Conformational Switching between ON and OFF States Proceeds through Multiple Pathways in the SAM-III Translational Riboswitch

Gene regulation by translational riboswitches relies on repeated and reversible conformational switching between ON (apo) and OFF (holo) states. The switching mechanism often involves significant structural rearrangement, and the role of the metal ions in this mechanism is unclear. Using molecular dynamics simulations, we studied the switching transition of a translational riboswitch that regulates the concentration of S-adenosyl methionine (SAM), the `universal methyl donor'. We show that Mg2+ ions are essential for stabilizing the ON-state, and the three-way junction exhibits a `breathing-like' dynamic interconversion between open and closed modes. The riboswitch has a low population of a holo-like READY (R) state with a partially organized SAM-binding pocket prior to ligand binding. Multiple intermediates are observed in the switching landscape, contributing to the gradual stepwise structural transition between the ON and R states, and proceeding through multiple competing pathways. Interestingly, Mg2+ ions influence the dominant pathway for the ON to R state transition, whereas the reverse transition shows a weak dependence. These results provide broad insights into the function of translational riboswitches, which repeatedly and rapidly respond to changes in metabolite concentrations, in contrast to transcriptional riboswitches.