Structural basis for transcription activation by Crl through tethering of σS and RNA polymerase

In bacteria, a primary σ factor associates with the core RNA polymerase (RNAP) to control most transcription initiation, while alternative σ factors are used to coordinate expression of additional regulons in response to environmental conditions. Many alternative σ factors are negatively regulated by anti-σ factors. In Escherichia coli, Salmonella enterica , and many other γ -proteobacteria, the transcription factor Crl positively regulates the alternative σ S regulon by promoting the association of σ S with RNAP without interacting with promoter DNA. The molecular mechanism for Crl activity is unknown. Here, we determined a single-particle cryo-electron microscopy structure of Crl-σ S -RNAP in an open promoter complex with a σ S regulon promoter. In addition to previously predicted interactions between Crl and domain 2 of σ S (σ S ), the structure, along with p-benzoylphenylalanine crosslinking, reveals that Crl interacts with a structural element of the RNAP β’ subunit we call the β’-clamp-toe (β’CT). Deletion of the β’CT decreases activation by Crl without affecting basal transcription, highlighting the functional importance of the Crl-β’CT interaction. We conclude that Crl activates σ S -dependent transcription in part through stabilizing σ S -RNAP by tethering σ S and the β’CT. We propose that Crl, and other transcription activators that may use similar mechanisms, be designated σ-activators. Significance Statement In bacteria, multiple σ factors can bind to a common core RNA polymerase (RNAP) to alter global transcriptional programs in response to environmental stresses. Many γ-proteobacteria, including the pathogens Yersinia pestis, Vibrio cholera, Escherichia coli , and Salmonella typhimurium , encode Crl, a transcription factor that activates σ S -dependent genes. Many of these genes are involved in processes important for infection, such as biofilm formation. We determined a high-resolution cryo-electron microscopy structure of a Crl-σ S -RNAP transcription initiation complex. The structure, combined with biochemical experiments, shows that Crl stabilizes σ S -RNAP by tethering σ S directly to the RNAP.