Mg2+-dependent folding of a Diels-Alderase ribozyme probed by single-molecule FRET analysis |
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Authors: | Kobitski Andrei Yu Nierth Alexander Helm Mark Jäschke Andres Nienhaus G Ulrich |
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Affiliation: | Andrei Yu. Kobitski, Alexander Nierth, Mark Helm, Andres Jäschke, and G. Ulrich Nienhaus |
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Abstract: | Here, we report a single-molecule fluorescence resonance energy transfer (FRET) study of a Diels-Alderase (DAse) ribozyme, a 49-mer RNA with true catalytic properties. The DAse ribozyme was labeled with Cy3 and Cy5 as a FRET pair of dyes to observe intramolecular folding, which is a prerequisite for its recognition and turnover of two organic substrate molecules. FRET efficiency histograms and kinetic data were taken on a large number of surface-immobilized ribozyme molecules as a function of the Mg2+ concentration in the buffer solution. From these data, three separate states of the DAse ribozyme can be distinguished, the unfolded (U), intermediate (I) and folded (F) states. A thermodynamic model was developed to quantitatively analyze the dependence of these states on the Mg2+ concentration. The FRET data also provide information on structural properties. The I state shows a strongly cooperative compaction with increasing Mg2+ concentration that arises from association with several Mg2+ ions. This transition is followed by a second Mg2+-dependent cooperative transition to the F state. The observation of conformational heterogeneity and continuous fluctuations between the I and F states on the ~100ms timescale offers insight into the folding dynamics of this ribozyme. |
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