Beyond Mistranslation: Expanding the Role of Aminoacyl-tRNA Synthetases towards the Maintenance of Cellular Viability DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Kyle Phillip Mohler Graduate Program in Microbiology The Ohio State University 2017 Dissertation Committee: Professor Michael Ibba, Advisor Professor Juan Alfonzo Professor Irina Artsimovitch Professor Kelly Wrighton Copyrighted by Kyle Phillip Mohler 2017 Abstract When the sequence of amino acids in a newly synthesized protein is not the same as the genetically encoded sequence, a gene is said to have been mistranslated. Alterations in protein sequence resulting from errors in genome maintenance and expression occur with low frequency. The next step in gene expression, protein synthesis, offers the greatest opportunity for errors, with mistranslation events routinely occurring at a frequency of ~1 per 10,000 mRNA codons translated. The translation of genetic information into functional proteins is a multistep process. Aminoacyl-tRNA synthetases (aaRSs) provide the cell with substrates for protein synthesis by correctly pairing amino acids with their cognate tRNAs. Aminoacylation occurs in a two-step reaction. First, cognate amino acid is activated within the catalytic domain to form an aminoacyl-adenylate (aa-AMP). The activated amino acid is then transferred to the 3’ OH of the terminal adenosine on the tRNA acceptor stem of its cognate tRNA, forming an aminoacyl-tRNA (aa-tRNA). Phenylalanyl-tRNA synthetase (PheRS), for example, is responsible for pairing phenylalanine (Phe) with tRNAPhe. Mispaired aa-tRNA species occasionally arise due to a lack of adequate amino acid discrimination within the PheRS active site, resulting in the synthesis of misacylated Tyr- tRNAPhe. The ability of PheRS to misacylate tRNAPhe with non-cognate amino acid makes ii tRNA proofreading (“editing”) mechanisms of PheRS essential to maintaining the accuracy of translation. In eukaryotes, amino acid starvation activates the protein kinase Gcn2p, which leads to changes in gene expression and protein synthesis as part of a global stress response. The signal for Gcn2p activation is deacylated tRNA, which accumulates when tRNA aminoacylation is limited either through lack of substrates or inhibition of synthesis. While the primary role of aaRS editing is to prevent misaminoacylation, here we describe conditions under which editing of non-cognate aa-tRNA is also required for proper detection of amino acid starvation by Gcn2p. In order to directly determine the identity of amino acids misacylated to specific tRNAs, we developed a tRNA pulldown method which allows determination of global aminoacylation profile for a given tRNA isoacceptor. Applying this technology, we identified that PheRS lacking quality control mechanisms allowed for accumulation of Tyr- tRNAPhe (5%) but not deacylated tRNAPhe during amino acid starvation. As with the bacterial stringent response, we found that accurate monitoring of amino acid starvation in yeast is dependent on aaRS-mediated translation quality control to ensure proper accumulation of deacylated tRNA species. Our data reveal a critical function for aaRS- editing in stress responses that is independent of their role in preventing mistranslation. With regards to mistranslation, cumulative translational error rates have been determined at the organismal level, yet the extent of codon specific error rates and the spectrum of misincorporation error from system to system remain less explored. To address this deficiency, we have developed a technique for the quantitative analysis of amino acid iii incorporation that provides the sensitivity necessary to detect mistranslation events during translation of a single codon at frequencies as low as 1 in 10,000 for all 20 proteinogenic amino acids, as well as non-proteinogenic and modified amino acids. Implementation of high resolution methods to determine global baselines for codon specific misincorporation events is required to shed light on these important metrics and would ultimately constitute an essential resource for the study of translation. If we consider the full spectrum of mistranslation presented herein, it becomes clear that some organisms tolerate substantial misincorporation events and that these events have the potential to confer selective fitness advantages at the organismal level. The technical advances described in Chapter 2 provide the opportunity to substantially broaden our understanding of the role of mistranslation by allowing measurement of the rates of both misaminoacylation and mistranslation in vivo. Accurate error rate measurements will provide a means to properly establish the relevance of mistranslation in physiological