l l o w i n g were o b t a i n e d f r o m Sigma Chemical Company: Cacodyl ic a c i d , d i e t h y l p y r o c a r b o n a t e , b o r i c a c i d , bromophenol b l u e , x y l e n e c y a n o l , sodium d o d e c y l s u l f a t e , i m i d a z o l e, sodium m e t a p e r i o d a t e , rhamnose, l y s i n e , 2-mercaptoe t h a n o l , h y p o x a n t h i n e , t h e d i n u c l e o t i d e monophosphate ApG, and t h e n u c l e o s i d e 3 ' monophosphates and n u c l e o s i d e 5 ' monophosphates (AMP, CMP, GMP, UMP, and I M P ) . 5.2.2 Enzvmatic Cleavacle o f tRNA 5.2.2.1 R i b o n u c l e a s e T, R i b o n u c l e a s e T, f r o m A s p e r g i 7 7 o s o r y z a e was u s e d t o c l e a v e t h e tRNA a n t i c o d o n s p e c i f i c a l l y a f t e r g u a n i n e r e s i d u e s , t h e r e b y y i e l d i n g a 5 ' h y d r o x y l g r o u p and a 3 ' p h o s p h a t e a t t h e c l e a v a g e s i t e . I n a m o d i f i c a t i o n o f t h e method o f Carbon e t a 7 . ( r e f . 18), t h e r e a c t i o n was c a r r i e d o u t i n a 20 mM c a c o d y l i c a c i d b u f f e r (pH 6.5) w i t h 10 mM MgC1,. G e n e r a l l y , 500 pg o f t R N A were c l e a v e d i n a 500 p l r e a c t i o n m i x t u r e c o n t a i n i n g 50 u n i t s T, [lo0 u n i t s T,/mg tRNA]. The r e a c t i o n m i x t u r e was made w i t h o u t RNase-T, p r e s e n t and was p r e - i n c u b a t e d f o r 5 m i n u t e s a t 4°C. A f t e r a d d i n g t h e enzyme

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and i n c u b a t i n g f o r 45 minutes a t 4"C, the r e a c t i o n was stopped by adding 5 p1 d i e t h y l p y r o c a r b o n a t e with vigorous v o r t e x i n g . The r e a c t i o n mixture was then e x t r a c t e d with 500 p1 o f phenol s a t u r a t e d with 100 mM sodium a c e t a t e (pH 4 . 8 ) . A f t e r v o r t e x i n g f o r 1 minute, the mixture was c e n t r i f u g e d a t 12,400 x g i n a microcent r i f u g e f o r 5 minutes. The aqueous phase was removed and the phenol was r e e x t r a c t e d w i t h 500 p1 of water. The aqueous phases were combined and the t R N A fragments were p r e c i p i t a t e d with 10% (v/v) 3 M sodium a c e t a t e (pH 4 . 8 ) and 2.5 volumes o f ethanol followed by s t o r a g e a t minus 70°C f o r 1 hour o r a t minus 20°C f o r a t l e a s t 4 hours. The cleavage fragments were then p u r i f i e d by PAGE ( d e s c r i b e d be1 ow). 5.2.2.2

Muna Bean Nuclease

Mung bean nuclease was used t o c l e a v e t R N A molecules a f t e r u r i d i n e r e s i d u e s i n the anticodon following the procedure of Beauchemin e t a 7 . ( r e f . 1 9 ) . In a r e a c t i o n mixture c o n t a i n i n g 30 mM sodium a c e t a t e (pH 4 . 5 ) , 50 mM NaCl , and 1 mM ZnSO, , 500 pg of t R N A ( 1 mg/ml) were cleaved with 25 u n i t s Mung bean n u c l e a s e (50 units/mg t R N A ) a t 37°C f o r 15 minutes. Phenol e x t r a c t i o n and ethanol p r e c i p i t a t i o n were performed a s d e s c r i b e d above. The cleavage fragments were then p u r i f i e d by PAGE ( d e s c r i b e d below).

5.2.3

Polvacrvlamide Gel ElectroDhoresis T r a n s f e r RNA fragments were p u r i f i e d by d e n a t u r i n g PAGE. The a n a l y t i c a l g e l s were 14 cm wide, 16 cm long, and 0.75 mm t h i c k , and were prepared a s follows: To 20 ml of gel s o l u t i o n [15% acrylamide, 0.75% bis-acrylamide, 8 M urea, 100 mM T r i s - b o r a t e (pH 8 . 3 ) , and 2 . 5 mM EDTA] was added 200 p1 of 10% ammonium p e r s u l f a t e and 20 p1 of TEMED t o s t a r t t h e polymerization p r o c e s s . This s o l u t i o n was poured t o the top of t h e g l a s s p l a t e s and allowed t o polymerize f o r one hour with a 15 well sample comb i n s e r t e d approximately 1 cm. The comb was removed and the sample w e l l s were thoroughly r i n s e d with water. The gel was pre-run a t 800 v o l t s f o r one hour i n a running b u f f e r of 100 mM T r i s - b o r a t e (pH 8 . 3 ) and 2.5 mM EDTA without c o o l i n g . The t R N A was d i s s o l v e d i n a sample b u f f e r of 8 M urea, 15% s u c r o s e , and 0.05% bromophenol b l u e and xylene cyanol Five pl of sample were loaded per we1 1 . The gel was run a t 800 v o l t s u n t i l the xylene cyanol marker was approximately 2 cm from the bottom of the gel ( c a . 3 h o u r s ) . The

.

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gel was removed from t h e g l a s s p l a t e s and s t a i n e d f o r 5 minutes i n a s o l u t i o n o f 0.2% methylene blue, 3.9% sodium a c e t a t e , and 2% acetic acid. The g e l was then d e s t a i n e d w i t h s e v e r a l changes o f water. A f t e r t h e bands were i d e n t i f i e d , t h e y were e x c i s e d and c u t 5 The t R N A was then e l u t e d and r e c o v e r i n t o small p i e c e s ( ~ mm2). ed by a m o d i f i c a t i o n o f t h e method o f Maxam and G i l b e r t ( r e f . 20). The g e l s l i c e s were suspended i n j u s t enough e l u t i o n b u f f e r (500 mM ammonium acetate, 10 mM MgCl,, 0.1% SDS, and 100 pM EDTA) t o cover t h e s l i c e s . T h i s suspension was r o c k e d on a r o c k e r p l a t f o r m The f o r 12-18 hours t o e l u t e t h e t R N A from t h e g e l s l i c e s . e l u t i o n b u f f e r was then p i p e t t e d o f f and t h e t R N A was p r e c i p i t a t e d w i t h 3 volumes o f ethanol and s t o r e d a t minus 20°C u n t i l i t was needed. 5.2.4 E n d - l a b e l l i n a RNA 5.2.4.1 5' End-labellina with r - P Z P l A T P T,-polynucleotide k i n a s e was used t o l a b e l t h e 5 ' end o f t R N A fragments w i t h ["PI u s i n g 7-[32P]ATP f o l l o w i n g methods s i m i l a r t o those o f D o n i s - K e l l e r e t a l . ( r e f . 21) and Cameron e t a l . ( r e f . 22). T h i s procedure ( a l o n g w i t h nuclease P, d i g e s t i o n and 2D-TLC, see below) was used t o i d e n t i f y t h e 5 ' n u c l e o t i d e o f t h e t R N A fragments c r e a t e d by enzymatic cleavage (see above). The r e a c t i o n was c a r r i e d o u t a t e i t h e r pH 6.9 o r pH 7.6 depending on t h e s t a t e o f the 5' nucleotide. I f t h e 5 ' n u c l e o t i d e had a t e r m i n a l phosphate group, t h e r e a c t i o n was c a r r i e d o u t a t pH 619. A t t h i s pH, t h e enzyme t r a n s f e r s t h e 5 ' t e r m i n a l phosphate o f t h e RNA t o ADP p r e s e n t i n t h e r e a c t i o n m i x t u r e . Since t h e pH i s , 6.9, t h e k i n a s e a c t i v i t y o f t h e enzyme a l s o a c t s t o r e p l a c e t h e removed phosphate u s i n g t h e T - [ ~ ~ P ] A T P p r e s e n t ( r e f . 23). Therefore, t h i s r e a c t i o n served t o exchange t h e 5 ' t e r m i n a l phosphate o f t h e RNA fragment w i t h a [32P] l a b e l . When t h e RNA t o be l a b e l l e d had a t e r m i n a l h y d r o x y l group, t h e l a b e l l i n g r e a c t i o n was c a r r i e d o u t a t pH 7.6 where t h e enzyme a c t s e x c l u s i v e l y as a k i n a s e ( r e f . 22). I n t h i s instance, the r e a c t i o n was r u n i n t h e same r e a c t i o n m i x t u r e w i t h t h e e x c e p t i o n of t h e pH o f t h e b u f f e r . Ethanol p r e c i p i t a t e d RNA samples (0.050.2 u n i t s ) were c o l l e c t e d by c e n t r i f u g a t i o n a t 12,400 x g f o r 15 minutes i n a m i c r o c e n t r i f u g e a t 4°C. The e t h a n o l was decanted

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and the RNA samples were dried under a vacuum. The RNA samples mM imidazole, 10 mM MgCl,, 5 mM d i t h i o t h r e i t o l (DTT), 0.1 mM EDTA, 30 pM A D P , 10 pCi T - [ ~ ~ P ] A T P (>3,000 Ci/mmol), and 1 u n i t of T,polynucleotide kinase. This mixture was incubated a t 37°C f o r 2 hours. The reaction was stopped by adding 10 pl of electrophore s i s sample buffer. Labelled RNA was then purified by PAGE, eluted, and digested with nuclease P, as described below. The labelled nucleotide was then i d e n t i f i e d by 2D-TLC as a l s o desc r i bed be1 ow. 5.2.4.2 3 ' End-Labellinq With 5'-r32plDCD T,-polynucleotide kinase and T - [ ~ ~ P ] A T P were used t o add a [''PI label on the 5 ' s i d e of cytidine 3 ' monophosphate (Cp). Following the method of Keith ( r e f . 24), 100 pCi of 7-[32P]ATP (>3,000 Ci/mmole) were dried under a vacuum in an Eppendorf microcentrifuge tube. This ATP was suspended in 20 p1 o f a reaction mixture containing 50 mM Tris-HC1 (pH 7.6), 10 mM MgCl,, 10 mM DTT, 50 pg BSA/ml , 6 mM mononucleotide Cp, and 5 u n i t s of T,polynucleotide kinase. The reaction mixture was incubated a t 37°C f o r 1 hour, and the reaction was then stopped by heating t o 65°C f o r 3 minutes. The 5'-[32P]pCp was stored a t minus 20°C until i t was used. T,-RNA l i g a s e was used t o label the 3 ' end of RNA fragments with the 5'[32P]pCp as described by Bruce and Uhlenbeck ( r e f . 25). This procedure (along with ribonuclease T, digestion and 2D-TLCr see below) was used t o i d e n t i f y the 3 ' nucleotide of the t R N A fragments generated by enzymatic cleavage. RNA, which had been dephosphorylated with T,-polynucleotide kinase a t pH 6.0 (described below) , was ethanol precipitated, collected by centrifugation, and dried under vacuum. To each dried t R N A sample, a 10 p1 reaction mixture was added which contained 50 mM HEPES buffer (pH 7 . 6 ) , 125 pM ATP, 20 mM MgCl,, 3.3 mM DTT, 10 pg B S A / m l , 50 pCi 5'-[3*P]pCp, and 1.5 u n i t s of T,-RNA l i g a s e . The reaction mixture was incubated a t 4°C overnight and the reaction was stopped by adding 10 p l of electrophoresis sample buffer. The fragments were purified by PAGE a n d eluted as described above. After hydrolysis with ribonuclease T, (described below), the 3 ' - labelled nucleotides were then determined by 2D-TLC as a l s o described below.

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5.2.5 H v d r o l v s i s o f t R N A 5.2.5.1 Nuclease P, Nuclease P 1 from P e n i c i l l i u m c i t r i n u m was used t o d i g e s t t R N A fragments t o t h e 5 ' monophosphates f o l l o w i n g a m o d i f i c a t i o n o f t h e D r i e d t R N A samples were method o f S i l b e r k l a n g e t a l . ( r e f . 26). suspended i n 10 p1 o f a r e a c t i o n m i x t u r e which was 50 mM i n amThe monium a c e t a t e (pH 5.3) and c o n t a i n e d 1 pg o f nuclease P, r e a c t i o n m i x t u r e was incubated a t 37°C f o r 2 hours a f t e r which i t was e i t h e r f r o z e n a t minus 20°C f o r l a t e r use o r was a p p l i e d d i r e c t l y t o a TLC sheet f o r r e s o l u t i o n o f t h e n u c l e o s i d e monophosphates (described below).

.

R i bonucl ease T, 5.2.5.2 Ribonuclease T, from A . o r y z a e was used t o d i g e s t t R N A f r a g ments t o t h e 3 ' monophosphates a c c o r d i n g t o a m o d i f i c a t i o n o f t h e method o f N i s h i k u r a and De R o b e r t i s ( r e f . 27). D r i e d t R N A samples were suspended i n 10 p1 o f a r e a c t i o n m i x t u r e which was 50 mM i n HEPES b u f f e r (pH 4.5) and contained 0 . 1 u n i t o f r i b o n u c l e a s e T,. The r e a c t i o n m i x t u r e was incubated a t 37°C f o r 2 hours a f t e r which i t was e i t h e r f r o z e n a t minus 20°C f o r l a t e r use o r was a p p l i e d d i r e c t l y t o a TLC sheet f o r r e s o l u t i o n o f t h e n u c l e o s i d e monophosphates (described below). I n t h e case o f r i bonuclease T, d i g e s t i o n , i t should be noted t h a t 2D-TLC problems can be encountered w i t h t h e f o r m a t i o n o f 2 ' - 3 ' c y c l i c mononucleotides, b u t such problems can be overcome by t r e a t m e n t under a c i d c o n d i t i o n s ( r e f . 28). 5.2.6

Two-Dimensional Thin Laver ChromatoaraDhv Two-dimensional-TLC was c a r r i e d o u t a c c o r d i n g t o t h e p r o t o c o l o f Nishimura ( r e f . 2 9 ) . A f t e r digestion o f the tRNA t o e i t h e r the 5 ' o r 3 ' monophosphates w i t h nuclease P, o r r i b o n u c l e a s e T,, r e s p e c t i v e l y , t h e r e a c t i o n m i x t u r e was s p o t t e d o n t o t h e c o r n e r o f a TLC square (6.7 x 6.7 cm) o f 0 . 1 mm c e l l u l o s e pre-coated on plastic. Each sample was s p o t t e d 2 pl a t a t i m e and d r i e d a f t e r each a p p l i c a t i o n . A f t e r l o a d i n g a l l o f t h e i n d i v i d u a l sample, 2 p1 o f each o f t h e u n l a b e l l e d monophosphate standards ( s a t u r a t e d s o l u t i o n s ) were s p o t t e d on t h e square. The monophosphates were then r u n i n t h e f i r s t dimension [66% i s o b u t y r i c a c i d : 1%ammonia: 33% water] u n t i l t h e s o l v e n t f r o n t was near t h e t o p o f t h e TLC sheet (ca. 1 hour). The sheet was then removed and a l l o w e d t o a i r

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dry overnight. The second dimension [18% HC1: 68% i s o p r o p a n o l : 14% water] was r u n u n t i l t h e s o l v e n t f r o n t was n e a r t h e t o p o f t h e The sheet was a g a i n a l l o w e d t o a i r d r y and sheet ( c a . 2 hours). The s p o t s c o r r e s p o n d i n g t o was then i l l u m i n a t e d w i t h a UV lamp. t h e u n l a b e l l e d standards were marked and t h e squares were t h e n s u b j e c t e d t o autoradiography. A f t e r development o f t h e a u t o r a d i o graph, t h e r a d i o a c t i v e spots were i d e n t i f i e d by comparison w i t h t h e standards. Q u a n t i t a t i n g t h e degree o f a p a r t i c u l a r m o d i f i c a t i o n can then be accomplished by s c r a p i n g and e x t r a c t i n g s p o t s o f i n t e r e s t and s u b j e c t i n g t h e s e t o Cerenkov c o u n t i n g . By m o n i t o r i n g t h e t i m e course of t h e r e a c t i o n , t h e disappearance o f t h e s u b s t r a t e and appearance o f t h e corresponding p r o d u c t can be f o l l o w e d . 5.2.7

Periodate Oxidation Sodium m e t a p e r i o d a t e and l y s i n e were used a c c o r d i n g t o t h e method o f K e i t h and Gilham ( r e f . 30) t o c l e a v e t h e base f r o m t h e 3 ' end o f t R N A fragments t o generate a 3 ' phosphate. T h i s was done t o b l o c k t h a t end o f t h e fragment f r o m r e a c t i o n i n t h e subsequent l i g a t i o n r e a c t i o n . The t R N A fragment t o be m o d i f i e d was suspended i n 300 p l w a t e r i n a 1.5 m l Eppendorf t u b e and covered w i t h f o i l . Sodium m e t a p e r i o d a t e (30 p l , 100 mM; f r e s h l y prepared) was added Rhamnose (4 p l , and i n c u b a t e d f o r 60 minutes i n t h e d a r k a t 4°C. 1 M) was t h e n added and i n c u b a t e d f o r 20 minutes a t room temperat u r e t o i n a c t i v a t e t h e excess p e r i o d a t e . L y s i n e (100 p l , 1 M, pH 8.9) was t h e n added and i n c u b a t e d 45 minutes a t 45°C t o c l e a v e t h e 3 ' base from t h e tRNA fragment. The fragment was t h e n e t h a n o l precipitated. 5.2.8

5 ' PhosDhorvlation T, - p o l y n u c l e o t i d e k i n a s e and ATP were used t o p h o s p h o r y l a t e t h e 5 ' h y d r o x y l group o f t R N A fragments u s i n g t h e methods o f 22). The D o n i s - K e l l e r e t a l . ( r e f . 21) and Cameron e t a l . ( r e f . t R N A fragment t o be phosphorylated was suspended i n 10 p1 o f a r e a c t i o n m i x t u r e which c o n t a i n e d 50 mM Tris-HC1 (pH 7.6), 10 mM MgCl,, 10 mM DTT, 50 pg BSA/ml, and 10 mM ATP. F i v e u n i t s o f T,p o l y n u c l e o t i d e k i n a s e were added and t h e r e a c t i o n m i x t u r e was i n c u b a t e d f o r 2 hours a t 37°C. The r e a c t i o n was stopped by h e a t i n g t h e m i x t u r e t o 80°C f o r 2 minutes, a f t e r which t h e t R N A was p r e c i p i t a t e d (as above).

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DePhosPhorvl a t i on Dephosphorylation o f 3' RNA phosphate was accomplished u s i n g T,-polynucleotide k i n a s e a t pH 6.0. Using t h e method o f Carbon e t a 7 . ( r e f . 31), t h e t R N A fragment was suspended i n a s m a l l amount o f b u f f e r c o n t a i n i n g 100 mM i m i d a z o l e (pH 6.0), 10 mM MgCl,, 10 mM 2-mercaptoethanol , and 4 mM ATP. T, - p o l y n u c l e o t i d e k i n a s e was then added t o a f i n a l c o n c e n t r a t i o n o f 1 u n i t l p g tRNA, and t h e m i x t u r e was incubated f o r 2 hours a t 37°C. The r e a c t i o n was stopped by h e a t i n g a t 80°C f o r 2 minutes. The r e a c t i o n volume was then brought t o 100 p1 w i t h water and was e x t r a c t e d w i t h an equal volume o f phenol s a t u r a t e d w i t h 0.1 M sodium a c e t a t e (pH 4.5). The phenol was r e e x t r a c t e d w i t h 100 p1 o f water and t h e aqueous phases were combined b e f o r e ethanol p r e c i p i t a t i o n . The t R N A was then washed several times w i t h 70% e t h a n o l t o remove t r a c e s o f phenol which c o u l d i n t e r f e r e w i t h subsequent r e a c t i o n s .

5.2.9

5.2.10 R e n a t u r a t i o n and Reannealina o f t R N A Denatured tRNA may be r e t u r n e d t o i t s n a t i v e c o n f o r m a t i o n by h e a t i n g i t t o a h i g h temperature and t h e n a l l o w i n g i t t o cool s l o w l y . I t i s a l s o p o s s i b l e t o reanneal two t R N A " h a l f " molecules by t h i s method so t h e y may combine t o form an " a n t i c o d o n - d e p r i v e d " t R N A ( r e f . 32). I n t h i s case, equal q u a n t i t i e s o f t h e two h a l f molecules were suspended i n 50 mM Tris-HC1 (pH 7.6) and 20 mM MgC1, t o a c o n c e n t r a t i o n o f a p p r o x i m a t e l y 1 A,, u n i t / 1 0 0 p1 o f solution. T h i s s o l u t i o n was heated t o 70°C f o r 5 m i n u t e s i n a water b a t h a f t e r which t h e h e a t i n g u n i t was t u r n e d o f f t o a l l o w The t R N A was t h e n removed f r o m t h e w a t e r t o cool s l o w l y t o 45". t h e water b a t h and allowed t o cool a t room temperature f o r an a d d i t i o n a l 30 minutes. The t R N A was then ethanol p r e c i p i t a t e d and k e p t a t minus 20°C u n t i l i t was used. Radio1 a b e l l i n a 01 i a o n u c l e o t i d e s D i n u c l e o t i d e monophosphates t o be 1 i g a t e d i n t o r e c o n s t r u c t e d t R N A were 5' e n d - l a b e l l e d w i t h T,-polynucleotide k i n a s e and 7[32P]ATP ( r e f . 22). T h i s was done t o l a b e l t h e n u c l e o t i d e o f i n t e r e s t i n t h e t R N A a n t i c o d o n so i t c o u l d be m o n i t o r e d d u r i n g subsequent m o d i f i c a t i o n s t u d i e s i n v i t r o . A f t e r d r y i n g 100 p C i o f 7-[32P]ATP (>3,000 Ci/mmol) under a vacuum, i t was resuspended i n 3 p l o f r e a c t i o n b u f f e r [lo0 mM Tris-HC1 (pH 7.6), 20 mM MgCl,, 20 mM DTT, and 100 pg B S A / m l ] and 2 p l o f d i n u c l e o t i d e monophosphate

5.2.11

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.

T, - p o l y n u c l e o t i d e k i n a s e was added t o a f i n a l concentra(60 pM) t i o n o f 150 u n i t s / m l o f r e a c t i o n volume and i n c u b a t e d f o r 2 hours a t 37°C. The r e a c t i o n was t e r m i n a t e d by h e a t i n g t o 80°C f o r 2 minutes and t h e r e a c t i o n m i x t u r e was used i m m e d i a t e l y i n t h e subsequent 1 i g a t i o n r e a c t i o n ( d e s c r i b e d below).

5.2.12 Liaation F o l l o w i n g procedures s i m i l a r t o those o f England and Uhlenbeck ( r e f . 33), T,-RNA l i g a s e was used t o c o v a l e n t l y j o i n 2 t R N A fragments. T h i s l i g a s e enzyme r e q u i r e s a 3 ' phosphate and a 5 ' h y d r o x y l group f o r l i g a t i o n t o occur. The reannealed t R N A was suspended i n 2 p1 of b u f f e r [50 mM HEPES (pH 7.6), 120 pM ATP, 20 mM MgCl,, 3.3 mM DTT, and 10 p g B S A / m l ] and 6 p1 o f d i n u c l e o t i d e monophosphate l a b e l 1i n g r e a c t i o n m i x t u r e ( d e s c r i b e d above). Two u n i t s o f T,-RNA l i g a s e were added and i n c u b a t e d a t 4°C f o r 18-24 hours. The r e a c t i o n was stopped by a d d i t i o n o f 10 p l o f sample b u f f e r f o r e l e c t r o p h o r e s i s (8 M urea, 30% sucrose, 0.1% bromophenol blue, and 0.1% x y l e n e c y a n o l ) . The l i g a t e d t R N A was t h e n p u r i f i e d by PAGE. 5.2.13 I n V i t r o M o d i f i c a t i o n o f t R N A Human promyel o c y t i c 1eukerni a (HL-60) c e l l s grown in suspens i o n c u l t u r e were harvested b y low-speed c e n t r i f u g a t i o n i n 50 m l c o n i c a l c e n t r i f u g e tubes a t 200 x g f o r 10 m i n u t e s ( r e f . 34). C e l l p e l 1e t s were combined a f t e r resuspendi ng i n ice-col d phosphate b u f f e r e d s a l i n e (PBS). The c e l l s were t h e n washed one a d d i t i o n a l t i m e w i t h c o l d PBS and c o l l e c t e d by c e n t r i f u g a t i o n . The c e l l s were resuspended i n 5 m l o f c o l d B u f f e r A [ l o mM T r i s HC1 (pH 7.4), 10 mM MgCl,, 1 mM EDTA, 0.5 mM DTT, and 10% g l y c e r o l ] and homogenized i n an ice-col d, ground-gl ass t i s s u e g r i n d e r u n t i l most o f t h e c e l l s were broken (as determined by t r y p a n b l u e exclusion). The homogenate was c e n t r i f u g e d a t 500 x g f o r 10 minutes, and t h e r e s u l t i n g s u p e r n a t a n t was t h e n c e n t r i f u g e d a t 18,000 x g f o r 30 minutes. The 18,000 x g s u p e r n a t a n t was used immediately as t h e source o f tRNA-hypoxanthine r i b o s y l t r a n s f e r a s e . The assay was a m o d i f i c a t i o n o f t h e procedure o f E l l i o t t and Trewyn ( r e f . 34). The standard r e a c t i o n m i x t u r e c o n t a i n e d 20 mM Tris-HC1 (pH 7.4), 90 mM KC1, 3 mM MgCl,, 0.3 mM EDTA, 0.5 mM DTT, 10 pM hypoxanthine, a l o n g w i t h r e c o n s t r u c t e d [32P]-1abe11ed y e a s t

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and 100 p1 o f enzyme e x t r a c t i n a t o t a l volume o f 200 p l . I n c u b a t i o n was a t 37OC f o r 0-90 minutes. The t R N A was p r e c i p i t a t e d w i t h sodium a c e t a t e and ethanol as d e s c r i b e d above and was c o l l e c t e d by c e n t r i f u g a t i o n a t 12,400 x g f o r 15 m i n u t e s i n a m i c r o c e n t r i f u g e , a f t e r which t h e e t h a n o l was decanted and t h e t R N A was d r i e d under a vacuum. The t R N A was d i g e s t e d w i t h 1 pg o f nuclease P, from P . c i t r i n u m i n 10 p1 o f 50 mM ammonium a c e t a t e The l a b e l l e d 5 ' monophosb u f f e r (pH 5.3) f o r 2 hours a t 37°C. phates were then i d e n t i f i e d by PD-TLC as d e s c r i b e d above. tRNAAla,

5.3

RESULTS AND DISCUSSION The scheme f o r r e c o n s t r u c t i o n o f y e a s t t R N A A L a i s o u t l i n e d i n F i g u r e 5.1. The o b j e c t i v e was t o remove i n o s i n e f r o m t h e f i r s t p o s i t i o n o f t h e a n t i c o d o n and t o r e p l a c e i t w i t h adenosine l a b e l l e d on i t s 5 ' s i d e w i t h a ["PI molecule. Adenosine was s e l e c t e d

u

U

P I1 I G C

(A1

I81

ICI

pericdate

>-

>-

TI-kinase F UOH

(D)

oxidation F

I1

I1 PC

HOC

I1 PC

reanneal

(E)

v

U

P 32P-ATP TI-kinase

u A'

F i g u r e 5.1

U

F I1 0

C

Schematic procedure f o r t h e c o n s t r u c t i o n o f t h e AGC

B155

Figure 5.1 (continued) "anticodon-substituted'' yeast t R N A A L a . The t R N A was reconstructed v i a enzymatic cleavage and l i g a t i o n as

in

the f i r s t t R N A was

inosine; F , pseudouridine. for the replacement because a l l t R N A genes sequenced t o date have adenosine in the position corresponding t o inosine in the mature t R N A ( r e f . 35), and i n v i t r o studies have indicated a hypoxanthine for adenine exchange reaction t o generate inosine in the mature macromolecule ( r e f . 34). The f i r s t s t e p i n the reconstruction was the enzymatic cleavage of the t R N A t o generate the appropriate half molecules (Figure 5.1). Mung bean nuclease was used t o generate the 5' half molecule ( A ) , w i t h the t R N A fragments being purified by PAGE in 15% acrylamide with 8 M urea. A typical

Figure 5.2

Polyacrylamide gel electrophoresis of yeast t R N A A L a

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Figure 5.2 ( c o n t i n u e d ) " h a l f " molecules generated w i t h Mung bean nucl ease. The ha1 f molecules, generated by enzymatic cleavage w i t h Mung bean nuclease (50 u n i t s / m g tRNA), were s e arated by electrophoresis on a 15% polyacrylamide gel containing M urea.

rp

c

Figure 5.3 Polyacrylamide gel e l e c t r o horesis of e a s t t R N A A L a "half" molecules generated w i t h ri bonuc ease T1. TKe half molecul e s , generated by enzymatic cleavage w i t h ri bonucl ease T1 (100 units/mg t R N A , were separated by electrophoresis on a 15% polyacrylamide ge containing 8 M urea.

t

cleavage pattern is shown in Figure 5.2. Ribonuclease T, was used t o generate the 3 ' half molecule indicated as (B) in Figure 5.1. Figure 5.3 represents a typical T, cleavage pattern a f t e r separation by PAGE, and i t is c l e a r t h a t an excellent yield was obtained. The Mung bean nuclease r e s u l t (Figure 5.2) points out one o f the d i f f i c u l t i e s associated with recombinant RNA technology, where preparation o f the necessary RNA fragments re1 i e s on nucleases with only nucl eoti de and/or gross s t r u c t u r e speci f i ci t y ; t h e r e a r e

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no RNA-speci f i c " r e s t r i c t i o n - t y p e " enzymes a v a i l a b l e . Therefore, t o p r e p a r e l a r g e fragments, i t i s necessary t o p e r f o r m h y d r o l y s e s under l i m i t i n g c o n d i t i o n s which o f t e n r e s u l t i n l o w y i e l d s . perhaps due t o Ribonuclease T, i s an e x c e p t i o n ( F i g u r e 5.3), greater s p e c i f i c i t y o r purity. While t h e y i e l d o f t h e 5 ' h a l f molecule ( A ) was n o t h i g h ( F i g u r e 5.2), t h e use o f Mung bean nuclease was s t i l l p r e f e r a b l e t o an a l t e r n a t i v e method (two c y c l e s of p e r i o d a t e o x i d a t i o n / a l k a l i n e phosphatase t r e a t m e n t o f t h e T, 5 ' h a l f molecule; see r e f . 16) where c o n t a m i n a t i n g nucleases, t h e need t o r e p l a c e t h e 5 ' phosphate p r i o r t o l i g a t i o n , and l o s s o f t h e t e r m i n a l 5 ' phosphate become p r o b l e m a t i c . A f t e r e l u t i o n from t h e g e l , a small p o r t i o n o f each o f t h e fragments was 5 ' o r 3 ' e n d - l a b e l l e d t o determine t h e t e r m i n a l n u c l e o t i d e a t t h e cleavage s i t e . Using [32P]pCp and T,-RNA l i g a s e t o 3 ' end-label t h e Mung bean fragments, o r [32P]ATP and T,-polyn u c l e o t i d e k i n a s e t o 5 ' end-label t h e T, fragments, t h e fragments were again p u r i f i e d by PAGE. These l a b e l l e d fragments were e l u t e d and h y d r o l y z e d c o m p l e t e l y t o t h e monophosphate l e v e l u s i n g e i t h e r f o r 3 ' monophosphates o r nuclease P, f o r 5 ' r i b o n u c l e a s e T, monophosphates. The l a b e l l e d monophosphates were t h e n i d e n t i f i e d by PD-TLC u s i n g cochromatography w i t h n u c l e o s i d e monophosphate standards. As expected, t h e t o p band i n t h e bottom s e t o f Mung bean fragments (band A, F i g u r e 5.2) y i e l d e d u r i d i n e as t h e o n l y l a b e l l e d monophosphate i n d i c a t i n g i t was t h e d e s i r e d fragment [ ( A ) i n F i g u r e 5.11 f o r r e c o n s t r u c t i o n . The fragment j u s t below (A) i n t h i s g e l a l s o y i e l d e d u r i d i n e as t h e 3 ' end n u c l e o t i d e , b u t t h i s fragment corresponds t o cleavage a f t e r u r i d i n e i n p o s i t i o n 32 and n o t 33 as needed. The t o p band i n t h e T, d i g e s t i o n p a t t e r n (band B , F i g u r e 5.3) y i e l d e d a c y t i d i n e as t h e o n l y l a b e l l e d monophosphate i n d i c a t i n g i t was t h e c o r r e c t fragment [(B) i n F i g u r e 10.11. A f t e r i d e n t i f i c a t i o n , t h e 3 ' h a l f molecule was prepared f o r t h e l i g a t i o n r e a c t i o n as shown i n F i g u r e 5.1. The 5 ' end o f f r a g ment B was phosphoryl a t e d w i t h ATP and T, - p o l y n u c l e o t i de k i nase t o The n e x t s t e p y i e l d a r e a c t i v e end f o r t h e l i g a t i o n r e a c t i o n (C). was t o s h o r t e n t h e 3 ' end by one n u c l e o t i d e u s i n g t h e p e r i o d a t e o x i d a t i o n method ( D ) . The reason f o r t h i s n u c l e o t i d e removal was t o generate a 3 ' phosphate t o b l o c k t h i s end from b e i n g r e a c t i v e

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i n the final ligation reaction. By u s i n g the 3 ' phosphatase a c t i v i t y of T, -polynucl eoti de k i nase ( r e f . 22) r a t h e r than a1 kal i n e phosphatase, the 5 ' phosphate i s preserved. The two half molecules were then reanneal led t o y i e l d an "anticodon-deficient'' tRNA (E). The dinucleotide monophosphate ( F ) was prepared f o r l i g a t i o n by adding a 5'[32P] label using T,-polynucleotide k i n ase*, a f t e r which the anticodon-deficient t R N A ( E ) and labelled dinucleotide (G) were ligated u s i n g T,-RNA l i g a s e . This resulted i n an i n t a c t yeast t R N A A I a molecule being generated which cont a i ned a [' P] -1 abel 1 ed adenosine (the precursor of i nosi ne) i n the f i r s t position of the anticodon. The products of this l i g a tion reaction were separated by PAGE and visualized by autoradiography (Figure 5 . 4 ) . As can be seen i n Figure 5.4, performing a "one-step'' l i g a t i o n reaction ( i . e . , joining the dinucleotide t o the 5 ' and 3' h a l f molecules i n a s i n g l e reaction mixture) has the potenti a1 t o y i el d mu1 t i p l e products. The reconstructed t R N A containing a 7 nucleotide loop corresponding t o the i n s e r t i o n of a s i n g l e dinucleotide (indicated by the arrow i n Figure 5.4) was then eluted from the gel. The reason multiple products a r e generated i s t h a t more than one dinucleotide can be added by the ligase since the dinucleotide has a 5 ' phosphate and a 3 ' hydroxyl group. An a l t e r n a t i v e approach would be t o perform a "two-step" 1 igation reaction, whereby the dinucleotide would have a phosphate a t b o t h ends and would be ligated t o the reannealed 5 ' half Following another PAGE i s o l a t i o n s t e p (which molecule f i r s t . diminishes product y i e l d ) , the 3 ' phosphate would have t o be removed and another l i g a t i o n reaction run. The additional l i g a s e reaction also has the potential t o reduce the product y i e l d s i g n i f i c a n t l y , since T,-RNA ligase can a c t i n t h e reverse direct i o n as well ( r e f . 6 ) , i . e . , i t can a c t as a nuclease. Another benefit of the one-step ligation i s t h a t duplex RNA i s much more r e s i s t a n t t o nuclease digestion than s i n g l e stranded RNA, so product recovery generally increases s i g n i f i c a n t l y when more of the manipulations a r e carried o u t w i t h duplex material. Therefore, i n most cases, the one-step procedure i s preferable. Another valuable feature of T,-RNA l i g a s e not addressed i n *A PseT mutant T,-polynucleotide kinase l a c k i n g t h e 3 ' phosphatase a c t i v i t y ( r e f . 2 2 ) i s commercially a v a i l a b l e , and i t i s i n v a l u a b l e when 3 ' p h o s p h a t e r e m o v a l must b e p r e v e n t e d .

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Figure 5.4 Autoradiograph o f r e c o n s t r u c t e d y e a s t t R N A A t a . The p r o d u c t s o f t h e l i g a t i o n r e a c t i o n i n which t h e 5 ' 3 2 P ] - l a b e l l e d d i n u c l e o t i de *pApG was c o v a l e n t l y ins.erted 1 n t o t e reanneal ed "anticodonydeprived." t R N A were p u r i f i e d by d e n a t u r i n g PAGE. The tRNA and d i n u c l e o t i d e were J o i n e d u s i n T,-RNA li ase. The arrow i n d i c a t e s t h e r e c o n s t r u c t e d tRNAALaw i t % one l a b e l y e d d i n u c l e o t i d e i n s e r t e d i n t o t h e anticodon.

k

t h e experimental p r o t o c o l s o u t 1 ined is i t s broad s u b s t r a t e s p e c i f i c i t y f o r donor and a c c e p t o r molecules ( r e f s . 25, 33, 3 7 ) . T h i s a t t r i b u t e offers the possiblity o f incorporating modified or a1 t e r e d n u c l e o t i d e s i n t o s e l e c t e d s i t e s (discussed i n r e f . 13),

B160

and t h i s could be very useful f o r many s t u d i e s . Other methods currently avai 1 able f o r generating t R N A macromolecules do not o f f e r t h i s unique capability. Under most circumstances, the best y i e l d of anticodon reconstructed t R N A t h a t one could expect from the s e r i e s of procedures described above would be 0 . 1 t o 5% of the s t a r t i n g material, although recoveries as h i g h as 60% have been reported f o r another t R N A species ( r e f . 10). However, w i t h the h i g h s p e c i f i c a c t i v i t y of the [ 3 z P ] u t i l i z e d , s u f f i c i e n t material can be obtained t o perform numerous experiments i n v i t r o or i n v i v o . I t should be noted, however, t h a t similar manipulations w i t h other portions of the macromolecule (outside the anticodon loop) would l i k e l y generate even 1 ower y i e l d s . The reconstructed yeast t R N A A L a was used in an i n v i t r o assay f o r tRNA-hypoxanthine ri bosyl transferase a c t i v i t y . The reconstructed t R N A was incubated f o r 60 minutes with hypoxanthine and a crude cytosol i c enzyme preparation from HL-60 promyel ocyti c 1 eukemi a c e l l s . Transfer RNA was then ethanol preci p i t a t e d and digested t o the 5 ' monophosphates with nuclease P , . The monophosphates were separated by 2D-TLC and the labelled nucleotides detected by autoradiography. As shown i n Figure 5.5, the control (unreacted) t R N A yielded only 1 abel led adenosine monophosphate [5(A)], whereas the modified reconstructed t R N A yielded predominately inosine monophosphate [5(B)]. In separate experiments, a lower conversion of A,, t o I,, occurred when the reaction time was shortened t o 30 minutes, and similar r e s u l t s were obtained using an enzyme e x t r a c t from human T-lymphoblasts and a reconstructed alanine t R N A from E . c 0 7 i (data n o t presented). Using methodology s i m i l a r t o t h a t described above f o r yeast t R N A A L a a variety of pure tRNAs have been subjected t o s i t e directed reconstruction, and published studies i n this area a r e summarized i n Table 5.1. While only 13 tRNAs from y e a s t and E . c o l i have been manipulated i n this fashion, more than 150 variant product tRNAs have been generated. In f a c t , when multiple nucleot i d e s a r e removed from the anticodon, or some other s i t e i n the t R N A , i t i s possible t o r e i n s e r t numerous combinations of nucleot i d e s , and t h a t i s a d i s t i n c t advantage over other a l t e r n a t i v e methods ( e . g . , individual mutant i s o l a t i o n by site-directed mutagenesis a t the level of a t R N A gene).

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To date, most analyses of modified nucleoside biosynthesis in the anticodon loop of reconstructed tRNAs have been performed i n v i v o (in X e n o p u s l a e v i s oocytes; reviewed in refs. 14, 38), but as described above (also see ref. 28), analyses i n v i t r o are plausible as well. The main advantage o f the i n v i v o (oocyte) system over an i n v i t r o system is that it allows one to examine modifications where several enzymes are probably involved; e . g . , the

Figure 5.5 Autoradiograph of 5 ' nucl eosi de monophos hates separated by two-dimensional thin layer chromatography. econstructed yeast tRNAAla was used as a substrate for tRNA-hypoxanthine ribosyltransferase in an i n v i t r o assay. The reconstructed tRNA was incubated at 37°C for 60 minutes with an enzyme preparatien from human HL-60 cells as described in "Materials and Methods Transfer RNA was collected by ethanol preci itation and hydrolyzei mono hosphates. The with nuclease P, from P. c i t r i n u m to the nucleoside monophosphates were separated by 2D-.LC according to the procedure of Nishimura (ref. 27), after which the labelled nucleotide was determined by autoradiography. Identification of the 1 abel led 5' nucl eoside monophosphate was made by compari son with known standards AMP and IMP). A) Control (unreacted) tRNAAta. B) Modified tR AAla. hypermodified nucleosides found in positions 34 and 37 of tRNA. Since the biosynthesis of these unusual modifications could require mu1 ti-enzyme complexes to form, defining the reaction conditions i n v i t r o could be problematic. Even in less complex cases such as the inosine modification described above, identifying the reaction conditions can be difficult, since in the inosine case, the modification was assumed t o occur by a macromolecular deamination until the insertion of preformed hypoxanthine was

ti

8

k

P

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demonstrated ( r e f . 34). A summary o f t h e anticodon m o d i f i c a t i o n s and m o d i f i c a t i o n enzymes e v a l u a t e d u s i n g recombinant RNA techniques i s presented i n Table 5.2. While t h e d e t a i l s o f any p a r t i c u l a r r e c o n s t r u c t i o n necessary f o r such s t u d i e s may vary, t h e o v e r r i d i n g p r i n c i p l e s a r e g e n e r a l l y t h e same, i . e . , t o make use o f n u c l e o t i d e - s p e c i f i c nucl eases a c t i n g on s i n g l e stranded RNA t o generate a n t i codondef i c i e n t macromolecules l a c k i n g one o r more n u c l e o t i d e s and then l i g a t i n g a monomer o r o l i g o m e r i n t o t h e a n t i c o d o n w i t h a ["PI l a b e l a d j a c e n t t o t h e n u c l e o s i d e o f i n t e r e s t . Under t h e b e s t o f circumstances, merely opening t h e a n t i c o d o n w i t h a s i n g l e , h i g h l y speci f i c n u c l ease ( e . g . , 1, ) , rep1 a c i ng t h e phosphate w i t h [' P I , and l i g a t i n g may a l l o w a d e s i r e d a n a l y s i s t o be performed f o l l o w However, numerous i n g p u r i f i c a t i o n o f t h e p r o d u c t by PAGE. d e t a i l s about enzyme s p e c i f i c i t y can be gained by t h e s y s t e m a t i c replacement o f n e i g h b o r i n g n u c l e o t i d e s d u r i n g t h e r e c o n s t r u c t i o n ( r e f . 14), so such p o s s i b i l i t i e s should n o t be overlooked. The i n f l u e n c e o f anticodon sequence on t h e p o t e n t i a l o f a c h i m e r i c t R N A t o s e r v e as a s u b s t r a t e f o r a g i v e n m o d i f i c a t i o n has been examined i n many cases c i t e d i n Table 5.2. The r e s u l t s i n d i c a t e t h a t most o f t h e m o d i f i c a t i o n enzymes r e c o g n i z e n o t o n l y s p e c i f i c n u c l e o t i d e s w i t h i n t h e anticodon loop, b u t a l s o more general f e a t u r e s o f t h e t R N A molecule. Thus, t h e r e c o g n i t i o n of a t R N A by a g i v e n m o d i f i c a t i o n enzyme m i g h t be as complex as t h a t f o r r e c o g n i t i o n by t h e cognate aminoacyl-tRNA synthetase. No general r u l e s can y e t be d e f i n e d t h a t would a l l o w one t o p r e d i c t a g i v e n s p e c i f i c i t y f o r t h e v a r i o u s m o d i f i c a t i o n enzymes s t u d i e d t o date. FUTURE PERSPECTIVES The method d e s c r i b e d here f o r t h e c o n s t r u c t i o n o f c h i m e r i c tRNAs has d e a l t w i t h t h e recombination o f l a r g e fragments d e r i v e d from n a t u r a l t R N A macromolecules. A t t h e p r e s e n t time, t h i s method appears t o be more u s e f u l t h a n step-wise chemical methods ( i n c l u d i n g l i g a t i o n o f s h o r t s y n t h e t i c fragments), s i n c e t h e chemical techniques a r e e x c e e d i n g l y t e d i o u s and t i m e consuming However, technology i n t h i s area i s e v o l v i n g ( r e f s . 69, 70). r a p i d l y , so i t may be p o s s i b l e t o f o r e s e e automated RNA syntheses i n t h e f u t u r e ( r e f . 71). T h i s w i l l be e s p e c i a l l y v a l u a b l e so l o n g as s e l e c t e d m o d i f i d e d nucleosides can be i n c o r p o r a t e d d u r i n g t h e 5.4

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TABLE 5 . 1 S t u d i e s Employing Recombinant RNA Techniques f o r t h e S i t e D i r e c t e d Rep1acement o f Nucl e o t i d e s i n tRNA1

t R N A (anticodon)

Source

tRNAA I a ( I G C ) tRNAArg (ICG) t R N A A S p(GUC)

Yeast Yeast Yeast

t R N A C y S (GCA) t RNAL (UAG) tRNAi (CAU) t R N A f t (CAU)

Yeast Yeast Yeast

t RNAP

(G, AA)

t RNAP h tRNAS tRNAT tR N A T p t RNAT y

(GAA) (IGA) (mo5 UGU) (C, GA) (G$A)

E. c o l i

Yeast E. c o l i

Yeast Yeast Yeast YpaSt

S i tez AC Loop AC Loop AC Loop T Stem/Loop AC Loop AC Loop AC Loop AC Loop AA Stem AC Loop AC Stem AC Loop AC Loop AC Loop AC Loop AC Loop D Loop

References

15,16,39 32,40,41 18,31,40-42 43 44 31 19 11,45-49 50,51 12,28,40,41,52-60 61 41,62 40,41 63 40,41 12,64-66 67,68

' T h e t a b l e does not include a complete reference l i s t o f a77 biochemical/biophysical studies performed w i t h t h e various c h i m e r i c t R N A s , s i n c e t h i s a s p e c t was r e v i e w e d r e c e n t l y ( r e f . 1 3 ) . See T a b l e 5 . 2 f o r a d d i t i o n a l information concerning a n t i codon m o d i f i c a t i o n .

2The stem and l o o p a b b r e v i a t i o n s f o r t h e t R N A s i t e s r e c o n s t r u c t e d anticodon; AA, aminoacyl; T, (ribo)thymia r e as f o l l o w s : AC, dine; D, d i h y d r o u r i d i n e . s y n t h e t i c procedure. U n t i l such time, RNA r e c o m b i n a t i o n employing T,RNA l i g a s e w i l l r e t a i n c o n s i d e r a b l e importance, s i n c e t h e s t e p w i s e chemical methods may n o t o f f e r a convenient way t o i n t r o d u c e h i g h l y m o d i f i e d n u c l e o t i d e s , o r t h e s i t e - s p e c i f i c ["PI label, i n t o t h e i n t e r i o r o f t h e macromolecule. While c e r t a i n d i f f i c u l t i e s s t i l l remain w i t h t h e recombinant RNA technology d e s c r i b e d here, t e c h n i c a l improvements appear t o be forthcoming. The problem o f o b t a i n i n g t R N A fragments i n good y i e l d due t o t h e l a c k o f a p p r o p r i a t e RNA r e s t r i c t i o n - t y p e enzymes

B164

may be overcome by DNA a s s i s t e d methods. For example, h y b r i d i z a t i o n w i t h DNA oligomers may a l l o w s e l e c t e d s i t e s i n t h e t R N A macromol e c u l e t o be exposed f o r n u c l ease c l eavage (reviewed i n r e f . 13). A l t e r n a t i v e l y , s y n t h e t i c DNA o l i g o m e r s may be used t o i n t e r f e r e w i t h t h e enzymatic cleavage o f t R N A i n u n d e s i r e d l o c a t i o n s ( r e f . 72). A technique t h a t on t h e s u r f a c e appears t o be a t t r a c t i v e f o r t R N A m o d i f i c a t i o n research i n v o l v e s t h e i n v i t r o t r a n s c r i p t i o n o f TABLE 5.2 M o d i f i c a t i o n Reactions and Enzymes Examined U s i n g Reconstructed tRNAs

Modification

Enzymati c A c t i v i t y

A,,

---->

I,,

G34

---->

434

434

---->

glYCOSY1-434

---->

2 ' -0-me-G,

---->

m l G,

tRNA-hypoxanthine r i bosyl t r a n s f e r a s e tRNA-guanine r i bosyl t r a n s f e r a s e tRNA-queuosi ne glycosyltransferase tRNA-guanosine,, 2'-O-methyl t r a n s f e r a s e tRNA-guanosi ne, N1 -methyl t r a n s f e r a s e u n c h a r a c t e r i zed r e a c t i o n sequence tRNA-adenosine,, N6 - t h r e o n y l carbamoyltransferase tRNA-adenosine,,

G,

,

G, m1 G,

---->

,

Y,

A,,

---->

PA3,

A,,

---->

i6A,,

,

References

32 ,42 18,31,40 31,40 41 28,54 28 46

46

N6-isopentenyltransferase U, U,

,

---->

mcm5U,,

---->

mcm5s2 U,

,

u n c h a r a c t e r i zed r e a c t i o n sequence u n c h a r a c t e r i zed r e a c t i o n sequence

18,32 18,32

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c l o n e d o r s y n t h e s i z e d t R N A genes u s i n g t h e SP6 o r 17 b a c t e r i o p h a g e RNA polymerase (discussed i n r e f . 13). E i t h e r t h e complete gene o r a fragment c o u l d be t r a n s c r i b e d , and i t s h o u l d be p o s s i b l e t o generate microgram o r even m i 11 igram amounts o f t h e t h e c h i m e r i c tRNA ( r e f . 7 ) . These types o f y i e l d s a r e o u t o f t h e r e a l m o f p o s s i b i l i t y using the anticodon reconstruction techniques described i n t h i s c h a p t e r . However, w i t h t h e i n v i t r o t r a n s c r i p t i o n method, t h e RNA p r o d u c t would be c o m p l e t e l y d e v o i d o f m o d i f i e d nucleosides, and t h i s c o u l d l e a d t o problems f o r c e r t a i n t y p e s o f studies. T r a n s f e r RNA i s unique by i t s h i g h degree o f m o d i f i c a t i o n (10-25% o f t h e n u c l e o s i d e s a r e m o d i f i e d i n e u k a r y o t i c t R N A ) , so t h e t o t a l l y unmodified macromolecule c o u l d be b i o l o g i c a l l y i r r e l e v e n t and/or v e r y u n s t a b l e i f used f o r m o d i f i c a t i o n s t u d i e s in vitro or in vivo. L i g a t i n g s y n t h e t i c (unmodified) h a l f molecul e s t o n a t u r a l ( m o d i f i e d ) h a l f molecules may o f f e r a compromise However, t o t a l l y u n m o d i f i e d t R N A P has approach i f necessary. been shown t o be a s u b s t r a t e f o r a m i n o a c y l a t i o n i n v i t r o ( r e f . 7 ) , so t h i s s u b j e c t i s s t i l l open t o c o n j e c t u r e . Although t h e d a t a p r e s e n t e d i n t h i s c h a p t e r d e a l t e x c l u s i v e l y w i t h t h e i n o s i n e m o d i f i c a t i o n r e a c t i o n i n v i t r o , s i m i l a r analyses Future have been performed i n X e n o p u s oocytes ( r e f s . 32, 42). s t u d i e s o f t R N A m o d i f i c a t i o n r e a c t i o n s , e s p e c i a l l y t h o s e generati n g t h e h y p e r m o d i f i e d n u c l e o s i d e s i n p o s i t i o n s 34 and 37, a r e l i k e l y t o employ p r e d o m i n a n t l y t h e i n v i v o system as w e l l , s i n c e t h e r e a c t i o n c o n d i t i o n s a r e p r e d e f i n e d by t h e c e l l (reviewed i n refs. 14, 38). The i n t e r p l a y between t h e v a r i o u s m o d i f i c a t i o n s i n t h e a n t i c o d o n l o o p o f tRNAs ( e . g . , t h o s e i n p o s i t i o n s 34 and 37) i s b e g i n n i n g t o be e l u c i d a t e d , and t h i s s h o u l d c o n t i n u e t o be a m a j o r area o f r e s e a r c h i n t h e f u t u r e . I t a l s o seems p r o b a b l e t h a t recombinant RNA technology w i l l remain a t t h e f o r e f r o n t as s t r u c t u r e - f u n c t i o n r e l a t i o n s h i p s i n v o l v i n g these complex m o d i f i cations are investigated. SUMMARY C u r r e n t know1 edge concerning t h e b i o s y n t h e s i s o f modi f ied nucleosides i n the anticodon loop o f eukaryotic tRNA i s l i m i t e d due t o t h e l a c k o f a p p r o p r i a t e s u b s t r a t e s f o r s t u d y i n g t h e s e macromolecular m o d i f i c a t i o n r e a c t i o n s i n v i t r o and i n v i v o . In t h i s chapter, we have d e s c r i b e d recombinant RNA t e c h n o l o g y which

5.5

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allows one to generate the required tRNA substrates i n v i t r o by replacing specific nucleotides within the anticodon loop. For the purposes of illustration, yeast tRNAALa with the anticodon IGC was reconstructed to contain the anticodon AGC, and this tRNA was used as a substrate for the A,, to I,, modification catalyzed i n v i t r o by the tRNA-hypoxanthine ribosyltransferase from cultured human promyelocytic leukemia cells. The key to utilizing this technology is based on placing a ['*PI label adjacent to the nucleoside of interest, so the modification reaction can be monitored. While the modification reaction illustrated was performed i n v i t r o , studies i n v i v o are also possible; for example, by injecti ng the reconstructed, [32 PI -1 abel 1 ed tRNA i nto the nucleus or cytoplasm of X e n o p u s l a e v i s oocytes. Published recombinant RNA studies are summarized at the end of the chapter, as are investigations employing reconstructed tRNAs to examine specific modification reactions i n v i t r o and i n v i v o . The site directed replacement o f one or more nucleotides in pure tRNA isoacceptors has already led to new insights into the mechanism and specificity o f several modification enzymes acting at positions 3 4 and 37 in the anticodon loop, and similar technology should prove useful for future analyses of the nature and role of these important modifications. 5.6 ACKNOWLEDGEMENTS Supported in the United States by grant AFOSR-85-0003 (RWT) from the Air Force Office of Scientific Research, Department of Defense, and National Service Award T32-CA-09498-02 (KAK) from the National Cancer Institute, Department of Health and Human Services. Supported in Belgium by a grant (HG) from Fonds National de la Recherche Fondamental Collective (F.R.F.C.). Supported internationally by grant RG.86/0140 (RWT,HG) from the North Atlantic Treaty Organization. The assistance of Louis Droogmans and Etienne Haumont in realizing the reconstruction of chimeric t R N A A L * is acknowledged and most appreciated. 5.7 1.

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and U. Z. L i t t a u e r , Covalent j o i n i n o f phenyla l a n i n e t R N A h a l f - m o l e c u l e s by T4-RNA l i g a s e , !roc. Natl. Acad. S c i . USA, 71 (1974) 3741-3745. K. Nishikawa, B.L. Adams and S.M. Hecht, Chemical e x c i s i o n of a u r i n i c a c i d s from RNA: A s t r u c t u r a l 1 m o d i f i e d y e a s t tRNAPle, J . Am. Chem. SOC 104 (1982) 326-528 K. Nishikawa and S. M: Hecht, A s t r u c t u r a l l y m o d i f i e d e a s t tRNA-Phe w i t h s i x n u c l e o t i d e s i n t h e a n t i c o d o n l o o p r a c k s s i n i f i c a n t phen l a l a n i n e acceptance, J. B i o l . Chem., 257

57. G. Kaufmann 58. 59.

(1882) 10536-10538 60. W. L. W i t t e n b e r g a i d 0. C. Uhlenbeck, S p e c i f i c replacement o f

f u n c t i o n a l group o f u r i d i n e - 3 3 i n y e a s t p h e n y l a l a n i n e tRNA, Biochemistry, 24 1985) 2705-2712. 61. A. S. B o u t o r i n , K. Vassilenko, M. M. Baklanov and Y . S. Nechaev, R e c o n s t r u c t i o n o f tRNA-Phe molecules from the fragments b l i n k a g e w i t h T4-RNA l i g a s e i n double-stranded r e g i o n s , F E B ~L e t t , 165 (1984 93-96. 62. A. G. Bruce, J . F. A t k i n s and F. Gesteland, t R N A a n t i c o d o n replacement ex e r i m e n t s show t h a t ribosomal frame s h i f t i n g d o u b l e t decoding, Proc. N a t l . Acad. S c i . can be caused USA, 83 (1986 5862-5066 63. T. Hasegawa, Murao a n i H. I s h i k u r a , Enzymatic s y n t h e s i s o f anticodon-deleted and r e l a c e d E . s u b t i l i s tRNA-Thr and t h e i r amino a c i d acceptor, Nuceeic Acids Res. Symp. S e r i e s No. 15,

1. .

b.

1

(1984) 121-124. 64. L. Bare and O.C. Uhlenbeck, substituted yeast-tyrosine

Aminoac l a t i o n o f a n t i c o d o n l o o tRNA, g i o c h e m i s t r y , 24 (1985p

2354-2360. 65. L. Bare, A. G. Bruce, R. Gesteland and 0. C. Uhlenbeck, U r i dine-33 i n y e a s t t R N A i s n o t e s s e n t i a l f o r amber suppression, Nature, 305 (1983) 554-556.

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a c y l a t i o n and conformation, J . Biochem. (Tokyo), 97 (1985) 29-36. T. Ohyama, K. Nishikawa and S. Takemura, S t u d i e s on T . o t i l i s t R N A T v r v a r i a n t s w i t h e n z y m a t i c a l l y a1 t e r e d D-loop sequences, 11. R e l a t i o n s h i p between t e r t i a r s t r u c t u r e and t y r o s i n e acce tance, J . Biochem. {Tokyo), 98 1986) 859-866 E. OEtsuka, S. Tanaka Tanaka, Miyake, A. F. Markham, E. Nakagawa, T. Wakabayashi, Y . Taniyama, S. Nishikawa, R. Fukumoto, R., Uemura, H . , Doi, T., Tokunaga, and M. Ikehara, T o t a l s y n t h e s i s o f a RNA m o l e c u l e w i t h sequence i d e n t i c a l t o t h a t o f E . c o l i form l m e t h i o n i n e t R N A , Proc. N a t l . Acad. S c i . USA, 78 (1981) 5493-{497. D. Wang e t a l . , Total synthesis o f yeast alanine transfer r i b o n u c i e i c a c i d , S c i e n t i a S i n i c a S e r i e s B, 26 (1983) 464481. N. Usman, K. K. O g i l v i e , K. Nicoghosian and R. J . Cedergren, Automated s t e p wise s o l i d phase chemical s y n t h e s i s o f l o n g o l i g o - r i b o n u c l e o t i d e s u s i n g Z ' - O - s i l l r i b o n u c l e o s i d e 3'-0hosphoramidites: The s y n t h e s i s o f t i e 4 3 - n u c l e o t i d e l o n g ! ' - h a l f molecule o f E . c o l i formyl-Met tRNA. Submitted. N. Beauchemin, J . Pa u e t t e and R. Cedergren, S i t e - d i r e c t e d r o t e c t i o n o f RNA j u r i n g nuclease d i g e s t i o n , Biochem. C e l l i i o l , 65 (1987) i n press.

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CHAPTER 6 TRNA AND TRNA-LIKE MOLECULES: STRUCTURAL PECULIARIT I E S AND BIOLOGICAL RECOGNITION RAJIV L

. JOSH1

and ANNE-LISE HAENNI

I n s t i t u t Jacques N o n o d * . 2 Place Jussieu. 75251 Paris Cedex 05. France .

TABLE OF CONTENTS 6.1 I n t r o d u c t i o n B174 6.2 tRNAs i n T r a n s l a t i o n . An Overview . . . . . . . . . . B174 6.3 tRNAs and tRNA-Like RNAs I n v o l v e d i n O t h e r F u n c t i o n s B176 6.3.1 tRNAs and B i n d i n g o f Amino A c i d s t o Acceptor Molecules . . . . . . . . . . . . . . . . . . B176 B i n d i n g t o an Acceptor P r o t e i n 8177 6.3.1.1 B i n d i n g t o Glycans . . . . . . . . B178 6.3.1.2 Binding t o Phosphatidylglycerol B179 6.3.1.3 6.3.2 t R N A i n C h l o r o p h y l l S y n t h e s i s . . . . . . . . 8179 6.3.3 tRNAs and the Synthesis of Guanylic A c i d D e r i v a t i v e s . . . . . . . . . . . . . . 8180 tRNAs as P r i m e r s i n Reverse T r a n s c r i p t i o n . . 8181 6.3.4 tRNAs and t h e U b i q u i t i n - and ATP-Dependent 6.3.5 8181 System 6.4 tRNA-Like S t r u c t u r e s i n P l a n t V i r a l RNAs 8183 6.4.1 S t r u c t u r a l Aspects 8183 8188 6.4.2 Model P a r t n e r s f o r tRNA-Specific P r o t e i n s 6.5 tRNA-Like Features i n O t h e r N u c l e i c A c i d s 8188 8189 6 . 5 . 1 Bacteriophage-Encoded 'Species I' RNA 6.5.2 RNA o f D e f e c t i v e I n t e r f e r i n g P a r t i c l e s o f Sindbis Virus B189 Polyoma V i r u s DNA 8190 6.5.3 6.5.4 h i s Operon mRNA B190 B190 6.5.5 Threonyl-tRNA Synthetase mRNA 8190 6.5.6 Glycyl-tRNA Synthetase Gene 6.6 F u t u r e Prospects and Impact 8191 8191 6.7 Summary . . . . . . . . . . . . . . . . . . . . . . . 8191 6.8 Acknowledgements 6.9 References . . . . . . . . . . . . . . . . . . . . . . B192

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................... ....... .............. .. ...... .... ................ .............. ............... . . . . . . . . ......... .............

