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19 “Antiparallel” Loops and Bacterial Nucleoid Structure

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Chapter 18

Transcriptional Regulation

by Nucleoid-Associated Proteins at Complex

Promoters in Escherichia coli

Douglas F. Browning, David C. Grainger, Meng Xu,

and Stephen J.W. Busby

Abstract  The expression of different Escherichia coli transcription units is

tightly regulated at the level of transcription initiation. Promoter strength is

fixed by DNA sequence elements, and changes in promoter activity are primarily

modulated by a combination of sigma factors and transcription factors, whose

activities are controlled by the growth environment. These factors all operate in

the context of bacterial chromatin which plays a key role in the expression of

many transcription units. Here we describe how IHF and FIS intervene directly

at some complex Escherichia coli promoters to bring about different regulatory

outcomes. At the nir operon promoter, the binding of IHF and FIS together

makes expression co-dependent on two transcription activators that are triggered by two different environmental signals. We discuss three different mechanisms by which FIS represses promoter activity, thereby down-regulating gene

expression during rapid growth. At the nrf operon promoter, FIS behaves as a

conventional repressor, at the ogt and acs promoters, FIS displaces the essential

activator, whilst, at the dps promoter, FIS jams RNA polymerase containing s70

in an inactive complex. In each of the three cases, derepression occurs when FIS

levels drop, as cell growth slows in response to nutrient limitation. Genomic

studies of the distribution of IHF and FIS across the Escherichia coli chromosome suggest that they intervene at many intergenic regulatory regions, and that

there may be little or no distinction between some nucleoid-associated proteins

and transcription factors.

Keywords  Nucleoid • nucleoid-associated proteins • transcription initiation

• promoter • RNA polymerase • activation • repression • integration host factor

(IHF) • factor for inversion stimulation (FIS) • histone-like nucleoid structuring

protein (H-NS)

D.F. Browning, D.C. Grainger, M. Xu, and S.J.W. Busby (*)

School of Biosciences, University of Birmingham, Birmingham, B15 2TT, UK

e-mail: s.j.w.busby@bham.ac.uk

R.T. Dame and C.J. Dorman (eds.), Bacterial Chromatin,

DOI 10.1007/978-90-481-3473-1_18, © Springer Science+Business Media B.V. 2010



D.F. Browning et al.

18.1 Transcription Regulation in Escherichia coli

Gene expression in E. coli is primarily controlled at the level of transcription initiation,

the point at which RNA synthesis begins. The enzyme responsible for RNA synthesis

is RNA polymerase and, predictably, it is the target for many regulatory factors. It

is estimated that there are ~3,000–8,000 RNA polymerase molecules per E. coli K-12

cell, according to growth conditions, which must be distributed between ~2,500 transcription units (Ishihama 1997; Karp et al. 2007). Everything we know about bacterial transcription tells us that this distribution is not even and, thus, the cell has to

regulate the binding of RNA polymerase across its chromosome prudently.

The potential activity of any promoter is set by its DNA sequence elements

whilst changes in response to the environment are principally mediated by transcription factors and sigma factors (Miroslavova and Busby 2006; Browning and

Busby 2004). The E. coli genome encodes over 250 transcription factors that exert

their effects by binding at specific promoters and activating or repressing transcription (Perez-Rueda and Collado-Vides 2000). The activities of most transcription

factors are regulated in response to environmental cues, usually by ligand binding,

by covalent modification, or by changes in their level. A small number of transcription factors, termed “global” regulators (e.g. the cyclic AMP receptor protein,

CRP), influence the expression of a large number of transcription units. Conversely,

a large number of “specific” transcription factors (e.g. the Lac repressor) each

affect the expression of a small number of transcription units. The expression of

many transcription units is regulated by a combination of both global and specific

transcription factors and this allows bacteria to differentially regulate the gene

expression in response to combinations of different environmental stimuli.

