Abstract
Riboswitches are highly structured RNA molecules that control genetic expression by altering their structure as a function of metabolite binding. Accumulating evidence suggests that riboswitch structures are highly dynamic and perform conformational exchange between structural states that are important for the outcome of genetic regulation. To understand how ligand binding influences the folding of riboswitches, it is important to monitor in real time the riboswitch folding pathway as a function of experimental conditions. Single-molecule FRET (sm-FRET) is unique among biophysical techniques to study riboswitch conformational changes as it allows to both monitor steady-state populations of riboswitch conformers and associated interconversion dynamics. Since FRET fluorophores can be attached to virtually any nucleotide position, FRET assays can be adapted to monitor specific conformational changes, thus enabling to deduce complex riboswitch folding pathways. Herein, we show how to employ sm-FRET to study the folding pathway of the S-adenosylmethionine (SAM) and how this can be used to understand very specific conformational changes that are at the heart of riboswitch regulation mechanism.
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Acknowledgements
We thank members of the Penedo and Lafontaine laboratories for critical reading of the manuscript and the National Sciences and Engineering Research Council of Canada (NSERC) for financial support. D.A.L. is a Fonds de Recherche Santé Québec Senior Scholar.
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Boudreault, J., Perez-Gonzalez, D.C., Penedo, J.C., Lafontaine, D.A. (2015). Single-Molecule Approaches for the Characterization of Riboswitch Folding Mechanisms. In: Leblanc, B., Rodrigue, S. (eds) DNA-Protein Interactions. Methods in Molecular Biology, vol 1334. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2877-4_6
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DOI: https://doi.org/10.1007/978-1-4939-2877-4_6
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