UC Santa Cruz

Over the past two decades, the hammerhead ribozyme has been extensively studied leading to many new insights regarding the biochemical and structural properties of catalytic RNAs. Despite these insights, the role metal ions in catalysis is still of great debate. We have obtained a 1.55Å crystal structure of a hammerhead ribozyme from Schistosoma mansoni in conditions that permit detailed observation of Na⁺ ions binding in the ribozyme's active site. A Na⁺ ion binds to the N7 of G10.1 and the adjacent A9 phosphate in a manner identical to that previously observed for divalent cations. A second Na⁺ ion binds to the Hoogsteen face of G12, the general base in the hammerhead ribozyme cleavage reaction, thereby potentially dissipating the negative charge of the catalytically active enolate form of the base. We have also obtained a 2.2Å crystal structure of a hammerhead ribozyme variant containing adenine substituted for the general base that displays a structurally perturbed active site. Our results suggest that Na⁺ directly and specifically substitutes for divalent cations in the hammerhead active site. Additionally, we suggest that hammerhead ribozyme substituted with adenine for the general base may compete between two conformational states: an active state in which A12 is situated for deprotonation of the 2'H of C17; and an inactive state in which A12 makes a non-canonical base pair A9, causing a perturbation in the active site, disrupting metal ion binding. These results permit us to estimate the contribution that structural perturbation has on the catalytic activity of other hammerhead ribozyme variants containing nucleobase substitutions for the general base.