Directional co-transcriptional folding and pausing create kinetic checkpoints for riboswitch-controlled gene expression

Transcriptional riboswitches typically regulate gene expression by sensing small metabolites or ions that cannot be fluorophore-labeled without perturbing recognition, limiting direct observation of ligand binding during transcription. The GlyQS T-box riboswitch instead senses the uncharged 3′ end of a macromolecular ligand, tRNAGly, enabling visualization of co-transcriptional ligand binding. Using single-molecule fluorescence microscopy, we show that tRNAGly dynamically samples discrete structural domains of the nascent riboswitch as transcription proceeds. Productive recognition depends on a hierarchical 5′-to-3′ folding pathway shaped by transcription rate and pausing. Pausing enhances tRNA anchoring, stabilizes the riboswitch–tRNA complex, and selectively promotes RNA polymerase readthrough when an uncharged 3′ end is presented. These results support a kinetic checkpoint model in which early recruitment increases local ligand concentration, while pause-mediated stabilization converts transient encounters into committed regulatory decisions. More broadly, our findings identify directional co-transcriptional folding and transcriptional pausing as principles synergistically enabling selective and robust riboswitch function.