Involvement of a Specific Metal Ion in the Transition of the Hammerhead Ribozyme to Its Catalytic Conformation

Abstract

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...

Significant activity of a modified ribozyme with N7‐deazaguanine at G10.1: the double‐metal‐ion mechanism of catalysis in reactions catalysed by hammerhead ribozymes

“…In this study, phosphorothioate substitution at P9 inhibited cleavage, a result that suggested the importance of the P9 oxygen as a metalbinding site. This suggestion was later supported by Peracchi et al in a study that involved the speci®c stereoselective introduction of a phosphorothioate moiety at the P9 position (Peracchi et al 1997). An Rp-but not a Sp-phosphorothioate reduced the cleavage rate 1000-fold, but the original rate was restored by the subsequent addition of a low concentration of Cd 2 ions, which are thiophilic.…”

“…By contrast, and in agreement with previous ®ndings (Ruffner et al 1990;Tuschl & Eckstein 1993;Peracchi et al 1996), k cat was reduced signi®cantly by the replacement of G 10.1 with 7-deazaguanine; the k cat of 0.0064 min À1 for 7-deaza-R34 was considerably lower than that of 0.19 min À1 for R34. However, the extent of the reduction (30-fold) caused by the removal of N7 of G 10.1 was much smaller than that (1000-fold reduction) caused by the introduction of the Rp-phosphorothioate at the P9 position (Peracchi et al 1997). This result suggests that, even though binding of Mg 2 to N7 of G 10.1 is catalytically important (Peracchi et al 1998;Wang et al 1999), it is not absolutely indispensable.…”

“…In particular, they can bind between the N7 of G 10.1 and the pro-Rp oxygen (P9 oxygen) of the phosphate moiety of A 9 , modulating the activity of the ribozyme. Almost all crystallographic and biochemical studies to date have suggested that N7 of G 10.1 and the pro-Rp oxygen of the phosphate moiety of A 9 are ligated with a metal ion (the conserved P9 metal) and that this bound metal ion is catalytically important (Buzayan et al 1990;Ruffner & Uhlenbeck 1990;Ruffner et al 1990;Tuschl & Eckstein 1993;Pley et al 1994;Scott et al 1995Scott et al , 1996Peracchi et al 1996Peracchi et al , 1997Murray et al 1998a, b;Wang et al 1999;Yoshinari & Taira 2000). In some cases, another metal-binding site has been found close to G 5 and replacement here of Mg 2 by Tb 3 inhibited the cleavage reaction (Feig et al 1998).…”

Probing the Role of Metal Ions in RNA Catalysis: Kinetic and Thermodynamic Characterization of a Metal Ion Interaction with the 2‘-Moiety of the Guanosine Nucleophile in the Tetrahymena Group I Ribozyme

“…Oligonucleotides were made by solid phase synthesis and supplied by the Protein and Nucleic Acid Facility at Stanford University or were gifts from L. Beigelman (Ribozyme Pharmaceuticals Inc.). Oligonucleotide substrates (Chart 1) were 5′-end-labeled using [γ- 32 P]ATP and T4 polynucleotide kinase and purified by electrophoresis on 24% nondenaturing polyacrylamide gels, as described previously (36,37). Oligonucleotides for inhibition studies were purified by ionexchange HPLC.…”

“…Specific metal ion ligands have been identified by metal ion specificity switch experiments (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32). These experiments exploit the preference of nitrogen and sulfur ligands to interact more strongly with soft metal ions, such as Mn 2+ , than with hard metal ions, such as Mg 2+ .…”