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Structure and Function of Protein-Only Rnase P by Michael J. Howard a Dissertation Submitted in Partial Fulfillment of the Requ

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Structure and Function of Protein-Only Rnase P by Michael J. Howard a Dissertation Submitted in Partial Fulfillment of the Requ Structure and Function of Protein-only RNase P by Michael J. Howard A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Biological Chemistry) in the University of Michigan 2015 Doctoral Committee: Professor Carol A. Fierke, Chair Professor David. R. Engelke Associate Professor Patrick J. O’Brien Associate Professor Bruce A. Palfey Professor Nils G. Walter Dedication To those in an honest pursuit to understand how nature works. ii Acknowledgements My time as a graduate student has been enjoyable, and this is in large part due to my mentor Carol Fierke, who granted me with freedom to make valuable mistakes but did not let me stray too far. Her breadth of scientific knowledge, patience, and compassion are a continual source of inspiration. I would also like to thank past and present members of the Fierke lab for creating a great work environment. I particularly would like to thank members of the RNase P group for help, reagents, comments, and discussions (Wan Lim, Xin Liu, Yu Chen, Bradley Klemm, and Nancy Wu). Also great thanks to Markos Koutmos who has been almost like a second mentor to me. Chapter 2 would not have been possible without his expertise. Additionally, I would like to thank the Biological Chemistry department, both administrators and faculty members, for creating a great environment for learning. I would also like to acknowledge my family and friends for their support. In particular my father’s skepticism, my mother’s strong work ethic, and their shared reverence for nature are attributes that have shaped my life and brought me here. Furthermore, I would like to acknowledge a friend of the family, Riley Mahar, whose passion for science and critic thinking really sparked my interest in science. And my fiancé, Ashley Konal, who shares my scientific curiosity and will partake in discussions about my ideas concerning enzymes, evolution, and life. Thanks for all the love, support, and encouragement. iii Table of Contents Dedication ........................................................................................................................ii Acknowledgements ......................................................................................................... iii List of Figures ................................................................................................................. vii List of Tables ...................................................................................................................ix Abstract ........................................................................................................................... x Chapter 1 Introduction ..................................................................................................... 1 Background ................................................................................................................. 1 RNA-Based RNase P .................................................................................................. 2 Discovery of Protein-only RNase P.............................................................................. 4 The Evolution of PRORP ............................................................................................. 5 Conclusions and Significance ...................................................................................... 9 Objectives .................................................................................................................. 10 References ................................................................................................................ 11 Chapter 2 Mitochondrial Ribonuclease P Structure Provides Insight into the Evolution of Catalytic Strategies for Precursor-tRNA 5’-Processing ................................................. 13 Abstract ..................................................................................................................... 13 Background ............................................................................................................... 14 Results ....................................................................................................................... 15 Global architecture of PRORP1 ............................................................................. 15 The PPR Domain is Important for Activity and Substrate Affinity ........................... 15 Central Domain ...................................................................................................... 18 Metallonuclease domain ........................................................................................ 19 Metal Activation ...................................................................................................... 21 Discussion ................................................................................................................. 23 Comparison with RNA-based RNase P .................................................................. 24 Methods ..................................................................................................................... 26 Appendix A. ............................................................................................................... 27 Expression and Purification .................................................................................... 27 iv Mitochondrial pre-tRNACys Synthesis and Radio-Labeling ..................................... 28 RNase P Cleavage Assays .................................................................................... 28 Fluorescent Polarization Binding Assays ............................................................... 28 Crystallization and Crystal Harvesting .................................................................... 28 Data Collection and Structure Determination ......................................................... 29 References ................................................................................................................ 36 Chapter 3 Mechanistic Studies Reveal Similar Catalytic Strategies for Phosphodiester Bond Hydrolysis by Protein-only and RNA-dependent Ribonuclease P ........................ 39 Abstract ..................................................................................................................... 39 Background ............................................................................................................... 40 Results ....................................................................................................................... 42 Single- and Multiple-turnover kinetics of PRORP1-catalyzed pre-tRNA Cleavage. 42 PRORP1 Magnesium Dependence ........................................................................ 45 Metal Dependence of Aspartate to Alanine Mutants .............................................. 48 pH Dependence ..................................................................................................... 52 Discussion ................................................................................................................. 55 A minimal kinetic mechanism reveals product release is not rate-limiting at neutral pH .......................................................................................................................... 55 Evidence for a 2-metal ion mechanism .................................................................. 56 D474 and D475 enhance the active site metal ion affinity ...................................... 57 pH Dependence reveals a single ionization important for catalysis........................ 57 Convergence of enzyme mechanism ..................................................................... 58 Methods ..................................................................................................................... 60 Enzyme Preparation ............................................................................................... 60 Pre-tRNA Preparation ............................................................................................ 61 Enzyme assays ...................................................................................................... 61 Metal Dependence ................................................................................................. 62 Metal Dependence of Mutants ............................................................................... 62 pH Dependence ..................................................................................................... 63 Anisotropy Binding Assays ..................................................................................... 64 References ................................................................................................................ 65 Chapter 4 Kinetic characterization of plant protein-only RNase P isozymes reveals overlapping substrate specificities and varying cleavage fidelities ................................ 68 Abstract ..................................................................................................................... 68 Background ............................................................................................................... 69 v Results ....................................................................................................................... 72 PRORPs catalyze cleavage of most pre-tRNA substrates with uniform efficiency . 72 PRORPs bind individual pre-tRNAs similarly, regardless of pre-tRNA origin ........
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