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

Transcription initiation is a key checkpoint and highly regulated step of gene expression. The sigma (σ) subunit of RNA polymerase (RNAP) controls all transcription initiation steps, from recognition of the −10/−35 promoter elements, upon formation of the closed promoter complex (RPc), to stabilization of the open promoter complex (RPo) and stimulation of the primary steps in RNA synthesis. The canonical mechanism to regulate σ activity upon transcription initiation relies on activators that recognize specific DNA motifs and recruit RNAP to promoters. This mini-review describes an emerging group of transcriptional regulators that form a complex with σ or/and RNAP prior to promoter binding, remodel the σ subunit conformation, and thus modify RNAP activity. Such strategy is widely used by bacteriophages to appropriate the host RNAP. Recent findings on RNAP-binding protein A (RbpA) from Mycobacterium tuberculosis and Crl from Escherichia coli suggest that activator-driven changes in σ conformation can be a widespread regulatory mechanism in bacteria.

The σ2 domain harbors the highly conserved regions 1.2, 2.1, 2.2, 2.3, and 2.4 that are essential for binding to RNAP βclamp, for recognition of the −10 element, and for melting of promoter DNA (Feklistov and Darst, 2011;Zhang et al., 2012). About 73% of the σ2 contact surface with ssDNA of the −10 element is formed by region 2.3 residues. In addition, residues in region 1.2 contact with the T −7 base of the −10 element (Feklistov and Darst, 2011) and control recognition of the −10 element allosterically (Zenkin et al., 2007;Morichaud et al., 2016). The σ 70 NCR interacts with promoter DNA at positions -16/-17 (R157 Eco ) and is implicated in DNA unwinding (Narayanan et al., 2018). The σ 70 NCR/β interaction facilitates promoter escape (Leibman and Hochschild, 2007). Domain σ4 interacts with the β-flap domain of core RNAP and harbors a helixturn-helix DNA binding domain that recognizes the −35 motif. The σ3 and σ4 subunits are connected by a weakly structured linker (region 3.2) that fills the RNA exit channel and is ejected upon the initial RNA synthesis (Zhang et al., 2012;Li et al., 2020). Most bacterial promoters recognized by group 1 and 2 σ subunits belong to the −10/−35 class and contain the −10 and also the −35 elements. The extended −10 class of promoters (∼20% in E. coli) contains the extended −10 motif (T −17 R −16 T −15 G −14 ; R = purine) that is located one base upstream of the −10 element (Keilty and Rosenberg, 1987;Burr et al., 2000;Mitchell et al., 2003) and interacts with the σ3 domain (Barne et al., 1997) (Figure 1B). It has been shown that the extended −10 motif bypasses the requirement of the σ4/−35 element interaction (Kumar et al., 1993). However, σ4 per se is essential for transcription initiation by σ B -MtbRNAP at the extended −10 promoters .
As a general rule, the principal σ subunit, the cellular concentration of which exceeds that of core RNAP (Gaal et al., 2006) should recognize and bind to promoter DNA only in the context of RNAP holoenzyme. Free σ should be devoid of DNA binding activity that might inhibit transcription. Data from structural and biophysical studies suggest that free group 1 and 2 σ subunits adopt a "closed" inactive conformation in which the spatial arrangement of domains σ2 and σ4 is incompatible with promoter DNA binding. Binding to core RNAP induces or stabilizes an "open, " active σ conformation, optimal for promoter binding (Callaci et al., 1999;Schwartz et al., 2008;Vishwakarma et al., 2018). Canonically, RNAP activity at promoters is regulated through DNA-binding transcription factors (Browning and Busby, 2016) that recognize and bind to specific motifs on dsDNA (DB-TFs) and influence the initiation pathway steps after promoter binding ( Figure 1A). A number of proteins, called σ-regulators in this review, have evolved to tune the structure of the σ/core RNAP interaction, thus altering RNAP promoter selectivity and activity globally. These RNAPbinding transcription factors (RPB-TFs) bind to RNAP before the RNAP-promoter complex formation upon RNAP assembly. Consequently, RPB-TFs can influence all the ensuing steps of initiation and in some cases, also elongation and termination (Figures 1A,D). These proteins can be divided in two groups: (1) σ-activators (RbpA, Crl, GcrA, and GrgA) that target the σ2 domain and consequently its interaction with the −10 element, and (2) σ-repressors (Gp39, AsiA, P7, and Scc4) that target the σ4 domain and consequently its interaction with −35 element ( Table 1). All σ-repressor, but one, are phage-encoded proteins that appropriate the host transcriptional machinery during infection (Tabib-Salazar et al., 2019).

