Structural and functional features of Crl proteins and identification of conserved surface residues required for interaction with the RpoS/σS subunit of RNA polymerase

Abstract

In many γ-proteobacteria, the RpoS/σS sigma factor associates with the core RNAP (RNA polymerase) to modify global gene transcription in stationary phase and under stress conditions. The small regulatory protein Crl stimulates the association of σS with the core RNAP in Escherichia coli and Salmonella enterica serovar Typhimurium, through direct and specific interaction with σS. The structural determinants of Crl involved in σS binding are unknown. In the present paper we report the X-ray crystal structure of the Proteus mirabilis Crl protein (CrlPM) and a structural model for Salmonella Typhimurium Crl (CrlSTM). Using a combination of in vivo and in vitro assays, we demonstrated that CrlSTM and CrlPM are structurally similar and perform the same biological function. In the Crl structure, a cavity enclosed by flexible arms contains two patches of conserved and exposed residues required for σS binding. Among these, charged residues that are likely to be involved in electrostatic interactions driving Crl-σS complex formation were identified. CrlSTM and CrlPM interact with domain 2 of σS with the same binding properties as with full-length σS. These results suggest that Crl family members share a common mechanism of σS binding in which the flexible arms of Crl might play a dynamic role.

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

“…Our results provide insights into the mechanisms by which Crl promotes Eσ S assembly. The Crl-Eσ S -dps-RPo cryo-EM structure is consistent with, and expands upon, previous information about the interactions of Crl with σ S 2 (26,28,34,35,40), and includes an interaction with the RNAP β'CT. To our knowledge, this region of core RNAP has not been previously described as a binding determinant for transcription factors and may represent a target for other uncharacterized transcription factors.…”

“…As previously reported, Crl is small arc-shaped protein with a shallow concave surface composed of four antiparallel β-strands and flanked by intervening loops (34,35). This cavity makes extensive electrostatic, polar, and hydrophobic interactions with helix α2 (A73 to R85) of σ S , which resides within conserved region σ S 1.2 (3) (SI Appendix, Fig.…”

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

“…A large collection of genetic and biochemical data have highlighted the importance of Crl in σ S -mediated transcription in bacteria that encode σ S (Cavaliere and Norel, 2016). Crl was demonstrated to directly activate σ S -mediate transcription both in vitro and in vivo (Banta et al, 2013;Banta et al, 2014;Cavaliere et al, 2014;Cavaliere et al, 2015;England et al, 2008;Monteil et al, 2010a;Pratt and Silhavy, 1998;Typas et al, 2007a), and Crl-null Salmonella and E. coli strains displayed impaired biogenesis of curli (important for host cell adhesion and invasion as well as formation of biofilm), increased sensitivity to H 2 O 2 stress, and reduced virulence due to decrease expression of several σ S -regulated genes (Arnqvist et al, 1992;Barnhart and Chapman, 2006;Monteil et al, 2010a;Robbe-Saule et al, 2008;Robbe-Saule et al, 2006).…”

“…Crl has been demonstrated to interact with σ S 2 and probably also RNAP core enzyme (England et al, 2008), but whether or how it interacts with DNA remains elusive. Although crystal and NMR structures of Crl are available (Banta et al, 2014;Cavaliere et al, 2014;Cavaliere et al, 2015), it is still unclear how Crl interacts with σ S - (Boyaci et al, 2019;Ruff et al, 2015). Conventional bacterial transcription activators such as E. coli CAPs (Browning and Busby, 2016;Liu et al, 2017), C. cresentus GcrA (Wu et al, 2018), Chlamydia trachomatis GrgA (Bao et al, 2012), and Mycobacteria RbpA and CarD (Bae et al, 2015;Hubin et al, 2015) modulate the intermediate steps during RPo transition through simultaneously engaging RNAP holoenzyme and promoter DNA, thereby strengthening the interaction between RNAP and promoter DNA to promote transcription (Browning and Busby, 2016).…”

High-Resolution Crystal Structure of RpoS Fragment including a Partial Region 1.2 and Region 2 from the Intracellular Pathogen Legionella pneumophila

“…Asp135, Pro136, and Glu137 from EcRpoS region 2 with the DPE motif. In particular, the charge of Glu137 in the DPE motif is crucial for Crl recognition [27].…”