Molecular Recognition of Inhibitors, Metal Ions and Substrates by Ribonuclease P.

Abstract

Ribonuclease P (RNase P) is a divalent metal ion-dependent endonuclease that catalyzes cleavage of the 5’ leader of precursor transfer RNA (pre-tRNA), an essential tRNA processing step in all domains of life. Bacterial RNase P is a potential antibacterial target because it is essential for cell survival and varies in composition from its eukaryotic counterparts. Bacterial RNase P contains a catalytic RNA (P RNA) with one protein subunit while eukaryotic nuclear RNase P has multiple protein subunits with a catalytic RNA core and the human mitochondrial RNase P (mtRNase P) consists solely of protein subunits. In this dissertation research, first a novel fluorescence polarization (FP) assay was developed to facilitate rapid and real-time measurements of RNase P activity. This new FP assay was optimized for high-throughput screening to search for new inhibitors of bacterial RNase P in small molecule (2,880 compounds) and natural product extracts (22,720 samples) libraries. A new RNase P inhibitor was identified from the screen. Second, to test the biochemical role and metal ion binding function of a carbonyl oxygen (oxygen-4, O4) in the universally conserved bulged uridine (U51) of P RNA, RNase P with single atom modifications of 4-thiouridine, 4-deoxyuridine, 3-methyluridine and an abasic site were prepared and analyzed. Binding data demonstrate that the O4 of U51 enhances pre-tRNA affinity in a divalent metal-ion dependent fashion. In addition, kinetic data suggest that U51 enhances pre-tRNA cleavage by interacting with a magnesium ion stabilizing an active enzyme-substrate conformation. Third, a library of human mitochondiral pre-tRNAs and high purity human mtRNase P was prepared for studying the substrate specificity of human mtRNase P. Single-turnover cleavage data demonstrate that the MRPP3 subunit of human mtRNase P is catalytically active alone in vitro and that the MRPP1∙MRPP2 subcomplex increases the cleavage rate and cleavage site fidelity. Overall, the work presented in this dissertation has provided a new real-time and high-throughput methodology to assay RNase P activity in vitro, enhanced our understanding of how bacterial RNase P recognizes inhibitors and metal ions, and laid the foundation for elucidating substrate recognition by the newly identified protein-only human mtRNase P.