BACKGROUND: The 2'-hydroxyl of U preceding the cleavage site, U(-1), in the Tetrahymena ribozyme reaction contributes 10(3)-fold to catalysis relative to a 2'-hydrogen atom. Previously proposed models for the catalytic role of this 2'-OH involve coordination of a catalytic metal ion and hydrogen-bond donation to the 3'-bridging oxygen. An additional model, hydrogen-bond donation by the 2'-OH to a nonbridging reactive phosphoryl oxygen, is also consistent with previous results. We have tested these models using atomic-level substrate modifications and kinetic and thermodynamic analyses. RESULTS: Replacing the 2'-OH with -NH(3)(+) increases the reaction rate approximately 60-fold, despite the absence of lone-pair electrons on the 2'-NH(3)(+) group to coordinate a metal ion. Binding and reaction of a modified oligonucleotide substrate with 2'-NH(2) at U(-1) are unaffected by soft-metal ions. These results suggest that the 2'-OH of U(-1) does not interact with a metal ion. The contribution of the 2'-moiety of U(-1) is unperturbed by thio substitution at either of the nonbridging oxygens of the reactive phosphoryl group, providing no indication of a hydrogen bond between the 2'-OH and the nonbridging phosphoryl oxygens. In contrast, the 10(3)-fold catalytic advantage of 2'-OH relative to 2'-H is eliminated when the 3'-bridging oxygen is replaced by sulfur. As sulfur is a weaker hydrogen-bond acceptor than oxygen, this effect suggests a hydrogen-bonding interaction between the 2'-OH and the 3'-bridging oxygen. CONCLUSIONS: These results provide the first experimental support for the model in which the 2'-OH of U(-1) donates a hydrogen bond to the neighboring 3'-bridging oxygen, thereby stabilizing the developing negative charge on the 3'-oxygen in the transition state.