Previous crystallographic and biochemical studies of the hammerhead ribozyme suggest that a metal ion is ligated by the pro-R p oxygen of phosphate 9 and by N 7 of G10.1 and has a functional role in the cleavage reaction. We have tested this model by examining the cleavage properties 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 concentrations of Cd 2؉, a thiophilic metal ion. These results strongly suggest that this bound metal ion is critical for catalysis, despite its location ϳ20 Å from the cleavage site in the crystal structure. Analysis of the concentration dependence suggests that Cd 2؉ binds with a K d of 25 M in the ground state and a K d of 2.5 nM in the transition state. The much stronger transition state binding suggests that the P9 metal ion adopts at least one additional ligand in the transition state and that this metal ion may participate in a large scale conformational change that precedes hammerhead cleavage.The catalytic cleavage of an RNA phosphodiester bond to a 2Ј,3Ј-cyclic phosphate by the hammerhead ribozyme requires the participation of divalent metal ions. McKay and co-workers (1) observed a single bound metal ion when Mn 2ϩ or Cd 2ϩ was soaked into hammerhead crystals, with the metal ion in close proximity to the pro-R p -oxygen of the P9 phosphate and N 7 of the guanine base at position 10.1 (Fig. 1). Previous biochemical experiments suggested that both of these groups were important in hammerhead cleavage: the P9 pro-R p -oxygen was identified in phosphorothioate interference experiments (2, 3), and a role for a purine at position 10.1 was implicated in nucleotide substitution experiments (4, 5). More recently, the N 7 of this purine was implicated by the ability of guanine, but not 7-deazaguanine, to efficiently rescue the activity of a ribozyme with an abasic nucleotide at position 10.1 (6).These results, taken together, support a model in which binding of a metal ion to the P9 pro-R p -oxygen and the N 7 of G10.1 affects catalysis. However, the metal ion site identified in the crystal structure is ϳ20 Å from the cleavage site phosphodiester, with no obvious connection to this site. In addition, there are no data directly demonstrating a functional role for the structurally identified metal ion, nor are there quantitative data that indicate how important this metal ion might be for catalysis. Finally, coordination of a metal ion to the pro-S poxygen of P9 rather than the pro-R p -oxygen was suggested from crystallographic data with a different ribozyme construct (7).We have therefore tested this model and quantitated the functional consequences of perturbing this site by substituting the pro-R p -and pro-S p -phosphoryl oxygen atoms at position P9 with sulfur and following catalysis in the presence and absence of Cd 2ϩ , a thiophilic metal ion. The results provide strong support for the model and indicate that a metal ion co...

The effect of introducing a phosphorothioate at the hammerhead cleavage site was investigated using a kinetically well-characterized hammerhead. In buffers containing Mg ion, the RP-phosphorothioate isomer cleaved 2000- to 80 000-fold slower than the SPisomer or the unmodified RNA substrate. Addition of low concentrations of several thiophilic metal ions, especially Cd2+, to these reactions is sufficient to fully restore the cleavage rate of the RPsubstrate without affecting cleavage rate of the all-oxygen or SPsubstrate. Thus, a model proposing coordination of a divalent metal ion to the pro-R oxygen at the hammerhead cleavage site appears justified.

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