Observation of SAM-VI Riboswitch Dynamics Using Single-Molecule FRET

Riboswitches regulate gene expression through intricate dynamic conformational transitions, with divalent cation Mg²⁺ and their ligands playing pivotal roles in this process. The dynamic structural mechanism by which the S-adenosyl-L-methionine (SAM) responsive SAM-VI riboswitch (riboSAM) regulates the downstream SAM synthase gene translation remains unclear. In this study, we employed position-selective labeling of RNA (PLOR) to incorporate Cy3-Cy5 into designated positions of riboSAM, applying single-molecule Förster resonance energy transfer (smFRET) method to track its conformational switches in response to Mg²⁺ and SAM. smFRET analysis revealed that in the absence of Mg²⁺ and ligand, riboSAM predominantly adopted a translation-activating <i>apo</i> conformation. Physiological concentrations of Mg²⁺ induced riboSAM to fold into dynamic <i>transit-p</i> and <i>holo-p</i> states, creating a transient and structurally pliable binding pocket for ligand binding. SAM binding locks the dynamic <i>transit-p</i> and <i>holo-p</i> states into their final stable <i>transit</i> and <i>holo</i> conformations through conformational selection, turning off downstream cis-gene expression and completing feedback regulation of cellular SAM concentration. The observed synergistic regulatory effect of Mg²⁺ ions and ligand on riboSAM's conformational dynamics at single-molecule resolution provides new mechanistic insights into gene regulation by diverse riboswitch classes.