NMR spectroscopic approaches to the mechanism of action of hammerhead ribozyme
Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aza-Aoba, Aoba-ku, Sendai 980-8578, Japan
Hammerhead ribozyme (HHRz) is a catalytic RNA which promotes a site-specific cleavage of RNA strand. At the early stage of our study, HHRz had though as a metallo-enzyme. Therefore, our group studied the HHRz-metal interaction as an initial theme for a mechanistic study. At that time, there had been several crystal structure of HHRz, and a possible metal ion-binding motif was identified. Therefore, we first focused on this motif for the following points. 1) Does this motif truly work as a metal ion-binding motif in solution? 2) Is this motif a functional module independently from HHRz? 3) Do metal cations bind with nucleobase through inner-sphere coordination?
For these points, we chemically synthesized site-specifically 15N-labeled RNA oligomers which mimic the metal ion-binding motif of HHRzs. From the Cd+-titration experiments with 15N NMR spectroscopy (Tanaka et al. JACS, 2002, 124, 4595) and the theoretical calculations (Sychrovský et al. JACS, 2004, 126, 663), it was found that the answers to the above three questions were all "yes". Unfortunately, from the phylogenetic studies, catalytic roles of this metal at G10.1 became questionable. However, new crystal structure solved by Scott's group (Cell, 2006, 126, 309) shed light on this motif again, because G12 in this motif was found to be responsible for the deprotonation of 2'-OH at the cleavage site. Therefore, we established a novel site-specific labeling technique. So far, we confirmed that G10.1-labeld HHRz was surely produced, and the preparation of further labeled HHRzs and the mechanistic studies are underway.
This works was supported by Human Frontier Science Program.