sign in sign up
<<
Home , Protein biosynthesis

Protein Biosynthesis in NATO ADVANCED STUDY INSTITUTES SERIES

A series of edited volumes comprising multifaceted studies of contem• porary scientific issues by some of the best scientific minds in the world, assembled in cooperation with NATO Scientific Affairs Division.

Series A: Sciences Recent Volumes in this Series Volume 32 - The Blood - Retinal Barriers edited by Jose G. Cunha-Vaz

Volume 33 - Photoreception and Sensory Transduction in Aneural Organisms edited by Francesco Lenci and Giuliano Colombetti

Volume 34 - Lasers in Biology and Medicine edited by F. Hillenkamp, R. Pratesi, and C. A. Sacchi

Volume 35 - Environmental Physiology of Fishes edited by M. A. Ali

Volume 36-The Prostaglandin System: Endoperoxides, Prostacyclin, and Thromboxanes edited by F. Berti and G. P. Velo

Volume 37- Active Defense Mechanisms in Plants edited by R. K. S. Wood

Volume 38 - Growth edited by Claudio Nicolini

Volume 39 - Drug Development, Regulatory Assessment, and Postmarketing Surveillance edited by William M. Wardell and Giampaolo Velo

Volume 40-Chromosome Damage and Repair edited by Erling Seeberg and Kjell Kleppe

Volume 4 J -- Biosynthesis in Eukaryotes edited by R. Perez-Bercoff

This series is published by an international board of publishers in con• junction with NATO Scientific Affairs Division

A Life Sciences Plenum Publishing Corporation B Physics London and New York C Mathematical and D. Reidel Publishing Company Physical Sciences Dordrecht, Boston, and London D Behavioral and Sijthoff & Noordhoff International Social Sciences Publishers E Applied Sciences Alphen aan den Rijn, The Netherlands, and Germantown, U.S.A. Protein Biosynthesis in Eukaryotes

Edited by R. Perez-Bercoff The Institute of Virology University of Rome Rome, Italy

PLENUM PRESS • NEW YORK AND LONDON Published in cooperation with NATO Scientific Affairs Division Library of Congress Cataloging in Publication Data NATO Advanced Study Institute on Protein Biosynthesis in Eukaryotes (1980: Maratea, Italy) Protein biosynthesis in eukaryotes. (NATO advanced study institute series. Series A, Life sciences; v. 41) "Proceedings of a NATO Advanced Study Institute on Protein Biosynthesis in Eukaryotes, held in Maratea, Italy, September 7 -17, 1980" - T.p. verso. Bibliography: p. Includes index. 1. Protein biosynthesis-Congresses. 2. Eukaryotic cells-Congresses. I. Perez-Bercoff, R. II. Title. III. Series. QP551.N36 1980 574.19'296 81-22720 ISBN 978-1-4684-4126-0 ISBN 978-1-4684-4124-6 (eBook) AACR2 DOI 10.1007/978-1-4684-4124-6

Proceedings of a NATO Advanced Study Institute on Protein Biosynthesis in Eukaryotes, held in Maratea, Italy, September 7-17, 1980.

© 1982 Plenum Press, New York Softcover reprint of the hardcover 1st edition 1982 A Division of Plenum Publishing Corporation 233 Spring Street, New York, N.Y. 10013 All rights reserved No part of this book may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise, without written permission from the Publisher PROTEIN BIOSYNTHESIS IN EUKARYOTES

( or "HOW DIVERGENT THEIR WAYS MAY BE OF E. co U AND ELEPHANTS ... " )

Protein biosynthesis is the most complex biological process known to occur at the cellular level: Even if we disregard the directly related steps which precede the initiation of (e.g. trans• cription~ ~ "capping", and splicing of messenger RNA, among others), or those that usually take place after a protein has been made (e.g. cleavage, modification, maturation), the synthesis of the simplest still constitutes the most formidable task a cell is faced with. .. . vi PREFACE

