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Functional Studies of Transfer Rna Interactions in The FUNCTIONAL STUDIES OF TRANSFER RNA INTERACTIONS IN THE RIBOSOME DISSERTATION Presented in Partial Fulfillment of the Requirements for The Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Sarah Elizabeth Walker, B.S. ***** The Ohio State University 2008 Dissertation Committee: Professor Kurt Fredrick, Advisor Approved by Professor Juan Alfonzo Professor Mark Foster ___________________________ Advisor Professor Michael Ibba Graduate Program in Microbiology ABSTRACT During translation, tRNAs must move rapidly to their adjacent sites on the ribosome, while maintaining precise pairing with mRNA. In this work, the interactions of tRNA with the ribosome were examined in order to gain insights into the mechanisms by which the ribosome maintains the correct reading frame, and those interactions which contribute to rapid translocation. Mutant tRNAs with expanded anticodon loops have been isolated, which are capable of decoding a four-nucleotide sequence of mRNA. Using an in vitro assay with purified components, the position of mRNA was mapped with respect to the ribosome to determine the molecular basis by which these expanded-anticodon loop tRNAs recognize and position a quadruplet frameshift sequence. The results of these experiments suggest a new model for frameshift suppression by tRNAs with expanded anticodon loops, and provide insights into how the ribosome normally maintains the reading frame (Chapter 2). Movement of tRNAs through the ribosome is thought to take place in a stepwise manner, with hybrid intermediates. This work demonstrates that movement of tRNA into one of these hybrid-tRNA binding sites, the P/E site, destabilizes the mRNA (Chapter 3). In order to determine how these hybrid states relate to kinetically defined events of ii translocation, substitutions in tRNA or rRNA were examined which inhibited movement into either the P/E site or the A/P site. These data suggest that movement of tRNA into the P/E and A/P sites are separable events and allow tRNA movements with respect to both subunits to be integrated into a kinetic model for translocation (Chapter 4). iii Dedicated to H. Norman Walker iv ACKNOWLEDGMENTS I would first like to express my gratitude to Kurt Fredrick for his time and dedication as an advisor, optimistic attitude, and commitment to thoroughly scrutinizing lab work, presentations, and writing. I feel fortunate to have been the first student to join the lab and to be given so many opportunities to pursue interesting projects, present data, and learn different techniques. I am also appreciative of my committee: Juan Alfonzo, Mark Foster, and Michael Ibba, for holding me to high standards during committee meetings and classes, and for all their time and support. In addition, I thank Dr. Ibba for discussion of the results in Chapter 4, and the members of his and Karin Musier-Forsyth’s labs for thoughtful questions and suggestions during journal club and joint group meetings. In particular, Hervé Roy assisted with the stopped-flow and fluorometer, and provided an endless supply of tRNA –CA nucleotidyl transferase. I also thank Barry Cooperman (U Pennsylvania) and his lab (especially Dongli Pan) for hosting me in Philadelphia, sharing advice on measuring the GTPase activity of EF-G, and performing and editing kinetic modeling on the model proposed in Chapter 4. Dr. Cooperman also made important suggestions in revising the PNAS manuscript. v Thanks is also given to current and former members of the Fredrick lab for sharing data and for assistance on various projects: specifically Nimo Abdi for constructing the SQZ10 strains (Chapter 3); Kevin McGarry for performing Quikchange mutagenesis and collaborating to collect a full data set on our overlapping projects (as noted and discussed in Chapter 3 and published in Molecular Cell); Ichi Shoji for taking on the reverse translocation project and for performing kinetic modeling on the results of Chapter 4 (published in Molecular Cell and PNAS); Xiaofen Zhong for developing an in vitro translation system used to compare activity of mutant ribosomes (data not discussed). I also thank Daoming Qin for discussions, critical reviews, and for sharing his story about pulling the safety shower, and Aishwarya Devaraj and Sean McClory for their friendship over the past year. I am grateful for my friends in Ohio and Texas and mine and Joe’s families, who were a source of understanding and encouragement. Finally, I thank Joseph Barbi for critical review of presentations and writing, and continuous encouragement and support throughout graduate school: scientific, technical, and otherwise. The following provided reagents: Haru Asahara (UC-Santa Cruz) provided the plasmid encoding tRNAAla2 and Catherine Squires and Selwyn Quan (Tufts U.) provided strain SQZ10. Financial support was provided by NIH grant RO1 GM072528 (Kurt Fredrick), a Herta Camerer Gross Graduate Research Fellowship from the OSU College of Biological Sciences, and a teaching assistantship and travel funds from the OSU Department of Microbiology. vi VITA April 14, 1980 .................................Born–Victoria, Texas, United States 2002.................................................B. S. Biology, Texas A&M University-Corpus Christi 2003.................................................Research Technician, University of Missouri 2003-present....................................Graduate Teaching and Research Associate, The Ohio State University PUBLICATIONS 1. Walker, S. E., Shoji, S., Pan, D., Cooperman, B.S., and K. Fredrick. 2008. Role of hybrid-tRNA binding states in ribosomal translocation. PNAS, 105, 9192-9197. 2. Walker, S. E. and K. Fredrick. 2008. Preparation and evaluation of acylated tRNAs. Methods 44: 81-86. 3. Shoji, S., Walker, S.E., and K. Fredrick. 2006. Reverse translocation of tRNA in the ribosome. Mol. Cell 24, 931-942. 4. Walker, S.E. and K. Fredrick. 2006. Recognition and positioning of mRNA in the ribosome by tRNAs with expanded anticodons. J. Mol. Biol. 360: 599-609. 5. McGarry, K.G.*, Walker, S.E.*, Wang, H. and K. Fredrick. 2005. Destabilization of the P site codon-anticodon helix results from movement of tRNA into the P/E hybrid state. Mol. Cell 20, 613-622. (*Authors contributed equally) vii FIELDS OF STUDY Major Field: Microbiology viii TABLE OF CONTENTS Page Abstract............................................................................................................................... ii Dedication.......................................................................................................................... iv Acknowledgments............................................................................................................... v Vita.................................................................................................................................... vii List of Tables ................................................................................................................... xiii List of Figures.................................................................................................................. xiv List of Symbols/Abbreviations ....................................................................................... xvii Chapters: 1. Introduction............................................................................................................. 1 1.1. Overview of translation.................................................................................... 1 1.2. Ribosome structure .......................................................................................... 4 1.3. tRNA interactions during translation............................................................. 13 1.4. Models of translocation.................................................................................. 16 1.4.1. Thermodynamics of translocation ....................................................... 16 1.4.2. Kinetics of translocation...................................................................... 17 1.4.3. Structural basis for translocation......................................................... 21 2. Recognition and positioning of mRNA within the ribosome by tRNAs with expanded anticodons............................................................................................. 24 2.1. Introduction.................................................................................................... 24 2.2. Materials and Methods................................................................................... 28 2.2.1. Reagents .............................................................................................. 28 2.2.2. Toeprinting .......................................................................................... 29 ix 2.2.3. Filter Binding ...................................................................................... 29 2.3. Results............................................................................................................ 30 Ala2 Ala2 2.3.1. Positioning of mRNA by tRNA ACCG and tRNA GCCG bound to the P site ............................................................................................................. 30 Ala2 2.3.2. Translocation of tRNA ACCG from the A site to the P site.................... 38 2.3.3. Translocation of ASL Ala1B and ASL Val1 from the A site to the P site €ACCG ACCG€ ............................................................................................................. 42 2.3.4. Codon dependence of ASL
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