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 (σS2), the structure, along with p-benzoylphenylalanine cross-linking, reveals that Crl interacts with a structural element of the RNAP β′-subunit that 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 σS2 and the β′CT. We propose that Crl, and other transcription activators that may use similar mechanisms, be designated σ-activators.

Crl activates transcription by stabilizing active conformation of the master stress transcription initiation factor

“…We point out that Cartagena and coworkers recently also reported a cryo-EM structure of a transcription activation complex comprising the E. coli RNAP core enzyme, Salmonella enterica serovar Typhimurium Crl and σ S (Cartagena et al, 2019). Our structure of E. coli Crl–TAC agrees well with this chimeric complex structure, and both structures show that Crl engages σ S -RNAP through a large interface with σ S 2 and a small interface with the RNAP core enzyme.…”

Structural Insights into Transcription Initiation from De Novo RNA Synthesis to Transitioning into Elongation

“…coli STIC crystals grow to full size in about 1 week and remain enzymatically active while present in the crystallization drops ( Liu et al., 2016 ). Crystallized STICs ( Figure 1 A) demonstrate essentially the same architecture as the STIC in solution ( Figure 1 B) ( Cartagena et al., 2019 ), but lack the contacts between the σ 4 domain and the promoter −35 element due to crystal packing. Compared with the STIC in solution, crystallized STICs show slightly tighter clamping by the pincers and a more localized trigger loop (TL) insertion domain.…”

The σ Subunit-Remodeling Factors: An Emerging Paradigms of Transcription Regulation

“…Crl binds to stationary phase σ S and activates σ S -RNAP-mediated transcription, independently of the promoter sequence (Pratt and Silhavy, 1998;Bougdour et al, 2004). Although Crl does not bind to σ 70 because of the steric clash with σ 70 -NCR (Cartagena et al, 2019) it can activate σ 70 -dependent transcription (Gaal et al, 2006). As observed for RbpA, Crl facilitates transcription initiation by stabilizing the σ S -RNAP holoenzyme and stimulating RPo formation (Banta et al, 2013;Xu et al, 2019).…”

“…As observed for RbpA, Crl facilitates transcription initiation by stabilizing the σ S -RNAP holoenzyme and stimulating RPo formation (Banta et al, 2013;Xu et al, 2019). Two high resolution cryo-EM-based structures of Crl-σ S -RNAP RPo have been recently described (Cartagena et al, 2019;Xu et al, 2019). Cartagena et al, suggested that Crl stabilizes σ S -RNAP by tethering σ S directly to RNAP though contacts with the β -clamp-toe domain (β -CT, residues 144-179).…”

“…It is not known whether Crl plays any role in promoter escape. However, the finding that the β -CT/σ 70 -NCR interaction antagonizes the σ2/β -clamp interaction and facilitaes promoter escape (Leibman and Hochschild, 2007) suggests this possibility (Banta et al, 2013;Cartagena et al, 2019).…”