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The Modification State of Elongation Factor-P in Bacillus Subtilis and Pseudomonas

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The Modification State of Elongation Factor-P in Bacillus Subtilis and Pseudomonas The Modification State of Elongation Factor-P in Bacillus subtilis and Pseudomonas aeruginosa Thesis Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University By Sarah Beth Tyler Graduate Program in Chemistry The Ohio State University 2015 Thesis Committee: Dr. Michael Ibba, Advisor Dr. Karin Musier-Forsyth, Advisor Dr. Ross Dalbey Copyright by Sarah Beth Tyler 2015 Abstract In order for life to function properly, proteins must be correctly translated. One instance that poses as a threat to translation is stretches of poly prolines in the transcript. Because of the pyrrolidine ring in proline, proline is a poor imino acceptor and donor, and a string of three or more prolines in a row can cause the ribosome to pause. The translation factor Elongation Factor-P (EF-P) binds to the ribosome and stimulates bond formation between the prolines, alleviating the pause and allowing translation to continue. EF-P is post translationally modified on a conserved residue, and while EF-P or an EF-P homolog is found in all domains of life, there is variety in the post-translational modifications from one bacterial organism to another. Thus far, it has been discovered that EF-P in E. coli is modified with (R)-ß-Lysine, with dTDP-Rhamnose in P. aeruginosa, and a hypusine in the eukaryotic homolog, eIF5A. These modifications currently known in bacteria only represent a small amount of bacteria, and the question of what other modifications are remains unanswered. It has been confirmed by mass spectrometry that Bacillus subtilis EF-P is post-translationally modified. The identity of this modification however, still remains elusive. In this work, methods including mass spectrometry, isoelectric focusing, and in vitro reactions are used to probe what the modification on B. subtilis EF-P is. Besides getting closer to understanding what the modification on EF-P is, these techniques are also used to reveal potential regulation of EF-P. Finally, this work ii addresses some of the experiments performed in identifying the modification on P. aeruginosa EF-P, including the synthesis of the substrate for modification of EF-P, dTDP-rhamnose. iii This document is dedicated to my parents, Ann and Steven Tyler, who have instilled in me a deep appreciation for education. iv Acknowledgments I would like to thank my advisor, Dr. Michael Ibba for mentoring me during the time I was in his lab. He is a good leader and a scientist whom I wish to emulate. I would also like to acknowledge and thank Andrei Rajkovic for all of his training and guidance, Tammy Bullwinkle, Kyle Mohler, and Sara Elgamal for their insight in experiments, and the rest of Ibba lab for their input and support. Lastly, I would like to acknowledge The Ohio State Chemistry and Biochemistry Program and the Chemistry and Biochemistry Interface Program for acceptance into the program and for the funding of my research. v Vita June 2009 .......................................................East High School 2013................................................................B.S. Chemistry, University of Utah 2013 to present ..............................................CBIP Trainee, Department of Chemistry and Biochemistry, The Ohio State University Fields of Study Major Field: Chemistry vi Table of Contents Abstract ...................................................................................................................................................... ii Acknowledgments .................................................................................................................................. v Vita............................................................................................................................................................... vi Fields of Study ......................................................................................................................................... vi Table of Contents .................................................................................................................................. vii List of Tables ............................................................................................................................................. x List of Figures .......................................................................................................................................... xi Chapter 1. Introduction ....................................................................................................................... 1 1.1. Aminoacyl-tRNA Synthetases .................................................................................................... 1 1.1.2. Alternative Synthetase Functions ........................................................................................ 3 1.2. Translation Machinery ................................................................................................................. 4 1.2.1. Initiation, Elongation, and Termination ............................................................................. 5 1.2.2. Regulation by mRNA .................................................................................................................. 8 1.3. Elongation Factor-P ....................................................................................................................... 9 1.3.1. eIF5A ............................................................................................................................................. 11 vii 1.3.2. Modification Pathways of EF-P and eIF5A ..................................................................... 12 1.3.3. Bacterial responses to ∆EF-P .............................................................................................. 15 Chapter 2. Materials and Methods ................................................................................................ 16 2.1. Bacterial Strains and Plasmids ............................................................................................... 16 2.2. Growth Curves .............................................................................................................................. 18 2.3. Purification of EF-P ..................................................................................................................... 18 2.4. Isoelectric focusing and Western Blotting as a Tool ...................................................... 20 2.5. In Vitro aminoacylation of EF-P ............................................................................................. 21 2.6. Glycoprotein Stain ....................................................................................................................... 22 2.7. Lectin Binding Assays ................................................................................................................ 22 2.8. [14C] dTDP-Rhamnose Synthesis ........................................................................................... 23 Chapter 3. Results ................................................................................................................................ 25 3.1. Modification Status of EF-P in Bacillus subtilis................................................................. 25 3.2. Mass Spectrometry ..................................................................................................................... 26 3.2. Characterization of B. subtilis EF-P by Isoelectric Focusing ...................................... 30 3.3. In-vitro assays probing the identity of the modification substrate ......................... 32 3.4. Glucose as a Potential Regulator ........................................................................................... 35 3.5. EF-P in P. aeruginosa.................................................................................................................. 38 viii 3.5. Glycoprotein Staining ................................................................................................................ 38 3.6. Lectin Binding Assays ................................................................................................................ 39 3.7. [14C]Rhamnose Synthesis ......................................................................................................... 40 Chapter 4. Future Work ..................................................................................................................... 43 4.1. Identifying potential modifying enzyme in B. subtilis ................................................... 43 References. ............................................................................................................................................. 46 ix List of Tables Table 1. A list of the B. subtilis strains used in this work. ............................................... 16 Table 2. P. aeruginosa strains used in this work .............................................................. 17 x List of Figures Figure 1. Translation cycle ................................................................................................. 8 Figure 2. Comparison of EF-P to tRNA ........................................................................... 11 Figure 3. Structure of Human eIF5A compared to EF-P .................................................. 12 Figure 4. Different modification pathways of EF-P and homolog eIF5A ........................ 14 Figure 5. Exemplary Mass spectrometry data..................................................................
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