Single-Molecule FRET Kinetics of the Mn Riboswitch: Evidence for Allosteric Mg Control of "Induced-Fit" vs "Conformational Selection" Folding Pathways.

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, Mn, 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 Mn 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 Mg to a nonselective metal ion binding site. Of particular kinetic interest, such a Mg "prefolded" conformation of the riboswitch is shown to exhibit a significantly increased affinity for Mn 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 Mg-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 Mg binding.