INVOLVEMENT OF A SPECIFIC METAL-ION IN THE TRANSITION OF THE HAMMERHEAD RIBOZYME TO ITS CATALYTIC CONFORMATION

Previous crystallographic and biochemical studies of the hammerhead ri bozyme suggest that a metal ion is ligated by the pro-R-p oxygen of ph osphate 9 and by N-7 of G10.1 and has a functional role in the cleavag e reaction, We have tested this model by examining the cleavage proper ties of a hammerhead containing a unique phosphorothioate at position 9. The R-p-, but not S-p-, phosphorothioate reduces the cleavage rate by 10(3)-fold, and the rate can be fully restored by addition of low c oncentrations of Cd2+, a thiophilic metal ion. These results strongly suggest that this bound metal ion is critical for catalysis, despite i ts location similar to 20 Angstrom from the cleavage site in the cryst al structure. Analysis of the concentration dependence suggests that C d2+ binds with a K-d of 25 mu M in the ground state and a K-d of 2.5 n m in the transition state, The much stronger transition state binding suggests that the P9 metal ion adopts at least one additional ligand i n the transition state and that this metal ion may participate in a la rge scale conformational change that precedes hammerhead cleavage.