RbpA
RbpA is a ∼14-kDa protein specific to Actinomycetes sp. RbpA was discovered in Streptomyces coelicolor as a protein that is associated with the RNAP holoenzyme (Paget et al., 2001) and is required for rapid growth and confers basal levels of rifampicin resistance (Newell et al., 2006). Later studies in Mtb described RbpA as a σ-specific transcriptional activator implicated in the stress response (Hu et al., 2012(Hu et al., , 2014 and essential for growth (Forti et al., 2011). RbpA binds to group 1 and group 2 σ subunits (σ A and σ B in Mtb;σ HrdB and σ HrdA in S. coelicolor), but not to group 3 and group 4 σ subunits (Bortoluzzi et al., 2013;Tabib-Salazar et al., 2013;Hu et al., 2014). RbpA exerts multiple effects on transcription initiation. It stabilizes σ interaction with core MtbRNAP, promotes DNA melting, stabilizes RPo, and accelerates promoter escape (Hu et al., 2012(Hu et al., , 2014Perumal et al., 2018). However, a recent study on the σ A -MtbRNAP holoenzyme suggests that RbpA inhibits promoter escape (Jensen et al., 2019). This discrepancy indicates that RbpA effect on transcription might be promoterspecific. RbpA structure comprises an unstructured N-terminal tail (NTT), a central RbpA core domain (RCD), and a C-terminal region called the σ-interacting domain (SID) ( Figure 1C). RCD and SID are connected by a flexible loop called the basic linker (BL) (Bortoluzzi et al., 2013;Tabib-Salazar et al., 2013;Hubin et al., 2015). RbpA interacts with σ2 via its SID, whereas BL (R79) interacts with promoter DNA upstream of the −10 element (Hubin et al., 2017). RbpA-SID interacts with three σ2 regions: NCR, 1.2, and 2.3 ( Figure 1C). RbpA tethers σ A to core RNAP via the β -Zinc-binding domain (Hubin et al., 2015(Hubin et al., , 2017. Recent cryo-EM structures of Mtb RPo ( Figure 2C) showed that RbpA-NTT threads through the RNA exit channel into the active site cleft and interacts with the 3.2 region of σ A and the DNA template strand at position −5 (Boyaci et al., 2019). However, RbpA-SID is sufficient for partial transcription activation (Hubin et al., 2015). The complex network of interactions between RbpA and key structural modules of RNAP explains why RbpA affects different steps of initiation, from RPo formation to promoter escape. Recent single-molecule Förster resonance energy transfer (smFRET) study showed that Mtb σ B adopts a closed, inactive conformation (∼50 Å distance between σ2 and σ4) even after assembly of the σ B -RNAP holoenzyme . During holoenzyme assembly, RbpA stabilizes (or induces) the open conformation of σ B (∼83 Å distance between σ2 and σ4), required for its tight binding to core MtbRNAP and to promoter DNA (Figure 2A). Thus, RbpA acts as a chaperone to promote holoenzyme formation. This finding suggests that in the absence of RbpA, part of the σ-core RNAP interface cannot be formed, thus explaining the low stability of the σ A and σ B MtbRNAP holoenzymes (Hu et al., 2012(Hu et al., , 2014. On the basis of the high structural similarity between σ A and σ B we propose that the same activation mechanism works also for σ A . This conclusion is supported by the cryo-EM structure of Mycobacterium smegmatisσ A -RNAP holoenzyme lacking electron density for the domain σ4. This indicates that σ fluctuates between different conformational states (Kouba et al., 2019). The smFRET study on σ B -MtbRNAP also explains why RbpA is essential for transcription initiation at the −10/−35 promoters and dispensable at the extended −10 promoters (Hu et al., 2012(Hu et al., , 2014Perumal et al., 2018). Indeed, RPo formation at the −10/−35 promoters requires the distance between domains σ2 and σ4 to match the distance between the −10 and −35 elements. This condition is dispensable for RPo formation at the extended −10 promoter. Therefore, regulation of the σ conformational state by RbpA allows modulating RNAP promoter selectivity .

Crl
Crl is a ∼16-kDa protein from γ-proteobacteria, initially identified in E. coli as an activator of genes implicated in curli fimbriae production (Arnqvist et al., 1992). 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   Based on structure and hydrogen-deuterium exchange mass spectrometry analysis of σ S conformation, Xu et al. (2019) suggested that Crl acts as a chaperone that facilitates the σ S -RNAP holoenzyme assembly mainly by modifying σ2 conformation, but not through its contacts with the β -clamp. In addition, interaction of Crl residue R51 with residues D135 and E137 in the "specificity loop" of region 2.3 of σ ( Figure 1C) stabilizes its optimal conformation for binding to the −10 element ssDNA. This interaction promotes RPo formation. 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).

