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Regulation of Alternative Sigma Factor Use

Soa Osterberg, Teresa del Peso-Santos, and Victoria Shingler
Department of Molecular Biology, Umea University, 901 87 Umea, Sweden; email: victoria.shingler@molbiol.umu.se

Annu. Rev. Microbiol. 2011.65:37-55. Downloaded from www.annualreviews.org by Universidade Federal do Rio Grande do Sul on 03/27/12. For personal use only.

Annu. Rev. Microbiol. 2011. 65:3755 First published online as a Review in Advance on May 31, 2011 The Annual Review of Microbiology is online at micro.annualreviews.org This articles doi: 10.1146/annurev.micro.112408.134219 Copyright c 2011 by Annual Reviews. All rights reserved 0066-4227/11/1013-0037$20.00

Keywords
transcription, antisigma factors, ppGpp, DksA, Crl

Abstract
Alternative bacterial sigma factors bind the catalytic core RNA polymerase to confer promoter selectivity on the holoenzyme. The different holoenzymes are thus programmed to recognize the distinct promoter classes in the genome to allow coordinated activation of discrete sets of genes needed for adaptive responses. To form the holoenzymes, the different sigma factors must be available to compete for their common substrate (core RNA polymerase). This review highlights (a) the roles of antisigma factors in controlling the availability of alternative sigma factors and (b) the involvement of diverse regulatory molecules that promote the use of alternative sigma factors through subversion of the domineering housekeeping 70 . The latter include the nucleotide alarmone ppGpp and small proteins (DksA, Rsd, and Crl), which directly target the transcriptional machinery to mediate their effects.

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Contents
INTRODUCTION . . . . . . . . . . . . . . . . . . THE TRANSCRIPTION CYCLE AND DEPLOYMENT OF SIGMA FACTOR DOMAINS . . . . . . . . . . . . . The Sigma Cycle . . . . . . . . . . . . . . . . . . The 70 Family . . . . . . . . . . . . . . . . . . . . The 54 Family . . . . . . . . . . . . . . . . . . . . ANTISIGMA FACTORS AND THEIR ANTAGONISTS . . . . . . . . . Counteracting Physicochemical Assaults . . . . . . . . . . . . . . . . . . . . . . . . Responses to Iron Limitation . . . . . . . Partner Switching and Sigma Factor Mimicry Mechanisms . . . . . . . . . . . Checkpoint Coupling to Organelle Biogenesis . . . . . . . . . . . . . . . . . . . . . . PROMOTION OF ALTERNATIVE SIGMA FACTOR ACTIVITY THROUGH SUBVERSION OF 70 . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alternative Sigma Factors Are Not Created Equal . . . . . . . . . . . . . . . . . . The Bacterial Alarmone ppGpp and Its Cohort DksA . . . . . . . . . . . . . . . . ppGpp and Sigma Factor Competition . . . . . . . . . . . . . . . . . . . . Modulation of Sigma Factor Usage Through Diversion of 70 . . . . . . . CONCLUSIONS . . . . . . . . . . . . . . . . . . . .
Core RNA polymerase (core-RNAP): the multisubunit catalytic machinery of bacterial RNA synthesis Sigma factor (): a dissociable subunit of bacterial RNAP that binds with 1:1 stoichiometry to coreRNAPs and is essential for initiation of transcription from promoters by the resulting RNAP holoenzyme

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39 39 39 41 41 42 42 44 45

46 46 46 47 48 49

INTRODUCTION
The temporal and conditional control of transcription initiation is a primary access point for regulating gene expression in all domains of life. In eubacteria, the evolutionarily conserved 380-kDa catalytic core RNA polymerase (core-RNAP; subunit composition consists of 2 ) can accurately synthesize RNA and terminate transcription at appropriate sites. However, promoter DNA recognition and initiation of transcription is dependent on a dissociable sixth subunit, namely a sigma factor (). Association of a given sigma factor with
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core-RNAP dictates the DNA-binding specicity of the resulting holoenzyme (-RNAP) by providing the majority of determinants for recognition of promoter DNA motifs. All bacterial species have a housekeeping sigma factor responsible for transcription from the majority of promoters, and most encode additional alternative sigmas used to redirect RNAP to sets of genes required for adaptive responses (30, 37). Thus, the trademark ability of most bacteria to adapt to changing ecological conditions is underpinned by highly regulated dynamic changes in the functional pools of the different -RNAPs that dictate when, and to what extent, the different promoter classes in the genome can be occupied. Because promoter-binding by a given RNAP holoenzyme is a prerequisite for correct transcriptional initiation, the composition of the holoenzyme pool provides the background against which other promoter-output modulating factors must act. These include classical DNA-binding transcriptional regulators (repressors and activators) and regulatory molecules such as the nucleotide guanosine tetraphosphate (ppGpp) and proteins such as DksA that directly target the active site of RNAP to modulate its performance at promoters (reviewed in Reference 33). The repertoire of alternative sigma factors used to globally alter and coordinate transcriptional responses to changing cellular demands varies widely between different species and generally reects the lifestyle of the bacterium. For example, dedicated intracellular pathogens that thrive in a relatively constant environment frequently possess only a single sigma factor (e.g., Mycoplasma genitalium). The gut commensal Escherichia coli has 7 sigmas, whereas soil and water bacteria, which are exposed to a plethora of uctuating physicochemical and nutritional stresses in their natural environments, possess many morereaching an excess of a remarkable 60 alternative sigma factors in Streptomyces coelicolor (30). The roles of alternative sigma factors in counteracting stress, during biogenesis of extracellular appendages, and in developmental programs such as spore formation are

