Structure of the RNA Polymerase Assembly Factor Crl and Identification of Its Interaction Surface with Sigma S

Abstract

bBacteria utilize multiple sigma factors that associate with core RNA polymerase (RNAP) to control transcription in response to changes in environmental conditions. In Escherichia coli and Salmonella enterica, Crl positively regulates the S regulon by binding to S to promote its association with core RNAP. We recently characterized the determinants in S responsible for specific binding to Crl. However, little is known about the determinants in Crl required for this interaction. Here, we present the X-ray crystal structure of a Crl homolog from Proteus mirabilis in conjunction with in vivo and in vitro approaches that probe the Crl-S interaction in E. coli. We show that the P. mirabilis, Vibrio harveyi, and E. coli Crl homologs function similarly in E. coli, indicating that Crl structure and function are likely conserved throughout gammaproteobacteria. We utilize phylogenetic conservation and bacterial two-hybrid analyses to predict residues in Crl important for the interaction with S . The results of p-benzoylphenylalanine (BPA)-mediated UV cross-linking studies further support the model in which an evolutionarily conserved central cleft is the surface on Crl that binds to S . Within this conserved binding surface, we identify a key residue in Crl that is critical for activation of E S -dependent transcription in vivo and in vitro. Our study provides a physical basis for understanding the S -Crl interaction.

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

“…Altogether, the interaction between Crl and σ S forms an interfacial area of 785 Å 2 (41) and is completely consistent with previous analyses of Crl-s S interactions (26,34,38,40). In summary, our structure: 1) confirms the s S residues previously proposed to interact with Crl based on genetic and biochemical data; 2) identifies additional residues in s S and Crl involved in the intermolecular interaction; 3) reveals that 6 out of 11 (55%) of the residues of σ s contacting Crl are not conserved in σ 70 (Fig.…”

“…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.…”

“…D135 makes favorable electrostatic interactions with Crl R51, which is absolutely conserved among Crl homologs (40). Substitutions Crl R51A or R51K were totally defective in Crl function in vitro and in vivo (34). D135 and E137 of s S , along with P136, have been referred to as the DPE motif ( Fig.…”

Structural, functional, and genetic analyses of the actinobacterial transcription factor RbpA

“…Interacting with the primary σ-NCR is a property that RbpA shares with the unrelated Chlamydia trachomatis transcription factor GrgA, which binds to the Chlamydia trachomatis σ 66 -NCR (20). Moreover, although structurally distinct from RbpA, the holoenzyme assembly factor Crl from enteric bacteria interacts with the equivalent region of the group 2 σ-factor σ S (21,22). The cocrystal structure of RbpA-σ A 2 represents the first structure, to our knowledge, of an activator (or any protein) interacting with the NCR of a housekeeping-σ.…”

The essential activities of the bacterial sigma factor

“…These include the essential mycobacterial activator RbpA (Paget et al 2001;Tabib-Salazar et al 2013;Hubin et al 2015), GrgA from Chlamydia trachomatis (Bao et al 2012), Crl from E. coli (Pratt and Silhavy 1998), and RsoA from B. subtilis (MacLellan et al 2008). Crl stimulates S -dependent transcription at least in part by interacting with S and promoting its assembly into holoenzyme (Banta et al 2013(Banta et al , 2014. RsoA, which possesses some sequence similarity to , interacts with the amino terminus of the ECF-like factor SigO and displays weak interaction activity with the clamp helices region of the RNAP ␤= subunit (MacLellan et al 2009;Xue et al 2016).…”