RIBONUCLEASE-P (RNASE-P) RNA IS CONVERTED TO A CD2-RIBOZYME BY A SINGLE RP-PHOSPHOROTHIOATE MODIFICATION IN THE PRECURSOR TRANSFER-RNA AT THE RNASE-P CLEAVAGE SITE()

To study the cleavage mechanism of bacterial Nase P RNA, we have synth esized precursor tRNA substrates carrying a single Rp- or Sp-phosphoro thioate modification at the RNase P cleavage site, Both the Sp- and th e Rp-diastereomer reduced the rate of processing by Escherichia coli R Nase P RNA at least 1000-fold under conditions where the chemical step is rate-limiting, The Rp-modification had no effect and the Sp-modifi cation had a moderate effect on precursor tRNA ground state binding to RNase P RNA. Processing of the Rp-diastereomeric substrate was largel y restored in the presence of the ''thiophilic'' Cd2+ as the only diva lent metal ion, demonstrating direct metal ion coordination to the (pr o)-Rp substituent at the cleavage site and arguing against a specific role for Mg2+-ions at the pro-Sp oxygen. For the Rp-diastereomeric sub strate, Hill plot analysis revealed a cooperative dependence upon [Cd2 +] of n(H) = 1.8, consistent with a two-metal ion mechanism. In the pr esence of the Sp-modification, neither Mn2+ nor Cd2+ was able to resto re detectable cleavage at the canonical site, Instead, the ribozyme pr omotes cleavage at the neighboring unmodified phosphodiester with low efficiency, Dramatic inhibition of the chemical step by both the Rp- a nd Sp-phosphorothioate modification is unprecedented among known riboz ymes and points to unique features of transition state geometry in the RNase P RNA-catalyzed reaction.