The structural features of M1 RNA and C5 protein (the catalytic RNA subunit and the protein subunit, respectively, of RNase P from E. coli) which are required for catalysis and the accurate cleavage of tRNA precursor molecules will be investigated by utilizing subunits of the enzyme that have been altered by chemical and enzymatic methods. In addition, nucleotide sequences of the analog of M1 RNA from several eucaryotic organisms will be determined using a technique based on the PCR reaction in order to test a universal model of the secondary structure of these RNAs. Subunits of the enzyme, in combination with certain modified substrates, will be used to improve the yield of enzyme-substrate complexes, and, thereby, to facilitate identification and study by chemical techniques of the active site of the enzyme. Model substrates with phosphorothioates located at the site of cleavage by the enzyme will be used to test predictions concerning the reaction mechanism. Model substrates will also be synthesized to elaborate the features of substrates that determine the site of cleavage by the enzyme. Attempts to crystallize RNase P from E. coli will proceed concurrently.
