title: NMR-spectroscopic characterization of phosphodiester bond cleavage catalyzed by the minimal hammerhead ribozyme.
abstract: In order to relate the conformational dynamics of the hammerhead ribozyme to its biological function the cleavage reaction catalyzed by the hammerhead ribozyme was monitored by time-resolved nuclear magnetic resonance (NMR) spectroscopy. For this purpose, the two nucleosides around the scissile phosphodiester bond were selectively (13)C labelled in multi-step organic syntheses starting from uniformly (13)C-labelled glucose. The phosphoamidites were incorporated using phosphoamidite chemistry in the hammerhead substrate strand. In addition, the 2'-OH group on the 5'-side of the hammerhead substrate strand was labelled with a photolabile protecting group. This labelling strategy enabled a detailed characterisation of the nucleotides around the scissile phosphodiester bond in the ground state conformation of the hammerhead ribozyme in the absence and presence of Mg(2+) ions as well as of the product state. Photochemical induction of the reaction in situ was further characterized by time-resolved NMR spectroscopy. The detailed structural and dynamic investigations revealed that the conformation of the hammerhead ribozyme is significantly affected by addition of Mg(2+) leading to an ensemble of conformations where dynamic transitions between energetically similar conformations occur on the ms-timescale in the presence of Mg(2+). The dynamic transitions are localized around the catalytic core. Cleavage from this ensemble cannot be described by mono-exponential kinetics but follows bi-exponential kinetics. A model is described to take into account these experimental data.
authors.affiliation: Institute for Organic Chemistry and Chemical Biology, Center for Biomolecular Magnetic Resonance, Johann Wolfgang Goethe-University, Frankfurt, Germany.
authors.forename: Boris
authors.initials: B
authors.lastname: Fürtig
authors.forename: Christian
authors.initials: C
authors.lastname: Richter
authors.forename: Peter
authors.initials: P
authors.lastname: Schell
authors.forename: Philipp
authors.initials: P
authors.lastname: Wenter
authors.forename: Stefan
authors.initials: S
authors.lastname: Pitsch
authors.forename: Harald
authors.initials: H
authors.lastname: Schwalbe
mesh_terms: D002247:Carbon Isotopes; D002384:Catalysis; D020134:Catalytic Domain; D002413:Cations, Divalent; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D009690:Nucleic Acid Conformation; D009705:Nucleosides / Q000737:chemistry*; D010755:Organophosphates / Q000737:chemistry*; D010777:Photochemistry; D016337:RNA, Catalytic / Q000737:chemistry*
chemical_list: D002247:Carbon Isotopes; D002413:Cations, Divalent; D009705:Nucleosides; D010755:Organophosphates; D016337:RNA, Catalytic; C427204:hammerhead ribozyme; D008274:Magnesium