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most familiar from studies of the model organisms E. coli and Bacillus subtilis. However, these represent just a limited subset of the myriad physiological processes controlled by alternative sigma factors, which extend to pivotal roles in other development programs, e.g., production of aerial hyphae by S. coelicolor, regulation of photosynthesis and circadian rhythms in cyanobacteria, and control of transcription by bacteria-like RNAP in plant plastids (30, 37). The ability of sigma factors to capture coreRNAP to form a holoenzyme is determined by their free concentrations and afnity for coreRNAP. To accommodate the intermittent and environment-specic requirement for alternative sigma factors, bacteria have evolved sophisticated regulatory systems to control their production, activity, and availability. In this review we rst provide a brief overview of the interactions of sigma factors with core-RNAP and promoters as a preface to highlighting how these critical interfaces are exploited by antisigma factors. Second, we focus on mechanisms by which targeting of the housekeeping sigma and/or its holoenzyme by regulatory molecules can provide a more general strategy to promote the use of many alternative sigma factors.

a
Initiation Elongation

Stochastic release Promoter engagement

Termination

RNA

Competition

Sigma binding
Core-RNAP

b
R+P RPc RPi

NTPs RPo

NTPs RPinit RPE

Figure 1 The sigma cycle allows reprogramming of core RNA polymerase (core-RNAP). (a) Schematic illustration of the transcription cycle in which sigma factors compete for association with core-RNAP to direct the holoenzyme to engage promoters. (b) Simplied schematic of the multiple reversible steps of transcriptional initiation: -RNAP (R) binds promoter DNA (P) to form the initial closed complex (RPc ), which, through sigma-assisted formation of a number of unstable intermediate complexes (RPi ), eventually leads to the open complex (RPo ), which is competent to initiate transcription. Note that reversible steps of initiation end as the elongation complex (RPE ) escapes the promoter.

THE TRANSCRIPTION CYCLE AND DEPLOYMENT OF SIGMA FACTOR DOMAINS The Sigma Cycle
Within the holoenzyme, the sigma factor makes multiple and extensive contacts with coreRNAP and plays an active role in initial promoter engagement to form a closed promoter complex and in subsequent steps of DNA melting to form the open promoter complex required for transcriptional initiation (Figure 1). Tight -core-RNAP association is sequentially broken prior to promoter escape of RNAP into the elongation mode (66). However, complete detachment of the sigma is not a prerequisite for transcriptional elongation per se, and a partially attached sigma can cause elongation

stalling by binding promoter-element mimics within DNA. Nevertheless, the majority of sigma factors are rapidly, albeit stochastically, released during elongation (65, 74) to join the pool of free sigma for competitive association with core-RNAP. The release of sigma during each round of transcription provides the central mechanism for reprogramming the levels of the alternative -RNAP holoenzyme pools and thus cognate promoter occupancy.

-RNAP: holoenzyme RNA polymerase RNAP: RNA polymerase Guanosine tetraphosphate (ppGpp): the effector molecule of the stringent response; used here to also encompass pppGpp

The 70 Family
With the exception of homologs of E. coli 54 (see below), all alternative sigmas belong to the extensive 70 family, named after the
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1.1

3.2 loop DNA Core melting binding 70

4.2 4.1 3.1 3.0 2.4 2.3 2.2 2.1

DNA-binding inhibition
1.2

3.2

1.1 N

NCR
TTGACA TGn TATAAT GGGnnn

UP element

35 RpoN box

Ext. 10

10

Discriminator

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b
54

Region III

II

DNA interactions
TTGGCACG TTGC

Core binding

bEBP interactions

24
Figure 2

12

Sigma factor domains and their functions. (a) For 70 proteins, roles of the conserved subregions within the 2 , 3 , and 4 globular domains as described in the text are highlighted. NCR indicates the location of a nonconserved region. Consensus for the 35 hexamer (35 to 30), the extended 10 element (Ext.; 15 to 13), the 10 hexamer (12 to 7), and discriminator DNA (6 to 1, with an optimal GGG 6 to 4), relative to the transcriptional +1 start, are taken from References 33 and 56. (b) For 54 proteins, consensus for the 24 (27 to 20) and 12 (15 to 12) elements, which encompass almost invariant GG and GC recognition motifs (underlined), are taken from Reference 78. Abbreviation: bEBP, bacterial enhancer-binding protein.

Antisigma factor: any agonist that through binding to a sigma factor inhibits its ability to associate with core-RNAP ECF: extracytoplasmic function

housekeeping 70 of E. coli (also known as D ; A in B. subtilis and many other species). A dual (or sometimes triple) naming system for E. coli sigmas is prevalent in the current literature; therefore, after their rst introduction we use only numerical or gene name superscripting for E. coli sigmas. Extensive biochemical, genetic, and structural analysis has underscored the roles of different domains of housekeeping sigmas in providing four of the ve known interactions that occur with the promoter DNA (Figure 2a). At some promoters, a fth interaction is provided by the -subunits of core-RNAP at an AT-rich UP-element DNA (reviewed in Reference 33). The most
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conspicuous 70 -promoter recognition elements are the 35 and 10 hexamers that are contacted by the 4 and 2 domains of 70 , respectively (17, 66). Subregion 1.2 within the 2 domain can also provide promoter contacts through the discriminator DNA downstream of the 10 element (34). At extended 10 promoters, which frequently lack a discernable 35 element, additional promoter contacts are provided by interaction between the 3 domain and DNA just upstream of the 10 element. A nonconserved region of highly variable length intersperses the 2 domain of some housekeeping sigmas. For E. coli 70 , this region has been implicated in assisting dissociation of the sigma factor to alleviate pausing during the early stages of elongation (54). The 70 family of proteins is divided into subgroups based on phylogenic relations and differential possession of the four conserved domains (2 , 3 , 4 , and region 1.1) (Figure 2a) (reviewed in References 30 and 68). Group 1 comprises housekeeping sigmas that possess all four domains, including the group-specic region 1.1, which is involved in autoinhibition of DNA binding by free sigmas. Group 2 sigmas, represented by the E. coli stationary/stress factor 38/S , are related most closely to Group 1 but are dispensable for growth. Group 3 sigmas, which are more distantly related to Group 1 (e.g., E. coli 28/F/FliA and 32/H ; B. subtilis F ), also possess all three globular domains and usually control regulons in response to developmental checkpoints or heat shock. The 3 domains of E. coli 28 and 32 interact with composite extended 10/10 elements that account for the unusually long consensus recognition elements of their cognate promoters (50, 51). The nal and most divergent group of sigmas is Group 4the extracytoplasmic function (ECF) subfamily, so named because most respond to signals arising from the extracytoplasmic environment (e.g., E. coli 24/E and FecI ). Group 4 represents the most stripped-down version of sigmas, possessing the only two key sigma domains (2 and 4 ) that are structurally conserved even among the most divergent family members.

