A hydrogen-bonding triad stabilizes the chemical transition state of a group I ribozyme Original Research Article

Background The group I intron is an RNA enzyme capable of efficiently catalyzing phosphoryl-transfer reactions. Functional groups that stabilize the chemical transition state of the cleavage reaction have been identified, but they are all located within either the 5′-exon (P1) helix or the guanosine cofactor, which are the substrates of the reaction. Functional groups within the ribozyme active site are also expected to assist in transition-state stabilization, and their role must be explored to understand the chemical basis of group I intron catalysis. Results Using nucleotide analog interference mapping and site-specific functional group substitution experiments, we demonstrate that the 2′-OH at A207, a highly conserved nucleotide in the ribozyme active site, specifically stabilizes the chemical transition state by ~2 kcal mol−1. The A207 2′-OH only makes its contribution when the U(−1) 2′-OH immediately adjacent to the scissile phosphate is present, suggesting that the 2′-OHs of A207 and U(−1) interact during the chemical step. Conclusions These data support a model in which the 3′-oxyanion leaving group of the transesterification reaction is stabilized by a hydrogen-bonding triad consisting of the 2′-OH groups of U(−1) and A207 and the exocyclic amine of G22. Because all three nucleotides occur within highly conserved non-canonical base pairings, this stabilization mechanism is likely to occur throughout group I introns. Although this mechanism utilizes functional groups distinctive of RNA enzymes, it is analogous to the transition states of some protein enzymes that perform similar phosphoryl-transfer reactions