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Control of transcription by nucleoid proteins Sarah M McLeod* and Reid C Johnson*† Nucleoid proteins are a group of abundant DNA binding proteins that modulate the structure of the bacterial chromosome. They have been recruited as specific negative and positive regulators of gene transcription and their fluctuating patterns of expression are often exploited to impart an additional level of control with respect to environmental conditions. Addresses *Department of Biological Chemistry, School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095-1737, USA † Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA 90095, USA; e-mail: [email protected] Correspondence: Reid C Johnson

are essential for viability, although certain combinations of knockouts can be highly detrimental and, in some cases, result in nonviability. Some of these proteins share structural homology and display overlapping functions. The amino acid sequences of the HU and IHF subunits are at least 45% identical with each other, and the two proteins have very similar 3-D structures [3]. StpA shares 58% amino acid identity with H-NS, and both proteins have an amino-terminal oligomerization domain that is connected by a flexible linker to a carboxy-terminal DNA-binding domain [4–6]. Moreover, StpA has been shown to be able to substitute for H-NS in a subset of H-NS-regulated reactions, and StpA and H-NS can form active heterodimers.

Current Opinion in Microbiology 2001, 4:152–159 1369-5274/01/$ — see front matter © 2001 Elsevier Science Ltd. All rights reserved. Abbreviations α-CTD carboxy-terminal domain of RNA polymerase α subunit CRP cAMP receptor protein Dps DNA-binding protein from starved cells Fis factor for inversion stimulation H-NS histone-like nucleoid-structuring protein IHF integration host factor StpA suppressor of td mutant phenotype A

Introduction The 1.5 mm DNA chromosome of Escherichia coli is condensed about 1000-fold into the nucleoid by constrained supercoiled loops, random coiling, and binding of cations and proteins (reviewed in [1]). The protein composition of the nucleoid is dominated by a small group of polypeptides, referred to here as nucleoid proteins, which together coat up to half of the chromosome [2••]. Unlike eukaryotic histones, bacterial nucleoid proteins form unstable complexes with DNA. These proteins can profoundly affect the local as well as global structure of the chromosome, but the precise role of each protein in chromosome condensation has yet to be established. On the other hand, information regarding the function of these proteins in specific DNA reactions such as the regulation of transcription and DNA recombination is rapidly accumulating. In this review, we discuss recent data on the roles of nucleoid proteins as global regulators of transcription. After a discussion on the general features of the major nucleoid proteins in E. coli, we focus on a few recent examples of transcriptional repression, activation and combinatorial control by nucleoid proteins for which both in vivo and in vitro mechanistic information is available.

Properties of nucleoid proteins The major nucleoid proteins in E. coli are HU, integration host factor (IHF), histone-like nucleoid-structuring protein (H-NS), suppressor of td mutant phenotype A (StpA), factor for inversion stimulation (Fis), and DNA-binding protein from starved cells (Dps) (Table 1). None of these proteins

All nucleoid proteins bind DNA nonspecifically with physiologically significant affinities, but some show a preference for a particular DNA sequence or structure (Table 1). Fis preferentially binds to a 15 bp degenerate consensus core sequence, although it can contact up to 27 bp of DNA, depending on the flexibility of the flanking sequences [7,8]. IHF binds to a 35 bp site that comprises a 3′ conserved domain and a 5′ AT-rich domain [9]. Binding selection experiments on the 5′ portion of the IHF-binding site showed that sequence variations in this region can result in 100-fold differences in affinity [10]. Likewise, substitutions of basic residues that contact DNA within the 5′ AT-rich domain result in mutants that display reduced DNA binding and bending [11]. The related HU displays little preference for sequence but selectively binds to DNA-containing kinks, gaps or fourway junctions [12]. H-NS binding regions are almost always characterized by intrinsically curved sequences such as AT-rich segments [4–6]. In some cases, H-NS is believed to spread along DNA from the initial nucleation site and function as a regional silencer by altering the local DNA topology or by directly interfering with RNA polymerase interaction at a nearby promoter region. The precise mode of DNA binding by H-NS remains to be determined; oligomerization is clearly important, but the functional oligomeric state of the protein varies widely, depending on experimental conditions [13,14].

Expression and localization of nucleoid proteins Although the levels of many of the major DNA-binding proteins have been individually determined by various laboratories, Ishihama and co-workers [15••] have recently collectively measured the levels of 12 of the most abundant DNA-binding proteins in rapidly growing and stationary phase E. coli cells. In general agreement with previously reported values, these measurements indicate that Fis, HU, IHF, StpA, and H-NS are the most abundant nucleoid proteins in exponential phase (see Table 1). During stationary phase, the protein composition of the nucleoid changes, and primarily Dps, IHF and HU are

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Table 1 Properties of the major nucleoid proteins. Protein

Quaternary structure*

DNA target†

Exponential phase expression‡

Stationary phase expression‡

Additional functions§

References#

Fis

Homodimer (X-ray)

GNtYAaWWWtTRaNC

25,000–40,000

Very low

DNA recombination, replication

[16]

HU

Heterodimer (X-ray, NMR)

Kinked, gapped, threeor four-way junctions

15,000–30,000

5000–10,000

DNA replication, recombination

[3]

IHF

Heterodimer (X-ray)

WATCAANNNNTTR

6000–17,000

27,500

DNA recombination, replication

[3,40]

H-NS

Dimer-oligomer (NMR-partial)

Curved

10,000

4000

Site-specific recombination [4–6]

StpA

Dimer-oligomer?

