Well-conserved tandem G center dot A pairs and the flanking C center dot Gpair in hammerhead ribozymes are sufficient for capture of structurally and catalytically important metal ions

An important metal ion in reactions catalyzed by hammerhead ribozymes is th e 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 cataly tically important tandem G .A pairs in the core region, and the P9-G10.1 mo tif captures the P9 metal ion, which is most probably a Mg2+ ion under phys iological conditions. In this study, we used H-1, P-31, and C-13 NMR spectr oscopy to examine whether this motif by itself, in the absence of other cat alytic loops, might be sufficient to capture structurally and catalytically important metal ions in solution. We deduced that the P9-G10.1 motif was a ble 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 a tom of A6 (P/A6), C8 of G7 (C8/G7), and H8 of G7 (HB/G7) in the P9-G10.1 mo tif of a model duplex, designated GA10, were selectively perturbed during t itration up to a 1:1 molar ratio of Mg2+ ions and the P9-G10.1 motif. We ne xt 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 Acid s 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+ i ons and that hammerhead ribozymes utilize this intrinsic metal-binding prop erty of the P9-G10.1 motif for catalysis.