Studies on Escherichia coli RNase P RNA with Zn(2+) as the catalytic cofactor

We demonstrate, for the first time, catalysis by Escherichia coli ribonuclease P (RNase P) RNA with Zn(2+) as the sole divalent metal ion cofactor in the presence of ammonium, but not sodium or potassium salts. Hill analysis suggests a role for two or more Zn(2+) ions in catalysis. Whereas Zn(2+) destabilizes substrate ground state binding to an extent that precludes reliable K(d) determination, [Formula: see text] and Sr(2+) in particular, both unable to support catalysis by themselves, promote high-substrate affinity. Zn(2+) and [Formula: see text] substantially reduce the fraction of precursor tRNA molecules capable of binding to RNase P RNA. Stimulating and inhibitory effects of Sr(2+) on the ribozyme reaction with Zn(2+) as cofactor could be rationalized by a model involving two Sr(2+) ions (or two classes of Sr(2+) ions). Both ions improve substrate affinity in a cooperative manner, but one of the two inhibits substrate conversion in a non-competitive mode with respect to the substrate and the Zn(2+). A single 2′-fluoro modification at nt −1 of the substrate substantially weakened the inhibitory effect of Sr(2+). Our results demonstrate that the studies on RNase P RNA with metal cofactors other than Mg(2+) entail complex effects on structural equilibria of ribozyme and substrate RNAs as well as E·S formation apart from the catalytic performance.