Structural basis for transcription activation by Crl through tethering of σ^S and RNA polymerase

Abstract

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 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.

Diverse molecular mechanisms of transcription regulation by the bacterial alarmone ppGpp

“…This extensive network of interactions allows MglA‐SspA to fasten the σ 70 factor to the core enzyme and also enhances binding of the holoenzyme to virulence promoters which have nonoptimal −35 RNAPσ 70 binding elements (Figure 3b). Regulators that aid in the recruitment of specific RNAP holoenzymes through σ factor tethering have recently been referred to as σ‐activators (Cartagena et al., 2019; Chen et al., 2021). Notably, the Ftu RNAPσ 70 ‐(MglA‐SspA) complex with bound PigR and virulence promoter DNA revealed an unexpected role for PigR in the stabilization of RNAP on the promoter; not only does it bind the PRE site to help anchor the complex to the promoter but it also recruits the RNAP α C‐terminal domains (αCTDs) to ATrich Upstream (UP) elements that flank the PRE (Travis et al., 2021) (Figure 3b,c).…”

“…This extensive network of interactions allows MglA-SspA to fasten the σ 70 factor to the core enzyme and also enhances binding of the holoenzyme to virulence promoters which have nonoptimal −35 RNAPσ 70 binding elements (Figure 3b). Regulators that aid in the recruitment of specific RNAP holoenzymes through σ factor tethering have recently been referred to as σ-activators (Cartagena et al, 2019;Chen et al, 2021).…”

A negative feedback loop is critical for recovery of RpoS after stress inEscherichia coli

“…Unlike canonical transcriptional regulators, this 16-kDa protein does not bind to DNA at promoters but rather binds and specifically stabilizes the RNA polymerase-RpoS sigma factor holoenzyme. As highlighted in recent structures of the complex of RNAP-RpoS-Crl-promoter DNA, Crl plays a role in promoting the formation of the complex and increasing the transcription of the genes that are under the control of RpoS [18][19][20][21]. In the absence of Crl, transcription of some RpoS-controlled genes is diminished, primarily when RpoS levels are low [22][23][24].…”