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INTRODUCTION tRNAs p r o v i d e t h e c o r r e c t ami no a c i d s in mRNA-dependent ribosomal p r o t e i n s y n t h e s i s v i a codon-anticodon i n t e r a c t i o n . I n a d d i t i o n t o t h i s fundamental r o l e i n t r a n s l a t i o n , tRNAs o r tRNAl i k e RNAs f u l f i l l s e v e r a l o t h e r f u n c t i o n s i n v a r i o u s c e l l u l a r processes. C e r t a i n p l a n t v i r u s RNA genomes a l s o c o n t a i n a t R N A l i k e s t r u c t u r e a t t h e i r 3 ' end and tRNA-like f e a t u r e s have been d e s c r i b e d i n many v i r a l and c e l l u l a r n u c l e i c a c i d s . Our p r e s e n t knowledge o f t R N A - l i ke molecules a r e reviewed h e r e w i t h m a j o r focus p l a c e d on s t r u c t u r a l p e c u l i a r i t i e s and b i o l o g i c a l r e c o g n i tion. I n t h i s review, we s h a l l c o n s i d e r as t R N A s a l l molecules t h a t can f u l f i l l an amino a c i d donor f u n c t i o n i n mRNA-dependent r i b o somal p r o t e i n s y n t h e s i s , and as t R N A - l i k e R N A s molecules t h a t a p p a r e n t l y resemble tRNAs b u t cannot a c t as tRNAs and possess a d i f f e r e n t s p e c i a l i z e d f u n c t i o n ; we s h a l l d e s i g n a t e as t R N A - 1 i k e s t r u c t u r e s domains i n n u c l e i c a c i d s t h a t share c e r t a i n r e c o g n i t o r y p r o p e r t i e s w i t h tRNAs, and as t R N A - l i k e f e a t u r e s s t r u c t u r a l elements i n n u c l e i c a c i d s t h a t a r e r e m i n i s c e n t o f tRNAs. 6.1

6.2

tRNAs I N TRANSLATION, AN OVERVIEW Understanding t h e s t r u c t u r e and f u n c t i o n o f tRNAs i n v o l v e d i n p e p t i d e c h a i n i n i t i a t i o n and e l o n g a t i o n has been a m a t t e r o f e x t e n s i v e i n v e s t i g a t i o n s f o r t h e l a s t two decades ( f o r a r e v i e w see r e f . 1). The p r i m a r y s t r u c t u r e o f a l a r g e number o f tRNAs has been e l u c i d a t e d and i n general t h e n u c l e o t i d e sequences can be f o l d e d i n t o a common ' c l o v e r l e a f ' - l i k e secondary s t r u c t u r e as schematized i n F i g . 6.1A. However, o u r knowledge about t h e t h r e e dimensional f o l d i n g o f t R N A molecules i s r a t h e r l i m i t e d s i n c e t h e s t r u c t u r e of o n l y a few tRNAs has been e l u c i d a t e d by X-ray d i f f r a c t i o n and o t h e r b i o p h y s i c a l and biochemical methods. A g l o b a l 'L'-shaped s t r u c t u r e has been found f o r t h e tRNAs examined ( F i g . 6.1B) and i t i s assumed t h a t a l l tRNAs adopt t h i s common 'L'-shaped c o n f o r mation.

__---_______---_-_ *The s i t 6

l n s t i t u t Jacques P a r i s VII".

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U n i v e r -

B175

I t i s most s t r i k i n g t h a t a p p a r e n t l y ' m i n o r ' s t r u c t u r a l p e c u l i a r i t i e s o f i n i t i a t o r tRNAYet , as compared t o a l l e l o n g a t o r tRNAs, r e s u l t i n c o m p l e t e l y d i f f e r e n t r e c o g n i t o r y and f u n c t i o n a l p r o p e r t i e s : r e c o g n i t i o n by t h e t r a n s f o r m y l a s e i n p r o k a r y o t e s , complex f o r m a t i o n w i t h p e p t i d e c h a i n i n i t i a t i o n f a c t o r s , b i n d i n g

A

B 3l

T

3'

I

D

0

AC

F i g u r e 6 . 1 General schematic y e p r e s e n t a t i o n o f t R N A s t r u c t u r e i n t h e c l o v e r l e a f (A) o r i n t h e L'-shaped c o n f i g u r a t i o n ( B ) . Aa = acceptor stem; T = T stem and loop; V = v a r i a b l e stem avd l o o ; Ac = a n t i c o d o n stem and loop; D = D stem and l o o p . The aminoacyy RNA domain c o r r e s onds t o t h e s t r u c t u r e formed by t h e c o n t i n u o u s and t h e s t a c k i n g o f t e a c c e p t o r stem on t h e T stem and loop, a n t i c o d o n RNA domain t o t h e s t r u c t u r e formed by t h e s t a c k i n g o f t h e D stem and loop on t h e a n t i c o d o n stem and l o o p .

6

t o t h e P s i t e on t h e ribosome i n t h e process o f p e p t i d e c h a i n i n i t i a t i o n and i n a b i l i t y t o f u n c t i o n as e l o n g a t o r tRNA. Concerni ng e l o n g a t o r tRNAs , t h e r e e x i s t s e v e r a l examples o f 'minor' s t r u c t u r a l ' d e v i a t i o n s ' t h a t r e s u l t i n completely d i f f e r e n t p r o p e r t i e s o f r e c o g n i t i o n by s p e c i f i c p r o t e i n s and/or a1 low 'abnormal ' codon-anticodon i n t e r a c t i o n s l e a d i n g t o m i s r e a d i n g , suppression o r f r a m e s h i f t . More r e c e n t l y , i t has appeared t h a t c e r t a i n m i t o c h o n d r i a 1 tRNAs ( r e f . 2 and r e f s . t h e r e i n ) possess q u i t e p e c u l i a r s t r u c t u r e s l a c k i n g t h e e n t i r e D o r t h e T and V stems and l o o p s . I t would be i n t e r e s t i n g t o compare t h e o v e r a l l s t r u c t u r e of some o f t h e s e

B176

tRNAs by b i o p h y s i c a l methods and t o i n v e s t i g a t e i n d e t a i l t h e i r r e c o g n i t o r y and f u n c t i o n a l p r o p e r t i e s .

6.3

tRNAs AND tRNA-LIKE RNAs INVOLVED I N OTHER FUNCTIONS I n a d d i t i o n t o t h e i r r o l e as donors o f aminoacyl r e s i d u e s f o r c l a s s i c a l mRNA- and r i bosome-dependent p r o t e i n s y n t h e s i s , a wide v a r i e t y o f o t h e r f u n c t i o n s a r e performed by c e r t a i n tRNAs and tRNA-1 ike RNAs i n c e l l u l a r systems. When aminoacylated species p e r f o r m such f u n c t i o n s , t h e ami noacyl m o i e t y is general l y t r a n s f e r r e d t o an a c c e p t o r molecule, as i n t h e case o f a c c e p t o r p r o t e i n s , o f glycans and o f phosphat i d y l g l y c e r o l . An e x c e p t i o n t o t h i s scheme i s t h e involvement o f a glutamyl-tRNA i n c h l o r o p h y l l s y n t h e s i s . I n o t h e r i n s t a n c e s , t h e uncharged species i s t h e a c t i v e f o r m as i n t h e p r o d u c t i o n o f p a r t i c u l a r guanyl ic a c i d d e r i v a t i v e s and i n DNA s y n t h e s i s medi a t e d by t h e r e v e r s e t r a n s c r i p t a s e . F i n a l l y , i n t h e case o f t h e u b i q u i tin-dependent system, i t seems l i k e l y t h a t a t l e a s t i n one case t h e t R N A must be aminoacylated t o be o p e r a t i v e . I n t h e few cases where t h e n u c l e o t i d e sequence of t h e tRNAs i n v o l v e d i n these n o n - c l a s s i c a l r e a c t i o n s has been e s t a b l i s h e d , i t appears t h a t g e n e r a l l y a c e l l u l a r i s o a c c e p t o r t R N A t h a t i s a l s o a c t i v e i n c l a s s i c a l p r o t e i n s y n t h e s i s i s r e s p o n s i b l e f o r t h e nonc l a s s i c a l r e a c t i o n . T h i s i s however, n o t t h e case o f t h e t R N A G l v t h a t donates i t s g l y c y l r e s i d u e f o r i n t e r p e p t i d e b r i d g e f o r m a t i o n o f p e p t i d o g l y c a n s and i t i s l i k e l y t h a t t h e o t h e r tRNAs i n v o l v e d i n s i m i l a r i n t e r p e p t i d e b r i d g e f o r m a t i o n a l s o have evolved a s p e c i f i c t R N A species r e s p o n s i b l e f o r t h i s f u n c t i o n . I t may a l s o n o t be t h e case o f t h e t R N A G L u i n v o l v e d i n c h l o r o p h y l l s y n t h e s i s . T h i s s e c t i o n reviews o u r p r e s e n t knowledge o f t h e tRNAs i n v o l v e d i n these non-cl a s s i c a l r e a c t i o n s . 6.3.1

tRNAs and B i n d i n a o f Amino Acids t o Acceotor Molecules I t has been known f o r over 20 y e a r s t h a t c e r t a i n aminoacyl-tRNAs can donate t h e i r amino a c i d f o r c o v a l e n t attachment i n v a r i o u s b i o l o g i c a l processes d i s t i n c t f r o m cl a s s i c a l p r o t e i n s y n t h e s i s . The enzymes r e s p o n s i b l e f o r t h e s e r e a c t i o n s a r e termed aminoacyl - t R N A t r a n s f e r a s e s ; t h e y r e q u i r e n e i t h e r ribosomes, GTP n o r mRNAs. The t r a n s f e r r e a c t i o n s can be c l a s s i f i e d on t h e b a s i s o f t h e n a t u r e o f t h e acceptor m o l e c u l e t o which t h e aminoacyl

moiety i s transferred: the acceptor molecule can be a preformed protein, glycans o r phosphatidylglycerol

.

B i n d i n s t o an AccePtor Protein The b i n d i n g of an amino acid residue t o the NH, terminus of i s catalyzed by s p e c i f i c s p e c i f i c proteins v i a aminoacyl-tRNAs aminoacyl-tRNA protein transferases ( f o r reviews see r e f s . 3 , 4 ) . To date the following amino acids have been reported t o be i n volved in such t r a n s f e r reactions: leucine, phenylalanine and methionine i n gram-negative bacteria, and arginine in eukaryotes. The arginyl-tRNA protein t r a n s f e r a s e from r a t 1 i v e r catalyzes the t r a n s f e r of argi n i ne from argi nyl - t R N A t o the NH, -terminal a s p a r t i c acid residue of bovine serum albumin and of several other protei ns such as bovi ne t h y r o g l obul i n , porci ne p-me1 anotropi n and human-type angiotensin 11. Similarly, the leucyl-, phenylalanylt R N A protein t r a n s f e r a s e from E s c h e r i c h i a c07 i catalyzes the t r a n s f e r of leucine or phenylalanine from their respective t R N A t o the NH, -terminal argi ni ne residue of a-casei n . The same enzyme also catalyzes the t r a n s f e r of methionine from methionyl-tRNA, the non-initiator species being preferred t o the i n i t i a t o r species. Using synthetic peptides, i t was shown t h a t t h e arginyl-tRNA protein transferase requires t h a t the NH,-terminal residue of the acceptor species be a dicarboxylic acid, a s p a r t i c acid o r glutamic acid, whereas the f. c o 7 i enzyme requires lysine, arginine or h i s t i d i n e i n the NH, position of the acceptor peptide. Consequently, the nature of the NH,-terminal residue of the acceptor protein i s determinant f o r both transferases. The nature of the aminoacyl residue donated d u r i n g t r a n s f e r i s an absol Ute determi nant. T h u s , E . co 7 i phenyl a1 anyl - t R N A V a 1 mi scharged u s i n g N e u r o s p o r a c r a s s a phenyl a1 anyl - t R N A synthetase donates i t s phenyl a1 anyl moiety, whereas valyl - t R N A V a does not. I f previously acetylated, phenylalanyl-tRNA o r arginyl-tRNA no longer d o n a t e t h e i r modified aminoacyl residue. The influence of the polynucleotide moiety i s r e f l e c t e d by the f a c t t h a t Met-tRNA, i s preferred t o Met-tRNA,, and t h a t the phenylalanine bound t o the 3 ' pentanucleotide of i t s t R N A cannot be donated. I t a l s o appears t h a t the d i f f e r e n t isoacceptor tRNAs of phenylalanine and leucine can a l l function in the t r a n s f e r reaction.

6.3.1.1

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6.3.1.2 B i n d i n g t o Glvcans I n b a c t e r i a l c e l l w a l l s ( f o r a r e v i e w see r e f . 5) c e r t a i n aminoacyl-tRNAs p r o v i d e t h e i r aminoacyl-moiety f o r t h e f o r m a t i o n o f t h e i n t e r p e p t i d e b r i d g e s t h a t l i n k t h e g l y c a n s t r a n d s t o form t h e 3-dimensional s t r u c t u r e o f t h e p e p t i d o g l y c a n s (mureins). The interpeptide bridge i s a pentaglycine i n Staphylococcus aureus ( r e f . 6 ) , i t i s composed o f g l y c i n e and s e r i n e i n 5 . e p i d e r m i d i s ( r e f . 7), o f t h r e o n i n e and a l a n i n e i n M i c r o c o c c u s r o s e u s ( r e f . 8 ) and o f a s i n g l e a l a n i n e r e s i d u e i n A r t h r o b a c t e r c r y s t a 7 7 o p o i e t e s ( r e f . 9). I n a l l cases t h e p e p t i d o g l y c a n synthetase, a p a r t i c u l a t e enzyme, c a t a l y z e s t h e t r a n s f e r o f t h e aminoacyl m o i e t y o f t h e corresponding aminoacyl-tRNA t o t h e g l y c a n v i a a l i p i d i n t e r med ia t e For each amino a c i d i n v o l v e d i n i n t e r p e p t i d e b r i d g e f o r mation, an unusual t R N A species seems t o e x i s t t h a t i s s p e c i f i c a l l y adapted f o r t h i s f u n c t i o n ( a t l e a s t i n v i t r o ) and i s n o t a c t i v e i n mRNA-dependent p r o t e i n s y n t h e s i s . I n S. e p i d e r m i d i s f o r instance, a l l t h e tRNAs acceptor o f g l y c i n e can p a r t i c i p a t e i n p e p t i d o g l y c a n s y n t h e s i s , b u t one o f them i s i n a c t i v e i n mRNAdependent p r o t e i n s y n t h e s i s . By o u r d e f i n i t i o n , t h i s species should be considered as a tRNA-like RNA. The n u c l e o t i d e sequence o f t h e two c l o s e l y r e l a t e d t R N A G L y species t h a t p a r t i c i p a t e i n i n t e r p e p t i d e b r i d g e f o r m a t i o n has been e s t a b l i s h e d . These tRNAs d i f f e r from c l a s s i c a l tRNAs i n having t h e sequence GUGC i n p l a c e o f GT$C which may account f o r t h e i r f a i l u r e t o b i n d t o ribosomes. The D-loop c o n t a i n s an unusual number o f unmodified u r i d i n e r e s i d u e s and l a c k s t h e sequence G ( o r 2'-0MeG)-G commonly found i n this l o o p ( r e f . 10). The same s i t u a t i o n e x i s t s w i t h r e s p e c t t o t h e tRNAs acceptor o f s e r i n e : a l l a r e a c t i v e i n p e p t i d o g l y c a n s y n t h e s i s , b u t one o f them (which would correspond t o a tRNA-like RNA) i s i n a c t i v e i n mRNA-dependent p r o t e i n s y n t h e s i s ( r e f . 7 ) . The sequence o f t h i s unusual t R N A S e r has n o t been e s t a b l i s h e d . L i k e wise, t h e sequence o f t h e o t h e r tRNAs i n v o l v e d i n t h e f o r m a t i o n o f o t h e r b a c t e r i a l c e l l w a l l s i s unknown. I n i t i a t i o n o f i n t e r p e p t i d e b r i d g e f o r m a t i o n occurs b y aminoa c y l a t i o n o f t h e c-amino group i n a l y s i n e r e s i d u e o f one g l y c a n s t r a n d ( t h e g l y c a n s t r a n d c o n t a i n s t h e t e t r a p e p t i d e L-alanyl-Disoglutaminyl-L-lysyl-D-alanine) and extends v i a s e q u e n t i a l aminoa c y l a t i o n o f a-amino groups u n t i l t h e f i n a l amino a c i d forms a

.

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peptide bond with the D-alanine residue of an adjacent glycan strand. Binding of a specific amino acid to an acceptor protein or to glycans thus presents an additional unusual feature. Whereas in classical protein synthesis elongation proceeds from the NH, terminus towards the COOH terminus, in the two situations just reviewed, elongation proceeds in the opposite direction, namely from the COOH towards the NH, terminus. 6.3.1.3 Bindins to Phosohatidvlslvcerol Among the lipids contained in the membranes of certain bacteria are lysylphosphatidylglycerol and alanylphosphatidylglycerol; the former compound is found in S a c c h a r o m y c e s a u r e u s , B a c i l l u s m e g a t e r i u m , B . c e r e u s and C l o s t r i d i u m w e l c h i i , whereas the latter i s found in C. w e l c h i i (ref, 11). These compounds are produced v i a aminoacylation of phosphatidylglycerol , the amino acid donor being the corresponding aminoacyl -tRNA (for a review see ref. 12). Here, a transesterification reaction occurs in which the aminoacyl group engaged in an ester linkage with the ribose of the 3'-terminal adenosine in the tRNA is transferred to the diol system of the glyceryl moiety of phosphatidylglycerol. The transesterification reaction is mediated by specific transferases (phosphatidylglycerol synthetases) that are associated with the cytoplasmic membrane. The enzyme recognizes both the aminoacyl and the polyribonucleotide moieties of the aminoacyl-tRNA (refs. 13, 14) . Indeed, neither acetylalanyl- or lactyl-tRNA , nor alanyltRNACvS is substrate of the transferase. Furthermore, the 3'terminal "half-molecule" of alanyl-tRNA is a poor substrate of the transesterification reaction. 6.3.2 tRNA in Chloroohvll Synthesis The synthesis of chlorophyll requires a-aminolevul inic acid (DALA) which is produced from glutamate. In barley and C h l a m y d o m o n a s , a chloroplast tRNACLu is one of the components required for the reduction of glutamate to gl utamate-1-semialdehyde which is i n turn converted to DALA (ref. 15). The reduction reaction takes place *at the level of a glutamyl-tRNA and is catalyzed by a dehydrogenase, NADPH serving as cofactor. The dehydrogenase is highly selective and must recognize specific features i n the glutamyl-tRNA that serves as intermediate in DALA synthesis. The

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sequence o f t h e t R N A i s c h a r a c t e r i z e d by a h i g h l y modi f ied anticodon; i t i s t h e on y one o f t h r e e t R N A G L U o f ch o r o p l a s t o r i g i n capable o f s u p p o r t ng DALA s y n t h e s i s . 6.3.3

tRNAs and t h e Svnthesis o f G u a n v l i c A c i d D e r i v a t i v e s The mechanism whereby b a c t e r i a adapt t h e i r metabol ism t o amino a c i d s t a r v a t i o n i s known as t h e " S t r i n g e n t response". Our p r e s e n t view on s t r i n g e n t response d e r i v e s m a i n l y f r o m g e n e t i c and biochemical s t u d i e s u s i n g E . coli and more r e c e n t l y B . sobtilis ( f o r reviews see r e f s . 16-19). The s t r i n g e n t response r e g u l a t e s n o t o n l y t h e b i o s y n t h e s i s o f s t a b l e RNA (rRNA and t R N A ) , b u t a l s o t h e b i o s y n t h e s i s o f membranes and o f many enzymes i n v o l v e d i n a c t i v e t r a n s p o r t and p r o t e i n degradation. "Relaxed" mutants (re7 A- s t r a i n s ) do n o t e x e r t t h i s s t r i n g e n t c o n t r o l o v e r s t a b l e RNA s y n t h e s i s . D u r i n g amino a c i d s t a r v a t i o n o f re7 A+ E . c o l i s t r a i n s , uncharged tRNAs accumulate t h a t b i n d on t h e ribosomal A s i t e t o t h e mRNA b e a r i n g t h e a p p r o p r i a t e codon, and as a consequence elonga t i o n o f p o l y p e p t i d e chains i s d r a m a t i c a l l y reduced. T h i s " i d l i n g " r e a c t i o n i s communicated by t h e ribosomes t o t h e s t r i n g e n t f a c t o r whose a c t i v i t y i s t h e r e b y s t i m u l a t e d . T h i s f a c t o r (coded by t h e re7 A gene) i s a s s o c i a t e d t o t h e ribosomes and c a t a l y z e s t h e s y n t h e s i s o f two unusual n u c l e o t i d e s , t h e "magic s p o t s " (MS), ppGpp and pppGpp, i n an ATP-dependent manner. The MS appear t o be r e s p o n s i b l e f o r many e f f e c t s i n t h e c e l l . They b r i n g about t h e s t r i n g e n t response by m o d i f y i n g t h e RNA polymerase such t h a t i t no l o n g e r t r a n s c r i b e s genes f o r r R N A and tRNA; t h e y s t i m u l a t e t h e s y n t h e s i s o f enzymes i n v o l v e d i n amino a c i d s y n t h e s i s ; t h e y gene r a l l y e x e r t a d i r e c t i n h i b i t o r y a c t i o n on c e r t a i n enzymes. Experiments have been performed t o e v a l u a t e t h e s t r u c t u r a l requirements o f t h e tRNA f o r MS s y n t h e s i s . The t R N A must c o n t a i n t h e complete -CCA end and an i n t a c t t e r m i n a l r i b o s e r e s i d u e w i t h a f r e e 3 ' -OH group. Thus n e i t h e r tRNA-CCAoxi n o r tRNA-CCAoxi-red can s u p p o r t (p)ppGpp s y n t h e s i s and tRNA-CCC o r tRNA-CCCA induce lower (p)ppGpp s y n t h e s i s than normal t R N A . The t R N A fragment T$CG can r e p l a c e uncharged t R N A i n a c t i v a t i n g t h e s t r i n g e r t t f a c t o r and i t competes w i t h t h e b i n d i n g o f uncharged t R N A t o t h e A s i t e on t h e ribosome i n t h e s t r i n g e n t r e a c t i o n . I t i s as y e t u n c l e a r whether t h e s t r i n g e n t f a c t o r a c t i v e l y promotes b i n d i n g o f t h e t R N A

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i n a manner s i m i l a r t o t h e EF-Tu-GTP mediated b i n d i n g o f aminoacyl-tRNAs t o t h e A s i t e o f ribosomes. 6.3.4

tRNAs as Primers i n Reverse T r a n s c r i D t i o n I t has been known f o r o v e r 15 y e a r s t h a t a s p e c i f i c h o s t t R N A serves as p r i m e r f o r t h e s y n t h e s i s o f DNA complementary t o t h e RNA genome o f r e t r o v i r u s e s ( r e f . 20; f o r reviews see r e f s . 21-23). Indeed, r e t r o v i r u s e s s e l e c t i v e l y encapsidate c e l l u l a r tRNAs. These can be d i s s o c i a t e d from t h e 35s v i r a l genome by h e a t i n g t h e RNA: a l l b u t t h e t R N A p r i m e r f o r DNA s y n t h e s i s a r e d i s s o c i a t e d a t 60°C, whereas t h e t R N A p r i m e r i s o n l y r e l e a s e d a t 90°C. The n a t u r e o f t h e t R N A p r i m e r i s d i f f e r e n t f o r d i f f e r e n t r e t r o v i r u s e s . I t i s a t R N A T r p i n a v i a n sarcoma and l e u k o s i s v i r u s e s such as Rous sarcoma v i r u s and a v i a n m y e l o b l a s t o s i s v i r u s ( r e f . 24), i t i s a t R N A P r O i n murine leukemia v i r u s e s and i n s e v e r a l o t h e r mammal ian v i ruses such as mol oney m u r i ne 1eukemi a v i r u s ( r e f . 25), and i t i s a t R N A L v S i n mouse mammary tumor v i r u s ( r e f s . 26,27). I n a l l t h r e e cases, t h e n a t u r e o f t h e t R N A p r i m e r has been e s t a b l i s h e d . I n AMV i t has been demonstrated ( r e f . 25) t h a t t h e r e g i o n l o c a t e d 100 n u c l e o t i d e s f r o m t h e 5 ' end o f t h e v i r a l genome i s complementary t o t h e 17 n u c l e o t i d e s t h a t a r e 3 ' - t e r m i n a l i n t R N A T r p . Likewise, 17 n u c l e o t i d e s from t h e 3 ' end o f t R N A P r 0 a r e complementary t o a corresponding r e g i o n i n murine r e t r o v i r u s RNA ( r e f . 25); one t R N A p r i m e r i s found a s s o c i a t e d p e r v i r a l 35s RNA. The p r i m e r tRNA shows a h i g h a f f i n i t y f o r t h e r e v e r s e t r a n s c r i p t a s e w i t h which i t forms a s t a b l e complex. I t appears t h a t t h e enzyme can p a r t i a l l y unwind t h e a c c e p t o r stem o f t h e t R N A ( r e f . 28), and t h i s f i r s t s t e p may then ensure b i n d i n g o f t h e t R N A t o t h e corresponding p o s i t i o n o f t h e v i r a l genome mediated by t h e enzyme. Whereas t h i s model may apply i n t h e case o f t R N A T r p and AMV RNA, i t i s p o s s i b l e t h a t another enzyme t r i g g e r s a n n e a l i n g o f t R N A P r o t o murine v i r a l RNAs ( r e f . 29). 6.3.5

tRNAs and t h e U b i a u i t i n - and ATP-DeDendent System I n eukaryotic c e l l s , the selective turnover o f short-lived non-lysosomal p r o t e i n s i s under t h e c o n t r o l o f u b i q u i t i n and ATP ( r e f s . 30,31). I n t h i s system u b i q u i t i n becomes c o v a l e n t l y l i n k e d t o t h e s u b s t r a t e p r o t e i n , and i n t h e r e s u l t i n g u b i q u i t i n - p r o t e i n conjugate, t h e p r o t e i n becomes t a r g e t e d f o r a t t a c k by s p e c i f i c

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p r o t e i nases. The cascade o f r e a c t i o n s in v o l ved i n t h i s s e l e c t i v e d e g r a d a t i o n o f t a r g e t e d p r o t e i n s has been l a r g e l y worked o u t u s i n g rabbit reticulocyte lysates. I t has been d e m o n s t r a t e d ( r e f . 32) t h a t i n a d d i t i o n t o u b i q u i t i n and ATP, a tRNAHis f r o m r e t i c u l o c y t e l y s a t e s , c a l f l i v e r o r mouse c e l l s r e s t o r e s p r o t e o l y t i c a c t i v i t y t o a l y s a t e whose tRNAs have p r e v i o u s l y been e l i m i n a t e d b y r i b o n u c l e a s e t r e a t m e n t . R a t h e r s u r p r i s i n g l y , t h e r e q u i r e m e n t f o r tRNAHis i n t h e u b i q u i t i n and ATP-dependent p r o t e o l y t i c system appears t o be s u b s t r a t e s p e c i f i c : if t h e tRNA i s destroyed by ribonuclease treatment, b o v i n e serum a l b u m i n d e g r a d a t i o n i s a l m o s t c o m p l e t e and t h i s e f f e c t can be r e s t o r e d b y t h e a d d i t i o n o f tRNAHi s . On t h e o t h e r hand, r i b o n u c l e a s e t r e a t m e n t a f f e c t s g l o b i n and lysozyme deg r a d a t i o n b y o n l y 40% and 10-20%, r e s p e c t i v e l y . F u r t h e r e x p e r i ments ( r e f . 33) have c o n f i r m e d t h a t t R N A H i S i s r e q u i r e d f o r t h e ubiquit i n a t i o n o f c e r t a i n protein substrates but n o t o f others. Indeed, r i b o n u c l e a s e s i n h i b i t c o n j u g a t i o n o f r a d i o l a b e l l e d u b i q u i t i n t o o n l y c e r t a i n p r o t e i n s and t h i s i n t u r n i n h i b i t s t h e ATP-dependent d e g r a d a t i o n o f t h e s e p r o t e i n s ; a d d i t i o n o f tRNA restores both conjugation, and u b i q u i t i n - and ATP-dependent p r o t e o l y s i s o f these substrates. I n contrast, ribonucleases a c c e l e r a t e d e g r a d a t i o n o f o t h e r s u b s t r a t e p r o t e i n s (lysozyme, RNAse A, a - c a s e i n and 8 - l a c t o g l o b u l i n ) and do n o t i n h i b i t conj u g a t i o n o f u b i q u i t i n t o these p r o t e i n s . Consequently, a t l e a s t two d i s t i n c t u b i q u i t i n a t i o n pathways c o - e x i s t i n a r e t i c u l o c y t e l y s a t e : a tRNA-dependent and a tRNA-independent pathway, i n h i b i t i o n o f one pathway a c c e l e r a t i n g t h e o t h e r . These r e s u l t s do n o t r u l e o u t t h e p o s s i b i l i t y t h a t tRNA s p e c i e s o t h e r t h a n tRNAHiS m i g h t be r e q u i r e d f o r t h e u b i q u i t i n - and ATP-dependent d e g r a d a t i o n o f o t h e r p r o t e i n s u s b s t r a t e s (See b e l o w ) . I n eukaryotic c e l l s , the r a t e o f p r o t e i n degradation i n c r e a s e s as g r o w t h r a t e and p r o t e i n s y n t h e s i s d e c r e a s e . T h i s can be c o r r e l a t e d t o t h e o b s e r v a t i o n s t h a t i n Chinese h a m s t e r o v a r y (CHO) c e l l s p r o t e i n degradation i s induced i n t h e presence o f h i s t i d i n o l , an i n h i b i t o r o f tRNAHiS a m i n o a c y l a t i o n . I n a CHO c e l l l i n e w i t h a t s histidyl-tRNA synthetase, p r o t e i n degradation increases a t t h e n o n - p e r m i s s i v e t e m p e r a t u r e , and i n r a t l i v e r p r o t e i n d e g r a d a t i o n i n c r e a s e s when s p e c i f i c amino a c i d s such as h i s t i d i n e o r a l a n i n e a r e l a c k i n g . Thus, t h e l e v e l o f s p e c i f i c u n c h a r g e d

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tRNAs appears t o r e g u l a t e t h e r a t e o f non lysosomal p r o t e i n degradation. More r e c e n t d a t a i n d i c a t e ( r e f . 34) t h a t t R N A i s r e s p o n s i b l e f o r t h e a d d i t i o n o f a r g i n i n e t o t h e NH, terminus o f a c i d i c prot e i n s , most 1ik e l y c a t a l y z e d by a r g i nyl-tRNA-protein t r a n s f e r a s e (see s e c t i o n 6.3.1.1). Such N-terminal m o d i f i e d p r o t e i n s become t a r g e t e d f o r d e g r a d a t i o n by t h e u b i q u i t i n system. 6.4

t R N A - L I K E STRUCTURES I N PLANT V I R A L RNAs The 3 ' - t e r m i n u s of t h e RNA genomes o f a number o f p l a n t v i r u s e s can be aminoacylated i n v i t r o w i t h a s p e c i f i c amino a c i d by t h e cognate aminoacyl-tRNA s y n t h e t a s e i n c o n d i t i o n s s i m i l a r t o those l e a d i n g t o t h e a m i n o a c y l a t i o n o f tRNAs. The l i s t o f p l a n t v i r u s e s whose RNAs can be aminoacylated i n v i t r o i s p r e s e n t e d i n Table 6.1. These v i r a l RNAs a l s o i n t e r a c t i n v i t r o w i t h s e v e r a l o t h e r tRNA-specific enzymes. They are, f o r i n s t a n c e , r e c o g n i z e d by t h e t R N A n u c l e o t i d y l t r a n s f e r a s e t h a t can r e p a i r t h e 3 ' - t e r m i n a l -CCA end, and by t h e p e p t i d e c h a i n e l o n g a t i o n f a c t o r (EF-Tu o r EF-la) w i t h which t h e aminoacylated v i r a l RNAs can form s t a b l e complexes i n t h e presence o f GTP. These v i r a l RNA genomes t h u s d i s p l a y tRNA-1 ike r e c o g n i t o r y p r o p e r t i e s and t h e s t r u c t u r e a t t h e i r 3 ' end has been r e f e r r e d t o as tRNA-like s t r u c t u r e . The f u n c t i o n o f t h e s e tRNA-like p r o p e r t i e s o f p l a n t v i r a l RNA genomes s t i l l remains t o be e l u c i d a t e d ( f o r reviews see r e f s . 35, 36).

6.4.1

S t r u c t u r a l Aspects When t h e n u c l e o t i d e sequences o f t h e 3 ' - r e g i o n s o f t h e v i r a l RNAs were determined t o e s t a b l i s h s t r u c t u r a l analogy w i t h tRNA, i t came as a s u r p r i s e t h a t none o f them c o u l d be f o l d e d i n t o t h e c l a s s i c a l c l o v e r l e a f p a t t e r n o f tRNAs, and f o r many y e a r s t h e q u e s t i o n remained as t o t h e s t r u c t u r a l f e a t u r e s o f t h e v i r a l tRNA-1 ike r e g i o n s u n d e r l y i n g r e c o g n i t i o n b y t R N A - s p e c i f i c p r o t e i n s . Moreover, i t was q u i t e s t r i k i n g t h a t t h e s e v i r a l tRNA-like r e g i o n s a r e d e v o i d o f modi f i ed n u c l e o t i des. Recently, t o o b t a i n more i n s i g h t i n t o t h e s t r u c t u r e o f t h e tRNA-like r e g i o n s i n v i r a l RNAs, two approaches have been used. F i r s t , t h e minimum l e n g t h o f t h e v i r a l RNAs r e q u i r e d f o r aminoa c y l a t i o n was determined t o d e f i n e t h e s i z e o f t h e tRNA-like

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TABLE 6.1 A m i n o a c y l a t i o n C a p a c i t y o f P l a n t V i r a l RNA Genomes I n V i t r o ~

Ami no a c i d bound

V i r u s group

Source o f v i r a l RNAs

Tymovi r u s

T u r n i p ye1 l o w mosaic v i r u s TYMV) Cacao y e l l o w mosaic v i r u s ( YMV) E g p l a n t m o s a i c v i r u s (EMV) O&ra m o s a i c v i r u s IOMV) W i l d cucumber m o s a i c v i r u s (WCMV)

Val i n e Valine Val ine Val ine Valine

Tobamovi r u s

Tobacco m o s a i c v i r u s (TMV) Cucumber r e e n m o t t l e m o s a i c virus (C~MMV) Sun hemp m o s a i c v i r u s (SHMV)*

H i s t i d i ne

Brome m o s a i c v i r u s (BMV) Broad bean m o t t l e v i r u s (BBMV) Cowpea c h l o r o t i c m o t t l e v i r u s (CCMV)

T y r o s i ne T y r o s i ne

Cucumovi r u s

Cucumber mosaic v i r u s (CMV)

T y r o s i ne

Hordei v i r u s

Bar1 e y s t r i p e mosaic v i r u s (BSMV)

T y r o s i ne

Bromovi r u s

*Identical

d

H i s t i d i ne Val ine

T y r o s i ne

t o c o w p e a s t r a i n o f TMV ( C c T M V ) .

r e g i o n s ( r e f s . 37-39). Second, b a s e - p a i r i n g s w i t h i n t h e tRNA-1 ike r e g i o n s were s t u d i e d u s i n g c h e m i c a l and b i o c h e m i c a l approaches: t h e a c c e s s i b i l i t y o f t h e d i f f e r e n t bases t o c h e m i c a l r e a g e n t s was examined and p a r t i a1 d i g e s t i o n s b y s p e c i f i c n u c l eases were p e r formed ( r e f s . 40-44). From t h e s e s t u d i e s t h e c o n c l u s i o n emerged ( f o r r e v i e w s see r e f s . 36,45) t h a t t h e t R N A - l i k e r e g i o n s i n t h e v i r a l RNAs t h a t can be a m i n o a c y l a t e d w i t h v a l i n e (TYMV), t y r o s i n e (BMV) o r h i s t i d i n e (TMV) a d o p t c o n f o r m a t i o n s r e m i n i s c e n t o f t h e ' L ' - s h a p e o f tRNAs i n c o m p l e t e l y u n c o n v e n t i o n a l manners ( F i g . 6.2). The e x i s t e n c e o f ' p s e u d o k n o t s ' ( F i g . 6.3) i n t h e f o l d i n g o f t h e t R N A - l i k e r e g i o n s i s r e m a r k a b l e ( f o r a r e v i e w see r e f . 4 6 ) . The appearance o f a ' p s e u d o k n o t ' i n t h e f o r m a t i o n o f t h e a c c e p t o r stem i n t h e t R N A - l i k e s t r u c t u r e o f TYMV RNA i s i l l u s t r a t e d i n F i g . 6.4. The a c c e p t o r stem i n t h e t R N A - l i k e s t r u c t u r e o f TMV RNA i s formed i n a s i m i l a r manner. I n t h e c a s e o f BMV RNA a l s o , a ' p s e u d o k n o t ' appears d u r i n g t h e f o r m a t i o n o f t h e a m i n o a c y l RNA domain ( F i g . 6 . 5 ) .

B185

ucq

UUGA AG.CC C*G G*C

G U.A COG U.A G*C U-A C-G

c

C

2

A A

u AGGCU,, U Uc AA G AGGGGUUC G

....UP, .....

cc

U

TYMV RNA

G.U C.G U.A U.A G*C C G AUA

A U C

BMV R N A

tauA TMV R N A G C GUU

F i g u r e 6.2 L ' -shaped r e p r e s e n t a t i o n o f t h e tRNA-1 ike r e g i o n s o f p l a n t v i r a l RNAs. I

More r e c e n t l y , b a s e - p a i r i n g s between l o o p s and d i s t a n t l y 1 ocated r e g i o n s 1e a d i ng t o ' pseudoknots ' were a1 so d e s c r i bed i n o t h e r r e g i o n s o f v i r a l RNAs ( r e f . 4 7 ) , i n 16s ribosomal RNA ( r e f . 48), i n c e r t a i n m i t o c h o n d r i a 1 i n t r o n s , i n f u n g i ( r e f . 49) and i n 5s RNA ( r e f . 50). I t i s c o n c e i v a b l e t h a t r e g i o n s i n RNAs where such basep a i r i n g s can occur m i g h t adopt e i t h e r a ' t i g h t ' o r a ' l o o s e ' conformation t o r e g u l a t e i n t e r a c t i o n w i t h s p e c i f i c p r o t e i n s . The s t u d i e s b e a r i n g on t h e tRNA-like r e g i o n s i n v i r a l RNAs c l e a r l y

B186

A

B

I

b

I I

F i g u r e 6.3 'Pseudoknots' i n t h e f o l d i n g o f RNA molecules. I f bases (open c i r c l e s ) i n l o o p b a r e complementar fo bases ( c l o s e d c i r c l e s ) i n a d i s t a n t l y located r e g i o n c (A), t e i r p a l r i n g leads t o t h e f o r m a t i o n of a 'pseudoknot' (6) i n which t h e new basep a i r s a r e stacked on t h e base-pairs o f stem a f h a t i,s t h u s extended. The RNA molecule c o u l d adopt e i t h e r a l o o s e (A) o r a ' t i g h t ' (B) conformation.

K

A

B

3'

c

3'

3'

II

IV

111

F i g u r e 6.4 Formation o f t h e acceptor stem i n t h e tRNA-like s t r u c t u r e o f TYMV RNA represented i n a c o n f i g u r a t i o n r e c a l l i n g t h e c l o v e r l e a f . I I - I V c o r r e s ond t o stems and l o o s T, a n t i c o d o n and D o f tRNAs r e s p e c t i v e l y . T r e e bases (open c i r c es) o f l o o p I a r e

I:

Y

B187

Figure 6.4 ( c o n t i n u e d ) corn lementary of t h r e e bases (closed c i r c l e s ) d i s t a n t l y located (A!. t h e i r p a i r i n g i s shpwn in (B). The acce t o r stem i s formed by sticking of these 'new base-pairs on the l a s e - p a i r s of stem I ( C ) . Two s h o r t bridges of 4 and 3 nucleotides connect t h e two s i d e s of the a c c e p t o r stem of t h i s v i r a l RNA .

A

F i ure 6 . 5

( A ) Schematic 'L'-shaped r e p r e s e n t a t i o n o f . the t R N A l i f e region of BMV RNA. Pairing between bases (open c i r c l e s ) of loop e and complementary bases (closed c i r c l e ? of region a i s involved i n the formation of the aminoacyl RNA omain, which thus contains a s h o r t bridge o f 2 n u c l e o t i d e s t h a t l i n k s the two p a r t s o f the stem composing the aminoacyl RNA domain. ( B ) Non t R N A - l i k e configuration t h a t t h e 3 ' region of BMV RNA could adopt i f regions a and e a r e not base-paired.

demonstrate t h a t base-pai r i n g s between loops and d i s t a n t l y 1 ocated regions i s fundamental f o r recognition by tRNA-specific p r o t e i n s . I t should a l s o be mentioned t h a t the RNAs of c e r t a i n viruses ( i . e . tomato aspermy virus, a cucumovirus, and tobacco r a t t l e virus, a t o b r a v i r u s ) can be adenylated a t t h e i r 3 ' terminus by t h e t R N A nucleotidyl t r a n s f e r a s e b u t cannot be aminoacylated i n v i t r o ( r e f s . 51, 52). Furthermore, sequencing of s a t e 1 1 i t e tobacco n e c r o s i s virus (STNV) RNA has revealed t h a t i t s 3 ' region can be folded i n t o a c l o v e r l e a f s t r u c t u r e w i t h an anticodon f o r AUG i n an a p p r o p r i a t e p o s i t i o n ( r e f . 53). However, i t i s n o t known whether STNV RNA can be charged w i t h methionine o r w i t h any o t h e r amino acid.

B188

6.4.2

Model P a r t n e r s f o r tRNA-SDecific P r o t e i n s I t i s i n t e r e s t i n g t o p o i n t o u t some s t r i k i n g s t r u c t u r a l d i f f e r e n c e s between p l a n t v i r a l tRNA-1 ike s t r u c t u r e s and tRNAs. A t f i r s t glance, t h e f o l d i n g o f t h e tRNA-like r e g i o n o f TYMV RNA may l o o k q u i t e s i m i l a r t o t h a t o f t R N A s . However, a fundamental d i f f e r e n c e e x i s t s i n t h e f o r m a t i o n o f t h e a c c e p t o r stem. I n tRNAs, t h e 5 ' p a r t o f t h e molecule p a r t i c i p a t e s i n t h e f o r m a t i o n o f t h e a c c e p t o r stem whereas i n t h e v i r a l RNA t h e a c c e p t o r stem i s formed by f o l d i n g o f o n l y t h e 3 ' p a r t , w i t h o u t involvement o f t h e 5 ' p a r t o f t h e tRNA-like r e g i o n . Therefore, t h e aminoacyl RNA domain c o n s t i t u t e s an independent r e g i o n i n t h e tRNA-like s t r u c t u r e o f TYMV RNA. I n t h e tRNA-like s t r u c t u r e o f TMV RNA, t h e aminoacyl RNA domain i s comparable t o t h a t o f TYMV RNA. However, t h e a n t i c o d o n RNA domain i s formed i n a c o m p l e t e l y d i f f e r e n t manner, by a s i n g l e l o n g stem and loop, whereas i n TYMV RNA i t i n v o l v e s two d i s t i n c t stems and loops. F i n a l l y , t h e f o l d i n g o f t h e tRNA-like r e g i o n o f BMV RNA r e v e a l s a completely o r i g i n a l way o f r e a c h i n g an 'L'-shaped conformation i n which no element o f t h e c l o v e r l e a f p a t t e r n o f tRNAs can be d i s t i n g u i s h e d . Because o f these p e c u l i a r f o l d i n g s , these v i r a l RNAs c o n s t i t u t e u s e f u l model s u b s t r a t e s t o e l u c i d a t e t h e RNA c o n f o r m a t i o n a l requirements o f c e r t a i n tRNA-specific enzymes. Indeed, i t has been e s t a b l i s h e d u s i n g these v i r a l RNAs t h a t t h e aminoacyl RNA domain suffices f o r interaction with the tRNA nucleotidyl transferase ( r e f s . 37-39) and w i t h t h e p e p t i d e c h a i n e l o n g a t i o n f a c t o r EF-Tu o r EF-lo ( r e f s . 54, 55). Moreover, t h e f o l d i n g o f t h e tRNA-like r e g i o n s i n t h e v i r a l RNAs s t r o n g l y suggests t h a t an 'L'-shaped conformation i s r e q u i r e d f o r r e c o g n i t i o n by aminoacyl -tRNA synthetases. I n t h i s r e s p e c t , a more d e t a i l e d s t u d y o f t h e i n t e r a c t i o n o f p l a n t v i r a l RNAs w i t h aminoacyl-tRNA synthetases m i g h t shed some l i g h t on t h e s p e c i f i c i t y o f r e c o g n i t i o n o f tRNAs by these enzymes. tRNA-LIKE FEATURES I N OTHER NUCLEIC ACIDS On t h e b a s i s o f p o s s i b l e f o l d i n g and / o r sequence homologies w i t h canonical tRNAs, tRNA-1 ike f e a t u r e s have been d e s c r i b e d f o r v a r i o u s r e g i o n s o f many o t h e r v i r a l o r c e l l u l a r n u c l e i c a c i d s .

6.5

B189

These examples a r e reviewed below. Bacterioohaae-Encoded 'SDecies I' RNA D u r i n g i n f e c t i o n o f E . c o l i w i t h c e r t a i n DNA bacteriophages (T2, T4 o r T6), t h e r e appears a p h a g e - s p e c i f i c s t a b l e RNA d e s i g nated 'Species I' o f 140 n u c l e o t i d e s whose 3'-end t e r m i n a t e s by t h e sequence -CCA. Two p a r t s o f t h i s tRNA, n u c l e o t i d e s 46 t o 67 and 87 t o 140, b o t h o f which resemble about one h a l f o f a t R N A molecule, can be f o l d e d i n t o a c l o v e r l e a f c o n f i g u r a t i o n ( r e f . 56). The f u n c t i o n o f t h i s RNA remains unknown. 6.5.1

RNA o f D e f e c t i v e I n t e r f e r i n a P a r t i c l e s o f S i n d b i s V i r u s I t i s a w e l l known phenomenon t h a t s e r i a l h i g h m u l t i p l i c i t y passages o f DNA o r RNA v i r u s e s i n animal c e l l c u l t u r e s l e a d t o v i r u s p o p u l a t i o n s e n r i c h e d i n p a r t i c l e s designated d e f e c t i v e i n t e r f e r i n g (DI) p a r t i c l e s . Usually, t h e n u c l e i c a c i d contained i n t h e D I p a r t i c l e s i s much s m a l l e r t h a n t h e v i r a l genome, b u t i t c o n t a i n s a t l e a s t t h e s i g n a l s f o r r e p l i c a t i o n and e n c a p s i d a t i o n . Thus, t h e D I p a r t i c l e s m u l t i p l y o n l y i n t h e presence o f t h e standard v i r u s and due t o i t s small s i z e , t h e n u c l e i c a c i d o f t h e D I p a r t i c l e s r e p l i c a t e s more e f f i c i e n t l y than t h a t o f t h e s t a n d a r d v i rus. I t was observed t h a t t h e RNA e x t r a c t e d f r o m c e r t a i n D I p a r t i c l e s o f S i n d b i s v i r u s c o n t a i n s a t i t s 5 ' end a r e g i o n t h a t i s remarkably s i m i l a r t o c e l l u l a r t R N A A s P as r e v e a l e d by cDNA sequencing ( r e f . 57). Although t h i s r e g i o n l a c k s t h e n i n e 5 ' - t e r m i n a l n u c l e o t i d e s o f t h e a c c e p t o r stem o f t R N A A S p , i t d i f f e r s by o n l y two bases from t h i s t R N A . The 3 ' -CCA end i s n o t f r e e , b u t i s c o v a l e n t l y bound t o t h e remainder o f t h e S i n d b i s v i r u s - s p e c i f i c RNA o f t h e D I p a r t i c l e , and consequently cannot be aminoacylated. One does n o t understand how such a sequence appeared i n t h e RNA o f t h e D I p a r t i c l e s and what r o l e i t m i g h t p l a y i n D I p a r t i c l e p r o p a g a t i o n s i n c e i t i s absent from t h e RNA o f t h e s t a n d a r d v i r u s . I t i s w o r t h m e n t i o n i n g here t h e e a r l y o b s e r v a t i o n s t h a t t h e RNAs o f two animal v i r u s e s (Mengo v i r u s and encephalomyocardi t i s v i r u s ) c o u l d b i n d h i s t i d i n e and s e r i n e i n v i t r o r e s p e c t i v e l y . Since t h e genome of these v i r u s e s t e r m i n a t e s a t t h e 3 ' end by a poly(A) sequence, i t seems obvious t h a t a m i n o a c y l a t i o n can o n l y occur a t some i n t e r n a l s i t e o f fragmented RNA ( f o r a r e v i e w see r e f . 35). 6.5.2

B190

6.5.3

Polvoma v i r u s DNA I n a d d i t i o n t o t h e s e examples o f r e g i o n s i n v i r a l RNAs c o n t a i n i n g f e a t u r e s o f tRNAs, a n o t h e r example has been p r o v i d e d b y sequence a n a l y s i s o f Polyoma v i r u s DNA: t w o r e g i o n s o f t h e genome (between p o s i t i o n s 715-799 and between p o s i t i o n s 5160-5231) can each be made t o f o l d i n t o a c l o v e r l e a f - 1 ik e manner ( r e f s . 58,59). 6.5.4

h i s ODeron mRNA

I t i s w e l l e s t a b l i s h e d t h a t i n t h e b a c t e r i a l operons f o r t h e b i o s y n t h e t i c pathways o f c e r t a i n amino a c i d s , one o f t h e c o n t r o l s a t t h e t r a n s c r i p t i o n l e v e l i s achieved by a t t e n u a t i o n , i . e . t r a n s l a t i o n a l c o n t r o l o f t r a n s c r i p t i o n t e r m i n a t i o n . The 5 ' l e a d e r r e g i o n o f t h e mRNA o f t h e operon encodes a s m a l l p e p t i d e r i c h i n t h e amino a c i d whose s y n t h e s i s i s s p e c i f i e d b y t h e operon. Dependi n g on t h e e f f i c i e n c y o f t r a n s l a t i o n o f t h i s l e a d e r mRNA and t h e r e f o r e on t h e amount o f t R N A e s t e r i f i e d w i t h t h e amino a c i d s p e c i f i e d b y t h e operon, t h e l e a d e r r e g i o n adopts e i t h e r a s t r u c t u r e ( a t t e n u a t o r ) t h a t provokes t e r m i n a t i o n o f t r a n s c r i p t i o n a t t h e end o f t h e l e a d e r r e g i o n , o r a n o t h e r p o s s i b l e s t r u c t u r e ( a n t i a t t e n u a t o r ) t h a t a l l o w s t r a n s c r i p t i o n t o proceed i n t o t h e s t r u c t u r a l genes o f t h e operon. The l e a d e r r e g i o n o f t h e h i s operon mRNA i n S a l m o n e 7 7 a t y p h y m u r i u m can a d o p t t w o a1 t e r n a t i v e c o n f i g u r a t i o n s ( r e f . 60): one o f them i s r e m i n i s c e n t o f t h e c l o v e r l e a f s t r u c t u r e o f t R N A w i t h stems and l o o p s analogous t o t h o s e found i n tRNAH s . I t has been suggested t h a t enzymes s p e c i f i c o f tRNAHiS m i g h t a l s o i n t e r a c t w i t h t h e l e a d e r r e g i o n o f t h e mRNA and m o d u l a t e t h e s t a b i l i t y o f t h i s configuration t o e i t h e r favour o r hinder e a r l y transc r i p t i o n termination.

6.5.5

Threonvl-tRNA S v n t h e t a s e mRNA E x p r e s s i o n o f t h e E . c o 7 i threonyl-tRNA s y n t h e t a s e gene i s regulated a t the translational level. Interestingly, a cloverleafl i k e f o l d i n g has been proposed f o r t h e 5 ' r e g i o n o f t h e mRNA c o d i n g f o r t h e threonyl-tRNA s y n t h e t a s e ( r e f . 61). I t has been p o s t u l a t e d t h a t t h e threonyl-tRNA s y n t h e t a s e c o u l d b i n d t o t h e 5 ' r e g i o n o f i t s own mRNA and t h e r e b y i n h i b i t t r a n s l a t i o n . 6.5.6

Glvcvl-tRNA S v n t h e t a s e Gene F i n a l l y , i t i s noteworthy t h a t i n t h e E . c o l i glycyl-tRNA s y n t h e t a s e gene ( r e f . 62) t h e r e e x i s t s a 12 n u c l e o t i d e - l o n g

B191

sequence s i m i l a r t o t h e one found i n t h e a n t i c o d o n r e g i o n o f E . c o l i t R N A t L y . I t has been proposed t h a t glycyl-tRNA synthetase m i g h t b i n d t o t h i s sequence a t t h e l e v e l o f t h e DNA, t h e r e b y r e g u l a t i n g t h e expression o f i t s gene. I t i s i m p o r t a n t t o p o i n t o u t t h a t i n a l l t h e s e examples o f tRNA-like f e a t u r e s i n v a r i o u s n u c l e i c a c i d s , i t i s n o t known whether t h e y can i n t e r a c t w i t h tRNA-specific enzymes. Indeed, these f e a t u r e s have so f a r been d e s c r i b e d s o l e l y on t h e b a s i s o f sequence analogy w i t h tRNAs and/or p o s s i b l e c l over1 eaf-1 ike f o l d i n g . T h i s c o n t r a s t s w i t h t h e tRNA-like s t r u c t u r e s i n p l a n t v i r a l RNA genomes whose presence was d i s c o v e r e d because o f t h e enzymatic r e a c t i o n s t h a t these RNAs c o u l d undergo w i t h c e r t a i n tRNA-specific p r o t e i n s .

6.6

FUTURE PROSPECTS AND IMPACT The p r i n c i p l es t h a t determine speci f ic i t y in RNA-protei n i n t e r a c t i o n s as w e l l as t h e m o l e c u l a r mechanisms i n v o l v e d i n t h e f u n c t i o n o f r i b o n u c l e o p r o t e i n complexes a r e as y e t o n l y p o o r l y understood. I n t h i s r e s p e c t , s t u d i e s focused on tRNAs a r e p a r t i c u l a r l y i n t e r e s t i n g because o f t h e g r e a t number o f macromolec u l e s w i t h which t h e y can i n t e r a c t . I t i s e v i d e n t f r o m t h e p r e s e n t r e v i e w t h a t a p a r a l l e l i n v e s t i g a t i o n o f tRNAs and c e r t a i n t R N A l i k e molecules i n view o f c o r r e l a t i n g s t r u c t u r a l p e c u l i a r i t i e s w i t h b i o l o g i c a l r e c o g n i t i o n and/or s p e c i a l i z e d f u n c t i o n m i g h t be e x t r e m e l y h e l p f u l i n d e f i n i n g these p r i n c i p l e s .

6.7

SUMMARY One o f t h e key concepts i n m o l e c u l a r b i o l o g y has been t h a t s i m i l a r s t r u c t u r e s d i s p l a y s i m i l a r p r o p e r t i e s and f u l f i l l s i m i l a r f u n c t i o n s . A c l o s e l o o k i n t o t h e s t r u c t u r e , r e c o g n i t i o n and f u n c t i o n s o f t R N A and o f some tRNA-like molecules r e v e a l s a much g r e a t e r c o m p l e x i t y i n understanding s t r u c t u r e - f u n c t i o n r e l a t i o n s h i p s i n n u c l e i c a c i d s as w e l l as i n t h e i r b i o l o g i c a l r e c o g n i t i o n t h a n has p r e v i o u s l y been surmised. 6.8

ACKNOWLEDGEMENTS We thank F. C h a p e v i l l e f o r h i s c o n s t a n t encouragements. T h i s work was supported i n p a r t by a g r a n t from t h e " E c o l e P r a t i q u e des Hautes Etudes".

B192

6.9

1. 2. 3.

4.

5. 6.

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GUY DIRHEIMER and ROBERT P

. MARTIN

I n s t i t u t de Biologie Uoleculaire e t C e l l u l a i r e U n i v e r s i t e L o u i s P a s t e u r . 15 r u e R e n e D e s c a r t e s . bourg. France

d u CNRS and 67084 S t r a s -

TABLE OF CONTENTS Introduction 7.1 Is01 a t i o n of mi tochondri a1 tRNAs . . . . . . . . . 7.2 S a c c h a r o m y c e s c e r e v i s i a e Mitochondrial tRNAs 7.3 7.3.1 t R N A I s o a c c e p t o r s and T h e i r Coding O r i g i n 7.3.2 Structures . . . . . . . . . . . . . . . Modified Nucleosides . . . . . . . . . . 7.3.3 7.3.4 Codon Reading P a t t e r n s T o r u l o p s i s g l a b r a t a Mitochondrial tRNAs 7.4 7.4.1 Structures 7.4.2 Codon Reading P a t t e r n s . . . . . . . . . N e u r o s p o r a c r a s s a Mitochondrial tRNAs . . . . . . 7.5 Structures 7.5.1 7.5.2 Modified Nucleosides 7.5.3 Codon Reading P a t t e r n s A s p e r g i l l u s n i d u l a n s Mitochondrial tRNAs . . . . 7.6 Structures . . . . . . . . . . . . . . . 7.6.1 7.6.2 Codon Reading P a t t e r n s . . . . . . . . . S c h i z o s a c c h a r o m y c e s pombe Mitochondrial tRNAs . . 7.7 7.7.1 Structures 7.7.2 Modified Nucleosides P o d o s p o r a a n s e r i n a Mitochondrial tRNAs ..... 7.8 Structures 7.8.1 Codon Reading P a t t e r n s 7.8.2 T e t r a h y m e n a p y r i f o r m i s Mitochondrial tRNAs 7.9 Structures 7.9.1 Modified Nucleosides . . . . . . . . . . 7.9.2 Codon Reading P a t t e r n s . . . . . . . . . 7.9.3

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. . B200 . . . . B201 . . B201 . . 8204 . . 8206 . . . . . . . . . . . B208 . . . . . . . B214 . . . . . . . . . . . . B214 . . B216 . . 8216 . . . . . . . . . . . . 8216 . . . . . . . . . . . . B217 . . . . . . . . . . . B217 . . 8218 . . B219 . . B219 . . 8220 . . . . . . . . . . . . 8220 . . . . . . . . . . . . B221 . . 6223 . . . . . . . . . . . . 8223

. . . . . . . . . . . 8223 . . . . . 8224 . . . . . . . . . . . . . . . . . B224 . .

B225

. . 8225

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7.10

7.11

7.12

7.13

... ........ . . Mosquito Mitochondrial tRNAs . . . . 7.11.1 S t r u c t u r e s . . . . . . . . 7.11.2 Modified Nucleosides . . . 7.11.3 Codon Reading P a t t e r n s . . D r o s o p h i l a Mitochondrial tRNAs . . . 7.12.1 S t r u c t u r e s . . . . . . . . 7.12.2 Codon Reading P a t t e r n s . . P a r a m e c i u m Mi tochondrial tRNAs 7.10.1 S t r u c t u r e s 7.10.2 Codon Reading P a t t e r n s

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7.17 7.18 7.19

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Mitochondrial tRNAs . Structures . . . . . . . . Codon Reading P a t t e r n s . . . Mammal ian Mi tochondrial tRNAs 7.14.1 S t r u c t u r e s . . . . . . . . . 7.14.2 Modified Nucleosides . 7.14.3 Codon Reading P a t t e r n s . . 7.14.4 Tumors Cell Mi tochondri a1 tRNAs A s c a r i s suum Mi tochondrial tRNAs . . . . 7.15.1 Structures . . . . . . . . . . 7.15.2 Codon Reading P a t t e r n s . . . . Plant Mitochondrial tRNAs . . . . . . . 7.16.1 S t r u c t u r e s . . . . . . . . . . ... 7.16.2 Modified Nucleosides ... 7.16.3 Codon Reading P a t t e r n s General Remarks and Conclusions . . . . Acknowledgements . . . . . . . . . . . . References . . . . . . . . . . . . . . .

Xenopus leavis

7.13.1 7.13.2

7.14

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. . . . . . . B228 . . . . . . . B228 . . . . . . . 8230 . . . . . . . 8232 . . . . . . . 8232 . . . . . . . B232 . . . . . . . 8233 . . . . . . . B233 . . . . . . . 8236 . . . . . . . B236 . . . . . . . B238 . . . . . . . B241 . . . . . . . B241 . . . . . . . 8243 . . . . . . . 8243 . . . . . . . B244 . . . . . . . 8245 . . . . . . . 8246 . . . . . . . B247 . . . . . . . B252 . . . . . . . 8253

INTRODUCTION Mitochondria (along with c h l o r o p l a s t s ) occupy a unique p o s i t i o n among c e l l u l a r o r g a n e l l e s because they possess a s e p a r a t e genome and a l l the machinery f o r t r a n s c r i b i n g and t r a n s l a t i n g t h e i r g e n e t i c information . Research i n molecular biology of mitochondria during the p a s t decade has provided a number of unexpected r e s u l t s about t h e way in which the mitochondria1 DNA (rnt DNA) i s expressed . These r e s u l t s have c o n t r i b u t e d novel i n s i g h t s i n t o gene expression mechanisms ( e . g . intron splicing. 7.1

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codon r e c o g n i t i o n , g e n e t i c code) and have s u b s t a n t i a l l y lowered our assessment of the minimal requirements f o r f u n c t i o n a l components of a g e n e t i c system. The m t DNA i s f u n c t i o n a l l y conservative, encoding b a s i c a l l y the same genes i n a l l eucaryotes examined t o d a t e . Although most of the mi tochondrial p r o t e i n s a r e synthesized from n u c l e a r gene products, a few a r e specified by mitochondrial genes ( f o r r e c e n t reviews, see r e f s . 1-6). These include the l a r g e r t h r e e s u b u n i t s of cytochrome oxidase, apocytochrome b , two t o three subunits o f t h e membrane p a r t of the ATPase complex and one s u b u n i t of t h e non-membrane F, p a r t ( i n p l a n t mitochondria o n l y ) . In a d d i t i o n , t h e r e a r e several unassigned reading frames ( r e f e r r e d t o a s URFs). Some of t h e intron-encoded reading frames ( n o t found i n animal m t DNA) code f o r maturases involved i n i n t r o n s p l i c i n g . Of the e i g h t URFs found i n mammalian m t DNA, seven have r e c e n t l y been assigned t o NADH-ubiquinone oxidoreductase subunits and the e i g h t h t o subunit 8 of ATPase ( r e f s . 7,8). V i r t u a l l y a l l of the p r o t e i n components of t h e mitochondrial t r a n s l a t i o n machinery a r e coded f o r by nuclear genes, t h e only exception b e i n g a mitoribosomal small s u b u n i t p r o t e i n ( t h e varl p r o t e i n i n Saccharomyces c e r e v i s i a e and T o r u l o p s i s g l a b r a t a and a S5-like p r o t e i n i n Neurospora crassa). In c o n t r a s t , t h e RNA components of t h e t r a n s l a t i o n apparatus, i . e . a f u l l s e t of tRNAs and two rRNAs, a r e t r a n s c r i b e d from the m t DNA. In a d d i t i o n , p l a n t mitochondrial genomes a l s o code f o r a 5 s rRNA not found i n non-plant mitoribosomes ( r e f . 9 ) . F i n a l l y , a novel gene on Saccharomyces c e r e v i s i a e m t DNA, coding f o r a 9s RNA necessary f o r t R N A synthesis, has been reported ( r e f . 10).

