The role of magnesium ions and 2'-hydroxyl groups in the VS ribozyme-substrate interaction.

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