A unique mechanism for RNA catalysis: the role of metal cofactors in hairpin ribozyme cleavage Original Research Article

Background: Ribozymes are biological catalysts that promote the hydrolysis and transesterification of phosphate diesters of RNA. They typically require divalent magnesium ions for activation, although it has proven difficult to differentiate structural from catalytic roles for the magnesium ions and to identify the molecular mechanism of catalysis. Direct inner-sphere coordination is usually invoked in the catalytic step, although there is no evidence to support the generality of such a pathway for all ribozymes. Results: We studied the catalytic pathway for the hairpin class of ribozyme. The substitutionally inert transition metal complex cobalt hexaammine [Co(NH3)63+) was shown to be as active as Mg2+(aq) in promoting hairpin ribozyme activity, demonstrating that inner-sphere pathways are not used by this class of ribozyme. These results were confirmed by studies with RP- and SP-phosphorothioate substrate analogs which show a similar reactivity to that of the native substrate towards the magnesium-activated ribozyme. Monovalent cations enhance the activity of Co(NH3)63+-promoted reactions, but inhibit Mg2+-activated catalysis, demonstrating a requirement for hydrated cations at several key sites in the ribozyme. Conclusions: These results provide clear support for a model of RNA catalysis that does not involve direct coordination of magnesium to the phosphate ester, nor activation of a bound water molecule. A mechanism in which catalysis is carried out by functional groups on the RNA ribozyme itself is possible; such functional groups are likely to have pKa values that are appropriate for carrying out this catalysis. The metal cofactor would then serve to define the architecture of the catalytic pocket and contribute to the stabilization of transient species, as has been described earlier. Hydrolytic pathways in nucleic acid reactions are apparently more diverse than was previously thought, and the hairpin ribozyme falls into a mechanistically distinct class from the Tetrahymena and the hammerhead ribozymes.