Ribonuclease P is the processing endoribonuclease responsible for the accurate cleavage of tRNA precursor molecules to form their mature 5' termini. Since the discovery of its catalytic activity, the RNA subunit of eubacterial Rnase P has been the focus of intense study. In contrast, much less is known about the RNA subunit of eucaryotic Rnase P. The main thrust of present application is to develop more detailed knowledge about the structure of the RNA subunit of the eucaryotic enzyme and to learn the manner in which the RNA subunit contributes to catalytic function.
The specific aims of the present application are two-fold. I. Additional eucaryotic RNA subunit sequences will be determined in order to refine the proposed secondary structure model. Primers complementary to the most conserved sequence elements of the known eucaryotic RNA subunits will be used to clone the corresponding RNAs from other eucaryotic sources. These RNA sequences will guide the development of specific hypotheses about eucaryotic RNase P RNA structure that may be tested. II. The secondary and tertiary structures of the RNA subunit from representatives of the eubacterial and eucaryotic RNase P families will be characterized. Cross-linking probes attached to the RNA structure as well as solvent-based cleavage or modification reagents that react with the ribose or base moieties of the RNA structure will be employed to study the folded conformation of the RNA subunit of RNase P. These studies will permit an assessment of whether P RNAs share similarly folded structures in solution, and provide a first critical test of the universal secondary structure model proposed for all RNase P and related processing enzymes. As a long-term objective, basic knowledge will be gathered about the roles of RNA folding and RNA-protein interactions in such phenomena like RNA catalysis, RNA catalysis, RNA processing and transport, and translation. Since the human RNase P enzyme is antigenically related to a common target of autoantibodies found in sera from patients with autoimmune diseases, general knowledge of the eucaryotic RNase P holoenzyme may provide clues relevant to the potential role of RNase P and related enzymes in certain autoimmune disorders, such as systemic lupus erythematosus.