This review d e a l s w i t h the s t r u c t u r e and codon recognition p a t t e r n s of m t tRNAs. Nearly t h r e e hundred m t t R N A s t r u c t u r e s

from organisms of d i f f e r e n t phylogenetic o r i g i n s have been d e t e r mined, e i t h e r d i r e c t l y by RNA sequencing o r i n d i r e c t l y by gene sequencing. However, DNA sequencing does not show whether t h e gene i s actual l y t r a n s c r i b e d i n t o a functional t R N A , especi a1 l y s i n c e many animal m t tRNAs e x h i b i t a t y p i c a l c l o v e r l e a f s t r u c t u r e s . Furthermore, t h e gene sequence alone may not be s u f f i c i e n t t o deduce the s p e c i f i c i t y of a g i v e n t R N A because p o s t - t r a n s c r i p t i o n a1 base modification may i n f l u e n c e the codon recognition p a t t e r n ,

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and because the mitochondria1 g e n e t i c code d i f f e r s from the standard code. T h u s , a c e r t a i n ambiguity may e x i s t which can only be solved by d i r e c t sequencing of the t R N A (including d e t e r mination of modified r e s i d u e s ) , by studying i t s decoding propert i e s i n in v i t r o t r a n s l a t i o n systems, and by comparing the amino acid sequence o f mi tochondri a1 p r o t e i n s wi t h the nucl eoti de sequence of t h e i r genes. 7.2

I S O L A T I O N OF MITOCHONDRIAL t R N A s

Since the work of Wintersberger ( r e f . 11) one knows t h a t mitochondria contain t h e i r own tRNAs which a r e d i s t i n c t from t h e cytoplasmic tRNAs and coded f o r by t h e m t DNA. However, opinions have long been divergent a s t o the number of n a t i v e t R N A s p e c i e s w i t h i n the o r g a n e l l e . This came from t h e d i f f i c u l t i e s of i s o l a t i ng mi tochondri a not contami nated by cytopl asmj c materi a1 . For example, s i n c e the content of t R N A i n cytoplasm i s about 100 times higher than the content i n the mitochondria, a contamination by 1% o f c y t o s o l i c material doubles t h e amount o f t R N A i n the mitochond r i a l preparations. Therefore, a c a r e f u l p r e p a r a t i o n o f the organel 1 es i s necessary t o i sol a t e uncontaminated m t tRNAs ( r e f . 12). D i f f e r e n t types of col umn chromatographic systems have been described f o r the p u r i f i c a t i o n of e u b a c t e r i a l o r e u c a r y o t i c cytoplasmic tRNAs. However, they mostly s t a r t from r a t h e r l a r g e q u a n t i t i e s of t R N A ( a t l e a s t 10-50 mg) which i s not easy t o prepare from mitochondria. We t h e r e f o r e have adapted the twodimensional polyacrylamide gel e l e c t r o p h o r e t i c system o r i g i n a l l y described by Fradin e t al. ( r e f . 1 3 ) , which proved t o be s u i t a b l e f o r microscale i s o l a t i o n of S a c c h a r o m y c e s c e r e v i s i a e m t tRNAs ( r e f . 14). This method has a l s o been used f o r the i s o l a t i o n of m t tRNAs from various o t h e r sources. In the case of 5. c e r e v i s i a e m t tRNAs, more than h a l f of t h e s p e c i e s were c l e a r l y resolved by two-dimensional gel e l e c t r o phoresis whereas o t h e r t R N A s p o t s were confluent o r contained more than one s p e c i e s . In t h e l a t t e r case, preliminary column chromatography i n the RPC-5 system ( r e f . 15) was required (Figure 7 . 1 ) . Fractionation of 0.5 mg t o t a l m t t R N A yielded from 1 t o 12 pug of pure tRNAs, depending on t h e s p e c i e s . These q u a n t i t i e s a r e

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sufficient for analysis of their nucleotide sequence using postlabelling techniques (see G. Keith, Volume I, Chapter 3 ) . 7 . 3 5ACCHAROlYCfS CfREVZ5ZAf MITOCHONDRIAL t R N A s 7.3.1 tRNA IsoacceDtors and Their Codinq Oriqin The number of 5. c e r e v i s i a e mt tRNA species and their coding

origin were examined using two fractionation methods: RPC-5 col umn chromatography and two-dimensional gel electrophoresis (refs. 14, 16, 17). RPC-5 chromatography of mt tRNA (Figure 7.1) a1 lowed the characterization of the isoacceptors corresponding to 19 amino acids (cysteine was not tested). Mitochondria1 tRNA

FRACTION

Figure 7 . 1 mt tRNA.

NUMBER

RPC-5 chromatography (pH 4.5) o f uncharged total yeast

could not be aminoacylated with glutamine. However, we detected two tRNA peaks which could be charged with glutamic acid, one of which could be subsequently amidified by the mitochondria1 enzyme extract which probably contains a transamidase, and thus cor-

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responds t o tRNAGLn. Two-dimensional p o l y a c r y l amide g e l e l e c t r o p h o r e s i s r e s o l v e d m t t R N A i n t o 24-26 m a j o r s p o t s and a few m i n o r F o r i d e n t i f i c a t i o n o f t h e tRNAs i n t h e d i f ones ( F i g u r e 7 . 2 ) . f e r e n t s p o t s , t h e g e l s were s t a i n e d w i t h m e t h y l e n e b l u e , t h e tRNAs e x t r a c t e d f r o m t h e e x c i s e d s p o t s and a m i n o a c y l a t e d u s i n g a m i t o c h o n d r i a l enzyme e x t r a c t ( r e f . 14). T a b l e 7 . 1 shows t h a t t h e number o f t R N A i s o a c c e p t o r s s e p a r a t e d b y RPC-5 c h r o m a t o g r a p h y i s h i g h e r t h a n f o u n d b y t w o - d i m e n s i o n a l g e l e l e c t r o p h o r e s i s . T h i s i s due t o t h e h i g h e r s e n s i t i v i t y o f RPC-5 chromatography t o tRNA t e r t i a r y s t r u c t u r e and p o s t - t r a n s c r i p t i o n a l base m o d i f i c a t i o n .

I:Scr I 2 : SrrZ

-

J : 8rrJ 4: Leu 5 :Tyr

2

6 : Met I f 1 7 ALU K : ALI) 9 ThrZ*J 10 T r p 1 1 : Lys 2 12 : Metlml / J : Cln 14 : L L c t T h r I 16 : ArgPtVoLZ-J 17 , VUL I 18 . Phc 19 : Phe ZO ' H i s 2/.1sn 22 : P r o ZJ : P r o 2 4 Glu ~

0 zo

Z5 :G l y 26 I Asp

2s : G l y

x

:

LYSf

C :Argl

F i g u r e 7.2 I d e n t i f i c a t i o n o f t h e resolved yeast m t tRNA species c o r r e s p o n d i n g t o 20 amino a c i d s , e x c e p t c y s t e i n e . F o r example, one tRNA can g i v e t w o o r more peaks i n t h e RPC-5 column chromatography i f i t s c o n f o r m a t i o n i s n o t s t a b l e . This i s t h e c a s e o f t R N A P h e w h i c h gave 2-3 peaks on t h e RPC-5 column ( F i g u r e 7.1) a1 though t h e n u c l e o t i d e sequences o f t h e s e i s o a c c e p t o r s p r o v e d t o be i d e n t i c a l , i n c l u d i n g p o s t - t r a n s c r i p t i o n a l modifications. On t h e o t h e r hand, RPC-5 c h r o m a t o g r a p h y can a l s o separate isoacceptors d i f f e r i n g by o n l y p o s t - t r a n s c r i p t i o n a l m o d i f i c a t i o n s . T h i s i s t h e case o f t R N A ; e r and t R N A g e r w h i c h o n l y d i f f e r b y t h e r e s i d u e a t t h e 5 ' end o f t h e a n t i c o d o n stem ( p o s i 1

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Table 7.1 Nwiihcr o f isoacceptors compared to thc numher of c o d o n s Nunilxr o f isoacceptors fractionated by Aniirio acid

Ala Arg Asn Asp

Glu + Gin GiY His

Ile Leu LYS

Met Phe Pro Ser Thr

TrP TYr Val Total

two-dimensional clcctrophoresis

2

2 1 1 2 2 I I

2 2 2

2 3 2 1

1 2-3 30-32

RPC-5 chromatography

3 2 1

1 1+1 2

2 1

3 2 3 2-3 2 3 3 3 3 3 42

Nurnbcr of codons (genetic code)

4

6 2 2 2+2 4 2 3 6 2 1

2 4 6 4 1

2 4

tion 27 in the standard cloverleaf): U27 (tRNASer) is modified to 11, in tRNASer (ref. 18). Therefore, the number of isoacceptors found by two-dimensional gel electrophoresis better reflects the number of tRNA genes on mt DNA. To determine the coding origin of the mt tRNAs, [32P]-mt tRNA was hybridized to mt DNA; the tRNAs were then eluted from the hybrids and analyzed by 2D electrophoresis. Only one spot was lacking in the hybridized mt tRNA pattern (Figure 7.3) compared to the control which consisted of non-hybridized mt tRNA (ref. 17). It corresponds to one of the two lysine isoacceptors found in yeast mitochondria. We could show by sequence determination that

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i t i s a c y t o p l a s m i c species which i s i m p o r t e d i n t o t h e mitochondr i a b u t which i s n o t a c y l a t e d by t h e m i t o c h o n d r i a l aminoacyl-tRNA synthetase ( r e f . 19). We t h e r e f o r e hypothesized t h a t i t may have a r e g u l a t o r y f u n c t i o n t o c o o r d i n a t e d i v i s i o n o f c e l l and mitochond r i a . An a l t e r n a t i v e hypothesis c o u l d be a p r i m e r f u n c t i o n f o r e i t h e r rep1 ic a t i o n o f mt-DNA o r r e v e r s e t r a n s c r i p t i o n o f m i tochond r i a l i n t r o n s . F i n a l l y , S o i d l a ( r e f . 20) proposed t h a t t h i s t R N A may s e r v e as a guide-RNA i n t h e s p l i c i n g o f y e a s t m i t o c h o n d r i a l precursors. The f u n c t i o n o f t h i s t R N A L y S i n y e a s t m i t o c h o n d r i a deserves f u r t h e r i n v e s t i g a t i o n s .

7.3.2

Structures The s t r u c t u r e s o f 17 y e a s t m i t o c h o n d r i a l tRNAs were d e t e r mined i n o u r l a b o r a t o r y ; t h e y a r e t h e f o l l o w i n g : tRNA;;g and tRNAft;g ( r e f . 21)*, t R N A C L y ( r e f . 21), t R N A H i S ( r e f . 22), t R N A I l e ( r e f . 23), t R N A L e U ( r e f . 21), t R N A L y s ( r e f . 21). tRNA:et (ref. 23), tRNAYet ( r e f . 24), t R N A P h e ( r e f . 25), t R N A P r O ( r e f . 21), tRNA;;; ( r e f . 18), two tRNAs;;; ( r e f . 18), tRNA:;; ( r e f . 26), t R N A T r p ( r e f . 27) and t R N A T y r ( r e f . 28). The complete s e t o f m t t R N A genes has been sequenced, essent i a l l y i n t h e l a b o r a t o r i e s o f N.C. M a r t i n and A. T z a g o l o f f ; t h e y t R N A A L a ( r e f s . 29, 30), tRNA$;g ( r e f s . 29,31), tRNAft;: are : ( r e f s . 29, 31, 32), t R N A A S n ( r e f . 33), t R N A A s p ( r e f s . 16, 29, 31), t R N A C y S ( r e f s . 34, 35), t R N A G t n ( r e f . 36), t R N A G L u ( r e f s . 16, 37), t R N A G L y ( r e f s . 29, 31, 38), t R N A H i s ( r e f s . 34, 35), t R N A I L e ( r e f . 30), t R N A L e U ( r e f . 36), t R N A L y S ( r e f s 29, 31), tRNA:et (ref. ( r e f . 39), t R N A P h e ( r e f . 40), t R N A P r o ( r e f . 39), 33), t R N A Y e t tRNA;;; ( r e f s . 29, 31, 32), tRNA;;; ( r e f s . 40, 41), tRNA::; (ref. ( r e f . 35), t R N A T r p ( r e f . 16, 43), t R N A T y r ( r e f s . 30, 42), tRNA:;; 44, 451, t R N A v a L ( r e f s . 38, 42). The main f e a t u r e s t h a t can be deduced from t h e s e sequences are the following: - T h e i r G+C c o n t e n t i s v e r y low and v a r i e s from 18% (tRNAft;&) t o 41% ( t R N A T y r , t R N A H i s ) . -They c o n t a i n o n l y a few m o d i f i e d n u c l e o s i d e s .

* I n t h i s paper, ue s h a l l o n l y i n d i c a t e t he anticodons of t h e tRNAs corresponding t o t h e s i x codonf a m i l i e s ( a r g i n i n e and s e r i n e ) and t o t h r e o n i n e .

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F i g u r e 7.3 Se a r a t i o n o f [ 3 2 P ] - i n v i v o l a b e l e d y e a s t m t tRNAs by two-dimensionaf p o l acrylamide g e l e l e c t r o p h o r e s i s . I n the l e f t g e l autorachograms show t h e f r a c t i o n a t i o n o f tRNAs i s o f%!isrfrom u r i f i e d m i t o c h o n d r i a (A) and o f tRNAs e l u t e d from m i t o c h o n d r i a f t R N A / m i t o c h o n d r i a l DNA h y b r i d s (B). (A' , B ' ) Schemat i c drawings o f t h e s t a i n e d g e l s . Spots c o n t a i n i n g l e s s than 1% o f t h e t o t a l r a d i o a c t i v i t y (dashed c i r c l e s ) .

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- U n l i k e mammalian m t tRNAs, t h e y can be f o l d e d i n t o o r t h o d o x cloverleaves. However, e x c e p t i o n s t o r e s i d u e s w h i c h a r e in v a r i a n t o r semi -in v a r i a n t i n t h e s t a n d a r d c l o v e r 1 e a f a r e f o u n d f o r some s p e c i e s ; f o r example, i n tRNAgt;, t R N A T v r , and t R N A P r o ( F i g u r e 7.4) -Some m t tRNAs show o r i g i n a l s t r u c t u r a l f e a t u r e s w h i c h may have f u n c t i o n a l i m p l i c a t i o n s , f o r example: (i) tRNAPhe, t R N A L v S , and t R N A i e t have a n u c l e o t i d e w h i c h i s e x c l u d e d f r o m base p a i r i n g g i v i n g a b u l g e i n t h e T$C stem ( F i g u r e 7.5), and (ii)tRNA:;; has an unusual a n t i c o d o n l o o p composed o f 8 r e s i d u e s (see F i g u r e 7 . 7 ) . I n t e r m s o f o v e r a l l sequence s i m i l a r i t i e s t h e 5. c e r e v i s i a e m t tRNAs show l i t t l e resemblance t o t h e i r p r o c a r y o t i c and e u c a r y o t i c c y t o p l a s m i c c o u n t e r p a r t s . However, some m t tRNAs show s t r u c t u r a l f e a t u r e s w h i c h a r e o t h e r w i s e u n i q u e t o p r o c a r y o t i c tRNAs: f o r example, (i)t h e tRNATvr has a l a r g e v a r i a b l e l o o p (14 n u c l e o t i d e s ) s i m i l a r t o t h a t o f p r o c a r y o t i c tRNAsTvr (13 n u c l e o t i d e s ) , b u t d i f f e r e n t f r o m t h o s e o f c y t o p l a s m i c tRNATvr (5 n u c l e o t i d e s ) ; and (ii)t h e t R N A Y e t l a c k s t h e Watson-Crick b a s e - p a i r a t t h e end o f t h e a c c e p t o r stem and has a T$CA sequence i n l o o p I V . However, R, base w h i l e p r o c a r y o t i c i n i t i a t o r tRNAs l a c k t h e t y p i c a l Y,,: p a i r i n t h e i r D-stem h a v i n g A,, :U, , y e a s t m t t R N A Y e t has U,, :A2,. 7.3.3

M o d i f i e d Nucl e o s i d e s The 5. c e r e v i s i a e m t tRNAs c o n t a i n a l o w number ( f r o m 6 t o 9) o f m o d i f i e d n u c l e o s i d e s , w h i c h i s a c h a r a c t e r i s t i c o f m t tRNAs, whatever t h e i r o r i g i n . B e s i d e s T ( i n p o s i t i o n 54), $ ( p o s i t i o n s 27, 28, 31, 39 and 55) and D (one o r t w o r e s i d u e s i n t h e D-loop) which a r e p r e s e n t i n a l l t h e s t r u c t u r e s (except i n t R N A P h e which has no T ) , o n l y 3 o t h e r p o s i t i o n s o f t h e tRNA c l o v e r l e a f a r e sometimes m o d i f i e d . These a r e : (i) r e s i d u e 26, w h i c h i s m 2 G i n t R N A P r o , tRNATvr, tRNA:;;, and m$G i n t R N A G L y , tRNA;,':, tRNAPhe and tRNALe"; t h i s i s a e u c a r y o t i c f e a t u r e ( r e f . 46). (ii) r e s i d u e 37 w h i c h i s m o d i f i e d i n 15 o u t o f 17 tRNAs m o s t l y t o m1G b u t a l s o t o t 6 A o r i 6 A . I n contrast, and o f t R N A g t r , i s an r e s i d u e 37 o f t h e m i n o r tRNA;;! u n m o d i f i e d A; (iii) t h e wobble p o s i t i o n o f t h e a n t i c o d o n . The tRNAs w h i c h

A

p.

-

.

1 8 nucleotides i n the anticodon loop i n t R N A l h r

I

~~

Figure 7.4.

Exceptions to the invariant and semi-invariant nucleosides of tRNAs found in yeast mt tRNAs.

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u u A

c u u c c I

I

I

I

G A 'U '

I

A

G

G

A

tHNAMet m

A

tKNALYs

G

tRNAPhe

C

T Y

u c A C

G

I

I

G

C

A

C

C

I

I

I

U, ,G U

G C T

Y

u u A U

A

1

1

A

U

C

U

1

1

?,A U

C 1

G C U

Y

F i g u r e 7.5 N u c l e o t i d e s excluded from b a s e - p a i r i n g i n t h e T$C stem o f y e a s t m t tRNAs. r e c o g n i z e a E-codon f a m i l y ending i n a p u r i n e have a m o d i f i e d u r i d i n e showing m i g r a t i o n p r o p e r t i e s i d e n t i c a l t o t h o s e o f 5 - c a r b o x y m e t h y l ami n o m e t h y l u r i d i n e (cmnm5 U) ( F i g u r e 7.6), which has been i d e n t i f i e d i n t R N A G i v o f B a c i l l u s s u b t i l i s ( r e f . 47). The a n a l y s i s o f t h e e x a c t n a t u r e o f t h i s m o d i f i e d u r i d i n e i s c u r r e n t l y i n progress. I s o a c c e p t o r s d i f f e r i n g by o n l y p o s t - t r a n s c r i p t i o n a l m o d i f i c a t i o n s were a l s o found. For example, t h e r e s i d u e a t p o s i t i o n 27 o f t h e two tRNAsSer i s e i t h e r U ( t R N A j e r ) o r $ ( t R N A s e r ) , and t h e r e s i d u e a t p o s i t i o n 72 i n tRNA7.t i s e i t h e r U o r $ ( r e f s . 18, 24). 7.3.4

Codon Readina P a t t e r n s M i t o c h o n d r i a l p r o t e i n s y n t h e s i s uses a r e s t r i c t e d number o f m t DNA-coded tRNAs (24 species), which i s f a r below t h e minimal

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I

Ribose

cmnm5 u

Figure 7.6 Structure of 5-carboxymethylaminomethyluridine (cmnm5U)

G

*

A pG - C U - A-70 A - U A - U 5-A - U U - A A - U-" : 6 IJ u GAUUC D A A '?A I I I I CU$AG UUUA I

G

D

2

I

I

I

AAAU 2J.n?G V-A A-U U-A G

Figure 7.7

u

u

A A ~

T

w

A c-u

U-45

- C-40

Structure of yeas, mt tRNA

i r \G *

number of tRNAs, i . e . 32, necessary to translate all the codons in the genetic code according to the wobble hypothesis (ref. 48). This deficit is not overcome by the import into the organelle of nuclear-coded tRNA species (refs. 17, 19). From the sequence determination of 6 N e u r o s p o r a c r a s s a mi tochondri a1 tRNAs (ref. 49), the following codon recognition rules have been proposed: the tRNAs recognizing a 4-codon family have an unmodified U in the wobble position of their anticodon, whilst tRNAs which recognize a

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This allows 2-codon f a m i l y e n d i n g i n a p u r i n e have a m o d i f i e d U. d i s c r i m i n a t i o n i n t h e m i x e d f a m i l i e s between codons e n d i n g i n a From t h e d e t e r m i n a t i o n p u r i n e and codons e n d i n g i n a p y r i m i d i n e . o f t h e y e a s t m t t R N A gene sequences and f r o m o u r d e t e r m i n a t i o n s o f t h e p r i m a r y s t r u c t u r e s o f 17 m t tRNAs, i t appears t h a t t h e same r u l e s a r e a l s o o p e r a t i v e i n y e a s t m i t o c h o n d r i a ( r e f s . 21, 50). Among t h e 17 S . c e r e v i s i a e m t tRNAs we have sequenced, t R N A G 1 y , t R N A P r O , tRNA;;; and tRNA::;, whose amino a c i d s use 4codon f a m i l i e s (GGN, CCN, UCN, and CUN, r e s p e c t i v e l y ) a l l c o n t a i n In an u n m o d i f i e d U i n t h e f i r s t p o s i t i o n o f t h e a n t i c o d o n . c o n t r a s t , tRNA;;g, tRNA:;;:, t R N A L y S and t R N A T r p , which recognize 2-codon f a m i l i e s e n d i n g i n a p u r i n e (AGR, UUR, AAR, and UGR, r e s p e c t i v e l y ) a l l c o n t a i n a modifed U i n t h e wobble p o s i t i o n . Moreover, t h i s m o d i f i e d u r i d i n e appears t o be t h e same i n t h e s e 4 I t i s most l i k e l y t o be cmnm5U (see above). Murao and tRNAs. I s h i k u r a ( r e f . 47) have shown t h a t i n r i b o s o m e b i n d i n g e x p e r i ments, cmnm5U r e c o g n i z e s b o t h A and G, and t o a l e s s e r e x t e n t , However, p r o t o n NMR a n a l y s e s o f d i f f e r e n t m o d i f i e d a l s o U. u r i d i n e s ( r e f . 51) s u g g e s t t h a t cmnm5U i n t h e f i r s t p o s i t i o n o f t h e a n t i c o d o n s h o u l d be p r e f e r e n t i a l l y i n t h e C3'-endo conformation. T h i s enhances t h e r i g i d i t y o f t h e a n t i c o d o n , so as t o p r o h i b i t t h e m i s r e c o g n i t i o n o f codons t e r m i n a t i n g i n a p y r i m i d i n e . Such a r i g i d i t y o f cmnm5U w o u l d a l s o n o t f a v o r t h e p a i r i n g w i t h G. This could explain the strong bias against u t i l i z a t i o n o f G i n the 3 r d p o s i t i o n o f m i x e d codon f a m i l i e s e n d i n g i n a p u r i n e i n y e a s t m i t o c h o n d r i a1 p r o t e i n c o d i n g genes. The p r e s e n c e o f an u n m o d i f i e d u r i d i n e i n t h e w o b b l e p o s i t i o n o f tRNAs w h i c h r e a d 4-codon f a m i l i e s i s unusual and c o n t r a s t s w i t h t h e s i t u a t i o n i n b o t h p r o k a r y o t i c and e u k a r y o t i c c y t o p l a s m i c tRNAs, i n w h i c h a u r i d i n e i n t h e f i r s t p o s i t i o n o f t h e a n t i c o d o n i s a l w a y s m o d i f i e d . The o n l y e x c e p t i o n t o t h i s i s a y e a s t c y t o w h i c h has been shown t o r e a d a l l 4 codons o f t h e p l a s m i c tRNA:;;, CUN l e u c i n e f a m i l y ( r e f . 5 2 ) . C r i c k , i n h i s wobble hypothesis ( r e f . 48), n o t e d t h a t U:U and U:C base p a i r i n g s were p o s s i b l e , b u t d i s c o u n t e d them on t h e b a s i s t h a t t h e y were t o o c l o s e and w o u l d cause m i s r e a d i n g i n t h e m i x e d codon f a m i l i e s , G r o s j e a n e t al. ( r e f . 53) have examined t h e s t a b i l i t y o f a number o f a n t i c o d o n a n t i c o d o n i n t e r a c t i o n s and c o n c l u d e d t h a t t h e o n l y s t a b l e non-

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wobble p a i r s i n v o l v e U p a i r i n g w i t h e i t h e r U o r C. Thus, an unmodified u r i d i n e i s t h e o n l y base t h a t can p o s s i b l y f o r m a more o r l e s s s t a b l e p a i r w i t h a l l 4 n u c l e o t i d e s i n t h e wobble p o s i t i o n of t h e codon. The o n l y e x c e p t i o n t o t h i s r u l e i s p r o v i d e d by t h e which belongs t o t h e 4-codon f a m i l y CGN, b u t has y e a s t m t tRNA:;;! an unmodified A i n t h e f i r s t p o s i t i o n o f t h e a n t i c o d o n ( r e f . 21). The presence o f an unmodified A i n t h i s p o s i t i o n has never been I n f a c t , A i s always m o d i f i e d found i n any n o n - o r g a n e l l a r t R N A . t o i n o s i n e , except i n E . c o l i tRNA:;,” where i t s m o d i f i c a t i o n i s o f I n t h e wobble h y p o t h e s i s , i n o s i n e unknown s t r u c t u r e ( r e f . 5 4 ) . p a i r s w i t h A, U and C, whereas an unmodified A p a i r s w i t h U. T h i s recognizes o n l y t h e CGU codon. An exmeans t h a t t h e m t tRNA:;g a m i n a t i o n o f t h e frequency o f u t i l i z a t i o n o f CGN codons i n y e a s t m i t o c h o n d r i a l p r o t e i n coding genes r e v e a l e d t h a t none o f t h e genes which code f o r s t r u c t u r a l p r o t e i n o f t h e r e s p i r a t o r y c h a i n uses CGN codons, b u t e x c l u s i v e l y AGR codons f o r a r g i n i n e . However, i n t h e v a r l ribosomal p r o t e i n gene, b o t h CGU and CGG a r e used (one u t i l i z a t i o n each). I n i n t r o n i c open r e a d i n g frames c o d i n g f o r maturases o r i n f r e e - s t a n d i n g unassigned r e a d i n g frames, t h e CGN codons a r e more r e p r e s e n t e d (18 u t i 1i z a t i o n s ) , b u t t h e i r frequency Nevertheremains low when compared t o t h e u t i l i z a t i o n o f AGR. l e s s , t h e u t i l i z a t i o n o f some CGA, CGG and CGC codons r a i s e s t h e q u e s t i o n o f t h e mechanism o f t h e i r r e c o g n i t i o n by m t tRNA:;e. In t h i s case, a “two o u t o f t h r e e ” mechanism ( r e f . 55) remains t h e most p l a u s i b l e one, e s p e c i a l l y s i n c e t h e ACG/CGN i n t e r a c t i o n c o n t a i n s 2 p a i r s C:G a b l e t o secure s t a b i l i t y . I n a d d i t i o n , y e a s t m i t o c h o n d r i a do n o t c o n t a i n any o t h e r t R N A a b l e t o compete w i t h tRNA:;;! f o r t h e decoding o f CGN. I t should a l s o be emphasized t h a t t h e l o w frequency o f u t i l i z a t i o n o f CGN codons compared t o AGR i n m i t o c h o n d r i a l genes i s a m i n o r s p e c i e s when p a r a l l e l s t h e f a c t t h a t t h e tRNA:;;! compared t o tRNA1;g. Such a s i t u a t i o n suggests t h e e x i s t e n c e o f a mechanism which a l l o w s a d a p t a t i o n o f t h e l e v e l s o f e x p r e s s i o n o f t h e two i s o a c c e p t o r s t o t h e i r r e s p e c t i v e u t i l i z a t i o n i n m i t o chondri a1 p r o t e i n s y n t h e s i s . Besides t h e unusual r u l e s governing codon-anti codon in t e r a c t i o n i n m i t o c h o n d r i a , t h e y e a s t m i t o c h o n d r i a l t r a n s l a t i o n code shows d e v i a t i o n s f r o m t h e standard code. The most s t r i k i n g

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example i s t h a t t h e UGA t r i p l e t , i n s t e a d o f b e i n g a s t o p codon, i s used t o s p e c i f y t r y p t o p h a n . T h i s has f i r s t been shown by c y t o chrome oxidase s u b u n i t 2 gene sequence-protein sequence comparison ( r e f . 56, 57). T h i s change i n t h e g e n e t i c code i s accomodated by t h e presence o f an a n t i c o d o n U*CA i n t h e y e a s t m t t R N A T r p ( r e f . 27). I t remained t o be demonstrated t h a t t h e m t t R N A T r p can Using t h e X e n o p u s o o c y t e m i c r o i n j e c e f f e c t i v e l y t r a n s l a t e UGA. t i o n system, we have shown t h a t t h i s tRNA, when i n j e c t e d t o g e t h e r w i t h r a b b i t 8 - g l o b i n mRNA and E . c o l i tryptophanyl-tRNA s y n t h e t a s e (which i s needed t o charge t h e m t t R N A T r p i n t h e o o c y t e c y t o plasm), suppresses UGA t e r m i n a t i o n e f f i c i e n t l y , t h u s l e a d i n g t o t h e p r o d u c t i o n o f a 8 - g l o b i n - r e l a t e d readthrough p r o d u c t ( r e f . 58). Whereas t h e use o f UGA as a codon f o r t r y p t o p h a n i s found i n m i t o c h o n d r i a from v a r i o u s sources ( f u n g i , protozoan, i n s e c t s , mammals), t h e r e a r e o t h e r exceptions t o t h e s t a n d a r d g e n e t i c code which a r e r e s t r i c t e d t o m i t o c h o n d r i a o f a g i v e n s p e c i e s . I n S. c e r e v i a s i e , such an e x c e p t i o n has been deduced f r o m t h e observat i o n t h a t i n t h e ATPase p r o t e o l i p i d amino a c i d sequence, t h e t h r e o n i n e r e s i d u e a t p o s i t i o n 46 i s encoded by t h e CUA codon This (which s p e c i f i e s l e u c i n e i n t h e s t a n d a r d code) ( r e f . 5 9 ) . unexpected amino a c i d assignment f o r CUA was c o n f i r m e d by t h e i s o l a t i o n o f an unusual m t threonine-tRNA (and i t s gene) b e a r i n g an unorthodox anticodon l o o p o f 8 n u c l e o t i d e s w i t h an a n t i c o d o n UAG ( r e f s . 26, 42) ( F i g u r e 7 . 7 ) . The f i n d i n g t h a t t h e u r i d i n e r e s i d u e o f t h e UAG a n t i c o d o n o f t h i s t R N A i s u n m o d i f i e d and t h e f a c t t h a t y e a s t m i t o c h o n d r i a l a c k a CUN-specific t R N A L e U , s t r o n g l y n o t o n l y t r a n s l a t e s CUA b u t a l s o t h e suggest t h a t t h e tRNA::,: t h r e e o t h e r t r i p l e t s o f t h e CUN f a m i l y . does n o t seem t o be c l o s e l y r e l a t e d t o The y e a s t m t tRNA::; any o t h e r threonine-tRNA, n e i t h e r from p r o c a r y o t e s n o r from eucaryotes (cytoplasm and m i t o c h o n d r i a ) , s i n c e i t shows r a t h e r poor sequence homology w i t h known tRNAsT from e i t h e r c l a s s ( r e f . 26). I n t e r e s t i n g l y , t h e h i g h e s t homology (59%) i s observed w i t h t h e m t tRNA:,'; from N . c r a s s a ( r e f . 49). On t h e b a s i s o f t h i s originates resemblance, we have hypothesized t h a t y e a s t m t tRNA:;; from an a n c e s t r a l m t leucine-tRNA gene h a v i n g an a n t i c o d o n UAG and a normal s i z e f o r t h e " c o r r e s p o n d i n g l o o p . A u r i d i n e - i n s e r t i n g

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m u t a t i o n i n t h e a n t i c o d o n l o o p o f t h i s t R N A would have r e s u l t e d i n a change i n i t s a m i n o a c y l a t i o n s p e c i f i c i t y . T h i s would e x p l a i n how m t tRNAb,'; became a t h r e o n i ne-i n s e r t i ng species i n response t o CUN codons i n t h e p r e s e n t day y e a s t m i t o c h o n d r i a . Such a hypothes i s i s c o n s i s t e n t w i t h e a r l i e r f i n d i n g s ( r e f . 60) showing t h a t a m u t a t i o n a l change w i t h i n t h e CCA a n t i c o d o n o f E . c o l i t R N A T r p (su'7 m u t a t i o n ) n o t o n l y a l t e r s t h e tRNA t o a UAG suppressor, b u t a l s o provokes t h e l o s s o f t r y p t o p h a n a c c e p t i n g s p e c i f i c i t y and t h e a c q u i s i t i o n o f g l u t a m i n e a c c e p t o r a c t i v i t y . The m u t a t i o n g i v i n g r i se t o a 1e u c i n e - t o - t h r e o n i ne change i n m i t o c h o n d r i a1 p r o t e i ns from y e a s t would o n l y have a r i s e n a t a l a t e stage i n m i t o c h o n d r i a e v o l u t i o n s i n c e t h e CUN codons s p e c i f y l e u c i n e and n o t t h r e o n i n e i n a l l o t h e r known m i t o c h o n d r i a l systems (except i n t h e c l o s e l y T h i s change m i g h t r e l a t e d y e a s t T o r u 7 o p s i s g l a b r a t a ; see 7.4). have s u r v i v e d s i n c e (i)t h e CUN codons a r e i n f r e q u e n t l y used i n y e a s t m i t o c h o n d r i a l genes, and (ii)t h e m t DNA-coded p r o t e i n s would n o t have been a f f e c t e d f u n c t i o n a l l y by t h i s change. F i n a l l y , a t h i r d v a r i a t i o n i n the 5 . cerevisiae mitochondrial g e n e t i c code concerns t h e usage o f t h e codon AUA. I t has been suggested t h a t AUA (a codon f o r i s o l e u c i n e i n t h e s t a n d a r d code) i s t r a n s l a t e d i n t o m e t h i o n i n e based on t h e comparison o f t h e rnt ribosomal p r o t e i n v a r l gene sequence w i t h t h e amino a c i d composit i o n o f t h e p u r i f i e d p r o t e i n ( r e f . 61). I n o r d e r t o g a i n an i n s i g h t i n t o t h e m o l e c u l a r b a s i s o f AUA r e c o g n i t i o n i n y e a s t m i t o c h o n d r i a , we have i s o l a t e d and sequenced t h e m t tRNAMmet and t R N A l L e ( r e f . 23). The m t t R N A 1 L e has an a n t i c o d o n GAU f o r t r a n s l a t i o n o f t h e AUC and AUU i s o l e u c i n e codons and t h e m t tRNAMmet has an a n t i c o d o n CAU ( t h e C i s unmodified) f o r decoding AUG. Thus, i f o n l y t h e r e s p e c t i v e a n t i c o d o n sequences a r e cons i d e r e d , n e i t h e r t R N A 1 L e n o r t R N A i e t should be a b l e t o r e c o g n i z e t h e AUA codon. However, r e s u l t s o f i n v i t r o p r o t e i n s y n t h e s i s u s i n g Tobacco Mosaic V i r u s (TMV) mRNA i n d i c a t e d t h a t t h e m t t R N A Z e t i n s e r t s m e t h i o n i n e i n t o t h e TMV c o a t p r o t e i n i n response t o AUA ( r e f s . 23, 62). How can t h e r e a d i n g o f AUA be achieved by t h i s t R N A ? T h i s c o u l d be due t o t h e unique s t r u c t u r a l f e a t u r e o f t h e m t t R N A I e t , a n u c l e o t i d e b u l g i n g o u t from base p a i r i n g s i n t h e T$C stem (see 7.2.2 and F i g u r e 7.5), which c o u l d i n f l u e n c e t h e decoding p r o p e r t i e s o f t h i s t R N A . Structural features outside o f

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t h e a n t i c o d o n a r e known t o i n f l u e n c e t h e d e c o d i n g p r o p e r t i e s o f a C i n t h e wobble p o s i t i o n . F o r example, i n t h e c a s e o f t h e E . c o i o p a l s u p p r e s s o r t R N A T r p ( a n t i c o d o n CCA), a sequence a1 t e r a t i o n n It s t h e D-stem c o n f e r s t h e a b i l i t y t o s u p p r e s s UGA ( r e f . 6 3 ) . tempting t o speculate t h a t t h e e x t r a unpaired n u c l e o t i d e i n t h e T$C stem o f t h e m t tRNA:" s i m i l a r l y enhances C:A wobble, t h u s p e r m i t t i n g t h e d e c o d i n g o f b o t h AUG and AUA. TORULOPSIS GLABRATA MITOCHONDRIAL tRNAs The T o r u l o p s i s g l a b r a t a m i t o c h o n d r i a 1 genome (19kb) d i f f e r s Howi n s i z e f r o m t h e S a c c h a r o m y c e s c e r e v i s i a e m t DNA (80 k b ) . e v e r , d e s p i t e t h i s l a r g e s i z e d i f f e r e n c e and a d i f f e r e n t gene o r g a n i z a t i o n , t h e t w o y e a s t m t DNAs c o n t a i n t h e same b a s i c gene C l a r k - W a l k e r e t a l . ( r e f . 64) complement, i n c l u d i n g t R N A genes. have i d e n t i f i e d 23 tRNA genes i n T . g l a b r a t a m t DNA t h a t a r e l o c a t e d i n s i x o f t h e n i n e i n t e r g e n i c r e g i o n s between t h e l a r g e rRNA genes. The i d e n t i t y and number o f tRNA genes have been deduced b y comparison w i t h t h e 5. c e r e v i s i a e m t tRNA sequences, as base m a t c h i n g r a n g e s f r o m 85% ( f o r t R N A C L U ) t o 100% ( f o r tRNA;;!), and a n t i c o d o n s f o r homologous tRNAs a r e i d e n t i c a l . The m t t R N A complements a r e t h e same i n t h e t w o y e a s t s , e x c e p t t h a t t h e r e i s o n l y one a r g i n i n e t R N A gene i n t h e T . g l a b r a t a m t genome ( c o d i n g f o r tRNA;;:). 7.4

7.4.1

Structures From t h e gene s e q u e n c i n g d a t a o f C l a r k - W a l k e r e t a l . ( r e f . 64), i t appears t h a t t h e 23 T . g l a b r a t a m t tRNAs can be f o l d e d i n t o o r t h o d o x c l o v e r l e a f s i n c l u d i n g m o s t o f t h e i n v a r i a n t and semi-invariant residues. Most o f t h e e x c e p t i o n s t o t h e common f e a t u r e s o f t h e standard tRNA c l o v e r l e a f are those a l s o found i n S. c e r e v i s i a e m t tRNAs (see 7.3.2); f o r example: - tRNAPrO has an A, i n s t e a d o f t h e i n v a r i a n t U,; - t h e i n v a r i a n t GG i n p o s i t i o n s 18-19, and UUC ( c o r r e s p o n d i n g t o r e s i d u e s T$C) i n p o s i t i o n s 54-56, are replaced i n tRNAAsp b y AG and UGC, r e s p e c t i v e l y ; - t h e s e m i - i n v a r i a n t p u r i n e 2 1 i s r e p l a c e d b y C,, i n t R N A C y S . The l o o p s and stems a r e o f c l a s s i c a l l e n g t h s i n a l m o s t a l l tRNAs, e x c e p t i n t R N A C y S w h i c h can be r e p r e s e n t e d w i t h 6 base p a i r i n g s i n t h e a n t i c o d o n stem. I n a d d i t i o n , some tRNAs l a c k a

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Watson-Crick p a i r i n g a t the end of the amino acid stem ( t R N A G L y ) , of the D-stem ( t R N A V a L , t R N A L e U , tRNA;:: , tRNA;;; , t R N A P r o , t R N A A L a ) , and of the anticodon stem ( t R N A L e U , tRNA:;;). The unusual structural features found i n some 5 . c e r e v i s i a e m t tRNAs are also found i n the T . g l a b r a t a counterparts. For example, the m t t R N A P h e and t R N A E e t have a bulged-out residue i n the stem of the T@C arm. However, unlike the S. c e r e v i s i a e m t t R N A L y s (which also has an outloop in t h i s stem), this i s not the case i n the T . g l a b r a t a m t t R N A L y S . The structure of the T . g l a b r a t a m t t R N A H i s can be represented i n two ways, w i t h a bulged-out residue i n e i t h e r the 0-stem or the anticodon stem (Figure 7.8). F i n a l l y , another exceptional feature i s found i n the tRNA:;; which has an anticodon l o o p o f 9 nucleotides ( i n 5 . c e r e v i s i a e , t h i s t R N A has 8 nucleotides i n the loop).

&

A U

A

uc

I

I

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u u

A

A

U A A A

A

u I

I

I

C-G G-C C-G

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cuuu

G

I

I

C UAA G

U I A

G

P

U-A A-U A-U A-U U-A UA-U GAU

U

A G

U

C

u G

U

G

m A-U C-G G-C

Figure 7.8 glabrata m t

Two possible secondary structures f o r t R N A H i S (deduced from the gene).

Torulopsis

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7.4.2

Codon Readins P a t t e r n s The expanded codon r e a d i n g p a t t e r n s f o u n d i n S. c e r e v i s i a e m i t o c h o n d r i a (see 7.3.4) a l s o a p p l y t o T . g l a b r a t a s i n c e homol o g o u s m t tRNAs o f t h e two y e a s t s have i d e n t i c a l a n t i c o d o n s ( r e f . 64). However, no a r g i n i n e t R N A gene c o r r e s p o n d i n g t o t h e f o u r codon f a m i l y CGN was f o u n d i n m t DNA. T h i s means t h a t e i t h e r CGN codons a r e n o t used a t a l l i n m t genes o r t h a t a CGN-reading t R N A ( i f needed i n m i t o c h o n d r i a 1 p r o t e i n s y n t h e s i s ) i s i m p o r t e d f r o m t h e c y t o p l asm. Based on p r o t e i n c o d i n g genes sequence d a t a , t h e g e n e t i c code used i n m i t o c h o n d r i a o f t h e two y e a s t s s h o u l d a l s o be t h e same, w i t h UGA b e i n g a codon f o r t r y p t o p h a n , t h e CUN codons f a m i l y b e i n g t r a n s l a t e d b y tRNA::;, and AUA b e i n g t r a n s l a t e d i n t o m e t h i o n i n e . NEUROSPORA CRASSA MITOCHONDRIAL tRNAs A p p r o x i m a t e l y 80% o f t h e N e o r o s p o r a c r a s s a m t DNA (60kb) has been sequenced. O f t h e 27 tRNA genes i n m t DNA, 23 a r e c l u s t e r e e Two o f them, on e i t h e r s i d e o f t h e l a r g e r R N A gene ( r e f s 6, 6 5 ) . t h e i n i t i a t o r and e l o n g a t o r t R N A M e t genes, a r e d u p l i c a t e d . Thus, t h e r e a r e 25 d i f f e r e n t t R N A genes i n N . c r a s s a m i t o c h o n d r i a , w h i c h i s i n agreement w i t h t h e number o f m t tRNA s p e c i e s s e p a r h l e d b y t w o - d i m e n s i o n a l p o l y a c r y l amide g e l e l e c t r o p h o r e s i s ( r e f . 66).

7.5

7.5.1

Structures 2 1 t R N A genes f r o m t h e l a r g e r R N A gene r e g i o n have been sequenced ( r e f . 65). A t R N A C y s gene, l o c a t e d n e a r t h e c y t o c h r o m e o x i d a s e s u b u n i t 1 gene, has a l s o been sequenced r e c e n t l y ( r e f . 67). The sequences o f 12 d i f f e r e n t t R N A s p e c i e s have been d e t e r mined a t t h e RNA l e v e l b y RajBhandary and h i s c o w o r k e r s , i n c l u d i n g t h e i n i t i a t o r tRNA ( r e f . 68) - w h i c h was t h e f i r s t n u c l e i c a c i d t o be sequenced f r o m m i t o c h o n d r i a -, t h e t y r o s i n e t R N A ( r e f . 69), and t h e alanine, glutamine, leucine-1, leucine-2, threonine, t r y p t o p h a n and v a l i n e tRNAs ( r e f . 49). The f o l l o w i n g unusual s t r u c t u r a l f e a t u r e s a r e f o u n d i n t h e s e t RNAs : - Unlike the 5 . c e r e v i s i a e m t tRNAYet, the N. crassa m t i n i t i a t o r tRNA has a t y p i c a l Watson-Crick base p a i r a t t h e end o f t h e a c c e p t o r - s t e m and l a c k s t h e t y p i c a l T$C sequence

B217

i n loop I V . The corresponding sequence, however, i s UGC whereas i t i s AU(or $ ) C i n c y t o p l a s m i c i n i t i a t o r s . Thus, u n l i k e the yeast m t i n i t i a t o r , the N . crassa m t tRNAYet resembles c y t o p l asmi c in i t i a t o r s r a t h e r t h a n p r o c a r y o t i c ones. Both m i t o c h o n d r i a l i n i t i a t o r s have a p a i r U,, :A2,, i n s t e a d o f A,, :U24 found i n e u b a c t e r i a l i n i t i a t o r s . - Like procaryotic, but c o n t r a r i l y t o eucaryotic cytoplasmic t y r o s i n e tRNAs, t h e N . c r a s s a rnt t R N A T v r has a l a r g e v a r i a b l e arm. - Both t R N A Y e t and t R N A T h r c o n t a i n A,, ( i n t h e D-loop) and G5, ( i n l o o p I V ) i n s t e a d o f h a v i n g t h e i n v a r i a n t G,, and *55

*

lacks the invariant purine i n position 3'adjacent instead. t o t h e a n t i c o d o n ( p o s i t i o n 37) and has U, - t R N A T v r l a c k s t h e sequence A 1 4 - p u r i n e 1 5 which i s r e p l a c e d by u, 4 -u, 5 - t R N A C v S has two unusual s t r u c t u r a l f e a t u r e s : (i)r e s i d u e A, r e p l a c e s t h e i n v a r i a n t U, and (ii)t h e r e a r e two nucleot i d e s ( i n s t e a d o f one) between t h e D- and a n t i c o d o n arms. - F i n a l l y , base couples i n t h e a n t i c o d o n stem o f s e v e r a l tRNAs a r e non-Watson-Cri ck p a i r s ( i n t R N A T , -Val , -Trp, and -Cys). -

tRNAALa

-

7.5.2

M o d i f i e d n u c l eosides The m o d i f i e d n u c l e o s i d e c o n t e n t o f N . c r a s s a m t t R N A i s a t l e a s t two t i m e s l o w e r than t h a t o f c y t o p l a s m i c tRNA. W h i l e mlA, m 7 G and r i b o s e - m e t h y l a t e d n u c l e o s i d e s do n o t occur i n m t t R N A , D, T, $ , m l G , mZG, m:G, m5C, and t 6 A a r e found ( r e f . 66). D, T, and $ a r e p r e s e n t i n a1 1 t h e sequenced species, except i n tRNA?.' and t R N A T h r which l a c k T ( r e f s 49, 68, 69). A m o d i f i e d u r i d i n e (U*) i s found i n t h e wobble p o s i t i o n o f tRNAs c o r r e s p o n d i n g t o twocodon f a m i l i e s ending i n a p u r i n e ( r e f . 49). 7.5.3

Codon Readina P a t t e r n s Based on t h e s t r u c t u r e s o f N . c r a s s a m t tRNAs, Heckman e t a l . ( r e f . 49) were t h e f i r s t t o show t h a t m t tRNAs use a s i m p l i f i e d mechanism f o r codon r e a d i n g which a l l o w s m i n i m i z a t i o n o f t h e number o f t R N A species r e q u i r e d f o r m i t o c h o n d r i a l p r o t e i n synt h e s i s . I n summary, N . c r a s s a m t tRNAs c o n t a i n i n g an u n m o d i f i e d U

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i n the f i r s t p o s i t i o n of the anticodon a r e capable of reading a l l f o u r codons of a four-codon family, whereas t h o s e containing a modified U a r e r e s t r i c t e d t o reading codons ending i n A o r G ( f o r Such an unusual codon reading f u r t h e r d i s c u s s i o n , see 7.3.4). pattern i s a l s o used i n y e a s t ( r e f s . 21, 50) and mammalian ( r e f . 70) mitochondria and most probably i s a general f e a t u r e i n mitochondria. Since there a r e 8 four-codon boxes i n the genetic code, t h e expanded codon-reading a b i l i t y of c e r t a i n m t tRNAs reduces the t o t a l number of tRNAs needed i n p r o t e i n s y n t h e s i s from a minimum of 32 i n procaryotic and e u c a r y o t i c cytoplasmic systems t o 24 i n mitochondria. This conclusion would explain why mitochondria i n general contain a much smaller number of t R N A s p e c i e s compared w i t h o t h e r systems ( r e f . 49). The anticodon sequence of the N . c r a s s a mitochondria1 t r y p t o phan t R N A i s U*CA and not CCA o r CmCA a s i n tryptophan tRNAs from prokaryotes o r from eukaryotic cytoplasm. Because a t R N A w i t h an anticodon U*CA i s expected t o read the codon UGA a s well a s t h e usual tryptophan codon UGG, this suggests t h a t i n N . c r a s s a mitochondria (as i n y e a s t and i n human mitochondria) UGA i s a codon f o r tryptophan and not a signal f o r chain t e r m i n a t i o n . F i n a l l y , the anticodon sequences of the two l e u c i n e tRNAs i n d i c a t e t h a t N . c r a s s a mitochondria use both f a m i l i e s of l e u c i n e codons, UUR and C U N (R = A o r G; N = U , C , A o r G) f o r l e u c i n e ( r e f . 49), i n c o n t r a s t t o y e a s t mitochondria i n which the CUN family i s t r a n s l a t e d i n t o threonine ( r e f s . 26, 42).

MITOCHONDRIAL tRNAs The filamentous, o b l i g a t e a e r o b i c ascomycete A s p e r g i l l u s n i d u l a n s has a 32 kb mitochondria1 genome which i s i n t e r m e d i a t e i n s i z e between the genomes of t h e two o t h e r groups of ascomycetes, 5. c e r e v i s i a e (80 kb) and N . c r a s s a (60 k b ) on one hand, and T o r u l o p s i s g l a b r a t a and S c h i z o s a c c h a r o m y c e s pombe (19 kb) on t h e o t h e r hand. The group of Kuntzel ( r e f s . 71, 72) has sequenced a 14 k b segment containing 24 t R N A genes. Of t h e s e , 21 a r e organized i n two c l u s t e r s located on e i t h e r side of the l a r g e rRNA gene. The t R N A C y s and t R N A A S n genes a r e found i n d u p l i c a t e c o p i e s , each separated by about 8 kb of the genome ( r e f . 73). 27-28 t R N A genes a r e present on m t DNA. The u n c e r t a i n t y over the p r e c i s e

7.6

ASPERGZLLOS NZDULANS

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number of t R N A genes on m t DNA d e r i v e s from t h e f a c t t h a t a CGN decoding t R N A A r g has n o t y e t been l o c a t e d . As CGN codons e x i s t i n s e v e r a l p r o t e i n coding genes, t h i s t R N A i s presumably p r e s e n t , p o s s i b l y i n one o f t h e s h o r t segments o f m t DNA so f a r unsequenced ( r e f . 73). The 27-28 t R N A genes code f o r 25-26 d i f f e r e n t t R N A species. These i n c l u d e two i s o a c c e p t o r s f o r each l e u c i n e , s e r i n e , and g l y c i n e , and t h r e e i s o a c c e p t o r s f o r m e t h i o n i n e ( r e f s . 71, 72, 73). 7.6.1

Structures Most o f t h e c l o v e r l e a f s t r u c t u r e s have t h e normal p a t t e r n o f i n v a r i a n t and semi -in v a r i a n t bases common t o p r o c a r y o t i c and e u c a r y o t i c c y t o p l a s m i c tRNAs. However, some unusual f e a t u r e s should be noted. The i n v a r i a n t r e s i d u e U, i s r e p l a c e d by an A i n tRNAPhe and by a G i n t R N A P r o . I n tRNAS,;;, the nucleotide a t Three e x c e p t i o n s were found i n t h e A,,p o s i t i o n 9 i s deleted. Pu,, sequence: G,,-G,, i n t R N A P r O , A,,-U,, i n tRNA;,e; and t R N A T y r . The sequences A,,-G,, i n s t e a d o f G,,-G,, i s found t w i c e ( i n t R N A T h r and t R N A ; e t ) . These two tRNAs a l s o have a G,, instead o f t h e $,, i n t h e sequence T$C o f l o o p I V . Another e x c e p t i o n i s a U,, i n tRNAPro and tRNATYr i n s t e a d o f t h e c l a s s i c a l Pu,, . The t e r t i a r y i n t e r a c t i o n Pu,,:Py,, i s n o t p o s s i b l e i n t R N A T h r and t R N A P r o which show r e s p e c t i v e l y A:A and G:G i n t h e s e p o s i t i o n s . F i n a l l y , A,, i s found i n l o o p I V o f t R N A A L a i n p l a c e o f U ( o r r T ) and t h e p a i r A,, :U6, r e p l a c e s t h e p a i r G,, :C6, i n t h r e e cases (tRNA7 y , tRNA; y , tRNA: ) The l e u c i n e and s e r i n e tRNAs e x h i b i t l o n g extra-arms. This i s a l s o t h e case w i t h t R N A T y r making t h i s t R N A resemble t h e e u b a c t e r i a l tRNAsTyr. The sequences o f A . n i d u l a n s rnt tRNAs a r e c l e a r l y r e l a t e d t o those o f t h e corresponding species i n N . crassa m i t o c h o n d r i a ( f r o m 76% t o 87% s i m i l a r i t i e s between m t tRNAs o f t h e two ascomycetes). I n c o n t r a s t , s i m i l a r i t i e s t o t h e y e a s t m t sequences a r e d i s t i n c t l y l o w e r ( r e f s . 71, 72).

.

7.6.2

Codon Readina P a t t e r n s As i n y e a s t and N . crassa m i t o c h o n d r i a , t h e four-codon f a m i l i e s a r e most p r o b a b l y read by a s i n g l e tRNA w i t h an unmodified U i n t h e wobble p o s i t i o n . The presence o f two tRNAsGLv w i t h d i f f e r -

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e n t anticodons UCC and ACC appears t o be an e x c e p t i o n t o t h i s r u l e . The tRNA;Lr should o n l y read t h e codon GGU. Both tRNAs show 83% sequence homology. However, t h e r e i s no evidence t h a t b o t h genes a r e t r a n s c r i b e d i n t o f u n c t i o n a l tRNAsGLy, They There a r e t h r e e tRNAsMet i n A . n i d u l a n s m i t o c h o n d r i a . show a r a t h e r l o w degree o f homology, except f o r t h e i r anticodon. The i s o a c c e p t o r 1 and 2 seem t o be f u n c t i o n a l l y analogous t o N . c r a s s a m i t o c h o n d r i a l i n i t i a t o r and e l o n g a t o r tRNAsMet, respect i v e l y , by t h e i r s i g n i f i c a n t sequence homologies. The f u n c t i o n o f t h e t h i r d t R N A M e t species i s unknown ( r e f . 71). The cytochrome oxidase s u b u n i t 3 gene s t a r t s w i t h t h e codon GUG which i s o c c a s i o n a l l y used as an i n i t i a t i o n codon i n b a c t e r i a ( r e f s . 72, 74). However, i t i s n o t known whether t h i s unusual i n i t i a t i o n codon i s t r a n s l a t e d by one o f t h e 3 tRNAsMet o r by t h e t R N A V a L The i n t e r n a l v a l i n e codon GUG i s used o n l y once ( i n URF A), b u t i s absent from a l l o t h e r sequenced p r o t e i n coding genes ( r e f . 73). The t R N A T r p has an anticodon UCA and i s b e l i e v e d t o t r a n s l a t e t h e codon UGA (a s t o p codon i n t h e " u n i v e r s a l " code), as i n a l l o t h e r m i t o c h o n d r i a l systems, except p l a n t m i t o c h o n d r i a ( r e f . 7 1 ) .

.

7.7

SCHZZOSACCHARONYCES PONS€ MITOCHONDRIAL tRNAs

S i m i l a r l y t o t h e m i t o c h o n d r i a l genome o f r . g l a b r a t a , t h e m i t o c h o n d r i a l genome o f s. pombe i s small and has o n l y 19.43 kb. I t has been c o m p l e t e l y sequenced by Lang and coworkers ( r e f s 7580) and found t o c o n t a i n 25 t R N A genes ( i n s t e a d o f 24 i n 5. c e r e v i s i a e mitochondria). I t c o n t a i n s t h e same number o f i s o acceptors as S. c e r e v i s i a e , except f o r l e u c i n e where 2 tRNAs a r e found and f o r t h r e o n i n e where o n l y one i s found. Furthermore, two p o s s i b l e t R N A l I e a r e coded f o r by t h e m t DNA.

7.7.1

Structures The m a j o r exceptions t o t h e common f e a t u r e s of t h e standard t R N A c l o v e r l e a f a r e t h e f o l l o w i n g : I n two tRNAs, t h e i n v a r i a n t U, is i s r e p l a c e d by an A ( t R N A P r o , t R N A T y r ) . The sequence A,,-Pu,, n o t p r e s e n t i n t R N A P r O (U,,-A,,), t R N A T y r ( U , 4 - G 1 5 ) and t R N A C y S , t h e l a t t e r having o n l y a s h o r t , 4 n u c l e o t i d e - l o n g D-loop ( F i g u r e The G,,-G,, sequence i s p r e s e n t i n a l l m t tRNAs, except i n 7.9). tRNAAsp (G18-U19). The same is t r u e f o r Pu,, which i s , however,

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not found i n t R N A C v S , t R N A P r a , and t R N A T v r which have U,, . The i n v a r i a n t n u c l e o t i d e U,, i s present i n 24 tRNAs, the only except i o n b e i n g t R N A G L u which has a C 3 3 . The absence o f an U i n 5 ’ t o the anticodon i s r a r e l y encountered i n tRNAs, except i n v e r t e b r a t e and p l a n t cytoplasmic i n i t i a t o r tRNAsMetwhich a l s o have a C S 3 . The base p a i r G 5 , : C 6 , i s g e n e r a l l y p r e s e n t i n s. pombe m t tRNAs, except i n t R N A A L a ( U s 3 : A 6 , ) and i n t R N A A s p ( A 5 3 : A 6 , ) . In t R N A A S p , t h e sequence UUC (corresponding t o the conserved sequence T$C i n loop IV) i s replaced by CUC. The loops and stems a r e of the c l a s s i c a l length i n most of t h e S. pombe m t tRNAs. As i n p r o c a r y o t i c t R N A s T v r , the t R N A T v r has a long extra-arm (15 n u c l e o t i d e s ) . The D-loop s i z e ranges from 6 t o 12 n u c l e o t i d e s , except i n t R N A C v s . The secondary s t r u c t u r e of t h i s t R N A has another p e c u l i a r i t y s i n c e i t can be drawn w i t h a 4 base-paired D-stem and with a 7 base-paired a n t i codon stem (Figure 7 . 9 ) . The tRNA;;e can a l s o be drawn w i t h a 7 base-paired anticodon stem whereas the t R N A n e t could have a 6 base-paired anticodon stem having A,, o p p o s i t e t o U 4 & . 7.7.2 Codon Readina P a t t e r n s The 25 5 . pombe m t DNA-coded tRNAs a r e s u f f i c i e n t f o r t r a n s l a t i n g a l l the codons assuming t h a t t h e expanded codon reading p a t t e r n found i n 5. c e r e v i s i a e and N . c r a s s a mitochondria a l s o apply t o s. p o m b e . However, f o u r codon reading p a t t e r n s d i f f e r between S. pombe and S . c e r e v i s i a e . (i) The 5. pombe m t t R N A T r p has an anticodon CCA ( l i k e cytoplasmic and procaryotic t R N A s T r p ) , and not U*CA a s i n a l l fungal and animal mitochondria1 t R N A s T r p . In t h e ubiquitous m t DNA-encoded p r o t e i n s found i n S. p o m b e , tryptophan i s s p e c i f i e d e x c l u s i v e l y by TGG. However, 3 TGA codons a r e found i n two o u t o f t h r e e i n t r o n i c Urfs and i n a f u r t h e r u n i d e n t i f i e d reading frame, Urf a . Since i n case of t h e Urf i n the second cox1 i n t r o n , two TGA codons a r e a t i d e n t i c a l p o s i t i o n s of a h i g h l y homologous i n t r o n i c Urf of A s p e r g i l l u s n i d u l a n s , one can assume t h a t they a r e a l s o t r a n s l a t e d i n t o tryptophan. The p r o p e r t i e s o f t R N A T r p were checked i n the r a b b i t r e t i c u l o c y t e s p r o t e i n synthesizing system and we could show ( r e f . 80) t h a t , although i t has a CCA anticodon,

B222

t h i s tRNA i s a b l e t o t r a n s l a t e UGA i n t o t r y p t o p h a n , b u t w i t h a 10-20 t i m e s l e s s e r e f f i c i e n c y t h a n t h e 5. c e r e v i s i a e m t tRNATrp ( r e f . 58).

G

U G-C A-U U- A A-U A-U U-A G-C 'A U UACUC U U ~ I l ~l I 1 ~ A AUGAGU C I l l (I U U U CUGAA A A----U U,---A

,

u+G Gtu A-U A- U U-A

U U

A A

GCA

Figure 7.9 S t r u c t u r e o f S c h i z o s a c c h a r o m y c e s pombe m t (deduced f r o m t h e gene).

tRNACvS

(ii) A comparison o f h i g h l y c o n s e r v e d r e g i o n s o f m i t o c h o n d r i a1 p r o t e i n s i n d i c a t e s t h a t AUA s p e c i f i es isol e u c i ne i n 5. pombe r a t h e r t h a n m e t h i o n i n e ( w h i c h i s t h e case i n animal and y e a s t m i t o c h o n d r i a ) . The t R N A t h a t c l e a r l y decodes i s o l e u c i n e has an a n t i c o d o n GAU w h i c h w o u l d r e c o g n i z e b o t h AUC and AUU. The A U A - s p e c i f i c t R N A t L e i s most p r o b a b l y coded f o r b y one o f t h e t h r e e tRNA genes

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(iii)

(iv)

having a "methionine" CAU anticodon. The C-residue of i t s anticodon might then be modified i n such a way a s t o prevent C:G wobble, while allowing C:A wobble. As i n N . c r a s s a mitochondria, there a r e 2 tRNAsLe", one with an UAA anticodon and the o t h e r with an UAG a n t i codon. T h u s , c o n t r a r i l y t o s. c e r e v i s i a e mitochondria, the C U N family i s read a s l e u c i n e r a t h e r than t h r e o n i n e . F i n a l l y , the S. pombe CGN-reading m t t R N A A r g has an anticodon UCG (and not ACG l i k e i n S. c e r e v i s i a e ) and thus uses U:N ( N = A , G , C , o r U) wobble f o r decoding the 4 codons of t h e a r g i n i n e CGN family.

