Structural and functional basis of the universal transcription factor NusG pro-pausing activity in Mycobacterium tuberculosis
NusG can either suppress RNAP pausing in E. coli or promote pausing in B. subtilis. All NusG family proteins bind RNAP by contacts of the NusG N-terminal domain (NGN) to anti-parallel alpha-helices on the conserved RNAP clamp domain. B. subtilis–like NusG exhibit pause enhancement via sequence specific binding of the NGN to a TTnTTT motif in the non-template DNA strand (nt-DNA) whereas E. coli–like NusGs lack affinity for nt-DNA bases. Little is known about pausing and NusG in the major human pathogen Mycobacterium tuberculosis (Mtb). Using biochemistry, and cryo-EM, we compared Mtb NusG to E. coli NusG with the same RNAP and in the same sequence context to decipher the structural and mechanistic bases of pro- vs anti-pausing NusG. We established that Mtb NusG promotes pausing by stabilizing a translocation-inhibited, swiveled PEC and interacting non-specifically with the nt-DNA. More specifically, Mtb NusG contacts the Beta-protrusion extensively creating a gap between NusG and the Beta-gate loop into which the single-stranded nt-DNA wedges forcing the –6 base to flip into a hydrophobic pocket. In contrast, E. coli NusG suppresses the same pause by contacting the Beta-protrusion and the Beta-gate loop to eliminate the gap between NusG and Beta-gate loop, which reverses swiveling and favors forward translocation. Further comparison of Mtb, E. coli, B. subtilis, and M. smegmatis NGNs using CRISPR-mediated mycobacterial genetics suggest that the pro-pausing NusG-NGN activity is required for robust cell growth of mycobacteria. The data shown herein represent original, and unprocessed biochemistry gels, western blot gels, and spot assay plates used to generate the main and supplemental figures in our Delbeau et al Molecular Cell paper. All gels and plates are labelled accordingly to help viewers identify and understand the important parts. These data were collected in triplicate using methods described in Delbeau et al.