The E. coli genome encodes seven sigma factors that exert their effects by binding

to RNA polymerase, thereby steering it to specific subsets of promoters. Sigma factors thus play a pivotal role in managing the chromosome-wide distribution of the

transcriptional machinery (Helmann and Chamberlin 1988; Murakami and Darst

2003). E. coli, like all bacteria, contains one major s factor (s70), responsible for the

recognition of most promoters. Each of the six alternative s factors is responsible for

transcription of a subset of genes, usually in response to a stress. Thus, for example,

the stationary phase s factor (s38) controls the expression of many proteins needed

for the long-term survival of non-growing cells (Ishihama 1997).

Some promoters are active in the absence of additional factors and when the genes

under their control are not required, they are silenced by transcription repressors.

Repressor proteins reduce transcription initiation at target promoters and the textbook

view is that this is simple to understand. Thus, at some promoters, a single repressor is

involved and its binding prevents promoter recognition by RNA polymerase (Choy and

Adhya 1996). In these instances, the repressor binding site is located at, or close to, the

core promoter elements. Note that, in some cases, the repressor may not prevent binding

of RNA polymerase but, rather, interferes with post-recruitment steps in transcription

initiation, sometimes ‘jamming’ RNA polymerase and preventing it from initiating or

elongating a transcript (Rojo 2001). At some other promoters, multiple repressor molecules bind to promoter-distal sites, and repression is caused by DNA looping, which

shuts off transcription initiation within the looped domain (Semsey et al. 2005).

18  Transcriptional Regulation by Nucleoid-Associated Proteins


Most promoters lack a good match to the consensus elements for RNA polymerase binding, and many of these require ancillary proteins, known as transcription

activators, to function. At some promoters, activation of transcription is simple, and

involves the action of a single activator (Busby and Ebright 1994; Rhodius and

Busby 1998). Two general mechanisms are used for ‘simple’ activation. In most

cases, the activator binds to a target located immediately upstream of the promoter

elements and recruits RNA polymerase by directly interacting with a target usually

in the RNA polymerase a or s subunits. In a small number of cases, the activator

alters the conformation of the target promoter, to enable the interaction of RNA

polymerase with the promoter -10 and -35 elements (Brown et al. 2003).

Most bacterial promoters are regulated by more than one transcription factor and

this permits regulatory input from multiple environmental cues (Martinez-Antonio

and Collado-Vides 2003). Different mechanisms have evolved for integrating the

effects of different transcription factors at such complex promoters. Thus, at promoters that are co-dependent on two or more activators, several complicated

mechanisms have been discovered, involving the repositioning of one activator by

another, independent activator-RNA polymerase contacts, or anti-repression by an

activator (Barnard et al. 2004; Browning and Busby 2004). For mechanisms involving repositioning, the role of the secondary activator is to reposition the primary

activator from a location where it is unable to activate transcription, to a location

where it can activate transcription. This repositioning can involve either shifting the

primary activator from one DNA site to another, or altering the conformation of the

DNA to allow the primary activator to interact with RNA polymerase. For example,

at the E. coli malK promoter, CRP repositions MalT from a non-productive high

affinity binding site to a location where it can interact with RNA polymerase

(Richet et al. 1991). In contrast, at the narG promoter, IHF induces a DNA bend

that permits upstream-bound NarL to activate transcription (Schroder et al. 1993).

A different mechanism operates at promoters where two activators must each make

contact with RNA polymerase for transcription. In these cases, one activator-RNA

polymerase contact is insufficient. In most cases studied to date, the two activators

bind independently at the target promoter. For example, at the E. coli ansB promoter, activation depends on CRP and FNR, bound independently at positions -91

and -41 respectively, making separate contacts with RNA polymerase (Scott et al.

1995). Finally, in some cases, the role of the second activator is not to activate

directly, but rather to prevent the action of a repressor that is interfering with the

function of the primary activator. In these cases, the second activator behaves as an

anti-repressor rather than a true activator (Browning and Busby 2004).

