The structural features of the catalytic RNA subunit and the protein subunit of RNAase P from E. coli, which are required for catalysis and the accurate cleavage of tRNA precursor molecules, will be investigated by utilizing subunits of enzyme that have been altered by chemical enzymatic or genetic methods. Derivatives of the catalytic subunit or altered forms of the RNase P holoenzyme with Km or k cat values, different from those of the wild type will be identified. In particular, subunits of the enzyme, in combination with modified substrates, will be used to improve the yield of enzyme-substrate complexes, and thereby facilitate the identification and study by chemical techniques of the active site of the enzyme. Model substrates will be synthesized to determine the features of the substrates which are essential for cleavage by the enzyme. Hybrid enzymes, composed of subunits from different organisms, will also be used to determine the role played by each subunit in the catalytic activity of the holoenzyme in vitro. Attempts to crystallize the RNase P from E. coli will proceed concurrently. The importance of the processing of RNA in the biosynthesis of RNA molecules and in the regulation of gene expression during the cell cycle of simple organisms and during differentiation of higher organisms is well established. It is apparent that the endo and exonucleolytic cleavages, ligations, modification and addition of nucleotides, which RNA molecules undergo are not biochemical curiosities, but are essential to the normal function and expression of all genomes. Many of these reactions involve novel mechanisms of catalysis and/or novel aggregates of macromolecules which either directly govern processing reactions or in some way act as cofactors. Dr. Altman originally discovered that RNA is an essential component of RWase P. After the discovering of self-splicing introns he showed that RNA above could perform the catalytic function.
