Protein Biosynthesis in Eukaryotes

vi The word ppotein, coined one and a half century ago from the 1TpOTE:toa ("proteios" = of primary importance), underlines the "primary importance" ascribed to proteins 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 enzymes known to split a protein into its constituent amino acids were thought to be able, under adequate conditions, to reconstitute the peptide bond. 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 gene.

Section I: The Protein Synthesizing Machinery of Eukaryotes.- 1: Structure and Function of tRNA and Aminoacyl tRNA Synthetases.- I. Aminoacylation.- A. Stoichiometry and Energetics.- B. Structure of Aminoacyl-tRNA.- C. Mechanism of Aminoacylation.- II. Structure of tRNA.- A. Multiplicity and Location of tRNA in the Cell.- B. Primary and Secondary Structure.- C. Tertiary Structure.- III. Structure of Aminoacyl tRNA Synthetase.- A. Multiplicity and Cellular Location.- B. Protein Structure.- C. Multi-Enzyme Complexes.- IV. Specificity of Aminoacylation.- A. Importance of Specificity.- B. Amino Acid Recognition.- C. tRNA Recognition.- V. Codon-Anticodon Recognition.- A. Codon Translation in the Cytoplasm.- B. Codon Translation in the Mitochondria.- C. Codon Translation in vitro.- VI. tRNA Recognition by the Eukaryotic Protein Synthesis System.- A. Initiation and Elongation Factors.- B. Rihosomes.- VII. Other Functions of tRNA.- A. tRNA-Like Structure in Viral RNA.- B. Primer for Reverse Transcriptase.- C. Aminoacyl-tRNA Protein Transferase.- D. Regulatory Functions.- VIII. tRNA Biosynthesis.- Acknowledgement.- References.- Appendix: Table 2. Published tRNA Sequences as for August 1, 1980.- 2: The Structure of Eukaryotic Ribosome.- I. General Characteristics of Eukaryotic Ribosomes.- II. Isolation and Characterization of Eukaryotic Ribosomal Proteins.- III. Primary Structure of Eukaryotic Ribosomal Proteins.- IV. RNA-Protein Interactions in Eukaryotic Ribosomes.- A. 5S rRNA.- B. E.8S rRNA.- C. E. coli 5S rRNA.- D. tRNA.- V. CODA.- References.- 3: The Initiation Factors.- I. Identification of the Initiation Factors.- II. Physical Characterization.- III. Covalent Modifications.- IV. Cellular Levels and Biogenesis.- V. Pathway of Initiation.- A. Dissociation of Ribosomes into Subunits.- B. Ternary Complex Formation.- C. Ternary Complex Binding to to 40S Subunits.- D. Binding of mRNA to 40S Subunits.- E. Junction of the 60S Subunit and Formation of the 80S Initiation Complex.- VI. Molecular Mechanism of Initiation.- A. mRNA-Ribosome Interaction.- B. Specific Factors for mRNA?.- C. Ribosomal Sites for Initiation.- Acknowledgements.- References.- Section II: On the Importance of Being Spliced.- 4: Messenger RNA Structure and Biosynthesis.- I. Determination of mRNA secondary structure.- II. Messenger RNA Processing: Historical Background.- III. Modified Nucleotides. — CAP structure.- IV. Sites of Transcriptional Initiation of mRNA.- V. Splicing.- VI. Order of Processing Reactions.- References.- 5: SV40 as a Model System for the Study of RNA Transcription and Processing in Eukaryotie Cells.- I. SV40 as a Model System.- II. Initiation of Transcription Of SV40 DNA Late After Infection.- III. The SV40 Minichromosome.- IV. Splicing of SV40 Late mRNA.- V. Mapping the “Leader” and the “Body” of the Viral mRNAs by Electron Microscopy.- A. Analysis of the DNA-RNA Hybrids.- B. Analysis of the R-Loop Structures.- VI. Models for Joining the “Leader” to the Coding Sequences.- VII. Models for Splicing of mRNA.- A. Splicing Intermediates.- Conclusions.- Acknowledgements.