7.8 PODOSPORA ANSERINA MITOCHONDRIAL t R N A s P o d o s p o r a a n s e r i n a i s a l s o a filamentous ascomycete. Its mitochondria1 genome c o n s i s t s of a 94 k b c i r c u l a r DNA molecule ( r e f . 8 1 ) . During senescence s p e c i f i c r e g i o n s (termed sen DNA) of t h e m t DNA a r e e x c i s e d , l i g a t e d and a m p l i f i e d . Cummings e t a 7 . ( r e f . 82) have cloned 3 sen DNAs. In t h e Q sen DNA, a t R N A C Y s sequence was d e t e c t e d , whereas p sen DNA contained t h e sequences f o r t R N A A S p , tRNA;;; , t R N A T r p and t R N A V a L . The t R N A A s p and tRNA;;; a r e more than 70% homologous t o e q u i v a l e n t m t tRNAs from A s p e r g i l J u s whereas v a l i n e and tryptophan tRNAs a r e more homologous t o t h e i r N e u r o s p o r a c o u n t e r p a r t s ( p a r t i c u l a r l y t h e tryptophan t R N A which shows more than 90% s i m i l a r ity) . Structures There a r e s e v e r a l unusual f e a t u r e s i n the sequences of two of the f i v e known m t tRNAs which a r e exceptions t o the i n v a r i a n t and semi-invariant nucleotides: tRNA;;; and t R N A C y S have an A, i n s t e a d of U, . tRNA;,e: has a sequence A,,-U, i n s t e a d of A, P u , , . F i n a l l y , tRNA;;; has o n l y f o u r Watson-Crick p a i r s i n t h e anticodon stem and t R N A C v S has a bulged-out r e s i d u e and s i x p a i r e d n u c l e o t i d e s i n t h i s stem. 7.8.1

,-

7.8.2

Codon Readinq P a t t e r n s The t R N A T r p has an anticodon UCA complementary t o both UGA and UGG. T h u s , P. a n s e r i n a most probably uses UGA f o r tryptophan r a t h e r than chain t e r m i n a t i o n .

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7.9

TETRAHYMENA P Y R I F O R M I S

MITOCHONDRIAL tRNAs

The c i 1 i a t e p r o t o z o a n T e t r a h y m e n a p y r i f o r m i s m i t o c h o n d r i a l DNA (55 kb) i s a l i n e a r d u p l e x m o l e c u l e ( l i k e i n P a r a m e c i u m ) w h i l e m t DNAs f r o m a l l o t h e r organisms a r e c l o s e d c i r c u l a r m o l e c u l e s . I t codes f o r o n l y 10 t R N A s p e c i e s ( r e f . 83) w h i l e a b o u t 26 n u c l e a r DNA-encoded tRNAs a r e i m p o r t e d f r o m t h e c y t o p l a s m . The genes e n c o d i n g tRNAH i s , tRNAG u , tRNAP , tRNAT p and a d u p l ic a t e d t R N A ' v r gene have been l o c a t e d a t t h e t e r m i n a l r e p e a t segments o f m t DNA ( r e f . 8 4 ) . T h r e e tRNAsLeu and t w o tRNAsMet a r e a l s o e x p e c t e d t o be on t h e m t DNA ( r e f s . 83, 85), b u t t h e y have n o t y e t been l o c a t e d . The t h r e e tRNAsLeu c o u l d be d e r i v e d f r o m t h e same gene and p o s t - t r a n s c r i p t i o n a l l y m o d i f i e d ( r e f . 8 3 ) . 7.9.1

Structures T h r e e m t t R N A genes have been sequenced ( r e f . 86). They code f o r t R N A P h e , t R N A H i S , and t R N A T r p . On t h e o t h e r hand, t w o m t DNAencoded tRNAs sequences ( t R N A P h e , t R N A T v P ) have been d e t e r m i n e d a t These tRNAs have a r a t h e r h i g h G+C t h e RNA l e v e l ( r e f . 8 7 ) . content: 48.7% f o r t R N A P h e , 50.6% f o r t R N A T v r , 47% f o r tRNAHiS and 49% f o r t R N A T r p . T h i s i s u n e x p e c t e d s i n c e T e t r a h y m e n a m t DNA i s e x t r e m e l y r i c h i n A+T, w i t h an a v e r a g e o f o n l y 23% G+C. The T e t r a h y m e n a m t tRNAs show s t a n d a r d s t r u c t u r a l f e a t u r e s . The o n l y e x c e p t i o n s a r e t h e f o l l o w i n g : t R N A P h e has a sequence U 5 4 - C 5 5 . T h i s t R N A a l s o has an a d d i t i o n a l r e s i d u e (U) a t i t s 5't e r m i n u s t h a t c o u l d p o t e n t i a l l y e x t e n d t h e a m i n o a c y l stem b y f o r m i n g an a d d i t i o n a l base p a i r w i t h t h e A i n t h e f o u r t h p o s i t i o n f r o m t h e 3 ' end. The tRNAHiS has a l s o 8 base p a i r s i n t h e amino a c i d stem, b u t t h i s i s a g e n e r a l f e a t u r e f o r tRNAsHis. F i n a l l y , as o t h e r m t tRNAsTvr ( e x c e p t i n a n i m a l m i t o c h o n d r i a ) , t h e T e t r a h y m e n a m t t R N A T v r has a l o n g e x t r a - a r m ( l i k e t h e e u b a c t e r i a l tRNAsTvr). Sequence comparisons o f t h e T e t r a h y m e n a m t tRNA genes w i t h t h e c o r r e s p o n d i n g genes f r o m o t h e r s o u r c e s r e v e a l t h a t t h e T e t r a hymena m t tRNA genes have h i g h sequence s i m i l a r i t y t o t h e €. c o l i t R N A genes; s i m i l a r i t y t o E . c o l i i s i n v a r i a b l y h i g h e r t h a n t o t h e c o r r e s p o n d i n g m i t o c h o n d r i a 1 genes f r o m o t h e r e u k a r y o t e s . However, t h e T e t r a h y m e n a t R N A P h e gene shows e x t r a o r d i n a r y sequence s i m i l a r i t y t o t h e c o r r e s p o n d i n g mammalian n u c l e a r tRNA ( r e f . 86).

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7.9.2

M o d i f i e d Nucl eosi des S i n c e o n l y two tRNAs have been determined a t t h e RNA l e v e l ( r e f . 87), i n f o r m a t i o n concerning t h e m o d i f i e d n u c l e o s i d e s i s scarce. However, queuosine occupies t h e wobble p o s i t i o n o f t R N A T v r as i t does i n E . c o 7 i and mammalian c y t o p l a s m i c tRNAsTvr. m 2 G and m$G r e s i d u e s occupy r e s p e c t i v e l y p o s i t i o n s 10 and 26. This i s a c h a r a c t e r i s t i c o f e u k a r y o t i c c y t o p l a s m i c tRNAs ( r e f s . However, s e v e r a l f u n g a l m t tRNAs a l s o have m $ G a t 46, 88). p o s i t i o n 26. The o t h e r m o d i f i e d n u c l e o s i d e s found a r e D, $, T , Um and a m o d i f i e d A. t R N A T v r c o n t a i n s 11 m o d i f i e d n u c l e o s i d e s which i s a r a t h e r h i g h degree o f m o d i f i c a t i o n f o r a m i t o c h o n d r i a 1 t R N A . 7.9.3

Codon Readina P a t t e r n s The t R N A P h e , t R N A T v r , and tRNAHiS have anticodons correspondi n g t o t h e u n i v e r s a l codons whereas t h e t R N A T r p has an a n t i c o d o n UCA which should a l l o w t r a n s l a t i o n o f t h e codon UGA i n a d d i t i o n t o t h e standard UGG t r y p t o p h a n codon. 7.10

PARAMECIUM MITOCHONDRIAL tRNAs

7.10.1 S t r u c t u r e s L i k e t h e T e t r a h y m e n a m t DNA, t h e P a r a m e c i u m one i s a l s o a l i n e a r duplex molecule. Only t h e t R N A T v r gene from P a r a m e c i u m p r i m a u r e l i a ( r e f . 89) and t h e t R N A T r p and t R N A T v r gene from P a r a m e c i u m t e t r a a u r e l i a have been sequenced ( r e f s . 90, 91). They p r e s e n t a1 1 t h e i n v a r i a n t and s e m i - i n v a r i a n t n u c l e o t i d e s o f t h e standard t R N A c l o v e r l e a f . L i k e fungal m t tRNAsTvr, P a r a m e c i u m m t t R N A T v r has a l a r g e extra-arm, whereas D r o s o p h i l a , X e n o p u s and mammalian m t tRNAsTvr have a s h o r t extra-arm. Thus, m t tRNAsTvr from u n i - and p l u r i c e l l u l a r organisms c l e a r l y d i f f e r i n t h e l e n g t h s o f t h e i r extra-arm: P a r a m e c i u m p r i m a u r e 7 i a t R N A T v r shows 100% homology w i t h P a r a m e c i u m t e t r a a u r e l i a t R N A T v r . With E . c o 7 i t R N A T v r , s i m i l a r i t j drops t o 60% whereas w i t h y e a s t m t t R N A T v r i t i s about 50%. However, w i t h T e t r a h y m e n a p y r i f o r m i s t h e homology i s 80%. The s i m i l a r i t i e s a r e l e s s c l e a r c u t w i t h t R N A T r p where t h e resemblance w i t h t h e c o u n t e r p a r t from E . c o 7 i and from y e a s t m i t o c h o n d r i a i s almost t h e same (68 and 67%).

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7 . 1 0 . 2 Codon Readi n q P a t t e r n s F o l l o w i n g t h e d i s c o v e r y t h a t f u n g a l and mammalian m t tRNAsTrp t r a n s l a t e t h e o p a l codon UGA, S e i l hamer e t a 7 . ( r e f . 91) sequenced t h e t R N A T r p gene f r o m P a r a m e c i u m t e t r a a u r e l i a . They f o u n d t h a t i t s a n t i c o d o n UCA s h o u l d r e c o g n i z e b o t h UGA and UGG codons. 7.11 MOSQUITO MITOCHONDRIAL tRNAs 7.11.1 S t r u c t u r e s N i n e m o s q u i t o ( A e d e s a 7 b o p i c t u s ) m t tRNAs have been sequenced. They a r e tRNA;;g ( r e f . 9 2 ) , tRNAASp ( r e f . 9 2 ) , t R N A G L u ( r e f . 93), tRNAGlY ( r e f . 9 3 ) , t R N A I L e ( r e f . 9 2 ) , tRNALys ( r e f . 9 4 ) , t R N A n e f ( r e f . 95), tRNA;;; ( r e f . 9 6 ) , t R N A V a L ( r e f . 9 3 ) . The gene sequences o f 9 tRNAs f r o m t h e same o r g a n e l l e a r e a l s o known: tRNAA'a ( r e f s . 93, 9 7 ) , tRNA;;! ( r e f s . 93, 9 7 ) , tRNAAsn ( r e f s . 93, 97), t R N A G L u ( r e f . 9 3 ) , t R N A C L y ( r e f . 9 3 ) , tRNALeU ( r e f . 9 8 ) , tRNAPhe ( r e f . 93), tRNA;;; ( r e f s . 93, 97) and t R N A V a L ( r e f . 9 8 ) . T h i s makes a t o t a l o f t h i r t e e n d i f f e r e n t m t tRNAs. A tRNACLy subspecies, d i f f e r i n g o n l y from t h e m a j o r t R N A G L y b y u n i d e n t i f i e d U and G d e r i v a t i v e s ( a t p o s i t i o n s 27 and 2 8 ) , has a l s o been sequenced ( r e f . 9 3 ) . They a l l show a h i g h A t U c o n t e n t w i t h t R N A G L u showing t h e h i g h e s t A+U c o n t e n t (88%). T h i s t R N A G L " has t w o stems l a c k i n g G:C p a i r s and t w o c o n t a i n i n g o n l y one G:C p a i r each. A e d e s m t tRNAs l a c k a number o f o t h e r w i s e h i g h l y c o n s e r v e d n u c l e o t i d e s . S i x o f tRNAAla, tRNA;;;!, tRNAASn, tRNAGLy, them do n o t c o n t a i n t h e U,: t R N A V a L , w h i c h have A, and tRNA;;; w h i c h has G,. The A,, i s f o u n d i n 11 tRNAs b u t n o t i n tRNA;;; and tRNA;;:. I t i s f o l l o w e d by a Pu,, o n l y i n 6 o u t o f t h e 13 tRNAs. The G,,-G,, sequence i s always l a c k i n g , whereas t h e r e a r e o n l y 2 e x c e p t i o n s t o t h e Pu,, (tRNALyS has a C,,, tRNAGLy has a U 2 , ) . The Py,, i s present The U, and Pu,, however, e x c e p t i n t R N A A S p w h i c h has an A32. a r e always present. The G,,:C, p a i r i n g i s o n l y r a r e l y present. i s p r e s e n t i n a l l except two cases. F i n a l l y , a Py, a n t i c o d o n - and T#C-stem The s i z e s o f t h e a c c e p t o r - , D-, f o l l o w t h e g e n e r a l r u l e b y h a v i n g 7, 3-4, 5 and 6 base p a i r s , respectively. The s i z e o f t h e D and T$C l o o p i s , however, v e r y v a r i a b l e . The s i z e o f t h e D-loop r a n g e s f r o m 4 t o 7 n u c l e o t i d e s w h i c h l a c k s a c o n v e n t i o n a l D-arm. w i t h t h e e x c e p t i o n o f tRNA;;; o n l y 67 n u c l e o t i d e s l o n g . O n l y 10 n u c l e o T h i s makes t h i s tRNA;;;

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t i d e s a r e p r e s e n t between t h e amino a c i d stem and t h e a n t i c o d o n stem making i t d i f f i c u l t t o imagine a s t a b l e stem and l o o p s t r u c t u r e , as a l o o p should have a t l e a s t 3-4 n u c l e o t i d e s . T h i s would g i v e a 2 A:U base-paired D stem t h e s t a b i l i t y o f which i s questionable. However, compensatory mechanisms by t h e e l o n g a t e d anticodon stem and t h e o v e r l y l o n g T$C l o o p ( n i n e r e s i d u e s ) , as proposed by De B r u i j n and Klug ( r e f . 99) f o r b o v i n e m t tRNA;;; c o u l d l e a d t o a t e r t i a r y s t r u c t u r e resembling t h a t o f standard tRNAs. The s i z e o f t h e T$C l o o p o f mosquito m t tRNAs i s comprised between 3 n u c l e o t i d e s ( i n tRNAljg;j and t R N A P h e ) and 9 n u c l e o t i d e s F i n a l l y , t h e e x t r a - l o o p i s always s h o r t (4-5 ( i n tRNA;;;). nucl e o t i des) . 7.11.2 M o d i f i e d Nucleosides The m o d i f i c a t i o n s t a t u s o f mosquito m t tRNAs as deduced from t h e 9 m t tRNAs sequenced a t t h e RNA l e v e l i s q u i t e homogenous: p o s i t i o n 9 (mlA o r m l G ) i s m o d i f i e d i n 8 cases, p o s i t i o n s 28 ($) and 37 ( t 6 A ) i n 7 cases and p o s i t i o n s 55 ($) i n 5 cases. The o n l y encountered m o d i f i e d n u c l e o t i d e s a r e m l A , m l G , m2G, t 6 A , U*, $, Cm and $in. $ i s l o c a t e d a t 11 d i f f e r e n t p o s i t i o n s whereas mlA and m1G a r e o n l y found a t p o s i t i o n 9, t 6 A a t p o s i t i o n 37 and Cm and $m a t p o s i t i o n 39. F i n a l l y , A e d e s m t tRNAs l a c k D i n t h e D-arm and T i n t h e T$C arm. A s t r i k i n g feature are t h e s i x $ residues i n tRNAV* A t R N A G L y * subspecies h a v i n g as o n l y d i f f e r e n c e from t R N A G ' y u n i d e n t i f i e d U and G d e r i v a t i v e s a t p o s i t i o n s 27 and 28, has a l s o been sequenced ( r e f . 93). A m o d i f i e d U ( c a l l e d U*) i n t h e f i r s t p o s i t i o n o f t h e a n t i c o d o n which c o u l d r e c o g n i z e o n l y t h e p u r i n e o f two-codon f a m i l i e s ending i n a p u r i n e has been found i n t R N A L y S ( r e f . 94), t R N A G ' U , and t R N A G l n ( r e f . 93). I t must be emphasized t h a t mlA a t p o s i t i o n 9 has never been d e s c r i b e d i n non-mt tRNAs ( r e f . 88) and no Cm was d e s c r i b e d a t p o s i t i o n 39. However, nucleosides m e t h y l a t e d on t h e r i b o s e have a1 ready been found i n t h i s p o s i t i o n i n non-mi t o c h o n d r i a1 tRNAs ( r e f . 88).

'.

7.11.3 Codon Readina P a t t e r n s The anticodons o f t h e 9 mosquito m t tRNAs a l l o w i n f e r e n c e of some r u l e s concerning t h e codon r e a d i n g p a t t e r n s i n t h i s

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organelle. As i n f u n g a l m i t o c h o n d r i a , m t tRNAs r e c o g n i z i n g twocodon f a m i l i e s e n d i n g i n A o r G have a m o d i f i e d U (U*) i n t h e wobble p o s i t i o n , and tRNAs r e c o g n i z i n g f o u r - c o d o n f a m i l i e s have an F u r t h e r m o r e , as deduced f r o m A e d e s m t unmodified U ( r e f . 93). p r o t e i n c o d i n g genes, UGA codes f o r t r y p t o p h a n . F i n a l l y , AGA, n o r m a l l y an a r g i n i n e codon, codes f o r s e r i n e ( r e f . 9 3 ) . This s i t u a t i o n i s u n i q u e and was o n l y f o u n d i n i n s e c t m i t o c h o n d r i a ( s e e The meaning o f t h e AGG codon, i f used i n A e d e s also 7.12). m i t o c h o n d r i a , i s unknown.

7.12 7.12.1

D R O S O P H I L A MITOCHONDRIAL tRNAs

Structures The D r o s o p h i l a m i t o c h o n d r i a 1 genome has 19.5 k b . Twenty-two t R N A genes have been i d e n t i f i e d and sequenced i n D r o s o p h i l a y a c u b a by C l a r y e t a l . ( r e f s . 100-105) and seven i n D r o s o p h i l a m e l a n o These a r e t R N A A L a ( r e f . 1 0 5 ) , t R N A * s n g a s t e r ( r e f s . 102, 1 0 6 ) . ( r e f . 1 0 5 ) , t R N A A S p ( r e f . 1 0 1 ) , tRNA,A;;! ( r e f . 1 0 5 ) , tRNACys ( r e f . 1 0 4 ) , t R N A G l n ( r e f . 1 0 3 ) , t R N A G l U ( r e f s . 101, 1 0 5 ) , t R N A G t y ( r e f . 102), tRNAH ( r e f . 100) , t R N A ' I ( r e f . 103) , tRNA:;: (ref. loo), tRNA:;," ( r e f . 101), t R N A L y S ( r e f . 101), tRNAMet ( r e f . 103), t R N A P h e ( r e f . 1 0 5 ) , t R N A P r 0 ( r e f . l o o ) , tRNA;;: ( r e f s . 100, 1 0 2 ) , tRNA;;: ( r e f . 105), tRNAThr ( r e f . l o o ) , tRNATrp ( r e f . 104), t R N A T y r ( r e f . 1 0 4 ) , t R N A V a t ( r e f . 103) f r o m D . y a c u b a and tRNAs f o r Asp, Cys, Leu(UAA), Lys, T r p and Tyr f r o m D . m e l a n o g a s t e r ( r e f . 1 0 6 ) . A t R N A G L y f r o m t h e same o r g a n i s m was a l s o sequenced by C l a r y e t a l . ( r e f . 1 0 2 ) . The D r o s o p h i l a m t tRNAs a l l show a v e r y h i g h A+U c o n t e n t . The m t t R N A A S p and t R N A G L " , w h i c h have an A+U c o n t e n t o f 91%, a r e t h e most e x t r e m e r e p o r t e d examples o f t h i s g e n e r a l f e a t u r e o f m t tRNAs. S e v e r a l g e n e r a l f e a t u r e s o f t R N A s t r u c t u r e s a r e more or l e s s conserved : U, o c c u r s i n a l l o f them e x c e p t i n tRNA;;; w h i c h has G,, and i n tRNA$;p, t R N A A S n , and tRNAVal w h i c h have A,. The A,,-Pu,, i s o f t e n r e p l a c e d b y A 1 4 - U 7 5 , once b y G 1 4 Pu,, (tRNA;;I;), and once b y U,,-U,, (tRNA1;l). The G,,-G,, sequence i s n e v e r f o u n d i n t h e D-loop. The A,, i s m o s t l y found, w h i c h makes them r e s e m b l e more eucaeven i n tRNA:;," and tRNA:;; r y o t i c c y t o p l a s m i c t h a n p r o c a r y o t i c tRNAsLeu w h i c h have a G,,

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( r e f . 107). However, U,, and C,, a r e a l s o found t w i c e each. A P U 2 6 , a l t h o u g h n o t considered as an i n v a r i a n t n u c l e o t i d e , i s found i n a l l D r o s o p h i l a m t tRNAs. Py,, i s found w i t h 2 e x c e p t i o n s , and Pu,, a r e always found. The TTCPuA (corresponding whereas U, t o t h e T$CPuA sequence) i s never found, b e i n g m o s t l y r e p l a c e d by A+U-ri ch sequences. The Pu,,:Py4, t e r t i a r y i n t e r a c t i o n can f u n c t i o n i n 19 o u t o f t h e 22 tRNAs. The G S 3 : C b , p a i r i n g i s o n l y found i n 5 o u t o f 22 tRNAs. F i n a l l y , t h e P y 6 0 i s m o s t l y conserved, except i n 4 cases. The s i z e s o f t h e stems f o l l o w t h e general r u l e , except i n t R N A C v S and t R N A P h e where t h e T$C stem has o n l y 4 base p a i r s and which has no D-stem. Both t h e D-loop and T$C-loop a r e i n tRNA;;; v e r y v a r i a b l e i n s i z e . The 0-loop has from 3 ( t R N A P r O ) t o 8 nucleotides ( t R N A V a 1 ) . The T$C-loop o f D r o s o p h i l a m t tRNAs has (tRNACyS, a l s o a v a r i a b l e l e n g t h r a n g i n g from 3 n u c l e o t i d e s , tRNA;;g) t o 8 n u c l e o t i d e s ( t R N A L y S ) . The e x c e p t i o n i s tRNA;;; which has 11 n u c l e o t i d e s between t h e amino a c i d and t h e a n t i c o d o n stem, b u t these n u c l e o t i d e s cannot be arranged t o form a s t r u c t u r e resembling t h e usual D-arm o f o t h e r tRNAs ( F i g u r e 7.10). As mentioned by C l a r y and Wolstenholme ( r e f . 105), t h i s t R N A i s p e c u l i a r and can be f o l d e d i n t o a s t r u c t u r e w i t h normal aminoacyl and a n t i c o d o n stems and w i t h an a n t i c o d o n l o o p o f 7 n u c l e o t i d e s . The T$C-arm c o u l d comprise e i t h e r a stem o f f i v e base-pairs and a l o o p o f n i n e n u c l e o t i d e s o r a s i x n u c l e o t i d e - p a i r e d stem and a seven-nucleotide l o o p . The v a r i a b l e l o o p c o u l d have s i x nucleot i d e s . However, A, , A, and G, c o u l d p a i r w i t h C, , U, and U40 i n t h e v a r i a b l e l o o p which would r e s u l t i n an extended a n t i c o d o n stem ( F i g u r e 7.10). Seven m t tRNAs can be compared between D . y a c u b a and D . m e l a n o g a s t e r (Asp, Cys, Gly, Leu ( U A A ) , Lys, Trp and T y r ) . There a r e r e s p e c t i v e l y 5, 3, 1, 2, 1, 9, and 2 n u c l e o t i d e d i f f e r e n c e s between these tRNAs i n t h e two organisms. The most i m p o r t a n t d i f f e r e n c e s a r e t h e s i z e o f t h e D-loop i n tRNAsAsp and t h e s i z e o f t h e T$C-loop i n tRNAsTrp and tRNAsTyr which d i f f e r by one nucleot i d e , b e i n g always s m a l l e r i n D . m e l a n o g a s t e r . The s i m i l a r i t i e s t o mouse m t tRNAs a r e o f t h e average o f 50% w i t h extreme values ( o n l y 32%). With A e d e s a l b o p i c t u s tRNAi;g however, f o r tRNA;;$ t h e homology o f t h i s t R N A i s o f 84%.

,

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7.12.2 Codon Readina P a t t e r n s I n D . m e l a n o g a s t e r t h e CO 11, CO I11 and ATPase 6 genes s t a r t w i t h AUG codons, URF A6L and URF2 s t a r t w i t h AUU whereas CO I s t a r t s w i t h AUAA ( r e f , 106). The same was f o u n d i n D . y a c u b a ( r e f . 104). I f a +1 r i b o s o m a l f r a m e s h i f t o c c u r r e d i n t h i s r e g i o n i t w o u l d a l l o w t h e t r i p l e t AUA t o f u n c t i o n as an i n i t i a t i o n codon. The o n l y t R N A M e t gene sequenced f r o m D . y a c u b a m t DNA has a normal a n t i c o d o n l o o p o f seven n u c l e o t i d e s ( r e f . 1 0 3 ) . However, t h e r e i s a U 5 ' t o t h e CAU a n t i c o d o n . T h i s U may be f u n c t i o n a l i n a l l o w i n g t h e t R N A M e t t o r e a d t h e f o u r l e t t e r sequence AUAA as a s i n g l e codon, as s u g g e s t e d b y De B r u i j n ( r e f . 106). The o t h e r genes sequenced i n D . y a c u b a (CO 11, CO 111, c y t b URF4 and 4L) s t a r t w i t h AUG, whereas URFZ, URF5, URF6, and URF A6L s t a r t w i t h AUU and URFl w i t h AUA. I n t h e " u n i v e r s a l " g e n e t i c code, AUA s p e c i f i e s i s o l e u c i n e . The genes i n a D r o s o p h i l a m t DNA f r a g m e n t sequenced b y De B r u i j n ( r e f . 106) c o n t a i n o n l y one i n t e r n a l AUG, b u t a t o t a l o f 82 AUA codons ( r e f . 1 0 4 ) . When compared w i t h b o v i n e m t DNA, 39 AUA a l i g n up w i t h m e t h i o n i n e and o n l y 5 w i t h i s o l e u c i n e . T h e r e f o r e , D r o s o The p h i l a m i t o c h o n d r i a p r o b a b l y use AUA t o code f o r m e t h i o n i n e . U G-C A-U A-U G-C U-A A-U

cu

A A

C I

G A

I I

GI GU

c U

u

A50

A-U G-C A30 A

G C U

F i g u r e 7.10 S t r u c t u r e o f t h e D . y a k u b a m t tRNA;:: i t s gene sequence).

(deduced f r o m

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s t r u c t u r e o f t h e a n t i c o d o n o f t R N A M e f (CAU) p e r m i t s decoding o f t h e AUA (see 7.3.4), whereas t h e s t r u c t u r e o f t R N A l l e (GAU) would not. I n t e r n a l UGA codons (which a r e t h e opal t e r m i n a t i o n codon i n t h e " u n i v e r s a l " code) a r e found i n D . y a c u b a p o l y p e p t i d e genes. T h i r t y - o n e o u t of 47 UGA codons correspond i n p o s i t i o n t o t r y p t o phan s p e c i f i e d by UGA o r UGG i n t h e e q u i v a l e n t mouse genes. I n D . m e l a n o g a s t e r , t h e p r o t e i n coding genes c o n t a i n 45 i n t e r n a l UGA codons, 35 o f which a l i g n w i t h t r y p t o p h a n codons i n t h e correspond i n g bovine sequences ( r e f . 106). I n addition, a potential tRNATrp w i t h an a n t i c o d o n UCA has been i d e n t i f i e d i n b o t h D . y a c u b a and D . m e l a n o g a s t e r f u r t h e r s u p p o r t i n g t h e view t h a t , as i n t h e f u n g a l and mammalian m i t o c h o n d r i a l g e n e t i c code, UGA a l s o s p e c i f i e s t r y p t o p h a n i n t h e D r o s o p h i l a m i t o c h o n d r i a l g e n e t i c code. I n D . y a c u b a , AGA (which codes f o r a r g i n i n e i n t h e " u n i v e r s a l " code), i s used t o s p e c i f y an amino a c i d i n seven p o l y p e p t i d e However, these AGA codons never correspond i n genes id e n t i f ied. p o s i t i o n t o codons which s p e c i f y a r g i n i n e i n t h e e q u i v a l e n t AGA t r i p l e t s p o l y p e p t i d e genes from mouse, y e a s t and p l a n t . correspond t o t w i c e as many s e r i n e - s p e c i f y i n g codons (a t o t a l o f 14) than t o codons s p e c i f y i n g o t h e r amino a c i d s (seven each f o r a l a n i n e and asparagine) ( r e f . l o o ) , which i s c o n s i s t e n t w i t h t h e view t h a t i n t h e D r o s o p h i l a m i t o c h o n d r i a l g e n e t i c code AGA speci f i e s s e r i n e ( r e f s . 101, 104, 106). However, no t R N A w i t h a corresponding UCU anticodon has been found up t o now. Thus, t h e tRNA;;; should r e c o g n i z e t h e t h r e e codons AGC, AGU and AGG. Whether t h e a r g i n i n e codon AGG a l s o codes f o r s e r i n e i s n o t known, as i t i s n o t used i n t h e sequenced m t p o l y p e p t i d e genes. F i n a l l y , t h e CUU anticodon o f t R N A L y s i n D r o s o p h i l a mitochonHowever, d r i a would be expected t o r e c o g n i z e o n l y t h e codon AGG. t h e codon AAA ( a l s o expected t o s p e c i f y l y s i n e ) i s used much more I f t h e r e i s o n l y a s i n g l e t R N A L y S gene i n f r e q u e n t l y t h a n AAG. D r o s o p h i l a m i t o c h o n d r i a , then a C-A wobble, s i m i l a r t o t h e one p o s t u l a t e d f o r m t tRNA:et i n S . cerevisiae mitochondria ( r e f . 2 3 ) , should a l s o be used.

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7.13

XENOPUS L A E V Z S MITOCHONDRIAL tRNAs

7.13.1 S t r u c t u r e s The c o m p l e t e sequence o f t h e 17,553 n u c l e o t i d e s o f t h e X e n o p o s l a e v i s m i t o c h o n d r i a l genome has been d e t e r m i n e d ( r e f . 108). I t encodes 22 tRNAs w i t h o n l y one tRNA;;;! and one t R N A M e f . C o n c e r n i n g t h e i n v a r i a n t and s e m i - i n v a r i a n t n u c l e o t i d e s , 16 tRNAs one has a C,, and one a G,. The A-Pu have a U,, f o u r have an A,, i n p o s i t i o n s 14-15 i s a b s e n t i n t R N A A S n , t R N A C V S , and t R N A G L y and tRNA;;;, t h e G-G i n p o s i t i o n s 18-19 i s o n l y p r e s e n t f o u r t i m e s . The Py i n p o s i t i o n 32 i s more conserved, s i n c e o n l y t R N A A t a has a G32. The same h o l d s f o r U, which i s o n l y r e p l a c e d b y C i n tRNA:,"," and t R N A H e t . L e t us r e c a l l t h a t t h e c y t o p l a s m i c i n i t i a t o r tRNAs f r o m mammals, amphibians, f i s h e s , i n s e c t s and p l a n t s a l s o The m o s t c o n s e r v e d n u c l e o t i d e i s t h e have a C i n p o s i t i o n 33. p u r i n e i n p o s i t i o n 37, a f t e r t h e a n t i c o d o n , w h i c h i s p r e s e n t i n t h e 22 tRNAs. Seventeen tRNAs l a c k t h e sequence rT(U)-$-C-Pu-A in l o o p I V . F i n a l l y , t h e p y r i m i d i n e i n p o s i t i o n 60 i s a b s e n t i n f o u r t RNAs The s i z e o f t h e stems i s r e g u l a r , e x c e p t f o r t h e D-stem w h i c h c a n n o t be b u i l t up i n t R N A A s n where o n l y one base p a i r can be tRNA;;: has o n l y 10 n u c l e o t i d e s between t h e amino a c i d formed. stem and t h e a n t i c o d o n stem ( v e r s u s 16-21 n u c l e o t i d e s i n " c l a s s i c a l " tRNAs) and c a n n o t f o r m a stem-looped D-arm ( f o r d i s c u s s i o n see 7.12). On t h e o t h e r s i d e , t w o tRNAs c o u l d have a f i v e base p a i r e d D-stem ( t R N A T v r and t R N A L y S ) . The D-loop c o n t a i n s f r o m 4 t o 10 n u c l e o t i d e s ( e x c e p t i n tRNA;;: as s t a t e d above). The a n t i c o d o n l o o p has t h e c l a s s i c a l shape, whereas t h e e x t r a - l o o p i s a l w a y s s h o r t (3-5 n u c l e o t i d e s ) . F i n a l l y , t h e l o o p I V i s q u i t e c l a s s i c a l (7 n u c l e o t i d e s ) , e x c e p t i n t R N A C y s and t R N A G 1 y where i t c o n t a i n s o n l y 6 n u c l e o t i d e s .

.

7.13.2 Codon Readina P a t t e r n s A l l 13 open r e a d i n g frames o f t h e a m p h i b i a n m i t o c h o n d r i a 1 genome b e g i n w i t h AUG. The AUA codons w h i c h code a l s o f o r m e t h i o n i n e , a r e o b s e r v e d o n l y i n t e r n a l l y . The t R N A n e f has a CAU a n t i codon. Thus a wobble between C and A s h o u l d t a k e p l a c e as w i t h 5. c e r e v i s i a e m t tRNA:'t ( r e f . 23). X . l a e v i s uses UAA as a s t o p codon t w e l v e t i m e s . I n a d d i t i o n , i t employs t h e AGA s t o p codon

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once ( n o r m a l l y a codon f o r a r g i n i n e ) . The codon AGG i s n o t used as a t e r m i n a t o r . I n a d d i t i o n , b o t h UGG and UGA code f o r t r y p tophan. I n X . l a e v i s m i t o c h o n d r i a l genes, t h e r e i s an o v e r a l l p r e f e r e n c e f o r codons ending i n U i n t h e two codon f a m i l i e s whereas i n mammals t h e p r e f e r e n c e i s i n codons ending w i t h C. Thus, G:U wobble i s p r e f e r r e d t o t h e normal G:C r e a d i n g o f t h e codon t h i r d p o s i t i o n . MAMMALIAN MITOCHONDRIAL tRNAs The complete sequence of t h e 16,569 base p a i r human m i t o c h o n d r i a l genome was p u b l i s h e d i n 1981 by Anderson e t a 1 . ( r e f . 109). I t was f o l l o w e d by t h e complete n u c l e o t i d e sequences o f t h e bovine and mouse m i t o c h o n d r i a l genomes ( r e f s . 110, 111) which have r e s p e c t i v e l y 16,338 and 16,295 n u c l e o t i d e s . These f o l l o w e d s e v e r a l d e t e r m i n a t i o n s o f p a r t i a l sequences o f human, b o v i n e and mouse m i t o c h o n d r i a1 genomes, which p e r m i t t e d t h e sequence o f On t h e s e v e r a l m t t R N A genes ( r e f s . 112-114) t o be o b t a i n e d . o t h e r hand, a l l t h e r a t m t t R N A genes have been sequenced ( r e f s . 115-123). I n p a r a l l e l , De B r u i j n e t a ] . ( r e f . 124) and A r c a r i and Brownlee ( r e f . 125) sequenced t h e b o v i n e and human tRNA:;;. Seven r a t l i v e r o r r a t hepatoma m t tRNAs have been sequenced by Randera t h e t a 7 . ( r e f s . 126-131). T a i r a e t a 7 . ( r e f . 132) a l s o compared m t t R N A sequences i n f o u r r a t tumors w i t h t h o s e of normal r a t liver. The complete s e t o f b o v i n e m i t o c h o n d r i a l tRNAs has been sequenced a t t h e RNA l e v e l by Roe e t a 7 . ( r e f . 133). As t h e r e a r e h i g h s i m i l a r i t i e s between t h e mammalian m t tRNAs we s h a l l t r e a t them t o g e t h e r . 7.14

7.14.1 S t r u c t u r e s Twenty-two t R N A genes have been i d e n t i f i e d i n human, b e e f and mouse m i t o c h o n d r i a . There i s one t R N A p e r amino a c i d , except f o r l e u c i n e and s e r i n e . Thus, t h e r e i s no t R N A A r g c o r r e s p o n d i n g t o t h e codons AGA and AGG and o n l y one t R N A M e t f o r b o t h i n i t i a t i o n and i n t e r n a l m e t h i o n i n e codons. No tRNAs have been shown t o be imported from t h e c y t o p l asm i n t o mammal ian m i t o c h o n d r i a ( r e f . 133). The sequences o f t h e mammalian m t tRNAs a r e unusual i n t h a t they a l l l a c k s e v e r a l o f t h e i n v a r i a n t and s e m i - i n v a r i a n t nucleot i d e s t o g e t h e r w i t h t h e stem and l o o p s i z e s o f t h e " c l a s s i c a l "

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tRNAs. The o n l y " c l a s s i c a l " t R N A i s m t tRNA:;," f r o m human, b o v i n e and mouse. The u r i d i n e i n p o s i t i o n 8 i s p r e s e n t i n 15 o u t o f 22 mammalA, i s p r e s e n t i n f i v e tRNAs, and C, and G, i n one i a n m t tRNAs. each. No g e n e r a l r u l e can be f o u n d c o n c e r n i n g t h e A-Pu i n p o s i t i o n s 14-15; h a l f o f t h e tRNAs have t h i s sequence ( m o s t l y A-A). The G-G sequence i n p o s i t i o n s 18-19 i s o n l y f o u n d i n tRNA:;,", t R N A G L n and tRNA:::. The Pu,, i s more g e n e r a l l y p r e s e n t , e x c e p t tRNA;;; and tRNALyS. Three i n a l l mammalian t R N A C y S , tRNA;,";, n u c l e o t i d e s have conserved, a l m o s t w i t h o u t e x c e p t i o n , their s t a n d a r d p o s i t i o n . These a r e a Py i n p o s i t i o n 32, a Pu i n p o s i which i s p r e s e n t w i t h t i o n 37 w h i c h a r e always p r e s e n t , and U, two e x c e p t i o n s : i t i s r e p l a c e d b y C i n a l l tRNAsHet ( w h i c h i s a l s o t h e case i n mammalian c y t o p l a s m i c i n i t i a t o r tRNAsMet) and i n The Py, i s present except i n bovine m t mouse m t t R N A V a l . tRNAThr, tRNATrp, tRNACyS and tRNACLn, i n human tRNACyS and tRNAGLn, and i n mouse tRNAGL n . F i n a l l y , t h e sequence U-U-C-Pu-A is r a r e l y p r e s e n t in t h e mammal ian ( c o r r e s p o n d i n g t o T-$-C-Pu-A) m t tRNAs. As a l r e a d y s t a t e d , i t i s p r e s e n t i n t h e tRNAs:;,", but a l s o i n a l l tRNAsCLn and tRNAs::; and i n human and b o v i n e tRNACyS. C o n c e r n i n g t h e s i z e o f t h e stems and l o o p s , t h e amino a c i d stem and t h e a n t i c o d o n stem and l o o p have t h e c l a s s i c a l s i z e . The v a r i a b l e l o o p i s always s h o r t (3-5 n u c l e o t i d e s ) , even i n tRNAsLeU and tRNAsSer, w h i c h a r e known f o r h a v i n g a l o n g v a r i a b l e l o o p i n p r o c a r y o t e s and t h e e u c a r y o t i c c y t o p l a s m , and i n t R N A T y r ( a l o n g v a r i a b l e - a r m i s f o u n d i n tRNAsTyr f r o m e u b a c t e r i a and f r o m f u n g a l and p r o t o z o a n m i t o c h o n d r i a ) . The l e n g t h of t h e D-loop v a r i e s f r o m 3 t o 10 n u c l e o t i d e s . Sometimes i t has t h e same number o f n u c l e o t i d e s i n i s o a c c e p t i n g tRNAs f r o m t h e t h r e e mammals, b u t m o s t l y , i t d i f f e r s b y 1-2 n u c l e o t i d e s between human, b o v i n e and mouse i s o a c ceptors. T h i s i s a l s o t r u e f o r l o o p I V where even more d r a m a t i c d i f f e r e n c e s were f o u n d . O n l y seven tRNAs have t h e c l a s s i c a l number o f 7 n u c l e o t i d e s i n t h i s l o o p amongst a l l t h r e e mammalian m i t o c h o n d r i a , b u t t w o human m t tRNAs have o n l y 3 n u c l e o t i d e s i n t h i s l o o p (tRNA;;;! and t R N A T h r ) whereas t h e c o r r e s p o n d i n g tRNAs from b o v i n e and mouse have between 5 and 8 n u c l e o t i d e s i n t h a t loop. Most o f t h e tRNAsSer and one tRNAHiS have a l s o 8 n u c l e o t i d e s i n l o o p I V and human t R N A L y s has 9 n u c l e o t i d e s . F i n a l l y ,

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the most extreme c a s e i s tRNA;;; which l a c k s the D-arm. A large s c a l e i s o l a t i o n method f o r t h i s t R N A from bovine h e a r t mitochond r i a was developed by Ueda e t a l . ( r e f . 134). By m e l t i n g and nuclease d i g e s t i o n methods i t was suggested t h a t three e x t r a base p a i r s could occur i n t h e anticodon stem region with one adenosine r e s i d u e " p r o t r u d i n g " . The T-loop does not seem t o i n t e r a c t with o t h e r r e g i o n s ( F i g u r e 7.11). Three m t tRNAs genes from hominoid apes (chimpanzee, g o r i l l a , orangutan and gibbon) have been sequenced by Brown e t a l . ( r e f . 135). They a r e t R N A H i S , tRNA:;; and t R N A 2 ; ; . The comparison t o t h e i r human m t t R N A c o u n t e r p a r t s shows d i f f e r e n c e s i n a l l stem and loop r e g i o n s , t h e most important being l o c a t e d i n t h e Tq$C-loop of t R N A H i S (5 o u t of 7 n u c l e o t i d e s ) and between t h e a c c e p t o r stem and anticodon stem of t h e tRNA;;; (4 o u t of 5 n u c l e o t i d e s ) . A high v a r i a b i l i t y i s a l s o found i n p o s i t i o n s 42-47 of t h i s tRNA;;;. The sequences of mammalian m t tRNAs have been compared by Cantatore e t a l . ( r e f . 120) who c a l c u l a t e d t h e percentages of A C C G G-C A-U

A-U A-U A-U A-U U G - C ~G I

A

I

u

A

u

I

I

I

G * U A

~ ~ C C A U A

u

c

A

u c u

G-C C-G

>I: C U

G-C A-U A-U

A t 6A

G C U

Figure 7 . 1 1

S t r u c t u r e of the bovine m t tRNA;:;.

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homology between r a t , mouse, b o v i n e and human m t tRNAs. The homologies f o r 17 o u t o f 22 tRNAs r a n g e f r o m 84%-97% between r a t and mouse, f r o m 63%-97% between r a t and b e e f and f r o m 66%-97% I t must be emphasized t h a t , on t h e a v e r between r a t and human. age, i t i s t h e tRNAMet w h i c h i s t h e m o s t c o n s e r v e d m t t R N A between mammals. T h i s tRNA works b o t h i n i n i t i a t i o n and c h a i n e l o n g a t i o n and i s p r o b a b l y s u b m i t t e d t o h i g h f u n c t i o n a l c o n s t r a i n t s ( s e e 7.14.3). When t h e m t tRNAs f r o m r a t s a r e compared w i t h t h e sequences o f m t tRNAs f r o m A . n i d u l a n s o r S. c e r e v i s i a e , t h e d e g r e e o f s i m i l a r i t y d r o p s t o 32-50% w i t h y e a s t and t o 20-51% w i t h A . n i d u l a n s ( r e f . 120). W i t h E . c o l i tRNAs, t h e s i m i l a r i t y i s a b o u t t h e same: 28-50% ( a v e r a g e 39%), whereas w i t h c y t o p l a s m i c tRNAs f r o m y e a s t o r mammals i t i s even l o w e r ( a v e r a g e o f 33% and 38%, r e s p e c t i v e l y ) . 7.14.2 M o d i f i e d N u c l e o s i d e s I n a d d i t i o n t o t h e s e q u e n c i n g o f t h e m t tRNA genes, Roe e t al. have d e t e r m i n e d t h e sequence o f t h e 22 b o v i n e m t tRNAs a t t h e RNA l e v e l ( r e f . 133), and Randerath e t al. ( r e f s . 126-131) t h o s e o f s e v e r a l r a t l i v e r o r M o r r i s hepatoma 5123D m t tRNAs (tRNA:;;, tRNAASp, t R N A L y S , t R N A V a L , t R N A P h e , tRNATrp and tRNA();g) w h i c h p e r m i t t e d i d e n t i f i c a t i o n and l o c a t i o n o f t h e m o d i f i e d n u c l e o s i d e s . When an A i s p r e s e n t a t p o s i t i o n 9, i t i s m e t h y l a t e d t o m l A , a unique l o c a t i o n f o r t h i s m o d i f i e d n u c l e o t i d e u s u a l l y found i n e u k a r y o t i c c y t o p l a s m i c tRNAs a t p o s i t i o n s 14 and 58 and i n p r o c a r y o t i c tRNAs a t p o s i t i o n 22 ( r e f . 8 8 ) . I f p o s i t i o n 9 i s a G, i t w i l l be m o d i f i e d t o m l G . A g u a n o s i n e a t p o s i t i o n 10 may be m e t h y l a t e d t o m*G. $ i s f o u n d a t p o s i t i o n s 27, 32, o r 39. D i s found a t p o s i t i o n 20, and m 5 C a t p o s i t i o n 49 whereas an A a t p o s i t i o n 37, a f t e r and t R N A I t e o r t o t h e a n t i c o d o n , may be m o d i f i e d t o t 6 A i n tRNA2;: m s * i 6 A i n t R N A T r p and tRNAPhe. I f t h e r e i s a G i n p o s i t i o n 37, i t i s m o d i f i e d t o m1G. The U i n t h e wobble p o s i t i o n o f tRNATrp i s a l s o m o d i f i e d , p r o b a b l y i n a same manner i n a l l m t tRNAs r e c o g n i z i n g boxes o f two codons t e r m i n a t e d b y a p u r i n e . 7.14.3 Codon Readinci P a t t e r n s As e a r l y as 1979, B a r r e l 1 e t al. ( r e f . 136) b y c o m p a r i n g t h e human m t DNA sequence o f t h e cytochrome o x i d a s e s u b u n i t I 1 gene

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and the sequence of the corresponding beef h e a r t p r o t e i n , showed t h a t UGA i s used a s a tryptophan codon and not a s a termination codon. This comparison a l s o suggested t h a t AUA may be a methio nine r a t h e r than a i s o l e u c i n e codon. This was confirmed when the complete sequence of the human mitochondria1 genome was determined ( r e f . 109). The t R N A T r p has an anticodon UCA ( i n s t e a d of CCA i n non-mitochondria1 tRNAsTr p ) allowing t r a n s l a t i o n of both UGA and UGG.

Concerning i n i t i a t i o n codons, a l l t h e i d e n t i f i e d genes and some of t h e URFs use AUG a s an i n i t i a t i o n codon. However, t h r e e URFs use AUA, and URF2 (NDZ) uses AUU. Recently, Fearnley and Walker ( r e f . 185) have shown t h a t AUU has a dual coding function i n human mitochondria: i t serves a s an i s o l e u c i n e codon i n elongation of cytochrome b , b u t i t a l s o s p e c i f i e s t h e i n i t i a t o r methionine residue of the ND2 gene product. By c o n t r a s t , AUA encodes methionine both i n i n i t i a t i o n and i n elongation of mitochondrial p r o t e i n synthesis. However, only one t R N A M e gene, coding f o r a t R N A w i t h a CAU anticodon, i s apparent i n the comA possible p l e t e mi tochondrial DNA sequence ( r e f s . 109-111). explanation i s t h a t s p e c i f i c i t y f o r i n i t i a t i o n o r elongation i s conferred by a s p e c i f i c modification of t h e unique t R N A M e t ( s e e be1 ow). In a d d i t i o n t o t h e use of the c l a s s i c a l UAA and UAG terminat i o n codons, termination occurs a t AGA and AGG codons i n three cases (human COI and URF6, and bovine cytochrome b ) . AGR t r i p l e t s have been predicted t o be termination codons r a t h e r than a r g i n i n e codons a s i n the universal g e n e t i c code ( r e f . 7 0 ) . This i s based on the observation t h a t ( i ) only a r g i n i n e CGN codons a r e used in a l l t h e reading frames, ( i i ) AGA and AGG a r e only found a t t h e end of reading frames, ( i i i ) no gene coding f o r a t R N A t h a t could t r a n s l a t e the codons AGR has been found. The codon usages found i n r a t and mouse mitochondria a r e very s i m i l a r t o those i n human mitochondria. Minor exceptions concerning the use of i n i t i a t i o n and termination codons a r e however observed. For example, the URFl i n i t i a t i o n codon i s AUA i n human mitochondria and A U U i n mouse mitochondria. On t h e o t h e r hand, the COI termination codon i s AGA i n human mitochondria, whereas i t i s UAA i n mouse and r a t mitochondria. No termination codons AGA

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o r AGG a r e f o u n d i n mouse m i t o c h o n d r i a . A more s t r i k i n g d i f f e r e n c e was f o u n d i n mouse m i t o c h o n d r i a where b o t h URF3 and URF5 AUA, s t a r t w i t h AUC ( r e f . 111). Thus, t h e f o u r codons AUG, AUU, and AUC a r e used as i n i t i a t o r codons i n t h i s o r g a n e l l e . As the C i n the t h e o n l y tRNAMef f o u n d has a CAU a n t i c o d o n , wobble p o s i t i o n s h o u l d p a i r w i t h a l l f o u r bases i n t h e t h i r d p o s i t i o n o f t h e i n i t i a t i o n codon. T h i s c o u l d be o b t a i n e d b y a s p e c i f i c m o d i f i c a t i o n o f t h i s C ( r e f s . 137, 1 3 8 ) . Howeve r , t h i s same t R N A M e t s h o u l d a l s o r e c o g n i z e o n l y t h e i n t e r n a l AUG and AUA codons s p e c i f i c f o r m e t h i o n i n e b u t n o t i n t e r n a l AUU and AUC codons o f i s o l e u c i n e . Thus, t w o f o r m s o f a u n i q u e t R N A M e t c o u l d e x i s t : one w i t h a m o d i f i e d C i n t h e f i r s t p o s i t i o n o f t h e a n t i c o d o n w h i c h w o u l d f u n c t i o n as t h e tRNAYet a b l e t o r e c o g codons, and one w i t h a nonnize a l l f o u r AUN i n i t i a t i o n able m o d i f i e d C w h i c h w o u l d f u n c t i o n as t h e e l o n g a t o r tRNA;.t t o r e c o g n i z e i n t e r n a l AUG and AUA codons. A p e c u l i a r f i n d i n g was o b t a i n e d i n o r a n g u t a n m i t o c h o n d r i a where ACA ( w h i c h codes f o r t h r e o n i n e i n t h e " u n i v e r s a l " code) i s t h e f i r s t codon i n URF5. However, an i n - f r a m e c a n o n i c a l AUG s t a r t codon i s p r e s e n t t w o codons downstream f r o m ACA. I t may be t h a t AUG r a t h e r t h a n ACA i s used f o r i n i t i a t i o n o f t r a n s l a t i o n o f t h e URF5 p r o d u c t . F i n a l l y , t h e f o u r - c o d o n boxes i n t h e g e n e t i c code a r e each r e a d b y a s i n g l e tRNA w i t h U i n t h e f i r s t p o s i t i o n o f t h e a n t i codon and two-codon boxes t e r m i n a t e d b y a p u r i n e a r e r e a d b y a t R N A w i t h a U i n t h e wobble p o s i t i o n ( r e f s . 70, 133). This i s l i k e l y t o be a m o d i f i e d U* as i n o t h e r m i t o c h o n d r i a . L e t us emphasize t h a t mammalian m i t o c h o n d r i a use much more f r e q u e n t l y codons e n d i n g w i t h C (41% i n human, 26% i n mouse m i t o c h o n d r i a ) t h a n f u n g a l m i t o c h o n d r i a where codons e n d i n g i n C a r e v e r y r a r e l y used, o r even n o t used a t a l l ( f o r example, a r g i n i n e GCG, g l y c i n e GGC, and l e u c i ne CUC)

.

7.14.4 Tumors C e l l M i t o c h o n d r i a l tRNAs M i t o c h o n d r i a 1 tRNAs f r o m t u m o r s e x h i b i t a t r e n d t o w a r d u n d e r m o d i f i c a t i o n and u n d e r m e t h y l a t i o n . T h i s has f i r s t been shown b y C h i a e t a l . ( r e f . 139) who compared t h e p a t t e r n o f m a j o r and m o d i f i e d n u c l e o s i d e s o f m t tRNAs f r o m M o r r i s hepatomas 5123D and 7777 t o t h e one o f normal l i v e r . U n d e r m o d i f i c a t i o n was f o u n d t o

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be more p r e v a l e n t i n m t tRNAs from the r a p i d l y growing, h i g h l y malignant hepatoma 7777 than from the slowly growing, l e s s malignant hepatoma 5123D. The p a t t e r n of hypermodi f i e d nucl e o s i des between normal and tumor m t tRNAs was a l s o found t o be a l t e r e d . For example, hepatoma 5123D m t t R N A A S p was found t o have a G i n t h e anticodon wobble p o s i t i o n whereas i t s c o u n t e r p a r t from normal 1 i v e r has queuosine ( r e f s . 127, 130). In a d d i t i o n , m t tRNAs from tumor and normal c e l l s a l s o d i f f e r by base changes. Randerath and coworkers have compared t h e sequences of Morris hepatoma 5123D m t t R N A A r g , -Asp, -Leu (UAG), and -Val with t h e corresponding tRNAs i n normal 1 iver ( r e f s . 126131). For example, t h e m t t R N A V a L from normal l i v e r has U and G i n p o s i t i o n s 3 and 5 r e s p e c t i v e l y , whereas the hepatoma counterp a r t has predominantly G, and U, ( r e f . 131). The l i v e r m t tRNA:,'; has a U, whereas both U, and G, a r e found i n tumor ( r e f s . 126, 131). In the l i v e r m t t R N A E ; $ , only U,, was found whereas i n tumor t R N A , both U,, and G,, were found ( r e f . 128). However, no d i f f e r e n c e between normal l i v e r and hepatoma was found i n the m t t R N A P h e and t R N A T r p ( r e f . 131). Base changes between normal and tumor m t tRNAs a r e not only found a t the RNA l e v e l b u t a l s o i n the t R N A genes. T a i r a e t a l . have compared m t t R N A gene sequences i n f o u r r a t tumors (Morris hepatoma 5123D, Yoshida sarcoma, a s c i t e s hepatoma AH-130 and AH -7974) with t h o s e of normal r a t l i v e r ( r e f . 132). Whereas t h e gene sequences between l i v e r and 5123D tumors a r e i d e n t i c a l f o r t h e f i v e s t u d i e d t R N A genes (Ala, Asn, Cys, T r p , T y r ) , s i g n i f i c a n t changes of n u c l e o t i d e sequences were d e t e c t e d i n the genes f o r t R N A T y r , t R N A C v S and t R N A T r p of t h e t h r e e o t h e r tumor mitochond r i a . In the t R N A T y r gene of Yoshida sarcoma, two n u c l e o t i d e s (A, and Ub6) a r e d e l e t e d thus g i v i n g an a c c e p t o r stem c o n s i s t i n g of only s i x b a s e - p a i r s . In a s c i t e s hepatomas AH-130 and AH-7974, the t R N A T y r gene c a r r i e s a C t o G t r a n s v e r s i o n a t p o s i t i o n 60 (which i s a s e m i - i n v a r i a n t p o s i t i o n i n loop IV). The AH-130 m t t R N A C y s gene has an i n s e r t i o n of a d i n u c l e o t i d e CC g i v i n g a 10 n u c l e o t i d e long loop IV, which i s o u t s i d e t h e s i z e - r a n g e of t h i s loop, even i n mitochondria. In a d d i t i o n , two U t o C t r a n s i t i o n s a l s o occurred i n t h i s loop ( s e e Figure 7.12). In both AH-130 and AH-7974 t R N A C v S genes, an A t o T t r a n s v e r s i o n occurred a t the semi-in-

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v a r i a n t position 21. F i n a l l y , the t R N A T r p gene from AH-7974 carries a deletion of a C residue a t position 4 1 (anticodon stem). This deletion m i g h t result i n formation of two alternative unusual anticodon-stem structures (see Figure 7 . 1 2 ) . From these results, i t appears t h a t a l l types of mutations are f o u n d i n tumor m t tRNAs genes: deletions, insertions, base transversions and transitions. However, Taira e t a 7 . ( r e f . 132) have proposed t h a t the products of these mutated genes are a p p a r ently functional tRNAs, for the following reasons: ( i ) i f they were non-functional , mi tochondrial protein synthesis would be inhibited, because the m t DNA has a single gene for each t R N A species; and ( i i ) a large s e t of non-tumor mammalian m t tRNAs have unusual cloverleaf structures, b u t nevertheless are functional. However, i t i s not known whether these mutated t R N A genes are fully f u n c t i o n a l in mitochondria1 protein synthesis. The mutations of tumor cells m t tRNAs could lower f i d e l i t y (or lower veloc i t y ) of translation w h i c h may influence cell function. Rat liver tRNA cys

Asdte hepatma AH-130 tRNACYs

TY loop

TY loop

ccu CAUCU I I I I I

GUA G A G

C

CAUCU

U

1 1 1 l 1

GUAGA

A A Rat liver tRNAT'P anticodon stem

cccc

C C

GA A c

Two possibilitles of secondary structure of Asdte hepatoma AH-7974 tRNATrP anticodon stem

AG A A-U

A G A A-U

AG A U A-U G-C

lJ

A C U

C-G

A A

U C A

C U

G-C _ _ C-G

C-G A A

U C A

Figure 7 . 1 2 tRNACvS

and m t

hepatoma.

C U

A A

U C A

Comparison of partial sequences of r a t liver m t t R N A T r p with the same tRNAs originating from ascite

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A S C A R I S SUUN MITOCHONDRIAL t R N A s 7.15.1 Structures The complete nucleotide sequence o f the p a r a s i t i c nematode worm A s c a r i s suum m t DNA has been recently determined by Wolstenholme e t a l . ( r e f . 140). The molecule i s 14,284 base p a i r s long. I t does n o t contain sequences t h a t can be folded i n t o the charact e r i s t i c secondary s t r u c t u r e of known tRNAs. However, between many o f the d i f f e r e n t protein a n d rRNA genes a r e found 2 1 sequences t h a t have a common potential secondary s t r u c t u r e resembling t R N A cloverleaves i n which the T$C arm and the variable arm are replaced by a loop of between 4 and 12 nucleotides (called TV rep1 acement loop by the authors). Each of these t R N A cloverleaves, which contain from 51 t o 62 nucleotides (without the 3'CCA), has a c l a s s i c a l aminoacyl stem of 7 base pairs, a D-stem of 3-4 base p a i r s , a D-loop o f between 5 and 9 nucleotides and a c l a s s i c a l anticodon stem and loop (5 base p a i r s

7.15

U G.U A-U C-G A-U A-U A-U

U G.U

C A U A

A

u

U.G C-G U-A

u .G

G U

u U

PGUP

U

GUUGU

U U

C C U * A I 1

A U-A G-C U-A G-C U-A U

C-G A-U G-C G.U U-A A

U

A G A

A

I

G G A G A U U

C U

u"

A A

cu

B

Figure 7.13 Structure o f the A s c a r i s suum m t t R N A ; Z i (B) deduced from t h e i r gene sequences. tRNA;;;

(A) and

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and 7 n u c l e o t i d e s ) ( f o r an example see F i g u r e 7.13). A further in s i n g l e s t r u c t u r e ( r e s e m b l i n g t h e mammalian m t tRNA;;;) w h i c h t h e D-arm i s r e p l a c e d by a l o o p o f 5 n u c l e o t i d e s (D-arm replacement loop) and t h a t c o n t a i n s a UCU a n t i c o d o n , i s a l s o f o u n d ( F i g u r e 7.13). The a u t h o r s p r e s e n t s t r o n g arguments i n favor o f t h e f a c t t h a t these b i z a r r e s t r u c t u r e s represent t h e c o m p l e t e s e t o f t R N A genes o f t h e A . s u u m m i t o c h o n d r i a 1 genome ( r e f . 140). I n p a r t i c u l a r , t h e s e tRNAs p r e s e n t a l l t h e anticodons necessary t o trans1a t e t h e m i t o c h o n d r i a1 g e n e t i c code o f A . s u u m . These tRNAs show a 100% c o n s e r v a t i o n o f r e s i d u e s U,, A,, A,,, Py,, , U, , and Pu,, A p u r i n e , l o c a t e d i n p o s i t i o n 2 o f t h e TV r e p l a c e m e n t l o o p , i s a l s o f o u n d i n a l l tRNAs. The Pu,, i s present p a i r . The e x c e p t i n 4 tRNAs. The same i s t r u e f o r t h e Py,, :Pu,, are o t h e r i n v a r i a n t n u c l e o t i d e s o f t h e D l o o p , Pu,, and G1,-G1,, a b s e n t . T h i s i s p r o b a b l y n o t i m p o r t a n t because t h e s e n u c l e o t i d e s n o r m a l l y p a i r w i t h Py, and w i t h t h e $ 5 5 - C 5 b sequence w h i c h a r e a b s e n t i n A . s u u m m t tRNAs. The e x c e p t i o n i s tRNA;;; which i s t h e s h o r t e s t t R N A e v e r sequenced (54 n u c l e o t i d e s ) where even t h e T$C arm has o n l y 10 n u c l e o t i d e s : a t h r e e - b a s e p a i r e d stem and f o u r n u c l e o t i d e loop. Thus, t h e absence o f t h e D-loop i n t h i s tRNA i s n o t f u l l y compensated b y a c l a s s i c a l T$C-arm. To see i f b i z a r r e m t tRNAs a r e l i m i t e d t o p a r a s i t i c and p a r t i a l l y a n a e r o b i c organisms l i k e A . s u u m , Wolstenholme e t a 7 . ( r e f . 140) have a l s o sequenced a segment o f t h e m t DNA f r o m t h e obligate f r e e - l i v i n g s o i l nematode C a e n o r h a b d i t i s e l e g a n s , an aerobe. T h i s segment i n c l u d e s s i x tRNAs genes, f o r a s p a r t i c a c i d , c y s t i n e , g l y c i n e , h i s t i d i n e , m e t h i o n i n e and t h r e o n i n e . Each o f t h e s e t R N A genes c o u l d be f o l d e d i n t o s e c o n d a r y s t r u c t u r e s s i m i l a r t o t h o s e o f t h e c o r r e s p o n d i n g A . s u u m m t tRNAs, w i t h no T$C-arm. O v e r a l l sequence s i m i l a r i t i e s r a n g e f r o m 58% ( t R N A A S p ) t o 85% (tRNAHef ) . 90% o f t h e d i f f e r e n c e s a r e s u b s t i t u t i o n s and 10% d e l e t i o n s - i n s e r t i o n s w h i c h o c c u r o n l y i n D l o o p and TV r e placement l o o p . Most o f t h e s u b s t i t u t i o n s i n t h e stems i n v o l v e changes such t h a t t h e base p a i r i n g s a r e s t i l l p o s s i b l e , t h u s m a i n t a i n i n g secondary s t r u c t u r e s t a b i l i t y . These d a t a s t r e n g t h e n t h e h y p o t h e s i s t h a t t h e s e genes c o r r e s p o n d t o f u n c t i o n a l tRNAs and i n d i c a t e t h a t t h e s e tRNAs a r e c h a r a c t e r i s t i c t o nematode m i t o c h o n d r i a.

.

