Role of a hairpin-stabilized pause in the Escherichia coli thiC riboswitch function

Transcriptional pauses have been reported in bacterial riboswitches and, in some cases, their specific positioning has been shown to be important for gene regulation. Here, we show that a hairpin structure in the Escherichia coli thiamin pyrophosphate (TPP) thiC riboswitch is involved in transcriptional pausing and ligand sensitivity. Using in vitro transcription kinetic experiments, we show that all three major transcriptional pauses in the thiC riboswitch are affected by NusA, a transcriptional factor known to stimulate hairpin-stabilized pauses. Using a truncated region of the riboswitch, we isolated the hairpin structure responsible for stabilization of the most upstream pause. Destabilization of this structure led to a weaker pause and a decreased NusA effect. In the context of the full-length riboswitch, this same mutation also led to a weaker pause, as well as a decreased TPP binding affinity. Our work suggests that RNA structures involved in transcriptional pausing in riboswitches are important for ligand sensitivity, most likely by increasing the time allowed to the ligand for binding to the riboswitch.