Ribonuclease P (RNase P) catalyzes 5'end maturation of precursor tRNA (pre-tRNA) to form tRNA, an essential component of protein synthesis. RNase P is found in all domains of life, but the composition of this indispensable enzyme varies from a RNA-protein heterodimer in bacteria to a complex of three proteins in human mitochondrial RNase P (mtRNase P). These enzymes provide an ideal system for defining catalytic features that distinguish RNA- and protein-based catalysis. Furthermore, the distinct subunit compositions highlight the potential of bacterial RNase P as a novel antibiotic target. In mitochondria, mutations in (mt)tRNA and mtRNase P subunits have been linked to a number of diseases, including neurodegeneration, X-linked mental retardation, myocardial infarction, coronary artery disease as well as mitochondria dysfunction which manifests clinically as MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like symptoms), progressive external opthalmoplegia and/or diabetes. Analysis of the in vivo and in vitro function of mtRNase P will provide insight into mitochondrial tRNA processing pathways and their role in mitochondria biogenesis and dysfunction. Thus, investigation of RNase P structure and function has the potential for wide-ranging impact on a variety of health issues, from improving antibacterial therapeutics to characterization of the biological pathways linked to the pathogenesis of multiple mitochondrial diseases. This proposal consists of two primary objectives. First, we propose to develop biophysical methods, including single molecule fluorescence spectroscopy and NMR spectroscopy (in collaboration with Professors Al-Hashimi and Walter) to investigate two hallmark features of large RNA molecules, such as the RNase P RNA subunit: dynamic RNA-metal interactions that exchange between diffusive, inner-sphere, and outer- sphere contacts;and conformational plasticity that is central to RNA function, including substrate recognition and catalysis. In applying these methods to bacterial RNase P we aim to: (1) explore the changes in structure and dynamics that occur in RNase P throughout the catalytic cycle;and (2) delineate the structure and interactions within proposed metal ion binding sites in RNase P. Second, we will identify the strategies employed by the newly discovered protein-based mtRNase P to achieve catalysis and substrate recognition. In particular, we explore the function of MRPP3 using mutagenesis, metal substitution and kinetic analysis to elucidate mechanistic features of this member of a novel family predicted to have metal-dependent nuclease activity. Finally, we will examine determinants of pre-tRNA recognition and the role of defects in mtRNase P processing in the pathophysiological mechanisms of human mitochondrial tRNA mutations. These studies will significantly enhance our understanding of the structure and function of these two distinct classes of RNase P enzymes and their homologues, develop methods useful for studying similar enzymes, and provide fundamental insights into the nature of biological catalysis.

Public Health Relevance

RNase P is essential for the formation of tRNA, which plays a key role in protein synthesis. Due to this essential role in life, RNase P has potential medical applications as a novel antibiotic target. Furthermore, errors in tRNA processing in mitochondria are linked to multiple mitochondrial dysfunction diseases. The studies in this proposal will provide insight into the development of inhibitors of bacterial RNase P and yield important information about tRNA processing pathways and the function of mitochondrial RNase P that will impact our understanding of mitochondrial-related disorders.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM055387-15
Application #
8209006
Study Section
Macromolecular Structure and Function E Study Section (MSFE)
Program Officer
Anderson, Vernon
Project Start
1997-01-01
Project End
2014-12-31
Budget Start
2012-01-01
Budget End
2012-12-31
Support Year
15
Fiscal Year
2012
Total Cost
$301,396
Indirect Cost
$101,396
Name
University of Michigan Ann Arbor
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Klemm, Bradley P; Wu, Nancy; Chen, Yu et al. (2016) The Diversity of Ribonuclease P: Protein and RNA Catalysts with Analogous Biological Functions. Biomolecules 6:
Kim, Laura Y; Thompson, Peter M; Lee, Hyunna T et al. (2016) The Structural Basis of Actin Organization by Vinculin and Metavinculin. J Mol Biol 428:10-25
Karasik, Agnes; Shanmuganathan, Aranganathan; Howard, Michael J et al. (2016) Nuclear Protein-Only Ribonuclease P2 Structure and Biochemical Characterization Provide Insight into the Conserved Properties of tRNA 5' End Processing Enzymes. J Mol Biol 428:26-40
Howard, Michael J; Karasik, Agnes; Klemm, Bradley P et al. (2016) Differential substrate recognition by isozymes of plant protein-only Ribonuclease P. RNA 22:782-92
Mustoe, Anthony M; Liu, Xin; Lin, Paul J et al. (2015) Noncanonical secondary structure stabilizes mitochondrial tRNA(Ser(UCN)) by reducing the entropic cost of tertiary folding. J Am Chem Soc 137:3592-9
Bartke, Rebecca M; Cameron, Elizabeth L; Cristie-David, Ajitha S et al. (2015) Meeting report: SMART timing--principles of single molecule techniques course at the University of Michigan 2014. Biopolymers 103:296-302
Howard, Michael J; Klemm, Bradley P; Fierke, Carol A (2015) Mechanistic Studies Reveal Similar Catalytic Strategies for Phosphodiester Bond Hydrolysis by Protein-only and RNA-dependent Ribonuclease P. J Biol Chem 290:13454-64
Wang, Ningkun; Lodge, Jean M; Fierke, Carol A et al. (2014) Dissecting allosteric effects of activator-coactivator complexes using a covalent small molecule ligand. Proc Natl Acad Sci U S A 111:12061-6
Liu, Xin; Chen, Yu; Fierke, Carol A (2014) A real-time fluorescence polarization activity assay to screen for inhibitors of bacterial ribonuclease P. Nucleic Acids Res 42:e159
Daniels, Kyle G; Tonthat, Nam K; McClure, David R et al. (2014) Ligand concentration regulates the pathways of coupled protein folding and binding. J Am Chem Soc 136:822-5

Showing the most recent 10 out of 26 publications