Transcription activation is established through extensive protein–protein and protein–DNA interactions that allow an activator to engage and remodel RNA polymerase. SoxS, a global transcription activator, diversely regulates subsets of stress response genes with different promoters, but the detailed SoxS-dependent transcription initiation mechanisms remain obscure. Here, we report cryo-EM structures of three SoxS-dependent transcription activation complexes (SoxS-TACI, SoxS-TACII and SoxS-TACIII) comprising of Escherichia coli RNA polymerase (RNAP), SoxS protein and three representative classes of SoxS-regulated promoters. The structures reveal that SoxS monomer orchestrates transcription initiation through specific interactions with the promoter DNA and different conserved domains of RNAP. In particular, SoxS is positioned in the opposite orientation in SoxS-TACIII to that in SoxS-TACI and SoxS-TACII, unveiling a novel mode of transcription activation. Strikingly, two universally conserved C-terminal domains of alpha subunit (αCTD) of RNAP associate with each other, bridging SoxS and region 4 of σ70. We show that SoxS interacts with RNAP directly and independently from DNA, remodeling the enzyme to activate transcription from cognate SoxS promoters while repressing transcription from UP-element containing promoters. Our data provide a comprehensive summary of SoxS-dependent promoter architectures and offer new insights into the αCTD contribution to transcription control in bacteria.

Rob, which serves as a paradigm of the large AraC/XylS family transcription activators, regulates diverse subsets of genes involved in multidrug resistance and stress response. However, the underlying mechanism of how it engages bacterial RNA polymerase and promoter DNA to finely respond to environmental stimuli is still elusive. Here, we present two cryo-EM structures of Rob-dependent transcription activation complex (Rob-TAC) comprising of Escherichia coli RNA polymerase (RNAP), Rob-regulated promoter and Rob in alternative conformations. The structures show that a single Rob engages RNAP by interacting with RNAP αCTD and σ70R4, revealing their generally important regulatory roles. Notably, by occluding σ70R4 from binding to -35 element, Rob specifically binds to the conserved Rob binding box through its consensus HTH motifs, and retains DNA bending by aid of the accessory acidic loop. More strikingly, our ligand docking and biochemical analysis demonstrate that the large Rob C-terminal domain (Rob CTD) shares great structural similarity with the global Gyrl-like domains in effector binding and allosteric regulation, and coordinately promotes formation of competent Rob-TAC. Altogether, our structural and biochemical data highlight the detailed molecular mechanism of Rob-dependent transcription activation, and provide favorable evidences for understanding the physiological roles of the other AraC/XylS-family transcription factors.

GlnR, an OmpR/PhoB subfamily protein, is an orphan response regulator that globally coordinates the expression of genes responsible for nitrogen, carbon and phosphate metabolism in actinobacteria. Although much efforts at biochemical and genetic analyses have been made on the mechanism of GlnR-dependent transcription activation, it still remains unclear owing to lacking the structure of GlnR-dependent transcription activation complex (GlnR-TAC). Here, we report a crystal structure of a binary complex including a C terminal DNA binding domain of GlnR (GlnR_DBD) and its regulatory cis-element DNA, and a cryo-EM structure of GlnR-TAC comprising of Mycobacterium tuberculosis RNA polymerase, GlnR, and a promoter containing four well-characterized conserved GlnR binding sites. These structures show four GlnR protomers coordinately engage promoter DNA in a head-to-tail manner, with two N-terminal receiver domains of GlnR (GlnR-RECs) jointly act as a bridge to connect RNAP αNTD with the upstream GlnR_DBD. GlnR-TAC is stabilized by complex protein-protein interactions between GlnR and the conserved beta flap, σAR4, alphaCTD, alphaNTD domains of RNAP. These are in good agreement with our mutational and kinetic single-molecule fluorescence assays. Altogether, our results reveal a general transcription activation mechanism for the global regulator GlnR and other OmpR/PhoB subfamily proteins, and present a unique mode of bacterial transcription regulation.