Conformational changes are often required for the biological function of RNA molecules. In the Tetrahymena group I ribozyme reaction, a conformational change has been suggested to occur upon binding of the oligonucleotide substrate (S) or the guanosine nucleophile (G), leading to stronger binding of the second substrate. Recent work showed that the two substrates are bridged by a metal ion that coordinates both the nonbridging reactive phosphoryl oxygen of S and the 2′-OH of G. These results suggest that the energy from the metal ion[bull]substrate interactions is used to drive the proposed conformational change. In this work, we provide an experimental test for this model. The results provide strong support for the proposed conformational change and for a central role of the bridging metal ion in this change. The results from this work, combined with previous data, allow construction of a two-state model that quantitatively accounts for all of the observations in this and previous work. This model provides a conceptual and quantitative framework that will facilitate understanding and further probing of the energetic and structural features of this conformational change and its role in catalysis.

Although the hammerhead reaction proceeds most efficiently in divalent cations, cleavage in 4 M LiCl is only ∼10-fold slower than under standard conditions of 10 mM MgCl2 (Murray et al., Chem Biol, 1998, 5:587–595; Curtis & Bartel, RNA, 2001, this issue, pp. 546–552). To determine if the catalytic mechanism with high concentrations of monovalent cations is similar to that with divalent cations, we compared the activities of a series of modified hammerhead ribozymes in the two ionic conditions. Nearly all of the modifications have similar deleterious effects under both reaction conditions, suggesting that the hammerhead adopts the same general catalytic structure with both monovalent and divalent cations. However, modification of three ligands previously implicated in the binding of a functional divalent metal ion have substantially smaller effects on the cleavage rate in Li+ than in Mg2+. This result suggests that an interaction analogous to the interaction made by this divalent metal ion is absent in the monovalent reaction. Although the contribution of this divalent metal ion to the overall reaction rate is relatively modest, its presence is needed to achieve the full catalytic rate. The role of this ion appears to be in facilitating formation of the active structure, and any direct chemical role of metal ions in hammerhead catalysis is small.