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The 54 Family
This second class of sigma factors is uniquely represented by orthologs of E. coli 54/N , which directs recognition of distinct promoter motifs located at positions 24 and 12 relative to the transcriptional start (Figure 2b). Although initially identied for their role in nitrogen assimilation, 54 proteins are widely distributed in bacteria and are utilized in coordinating many different physiological processes ranging from utilization of alternative carbon sources, through assembly of motility organs, to production of extracellular alginate. Although 54 proteins need to perform many of the same functions as other sigmas, they bear no primary sequence similarity to 70 proteins and regulate transcription by a different mechanism. A key feature of 54 -RNAP that contrasts other holoenzymes is its complete inability to spontaneously isomerize (melt) DNA to form open promoter complexes. This step strictly requires assistance from mechanotranscriptional activators (also known as bacterial enhancer binding proteins, or bEBPs) that utilize ATP hydrolysis to drive conformational changes for this transition (reviewed in Reference 78). Genetic and biochemical data on the roles of the three main subregions of 54 (regions I to III, Figure 2b) have recently been augmented by structural analysis and cryoelectron microscopy reconstructions (12, 42). Region I mediates weak contact with the 12 promoter element and with core-RNAP such that it occludes loading of promoter DNA into the active site. The varying region II links regions I and III, which makes the main contacts with the 24 and 12 promoter elements and with coreRNAP (Figure 2b). Activation by an obligatory activator serves two functions. First, relocating region I from an inhibitory conformation allows entrance of the DNA. Second, facilitating correct promoter DNA-54 alignment allows for open complex formation (12). Because of the unique properties imparted by 54 (e.g., unusual promoter recognition, the ability to bind DNA in the absence of core-RNAP, and the necessity for ATP-utilizing mechanoactivators),

54 -dependent transcription is considered a second paradigm of bacterial transcription. Although the 54 and the 70 family members lack sequence identity, they do bind overlapping surfaces of core-RNAP, and performance of 54 -RNAP is affected by the same mobile modules of the core-RNAP - and -subunits that inuence 70 -dependent transcription, albeit with different regulatory outcomes (21, 88). Hence, 54 is not exempt from fundamental regulatory mechanisms that involve competitive association with core-RNAP.

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ANTISIGMA FACTORS AND THEIR ANTAGONISTS

Modulating the levels and/or activities of different sigma factors, and consequently the levels of cognate RNAP holoenzymes, provides a simple yet versatile means to control the basal-line occupancy of distinct promoter classes. Mechanisms known to modulate the activities of sigmas are diverse and include phosphorylationactivated binding to a partner protein that tags the sigma for destruction (e.g., interaction between the E. coli response regulator RssB and 38 ) (89), proteolytic cleavage of inactive prosigmas to remove inhibitory Nterminal extensions (e.g., B. subtilis proE and proK ) (41), and signal-cued use of alternative start codons to generate high-molecularweight variants that are vulnerable to rapid proteolytic turnover (e.g., S. coelicolor R and its Mycobacterium ortholog) (49). For some sigmas, protein levels are controlled at all steps of gene expressionfrom transcription initiation, through mRNA stability and control of translational efciency by small noncoding RNAs, to signal-responsive proteolytic degradation (e.g., E. coli 38 and 32 ) (31, 38). In many other cases, however, the primary level of control involves sequestering by antisigma factors to prevent their association with core-RNAP. The following sections provide an overview of selected sigmas that illustrate different systems that control the release of sigma factor activities when they are needed.
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Counteracting Physicochemical Assaults

Coantisigma factor: a factor that acts in concert with a partner to bind and sequester a sigma factor and thereby prevent association with core-RNAP

