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Probing the Regulation of Elongation Factor P-Mediated Translation Thesis Presented in Partial Fulfillment of the Requirements Probing the Regulation of Elongation Factor P-Mediated Translation Thesis Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University By Mengchi Wang, B.S. Graduate Program in Microbiology The Ohio State University 2013 Thesis Committee: Dr. Michael Ibba, Advisor Dr. Kurt Fredrick Dr. Irina Artsimovitch Copyright by Mengchi Wang 2013 ABSTRACT Elongation factor P (EF-P) is a universally conserved bacterial translation factor homologous to eukaryotic/archaeal initiation factor 5A. However, the mechanism by which EF-P regulates certain translation process is still largely unclear. Previous studies show that EF-P facilitates peptide bond formation in vitro. The crystal structures of EF-P revealed that the protein contains three domains and an overall structure that mimics a tRNA, binding between the P-site and E- site of the ribosome during translation. However, only a limited number of proteins are affected by the loss of EF-P. In E. coli and Salmonella, deletion of the efp gene results in pleiotropic phenotypes, including increased susceptibility to numerous cellular stressors. Based on these results, we hypothesize that EF-P mediates translation of a subset of mRNAs, which share common characteristics in their sequences. We conducted an unbiased in vivo investigation of the specific targets of EF-P by employing stable isotope labeling of amino acids in cell culture (SILAC) to compare the proteomes of wild-type and efp mutant Salmonella. We found that metabolic and motility genes are prominent among the subset of proteins with decreased production in the efp mutant. Furthermore, particular tripeptide motifs are statistically overrepresented among the proteins downregulated in efp mutant strains. These include PPP, PPG, APP and YIRYIR, all of which were confirmed to induce EF-P dependence by a translational fusion assay. Notably, we found that many proteins containing these identified motifs are not misregulated in an EF-P-deficient background, suggesting that the factors that ii govern EF-P-mediated regulation are complex. The possibility of a structural feature that underlies EF-P mediated translation is discussed. In summary, this work established a productive bioinformatics strategy for screening EF-P target motifs. The discoveries made in this study help provide important clues regarding the mechanism of EF-P regulated translation. iii ACKNOWLEDGEMENTS I would like to express my deepest gratitude to my advisor, Dr. Michael Ibba for his trust, support and guidance in every step along the way. He personified the aptitude of a brilliant scientist, the charisma of a true leader and provided to me wise mentorship that inspires me to live up to my full potential in research and my career. I am also indebted to my advisory committee members Dr. Kurt Fredrick and Dr. Irina Artsimovitch for their unreserved support and insightful critiques on my project. I am grateful for my friends and fellow lab members, in particular, Dr. Hervé Roy for guiding me into the lab and helping me start the project, Dr. Tammy Bullwinkle and Medha Raina for their keen advice on my experiments, Dr. Assaf Katz and Andrei Rajkovic for their helpful insights, Sara Elgamal and Jing Li for their great editorial help with this thesis, and finally, James Bardeen and Ellen Bardeen for their unconditional love and warm friendship. Last but not the least, this work is not possible without the collaborative efforts with our colleagues at Dr. William Navarre’s Lab in University of Toronto, especially Steven Hersch. iv VITA June 2006...................................Nanjing Jinling High School, China July 2010....................................B.S. Biology, Nanjing Agricultural University, China. 2010 to present ..........................Graduate Research Assistant, Department of Microbiology, The Ohio State University PUBLICATIONS Hersch, S. J.*, Wang, M.*, Zou, S. B., Moon, K. M., Foster, L. J., Ibba, M. & Navarre, W. W. (2013) Divergent Protein Motifs Direct Elongation Factor P-Mediated Translational Regulation in Salmonella enterica and Escherichia coli, mBio. 4. * Co-first Author. FIELDS OF STUDY Major Field: Microbiology v TABLE OF CONTENTS ABSTRACT ................................................................................................................... ii ACKNOWLEDGEMENTS .......................................................................................... iv VITA .............................................................................................................................. v PUBLICATIONS ........................................................................................................... v FIELDS OF STUDY...................................................................................................... v TABLE OF CONTENTS .............................................................................................. vi LIST OF TABLES ....................................................................................................... vii LIST OF FIGURES ................................................................................................... viii CHAPTER 1. INTRODUCTION .................................................................................. 