Single-Molecule FRET Kinetics of the Mn2+ Riboswitch: Evidence for Allosteric Mg2+ Control of “Induced-Fit” vs “Conformational Selection” Folding Pathways

Author
Abstract
Gene expression in bacteria is often regulated dynamically by conformational changes in a riboswitch upon ligand binding, a detailed understanding of which is very much in its infancy. For example, the manganese riboswitch is a widespread RNA motif that conformationally responds in regulating bacterial gene expression to micromolar levels of its eponymous ligand, Mn2+, but the mechanistic pathways are poorly understood. In this work, we quantitatively explore the dynamic folding behavior of the manganese riboswitch by single-molecule fluorescence resonance energy transfer spectroscopy as a function of cation/ligand conditions. From the detailed analysis of the kinetics, the Mn2+ is shown to fold the riboswitch by a “bind-then-fold” (i.e., “induced-fit”, IF) mechanism, whereby the ligand binds first and then promotes folding. On the other hand, the data also clearly reveal the presence of a folded yet ligand-free structure predominating due to the addition of physiological Mg2+ to a nonselective metal ion binding site. Of particular kinetic interest, such a Mg2+ “prefolded” conformation of the riboswitch is shown to exhibit a significantly increased affinity for Mn2+ and further stabilization by subsequent binding of the ligand, thereby promoting efficient riboswitch folding by a “fold-then-bind” (i.e., “conformational selection”, CS) mechanism. Our results not only demonstrate Mg2+-controlled switching between IF and CS riboswitch folding pathways but also suggest a novel heterotropic allosteric control in the manganese riboswitch activity co-regulated by Mg2+ binding.
Year of Publication
2019
Journal
The Journal of Physical Chemistry B
Volume
123
Number of Pages
2005-2015
Date Published
2019-02
ISSN Number
1520-6106
URL
https://pubs.acs.org/doi/full/10.1021/acs.jpcb.8b11841
DOI
10.1021/acs.jpcb.8b11841
JILA PI
Journal Article