THE EVOLUTIONARILY CONSERVED ARGININE ATTENUATOR PEPTIDE REGULATES THE MOVEMENT OF RIBOSOMES THAT HAVE TRANSLATED IT Zhong Wang B .S ., Fudan University, 1990 M. S., Institute of Microbiology, Chinese Academy of Sciences, 1993 A dissertation submitted to the faculty of the Oregon Graduate Institute of Science and Technology in partial fulfillment of the requirements for the degree Doctor of Philosophy in Biochemistry and Molecular Biology August 2000 The dissertation "The Evolutionarily Conserved Arginine Attenuator Peptide Regulates the Movement of Ribosomes That Have Translated It" by Zhong Wang has been examined and approved by the following Examination Committee: Matthew S. Sachs, Advisor Associate Professor - James M. creg&/ Professor Peter Zuber Professor Alan B. Sachs Associate Professor University of California, Berkeley ACKNOWLEDGMENTS I wish to express my deep gratitude to my thesis advisor, Dr. Matthew S. Sachs, who made my Ph.D. studies full of inspiration and enjoyment. Matt taught me how to do experiments step by step when I first came to the lab. And making cell- free translation extract with him in and out of the cold room is one of the greatest things in my graduate period. What impressed me most about Matt is his striving for perfection in every detail of the lab's scientific research. He is truly everything a wonderful thesis advisor could be. Matt's dedicated guidance and instructions are essential for the completion of this work. Many thanks to the members of the dissertation committee, Drs. James Cregg, Alan Sachs, and Peter Zuber. Special thanks to Dr. Adam Geballe at the Fred Hutchinson Cancer Research Center in Seattle. Adam and Alan have provided technical assistance and much input for this research. I also am indebted to the members of the Sachs lab, especially to my fellow graduate students Peng Fang and Anthony Gaba, who helped me a lot with this research and made the lab a big family. Thanks to the members of the labs of Drs. James Cregg, Michael Gold, and Gebretateos Woldegiorgis for their technical support and encouragement. Special thanks to Drs. Geoff and Joan Lin Cereghino in the Cregg lab for their help with my technical writing. Heartfelt thanks to our department administrators Nancy Christie and Terrie Hadfield for everything they have done for me at OGI. This work would have been quite impossible without the support of my dear wife, Guiqin Zhang. She truly understands what are the important things to me and appreciates my success. For that, I dedicate this thesis to her. TABLE OF CONTENTS ACKNOWLEDGMENTS .................................. iii TABLEOFCONTENTS ................................... iv ... LIST OF TABLES ..................................... viii LIST OF FIGURES ...................................... ix ABSTRACT .......................................... xii CHAPTER 1 INTRODUCTION .............................. 1 1.1 The Mechanism of Eukaryotic Translation .....................2 1.1.1 Initiation: Scanning Model ......................... 4 1.1.2 Elongation and Termination ........................ 11 1.2 Translational Control in Eukaryotes . Why Control Translation? ....... 14 1.3 Global and Specific Control of Translation .................... 15 1.3.1 Specific Control of Translation ...................... 15 1.3.2 Global Control of Translation ....................... 24 1.4 uORF-Mediated Translational Control ....................... 29 1.4.1 Coding Sequence-Independent uORFs .................. 29 1.4.2 Coding Sequence-Dependent uORFs ................... 33 1.4.3 Other uORFs That Have Important Biological Functions ....... 35 1.4.4 N. crassa arg-2 and S . cerevisiae CPA 1 ................. 36 CHAPTER 2 ARGININE-SPECIFIC REGULATION MEDIATED BY THE NEUROSPORA CRASSA ARG-2 UPSTREAM OPEN READING FRAME IN A HOMOLOGOUS. CELL-FREE IN VITRO TRANSLATION SYSTEM ..................... 42 2.1 Introduction ...................................... 42 2.2 Experimental Procedures ...............................44 2.2.1 Preparation of Templates Containing Wild-Type and Mutant arg-2 Sequences ...............................44 2.2.2 Preparation of Synthetic RNA Transcripts ................ 44 2.2.3 Preparation of Cell-Free Extracts for Translation ............ 46 2.2.4 Cell-Free Translation and Analyses of Translation Products ..... 47 2.3 Results .........................................48 2.3.1 Characterization of the N. crassa Cell-Free Translation System ... 48 2.3.2 Effects of Upstream Open Reading Frames on Translation ...... 55 2.4 Discussion .......................................62 CHAPTER 3 RIBOSOME STALLING IS RESPONSIBLE FOR ARGININE-SPECIFIC TRANSLATIONAL ATTENTION IN NEUROSPORA CRASSA ............................ 65 3.1 Introduction ...................................... 