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. Author manuscript; available in PMC: 2009 Apr 1.
Published in final edited form as: Curr Opin Microbiol. 2008 Mar 28;11(2):121–127. doi: 10.1016/j.mib.2008.02.016

Regulation of bacterial RNA polymerase σ factor activity: a structural perspective

Elizabeth A Campbell 1,*, Lars F Westblade 1, Seth A Darst 1
PMCID: PMC2386898  NIHMSID: NIHMS49043  PMID: 18375176

Summary

In bacteria, σ factors are essential for the promoter DNA-binding specificity of RNA polymerase. The σ factors themselves are regulated by anti-σ factors that bind and inhibit their cognate σ factor, and ‘appropriators’ that deploy a particular σ-associated RNA polymerase to a specific promoter class. Adding to the complexity is the regulation of anti-σ factors by both anti-anti-σ factors, which turn on σ factor activity, and co-anti-σ factors that act in concert with their partner anti-σ factor to inhibit or redirect σ activity. While σ factor structure and function is highly conserved, recent results highlight the diversity of structures and mechanisms that bacteria use to regulate σ factor activity, reflecting the diversity of environmental cues that the bacterial transcription system has evolved to respond to.

Introduction

In bacteria, gene expression is regulated primarily at the level of transcription initiation. All bacteria contain a single ∼400 kDa multi-subunit core RNA polymerase (RNAP) enzyme that is catalytically competent and can recognize DNA non-specifically, but requires an additional factor, σ, for promoter recognition and initiation [reviewed in [1-3]]. Most σ factors belong to the σ70-family [4], whose members contain at least two domains connected by flexible linkers: σ2, which binds the RNAP β′ subunit coiled-coil and the promoter −10 element, and σ4, which binds the RNAP β subunit flap and promoter −35 elements [5-8]. The σ70-family members share a high degree of sequence and structural conservation within domains σ2, and σ4 [4]. An additional σ family, σ54, does not share significant sequence similarity to the σ70 family and is functionally distinct.

All bacteria have at least one essential σ factor that serves to transcribe the genes required for cell viability, and most bacteria harbor alternative σ factors that transcribe operons in response to specific stimuli. The availability and activity of σ factors are controlled, in part, by at least two types of regulatory factors: 1) anti-σ factors that bind and inhibit their cognate σ factor, and 2) appropriators that alter the activity of a specific RNAP holoenzyme. Adding to the complexity is the regulation of the anti-σ factors themselves; either antagonistically by anti-anti-σ's, cooperatively by co-anti-σ's, or post-translationally by proteolysis. This review will focus on the structural details of the regulation of σ70-family members by anti-σ, anti-anti-σ, and co-anti-σ factors.

Regulation of Escherichia coli σ70 by σ70-binding proteins

The coordinated transcription of the bacteriophage T4 genome is dependent upon T4-encoded regulators that bind to and alter the specificity of the host Escherichia coli (Ec) RNAP [9]. One regulator expressed early during T4 infection, AsiA, binds specifically and tightly to σ704 [10-12], and both inhibits and co-activates transcription. Early during T4 infection, AsiA inhibits the activity of σ70-associated RNAP at host promoters dependent on the -35 element by binding the σ704 Helix-Turn-Helix motif (HTH) responsible for -35 element recognition [10,11,13]. Along with reducing transcription from host -35-dependent promoters, AsiA functions as an appropriator by deploying Ec RNAP holoenzyme to T4 middle promoters, where it acts in concert with the T4-encoded DNA-binding protein MotA to stimulate middle gene transcription [14,15].

To gain insight into the mechanism of AsiA, the solution structure of the σ704/AsiA complex was determined [16]. Structures of σ4 have been determined in many different contexts: alone [5], in complex with −35 element DNA [5], in RNAP holoenzyme [6-8], and with anti-σ factors [17,18]. The σ4 normally forms a structural core of three α-helices (residues 551-599 of Ec σ704; Figure 1a). Remarkably, the binding of AsiA rearranges σ704 so that the HTH motif is restructured into one continuous helix (Figure 1b). Not surprisingly, the interaction of AsiA with σ4 prevents the usual interaction of σ4 with the β-flap in the RNAP holoenzyme [19,20]. The displacement and restructuring of σ4 by AsiA are thought to reposition and display elements of σ4 for interaction with MotA [14,21].

Figure 1. The conformation of σ4 when bound to bacteriophage T4 AsiA or Rsd.

Figure 1

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The σ4's are shown as ribbon diagrams.

(A) On the left is Thermus aquaticus (Taq) σA4 alone [5], showing the native conformation of σ4.

