Book of Abstracts: Albany 2003

category image Albany 2003
Conversation 13
Abstract Book
June 17-21 2003

The Structure and Active Site of the VS Ribozyme

The VS ribozyme is one of the nucleolytic ribozymes, that catalyse the breakage of the phosphodiester bond at a particular site by a transesterification reaction in which the 2?-oxygen carries out a nucleophilic attack on the 3?-phosphorus. The reaction occurs at 2 min-1, which represents an RNA-mediated acceleration by a factor of ≥ 105. The ribozyme also carries out a highly efficient reverse (ligation) reaction.

The VS ribozyme is the largest of the nucleolytic ribozymes, and the only one for which there is no crystal structure. We have deduced the global structure of this ribozyme by a combination of electrophoretic and FRET approaches. The ribozyme is organised by two three-way helical junctions. We have deduced the structure of both junctions, and shown that they fold in response to the non-cooperative binding of Mg2+ ions. We have determined the dihedral angle between the two junctions, and thereby established the global structure of the five-helix ribozyme. We have deduced the general manner of binding of the substrate stem-loop, docked into the cleft formed between helices II and VI. In that position the cleaved phosphate can interact closely with the A730 loop. We have shown that the cleavage activity of the ribozyme is strongly impaired by most single substitutions of the four nucleotides of this loop. The cleavage rate is also strongly affected by the dihedral angle about helix III, consistent with the requirement for substrate interaction at both ends. Cleavage activity is especially sensitive to changes in A756 within the probable active site. Replacement by any other nucleotide leads to reduction in cleavage rate by three orders of magnitude, as does ablation of the base. The Watson-Crick edge of the base is particularly critical, notably the presence and position of the exocyclic amine group. This base is a good candidate for direct participation in the cleavage reaction.

Daniel A. Lafontaine
David G. Norman
Timothy J. Wilson
Aileen McLeod
David M. J. Lilley

Biochemistry Department
University of Dundee
Dundee DD1 5EH, UK

References and Footnotes
  1. D. A. Lafontaine, D. G. Norman and D. M. J. Lilley, EMBO J. 21, 2461-2471 (2002).
  2. D. A. Lafontaine, T. J. Wilson, D. G. Norman and D. M. J. Lilley, J. Molec. Biol. 312, 663-674 (2001).
  3. D. A. Lafontaine, T. J. Wilson, Z.-Y. Zhao and D. M. J. Lilley, J. Molec. Biol. 323, 23-34 (2002).