Abstract
RNA folds and balances between distinct conformational states for function. Riboswitches, a class of non-coding regulatory RNAs composed of a ligand-sensing domain and an expression platform, are known to control gene expression by folding into alternative conformations upon specific recognition of cellular cues. However, a molecular understanding of the dynamic interplay between the sensing domain and the expression platform that underlies riboswitch regulation remains elusive. Here, by developing and applying nucleic-acid-optimized chemical exchange saturation transfer (CEST) NMR spectroscopy, together with mutagenesis and functional measurements, we show that conformational kinetics of the riboswitch serves as a new layer of regulation, where ligand-dependent accessibility of a low-populated (~1%) and short-lived (~ 3ms) RNA state guides distinct co-transcriptional folding pathways to direct gene expression outcome. Our results provide an integrated molecular mechanism for transcriptional riboswitches and exemplify a new mode of ligand-dependent RNA regulation.