Structural and functional basis of the universal transcription factor NusG pro-pausing activity in Mycobacterium tuberculosis

Transcriptional pauses mediate regulation of RNA biogenesis. DNA-encoded pause signals trigger pausing by stabilizing RNA polymerase (RNAP) swiveling and inhibiting DNA translocation. The N-terminal domain (NGN) of the only universal transcription factor, NusG/Spt5, modulates pausing through contacts to RNAP and DNA. Pro-pausing NusGs enhance pauses, whereas anti-pausing NusGs suppress pauses. Little is known about pausing and NusG in the human pathogen Mycobacterium tuberculosis (Mtb). We report that MtbNusG is pro-pausing. MtbNusG captures paused, swiveled RNAP by contacts to the RNAP protrusion and nontemplate-DNA wedged between the NGN and RNAP gate loop. In contrast, anti-pausing Escherichia coli (Eco) NGN contacts the MtbRNAP gate loop, inhibiting swiveling and pausing. Using CRISPR-mediated genetics, we show that pro-pausing NGN is required for mycobacterial fitness. Our results define an essential function of mycobacterial NusG and the structural basis of pro- versus anti-pausing NusG activity, with broad implications for the function of all NusG orthologs. [Display omitted] •Cryo-EM reveals MtbPECs, like EcoPECs, are swiveled and half-translocated•MtbNusG is pro-pausing and promotes RNAP swiveling via contacts to the RNAP protrusion•Anti-pausing EcoNusG inhibits swiveling via contacts to the MtbRNAP gate loop•A pro-pausing NusG NGN is essential for robust mycobacterial growth NusG/Spt5, the only universal transcription factor, regulates transcript elongation by modulating RNAP pausing via contacts of its NGN domain to RNAP and DNA. Delbeau and colleagues describe how mycobacterial NusG NGN contacts that promote pausing and aid mycobacterial growth differ from contacts made by anti-pausing proteobacterial NusG.