The word ppotein, coined one and a half century ago from the 1TpOTE:toa ("proteios" = of primary importance), underlines the "primary importance" ascribed to from the time they were described as biochemical entities. But the unmatched compl~xity of the process involved in their biosynthesis was (understandably) overlooked. Indeed, protein biosynthesis was supposed to be nothing more than the reverse of protein degradation, and the same known to split a protein into its constituent amino acids were thought to be able, under adequate conditions, to reconstitute the . This oversimplified view persisted for more than 50 years: It was just in 1940 that Borsook and Dubnoff examined the thermodynamical aspects of the process, and concluded that protein synthesis could not be the reverse of protein degradation, such an "uphill task being thermody• namically impossible ••• • "

The next quarter of a century witnessed the unravelling of the basic mechanisms of protein biosynthesis, a predictable aftermath of the Copernican revolution in biology which followed such dramatic de• velopments as the discovery of the nature of the genetic material, the double helical structure· of DNA, and the determination of the ge• netic code. Our present understanding of the sophisticated mechan• isms of regulation and control is a relatively novel acquisition, and recent studies have shed some light into the structure and organi• zation of the eukaryotic .

First unravelled in bacterial systems, then in eukaryotes, the basic mechanisms of protein biosynthesis proved to be surprisingly similar in E. aoli and ~obo8aida elephantidaet , a circumstance that led to the enunciation, by the mid-sixties, of a unifYing theo• ry on bacteria and elephants: " •• . what holds true fop E. aoli ... " read the versicle of the Holy Scripture of those days. Later obser• vations, however, established that elephants find it difficult to march over the footsteps of bacteria, and apparently one day, several million years ago, they decided to make proteins their own way ..• So great a success crowned their search for originality, that last September we could devote a N.A.T.O. Advanced Study Institute to re• view and discuss our present understanding (and misunderstandings ••• ) of the mechanism of protein biosynthesis in eukaryotes ••.• This book is, indeed, the natural "fallout" of this N.A.T.O. Conference, and features the edited version of the course.

For a course it was, and held at the highest possible level thanks to the unlimited collabora:tion 9f our invited speakers: Their response to my original request to contribute "a fresh, original and interpretative Peview lecture" on topics I knew they had unchallenged command of, was equalled only by the interest of all participants. Needless to add, therefore, that all the merits of this book should be

t commonly named "elephants" •.• PREFACE vii accounted to their credit, while I take the blame for the imperfec• tions that might be found in it.

Thanks are due to the Italian National Research Council (CNR) , for its financial contribution, partly to defray the costs of pre• paring the edited manuscript for publication.

Finally, it is my pleasant duty to express, on behalf of all those who had the privilege of taking part at this N.A.T.O. Advanced Study Institute, my gratitude to the local authorities of Maratea, the Mayor Professor Sisini, and the Chairman of the Tourist Office, Mr. B. Vitolo, who so decisively contributed to make of our time in their blessed seaside resort an unforgettable experience.

R.perez-Bercoff Rome, November 1980 CONTENTS

SECTION I: THE PROTEIN SYNTHESIZING MACHINERY OF EUKARYOTES

Chapter 1: Structure and Function of tRNA and Aminoacyl tRNA Synthetases ...... 1 James OFENGAND

Introduct ion ...... 1 I. AMINOACYLATION A. Stoichiometry and Energetics ...... 2 B. Structure o£ Aminoacyl-tRNA ...... 3 C. Mechanism of Aminoacylation ...... 6 II. STRUCTURE OF tRNA A. Multiplicity and Location of tRNA in the Cell ...... •... 8 B. Primary and Secondary Structure ...... 10 C. Tertiary Structure ...... 19 III. STRUCTURE OF AMINOACYL tRNA SYNTHETASE A. Multiplicity and Cellular Location ...... 25 B. ...... 25 C. Multi- Complexes ...... 28 IV. SPECIFICITY OF AMINOACYLATION A. Importance of Specificity ...... 29 B. Recognition ...... 29 C. tRNA Re cognit ion ...... •...... 32 V. CODON-ANTICODON RECOGNITION A. Codon Translation in the ...... 37 B. Codon Translation in the Mitochondria ...... 41 C. Codon Translation in vivo ...... 42 VI. tRNA RECOGNITION BY THE EUKARYOTIC PROTEIN SYNTHESIS SYSTEM A. Initiation and Elongation Factors ...... 43 B. ...... •...... 44 VII. OTHER FUNCTIONS OF tRNA A. tRNA-Like Structure in Viral RNA ...... 45 B. Primer for ...... 46 C. Aminoacyl-tRNA Protein Transferase ...... 47 D. Regulatory Functions ...... 48

