Cotranscriptional RNA folding is widely assumed to influence the timely control of gene expression, but our understanding remains limited. In bacteria, the fluoride (F2)-sensing riboswitch is a transcriptional control element essential to defend against toxic F2 levels. Using this model riboswitch, we find that its ligand F2 and essential bacterial transcription factor NusA compete to bind the cotranscriptionally folding RNA, opposing each other's modulation of downstream pausing and termination by RNA polymerase. Single-molecule fluorescence assays probing active transcription elongation complexes discover that NusA unexpectedly binds highly reversibly, frequently interrogating the complex for emerging, cotranscriptionally folding RNA duplexes. NusA thus fine-tunes the transcription rate in dependence of the ligandresponsive higher-order structure of the riboswitch. At the high NusA concentrations found intracellularly, this dynamic modulation is expected to lead to adaptive bacterial transcription regulation with fast response times. [ABSTRACT FROM AUTHOR]