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Journal/Book: J Am Chem Soc
Published: 2009
Pages: 6261–6270
Volume: 131
Accession no.: 170

Metal-induced folding of Diels-Alderase ribozymes studied by static and time-resolved NMR spectroscopy.

Vijayalaxmi Manoharan, Boris Fürtig, Andres Jäschke, Harald Schwalbe
The metal ion-induced folding of the Diels-Alder ribozyme into a catalytically active form with a complex RNA pseudoknot has been characterized by static and time-resolved NMR spectroscopy. The conformations of two sequences from the Diels-Alder ribozyme family, A27 WT and G27 MUT, were studied in the absence and presence of metal ions and of ligand. The single nucleotide mutant G27 MUT in the absence of metal ions displayed conformational heterogeneity which greatly influences its folding trajectory. Time-resolved NMR experiments were applied using a sample-mixing technique to rapidly add Ca(2+) ions to induce folding in situ. The folding rates observed for the G27 MUT ribozyme differed by 3 orders of magnitude from the A27 WT folding rates determined previously by FRET experiments. A model based on the characterization of the free and metal-bound forms of the ribozymes is proposed to account for the difference in the folding rates of the two ribozymes. Evidence is provided that the reactivity is modulated due to local dynamics around the catalytic pocket for the G27 MUT ribozyme.
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Prof. Dr. Harald Schwalbe
Institut für Organische Chemie und Chemische Biologie
Johann Wolfgang Goethe Universität
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D-60438 Frankfurt am Main
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