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.

Viroids are small pathogenic circular single-stranded RNAs, present in two complementary sequences, named plus and minus, in infected plant cells. A high degree of complementarities between different regions of the RNAs allows them to adopt complex structures. Since viroids are naked non-coding RNAs, interactions with host factors appear to be closely related to their structural and catalytic characteristics. Avocado sunblotch viroid (ASBVd), a member of the family Avsunviroidae, replicates via a symmetric RNA-dependant rolling-circle process, involving self-cleavage via hammerhead ribozymes. Consequently, it is assumed that ASBVd plus and minus strands adopt similar structures. Moreover, by computer analyses, a quasi-rod-like secondary structure has been predicted. Nevertheless, secondary and tertiary structures of both polarities of ASBVd remain unsolved. In this study, we analyzed the characteristic of each strand of ASBVd through biophysical analyses. We report that ASBVd transcripts of plus and minus polarities exhibit differences in electrophoretic mobility under native conditions and in thermal denaturation profiles. Subsequently, the secondary structures of plus and minus polarities of ASBVd were probed using the RNA-selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) method. The models obtained show that both polarities fold into different structures. Moreover, our results suggest the existence of a kissing-loop interaction within the minus strand that may play a role in in vivo viroid life cycle.