- References.- 6: Messenger Ribonucleoprotein Particles.- I. Biological Properties.- A. Early Developments in Sea Urchin.- B. Differentiating Animal Cells.- C. Non-Differentiating or Terminally Differentiated Mammalian Cells.- II. Isolation and Composition.- III. Translation of mRNPs.- IV. Summary and Conclusions.- Acknowledgement.- References.- Section III: On Selecting the Right Messenger.- 7: Recognition of Initiation Sites in Eukaryotic mRNAs.- I. Characteristics of Initiation Regions in Eukaryotic Messenger RNAs.- II. Mechanisms which have been Proposed to explain Selection of Initiation Sites by Eukaryotic Ribosomes.- III. Evaluation of the “Scanning” Model for Initiation.- A. A Summary of the Evidence.- B. Variations on the Theme.- C. How Can the Exception be Explained.- IV. Questions and Speculations.- A. An Economical Message Might Initiate at the First and Second AUG.- B. Role of the 5?-Terminal Methylated Residues.- C. Determinants of Messenger Efficiency.- D. Translation of Viral Messages.- Acknowledgements/Notes.- References.- 8: A Closer Look at the 5? End of mRNA in Relation to Initiation.- I. Facilitating Effect of the CAP on mRNA translation at the Level of Ribosome Binding.- II. Detection of cap binding Protein by Chemical Cross-Linking to mRNA 5? End.- A. Cap-Binding Activity in Cell-Free Extracts.- B. Cap-Affinity of Initiation Factors.- III. Functional CAP Binding Proteins Purified by m GDP-Sepharose affinity chromatography.- IV. mRNA 5? Region Proximity to 18s Ribosomal RNA in Initiation Complexes.- Acknowle dgement.- References.- 9: Initiation Factor/mRNA Interactions and mRNA Recognition.- I. General Aspects of mRNA Recognition.- II. Approachs to the Study of mRNA/Initiation Factor Interactions.- III. Recognition of mRNA by eIF-.- A. eIF-2 Binds to mRNA.- B. The Untranslated Portion of mRNA and poly(A) Are Not Recognized by eIF-2.- C. Role of the 5?-Terminal Cap and Internal mRNA Sequences in Binding of eIF-2.- D. Specific Binding of eIF-2 to a 5?-Terminal Sequence Comprising the Ribosome-Binding Site.- E. Hole of mRNA Conformation.- F. Alteration of the 5?-Proximal HNA Conformation Induced by Binding of eIF-2.- G. Relationship Between Binding of eIF-2 and Binding of the Ribosome.- H. mRNA Competition for eIF-2 during Translation.- I. Interaction Between eIF-2 and Double-Stranded RNA.- J. Mutually Exclusive Binding of mRNA and Met-tRNAf for eIF-2.- K. eIF-2 and Initiation of Translation.- IV. Binding of other Initiation Factors to mRNA.- Conclusions.- Acknowledgements.- References.- 10: But Is the 5? End of mRNA Always Involved in Initiation?.- I. The Genomic RNA of Picornaviruses.- II. Evidence for More-Than-One Initiation Site in Picornavirus RNA.- III. Involvement of Internal Regions of Picornavirus RNA in Initiation.- IV. Studies on the Ribosome-Binding Sites of Mengovirus RNA.- V. “In Vitro Veritas”.- References.- Section IV: Synthesis and Processing of Proteins.- 11: Peptide Chain Elongation and Termination in Eukaryotes.- I. Binding of Aminoacyl-tRNA to the Ribosomes.- A. Characteristics of EF-1.- B. Assay of EF-1.- C. Purification of IF-1.- a. EF-1H.- b. EF-1L.- c. EF-1?.- D. Interaction of EF-1 with Guanosine Nucleotide and AA-tRNA.- E. Interaction of the Ternary Complex with Ribosomes.- F. Recycling of EF-1.- II. Peptide Bond Formation.- III. Translocation.- A. Elongation Factor 2.- a. Purification and Properties.- b. Assay.- c. Interactions of EF-2 with Guanosine Nucleotides and with Ribosomes.- d. The Inhibition of EF-2 Activity by Diphtheria Toxin.- IV. Termination.- References.