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7.15.2 Codon Readinq P a t t e r n s I n A . suum m t p r o t e i n genes a l l codons a r e used, except 4 t h a t end i n a C: CUC ( l e u c i n e ) , CCC ( p r o l i n e ) , UGC ( c y s t e i n e ) , CGC ( a r g i n i n e ) . The p r e f e r e n t i a l usage o f codons ending i n U i s a l s o a c h a r a c t e r i s t i c of fungal m i t o c h o n d r i a . As i n o t h e r m i t o chondria, UGA s p e c i f i e s t r y p t o p h a n r a t h e r t h a n f u n c t i o n i n g as a s t o p codon. The 22 anticodons found i n t h e m t tRNAs a r e unique and compatible w i t h codon usage i n A . suum m t DNA. Nineteen o f these anticodons correspond t o anticodons found i n tRNAs o f b o t h v e r t e b r a t e and d r o s o p h i l a m i t o c h o n d r i a . One o f t h e A . suum m t t R N A has an a n t i c o d o n UUU which corresponds t o t h e AAA and AAG l y s i n e codons ( t h i s i s a l s o t h e case i n v e r t e b r a t e m t tRNAsLvs whereas d r o s o p h i l a and mosquito m t tRNAsLvs have a CUU a n t i c o d o n ) . I n A . suum m i t o c h o n d r i a , a l l 4 AGN codons a r e used t o s p e c i f y serine. T h i s i s a unique s i t u a t i o n because i n t h e " u n i v e r s a l " code AGU and AGC s p e c i f y s e r i n e and AGA and AGG s p e c i f y a r g i n i n e . I n v e r t e b r a t e m i t o c h o n d r i a , o n l y AGU and AGC a r e used as s e r i n e codons whereas AGA and AGG a r e e i t h e r absent o r b e l i e v e d t o be s t o p codons. I n d r o s o p h i l a m i t o c h o n d r i a AGU, AGC and AGA s p e c i f y I n A . suum, t h e t r a n s l a t i o n o f s e r i n e , whereas AGG i s n o t used. t h e f o u r codons AGN i s presumably done by t h e t R N A h a v i n g a UCU anticodon and which corresponds t o s e r i n e . The p u t a t i v e m t t R N A A r g o f A . suum c o n t a i n s an a n t i c o d o n ACG which i s i n c o n t r a s t t o t h e UCG a n t i c o d o n o f a l l known metazoan m t tRNAsArg. T h i s should decode o n l y t h e codon CGU. However, tRNAsArg w i t h an a n t i c o d o n ACG have been r e p o r t e d f o r f u n g a l m i t o c h o n d r i a ( r e f s . 21, 32). 7.16 PLANT MITOCHONDRIAL tRNAs The p l a n t m i t o c h o n d r i a 1 genome has been shown t o c o n s i s t o f a number o f c i r c u l a r molecules d e r i v i n g from a master chromosome by recombination events, p l us a s e t o f small p l asmid-1 ike molecules ( r e f s . 141, 142). Together, t h e s e DNA molecules c o n s t i t u t e l a r g e genomes, t h e s i z e o f which d i f f e r depending on t h e p l a n t considered ( f r o m 200 t o 2500 kb) ( r e f s . 142-146). I n a d d i t i o n , p l a n t m i t o c h o n d r i a l genomes c o n t a i n c h l o r o p l a s t DNA i n s e r t i o n s sometimes h a r b o u r i n g c h l o r o p l a s t i c t R N A genes ( f o r reviews, see r e f s . 147, 148). For i n s t a n c e , t h e maize m t genome

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contains a 12 kb chloroplast DNA insertion ( r e f . 149) which carries chloroplast genes coding for t R N A V a L , t R N A t L e and the 5 ' ha1 f of t R N A A 1 a . The distinction between unexpressed pseudogenes present i n chloroplastic DNA insertions and functional plant m t t R N A genes i s therefore not evident. Some of these inserted chl oropl a s t genes are not transcri bed i n the mitochondria, However, recently a t R N A T r p gene was found in the mitochondrial genomes of wheat and maize. This gene was shown t o r e s u l t from a chloroplastic DNA insertion and t o be transcribed ( r e f . 150). A t R N A C v S found i n maize i s also a chloroplast DNA insertion and i s expressed i n the mitochondria ( r e f . 151). Structures The structures of 6 p l a n t m i t o c h o n d r i a l tRNAs have been published so f a r , namely tRNA:et ( r e f . 152), tRNA:et ( r e f . 152), t R N A P h e ( r e f . 153), t R N A P r o ( r e f . 154), t R N A T r p ( r e f . 155), and t R N A T v r ( r e f . 156). On the other hand, the sequences of 12 m t t R N A genes have been reported: maize t R N A A s p ( r e f . 157), t R N A C v S ( r e f . 151), t R N A H i s ( r e f . 158), B r a s s i c a o l e r a c e a tRNAh;; (most p r o b a b l y a pseudogene) ( r e f . 159), maize, O e n o t h e r a , wheat a n d lupine t R N A Y e t ( r e f s . 160-163), maize t R N A i e t ( r e f . 160), wheat t R N A P r O ( r e f . 154), maize tRNA;;; (ref. 151), and wheat t R N A T r p ( r e f . 150). All p l a n t tRNAs present the invariant and semi-invariant nucl eoti des found i n eubacteri a1 and cytopl asmi c tRNAs, being much more "classic" t h a n the m t tRNAs from other sources. The o n l y exception i s O e n o t h e r a t R N A Y e t which has a U,, instead of the invariant purine ( r e f . 161). This could mean t h a t the r a t e of evolution of p l a n t mitochondria i s less rapid t h a n the one of mitochondria from other sources or t h a t more constraints exist a t the level of the t r a n s l a t i o n a l machinery. The i n i t i a t o r t R N A M e t (or i t s gene) has been sequenced in mitochondria from several p l a n t s : bean, wheat, maize, lupine and O e n o t h e r a . They show a very high homology (from 88.3 t o 89.6%). The comparison of the t R N A T r p and t R N A P r o from wheat and bean show respectively 96% and 100% s i m i l a r i t i e s . Thus, contrarily t o the rnt tRNAs from fungi, the m t tRNAs from different plants seem t o share h i g h homologies. However, there i s an exception t o this 7.16.1

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r u l e s i n c e t h e m t t R N A i e t from bean ( r e f . 152) has l i t t l e sequence homology (40.7%) with a p u t a t i v e maize m t tRNA:et gene ( r e f . 1 6 0 ) . There i s u p t o now no e x p l a n a t i o n t o t h i s s u r p r i s i n g r e s u l t . The simi 1 a r i t i e s between p l a n t m t tRNAs and thei r chlorop l a s t i c c o u n t e r p a r t s i s h i g h : between 70% and loo%, In f a c t , one can d i s t i n g u i s h t h r e e major t R N A c l a s s e s i n h i g h e r p l a n t mitochondria ( r e f . 152). The f i r s t c l a s s , i n c l u d i n g bean m t t R N A i e t , bean m t t R N A T r p , maize m t t R N A H i S , wheat t R N A T r p , and t R N A C v S show a s t r o n g sequence homology (93-100%) with t h e i r c h l o r o p l a s t c o u n t e r p a r t s . C h l o r o p l a s t i n s e r t i o n s i n the mitochondria1 genome can e x p l a i n t h e o r i g i n of t h e s e tRNAs. B u t the p o s s i b i l i t y t h a t c h l o r o p l a s t and mitochondria may have had p r o k a r y o t i c a n c e s t o r s and t h a t t h e r a t e of divergence f o r each t R N A gene may be q u i t e d i f f e r e n t during e v o l u t i o n , g e n e r a t i n g t R N A s p e c i e s having v a r i o u s degrees o f homology with t h e i r c h l o r o p l a s t o r p r o k a r y o t i c counterp a r t s , cannot be excluded. The second c l a s s i n c l u d e s m t tRNAs showing lower sequence s i m i l a r i t i e s with t h e i r c h l o r o p l a s t c o u n t e r p a r t s . This class comprises bean m t t R N A Y e f , bean m t t R N A P h e , bean m t t R N A T v r , bean and maize m t t R N A P r o , the t h r e e p l a n t m t tRNAsnet and the maize m t t R N A S e r and m t t R N A A S p . All t h e s e tRNAs show about 70-76% sequence homology with t h e i r c h l o r o p l a s t c o u n t e r p a r t s . The maize m t t R N A i e t , with only about 43% sequence s i m i l a r i t i e s with i t s c h l o r o p l a s t c o u n t e r p a r t , cannot be included i n e i t h e r of the two previous c l a s s e s and should be considered a s a member of a t h i r d c l a s s among p l a n t m t t R N A s p e c i e s . P l a n t m t tRNAs d i s p l a y a l s o a high degree of s i m i l a r i t y with t h e i r p r o c a r y o t i c c o u n t e r p a r t s (between 61% and 83%). T h u s , i t i s not s u r p r i z i n g t h a t bean t R N A T v r has a p r o c a r y o t i c - l i k e l a r g e extra-loop (14 n u c l e o t i d e s ) and t h a t i n i t i a t o r methionine tRNAs have the c h a r a c t e r i s t i c f e a t u r e s of p r o c a r y o t i c t R N A Y e t : a nonpaired 5'-terminal r e s i d u e , a A , , -U2& base-pair and a T-$-C-Gsequence ( p o s i t i o n s 54-57). On the c o n t r a r y , the s i m i l a r i t i e s of plant mi tochondri a1 tRNAs with t h e i r fungal and mammal i a n mi tochondrial c o u n t e r p a r t s a r e r a t h e r low (35-66%). 7.16.2 Modified Nucl eosi des The percentage o f modified nucleosides of the s i x p l a n t m t tRNAs v a r i e s from 7% t o 11.8%. Up t o now, 12 d i f f e r e n t t y p e s of

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m o d i f i e d nucleosides have been determined $, T, D, acp3U, Cm, Gm, m l G , m2G, m;G, m7G, m 6 A and i 6 A ( o r m s 2 i 6 A ) . The s t r u c t u r e s o f two a d d i t i o n a l nucleosides ( i n p o s i t i o n s 34 and 47) and o f a m o d i f i e d A ( i n p o s i t i o n 37) have n o t y e t been determined. T,, and $5 a r e p r e s e n t i n a1 1 tRNAs. Gm, i s a1 so always p r e s e n t except i n a undermodified t R N A T v r i s o a c c e p t o r ( r e f . 156). D2, i s p r e s e n t i n 5 o u t o f t h e 6 tRNAs. The o t h e r p o s i t i o n s occupied by D a r e 17, 20:l and 48; $ may occupy p o s i t i o n s 27, 31, 38, 39 and 55, and m 2 G o r m $ G occupies p o s i t i o n 26. Cm i s found i n p o s i t i o n 34, m,G i n p o s i t i o n 46, acp3U i n p o s i t i o n 47 and m6A, i 6 A ( o r ms*i6A) and m l G i n p o s i t i o n 37. A l l these p o s i t i o n s a r e t h o s e u s u a l l y encountered in c y t o p l asmi c o r e u b a c t e r i a1 tRNAs ( r e f . 46). Codon Reading P a t t e r n s The anticodons o f p l a n t m t tRNAs corresponding t o 10 amino a c i d s a r e known up t o now. They a l l correspond t o codons o f t h e standard g e n e t i c code. Bean t R N A P r a has an u n m o d i f i e d U i n t h e wobble p o s i t i o n . I t would be i n t e r e s t i n g t o know whether t h i s t R N A behaves l i k e t h e o t h e r m i t o c h o n d r i a l tRNAs h a v i n g a noni f i t reads a l l f o u r m o d i f i e d U i n t h e wobble p o s i t i o n , i . e . pro1 ine codons. t R N A i e t has a CAU anticodon w i t h an u n m o d i f i e d C i n t h e wobble p o s i t i o n . Thus, i t should o n l y be a b l e t o t r a n s l a t e t h e AUG codon. I t must, however, be p o i n t e d o u t t h a t some o t h e r m t tRNAsMet a l s o t r a n s l a t e t h e codon AUA, which t h u s becomes a m e t h i o n i n e codon a l t h o u g h i n t h e " u n i v e r s a l " code AUA i s a codon f o r i s o l e u c i n e . These AUA-reading m e t h i o n i n e t R N A have a l s o a CAU anticodon. Thus, n o t h i n g can be p r e d i c t e d from t h e a n t i c o d o n sequence concerning t h e meaning o f AUA i n p l a n t m i t o c h o n d r i a . I t has been proposed t h a t i n p l a n t m i t o c h o n d r i a t r y p t o p h a n c o u l d be encoded n o t o n l y by t h e " u n i v e r s a l " codon UGG, b u t a l s o by CGG ( r e f s . 164-166) which codes f o r a r g i n i n e i n t h e " u n i v e r s a l " code. However, t h e sequenced bean t R N A T r p has a CmCA a n t i c o d o n complementary t o t h e usual T r p codon UGG, which s h o u l d n o t be a b l e t o read CGG ( r e f . 155). The p o s s i b i l i t y t h a t a n o t h e r tRNATrP w i t h a CCG anticodon e x i s t s i n p l a n t m i t o c h o n d r i a cannot be r u l e d o u t , However, i t should be p o i n t e d o u t t h a t i n soybean, o n l y one m t t R N A T r P has been d e t e c t e d a f t e r a m i n o a c y l a t i o n o f t o t a l m t tRNAs w i t h t r y p t o p h a n u s i n g m i t o c h o n d r i a l c h l o r o p l a s t o r E . c o l i enzymes

7.16.3

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and f r a c t i o n a t i o n on RPC-5 column ( r e f . 167). F i n a l l y , i n p l a n t mitochondria, UGA i s used a s a termination codon and not a s a tryptophan codon. 7.17

GENERAL REMARKS AND CONCLUSIONS In 1980, a correspondent from the j o u r n a l Nature q u a l i f i e d mitochondria a s b e i n g maverick o r g a n e l l e s because of t h e i r unique codon recognition p a t t e r n s and t h e i r b i z a r r e t R N A s t r u c t u r e s ( r e f .

168). As more mitochondrial genomes have now been sequenced, more and more weird m t t R N A s t r u c t u r e s have been uncovered, and one may wonder about the minimal s t r u c t u r a l requirements of a f u n c t i o n a l t R N A . When one goes from p l a n t m t tRNAs (which f u l f i l l a l l requirements of the standard c l o v e r l e a f ) t o nematode worm m t tRNAs (which a l l lack e i t h e r the D-arm o r t h e T$C-arm), one f i n d s a whole p a l e t t e of unusual s t r u c t u r a l f e a t u r e s . T h u s , m t t R N A s a s a group have unusual and highly v a r i a b l e s t r u c t u r e s . Almost any given m t t R N A (except from p l a n t ) has one o r more "odd" f e a t u r e s , including base changes in t h e D-, anticodon, and T$C-stems and loops. Often t h e s e changes a f f e c t r e s i d u e s implicated i n secondary o r t e r t i a r y i n t e r a c t i o n s , r e s u l t i n g i n a reduction ( o r destabi 1 i z a t i o n ) o f p o t e n t i a l higher o r d e r s t r u c t u r e . T h u s , m t tRNAs a r e apparently s t a b i l i z e d by fewer t e r t i a r y i n t e r a c t i o n s . Analysis of secondary and t e r t i a r y s t r u c t u r e of the highly atypi c a l mammalian m t t R N A S e r (which lacks the whole D-loop and has an unusual TgC-loop s t r u c t u r e ) has suggested t h a t i t has a l o o s e conformation w i t h t h e T$C-loop exposed ( r e f . 134). However, t h i s t R N A i s a b l e t o i n t e r a c t w i t h b a c t e r i a l elongation f a c t o r and GTP, i n d i c a t i n g t h a t the D-loop as well a s t h e s t r u c t u r e of the T$Cloop a r e not e s s e n t i a l f o r t e r n a r y complex formation ( r e f . 169). This t R N A could r e p r e s e n t a molecule stripped down t o i t s essence by having conserved the minimal requirements f o r function i n protein synthesis. Mitochondria1 tRNAs use a s i m p l i f i e d mechanism f o r codon reading, which m i n i m zes the number of required t R N A s p e c i e s f o r mitochondrial p r o t e n synthesis, i n apparent d e f i a n c e of t h e wobble r u l e s enunc ated by Crick ( r e f . 48). This expanded codon recognition p a t t e r n i s c o r r e l a t e d w i t h the presence of an unmodi f i e d uridine i n the f i r s t p o s i t i o n of t h e anticodon i n each of

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the s i n g l e tRNAs t h a t decode a given four-codon family. Two-codon boxes ending i n a purine a r e read by tRNAs having a modified u r i d i n e (most probably, cmnm5U i n y e a s t ) i n the wobble p o s i t i o n which prevents the t R N A from reading the codons e n d i n g i n C o r U . Since there a r e 8 four-codon boxes i n t h e g e n e t i c code, the t h e o r e t i c a l minimal number o f t R N A s p e c i e s needed f o r p r o t e i n s y n t h e s i s i s reduced from 32 i n non-mitochondria1 systems t o 24 i n mitochondria. I t i s not known whether this simple decoding mechanism represents a primitive way of reading the g e n e t i c code, o r whether i t evolved toward simp1 i c i t y a s a r e s u l t of unique s e l e c t i v e pressures within the mitochondria ( r e f . 6 ) . Slight d e v i a t i o n s from the minimal number of t R N A s p e c i e s a r e found w i t h i n t h e mitochondria from d i f f e r e n t organisms. For example, only 22 d i f f e r e n t tRNAs a r e found i n mammalian mitochondria, due t o the f a c t t h a t t h e r e i s only one methionine t R N A ( f o r both i n i t i a t i o n and elongation) and t h a t there i s no a r g i n i n e t R N A f o r Other examples a r e g i v e n i n Table 7 . 2 . decoding AGA and AGG. The number of t R N A genes in the mitochondrial genome c l e a r l y decreases from p l a n t s via fungi t o i n s e c t s and mammals ( s e e Table 7.2). The exception i s T e t r a h y m e n a where only 10 m t DNA-coded tRNAs a r e found, t h e remainder having t o be imported from the cytoplasm ( r e f s . 83, 8 6 ) . The m t DNA of the p a r a s i t i c f l a g e l l a t e s trypanosomes seems t o be devoid of any t R N A gene s i n c e tRNAs f a i l t o show up not only i n sequence a n a l y s i s , b u t a l s o i n hybridizat i o n s t u d i e s ( r e f . 170). This may r e f l e c t highly a t y p i c a l t R N A s t r u c t u r e s , e s p e c i a l l y s i n c e the two m t ribosomal RNAs from t h e s e protozoa a r e a l s o very unusual i n t h a t they a r e l e s s than 40% o f the s i z e of the corresponding E . c o l i rRNAs. Simpson e t a l . reported t h a t several a t y p i c a l cl over1 eaves can be formed from maxi-cicle sequences ( r e f . 171). However, t h e number of p u t a t i v e t R N A sequences a r e l i m i t e d and could not comprise a complete s e t of t R N A genes. Therefore, l i k e i n T e t r a h y m e n a , tRNAs may be imported from t h e cytoplasm. Such a s i t u a t i o n may be a general phenomenon i n protozoa ( r e f . 171). In S. c e r e v i s i a e mitochondria, a s i n g l e nuclear-coded t R N A i s imported from the cytoplasm ( r e f . 19), b u t i t seems not t o be d i r e c t l y involved i n mitochondrial protein s y n t h e s i s and i t s r o l e i n mitochondria i s unknown. The b a s i c question of how tRNAs (and n u c l e i c a c i d s i n g e n e r a l ) can

Table 7.2 Mitochondria1 IRNAs genes

Organism

Number

Remarks

Saccharomyces cerevisiae

24

two tRNAsArg (UCU and ACG)

Torulopsis glabrata

23

Only one tRNAArg (UCU)

Neurospora crassa

27

Duplicated genes for each initiator and elongator tRNAMet

Aspergillus nidulans

27-28

Only 1 tRNAArg (UCU). Duplicated genes for both tRNACYs and tRNAASn 2 isoacceptors for each leucine, serine and glycine (UCU and ACC) and 3 for methionine (CAU)

Schizosaccharomyces pombe

25

3 tRNA genes with a CAU anticodon, one of which could code for a tRNAlle

Tetrahymena pyriformis

10

Duplicated tRNATYr genes. 26 nuclear-codedtRNAs imported from the cytoplasm

Aedes

N.D.

Drosophila, Xenopus laevls Mammals

22

One tRNAMet and one tRNAArg (UCG)

Ascaris suum

22

One tRNAMet and one tRNAArg (ACG)

Plants

N.D. (>24)

N.D. not determined

E + (9

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p e n e t r a t e i n s i d e the o r g a n e l l e remains unsolved. An obvious hypothesis i s t h a t they a r e t r a n s p o r t e d i n a complex w i t h the corresponding ami noacyl - t R N A s y n t h e t a s e ( o r o t h e r p r o t e i n s ) which a r e t a r g e t e d i n t o mitochondria. Mitochondria have provided t h e f i r s t c l u e t o t h e non-univers a l i t y o f t h e g e n e t i c code. The most prominent change from the standard code i s t h e use of UGA t o s p e c i f y tryptophan ( r a t h e r than chain termination) i n a1 1 mi tochondri a1 systems, except p l a n t s . In o t h e r r e s p e c t s , the mitochondrial g e n e t i c code i s s l i g h t l y v a r i a b l e i n d i f f e r e n t mitochondrial systems. Table 7.3 summarizes i a b l e

7.3

Exceptions to the "universal" genetic code

CUN

AUA

Leu

lie

Arg

Saccharomyces cere visiae

Thr

Met

Arg

Torulopsis glabrata

Thr

Met

Arg

Neurospora crassa

Leu

lie

Arg

Aspergillus nidulans

Leu

Ile

Arg

Schizosaccharomyces pombe

Leu

lie

Arg

Podospora anserina

N.D.

Ile

Arg

Tetrahymena pyriformis

N.D.

lie

Arg

Paramecium

N.D.

N.D.

N.D.

Aedes

N.D.

N.D.

AGA : Ser AGG : N.D.

Drosophila

Leu

Met

AGA Ser AGA : N.U.

Xenopus laevis

Leu

Met

AGA : s l o p AGG : N.U.

Mammals

Leu

Met

stop

Ascaris suum

Leu

N.D.

Ser

Plants

Leu

N.D.

Arg

UQA "UNIVERSAL CODE

CQN

WQR

MITOCHONDRIA

N.D. not determined N.U. not used 'CGG has been proposed to code for Trp (see text).

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t h e p e c u l i a r i t i e s t h a t have been uncovered t o date. Even between " c l o s e l y - r e 1 a t e d " organisms 1 ike S a c c h a r o m y c e s c e r e v i s i a e and S c h i z o s a c c h a r o m y c e s p o m b e , t h e r e a r e v a r i a t i o n s i n codon usages. For example, t h e CUN codon f a m i l y i s r e a d as t h r e o n i n e i n s . c e r e v i s i a e whereas i t means l e u c i n e i n S. pombe (as i n a l l o t h e r m i t o c h o n d r i a 1 systems). T h i s l e u c i n e - t o - t h r e o n i n e change seems t o be a consequence o f an unusual s t r u c t u r a l f e a t u r e o f t h e s. cerevisiae mt tRNA:;; (for f u r t h e r d i s c u s s i o n , see 7.3.4). V a r i a t i o n s a l s o e x i s t i n t h e use o f i n i t i a t i o n and t e r m i n a t i o n sometimes t h e r e i s codons i n d i f f e r e n t m i t o c h o n d r i a l systems: o n l y one codon f o r each i n i t i a t i o n and t e r m i n a t i o n , sometimes A l l these d e v i a t i o n s from t h e t h e r e a r e s e v e r a l (Table 7 . 4 ) .

T a b l e 7.4 initiation and termination codon usage in rnitochondrla

initiation codons Termination codons (in addition to AUG) (in addition to UAA and UAG) Aspergillus nidulans

GUG

none

Drosophila

AUA, AUU, AUAA

none

Xenopus laevis

none

AGA

Mammals

AUA, AUU, (and AUC in mouse and rat)

AGA and AGG ( not found in mouse)

Plant

N.D.

UGA

N.D. not determined

s t a n d a r d code may r e f 1 e c t f a s t and v a r i a b l e sequence e v o l u t i on w i t h i n mitochondria. The tremendous sequence v a r i a b i l i t y o f m t tRNAs from d i f f e r -

B252

e n t systems should be emphasized. The m t t R N A sequences o f species t h a t d i v e r g e d o n l y 5 m i l l i o n y e a r s ago d i f f e r s i g n i f i c a n t l y whereas i d e n t i c a l nuclear-coded t R N A sequences o c c u r i n organisms whose 1ineages d i v e r g e d over 500 m i 11 i o n y e a r s ago ( r e f . 172). For example, t h e r e i s no d i f f e r e n c e i n t h e sequence o f c y t o p l a s m i c tRNAL y s between mouse and D r o s o p h i l a whereas t h e m t t R N A L v S from t h e two organisms d i f f e r i n no l e s s t h a n 40 p o s i t i o n s T h i s suggests t h a t m t t R N A genes evolved a t l e a s t 100 t i m e s f a s t e r than t h e i r n u c l e a r c o u n t e r p a r t s . However, t h e r a t e o f e v o l u t i o n can be slower i n m i t o c h o n d r i a o f one branch o f t h e e v o l u t i o n a r y t r e e than i n another ( r e f . 173). The f a c t o r s a f f e c t i n g a h i g h e r m u t a t i o n r a t e i n m t DNA than i n n u c l e a r DNA p r o b a b l y i n c l u d e (i) g r e a t e r exposure t o o x i d a t i v e damage and chemical carcinogens, (ii)a more e r r o r - p r o n e r e p l i c a t i o n system, (iii)a l e s s e f f i c i e n t e d i t i n g o f r e p a i r f u n c t i o n s , and ( i v ) a h i g h e r r a t e o f t u r n o v e r ( r e f s . 172-177). The p h i l o g e n y o f m t tRNAs and t h e endosymbiotic h y p o t h e s i s o f m i t o c h o n d r i a evol u t i on have been c a r e f u l l y examined and d i scussed by s e v e r a l authors ( r e f s . 175,178-184). No c o n c l u s i v e answer t o ,the o r i g i n o f m i t o c h o n d r i a can be b r o u g h t from t h e a n a l y s i s o f t R N A sequences. The s i g n i f i c a t i v e resemblance o f p l a n t m t tRNAs t o e u b a c t e r i a l tRNAs makes most e x p e r t s b e l i e v e t h a t t h e s e organe l l e s o r i g i n a t e d i n e v o l u t i o n as endosymbionts. However, f u n g a l and e s p e c i a l l y animal m t tRNAs a r e f a r from b e i n g t y p i c a l l y e u b a c t e r i a l . How then, can one r e c o n c i l e such an o r i g i n w i t h t h e f a c t t h a t m i t o c h o n d r i a l and e u b a c t e r i a l tRNAs a r e so d i f f e r e n t . I t has been proposed t h a t r a p i d and v a r i a b l e sequence divergence w i l l have tended t o obscure t h e p h y l o g e n e t i c a n c e s t r y o f m t DNA ( r e f s . 180-181). The g r e a t e r resemblance o f p l a n t m t tRNAs t o eub a c t e r i a l tRNAs may be due t o l e s s r a p i d e v o l u t i o n o f p l a n t m t DNA than o f f u n g a l and animal m t DNAs.

7.18

ACKNOWLEDGEMENTS T h i s work was supported by g r a n t s from t h e C e n t r e N a t i o n a l de l a Recherche S c i e n t i f i q u e (ATP 2080 and 4256) and from t h e Fondat i o n pour l a Recherche Medicale.

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7.19

1.

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H . Wintz, J.M. Grienenberger, J . H . Weil and D. M. Lonsdale, L o c a l i z a t i o n and n u c l e o t i d e sequence of two t R N A genes and a t R N A seudogene i n the maize mi tochondrial genome: Evidence f o r tge t r a n s c r i p t i o n o f a c h l o r o p l a s t gene i n mitochondria, Curr. Genet., 13 1988) 247-254. L. Marechal, P . u i l l e m a u t , J . M. Grienenberger, G . Jeannin and J.H. Weil, Sequences of i n i t i a t o r and elon a t o r methioi n e t R N A i n bean mitochondria. L o c a l i z a t i o n o the c o r r e s gonding genes on maize and wheat mitochondrial genomes, l a n t Mol. B i o l . , 7 (1986) 245-253. L. Marechal, P . Guillemaut, J . M. Grienenberger, G. Jeannin and J . H . Wei 1 , S t r u c t u r e o bean mi t o c h o n d r i a l t R N A P h e and l o c a l i z a t i o n of the tRNAJhe gene on the mi tochondrial genomes of maize and wheat, FEBS Lett., 184 (1985 289-293. P . Runeberg-Roos, J . M . Grienenberger, P. Guil emaut, L . Marechal, V: Gruber and J . H . Weil, L o c a l i z a t i o n sequence and e x p r e s s i o n of t h e ene codin f o r t R N A P r o (UGG) i n p l a n t (1987) 237-246. mitochondria, P l a n t Mo!. B i o l . , L. Marechal, P. Guillemaut, J . M. Grienenberger, G . Jeannin and J . H . Weil, Sequence and codon r e c o g n i t i o n of bean mitochondria and c h l o r o p l a s t t R N A T r p : evidence f o r a h i h degree of homolog , Nucleic Acids Res., 13 (1985) 4411-441:. L . Marechar, P . Guillemaut and J . H . Weil, Sequences of two bean mitochondria t R N A s T v r which d i f f e r i n the l e v e l of o s t - t r a n s c r i p t i o n a l m o d i f i c a t i o n and have a prokar o t i c Ti ke l a r e e x t r a - l o o p , P l a n t Mol . Biol . , 5 (1985) 347-3{1 T.D. P a r & s , G . Dougherty, C.S. Levings I11 and D . H . Timottiy I d e n t i f i c a t i o n of an a s p a r t a t e t R N A ene i n maize mitochon: d r i a l DNA, Curr. Genet., 9 (1985) 517-519 K. P . Iams, J . E. Heckman and J . H. S i n c i a i r , Sequence of histidyl-tRNA, r e s e n t a s a c h l o r o l a s t i n s e r t i n m t DNA of Z e a m a y s , P l a n t i o l . Biol ., 4 (1985p 225-233 M. Dron, C . Hartmann, A. Rode and M. Sevignab, Gene convers i o n a s a mechanism f o r divergence o f a c h l o r o p l a s t t R N A gene i n s e r t e d i n t h e mitochondrial enome of B r a s s i c a o l e r a c e a , Nuclei c Acids Res. 13 (1985) 8603-8610 T.D. Parks, G . Dougherty, C . S . Levings I11 and D.H. Timothy, enes i n the maize I d e n t i f i c a t i o n of two methionine t R N A mi tochondrial genome, P l a n t Physiol . , 76 (7984) 1079-1082 M . Gottschal k and A. Brenni ke, I n i t i a t o r methionine t k N A gene i n O e n o t h e r a mitochondria, Curr. Genet., 9 (1985) 165168. M.W. Gray and D.F. Spencer, Wheat mitochondrial DNA encodes a e u b a c t e r i a - l i k e i n i t i a t o r methionine t R N A , FEBS Lett., 161 (1983) 323-327. P. Borsuk, A . S i r k o and E . B a r t n i k , A methionine t R N A ene from 1 upi ne mitochondria, Nuclei c Acids Res. , 14 (1886) 7508. T. D. Fox and C . J . Leaver, The Z e a m a y s mitochondrial gene s u b u n i t I 1 has an i n t e r v e n i n coding cytochrome oxidase s e uence and does not c o n t a i n TGA codons, Cell 26 (19813 319-323 R . H i e s i l and A. Brennicke, Cytochrome o x i d a s e s u b u n i t I 1 ene i n mitochondria of O e n o t h e r a has no i n t r o n , EMBO J . , 2 1983) 2173-2178.

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168. 169. 170. 171.

172. 173. 174. 175.

176. 177.

178. 179. 180. 181. 182. 183.

184. 185.

L. Bonen, P. H. Boer and M. W. Gray, The wheat c y t o c h r o m o x i d a s e I1 gene has an i n t r o n . i n s e r t and t h r e e r a d i c a l amino a c i d changes r e l a t i v e t o maize, EMBO J., 11 (1984) 25312536. G.S. Swam and D. T . N. P i l l a y , C h a r a c t e r i z a t i o n o f G l y c i n e m a x c y t o p asmic, c h l o r o l a s t i c and m i t o c h o n d r i a l tRNAs and s nthetases f o r henyyal anine t r y p t o p h a n and t y r o s i n e . P f a n t S c i . L e t t . , $5 (1982) 73-94. From a c o r r e s p o n d e n t , M a v e r i c k m i t o c h o n d r i a , N a t u r e , 287 1980 9-10. Ge h a r d t - S i n g h and M. S p r i n z l , Ser-tRNAs f r o m b o v i n e m i t o c h o n d r i a f o r m t e r n a r y complexes w i t h b a c t e r i a1 e l on at i o n f a c t o Tu and GTP. N u c l e i c A c i d s Res., 14 (1986) 71357 188. R. Benne, M i t o c h o n d r i a l genes i n trypanosomes, Trends Genet., 1 1985) 117-121. L. Simpson A.M. Simpson, V.F. De l a Cruz, N. Neckelmann and M. Muhich, Genomic o r a n i z a t i o n and t r a n s c r i p t i o n o f m i t o c h o n d r i a l m a x i c i r c l e !NA i n t r v o a n o s o m i a l P r o t o z o a . i n F. Quagl ia r e 1 i a e t a l . (Eds), Achievements and p e r s p e c t i v e s o f m i t o c h o n d r a1 research, V o l . 11, B i o e n e s i s , E l s e v i e r S c i e n c e Pu l i s h e r s , Amsterdam, 1985, pp. 9 -110. W. M. Brow M. George and A. C. W i l s o n , R a p i d e v o l u t i o n o f animal m i h c h o n d r i a l DNA, Proc. N a t l . Acad. R. Cedergren and B. F. Lang, P r o b i n g f u n g a l m i t o c h o n d r i a1 e v o l u t i o n w i t h tRNA, B i o s stems, 18 (1985) 263-267. W.M. Brown, Mechanisms o r e v o l u t i o n o f a n i m a l m i t o c h o n d r i a l DNA, Annals NY Acad. S c i ., 361 (1981) 119-134. M. Hasegawa, R. Kikuno, T. M i y a t a and T. Yano, The o r i g i n and t h e e v o l u t i o n o f c e l l u l a r o r a n e l l e s , E n d o c y t o b i o l o g y , W a l t e r de G r u y t e r , B e r l i n , New ,or\, 2 (1983) 199-210. J.A. A l l e n and M.M. Combs, C o v a l e n t b i n d i n g o f p o l c y c l i c a r o m a t i c compounds t o m i t o c h o n d r i a l and n u c l e a r DNA, { a t u r e , 287 (1980) 244-245. F. Wund-Bisseret, B. Barraud-Hadidane and G . D i r h e i m e r , I n v i v o c o v a l e n t b i n d i ng o f ami n o f l u o r e n e d e r i v a t 1 ves t o m i t o c h o n d r i a l 1 i v e r DNA o f r a t s p r e t r e a t e d w i t h p h e n o b a r b i t a l , T o x i c o l og L e t t . , 32 (1986) 227-233. L. M a r g u f i s , O r i i n o f e u k a r y o t i c c e l l s , Y a l e U n i v e r s i t y Press, New Haven 1970). M. Hasegawa, T. ano and T. M i y a t a , Ph l o en o f t R N A and o r i g i n o f o r g a n e l 1es, Precambrian Res., 4! ?19$11) 81-98 M.W. Gray and W.F. D o o l i t t l e , Has t h e e n d o s y m b i o n t h y p i t h e s i s been p r o v e n ? , M i c r o b i o l Rev., 46 (1982) 1-42. M.W. Gra M i t o c h o n d r i a 1 genome d i v e r s i t and t h e e v o l u t i o n o f m i t o c l i n d r i a l DNA, Can. J. Biochem., (1982) 157-171 M. Hasegawa, T. Yano and T . M i y a t a , E v o l u t i o n a r y i m p l i c a t i o n s i n the self-replicating and p r o t e i n - s y n t h e s i z i n g m a c h i n e r y , J. Mol . E v o l , 20 (1984) 77-85. M.W. Gray, D. S a n k o f f and R.J. Ceder r e n , On t h e e v o l u t i o n a r y d e s c e n t o f o r g a n i s m s and o r g a n e l yes: a g l o b a l h y l o g e n y based on a h i g h c o n s e r v e d s t r u c t u r a l c o r e i n s m a l f s u b u n i t

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CHAPTER 8 THE MODIFIED NUCLEOTIDES I N RIBOSOMAL RNA OF MAN AND OTHER EUKARYOTES B.E.H.

MADEN

Department o f Biochemistry. University o f Liverpool. Liverpool L69 3BX. United Kingdom

P . 0 . Box 147.

TABLE OF CONTENTS 8.1 I n t r o d u c t i o n . . . . . . . . . . . . . . . . . . . . . 8.2 E a r l y Analyses 8.2.1 2 -0-Methyl Groups . . . . . . . . . . . . . . C h a r a c t e r i z a t i o n o f A1 k a l i - S t a b l e Sequences 8.2.2 i n Wheat Germ r R N A . . . . . . . . . . . . . . 8.2.3 Pseudouridine 8.3 M o d i f i e d N u c l e o t i d e s and t h e rRNA M a t u r a t i o n Pathway 8.3.1 Use o f C u l t u r e d C e l l s 8.3.2 Methyl L a b e l l i n g o f r R N A and Ribosomal P r e c u r s o r RNA i n HeLa C e l l s 8.3.3 E s s e n t i a l Role o f M e t h y l a t i o n i n r R N A Maturation . . . . . . . . . . . . . . . . . . Pseudouridine . . . . . . . . . . . . . . . . . 8.3.4 8.4 O l i g o n u c l e o t i d e Data 8.4.1 The M e t h y l a t e d N u c l e o t i d e Sequences i n HeLa C e l l r R N A and Ribosomal P r e c u r s o r RNA . . . . . 8.4.2 M e t h y l a t e d Sequences i n r R N A From O t h e r V e r t e b r a t e Sources . . . . . . . . . . . . . .

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M e t h y l a t i o n i n Yeast r R N A and Ribosomal P r e c u r s o r RNA . . . . . . . . . . . . . . . . 8.4.4 I m p l i c a t i o n s o f t h e O l i g o n u c l e o t i d e Data Locating the Methylated Nucleotides 8.5.1 M o d i f i e d N u c l e o t i d e s i n 5.8s r R N A . . . . . . 8.5.2 M e t h y l a t i o n Map o f X e n o p u s L a e v i s r R N A 8.5.3 Exact L o c a t i o n s o f t h e Methyl Groups i n 18s r R N A o f X e n o p u s Laevis and Man . . . . . . . Problems and Prospects . . . . . . . . . . . . . . . 8.6.1 The Methyl Groups i n 28s r R N A . . . . . . . . 8.6.2 Pseudouridine and Other M o d i f i e d N u c l e o t i d e s .

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8.6.3 Methylation S i t e s and Conformation: 18s rRNA 8.6.4 Closing Comments . . . . . . . . . . . . . . 8.7 Summary . . . . . . . . . . . . . . . . . . . . . . . . 8.8 Acknowledgements , .............. 8.9 References . . . . . . ..............

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INTRODUCTION

I t i s a curious and poor y understood f a c t t h a t some types of RNA molecules contain r e l a t i v l y large numbers of modified nucleot i d e s whereas other types of RNA contain few or none. Analysis of rRNA from a variety of eukaryotes has revealed the presence o f numerous modified nucleotides. These can be c l a s s i f i e d i n t o three groups: 2'-O-methylated nucleotides, various base-modified nucleotides, and pseudouridine residues. Among the vertebrates, rRNA from human c e l l s and from the frog, X e n o p u s l a e v i s , have been studied i n d e t a i l . Among the lower eukaryotes, rRNA from the yeast, S a c c h a r o m y c e s c a r 7 s b e r g e n s i s has been studied in detail and some data are available from other sources including plants. O f particular significance has been the recent determination of the exact locations of the 40 or so methyl groups i n 18s rRNA of X . 7 a e v i s and man. The picture t h a t i s beginning t o emerge from these studies i s t h a t the modified nucleotides f i t into the functional architecture of rRNA i n a precise and i n t r i c a t e manner. Moreover, most of the nucleotide modifications are made while the ribosomal sequences are within ribosomal precursor RNA in the nucleolus. Therefore, a fundamental aim of work in t h i s area of research i s t o unravel the detailed relationship between the modified nucleotides, rRNA maturation and the overall structure of rRNA. Current knowledge of the modified nucleotides in eukaryotic rRNA has come from the application of successive new techniques, several of which have been basic t o the development of broad areas i n molecular and c e l l biology. To i l l u s t r a t e the relationship between advances in general methods and in s p e c i f i c knowledge of the modified nucleotides, I have adopted a broadly historical approach, in which key references t o the major developments are cited and the main findings and conclusions are summarized. I have emphasized work on modified nucleotides in rRNA from ver-

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tebrates, but have also outlined data from other eukaryotes where they have contributed importantly to the overall picture. 8.2 EARLY ANALYSES 8.2.1 2I-O-Methvl Grouos 2'-O-methylated nucleotides in RNA were first identified by Smith and Dunn (ref. 1 ) . The discovery came from finding a small proportion of a1 kali-stable dinucleotides in a1 kal ine hydrolysates of RNA from a number of sources, including wheat embryo, rat liver microsomes and rat liver "soluble" RNA. Modern methods for fractionating cell ul ar RNA species were not then avai 1 able, but it is clear in retrospect that the "rnicrosomal fraction contained mainly rRNA whereas the "soluble" RNA fraction consisted largely of tRNA. From the chemical standpoint the analysis by Smith and Dunn was fundamental. The a1 kal i-stable dinucleotides were resolved into groups by a two-stage procedure o f paper chromatography The recovered di nucl eoti des were fol 1 owed by el ectrophoresis. subjected to degradation by phosphomonoesterase, phosphodiesterase, and the Whitfield procedure (ref. 2). The products were characterized by further paper chromatography and electrophoresis. The results established the general structure NxpNp for the alkali-stable dinucleotides, where Nx is a nucleoside with a blocked 2'-OH group, linked via its 3'-OH group through a standard phosphodiester bond to the adjacent nucleoside. Evidence on the existence and nature of the blocked 2'-OH group came from a number of observations: (i) The initial fact of alkali-stability implied the absence of a normal 2'-OH group, since this group participates in the mechanism of hydrolysis by alkali. (ii) Upon electrophoresis in borate buffer (at pH 9.2) the Nx nucleosides failed to form complexes with borate, whereas ordinary ribosides form complexes due to the presence of two c i s OH groups. (iii) The RF yalues of Nx on paper chromatography (in acid and alkaline solvents) were greater than for the standard ribosides or deoxyri bosides, implying the presence of a hydrophobic group. The absorption spectra of the Nx nucleosides were as for the corresponding standard nucleosides, indicating that the bases were probably unmodified. All these observations led to the tentative identification o f the Nx compounds as 2'-0-methyl "

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nucleosides. Smith and Dunn also noted that a1 kal i-stable dinucleotides appeared to be absent from alkaline hydrolysates of RNA from Aerobacter aerogenes and turnip-yellow mosaic virus. Hall (ref. 3) provided further evidence that the modified sugar in the Nx nucleosides is 2'-0-methyl ribose. Each of the four presumed 2'-0-methyl nucleosides was isolated from a large preparation of "soluble yeast RNA" following enzymic digestion. The compounds were characterized by methods which were fairly similar to those used by Smith and Dunn. In addition, the nucleosides were hydrolyzed by HC1, and in each case the recovered sugar was shown to co-migrate chromatographically with chemically synthesized 2'-0-methyl ribose. 8.2.2 Characterization of Alkali-Stable Seauences i n Wheat Germ

m.

Lane and coworkers (refs. 4-6) then made a detailed study of alkali-stable sequences i n wheat germ rRNA. This study was important both from the standpoint of methodology and from the results obtained. A1 kal ine hydrolysates of RNA were separated into mononucleotides, a1 kali-stable dinucleotides and small amounts of a1 kal i-stable trinucleotides by chromatography on DEAE cellulose (refs. 4-6). The separation method was suitable for large quantities of starting material ( > lg) and hence yielded several mg of a1 kal i -stab1 e materi a1 for analysis. The materi a1 i n the a1 kal i -stab1 e di nucl eoti de peak was dephosphoryl ated with a1 kal ine phosphatase and was fractionated into individual components or isomeric pairs of compounds by paper chromatography. The a1 kal i-stable trinucleotide peak was simi larly dephosphorylated and resolved into several distinct compounds. These various compounds were then digested with snake venom phosphodiesterase to yield products as indicated:- ------- > Nm + pN di nucl eoti des: NmpN --------> Nm + pNm + pN trinuc1eotides:Nmp Nmp N These products were in turn separated by paper chromatography using a borate-saturated developing system (system 3 of ref. 4 ) , in which the 2'-0-methylated compounds were clearly distinguished from their non-methyl ated counterparts due to complexi ng o f the latter with borate. This degradation procedure, which was a

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r e f i n e m e n t o f t h a t o r i g i n a l l y used by Smith and Dunn, enabled t h e s t r u c t u r e s o f a l l t h e a l k a l i - s t a b l e compounds t o be determined. A p p l i c a t i o n o f t h e procedure t o t h e a1 k a l i - s t a b l e components i n wheat germ rRNA l e d t o t h e f o l l o w i n g main c o n c l u s i o n s ( r e f s 5 (i)A l l 16 p o s s i b l e a l k a l i - s t a b l e d i n u c l e o t i d e s were and 6): p r e s e n t i n wheat germ rRNA, amounting t o g e t h e r t o 3% o f t h e t o t a l nucleotides. (ii) The r e l a t i v e amounts o f t h e f o u r 2'-O-methyl r i b o s i d e s d i f f e r e d d i s t i n c t l y from t h e r e l a t i v e amounts o f t h e four normal r i b o s i d e s , i m p l y i n g t h a t a non-random s e t o f nucleo(iii)The s e v e r a l a l k a l i - s t a b l e t r i n u c l e o t i d e s i s methylated. t i d e s were a l s o c h a r a c t e r i z e d ( r e f . 6); a l l o f t h e s e were found subsequently t o be i n 28s rRNA ( r e f . 7). ( i v ) Pseudouridine was a l s o p r e s e n t , i n a q u a n t i t y which was r o u g h l y equal t o t h e t o t a l number o f 2 ' -0-methyl groups. One o f t h e a1 k a l i-stab1 e tri n u c l eoA c o r r e l a t i o n was t i d e s c o n t a i n e d pseudouridine:- UmpGmp$p. p o i n t e d o u t ( r e f . 6) between t h e occurrence o f Z'-O-methyl r i b o s i d e s and p s e u d o u r i d i n e i n RNA species f o r which a n a l y t i c a l d a t a were then a v a i l a b l e .

8.2.3 Pseudouridine Pseudouridine was f i r s t c l e a r l y d e f i n e d as an e x t r a , unident i f i e d component i n h y d r o l y s a t e s o f RNA by Davis and A l l e n ( r e f . 8) and then by Smith and Dunn ( r e f . l), a l t h o u g h t h e f i r s t s i g h t i n g had been made e a r l i e r (peak l a b e l l e d ? i n r e f . 9 and quoted i n Cohn d e f i n i t i v e l y c h a r a c t e r i z e d t h e unknown r e f s . 10 and 11). compound as 5 - r i b o s y l u r a c i l ( r e f s . 10 and 11). I t s main chemical f e a t u r e s d e r i v e from t h e carbon-carbon g l y c o s i d i c bond 1 i n k i n g C-5 o f u r a c i l w i t h C-1' o f r i b o s e , and t h e consequent a l t e r e d o r i e n t a t i o n o f the pyrimidine ring. As mentioned above ( r e f . 6) and f u r t h e r d e s c r i bed be1 ow, pseudouri d i ne i s r e 1 a t i v e l y abundant i n e u k a r y o t i c rRNA.

8.3 MODIFIED NUCLEOTIDES AND THE rRNA MATURATION PATHWAY 8.3.1 Use o f C u l t u r e d C e l l s The s t u d i e s o u t l i n e d i n t h e p r e v i o u s s e c t i o n were c a r r i e d o u t w i t h l a r g e amounts o f m a t e r i a l , and t h e p r o d u c t s a r i s i n g from t h e v a r i o u s d e g r a d a t i o n procedures were recovered i n s u f f i c i e n t q u a n t i t i e s f o r d e t e c t i o n and c h a r a c t e r i z a t i o n by u l t r a v i o l e t a b s o r p t i o n o r comparable methods. These e a r l y s t u d i e s were, o f

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n e c e s s i t y , p r i m a r i l y chemical i n n a t u r e . The next advances were made by studying RNA metabolism i n c u l t u r e d c e l l s . Cultured c e l l s a f f o r d a number o f advantages f o r metabolic s t u d i e s . F i r s t , they comprise a homogeneous population of growing c e l l s . Second, r a d i o a c t i v e l a b e l l i n g o f n u c l e i c a c i d s i s r e a d i l y performed. T h i r d , rRNA and i t s n u c l e o l a r p r e c u r s o r molecules can be i s o l a t e d and f r a c t i o n a t e d r e l a t i v e l y e a s i l y . The major steps i n the maturation pathway f o r rRNA i n the n u c l e o l i of animal c e l l s were worked o u t l a r g e l y by t h e use of cultured c e l l s . 45s RNA i s the primary t r a n s c r i p t and common p r e c u r s o r t o 18s and 28s rRNA i n mammal i an cel Is, and 32s RNA i s an i n t e r m e d i a t e i n the maturation of 28s rRNA. Refs. 12 and 13 a r e e a r l y reviews. Nucleotide m o d i f i c a t i o n s f e a t u r e prominently i n the rRNA maturation pathway, and a l s o f e a t u r e d i n i t s e l u c i d a t i o n , a s summari zed i n t h e f o l 1 owing paragraphs. Methyl Labellina of rRNA and Ribosomal P r e c u r s o r RNA in HeLa C e l l s Brown and A t t a r d i ( r e f . 14) were among the f i r s t t o apply methyl l a b e l l i n g t o the study of n u c l e i c a c i d s i n animal c e l l s . They grew HeLa c e l l s (a l i n e of c u l t u r e d human c e l l s ) f o r two g e n e r a t i o n s i n E a g l e ’ s medium which had been prepared f r e e of methionine and supplemented with l%-methyl methionine. The RNA was then i s o l a t e d and 28s and 18s rRNA were s e p a r a t e d by s u c r o s e gradient centrifugation. The r e s u l t s showed i n c o r p o r a t i o n of methyl l a b e l i n t o both 28s and 18s r R N A , the 18s rRNA being l a b e l l e d t o a somewhat higher s p e c i f i c a c t i v i t y than 28s rRNA. P a r t i a l a n a l y s i s of the methylated components suggested t h a t most of the r a d i o a c t i v i t y was i n 2’-O-methyl r i b o s e with some l a b e l l i n g a1 so of speci f i c bases. Greenberg and Penman ( r e f . 15) then made the important discovery t h a t methylation t a k e s p l a c e a t t h e l e v e l of 45s r i b o somal p r e c u r s o r RNA i n the nucleolus. By using s h o r t pulse l a b e l l i n g with 1%-methyl methionine, followed by c e l l f r a c t i o n a t i o n and s u c r o s e g r a d i e n t c e n t r i f u g a t i o n of RNA, t h e y obtained evidence t h a t methylation t a k e s p l a c e on nascent c h a i n s of 45s RNA. During longer l a b e l l i n g p e r i o d s , o r following a chase with 8.3.2

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excess unl abel 1 ed methi oni ne, 1abel passes i n t o nucl eol a r 32s RNA and cytoplasmic 18s and 28s rRNA. S i m i l a r general conclusions were reached by Zimmerman and H o l l e r ( r e f . 1 6 ) , using s l i g h t l y d i f f e r e n t methods. When HeLa c e l l s were l a b e l l e d f o r 30 min. with 14C-methyl methionine, and actinomycin was added t o block f u r t h e r RNA s y n t h e s i s , the methyl l a b e l was chased i n t o 32s and 18s RNA. When actinomycin was added 5 minutes p r i o r t o 14C-methyl methionine no l a b e l was taken up i n t o 45s RNA, implying t h a t methylation o f 45s RNA i s c l o s e l y coupled t o s y n t h e s i s . However, methyl l a b e l was taken u p i n t o dimethyladenosine i n 18s rRNA under these c o n d i t i o n s ( r e f . 1 7 ) . This was t h e f i r s t evidence f o r a small group of l a t e m e t h y l a t i o n s i n rRNA m a t u r a t i o n , f u r t h e r discussed below ( s e c t i o n 1 7 . 4 . 1 ) . Wagner e t . a l . ( r e f . 18) then c a r r i e d o u t a chemical a n a l y s i s of the a1 kal i - s t a b l e sequences i n HeLa c e l l rRNA and i t s nucl eol a r precursors. The methyl -1 abel 1 i ng approach was appl i e d , i n cont r a s t t o the e a r l i e r s t u d i e s ( r e f s . 4-6) on l a r g e - s c a l e preparat i o n s of u n l a b e l l e d rRNA. A f t e r s e p a r a t i o n of the RNA s p e c i e s by s u c r o s e g r a d i e n t c e n t r i f u g a t i o n , a1 kal i n e h y d r o l y s i s was c a r r i e d o u t , followed by dephosphorylation and s e p a r a t i o n of the r e s u l t i n g compounds by chromatography on DEAE Sephadex. The recovered compounds were i d e n t i f i e d by t h e i r e l e c t r o p h o r e t i c m o b i l i t i e s a t pH3 and by cleavage with venom phosphodiesterase t o y i e l d the methyl-label l e d n u c l e o s i d e s . The important conclusions from t h i s work were: ( i ) t h e methylation p a t t e r n s of HeLa c e l l 18s and 28s rRNA d i f f e r e d from each o t h e r , ( i i ) t h e 2'-0-methylation p a t t e r n of 45s RNA resembled t h a t of an equimolar mixture of 18s and 28s rRNA and ( i i i ) t h e 32s p a t t e r n resembled t h a t of 28s rRNA. T h u s , t h e s e d a t a provided chemical confirmation of t h e evidence from RNA l a b e l l i n g k i n e t i c s and i n h i b i t o r s t u d i e s t h a t most of the methyl groups o f rRNA a r e added t o ribosomal p r e c u r s o r RNA i n the nucleolus. (Subsequent work has shown t h a t the e s t i m a t e s o f the abs o l u t e numbers of methyl groups i n this s t u d y were n o t q u i t e a c c u r a t e , b u t t h e general conclusions ( i ) t o ( i i i ) remain v a l i d , a s f u r t h e r d e t a i l e d i n s e c t i o n 8 . 4 . 1 , below). E s s e n t i a l Role of Methvlation i n rRNA Maturation A key q u e s t i o n which a r o s e from t h e s e s t u d i e s was whether the methylation of ribosomal p r e c u r s o r RNA plays an e s s e n t i a l r o l e i n 8.3.3

ribosome m a t u r a t i o n . T h i s q u e s t i o n was addressed i n a s e r i e s o f HeLa c e l l s were susexperiments by Vaughan e t . a l . ( r e f . 19). pended i n m e t h i o n i n e - f r e e medium f o r s e v e r a l hours and were then Analysis o f l a b e l l e d w i t h 14C u r i d i n e o r adenosine f o r 2.5 h r . RNA from t h e v a r i o u s c e l l f r a c t i o n s on sucrose g r a d i e n t s showed t h a t 45s RNA c o n t i n u e d t o be s y n t h e s i z e d and processed i n t o 32s RNA i n t h e nucleolus, b u t no newly formed rRNA appeared i n t h e cytoplasm. I n a p a r a l l e l c u l t u r e d e p r i v e d o f v a l i n e , an e s s e n t i a l amino a c i d which has no s p e c i a l r o l e i n rRNA m e t h y l a t i o n , r R N A c o n t i n u e d t o appear i n t h e cytoplasm a t a reduced r a t e (see a l s o The 45s and 32s RNA f r o m m e t h i o n i n e - d e p r i v e d r e f s . 20 and 21). c e l l s were s u b j e c t e d t o a1 k a l i n e h y d r o l y s i s f o l l o w e d by chromatography on DEAE c e l l u l o s e and were found t o be s e v e r e l y d e f i c i e n t i n 2'-0-methyl groups: 45s RNA possessed o n l y 20% o f t h e 2'-0m e t h y l a t i o n found i n c o n t r o l c e l l s , and 32s RNA possessed about 30-40% o f t h e normal l e v e l o f 2 ' - 0 - m e t h y l a t i o n . These r e s u l t s s t r o n g l y i m p l i e d t h a t m e t h y l a t i o n o f ribosomal p r e c u r s o r RNA was t h e f a c t o r l i m i t i n g ribosome m a t u r a t i o n d u r i n g m e t h i o n i n e d e p r i vation. Moreover, t h e continued p r o d u c t i o n o f 45s and 32s RNA t o g e t h e r w i t h t h e 1ack o f appearance o f c y t o p l a s m i c rRNA i n d i c a t e d t h a t t h e m e t h y l - d e f i c i e n t RNA was b e i n g degraded a t a r e l a t i v e l y I t was shown i n an acl a t e stage i n t h e m a t u r a t i o n pathway. t i n o m y c i n chase experiment t h a t a t l e a s t some o f t h e m e t h y l d e f i c i e n t RNA c o u l d be rescued by a d d i t i o n o f m e t h i o n i n e . 8.3.4

Pseudouridi ne The presence o f pseudouridine i n HeLa c e l l rRNA and ribosomal p r e c u r s o r RNA was r e p o r t e d by A t t a r d i and co-workers ( r e f s . 22 and 23). Thus, t h i s t y p e o f m o d i f i c a t i o n a l s o t a k e s p l a c e a t t h e l e v e l o f 45s RNA. The amounts o f pseudouridine i n h y d r o l y s a t e s o f t h e v a r i o u s RNA species were o f t h e o r d e r o f 1-2% o f t h e t o t a l n u c l e o t i d e s , t h e h i g h e s t r e l a t i v e c o n t e n t b e i n g i n 18s r R N A . R e f i n e d e s t i m a t e s o f t h e pseudouridine c o n t e n t o f r R N A a r e g i v e n l a t e r , below.

