Young K J, Gill F, Grasby J A
Department of Chemistry, Krebs Institute, University of Sheffield, Sheffield S3 7HF, UK.
Nucleic Acids Res. 1997 Oct 1;25(19):3760-6. doi: 10.1093/nar/25.19.3760.
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.
发夹状核酶是一种小催化RNA的例子，它以高度序列特异性的方式催化RNA的内切核酸酯交换反应。与所有其他小核酶（如锤头状核酶）一样，发夹状核酶需要二价金属离子辅助因子（通常是镁离子）的存在才能发生反应。为了研究镁离子在发夹状核酶催化反应中的作用，我们合成了两种差向异构修饰的底物，其中硫代磷酸酯取代了可断裂的磷酸二酯键。此前，伯克及其同事报道，Rp-硫代磷酸酯异构体未观察到硫代效应。我们观察到两种非对映硫代磷酸酯发夹状底物均不存在硫代效应。此外，我们报道惰性钴（III）配合物能够支持发夹状核酶反应，其效率与Mg2+相似，即使在存在乙二胺四乙酸（EDTA）的情况下也是如此。惰性钴配合物净电荷的变化不会改变观察到的反应速率。这些结果表明，金属离子在发夹状核酶催化反应中起被动作用，可能仅用于结构目的。这使发夹状核酶与其他小核酶（如锤头状核酶）处于不同的作用机制类别。
