bioRxiv2019
In bacteria, a primary {sigma} 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 {sigma} 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), the structure, along with p-benzoylphenylalanine crosslinking, reveals that Crl interacts with a structural element of the RNAP {beta} subunit 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 {sigma}S-RNAP by tethering {sigma}S and the {beta}CT. We propose that Crl, and other transcription activators that may use similar mechanisms, be designated {sigma}-activators.\n\nSignificance StatementIn bacteria, multiple {sigma} factors can bind to a common core RNA polymerase (RNAP) to alter global transcriptional programs in response to environmental stresses. Many {gamma}-proteobacteria, including the pathogens Yersinia pestis, Vibrio cholera, Escherichia coli, and Salmonella typhimurium, encode Crl, a transcription factor that activates {sigma}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-{sigma}S-RNAP transcription initiation complex. The structure, combined with biochemical experiments, shows that Crl stabilizes {sigma}S-RNAP by tethering {sigma}S directly to the RNAP.