The Mechanism of Phosphodiester Hydrolysis: Near In-line Attack Conformations in the Hammerhead Ribozyme

The hammerhead ribozyme is a small RNA molecule capable of self-cleavage at a specific site in the phosphodiester backbone. The mechanism of hydrolysis involves in-line nucleophilic attack by the 2‘-hydroxyl of residue 17 on the adjoining phosphorus of residue 1.1, resulting in the formation of a 2‘,3‘-cyclic phosphate ester on residue 17 (C17) and elimination of the 5‘-hydroxyl group of residue 1.1 (A1.1). Unconstrained molecular dynamics (MD) simulations on the recently solved crystallographic unmodified hammerhead ribozyme structure were performed in solution using two crystallographic Mg2+ ions. The simulations indicate that near in-line attack conformations (NACs), in which the distance of the 2‘-oxygen of C17 to the phosphorus of A1.1 is ≤3.25 A and the C17 2‘O−A1.1 P−A1.1 O5‘ angle of displacement is ≥150°, form approximately 18% of the simulation time. The motions leading to these catalytically competent conformations are discussed. Stems I and II of the hammerhead ribozyme structure, released fro...