The role of the cleavage site 2 '-hydroxyl in the Tetrahymena group I ribozyme reaction

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 o f this 2'-OH involve coordination of a catalytic metal ion and hydrogen-bon d donation to the 3'-bridging oxygen. An additional model, hydrogen-bond do nation by the 2'-OH to a nonbridging reactive phosphoryl oxygen, is also co nsistent with previous results. We have tested these models using atomic-le vel substrate modifications and kinetic and thermodynamic analyses. Results: Replacing the 2'-OH with -NH3+ increases the reaction rate similar to 60-fold, despite the absence of lone-pair electrons on the 2'-NH3+ grou p to coordinate a metal ion. Binding and reaction of a modified oligonucleo tide substrate with 2'-NH2 at U(-1) are unaffected by soft-metal ions. Thes e results suggest that the 2'-OH of U(-1) does not interact with a metal io n. The contribution of the 2'-moiety of U(-1) is unperturbed by thio substi tution at either of the nonbridging oxygens of the reactive phosphoryl grou p, providing no indication of a hydrogen bond between the 2'-OH and the non bridging phosphoryl oxygens. In contrast, the 103-fold catalytic advantage of 2'-OH relative to 2'-H is eliminated when the 3'-bridging oxygen is repl aced by sulfur. As sulfur is a weaker hydrogen-bond acceptor than oxygen, t his effect suggests a hydrogen-bonding interaction between the 2'-OH and th e 3'-bridging oxygen. Conclusions: These results provide the first experimental support for the m odel 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.