Cotranscriptional Folding of a Riboswitch at Nucleotide Resolution

RNAs can begin to fold immediately after emerging from RNA polymerase during transcription. Interactions between nascent RNAs and ligands during cotranscriptional folding can direct the formation of alternative RNA structures, a feature exploited by non-coding RNAs called riboswitches to make gene regulatory decisions. Despite their importance, cotranscriptional folding pathways have yet to be uncovered with sufficient resolution to reveal how cotranscriptional folding governs RNA structure and function. To access cotranscriptional folding at nucleotide resolution, we extend selective 2’-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq) to measure structural information of nascent RNAs during transcription. With cotranscriptional SHAPE-Seq, we determine how the B. cereus crcB fluoride riboswitch cotranscriptional folding pathway undergoes a ligand-dependent bifurcation that delays or promotes terminator formation via a series of coordinated structural transitions. Our results directly link cotranscriptional RNA folding to a genetic decision and establish a framework for cotranscriptional analysis of RNA structure at nucleotide resolution.