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Rationale for the Evolutionary Retention of Two

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Rationale for the Evolutionary Retention of Two RATIONALE FOR THE EVOLUTIONARY RETENTION OF TWO UNRELATED LYSYL-tRNA SYNTHETASES DISSERTATION Presented in Partial Fulfillment of the Requirements for The Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Sandro Fernandes Ataide, M.S. ***** The Ohio State University 2006 Dissertation Committee: Professor Michael Ibba, Adviser Professor Juan Alfonzo Approved by Professor Irina Artsimovitch Professor Mark Foster ______________________________ Adviser Graduate Program in Microbiology ABSTRACT Lysine insertion during coded protein synthesis requires lysyl-tRNALys, which is synthesized by lysyl-tRNA synthetase (LysRS). Two unrelated forms of LysRS are known: LysRS2, which is found in eukaryotes, most bacteria and a few archaea, and LysRS1, which is found in most archaea and a few bacteria. A detailed comparison of the amino acid recognition strategies of LysRS1 (Borrelia burgdorferi) and LysRS2 (Escherichia coli) was undertaken by studying the effects of lysine analogues on the aminoacylation reaction in vitro and in vivo. Also, based on comparisons of crystal structures and discrimination of lysine analogues by both LysRSs, the roles of the key residues in the active site of LysRS2 (lysS encoded) from E. coli were determined in vitro and in vivo. The differences in resistance to naturally occurring non-cognate amino acids suggest the distribution of LysRS1 and LysRS2 contributes to quality control during protein synthesis. LysRS1 and LysRS2 are not normally found together within one organism. From more than 250 publicly available genome sequences, the only instances where both LysRS1 and LysRS2 are found together are the Methanocarcineae in the archaea and certain Bacilli among the bacteria. It was shown that Methanosarcina barkeri LysRS1 and LysRS2 can together aminoacylate the rare tRNAPyl species, although the role of this ii in vitro activity remains unclear in vivo. In the pathogen Bacillus cereus, both forms of LysRS are also encoded, but genome sequence analysis does not identify tRNAPyl or any other components of the pyrrolysine insertion pathway. To investigate what role the two LysRSs might play in B. cereus, their RNA substrate specificities were investigated. It was found that in B. cereus the two different LysRSs together aminoacylate a small RNA of unknown function named tRNAOther, and that the aminoacylated product stably binds translation elongation factor Tu. In vivo analyses revealed that the class 2 LysRS was Other present both during and after exponential growth, while the class I enzyme and tRNA Other were predominantly produced during stationary phase. Aminoacylation of tRNA was also found to be confined to stationary phase, suggesting a role for this non-canonical tRNA in growth phase-specific protein synthesis. Analysis of the non-canonical Watson- Crick base pairs and a bulge in the acceptor stem of tRNAOther, present in the predicted secondary structure of tRNAOther, indicate the importance of these identity elements in recognition by the LysRS1:LysRS2 complex. The role of tRNAOther in B. cereus 14579 was investigated by the construction of a deletion strain. Comparison of the deletion strain with B. cereus wild type (wt) indicates that tRNAOther is not an essential gene but its absence de-regulates both the production of a Bactericin-Like Inhibitory Substance (BLIS) and the macrolide efflux protein conferring resistance against other bacterial macrolides. Also, other secondary metabolic effects were observed such as loss of resistance against certain compounds in the deletion strain. These results implicate tRNAOther in multiple regulatory functions that remain, as yet, uncharacterized. iii Dedicated to my family, especially my wife, Daniele, and my parents, Luiz and Maria Helena iv ACKNOWLEDGMENTS I wish to express my sincere gratitude to my advisor, Dr. Michael Ibba, for his superb guidance, generous support, encouragement and endless patience in correcting my stylist errors throughout the years. It has been an honor to work with such a talented scientist and excellent mentor. I would like to thank my committee members, Dr. Juan Alfonzo, Dr. Irina Artsimovitch, and Dr. Mark Foster, for their insight, support and time over the years. I would like to thank Corinne Hausmann for providing me with her support, valuable feedback, and most importantly her friendship throughout the years. I thank Jiqiang Ling for the insightful discussions and valuable feedback throughout the years. I thank the past and present members of the Ibba laboratory who I have worked with for providing a comfortable and intellectually stimulating environment: Mette Prætorius-Ibba, Jeffrey Levengood and Hervé Roy for all the support and