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

In bacteria, a primary a-factor associates with the core RNA polymerase (RNAP) to control most transcription initiation, while alternative sigma-factors are used to coordinate expression of additional regulons in response to environmental conditions. Many alternative a-factors are negatively regulated by anti-sigma-factors. In Escherichia coli, Salmonella enterica, and many other gamma-proteobacteria, the transcription factor Crl positively regulates the alternative sigma(s)-regulon by promoting the association of sigma(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-sigma(s)-RNAP in an open promoter complex with a sigma(s)-regulon promoter. In addition to previously predicted interactions between Crl and domain 2 of sigma(s) (sigma(s)(2)), the structure, along with p-benzoylphenylalanine cross-linking, reveals that Crl interacts with a structural element of the RNAP beta'-subunit that we call the beta'-clamp-toe (beta'CT). Deletion of the beta'CT decreases activation by Crl without affecting basal transcription, highlighting the functional importance of the Crl-beta'CT interaction. We conclude that Crl activates sigma(s)-dependent transcription in part through stabilizing a s RNAP by tethering sigma(s)(2) and the beta'CT. We propose that Crl, and other transcription activators that may use similar mechanisms, be designated sigma-activators.