ix x CONTENTS

VIII. tRNA BIOSYNTHESIS ....•...... •.....•.....•• 48 Acknowledgement ..•••.•..•....••••..•••••...... •.••..•.• 49 References .•.....•..•...... ••.....•.....•..•...... •..•.•.•. 49 Appendix: Table 2. Published tRNA Sequences as for August 1, 1980 ••.•...•.•....•.•.•...... •.....• 60

Chapter 2: The Structure of Eukaryotic ...•.•.••••...•• 69 Ira G. WOOL

Introduct ion ..•...••••.•••••..•.•..•••••.•.•.•...... •.. 69 I. GENERAL CHARACTERISTICS OF EUKARYOTIC RIBOSOMES .•..•••. 70 II. ISOLATION AND CHARACTERIZATION OF EUKARYOTIC RIBOSOMAL PROTEINS ...... •...... •.•.•..•...... 71 III. PRIMARY STRUCTURE OF EUKARYOTIC RIBOSOMAL PROTEINS •.... 75 IV. RNA-PROTEIN INTERACTIONS IN EUKARYOTIC RIBOSOMES A. 5S rRNA ..•.••...... •.•••.•..••.•.....••• 78 B. 5. 8s rRNA .••.•••••••..•.•••...•..•...... •...... •...••• 84 C. E. coli 5S rRNA ...... •...... •.. 86 D. tRNA ..•...••••••..•.••..•..•••...•.•...... •.•••••.... 88 V. CODA •...... •..•.•.••••.•.•.•.. 89 References ...... •.•....•...... •..•••••..•...... 90

Chapter 3: The Initiation Factors •••••••••.•.•••...... •.•• 97 John W. B. HERSHEY

Introduction ...••.•...... ••..••.•..•.•..•...•...... •• 97 I. IDENTIFICATION OF THE INITIATION FACTORS •••.•..••.•..•• 97 II. PHYSICAL CHARACTERIZATION .••.•...... ••...... •...••••.. 101 III. COVALENT MODIFICATIONS ...... •.....••••..•..••••..•... 106 IV. CELLULAR LEVELS AND BIOGENESIS ..•.••.....•..•...... 106 V. PATHWAY OF INITIATION A. Dissociation of Ribosomes into Subunits •.•...... •. 108 B. Ternary Complex Formation ...... ••...•...•..•..•...•..• 108 C. Ternary Complex Binding to 40s Subunits •..•.••••••••.. 109 D. Binding of mRNA to 40s Subunits ...... 110 E. Junction of the 60S Subunit and Formation of the 80S Initiation Complex ..•.•.....•..••••....•.•. 111 VI. MOLECULAR MECHANISM OF INITIATION A. mRNA-Ribosome Interaction ...•..••.•••...... •...... 112 B. Specific Factors for mRNA ? •••••.•.••••.•••.••••.•••• 113 C. Ribosomal Sites for Initiation ...... •.•..•.••.... 114 Acknowledgements ...... •.••...... •...... ••.••..••..•. 114 References ....•...... •...... •...•...... ••...... •.• 114

SECTION II: ON THE IMPORTANCE OF BEING SPLICED

Chapter 4: Messenger RNA Structure and Biosynthesis ...... • 119 Robert P. PERRY

Introduction ...... •...... •.....•...... •....•.. 119 CONTENTS xi

I. DETERMINATION OF mRNA SECONDARY STRUCTURE ...... 120 II. MESSENGER RNA PROCESSING: HISTORICAL BACKGROUND ...... 122 III. MODIFIED .- CAP STRUCTURE .....•.•...•.•.... 123 IV. SITES OF TRANSCRIPTIONAL INITIATION OF mRNA ...... 125 V. SPLICING ...... •...... 126 VI. ORDER OF PROCESSING REACTIONS ...... •.....•.....•. 131 References ...... •...... •.....•. 133