The Group 4 ECF sigmas encompass 60% of all sigma factors in bacteria (15) and are chiey associated with counteracting physical or chemical stresses or communicating the availability of iron. A common feature of most ECFs is that their activity is regulated by stoichiometric association with an antisigma factor, which is usually coexpressed through transcriptional coupling of the genes within an operon. This is the case for E. coli ECF 24 , which controls responses to membrane stress and represents one of the rare exceptions to the dispensable nature of alternative sigma factors (23). The 24 gene (rpoE) is cotranscribed with those of its antisigma factor RseA and its coantisigma factor RseB, which tightly tether 24 to the membrane in an inactive state (Figure 3a). The molecular details of the threecompartment proteolytic cascade that governs 24 availability have previously been extensively reviewed (4, 14, 36), so only an overview is given here. The DegS serine protease is both the molecular sensor of stress-induced misfolded proteins within the periplasm and the initiator of the RIP (regulated intramembrane proteolysis) cascade that releases 24 . Activation of the proteolytic activity of DegS results in cleavage of the antisigma RseA within its periplasmic region (site 1 cleavage), rendering it as a substrate for the metalloprotease RseP, which in turn processes RseA within its inner membrane spanning region (site 2 cleavage). The released cytoplamsic RseA/24 subcomplex still sequesters 24 but has an exposed tag for recognition by the adaptor protein SspB that directs the complex to ClpXP for nal processing to free 24 for association with core-RNAP. Similar RIP cascades likely control the availability of gram-negative 24 orthologs and other ECFs such as AlgU , which is involved in the production of alginate by Pseudomonas aeruginosa with devastating consequences for cystic brosis patients (reviewed in Reference 36). Likewise, although gram-positive bacteria lack a periplasm and the mechanistic details
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differ, a conceptually similar RIP cascade controls the stress responses mediated by B. subtilis W that is cotranscribed with its antisigma RsiW (36). Sequestering of a sigma to the membrane is an efcient means to couple availability to extracellular signals or those that result in alterations within the periplasmic compartment. A few ECFs, however, govern responses to intracellular stress and are consequently controlled by cytoplasmic antisigma factors. This is the case for the S. coelicolor R /RsrA (48) and the Rhodobacter sphaeroides E /ChrR (6) systems that control responses to damaging oxygen species. Irrespective of the cellular location, efcient sequestering requires the sigma/antisigma interactions to be tight and mask portions of key interfaces usually involved in interacting with core-RNAP (19). The cytoplasmic portion of E. coli RseA has been estimated to bind E with 300-fold-higher afnity than coreRNAP. Structural analysis has shown that sequestering involves the N-terminal domain of RseA, which sterically occludes the critical 2 and 4 domains of this ECF (18). Despite little primary sequence homology, an analogous domain with a common fold within ChrR likewise exploits the same interfaces in its interactions with E of R. sphaeroides (15). Bioinformatic searches indicate that the common domain of RseA and ChrR (ASD, antisigma domain) is fused to diverse signaling domains in >30% of all ECFs (i.e., 20% of all annotated sigma factor genes). Thus, manipulation of the geometry of the critical 2 and 4 domains likely underlies sequestering and release by cognate antisigma factors in many systems.

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Responses to Iron Limitation

In addition to stress responses, ECFs are also frequently involved in processes that ensure a sufciency of iron, which is often in limited environmental supply. For example, in Pseudomonas putida, 13 of its 19 ECFs appear to be dedicated to this essential element (61). This subgroup of ECFs is usually cotranscribed with a cognate antisigma factor to

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a
OmpC RseB

Stress Outer membrane OmpC* Periplasm 1 RseA DegS Sequestered ClpXP 3 RseP 2 SspB Inner membrane Cytoplasm Available

24/E

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b
Stress

c
Basal body

Hook

PhyR

domain

mimic

P P

FlgM

NepR Sequestered
Figure 3 Control of sigma factor availability by antisigma factors. (a) Schematic illustration of DegS/RseP RIP (regulated intramembrane proteolysis) protease cascade (all blue elements) that releases 24/E from sequestration at the membrane by its antisigma factor RseA and coantisigma factor RseB. Activity of the RIP cascade is triggered by stress that elicits misfolded proteins in the periplasm (such as OmpC ) to eventually release a RseA/24 subcomplex that is guided to the cytosolic ClpXP protease by SspB for nal trimming to release 24 to compete for core RNA polymerase (core-RNAP). The sequential cleavage sites (1 to 3) within RseA are indicated. (b) The antisigma factor NepR sequesters ECfG1 within the cytoplasm until stress signals result in the phosphorylation of the receiver domain of the anti-antisigma factor PhyR. Phosphorylation of PhyR exposes a sigma mimic domain of PhyR that recruits and sequesters the antisigma (NepR), thus leaving ECfG1 free to associate with core-RNAP. (c) The antisigma factor FlgM likewise sequesters FliA within the cytosol. However, in this instance, completion of the agella basal body allows export of partially unstructured FlgM, resulting in a pool of available 28 ready for holoenzyme formation.

EcfG1

28/FliA
Available Sequestered Available

control expression of genes involved in the uptake of iron-scavenging siderophoresrst through an outer membrane siderophore transporter and then from the periplasm to the cytosol via an ABC-type transporter. The most extensively studied siderophore signaling pathway is that of the Fec system for ferric citrate

uptake in E. coli, which represents a paradigm system for responses to a signal that can only enter the cell through transport. Signaling involves communication from the cell surface siderophore transporter (FecA) to the inner-membrane-anchored antisigma factor (FecR), which sequesters FecI to the
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Anti-antisigma factor: a factor that antagonizes or counteracts the activity of an antisigma factor

cytoplasmic side of the inner membrane. Cell surface binding of ferric citrate to the FecA transporter triggers structural changes that facilitate interaction between the N-proximal portion of FecA and the C-terminal portion of the antisigma FecR within the periplasm. FecR then transmits the signal across the inner membrane to its N-terminal portion to relieve inhibition of FecI activity in the cytoplasm compartment. The regions of FecA that are involved in transducing signals arising from siderophore transport are comparatively well understood; however, the details of the mechanisms that underlie the propagation of the ferric citrate-binding signal to the C-terminal portion of FecR, and from there through FecR to affect FecI activity, remain an open question (14). In some Fec-like systems the antisigma factor only has a negative effect on sigma activity and, upon receiving the activating signal, may well simply release the sigma for association with core-RNAP, as is typical of antisigma factors. However, for FecI /FecR and some other related systems, activation through the FecA transporter converts the antisigma factor from a negative to a positive regulator that stimulates the activity of the cognate sigma factor (63 and references therein). In these cases, the antisigma likely remains bound to the sigma to achieve this outcome. Genetic analysis with truncates and point mutations of FecI has demonstrated that FecR sequesters FecI only via interaction with its 4 domain, and that this interaction is required for FecI to function as a sigma factor (60). Sequestering solely via the 4 domain may be a reection of the relative unimportance of the 35 element (that is usually bound by a 4 domain) for FecI -dependent transcription (5). Rather than FecI release, signaling to the cytoplasmic N terminus of FecR stimulates FecI association with the -subunit of RNAP (59). Because portions of FecR, FecI , and stably interact simultaneously (59), and activation via FecR also promotes novel promoter interactions with DNA at +13 from the transcriptional start site (5), the activation model that emerges is one in which FecR remains a functional part of the transcriptional
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initiation complex. Tethering to the inner membrane via FecR would not necessarily impose a hindrance to transcriptional elongation because the sigma factor is released during (or shortly after) transcriptional initiation. However, it does pose the interesting conundrum of how the FecI -RNAP holoenzyme locates a target promoter from its restricted location.