insightful discussions; Sandy Dang for her patience to work with me and Theresa Rogers for her insightful comments and critical review. I thank my wonderful wife Daniele who has helped me throughout this entire process providing me with endless support. v This work was supported by a Pre-doctoral Fellowship from the American Heart Association and a grant from the National Institutes of Health. vi VITA December 14, 1976………………………Born – Mirandopolis, Brazil 1999………………………………………B.S. Chemistry, University of Campinas 1999 - 2001……………………………….M.S. Biochemistry, University of Sao Paulo 2002 – present…………………………….Graduate Teaching and Research Associate, The Ohio State University PUBLICATIONS 1. Ataide, S. F. and Ibba, M. (2006) Small Molecules – Big Players in the Evolution of Protein Synthesis. ACS Chem. Biol. 1:285-297. 2. Wang, S., Prætorius-Ibba, M., Ataide,S.F., Roy, H., and Ibba, M. (2006), Discrimination of cognate and non-cognate substrates at the active site of class I lysyl- tRNA synthetase. Biochemistry 45:3646-3552. 3. Prætorius-Ibba, M., Ataide, S.F., Hausmann, C., Levengood, J.D., Ling, J., Wang, S., Roy, H., and Ibba, M. (2006) Quality Control during aminoacyl-tRNA synthesis. Kem. Ind. 55:129-134 4. Ataide,S. F., Jester, B.C., Devine, K.M., Ibba, M. (2005) Stationary-phase expression and aminoacylation of tranfer-RNA-like small RNA. EMBO Reports 6:742- 747. 5. Ataide, S. F. and Ibba, M. (2004) Discrimination of cognate and noncognate substrates at the active site of class II lysyl-tRNA synthetase. Biochemistry 43:11836- 11841. vii 6. Levengood, J., Ataide, S. F.*, Roy, H., Ibba, M. (2004) Divergence in noncognate amino acid recognition between class I and class II lysyl-tRNA synthetases. J.Biol.Chem. 279:17707-17714. (* joint first author) 7. Polycarpo, C., Ambrogelly, A., Ruan, B., Tumbula-Hansen, D., Ataide, S. F., Ishitani, R., Yokoyama, S., Nureki, O., Ibba, M., Söll, D. (2003) Activation of the pyrrolysine suppressor tRNA requires formation of a ternary complex with class I and class II lysyl-tRNA synthetases. Mol. Cell 12:287-294. FIELDS OF STUDY Major Field: Microbiology viii TABLE OF CONTENTS Page Abstract……………………………………………………………………………………ii Dedication...………………………………………………………………………………iv Acknowledgments…………………………………………………………………………v Vita……………………………………………………………………………………....vii List of Tables…………………………………………………………………………….xii List of Figures…………………………………………………………………………...xiii List of Symbols/Abbreviations………………………………………………………….xvi Chapters: 1. Introduction………………………………………………………………………..1 1.1. Aminoacyl-tRNA synthetases and translation………………………………..4 1.2. Amino acid discrimination in aaRS active sites……………………………...7 1.3. Amino acid discrimination in editing sites………………………………….17 1.4. Heterogeneity in alternative aa-tRNA synthesis…………………………….21 1.5. Discrimination of small molecules through aaRS duplication……………...24 1.6. Class I Lysyl-tRNA synthetase……………………………………………...26 1.7. Class II Lysyl-tRNA synthetase……………………………………………..29 1.8. tRNALys……………………………………………………………………...32 2. Divergence in noncognate amino acid recognition between class I and class II lysyl-tRNA synthetases………………………………………………….36 2.1. Introduction………………………………………………………………….36 2.2. Materials and methods………………………………………………………38 2.2.1. Lysyl-tRNA synthetase purification………………………………38 2.2.2. Aminoacylation assays…………………………………………….39 2.2.3. Ki Determination…………………………………………………..40 ix 2.2.4. ATP-PPi exchange reaction……………………………………….40 2.2.5. In vivo growth inhibition…………………………………………..41 2.2.6. In vivo analysis of LysRS2 variants……………………………….41 2.2.7. Random mutagenesis of LysRS2 variants………………………...43 2.2.8. Detection of LysRS2 variants by immunoblotting………………..44 2.3. Results……………………………………………………………………….44 2.3.1. Inhibition of LysRS1 and LysRS2 catalyzed in vitro aminoacylation…………………………………………………….44 2.3.2. Active site homology plots………………………………………..49 2.3.3. Growth inhibition by L-lysine analogues…………………………51 2.3.4. Selection and characterization of LysRS2 variants……………….52 2.3.5. Discrimination of L-lysine analogues by LysRS2 variants……….56 2.3.6. Activation of AEC………………………………………………...58 2.3.7. Cell viability with LysRS2 variants……………………………….60 2.3.8. In vivo AEC resistance by LysRS2 variants………………………64 2.3.9. Screening for enhanced AEC resistance in LysRS2 variants……..68 2.3.10. Characterization of the protein level of LysRS2 variants in E. coli PAL∆S∆UTR ……………………………………………69 2.4. Discussion…………………………………………………………………...72 2.4.1. Comparison of amino acid discrimination by LysRS1 and LysRS2…………………………………………………………….72 2.4.2. LysRS1 displays a narrower substrate spectrum than LysRS2……76 2.4.3. Functional consequences of divergent recognition of non-cognate amino acids…………………………………………..78 2.4.4. Specific recognition of L-lysine in the LysRS2 active site………..79 2.4.5. Substrate specificity determinants in LysRS2…………………….83
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