- 12: Biosynthesis, Modifications, and Processing of Viral Polyprot eins.- I. The Proteolytic Processing of Picornavirus Proteins.- A. Picornavirus-directed Protein Synthesis.- a. Processing of NCVP1a.- b. NCVPlb, VPg, and Viral RNA Replication.- B. The Effect of Guanidine on the Processing of Viral Proteins.- C. Non-Uniform Synthesis and/or Accumulation of Poliovirus Proteins under Conditions of Restricted Polypeptide Chain Initiation and at Early Time after Infection.- D. Further Characterization of Protease Using Viral Proteins as Substrate: Studies in Cell-Free Systems.- II. Post-Transcriptional Modifications of Oncornavirus-Directed Proteins.- A. Protease Specific of RNA Tumor Viruses.- B. Synthesis and Processing of Viral Proteins in Friend Erythroleukemia Cell Lines.- C. Detection and Processing of Intermediates.- D. Modification and Processing of Viral Precursor Polypeptides during the Induced Differentiation of Friend Cells.- E. Synthesis and Processing of Viral Precursor Proteins under Conditions Inducing Terminal Differentiation.- F. Amplification of Translational Control of Gene Expression during the Differentiation of Friend Cells.- a. Inducers of Differentiation Inhibit Protein Synthesis.- b. Differential Effects of Inhibitors.- G. Effects of Inhibitors of Polypeptide Chain Initiation on the Modifications and Processing of Viral Polyproteins.- Conclusive Remarks.- References.- Section V: Inhibition of Protein Synthesis at Selected Levels.- 13: Action of Inhibitors of Protein Biosynthesis.- I. Translation of mRNA.- A. Inhibitors of Initiation.- B. Inhibitors of the Recognition of the Initiatior Substrate (Step A).- C. Inhibitors of mRNA Recognition (Step B).- D. Inhibitors of Subunit Joining (Step C1).- E. Inhibitors of Positioning in the Donor Site (Step C2).- F. Unclassified Inhibitors of Initiation.- II. Inhibitors of Elongation.- A. Compounds Interfering with Aminoacyl-tRNA Recognition.- a. Inhibitors of EF-1-dependent binding of Aminoacyl-tRNA.- b. Misreading Compounds.- B. Inhibition of Peptide Bond Formation (Step E).- C. Inhibitors of Translocation (Step F).- III. Inhibitors of Termination.- IV. GTP Analogs.- V. Selectivity of Inhibitors.- VI. Specificity.- References.- 14: Virus-Induced Shut-Off of Host Specific Protein Synthesis.- I. Physiological Regulation of Protein Synthesis at the Level of Translation.- II. Differential Inhibition of mRNA Translation by Hypertonic Initiation Block (HIB).- III. Comparison of the Effects of HIB and Viral iInfection on the Relative Synthesis of Individual Cellular Proteins in Host Cells.- IV. Competition Between Viral and Host mRNAS.- V. Effects of Nutritional Conditions on Virus-Induced Shut-Off.- VI. Role of Initiation Factors in sShut-Off of Host Protein Synthesis.- VII. Alteration in Phosphorylation State of Ribosomal and Cytoplasmic Proteins After HIB Treatment and Virus Infection.- VIII. Changes In Permeability of Cell Membranes After Virus Adsorption.- IX. Inhibition of Aminoacid Transport in Cells Upon Virus Infection.- X. Role of Cell Membrane in Mediating the Pleiotropic Response.- XI. Interaction Between Viral Proteins and Cellular Constituents.- Acknowledgement.- References.- Section VI: Mechanisms of Regulation and Control.- 15: The Cytoplasmic Control of Protein Synthesis.- Initial Remarks.- I. The Mechanism of Initiation: Outstanding Problems.- A. Are Additional Initiation Factors Required?.- B. Are Initiation Factors Specific with Respect to mRNA?.- C. The Recycling of Initiation Factors.- D. The Selection of the Initiation Site on mRNA.