Curved

12,500

4000–5000

RNA chaperone

[4–6]

Dps

Dodecamer (X-ray)

Nonspecific

500

15,000

Oxidative DNA damage protection

[18]

*Method used to determine an atomic structure is parenthetically denoted, if available. †Each of the proteins binds DNA nonspecifically with physiologically significant affinities. A preferred binding sequence or DNA structure is noted. Y = C or T, R = G or A, W = A or T, N = any base. ‡Fis, HU, and IHF are expressed in dimers per cell and Dps is

given in dodecamers per cell. H-NS and StpA are expressed as dimers per cell, but the functional binding form may be a tetramer or higher order. Data is collated from individual studies and [15••]. §Reactions other than transcription in which the nucleoid protein has been shown to function. #Reviews or papers containing general information are noted.

present. This fluctuation in the population of nucleoid proteins is thought to mediate global changes in nucleoid structure and activity. For example, Fis, which is the most abundant DNA-binding protein in rapidly growing cells, positively regulates the expression of a number of genes that are transcribed at high rates during rapid growth, such as the operons for stable RNAs (rRNAs and tRNAs) [16]. Fis also negatively regulates genes that are normally expressed under poor growth conditions or in stationary phase. Conversely, the rise in Dps levels as cells enter stationary phase is believed to alter transcription patterns and to protect the chromosome from oxidative damage [17]. The recent crystal structure of Dps shows that it is a dodecamer, analogous to ferritin, with a negatively charged hollow core that protects DNA from oxidative damage by sequestering iron ions, which are capable of generating free radicals [18].

varies in response to environmental cues, such as those that mediate responses to changes in temperature and osmolarity as well as genes affecting virulence. Recent gene profiling assays on ihf+ and ihf mutant cells by microarray analysis have revealed that IHF directly or indirectly affects the transcription of over 100 genes of widely varying function [20••]. At least 46 of these genes contain a documented or putative high affinity IHF-binding site and thus are believed to be directly controlled by IHF.

Recent immunofluorescence experiments on exponential and stationary phase cells showed that HU, IHF, H-NS, StpA, and Dps (in stationary phase cells) were distributed throughout the nucleoid [19•]. Surprisingly, Fis appeared to be localized within multiple densely stained foci (in exponential phase cells). The clustering of Fis at particular locations within the nucleoid could reflect its association with the upstream regulatory elements of the stable RNA operons.

Global regulation of gene expresison by nucleoid proteins Nucleoid proteins have been shown to function directly or indirectly, and often in combination, to control expression of a wide variety of genes that are integral for cell viability, such as those involved in protein synthesis and metabolism. They also regulate other important genes whose expression

Modes of transcriptional repression by nucleoid proteins Nucleoid proteins have been documented to directly repress transcription by several mechanisms. The most common method occurs when the repressor binds to the DNA within the recognition site for RNA polymerase and occludes the polymerase from the promoter. Fis is believed to repress a number of promoters including gyrA in this manner (Figure 1a) [16,21••]. Promoters repressed by Fis usually have multiple binding sites both upstream and downstream of the transcriptional start point, but the site overlapping the –35 or –10 elements has the greatest effect on repression. H-NS is also thought to repress transcription in this manner at many different promoters [6]. However, at the rrnB P1 promoter, RNA polymerase can still form an open complex when H-NS is bound within the –35 region [22•]. The transcription complex containing H-NS is only capable of synthesizing three-nucleotide-long abortive transcription products, leading Schröder et al. [22•] to propose that H-NS traps RNA polymerase in an altered complex that is incapable of proceeding into the elongation phase. The Fis protein appears to repress transcription at the gyrB promoter by a related mechanism, but in this case,

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Cell regulation

Figure 1

Figure 1 legend +1

(a)

–35 –10

gyrA

–16 to –68 Fis +1 –35 –10 –109 Fis

(b)

AT-rich silencer

CRP –60.5

H-NS

+1

Silencer

–35 –10

bglG

–27 Fis

–52 Fis

+1

Intrinsic bends

(c)

–274

gyrB

–62 Fis

–35 –10 H-NS

–46

–218

(d)