1 CHAPTER 2. MATERIALS AND METHODS .......................................................... 15 CHAPTER 3. PREDICTING SEQUENCE MOTIFS TARGETS OF EF-P ............... 21 CHAPTER 4. VERIFICATION OF PREDICTED EF-P TARGET MOTIFS. ............ 27 CHAPTER 5 EF-P TARGET: BEYOND SEQUENCE MOTIFS. .............................. 37 REFERENCES ............................................................................................................ 43 vi LIST OF TABLES Table 1. Bioinformatics analysis predicts EF-P regulated tripeptide motifs. ............... 25 Table 2. Verification of predicted EF-P dependent motifs. .......................................... 28 Table 3. Identity of the second amino acid has marginal influence on EF-P mediated translation.. ................................................................................................................... 36 Table 4. Motif targets and structural rigidity ............................................................... 40 vii LIST OF FIGURES Figure 1. Structure of the ribosome. .............................................................................. 3 Figure 2. Overview of bacterial translation. .................................................................. 4 Figure 3. The crystal structure of EF-P bound to the 70S ribosome. ........................... 11 Figure 4. A subset of proteins is significantly misregulated in Δefp Salmonella.. ...... 23 Figure 5. Target motifs verified by a fluorescent reporter. .......................................... 27 Figure 6. Translation of poly-proline motifs with defects in EF-P modification. ........ 29 Figure 7. Comparison of proteins identified in SILAC and those with EF-P target motifs ........................................................................................................................... 31 Figure 8. Tripeptide "R1 R2 R3" with Cα and Cβ positions ........................................ 40 viii CHAPTER 1. INTRODUCTION 1.1 The Mechanism of Translation 1.1.1 Overview of Translation Translation is the crucial step of protein synthesis where genetic information encoded on mRNA is converted to the corresponding polypeptide sequence. In bacteria, this takes place on the 70S ribosome and uses messenger RNA (mRNA) as the template and amino acids as substrates which are delivered by transfer RNAs (tRNA). There are four phases of translation namely initiation, elongation, termination and ribosome recycling. Additional factors are required for each of these four steps, and the accuracy and speed of translation are ensured by a fine-tuned cooperation of these components. (1, 2) 1.1.2 The Translation Machinery The ribosome consists of two subunits in all species. In bacteria, the 70S ribosome is constituted by a large (50S) and a small (30S) subunit, both of which contain three binding sites for tRNA, namely, the A (aminoacyl) site, the P (peptidyl) site and the E (exit) site. The 30S subunit plays roles in the fidelity of translation by binding to mRNA together with the anticodon stem-loops of tRNA and hence monitoring base pairing between the two. The 50S subunit binds to the acceptor arms of the tRNA and catalyzes peptide bond formation between the incoming amino acid on the A-site tRNA and the nascent peptide chain on the P-site tRNA. Understanding of the molecular mechanism by which the ribosome catalyzes 1 protein synthesis was considerably expanded by the solving of a series of high resolution crystal structures, starting with an archaeal 50S subunit from Haloarcula marismortui and a bacterial 30S subunit from Thermus thermophilus published in 2000, followed by structures of the 70S ribosome (3, 4, 5, 6), the bacterial 50S subunit (7), as well as the mobile elements of the 50S including the L1 or L7/L12 stalks that have been solved individually (8, 9). These results revealed the 70S ribosome (Figure 1) includes an interface between the 30S and the 50S subunits that consists mainly of RNA. The A (aminoacyl) site accepts the incoming charged tRNA, the P (peptidyl) site holds the nascent peptide chain, and the E (exit) site is where deacylated P-site tRNA locates after peptide bond formation before leaving the ribosome. Between the “head” and “body” of the 30S
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