An important metal ion in reactions catalyzed by hammerhead ribozymes is the so-called P9 metal ion (the metal ion captured by the phosphorus atom of A9 (P9) and N7 of G10.1 [P9-G10.1 motif]). Hammerhead ribozymes have catalytically important tandem G·A pairs in the core region, and the P9-G10.1 motif captures the P9 metal ion, which is most probably a Mg2+ ion under physiological conditions. In this study, we used 1H, 31P, and 13C NMR spectroscopy to examine whether this motif by itself, in the absence of other catalytic loops, might be sufficient to capture structurally and catalytically important metal ions in solution. We deduced that the P9-G10.1 motif was able to capture a Mg2+ ion in solution in the absence of any other part of a hammerhead ribozyme, because the chemical shift values of the phosphorus atom of A6 (P/A6), C8 of G7 (C8/G7), and H8 of G7 (H8/G7) in the P9-G10.1 motif of a model duplex, designated GA10, were selectively perturbed during titration up to a 1:1 molar ratio of Mg2+ ions and the P9-G10.1 motif. We next studied the binding of a Cd2+ ion to the P9-G10.1 motif and found that, in agreement with recent kinetic studies (Yoshinari and Taira, Nucleic Acids Res. 2000, 28, 1730-1742), a Cd2+ ion also bound to this motif. Finally, we conclude that the P9-G10.1 motif (a sheared-type G·A pair with a guanine residue on the 3' side of the adenine residue) with several flanking base pairs is sufficient for capture of divalent cations such as Mg2+ and Cd2+ ions and that hammerhead ribozymes utilize this intrinsic metal-binding property of the P9-G10.1 motif for catalysis.
ASJC Scopus subject areas
- Colloid and Surface Chemistry