Key features of σ ^S required for specific recognition by Crl, a transcription factor promoting assembly of RNA polymerase holoenzyme

Abstract

Bacteria use multiple sigma factors to coordinate gene expression in response to environmental perturbations. In Escherichia coli and other γ-proteobacteria, the transcription factor Crl stimulates σ Sdependent transcription during times of cellular stress by promoting the association of σ S with core RNA polymerase. The molecular basis for specific recognition of σ S by Crl, rather than the homologous and more abundant primary sigma factor σ 70 , is unknown. Here we use bacterial two-hybrid analysis in vivo and p-benzoylphenylalanine cross-linking in vitro to define the features in σ S responsible for specific recognition by Crl. We identify residues in σ S conserved domain 2 (σ S 2 ) that are necessary and sufficient to allow recognition of σ 70 conserved domain 2 by Crl, one near the promoter-melting region and the other at the position where a large nonconserved region interrupts the sequence of σ 70 . We then use luminescence resonance energy transfer to demonstrate directly that Crl promotes holoenzyme assembly using these specificity determinants on σ S . Our results explain how Crl distinguishes between sigma factors that are largely homologous and activates discrete sets of promoters even though it does not bind to promoter DNA.RNAP formation | transcription initiation | bacterial stress response | RpoS | curli fiber T ranscription initiation in bacteria requires the assembly of a sigma factor (σ) with the RNA polymerase (RNAP) catalytic core (E, composed of 2 α-subunits and one each of β, β′, and ω) to form RNAP holoenzyme (Eσ), which in turn recognizes promoter sequences (1) (reviewed in ref. 2). Multiple sigma factors compete for binding to core RNAP (reviewed in refs. 3,4), and each sigma factor controls a specific set of promoters.In Escherichia coli, which has seven sigma factors, σ 70 is the primary sigma, and σ S is important for certain stress responses and during the stationary phase of growth (5). Eσ S -dependent transcription initiation is regulated by σ S , whose concentration is itself regulated at the levels of transcription, translation, and protein stability (reviewed in ref. 6). Eσ S -dependent transcription is also activated by Crl (7), a small protein that increases expression of many stress response genes and those required for formation of amyloid curli fibers (which accounts for its name) involved in adhesion and biofilm formation (reviewed in refs. 2,6,8).The effect of Crl on σ S -dependent transcription in vivo is most pronounced during the transition into stationary phase (9). It has been proposed that Crl functions by increasing the concentration of Eσ S holoenzyme by facilitating assembly of σ S with core RNAP (10-12) because Crl's effects on transcription are greatest in vitro when the concentration of σ S is lowest, and overexpression of σ S complements a crl deletion in vivo (13). Effects of Crl have also been reported on postholoenzyme assembly steps including promoter binding (14) and open complex formation (12).Sigma factors contain several protease-resistant domains, e...

Structures of E . coli σ S -transcription initiation complexes provide new insights into polymerase mechanism

“…In E. coli and many γ-proteobacteria, a small protein called "Crl" was found to stimulate σ S -dependent transcription by promoting the formation of the σ S -RNAP holoenzyme (13,14). It was shown previously that the σ S -Crl interaction involves a general area on the surface of σ S including residues of Asp87, Asp135, Pro136, and Glu137 (15). This general area corresponds to the attachment face of the NCR to the primary σ factors (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

“…In practice, it appears that recruitment of RNAP to promoters is the dominant mechanism that leads to activation (Lee et al 2012). Other established mechanisms of transcription activation include promotion of holoenzyme formation (e.g., Crl (Banta et al 2013)), regulation of the isomerization from closed double-stranded DNA to singlestranded open complex (e.g., cI (Dove et al 2000)), and the enhancement of polymerase promoter escape (e.g., Btr (Gaballa et al 2012)). In some specific cases, certain promoters possess suboptimal spacing between the -35 and -10 recognition elements, and binding of an activator protein bends or twists the promoter DNA to establish an optimal spacing between these elements, thus permitting holoenzyme binding (Heldwein and Brennan 2001;Brown et al 2003).…”

“…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).…”