8.4 OLIGONUCLEOTIDE DATA 8.4.1 The M e t h v l a t e d N u c l e o t i d e Seouences i n HeLa C e l l r R N A and R i bosomal Precursor RNA The n e x t major advances came from a n a l y s i s o f t h e m e t h y l a t e d

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oligonucleotides released by enzymic digestion of rRNA and ribosomal precursor RNA. The methods which had been developed by Sanger and Brownlee for electrophoretic separation and sequence analysis of radioactive oligonucleotides (refs. 24, 25) were first applied to the modified nucleotides in rRNA by Fellner (ref. 26), who analyzed the methylated sequences in E . c o 7 f rRNA. E . c o 7 i rRNA contains a relatively small number of methyl groups, most of which occur on bases. (See also the chapter by Ebel in this treatise.) By contrast, the methylation patterns of HeLa cell rRNA and its nucleolar precursors are quite complex when analyzed at the oligonucleotide level. Successive stages i n the description and analysis of the methylated oligonucleotides in rRNA and its precursors from HeLa cells were reported in refs. 27-32, and some additional findings were reported later. The following account summarizes the main features and findings of the analysis. (i) Methods. Ref. 32 contains a full account of the methods that were employed for preparing rRNA and nucleolar RNA after i n v i v o labelling of HeLa cells with 14C-methyl methionine or 32P04, and a1 so the procedures for enzymic hydrolysis of RNA, separation of the ol igonucl eotides by two-dimensional electrophoresis ('fingerprinting') and characterization of the oligonucleotides. Two methodological points may be mentioned here. First, to obtain RNA that is radioactively labelled exclusively in its methyl groups it is necessary to carry out labelling in the presence of adenosine, guanosine (2 x 10-5M each) and sodium formate (10-2M). In the absence of these compounds, small amounts of carbon label enter the "one-carbon pool" and purine biosynthetic pathway. Cells labelled in the presence of purine nucleosides and formate yielded RNA with no trace of purine ring labelling, as shown by the complete absence of label in nonmethylated but abundant T, ribonuclease products such as Gp and APGP. Secondly, the procedure for purifying nucleol i and obtaining nucleolar RNA was developed for HeLa cells (ref. 33) and has worked well for these cells, but may not necessarily work well for other cell types. Moreover, the nucleolar processing pathway is slower in HeLa cells than in many other cell types; hence the steady-state levels of 45s and 32s RNA are higher and good yields of these molecules can be obtained. These considerations underlay

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the choice of HeLa c e l l s f o r the work in r e f . 32 as well as f o r many e a r l i e r studies, including those outlined i n section 8.3, above. ( i i ) Numbers of methyl aroutx in rRNA. When T, ribonuclease fingerprints of methyl labelled rRNA were f i r s t obtained ( r e f s . 2 7 , 28) i t was apparent t h a t many methylated oligonucleotides were approximately equally labelled. The simplest i n t e r p r e t a t i o n was t h a t each of these equally labelled products was present once per molecule of rRNA. However, Fellner ( r e f . 26) had o r i g i n a l l y inferred t h a t most of the methylated oligonucleotides i n f . c o 7 i rRNA occurred twice per molecule. (The frequencies were corrected l a t e r t o once per molecule: r e f . 34). Conversely, the numbers of methyl groups per HeLa rRNA molecule, assuming unimol a r frequenc i e s , were i n e x c e s s of the numbers previously estimated by Wagner e t . a 1 . ( r e f . 18). I t was c l e a r l y necessary t o resolve the question of the numbers of methyl groups. The following approach made t h i s possible ( r e f s . 29, 30). When 32P labelled rRNA was digested w i t h combined T, plus pancreatic ri bonucleases, not o n l y were the expected s t a n d a r d products obtained (Up, Gp, Cp and t r a c t s of A residues terminated by Up, Gp, or Cp) b u t also an array of e x t r a products. Nearly a l l of the extra products possess Z'-O-rnethylated nucleotides which confer resistance t o cleavage by the respective enzymes. Several of these extra products were completely separated from non-methylated products in fingerprints ( r e f . 29). The s t r u c t u r e s of these products were analyzed, and t h e i r mol ar y i e l d s were determi ned with reference t o the t o t a l 32P label in the f i n g e r p r i n t and the known s i z e s of the rRNA molecules. Several of the T, p l u s panc r e a t i c RNase products were indeed present once per RNA molecule. 14C methyl fingerprints were then prepared, and the molar yields of a l l the labelled products were determined with reference t o the products which had a1 ready been characterized in the P fingerprints. This gave an estimate of the t o t a l numbers of methyl groups of T, pl us pancreatic r i bonucl ease f i n g e r p r i n t s of rRNA ( r e f . 30). I t was then possible t o identify the T, plus panc r e a t i c RNase products as "derivatives" of the T, products ( r e f s . 32, 35). The r e s u l t s of t h i s correlation c l e a r l y indicate t h a t most of the T, products occur once per molecule of rRNA, with a

small number of s i t e s which a r e incompletely methylated. The numbers of methyl groups c a l c u l a t e d i n t h i s way a r e : 18s rRNA: approximately 47 28s rRNA: approximately 70 5.8s rRNA: 1.2 (One s i t e i n 5 . 8 s rRNA i s f r a c t i o n a l l y methylated.) ( i i i ) Comoarison of rRNA with ribosomal p r e c u r s o r RNA. Comparison of the methyl f i n g e r p r i n t s of rRNA with t h o s e of ribosomal p r e c u r s o r RNA ( r e f s . 28, 32) immediately confirmed t h e r e l a t i o n s h i p s between the r e s p e c t i v e molecules, which had been i n f e r r e d e a r l i e r from k i n e t i c l a b e l l i n g experiments and a n a l y s i s of a1 kal i - s t a b l e products ( r e f . 18). The f i n g e r p r i n t o f 45s RNA i s very s i m i l a r t o t h a t of 28s p l u s 18s rRNA; t h a t of pure 32s RNA i s the same a s t h a t of 28s p l u s 5 . 8 s rRNA ( s e e Fig. 8.1). Two a s p e c t s of t h i s r e s u l t r e q u i r e f u r t h e r comment. F i r s t , ribosomal p r e c u r s o r RNA c o n t a i n s e x t e n s i v e t r a n s c r i b e d s p a c e r s which a r e e l i m i n a t e d d u r i n g ribosome m a t u r a t i o n . Electron microscopy revealed t h a t t h e t r a n s c r i b e d s p a c e r s amount t o some 40% of the o v e r a l l l e n g t h o f HeLa c e l l 45s RNA ( r e f . 3 6 ) . Howe v e r , t h e methyl f i n g e r p r i n t s o f ribosomal p r e c u r s o r RNA do not c o n t a i n any e x t r a , methylated ol i g o n u c l e o t i d e s which cannot be accounted f o r i n t h e rRNA f i n g e r p r i n t s . T h u s , although t h e g r e a t m a j o r i t y of m e t h y l a t i o n s occur r a p i d l y on ribosomal p r e c u r s o r R N A , the methyl groups a r e a l l l o c a t e d w i t h i n the rRNA sequences i n t h e p r e c u r s o r molecules: t h e t r a n s c r i b e d s p a c e r s a r e unmethyl a t e d . This important conclusion w i l l be d i s c u s s e d l a t e r , below. Second, following the a n a l y s i s of Zimmerman ( r e f . 17, above), experiments were c a r r i e d o u t t o d i s t i n g u i s h between e a r l y and l a t e methylations ( r e f s . 31, 32). I t was found t h a t t h r e e charact e r i s t i c 18s methylated o l i g o n u c l e o t i d e s a r e a b s e n t from t h e 45s f i n g e r p r i n t , and a r e s e l e c t i v e l y l a b e l l e d i n 18s rRNA a f t e r RNA s y n t h e s i s has been blocked by actinomycin ( F i g u r e 8 . 2 ) . These l a t e methylations e v i d e n t l y comprise a small c l a s s of events which a r e d i s t i n c t from the many e a r l y m e t h y l a t i o n s , and probably occur a t about the time when t h e nascent small ribosomal s u b u n i t emerges from t h e nucleolus i n t o the cytoplasm. ( i v ) Nature of the methvlated seauences. Analysis of t h e methylated ol i g o n u c l e o t i d e s showed t h a t t h e g r e a t m a j o r i t y a r e 2 ' O-methylated and a few c o n t a i n methylated b a s e s . The f i r s t major

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data compilation was given in r e f . 32. Some additional data were obtained and a few corrections made in a f.urther compilation ( r e f . 37), which also contains data f o r several other vertebrate species (see below). Table 8.1 summarizes d a t a on the modified components i n HeLa c e l l rRNA, together w i t h d a t a from X e n o p u s and yeast (see also below). A l l of the 2'-O-methylations occur rapidly on 45s rRNA, with one exception i n the 28s sequence. There i s great d i v e r s i t y among the methylated sequences. A l l of the 16 possible 2'-O-methylated dinucleotides are present i n HeLa c e l l rRNA, most of them occurri n g in both 18s and 28s rRNA, i n d i f f e r e n t frequencies (Table 8 . 1 ) . A few oligonucleotides contain more t h a n one methyl group, e i t h e r on adjacent nucleotides (see note g t o Table 8.1) or separated by one or more unmethylated nucleotides (see r e f . 37 f o r detai 1 s ) . The l a t e methylations i n 18s rRNA a r e a l l base methylations. There i s a l s o a hypermodified nucleoside i n 18s rRNA, which was f i r s t characterized by Saponara and Enger ( r e f . 38) and designated 3- (3-ami no-3-carboxypropyl ) -1-methyl pseudouri d i ne (m1cap3$, sometimes a l s o abbreviated am$). This nucleoside receives both i t s methyl group and the 3-amino-3-carboxypropyl group from methionine (via S-adenosyl methionine) i n separate reactions, and therefore has the unusual property t h a t i t can be labelled w i t h l - 1 4 C or 214C labelled methionine as well as with methyl labelled methionine. These c h a r a c t e r i s t i c s enabled the t i m i n g of the various steps i n the modification of this nucleoside t o be deduced w i t h respect t o the overall rRNA maturation pathway ( r e f . 39). The methyl group i s added in the nucleolus whereas the bulkier a l i phatic group appears t o be added a f t e r 18s rRNA has l e f t the nucleolus, probably a t about the same time as the other l a t e base methyl ations. There a r e also 5 base methylations in 28s rRNA. These 28s base methyl groups a r e already present i n 4 5 s rRNA and t h e i r timing i s not e a s i l y distinguishable from the majority of 2'-0methylations, although there may be very s l i g h t delays (M.S.N. Khan and B . E . H . Maden, unpublished d a t a ) . However, one "semi1 a t e " methyl a t i on was detected: the sequence UmpGmp$p, indicated in note g of Table 8.1, i s incompletely methylated a t the level of

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RNA. This multiply modified component occurs in a large T, similarity between the methylation patterns of rRNA from these

45s

Figure 8.1. T, ribonuclease fingerprints of HeLa cell methyl labelled rRNA and nucleolar 45s and 32s RNA, showin the relationships between the methylation patterns of rRNA an] the nucleolar precursors. Spots above and to the left of the diagonal line in the key are better resolved in fingerprints obtained by the Such combined T, plus phosphatase procedure (ref. 32). fin er rints also show correspondence between the ribosomal and nuc!eo!?ar methylation patterns. Spots 29a and 36a are not seen in fingerprints of mature rRNA. Spot 36a is an incompletely modified form of the hypermodified mlcap311, spot see the text and Table 8.1). Spot 29a may be an incompletely mo ified form of the UmGm11, sequence. Reproduced from ref. 32.

d

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Fi ure 8.2. Fingerprints showing the late methylation sites in l8! rRNA, within T, products 30, 34, and 49. These oligonucleotides contain, respectively, the following methylated bases: m$A (two residues), m6A, m7G. The spots are absent from methyl fingerprints o f nucleol ar 18S-20s RNA (the immedi ate recursor to cytoplasmic RNA). They are selectively labelled in 1 S rRNA when methyl labelled methionine is added to cells 5 minutes after RNA

l

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Figure 8.2 (cant i n u e d ) synthesis has been blocked by actinomycin. There i s v i r t u a l l y no labelling of 28s rRNA under these condit i o n s , except for a very f a i n t , submolar component designated 2a. Reproduced from r e f . 32. several vertebrate sources ( r e f . 37). Various f u r t h e r ri bonuclease ol igonucleotide whose sequence was determined Eladari e t . a 7 . ( r e f . 40).

by

Methvlated Sequences in r R N A from Other Vertebrate Sources I t was of i n t e r e s t t o extend the analysis of methylated ol igonucleotides t o rRNA from other vertebrate sources. Accordi ngly, methyl -1 abel 1 ed rRNA was prepared from the fol1 owing sources: mouse L c e l l s , hamster C13 c e l l s , freshly cultured chick embryo fibroblasts and a l i n e of X . l a e v i s c e l l s . T 1 ribonuclease fingerprints were prepared, and revealed a very high level of similarity between the methylation patterns o f rRNA from these several vertebrate sources ( r e f . 37). Various f u r t h e r analytical procedures were carried o u t using 14C-methyl-1 abell ed or 32P-labe11ed material, and the data on the oligonucleotides were tabulated i n r e f . 37. (T, plus pancreatic RNase fingerprints also yielded useful data f o r X e n o p u s and chick; r e f . 41). The r e s u l t s of t h i s comparative analysis may be summarized as follows. 14C-methyl fingerprints of 18s rRNA from the three mammalian sources were indistinguishable. The chick and X e n o p u s 18s fingerprints resembled the mammalian fingerprints closely b u t differed in a few respects. (See Table 8.1 f o r a summary of Xenopus d a t a a t the mono- and dinucleotide l e v e l , and r e f . 37 f o r the ol igonucleotide d a t a . ) The various 28s methyl fingerprints a l s o differed only s l i g h t l y between species. Some of the d i f ferences between fingerprints appeared t o be due t o single base substitutions; others appeared t o be due t o the presence or absence of a methyl g r o u p a t a particular point in the sequence. (Both of these inferences were confirmed i n subsequent work: see below.) All of the differences were between 2'-O-methylated sequences. The base methyl ated sequences were identical between species.

8.4.2

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At about this time Choi and Busch (ref. 42) published a catalogue of T, 01 i gonucl eoti des from rat hepatoma (asci tes cell ) 18s rRNA. Their partial sequence data and quantification of methylated 01 igonucleotides were in practically complete agreement with our data for mammalian 18s rRNA (ref. 37), with the same numbers of the 16 different 2'-O-methylated dinucleotides as summarized for HeLa cell 18s rRNA in Tab1 e 8.1, 8.4.3 Methvlation in Yeast rRNA and Ribosomal Precursor RNA While the above work on vertebrate rRNA was in progress, Klootwijk and Planta carried out a similar analysis on the methylated ol igonucleotides in rRNA of the yeast, S a c c h a r o m y c e s c a r l s b e r g e n s i s (refs. 39, 43, 44, and 45). The general conclusions were very similar to those obtained from vertebrate rRNA, although there were many differences in detail. As in vertebrates, the majority o f methylations are 2'-O-substituents (ref. 44). These are added rapidly to ribosomal precursor RNA, with the exception of semi-late methylation of UmpGmp$p in the 28s sequence (ref. 45). There are, however, considerably fewer 2'-O-methyl groups in yeast rRNA than in vertebrate rRNA (Table 8.1). The base methylations are also similar, but not quite identical, to those in vertebrate rRNA. The timing of the base methylations is also similar with respect to the rRNA maturation pathway, the 18s base methylations occurring late in the pathway (ref. 45). 8.4.4 ImPlications of the Oliaonucleotide Data It was evident from the methyl fingerprints that rRNA methylation is a highly specific process. The great majority o f methylations, including all the 2'-O-methylations, occur rapidly upon ribosomal precursor RNA, and all of the methylation sites are within the ribosomal sequences of the precursor molecule. The oligonucleotide data imply that most of the methylation sites are highly conserved in rRNA from different vertebrates, but the numbers of 2'-O-methyl groups differ considerably between vertebrates and yeast (Table 8.1). Despite the highly specific patterns of the methyl fingerprints, sequence analysis of the methylated oligonucleotides revealed no common motif in the methylation sites at the level of primary structure; indeed, there is great diversity at this level among the methylated sequences.

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TABLE 8 . l a Modified Nucleotides i n 18s rRNA

The HeLa and X e n o p u s d a t a on methyl groups a r e summarized from t h e o l i g o n u c l e o t i d e d a t a i n r e f . 37 a n d the sequence d a t a i n r e f s . 61 and 62. The y e a s t ( S a c c h a r o m y c e s c a r l s b e r g e n s i s ) d a t a on methyl groups a r e summarized from the o l i g o n u c l e o t i d e d a t a i n ref. 44. HeLa X. l a e v i s S.carlsbergensis Notes A1 kal i -stab1 e d i n ucl eo t i des : UmpU UmpG UmpA UmpC Total Um

2 4 2 3 11

2 3 1 3

0

GmpU GmpG GmpA GmpC Total Gm

1 6 1 1 9

1 5 0 1 7

2 2 1 0

AmpU AmpG AmpA AmpC Total Am

2 4 2 13

5

4 2 4 2 12

2 1 4 1

CmpU CmpG CmpA CmpC

Total Cm Total 2'-O-methyl

9

1 1

a

0 2

a a

5

a

0

3 7

0 2 0 3 5

40

33

ia

1 1 1 2 7

1 1 1 2 7

1 1 0 2 6

b

37

46

14

d

1 2 1

1

0 2 3

a

Methylated bases: rn1cap3fl

m7G

m6A

m$A Total base methyl Pseudouridi ne (approx. 1 a. b.

c.

C

In HeLa c e l l 18s rRNA t h e r e a r e f o u r p a r t i a l 1 meth l a t e d UmpG, &npG, &PA, a n d s i t e s , one f o r each of t h e CmpC (see r e f . 62 f o r Thi s a b b r e v i a t i o n denotes -1methyl pseudouri d i n e ; see the t e x t . Each m$A c a r r i e s 2 methyl groups.

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TABLE 8 . l b M o d i f i e d N u c l e o t i d e s i n 28s rRNA HeLa

X . laevis

S.carlsbergensis

Notes

A1 k a l i-stab1 e d i nucleotides: UmpU UmpG UmpA UmpC T o t a l Um GmpU GmpG GmpA GmpC T o t a l Gm AmpU AmpG AmpA AmpC T o t a l Am

0 3

1

3 7 6

10

3 0

19 5 7 2 4

18

0 3 1 3 7

2 3 1 1 7

e

f f

5 9 3 1 18 5 7 2 4 18

f

14

5 2 3 3 13

A1 k a l i-stab1 e 01 igonucl e o t i des (2 ' -0-methyl groups)

7

7

8

9

T o t a l 2'0-methyl

65

63

37

f

1 2 2 5

1 2 2 5

2 2 2 6

h h

56

62

32

d

CmpU CmpG CmpA CmpC T o t a l Cm

4

3

3 4

Methyl a t e d bases:

m3 U mA mC T o t a l base methyl Pseudouri d i ne (approx. 1

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TABLE 8 . l c

Modified Nucleotides i n 5.8s rRNA GmpC Um G

Otler methyl Pseudouri di ne

1 0.2 0 2

1 0.4 0 2

0 0 0 1

i

Notes t o T a b l e 8.1 ( c o n ' t )

d.

e. f.

g.

h.

i.

The v a l u e s f o r pseudouridine a r e from r e f s . 61 and 65 (HeLa and X e n o p u s ) and 43 ( S a c c h a r o m y c e s ) , and a r e probably a c c u r a t e t o w i t h i n + 10% of the s t a t e d v a l u e s . I n c l u d e s . oiie dmpG. The p r e c i s e numbers o f the i n d i c a t e d d i n u c l e o t i d e s i n HeLa and Xenopus a r e s l i g h t 1 u n c e r t a i n from o l i g o n u c l e o t i d e d a t a , b u t the u n c e r t a i n t i e s a t f e c t t h e t o t a l numbers of methyl groups by o n l y about + 2. The a l k a l i - s t a b l e o l i g o n u c l e o t i d e s i n HeLa and X e n o p u s a r e : UmpGmpU, UmpGmp , and AmpGmpCmpA; i n S a c c h a r o y m c e s t h e y a r e : UmpGmpd, AmpGmp8 AmpCmpG, and AmpAmpU. The s i t e s of s u b i t i t u t i o n i n mA and mC i n HeLa and X e n o p u s 28s rRNA have not a l l been determined. Yeast 5 . 8 s d a t a a r e from S . c e r e v i s i a e , r e f . 46.

To account f o r t h e s e v a r i o u s f i n d i n g s i t was proposed ( r e f . 32) t h a t the s p e c i f i c i t y f o r methylation i s determined by conformation w i t h i n t h e rRNA sequences: i n p a r t i c u l a r , t h a t the many 2'-O-methylation s i t e s p r e s e n t some common conformational f e a t u r e t o a n u c l e o l a r enzyme o r a small number of enzymes which c a r r y o u t t h i s t y p e of methylation on ribosomal p r e c u r s o r R N A . I t was now becoming apparent t h a t f u r t h e r p r o g r e s s would r e q u i r e knowledge of the l o c a t i o n s of t h e methyl groups i n t h e complete primary s t r u c t u r e of rRNA. 8.5 LOCATING THE METHYLATED NUCLEOTIDES 8 . 5 . 1 Modified Nucleotides i n 5.8s rRNA A s t a r t t o l o c a t i n g t h e modified n u c l e o t i d e s i n rRNA came from the sequence a n a l y s i s of 5.8s rRNA. This small (160 nucleot i d e ) molecule i s non-covalently a t t a c h e d t o 28s rRNA and i s t r a n s c r i b e d a s p a r t of 45s ribosomal p r e c u r s o r R N A . I t becomes a s e p a r a t e e n t i t y a s a r e s u l t of e x c i s i o n o f the second i n t e r n a l t r a n s c r i b e d s p a c e r region (ITS 2) during the f i n a l m a t u r a t i o n of 32s t o 28s rRNA. When 28s rRNA i s i s o l a t e d by non-denaturing

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procedures, 5.8s r R N A can be r e l e a s e d subsequently by h e a t denat u r a t i o n o r comparable methods and recovered i n p u r e form. Because o f i t s r e l a t i v e l y small s i z e i t was t h e f i r s t f u n c t i o n a l r e g i o n o f t h e e u k a r y o t i c ribosomal t r a n s c r i p t i o n u n i t t o be s u b j e c t e d t o complete sequence a n a l y s i s . 5.8s sequences were r e p o r t e d f o r y e a s t ( S a c c h a r o m y c e s c e r e v i s i a e , r e f . 46), r a t ( r e f . 47), o t h e r v e r t e b r a t e sources ( r e f s . 48, 49) and o t h e r non-vert e b r a t e s . Ref. 50 g i v e s a summary o f c u r r e n t l y known 5.8s sequences. ( T h i s c o m p i l a t i o n a l s o i n c o r p o r a t e s c o r r e c t i o n s t o some small e r r o r s which appeared i n some o f t h e o r i g i n a l r e p o r t s . ) Each of t h e 5.8s sequences c o n t a i n s one o r more m o d i f i e d nucleotides. The v e r t e b r a t e 5.8s sequences c o n t a i n two 2 ' - 0 m e t h y l a t i o n s i t e s , one o f which i s f r a c t i o n a l l y m e t h y l a t e d , and two pseudouridines. Thus 5.8s rRNA, which i s f u n c t i o n a l l y p a r t o f 28s rRNA b u t i s separated from t h e r e s t o f t h e 28s sequence by ITS 2 i n ribosomal p r e c u r s o r RNA, c o n t a i n s s p e c i f i c r e c o g n i t i o n s i t e s f o r 2 ' - 0 - m e t h y l a t i o n and p s e u d o u r i d i n e f o r m a t i o n . Although t h e 5.8s and 28s sequences a r e separated from each o t h e r i n t h e p r i m a r y s t r u c t u r e o f ribosomal p r e c u r s o r RNA i t seems probable t h a t t h e y e s t a b l i s h t h e i r mutual i n t e r a c t i o n w h i l e t h e y a r e w i t h i n t h e p r e c u r s o r molecule. O l i g o n u c l e o t i d e d a t a show t h a t t h e p r i n c i p a l 2 ' - 0 - m e t h y l a t i o n s i t e i n HeLa c e l l 5.8s rRNA i s a l r e a d y m o d i f i e d w i t h i n ribosomal p r e c u r s o r RNA ( r e f s . 32, 51). I t i s an i n t e r e s t i n g and c u r r e n t l y unanswered q u e s t i o n whether m e t h y l a t i o n o f t h e 5.8s sequence occurs b e f o r e o r a f t e r i t s i n t e r a c t i o n w i t h t h e 28s sequence. T h i s p r i n c i p a l 5.8s m e t h y l a t i o n s i t e occurs i n a 10 nucleot i d e h a i r p i n l o o p near t h e m i d d l e o f t h e sequence, and i s conserved i n v e r t e b r a t e 5.8s rRNA ( r e f s . 47, 48). The c o r r e s p o n d i n g l o o p i n S a c c h a r o m y c e s 5.8s rRNA i s s m a l l e r and unmethylated. Comparative d a t a o f t h i s k i n d may p r o v i d e a s t a r t i n g p o i n t from which t o i d e n t i f y t h e s t r u c t u r a l determinants o f m e t h y l a t i o n sites. 8.5.2

M e t h v l a t i o n Map o f X e n o u u s 7 a e v i s rRNA By t h e l a t e 1970s i t had become c l e a r t o many workers t h a t sequenci ng 1arge RNA mol ecul es by t h e c l a s s i c a l procedures which were then a v a i l a b l e ( r e f s . 25, 34, 46) was l i k e l y t o be e x t r e m e l y

B285

l a b o r i o u s and a l s o e r r o r - p r o n e . Thus an impasse appeared t o have been reached i n t h e a n a l y s i s o f e u k a r y o t i c r R N A and i t s m o d i f i e d nucleotides. T h i s impasse was surmounted w i t h t h e a i d o f recomb i n a n t DNA. The DNA encoding t h e m a j o r r R N A species (rDNA) f r o m Xenopus l a e v i s was t h e f i r s t e u k a r y o t i c DNA t o be c l o n e d ( r e f s . 52, 53). The a v a i l a b i l i t y of c l o n e d X e n o p u s rDNA enabled a s e r i e s o f experiments t o be c a r r i e d o u t i n which t h e d i s t r i b u t i o n o f m e t h y l a t i o n s i t e s w i t h i n X e n o p u s 18s and 28s rRNA was e x p l o r e d . The e x p l o r a t i o n was c a r r i e d o u t by repeated a p p l i c a t i o n o f t h e f o l l o w i n g experimental p r o t o c o l ( r e f . 54). Methyl -1 abel l e d o r 32P-1abelled 18s o r 28s r R N A was h y b r i d i z e d t o c l o n e d fragments o f rDNA, o r t o s m a l l e r r e s t r i c t i o n fragments which had been p u r i f i e d from t h e clones. I n each h y b r i d i z a t i o n , o n l y p a r t o f t h e r R N A was represented by t h e rDNA c l o n e o r fragment, and was t h e r e f o r e bound i n t h e RNA:DNA h y b r i d . The n o n - h y b r i d i z e d r e g i o n o f RNA was removed by t r i m m i n g w i t h T, RNase. The h y b r i d i z e d r e g i o n was t h e n recovered by h e a t i n g t h e h y b r i d , and was analyzed by f i n g e r p r i n t i n g f o r i t s content o f methylated oligonucleotides (using t h e T, o r t h e combined T, p l u s p a n c r e a t i c RNase f i n g e r p r i n t i n g procedure). I n t h i s way each o f t h e m e t h y l a t e d o l i g o n u c l e o t i d e s i n Xenopus 18s r R N A was l o c a l i z e d t o one o f e i g h t r e g i o n s d e f i n e d by r e s t r i c t i o n s i t e s i n rDNA. S i m i l a r l y , each o f t h e m e t h y l a t e d o l i g o n u c l e o t i d e s i n 28s rRNA was l o c a l i z e d t o one o f eleven r e g i o n s d e f i n e d by r e s t r i c t i o n s i t e s i n rDNA. The r e s u l t s y i e l d e d a “ m e t h y l a t i o n map” o f X e n o p u s r R N A ( r e f . 54). The map y i e l d e d a u s e f u l overview o f t h e d i s t r i b u t i o n o f m e t h y l a t e d sequences a l o n g 18s and 28s rRNA. The d i s t r i b u t i o n i s non-uniform. I n 18s rRNA, 18 o f t h e 33 2’-O-methyl groups (54%) were found i n t h e 5 ’ 40% o f t h e molecule. The base m e t h y l a t i o n s i t e s were i n t h e 3 ’ h a l f o f t h e molecule. I n 28s r R N A t h e r e was a much lower m e t h y l a t i o n d e n s i t y i n t h e 5 ‘ r e g i o n , and 40 o u t o f t h e 68 o r so methyl groups (60%) were found i n t h e 3 ’ 30% o f t h e mol ecul e. Meanwhile Gerbi and co-workers had s t a r t e d t o e x p l o r e t h e d i s t r i b u t i o n of p h y l o g e n e t i c a l l y conserved and v a r i a b l e r e g i o n s along t h e rRNA sequences ( r e f . 55) and had a l s o c a r r i e d o u t some h y b r i d i z a t i o n experiments w i t h m e t h y l - l a b e l l e d r R N A ( a l t h o u g h n o t

B286

a t t h e l e v e l o f f i n g e r p r i n t i n g a n a l y s i s , r e f . 56). The r e s u l t s suggested t h a t m e t h y l a t i o n i s concentrated i n t o rRNA r e g i o n s which show h i g h p h y l o g e n e t i c sequence c o n s e r v a t i o n . To e x p l o r e f u r t h e r t h i s s u g g e s t i v e r e l a t i o n s h i p i t was n e c e s s a r y t o o b t a i n f u l l sequence d a t a w i t h t h e e x a c t l o c a t i o n s o f t h e rRNA m e t h y l g r o u p s . E x a c t L o c a t i o n s o f t h e M e t h y l Grouos i n 18s rRNA o f X e n o p o s l a e v i s and Man The n u c l e o t i d e sequence o f a c o m p l e t e X . l a e v i s gene e n c o d i n g 18s r R N A was d e t e r m i n e d b y S a l i m and Maden ( r e f . 5 7 ) . The m e t h y l groups were l o c a t e d i n t h e i n f e r r e d r R N A sequence, i n m o s t i n s t a n ces e x a c t l y , i n t h e r e m a i n i n g few i n s t a n c e s t o w i t h i n a few n u c l e o t i d e s ( t h e u n c e r t a i n t i e s b e i n g r e s o l v e d l a t e r , see b e l o w ) . The 18s gene sequence o f S a c c h a r o m y c e s c e r e v i s i a e had been d e t e r mined a s h o r t t i m e p r e v i o u s l y ( r e f . 58), a l t h o u g h t h e RNA m e t h y l g r o u p s i n S a c c h a r o m y c e s were n o t l o c a t e d . Comparison o f t h e t w o sequences r e v e a l e d e x t e n s i v e t r a c t s o f h i g h homology i n t e r s p e r s e d w i t h t r a c t s h a v i n g l i t t l e o r no homology. Most o f t h e X e n o p u s r R N A m e t h y l groups were f o u n d t o be l o c a t e d i n t h e h i g h l y cons e r v e d t r a c t s , as d e p i c t e d s c h e m a t i c a l l y i n F i g . 8.3, w h i c h i s f r o m r e f . 57. R e s u l t s f r o m f u r t h e r s e q u e n c i n g o f c l o n e d and u n c l o n e d x . l a e v i s rDNA i n d i c a t e d t h a t t h e X . l a e v i s 18s gene p o o l i s homogeneous, and hence t h a t 18s r R N A a l s o c o m p r i s e s a homogeneous A single, small c o r r e c t i o n t o p o p u l a t i o n o f m o l e c u l e s ( r e f . 59). t h e o r i g i n a l sequence was made ( r e f . 60) and a s e c o n d a r y s t r u c t u r e model was proposed ( r e f . 60, see b e l o w ) . The human 18s rDNA sequence was d e t e r m i n e d and t h e m e t h y l g r o u p s were l o c a t e d i n t h e i n f e r r e d 18s rRNA sequence ( r e f . 61). D e t a i l e d e v i d e n c e on t h e l o c a t i o n s o f t h e i n d i v i d u a l m e t h y l g r o u p s i n X . l a e v i s and human 18s r R N A was p u b l i s h e d i n r e f . 62. I n most i n s t a n c e s t h e l o c a t i o n s were e s t a b l i s h e d u n a m b i g u o u s l y by c o r r e l a t i o n o f d a t a from t h r e e l i n e s o f evidence: o l i g o n u c l e o t i d e d a t a ( r e f . 37 and f u r t h e r d a t a i n r e f . 62), t h e X e n o p o s m e t h y l a Some a d d i t i o n a l t i o n map ( r e f . 54) and t h e rDNA sequence d a t a . i n f o m a t i o n came f r o m t h e c a t a l o g u e o f m e t h y l a t e d 01 i g o n u c l e o t i d e s f r o m r a t 18s rRNA ( r e f . 42), w h i c h complemented o u r own d a t a . F i n a l y , Connaughton e t . a l . ( r e f . 63) c a r r i e d o u t a d i r e c t 8.5.3

X

x

5'

xx

X

Y

Y

x xxxx

x

200

A

400

x

x

X

x x x x x

a

X

X I

boo

0 0

X

800

1

1

x

x

x

x

x

x

@

X

x

X

0 m

1

1200

1000

1400

--

B

C

1600

1800

3'

D

Figure 8.3. Summary o f m e t h y l a t i o n s i t e s i n X . 7 a e v i s 18s rRNA and p a t t e r n o f homology with y e a s t (5. c e r e v i s i a e ) 18s rRNA. Up e r s e c t i o n : p l a i n a s t e r i s k s d e n o t e p o s i t i o n s o f 2'-0methyl roups a l o n g the X . 7 a e v i s 1 l S rRNA sequence- c i r c l e d a s t e r i s k s d e n o t e base methyl groups ?see a l s o F i g . 8 . 4 ) . Lower s e c t i o n : the h i 'h blocks i n d i c a t e r e g i o n s o f the x . l a e v i s sequence showing 85-100% homology w i t h y e a s t ; ow blocks i n d i c a t e r e g i o n s showing 7085% homology. A , B , C , D d e n o t e the r e g i o n s o f g r e a t e s t v a r i a b i l i t y between the two 18s sequences. Reproduced by permission from Nature, Vol. 291, No. 5812, p p . 205-208. Copyr i g h t ( c ) 1981, Macmillian J o u r n a l s Limited.

B

B288

sequence a n a l y s i s o f 18s r R N A from r a b b i t r e t i c u l o c y t e s u s i n g endl a b e l l i n g and p a r t i a l d e g r a d a t i o n methods, w i t h s e p a r a t i o n o f t h e p r o d u c t s on sequencing g e l s . Z'-O-methyl groups gave r i s e t o gaps a t s p e c i f i c p o i n t s i n the gels. By c o r r e l a t i n g t h e l o c a t i o n s o f these gaps w i t h known o l i g o n u c l e o t i d e d a t a ( r e f . 37) and sequence d a t a (e.g. r e f . 57) these authors i n f e r r e d t h e l o c a t i o n s o f 2'-0methyl groups i n 18s rRNA from r a b b i t r e t i c u l o c y t e s . The r e s u l t s a f f o r d e d a u s e f u l cross-check on t h e d a t a c o m p i l a t i o n i n r e f . 62, and enabled t h e l a s t few u n c e r t a i n t i e s i n t h e X e n o p u s and human d a t a t o be resolved, as d e t a i l e d i n r e f . 62. The Xenopus 18s sequence w i t h t h e l o c a t i o n s o f a l l t h e methyl groups as f i n a l l y i n f e r r e d ( r e f . 62) i s shown i n F i g . 8.4. In F i g . 8.5 t h e human sequence i s shown a l o n g w i t h s i t e s o r r e g i o n s where X e n o p u s d i f f e r s from t h e human sequence. Human 18s rRNA i s m e t h y l a t e d a t a l l t h e same l o c a t i o n s as i n X e n o p u s , and a l s o a t a few a d d i t i o n a l l o c a t i o n s , m a i n l y i n t h e 5 ' r e g i o n o f t h e sequence.

8.6 PROBLEMS AND PROSPECTS 8.6.1 The Methyl Groups i n 28s rRNA The t a s k o f l o c a t i n g a l l o f t h e m e t h y l a t i o n s i t e s i n 28s r R N A a t t h e n u c l e o t i d e sequence l e v e l has n o t y e t been completed. However, about 50 o f t h e 70 methyl groups have been l o c a t e d i n t h e Xenopus and human 28s sequences on t h e b a s i s o f v a r i o u s l i n e s o f a v a i l a b l e evidence (B.E.H.M., manuscript i n preparation). T h i r t y o f t h e 43 methyl groups i n y e a s t 28s rRNA were l o c a t e d i n t h e A c o n s i d e r a b l e number o f t h e s e occur a t sequence ( r e f . 64). homologous l o c a t i o n s i n t h e y e a s t and v e r t e b r a t e sequences, as w i l l be d e s c r i b e d elsewhere i n d e t a i l (B.E.H. Maden, i n preparat i o n ) . Meanwhile, t h e X e n o p u s 28s " m e t h y l a t i o n map" which was o b t a i n e d by h y b r i d i z a t i o n experiments ( r e f . 54) i s shown here ( F i g . 8.6) t o d e p i c t t h e heavy c l u s t e r i n g o f methyl groups i n t h e 3 ' r e g i o n o f t h e molecule. 8.6.2 Pseudouridine and Other M o d i f i e d N u c l e o t i d e s The approximate numbers o f p s e u d o u r i d i ne r e s i d u e s i n human, X e n o p u s and S a c c h a r o m y c e s r R N A a r e summarized on t h e bottom l i n e s The values i n r e f s . 6 1 and 65 were from base o f Table 8.1. composition a n a l y s i s f o l l o w e d by chromatographic s e p a r a t i o n o f

B289 m UACCUCCUUC AUCCUCCCAC UACCAUAUCC UUCUCUCAAA GAUUAACCCA UCCACCUCUA

60

m ACUACCCACC CCCCCUACAC UCAAACUCCG AAUCCCUCAU UAAAUCAGUU AUCCUUCCUU

120

UCAUCCCUCC AUCUCUUACU UCCAUAACUC U C C U A A U U C U C C U A A U A

CAUCCCCACC

180

ACCCCUCACC CCCAGGCAUC CGUCCAUUUA

UCACACCAAA ACCAAUCCCC CCCCCCCCCC

240

CCCCCCCCCC UUUCCUGAC-UAACC

UCGCCCCCAU C C C A C G U C C C CWGACCCCC

300

XbaI

XbaI

ACCAUACAUU CCCAUGUCUC CCCUAUCAAC UUUCGAUCCU ACUUUCUGCC CCUACCAUCC

360

m m UCACCACGCC UAACCCCGAA UCACCCUUCC AUUCCCCACA GCCACCCUCA CAAACGGCUA

420

r n m AUUACCCACU CCCGACCCCC CCACCUACUC

480

n m 111 CCACAUCCAA CCAACCCACC ACCCGCCCAA

..

.

..

. .

m ACCAAAAAUA A C A A U A C A C C U U U C C A CCCCCUCUAA UUCCAAUGAC UACACUUUAA HlCIt-1 .m nl .m. ..m AUCCUUUAAC CACCAUCUAU UGCACGCCAA CucuCCuCcc A C C A ~ C C C C C

.

-..

.

540

CUAAUUCCAC

600

m m U U A A A A A C C U CCUACUUGCA

UCUUCCCAUC

660

CACCUCCCCC UCCCCCGCCA CGCCACCUAC CCCCUCUCCC ACCCCCUGCC

UCUCGCCCCC

720

UCCCCCAUCC UCUUGACUCA GUCUCCCGCG CCCCCCAACC CUUUACUUUC A A A A A A U U A C

780

. . PStI

m . CUCCAAUAGC GUAUAUUAAA CUUC-

m

m

?,ma1 -

AGUCUUCCAA G C A C C C C C C C

uccccuccnu

ACUUCACCUA CCAAUAAUCC AAUACCACUC

840

CCGUUCUAUU UUCUUCCUUU UCCCAACUGC CCCCAUCAUU AAGACCCACC CCCCGCCCCA

900

UUCCUAUUCU CCCCCUACAC GUCAAAUUCU UCCACCGCCC CAACACCAAC C A A A C C C A A A

960

C C A U U U C C C A A C A A U G U U U U C A U U A A U C A A G A A C C A A A C U CCGACCUUCC AACACCAUCA

1020

GAUACCCUCG UACUUCCCAC C A U A A A C C A U CCCCACUACC CAUCCGCCCG CGUUAUUCCC

1080

AUGACCCCCC CACCAGCUUC CCCCAAACCA AAGUCUUUCC GUUCCCCGGC CACUAUCCUU

1140

CCAAACCUCA AACUUAAAGC AAUUGACCCA ACCGCACCAC CACCACUGCA CCCUGCGCCU

1200

M

m UAAUUUCACU CAACACCCCA AACCUCACCC CCCCCGGACA C C G A A A C C A U UCACACAUUC m m AUAGCUCUUU CUCCAUUCUC UCCCUCCUGC UCCAUCCCCC UUCUUACUUC GUCGACCCAU -1 m UUCUCUGGUU AAUUCCCAUA A C C A A C C A ~ C U C C A U C CUAACUAGUU ACCCCACCCC

1260

1320 1380

Hint-1

m CCCCGGUCCC CCUCCAACUU CUUACACCCA CAACUCCCGU UCACCCACAC CACAUCGACC

1440

m AAUAACACCU CUCUGAUCCC CUUACAUCUC CCCCCCUCCA CCCGCGCUAC ACUGAACGCA

1500

UCACCCUCUC UCUACCCUCC CCCCACACCU CCCCGUAACC CCCUCAACCC CCUUCGUGAU

1560

M ACCGAUCGCC CAUUCCAAUU AUUUCCCAUG AACCAC-CCACUAAG

UGCGCGUCAU

1620

m m AAGCUCCCCU UCAUUAACUC CCUCCCCUUU CUACACACCC CCCCUCCCUA CUACCGAUUC

1680

CAUCCUUUAC UCACCUCCUC GCAUCCCCCC CCCCCCCCUC CCCCACCCCC CUCCCCCAGC

1740

m M X C G A G A A C A CGAUCAAACU UCACUAUCUA GACCAACUAA AAClJCCUAAC AAGCUUUCCC

1800

MM UACCUCAACC UGCCCAACCA UCAUUA

1826

&RI

Figure 8.4. Nucleotide sequences of X . l a e v i s 18s rRNA, w i t h t h e l o c a t i o n s of the 2I-O-rneth 1 groups (rn) and base methyl g r o u p s (M). (Legend continued folfowing).

B290 m UACCUGCUUC AUCCUCCCAC UACCAUAUCC UUCUCUCAAA CAUUAACCCA UCCAUCUCUA C C

..

m

"

ACUACCCACG CCCCCUACAC UCAAACUCCC AAUCCCUCAU UAAAUCACUU AUCCUUCCUU

"

rn

UGCUCCCUCC CUCCUCUCCC ACUUCGAUAA CUCUCCUAAU UCUACACCUA A CUU A

- --

____

ACGGCCCCUC ACCCCCUUCC CCGGCCCGAU GCGUCCAUUU AUCACAUCAA A ____A C

mnl AUACAUCCCC

60 60 120 120 180 177

AACCAACCCC

240 229

CUCAGCCCCU CUCCCCCCCC CCCCCCCGCC CGGCCCCCCC CCCCUUUCGU CACUCUACAU

300 266

AACCUCGCCC CCAUCCCACC CCCCCCCUCG CCCCCACCAC CCAUUCCAAC CUCUGCCCUA U A U A C U

360 325

UCAACUUUCC AUCCUACUCC CCCUGCCUAC CAUCCUCACC ACCCCUCACC CCCAAUCACC cull u c A

420 385

m m m CUUCCAUUCC GGACAGCCAC CCUCACAAAC GCCUACCACA UCCAACCAAC CCAGCACGCC

480

c

--c

----- --- - ----_- ------

u

c

445

m

m

m

m

CCCAAAUUAC CCACUCCCCA CCCCCCCACG UACUCACGAA AAAUAACAAU ACACCACUCU G

540 505

m UUCGACGCCC UCUAAUUCCA

AUCACUCCAC UUUAAAUCCU UUAACCACGA UCCAUUGCAG U A

600 565

m CCCAAGUCUC CUGCCAGCAC CCCCGCUAAU UCCACCUCCA AUAGCGUAUA UUAAAGUUCC

"

m x.1. X.D.

UCCACUUAAA AAGCUCCUAC UUCGAUCUUC CCACCCCCCC GCCCCUCCGC CCCGACGCCA U A U c A CCCACCCCCC CUCCCCCCCC CUUGCCUCUC CCCGCCCCCU CGAUCCUCUU ACCUCAGUCU u c GA U U A

rnm

-

660 625 720 685 780 744

CCCCCCCCCC CCCAACCGUU UACUUUGAAA AAAUUACACU CUUCAAACCA CGCCCCACCC c u C

840 802

CCCUGCAUAC CGCAGCUACG AAUAAUCCAA UACCACCCCC CUUCUAUUUU CUUCGUUUUC

900 862

CCAACUCACC CCAUCAUUAA CACCCACCGC CCGGCCCAUU CGUAUUGCCC CCCUACACGU U G

960 922

CAAAUUCUUC CACCGCCCCA ACACCGACCA GACCCAAACC AUUUCCCAAC AAUGUUUUCA A A

1020 982

uc

uu

UUAAUCAACA ACCAAACUCC GAGCUUCGAA GACCAUCACA UACCCUCGUA GUUCCCACCA

1080 1042

UAAACGAUCC CCACCCCCCA UOCCCCCCCG UUAUUCCCAU CACCCCCCCG CCACCUUCCC UA C A

1140 1102

CCAAACCAAA GUCUUJCCCU UCCGGCGCCA CUAUCCUUCC AAACCUCAAA

CUUAAACCAA

1200 1162

UUCACCCAAC CCCACCACCA CCACUCCACC CUCCCCCUUA AUUUCACUCA ACACCCCAAA

1260 1222

CCUCACCCCC CCCCCACACC CACACCAUUC ACACAUUCAU ACCUCUUUCU CCAUUCCCUC A U

1320 1282

ti

m m CCUCCUCCUC CAUCCCCCUU CUUACUUCCU CCACCCAUUU CUCUCCUUAA UUCCGAUAAC

m

m

,

-__

CAACCACACU CUCCCAUCCU AACUACUUAC CCCACCCCCC ACCCGUCCCC CUCCCCCAAC

m

cuc

m

m

1440 1398

UUCUUACAGC CACAAGUCCC CUUCACCCAC CCCAGAUUCA GCAAUAACAC CUCUCUCAUC A C

1500 1458

CCCUUACAUC UCCCCCCCUC CACCCCCCCU ACACUCACUC CCUCAGCCUG UCCCUACCCU U AC A

1560 1518

ACGCCCCCAC CCGCCCCUAA CCCGUUGAAC CCCAUUCCUC AUCGCCAUCC CCCAUUGCAA c C A U C H A * UUAUUCCCCA UCAACGACGA AUUCCCACUA ACUCCCCCUC AUAACCUUCC GUUCAUUAAC U C

1620 1578 1680 1638

UCCCUGCCCU UUCUACACAC CCCCCCUCCC UACUACCGAU UCCAUCCUUU ACUCACGCCC U

1740 1698

UCCCAUCCCC CCCGCCCGCC UCCCCCCACC GCCCUCCCGG ACCCCUGACA ACACCCUCCA CC A A A

1800 1157

"

Y.1. X.b.

1380 1342

M

M M

ACUUGACUAU CUACACGAAG UAAAAGUCCU AACAACCUUU CCCUACCUCA ACCUCCCGAA

1860 1817

GGAUCAUUA

1869 1826

F i g u r e 8.5. N u c l e o t i d e sequence o f human 18s r R N A w i t h l o c a t i o n s o f methyl groups and comparison w i t h X e n o p u s . The f i r s t s u b s c r i p t l i n e shows t h e p o s i t i o n s a t which t h e X . 7 a e v i s se uen.ce d i f f e r s from t h a t of human ( o r from x). (legend c o n t i n u e d f o l y o w i n g ) .

B291

F i g u r e 8.4 l e g e n d c o n t i n u e d . R e s t r i c t i o n s i t e s i n rDNA which were used f o r ma p i n g t h e methyl groups t o w i t h i n s p e c i f i c regions of

rRNA r e f . !4) a r e shown as s u b s c r i p t s . S u p e r s c r i p t d o t s represent RNase cleava e s i t e s i n one such rRNA region, between nucleo2ides 500 and 6j5. The base modified n u c l e o t i d e s a r e : t h e hypermodified m'ca 3$ ( p o s i t i o n 1210), m7G (1597), m6A (1789) and two mqA r e s i d u e s (f.807, 1808). Reproduced from r e f . 62.

\

b o r e a l i s , i n d i c a t e d in the second sugscript line). Where e x t r a n u c l e o t i d e s occur i n the human sequence dashes a r e shown i n t h e aligned X . l a e v i s sequence. The l o c a t i o n s of Z'-O-methyl groups (m) and base methyl qroups ( M a r e i n d i c a t e d . A s t e r i s k s denote n u c l e o t i d e s which a r e 2 -0-methy a t e d i n human 18s rRNA b u t unmethylated i n X e n o p u s . The same base methyl groups occur a t homologous l o c a t i o n s i n the X e n o p u s and human 18s sequences, and a r e defined i n the l e end t o Figure 18.4. One o l i g o n u c l e o t i d e containing CmpC i n human 18s rRNA i s u n laced Reprinted by permission from Biochem. J . , Vol. 232, pp. 755-733: Copyright ( c ) 1985. F i u r e 8.5 l e g e n d c o n t i n u e d .

1

pseudouridine from u r i d i n e . The human 18s value obtained i n this way was i n very good agreement w i t h r a t 18s d a t a from oligonucleot i d e a n a l y s i s (38 pseudouridines, r e f . 42) , suggesting t h a t most The numbers of of the values i n r e f . 65 a r e f a i r l y a c c u r a t e . pseudouridines a r e f a i r l y s i m i l a r t o the numbers of 2'-O-methyl groups i n the r e s p e c t i v e rRNA s p e c i e s . There a r e about 95 pseudouri d i nes per human ribosome. Few of t h e s e pseudouridines have yet been l o c a t e d i n p u b l i s h ed primary s t r u c t u r e s of 18s o r 28s rRNA. This i s the g r e a t e s t unsolved problem a t the level of primary s t r u c t u r e determi,nation of rRNA from eukaryotes. Locating the pseudouridines w i l l provide i n s i g h t i n t o t h e r o l e of t h i s i n t r i g u i n g c l a s s of modified nucleotides i n the maturation and function of rRNA. Very r e c e n t l y we have succeeded i n c o r r e l a t i n g most of the pseudouridine-containing o l i g o n u c l e o t i d e s i n r e f . 42 w i t h s p e c i f i c l o c a t i o n s i n the mammal ian 18s sequence (Maden and Wakeman, manuscript i n preparat i o n ) . Further experiments d i r e c t e d toward l o c a t i n g the pseudouridines a r e i n progress i n our l a b o r a t o r y . 18s rRNA analyzed from a v a r i e t y of e u k a r y o t i c s p e c i e s c o n t a i n s N4-acetylcytidine i n an apparent y i e l d of 1.4 mol per mol of rRNA ( r e f . 66). A t t h e time of w r i t i n g this compound has not been l o c a t e d i n the 18s sequence.

,

X

x

9

x ;}x

x

x

x 9 x i l x

xxx ; z x

0.5kb

x x x x x x x x @X X X @ X X @ X X x x x x x x x x x x x x x x x } x @ x x x l x x l

x

Figure 8.6. General distribution of methyl groups in X . l a e v i s 28s rRNA. Methyl groups were identified as oligonucleotides within rRNA regions that hybridize between the indicated restriction sites in rDNA (ref. 54 . The analysis shows a heavy cluster of methyl groups in the 3 ’ region of 28s rRNA. T e precise locations o f individual methyl grou s within the indicated regions are currently under analysis (unpublished data of B.E.H.M.! Reprinted by permission from Nature, Vol. 288, No. 5788, pp. 293-296. Copyright (c) 1980; Macmi 1 1 i an Journals Limited

b

B293

8.6.3

Methvlation S i t e s and Conformation: 18s rRNA How do the modified nucleotides f i t i n t o the conformation of rRNA? This question has been addressed i n two ways f o r the methylated nucleotides in 18s rRNA. The f i r s t approach r e l a t e s t o secondary structure modelling. A number of attempts have been made t o construct secondary s t r u c t u r e models f o r eukaryotic 18s rRNA t o which the various published primary structures can be accommodated (e.g. r e f s . 60, 67-69). The various models agree i n many of the proposed interactions although some differences i n detail remain t o be resolved. In most cases the locations of the However, the X e n o p u s model methyl groups were n o t considered. ( r e f . 60) includes the methyl groups. The 5 ' one third of the molecule i s believed t o form a functional domain, i n which various segments from nucleotides 2253 i n t e r a c t w i t h t r a c t s between nucleotides 440 and 617 ( i n the X e n o p u s numbering system), the whole region forming an array of local hairpin arms and 1 onger range interactions. The model brings together many of the 2'-O-methylation s i t e s i n the f i r s t 620 nucleotides i n t o a rather complex conformational core. (Revisions are currently being made t o d e t a i l s o f the model i n ref. 60 t o allow a b e t t e r f i t w i t h the human 18s sequence: B . E . H . Maden, i n preparation.) The second approach involves d i r e c t p r o b i n g of the accessib i l i t y of specific features of rRNA t o enzymic or chemical probes, particularly w i t h methyl-labelled rRNA as substrate. Simple conformational hypotheses f o r rRNA methylation m i g h t envisage t h a t the methylation s i t e s are i n exposed regions of the molecule. Khan and Maden explored t h i s p o s s i b i l i t y some years ago, before the primary structure of 18s rRNA had been determined, by p r o b i n g the a c c e s s i b i l i t y of methylated sequences i n HeLa c e l l rRNA t o m i l d digestion by the single strand s p e c i f i c nuclease S,. An i n i t i a l s t u d y on 5.8s rRNA ( r e f . 70) showed t h a t the loop containi n g the principle methylation s i t e (see above) i s highly suscept i b l e t o S, nuclease digestion. The r e s u l t s from 18s rRNA were more complicated. After m i l d predigestion w i t h S, nuclease some o f the methylated o l igonucleot i d e s were almost eliminated from subsequent fingerprints whereas others were recovered i n good yields and had evidently n o t been

B294

a c c e s s i b l e t o S, nuclease ( r e f . 7 1 ) . In p a r t i c u l a r , the base methylation s i t e s were highly s u s c e p t i b l e t o S, nuclease, i n accord w i t h the f a c t t h a t t h e s e methylations occur l a t e d u r i n g ribosome maturation; hence t h e s e s i t e s would be expected t o be i n exposed l o c a t i o n s i n rRNA (and i n nascent ribosomes). By contrast, only some of t h e 2'-O-methylation s i t e s were s u s c e p t i b l e t o S, nucl e a s e whereas o t h e r s were r e s i s t a n t and were presumably "buried" i n the conformation. Re-examination of the S, d a t a i n the l i g h t of knowledge of t h e l o c a t i o n s of the methyl groups i n the primary s t r u c t u r e gives an i n d i c a t i o n a s t o which methylated regions a r e i n a c c e s s i b l e t o S, nuclease. The i n a c c e s s i b l e methylation s i t e s i n c l u d e several i n the 5 ' domain. For example the ol i gonucl e o t i des encompassing t h e methyl groups a t p o s i t i o n s 428, 462 and 468 (Fig. 8.5) in t h e human sequence (corresponding t o p o s i t i o n s 393, 427 and 433 i n X e n o p u s , F i g . 8.4) a r e i n a c c e s s i b l e t o S, nuclease, whereas those encompassing p o s i t i o n s 484, 512 and 517 i n the human sequence ( p o s i t i o n s 449, 477 and 482 i n X e n o p u s ) a r e in exposed regions. T h i s d i f f e r e n t i a l e f f e c t could not have been p r e d i c t e d from the secondary s t r u c t u r e model ( r e f . 60), which is e s s e n t i a l l y twodimensional. The d a t a suggest t h a t t h e r e a r e t e r t i a r y intera c t i o n s i n t h i s region whereby t h e methyl groups a t p o s i t i o n s 428, 462 and 468 a r e buried i n mature rRNA. A f u l l e r account of t h e s e c o r r e l a t i o n s i s i n preparation (B.E.H.M., unpublished d a t a ) . Further e x p l o r a t i o n s with conformational probes should a f f o r d unique o p p o r t u n i t i e s f o r continuing t o unravel the s t r u c t u r a l organization of rRNA, w i t h special r e f e r e n c e t o the p o s i t i o n s and c o n t r i b u t i o n s of the modified nucleotides i n the rRNA a r c h i t e c t u r e and the r i bosome assembly process. 8.6.4

Closinq Comments The thrust of t h i s account has been t o o u t l i n e how f i n d i n g s from successive experimental approaches have c o n t r i b u t e d t o the unfolding of knowledge of the modified n u c l e o t i d e s i n rRNA from man and o t h e r eukaryotes. The a n a l y s i s i s unfinished, b u t the avai 1 a b l e d a t a can be summari zed and provisional general i z a t i o n s can be drawn.

B295

Among t h e many thousands of n u c l e o t i d e s i n 45s rRNA, about 200 ( i n v e r t e b r a t e s ) a r e recognized and modified, probably by a l i m i t e d number of s p e c i f i c enzymes. All of the methylations and probably a l l of the o t h e r modifications occur i n t h e ribosomal sequences of the precursor molecule, and most of them occur i n regions where primary s t r u c t u r e i s highly (but not a b s o l u t e l y ) conserved among eukaryotes. The d i v e r s i t y of sequences which a r e 2'-O-methylated suggests t h a t t h e s p e c i f i c i t y f o r t h i s ty p e of A t least methylation i s determined by conformation ( r e f . 32). some of the methylations seem t o be i n regions where conformation may be more complex than i s evident from standard secondary s t r u c t u r e model s. Perhaps small i n t e r s p e c i e s v a r i a t i o n s between primary and hence t e r t i a r y s t r u c t u r e may determine the presence o r absence of p a r t i c u l a r methyl a t i on s i t e s . Given t h a t 2'-O-methylation i s i n i t i a t e d upon nascent 45s RNA ( r e f . 15), i t w i l l be of i n t e r e s t t o discover how l a r g e a region of t h e RNA i s required t o generate the conformation t h a t i s recognized by t h e methylase. Although h i t h e r t o i t has only been p o s s i b l e t o c o n j e c t u r e upon the r e l a t i o n s h i p between rRNA methylat i o n , f o l d i n g , and o t h e r i n t e r a c t i o n s d u r i n g ribosome maturation, i t may soon become p o s s i b l e t o approach these q u e s t i o n s d i r e c t l y by u s i n g recombinant DNA t o produce i n i t i a1 l y unmodi f i ed rRNA t r a n s c r i p t s ( r e f . 72), and then u s i n g t h e t r a n s c r i p t s i n assays f o r secondary modification and f u r t h e r s t e p s i n ribosome product i o n . T h i s approach i s c u r r e n t l y being explored i n our laboratory. 8.7

SUMMARY

Ribosomes of man and o t h e r v e r t e b r a t e s contain more than 200 modified n u c l e o t i d e s . In p a r t i c u l a r , ribosomes from human c e l l s contain about 110 2'-0-methylated n u c l e o t i d e s , a small number of base-modified nucleotides and about 95 pseudouridine r e s i d u e s . Approximately 80 of t h e modified n u c l e o t i d e s occur i n 18s rRNA i n the small ribosomal subunit; f o u r occur i n 5.8s rRNA and t h e rest (about 130) a r e i n 28s r R N A i n t h e l a r g e ribosomal s u b u n i t . All of t h e 2'-0-methyl groups a r e added t o ribosomal precursor RNA i n the nucleolus soon a f t e r t r a n s c r i p t i o n . Available d a t a suggest t h a t many o r a l l of the pseudouridine modifications a l s o t a k e

B296

p l a c e on r i b o s o m a l p r e c u r s o r RNA. Some o f t h e base m o d i f i c a t i o n s o c c u r l a t e d u r i n g r i b o s o m e m a t u r a t i o n . The e x a c t l o c a t i o n s o f a l l t h e m e t h y l groups i n t h e p r i m a r y s t r u c t u r e o f 18s r R N A f r o m X e n o p u s 7 a e v i s and man have been d e t e r m i n e d . The m e t h y l groups are widely b u t non-uniformly d i s t r i b u t e d , w i t h a major c l u s t e r o f 2'-O-methyl groups i n a s e c t i o n o f t h e 5 r e g i o n o f t h e m o l e c u l e w h i c h shows h i g h p h y l o g e n e t i c sequence c o n s e r v a t i o n . Some o f t h e s e m e t h y l groups a r e i n sequence t r a c t s w h i c h a r e r e l a t i v e l y i n a c c e s s i b l e t o S, n u c l e a s e , s u g g e s t i n g t h a t t h e y a r e b u r i e d w i t h i n complex t e r t i a r y s t r u c t u r e . P a r t i a l d a t a on t h e l o c a t i o n s o f t h e m e t h y l g r o u p s i n 28s rRNA a l s o i n d i c a t e c l u s t e r i n g i n p h y l o g e n e t i c a l l y c o n s e r v e d sequences, w i t h a m a j o r c l u s t e r i n t h e 3 ' r e g i o n o f t h e m o l e c u l e . O n l y a few p s e u d o u r i d i n e s i n rRNA have so f a r been l o c a t e d . C o m p l e t i o n o f t h e mapping o f t h e m o d i f i e d n u c l e o t i d e s i s t h e o u t s t a n d i n g problem i n t h e t o t a l d e t e r m i n a t i o n o f t h e p r i m a r y s t r u c t u r e o f r R N A f r o m human and o t h e r e u k a r y o t i c sources. A t t a i n m e n t o f t h i s o b j e c t i v e w i l l be a m a j o r s t e p towards understanding t h e h i g h l y s p e c i f i c m o l e c u l a r r e c o g n i t i o n p r o c e s s e s i n v o l v e d i n t h e m o d i f i c a t i o n s , and t h e i r b i o l o g i c a l r o l e i n r i b o s o m e b i o s y n t h e s i s and f u n c t i o n .

8.8

ACKNOWLEDGEMENTS I t h a n k a l l my f o r m e r c o l l e a g u e s who have c o n t r i b u t e d t o i m p o r t a n t p a r t s o f t h e work d e s c r i b e d i n t h i s c h a p t e r : i n part i c u l a r M. S a l i m , M.S.N. Khan and F.S. McCallum. The a u t h o r ' s work was s u p p o r t e d b y g r a n t s f r o m t h e M e d i c a l Research C o u n c i l . 8.9 1.

2. 3. 4.

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J . D. S m i t h and D. B. Dunn, An a d d i t i o n a l s u g a r

component o f r i b o n u c l e i c acids, Biochim. Biophys. Acta, 3 1 (1959) 573-575. P. R. W h i t f i e l d , A method f o r t h e d e t e r m i n a t i o n o f n u c l e o t i d e sequence i n p o l y r i b o n u c l e o t i d e s , Biochem. J . , 5 8 (1954) 390-396. R. H. H a l l , Method f o r i s o l a t i o n o f 2 ' - O - m e t h y l r i b o n u c l e o s i d e s and Nl-methyladenosine f r o m r i b o n u c l e i c a c i d , B i o c h i m . B i o p h y s . Acta, 6 8 1963) 278-283. H. S i n g h and B. Lane The s e p a r a t i o n , e s t i m a t i o n , and c h a r a c t e r i z a t i o n o f a1 k a l i - s t a b l e o l i g o n u c l e o t i d e s d e r i v e d r ib o n u c l e a t e p r e p a r a t i o n s , Canadian J . f r o m commerci a1 Biochem., 42 (1964) 87-93.

di .

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R. N. Nazar, T. 0. S i t z and H. Busch, S t r u c t u r a l a n a l y s i s o f mammal ian ribosomal r i bonucl e i c a c i d and i t s p r e c u r s o r s . N u c l e o t i d e sequence o f ribosomal 5 . 8 s r i b o n u c l e i c a c i d , J . B i o l Chem., 250 (1975 8591-8597. M. S . N. Khan and B.E. !I Maden, N u c l e o t i d e sequence r e l a t i o n s h i p s between v e r t e b r a t e 5 . 8 s ribosomal RNAs, Nucl. Acids Res., 4 (1977) 2495-2505. P. J . Ford and T. Mathieson, The n u c l e o t i d e sequences o f 5 . 8 s ribosomal RNA from X e n o p u s l a e v i s and X e n o p u s b o r e a l i s , Eur. J . Biochem., 87 (1978) 199-214. V . A. Erdmann and J . Wolters, C o l l e c t i o n of p u b l i s h e d 5S, 5 . 8 s and 4 . 5 s ribosomal RNA sequences, Nucl Acids Res., 14, Su p l ement (1986) r l - r 5 9 . B. E. Maden and J. S . Robertson, Demonstration o f t h e 5 . 8 s r i bosomal sequence i n HeLa c e l l ribosomal p r e c u r s o r RNA, J . Mol B i o l , 87 (1974 227-235. J . F. Morrow, S . N. ohen, A. C. Y. Chang, H. W . Boyer, H. M. Goodman and R. B. H e l l i n g , R e p l i c a t i o n and t r a n s c r i p t i o n o f eukar o t i c DNA i n E s c h e r i c h i a c o l i , Proc. Nat. Acad. S c i . U.S.A.. $1 (1974) 1743-1747. P.K. Wellauer, 1:B. Dawid, D. D. Brown and R: H. Reeder The m o l e c u l a r b a s i s f o r l e n t h h e t e r o e n e i t i n ribosomaf DNA from X e n o u s l a e v i s , J . Mo?. B i o l . , 185 (19$6) 461-486 B. E. H. kaden Meth l a t i o n map o f X e n o p u s l a e v i s ribosomal RNA, Nature, 2b8 (1986 293-296 R.L. Gourse and S . A. k e r b i FiAe s t r u c t u r e o f ribosomal RNA. 111. L o c a t i o n s o f e v o l u t i o n a r i l y conserved r e g i o n s w i t h i n ribosomal DNA, J . Mol B i o l , 140 (1980) 321-339. R. C. Brand and S . A. Gerbi, F i n e s t r u c t u r e o f ribosomal RNA. 11. D i s t r i b u t i o n o f m e t h y l a t e d sequences w i t h i n X e n o p u s l a e v i s rRNA, Nucl. Acids Res., 7 (1979) 1497-1511. M. S a l i m and B. E. H. Maden, N u c l e o t i d e sequence o f X e n o p u s gene sequence, l a e v i s 18s ribosomal RNA i n f e r r e d from Nature, 291 (1981) 205-208.

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Human 18s ribosomal RNA sequence i n f e r r e d from DNA se uence. V a r i a t i o n s i n 18s sequences and secondary modi c a t i o n atterns between v e r t e b r a t e s , B i ochem. J. , 232 (1985 725-73g B. E. H. Maden, I d e n t i f i c a t i o n o f t e l o c a t i o n s o f t h e methyl groups i n 18s ribosomal RNA from X e n o p u s 7 a e v i s and man, J. Mol. B i o l ., 189 (1986) 681-699. J. F. Connaughton, A . R a i r k e r , R. E. Lockard and A. Kumar, Primary s t r u c t u r e o f r a b b i t 18s ribosomal RNA b d i r e c t se uence a n a l y s i s , Nucl Acids Res. , 12 1984) 4731-4f45 Veldman, J. K l o o t w i j k , V.C.H.F. de e g t and R.J. P l a n t a r The p r i m a r y and secondary s t r u c t u r e o f y e a s t 26s rRNA, Nucl. Acids Res., 9 1981 6935-6952. D.G. Hughes an6 B. H. Maden, The p s e u d o u r i d i n e c o n t e n t s o f t h e ribosomal r i b o n u c l e i c a c i d s o f t h r e e v e r t e b r a t e species: numerical correspondence between p s e u d o u r i d i n e r e s i d u e s and Z’-O-methyl roups i s n o t always conserved, Biochem. J., 171

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Note added i n press. Since t h i s c h a p t e r was s u b m i t t e d t h e work on t h e l o c a t i o n s o f t h e 28s methyl g r o u s and on t h e p s e u d o u r i d i n e s .in 18s. rRNA, mentioned i n S e c t i o n s 8.g.l and 8.6.2, has been p u b l i s h e d i n t h e f o l l o w i n g references:

73. 74.

