Metal-induced folding of Diels-Alderase ribozymes studied by static and time-resolved NMR spectroscopy.
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.
Center for Biomolecular Magnetic Resonance, Institute of Organic Chemistry and Chemical Biology, Johann Wolfgang Goethe-University Frankfurt, Max-von-Laue-Strasse 7, D-60438 Frankfurt/Main, Germany.
Vijayalaxmi
V
Manoharan
Boris
B
Fürtig
Andres
A
Jäschke
Harald
H
Schwalbe
D055162:Biocatalysis / Q000187:drug effects; D002118:Calcium / Q000494:pharmacology; D018360:Crystallography, X-Ray; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008670:Metals / Q000378:metabolism / Q000494:pharmacology*; D009690:Nucleic Acid Conformation / Q000187:drug effects*; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism*; D013816:Thermodynamics; D013997:Time Factors
C518747:Diels-Alder ribozyme; D008024:Ligands; D008670:Metals; D016337:RNA, Catalytic; D002118:Calcium