The proposed program is a continuing analysis of the molecular biology of ribonuclease (RNase) P, a ribonucleoprotein enzyme that contains a catalytic RNA involved in tRNA processing. The general goals of the project are to understand the structure, mechanism of action and biological variation of this ribozyme. The specific elements of the proposed program are: 1. Continued analysis of RNase P secondary and tertiary structure using phylogenetic structure comparisons, chemical and enzymatic structure mapping, intramolecular crosslinking, and molecular dynamics computer modeling. 2. Continued study of the RNase P RNA-tRNA interaction using intermolecular crosslinking, high-resolution chemical footprinting, and mutational analysis. 3. A directed mutational inspection of RNase P RNA structure and mechanism, focusing on structural elements believed on the basis of previous results to be important in the reaction. 4. Continued characterization of thermally stable RNase P RNAs and the design of a simplified, model RNase P RNA based on stabilizing structural features identified in those thermophilic RNAs. 5. Characterization of novel types of eucaryal RNase P and comparative analysis of eucaryal RNase P RNA structure. 6. Continued comparative structural analysis of archaeal RNase P RNA. The program is health-related in that it will shed light on fundamental aspects of RNA function; including structure, the nature of RNA-RNA interactions, and RNA-mediated catalysis. RNA functions such as these are basic to many agents of pathological conditions, for instance viruses composed of RNA. RNA-based therapeutics, for instance ribozymes, offer enormous potential for new directions in treatment of disease. RNase P is an outstanding model for achieving an understanding of the many facets of RNA function.
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