Studies on Escherichia coli RNase P RNA with Zn2+ as the catalytic cofactor

We demonstrate, for the first time, catalysis by Escherichia coli ribonuclease P (RNase P) RNA with Zn21 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 Zn21 ions incatalysis.Whereas Zn21 destabilizessub-strate ground state binding to an extent that pre-cludes reliable Kd determination, Co(NH3)6 31 and Sr21 in particular, both unable to support catalysis by themselves, promote high-substrate affinity. Zn21 and Co(NH3)6 31 substantially reduce the fraction of precursor tRNA molecules capable of binding to RNase P RNA. Stimulating and inhibitory effects of Sr21 on the ribozyme reaction with Zn21 as cofactor could be rationalized by a model involving two Sr21 ions (or two classes of Sr21 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 Zn21. A single 20-fluoro modification at nt 1 of the substrate substantially weakened the inhibitory effect of Sr21. Our results demonstrate that the stud-ies on RNase P RNA with metal cofactors other than Mg21 entail complex effects on structural equilibria of ribozyme and substrate RNAs as well as ES formation apart from the catalytic performance