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 elemental pausing by stabilizing a half-translocated (RNA-not-DNA) state and by promoting RNAP swiveling that other factors can enhance. The universal transcription factor NusG (Spt5 in eukaryotes and archaea) N-terminal domain (NGN) modulates pausing through contacts to RNAP and DNA. Pro-pausing NusGs (e.g., Bacillus subtilis) enhance some pauses whereas anti-pausing NusGs (e.g., Escherichia coli) suppress some pauses. Little is known about pausing and NusG in the human pathogen Mycobacterium tuberculosis (Mtb). Using biochemistry and cryo-electron microscopy, we show that MtbNusG is a pro-pausing NusG that captures paused, swiveled RNAP by contacts to the RNAP protrusion and to a nontemplate strand-DNA wedge inserted between the NGN and the RNAP gate loop. On the other hand, we find that anti-pausing E. coli NGN contacts the RNAP gate loop to inhibit swiveling and pausing of MtbRNAP. Using CRISPR-mediated mycobacterial genetics, we show that a pro-pausing NGN is required to support robust mycobacterial growth. Our results define an essential function of NusG in mycobacteria and the structural basis of pro-vs. anti-pausing NusG activity with broad implications for NusG function in all domains of life.