Partner Switching and Sigma Factor Mimicry Mechanisms

Bacillus species provide prime examples of cascade production and compartmentalization of sigma factors, as well as other paradigms of how the activities of alternative sigmas can be controlled. Both the F forespore developmental program and the expression of the B stress regulon of B. subtilis are governed by analogous phosphorylation-dependent partner switch mechanisms (reviewed in References 35 and 41). In these systems, switching between alternative binding partners of the antisigma factorfrom the sigma factor to an anti-antisigma factoris the critical step that releases the sigma to perform its function. The activity of F is regulated by the serine kinase antisigma factor SpoIIAB and the anti-antisigma factor SpoIIAA, which are cotranscribed with the F gene (sigF) in the spoIIA operon. Structural determinations have highlighted the importance of the 3 domain of F for sequestration by its antisigma (SpoIIAB) (16). Dimeric SpoIIAB binds asymmetrically to a single molecule of F and occludes its 3 domain from interaction with core-RNAP. As a result of the asymmetric binding, one of the SpoIIAB protomers is more accessible for binding to the anti-antisigma factor (SpoIIAA) that, upon docking to SpoIIAB, displaces F to leave it free to associate with core-RNAP. SpoIIAB then phosphorylates its new partner SpoIIAA in a reaction that dissociates the ADP-bound form of SpoIIAB, which in turn associates with any unphosphorylated SpoIIAA to form a complex that inhibits both the kinase and antisigma activity of SpoIIAB (41, 62, and references therein).

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Binding partner switching is also an integral component of the general stress response in Alphaproteobacteria that lack an E. coli 38 or B. subtilis B ortholog. However, in these organisms, control involves the unusual anti-antisigma PhyR, which has a C-terminal response-regulator (RR) domain coupled to an ECF-like domain. Upon phosphorylation, Methylobacterium extorquens PhyR binds NepR, an antisigma factor that normally sequesters the ECF EcfG1 (Figure 3b) (26). Although the ECF-like domain of PhyR shares high homology with EcfG1 , it lacks critical residues that would be involved in DNA binding (82) and thus appears to serve as a pure mimic to entice NepR away to free EcfG1 to serve its duties. A recent structure of Caulobacter crescentus PhyR in its unphosphorylated state shows predictable structures for its component parts. However, extensive interactions between the RR domain and the ECF domain (2 linked to 4 ) force the 2 and 4 modules into a compact conformation that presumably prevents recognition by NepR until PhyR is phosphorylated (40). Based on the distinct structural components, and the fact that the ECF domain alone can act as an anti-antisigma, the authors propose that the RR domain can be considered as an antianti-antisigma factor. The counterpart sensor kinase(s) (or anti-antianti-antisigma factor) that would serve to phosphorylate PhyR, and thus initiate the whole cascade, remains to be identied. However, a variety of candidate periplasmic or cytoplasmic sensor kinases are encoded in the vicinity of phyR genes in different organisms (81).

Checkpoint Coupling to Organelle Biogenesis

Flagella are characteristically assembled in a stepwise manner through temporally controlled expression of their component parts. The hierarchical expression of agella genes can be achieved by diverse mechanisms but usually involves a master regulator that initiates the cascade and coupling of late agella gene expression to completion of the hook basal body

structure to form a developmental checkpoint (79). In most agellated bacteria, the key players in this developmental checkpoint are an antisigma factor (FlgM) and a specic sigma factor (E. coli FliA , B. subtilis D ) that is required for transcription of genes encoding the agella lament subunits and proteins involved in bacterial taxis. Upon completion of the hook basal body structure, FlgM can be secreted through the type III system housed within the basal body, thus releasing FliA for association with core-RNAP (Figure 3c). Hence, this mechanism uses secretion as a signal that cues completion of a functional active structure to ensure that lament and taxis proteins are expressed only when appropriate (43). NMR studies have established that FlgM is intrinsically partially disordered, with only the C-terminal half structured when in association with FliA and under molecular crowding conditions that would prevail in vivo (22, 24). The naturally unfolded state of FlgM (complete or partial) has been suggested to facilitate secretion of FlgM through the narrow hook basal body structure (22). Biochemical and genetic evidence that implicates multiple regions of FliA in its sequestration by the C-terminal of FlgM has been reinforced by crystallographic data from the Aquifex aeolicus FliA /FlgM complex, which shows a highly compact conformation of the 2 , 3 , and 4 domains of FliA , which masks its DNA-binding and core-RNAP association determinants (80). The FlgM and FliA genes are not encoded within an operon, rather the levels of FlgM are frequently under complex regulation that includes (a) dual promoter control of the gM gene, in which one promoter is FliA dependent and thus provides an autorepressing feedback circuitry; and (b) translational modulation of FlgM protein levels, which adjusts the relative FlgM:FliA ratios within the cell (reviewed in Reference 79). Recently, mathematic modeling and experimental rewiring of the gM and iA gene promoters have provided strong support for a previously proposed model in which FlgM secretion, in addition to enforcing the developmental checkpoint,
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functions as a proxy-measuring system that continually ne-tunes FlgM and FliA levels to provide a sensing mechanism that may also control agella numbers (77).

control. The following sections present evidence that global regulatory subversion of the household 70 to concomitantly enhance formation of alternative -RNAPs provides at least a partial solution to the problem.