L o c a t i o n s o f methyl groups i n 28s r R N A of clusterin i n t h e conserved c o r e of t h e molecule, J . Mol B i o l , 2 0 1 (!!988) 289-314 B.E.H. Maden and J . A. Wakeman, Pseudouridine d j s t r i b u t i o n i n mammalian 18s ribosomal RNA- a m a j o r c l u s t e r i n t h e c e n t r a l r e g i o n o f t h e molecule, Biochem. J . , 249 (1988) 459-464. B. E. H . Maden,

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CHAPTER 9 MODIFIED URIDINES I N THE F I R S T POSITIONS OF ANTICODONS OF TRNAS AND MECHANISMS OF CODON RECOGNITION S H I G E Y U K I YOKOYAMA and TATSUO MIYAZAWA Department o f Biophysics and Biochemistry, Faculty o f Science, University o f Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan

TABLE OF CONTENTS 9.1 Introduction 9.2 M a t e r i a l s and Methods . . . . . . . . . . . . . . . . . 9.2.1 M o d i f i e d N u c l e o t i d e s and N u c l e o s i d e s f r o m E . c o l i tRNAs 9.2.2 Chemically Synthesized Nucleosides and Nucleotides . . . . . . . . . . . . . . . . . . . . . 9.2.3 Proton NMR Spectroscopy . . . . . . . . . . . . 9.2.4 Conformati on Analyses . . . . . . . . . . . . . 9.3 R e s u l t s and D i s c u s s i o n 9.3.1 The P u c k e r i n g Equi 1 ib r i um o f Ribose Ring i s A f f e c t e d by t h e Two Types o f M o d i f i c a t i o n s of Uridine . . . . . . . . . . . . . . . . . . . . 9.3.2 S t a b i l i z a t i o n o f t h e C2‘-Endo Form i n pxo5U 9.3.3 S t a b i l i z a t i o n o f t h e C 3 ’ - E n d o Form i n pxm5s2U 9.3.4 Conformations o f M o d i f i e d U r i d i n e Residue i n t h e F i r s t P o s i t i o n of t h e A n t i c o d o n as Base-Paired w i t h t h e T h i r d L e t t e r o f Codon 9.3.5 C o r r e c t . a n d E f f i c i e n t Codon R e c o g n i t i o n by t h e Regul a t 1 on o f R i g i d i t y / F 1 e x i b i 11t y o f A n t i codon 9.4 Summary.. 9.5 F u t u r e Prospects and Impact 9 . 6 Acknowledgments . . . . . . . . . . . . . . . . . . . . 9.7 References.

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INTRODUCTION I n p r o t e i n b i o s y n t h e s i s , some t R N A s p e c i e s s t r i c t l y r e c o g n i z e o n l y one codon by Watson-Crick base p a i r s , w h i l e o t h e r t R N A s p e c i e s r e c o g n i z e two o r more synonymous codons i n t h e g e n e t i c code t a b l e . I n t h e m u l t i p l e r e c o g n i t i o n o f codons by t R N A species, wobble base p a i r s , as w e l l as Watson-Crick A - U and G - C

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pairs, are probably formed between the third letters of codons and the first letter of anticodon (ref. 1). Post-transcriptional modifications in the first position of anticodons of tRNAs may play important roles in the codon recognition (ref. 2 ) . In fact, uridine in the first position of anticodon [U(34)] is usually modified and two major types of modified uridines have been found (ref. 2). As for tRNAs specific to Gln, Lys and Glu (for two codons terminating in A or G), U(34) is always modified to a 5-methyl-2-thiouridine derivative (xm5s2U, Figure 9.1), e . g . 5-methyl aminomethyl-2-thiouridine (mnm5s2 U) in E s c h e r i c h i a co7 i tRNAs and 5 - m e t h o x y c a r b o n y l m e t h y l - 2 - t h i o u r i d i n e (mcm5s2 U) i n eukaryoti c tRNAs (ref. 2 ) . In the tri pl et-dependent binding to ribosomes and in the i n v i t r o protein synthesis, xm5s2U(34) primarily recognizes A as the third letter of the codon and the recognition of G is much less efficient (refs. 3, 4). By contrast, in tRNAs specific to Val, Ser, Thr and Ala (for four codons terminating in U, C, A or G), U(34) is modified to 5hydroxyuridi ne derivatives (xo5U, Figure 9.1), e . g . 5-carboxymethoxyuridine (cmo5U) in E . c o 7 i tRNAVaL (refs. 5 - 7 ) and 5methoxyuridine (mo5U) in B a c i l l u s s u b t i 7 i s tRNAVaL,tRNAThr and tRNAALa (refs. 8, 9). In the triplet-dependent binding to ribosomes (refs. 9-13) and also in the i n v i t r o protein synthesis (refs. 14, 15), xo5U(34) recognizes U in addition to A and G in the third position of the codon (the recognition of U is slightly less efficient than the recognition of A and G). For elucidating the conformational aspects as involved in the two types of codon recognition by tRNA species, we have made NMR analyses of modified uridines. Thus the conformational characteristics of x05U nucleotides have been found to be distinct from those of xm5s2U nucleotides. xo5U is much more "flexible" and xm5s2U is much more "rigid" than unmodified uridine. Accordingly, the post-transcriptional modifications of U(34) result in the regulation of the flexibilitylrigidity of the anticodon of tRNA species and allow the efficient and correct translations of codons in protein biosynthesis.

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I ;5 0/ c \ N / c \ H I

5

u 5

ho U [X = H ] 5 m o U [X = CH3] 5 cmo U [X = CH2COO-]

Ribose

5 2 xmsu 5 2

m s U [X=H]

I

5 2 + mnm s U [X = NH2CH31 m c m 5 s 2U [ X = C O O C H 3 ]

Ribose

Figure 9 . 1 Chemical structures of xm5szU and xo5U. 9 . 2 MATERIALS AND METHODS 9.2.1 Modified Nucleotides and Nucleosides from E . c o l i tRNAs

(i) Nucleoside 5'-monophosphates. Unfractionated tRNA was prepared from the phenol extract of E . c o l i 413 cells by the method of Zubay (ref. 16). This tRNA preparation was digested by nuclease P 1 (Yamasa Shoyu Co., Ltd., Choshi, Japan). The digest was applied to a Dowex 1 column (3 x 80 cm, formate form) and eluted with a linear gradient of formic acid (from 0 to 3 M). The fractions containing 5 - m e t h y l a m i n o m e t h y l - 2 - t h i o u r i d i n e 5'-monophosphate (pmnm5s2 U) was spotted on Whatman 3MM paper and developed with the solvent system of isobutyric acid : 0.5 M NH,OH (5:3, v/v). pmnm5szU was extracted from the spot and further applied to a Dowex 50 column (1 x 20 cm, Na' form) and eluted with water. The second peak in the elution profile contained pmnm5szU only. The first peak was found, by proton NMR spectroscopy, to contain 5-methyl ami nomethyl uridi ne 5'-monophosphate (pmnm5U) , which was a novel natural ly-occurring component of tRNA. The amount of pmnm5 U was only 20% of that of pmnm5s2U.

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(ii) Nucleosides. tRNA fractions partially purified by column chromatography were digested with bovine pancreatic ri bonuclease (EC 3.1.27.5) (Sigma), snake venom phosphodiesterase I (EC 3.1.4.1) (Worthington Biochemicals), and E . c o l i phosphomonoesterase (EC 3.1.3.1) (Sigma). Modified nucleosides were frac tionated by high-performance 1 iquid chromatography (HPLC) with an ODs-80TM column (Toyo Soda Co. Ltd.). A Shimadzu LC-6A HPLC system in combination with a photodiode array detector MCPD-350PC (Ohtsuka Denshi Co. Ltd.) was used. Elution was performed using a linear gradient of methanol from 3% to 20% in ammonium formate buffer (2.5 mM, pH 5.1). 9.2.2 Chemicallv Svnthesized Nucleosides and Nucleotides (i) 2-Thiouridine derivatives. A chemically synthesized sample of mnm5s2U was a generous gift from Prof. T. Ueda. 55Methyl-2-thiouridine (m5s2U) was prepared as in ref. 18. Methyl -2-thiodeoxyuridine (m5 s2dU) was synthesized from 5-methyl deoxyuridine (m5 dU) by 5'-tosylation, 5',2-cycl ization, 3'-acyla2tion, 2-thiolation with H,S, and 3'-deacylation (ref. 17). Thiouridine (szU) and 2-thiouridine 5'-monophosphate (pszU) were kindly provided by Dr. S. Higuchi. ( i i ) 5 - H y d r o x y u r i d i n e d e r i v a t i v e s . 5-Hydroxyuridine (ho5U) and 5-hydroxyuridine 5'-monophosphate (pho5U) were purchased from PL Biochemicals. mo5U and cmo5U were synthesized by methylation and carboxymethylation, respectively, of ho5U (refs. 6, 9). 5Methoxyuridine 5'-monophosphate (pmo5U) and 5-carboxymethoxyuridine 5'-monophosphate (pcmo5U) were synthesized by the phosphorylation of mo5U and cmo5U, respectively. (iii) Other nucleosides and nucleotides. Uridine (U), uridine 5'-monophosphate (pU) and 5-methyldeoxyuridine (m5 dU) were purchased from Yamasa Shoyu Co. Ltd. 5-Methyluridine (m5U) and 5methyl uridine 5'-monophosphate (pm5U) were purchased from PL Biochemical s.

Proton- N M R SoectroscoDv (i) Sample solutions. Nucleosides and nucleotides were dissolved in ,H,O at a concentration of 1 mM or less. The proton chemical shifts were found to depend only slightly on the sample concentration, indicating that the effect of molecular association was negligible i n this concentration range. The pH of the sample solutions was adjusted below 5.5 (pH meter reading). Under this 9.2.3

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pH c o n d i t i o n , t h e phosphate groups of nucleotides a r e monoanionic. The conformations of ribose-phosphate moieties i n monoanionic form, r a t h e r than i n d i a n i o n i c form, a r e expected t o be s i m i l a r t o those i n the polynucleotide chain. For t h e measurements of Nuclear Overhauser E f f e c t s , t h e sample s o l u t i o n i n an NMR tube was t r e a t e d w i t h c h e l a t i n g agents [tri-n-octylphosphine oxide (TOPO) and 2-thenoyl t r i f 1 uoroacetone (TAA)] t o remove paramagnetic impurities. The s o l v e n t water was evaporated from the NMR tube and replaced by ’H,O (99.85%) t r a n s f e r r e d through a vacuum l i n e . After degassing the 2H20 s o l u t i o n , the NMR tube was f i l l e d w i t h dry nitrogen gas. ( i i ) P r o t o n NMR s p e c t r a . The proton NMR s p e c t r a of t h e s e sample s o l u t i o n s were recorded w i t h a Bruker WH270 spectrometer (270 MHz) and a Bruker AM400 spectrometer (400 MHz). The probe temperature was control 1 ed w i t h i n 1 degree. Chemical shi f t s were measured from the i n t e r n a l standard of sodium 4,4-dimethyl-4silapentane-1-sul f o n a t e . As f o r the r i b o s e protons, chemical s h i f t s and spin-coupling c o n s t a n t s were determined w i t h i n 0 . 1 Hz by the s p e c t r a l simulation w i t h a computer program NMRSIM ( r e f . 17). When some proton resonances o f n u c l e o t i d e overlapped a t l e a s t p a r t i a l l y , the proton NMR s p e c t r a of n u c l e o t i d e i n t h e absence and i n t h e presence of a s h i f t reagent [Eu(NO,),] were simul taneously simulated w i t h t h e same s e t of s p i n-coup1 i ng constants. S p i n - l a t t i c e r e l a x a t i o n r a t e s were measured by t h e inversion recovery method. Nuclear Overhauser E f f e c t s (NOE) were observed by t h e gated decoupling method.

9.2.4

Conformation Analyses The puckering equilibrium of t h e r i b o s e r i n g moiety was analyzed by t h e use of v i c i n a l spin-coupling c o n s t a n t s ; t h e f r a c t i o n a l populations of the C.2”-endo form and the C 3 ’ - e n d o form (Figure 9.2) were obtained from the formulas J l , 2 , / ( J , , 2 , + J 3 , 4 r ) and J,, / ( J 1 , +J, , 4 , ) , r e s p e c t i v e l y . From t h e temperature dependence of t h e equilibrium constant [CZ’-endo]/[C3’-endo], t h e enthal py and entropy d i f f e r e n c e s between t h e s e two forms were determined t o g e t h e r w i t h t h e i r standard d e v i a t i o n s . The rotamer equilibrium about t h e C5’-C4’ bond (Figure 9.2) was a l s o analyzed by t h e use of v i c i n a l coupling c o n s t a n t s (Hz); t h e f r a c t i o n a l populations of t h e g g , g t , and t g forms were obtained from the formulas ( 1 3 . 7 - J 4 , 5 , - J 4 , 5 , , ) / 9 . 7 , ( J 4 , 5 , , - 2 - 0 ) / 9 - 7 ,

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and (J,,,5,-2.0)/9.7, respectively (ref. 18). The s y n - a n t i equilibrium about the Cl'-Nl bond was analyzed by the use of NOE and spin-lattice relaxation rates. For such analyses of NOE and relaxation data, the computer program (COFLEM) originally developed for the conformation analyses by the lanthanoid-probe method (refs. 19, 20) was modified to an extended version. RESULTS AND DISCUSSION 9.3.1 The Puckerina Eauilibrium of Ribose Rina is Affected bv the Two T w e s of Modifications of Uridine 9.3

(i)

Conformation

analyses

of

the

two

types

of

modified

pm05 U (5-methoxyuri di ne 5'-monophosphate) and pmnm5 s2 U (5-methylaminomethyl-2-thiouridine 5'-monophosphate) are taken as representative examples of the two types of modified uridine nucleotides (px05 U and pxm5s2U), which are concerned with two distinct types of codon recognition patterns. The 270-MI-l~ proton NMR spectra of pmo5U and pmnm5s2U (Figure 9.3) and unmodified pU were analyzed, and vicinal coupling constants, NOE and spin-lattice relaxation rates were measured at 23°C. On the basis of these data, the fractional populations were obtained for local conformers, namely the s y n and a n t i forms about the Cl'-Nl' bond, the g g , g t and t g forms about the C5'-C4' bond, and the C 2 ' - e n d o and C 3 ' - e n d o forms for the ribose ring (Figure 9.2) (refs. 16, 21, 22). (ii) T h e r o t a m e r e q u i l i b r i a a b o u t t h e C 1 ' - N l and C 5 ' - C 4 ' bonds a r e s i m i l a r among p U , pmo5U and p m n m 5 s 2 U . For the three uridine nucleotides, the a n t i form about the glycosidic Cl'-Nl bond is predominant as shown in Table 9.1. Similarly, the g g form about the exocyclic C5'-C4' bond is predominant in these three uridine nucleotides [especially, in pmnm5s ~ U , the fractional populations of the a n t i and gg form are as high as loo%!]. The a n t i form and g g form are predominant in almost all the naturally occurring pyrimidine nucleotides (ref. 16; S. Yokoyama e t al., unpublished). Thus, the rotamer equilibria about the Cl'-Nl and C5'-C4' bonds are not significantly affected by the two types of modification of uracil base (ref. 22). uridine

nucleot ides.

(iii)

T h e C 2 ' - e n d o f o r m and C 3 ' - e n d o f o r m o f r i b o s e r i n g a r e

In unmodified pU, the equilibrium constant of ribose ring puckering, K = [ C Z ' - e n d o ] /

n e a r l y e q u a l l y s t a b l e i n u n m o d i f i e d pU.

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[C3'-endo],

temperature

i s n e a r l y equal t o one (Table 9 . 1 ) . F u r t h e r , from t h e dependence of e q u i l i b r i u m c o n s t a n t s K , the e n t h a l p y

H5'

H5"

99

kc2/ u

Cd'

G-

G'

H 2'

C5'

I

03'

C2 '-endo

02'

C3 '-endo Figure 9 . 2

\

anti

Local conformations of u r i d i n e n u c l e o t i d e ( r e f . 3 6 ) .

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(and e n t r o p y ) d i f f e r e n c e s between t h e C Z ’ - e n d o f o r m and t h e c 3 ‘ 9.2. The e n t h a l p y d i f f e r e n c e f o r u n m o d i f i e d pU i s s m a l l as 0.1 k c a l / m o l , i n d i c a t i n g t h a t t h e C Z ’ - e n d o f o r m and C 3 ’ - e n d o f o r m a r e n e a r l y e q u a l l y s t a b l e ( r e f . 21). ( i v ) pmo5U p r e f e r s t h e C 2 ’ - e n d o f o r m r a t h e r t h a n t h e C 3 ‘ endo f o r m . The e q u i l i b r i u m c o n s t a n t o f r i b o s e r i n g p u c k e r i n g i s s i g n i f i c a n t l y a f f e c t e d b y t h e t w o t y p e s o f m o d i f i c a t i o n s ; K i s as l a r g e as 1.9 i n pmo5U w h i l e K i s as s m a l l as 0.3 i n pmnm5s2U a t 23°C ( r e f . 21). F o r pmo5U, t h e e n t h a l p y d i f f e r e n c e between t h e CP’-endo f o r m and C 3 ’ - e n d o f o r m i s o b t a i n e d as -0.72 k c a l / m o l ( T a b l e 9.1); t h e C 2 ‘ - e n d o f o r m i s r e m a r k a b l y more s t a b l e t h a n t h e C3’-endo f o r m ( r e f . 21). For another pxo5U-type n u c l e o t i d e , pcmo5U, t h e e n t h a l p y d i f f e r e n c e has been o b t a i n e d as -0.67 k c a l / mol ( r e f . 21). These t w o a r e t h e o n l y n a t u r a l l y - o c c u r r i n g p y r i m i d i n e n u c l e o t i d e s t h a t have been f o u n d t o t a k e t h e C 2 ‘ - e n d o f o r m as t h e predominant conformer. pmnm5s2U e x c l u s i v e l y t a k e s t h e C 3 ’ - e n d o f o r m . By (v) c o n t r a s t , f o r pmnm5s2U, t h e e n t h a l p y d i f f e r e n c e i s as l a r g e as 1.1 k c a l / m o l , i n d i c a t i n g t h a t t h e C 3 ’ - e n d o f o r m i s r e m a r k a b l y more s t a b l e t h a n t h e C 2 ‘ - e n d o f o r m ( r e f . 21). The C 3 ’ - e n d o f o r m i s a l s o p r e d o m i n a n t ( t h e f r a c t i o n a l p o p u l a t i o n as h i g h as 78%) ( r e f . 16) i n 5-methoxycarbonylmethyl-2-thiouridine, an xm5 s2 U - t y p e n u c l e o s i d e as f o u n d i n t R N A s p e c i e s f r o m y e a s t and mammals ( r e f . 2). (vi) R e l a t i v e s t a b i l i t y o f t h e C 3 ’ - e n d o f o r m o v e r t h e C2‘e n d o f o r m . The t w o t y p e s o f m o d i f i c a t i o n s o f u r i d i n e i n t h e f i r s t p o s i t i o n o f anticodon o f tRNA w i l l s i g n i f i c a n t l y a f f e c t t h e p u c k e r i n g e q u i l i b r i a o f t h e r i b o s e r i n g m o i e t y . The e n t h a l p y d i f f e r e n c e between t h e C 2 ’ - e n d o f o r m and t h e C 3 ’ - e n d o f o r m ( t h e r e l a t i v e s t a b i l i t y o f t h e C 3 ’ - e n d o form) i n pmnm5s2U i s h i g h e r b y 1.8 k c a l / m o l as compared t o t h a t i n pmo5U. Then, how do t h e s e m o d i f i cations r e g u l a t e r i g i d i t y l f l e x i b i l i t y o f u r i d i n e nucleotides? For i n v e s t i g a t i n g such r e g u l a t i o n mechanisms, t h e c o n f o r m a t i o n a l p r o p e r t i e s o f a v a r i e t y o f u r a c i l n u c l e o s i d e s and n u c l e o t i d e s have been e x t e n s i v e l y a n a l y z e d ( T a b l e 9.2). 9.3.2 S t a b i l i z a t i o n o f t h e C 2 ’ - e n d o f o r m i n 0x051J (i) B u l k y 5 - s u b s t i t u e n t i s i m p o r t a n t f o r t h e s t a b i l i t y o f t h e C 2 ‘ - e n d o f o r m . The C 2 ‘ - e n d o f o r m i s much more s t a b l e t h a n t h e e n d o f o r m have been o b t a i n e d as shown i n T a b l e

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C3'-endo f o r m i n pmo5U and pcmo5U (Table 9.2); t h e e n t h a l p y d i f f e r e n c e between t h e C3'-endo form and C P ' - e n d o f o r m ( t h e

1

5 2 prnnm s u

4.4

4.2

4.0

prnnrn 5 s 2 U + Eu(II1)

rl I

4.4

2

I

4.2

4.0

3.8

Chemical shift ( ppm)

2

F i g u r e 9.3 P r o t o n NMR s p e c t r a o f pmnm5s2U (5mM (5mM) + Eu(N0 ) 3 (1 mM) i n 2 H z 0 s o l u t i o n a t pH 3. and and 2 observed specffra and (b) s i m u l a t e d s p e c t r a ( r e f . 16).

YnmsszU "C; (a)

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relative stability of C2'-endo form) is about 0.8 kcal/mol higher than that in pU. Such remarkable stability of the C 2 ' - e n d o form TABLE 9 . 1 Fractional Populations (%) of Local Conformers at 23°C and Enthalpy (kcal/mol) and Entropy (e.u.) Differences Between the CZ'-endo Form and the C3'-endo Formatb PU

C5'-C4' bond

gg

gt tg Ribose ring

89 8 3

C2'-endo 47 C3'-endo 53 H 0.09(0.02) S 0.69(0.07)

pmnm5 s2 U

pm05 u

100 0 0

91 7 2

22 78 l.lO(0.05) 1.27(0.17)

65 35 -0.72(0.02) -1.27(0.07)

a S t a n d a r d d e v i a t i o n s a r e g i v e n in p a r e n t h e s e s . b F r o m refs. 16, 2 1 , 2 2 .

may not be accounted for by the electron affinities of 5-substituents (ref. 23). Further, in an analog of pxosU with x = H (pho5U, Figure 9.1), the relative stability of the C 2 ' - e n d o form is much less significant (as small as 0.28 kcal/mol). This indicates that the -CH20- moiety of the 5-substituent is important f o r the stabilization of the C2'-endo form. The steric effects of such bulky 5-substi tuents are probably responsible for the remarkable stability of the C 2 ' - e n d o form in pxosU. (ii) Interaction between the 5-substituent and 5'-phosphate g r o u p is i m p o r t a n t f o r t h e s t a b i l i t y o f t h e C 2 ' - e n d o f o r m . The

role of the 5'-phosphate group in the conformational characteristics of pxo5U has been studied by the comparison of the conformational stability of 5'-mononucleotides with the corresponding nucleosides (ref. 21). As for 5'-mononucleotides other than pxo5U, the relative stability of the CP'-endo form is slightly

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h i g h e r (by 0.22-0.28 k c a l / m o l ) than t h a t o f c o r r e s p o n d i n g nucleos i d e s (Table 9 . 2 ) . For pmo5U and pcmo5U, however, t h e r e l a t i v e s t a b i l i t y o f t h e C 2 ’ - e n d o form i s remarkably h i g h e r (by 1.1-1.3 k c a l / m o l ) t h a n t h a t o f mo5U and cmo5U ( r e f . 21). T h i s i n d i c a t e s a s i g n i f i c a n t i n t e r a c t i o n between t h e 5 - s u b s t i t u e n t and 5’-phosphate group o f pxo5U. Here, t h e oxygen atom by i t s e l f (as i n pho5U) does n o t appear t o i n t e r a c t d i r e c t l y w i t h t h e 5’phosphate group. Probably, i n pxo5U, t h e methyl o r methylene group o f t h e 5-subs t i t u e n t i n t e r a c t s w i t h t h e 5’-phosphate group and s t a b i l i z e s t h e C 2 ’ - e n d o form. (iii) 5 - S u b s t i t u e n t i s i n p r o x i m i t y t o 5 ’ - p h o s p h a t e g r o u p i n pcm05u. The p o s s i b l e i n t e r a c t i o n between t h e 5 - s u b s t i t u e n t and t h e 5’-phosphate group has been examined by t h e o b s e r v a t i o n o f t h e pH dependences o f p r o t o n chemical s h i f t s o f cmo5U and pcmo5U. The PKa v a l u e o f t h e t e r m i n a l c a r b o x y l a t e group o f pcmo5U (3.3) i s TABLE 9.2 E n t h a l p y D i f f e r e n c e s (kcal/rnol) and Entropy D i f f e r e n c e s Between t h e C 2 ’ - e n d o Form and t h e C 3 ‘ - e n d o Forma Enthal py difference U

u cm05 u ho5 u m05

mnm5 s2 U m5 s2 U 52

u

m5 U PU pmo5 u pcmo5U pho5 u pmnm5 s2 U ps2 u pm5U

0.37 0.58 0.43 -0.01 1.32 0.98 1.12 0.16 0.09 -0.72 -0.67 -0.28 1.10 0.87 -0.11

(0.03) (0.02) (0.01) (0.04) (0.07) (0.02) (0.02) (0.02) (0.02) (0.02) (0.04) (0.06) (0.05) (0.03) (0.02)

Entropy difference

0.86 1.26 1.03 -0.01 1.70 1.28 1.61

0.35 0.69 -1.27 -1.36 -0.81 1.27 1.22 0.31

(0.08) (0.06) (0.02) (0.11) (0.21) (0.05) (0.08) (0.04) (0.07) (0.07) (0.12) (0.19) (0.17) (0.08) (0.06)

aStandard deviations are given i n parentheses.

(e.u.)

Ref. 31 31 31 31 31 18 31 18 31,33 31,33 31 31 31,33 31 31

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s i g n i f i c a n t l y higher than t h a t of cmo5U (2.9) ( r e f . 21). Such a d i f f e r e n c e of 0 . 4 pKa u n i t i s c e r t a i n l y due t o an e l e c t r o s t a t i c e f f e c t of the n e g a t i v e l y charged 5'-phosphate group. T h u s , the 5s u b s t i t u e n t (-OCH,CO,-group) of pcmo5U i s i n f a c t found t o be i n c l o s e proximity t o the 5'-phosphate group. (iv) Coplanar conformation o f t h e 5 - s u b s t i t u t e d u r a c i l base i n pxo5U. The 5 - s u b s t i t u e n t s of mo5U and cmo5U probably l i e i n t h e same plane a s the u r a c i l base (Figure 9.4a) a s found f o r mo5U and methyl e s t e r of cmo5U (mcmo5U) i n c r y s t a l ( r e f s . 24, 25). The coplanar o r i e n t a t i o n of the 5-substi tuent of mcmo5U has been a s c r i b e d t o the p a r t i a l double bond c h a r a c t e r of the C5-0 bond ( r e f . 24). An a l t e r n a t i v e coplanar o r i e n t a t i o n of the 5-subs t i t u e n t (Figure 9.4b) i s probably much l e s s s t a b l e , because of s t r o n g s t e r i c r e p u l s i o n between the 4-carbonyl group of the u r a c i l base and t h e methyl o r s u b s t i t u t e d methylene group o f the 5substituent. T h u s , the 5 - s u b s t i t u e n t s of pxo5U n u c l e o t i d e s a r e

Figure 9.4 Schematic drawing t u e n t of pxo5 U .

of t h e o r i e n t a t i o n of t h e 5 - s u b s t i -

f i x e d i n the coplanar o r i e n t a t i o n a s found f o r t h o s e of xo5U nucleosides. (v) C o n f o r m a t i o n a l a s p e c t o f t h e s t a b i l i t y o f C 2 ' - e n d o f o r m . Furthermore i n pxo5U, t h e a n t i form about the Cl'-Nl bond and the

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g g form about t h e C5’-C4’

bond have been found t o be predominant as shown i n Table 9.1. Because o f t h e c o p l a n a r o r i e n t a t i o n o f t h e 5 - s u b s t i t u e n t o f u r a c i l base i n t h e a n t i form and t h e g g o r i e n m o i e t y o f t h e 5t a t i o n o f t h e 5’-phosphate group, t h e -CH,Os u b s t i t u e n t i s b r o u g h t i n c l o s e p r o x i m i t y t o t h e 5’-phosphate group i n pxo5U ( F i g u r e 9.4a). The i n t e r a c t i o n between t h e 5s u b s t i t u e n t and 5’-phosphate group w i l l be d i f f e r e n t between t h e C Z ‘ - e n d o form and t h e C 3 ’ - e n d o form, p r o b a b l y because o f t h e d i f f e r e n c e i n t h e d i h e d r a l a n g l e about t h e C l ’ - N l bond between these two forms ( r e f . 18). Thus, t h e C P ‘ - e n d o form i s remarkably more s t a b l e than t h e C 3 ’ - e n d o form i n pxo5U. 9.3.3

S t a b i l i z a t i o n o f t h e C 3 ’ - e n d o Form i n ~xm552U (i) 2 - T h i o c a r b o n y l g r o u p p l a y s a p r i m a r y r o l e i n t h e s t a b i l -

The C 3 ’ - e n d o f o r m i s much more i z a t i o n o f t h e C3’-endo f o r m . s t a b l e than t h e C P ‘ - e n d o form i n pmnm5s2U; t h e e n t h a l p y d i f f e r e n c e between t h e C 2 ‘ - e n d o form and t h e C J ’ - e n d o f o r m ( t h e r e l a t i v e s t a b i l i t y o f C 3 ’ - e n d o form) i s about 1.10 k c a l h o l h i g h e r than f o r pU (Table 9.2) ( r e f . 21). Furthermore, t h e p u c k e r i n g e q u i l i b r i a o f m o d i f i e d u r i d i n e n u c l e o s i d e s and n u c l e o t i d e s have been extens i v e l y analyzed ( r e f s . 16-18, 21, 22). Thus, t h e r e l a t i v e s t a b i l i t i e s o f t h e C 3 ’ - e n d o form o f 2 - t h i o u r i d i n e d e r i v a t i v e s , s2U (2t h i o u r i d i n e ) , pszU and m5s2U (5-methyl-2-thiouridine) a r e found t o be h i g h e r by 0.8 kcal/mol than those o f c o r r e s p o n d i n g u r i d i n e d e r i v a t i v e s (U, pU and m5U) (Table 9.2). S i m i l a r l y , t h e C3’-endO form i s predominant i n s2C ( 2 - t h i o c y t i d i n e ) and ps2C as compared w i t h C and pC, r e s p e c t i v e l y ( r e f . 16). I n contrast, the conform a t i o n a l p r o p e r t i e s o f s4U ( 4 - t h i o u r i d i n e ) and ps4U a r e much t h e same as those o f U and pU, r e s p e c t i v e l y ( r e f . 16). These i n d i c a t e that the 2-thio substitution o f the pyrimidine r i n g primarily c o n t r i b u t e s t o t h e s t a b i l i t y o f t h e C 3 ’ - e n d o form i n pxm5s2U. (ii) P - T h i o c a r b o n y l g r o u p i s v e r y b u l k y . T h i o c a r b o n y l group i s much b u l k i e r t h a n t h e carbonyl group; t h e C=S bond (0.17 nm) i s l o n g e r than t h e C=O bond (0.12 nm) and f u r t h e r m o r e , t h e van d e r Waals r a d i u s o f t h e s u l f u r atom (0.185 nm) i s much l o n g e r t h a n t h a t o f t h e oxygen atom (0.14 nm). Because o f t h e s t e r i c r e p u l s i o n between t h i s b u l k y t h i o c a r b o n y l group and t h e 5’-methylene group o r t h e 5‘-phosphate group, t h e f r a c t i o n a l p o p u l a t i o n o f t h e a n t i form i s a p p r e c i a b l y h i g h e r (96%) i n pmnm5s2U t h a n t h a t (89%)

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i n pU (Table 9.1) ( r e f . 22). The p o p u l a t i o n o f t h e a n t i form i s I n t h e a n t i form o f a l s o much h i g h e r i n m5s2U than i n m5U. xm5s2U, t h e 2 - t h i o c a r b o n y l group i s i n p r o x i m i t y o f t h e 2’-hyd r o x y l group o f t h e r i b o s e m o i e t y ( F i g u r e 9.5). (iii) T h e r e m a r k a b l e s t a b i l i t y o f t h e C 3 ‘ - e n d o f o r m is primarily d u e to t h e steric effect between t h e P-thiocarbonyl g r o u p a n d t h e 2 ’ - h y d r o x y l g r o u p . The abundance r a t i o s [ C 3 ’ e n d o ] / [ C Z ’ - e n d o ] i n m5s2U and m 5 U have been o b t a i n e d t o g e t h e r w i t h t h o s e o f corresponding 2’-deoxy analogs (m5s2dU and m5dU) ( r e f . 17). As f o r deoxyribunucleosides, m5 s2dU and m5 dU, t h e [ C 3 ’ - e n d o ] /[cP’e n d o ] r a t i o s (0.7 and 0.6, r e s p e c t i v e l y ) a r e n e a r l y t h e same. However, f o r r i b o n u c l e o s i d e s , m5szU and m5U, 2 - t h i o s u b s t i t u t i o n d r a s t i c a l l y a f f e c t s t h e conformation equi 1 ib r i a o f t h e r i b o s e r i n g ; t h e [ C 3 ’ - e n d o ] / [ C Z ’ - e n d o ] r a t i o o f m5s2U (2.7) i s much h i g h e r than t h a t o f m 5 U (1.1). T h i s i n d i c a t e s c l e a r l y t h a t t h e remarkable s t a b i l i t y o f C 3 ’ - e n d o form i s due t o t h e s t e r i c i n t e r a c t i o n between t h e b u l k y 2 - t h i o c a r b o n y l group and t h e 2 ’ - h y d r o x y l group ( F i g u r e 9.5) ( r e f . 1 7 ) . ( i v ) 5 - S u b s t i t u e n t a l s o c o n t r i b u t e s t o t h e s t a b i l i t y o f C3‘e n d o f o r m in x m 5 s 2 U . The r o l e o f t h e 5 - s u b s t i t u e n t i n xm5s2U n u c l e o t i d e s has been s t u d i e d from t h e a n a l y s i s o f t h e conforma t i o n a l e q u i l i b r i a o f t h e r i b o s e r i n g . Thus, t h e r e l a t i v e s t a b i l i t y o f t h e C 3 ’ - e n d o form o f mnm5s2U and pmnm5s2U a r e h i g h e r , by 0.2 kcal/mol, than those o f s2U and ps2U, r e s p e c t i v e l y (Table 9.2) ( r e f . 21). T h i s i n d i c a t e s t h a t t h e 5-methyl ami nomethyl group a1 so c o n t r i b u t e s t o t h e s t a b i l i t y o f t h e C 3 ’ - e n d o form. However, t h e p o s i t i v e charge o f t h e 5-methylaminomethyl group does n o t appear t o be i m p o r t a n t , s i n c e t h e u n i o n i z e d methoxycarbonylmethyl group o f mcm5s~Ua l s o c o n t r i b u t e s t o t h e s t a b i l i t y o f t h e C 3 ’ - e n d o f o r m ( r e f . 21). (v) L o n g - c h a i n 5 - s u b s t i t u e n t is r e q u i r e d f o r t h e s t a b i l i z a t i o n o f t h e C 3 ’ - e n d o f o r m . I t should be noted h e r e t h a t t h e 5methyl group s l i g h t l y s t a b i l i z e s t h e C 2 ‘ - e n d o f o r m r a t h e r than t h e C 3 ’ - e n d o form. As shown i n Table 9.2, t h e r e l a t i v e s t a b i l i t y o f t h e C 3 ’ - e n d o form i n m5s2U, m 5 U and pm5U a r e lower, by about 0.2 kcal/mol, than those i n s ~ U , U and pU, r e s p e c t i v e l y . Thus, longc h a i n 5 - s u b s t i t u e n t s such as t h e methylaminomethyl and methoxycarbonylmethyl groups a r e r e q u i r e d f o r t h e s t a b i l i z a t i o n o f t h e C3‘-endo form i n xm5s2U. The e x t r a o r d i n a r y s t a b i l i t y o f t h e C 3 ’ -

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Figure 9.5 Schematic drawings of t h e C 2 ' - e n d o form and form of m5s2U ( a ) and of m5s2dU (b) ( r e f . 1 7 ) .

C3'-endo

endo form i n x m 5 s 2 U i s due t o the c o l l a b o r a t i v e e f f e c t s of t h e 2thiocarbonyl group and the long-chain 5 - s u b s t i t u e n t . ( v i ) E f f e c t s o f 5 - s u b s t i t u e n t s a r e d i f f e r e n t b e t w e e n xm5 s 2 U and X O ~ U . The 5 - s u b s t i t u e n t s i n xm5s2U f u r t h e r s t a b i l i z e the C 3 ' e n d o form, whereas the 5 - s u b s t i t u e n t s i n xo5U s t a b i l i z e the C 2 ' endo form. Note t h a t t h e 5 - s u b s t i t u e n t (YCH,O- group) of the u r a c i l r i n g i n xo5U t a k e s a "coplanar" o r i e n t a t i o n . In c o n t r a s t , the methyl ami nomethyl chain i n m n m 5 s2 U i s o r i e n t e d "perpendi cul a r " In g e n e r a l , chain t o the u r a c i l r i n g plane ( r e f s . 26, 2 7 ) . s u b s t i t u e n t s having a methylene group d i r e c t l y bound t o an aromatic r i n g a r e found t o t a k e a perpendicular o r i e n t a t i o n ( r e f . 28). In such a conformation of m n m 5 s 2 U , s t e r i c e f f e c t s a r e not s i g n i f i c a n t between the 5-methylaminomethyl group and the 5 ' phosphate group ( r e f . 2 6 ) . Therefore, the c o n t r a s t between the e f f e c t s of the two types of 5 - s u b s t i t u e n t s a r i s e s from t h e d i f f e r e n c e i n the o r i e n t a t i o n o f the 5-substi tuents re1 a t i v e t o the uracil ring.

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9.3.4

Conformations o f M o d i f i e d U r i d i n e Residues i n t h e F i r s t P o s i t i o n o f t h e Anticodon as Base-Paired w i t h t h e T h i r d L e t t e r o f Codon (i) C o n f o r m a t i o n s o f m o d i f i e d U ( 3 4 ) a s b a s e - p a i r e d w i t h t h e From t h e analyses o f t h e c o n f o r m a t i o n s o f t h i r d l e t t e r o f codons. m o d i f i e d u r i d i n e s i n t h e f i r s t p o s i t i o n o f anticodons o f t R N A , t h e e f f e c t s o f t h e two types o f m o d i f i c a t i o n s o f u r i d i n e on t h e codon r e c o g n i t i o n p r o p e r t i e s o f tRNAs have been e l u c i d a t e d . For t h e f o r m a t i o n o f non-Watson-Crick base p a i r s , t h e base(s) should be d i s p l a c e d from t h e l o c a t i o n f o r t h e f o r m a t i o n o f a Watson-Crick base p a i r . Thus, c o n f o r m a t i o n a l " f l e x i b i l i t y " i s r e q u i r e d f o r t h e r e s i d u e i n t h e f i r s t p o s i t i o n o f t h e a n t i c o d o n o f t R N A species t h a t r e c o g n i z e more than one codon. I n f a c t , t h e conformation models o f xo5U(34) as base-paired w i t h A and U ( F i g u r e 9.6) and w i t h G i n t h e t h i r d p o s i t i o n o f codon have been found on t h e b a s i s o f t h e unusual s t a b i l i t y o f t h e C2'-endo form i n pxo5U ( r e f . 21). (ii) x o 5 U ( 3 4 ) . A p a i r . The xo5U(34).A base p a i r i s o f t h e standard Watson-Crick t y p e ( F i g u r e 9.6a). The codon.anticodon complex w i l l t a k e t h e A-RNA conformation w i t h t h r e e Watson-Crick base p a i r s . I n f a c t , i n t h e c r y s t a l o f y e a s t t R N A P h e ( r e f s . 2931), t h e conformation o f anticodon i s s i m i l a r t o a s t r a n d o f A-RNA duplex. Thus, t h e xo5U(34) r e s i d u e w i l l t a k e t h e C 3 ' e n d o form o f t h e r i b o s e r i n g and t h e G - form about t h e C3'-03' bond as shown i n F i g u r e 9.6a. (iii) x o 5 U ( 3 4 ) - U p a i r . The codon on t h e ribosome i s s e t i n t h e A-type RNA conformation on t h e ribosome, and t h e r e f o r e f o r t h e f o r m a t i o n o f non-Watson-Crick base p a i r s , t h e base i n t h e f i r s t p o s i t i o n o f t h e a n t i c o d o n needs t o be d i s p l a c e d from t h e l o c a t i o n f o r Watson-Crick p a i r s ( r e f . 1). For t h e f o r m a t i o n o f t h e xo5U-U p a i r , t h e r e q u i r e d displacement o f t h e s u b s t i t u t e d u r a c i l r i n g i s achieved by t h e c o n f o r m a t i o n change from t h e G - f o r m t o t h e G+ form about t h e C3'-03' bond o f xo5U(34). However, t h e c o n v e r s i o n from t h e G - form t o t h e G+ form about t h e C3'-03' bond has t o be accompanied by t h e c o n v e r s i o n from t h e C 3 ' - e n d o f o r m t o t h e ~ 2 ' endo form o f t h e r i b o s e r i n g . Surprisingly, j u s t t h i s CZ'-endo-G+ form i s s u i t a b l e f o r t h e f o r m a t i o n o f a xo5U-U p a i r ( F i g u r e 9.6b) ( r e f . 21). ( i v ) x o 5 U ( 3 4 ) - G p a i r . Two d i s t i n c t c o n f o r m a t i o n models have been found f o r t h e non-Watson-Crick xo5U(34).G p a i r . In one

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model, xo5U(34) t a k e s t h e C3'-endo-G- form, w h i l e , i n t h e o t h e r model, xo5 U(34) t a k e s t h e C2- 'endo'G+ form. However, t h e r e l a t i v e arrangement of t h e C4' atom o f N(35) and t h e C 1 ' atom o f t h e n u c l e o t i d e i n t h e t h i r d p o s i t i o n o f t h e codon i s k e p t t h e same among t h e m o l e c u l a r conformations f o r t h e f o u r t y p e s o f base p a i r s ( c f . F i g u r e 9.6). Thus, t h e l o c a t i o n s o f t h e second and t h i r d n u c l e o t i d e s o f t h e a n t i c o d o n and t h e t h r e e n u c l e o t i d e u n i t s o f t h e codon a r e n o t a f f e c t e d b y t h e conversions among t h e s e f o u r t y p e s o f base p a i r s ( r e f . 21). On t h e o t h e r hand, s t a b l e c o n f o r m a t i o n This models have n o t been o b t a i n e d f o r t h e p a i r xo5U(34) and C. i s c o n s i s t e n t w i t h t h e o b s e r v a t i o n t h a t xo5U(34) does n o t recogn i z e C i n t h e t h i r d p o s i t i o n o f t h e codon. 9.3.5

C o r r e c t and E f f i c i e n t Codon R e c o s n i t i o n bv t h e R e s u l a t i o n o f R i a i d i t v / F l e x i b i 1it v o f A n t i codon (i) R o l e of the m o d i f i c a t i o n of U to x o 5 U . Four types o f m o l e c u l a r conformations have j u s t been discussed f o r t h e base p a i r s o f xo5U(34) w i t h t h e t h i r d l e t t e r o f codons. Recall t h a t t h e CZ'-endo form i s remarkably s t a b l e i n t h e xo5U u n i t i t s e l f ( r e f . 21). T h i s i n d i c a t e s t h a t xo5U(34) r e c o g n i z e s U ( F i g u r e 9.6b) and G by t a k i n g t h e C Z ' - e n d o - G + form, i n a d d i t i o n t o A by t a k i n g t h e C3'-endo-G- form ( F i g u r e 9.6a). Note t h a t xo5U(34) has been found e x c l u s i v e l y i n t R N A species s p e c i f i c t o amino a c i d s The w i t h f o u r synonymous codons t e r m i n a t i n g i n U, C, A and G. m o d i f i c a t i o n o f U t o xo5U makes t h e a n t i c o d o n " f l e x i b l e " , and t h u s a l l o w s t h e t R N A species t o r e c o g n i z e t h r e e codons t e r m i n a t i n g i n U, A and G. Thus, t h i s t y p e o f m o d i f i c a t i o n c o n t r i b u t e s t o e f f i c i e n t t r a n s l a t i o n s o f codons ( r e f s . 21, 22). (ii) R o l e of the m o d i f i c a t i o n o f U to x m 5 s 2 U . By c o n t r a s t , i n t h e o t h e r t y p e o f m o d i f i e d u r i d i n e (xm5s2U), t h e C3'-endo form i s e x c l u s i v e l y s t a b l e ( r e f . 21). Because o f t h i s i n t r i n s i c s t a b i 1 it y o f t h e C3 ' - e n d o form, xm5 s2 U(34) forms stab1 e WatsonC r i c k base p a i r s w i t h A i n t h e t h i r d p o s i t i o n o f t h e codon. However, xm5s2U(34) may n o t form a non-Watson-Crick base p a i r w i t h U i n t h e t h i r d p o s i t i o n o f t h e codon, because o f t h e remarkable i n s t a b i l i t y o f t h e CZ'-endo form i n t h e xm5s2U u n i t i t s e l f . A c c o r d i n g l y , t h e a n t i c o d o n m o i e t y i s made " r i g i d " and t R N A species b e a r i n g xm5sZU(34) p r i m a r i l y r e c o g n i z e codons t e r m i n a t i n g i n A. T h i s i s c o n s i s t e n t w i t h t h e o b s e r v a t i o n t h a t xm5s2U(34) has been

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P

Figure 9 . 6 Base pairs of xo5U(34) w i t h A ( a ) and U (b) ( r e f . 2 1 ) .

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found i n t R N A species s p e c i f i c t o Gln, Lys and G l u w i t h two synonymous codons t e r m i n a t i n g i n A o r G. The m o d i f i c a t i o n s o f U t o xm5 s2 U does n o t a1 low misrecogni t i o n o f codons t e r m i n a t i n g i n U, and t h u s c o n t r i b u t e s t o c o r r e c t t r a n s l a t i o n o f codons.

9.4

SUMMARY P r o t o n NMR analyses have been made o f t h e c o n f o r m a t i o n a l c h a r a c t e r i s t i c s o f m o d i f i e d n u c l e o t i d e s as found i n t h e f i r s t p o s i t o n o f t h e anticodons o f tRNAs, namely t h e d e r i v a t i v e s o f 5methyl - 2 - t h i o u r i d i n e 5'-monophosphate (pxm5s2U) and d e r i v a t i v e s o f 5-hydroxyuri d i ne 5 ' -monophosphate (pxo5 U) I n pxm5 s2 U, t h e c 3 ' endo form i s remarkably more s t a b l e t h a n t h e CZ'-endo f o r m f o r t h e r i b o s e r i n g , because o f t h e e f f e c t s o f t h e b u l k y 2 - t h i o c a r b o n y l group and l o n g - c h a i n 5 - s u b s t i t u e n t . By c o n t r a s t , i n pxo5Ur t h e CZ'-endo form i s much more s t a b l e t h a n t h e C 3 ' - e n d o form, because of t h e i n t e r a c t i o n between t h e 5 - s u b s t i t u e n t and 5'-phosphate group. The e n t h a l p y d i f f e r e n c e s between t h e C2'-endo f o r m and t h e C 3 ' - e n d o form have been o b t a i n e d as 1.1, -0.7, and 0 . 1 kcal/mol f o r pxm5s2Ur pxo5U and unmodified u r i d i n e 5'-monophosphate, r e s p e c t i v e l y . xm5s*U i n t h e f i r s t p o s i t i o n o f t h e a n t i c o d o n t a k e s o n l y t h e C 3 ' - e n d o form t o r e c o g n i z e A as t h e t h i r d l e t t e r o f t h e codon, whereas x05U t a k e s t h e C2'-endo form as w e l l as t h e c 3 ' e n d o f o r m t o r e c o g n i z e U, A and G as t h e t h i r d l e t t e r o f t h e codon on t h e ribosome. The b i o l o g i c a l s i g n i f i c a n c e o f t h e m o d i f i c a t i o n s o f U t o xo5U/xmSs2U i s i n t h e r e g u l a t i o n o f t h e c o n f o r m a t i o n a l f l e x i b i l i t y l r i g i d i t y i n t h e f i r s t p o s i t i o n o f t h e a n t i c o d o n so as t o guarantee t h e e f f i c i e n t and c o r r e c t t r a n s l a t i o n o f codons i n protein biosynthesis.

.

9.5

FUTURE PROSPECTS AND IMPACT The b i o l o g i c a l s i g n i f i c a n c e o f t h e two types o f m o d i f i c a t i o n s o f u r i d i n e ( t o xm5s2U and xo5u) i n p o s i t i o n 34 o f t R N A i s t o c o n t r i b u t e t o t h e c o r r e c t and e f f i c i e n t t r a n s l a t i o n o f codons, through the r e g u l a t i o n o f the r i g i d i t y l f l e x i b i l i t y o f t h e f i r s t l e t t e r o f t h e anticodon. T h i s concept may be g e n e r a l i z e d t o o t h e r types o f m o d i f i e d u r i d i n e s . When t h e two synonymous codons t e r m i n a t i n g i n A and G a r e recognized by a t R N A species, t h e modified u r i d i n e i n the f i r s t p o s i t i o n o f t h e anticodon i s predict e d t o be " r i g i d " .

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The long-chain 5 - s u b s t i t u e n t o f xm5s2U i s a l s o found t o contribute t o the " r i g i d i t y " o f the nucleotide u n i t t h a t i s I n fact, f o r r e q u i r e d f o r t h e e x c l u s i v e r e c o g n i t i o n o f A and G. tRNASrg from yeast, which recognizes codons AGA and AGG, 5-methoxycarbonylmethyluridine (mcm5U) has been found i n t h e f i r s t p o s i t i o n o f t h e a n t i c o d o n ( r e f . 32). Furthermore, a novel modif i e d u r i d i n e from E . coli t R N A has been i d e n t i f i e d as 5-methylaminomethyluridine (mnm5U) by p r o t o n NMR analyses (S. Yokoyama, Z. Yamaizumi, S. Nishimura and T. Miyazawa, unpublished r e s u l t s ) . Probably, t h e t R N A A r g species t h a t recognizes codons AGA and AGG has t h i s mnm5U i n t h e f i r s t p o s i t i o n o f t h e anticodon, as expected from t h e analogy t o y e a s t t R N A S r g species b e a r i n g mcm5U. F u r t h e r , i n y e a s t m i t o c h o n d r i a , 5-carboxymethylaminomethyluridine (cmnm5U) has been found i n tRNA species t h a t recognizes two synonymous codons t e r m i n a t i n g i n A and G ( r e f . 33). These l o n g - c h a i n 5s u b s t i t u e n t s commonly have a methylene group d i r e c t l y a t t a c h e d t o t h e u r a c i l r i n g , and t h e r e f o r e a r e expected t o p r e v e n t t h e misr e c o g n i t i o n o f codons t e r m i n a t i n g i n U ( o r C) through t h e enhancement o f c o n f o r m a t i o n a l r i g i d i t y o f t h e anticodon. " R i g i d " m o d i f i e d u r i d i n e s have a l s o been found i n some tRNAs s p e c i f i c t o amino a c i d s w i t h f o u r synonymous codons, i n c l u d i n g 5carboxyme t hy 1 ami nomethyl u r i d i ne (cmnm5 U) in t RNAC y from 6. s u b t i l i s ( r e f . 34) and 5-carbamoylmethyluridine (ncm5U) i n t R N A V a L from y e a s t ( r e f . 35). These m o d i f i e d u r i d i n e s a r e p o s s i b l y i n v o l v e d i n t h e r e g u l a t i o n o f p r o t e i n s y n t h e s i s , through t h e d i s c r i m i n a t i o n o f t h e m a j o r codons and m i n o r codons among t h e f o u r synonymous codons. The s t e r i c i n t e r a c t i o n between t h e 2 - t h i o c a r b o n y l group and t h e 2 ' - h y d r o x y l group o f xm5s2U i s now found t o make t h e nucleot i d e u n i t extremely r i g i d . However, i f t h e 2-carbonyl group o f t h e u r a c i l r i n g i s n o t s u b s t i t u t e d by a 2 - t h i o c a r b o n y l group, t h e 2 ' - 0 - m e t h y l a t i o n o f t h e r i b o s e r i n g has a l s o been found t o s t a b i l i z e t h e C3'-endo form o f r i b o s e r i n g , because o f t h e s t e r i c i n t e r a c t i o n w i t h t h e 2-carbonyl group o f t h e u r a c i l base ( r e f s . 22, 36). F u r t h e r , t h e C3'-endo form i s a l s o r e p o r t e d t o be predominant i n t h e Z ' - o - m e t h y l c y t i d i n e (Cm) r e s i d u e o f CmpC ( r e f . 37). I t may be noted t h a t Cm i s found i n t h e f i r s t p o s i t i o n o f t h e a n t i c o d o n o f t R N A M e t (codon AUG), t R N A T r p (codon UGG) and

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tRNALeU (codons UUA and UUG). Such a modification of C will make the first letter of the anticodon rigid, and probably prevents the misrecognition of U (and/or' C). Recently, 2'-o-methyluridine (Um) has been found in the first position of tRNACLn species from T e t r a h y m e n a and mammals that recognize codons UAA/UAG or CAA/CAG (refs. 38, 3 9 ) . Probably, this Z'-O-methylation also contributes to the correct codon recognition by enhancing the conformational rigidity of this residue. Further, a novel modified uridine has been found i n the first position of anticodon of tRNALeu (codons UUA and UUG); this nucleotide is conformationally rigid because of the collaborative effects of the 2'-O-methylation and 5-substi tution (S. Yokoyama e t a / . , unpublished results). Two eukaryotic tRNALeU species that possibly recognize codons UUA and UUG also have 2'-O-methylated derivatives of uridine and cytidine, respectively (ref. 40). All these observations may be explained on the basis of our general pri nci pl e, In conclusion, the general concept of the regulation of rigiditylflexibility of the first letter of an anticodon by posttranscriptional modifications o f tRNA will certainly be useful for understanding the molecular mechanisms of the regulation of codon recognition in protein synthesis. This concept may also be extended to other types of modifications of nucleic acids. ACKNOWLEDGMENTS The authors are grateful to Dr. Susumu Nishimura and Dr. Ziro Yamaizumi of National Cancer Center Research Institute and Prof. Hisayuki Ishikura and Dr. Katsutoshi Murao o f Jichi Medical School for discussions and to Prof. Toru Ueda of Hokkaido University and Dr. Shigesada Higuchi of Mitsubishi Kasei Institute of Life Science for gifts of nucleosides. This work was supported, in part, by a Grant-in-Aid for Specially Distinguished Research (No. 60060004) from Ministry of Education, Science and Culture of Japan and a grant from Toray Science Foundation. 9.6

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H. s h i k u r a , Y. Yamada and S. N i s h i m u r a , S t r u c t u r e o f s e r i n e t R N A f r o m E s c h e r i c h i a c o l i . I. P u r i f i c a t i o n o f s e r i n e tRNA's w i t h d i f f e r e n t codon responses, B i o c h i m . B i o p h y s . Acta, 228 (1971) 471-481. K. Oda, F. Kimura, F. Harada and S. N i s h i m u r a , R e s t o r a t i o n o f Val i n e a c c e p t o r a c t i v i t y b y c o m b i n i n g 01 i g o n u c l e o t i d e f r a g ments d e r i v e d f r o m a B a c i l l u s s u b t i l i s r i b o n u c l e a s e d i g e s t o f E s c h e r i c h i a c o l i v a l i n e t r a n s f e r RNA, B i o c h i m . B i o p h y s . A c t a ,

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K. T a k e i s i, T. Takemoto, S. N i s h i m y r a and T. U k i t a , S e l e c t i v e u t i l i z a t i o n o f valyl-tRNA having a a r t i c u l a r coding s p e c i f i c i t y i n a r a b b i t h e m o g l o b i n s y n t l e s i z i n g system, Biochem. B i o p h y s . Res. Commun., 47 (1972) 746-752. T. Takemoto, K. T e k e i s h i , S. N i s h i m u r a and T. U k i t a , T r a n s f e r o f Val ine i n t o r a b b i t hemogl o b i n f r o m v a r i o u s is o a c c e p t i ng s p e c i e s o f v a l y l - t R N A d i f f e r i n g i n codon r e c o g n i t i o n , Eur. J . Biochem., 38 (1973) 489-496. S.K. M i t r a , F. L u s t i g , B. Akesson, T . Axberg, P. E l i a s and U. La e r k v i s t , R e l a t i v e e f f i c i e n c y o f a n t i c o d o n s i n r e a d i n t h e v a y i n e codons d u r i n p r o t e i n s y n t h e s i s i n v i t r o , J . i i o l . Chem., 254 (1979) 6387-6401.

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15. T . Samuelsson, P. Elias, F. Lustig, T . Axber G. Fdlsch, B. Akesson and U. Lagerkvist, Aberrations of t k classic codon reading scheme durin protein synthesis i n v i t r o , J. Biol. Chem., 255 (1980) 458!-4588. 16. S. Yokoyama, Z. Yamaizumi, S. Nishimura and T . Miyazawa, 1H NMR studies on the conformational characteristics of 2thiopyrimidine nucleotides found i n transfer RNAs, Nucleic Acids Res., 6 1979 2611-2626. 17. Y. Yamamoto, YoLoyama, T . Miyazawa K. Watanabe and S . Higuchi, NMR analyses on the molecuiar mechanism of the conformational rigidity of 2-thioribothymidine, a modified nucleoside i n extreme thermophile tRNAs, FEBS Lett., 157 (1983) 95-99. 18. K. Watanabe, S. Yokoyama, F. Hansske, H. Kasai and T . Miyazawa, CD and NMR studies on the conformational thermostability of 2-thioribothymidine found in the T C loop of thermo hile tRNA, Biochem. Biophys. Res. Commun., 91 (1979) 671-677 19. S. Yokoiama, F. Ina aki and T . Miyazawa, Advanced nuclear magnetic resonance ganthanide probe analyses of short-range conformational interrelations control lin ribonucleic acid structures, Biochemistr , 20 (1981) 2981-9988 20. S. Yokoyama, T. Oida, Uesugi, M. Ikehara and T. Miyazawa, NMR 1 anthanoid-probe anaiyses of conformational properties of 8,ZI-c cloadenosine 3'-monophos hate in aqueous solution, Bull. fhem. SOC. Japan, 56 (1983p 375-378. 21. S. Yokoyama, T . Watanabe, K. Murao, H. Ishikura, Z. Yamaizumi , S. Nishimura and T . Miyazawa, Molecular mechanism of codon recognition by tRNA species with modified uridine in the first position of the anticodon, Proc. Natl. Acad. Sci. USA, 82 (1985) 4905-4909. 22. S. Yokoyama and T . Miyazawa, Molecular conformations and codon recognition of transfer ribonucleic acids as anal zed by nuclear magnetic resonance, J. Mol . Structure, 126 (1685) 563-572. 23. W. Uhl, J . Reiner and H. G. Gassen, On the conformation of 5substituted uridines as studied b proton magnetic resonance, Nucleic Acids Res., 11 1983) 1167-1180 24. K. Morikawa, K. Torii, . Iitaka, M. Tsiboi and S. Nishimura, Crystal and molecular structure of the methyl ester of uridin-5-oxyacetic acid: A minor constituent of E s c h e r i c h i a c o l i tRNAs, FEBS Lett., 48 1974) 279-282. 25. W. Hillen, E . Egert, H . . Lindner, H. G. Gassen and H. Vorbruggen, 5-Methoxyuridine: the influence of 5-substituents on the keto-enol tautomerism o f the 4-carbonly group, J. Carb. Nucleos. Nucleot., 5 (1978) 23-32. 26. W. Hillen, E. Egert, H. J . Lindner and H. G. Gassen, Restriction or am lification of wobble recognition. The structure and the interaction of of 2-thio-!-methylaminomethyluridine odd uridines with the anticodon loop backbone, FEBS Lett., 94 1978) 361-364. 27 . Kasai, S . Nishimura, H. Vorbruggen and Y. Iitaka, Crystal and molecular structure of the acetonide of 5-methylaminomethyl-2-thiouridine: A minor constituent of E s c h e r i c h i a c o I i tRNAs , FEBS Lett. , 103 (1979) 270-273.

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CHAPTER 10 NATURALLY OCCURRING MODIFIED NUCLEOSIDES I N DNA MELANIE EHRLICH and XIAN-YANG ZHANG Department o f Biochemistry, Tulane Nedical School, New Orleans, LA 70112, U . S . A .

TABLE 10.1 10.2 10.3

10.4 10.5

10.6 10.7 10.8

OF CONTENTS Introduction H i g h l y M o d i f i e d Bacteriophage DNA M o d i f i e d Bases i n DNA f r o m B a c t e r i a and Lower Eukaryotes . . . . . . . . . . . . . . . . . . . . . . . . . Meth l a t i o n o f t h e DNA o f M i t o c h o n d r i a , C h l o r o p l a s t s , and h k a r y o t i c V i r u s e s D i s t r i b u t i o n o f m5C i n t h e N u c l e a r DNA o f H i g h e r P l a n t s and V e r t e b r a t e s The F u n c t i o n a l S i g n i f i c a n c e o f V e r t e b r a t e DNA Meth 1a t i o n : T r a n s c r i p t i o n , Chromatin S t r u c t u r e , DNA R e p r i c a t i o n and Repair, Cancer and Embryogenesis Summary References

. . . . . . . . . . . . . . . . . . . . . B327 . . . . . . . . . . 8328 8332

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INTRODUCTION To s t a t e t h a t DNA i s composed o f o n l y f o u r bases, adenine (A) t thymine (T), guanine (G) and c y t o s i n e (C) i s an o v e r - s i m p l i f i c a t i o n because most t y p e s o f DNA n a t u r a l l y c o n t a i n one t o t h r e e a d d i t i o n a l bases, m o d i f i e d forms o f A o r C. U s u a l l y , o n l y a m i n o r p o r t i o n o f t h e C o r A r e s i d u e s i s m o d i f i e d ( r e f s . 1-3). The e x c e p t i o n s a r e t h e DNA from c e r t a i n b a c t e r i o p h a g e s ( r e f . 4) and from some d i n o f l a g e l l a t e s ( r e f . 5 ) . One o r a n o t h e r o f a v a r i e t y o f m o d i f i e d p y r i m i d i n e s a r e found i n t h e s e phage DNAs and 5h y d r o x y m e t h y l u r a c i l i n d i n o f l a g e l l a t e DNA. I n c o n t r a s t , t h e o n l y m o d i f i e d bases found i n a1 1 o t h e r DNAs a r e 5-methyl cytosine (rn5C), N4-methylcytosine (m4C), and N6-methyladenine (m6A). The f u n c t i o n s o f t h e s e DNA m o d i f i c a t i o n s a r e d i v e r s e and range f r o m p r o t e c t i o n o f b a c t e r i a l h o s t DNA a g a i n s t a pathway f o r d e g r a d i n g f o r e i g n DNA t o c o n t r o l o f p r o k a r y o t i c and e u k a r y o t i c t r a n s c r i p t i o n a l a c t i v i t y .

