IDENTIFICATION OF PHOSPHATES INVOLVED IN CATALYSIS BY THE RIBOZYME RNASE-P RNA

The RNA subunit of ribonuclease P (RNase P RNA) is a catalytic RNA tha t cleaves precursor tRNAs to generate mature tRNA 5' ends. Little is k nown concerning the identity and arrangement of functional groups that constitute the active site of this ribozyme. We have used an RNase P RNA-substrate conjugate that undergoes rapid, accurate, and efficient self-cleavage in vitro to probe, by phosphorothioate modification-inte rference, functional groups required for catalysis. We identify four p hosphate oxygens where substitution by sulfur significantly reduces th e catalytic rate (50-200-fold). Interference at one site was partially rescued in the presence of manganese, suggesting a direct involvement in binding divalent metal ion cofactors required for catalysis. All s ites are located in conserved sequence and secondary structure, and po sitioned adjacent to the substrate phosphate in a tertiary structure m odel of the ribozyme-substrate complex. The spatial arrangement of pho sphorothioate-sensitive sites in RNase P RNA was found to resemble the distribution of analogous positions in the secondary and potential te rtiary structures of other large catalytic RNAs.