Protein Biosynthesis in Eukaryotes

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.