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

Abstract

AbstractIn 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 σS regulon by promoting the association of σS 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-σS-RNAP in an open promoter complex with a σS regulon promoter. In addition to previously predicted interactions between Crl and domain 2 of σS (σS), 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 σS-dependent transcription in part through stabilizing σS-RNAP by tethering σS and the β’CT. We propose that Crl, and other transcription activators that may use similar mechanisms, be designated σ-activators.Significance StatementIn 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 σS-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-σS-RNAP transcription initiation complex. The structure, combined with biochemical experiments, shows that Crl stabilizes σS-RNAP by tethering σS directly to the RNAP.