Cross talk between the +73/294 interaction and the cleavage site in RNase P RNA mediated cleavage.

To monitor functionally important metal ions and possible cross talk in RNase P RNA mediated cleavage we studied cleavage of substrates, where the 2'OH at the RNase P cleavage site (at -1) and/or at position +73 had been replaced with a 2' amino group (or 2'H). Our data showed that the presence of 2' modifications at these positions affected cleavage site recognition, ground state binding of substrate and/or rate of cleavage. Cleavage of 2' amino substituted substrates at different pH showed that substitution of Mg2+ by Mn2+ (or Ca2+), identity of residues at and near the cleavage site, and addition of C5 protein influenced the frequency of miscleavage at -1 (cleavage at the correct site is referred to as +1). From this we infer that these findings point at effects mediated by protonation/deprotonation of the 2' amino group, i.e. an altered charge distribution, at the site of cleavage. Moreover, our data suggested that the structural architecture of the interaction between the 3' end of the substrate and RNase P RNA influence the charge distribution at the cleavage site as well as the rate of cleavage under conditions where the chemistry is suggested to be rate limiting. Thus, these data provide evidence for cross talk between the +73/294 interaction and the cleavage site in RNase P RNA mediated cleavage. We discuss the role metal ions might play in this cross talk and the likelihood that at least one functionally important metal ion is positioned in the vicinity of, and use the 2'OH at the cleavage site as an inner or outer sphere ligand.