pH-dependent allosteric remodeling of a bacterial riboswitch couples alkaline activation to metal sensing

The widespread yybP-ykoY riboswitches control bacterial manganese (Mn) homeostasis by activating exporter expression in response to intracellular Mn²⁺ levels. The E. coli alx riboswitch distinctively couples Mn²⁺ sensing to cytoplasmic alkalinity, but the mechanism is unknown. We show that pH tunes the alx aptamer's conformational sampling to modulate Mn²⁺ sensitivity. Single-molecule FRET reveals that Mn²⁺ stabilizes a docked three-way-junction conformation, and alkaline pH shifts this equilibrium to sensitize metal-dependent folding. Molecular dynamics simulations identify a loop whose low-pH-induced base pairing perturbs the adjacent helix, predicted to allosterically disrupt the Mn²⁺-binding state. In vivo reporters indicate that both this loop and the Mn²⁺-binding core are required for optimal pH-dependent translational activation: replacing the core with the non-pH-responsive mntP sequence abolishes activation. These results define how RNA allosterically integrates orthogonal metal and proton cues to enable combinatorial environmental sensing during alkaline stress.