contexts by delineating how the level of mistranslation correlates with a cell’s ability to adapt, survive and thrive under different conditions. iv Dedication This document is dedicated to my family. Specifically to my mother (Shauna Welch), father (Paul Mohler), and to my siblings (Trent Mohler, Dylan Mohler, Holly Mohler, and Bryce Kerr) for their constant support during my education. v Acknowledgments This body of work would not have been possible without the help, guidance and support from so many people. First and foremost, I would like to thank my PhD advisor Dr. Michael Ibba for taking a risk. When I first met him, I didn’t have much to offer him in the way of evidence of past success, but I had plenty of examples of failure. For some reason, he took a chance on me. I am forever in his debt. His guidance, immeasurable patience, and belief in me throughout my graduate career has truly been the secret to my success. He has helped me to develop as a scientist and as I citizen. I truly cannot thank him enough. I am also thankful to my graduate committee, Dr. Juan Alfonzo, Dr. Irina Artsimovitch, and Dr. Kelly Wrighton for continually making themselves available for helpful discussions, critical comments and for sharing the most precious resource of all, time. I am especially indebted to Dr. Tammy Bullwinkle, and Dr. Medha Sharat for their friendship, guidance and support. They helped to make me the scientist I am today. I extend my sincere thanks to everyone who contributed to this dissertation work. Additionally, I would like to thank all the past and present members of Ibba lab for their help and support throughout the years. vi Vita 2006..........................................................Wadsworth High School 2011..........................................................B.S. Microbiology, The Ohio State University 2016..........................................................M.S. Microbiology, The Ohio State University 2013 to present ........................................Graduate Teaching/Research Associate, Department of Microbiology, The Ohio State University Publications Mohler, K., Ibba, M., “Mistranslation and the Stress Response. Nature Microbiology. (In Press) Mohler, K., Mann, R., Hopkins, K., Hwang, L., Reynolds, N., Polymenis, M., Faull, K., Ibba, M., “Translation quality control signals coordinate amino acid starvation response in Saccharomyces cerevisiae.” Nucleic Acids Res. 2017 Feb 7. doi: 10.1093/nar/gkx077. [Epub ahead of print] Mohler, K., Aerni, H.R., Gassaway, B., Ling, J., Ibba, M., Rinehart, J., “MS-READ: Quantitative Measurement of Amino Acid Incorporation.” BBA-General Subjects. 2017 Jan 24. doi: 10.1016/j.bbagen.2017.01.025. [Epub ahead of print] Mohler, K., Mann, R., Ibba, M., “Isoacceptor specific characterization of tRNA aminoacylation and misacylation in vivo.” Methods. 2017 Jan 15. 113:127-131. Walker, M., Mohler, K., Hopkins, K., Ibba, M., Phillip, M. 2015. “Novel compound heterozygous mutations expand the recognized phenotypes of FARS2-linked disease.” Journal of Child Neurology. 2016 Aug 31. 9:1127-37. vii Moghal A., Mohler K., Ibba M., “Mistranslation of the genetic code.” FEBS Letters. 2014. 588(23): 4305-10 Fields of Study Major Field: Microbiology viii Table of contents Abstract ............................................................................................................................... ii Dedication ........................................................................................................................... v Acknowledgments.............................................................................................................. vi Vita .................................................................................................................................... vii Table of contents ................................................................................................................ ix List of tables ...................................................................................................................... xv List of figures ................................................................................................................... xvi List of symbols and abbreviations .............................................................................. xviii Chapter 1 ............................................................................................................................. 1 1.1 Introduction .......................................................................................................... 2 1.2 Mechanisms of translational fidelity and error .................................................... 4 1.2.1 Amino acid selection