Chapter 5: sv40 as a Model System for the Study of RNA and Processing in Eukaryotic Cells •.. 137 Yosef ALONI

Introduct ion ...... •..•...... 137 I. sv40 AS A MODEL SySTEM ...... •...... •. 138 II. INITIATION OF TRANSCRIPTION OF sv40 DNA LATE AFTER .INFECTION ...... ••..•...•.....• 138 III. THE sv40 MINICHROMOSOME ...•...... ••...... •.• 141 IV. SPLICING OF sv40 LATE mRNA ...... •. 143 V. MAPPING THE "LEADER" AND THE "BODy1I OF THE VIRAL mRNAs.BY ELECTRON MICROSCOPY A. Analysis of the DNA-RNA Hybrids ...... •. 145 B. Analysis of the R-Loop Structures ....•...... 145 VI. MODELS FOR JOINING THE "LEADER" TO THE CODING SEQUENCES ..•....••.•...•...•...... •....••..... 148 VII. MODELS FOR SPLICING OF mRNA A. Splicing Intermediates ...... •.....•...... •••.... 153 Conclusions ..••.•...... •...... •..•..•..•...... 153 Acknowledgements ...... •...... ••...... •.. 153 References ...... •...... 154

Chapter 6: Messenger Ribonucleoprotein Particles ...... 157 John W. B. HERSHEY

Introduct ion ...... ••...... •...... 157 I. BIOLOGICAL PROPERTIES A. Early Developments in Sea Urchin ...... 158 B. Different iat ing Animal Cells ...... •...... 159 C. Non-Differentiating or Terminally Differentiated Mammalian Cells ...... •...... •..... 160 II. ISOLATION AND COMPOSITION ...... •...... 161 III. TRANSLATION OF mRNPs ...... 163 IV. SUMMARY AND CONCLUSIONS .....••.....•.....•...... 164 Acknowledgement ...... •...... 164 References ...... •...... •...... 164

SECTION III: ON SELECTING THE RIGHT MESSENGER

Chapter 7: Recognition of Initiation Sites in Eukaryotic mRNAs ...... •... 167 Marilyn KOZAK xii CONTENTS

Introduction .••...•.....•.•...... •.•...... •.•...•...... 167 I. CHARACTERISTICS OF INITIATION REGIONS IN EUKARYOTIC MESSENGER ...... 167 II. MECHANISMS WHICH HAVE BEEN PROPOSED TO EXPLAIN SELECTION OF INITIATION SITES BY EUKARYOTIC RIBOSOMES. 174 III. EVALUATION OF THE "SCANNING" MODEL FOR INITIATION· A. A Summary of the Evidence ...... 176 B. Variat ions on the Theme ...... 180 C. How Can the Exception be Explained ...... 181 IV. QUESTIONS AND SPECULATIONS A. An Economical Message Might Initiate at the First and Second AUG ...... 184 B. Role of the 5'-Terminal Methylated Residues ...... 185 C. Determinants of Messenger Efficiency ...... 186 D. Translation of Viral Messages ...... 188 Acknowledgements/Notes ...... 189 References ...... •.•...... • 189

Chapter 8: A Closer Look at the 5' End of rnRNA in Relation to Initiation ...... 199 Aaron J. SHATKIN

Int roduct ion ...... 199 I. FACILITATING EFFECT OF THE CAP ON rnRNA TRANSLATION AT THE LEVEL OF RIBOSOME BINDING ...... 200 II. DETECTION OF CAP BINDING PROTEIN BY CHEMICAL CROSS-LINKING TO rnRNA 5' END A. Cap-Binding Activity in Cell-Free Extracts .•...... 205 B. Cap Affinity of Initiation Factors ...... 206 III. F~CTIONAL CAP BINDING PROTEINS PURIFIED BY m GDP-SEPHAROSE AFFINITY CHROMATOGRAPHY ...... 208 IV. rnRNA 5' REGION PROXIMITY TO 18s RIBOSOMAL RNA IN INITIATION COMPLEXES ...... 212 Acknowledgements ...... 219 References ...... 219

