METAL-IONS PLAY A PASSIVE ROLE IN THE HAIRPIN RIBOZYME CATALYZED REACTION

The hairpin ribozyme is an example of a small catalytic RNA which cata lyses the endonucleolytic transesterification of RNA in a highly seque nce-specific manner, The hairpin ribozyme, in common with all other sm all ribozymes such as the hammerhead, requires the presence of a dival ent metal ion co-factor (typically magnesium) for the reaction to take place. To investigate the role of: magnesium ions in the hairpin cata lysed reaction we have synthesised two epimeric modified substrates in which a phosphorothioate replaces the scissile phosphodiester bond, p reviously, Burke and co-workers have reported that no thio-effect is o bserved with the Rp-phosphorothioate isomer, We observe the absence of a titio-effect with both diastereomeric phosphorothioate hairpin subs trates, Furthermore we report that inert cobalt (III) complexes are ca pable of supporting the hairpin ribozyme reaction, with a similar effi ciency to Mg2+, even in the presence of EDTA, Variation of the net cha rge on the inert cobalt complex does not change the observed rate of r eaction, 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 differ ent mechanistic class to other small ribozymes such as the hammerhead.