(B) Ec σ704 from the σ704/AsiA complex [16], with AsiA shown as a transparent blue worm. The fold of σ4 is altered (compare with A).

(C) Ec σ704 from the σ704/Rsd complex [30], with Rsd shown as a transparent green worm. Each view is oriented so that the recognition helices are aligned. The fold of σ4 is identical to σ4 alone (part A).

Compared to the alternative σ factors, σ70 has the highest affinity for RNAP [22] and is the most abundant σ factor throughout the Ec growth cycle [23]. A search for factors that enable alternative σ's to compete for binding to RNAP yielded a single polypeptide, Rsd, that specifically associates with σ70 [24]. The results of subsequent biochemical and genetic experiments suggest that both Rsd and its Pseudomonas aeruginosa homolog, AlgQ, a positive regulator of virulence [25,26], sequester σ70 in an inactive complex, thus allowing alternative σ's to exchange with RNAP [26-29]. Structural studies of the σ704/Rsd complex reveal that Rsd binds to residues of σ704 that are important for both RNAP and -35 element recognition. Unlike AsiA, however, this binding does not alter the structural core of σ704 (Figure 1c; [30]).

Regulation of σF activity during sporulation

In Bacillus species, σF initiates transcription of a cascade of forespore-specific σ factors responsible for the transcription of genes necessary for the morphological development of the spore [31]. The activity of σF is regulated by the anti-σ and serine kinase SpoIIAB (AB), and the anti-anti-σ SpoIIAA (AA) [32,33]. Key to the regulation of sporulation is the partner switching of AB from σF to AA. The structure of σF in complex with AB revealed that AB monomers form a symmetric homodimer that is in complex with a single σF molecule, giving rise to an asymmetric complex whereby the RNAP-binding determinants of σF are occluded [34]. Several structures of AB in complex with AA have now been solved [35]. These data provided structural evidence for the previously proposed docking model [34,36] that describes how AA induces σF release from the σF/AB complex (Figure 2a). In this model, one AB protomer of the asymmetric σF/AB2 complex (AB1 of Figure 2a; for more details see Figures 7 and 9 of ref. 32) is more accessible to AA. Upon docking to AB1, the AA molecule displaces σF due to electrostatic and steric interactions. The AA molecule can then be phosphorylated by the AB kinase activity and dissociates. This allows for another unphosphorylated AA (if available) to bind to the resulting AB(ADP), forming a stable complex that inhibits the AB kinase and anti-σ activities [37,38]. The model highlights the importance of the asymmetric binding of σF to the AB dimer.

Figure 2. Versatility of classical σ/anti-σ structures and mechanisms.

Figure 2

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(A) Superimposition of AB2 in complex with σF with AB2 in complex with AA [34,35]. This figure illustrates that the asymmetric binding of σF3 (blue surface representation, the other σ domains were present but disordered in the σF/AB2 crystal structure) to the AB2 results in one AB protomer (AB1, on the right, colored magenta) being more accessible for binding to AA (colored green). The nucleotides bound to AB are drawn in stick and the ATPase active site Mg2+ ions are drawn as green spheres.

(B) Structure of the Aae σ28(FliA)/FlgM complex [18]. FlgM (colored red) binds to and occludes the σ28 RNAP-binding domains (shaded gray) on σ282 (colored green) and σ284 (coloured yellow). σ283 is colored blue.

(C) Structure of the σE/RseA complex [17]. σE (surface representation) regions are colored σE2, green; σElinker, orange, σE4 yellow. Surfaces that bind RNAP are shaded gray. RseA, colored red, occludes both major RNAP binding surfaces and is sandwiched between the σE2 and σE4. RseB binds to RseA in the periplasm, forming a heterotetramer of RseA2:RseB2.

(D) The anti-σ ChrR is composed of two functionally and structurally modular domains [55*]. The structure illustrates that σE (colored similarly as σE (B)) interacts exclusively with the ChrR-ASD (colored bright pink). The second domain, ChrR-CLD (colored light pink), which is required to respond to 1O2, is connected to the ASD via a partially disordered linker (drawn as magenta dots). Both domains contain Zn2+ (shown as grey spheres): The ASD Zn2+ serves an essential structural function, although other functions have not been ruled out. The roles of both the ASD-Zn2+ and CLD-Zn2+ are currently under investigation (Tim Donohue, personal communication).