PROMOTION OF ALTERNATIVE SIGMA FACTOR ACTIVITY THROUGH SUBVERSION OF 70 Alternative Sigma Factors Are Not Created Equal
As highlighted in Figure 1, active transcription and the sigma cycle provide the means for reconstituting alternative -RNAP holoenzymes. However, as exemplied by the ndings in E. coli, alternative sigmas generally have lower afnity for core-RNAP than the housekeeping 70 , with the poorest (38 ) estimated to be approximately 10-fold lower (57). Moreover, even when presented with conditions that maximize the levels of active alternative sigmas, the cellular concentrations of alternative sigmas are greatly exceeded by those of 70 (29, 72, 75). The levels of E. coli 70 and coreRNAP are relatively constant over the growth curve and under different growth conditions. Although absolute values differ somewhat, the number of 70 molecules is consistently estimated to exceed that of core-RNAP by approximately threefold (29, 72, 75). Because much of the core-RNAP is employed in catalyzing RNA synthesis, competition between sigma factors to form a holoenzyme with the limited free coreRNAP will be erce. Many approaches, including articial manipulations of sigma factor levels and the use of sigma factor mutants that are altered in their afnity, and thus their competitiveness, for core-RNAP, have clearly established that sigma factor competition for core-RNAP limits output from promoters dependent on alternative sigmas such as 38 , 32 , and 54 (46, 53). This is likely the case for all alternative sigmas, which raises the question of how lowlevel and/or weak-afnity alternative sigma factors gain sufcient access to core-RNAP to drive transcription from promoters under their
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The Bacterial Alarmone ppGpp and Its Cohort DksA

The nucleotide ppGpp (also known as magic spot) is the primary mediator of the stringent response to amino acid starvation, where translational capacity is balanced to reduced demand through downregulation of transcription from tRNA and rRNA operon promoters (stringent 70 promoters). In addition to amino acid starvation, iron, carbon, and nitrogen limitations, as well as many environmental physicochemical stresses that reduce growth rate, cause induction of the intracellular levels of ppGpp (73). The rapid elevation of ppGpp levels during the hungry phase (just prior to the transition between exponential and stationary growth in rich media), or through articial manipulation of ppGpp levels under normally nonpermissive conditions, markedly enhances output from many promoters dependent on alternative sigmas (e.g., E. coli 38 , 32 , 24 , and 54 ) (20, 28, 46, 53, 84). It is now evident that ppGpp is the mediator of a far greater network that holistically redirects the global transcriptional capacity of the cell from genes for growth toward those for adaptive survival responses (32, 73). Targeting of the -RNAP by ppGpp specifically alters its performance at susceptible promoters to either decrease (e.g., stringent 70 promoters) or increase (e.g., some 70 promoters involved in amino acid transport or in virulence, and some specic 24 - and FliA dependent promoters) their activities (2, 20, 47, 67, 70, 71). The exact location and liganding residues for ppGpp within the active site cleft of core-RNAP remain elusive (86). Nevertheless, it appears clear that ppGpp binding to RNAP lowers the energy required for transition between intermediates in the pathway leading to open-complex formation.

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Depending on the rate-limiting step and relative stabilities of consecutive intermediates, lowering the energy required for conversion from one intermediate to the next would favor either the reverse or forward reactions in Figure 1b, leading to promoter-specic negative or positive outcomes (reviewed in Reference 33). The in vivo and in vitro effects of ppGpp at promoters are frequently amplied by DksA a member of a family of regulators that binds RNAP and accesses the active site through the secondary channel. DksA mediates long-range structural changes within RNAP that alter interaction with the 6 to +6 region at 70 promoters (11, 55, 76). Although it remains to be experimentally tested in most cases, there is no reason why DksA could not also affect the performance of any -RNAP, although the consequences might differ. DksA and ppGpp can have mutually independent and sometimes opposing effects (1, 3, 75, and references therein); however, DksA sensitizes RNAP to the cellular levels of ppGpp to account for their common coaction (70). In E. coli and P. putida, DksA levels are relatively constant (9, 75); therefore, it is the changing levels of ppGppthe herald of stressthat instigate proactive promoterspecic and global transcriptional responses that allow the cell to prepare for tough times ahead.

ppGpp and Sigma Factor Competition

As a global regulator, ppGpp by denition has pleiotropic effects in vivo, and thus many different mechanisms frequently converge to ultimately account for the total effect of ppGpp on output from a given promoter. These include promoter-specic effects on the performance of holoenzymes at kinetically susceptible promoters (see above), which in turn can initiate additional regulatory cascades through the gene products they encode. However, these regulatory consequences cannot account for the full effect of ppGpp in vivo. For example, ppGpp aids stability of S through production of antiadaptors to result in higher cellular levels of S