H-NS +46

+61

virF

HU +6.5

Examples of promoters repressed by nucleoid proteins. (a) A schematic of the E. coli gyrA and gyrB promoters denoting the Fis-binding regions [21••]. At gyrA (upper diagram), Fis binds to an extended region between –16 and –68 bp from the start of transcription (+1). Fis binding prevents RNA polymerase from productively interacting with the gyrA promoter. At gyrB (lower diagram), Fis binds to two sites upstream of the promoter. Fis inhibits transcription but does not interfere with RNA polymerase binding and promoter opening. (b) Key regulatory elements at the E. coli bgl promoter [28]. Transcriptional silencing regions are denoted with dashed lines where the AT-rich silencer region upstream of the promoter binds H-NS, and the downstream region is within the coding region for the bglG gene. CRP activates transcription through a site located at –60.5. Fis bound at –52 represses transcription by competing for binding with the activator, CRP. Fis has also been shown to repress transcription in vitro from a lower affinity site within the promoter region. (c) The Shigella sp. virF promoter. Thermoregulation is mediated by H-NS binding at low temperature to regions (represented by gray boxes) that extend from –218 to –274 and from –46 to +46 [23]. A region of intrinsic bending that extends from –40 to –80 (dotted line) undergoes a temperature-induced structural change that influences H-NS binding to both sites. (d) Repression loop formed at the gal promoter [26••]. An HU dimer binds to a site centered 6.5 bp downstream from the start of transcription from the P1 promoter. Two dimers of GalR bind to operators located upstream and downstream of the P1 and P2 promoters as shown. The presence of HU and DNA supercoiling are required to promote the interaction between GalR dimers. To account for the direction of DNA bending by HU relative to the helical phase of the two bound GalR dimers, a DNA crossover has been introduced into the model to align the carboxy-terminal regions of the GalR dimers [26••].

P1 P2

+53.5

GalR GalR

–60.5

assays performed at different temperatures, together with computer modeling, suggest that there is an intrinsic bend between the two H-NS sites in the virF promoter DNA, and that some sort of structural transition occurs around 32°C that antagonizes H-NS binding to both sites. A similar mechanism may occur at other promoters that are regulated by both H-NS and temperature such as at fimE and fimB [24].

the nucleoid protein is bound upstream of RNA polymerase [21••]. Fis binding to two sites centered at –62 and –109 does not interfere with RNA polymerase binding or promoter melting. Nevertheless, transcription from the promoter is severely inhibited, indicating that the open complex formed is not capable of supporting productive transcription.

Nucleoid proteins can also negatively regulate gene expression by competing for binding with a transcriptional activator. This model is illustrated at the bgl promoter, at which Fis binding to a site 52 bp upstream of the start of transcription has been shown by DNase I footprinting to displace the activator protein CRP (cAMP receptor protein) (Figure 1b) [25•]. Fis-mediated repression can be counteracted only if the CRP-binding site is mutated to improve cAMP–CRP binding to the bgl promoter.

H-NS is also capable of repressing transcription in response to environmental signals. An interesting example is the Shigella sp. virF promoter, which is activated at 37°C, but not at low temperatures (Figure 1c). Falconi et al. [23] have shown that H-NS represses transcription by cooperatively binding to supercoiled DNA at two regions at 28°C but not at 37°C. One of these regions overlaps the RNA-polymerase-binding site, whereas the other is located 160 bp upstream. Electrophoretic mobility

Additionally, nucleoid proteins can repress transcription by collaborating with a gene-specific repressor to form higherorder looped DNA structures. Thus far, this mechanism has been best described for control of the gal operon, in which the two operator sites bound by the GalR repressor are located over 50 bp upstream and downstream of the two promoters P1 and P2 (Figure 1d) [26••]. Repression of P2 is dependent on HU, both the upstream and downstream operators, and DNA supercoiling. An HU dimer

Current Opinion in Microbiology

Control of transcription by nucleoid proteins McLeod and Johnson

Figure 2 legend Examples of promoters activated by nucleoid proteins. (a) Activation by Fis at the E. coli rrnB P1 [36]. The Fis dimer is shown in dark gray bound at –71 contacting the α-CTD, which is bound at the adjacent UP element and tethered to the bulk of RNA polymerase (shown in light gray) through a flexible linker. Activation by Fis is countered by H-NS binding to a site at –25 [22••]. Additional H-NS binding sites are present in the upstream region (not shown). (b) Activation by Fis at the σ38-dependent proP P2 promoter of E. coli [35••]. The requirement for both Fis and σ38 for P2 promoter activity results in a brief period of transcription during late exponential growth phase. A four amino acid region on Fis, localized to only one subunit of the Fis homodimer near the helix-turn-helix DNA-binding region, is necessary for proP P2 transcription. Co-activation by CRP bound at –121.5 depends on Fis binding at –41 [47••]. Both Fis and CRP independently contact one of the α-CTDs of RNA polymerase. (c) Mechanism by which IHF stimulates transcription at the phage λ PL promoter [42]. DNA bending induced by IHF bound at –80 is believed to appropriately position an overlapping UP element to enable the binding of one or both α-CTDs. An intrinsic DNA bend at –50 also facilitates this interaction. (d) Mechanism by which IHF activates the E. coli ilvPG promoter through transmission of destabilized DNA [43]. IHF binds to a site centered at –92, which is just downstream from a region at which the DNA duplex is unwound (upper diagram). IHF binding and bending stabilizes the upstream region, causing the region around –10 to become destabilized (lower diagram).

