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

Background: The group I intron is an RNA enzyme capable of efficiently. cat alyzing phosphoryl-transfer reactions. Functional groups that stabilize the chemical transition state of the cleavage reaction have been identified, b ut they are all located within either the 5'-exon (P1) helix or the guanosi ne cofactor, which are the substrates of the reaction. Functional groups wi thin the ribozyme active site are also expected to assist in transition-sta te stabilization, and their role must be explored to understand the chemica l basis of group I intron catalysis. Results: Using nucleotide analog interference mapping and site-specific fun ctional group substitution experiments, we demonstrate that the 2'-OH at A2 07, a highly conserved nucleotide in the ribozyme active site, specifically stabilizes the chemical transition state by similar to 2 kcalmol(-1). The A207 2'-OH only makes its contribution when the U(-1) 2'-OH immediately adj acent to the scissile phosphate is present, suggesting that the 2'-OHs of A 207 and U(-l) interact during the chemical step. Conclusions: These data support a model in which the 3'-oxyanion leaving gr oup 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 a mine of G22. Because ail three nucleotides occur within highly conserved no n-canonical base pairings, this stabilization mechanism is likely to occur throughout group I introns. Although this mechanism utilizes functional gro ups distinctive of RNA enzymes, it is analogous to the transition states of some protein enzymes that perform similar phosphoryl-transfer reactions.