Characterizing the Landscape of Aminoacyl-tRNA Synthetase Protein Production in Bacillus subtilis By Darren John Parker B.S. Biochemistry University of Illinois, Urbana-Champaign, 2014 Submitted to the Department of Biology In partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY IN BIOLOGY at the MASSACHUSETTS INSTITUTE OF TECHNOLOGY September 2020 2020 Darren John Parker. All rights reserved. The author hereby grants MIT permission to reproduce and to distribute publicly paper and electronic copies of this thesis document in whole or in part in any medium known or hereafter created. Signature of the Author: __________________________________________________________ Darren J. Parker Department of Biology June 19, 2020 Certified by: ___________________________________________________________________ Gene-Wei Li Associate Professor of Biology Thesis Supervisor Accepted by: ___________________________________________________________________ Stephen Bell Uncas and Helen Whitaker Professor of Biology Investigator, Howard Hughes Medical Institute Co-Director, Biology Graduate Committee 2 Characterizing the Landscape of Aminoacyl-tRNA Synthetase Protein Production in Bacillus subtilis By Darren John Parker Submitted to the Department of Biology on June 19, 2020 in partial fulfillment of the requirements for the Degree of Doctor of Philosophy in Biology Abstract The phenotype of a cell is a consequence of both the identity of the genes in the genome and the magnitude of their expression into proteins. While the biochemical function of many proteins has been uncovered, for most it is unclear how important native protein abundances are for cell fitness. Furthermore, linking changes in abundances with downstream effects on enzymatic output, pathway function, and ultimately cell fitness is unexplored in nearly all cases. Here I use a model enzyme family, the aminoacyl tRNA synthetases (aaRS), to explore how sensitive Bacillus subtilis are to changes in aaRS production from the molecular to phenotypic level. This culmination of protein levels, functional output, and fitness, leads to a complete “fitness landscape” for the aaRS proteins and provides a framework for future study in quantitative biology. In Chapter I, I outline the conceptual questions explored in this thesis, review the current understandings of bacterial translation and aaRS function, and note the various regulatory strategies bacteria utilize to adapt to perturbations. In Chapter II, I find that the aaRS proteins are produced to optimize the growth rate of cells despite the presence of uncharged tRNAs. These native levels are positioned near a ‘fitness cliff’ as the underlying molecular processes of tRNA charging, translation, and regulation, are sensitive to reductions but not increases in synthetase production. In Chapter III, I complete the characterization of the aaRS fitness landscapes by exploring the source of the fitness defects of aaRS overproduction. In Chapter IV, I present a novel protocol for RNA-seq library preparation to reduce the cost and time associated with generating transcriptomic datasets. In Chapter V, using the aforementioned protocol, I characterize the transcriptomes of over 70 strains within the Escherichia coli single gene knockout collection. With the help of a colleague we find that strong selective pressures to induce genes involved in motility leads to a large amount of transcriptome heterogeneity within the collection. Finally, in Chapter VI, I discuss the results of my work, setting up future directions within the context of gene expression, bacterial physiology, and beyond. Thesis Supervisor: Gene-Wei Li Title: Associate Professor of Biology 3 Acknowledgements There are many people to thank for my being at this point…the common image of a scientist toiling away in a dark room, annoyed by any minor disturbance could not be farther from the truth. First and foremost, I have to thank Gene. I have yet to meet someone so intellectually capable yet so completely willing and eager to listen to the opinions of others. I always felt heard and respected, easily the two most important qualities in a mentor. I also admire the calming presence and patience he provides to myself and the lab. I would also like to majorly thank my undergraduate advisor, Auinash Kalsotra for inspiring me to continue on a scientific path. I was not even anticipating going to graduate school starting my last year of undergrad, but his enthusiasm for science, warm heart, and encouragement led me to aim high and continue to follow what I found interesting. I was incredibly fortunate to work with a great group of people during my last five years. First, I have to thank my long-time bay-mate and first colleague Jean-Benoît. Easily the smartest and hardest working person I have met at MIT, our numerous conversations, scientific or otherwise, were key in my development. I must also thank other members of the “founding core” of the Li Lab: Aaron, Grace, James, Lydia and Cassandra. Everyone brought a unique perspective to the lab, and I cherish the fact that I always felt comfortable discussing all elements of our work together. We had a lot of fun over the years, whether at Lab retreats, Halloween parties, or just coming up with shenanigans for the next event, and I will greatly miss those moments. There are many others within the MIT community I would like to acknowledge. Especially important to me was the culture of the 2nd floor of 68. Thanks to the awesome members especially in the Burge and Calo labs, I can safely say the 2nd floor has the best mix of high achieving work, fun people, and good times. I would never have expected to play beer pong against MIT professors mere feet from lab, but that happened and it was awesome. Also, as much as we joke about Bertucci’s I also really enjoyed RNA journal club which was, let’s be honest, another 2nd floor event. I hope that culture persists for years to come. I also would like to give a big thanks to Alan and members of the Grossman lab. The close interaction both in group meeting and outside was a huge part in setting the Li lab on its current course. I am sure I became annoying at some point, as I was coming up to ask questions at least once a day early on, so thank you all for your help. I also owe Alan my deepest gratitude for his excellent, frank feedback about science, careers, and life in lab. Life is not just work however, and I have to thank those that were part of the journey. First and foremost are those from my first true “home” 25 Cahvah (RIP) and 79 Putty; Nader, Sam, Charlie, Leanne and Jason. Whether it was Saturday morning cartoons, scientific discussions (i.e. gossip) or late night political debates, our apartment will forever be near to my heart. Second is my friends and members of various groups including DNA/Ribosome FC, the Ski trip squad, and the Microbios that semi-adopted me. 4 I was incredibly fortunate to have another group of friends to talk to nearly every day despite being 1,000 miles away. The Mofongo boys; Prath, Colin, Ross, and Ben, and Scott’s Tots; Jaydeep and Tania were all incredibly supportive of my journey and I would have gone crazy without them. To them I say, yes I am finally graduating college! Finally, I would be nowhere without my loving family. My mom, Jamie, and my dad, John have been extremely supportive throughout the entire process and gave the confidence to do whatever I wanted. My brothers, Adam, Lucas, and Jeremy were essential in recharging on my breaks and I am extremely grateful for all the fun we have had, whether in worlds real or virtual. I would also like to acknowledge and thank my Nanny and Papa for their absolute love and encouragement over the years. Having the feeling that if things aren’t going well I can turn to a loving and supportive environment is not something to take for granted. They are all equal members in the production of this thesis and completion of my Ph.D. 5 Table of Contents Abstract .........................................................................................................3 Acknowledgements .......................................................................................4 Table of Contents ..........................................................................................6 Figure Index ..................................................................................................8 Chapter I: Introduction ................................................................................11 Introduction to Quantitative Biology of Gene Expression ...................................... 12 High Abundance of Translation Machinery............................................................ 17 The Translation Cycle............................................................................................ 18 Elongation Kinetics & Codon Usage...................................................................... 21 Expression Stoichiometry of Translation Machinery .............................................. 24 Consequences of Translation Elongation Rate Reduction ....................................... 25 Aminoacyl-tRNA Synthetase Structure and Function............................................. 27 Biochemistry, Kinetics, and Inhibitors of aaRS Enzymes ....................................... 31 aaRS Gene Regulation ........................................................................................... 34 (p)ppGpp and the Stringent Response ...................................................................