ENZYMOLOGY OF RNA PROCESSING ENZYMES

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DESCRIPTION: Ribonuclease P (RNase P ) is a ribonucleoprotein that catalyzes the essential 5' maturation of precursor tRNA. While both the protein and RNA subunits are essential for in vivo activity, the bacterial RNase P RNA moiety exhibits catalytic activity in vitro. Mechanistic and structural investigations of this ribozyme are important because it is the only known RNA-containing nuclease that catalyzes multiple turnovers and is unchanged during catalysis. This proposal outlines a collaborative research effort between the Fierke and Christianson groups to probe the enzymology and structural biology of B. subtilis RNase P. Catalytic efficiency will be related to macromolecular structure through a combination of X-ray crystallography, kinetic and equilibrium analysis, and structural perturbation. Specifically, we aim to: (1) determine and refine the three-dimensional structure of the protein component of Bacillus subtilis RNase P; (2) investigate the catalytic mechanism of both RNase P and the 3', 5' exonuclease of DNA polymerase I using steady state and transient state kinetics to probe the dependence of catalysis on metal and phosphorothioate substitution, pH, isotopic composition of the solvent, and structural variants of the enzyme; (3) investigate the functional role of the protein component of RNase P by a combination of kinetics, footprinting, crosslinking and mutagenesis; (4) identify the position and role of essential magnesium ions by modification-interference experiments and phosphorothioate substitutions in the P RNA; (5) select RNAs that bind to the RNase P protein from libraries of fragments of RNase P RNA and completely random RNA, for use in investigating the structure of RNA-protein complexes by X-ray crystallography; and (6) explore the crystallization of a catalytically-active RNase P holoenzyme, especially using a circular RNA component.These proposed mechanistic and structure-function studies of RNase P will further our understanding of the catalytic modes used by ribozymes in comparison to protein catalysts, the relationship between RNA structure and function, and the specific interactions involved in RNA-RNA and RNA-protein binding. A fundamental understanding of these principles from in vitro experiments is a prerequisite for comprehending RNA catalysis in vivo, for designing mechanism-based drugs to inhibit RNase P and ribozymes in general, and for engineering endoribonucleases specific for particular RNA species, including the therapeutic use of ribozymes. Furthermore, since RNase P is an essential bacterial enzyme, this enzyme may represent a feasible target for novel antibiotics.