Chapter 9: /rnRNA Interactions and rnRNA Recognition ...... 223 Raymond KAEMPFER

I. GENERAL ASPECTS OF rnRNA RECOGNITION ...... 223 II. APPROACHS TO THE STUDY OF rnRNA/INITIATION FACTOR INTERACTIONS ...... 225 III. RECOGNITION OF rnRNA BY eIF-2 A. eIF-2 Binds to mRNA ...... 227 B. The Untranslated Portion of rnRNA and poly(A) Are Not Recognized by eIF-2 ...... 227 C. Role of the 5'-Terminal Cap and Internal rnRNA Sequences in Binding of eIF-2 ...... 228 CONTENTS xiii

D. Specific Binding of eIF-2 to a 51-Terminal Sequence Comprising the Ribosome-Binding Site ...... 229 E. Role of mRNA Conformat ion ...... 229 F. Alteration of the 51-Proximal RNA Conformation Induced by Binding of eIF-2 ...... 230 G. Relationship Between Binding of eIF-2 and Binding of the Ribosome ...... 232 H. mRNA Competition for eIF-2 during Translation ..•...... 234 I. Interaction Between eIF-2 and Double-Stranded RNA ..... 234 J. Mutually Exclusive Binding of mRNA and Met-tRNAf for e IF-2 ...... 236 K. eIF-2 and Initiation of Translation ...... 238 IV. BINDING OF OTHER INITIATION FACTORS TO mRNA ...... 240 Conclusions ...... 240 Acknowledgement s ...... 241 References ...... 241

Chapter 10: But Is the 51 End of mRNA Always Involved in Initiation ? ••••.••.••.•••••••.••••.•.•••..• 245 Raul PEREZ-BERCOFF

Introduction ...... 245 I. THE GENOMIC RNA OF PICORNAVIRUSES ...... •...... 246 II. EVIDENCE FOR MORE-THAW-ONE INITIATION SITE IN PICORNAVIRUS RNA ...... : ...... 246 III. INVOLVEMENT OF INTERNAL REGIONS OF PICORNAVIRUS RNA IN INITIATION ...... 249 IV. STUDIES ON THE RIBOSOME-BINDING SITES OF MENGOVIRUS RNA ...... 250 V. "IN VITRO VERITAS " ••••.••..•••.•.••••••.•..•••..•••• 250 References ...... 251

SECTION IV: SYNTHESIS AND PROCESSING OF PROTEINS

Chapter 11: Peptide Chain Elongation and Termination in Eukaryotes ...... 253 Nathan BROT

Introduction ...... 253 I. BINDING OF AMINOACYL-tRNA TO THE RIBOSOMES A. Characteristics of EF-1 ...... 254 B. Assay of EF-1 ...... 256 C. Purification of IF-1 ...... 256 a. EF-1 H...... 256 b. EF-1 JJ ••••••••••••••••••••••••••••••••••••••••••••• 256 c. EF-1 S...... •...... •...... 257 D. Interactlon of EF-1 with Guanosine and AA-tRNA ...... 258 E. Interaction of the Ternary Complex with Ribosomes ..... 259 F. Recycling of EF-1 ...... 260 xiv CONTENTS

II. PEPTIDE BOND FORMATION ...... •...... 261 III. TRANSLOCATION A. 2 ...... •...... •...... • 263 a. Purification and Properties ...... •...... 263 b. As s ay...... •..•.....•..•...... •...... 263 c. Interactions of EF-2 with Guanosine Nucleotides and with Ribosome s ...... •... 264 d. The Inhibition of EF-2 Activity by ••...... •..•...... •...... 266 IV. TERMINATION ...... •..••...... •...... •...... • 268 Reference s .••...... •...... •.....•.•..••.•..•... 269

Chapter 12: Biosynthesis, Modifications, and Processing of Viral Polyproteins ...... •. 275 Gebhard KOCH, Friedrich KOCH, John A. BILLELO, Eckard HILLER, Claudia SHARLI , GUnther WANECKE and Carsten WEHER

