Dynamic Pathway of Guanidine-III Riboswitch Folding Revealed by Single-Molecule FRET: Mg²⁺-Assisted Preorganization...

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 partially folded intermediate (I-state), and a folded pseudoknot structure (F-state)─with rapid interconversion in the absence of ligand. Mg²⁺ ions shift the conformational equilibrium toward the I- and F-states, reducing the reverse transition rates by up to 20-fold and enhancing guanidine binding affinity. Guanidine binding further suppresses the reverse transitions, kinetically trapping the riboswitch into its active folded state primarily through a conformational selection mechanism, with additional induced-fit contributions observed for the E-F transition. This work provides insight into the dynamic pathway by which the guanidine-III riboswitch integrates ionic and ligand cues, supporting its role in gene regulatory responses in bacteria.