Function and Metabolism of RNA

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DESCRIPTION (provided by applicant): Ribonuclease P is the ubiquitous endoribonuclease that cleaves pre-tRNAs to produce their mature 5'-ends. RNaseP is a particularly unusual enzyme because the catalytic center is composed not of protein, but of RNA. The proposed program will continue long-standing efforts by this laboratory to understand the biochemistry and biology of RNase P. The specific goals of the program are: 1. To determine the structure of the bacterial RNaseP using biochemical and crystallographic methods. Successful biochemical and phylogenetic analyses will be continued in order to refine and extend the structure model of RNase P. Atomic-level structure will be required to understand the chemical nature of the enzyme, however, and crystallographic analysis is underway with native and derived RNAs and holoenzymes. 2. To determine the role of bacterial RNaseP protein in holoenzyme structure and catalysis. The role of the bacterial RNaseP protein in structure and function of the holoenzyme is not clear. Biochemical and kinetic methods will be used to explore the interaction of the protein with the RNA and how that influences the interaction with substrate. 3. To determine the structure of the eukaryotic RNaseP and its components, with Schizosaccharomyces pombe as a model. The relatively simple structure and some properties of the S. pombe RNase PRNA indicate that it will be a fruitful model for the eukaryotic holoenzyme. 4. To determine the structure of the archaeal RNaseP and its components, with Sulfolobus acidocaldarius as a model. The properties of an archaeal RNase P, in comparison to the bacterial and eukaryal holoenzymes, will illuminate the universal properties of the enzyme. Archaea have homologs of eukaryal RNaseP proteins, so the properties of the archaeal enzyme will be relevant to understanding the eukaryotic enzyme. The hyperthermophilic character of this archaeal RNaseP may render it more amenable than the eukaryotic enzymes to procedures such as reconstitution from isolated components and crystallization for structure analysis.