binds to a discrete site that is centrally located between the two operator sites and promotes the formation of a repression loop that contains both gal promoters (Figure 1d). HU therefore functions exclusively as a DNA architectural protein by inducing an appropriate bend in the DNA to facilitate a GalR–GalR interaction that is otherwise too weak to form [27]. Another mechanism of repression, called transcriptional silencing, occurs when an extended DNA segment inhibits transcription from a location well removed from the promoter. The most studied cases involve the H-NS protein at the E. coli and Salmonella typhimurium proU operons and the E. coli bgl promoter. H-NS mediates repression by binding to extended regions that are remotely located either downstream or upstream of the target promoter (Figure 1b) [6,28,29]. Repression at proU appears to be independent of the phasing and positioning of these H-NS ‘silencer’ regions, suggesting that there is no direct contact between H-NS and RNA polymerase [30]. Moreover, insertion of an ectopic promoter between the proU promoter and the H-NS binding region results in repression of proU-directed transcription only, implying that silencing is promoter specific [31]. Evidence for oligomerization of H-NS binding along the DNA from the silencer to the promoter region has been obtained in the bgl system, in which the Lac or λ repressors relieve silencing of the bgl promoter when their respective binding sites are placed anywhere within this silencer region [32]. These high affinity binding repressors stimulate bgl transcription presumably by preventing H-NS from forming a repressing nucleoprotein complex along the DNA.

155

Figure 2 (a) β/β′

σ70

Fis –71

UP

rrnB

H-NS –25 CRP –121.5

(b)

β/β′

σ38

Fis –41

proP

–80 IHF

(c)

β/β′ σ70

–50

(d)

PL

IHF

ilvPG Supercoiled DNA Destabilized DNA –120

–10 –92

–50

ilvPG

–10

IHF –92

–50 Current Opinion in Microbiology

Modes of transcriptional activation by nucleoid proteins Most of the nucleoid proteins are also capable of directly or indirectly enhancing transcription. The Fis protein functions directly as a transcriptional activator by specifically interacting with RNA polymerase within a variety of promoter architectures. The interaction between Fis and RNA polymerase has been extensively characterized at rrnB and at proP. Fis binds to three sites upstream of the rrnB start site. The site centered at –71, immediately upstream of the UP element (an AT-rich DNA binding site for the carboxy-terminal domain of the α subunit of RNA polymerase [α-CTD]), promotes most of the Fis-dependent activation [33]. Conversely, at proP P2,

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Cell regulation

Figure 3 (a)

tyrT, facilitate open complex formation and promoter clearance, in addition to recruitment, by transmission of torsional energy within a DNA loop formed between Fis and RNA polymerase.

IHF –88

H-NS

FNR

+1

Fis +23

–41.5

–142 Fis

(b) –35 –10 IHF –85

tplP +1

TyrR TyrR

–220 CRP

TyrR

–250 CRP Current Opinion in Microbiology

Examples of combinatorial control involving nucleoid proteins. (a) A model of transcriptional repression of the E. coli nir promoter by Fis, IHF, and H-NS [44••]. IHF binds to a site at –88 and reverses the trajectory of DNA such that the DNA with Fis bound at –142 loops back on the ‘underneath’ side of RNA polymerase. Fis bound at +23 and H-NS bound between –68 and –83 also function to antagonize FNR-dependent transcription of nirP. (b) Activation of the Citrobacter freundii tpl promoter by TyrR, IHF, and CRP [46]. Three dimers of TyrR bind cooperatively to activate transcription through the TyrR dimer bound closest to the promoter. The cooperative assembly is facilitated by DNA bending induced by the IHF protein bound at –85 and two dimers of CRP bound at –220 and –250. FNR, activator for fumarate reductase and nitrate reductase expression.

