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1 Characterizing the Function of Alanyl-Trna Synthetase Activity In Characterizing the Function of Alanyl-tRNA Synthetase Activity in Microbial Translation Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Paul Michael Kelly, MS Graduate Program in Molecular, Cellular and Developmental Biology The Ohio State University 2020 Dissertation Committee: Dr. Michael Ibba, Advisor Dr. Kurt Fredrick Dr. Jane Jackman Dr. Natividad Ruiz 1 Copyrighted by Paul Michael Kelly 2020 2 Abstract Across all domains of life, mechanisms have evolved to ensure faithful translation of the genetic code. One integral step in the transition from a nucleic acid encoded-genome to functional proteins is the aminoacylation of tRNA molecules. To perform this activity, aminoacyl-tRNA synthetases (aaRSs) activate free amino acids in the cell forming an aminoacyl-adenylate before transferring the amino acid on to its cognate tRNA. These newly formed aminoacyl-tRNA (aa-tRNA) can then be used by the ribosome during mRNA decoding. In Escherichia coli, there are twenty aaRSs encoded in the genome, each of which correspond to one of the twenty proteinogenic amino acids used in translation. Given the shared chemicophysical properties of many amino acids, aaRSs have evolved mechanisms to prevent erroneous aa-tRNA formation with non-cognate amino acid substrates. Of particular interest is the post-transfer proofreading activity of alanyl-tRNA synthetase (AlaRS) which prevents the accumulation of Ser-tRNAAla and Gly-tRNAAla in the cell. Upon mis-activation of the non-cognate serine or glycine, AlaRS will transfer the amino acid to the 3’ end of tRNAAla where it will translocate into a distinct editing active site in the enzyme. Recognition of the non-cognate substrate will lead to hydrolysis of the amino acid from the tRNA leaving both substrates available for subsequent rounds of aminoacylation. It has been previously shown that disruption of the cytosolic and mitochondrial AlaRS editing domains in mice leads to neurodegeneration and embryonic ii lethality respectively. Here we show that perturbation of the E. coli AlaRS editing domain causes gross perturbation to the E. coli proteome causing a variety of pleiotropic phenotypes including growth defects, motility impairment, and antibiotic sensitivity. Furthermore, we have identified second-site suppressors within the AlaRS editing domain that alleviate these defects. This work highlights the importance of AlaRS fidelity in E. coli and characterizes novel elements within this aaRS editing domain. Given the essentiality for maintaining protein synthesis, aaRSs have been promising targets for therapeutic discovery. Leishmania major is a eukaryotic parasite that infects upwards of a million individuals a year. Current therapeutic options remain limited and have adverse effects on humans. While antibiotics are able to target prokaryotic cellular components specifically, developing therapies against eukaryotic pathogens is more challenging due to conservation between potential pathogen drug targets and their host counterparts. Previous studies have identified anti-fungal therapies that target pathogen aaRS with some degree of specificity, indicating that these essential enzymes could be developed as drug targets for a variety of infections. Using bioinformatic approaches, we have identified several aaRSs, including AlaRS in L. major that appear to be potential targets for anti-leishmanial therapies. Preliminary biochemical analysis have supported these in silico predictions and led to the identification of novel aaRS inhibitors in vitro. iii Dedication The body of work presented herein is dedicated to my Grandmothers (Patricia Kelly and Mary Sopira). This degree would have never been possible without their love, support, and continued belief in me. I hope I’ve made them proud. iv Acknowledgments First and foremost, I’d like to acknowledge my Mother (Donna Bittner), Father (Shawn Kelly), and Stepfather (Butch Bittner) for raising me in a home in which my aspirations were always supported and met with love and encouragement. They may have not understood what I was doing, but they were always the first to ask “How are your fishes doing?” when I was monitoring kidney regeneration in zebrafish, or “Are the E. coli growing?” while I was studying the role of prokaryotic translational fidelity (by the time you finish Chapter 3, you will see that the E. coli were in fact not always growing particularly well). Jokes apart, the pursuit of my academic ventures has rarely been easy and most of the time, incredibly isolating. Without the love and support of the many family and friends who have always been there for me, this journey would have likely ended long ago. I would also like to acknowledge my committee members, Drs. Kurt