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HIGHLY MODIFIED BACTERIOPHAGE DNA Some t y p e s of b a c t e r i a l viruses, although not most, have one of the DNA bases completely o r l a r g e l y replaced by a modified derivative (ref. 4). These highly modified phage DNAs almost always c o n t a i n pyrimidine m o d i f i c a t i o n s . The f i r s t of these t o be di scovered was 5-hydroxymethyl c y t o s i ne (hm5C) i n phage T2, T4, and T6 DNAs ( r e f . 6 ) . These e n t e r i c Escherichia c o y i phages, which were among the e a r l i e s t s u b j e c t s of i n t e n s i v e molecular b i o l o g i c a l i n v e s t i g a t i o n , a r e very highly modified, a f a c t which sometimes complicated t h e i r use a s experimental models. Not only do they have a hydroxymethyl group a t each C r e s i d u e , b u t a l s o , 75-100% o f t h e i r r e s u l t i ng hm5C r e s i d u e s a r e gl ucosyl a t e d ( r e f s . 7-9). The type of l i n k a g e of t h e glucose and t h e number of g l u c o s e m o i e t i e s per hm5C r e s i d u e depends upon t h e phage type ( r e f . 1 0 ) . In T2 phage DNA, 70% of t h e hm5C r e s i d u e s c o n t a i n an a-glucosyl moiety, 5% have a g e n t i o b i o s e (8-91 ucosyl -a-gl ucosyl) , and the rest a r e nonglucosylated. Phage T4 and T6 DNAs have the f o l l o w i n g d i s t r i b u t i o n s of t h e i r hm5C r e s i d u e s : T4, 70% a - g l u c o s y l a t e d , 30%-8glucosyl a t e d , and 0% nongl ucosyl a t e d ; T6, 3% 0-91 ucosyl a t e d , 72% 8-1,6-91 ucosyl -a-gl ucosyl a t e d ( g e n t i o b i o s e ) , and 25% nongl ucosyla t e d . The formation of t h e hydroxymethyl group i s c a t a l y z e d a t t h e deoxymononucl e o t i d e 1 eve1 by a phage-encoded dCMP hydroxymethylt r a n s f e r a s e p r i o r t o i n c o r p o r a t i o n i n t o the DNA ( r e f . 1 1 ) . The s y n t h e s i s of this phage DNA e x p l o i t s t h e h o s t ' s r e s o u r c e s by degrading h o s t DNA and dCTP and converting t h e r e s u l t i n g recycled dCMP a s well a s de nova synthesized dCMP t o hm5dCTP. This phosphorylation involves a phage-encoded deoxynucleoside monophosphate kinase and a host diphosphate kinase a s well a s the phage hydroxymethyl t r a n s f e r a s e ( r e f . 1 1 ) . A f t e r i n c o r p o r a t i o n of the hm5dCMP r e s i d u e s , the gl ucosyl a t i o n of t h e hydroxymethyl groups i nvol ves hmT-speci f i c a- and 8-gl ucosyl t r a n s f e r a s e s , which have some sequence s p e c i f i c i t y ( r e f . 1 2 ) . This g l u c o s y l a t i o n i s normally necessary t o prevent t h e degradation of these phage DNAs by E . c o l i nucleases a s well a s by phage-encoded n u c l e a s e s meant t o d e s t r o y h o s t DNA ( r e f s . 13-16). I f dCMP r e s i d u e s a r e i n c o r p o r a t e d i n t o phage DNA, the DNA w i l l be degraded by the T4-encoded endonuclease I 1 ( r e f . 1 6 ) . Therefore, hydroxymethyl a t i o n and glucos y l a t i o n of c y t o s i n e r e s i d u e s i n T-even phage p l a y an important 10.2

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r o l e i n t h e phage l i f e c y c l e by p r o t e c t i n g phage DNA f r o m degradat i o n w h i l e a l l o w i n g h o s t DNA t o be s p e c i f i c a l l y r e c o g n i z e d f o r such h y d r o l y s i s . I t a l s o f a c i l i t a t e s p h a g e - s p e c i f i c t r a n s c r i p t i o n l a t e i n i n f e c t i o n c a t a l y z e d by a phage-modified h o s t RNA p o l y merase ( r e f s . 4, 17, 18). L i k e many o t h e r types o f b a c t e r i o p h a g e DNAs i n c l u d i n g t h o s e which a r e n o t h i g h l y m o d i f i e d ( r e f s . 19, 20), T4 DNA c o n t a i n s m i n o r amounts o f m6A ( r e f . 21). T h i s m e t h y l a t i o n o f A r e s i d u e s i s c a t a l y z e d by a T4-induced, sequence-specific DNA methyl t r a n s f e r a s e ( r e f . 21). The p h y s i o l o g i c a l s i g n i f i c a n c e o f m i n o r amounts o f m6A o r m5C i n phage DNAs remains t o be d e t e r mined. E x t e n s i v e hydroxymethyl a t i o n occurs a t a n o t h e r p y r i m i d i n e r e s i d u e i n a s e t o f c l o s e l y r e l a t e d phage DNAs which a r e widespread i n s o i l ( r e f . 22). A few p a r t i a l l y homologous phages t h a t i n f e c t Bacillus subtilis, SPO1, ue, SP8, SP82G, 2C, and SP5, have t h e T r e s i d u e s i n t h e i r genomes c o m p l e t e l y r e p l a c e d by 5-hydroxym e t h y l u r a c i l (hm5U) r e s i d u e s ( r e f s . 11, 22) which a r e nonglucosy1 ated. L i k e t h e T-even phages, t h e s e phages hydroxymethylate a t t h e mononucleotide l e v e l , i n t h i s case, w i t h dUMP as t h e s u b s t r a t e . The r e a c t i o n i s s i m i l a r l y c a t a l y z e d by a phage-induced hydroxymethylase ( r e f . 23). To p r e v e n t i n c o r p o r a t i o n of dTTP, t h e phages encode a dTTPase ( r e f . 24) as w e l l as an i n h i b i t o r o f t h e h o s t b a c t e r i u m ' s t h y m i d y l a t e s y n t h e t a s e ( r e f . 2 3 ) . Channeling of p y r i m i d i n e s t o s y n t h e s i s o f hm5dUTP i s f a c i l i t a t e d by two phageinduced enzymes, a dCMP deaminase and a deoxynucleoside monophosphate k i n a s e ( r e f s . 4, 23). I n h i b i t i o n o f h o s t DNA r e p l i c a t i o n occurs a f t e r i n f e c t i o n i n i t i a l l y as t h e r e s u l t o f a phage-induced i n h i b i t o r o t h e r than t h e i n h i b i t o r o f t h y m i d y l a t e s y n t h e t a s e ( r e f . 22). U n l i k e t h e case f o r t h e T-even phages, h o s t DNA i s n o t e x t e n s i v e l y degraded and t h e hm5U-containing B . s u b t i l i s phages can have as much as 20% o f t h e i r hm5U r e p l a c e d by T w i t h o u t l o s i n g t h e i r v i a b i l i t y ( r e f . 25). Nonetheless, t r a n s c r i p t i o n i n t h e m i d d l e and l a t e stages o f t h e i n f e c t i o u s c y c l e r e q u i r e s hm*Uc o n t a i n i n g phage DNA sequences as w e l l as a phage-encoded regul a t o r y p r o t e i n r e p l a c i n g one of t h e h o s t RNA polymerase's sigma f a c t o r s ( r e f . 26). I n a d d i t i o n , a DNA-binding p r o t e i n which b i n d s s p e c i f i c a l l y t o c e r t a i n hm5U-containing sequences i s s y n t h e s i z e d ( r e f . 27).

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Another t y p e o f B. subtilis phage has a d i f f e r e n t s u b s t i t u e n t r e p l a c i n g t h e T r e s i d u e s of i t s DNA, namely u r a c i l (U) r e s i d u e s . J u s t as T r e s i d u e s a r e commonly found i n RNA ( t R N A ) a l t h o u g h t h e y a r e m o s t l y a s s o c i a t e d w i t h DNA, so U r e s i d u e s can be a normal c o n s t i t u e n t o f DNA. To date, g e n e t i c a l l y programmed f o r m a t i o n o f U r e s i d u e s i n DNA has been found o n l y i n phage PBSl and i t s v e r y c l o s e l y r e l a t e d d e r i v a t i v e PBS2 ( r e f s . 28, 29). PBS2 has been shown t o encode a dTMP 5'-phosphatase, which h e l p s p r e v e n t T r e s i d u e s from b e i n g i n c o r p o r a t e d i n t o phage DNA ( r e f . 30), and a dUMP kinase, which t o g e t h e r w i t h a h o s t n u c l e o s i d e diphosphate k i n a s e p r o v i d e s dUTP f o r phage DNA s y n t h e s i s . The phage d u r i n g i t s l i f e c y c l e has t o cope w i t h h o s t mechanisms f o r e x c l u d i n g advent i t i o u s U r e s i d u e s from DNA, namely, a h o s t dUTPase and a h o s t uracil-DNA g l y c o s y l a s e ( r e f . 31). The l a t t e r enzyme e x c i s e s t h e n o r m a l l y small amount o f m i s i n c o r p o r a t e d dUTP (dUMP r e s i d u e s ) as w e l l as U r e s i d u e s a r i s i n g from spontaneously o c c u r r i n g heatinduced deamination o f C r e s i d u e s ( r e f s . 32, 33). To c o u n t e r a c t t h e a c t i v i t y o f t h e s e enzymes, phage i n f e c t i o n induces t h e synt h e s i s o f i n h i b i t o r s o f t h e DNA-uracil e x c i s i o n pathway and o f dUTPase ( r e f s . 31, 34, 35). Another phage t h a t i n f e c t s B. subtilis has a c o m p l i c a t e d and unique t y p e o f m o d i f i c a t i o n i n i t s DNA. Phage SP15 DNA c o n t a i n s 60% o f i t s T r e s i d u e s r e p l a c e d by 5 - ( 4 ' ,5'-dihydroxypenty1)uracil (hpsU) ( r e f . 36) i n which a f i v e - c a r b o n s i d e c h a i n w i t h two hydroxyl groups i s bonded t o t h e 5 p o s i t i o n o f t h e p y r i m i d i n e , t h e same p o s i t i o n m o d i f i e d i n a l l t h e above-mentioned p y r i m i d i n e s . Attached t o one o f t h e h y d r o x y l groups i s a glucose m o e i t y and t o t h e o t h e r v i a a phosphodiester l i n k a g e i s a phosphoglucuronate ( r e f . 37). T h i s g l u c u r o n i c acid-l-phosphate m o i e t y i n t r o d u c e s a phosphate t h a t i s n o t p a r t o f t h e DNA's phosphodiester backbone. I t c o n f e r s e x t r a n e g a t i v e changes on t h e DNA and i s a p p a r e n t l y responsi b l e f o r t h e a1 k a l ine 1a b i 1i t y and 1ow me1ti ng temperature o f t h i s DNA ( r e f . 38). SP15 DNA i s t h e o n l y known DNA s t a b l y c o n t a i n i n g phosphates t h a t a r e n o t p a r t o f t h e phosphodiester backbone. The d i s t r i b u t i o n o f hp5U versus T r e s i d u e s i n SP15 DNA i s .nonrandom. How hp5U, which o n l y partially r e p l a c e s T i n t h i s DNA, i s i n t r o d u c e d i n t o s p e c i f i c p o s i t i o n s remains unknown ( r e f . 37). Less w e l l d e f i n e d c h e m i c a l l y i s t h e h y p e r m o d i f i e d T d e r i v a -

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t i v e which p a r t i a l l y r e p l a c e s T i n t h e B. subtilis phage S P l O DNA. The s y n t h e s i s o f t h i s DNA i n v o l v e s a novel pathway i n which hm5U r e s i d u e s ( f r o m hm5dUTP) a r e i n c o r p o r a t e d i n t o t h e DNA and t h e n is pyrophosphorylated. Subsequently, 15% of i t l o s e s t h e pyrophosphate and i s converted t o t h e hypermodified T r e s i d u e s and t h e r e s t i s converted t o T r e s i d u e s ( r e f . 39). A s i m i l a r pathway i s encoded by phage dW14 t h a t i n f e c t s Pseudomonas acidovorans. The DNA o f t h i s phage has h a l f o f i t s T r e s i d u e s r e p l aced by 5- (4-ami n o b u t y l ami nomethyl ) u r a c i 1 (a-putresc i n y l t h y m i n e ; putT) which c o n f e r s a p o s i t i v e charge on t h e r e s i d u e ( r e f . 4 ) . T h i s base i s r e s p o n s i b l e f o r t h e higher-than-expected m e l t i n g temperature o f t h e DNA and i t s a l k a l i l a b i l i t y ( r e f . 40). Both putT and T i n dW14 DNA a r i s e from hm5dUTP. A f t e r dW14 i n f e c t i o n , t h e h o s t s u f f e r s t h e gradual l o s s o f dTTP and t h e appearance o f hm5dUTP c a t a l y z e d by a phage-induced enzyme ( r e f . 41). T h i s hm5dUTP i s i n c o r p o r a t e d i n t o t h e DNA and, a t t h e p o l y n u c l e o t i d e l e v e l , pyrophosphorylated ( r e f . 42). As f o r t h e T r e s i d u e s o f SPlO, T r e s i d u e s i n dW14 a r e formed a t t h e DNA l e v e l f r o m 5[(hydroxymethyl)-0-pyrophosphoryl] u r a c i 1 r e s i d u e s ( r e f s . 41, 42). The d i s t r i b u t i o n o f putT, which o n l y p a r t i a l l y r e p l a c e s i t s corresponding unmodified m a j o r base, i s s e q u e n c e - s p e c i f i c ( r e f . 43) as i n t h e case of hp5U i n SP15 DNA. Two more examples o f unusual bases i n h i g h l y m o d i f i e d phage DNAs have been r e p o r t e d . One i s a C d e r i v a t i v e , 2,5-dihydroxy-4ami nopyrimi d i n e , (5-hydroxycytosi ne) , whi ch c o m p l e t e l y r e p l aces C i n t h e DNA o f phage N-17, whose h o s t i s Shigella flexneri ( r e f . 44). The o t h e r i s t h e o n l y r e p o r t e d example o f a h i g h l y m o d i f i e d DNA c o n t a i n i n g as a m a j o r base a p u r i n e d e r i v a t i v e . T h i s i s found i n cyanophage S-2L, which i n f e c t s t h e b l ue-green a l g a e S y n e c h o c o c c u s e l o n g a t u s . I t has been i d e n t i f i e d as 2-aminoadenine and c o m p l e t e l y r e p l a c e s A ( r e f . 45). The o n l y o t h e r example o f extens i v e , b u t n o t major, replacement o f a p u r i n e i n DNA i s found i n E . c o l i phage Mu DNA. T h i s phage encodes t h e c o n v e r s i o n o f 15% o f i t s A r e s i d u e s t o a-N-(9-~-0-2'-deoxyri b o f u r a n o s y l p u r i n-6-y1)glycinamide i n which an acetamido group i s on t h e N-6 o f adenine ( r e f . 46). A l l o f t h e above-mentioned phage DNAs have as t h e i r main m o d i f i e d base a d e r i v a t i v e n o t commonly found i n o t h e r DNAs. Only

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one o f t h e s e , hm5U, has been f o u n d i n any DNA o t h e r t h a n t h a t o f phage (see b e l o w ) . There i s one example o f a h i g h l y m o d i f i e d DNA, phage XP12 DNA, whose m o d i f i e d base i s a m a j o r component o f t h i s phage DNA and a m i n o r component o f many o t h e r DNAs. Phage XP12 was d i s c o v e r e d b y Tsong-Teh Kuo g r o w i n g on t h e p l a n t p a t h o g e n X a n t h o m o n a s oryzae ( r e f . 47). XP12 DNA has e s s e n t i a l l y a l l o f i t s C r e s i d u e s r e p 1 aced b y 5-methyl c y t o s i n e (m5C) ( r e f . 48-50). T h i s base i s a l s o f o u n d as a m i n o r component o f v e r y many t y p e s o f DNAs (see b e l o w ) . The m5C r e s i d u e s g i v e XP12 DNA a m e l t i n g t e m p e r a t u r e h i g h e r t h a n expected; t h e r e f o r e , t h e 5-methyl a t i o n i n c r e a s e s t h e s t a b i l i t y o f t h e h e l i x ( r e f . 49). The s o u r c e o f t h e m e t h y l g r o u p f o r XP12 D N A ' s m5C r e s i d u e s (and f o r T r e s i d u e s , i n g e n e r a l ) i s t h e 3-carbon o f s e r i n e r a t h e r t h a n t h e t h i o m e t h y l c a r b o n o f methi o n i n e as i n t h e f o r m a t i o n o f m5C as a m i n o r base i n DNA ( r e f . 49). M e t h y l a t i o n o f t h e C r e s i d u e s f o r XP12 DNA o c c u r s a t t h e m o n o n u c l e o t i d e l e v e l i n a r e a c t i o n c a t a l y z e d b y a phage-induced d e o x y c y t i d y l a t e m e t h y l t r a n s f e r a s e ( r e f . 51). XP12 i n f e c t i o n i n d u c e s t h e s y n t h e s i s o f a 5-methyldeoxyc y t i d i n e 5 ' - monophosphate k i n a s e , which, u n l i k e E . c o l i and X . oryzae m o n o n u c l e o t i d e k i n a s e s , can c a t a l y z e t h e p h o s p h o r y l a t i o n o f m5dCMP ( r e f . 52). The c o n v e r s i o n o f m5dCDP t o m5dCTP i s app a r e n t l y catalyzed by a r e l a t i v e l y n o n s p e c i f i c h o s t nucleoside d i p h o s p h a t e k i n a s e ( r e f . 52). I n t e r e s t i n g l y , XP12 u t i l i z e s t h e m5dCTP i n a u n i q u e pathway f o r p r o d u c t i o n o f T r e s i d u e s . I t i n d u c e s t h e f o r m a t i o n o f an mSdCTP deaminase t h a t g e n e r a t e s dTTP b y d e a m i n a t i o n o f p a r t o f t h e p o o l o f n e w l y c r e a t e d m5dCTP i n p h a g e - i n f e c t e d c e l l s ( r e f . 53). The phage a l s o i n d u c e s t h e synt h e s i s o f an e x o n u c l e a s e w i t h a p r e f e r e n c e f o r d o u b l e - s t r a n d e d DNA ( r e f . 5 4 ) . However, t h i s enzyme h y d r o l y z e s XP12 DNA and h o s t DNA e q u a l l y w e l l . T h i s c o n t r a s t s w i t h a h o s t e x o n u c l e a s e t h a t has a p r e f e r e n c e f o r s i n g l e - s t r a n d e d DNA and h y d r o l y z e s h o s t DNA much b e t t e r t h a n XP12 DNA ( r e f . 54, 55). Thus, some phage-induced enzymes i n v o l v e d i n m e t a b o l i s m o f h i g h l y m o d i f i e d phage DNAs may n o t have a s e l e c t i v i t y f o r t h e m o d i f i e d DNA, b u t r a t h e r may j u s t n o t be i n h i b i t e d by t h e m o d i f i c a t i o n . 10.3

MODIFIED BASES I N DNA FROM BACTERIA AND LOWER EUKARYOTES B a c t e r i a u s u a l l y c o n t a i n one o r more o f t h e f o l l o w i n g m e t h y l -

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ated bases i n t h e i r DNA: m6A, m5C, and t h e newly d i s c o v e r e d m4C ( r e f s . 3, 56-61). These m e t h y l a t e d bases u s u a l l y c o n s t i t u t e 0.010.7% o f t h e r e s i d u e s of t h e DNA, a l t h o u g h 2 mol% m6A i s p r e s e n t i n one b a c t e r i a l genome ( r e f s . 3, 61, 62). The r e l a t i v e frequency o f occurrence o f m i n o r amounts these m o d i f i e d bases i n b a c t e r i a l DNAs i s m6A > m5C > m4C ( r e f . 61). Also, i t i s o n l y m6A t h a t has been found a t l e v e l s > 0 . 4 mol% ( r e f s . 3, 61). These a r e t h e o n l y observed m o d i f i e d bases i n b a c t e r i a l DNA and a l t h o u g h t h e g r e a t m a j o r i t y o f s t u d i e d b a c t e r i a c o n t a i n a t l e a s t one o f them i n t h e i r genomes, some b a c t e r i a l DNAs have no d e t e c t a b l e m o d i f i e d bases ( r e f s . 3, 61). Much o f t h i s b a c t e r i a l DNA m e t h y l a t i o n i s p a r t o f r e s t r i c t i o n / m o d i f i c a t i o n systems ( r e f . 63). These i n v o l v e sequence-speci f i c adenine- o r c y t o s i ne-DNA methyl t r a n s f e r a s e s and corresponding sequence-speci f i c endonucl eases ( r e s t r i c t i o n endonucleases) R e s t r i c t i o n endonucleases general l y c l e a v e t h e same o l i g o n u c l e o t i d e recognized by t h e methyl t r a n s f e r a s e and w i 11 c l e a v e t h e sequence o n l y i n i t s unmethylated form. However, i n a t l e a s t one case, a r e s t r i c t i o n endonuclease c l e a v e s o n l y t h e a p p r o p r i a t e l y methylated r e c o g n i t i o n sequences r a t h e r than t h e unmethylated sequence ( r e f . 64). The restriction/modification systems s e r v e t o demarcate h o s t DNA as s e l f and a l l o w phage DNA which had i n f e c t e d a b a c t e r i a l s t r a i n w i t h a h e t e r o l o g o u s m o d i f i c a t i o n system o r o t h e r f o r e i g n DNA e n t e r i n g t h e b a c t e r i a l c e l l t o be recognized as n o n - s e l f and thereby, be degraded ( r e f s . 65, 66). However, t h e f u n c t i o n s o f t h e m o d i f i c a t i o n and r e s t r i c t i o n enzymes may not be l i m i t e d t o e x c l u s i o n o f f o r e i g n DNA. Also, some bact e r i a1 DNA methyl t r a n s f e r a s e s recogni ze s p e c i f i c DNA sequences f o r which no corresponding r e s t r i c t i o n enzyme e x i s t s i n t h e h o s t bacterium. The most w e l l s t u d i e d example o f t h e s e i s t h e DNA adenine m e t h y l a t i o n (dam) system o f E. coli which c o n v e r t s 5 ' GATC-3 ' sequences t o 5 ' -Gm6ATC-3 ' ( r e f . 67) Dam m e t h y l a t i o n d i r e c t s DNA mismatch r e p a i r as f o l l o w s ( r e f s . 68, 69). The r e c o g n i t i o n sequence i s a palindrome so t h a t n o r m a l l y b o t h s t r a n d s o f t h e DNA a r e s y m m e t r i c a l l y m e t h y l a t e d a t 5'-GATC-3' s i t e s . However, immediately a f t e r DNA s y n t h e s i s o n l y one s t r a n d , t h e t e m p l a t e s t r a n d , i s m e t h y l a t e d ( r e f s . 70, 71). T h i s hemim e t h y l a t i o n a l l o w s t h e nascent s t r a n d t o be d i s t i n g u i s h e d from t h e t e m p l a t e so t h a t t h e DNA mismatch r e p a i r system can p r e f e r e n t i a l l y

.

.

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e x c i s e a m i s i n c o r p o r a t e d base f r o m t h e t e m p l a t e s t r a n d . W i t h o u t a means t o d i s t i n g u i s h t h e t w o s t r a n d s , t h e nonmutant base o f t h e t e m p l a t e s t r a n d a t a DNA mismatch w o u l d be j u s t as l i k e l y t o be e x c i s e d as i s t h e m u t a n t , m i s i n c o r p o r a t e d base o f t h e n e w l y s y n t h e s i z e d s t r a n d ( r e f s . 68, 69). I n a d d i t i o n t o p a r t i c i p a t i n g i n DNA r e p a i r , dam m e t h y l a t i o n i n E. c o l i has been i m p l i c a t e d i n c o n t r o l l i n g t r a n s c r i p t i o n o f a few genes and i n r e g u l a t i n g t r a n s p o s i t i o n and p o s s i b l y DNA r e p l i c a t i o n ( r e f . 72). Dam-type m e t h y l a t i o n has been f o u n d i n v a r i o u s e n t e r i c b a c t e r i a ( r e f . 73) and m i g h t o c c u r i n some o t h e r t y p e s o f b a c t e r i a a l t h o u g h i t i s c l e a r l y n o t a common phenomenon ( r e f s . 3, 61, 7 4 ) . I t i s i n t e r e s t i n g t o n o t e t h a t t h e s i n g l e - s t r a n d d i s c o n t i n u i t i e s a s s o c i a t e d w i t h t h e n a s c e n t s t r a n d d u r i n g DNA r e p l i c a t i o n appear t o s e r v e t h e mismatch r e p a i r d i r e c t i n g f u n c t i o n o f dam-hemimethyl a t i o n i n Streptococcus pneumoniae, whose DNA i s d e v o i d o f dam m e t h y l a t i o n ( r e f . 7 5 ) . L i ke b a c t e r i a, some 1ower e u k a r y o t e s c o n t a i n modi f ied bases i n t h e i r genomes a1 t h o u g h u n l ike b a c t e r i a no c o r r e s p o n d i n g r e s t r i c t i o n endonucl eases have been r e p o r t e d i n t h e s e o r g a n i s m s . T h e r e f o r e , m o d i f i c a t i o n o f l o w e r e u k a r y o t i c DNAs, 1 i k e t h a t o f v e r t e b r a t e and h i g h e r p l a n t DNAs, p r o b a b l y s e r v e s some m a j o r f u n c t i o n ( s ) o t h e r t h a n e x c l u s i o n o f f o r e i g n DNA o r v i r u s e s . U n i c e l l u l a r e u k a r y o t e s can have m6A as t h e o n l y m i n o r m o d i f i e d b a s e (Tetrahymena thermophila m a c r o n u c l e a r DNA: r e f . 76, 77; O x y t r i c h a f a 1 l o x m a c r o n u c l e a r DNA: r e f . 78; Paramecium a u r e l i a m i c r o n u c l e a r and m a c r o n u c l e a r DNA: r e f . 79), m5C as t h e o n l y m o d i f i e d base ( C h l o r e l l a : r e f . 80), m6A p l u s m5C (Chlamydomonas r h e i n h a r d i : r e f . 81) o r no d e t e c t a b l e DNA m o d i f i c a t i o n ( S a c charomyces c e r e v i s i a e : r e f . 82). I n some d i n o f l a g e l 1a t e s , t h e r e i s a phenomenon n o v e l among e u k a r y o t e s , t h e f o r m a t i o n o f a m o d i f i e d base o t h e r t h a n m5C o r m6A. D i n o f l a g e l 1 a t e DNA has 4-19 m o l % 5 - h y d r o x y m e t h y l u r a c i 1 ( hm5U) depending on t h e s p e c i e s ( r e f . 5). The p h y s i o l o g i c a l s i g n i f i c a n c e o f t h e occurrence i n these u n i c e l l u l a r eukaryotes o f a r a t h e r l a r g e amount o f a DNA base o t h e r w i s e r e s t r i c t e d t o c e r t a i n bact e r i o p h a g e genomes (see above) o r t o f o r m a t i o n as a DNA l e s i o n r e s u l t i n g f r o m r a d i a t i o n damage ( r e f . 83) r e m a i n s t o be d e t e r mined. I n a d d i t i o n t o hm5U, m i n o r amounts o f m5C have been f o u n d

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i n t h e DNA o f two d i n o f l a g e l l a t e species and m6A i n one ( r e f . 84). I n mu1 t i c e l l u l a r eukaryotes, t h e o n l y demonstrated m o d i f i e d base i n t h e DNA i s m5C. I t i s w i d e l y d i s t r i b u t e d among organisms. T h i s base i s p r e s e n t as a m i n o r component i n t h e DNA o f t h e s l i m e mold Physarum polycephalum (1.1 mol% m5C: r e f s . 85, 86) and sea u r c h i n s ( 0.9 mol% m5C; r e f . 87). Although many f u n g i l a c k detect a b l e m o d i f i e d bases i n t h e i r DNA, some such as t h e zygomycete P h y c o m y c e s b 7 a k e s 7 e e a n u s and t h e basidomycete Coprinus cinereus have m5C as a m i n o r base i n t h e i r genome ( r e f . 88-90). Eukaryotes as d i v e r s e as t h e l a t t e r f u n g i , d i n o f l a g e l l a t e s , sea u r c h i n s , s l i m e molds, and v e r t e b r a t e s have t h e i r m5C r e s i d u e s p r e d o m i n a t e l y i n CpG d i n u c l e o t i d e s ( r e f s . 84, 91, 92, 88, 93, 1). In some i n s e c t s , DNA m o d i f i c a t i o n i s u n d e t e c t a b l e . Notably, no m5C has been found by chromatographic a n a l y s i s o f DNA d i g e s t s f r o m D r o s o p h i l a m e l a n o g a s t e r a t t h e l a r v a l o r a d u l t stages ( r e f s . 94, 95). However, m5C i s p r e s e n t i n t h e DNA o f c e r t a i n o t h e r i n s e c t spec i e s . The mealybug c o n t a i n s m5C as t h e o n l y d e t e c t a b l e m o d i f i e d base i n i t s DNA i n amounts dependent upon t h e sex as w e l l as t h e species and l i n e s o f these organisms ( r e f . 96). The observed d i f f e r e n c e s between t h e methyl a t i o n l e v e l s of v a r i o u s mealybug DNAs m i g h t be due t o a r e l a t i o n s h i p between DNA m e t h y l a t i o n and h e t e r o c h r o m a t i n i z a t i o n ( r e f . 96). C e l l s o f these i n s e c t s v a r y i n t h e i r c o n t e n t o f h e t e r o c h r o m a t i c o r supernumerary chromosomes i n a manner c o n s i s t e n t w i t h t h e h y p o t h e s i s t h a t i n c r e a s e d DNA methylat i o n i s a s s o c i a t e d w i t h DNA appearing i n t h e h e t e r o c h r o m a t i c f r a c t i o n ( r e f . 96). METHYLATION OF THE DNA OF MITOCHONDRIA, CHLOROPLASTS, AND EUKARYOTIC VIRUSES Higher p l a n t s and v e r t e b r a t e s i n v a r i a b l y c o n t a i n m5C as a m i n o r base i n t h e i r DNA and no o t h e r d e t e c t a b l e m o d i f i e d base ( r e f . 1). I n c o n t r a s t , t h e i r m i t o c h o n d r i a , c h l o r o p l a s t s ( i n t h e case o f p l a n t s ) , and t h e DNA v i r u s e s t h a t i n f e c t t h e s e c e l l s u s u a l l y c o n t a i n l i t t l e o r no DNA m e t h y l a t i o n . For example, polyoma v i r u s ( r e f . 97); adenovirus t y p e 2, t y p e s 5 and 12 ( r e f s . 98101), herpes s i m p l e x v i r u s t y p e 1 ( r e f . 102); herpes s a i m i r i v i r u s ( r e f . 99); and SV40 ( r e f . 101) do n o t c o n t a i n d e t e c t a b l e m o d i f i e d bases d e s p i t e m5C b e i n g p r e s e n t i n t h e i r h o s t DNAs. However, i n 10.4

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tegration of a number of the above DNAs as well as of retroviral provi ral DNA into host genomes in a transcriptional ly repressed form (but not in the transcriptionally active form) is associated with extensive DNA methylation to give m5CpG sites (refs. 104, 105, 106, 107, 108, 109). Although i n f e c t i o u s viral eukaryotic DNAs seem to be generally unmethylated, several exceptions have been reported. DNA from virions of frog virus 3 contains considerable amounts of m5C (ref. 110). Also, viral extrachromosomal DNA from another member of the iridovirus virus family, fish lymphocytosis disease virus, is methylated at 75% of its CpG dinucleotides when isolated from infected tissues (ref. 111). Evidence has been presented for a very slight degree of CpG methyl ation of extrachromosomal human papi 1 loma viral DNA isolated from warts (ref. 112). The extrachromosomal viral DNA in Shope papi 1 loma vi rus-i nfected neoplasms is more highly, but variably, methylated at CpG sites (ref. 113). The only report of a eukaryotic virus associated with a DNA modification other than m5C is a virus called NC-lA, which infects a chlorella-like green alga (ref. 114). This viral DNA contains mA in 5'-GANTC-3' sequences assoc ated with a restriction-modification type system. Its DNA has a high level of m5C (7 mol%) as well as of m6A (7 mol%). Analyses of organelle DNA indicate little or no methylation Chloroplasts from tobacco leaves were reported to contain no detectable m5C in their DNA although the detection limits in this analysis were not indicated (ref. 115). Mitochondria1 DNA from Paramecium a u r e l i a contains (0.1 mol% m5C or m6A; the nuclear DNA also has no detectable m5C but has 2.5 mol% m6A (ref. 79). Less than 0.1 and 0.05 mol% m5C was detectable in the mitochondrial DNA of frog and cultured human (HeLa) cells, respectively (ref. 116). Examination of mitochondria1 DNA from various cultured mammalian cell lines indicated that it contains one-fourth to onefourteenth the m5C content of nuclear DNA (ref. 117). Similarly, we found a low level of m5C (0.14 mol%) in DNA from a mitochondrial fraction of human placenta (ref. 118). Restriction analysis of this DNA indicated (5% contamination with bulk nuclear DNA. However, in these studies of organelle DNA, covalently closed circular DNA is isolated from partially purified subcellular fractions which might be contaminated with small circular DNAs.

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These may c o n t a i n c e r t a i n n u c l e a r DNA sequences t h e r e b y i n t r o ducing contaminating m5C r e s i d u e s . In t h i s regard r e s t r i c t i o n a n a l y s i s of t h e s e o r g a n e l l e DNAs i s l e s s l i a b l e t o f a l s e p o s i t i v e r e s u l t s . We found no d e t e c t a b l e methylation a t 5'-CCGG-3' s i t e s i n human p l a c e n t a l DNA by comparing HpaII and MspI d i g e s t s ( r e f . 119). S i m i l a r l y , no evidence f o r CpG methylation of wheat, y e a s t , Neurospora, r a t , o r c a l f mitochondria1 DNA was found by r e s t r i c t i o n a n a l y s e s ( r e f s . 120, 121). In c o n t r a s t , the u n i c e l l u l a r green a l g a Chlamydomonas methylates almost h a l f o f t h e C r e s i d u e s i n i t s c h l o r o p l a s t DNA i n t h e m a t e r n a l l y derived b u t n o t the p a t e r n a l l y derived c h l o r o p l a s t DNA i n zygotes ( r e f s . 122, 123, 1 2 4 ) . The b i o l o g i c a l impact of t h i s methylation i s u n c l e a r . D I S T R I B U T I O N OF m5C I N THE NUCLEAR DNA OF HIGHER PLANTS AND VERTEBRATES All studied higher p l a n t s c o n t a i n m5C a s a minor b u t cons i d e r a b l e c o n s t i t u e n t of t h e i r n u c l e a r DNA ( 2.3-7.1 mol%) just a s a l l v e r t e b r a t e genomes appear t o have m5C ( 0.7-2.8 mol%) a s t h e i r only minor base ( r e f s . 1, 1 2 5 ) . P l a n t DNAs can have up t o 33% of 10.5

t h e i r C r e s i d u e s methylated (tobacco: r e f . 125) and even higher percentages of the C r e s i d u e s i n c e r t a i n s a t e l l i t e DNAs (melon: r e f . 126; b l u e b e l l : r e f . 127). The d i s t r i b u t i o n o f m5C r e s i d u e s i s sequence-specific ( r e f . 128) with methylation o c c u r r i n g predominately a t CpG o r CpNpG (N i s any base) sequences ( r e f s . 129, 127, 130, 131). Although p l a n t DNA methylation has been s t u d i e d r e l a t i v e l y l i t t l e , there i s a t l e a s t one example of t i s s u e - s p e c i f i c d i f f e r e n c e s i n p l a n t DNA methylation ( r e f . 1 3 2 ) . This observed t i s s u e - s p e c i f i c change i n DNA methylation o f t h e z e i n gene i n d i c a t e s t h a t gene demethylation might, i n some c a s e s , p o s i t i v e l y c o n t r o l gene expression ( r e f . 132). A s t u d y of i n vivo methylation of the transforming plasmid fragment (T-DNA) from Agrobacterium tumefaciens, a b a c t e r i um oncogeni c f o r d i c o t y l donous p l a n t s , s u g g e s t s t h a t methylation of T-DNA i n s i d e p l a n t c e l l s might r e g u l a t e i t s t r a n s c r i p t i o n ( r e f . 133). Another proposed r o l e f o r p l a n t DNA methylation which has experimental s u p p o r t i s t h a t i t might d e c r e a s e t r a n s p o s i t i o n by t r a n s p o s a b l e elements r e s i d e n t i n p l a n t nuclei ( r e f . 134). In c o n s i d e r i n g t h e r o l e s of DNA m e t h y l a t i o n , the mode of f o r -

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mation of m5C residues should be evaluated. Methylation is catalyzed by a cytosine DNA methyl transferase with S-adenosylmethionine and DNA as the substrates after DNA replication (refs. 135-137). The methyl atable ol igonucleotide sequences (CpG and CpNpG) have dyad symmetry, that is, in the 5' ---> 3' direction the same sequence is present on both strands. This facilitates the recognition of plant DNA methylated in only one strand so that such hemimethylated DNA serves as the best DNA substrate for the plant's DNA methyl transferase(s) enzyme(s) (ref. 137). Methylation of hemimethylated DNA is referred to as maintenance methylation because it serves to conserve the pattern of DNA methylation that existed before DNA replication converted a bifilarly (symmetrically) methylated to a hemimethylated site (ref. 1, 127). I n contrast, increases in net DNA methylation that can help establish tissue-specific patterns of DNA methylation or genetic polymorphisms in the population are catalyzed in a de n o w methylation reaction, that is, i n a reaction utilizing a bifilarly unmethylated DNA sequence as the substrate. Similar considerations pertain to vertebrate DNA methylation and vertebrate DNA methyltransferases except that i n this case the site of methylation is predominantly or only CpG (ref. 1). Vertebrate DNAs contain only approximately one-fourth the expected frequency of CpG dinucleotide (ref. 138). Because the majority of CpG sites are methylated and CpG in the predominant site of vertebrate DNA methylation (refs. 2, 104, 139), the limitation on the frequency of mCpG sites is postulated to be due to the accumulation of m5C ---> T transition mutations (refs. 140-144). Both m5C and C residues can deaminate in a heat-induced reaction which should also occur, although to a lower extent, at physiological temperature (refs. 32, 33). Deamination o f an m5C residue yields a T residue which is a normal constituent of DNA unlike the C deamination product, U, which should be efficiently excised from DNA (refs. 32, 145). Furthermore, heat-induced deamination of m5C residues in single-stranded DNA in a physiological buffer occurs at a faster rate than for the analogous C residues (refs. 146, 33). For vertebrates, the extent and pattern of DNA methylation has been shown in many studies to be tissue-specific although, i n

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all cases, CpG dinucleotides appear to be the predominant site of methylation (refs. 1, 147-150, 1 5 2 ) . Generally, the majority of CpG dinucleotides in vertebrate DNA are methylated a1 though the percentage o f unmethylated CpG sites is considerable and varies from tissue to tissue (refs. 1, 150). Not only can the level of DNA methylation differ as much as 30% from one tissue to another and the patterns of DNA methylation vary much within normal cells of an organism, but also, as described below, the amount and distribution of genomic m5C can be highly altered upon tumorigenesis or oncogenic transformation (refs. 153-155). A1 though m5C is generally enriched in heterochromatin-associated DNA in vertebrates, much of it is also in single copy, moderately repeated, and interspersed repeated DNA fractions (refs. 156, 1 5 0 ) . The distribution of unmethylated CpG dinucleotides also shows a tendency toward clustering (refs. 157-159). Some DNA subfractions, like the DNA sequences encoding ribosomal RNA (rDNA) are either unusually m5C-rich (rDNA in fish and amphibi a) or m5C-poor (rDNA i n mammal s , repti 1 es, and bi rds) depending on the type of organism (ref. 141). The methylation status of a DNA sequence can change greatly depending upon whether it is chromosomal or extrachromosomal (refs. 150, 161) or upon its chromosomal location (ref. 139). THE FUNCTIONAL SIGNIFICANCE OF VERTEBRATE DNA METHYLATION: TRANSCRIPTION, CHROMATIN STRUCTURE, DNA REPLICATION AND REPAIR, CANCER AND EMBRYOGENESIS Vertebrate DNA methylation appears to be involved in controlling expression of certain genes. There are many examples of correlations of naturally occurring DNA methyl at ion and the inhibition of gene expression in mammalian and avian genomes (refs. 162-170; and reviewed in ref. 1; 172, 173). In a number of cases it is clear that genetically programmed, decreases in methylation (demethylation) of DNA sequences cannot be sufficient to activate gene expression although they might be necessary for turning on transcription (ref. 174-177). A1 though the mechanisms for methylation-directed transcription control remain to be established, two types of studies have provided insight into this phenomenon. In the first of these, evidence has been presented 10.6

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that a high density of m5C residues affects how DNA is packaged in nucl eosomes and supranucleosome particles (ref. 178, 179). In the second line of experimentation, a DNA-binding protein has been i sol ated from human pl acental nuclei , whi ch speci fi cal ly binds to certain 20-base-pai r DNA sequences only when they are methyl ated at their 2 or 3 CpG sites (refs. 180-183). The high degree of sequence-speci fi ci ty and methyl ati on-dependence of this bi ndi ng reaction suggests that this protein, methylated DNA-binding protein (MDBP), might be a novel transcription regulatory protein specifically responsive to local DNA methylation. Some of the most striking correlations between DNA methylation and silencing of gene expression have been made for mammalian and avian viral genomes or for proviral DNA integrated into mammal i an genomes where they can become methyl ated (refs. 105, 184-192). In some cases, the lack of DNA methylation was shown not to be sufficient for transcriptional activity although evidence suggests that de novo methylation is part of the mechanism for turning off viral genes in the host genome or for keeping them inactive (refs. 193-195). I n v i t r o DNA methylation of a viral promoter (refs. 196, 197) or of the whole proviral DNA (ref. 198) eliminated most or all o f its activity. In one case, methylation caused a shutdown in expression only after a prolonged delay (ref. 199). Demethylation of viral DNAs by 5-azacytidine, which is a pleiotropic antimetabolite that is the strongest known inhibitor of i n v i v o DNA methylation (refs. 200, 201), activates the expression of many viral DNA genomes that have become methylated upon integration into the host genome (refs. 106, 202-205). Similarly, 5-azacytidine or 5-aza-Zt-deoxycytidine treatment turned on expression of many vertebrate genes and induced differentiation in cultured cells (refs. 206-218). Furthermore, d e n o v o methylation of the human 7-globin promoter region prevented its transcription upon introduction into mouse L eel Is, whereas methylation of most of its protein-coding DNA sequence did not (ref. 219). This suggests that methylation at many sites in genes has no effect upon gene expression but at other sites does. This is consistent with our finding that methylation changes during the course of human development at >10 x 106 sites per haploid genome (ref. 150), an extent of change far too great for much of it to be

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invo ved directly in controlling gene expression. Similar results have been found for other mammals (ref. 220). As these results pred ct, a number of tissue-specific differences i n DNA methylation patterns have been found which cannot be correlated with transcriptional activity (refs. 174, 221-223) or which may be consequences rather than causes of transcriptional activation (refs. 224, 225). There are several lines of evidence suggesting that vertebrate DNA methylation plays an important role in determining the structure of chromatin. Satellite DNAs from mammalian tissues, which are found in highly condensed chromatin (heterochromatin), often have a higher m5C content than the low-copy fraction of DNA (refs. 150, 151, 226-230). Heterochromatin is often associated with centromeres. By analysis for immunoreactive sites on metaphase chromosomes from various mammals, m5C residues have been found to be highly enriched in centromeric regions (refs. 231, 232). The distribution of such immunoreactive m5C sites changes during meiosis (ref. 233). Treatment of cell s with 5-azacytidine induces decondensation of heterochromatin (refs. 234-236) as well as the formation of fragile sites and sister-chromatid exchanges (refs. 235-238). In the mealybug, a high mC content in male DNA was correlated with the presence of a paternally derived, genetically inactive facultative heterochromatin (ref. 96). That the methylation status of satellite DNA is physiologically important is suggested by the much higher level of methylation o f several mammalian satellite DNAs in embryoblast (primitive ectoderm-derived) and adult somatic ti ssues than in the extraembryonic trophobl ast and primitive endoderm derivatives or in gametes (refs. 151, 239-241). Not only has DNA methylation been implicated in higher order chromatin structure, but also, m5C residues have been shown to be preferentially associated with nucleosome core, histone H1-associated chromatin (refs. 178, 242). In addition, DNA cytosine methylation can have a large influence on the conformation of the DNA helix, such as, favoring the Z-DNA conformation (ref. 243). These findings suggest that DNA methylation critically influences the condensation of DNA i n chromatin and of certain satellite DNAs in heterochromatin and also may

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subtly affect local chromatin structure. As mentioned above, this, in turn, could be related to the transcriptional activity of DNA and, also, it could affect the ability of chromatin to undergo various types o f recombi nation. DNA methylation has been linked to another form of chromatin condensation namely, the heterochromatinization and transcriptional inactivation of one of the two X chromosome in female mammals. Much evidence suggests that DNA methylation is intimately involved i n this phenomenon (refs. 244-248, 169). Similarly, DNA methylation has been implicated in the late timing of replication of the inactive X chromosome during S phase (refs. 249, 250, 251). It is also possible that DNA methylation plays a role in controlling the initiation of DNA replication as it appears to in Escherichia c o l i (ref. 72). A n additional analogy to bacterial DNA is suggested by evidence that hemimethylation (at C residues) of CpG sites directs mismatch repair in mammalian cells (ref. 252) as does hemimethylation (at A residues of 5'-GATC-3' sites) i n E . c o l i (ref. 75). As described above, there are extensive tissue-specific differences in the amount and distribution of m5C in vertebrate DNA and some of these, probably only a small percentage, are involved i n controlling gene expression. This indicates that DNA methylation probably plays a major role i n guiding differentiation. It is informative, therefore, to compare the methylation status of specific DNA sequences in gametes and somatic cells. A number of vertebrate genes have been shown to be much more methylated at tested CpG sites in sperm DNA than in one or more adult somatic tissue DNAs (refs. 168, 173, 221, 253-255). On the other hand, human sperm DNA has a rather low overall m5C content compared to somatic tissues, which seems to be due to hypomethylation of satellite DNA sequences, as mentioned above, (ref. 220) as well as to hypomethylation of non-satellite (including low-copy-number) sequences which are speci fical ly undermethyl ated in sperm (refs. 158, 159) and probably also i n oocytes (ref. 240). The latter DNA sequences show similar hypermethylation at both 5'-CCGG-3' and 5'-GCGC-3' sites over a long region in all human adult somatic tissues i n contrast to their hypomethyl ation in sperm and intermediate level of methylation in placenta (refs.

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158, 159). The unusual m e t h y l a t i o n p a t t e r n i n t h e s e sequences i n p l a c e n t a i s c o n s i s t e n t w i t h i t s v e r y d i s t i n c t c e l l l i n e a g e and i t s DNA's r a t h e r low o v e r a l l m5C c o n t e n t ( r e f s . 150, 256). The r e s u l t s w i t h sperm DNA suggest t h a t hypomethylation o f many d i s p a r a t e DNA sequences may be necessary f o r gametogenesis o r e a r l y embryogenesis. T h i s may be f o l l o w e d by an i n c r e a s e i n m e t h y l a t i o n o f these sequences e a r l y i n development y i e l d i n g s i m i l a r hypermethylation i n a l l tissues destined t o g i v e r i s e t o the adult. I n a d d i t i o n t o these DNA sequences hypomethylated s p e c i f i c a l l y i n gametes, t h e r e a r e o t h e r a t y p i c a l l y undermethylated DNA sequences which appear t o be undermethylated i n a77 c e l l populat i o n s o f an organism. They a r e sometimes found i n 5 ' gene r e g i o n s n e x t t o a h i g h l y m e t h y l a t e d r e g i o n w i t h i n t h e gene ( r e f . 157). T h i s asymmetri c a l p l acement o f m5CpG s i t e s a1 ong t h e chromosome m i g h t be one s i g n a l f o r i d e n t i f i c a t i o n o f c e r t a i n genes as t r a n scriptional units. A s d e s c r i b e d above, DNA m e t h y l a t i o n i n v e r t e b r a t e s may h e l p i n d i v e r s e ways t o r e g u l a t e macromolecular s y n t h e s i s and d i f f e r e n t i a t i o n . Therefore, derangements i n normal DNA m e t h y l a t i o n c o u l d have p l e i o t r o p i c consequences t o c e l l u l a r p h y s i o l ogy. Indeed, t h e r e i s evidence suggesting t h a t a b n o r m a l i t i e s i n DNA m e t h y l a t i o n a r e i n v o l v e d i n carcinogenesis. Some o f t h i s evidence comes from a n a l y s i s o f d e m e t h y l a t i o n caused b y c a r c i n o g e n i c agents ( r e f s . 165, 205, 257-264). Furthermore, many o f t h e v i r a l DNAs and p r o v i r u s e s whose expression can be c o n t r o l l e d by DNA m e t h y l a t i o n a r e oncogenic v i r u s e s ( r e f s . 106, 107, 185, 187, 196-198, 202, 205). A l s o , s t u d i e s o f human tumors, w i t h o u t t h e p o s s i b l y comp l i c a t i n g intermediacy o f c e l l c u l t u r e , i n d i c a t e a r e l a t i o n s h i p between a1 t e r a t i o n s i n DNA m e t h y l a t i o n and c a r c i n o g e n e s i s . Examination of 23 human c o l o n neoplasms and normal a d j a c e n t i n t e s t i n a l mucosa by Feinberg, V o g e l s t e i n and coworkers ( r e f s . 154, 265, 266) r e v e a l e d t h a t some d i v e r s e genes have a h i g h probabi 1 it y o f becomi ng hypomethyl a t e d d u r i n g f o r m a t i o n o f c o l o n tumors. T h i s was seen i n genes l i k e t h a t o f 7-globin, whose expression ought t o be i r r e l e v a n t t o t h e tumor as w e l l as i n an oncogene, whose m e t h y l a t i o n m i g h t o r m i g h t n o t be o f p h y s i o l o g i c a l s i g n i f i c a n c e i n t h i s tumorigenesis ( r e f s . 154, 266). The hypo-

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methylation of these genes showed much heterogeneity i n its pattern but clearly was not random. In a collaborative study involving C. Gehrke, K. Kuo, A . Feinberg, and one of us (M. Ehrl ich), we have recently demonstrated that the demethylation n these colon tumors, which are known to arise from the essential Y homogeneous cell population of intestinal mucosa, is part of a genome-wide demethylation (ref. 155). Simi 1 arly, chemical ly induced hepatocarcinomas in rats had a lower m5C content than did normal rat liver (ref. 267). Furthe more, despite a wide range of genomic m5C contents, (0.35-1.03% mol% m5C), a statistically significant difference was found in the distribution of DNA methylation levels in various human malignancies, especially in metastases, as compared to that in benign neoplasms or normal human tissues (ref. 153). The percentages of DNA samples with <0.80 mol% m5C or >0.84 mol% m5C were strikingly different for malignancies and for normal tissues or benign tumors (ref. 153). These differences suggest that either hypomethylation often accompanied tumorigenesis or that many of these malignancies were derived from atypical, minor populations of cells with relatively low m5C contents. In the study o f colon tumors mentioned above, we can conclude that decreases in DNA methylation occurred because the tumors are known to arise from the predominant cell type of intestinal mucosa and methylation of tumor DNA was compared to that of the adjacent normal mucosa (ref. 155). The m5C content of neoplastic samples should reflect the percentage of neoplastic cells in the sample, the type of cells which gave rise to the neoplasm, and any changes in DNA methylation which occur during the early stages of oncogenic transformation or during tumor progression. We have proposed that tumor progression with its attendant continually generated cell ul ar diversity is often accompanied by extensive replacement of m5C residues in DNA with cytosine residues (ref, 153). Studies of cultured murine cell s and transplantable tumors support this hypothesis (refs. 260, 261, 268). Hypomethylation of DNA during tumor progression could provide epigenetic changes of the type associated with normal differentiation. It could help establish or mai ntai n transcriptional activity or 1 ead to cancer-re1ated chromosomal rearrangements, gene amplification, and a1 terations in

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chromosome conformation. The resulting changes in expression or replication of the genome might be an important component in the diversification of tumor cells, which allows them to successfully exploit thei r hosts. 10.7 SUMMARY

Most DNAs contain minor amounts of 5-methylcytosine (rn5C), N6-methyladeni ne (m6A), or N4-methyl cytosine (in"). Any one or more of these can be found in most bacterial DNAs. Some lower eukaryotes have m5C, m6A or both as a minor base in their genomes. In the DNA of all studied vertebrates and higher plants, m5C, and only m5C, is the naturally occurring modified base. The levels and patterns of m5C in vertebrate genomes are ti ssue-speci fi c. Some, but clearly not most, vertebrate DNA methylation has been linked to the regulation of gene transcription. As for m6A in the Escherichia c o l i genome, m5C in the DNA of eukaryotes may function in controlling DNA repair, replication, and rearrangements as well as in control of transcription. However, although bacterial DNA methylation is often involved in r e s t r i c t i o n - m o d i f i c a t i o n systems, there is no evidence for this function in the case of vertebrate DNA methylation. Vertebrate DNA methylation may, however, be an important determinant of chromatin structure. In addition to methylated bases present as minor DNA components, other modified bases are major components of certain bacteriophage genomes. These have one of their bases, usually a pyrimidine, largely or completely replaced by the modified derivative. The derivatives include gl ucosyl ated 5-hydroxy-methylcytosine, uracil, 5-hydroxymethyluracil, 5-(4-aminobutylaminomethy1)uraci 1 , and gl ucosyl ated and gl ucuroni c aci d-l-phosphorylThese major modifications ated 5-(4',5'-dihydroxypenty1)uracil. of phage genomes appear to help, sometimes in subtle ways, the virus to distinguish its genome from that of the host in its exploitation of the host cell and to evade host DNA restriction systems . 10.8

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213. 214.

215. 216.

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1

1

e

a

\

B362

266. 267.

A: P. Feinberg and B. V o g e l s t e i n , Hypomethylation d i s t i n g u i s h e s enes o f some human cancers from t h e i r normal c o u n t e r p a r t s , j a t u r e , 301 (1983) 89-92 J . N . Lape r e and F. F. Becker, 5 - M e t h y l c y t o s i r e c o n t e n t o f n u c l e a r D i A d u r i n g chemical he a t o c a r c i n o g e n e s i s and i n carcinomas which r e s u l t , Biochem iophys. Res. Commun., 87 (1979) 698-705. W.-L. Hsiao, S. G a t t o n i - C e l l i , and I . B. W e i n s t e i n E f f e c t s o f 5 ' - a z a c y t i d i n e on t h e p r o g r e s s i v e n a t u r e o f C e l i Transf o r m a t i o n , Molec. C e l l . B i o l . , 5 (1985) 1800-1803.

ts

268.

B363

INDEX - PART B Aminolevulinic acid; B181 Antibody specificity; B133 Anticodon region; B45, B145 Antiserum, preparation of; B 130 Archaebacteria; B 16 Ascaris suum mitochondrial tRNAs; B199 Aspergillus nidulans mitochondrial tRNAs; B220 Bacillus subtilis; B36 Bacteriophage DNA; B330 Cancer; B102, B341 Chlorophyll synthesis; B 181 Chromatography, RPC-5; B77, B203 Codon families; B43 Codon reading patterns; B199, B210 Codon usage; B111, B120 Codon usage, tables; B22, B113 Codon recognition; B305, B321 Decoding properties; B117 Dictyostelium discoideum; B36, B72, B78 DNA methylation; B329 DNA repair; B336 Drosophila melanogaster; B36, B101, B112 Drosophila mitochondrial tRNAs; B230 Gel electrophoresis; B80, B149, B204 ELISA, competitive; B131, B135 Elongation factor; B185 Enthalpy; B3 15 Epoxy Q (oQ); B74, B76 Erythroleukemic cells (F46 cells); B78, B97 Escherichia coli; B71, B79, B96, B305, B307 Eucaryotes; B79, B89 Genetics of tRNA modifying enzymes; B25, B93 GI ycans; B 180 Glycyl-tRNA synthetase gene; B 192 Hapten-protein conjugates; B 128 his operon mRNA; B192 Hydrolysis, tRNA; B 152 Immunoassay, solid phase; B 127 Inosine content, tRNA; B127 Inosine biosynthesis; B145 Inosine, quantitation; B127 Iron limitation; B79 Iron transport; B34 Leukemia; B85, B155 Ligation; B 155 Lymphomas; B85 Mammalian mitochondrial tRNA; B235

B364

Methylation (rRNA); B269, B276 Methylation, DNA; B337 Mitochondrial tRNAs; B 199 Mitochondria1 tRNAs, Aspergillus nidulans; B 2 2 0 n eurospora crassa; 8 2 1 1 , B218 paramecium, B 2 2 7 Podospora anserina; 8 2 2 5 Saccharomyces Cerevisiae; 8 2 0 3 Schizosaccharomyces pombe; 8 2 2 2 Tetrahymena pyriformis; B226 Torulopsis glabrata; B216 tumor cell; B240 Xenopus Leavis; 8234 Modified nucleosides, in tRNA; B 14 synthesis; B20 function; B37 Modifying enzymes, regulation on translational efficiency; B38 Modifying enzymes, tRNA Mosquito tRNA; B228 NMR; B307 Nuclease P1; B152 Periodate oxidation; B153 Phosphatidylglycerol; B 18 1 Phosphatidylglycerol synthetase; B 18 1 Plant mitochondria1 tRNA; B245 Protein synthesis; B118, B305 Pseudoknots; B187 Pseudouridine (rRNA); B271 Pteridines; B93 Q-base modification; B36 Queuine; B69, B l l l Queuosine, biosynthesis; B69 function; B69 occurrence; B72 Radiolabelling; B 150 Reading frames; B 199 Reversed-phase HPLC; B77 Ribonuclease T2; B152 Ribosomal precursor RNA; B272 Ribosomal RNA; B71; B267 Ribosomal RNA, HeLa; B274 Ribosomal RNA, rRNA Xenopus; B284 RNAs, cytosolic; B14 Salmonella typhimurium; B35, B70 Thin-layer chromatography; B 152 Threonyl-RNA synthetase; B192

B365 Transcription, reverse; B183 tRNA, eubacterial; B15 archaebacterial, B16 conformation; B46 mutations; B29 tRNA-guanine-transglycosylase; tRNAs, mitochondrial; B 19 tRNAs, organelle; B18 tRNAs, plant; B81 Ubiquitin; B 183 Uridines, modified; B30.5 Virus, polyoma DNA; B192 Viruses, methylation; B337 Viruses, plant RNA; B185 Vitamin B12 (Cobalamine); B74 Wheat germ; B270 Wobble position; B42 Xiphophorine fishes, Q; B84 Yeast tRNA; B282

B93

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Detectors in Gas Chromatography by J. SevEik

Volume 5

Instrumental Liquid Chromatography. A Practical Manual on High-Performance Liquid Chromatographic Methods (see also Volume 27) by N.A. Parris

Volume 6

Isotachophoresis. Theory, Instrumentation and Applications by F.M. Everaerts, J.L. Beckers and Th.P.E.M. Verheggen

Volume 7

Chemical Derivatization in Liquid Chromatography by J.F. Lawrence and R.W. Frei

Volume 8

Chromatography of Steroids by E. Heftmann

Volume 9

HPTLC -High Performance Thin-Layer Chromatography edited by A. Zlatkis and R.E. Kaiser

Volume 10

Gas Chromatography of Polymers by V.G. Berezkin, V.R. Alishoyev and I.B. Nemirovskaya

Volume 11

Liquid Chromatography Detectors (see also Volume 33) by R.P.W. Scott

Volume 12

Affinity Chromatography by J. TurkovP

Volume 13

Instrumentation for High-Performance Liquid Chromatography edited by J.F.K. Huber

Volume 14

Radiochromatography. The Chromatography and Electrophoresis of Radiolabelled Compounds by T.R. Roberts

Volume 15

Antibiotics. Isolation, Separation and Purification edited by M.J. Weinstein and G.H. Wagman

B368 Volume 16

Porous Silica. Its Properties and Use as Support in Column Liquid Chromatography by K.K. Unger

Volume 17

76 Years of Chromatography - A Historical Dialogue edited by L.S. Ettre and A. Zlatkis

Volume 18A

Electrophoresis. A Survey of Techniques and Applications. Part A: Techniques edited by Z. Deyl

Volume 18B

Electrophoresis. A Survey of Techniques and Applications. Part B: Applications edited by Z. Deyl

Volume 19

Chemical Derivatization in Gas Chromatography by J. Drozd

Volume 20

Electron Capture. Theory and Practice in Chromatography edited by A. Zlatkis and C.F. Poole

Volume 21

Environmental Problem Solving using Gas and Liquid Chromatography by R.L. Grob and M.A. Kaiser

Volume 22A

Chromatography. Fundamentals and Applications of Chromatographic and Electrophoretic Methods. Part A: Fundamentals edited by E. Heftmann

Volume 22B

Chromatography. Fundamentals and Applications of Chromatographic and Electrophoretic Methods. Part B: Applications edited by E. Heftmann

Volume 23A

Chromatography of Alkaloids. Part A: Thin-Layer Chromatography by A. Baerheim Svendsen and R. Verpoorte

Volume 23B

Chromatography of Alkaloids. Part B: Gas-Liquid Chromatography and High-Performance Liquid Chromatography by R. Verpoorte and A. Baerheim Svendsen

Volume 24

Chemical Methods in Gas Chromatography by V.G. Berezkin

Volume 25

Modern Liquid Chromatography of Macromolecules by B.G. Belenkii and L.Z. Vilenchik

Volume 26

Chromatography of Antibiotics. Second, Completely Revised Edition by G.H. Wagman and M.J. Weinstein

Volume 27

Instrumental Liquid Chromatography. A Practical Manual on High-Performance Liquid Chromatographic Methods. Second, Completely Revised Edition by N.A. Parris

Volume 28

Microcolumn High-Performance Liquid Chromatography by P. Kucera

Volume 29

Quantitative Column Liquid Chromatography. A Survey of Chemometric Methods by S.T. Balke

B369 Volume 30

Microcolumn Separations. Columns, Instrumentation and Ancillary Techniques edited by M.V. Novotny and D. Ishii

Volume 31

Gradient Elution in Column Liquid Chromatography. Theory and Practice by P. Jandera and J. ChurhEek

Volume 32

The Science of Chromatography. Lectures Presented at the A.J.P. Martin Honorary Symposium, Urbino, May 27-31,1985 edited by F. Bruner

Volume 33

Liquid Chromatography Detectors. Second, Completely Revised Edition by R.P.W. Scott

Volume 34

Polymer Characterization by Liquid Chromatography by G. Glockner

Volume 35

Optimization of Chromatographic Selectivity. A Guide to Method Development by P.J. Schoenmakers

Volume 36

Selective Gas Chromatographic Detectors by M. Dressler

Volume 37

Chromatography of Lipids in Biomedical Research and Clinical Diagnosis edited by A. Kuksis

Volume 38

Preparative Liquid Chromatography edited by B.A. Bidlingmeyer

Volume 39A

Selective Sample Handling and Detection in High-Performance Liquid Chromatography. Part A edited by R.W. Frei and K. Zech

Volume 39B

Selective Sample Handling and Detection in High-Performance Liquid Chromatography. Part B edited by K. Zech and R.W. Frei

Volume 40

Aqueous Size-Exclusion Chromatography edited by P.L. Dubin

Volume 41A

High-Performance Liquid Chromatography of Biopolymers and Biooligomers. Part A: Principles, Materials and Techniques by 0. Mike3

Volume 41B

High-Performance Liquid Chromatography of Biopolymers and Biooligomers. Part B: Separation of Individual Compound classes by 0. Mikei

Volume 42

Quantitative Gas Chromatography for Laboratory Analyses and OnLine Process Control by G. Guiochon and C.L. Guillemin

Volume.43

Natural Products Isolation. Separation Methods for Antimicrobials, Antivirals and Enzyme Inhibitors edited by G.H. Wagman and R. Cooper

B370 Volume 44

Analytical Artifacts. GC, MS, HPLC, TLC and PC by B.S. Middleditch

Volume 45A

Chromatography and Modification of Nucleosides. Part A: Analytical Methods for Major and Modified Nucleosides - HPLC, GC, MS, NMR, UV and FT-IR edited by C.W. Gehrke and K.C.T. Kuo

Volume 45B

Chromatography and Modification of Nucleosides. Part B: Biological Roles and Function of Modification edited by C.W. Gehrke and K.C.T. Kuo