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

Abstract

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 pathogenMycobacterium tuberculosis (Mtb). Using biochemistry and cryo-electron microscopy, we show thatMtbNusG 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-pausingE. coliNGN contacts the RNAP gate loop to inhibit swiveling and pausing ofMtbRNAP. 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.

DSIF factor Spt5 coordinates transcription, maturation and exoribonucleolysis of RNA polymerase II transcripts

“…Additionally, Spt5 can also control pausing independently of NELF 60,145,146 , which might represent a more ancient regulatory mechanism. Indeed, the NGN domain of Spt5 facilitates Pol II pausing by interacting with the non-template strand of DNA, reminiscent of the mechanism described for NusG, the Spt5 homologue in bacteria 62,147,148 .…”

An ensemble of interconverting conformations of the elemental paused transcription complex creates regulatory options

“…In contrast, available evidence indicates that both swiveling and half translocation are mechanistically important for pausing. Both states are catalytically inactive, represent the majority of some ePEC populations, and can be stabilized by PHs or pause-enhancing regulators ( 6 , 8 , 9 ). Swiveling kinks the BH toward the active site where it occludes DNA translocation and NTP binding and inhibits TL folding ( 6 , 9 ).…”

“…Both states are catalytically inactive, represent the majority of some ePEC populations, and can be stabilized by PHs or pause-enhancing regulators ( 6 , 8 , 9 ). Swiveling kinks the BH toward the active site where it occludes DNA translocation and NTP binding and inhibits TL folding ( 6 , 9 ). t-DNA translocation in the half-translocated ePEC appears to be the final energy barrier to pause escape ( 19 ).…”

“…Conformational fluctuations in the complex, multidomain RNAP, modulated by RNA–DNA sequence, govern this competition and ePEC lifetime. The ePEC can rearrange into longer-lived pauses by backtracking of the RNA and DNA chains, by interactions with nascent RNA secondary structures (pause hairpins; PHs), or by interactions with diffusible regulators (e.g., NusA or propausing NusG/Spt5) ( 6 – 9 ). RNA structures and regulators also can shorten or suppress elemental pausing (e.g., HK022 put RNA and λQ antiterminators or antipausing NusG/Spt5) ( 10 – 14 ).…”

Compensatory evolution in NusG improves fitness of drug-resistant M. tuberculosis

“…Among other hits, we found that the universally conserved transcription factor NusG is crucial for the fitness of RifR Mtb. In contrast to its role in Escherichia coli, Mtb NusG has an essential RNAP pro-pausing function mediated by distinct contacts with RNAP and the DNA 13 . We find this pro-pausing NusG-RNAP interface to be under positive selection in clinical RifR Mtb isolates.…”