Observation of coordinated cotranscriptional RNA folding events

RNA begins to fold as it is transcribed by an RNA polymerase. Consequently, RNA folding is constrained by the direction and rate of transcription. Understanding how RNA folds into secondary and tertiary structures therefore requires methods for determining the structure of cotranscriptional folding intermediates. Cotranscriptional RNA chemical probing methods accomplish this by systematically probing the structure of nascent RNA that is displayed from RNA polymerase. Here, we have developed a concise, high-resolution cotranscriptional RNA chemical probing procedure called Transcription Elongation Complex RNA structure probing-Multilength (TECprobe-ML). We validated TECprobe-ML by replicating and extending previous analyses of ZTP and fluoride riboswitch folding, and mapped the folding pathway of a ppGpp-sensing riboswitch. In each system, TECprobe-ML identified coordinated cotranscriptional folding events that mediate transcription antitermination. Our findings establish TECprobe-ML as an accessible method for mapping cotranscriptional RNA folding pathways.