The Role of Magnesium Ions and 2′-Hydroxyl Groups in the VS Ribozyme–Substrate Interaction

Abstract

The minimal substrate of the trans-cleaving Neurospora VS ribozyme has a stem-loop structure and interacts with the ribozyme by RNA tertiary interactions that remain only partially defined. The magnesium ion dependence of the catalytic parameters of a trans-cleaving VS-derived ribozyme were studied. The turnover number of the catalytic RNA was found to depend on the binding of at least three magnesium ions, with an apparent magnesium ion dissociation constant of 16 mM, but K M was observed to be metal ion independent in the millimolar range. To address the role of 2′-hydroxyl groups of the VS substrate RNA in interactions with the ribozyme, 23 altered substrates, each with a single 2′-deoxyribonucleoside substitution, were synthesised and their kinetic properties in the VS ribozyme reaction were analysed. The removal of five 2′-hydroxyl groups, at positions G620, A621, U628, C629 and G630 inhibited the reaction, whereas at two sites, G623 and A639, reaction was stimulated by the modification. Substitution of G620 with a 2′-deoxynucleoside was expected to inhibit the reaction, in line with the critical role of this 2′-hydroxyl group in the transesterification reaction. Altered substrates in which a 2′- O-methyl nucleoside replaced A621, U628, C629 and G630 were prepared and characterised. Although removal of the hydroxyl group of A621 inhibited the turnover number of the ribozyme significantly, this activity was recovered upon 2′- O-methyl adenosine substitution, suggesting that the 2′-oxygen atom of this nucleoside forms an important contact within the ribozyme active site. A cluster of residues within the loop region of the substrate, were more modestly affected by 2′-deoxynucleoside substitution. In two cases, magnesium binding was impaired, suggesting that stem-loop I is a possible magnesium ion binding site.