under stress conditions (reviewed in Reference 8). Nevertheless, a S promoter that is not dependent directly on ppGpp still requires ppGpp for activity in vivo even when reduced S levels are compensated for by ectopic expression (46, 52). Likewise, although the levels of 54 are constant irrespective of the presence or absence of ppGpp and/or DksA, the activities of 54 promoters that are not enhanced directly by either factor in vitro are still greatly stimulated by the presence of these molecules in vivo (9, 10, 53, 84). These ndings demand an alternative explanation for the action of ppGpp. In E. coli, elevated intracellular levels of ppGpp result in decreased association of 70 and core-RNAP (but not decreased 70 levels per se), so that less 70 -RNAP is available to occupy cognate 70 promoters (32, 39). In addition, a proteomic approach has shown that underproduction of 70 -RNAP essentially mimics the stringent response (58). Separation and immunological detection of free and core-RNAP-associated sigma has been used to demonstrate that elevated ppGpp, which decreases 70 -RNAP levels, concomitantly results in increased 38 -RNAP and 32 -RNAP holoenzyme levels (46). Although not experimentally tested, this would be anticipated to also be the case for other -RNAPs. These data, together with the nding that the requirement for ppGpp to achieve efcient 38 - and 54 dependent transcription can be simply bypassed using 70 mutants that are defective in their ability to compete for binding to core-RNAP (10, 46, 53, 84), make a convincing case for the idea that ppGpp plays a determining role in the outcome of sigma factor competition to favor holoenzyme formation with alternative sigmas. Whereas the importance of ppGpp in vivo is clear, it remains to be resolved if ppGpp and/or DksA can actively alter sigma factor competition, or if their effects occur indirectly (passively) through 70 -dependent transcription. An active role has been suggested based on the observation that ppGpp enhanced transcription from a 32 -dependent promoter only under conditions of competition with 70 in
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vitro (46). However, such stimulation was not found in similar experiments with 54 in competition with 70 , neither in the presence nor in the absence of ppGpp and/or DksA (10, 53, 83). In addition to altering sigma factor competition, 70 - and core-RNAP - and -bypass mutants also functionally mimic the action of ppGpp and DksA by further destabilizing the notoriously unstable open complexes of rRNA operon promoters (7, 83). The latter property suggests a passive mechanism by which these regulatory molecules could alter sigma factor competition. A unifying model has been proposed that would explain the properties of bypass mutants and enhanced performance by any alternative sigma in the presence of ppGpp (10, 83). This model, like many before it, invokes passive regulation through the consequences of the negative action of ppGpp and DksA at the seven powerful stringent 70 -rRNA operon promoters. In E. coli, the activities of these 70 promoters sequester approximately 60%70% of the transcriptional machinery during rapid growth in rich media (13). Under these conditions, where ppGpp levels are low, much of the core-RNAP is occupied in catalysis of the transcripts from these powerful promoters, leaving little available for association with any sigma factor. This would lead to low levels of alternative holoenzymes and consequent low occupancy and output from promoters under their control. Under slow growth and/or stress conditions that elicit high levels of ppGpp, however, the potent downregulation of transcription from the 70 -rRNA operon promoters would lead to increased levels of core-RNAP available for holoenzyme formation. As a consequence, alternative -RNAP levels would increase even in the absence of a change in sigma levels, leading to enhanced promoter output from cognate promoters. Within this model, decreased 70 availability and 70 -RNAP mutants would mimic high levels of ppGpp by reducing transcription from the powerful stringent 70 -rRNA operon promoters and by altering core-RNAP to sigma factor association to favor alternative holoenzyme
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formation. A prediction from this model is that low-afnity promoters that have holoenzyme binding as a rate-limiting step would be more susceptible to loss of these regulatory molecules than high-afnity counterpartsa prediction that has been experimentally veried to be the case for 54 -dependent transcription (9, 10).

Modulation of Sigma Factor Usage Through Diversion of 70

The model outlined above does not exclude, nor is it incompatible with, the possible existence of unknown factor(s) that may additionally contribute to the in vivo requirement for ppGpp and DksA. Analogous to the discovery of the role of DksA in ppGpp-mediated regulation, it cannot be ruled out that some other protein(s) may facilitate ppGpp-mediated regulation of alternative -RNAPs or aid their formation in vivo. On the contrary, the ppGpptriggered reduction of 70 -RNAP levels (but not those of 70 itself ) demands that 70 is diverted to prevent its association with coreRNAP. The answer to how this is achieved is not currently fully understood. However, as described below, the Rsd (regulator of sigma D) protein is likely a major contributor, and factors such as the Crl protein are also potentially involved. Rsd was initially identied through a search for factors that might allow alternative sigma factors to compete for limiting core-RNAP in E. coli (45). By forming 1:1 complexes with 70 , Rsd specically sequesters free 70 and can also actively remove 70 from 70 -RNAP in vitro (44, 87). Biochemical and genetic studies of Rsd and its AlgQ homolog have shown that these proteins sequester 70 primarily through interactions with the 4 domain (which contacts 35 elements), although other contact points with 70 -RNAP are also involved (25, 44). Structural studies of Rsd in complex with 4 of 70 have revealed that Rsd binding occludes residues critical for 4 /core-RNA interactions (69). By binding 70 and occluding association with core-RNAP, Rsd falls under the denition of an antisigma factor. However, this denition

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seems inappropriate because Rsd can be vastly overexpressed without compromising growth. Transcription of the Rsd gene is directed by the activities of two promoters, and is partially under ppGpp control, leading to elevated levels of Rsd when competition for coreRNAP would be at its highest (45, 72). The idea that Rsd might facilitate formation of alternative holoenzymes via reducing the availability of 70 has been spurred by the ndings that overexpression of Rsd results in increased output from some 54 -, 38 -, and 32 -dependent promoters (46, 53, 64), and because addition of Rsd can facilitate sigma factor exchange in vitro (L. Holmfeld & V. Shingler, unpublished data). Consistent with the idea that Rsd could facilitate access of alternative sigmas to coreRNAP, the naturally elevated levels of Rsd in stationary-phase E. coli sequester a signicant portion (25%) of 70 (72). However, it should be emphasized that Rsd null mutants have minimal effects on 38 - and 54 -dependent promoter outputs that are enhanced by overexpression of Rsd (9, 64), suggesting that Rsd does not act alone to bring about these regulatory events. An intriguing nding from structural studies of the Rsd/4 complex is that a network of interactions connects the binding interface with other potential binding cavities located on the surface of Rsd. Although some of these interactions may be involved in recognition of 70 -RNAP, this observation raises the possibility of functional coupling of Rsd/70 binding with binding of some as yet unknown protein and/or small regulatory molecule (69). If this is indeed the case, identication of such an entity would surely further our understanding of the physiological role of Rsd. The E. coli Crl protein binds 38 and preferentially favors 38 in competition with 70 for core-RNAP, presumably by facilitating 38 RNAP holoenzyme formation (27, 85). The Crl protein is restricted in its genome distribution and the global regulatory effect of Crl is limited to promoters of the 38 regulon (85). This makes it unlikely that Crl has any signicant effects on the levels of other -RNAP holoenzymes. Nevertheless, it does pose an alternative

