Mg²⁺ Shifts Ligand-Mediated Folding of a Riboswitch from Induced-Fit to Conformational Selection

Abstract

Bacterial riboswitches couple small-molecule ligand binding to RNA conformational changes that widely regulate gene expression, rendering them potential targets for antibiotic intervention. Despite structural insights, the ligand mediated folding mechanisms of riboswitches are still poorly understood. Using single molecule FRET, we here have investigated the folding mechanism of an H-type pseudoknotted preQ1 riboswitch in dependence of Mg2+ and three ligands of distinct affinities. We show that in the absence of Mg2+, both weakly and strongly bound ligands promote pseudoknot docking through an induced-fit mechanism. By contrast, addition of as low as 10 µM Mg2+ generally shifts docking toward conformational selection by stabilizing a folded-like conformation prior to ligand binding. Supporting evidence from transition state analysis further highlights the particular importance of stacking interactions during induced-fit, and of specific hydrogen bonds during conformational selection. Our mechanistic dissection provides unprecedented insights into the intricate synergy between ligand- and Mg2+-mediated RNA folding.

Superior cellular activities of azido- over amino-functionalized ligands for engineered preQ1riboswitches inE.coli

“…The faster on-rates for ligands 3 and 4 are consistent with the possibility for specific electrostatic interactions between the additional ammonium moieties and the phosphate backbone, and more generally, with an increase in local concentration due to improved electrostatic interactions with the negatively charged RNA. They are also consistent with an earlier proposed induced-fit binding mode of the preQ 1 class-I riboswitch [39,40]. …”

An integrated perspective on RNA aptamer ligand-recognition models: clearing muddy waters

“…In low Mg 2+ conditions, this small pseudoknot-based aptamer exists primarily in its unfolded state, and Suddala et al showed that it binds the ligand entirely through an IF mechanism. 84 The folding rate increased with PreQ 1 concentration. As the background Mg 2+ concentration was increased, however, the pseudoknot formed, much like the case of metX, and ligand binding decreased the unfolding rate of this structure, the kinetic hallmark of CS.…”

“…85 However, the presence of the ligand still enhanced the folding rate in these conditions. 84 The kinetics of PreQ 1 -I are notable for two reasons: first, it exhibits a clear transition from distinctively IF kinetics in one environment to a hybrid mechanism in another. Second, the mechanism in the presence of Mg 2+ cannot be simply categorized.…”

Single‐Molecule Monitoring of the Structural Switching Dynamics of Nucleic Acids through Controlling Fluorescence Blinking

“…[47,48] This is the smallest known riboswitch, and its folding process has attracted attention as am odel system to understand riboswitch folding. [49] Its aptamer domain specifically recognizes 7-aminomethyl-7-deazaguanine (preQ 1 )a nd controls gene expression through ligandmediated conformational switching.U sing NMR spectroscopy,M icura and co-workers demonstrated that the riboswitch adopts two different coexisting stem-loop structures,f old A and fold B (Figure 3a). [48] Thee quilibrium of this bistable stem-loop segment were shown to shift to the fold B structure in the presence of preQ 1 .W eintroduced ATTO 655 between positions A46 and A47 so as to bury ATTO 655 in the stem in the fold A structure while leaving it exposed to the solvent in the fold B structure.…”