Differential role of the intermolecular base-pairs G292-C75 and G293-C74 in the reaction catalyzed by Escherichia coli RNase P RNA

Abstract

We present a systematic investigation of the thermodynamic and kinetic role of the intermolecular G292-C75 and G293-C74 Watson-Crick base-pairs in the reaction catalyzed by Escherichia coli RNase P RNA. Single turnover kinetics were analyzed for wild-type RNase P RNA and two variants with a single G to C exchange (C292 or C293), either acting on wild-type precursor tRNA (ptRNA) or derivatives carrying a complementary change at the tRNA 3′-end (G74CA or CG75A). Ground state binding of tRNA was studied using three different methods, including a novel fluorescence-based assay measuring equilibrium binding. We conclude that: (1) the role of the G293-C74 interaction is essentially confined to Watson-Crick base-pairing, with no indication for crucial tertiary contacts involving this base-pair; (2) the G293-C74 pair, although being as important for ptRNA ground state binding as G292-C75, is much less crucial to catalytic performance than the G292-C75 pair; (3) disruption of the G292-C75 base-pair results in preferential destabilization of enzyme transition-state complexes; and (4) the identity of the G292-C75 pair, as part of the higher-order structural context consisting of coplanar G292-C75-A258 and G291-G259-A76 triples, contributes to high affinity binding of ptRNA and catalytic efficiency.