Dynamic Pathway of Guanidine-III Riboswitch Folding Revealed by Single-Molecule FRET: Mg ²⁺ -Assisted Preorganization and Ligand-Induced Kinetic Trapping

Riboswitches are structured RNA elements that regulate gene expression by sensing and binding small molecules. The guanidine-III riboswitch, a critical bacterial regulator responding to guanidine toxicity, undergoes precise conformational changes that remain poorly characterized at a dynamic, mechanistic level. In this study, we employed single-molecule Förster Resonance Energy Transfer (smFRET) coupled with molecular dynamics (MD) simulations to delineate how the guanidine-III riboswitch transitions among distinct conformational states. We identify three principal states ― an extended (E-state), a compacted prefolded intermediate (P-state), and a folded pseudoknot structure (F-state) ― with rapid interconversion in the absence of ligand. Magnesium ions significantly stabilize intermediate states via a cooperative, preorganization mechanism, enhancing ligand binding efficiency. Binding of guanidine drastically suppresses the reverse transitions, kinetically trapping the riboswitch into its active folded state primarily through a conformational selection mechanism, with additional contributions from induced-fit dynamics. This work illuminates the unique dynamic pathway by which the guanidine-III riboswitch integrates ionic and ligand cues, ensuring precise gene regulatory responses in bacteria.