Abstract

Riboswitches function through cotranscriptional conformation switching governed by cognate ligand concentration, RNA folding and transcription elongation kinetics. To investigate how these parameters influence riboswitch folding, we developed a novel vectorial folding assay (VF) in which the superhelicase Rep-X sequentially liberates the RNA strand from a heteroduplex in a 5-to-3 direction, mimicking the nascent chain emergence during transcription. The RNA polymerase (RNAP)-free VF recapitulates the kinetically controlled cotranscriptional folding of a ZTP riboswitch, whose activation is favored by slower transcription, strategic pausing, or a weakened transcriptional terminator. New methods to observe positions and local rates of individual helicases show an average Rep-X unwinding rate similar to bacterial RNAP elongation (~60 nt/s). Real-time single-molecule monitoring captured folding riboswitches in multiple states, including an intermediate responsible for delayed terminator formation. These methods allow observation of individual folding RNAs as they occupy distinct folding channels within the landscape that controls gene expression and showed that riboswitch fate control is encoded in its sequence and is readily interpreted by a directionally moving protein even in the absence of an RNA polymerase.