Structure-Function Relationships of Human Initiator tRNA Mutants and Attempted Regulated Expression of tRNA Genes in Yeast by Dawn C. Farruggio B. S. University of Chicago 1987 Submitted to the Department of Biology in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy at the Massachusetts Institute of Technology September 1995 © 1995 Massachusetts Institute of Technology All rights reserved Signature of Author /' bC: Department of Biology / v , / Cerified by Uttam L. RajBhandary Thesis Supervisor Accepted by ___A_ - 7,- Frank Solomon Chairman.___-- of Departmental Graduate Committee MASSACHUSETTS INSTITUTE OF TECHNOLOGY AUG 1 0 1995 LIBRARIES 1 Scentp Structure-Function Relationships of Human Initiator tRNA Mutants and Attempted Regulated Expression of tRNA Genes in Yeast by Dawn C. Farruggio Submitted to the Department of Biology in September 1995 in partial fulfillment of the requirements for the degree of Doctor of Philosophy Abstract Initiator tRNAs fulfill a special role in initiation of protein synthesis. This requires the initiator tRNA to make specific interactions with several components of the protein synthesis machinery. The specificity of these interactions is thought to be determined by conserved sequences within the tRNA, which are unique to the initiator tRNA species. Part I of this thesis describes a system for obtaining large quantities of wildtype and mutant human initiator tRNAs for use in biochemical assays. I have over- expressed wildtype and mutant (G1:C72, T33, T54C60) human initiator tRNAs in S. cerevisiae. Mutants with changes in the anticodon stem of the human initiator tRNA, A29T3 1:T39T41, were not produced in yeast, although the genes are transcribed by RNA Polymerase III. The wildtype and the G1:C72 mutant human initiator tRNAs were purified and their affinities for initiation factor eIF-2 determined by nitrocellulose filter binding assays. The G1:C72 mutant displayed a 10-17 fold decrease in affinity for eIF-2 compared to wildtype tRNA. Part II of this thesis addresses the question of whether tRNA genes, which are normally transcribed by RNA polymerase III (Pol III), can be produced from an RNA polymerase II (Pol II) promoter. To test for Pol II production of tRNAs in S. cerevisiae, I made use of two genes which are not transcribed by Pol III in yeast. The human initiator gene is not usually transcribed in yeast in the context of its own flanking sequences, however I found that a dimeric version of the human initiator gene was activated for Pol III transcription in some sequence contexts. A yeast amber suppressor tyrosine tRNA with a A53T61 change in its internal promoter is not transcribed by Pol III. Transcripts of this gene were made from an inducible Pol II promoter in a regulated manner, but were not processed to mature tRNA. The Pol II transcripts were polyadenylated and were found in both the nuclear and cytoplasmic cell compartments. T7 RNA polymerase transcripts containing the tRNA sequences could be processed to mature tRNA in vitro, even if capped and polyadenylated. Possible explanations for the lack of processing of the Pol II tRNA transcripts are discussed. Thesis supervisor: Uttam L. RajBhandary Title: Professor of Biology 2 Acknowledgments I would like to thank my advisor, Tom RajBhandary, for all the guidance and assistance he has provided to me throughout the course of this work and in the preparation of this thesis. I would also like to especially thank Harold Drabkin and Ming Chow for giving generously of their time, experience, and reagents, as well as for many interesting discussions of Star Trek, politics and science. Thanks are due to all members of the RajBhandary laboratory for their support and suggestions. In particular I would like to thank Ho-Jin Park and Louise Hancox, for their proofreading services as well as scientific discussions. Also Dev Mangroo, Xin-Qi Wu, Umesh Varshney, Shihong Li, Chan-Ping Lee, and Mike Dyson deserve thanks for their contributions of advice and reagents. My thanks also to Gobind Khorana and the members of his laboratory for their encouragement, advice, and many lively seminars. This work would not have been possible without the support of the many friends I have made at MIT, too numerous to be named here, for discussions, encouragement, and assistance in finding what was needed. I would like to acknowledge my parents, Betty and Jerry Farrell, for the encouragement and assistance they have given me. Finally, and most of all, I would like to thank my husband, Tony, for his unwavering belief that it was possible, his