Fis-dependent activation is mediated through a site at –41, overlapping the –35 RNA-polymerase-recognition element [34]. At both rrnB P1 and proP P2, Fis activates transcription from a four amino acid surface-exposed ridge located adjacent to the helix-turn-helix DNA-binding region of the Fis homodimer [35••,36]. This region is believed to specifically interact with the α-CTD (Figure 2a,b). Fis has been shown to aid in recruitment of RNA polymerase, but there is evidence that it also stimulates steps subsequent to polymerase binding [33,35••,37]. Through studies at rrnB P1 employing mutant polymerases, Bartlett et al. [38••] show that Fis activates steps after closed-complex formation as manifested by a lower apparent binding constant for the initiating nucleotide. Muskhelishvili et al. [37,39] suggest that the three required Fis-binding sites, which are in helical register at

Whereas the Fis protein directly contacts RNA polymerase to stimulate transcription, other nucleoid proteins are thought to enhance transcription by primarily acting as architectural factors. IHF stimulates transcription in this manner by several distinct mechanisms. Some of the best studied examples of activation are at σ54-dependent promoters, at which IHF positions a DNA-bound NtrC family activator such that it can directly contact RNA polymerase (reviewed in [40]). Recently, IHF has also been shown to correctly position an upstream α-CTDbinding site by introducing a bend in the DNA at the Pseudomonas putida Pu promoter [41]. This bending of the DNA by IHF stimulates recruitment of RNA polymerase by allowing a more favorable RNA-polymerase–DNA interaction. IHF appears to be functioning similarly at the phage λ PL promoter, at which it enables RNA polymerase interaction with a UP element centered about 90 bp upstream of the start site (Figure 2c) [42]. The ATrich domain of the IHF-binding site overlaps with the α-CTD-binding sites within the UP element. An additional flexible DNA region located at –50 between the IHF- and polymerase-binding site regions is also required and presumably contributes to the correct positioning of the α-CTD-binding site. Additionally, IHF has been found to be able to substitute for CRP at the malT promoter by facilitating upstream DNA contact with RNA polymerase. Mutants of IHF that display enhanced transcriptional activity in the malT system have been isolated, and these contain substitutions that reduce DNA bending. Engelhorn and Geiselmann [11] postulate that the IHF-induced bending at malT is too severe with wildtype IHF but is optimal with the mutant forms. These mutant IHF proteins are not generally more active for promoting transcription, as they display reduced activity in the λ PL system. Whereas these mechanisms involve DNA bending by IHF to position promoter elements, IHF has also been shown to directly activate transcription in an entirely different manner. At the E. coli ilvPG promoter, IHF binds to a sequence centered at –92 bp from the start of transcription (Figure 2d) [43]. An AT-rich region, in which the DNA duplex is destabilized by superhelical stress, is located just upstream of the IHF site. IHF binding stabilizes this region, and the resulting increase in DNA supercoiling is transmitted downstream to the region of the –10 recognition element to lower the activation energy for open complex formation.

Combinatorial control Although many investigations of transcriptional control exerted by nucleoid proteins have focused on the mechanism of regulation by one of these proteins, it is

Control of transcription by nucleoid proteins McLeod and Johnson

becoming increasingly evident that these proteins usually do not work in isolation. As noted above, nucleoid proteins often act in concert to repress or activate transcription with other nucleoid proteins and global or gene-specific transcriptional regulators. The nir promoter is an example of a complex system in which Fis, IHF, and H-NS collaborate to create a higher-order nucleoprotein complex that forms an inhibitory promoter architecture (Figure 3a) [44••]. IHF and Fis effectively repress transcription in the absence of H-NS. However, IHF is necessary for Fis-dependent repression. IHF binding induces a bend at –88 from the start of transcription that is proposed to position upstream sequences away from the promoter. This inhibitory bend could serve to prevent RNA polymerase from making additional contacts with the promoter DNA and to position the upstreambound Fis dimer such that Fis could either stabilize the repression structure or make an inhibitory contact with RNA polymerase. Similarly, binding of Fis and CRP to multiple sites is proposed to form different complexes that alternatively repress overlapping divergent promoters to control crp expression [45]. Likewise, multiple nucleoid proteins can also function to co-activate transcription. As noted above, a nucleoid protein can act as an architectural element to position the primary regulator to contact RNA polymerase. A more complex example of IHF activation in collaboration with the NtrC family of activators occurs at the Citrobacter freundii tpl promoter. DNA bends promoted by IHF and cAMP–CRP proteins allow three dimers of the activator protein TyrR to interact with each other and directly activate transcription (Figure 3b) [46]. The TyrR site located closest to the promoter has the weakest binding affinity of the three but is absolutely required for TyrR-dependent activation of the tpl promoter. Deletion of either of the other two TyrR binding sites, the IHF site, or CRP sites reduce activation, implying that the higher-order nucleoprotein complex is necessary for proper stimulation by the promoter-proximal TyrR molecule. In other scenarios of transcriptional activation, two or more factors directly contact RNA polymerase. Fis and the cAMP–CRP have been shown to activate transcription synergistically at the proP P2 promoter [47••]. In this case, each activator contacts a different α-CTD to stimulate transcription (Figure 2b). Transcriptional activation by the upstream regulator (CRP), however, requires binding of the downstream Fis dimer. Another method of cooperation results in promoter selectivity for a particular sigma factor. This has been observed at the growth phase regulated promoters osmY and csiD that are primarily expressed in vivo with the stationary phase sigma factor, σ38. At osmY, IHF, CRP, and LRP inhibit transcription by the σ70 form of RNA polymerase (Eσ70), but not Eσ38 [48••]. Conversely, at the csiD promoter, in vitro transcription reactions with a supercoiled template show that cAMP–CRP activates

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transcription equally well with Eσ70 and Eσ38, whereas addition of H-NS results in preferential transcription with Eσ38 [49].

