CR-UK Nucleic Acid Structure Group, MSI/WTB complex, University of Dundee, Dundee DD1 5EH, UK d.m.j.lilley@dundee.ac.uk The nucleolytic ribozymes are catalytic RNA molecules that generate site-specific cleavage by means of a transesterification reaction involving the 2’ and 5’ O atoms. We have made a study of two of these, the hairpin and VS ribozymes. The hairpin ribozyme folds to generate an intimate loop-loop interaction to create the local environment in which catalysis can proceed. By means of FRET we can observe individual hairpin ribozyme molecules as they undergo multiple cycles of cleavage and ligation, and measure the rates of the internal reactions. On average, the cleaved ribozyme undergoes several docking-undocking events before a ligation reaction occurs. On the basis of these experiments, we have explored the role of the nucleobases G8 and A38 in the catalysis. Both cleavage and ligation reactions are pH dependent, corresponding to the titration of a group with pKA = 6.2. We have used a novel ribonucleoside in which these bases are replaced by imidazole to investigate the role of acid-base catalysis in this ribozyme. We observe significant rates of cleavage and ligation, and a bell-shaped pH dependence for both. The VS ribozyme is the largest of the nucleolytic ribozymes, and the only one for which there is no crystal structure. The ribozyme consists of five helical sections organised by two three-way junctions, each of which undergo metal ion-induced folding. Using a ‘divide and conquer’ approach based principally on the analysis of component junctions by FRET, we deduced the global structure of the ribozyme. We have now solved the structure of the complete ribozyme at low resolution using small-angle X-ray scattering in solution. The binding of the substrate stem-loop generates a catalytically-productive interaction with the A730 loop active site. We have identified two critical nucleotides in the catalytic process; A756 within the A730 loop, and G638 in the substrate internal loop. Mutation or functional group substitution of either nucleobase leads to > 1,000-fold impairment of catalytic activity, while leaving the structure and binding to the ribozyme unaltered. The pH dependencies of the rate of cleavage of substrate with guanine, adenine, 2,6-diaminopurine or inosine at position 638 are fully consistent with a mechanism in which G638 and A756 act in concert in general acid-base catalysis. The proposed mechanism of the VS and hairpin ribozymes, together with the manner of the generation of the active sitea and their topology, are strikingly similar each other. This has probably arisen by convergent evolution. M.K. Nahas, T.J. Wilson, S. Hohng, K. Jarvie, D.M.J. Lilley and T. Ha Observation of internal cleavage and ligation reactions of a ribozyme Nature Struct. Molec. Biol. 11, 1107-1113 (2004). Z. Zhao, A. McLeod, S. Harusawa, L. Araki, M. Yamaguchi, T. Kurihara and D. M. J. Lilley Nucleobase participation in ribozyme catalysis. J. Amer. Chem. Soc. 127, 5026-5027 (2005). T. J. Wilson, J. Ouellet, Z. Zhao, S. Harusawa, L. Araki, T. Kurihara and D. M. J. Lilley Nucleobase catalysis in the hairpin ribozyme. RNA 12, 980-987 (2006). T. J. Wilson, A. C. McLeod and D. M. J. Lilley A guanine nucleobase important for catalysis by the VS ribozyme EMBO J. 26, 2489-2500 (2007).