Flagellar biosynthesis and the σ28/FlgM complex

In enteric bacteria, the flagellar regulon is divided into a three-tiered transcriptional hierarchy composed of early, middle and late genes. The coordinated assembly of the flagellum is tightly regulated [39], and central to this regulation is the σ/anti-σ pair σ28/FlgM. In response to a plethora of stimuli, the flagellar early genes activate transcription of middle genes. The middle genes encode the components of the hook-basal body (HBB) flagellar substructure, as well as two transcriptional regulators: σ28 (encoded by the fliA gene), which directs transcription of the extracellular late gene products, and FlgM, a negative regulator of σ28. FlgM binds to and inhibits σ28 until completion of the HBB; thereby preventing the expression of the extracellular late gene products prior to the completion of the HBB export apparatus. Once the HBB is completed, FlgM is secreted out of the cell by the HBB, allowing σ28 to transcribe the late extracellular gene products.

Biochemical and genetic analyses of the σ28/FlgM complex have now been complemented with the crystal structure of the σ28/FlgM complex from Aquifex aeolicus [18]. In this structure, a single FlgM molecule, composed of three α-helices, wraps around a compact σ28 molecule (Figure 2b). As a consequence, FlgM sterically occludes the RNAP-binding determinants of σ28 and stabilizes extensive interdomain contacts within σ28 which mask the σ28 promoter-binding determinants.

The extensive interdomain contacts observed within σ28 led to the proposal that the conformation of σ28 observed in complex with FlgM is stable in solution even in the absence of FlgM, and this proposal was confirmed experimentally using a disulfide crosslinking approach [40]. These findings suggest that the DNA-binding determinants for all σ70-family members may be masked by interdomain contacts in free σ. Core RNAP binding induces large rearrangements of the σ domains, resulting in the exposure of the DNA-binding determinants [18,41].

Response to misfolded proteins in the periplasm: σE, RseA and RseB

In Ec, response to cell envelope damage is mediated by a Group IV σ factor, σE, which is held in an inactive state by interactions with the cytoplasmic domain of the inner membrane-anchored anti-σE factor, RseA. Group IV σ factors are alternative σ's that direct transcription of regulons in response to environmental conditions [42]. The σE/RseA interaction is also stabilized by the perisplasmic co-anti-σ RseB [43,44]. Release of σE from RseA is induced by the presence of misfolded proteins in the periplasm, and is governed by a complex signal transduction pathway involving the regulated proteolysis of RseA [45]. The crystal structure of the cytoplasmic domain of RseA in complex with σE, combined with biochemical studies and molecular modeling, revealed that RseA functions as an anti-σ factor by sterically occluding the two major σE RNAP-binding determinants (Figure 2c; [17]).

Recently, structural and biochemical analyses of the co-anti-σE RseB, a homodimer, show that RseB is composed of two domains; a larger N-terminal domain (D1) linked to a smaller C-terminal domain (D2) [46,47]. Interestingly, the fold of D1 is similar to a class of lipoprotein transporters that have a deep, open hydrophobic β-barrel purported to bind lipids, suggesting that binding of lipids may play a role in the signal transduction cascade activating σE. This adds to the complexity of the signaling pathway controlling σE activity [45] in ways that have yet to be worked out.

Zinc anti-σ factors

Zinc anti-σ (ZAS) factors bind both Zn2+ and their cognate σ factor, and appear to be responsible for regulating the activity of a subset of Group IV σ factors. In Streptomyces coelicolor, the regulatory system that allows the detection and response to thiol-oxidative stress is orchestrated by the σR/RsrA complex [48]. Under reducing conditions, σR is held in an inactive state by RsrA, a member of the ZAS family. In response to thiol-oxidative stress, RsrA releases the Zn2+ ion, which promotes disulphide bond formation between two cysteine residues, Cys11 and Cys44 (formerly coordinated with the Zn2+), leading to a conformational change of RsrA that induces the release of σR [49*-51], enabling σR to direct the transcription of genes that help mitigate the effects of thiol-oxidative stress [52].

The reactive oxygen species singlet oxygen (1O2) is a toxic byproduct of photosynthesis. In the photosynthetic bacterium Rhodobacter sphaeroides (Rsp), a Group IV σ factor, σE, is responsible for the transcription of genes in response to 1O2 [53]. During the absence of 1O2, σE is held in an inactive 1:1 complex with the ZAS-family member ChrR [54], but upon sensing 1O2, ChrR releases σE. Structural studies of the σE/ChrR complex reveal a number of features pertinent to the regulation of the σE/ChrR complex [55*]; Figure 2d). First, ChrR comprises two structural domains: an N-terminal domain that interacts with σE (termed ASD, for Anti-σ Domain), and a C-terminal β-barrel domain with a Cupin fold (termed CLD, for Cupin-Like Domain; [56]. Second, the ChrR-ASD inhibits σE activity by occluding the RNAP binding sites in σE2 and σE4. Third, like AsiA, ChrR rearranges the structure of σE4, but the details of the rearrangement are different. Finally, the ASD coordinates a structural Zn2+, which is essential for maintaining the overall fold of the ASD by mediating interactions between the N-terminal loop and the first three α-helices. The ChrR-ASD is sufficient to bind and inhibit σE, but will not release σE in response to 1O2. Subsequent experiments revealed that ChrR 1O2 sensitivity is dependent on the CLD. A second Zn2+ ion coordinated by the CLD may play a role in the 1O2 response [55*].

