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Mechanistic Insights Into a Reverse Polymerase

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Mechanistic Insights Into a Reverse Polymerase Mechanistic insights into a reverse polymerase DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Brian A Smith Graduate Program in Biochemistry The Ohio State University 2012 Dissertation Committee: Jane E Jackman, Advisor Kurt Fredrick Thomas Magliery Richard Swenson Copyright by Brian A. Smith 2012 Abstract tRNAHis guanylyltransferase (Thg1) is a highly conserved enzyme found in all domains of life including higher eukaryotes. Thg1 catalyzes the incorporation of a single His guanosine residue to the -1 position (G-1) of tRNA , using an unusual 3'-5' nucleotidyl transfer reaction. G-1 serves as a necessary recognition element for histidyl-tRNA synthetase and is conserved among nearly all tRNAHis species. Thg1 is the only known enzyme that adds nucleotides in the 3'-5' direction and shares no identifiable sequence homology to any other known enzyme, thus its molecular mechanism is unknown. However, the high resolution crystal structure of Thg1 reveals remarkable structural similarities between canonical DNA/RNA polymerases and eukaryotic Thg1, suggesting that 3'-5' and 5'-3' nucleotide addition reactions share a common evolutionary origin. G-1 addition occurs via a complex mechanism involving three steps. In the first step, Thg1 activates the 5' end of tRNAHis by formation of an adenylylated intermediate via a 5'--5' phosphoanhydride bond (App-tRNAHis). Subsequently, Thg1 utilizes the 3'- OH of GTP to attack the intermediate in a nucleotidyltransfer step yielding AMP and resulting in the addition of a single GTP to the 5' end of the tRNA. In the final step Thg1 removes pyrophosphate from the G-1 residue in a pyrophosphatase step yielding mature His G-1-containing tRNA . A complete understanding of this complex reaction mechanism requires isolation and characterization of each catalytic step individually. ii I used transient kinetic assays to measure the pseudo-first order forward rate constants for each step of this reaction (kaden, kntrans and kppase). Using this kinetic framework in conjunction with data from the crystal structure of nucleotide-bound Thg1, we identified conserved residues involved in nucleotide binding and the adenylylation step of the G-1 addition reaction. A kinetic investigation of the third, pyrophosphate removal, step of the reaction reveals base pair-dependent effects on the rate of this step that affect the ability of the enzyme to add multiple nucleotides to some tRNA substrates. We also identified residues that when altered to alanine, cause severe defects in tRNA binding which are localized to a region distant from the putative active site, suggesting the possibility of structural rearrangements induced upon tRNA binding. iii Dedication This document is dedicated to my loving wife Amanda, and my parents Mary and Russell iv Acknowledgments First and foremost I would like to thank my advisor Dr. Jane Jackman for her unbelievable patience and guidance. In addition to being an incredible mentor I think of Jane as true friend (I couldn’t have done it without you). I would also like to thank our technician Maria Abad-de-Partida for all her hard work and making everyone’s lives in the Jackman lab so much easier. Furthermore I would like to thank all the members of the lab, Bhalchandra Rao (thanks for standing in my wedding), Bill Swinehart, Yicheng Long, Jeremy Henderson, Krishna Patel, and everyone else I’ve had the honor of working with. I will truly cherish our friendships for the rest of my life. I would also like to thank my wife Amanda for always standing by my side (I know this was a challenge for you too). v Vita 1999-2004 ......................................................Kalamazoo Valley Community College 2004-2007 ......................................................B.S Chemistry, Grand Valley State University 2007 to present ..............................................Graduate Teaching and Research Associate, Department of Biochemistry, The Ohio State University Publications Smith, B.A. and J.E. Jackman, Kinetic analysis of 3'-5' nucleotide addition catalyzed by eukaryotic tRNAHis guanylyltransferase. Biochemistry, 2012. 51(1): p. 453-65. Samantha J. Hyde, Brian E. Eckenroth, Brian A. Smith, William A. Eberley, Nicholas H. Heintz, Jane E Jackman, and Sylvie Doublie, From the Cover: tRNAHis guanylyltransferase (THG1), a unique 3'-5' nucleotidyl transferase, shares unexpected structural homology with canonical 5'-3' DNA polymerases. Proc Natl Acad Sci U S A, 2010. 107(47): p. 20305-10. David A. Leonard, Andrea M. Hujer, Brian A. Smith, Kyle Schneider, Christopher R. Bethel, Kristine M. Hujer, Robert A. Bonomo. The role of OXA-1 β-lactamase aspartate 66 in stabilization of the active site carbamate and substrate turnover. 2008 Biochem. J. 410, 455-462. Fields of Study Major Field: Biochemistry vi Table of Contents Abstract ............................................................................................................................... ii Dedication .......................................................................................................................... iv Acknowledgments............................................................................................................... v Vita ..................................................................................................................................... vi Publications ........................................................................................................................ vi Fields of Study ................................................................................................................... vi Table of Contents .............................................................................................................. vii List of Tables .................................................................................................................... xii List of Figures .................................................................................................................. xiii Chapter 1: Introduction ...................................................................................................... 1 1.1 G-1 residue acts as a critical element identity ............................................................ 4 1.2 The yeast tRNAHis guanylyltransferase (Thg1) ......................................................... 6 1.3 Covalent nucleotidyl transferase enzymes ................................................................ 8 1.4 Thg1 is a 3'-5' reverse polymerase ............................................................................ 9 1.5 TLPs in bacteria and archaea .................................................................................. 11 vii 1.6 tRNA 5′ editing ....................................................................................................... 13 1.7 Thg1 structure ......................................................................................................... 15 1.8 Two metal ion catalysis ........................................................................................... 18 1.9 Nucleotide bound hThg1 ......................................................................................... 19 Chapter 2: Kinetic analysis of 3'-5 nucleotide addition catalyzed by eukaryotic tRNAHis guanylyltransferase (Thg1) ............................................................................................... 22 2.1 Introduction ............................................................................................................. 22 2.2 Experimental methods ............................................................................................. 23 2.2.1 Nucleotides and reagents .................................................................................. 23 2.2.2 Thg1 expression and purification ..................................................................... 23 2.2.3 Adenylylation assay .......................................................................................... 24 2.2.4 Nucleotidyl transfer assays ............................................................................... 26 2.2.5 Pulse chase nucleotidyl transfer assays ............................................................ 27 2.2.6 Pyrophosphate removal assays ......................................................................... 28 2.2.7 Preparation and analysis of SceThg1 alanine variants ..................................... 29 2.3 Results ..................................................................................................................... 30 2.3.1 Analysis of the activation step .......................................................................... 30 2.3.2 Single-turnover measurement of nucleotidyl transfer ...................................... 33 2.3.3 Pyrophosphate removal is stimulated by the addition of GTP ......................... 38 viii 2.3.4 Application of kinetic framework to identify catalytic residues that participate in G-1 addition ............................................................................................................ 42 2.3.5 Two metal-ion catalysis may be used for all three chemical steps of G-1 addition ...................................................................................................................... 44 2.3.6 K44 and N161 participate in the adenylylation step ........................................
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