Introduct ion ...... •.•...... •...... •...... 275 I. THE PROTEOLYTIC PROCESSING OF PICORNAVIRUS PROTEINS A. Picornavirus-directed Protein Synthesis .•...... 277 a. Processing of NCVP 1a ...... •...... •...... 278 b. NCVP1b, VPg, and Viral RNA Replication ...... 280 B. The Effect of Guanidine on the Processing of Viral Prot eins ...... •...... ••.•... 281 C. Non-Uniform Synthesis and/or Accumulation of Poliovirus Proteins under Conditions of Restricted Polypeptide Chain Initiation and at Early Time after Infection ..... 284 D. Further Characterization of Using Viral Proteins as Substrate: Studies in Cell~Free Systems ... 285 II. POST-TRANSCRIPTIONAL MODIFICATIONS OF ONCORNAVIRUS-DlRECTED PROTEINS A. Protease Specific of RNA Tumor ...... 288 B. Synthesis and Processing of Viral Proteins in Friend Erythroleukemia Cell Lines ....•...... 290 C. Detection and Processing of Intermediates .....•..•..... 292 D. Modification and Processing of Viral Precursor Poly• during the Induced Differentiation of Friend Cells ...... •...... •. 293 E. Synthesis and Processing of Viral Precursor Proteins under Conditions Inducing Terminal Differentiation ....• 294 F. Amplification of Translational Control of Gene Expres- sion during the Differentiation of Friend Cells ...... 295 a. Inducers of Differentiation Inhibit Protein Synthesis ...... •..... 295 b. Differential Effects of Inhibitors ...... 296 G. Effects of Inhibitors of Polypeptide Chain Initiation on the Modifications and Processing of Viral Polyproteins ...... •. 297 Conclusive Remarks ...... 300 References ...... 302 CONTENTS xv

SECTION V: INHIBITION OF PROTEIN SYNTHESIS AT SELECTED LEVELS

Chapter 13: Action of Inhibitors of Protein Biosynthesis ...... 311 David VAZQUEZ, Eulalio ZAERA, HUMBERTO DOLZ, and Antonio JIMENEZ

Introduction •...... •...... 311 1. TRANSLATION OF mRNA A. Inhibitors of Initiation ...... •...... •...•...•..... 312 B. Inhibitors of the Recognition of the Initiatior Substrate (Step A) •...... ••...... •. 313 C. Inhibitors of mRNA Recognition (Step B) ...... •...... 315 D. Inhibitors of Subunit Joining (Step C1) ...... 316 E. Inhibitors of Positioning in the Donor Site (Step C2 ) .. 316 F. Unclassified Inhibitors of Initiation •...•.....•.•..... 317 II. INHIBITORS OF ELONGATION A. Compounds Interfering with Aminoacyl-tRNA Recognition .. 318 a. Inhibitors of EF-1-dependent binding of Aminoacyl-tRNA ...... •...... 319 b. Mi sreading Compounds ...... •...... 321 B. Inhibition of Peptide Bond Formation (Step E) ....•..... 321 C. Inhibitors of Translocation (Step F ) ...... 323 III. INHIBITORS OF TERMINATION ...... •...... 332 IV. GTP ANALOGS ...... •...... •...... 332 V. SELECTIVITY OF INHIBITORS ..•.....•.•...... •.... 333 VI. SPECIFICITY ...... •...•. 333 References ...... •...... •...... •...... 334

Chapter 14: -Induced Shut-Off of Host Specific Protein Synthesis ...... 339 Friedrich KOCH, Gebhard KOCH and Joachim KRUPPE

Introduction .....•...... ••.....•...... 339 I. PHYSIOLOGICAL REGULATION OF PROTEIN SYNTHESIS AT THE LEVEL OF TRANSLATION ...... 343 II. DIFFERENTIAL INHIBITION OF mRNA TRANSLATION BY HYPERTONIC INITIATION BLOCK (HIB) ...... 345 III. COMPARISON OF THE EFFECTS OF HIB AND VIRAL INFECTION ON THE RELATIVE SYNTHESIS OF INDIVIDUAL CELLULAR PROTEINS IN HOST CELLS •..•...... •...... 347 IV. COMPETITION BETWEEN VIRAL AND HOST mRNAs ...... 349 V. EFFECTS OF NUTRITIONAL CONDITIONS ON VIRUS-INDUCED SHUT-OFF ...... •...... 350 VI. ROLE OF INITIATION FACTORS IN SHUT-OFF OF HOST PROTEIN SYNTHESIS ...... 350 VII. ALTERATION IN STATE OF RIBOSOMAL AND CYTOPLASMIC PROTEINS AFTER HIB TREATMENT AND VIRUS INFECTION ...... 351 VIII. CHANGES IN PERMEABILITY OF CELL MEMBRANES AFTER VIRUS ADSORPTION ...... •...... 352 xvi CONTENTS