65 3.2 Materials and Methods ................................ 68 3.2.1 Preparation of Templates Containing Wild-Type and Mutant arg-2 Sequences ............................... 68 3.2.2 Preparation of Synthetic RNA Transcripts ................ 69 3.2.3 Cell-Free Translation and Analyses of Translation Products ..... 69 3.2.4 Preparation of 5' 32P-LabeledPrimers for Toeprinting and Sequencing Reactions ............................ 69 3.2.5 Primer Extension Inhibition (Toeprint) Assays ............. 70 3.3 Results .........................................71 3.3.1 Translational Arrest Mediated by the arg-2 uORF and Arg ..... 71 3.3.2 The Effects of Limiting Protein Synthesis ................ 77 3.3.3 The Effects of uORF Mutations on the Distribution of Ribosomes ................................... 81 3.3.4 The Effect of Arg is Rapid ......................... 85 3.4 Discussion ....................................... 88 3.4.1 Possible Mechanisms for Arg-Specific Translational Control ..... 89 3.4.2 Relation of arg-2 uORF Regulation to Other Regulatory Phenomena .................................. 93 CHAPTER 4 THE EVOLUTIONARILY CONSERVED EUKARYOTIC ARGININE ATTENUATOR PEPTIDE REGULATES THE MOVEMENT OF RIBOSOMES THAT HAVE TRANSLATED IT ... 95 4.1 Introduction ......................................95 4.2 Materials and Methods ................................98 4.2.1 Construction of Templates for RNA Synthesis ............. 98 4.2.2 Cell-Free Translation of RNA and Analyses of Translation Products .................................... 98 4.2.3 Primer Extension Inhibition (Toeprint) Assays ............ 101 4.3 Results ........................................ 102 4.3.1 Effects of Reducing the Distance Between the uORF Termination Codon and the Downstream Initiation Codon ..... 102 4.3.2 Effects of Altering the uORF Termination Codon .......... 105 4.3.3 Effects of Fusing the arg-2 uORF Peptide Directly to Luciferase .................................. 105 4.4 Discussion ...................................... 116 CHAPTER 5 A HIGHLY CONSERVED MECHANISM OF RIBOSOME STALLING MEDIATED BY FUNGAL ARGININE ATTENUATOR PEPTIDES THAT APPEARS INDEPENDENT OF THE CHARGING STATUS OF ARGINYL-tRNAs ........................ 120 5.1 Introduction .....................................120 5.2 Experimental Procedures .............................. 124 5.2.1 Templates for RNA Synthesis ...................... 124 5.2.2 Cell-Free Translation and Primer Extension Inhibition (Toeprint) Analyses ............................ 127 5.2.3 Measurement of tRNA Aminoacylation ................ 128 5.3 Results ........................................ 130 5.3.1 AAP-Mediated Arg-Specific Translational Attenuation in Three Cell-Free Translation Systems .................. 130 5.3.2 Ribosomal Stalling in High Arg Is Mediated by the Wild- Type CPAI and arg-2 AAPs ....................... 131 5.3.3 Arg-Specific Regulation Appears Independent of the Charging Status of Arginyl-tRNAs ................... 138 5.4 Discussion ...................................... 143 CHAPTER 6 CONCLUSIONS AND RJTURE DIRECTIONS ......... 148 6.1 Summary of Research ............................... 148 6.1.1 Development of an Amino Acid-Dependent Cell-Free Translation System in Which the Arginine-Specific Regulation of N. crassa arg-2 Is Fully Reconstituted ........ 148 6.1.2 Introduction of a Sensitive Assay Called "Toeprinting" (Primer Extension Inhibition) into This Cell-Free Translation System ... 149 6.1.3 Investigation of the Requirements for N. crassa arg-2 uORF Function in Arg-Specific Regulation .................. 150 6.1.4 Investigation of the Generality of This AAP-Mediated Regulation, Through a Collaborative Effort with Fellow Student Anthony Gaba .......................... 150 6.2 Future Directions .................................. 151 6.2.1 Do Arg and AAP Interact with Each Other to Cause Ribosomal Stalling? ................................... 151 6.2.2 What Is the Target(s) for AAP and Arg? ............... 152 6.2.3 Applications of Toeprinting in Vitro for Studying Other Events of Translation ........................... 153 LITERATURECITED ................................... 155 BIOGRAPHICAL SKETCH ................................ 191 vii LIST OF TABLES 1.1 Translation Factors and Their Functions ..................... 25 1.2 uORFs That Are Under Investigation for Their Possible Roles in Translational Control .................................37 4.1 Firefly Luciferase Constructs Used in This Study ............... 100 5.1 Firefly LUC Constructs Used in This Study and Their Regulation by Arg in Different Extracts ............................. 125 viii LIST OF FIGURES A diagram of structural elements in eukaryotic rnRNAs that can influence the process of translation and translational control