Relationship between 2'-hydroxyls and magnesium binding in the hammerhead RNA domain: a model for ribozyme catalysis

The use of deoxyribonucleotide substitution in RNA (mixed RNA/DNA polymers) permits an evaluation of the role of 2'-hydroxyl groups in ribozyme catalysis. Specific deoxyribonucleotide substitution at G9 and A13 of the ribozyme decreases the catalytic activity (kcat) of the ribozyme by factors of 14 and 20, respectively. The reduction of the reaction rate concomitant with the absence of these 2'-OHs or the 2'-OH of the substrate U7 position can be partially compensated by increasing the Mg2+ concentration above 10 mM. The KMg of the all-RNA ribozyme is 5.3 mM, and the lack of either of the three influential 2'-OHs increases this value by a factor of approximately 3. These and other reaction constants for the ribozyme and the deoxy-substituted analogues have been determined by assuming a three-step mechanism. The data presented here provide the basis for the formulation of a molecular model of ribozyme activity.