Conclusions The role of nucleoid proteins in controlling gene expression has become increasingly recognized over the past few years. They can modulate transcription in response to environmental cues by a variety of mechanisms, often in concert with each other and with gene-specific regulatory factors. In most cases, their ability to alter DNA structure is central to their activity in specific reactions, but clear examples of direct interactions with RNA polymerase also exist. As more experiments are performed using microarray technology to profile gene expression patterns, examples of direct and indirect contributions of nucleoid proteins in global control will no doubt become more widespread.

Acknowledgements Work in the authors’ laboratory is supported by National Institutes of Health grant GM38509.

References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:

• of special interest •• of outstanding interest 1.

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2. ••

Ishihama A: Modulation of the nucleoid, the transcription apparatus, and the translation machinery in bacteria for stationary phase survival. Genes Cells 1999, 4:135-143. This recent review discusses how the fluctuating patterns of nucleoid proteins, sigma subunits and factors that regulate translation effect chromosome activity and structure. This is the first time that these important factors have been collectively discussed and compared over the entire growth cycle of the cell. 3.

Nash HA: The HU and IHF proteins: accessory factors for complex protein–DNA assemblies. In Regulation of Gene Expression in Escherichia coli. Edited by Lynch AS, Lin ECC. Austin, Texas: RG Landes Company; 1996:149-179.

4.

Dorman CJ, Hinton JC, Free A: Domain organization and oligomerization among H-NS-like nucleoid-associated proteins in bacteria. Trends Microbiol 1999, 7:124-128.

5.

Williams RM, Rimsky S: Molecular aspects of the E. coli nucleoid protein, H-NS: a central controller of gene regulatory networks. FEMS Microbiol Lett 1997, 156:175-185.

6.

Atlung T, Ingmer H: H-NS: a modulator of environmentally regulated gene expression. Mol Microbiol 1997, 24:7-17.

7.

Hengen PN, Bartram SL, Stewart LE, Schneider TD: Information analysis of Fis binding sites. Nucleic Acids Res 1997, 25:4994-5002.

8.

Pan CQ, Finkel SE, Cramton SE, Feng JA, Sigman DS, Johnson RC: Variable structures of Fis–DNA complexes determined by flanking DNA-protein contacts. J Mol Biol 1996, 264:675-695.

9.

Goodrich JA, Schwartz ML, McClure WR: Searching for and predicting the activity of sites for DNA binding proteins: compilation and analysis of the binding sites for Escherichia coli integration host factor (IHF). Nucleic Acids Res 1990, 18:4993-5000.

10. Goodman SD, Velten NJ, Gao Q, Robinson S, Segall AM: In vitro selection of integration host factor binding sites. J Bacteriol 1999, 181:3246-3255.

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11. Engelhorn M, Geiselmann J: Maximal transcriptional activation by the IHF protein of Escherichia coli depends on optimal DNA bending by the activator. Mol Microbiol 1998, 30:431-441. 12. Pinson V, Takahashi M, Rouviere-Yaniv J: Differential binding of the Escherichia coli HU, homodimeric forms and heterodimeric form to linear, gapped and cruciform DNA. J Mol Biol 1999, 287:485-497.

26. Lewis DE, Geanacopoulos M, Adhya S: Role of HU and DNA •• supercoiling in transcription repression: specialized nucleoprotein repression complex at gal promoters in Escherichia coli. Mol Microbiol 1999, 31:451-461. HU and GalR cooperate to form a repression loop within the gal regulatory region. Formation of this repressing complex requires supercoiled DNA and HU, which bind to a specific site and bends the DNA, allowing an interaction between two dimers of the GalR protein separated by approximately 100 bp.

13. Ceschini S, Lupidi G, Coletta M, Pon CL, Fioretti E, Angeletti M: Multimeric self-assembly equilibria involving the histone-like protein H-NS. A thermodynamic study. J Biol Chem 2000, 275:729-734.

27.

14. Smyth CP, Lundback T, Renzoni D, Siligardi G, Beavil R, Layton M, Sidebotham JM, Hinton JC, Driscoll PC, Higgins CF et al.: Oligomerization of the chromatin-structuring protein H-NS. Mol Microbiol 2000, 36:962-972.

28. Mukerji M, Mahadevan S: Characterization of the negative elements involved in silencing the bgl operon of Escherichia coli: possible roles for DNA gyrase, H-NS, and CRP–cAMP in regulation. Mol Microbiol 1997, 24:617-627.

