Ribonuclease P (RNase P) catalyzes the maturation of the 5' end of precursor tRNA (pre-tRNA) to form tRNA, a component essential for the synthesis of proteins. RNase P from bacteria is composed of an RNA subunit that catalyzes pre-tRNA cleavage in vitro and a protein component that is essential for activity in vivo and enhances binding of the pre-tRNA substrate. In contrast, RNase P in eukaryotes contains one RNA and multiple protein subunits. We propose to investigate the function of the bacterial RNase P using a combination of biochemical and structural techniques. Specifically, we aim to: (1) explore the structure and dynamics of RNase P using time resolved fluorescence resonance energy transfer techniques to measure distances and mobility; (2) investigate substrate recognition in bacterial RNas P by determining the thermodynamics and function of PRNA-pre-tRNA and pre-tRNA-P protein contacts in RNase P and investigating the cleavage of novel RNA substrates; (3) investigate the position and functions of metals bound to RNase P for both catalysis and substrate recognition; and (4) delineate the position of metal ion binding sites in RNase P and the structure of isolated helices by NMR spectroscopic analysis. Our long term goal is to further understand (1) the mechanisms of catalysis used by ribozymes as compared to protein enzymes, and (2) the structure and energetics of RNA binding proteins and protein/RNA complexes. RNase P is a unique enzyme to investigate catalytic strategies and substrate recognition since the active site is near the protein-RNA interface. This unique collaboration between the protein and RNA subunits may provide insight into the evolution from RNA to protein catalysts. RNase P is an essential enzyme as tRNA maturation is required for protein synthesis. RNase P has potential medical applications as a novel antibiotic target since it is an essential enzyme and the eukaryotic and prokaryotic enzymes have different subunit composition. The structural and functional studies proposed here should provide insight into the development of active site-directed inhibitors of bacterial RNase P from target organisms such as S. aureus and Bacillus anthracis. ? ?
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