PNNL

Abstract

The hammerhead ribozyme is a small, self-cleaving catalytic RNA which activates a site-specific phosphodiester bond cleavage reaction. The folding pathway of the hammerhead ribozyme is critical to its catalytic activity, which has been known to depend on the metal ion concentration (DeRose, V.J., 2003, Curr. Op. Struct. Biol. 13, 317-324). Recently, it has been proposed that an extended hammerhead ribozyme that contains putative intramolecular loop-loop interactions between two helical regions has enhanced reactivity, and at lower cation concentrations (Khorvona, A., Lescoute, A., Westhof, E., Jayasena, S.D., 2003, Nat. Struc. Biol. 10, 708-712). In order to investigate potential tertiary interactions between the two loops as well as the folding pathway of the hammerhead ribozyme, site-directed spin labels have been introduced to stems I and II. Tertiary interactions can be predicted by measuring inter-spin distances (Kim, N.-K. Murali, A., DeRose, V.J., Submitted) with increasing metal concentration, and the distance distributions between two sites in the internal loops can be obtained using pulsed EPR techniques (DEER, double electron electron resonance), with which inter-spin distances can be measured up to 60Å. (Schiemann, O., Weber, A., Edwards, T.E., Prisner, T.F., Sigurdsson, S.T., J. Am. Chem. Soc. 2003, 125, 3434-3435)