assistance with problems related to doing science, and the many late nights he kept me company at the lab. 3 Table of Contents Abstract 2 Acknowledgments 3 Table of Contents 4 List of Figures 9 General abbreviations 13 Part I: Structure-Function Relationships of Human Initiator tRNA Mutants 1. Introduction 1.1 tRNA and its role in initiation of protein synthesis 16 1.2 The prokaryotic system 19 1.3 The eukaryotic system 23 1.4 Eukaryotic initiation factor 2 (eIF-2) 27 1.5 Eukaryotic elongation factor la (eEF-lct) 29 1.6 In vivo studies of eukaryotic initiator tRNAMet 30 1.7 In vitro studies of eukaryotic initiator tRNAMet 31 2. Results 2.1. Production of wildtype and mutant human initiator tRNAs in yeast 33 2.1.1. System for expression of the human initiator tRNA in yeast 2.1.2 Over-expression from plasmid pMP78-1 2.1.3 The human initiator tRNAs are aminoacylated in vivo in yeast 2.2 The anticodon stem mutant A29T31 :T39T41 49 2.2.1 The A29T31:T39T41 mutant is made in HeLa cell extracts and is stable 2.2.2 Additional changes in the A29T31 :T39T41 human initiator tRNA gene do not allow full expression of this mutant tRNA in yeast 2.3 Purification of WT and G1 :C72 mutant human initiator tRNA 70 2.3.1 Growth of yeast strains expressing the human initiator tRNA by fed- batch fermentation 2.3.2 Isolation of human tRNA expressed in yeast 2.4 Binding of human initiator Met-tRNA to eIF-2 84 2.4.1 Analysis of GDP in eIF-2 samples 2.4.2 Determination of assay conditions for binding of initiator Met-tRNA to eIF-2 2.4.3 Determination of dissociation constants for binding of human initiator tRNA to eIF-2 4 2.5 Binding of human initiator Met-tRNA to eEF- la 100 3. Discussion 3.1 Over-expression and purification of the wildtype and mutant human initiator tRNAs 108 3.2 Aminoacylation of human initiator tRNA in vivo in yeast 109 3.3 A29T31 :T39T41 mutant human initiator tRNAs 109 3.4 Comparison of binding affinities of eIF-2 for wildtype and mutant human initiator tRNAs 110 Part II: Attempted Regulated Expression of tRNA Genes in Yeast 4. Introduction 4.1 Eukaryotic nuclear RNA polymerases 114 4.1.1 RNA polymerase I 4.1.2 RNA polymerase II 4.1.3 RNA Polymerase HI 4.2 tRNA processing in eukaryotes 116 4.3 Switching the promoter specificity of genes 119 4.4 Regulated expression of tRNA genes 121 5. Results 5.1 Constructs with the human initiator gene under the control of a constitutive promoter 123 5.1.1 Monomeric tRNA gene constructs 5.1.2 Dimeric tRNA gene constructs 5.2 Constructs with the human initiator gene under the control of inducible Pol II promoters 133 5.3 Amber suppressor tRNATYr constructs 158 5.3.1 Suppression by tRNAam G53C61 5.3.2 Pol II transcripts of tRNAam A53T61 are made but are not processed to mature tRNA 5.3.3 Analysis of SctRNAam* Pol II transcripts 5.3.4 T7 transcripts of SctRNAam can be processed in vitro to give a tRNA- like product 5.3.5 Pol II specific features, such as the presence of a 5' cap and poly(A) tail, do not interfere with processing of pGAcen-dY*f T7 transcripts in vitro 5.3.6 Fractionation of yeast cells indicates that SctRNAam transcripts are 5 present in nuclei and in cytoplasm 5.3.7 "Minimal" constructs yield smaller Pol II SctRNAam transcripts which are still not processed in vivo 6. Discussion 6.1 Human initiator tRNA constructs 204 6.2 Amber suppressor tRNA gene constructs 205 6.3 Processing of tRNA from Pol II transcripts 206 6.3.1 Length 6.3.2 Pol II specific features 6.3.3 Localization 6.4 Processing of transcripts obtained from polymerase switching experiments 208 Part II: Appendices Appendix A: Materials and Methods A.1 Materials 211 A.2 Strains used in this work 211 A.3 Oligonucleotides 212 A.4 Plasmids 214 A.5 DNA protocols 217 A.5.1 Plaque hybridization A.5.2 Purification of DNA from low melting agarose A.5.3 Double-stranded DNA sequencing A.6 E. coli procedures 218 A.6.1 Preparation of E.coli competent cells A.6.2 Plasmid DNA mini-preps from E. coli A.6.3 Large scale plasmid preparations A.7 Yeast procedures 220 A.7.1 Yeast transformations A.7.2 Growth on plates of HEY301-129 strains A.7.3 Growth of yeast in liquid media A.7.4 Isolation of yeast DNA A.7.5 Preparation of total yeast tRNA A.7.6 Preparation of total yeast RNA and poly(A)+ RNA A.7.7 Fractionation of yeast cells 6 A.7.8 Fed-batch fermentation of yeast A.7.8 Large-scale tRNA extraction A.8 Southern blotting 223 A.9 RNA procedures 224 A.9.1 Formaldehyde agarose gel electrophoresis of RNA A.9.2 Northern blots A.9.3 Primer extension analysis A.9.4 S1 Nuclease protection assay A.10 tRNA purification procedures 226 A.