Abstract
Riboswitches are non-coding RNA motifs that regulate gene expression in response to ligand binding. The glycine tandem riboswitch (GTR) contains two glycine aptamers that interact extensively, driving conformational changes in the downstream expression platform to control gene expression. Despite numerous studies, the role of glycine and RNA folding pathways in co-transcriptional regulation remains unclear. Here, we integrate single-molecule kinetic analysis, co-transcriptional RNA structure probing, and modeling to reveal that the GTR processes multiple molecular inputs sequentially, guided by polymerase pausing. Our findings elucidate its stepwise 5'-to-3' folding pathway and demonstrate how sequential glycine binding to each aptamer, K(+) binding to a kink-turn, non-native folding intermediates, inter-aptamer docking driving binding site pre-organization, and modulation by transcription factor NusA collectively orchestrate co-transcriptional gene regulation. These results support a model wherein glycine binding cooperativity arises through non-equilibrium mechanisms, rather than a classical concerted model.