15. Ali Azam T, Iwata A, Nishimura A, Ueda S, Ishihama A: Growth phase•• dependent variation in protein composition of the Escherichia coli nucleoid. J Bacteriol 1999, 181:6361-6370. The authors collectively measured the intracellular concentrations of 12 of the most abundant DNA binding proteins by quantitative immunoblotting of logarithmic and stationary phase E. coli cells. This is the first paper to collectively measure the relative abundance of these proteins.

29. Gowrishankar J, Manna D: How is osmotic regulation of transcription of the Escherichia coli proU operon achieved? A review and a model. Genetica 1996, 97:363-378.

16. Finkel SE, Johnson RC: The Fis protein: it’s not just for DNA inversion anymore. Mol Microbiol 1993, 6:3257-3265. 17.

Martinez A, Kolter R: Protection of DNA during oxidative stress by the nonspecific DNA-binding protein Dps. J Bacteriol 1997, 179:5188-5194.

18. Grant RA, Filman DJ, Finkel SE, Kolter R, Hogle JM: The crystal structure of Dps, a ferritin homolog that binds and protects DNA. Nat Struct Biol 1998, 5:294-303. 19. Azam TA, Hiraga S, Ishihama A: Two types of localization of the • DNA-binding proteins within the Escherichia coli nucleoid. Genes Cells 2000, 5:613-626. The authors use indirect immunofluorescence microscopy to localize nucleoid proteins and components of transcription and translation machinery. HU, H-NS, IHF, StpA and Dps are found to be distributed uniformly over the entire nucleoid, whereas Fis forms specific loci within the nucleoid. 20. Arfin SM, Long AD, Ito ET, Tolleri L, Riehle MM, Paegle ES, •• Hatfield GW: Global gene expression profiling in Escherichia coli K12. The effects of integration host factor. J Biol Chem 2000, 275:29672-29684. This paper reports the first example of DNA microarray analysis with a nucleoid protein. The gene expression patterns of ihf+ and ihf E. coli strains are profiled.

Geanacopoulos M, Vasmatzis G, Lewis DE, Roy S, Lee B, Adhya S: GalR mutants defective in repressosome formation. Genes Dev 1999, 13:1251-1262.

30. Jordi BJ, Fielder AE, Burns CM, Hinton JC, Dover N, Ussery DW, Higgins CF: DNA binding is not sufficient for H-NS-mediated repression of proU expression. J Biol Chem 1997, 272:12083-12090. 31. Ueguchi C, Mizuno T: The Escherichia coli nucleoid protein H-NS functions directly as a transcriptional repressor. EMBO J 1993, 12:1039-1046. 32. Caramel A, Schnetz K: Lac and lambda repressors relieve silencing of the Escherichia coli bgl promoter. Activation by alteration of a repressing nucleoprotein complex. J Mol Biol 1998, 284:875-883. 33. Bokal AJ, Ross W, Gourse RL: The transcriptional activator protein FIS: DNA interactions and cooperative interactions with RNA polymerase at the Escherichia coli rrnB P1 promoter. J Mol Biol 1995, 245:197-207. 34. Xu J, Johnson RC: Fis activates the RpoS-dependent stationaryphase expression of proP in Escherichia coli. J Bacteriol 1995, 177:5222-5231. 35. McLeod SM, Xu J, Cramton SE, Gaal T, Gourse RL, Johnson RC: •• Localization of amino acids required for Fis to function as a class II transcriptional activator at the RpoS-dependent proP P2 promoter. J Mol Biol 1999, 294:333-346. The region on Fis that contacts the α-CTD to activate transcription at proP P2 is localized to a single loop near the DNA-binding region. This is the same patch that is responsible for activation at the rrnB P1 promoter, despite having different promoter architecture.

21. Schneider R, Travers A, Kutateladze T, Muskhelishvili G: A DNA •• architectural protein couples cellular physiology and DNA topology in Escherichia coli. Mol Microbiol 1999, 34:953-964. This paper shows that Fis reduces the amount of DNA gyrase in the cell by repressing transcription of both the gyrA and gyrB genes. Fis binding to the gyrA and gyrB promoters inhibits transcription initiation through different mechanisms.

36. Bokal AJ, Ross W, Gaal T, Johnson RC, Gourse RL: Molecular anatomy of a transcription activation patch: FIS–RNA polymerase interactions at the Escherichia coli rrnB P1 promoter. EMBO J 1997, 16:154-162.

22. Schröder O, Wagner R: The bacterial DNA-binding protein H-NS • represses ribosomal RNA transcription by trapping RNA polymerase in the initiation complex. J Mol Biol 2000, 298:737-748. The H-NS protein is shown to inhibit transcription at rrnB P1 by trapping RNA polymerase in a nonproductive complex. H-NS allows the polymerase to form an open complex, but only three-nucleotide-long products are predominantly formed.

