Helix P4 is a divalent metal ion binding site in the conserved core of theribonuclease P ribozyme

The ribonuclease P ribozyme (RNase P RNA), like other large ribozymes, requ ires magnesium ions for folding and catalytic function; however, specific s ites of metal ion coordination in RNase P RNA are not well defined. To iden tify and characterize individual nucleotide functional groups in the RNase P ribozyme that participate in catalytic function, we employed self-cleavin g ribozyme-substrate conjugates that facilitate measurement of the effects of individual functional group modifications. The self-cleavage rates and p H dependence of two different ribozyme-substrate conjugates were determined and found to be similar to the single turnover kinetics of the native ribo zyme. Using site-specific phosphorothioate substitutions, we provide eviden ce for metal ion coordination at the pro-R-p phosphate oxygen of A67, in th e highly conserved helix P4, that was previously suggested by modification- interference experiments. In addition, we detect a new metal ion coordinati on site at the pro-S-p phosphate oxygen of A67. These findings, in combinat ion with the proximity of A67 to the pre-tRNA cleavage site, support the co nclusion that an important role of helix P4 in the RNase P ribozyme is to p osition divalent metal ions that are required for catalysis.