| Popis: |
The hairpin ribozyme catalyzes site-specific cleavage of an RNA substrate using a magnesium-dependent transphosphorylation mechanism. Here, we describe experiments designed to test the importance of ribose 2'-hydroxyl groups for ribozyme function. Ribozymes for this work were synthesized in two segments using solid-phase RNA phosphoramidite chemistry. 2'-Deoxyribonucleotides were systematically introduced at each of the 50 positions within the ribozyme, and the catalytic activity of the resulting mixed RNA-DNA polymers was measured. Deletion of the 2'-hydroxyl group at each of four sites (A10, G11, A24, and C25) was found to result in severe inhibition of cleavage activity (kcat/KM decreased by 100- to 1000-fold), although KM measurements and mobility-shift assays showed that substrate binding was not affected. Identical results were obtained upon substitution of these ribonucleotides with 2'-O-methyl derivatives. Inhibition by 2'-modified sugars at G11 or A24 was rescued by increased Mg2+ concentrations, suggesting that these 2'-hydroxyls may function in magnesium binding. Our results demonstrate that the 2'-hydroxyl groups at A10, G11, A24, and C25 provide essential functions for catalysis, possibly forming important tertiary contacts or magnesium coordination sites that are necessary for active site architecture. |