IX. INHIBITION OF AMINOACID TRANSPORT IN CELLS UPON VIRUS INFECT I ON ...... • ...... 354 X. ROLE OF CELL MEMBRANE IN MEDIATING THE PLEIOTROPIC RESPONSE .•.••.••••...... 356 XI. INTERACTION BETWEEN VIRAL PROTEINS AND CELLULAR CONSTITUENTS ...... •...... 357 Acknowledgement ..•...... ~ .... 357 References ...... 357

SECTION VI: MECHANISMS OF REGULATION AND CONTROL

Chapter 15: The Cytoplasmic Control of Protein Synthesis ...... 363 Richard J. JACKSON

Initial Remarks ...... 363 I. THE MECHANISM OF INITIATION: OUTSTANDING PROBLEMS A. Are Additional Initiation Factors Required 7 •••••••••• 365 B. Are Initiation Factors Specific with Respect to mRNA 7 •••••••••••••••••••••••••••••••••••• 366 C. The Recycling of Initiation Factors ...... 366 D. The Selection of the Initiation Site on mRNA ..•...... 367 E. What is the Role of ATP in Initiation 7 •••••••••••••• 368 II. CONTROL OF TRANSLATION: SPECIFIC EFFECTS A. Poliovirus and Vaccinia Infection ...... 371 B. Heat-Shock in Drosophila ...... 372 C. Untranslated mRNAs in Eggs ...... 373 D. Untranslated mRNAs in Somatic Cells ...... 374 III. CONTROL OF INITIATION IN RETICULOCYTE SYSTEMS A. Control in Intact Cells ...... 377 B. Control in Cell-Free Systems ...... 378 IV. HAEMIN CONTROLS eIF-2 PHOSPHORYLATION A. Mechanism of Action of the Haem-Controlled Inhibitor .. 380 B. Is the Function of eIF-2 Impaired by Phosphorylation 7 ••••••••••••••••••••••••••••••••• 384 C. eIF-2 ...... 386 D. Anti-Inhibitor Proteins ...... 387 E. The Activation of the Haem-Controlled Inhibitor ...... 388 F. High-Pressure and High Temperature Effects ...... 390 V. CONTROL BY DOUBLE-STRANDED RNA A. The d-s RNA-Activated eIF-2 ...... 391 B. The d-s RNA-Activated Oligoisoadenylate Synthetase .... 392 VI. CONTROL BY SUGAR AND REDUCING AGENTS A. Introduction: The Nature of the Problem ...... 394 B. Properties of the Gel-Filtere'd Lysates and 2': 5' ADP Lysates ...... •..... 395 C. Protein Synthesis in Gel-Filtered Lysates ...... •... 397 D. Protein Synthesis in 2':5' ADP Lysates ...... 401 E. Phosphorylation of eIF-2 Is Controlled by Reducing Agent s ...... 405 F. The Effect of Oxidised Glutathione ...... 408 CONTENTS xvii

VII. ARE RETICULOCYTE CONTROL MECHANISMS RELEVANT TO OTHER CELLS ...... •..••••...•...... 409 Acknowledgement ...•...... •.•...... '...... •••.•..•...... 413 Ref'erences ...... •...... •....•...... 413

Chapter 16: Regulation of eIF-2 Activity and Initiation of Protein Synthesis in Mammalian Cells ...... 419 Naba K. GUPTA

