Metal ions play a passive role in the hairpin ribozyme catalysed reaction.

Abstract

The hairpin ribozyme is an example of a small catalytic RNA which catalyses the endonucleolytic transesterification of RNA in a highly sequence-specific manner. The hairpin ribozyme, in common with all other small ribozymes such as the hammerhead, requires the presence of a divalent metal ion co-factor (typically magnesium) for the reaction to take place. To investigate the role of magnesium ions in the hairpin catalysed reaction we have synthesised two epimeric modified substrates in which a phosphorothioate replaces the scissile phosphodiester bond. Previously, Burke and co-workers have reported that no thio-effect is observed with the Rp-phosphorothioate isomer. We observe the absence of a thio-effect with both diastereomeric phosphorothioate hairpin substrates. Furthermore we report that inert cobalt (III) complexes are capable of supporting the hairpin ribozyme reaction, with a similar efficiency to Mg2+,even in the presence of EDTA. Variation of the net charge on the inert cobalt complex does not change the observed rate of reaction. These results suggest that metal ions play a passive role in the hairpin ribozyme catalysed reaction and are probably required for structural purposes only. This places the hairpin ribozyme in a different mechanistic class to other small ribozymes such as the hammerhead.