Published online 1 December 2004
Nucleic Acids Research, Vol. 32 No. 21 © Oxford University Press 2004; all rights reserved
The role of phosphate groups in the VS ribozyme–substrate interaction
Centre for Chemical Biology, Department of Chemistry, Krebs Institute, University of Sheffield, Sheffield S3 7HF, UK and 1 Department of Molecular Biology and Biotechnology, Krebs Institute, University of Sheffield, Sheffield S10 2TN, UK
* To whom correspondence should be addressed. Tel: +44 0 114 2229478; Fax: +44 0 114 2229346; Email: j.a.grasby{at}sheffield.ac.uk
Received September 22, 2004; Revised October 20, 2004; Accepted November 5, 2004
The VS ribozyme trans-cleavage substrate interacts with the catalytic RNA via tertiary interactions. To study the role of phosphate groups in the ribozyme–substrate interaction, 18 modified substrates were synthesized, where an epimeric phosphorothioate replaces one of the phosphate diester linkages. Sites in the stem–loop substrate where phosphorothioate substitution impaired reaction cluster in two regions. The first site is the scissile phosphate diester linkage and nucleotides downstream of this and the second site is within the loop region. The addition of manganese ions caused recovery of the rate of reaction for phosphorothioate substitutions between A621 and A622 and U631 and C632, suggesting that these two phosphate groups may serve as ligands for two metal ions. In contrast, significant manganese rescue was not observed for the scissile phosphate diester linkage implying that electrophilic catalysis by metal ions is unlikely to contribute to VS ribozyme catalysis. In addition, an increase in the reaction rate of the unmodified VS ribozyme was observed when a mixture of magnesium and manganese ions acted as the cofactor. One possible explanation for this effect is that the cleavage reaction of the VS ribozyme is rate limited by a metal dependent docking of the substrate on the ribozyme.