Role of metal ions in the hydrolysis reaction catalyzed by RNase P RNA from Bacillus subtilis

Precursor tRNA (ptRNA) substrates carrying a single Rp or Sp-phosphorothioa te modification at the RNase P cleavage site were used as tools to study th e cleavage mechanism of RNase P RNA from Bacillus subtilis. Both the Sp and the Rp-diastereomer reduced the rate of processing at least 10(4)-fold und er conditions where the chemical step is essentially rate-limiting. Neither the Rp nor the Sp-phosphorothioate modification affected ptRNA ground stat e binding to B. subtilis RNase P RNA. Processing of the Rp-diastereomeric p tRNA could be restored in the presence of Mn2+ or Cd2+, demonstrating direc t metal ion coordination to the pro-RF oxygen during catalysis. With Cd2+, processing required the presence of another metal ion, such as Ca2+ or Mg2, to mediate substrate binding. This is in contrast to Escherichia coli RNa se P RNA, which promotes cleavage of Rp-diastereomeric ptRNA in the presenc e of Cd2+ as the sole divalent metal ion. Analysis of [Cd2+]-dependent proc essing of the Rp-diastereomeric substrate by B. subtilis RNase P RNA was co nsistent with the involvement of at least two metal ions in catalysis. The presence of two catalytic metal ion binding sites is also supported by the inhibition mode of Ca2+ on cleavage of unmodified ptRNA. In the presence of an Sp-phosphorothioate modification at the scissile bond, neither Mn2+ nor Cd2+ were able to restore significant cleavage at this location. Instead, the ribozyme promotes cleavage at the neighboring unmodified phosphodiester with low efficiency. Unaffected ground state binding of the Sp-diastereome ric ptRNA but a greater than or equal to 10(4)-fold reduced hydrolysis rate may indicate a crucial role of the pro-Sp oxygen in transition state stabi lization or may be attributed to steric exclusion of catalytic metal ions. Based on our comparative analyses of B, subtilis and E. coli RNase P RNA, e ach representing the main structural subtypes of bacterial RNase P RNA, com mon features in terms of active site constraints and role of catalytic meta l ions can now be formulated for bacterial RNase P RNAs. On the other hand, substantial and unexpected differences with respect to the overall metal i on requirements and tRNA binding modes have been observed for the two catal ytic RNAs. (C) 1999 Academic Press.