Exocyclic Amine of the Conserved G·U Pair at the Cleavage Site of the Tetrahymena Ribozyme Contributes to 5‘-Splice Site Selection and Transition State Stabilization

Abstract

A phylogenetically conserved guanine.uracil (G.U) pair defines the 5'-exon/intron boundary of precursor RNAs containing group I introns. In this wobble base pair, the G forms two hydrogen bonds with U in a base pairing geometry shifted from that of a canonical Watson-Crick pair. On the basis of thermodynamic measurements of synthetic base pair analogs (inosine, diaminopurine riboside, guanosine, or adenosine paired with U, C, or isocytidine) in place of the G.U pair, we have previously reported that the N2 exocyclic amine of the G is important for docking the 5'-exon into the active site of the Tetrahymena ribozyme [Strobel, S. A., & Cech, T. R. (1995) Science 267, 675-679]. Here we describe kinetic characterization of ribozyme-substrate combinations containing the same series of analogs. By measuring the rate constants of 5'-exon miscleavage (cleavage at incorrect phosphates), we demonstrate that the 5'-exon/intron boundary is primarily defined by the exocyclic amine of the G. The amine makes its contribution (2.5 kcal.mol-1) in the context of all three wobble pairs tested but fails to make a significant contribution (< 0.8 kcal.mol-1) when presented in a Watson-Crick base pairing geometry. We also demonstrate that the exocyclic amine makes a modest contribution to chemical transition state stabilization (1.0 kcal.mol-1 relative to an inosine-U pair). The majority of this transition state contribution (0.7 kcal.mol-1) is independent of that contributed by the 2'-hydroxyl of the neighboring U. This argues against the model in which substantial transition state stabilization is derived from a water molecule bridging between the exocyclic amine of G and the 2'-hydroxyl of U. Instead it suggests that the tertiary interaction between the exocyclic amine and its hydrogen bonding partner in the active site is slightly improved during the chemical transition. We conclude that the exocyclic amine of G is the primary contributor to many characteristics of reactivity that have been ascribed to the conserved G.U pair, including stabilization of the chemical transition state and definition of the 5'-exon/intron boundary.