Surprisingly, the fold of the ChrR-ASD is similar to the portion of RseA that interacts with Ec σE [17]. Spurred by these findings, the authors used an exhaustive bioinformatic analysis to define the conservation of the ASD. A total of 1,264 open reading frames adjacent to Group IV σ factors were found to contain an ASD, implying that this structural motif has been widely conserved in bacteria as a σ binding domain (Figure 3; [55*]).

Figure 3. Structural conservation of the N-terminal domains of ECF anti-σ's.

Figure 3

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(top) Structural alignments of the N-terminal domains of RseA and ChrR reveal a similar fold for the first three helices (termed the ASD). Both molecules are rendered as α-carbon backbone worms, with RseA-ASD colored green and ChrR-ASD colored pink. The ChrR-Zn2+ is rendered as a grey sphere. (bottom) Sequence alignments were generated from the structural alignment and optimized using known Zn2+ ligands and secondary structure predictions [55*]. The α-helices from the anti-σ crystal structures are shown schematically above (ChrR, pink cylinders [55*]) and below (RseA, green cylinders [17]) the sequences.

Conclusions

The regulation of many key biological processes in bacteria is effected by the interplay between σ factors and their cognate anti-σ factors. The structure/function studies highlighted in this review indicate that anti-σ's function by directly occluding important core RNAP binding determinants of their cognate σ, as well as by holding the flexibly-linked σ domains in relative orientations that are not compatible with holoenzyme formation and/or promoter binding. This is achieved by multipartite interactions, where the anti-σ interacts with two or more of the σ structural domains simultaneously. Thus, ChrR, FlgM, and RseA not only obscure the core binding determinants of their cognate σ factors, they also tie up the σ's in a conformation that could not form holoenzyme. Although not visualized in the structure, genetic studies indicate that SpoIIAB simultaneously binds three structural domains of σF [57], suggesting SpoIIAB operates though this mechanism as well.

The multipartite interactions between σ's and anti-σ's may appear redundant. However, because all the domains of σ interact with core RNAP [2,6,8], occlusion of only one domain by an anti-σ could allow the other domains of σ to bind to RNAP. Although unlikely to allow holoenzyme function, such complexes would be undesirable since they would titrate functional core RNAP away from the transcription cycle. On the other hand, appropriators like T4-AsiA might be expected to interact with only a single domain of σ (such as σ4) so that they can function as a component of the RNAP holoenzyme.

All σ70 family members contain at least two main domains (σ2 and σ4) that are highly conserved structurally despite significant sequence variability [17]. The σ2 domain forms the primary binding interface with core RNAP, recognizes the −10 promoter element, and plays a key role in melting the promoter DNA to form the open complex, while σ4 recognizes the −35 element and serves as a target for transcription activators [3]. Thus, these two domains perform all of the most critical σ factor functions and represent the elements for basic function common to all σ factors. The structural conservation of these domains between all members of the σ70 family indicates that functional constraints allow little room for structural evolution of σ factors.

Unlike the σ factors, the anti-σ's are highly diverse in sequence and structure (Figure 2). Alternative σ's must modulate the activity of a common core transcriptional apparatus in response to essentially all possible environmental conditions that a bacterium may encounter. The diversity of anti-σ structure and regulation reflects the wide variety of stimuli that anti-σ's respond to, and the wide variety of regulatory mechanisms that control their function. The evolution of structurally and functionally diverse anti-σ's provides much more flexibility for regulation of transcription initiation. Thus, the functional and structural diversity of anti-σ's reflect the need of bacteria to relay a wide variety of environmental cues to the core transcriptional apparatus via regulation of the structurally conserved σ factors.

Acknowledgments

We owe special thanks to William Lane for assistance in making Figure 1. We thank past and present members of the Darst Laboratory, as well as our many collaborators. We apologise to all our colleagues whose work we could not discuss due to space limitations. Work in the Darst Laboratory was funded by the National Institutes of Health. Figures were generated using Pymol (Delano WL: The Pymol Molecular Graphics System. Url: http://www.pymol.org.

Footnotes

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