AN UNUSUAL PH-INDEPENDENT AND METAL-ION-INDEPENDENT MECHANISM FOR HAIRPIN RIBOZYME CATALYSIS

Background: Hairpin ribozymes (RNA enzymes) catalyze the same chemical reaction as ribonuclease A and yet RNAs do not usually have functiona l groups analogous to the catalytically essential histidine and lysine sidechains of protein ribonucleases. Some RNA enzymes appear to recru it metal ions to act as Lewis acids in charge stabilization and metal- bound hydroxide for general base catalysis, but it has been reported t hat the hairpin ribozyme functions in the presence of metal ion chelat ors, This led us to investigate whether the hairpin ribozyme exploits a metal-ion-independent catalytic strategy. Results: Substitution of s ulfur for nonbridging oxygens of the reactive phosphate of the hairpin ribozyme has small, stereospecific and metal-ion-independent effects on cleavage and ligation mediated by this ribozyme. Cobalt hexammine, an exchange-inert metal complex, supports full hairpin ribozyme activi ty, and the ribozyme's catalytic rate constants display only a shallow dependence on pH. Conclusions: Direct metal ion coordination to phosp hate oxygens is not essential for hairpin ribozyme catalysis and metal -bound hydroxide does not serve as the general base in this catalysis. Several models might account for the unusual pH and metal ion indepen dence: hairpin cleavage and ligation might be limited by a slow confor mational change; a pH-independent or metal-cation-independent chemical step, such as breaking the 5' oxygen-phosphorus bond, might be rate d etermining; or finally, functional groups within the ribozyme might pa rticipate directly in catalytic chemistry. Whichever the case, the hai rpin ribozyme appears to employ a unique strategy for RNA catalysis.