Book of Abstracts: Albany 2007
June 19-23 2007
The structure and catalytic mechanism of the VS ribozyme
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. Calculated electron density maps are fully consistent with a coaxial stack of helices IV, III and VI, with helices II and V radiating away from this axis to create the substrate binding site. The substrate is bound close to the centre of the ribozyme.
The binding of the substrate stem-loop generates a catalytically-productive interaction with the A730 loop active site. A756 is a critical nucleotide in the catalytic process; substitution of the nucleobase by imidazole results in a ribozyme that is active in both cleavage and ligation reactions. We have now identified a second candidate catalytic nucleobase. Mutation or functional group substitution of G638 leads to 10,000-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 ribozyme, together with the manner of the generation of the active site and its topology, is strikingly similar to that of the hairpin ribozyme,. This has probably arisen by convergent evolution.
Timothy J. Wilson
CR-UK Nucleic Acid Structure Group, MSI/WTB complex, University of Dundee, Dundee DD1 5EH, UK