title: High-pressure analysis of a hammerhead ribozyme from Chrysanthemum chlorotic mottle viroid reveals two different populations of self-cleaving molecule.
abstract: 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.
authors.affiliation: Acides Nucléiques et Biophotonique, CNRS FRE 3207, UPMC Université Paris 06, France.
authors.forename: Hussein
authors.initials: H
authors.lastname: Kaddour
authors.forename: Jacques
authors.initials: J
authors.lastname: Vergne
authors.forename: Guy
authors.initials: G
authors.lastname: Hervé
authors.forename: Marie-Christine
authors.initials: MC
authors.lastname: Maurel
mesh_terms: D000818:Animals; D001483:Base Sequence; D031202:Chrysanthemum / Q000821:virology*; D006863:Hydrogen-Ion Concentration; D006874:Hydrostatic Pressure; D008274:Magnesium / Q000737:chemistry; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009997:Osmotic Pressure; D010942:Plant Viruses / Q000201:enzymology* / Q000235:genetics*; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014772:Viroids / Q000201:enzymology* / Q000235:genetics*
chemical_list: D016337:RNA, Catalytic; C427204:hammerhead ribozyme; D008274:Magnesium