38. Bartlett MS, Gaal T, Ross W, Gourse RL: Regulation of rRNA •• transcription is remarkably robust: FIS compensates for altered nucleoside triphosphate sensing by mutant RNA polymerases at Escherichia coli rrn P1 promoters. J Bacteriol 2000, 182:1969-1977. Mutant RNA polymerases are identified that, because of a requirement for higher concentrations of initiating nucleoside triphosphates (NTPs), form unstable promoter complexes. Fis activates these mutant RNA polymerases to a greater extent than it activates wild-type polymerase, suggesting that Fis stimulates transcription in part by lowering the apparent affinity of RNA polymerase for initiating NTPs.

23. Falconi M, Colonna B, Prosseda G, Micheli G, Gualerzi CO: Thermoregulation of Shigella and Escherichia coli EIEC pathogenicity. A temperature-dependent structural transition of DNA modulates accessibility of virF promoter to transcriptional repressor H-NS. EMBO J 1998, 17:7033-7043. 24. Olsen PB, Schembri MA, Gally DL, Klemm P: Differential temperature modulation by H-NS of the fimB and fimE recombinase genes which control the orientation of the type 1 fimbrial phase switch. FEMS Microbiol Lett 1998, 162:17-23. 25. Caramel A, Schnetz K: Antagonistic control of the Escherichia coli • bgl promoter by FIS and CAP in vitro. Mol Microbiol 2000, 36:85-92. This paper describes how the Fis protein represses transcription of the bgl promoter. Fis binds to two sites. One site overlaps the recognition site for RNA polymerase. At the other site, Fis competes for binding with the transcriptional activator protein, CRP.

37.

Muskhelishvili G, Buckle M, Heumann H, Kahmann R, Travers A: FIS activates sequential steps during transcription initiation at a stable RNA promoter. EMBO J 1997, 16:3655-3665.

39. Travers A, Muskhelishvili G: DNA microloops and microdomains: a general mechanism for transcription activation by torsional transmission. J Mol Biol 1998, 279:1027-1043. 40. Goosen N, van de Putte P: The regulation of transcription initiation by integration host factor. Mol Microbiol 1995, 16:1-7. 41. Bertoni G, Fujita N, Ishihama A, de Lorenzo V: Active recruitment of sigma54–RNA polymerase to the Pu promoter of Pseudomonas putida: role of IHF and alphaCTD. EMBO J 1998, 17:5120-5128. 42. Giladi H, Koby S, Prag G, Engelhorn M, Geiselmann J, Oppenheim AB: Participation of IHF and a distant UP element in the stimulation of the phage lambda PL promoter. Mol Microbiol 1998, 30:443-451.

Control of transcription by nucleoid proteins McLeod and Johnson

43. Sheridan SD, Benham CJ, Hatfield GW: Activation of gene expression by a novel DNA structural transmission mechanism that requires supercoiling-induced DNA duplex destabilization in an upstream activating sequence. J Biol Chem 1998, 273:21298-21308. 44. Browning DF, Cole JA, Busby SJ: Suppression of FNR-dependent •• transcription activation at the Escherichia coli nir promoter by Fis, IHF and H-NS: modulation of transcription initiation by a complex nucleoprotein assembly. Mol Microbiol 2000, 37:1258-1269. This is a good example of combinatorial control exerted by multiple nucleoid proteins. The authors show that FNR-dependent activation of the nir promoter is repressed by a collaboration of Fis, IHF and H-NS. 45. Gonzalez-Gil G, Kahmann R, Muskhelishivili G: Regulation of crp transcription by oscillation between distinct nucleoprotein complexes. EMBO J 1998, 17:2877-2885. 46. Bai Q, Somerville RL: Integration host factor and cyclic AMP receptor protein are required for TyrR-mediated activation of tpl in Citrobacter freundii. J Bacteriol 1998, 180:6173-6186.

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47. ••

McLeod SM, Xu J, Johnson RC: Co-activation of the RpoSdependent proP P2 promoter by Fis and cyclic AMP receptor protein. J Bacteriol 2000, 182:4180-4187. This paper describes the co-activation of the proP P2 promoter by both Fis and CRP in vivo and in vitro. Each activator is shown to directly interact with one of the α-CTD subunits of RNA polymerase. This is one of the first examples of combinatorial control in which the nucleoid protein directly contacts the RNA polymerase. 48. Colland F, Barth M, Hengge-Aronis R, Kolb A: Sigma factor •• selectivity of Escherichia coli RNA polymerase: role for CRP, IHF and Lrp transcription factors. EMBO J 2000, 19:3028-3037. The proteins CRP, Lrp and IHF are shown to preferentially inhibit transcription by Eσ70 as compared with Eσ38. This is a good example of how nucleoid proteins can cooperate to finely tune expression of a particular promoter. 49. Marschall C, Labrousse V, Kreimer M, Weichart D, Kolb A, Hengge-Aronis R: Molecular analysis of the regulation of csiD, a carbon-starvation-inducible gene in Escherichia coli that is exclusively dependent on σS and requires activation by cAMPCRP. J Mol Biol 1998, 276:339-353.