Fredrick, Jane Jackman, and Natacha Ruiz for their continued guidance over the last few years. Their advice has been invaluable towards the completion of my dissertation. To my lab mates, words cannot describe how fortunate I feel to have gotten to work with you all. The friendships made over the last six years are some of the best I’ve ever had, and I consider you all like family (albeit a very loud and dysfunctional one). The dynamic of the lab has changed over the years and I am grateful for everyone who has been v a part of this journey. Early on, I had the great fortune to learn from Drs. Tammy Bullwinkle and Kyle Mohler. They taught me how to think about science in new and interesting ways and I would not be the scientist I am today without their tutelage. The lab then transitioned to what I consider the “core” years when I got to witness the creativity and tenacity of Drs. Annie Witzky, Becky Steiner, and Rodney Tollerson II on a daily basis. The best way to describe Annie, Becky, and Rodney is that they are “winners,” and that winning mindset continues to inspire me as I plan for the next chapter of my life. Most recently, my role in the lab has changed to that of a mentor for our younger graduate students. I have had the opportunity to witness Nien-Ching Han, Aru Kavoor, and Mary Cranley grow as researchers and begin to find their scientific voice. Of the many great things that have happened in the lab, knowing that I may have played any role in their development is one of my proudest achievements. To any other lab members that I didn’t mention by name (sorry, I figured I should wrap this up because I’m sure Mike is already starting to doze off), just know that you have all contributed to this work and I am grateful for the time we have spent together. Finally, I owe my greatest debt to Dr. Michael Ibba because without him, this body of work would not exist. Throughout my life, I have been plagued with unrelenting self- doubt. Mike has always been there to support and encourage me even when I had given up on myself. For the first few years, I was convinced that I had “tricked” Mike into letting me join his lab. I couldn’t figure out how I could possibly be good enough to get to work with these amazing people and I kept waiting for the day that he would realize his mistake and I would finally be asked to leave. Well, that day never came and here I am ready to vi move on to the next stage of my life, knowing that I provide value and without any fear of my inadequacies. Mike is the greatest mentor I could have ever asked for, he has changed the trajectory of my life, and I am eternally grateful. vii Vita 2008 ................................................. Taylor Allderdice High School 2010 ................................................. Community College of Allegheny County 2012 ................................................. B.S. Biology, Robert Morris University 2014 ................................................. M.S. Biology, Indiana University of Pennsylvania 2014 to present ................................. Graduate Teaching and Research Associate, Molecular, Cellular and Developmental Biology, The Ohio State University Publications Kelly, P., Hadi-Nezhad, F., Liu, D., Lawrence, T., Linington, R., Ibba, M., Ardell, D., (2020) Targeting tRNA-Synthetase Interactions towards Novel Therapeutic Discovery Against Eukaryotic Pathogens. PLoS Negl. Trop. Dis. 14(2): e0007983 Kelly, P., Backes, N., Mohler, K., Buser C., Kavoor, A., Rinehart, J., Phillips, G., Ibba. M., (2019) Alanyl-tRNA Synthetase Quality Control Prevents Global Dysregulation of the Escherichia coli Proteome. mBio. 10 (6) Kelly, P., Ibba, M., (2018) Aminoacyl-tRNA Quality Control Provides a Speedy Solution to Discriminate Right from Wrong. J. Mol. Biol. 430(1). viii Fields of Study Major Field: Molecular, Cellular, and Developmental Biology ix Table of Contents Abstract .......................................................................................................................... ii Dedication ..................................................................................................................... iv Acknowledgments ...........................................................................................................v Vita ............................................................................................................................. viii List of Tables................................................................................................................xiv List of Figures ............................................................................................................... xv List of Symbols and Abbreviation ..............................................................................
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