NMR spectroscopic investigations of the roles of the metal ion at A9/G10.1 site in hammerhead ribozymes.

Abstract

Most hammerhead ribozymes have metal ion-binding sequences which are composed of the sheared type G12-A9 pair and the G10.1-C11.1 base-pair. However, in some hammerhead ribozymes, the G10.1-C11.1 base-pair is substituted with the A10.1-U11.1 base-pair. Here, we studied structural features of this altered motif, by using NMR spectroscopy. For this purpose, we have synthesized a model RNA oligomer, UGAA10:rGGAUGAAUCC which mimics the altered motif. From a 2-dimensional (2D) 1H-1H NOESY spectrum, we were able to trace sequential NOEs between base protons and anomeric protons (H1'), and assigned these resonances. It was also found that G5 and A6 formed a sheared type G-A pair from the imino proton resonance of G5. Observation of the imino proton resonance of U4 suggested that U4 forms a base-pair with A7. These structural features of the altered motif of UGAA10 are similar to the common metal ion-binding motif with G12-A9 and G10.1-C11.1.