Functional involvement of G8 in the hairpin ribozyme cleavage mechanism

The catalytic determinants for the cleavage and ligation reactions mediated by the hairpin ribozyme are integral to the polyribonucleotide chain. We d escribe experiments that place G8, a critical guanosine, at the active site , and point to an essential role in catalysis. Cross-linking and modeling s how that formation of a catalytic complex is accompanied by a conformationa l change in which N1 and O6 of G8 become closely apposed to the scissile ph osphodiester. UV cross-linking, hydroxyl-radical footprinting and native ge l electrophoresis indicate that G8 variants inhibit the reaction at a step following domain association, and that the tertiary structure of the inacti ve complex is not measurably altered. Rate-pH profiles and fluorescence spe ctroscopy show that protonation at the NI position of G8 is required for ca talysis, and that modification of O6 can inhibit the reaction. Kinetic solv ent isotope analysis suggests that two protons are transferred during the r ate-limiting step, consistent with rate-limiting cleavage chemistry involvi ng concerted deprotonation of the attacking 2'-OH and protonation of the 5' -O leaving group. We propose mechanistic models that are consistent with th ese data, including some that invoke a novel keto-enol tautomerization.