Spectroscopic analyses of the interaction between hammerhead ribozymes and metal ions

Abstract

Biologically active RNA molecules utilize metal ions to fold into specific conformations or to form a catalytic center. Hammerhead ribozymes also contain a metal ion-binding motif in their conserved core region. This motif and bound metal ions have recently been focused on from the viewpoint of whether the metal ions are a catalytic metal or a structural metal. We studied the interaction between metal ions and an RNA oligomer, r(GGACGAGUCC), which mimics the metal ion-binding motif of hammerhead ribozymes, using NMR spectroscopy in solution. Using 15N-labeled RNA oligomers, the chemical shift of N7 of the G7 residue [N7(G7)] in the metal ion-binding motif was specifically perturbed upon the addition of Cd(II). It was also found that the 31P resonance of the phosphate of the A9 residue showed the largest perturbation of all the 31P resonances during titration. These data indicate that divalent cations can bind to the metal ion-binding motif in solution, and that the binding sites for cations are N7(G7) and the phosphate of A9. More importantly, the metal ion-binding motif is an independent functional module that can capture divalent cations without the assistance of other conserved residues in hammerhead ribozymes.