The biogenesis of tRNA from primary gene transcripts to their final functional form in E. coli, yeast and human cells will be investigated and, in particular, the function of Rnase P, a ribonucleoprotein essential for the processing of the 5' termini of tRNAs, will be examined in detail. The function in vivo of mutants in both subunits of Rnase P from E. coli will be examined by complementation in backgrounds mutant for either the rnpA or rnpB genes. These experiments will verify the results of experiments in vitro in which the function of mutants in both subunits of the enzyme and the substrate have been made and assayed based on their proposed location in a three dimensional model of the RNA subunit of Rnase P and the crystal structure of the protein subunit of Rnase P from B. subtilis. Cross-linking experiments with substrates and enzymatic subunits will also be used to test aspects of the structural models and theories about enzyme-substrate interactions. Genetic methods will be used to assay both RNA-protein interactions in Rnase P and to search for new substrates for RNAse P in E. coli and S. cerevisiae. Characterization of the protein subunits of Rnase P from both yeast and human cells will be completed. Particular subunits, especially from the human enzyme, will be tagged with antigenic and fluorescent markers to facilitate both the purification of the enzyme complexes and studies of the localization of the protein subunits in cells and tissue culture. Reconstitution of the enzyme from isolated subunits will be attempted to facilitate functional studies of individual mutated subunits. Inactivation of in vivo of genes coding for drug resistance in bacteria, for regulation of viral function in a herpes virus in mammalian cells, and of the protein subunit of Rnase P in bacteria will be pursued using the concerted action of small, custom designed RNAs called external guide sequences (EGS) and Rnase P.
