NusG-dependent RNA polymerase pausing is a common feature of riboswitch regulatory mechanisms

Transcription by RNA polymerase is punctuated by transient pausing events. Pausing provides time for RNA folding and binding of regulatory factors to the paused elongation complex. We previously identified 1600 NusG-dependent pauses throughout the Bacillus subtilis genome, with ∼20% localized to 5′ leader regions, suggesting a regulatory role for these pauses. We examined pauses associated with known riboswitches to determine whether pausing is a common feature of these mechanisms. NusG-dependent pauses in the fmnP, tenA, mgtE, lysP and mtnK riboswitches were in strategic positions preceding the critical decision between the formation of alternative antiterminator or terminator structures, which is a critical feature of transcription attenuation mechanisms. In vitro transcription assays demonstrated that pausing increased the frequency of termination in the presence of the cognate ligand. NusG-dependent pausing also reduced the ligand concentration required for efficient termination. In vivo expression studies with transcriptional fusions confirmed that NusG-dependent pausing is a critical component of each riboswitch mechanism. Our results indicate that pausing enables cells to sense a broader range of ligand concentrations for fine-Tuning riboswitch attenuation mechanisms. Graphical abstract.