- E. What is the Role of ATP in Initiation?.- II. Control of Translation: Specific Effects.- A. Poliovirus and Vaccinia Infection.- B. Heat-Shock in Drosophila.- C. Untranslated mRNAs in Eggs.- D. Untranslated mRNAs in Somatic Cells.- III. Control of Initiation in Reticulocyte Systems.- A. Control in Intact Cells.- B. Control in Cell-Free Systems.- IV. Haemin Controls eIF-2 Phosphorylation.- A. Mechanism of Action of the Haem-Controlled Inhibitor.- B. Is the Function of eIF-2 Impaired by Phosphorylation?.- C. eIF-2 Phosphatases.- D. Anti-Inhibitor Proteins.- E. The Activation of the Haem-Controlled Inhibitor.- F. High-Pressure and High Temperature Effects.- V. Control by Double-Stranded RNA.- A. The d-s RNA-Activated eIF-2 Kinase.- B. The d-s RNA-Activated Oligoisoadenylate Synthetase.- VI. Control by Sugar Phosphates and Reducing Agents.- A. Introduction: The Nature of the Problem.- B. Properties of the Gel-Filtered Lysates and 2?: 5? ADP Lysates.- C. Protein Synthesis in Gel-Filtered Lysates.- D. Protein Synthesis in 2?: 5? ADP Lysates.- E. Phosphorylation of eIF-2 Is Controlled by Reducing Agents.- F. The Effect of Oxidised Glutathione.- VII. Are Reticulocyte Control Mechanisms Relevant to Other cells.- Acknowledgement.- References.- 16: Regulation of eIF-2 Activity and Initiation of Protein Synthesis in Mammalian Cells.- Operational Definitions.- I. Co-eIF-2A.- A. Requirement of Co-eIF-2A in Protein Synthesis.- B. Mechanism of Interaction of Co-eIF-2A with eIF-2.- C. Stoichiometry of Co-eIF-2A Binding to eIF-2.- D. Co-eiF-2A Confers Stability to the Ternary Complex.- II. Co-eIF-2B (TDF: Ternary Complex Dissociating Factor.- A. Millipore Filtration Assay for Met-tRNA Binding to Ribosomes (40S and 40S + 60S).- III. Co-eIF2C. b.- IV. eIF-2 KINASE.- V. sRF.- Conclusions.- Acknowledgements.- References.- 17: Messenger RNA Competition.- I. mRNA Discrimination vs. mRNA Competition.- II. Translational Competition Between ?- and ?-Globin mRNAs.- A. ?- and ?-Globin mRNAs Differ in Amount and Rate of Initiation of Translation.- B. Demonstration of mRNA Competition.- C. eIF-2 Is a Target of mRNA Competition.- D. The Effect of Salt on mRNA Competition.- E. The Effect of Salt on Binding of mRNA to eIF-2.- F. Involvement of Other Initiation Factors.- G. The High Affinity of ?-Globin mRNA for eIF-2.- III. Translational Competition Between Host and Viral mRNAs.- A. eIF-4B Is a Target of Competition.- B. eIF-2 Is a Target of Competition.- IV. Regulation by mRNA Competition.- A. The Role of eIF-2.- B. Differentiation and mRNA Competition.- References.- 18: Interferon Action: Control of RNA Processing, Translation and Degradation.- I. Survey of Interferons.- A. Assay.- B. Induction.- C. Interferon mRNA.- D. Control of Interferon Synthesis.- E. Mass Production of Human Interferons.- F. Isolation of Human Interferon Genes.- G. Isolation and Structure of Interferons.- H. Interferon Action: Establishment of the Antiviral State.- II. The Interferon-Induced Translational Regulation.- A. The (2?–5?) (A)n Synthetase-RNase L System.- a. (2?–5?) (A)n Synthetase.- b. Phosphodiesterase Degrading (2?–5?) (A)n.- c. RNase L.- B. Protein Kinase.- C. Double-Stranded RNA Does Not Have to be “Free” to Activate the Latent Enzymes.- D. Possible Rationale for the Multiple Roles of Double-Stranded RNA in Interferon Induction and action.- E. Impairment of Exogenous mRNA Translation: The tRNA Effect.- III. Messenger RNAs and Proteins Induced by Interferon.- IV. Conclusions.- Footnotes.- Acknowledgement.- References.- The Maratea Conferende: List of Participants.