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Research Paper
NMR-spectroscopic characterisation of phosphodiester bond cleavage catalyzed by the minimal hammerhead ribozyme
Boris Fürtig, Christian Richter, Peter Schell, Philipp Wenter, Stefan Pitsch and Harald Schwalbe
volume 5 | issue 1
january/february/march 2008Subscribe to this journal for $59/year
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 13C labelled in multi-step organic syntheses starting from uniformly 13C-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 Mg2+ 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 Mg2+ leading to an ensemble of conformations where dynamic transitions between energetically similar conformations occur on the ms-timescale in the presence of Mg2+. 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
Boris Fürtig
Max F. Perutz Laboratories; Vienna, Austria
Christian Richter
Institute for Organic Chemistry and Chemical Biology; Center for Biomolecular Magnetic Resonance; Johann Wolfgang Goethe-University; Frankfurt, Germany
Peter Schell
Institute for Organic Chemistry and Chemical Biology; Center for Biomolecular Magnetic Resonance; Johann Wolfgang Goethe-University; Frankfurt, Germany
Philipp Wenter
École Polytechnique Fédérale Lausanne; Laboratory of Nucleic Acid Chemistry; Lausanne, Switzerland
Stefan Pitsch
École Polytechnique Fédérale Lausanne; Laboratory of Nucleic Acid Chemistry; Lausanne, Switzerland
Harald Schwalbe
Institute for Organic Chemistry and Chemical Biology; Center for Biomolecular Magnetic Resonance; Johann Wolfgang Goethe-University; Frankfurt, Germany