Gourse, R. L. | bioRxiv Science

bioRxiv2019

E. coli TraR allosterically regulates transcription initiation by altering RNA polymerase conformation and dynamics

TraR and its homolog DksA are bacterial proteins that regulate transcription initiation by binding directly to RNA polymerase (RNAP) rather than to promoter DNA. Effects of TraR mimic the combined effects of DksA and its cofactor ppGpp. How TraR and its homologs regulate transcription is unclear. Here, we use cryo-electron microscopy to determine structures of Escherichia coli RNAP, with or without TraR, and of an RNAP-promoter complex. TraR binding induced RNAP conformational changes not seen in previous crystallographic analyses, and a quantitative analysis of RNAP conformational heterogeneity revealed TraR-induced changes in RNAP dynamics. These changes involve mobile regions of RNAP affecting promoter DNA interactions, including the {beta}lobe, the clamp, the bridge helix, and several lineage-specific insertions. Using mutational approaches, we show that these structural changes, as well as effects on {sigma}70 region 1.1, are critical for transcription activation or inhibition, depending on the kinetic features of regulated promoters.

biophysics↗

bioRxiv2019

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

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.

biophysics↗