GcrA
GcrA (173-aa) is a transcription factor from Caulobacter crescentus that is well conserved in α-proteobacteria. GcrA forms a stable complex with σ A -RNAP, recruits RNAP to methylated (m 6 A) promoters, and activates the expression of ∼200 genes that play an important role in cell cycle regulation during swarmer-tostalked cell transition (Holtzendorff et al., 2004;Haakonsen et al., 2015). Analysis of the promoter binding kinetics demonstrated that GcrA increases RNAP affinity for the promoter and the rate of RPc isomerization to RPo (Haakonsen et al., 2015). GcrA is composed of two domains: the N-terminal DNA-binding domain (GcrA-DBD, residues 1-45) that recognizes methylated promoter DNA, and the C-terminal σ-interacting domain (GcrA-SID, residues 108-173) that binds to σ2 (Figure 1C). GcrA-DBD and GcrA-SID are connected by an unstructured linker (residues 46-107). Recent crystal structures of the GcrA-SID-σ A complex and the GcrA-DBD-DNA complex revealed details of its interactions with RNAP and the promoter (Wu et al., 2018). Structural studies on the full length protein and its complex with RNAP are now needed to decipher GcrA mechanism of action.

GrgA
GrgA (ORF CTL0766, 288-aa) is a transcription factor from the human pathogen Chlamydia trachomatis. GrgA activates σ A -(also known as σ 66 ) and σ 28 -dependent transcription by interacting with σ-NCR and binding to DNA in a non-sequencespecific manner (Bao et al., 2012;Desai et al., 2018). The GrgA binding site on σ A was mapped to residues 269-316 (Bao et al., 2012) (Figure 1C). The detailed mechanism of GrgA action and its role in gene regulation remain obscure. GrgA is specific to Chlamydia species, and has not been found in any other organism. Therefore, it might be a good target for developing highly selective anti-chlamydial drugs .

Gp39 of Phage P23-45
Gp39, a ∼16-kDa protein encoded by the Thermus thermophilus phage P23-45, binds to the host σ A -RNAP holoenzyme and inhibits transcription from −10/−35 class promoters. Transcription of the middle and late promoters of P23-45, which belong to the extended −10 class, is less affected (Berdygulova et al., 2011;Tagami et al., 2014). Gp39 blocks transcription initiation probably at the step of RPc formation that depends on the σ4/−35 element contact. Besides its effect on initiation, gp39 also displays anti-termination activity (Berdygulova et al., 2012) suggesting that the σ subunit is not essential for its binding to RNAP. The crystal structure of the σ A -RNAP-gp39 complex (Tagami et al., 2014) reveled that gp39 binds to the RNAP βflap and to the σ4 domain and induces a ∼45 Å displacement of the σ4 relative to its default position in the RNAP holoenzyme (Figures 2B,D). This conformational change in the σ A subunit explains the selectivity of the RNAP-gp39 complex toward the extended −10 promoters.

AsiA of Phage T4
AsiA is 90-aa protein of the E. coli phage T4. AsiA employs a mechanism called σ appropriation to reprogram the host RNAP. AsiA forms a stable complex with σ 70 before holoenzyme assembly (Hinton et al., 1996;Hinton and Vuthoori, 2000) and thus inhibits transcription from the −10/−35 class promoters. Conversely, transcription from the extended −10 promoters is less affected (Severinovaa et al., 1998). At the same time, AsiA acts as a co-activator of the phage activator protein MotA, required for binding to the T4 middle promoter. The σ appropriation complex, which includes σ 70 , RNAP, AsiA and MotA, recognizes the MotA-box that replaces the −35 element at the T4 middle promoters. NMR solution structures of the AsiA-σ4 complex demonstrated that AsiA remodels σ4 making impossible its binding to the −35 element and its interaction with β-flap (Simeonov et al., 2003;Lambert et al., 2004). A recent cryo-EM structure of the σ 70 -RNAP-AsiA-MotA RPo revealed the detailed mechanism of σ appropriation . AsiA binds to and remodels the structure of the σ region 3.2 and σ4, displaces σ4, and takes its place. This allows MotA recruitment and RPo formation. In addition, AsiA interaction with upstream dsDNA stabilizes RPo.

