Title
NMR-spectroscopic characterisation of phosphodiester bond cleavage catalyzed by the minimal hammerhead ribozyme
Author
Boris Fürtig, Christian Richter, Peter Schell, Philipp Wenter, Stefan Pitsch, Harald Schwalbe
Year
2008
Journal
RNA Biology
Abstract
In order to relate the conformational dynamics of the hammerhead ribozyme to its biological function the cleavage reaction catalysed by the hammerhead ribozyme was monitored by timeresolved 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 characterised 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 monoexponential kinetics but follows bi-exponential kinetics. A model is described to take into account these experimental data.
Instrument
J-720
Keywords
Circular dichroism, Secondary structure, Ligand binding, Thermal stability, Thermodynamics, Biochemistry