Introduct ion ...... ••...... •...... 419 Operat ional Definitions ...... •.....•..•.•.... 419 I. Co-eIF-2A A. Requirement of Co-eIF-2A in Protein Synthesis ...... 421 B. Mechanism of Interaction of Co-eIF-2A with eIF-2 ...... 422 C. Stoichiometry of Co-eIF-2A Binding to eIF-2 ..•.•.....•. 423 D. Co-eIF-2A Confers Stability to the Ternary Complex ..... 423 II. Co-eIF-2B (TDF: TERNARY COMPLEX DISSOCIATING FACTOR A. Millipore Filtration Assay for Met-tRNAf Binding to Ribosomes ( 40s and 40s + 60S ) ...... •.... 425 III. Co-eIF2C ....•...... •.. 426 IV. eIF-2 KINASE ...... ••...... •...... 428 V. sRF ...... •...... •.....•...... 431 Conclusions ...... •...... 435 Acknowledgement s ...... •...... •...... 437 References ...... •...... •...... 437

Chapter 17: Messenger RNA Competition .....•...... •...... •..... 441 Raymond KAEMPFER

I. mRNA DISCRIMINATION vs. mRNA COMPETITION ...... 441 II. TRANSLATIONAL COMPETITION BETWEEN a- and S-GLOBIN mRNAs A. a- and S-Globin mRNAs Differ in Amount and Rate of Initiation of Translation .....•.....•..•..•...... 442 B. Demonstration of mRNA Competition ...... •...... 443 C. eIF-2 Isa Target of mRNA Competition •....•...... 443 D. The Effect of Salt on mRNA Competition ...... •..• 445 E. The Effect of Salt on Binding of mRNA to eIF-2 ...... 446 F. Involvement of Other Initiation Factors ...... 448 G. The High Affinity ofS-Globin mRNA for eIF-2 ...... 450 III. TRANSLATIONAL COMPETITION BETWEEN HOST AND VIRAL mRNAs A. eIF-4B Is a Target of Competition ...... •....•. 450 B. eIF-2 Is a Target of Competition .....•...... •.. 451 IV. REGULATION BY mRNA COMPETITION A. The Role of eIF-2 ...... •.....•...... 452 B. Differentiation and mRNA Competition .•...... •.•..•..... 455 References ...... 455

Chapter 18: Interferon Action: Control of RNA Processing, Translation and Degradation ...... 459 Peter LENGYEL xviii CONTENTS

Introduction .•.••...•.••..•.•.•••.•....•...••....•..•...... 459 I. SURVEY OF INTERFERONS A. Assay •.•...... •.•...... •...•..•...... •. 460 B. Induction .....•....•...... •.•....•...... •...•..... 460 C. Interferon mRNA ..•...... •...... 461 D. Control of Interferon Synthesis •....•..•...... •.•..... 461 E. Mass Production of Human Interferons ...•.•.....•....•.• 462 F. Isolation of Human Interferon •...... ••.• 463 G. Isolation and Structure of Interferons ...... ••..... 464 H. Interferon Action: Establishment of the Antiviral State .•.•.•.•...•...... •.•...... • 465 II. THE INTERFERON-INDUCED A. The (2'-5') (A) Synthetase-RNase L System ...... 467 a. (2'-5') (A) §ynthetase ...... 468 b. Phosphodies~erase Degrading (2'-5') (A) ...... •. 469 c. RN ase L ...•...... •...... •..... r: ...... 469 B. Protein Kinase .....•...••...... ••....•.•..••.•.•.....•. 471 C. Double-Stranded RNA Does Not Have to be "Free" to Activate the Latent Enzymes ...... •..•.... ; •...... 472 D. Possible Rationale for the Multiple Roles of Double-Stranded RNA in Interferon Induct.ion and action. 473 E. Impairment of Exogenous mRNA Translation: The tRNA Effect ...... •...•...... '.' ....•..•...... • 474 III. MESSENGER RNAs AND PROTEINS INDUCED BY INTERFERON ...... 475 IV. Conclusions ...... •...... 475 Footnotes ...•...... •.•...... 477 Acknowledgement s ...... ••...... •.. 477 References ...... •...... •..•..•...... 477

The MARATEA Conferenc'e: List of Participants ...... •...... 485

INDEX ...... '" ...... a .... " ...... " ...... II ...... 493