Investigation of the RNA-Protein Interactions in Bacterial Ribonuclease P (RNase P).

Abstract

Ribonuclease P (RNase P) is a highly conserved ribonucleoprotein (RNP) that catalyzes the cleavage of the 5’ leader off of all precursor tRNA (pre-tRNA) molecules in a metal dependent manner. Given the RNP nature of this essential ribozyme, discerning how the RNA and protein components of RNase P work together to perform this critical reaction is key to understanding how the enzyme works. The work presented here enhances current knowledge of RNase P substrate recognition, assigns additional functional roles to the required protein, and characterizes RNase P RNA – metal interactions in RNase P on a molecular level. Genomic analysis reveals that particular nucleotides are preferred in the 5’ leader sequence of pre-tRNAs, a region of the substrate proposed to interact with the protein component of RNase P. To test if this preference has relevance to substrate binding or processing, a series of affinity and single turnover measurements with pre-tRNAs substrates that varied the nucleotide identity at a particular 5’ leader position were undertaken. These studies reveal the first observed sequence specific interaction between a nucleotide in the 5’ leader of pre-tRNA and RNase P. Further investigations with B. subtilis RNase P protein mutants identified and characterized the specific contact between the 5’ leader and the protein. The protein contribution to catalysis was further studied with a combination of affinity studies and transient kinetic techniques. These investigations identify a number of roles for the most conserved region of the bacterial protein including stabilizing the RNP structure, and enhancing a kinetically important metal dependent conformational change. Additionally, a combination of nuclear magnetic resonance (NMR) and extended x-ray absorption fine structure (EXAFS) were used to identify and characterize an inner-sphere metal binding site in the putative active site of this metallo-ribozyme. Overall, the work presented in this thesis has advanced the understanding of how RNA, protein, and metals work synergistically to perform a fundamental biological process in the essential RNP and enzyme RNase P.