P7 of Phage Xp10
P7 is a small, ∼ 8-kDa, globular protein encoded by the lytic bacteriophage Xp10 that infects the Gram-negative bacterium Xanthomonas oryzae, which causes rice blight. At a later stage of infection, P7 shuts off the host gene transcription in favor of phage gene transcription by the Xp10 singlesubunit RNAP (Nechaev et al., 2002;Liu et al., 2014). P7 forms a stable complex with the host σ 70 -RNAP holoenzyme and inhibits RPo formation at −10/−35 promoters and to a lesser extent, at the extended −10 promoters. Luminescence resonance energy transfer (LRET) measurements demonstrated that in the P7-σ 70 -RNAP complex, the σ 70 subunit adopts a closed or partially closed conformation (Nechaev et al., 2002). The finding that P7 also binds to RNAP harboring the structurally distinct σ 54 (Brown et al., 2016) suggests that the σ subunit is not essential for its interaction with RNAP. Indeed, P7 can also modulate post-initiation steps of transcription, such as pausing and intrinsic termination (Nechaev et al., 2002;Zenkin et al., 2015You et al., 2019. A recently solved cryo-EM structure of P7 in the elongation complex  reveled that P7 binds to the RNA-exit channel at the place of σ4, and thus makes impossible the formation of the "open" σ conformation essential for RPo formation at −10/−35 class promoters. The lack of σ4-RNAP contact should decrease the overall stability of the holoenzyme, thus explaining the dissociation of σ from the P7-RNAP complex observed in biochemical experiments (Liu et al., 2014). It has been proposed that P7 induces the closed conformation of the RNAP clamp, and thus inhibits RPo formation . However, it is unlikely that such mechanism takes place at −10/−35 promoters. Indeed, according to the P7-RNAP complex structure, P7 should inhibit the interaction of σ4 with the −35 element, which is required for initial RNAP binding to the promoter (RPc formation). Clamp closing starts to play a role during RPc isomerization to RPo, the step following recognition of the −35 element. Thus, it is more likely that P7-mediated σ 70 remodeling inhibits the σ4/−35 element interaction and consequently RPc formation, as observed for the σ 54 -RNAP holoenzyme (Brown et al., 2016). However, P7induced clamp closing might play a role when the −35 element recognition is bypassed.

Scc4 (CT663) From Chlamydia trachomatis
Scc4 (ORF CT663) is a ∼15-kDa protein from the human pathogen C. trachomatis. Scc4 forms a heterodimer with Scc1, and both are type III secretion chaperons implicated in the regulation of cell growth and intracellular infection (Hanson et al., 2015). Scc4 was identified in a two-hybrid screen for regulators that interact with C. trachomatis RNAP β-FLAP (Rao et al., 2009). Scc4 binds to RNAP β-FLAP tip helix and also interacts with the σ4 domain of the principal σ A subunit. It can also interact with the σ4 domain of E. coli σ 70 that exhibits 60% amino acid identity with the σ4 of σ A , but does not interact with the σ4 of C. trachomatis σ 28 (Group 3). Scc4 inhibits transcription initiation from −10/−35 class promoters, but not from extended −10 type promoters. Although structural studies are needed to determine the mechanism of inhibition, on the basis of similarities with the mechanism of action of the phage proteins we hypothesize that Scc4 disrupts the σ4/Flap interaction and prevents RPo formation at −10/−35 promoters.

CONCLUSION
We can draw two basic principles of transcription regulation by RPB-TFs: positive regulation through strengthening of σ2/β -clamp/−10 element interactions, and negative regulation through weakening of σ4/β-flap/−35 element interactions. All contacts made by the three σ-activators RbpA, Crl and GcrA overlap and are clustered in four σ regions (σ1.2, σ-NCR, σ2.1 and σ2.3) that are responsible for core RNAP binding and −10 element recognition/melting ( Figure 1C). Consequently, all these activators act through a similar mechanism. They strengthen σ/RNAP interaction and stimulate RPo formation, the rate limiting step in transcription initiation. The only exception is GrgA the binding site of which was mapped entirely to σ NCR and thus may have a different mechanism of action. However, in the absence of a detailed biochemical and structural characterization, it cannot be excluded that GrgA contacts other regions besides σ -NCR. At least for RbpA, the stimulation of the "closed-to-open" transition is part of the σ activation mechanism required for efficient transcription initiation at the −10/−35 class promoters, but not at the extended −10 class promoters . It remains to be explored whether Crl, GcrA and GrgA can affect the relative movement of the σ2 and σ4 domains. Remarkably, all σ-repressors mentioned here act as antagonists to RbpA-type activation by destabilizing the σ4/β-flap interaction, and should favor the "open-to-closed" transition in the σ subunit. Consequently, σ-repressor-modified RNAP cannot initiate transcription at the −10/−35 class promoters, but only at the extended −10 class promoters (Figure 2A,B).
The σ-activators and σ-repressors illustrate how σ conformational dynamics, controlled by contacts with core RNAP, can be used for fine-tuning transcription in a lineagespecific manner. Considering the huge diversity in lifestyles of bacterial species, the number of the currently known σ-regulators of bacterial origin is strikingly low. The reason might be that most of these proteins are of small size and are not easy to detect. Yet, their discovery in pathogenic bacteria may offer new targets for developing pathogen-specific drugs. We expect that the number of the described σ-regulators and the diversity of regulatory mechanisms will continue to grow.

AUTHOR CONTRIBUTIONS
KB and RV wrote the manuscript. Both authors contributed to the article and approved the submitted version.