High‐pressure analysis of a hammerhead ribozyme from Chrysanthemum chlorotic mottle viroid reveals two different populations of self‐cleaving molecule

The activity of the full‐length hammerhead ribozyme requires a tertiary interaction between its distal loops leading to the closure of the molecule and its stabilization in the active conformation. In this study, the conformational changes accompanying the cis‐cleavage reaction of Chrysanthemum chlorotic mottle viroid hammerhead ribozyme were investigated by high‐pressure experiments on the complete cleavage reaction. Two activation volumes (ΔV≠) were measured, pointing to the presence of two different populations of molecules corresponding to fast‐cleaving and slow‐cleaving ribozymes in the reaction mixture. The fast population, with a small ΔV≠ of 2.6 mL·mol−1, most likely represents molecules in the near‐active conformation, whereas the slow population, with a larger ΔV≠ of 11.6 mL·mol−1, represents molecules that need a larger conformational change to induce activity. In addition, pH‐dependence experiments suggest that the group whose deprotonation is required for activity intervenes in the formation of the transition state or in the chemistry of the reaction, but not in the conformational change that precedes it. Two different populations of fast and slow self‐cleaving hammerhead ribozymes were revealed using the high pressure approach. The fast population, with a small ΔV≠ = 2.6 mL·mol−1, represents most likely molecules in near‐active conformation while the slow population, with a larger ΔV≠ = 11.6 mL·mol−1, represents molecules that need a larger conformational change to induce activity.