scenario to specic 70 sequestration to at least partially account for reciprocal alterations in 70 -RNAP versus alternative -RNAP holoenzyme levels. Given the large number of genes of unknown function in E. coli and other bacteria, it is certainly plausible that analogous facilitators of other alternative holoenzymes exist but have eluded detection.

CONCLUSIONS
Regulation of alternative sigma factor activity is usually complex, with multiple tiers of control to regulate both their expression levels and their activities. One major mechanism is sequestering by an antisigma factor, which provides systems for signal-specic control of sigma factor availability and thus the activity of promoters they regulate. Where known in any depth, these systems are exquisitely attuned to both the type of signal (i.e., the compartment from which the signal arises) and the constraints imposed by the nature of the signal (e.g., the need for iron transport into the cell). However, for many alternative sigmas, particularly those of the extensive ECF Group 4 family, only a few have been studied in any detail. In many cases neither the signal nor the factor(s) that controls their activity is known, which severely curtails understanding their role in microbial physiology. Given the novel mechanisms that have recently been identied by studying new members of this familysuch as the use of alternative start codons to generate proteolytically vulnerable variants of S. coelicolor R (49) and molecular mimicry of EcfG1 in Alphaproteobacteria (26)it is not unreasonable to expect the repertoire of mechanisms that can control sigma factor availability to continue to expand. Sequestering by an antisigma factor both protects the sigma factor from proteolytic attack and provides immediate availability upon demand. Genetic and structural studies of sigma/antisigma interactions have identied repeated themes within sequestering mechanisms, namely manipulation of the geometry of the key globular sigma domains (2 , 3 , and/or 4 ) to mask critical regions involved in
www.annualreviews.org Control of Sigma Factor Use 49

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interaction with core-RNAP and promoter DNA. Likewise, similar studies have also started to unravel and test models of how antisigma/anti-antisigma interactions break these interactions to free sigmas to perform their function. In addition to dedicated signaling pathways, the activities of many E. coli alternative sigma factors can be coordinately stimulated by the global regulator ppGpp, and this is likely to also be the case in other organisms. The adoption

of ppGpp to stimulate the activities of alternative sigmas is perhaps not surprising because stresses that elicit ppGpp synthesis overlap greatly with those that cue the need for alternative sigmas. Much evidence has accumulated that this stimulatory effect occurs through a role of ppGpp in determining the outcome of sigma factor competition for limiting core-RNAP to holistically favor association of alternative sigmas over 70 . However, much remains to be learned about how this is brought about.

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SUMMARY POINTS 1. The transcription cycle provides the means to rapidly strip off the sigma factor to generate naked core-RNAP ready for reprogramming by any available sigma. However, the activities and availability of most sigma factors are intricately controlled. 2. Antisigma factors manipulate the geometry of key regions of sigma factors to prevent their interaction with core-RNAP. Cognate dedicated signal transduction pathways that release the activities of sigma factors present a dazzling array of mechanismsfrom sophisticated protease cascades, through sigma factor mimicry and partner switching, to the conceptually simple but elegant solution of secretion of an antisigma factor to link activity to organelle biogenesis. 3. When free to interact with core-RNAP, all alternative sigma factors must compete ercely with 70 (and each other) for a limited amount of core-RNAP in order to direct transcription from the promoters they control. 4. Alternative sigma factors are aided in their battle against 70 by the alarmone ppGpp through mechanisms that divert 70 or otherwise counteract its association with core-RNAP.

FUTURE ISSUES 1. Has the repertoire of mechanisms that can control the availability of alternative sigma factors reached its limit, or are there future surprises ahead? 2. Do ppGpp and DksA affect transcription mediated by all -RNAP holoenzymes? 3. Does the effect of ppGpp and DksA on sigma factor competition operate purely passively, or is there an active component involved? 4. How is the dominating 70 subdued to allow alternative sigma factors sufcient access to limited core-RNAP? Is Rsd the only answer, or do other 70 sequesters exist? Does Rsd act in concert with a coregulator and/or with Crl-like facilitators of alternative holoenzyme formation?

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DISCLOSURE STATEMENT
The authors are not aware of any afliations, memberships, funding, or nancial holdings that might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS
Apologies are due to all researchers whose original contributions could not be cited due to space limitations. Our work is supported by the Swedish Research Council (grant number 621-20083557 to V.S.) and the European Molecular Biology Organization through a Long Term Research Fellowship (grant number 540-2009 to T. del P.-S.). LITERATURE CITED
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26. A conceptually new way to entice an antisigma from its target sigma.

32. An elegant approach to address the difcult problem of determining the relative proportions of free versus DNA-bound -RNAP.

34. Claries the identity of a previously unappreciated promoter recognition element that can be exploited to regulate promoter output.

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www.annualreviews.org Control of Sigma Factor Use 46. First extensive compilation of evidence that ppGpp plays a determining role to favor holoenzyme formation with alternative s (see also Reference 53).

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49. A conceptually new way to control sigma factor activity, with new insights into regulator system dynamics.

53. First extensive compilation of evidence that ppGpp plays a determining role to favor holoenzyme formation with alternative sigmas (see also Reference 46).

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70. The advent of understanding the role of DksA, especially its role in ppGpp-mediated regulation.

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