18.2 Nucleoid-Associated Proteins Can Participate at Promoters

The mechanisms of transcriptional regulation, described above, all operate in the

context of the bacterial chromosome, which is folded into a compact structure,

known as the nucleoid. Clearly, the distribution of RNA polymerase between different ­regulatory regions will be affected by this compaction. Most attention has


D.F. Browning et al.

focused on the nucleoid-associated proteins that, alongside supercoiling and

macromolecular crowding, induced by RNA and other proteins, are involved in

­maintaining compaction (Thanbichler et al. 2005; Dame 2005). In a seminal study,

Akira Ishihama and colleagues defined 12 different nucleoid-associated proteins:

HU, H-NS, StpA, FIS, IHF, CbpA, CbpB, Dps, Lrp, DnaA, Hfq and IciA (Talukder

et al. 1999). Most of these induce conformational changes in DNA upon binding

in vitro (e.g. bending or bridging) but, in many cases, the functional significance of

the changes in vivo are unclear. All of these proteins bind DNA non-specifically,

but some also have higher affinity for specific sequences. In their study, Ishihama

and colleagues raised antibodies against each of the 12 nucleoid-associated proteins,

and their levels were quantified during different stages of cell growth. Whilst the

levels of each different nucleoid-associated protein varied throughout growth, each

of the nucleoid-associated proteins was present at over 10,000 copies per cell at

some stage (with the exception of Lrp, DnaA and IciA). The most dramatic changes

during cell growth were found with FIS, Dps and CbpA. Rapidly growing cells

contain over 50,000 molecules of FIS and near zero levels of Dps and CbpA. When

cell growth slows in post-exponential phase, FIS levels drop and Dps levels rise.

When cells stop growing and arrive in stationary phase, FIS levels are near zero and

maximum amounts of Dps (>20,000 molecules) are found. At this point, CbpA is

induced and reaches ~12,000 molecules per cell. Figure 18.1 illustrates the changes

in the levels of several of the nucleoid-associated proteins as E. coli cells pass from

exponential to stationary phase and summarises some of the principal properties of

each factor.

An important point is that many of the abundant nucleoid-associated proteins also

act as transcription factors (McLeod and Johnson 2001), and many of these functions are described in this section of this book. These proteins regulate transcription

initiation by binding to specific sites at target promoters. Thus both IHF and FIS can

function to activate transcription initiation, behaving as ‘simple’ transcription factors

by recruiting RNA polymerase to target promoters. However, both IHF and FIS have

also been reported to activate transcription by binding upstream of target promoters

and inducing conformation changes in the downstream sequence. Thus, IHF binding

upstream of the ilvG promoter alters the conformation of the ilvG -10 element,

thereby activating transcription (Sheridan et al. 2001), and similar effects have been

reported during the activation of the leuV promoter by FIS (Opel et  al. 2004).

Opening of the DNA duplex by negative supercoiling plays a key role in this process

and is often referred to as supercoiling-induced DNA duplex destabilization.

At other promoters, nucleoid-associated proteins facilitate the action of activators

or repressors. Hence at many promoters whose activity depends on a direct interaction

between an upstream-bound transcription activator and RNA polymerase containing s54, DNA bending induced by IHF binding between the activator and RNA

polymerase is essential to ensure that the interaction occurs (Wigneshweraraj et al.

2008). Similarly, HU binding is required for GalR-dependent repression of galETK

expression. In this case, HU-induced DNA bending permits an interaction between

upstream-bound and downstream-bound GalR dimers that is essential for efficient

repression (Roy et al. 2005) (Chapter 17).

18  Transcriptional Regulation by Nucleoid-Associated Proteins

Molecules per cell


















HU binds non-specifically to bend

and compact the DNA at low

concentrations whilst coating the

DNA to form filaments at high



FIS binds to a degenerate AT rich

DNA target site, bending the DNA

by 50 to 90 degrees. FIS binding

sites are often clustered in

sections of non-coding DNA.







H-NS binds to AT rich DNA and

can form bridges between distal

segments of the chromosome,

looping the intervening DNA.

binds DNA with a


comparatively high degree of

sequence specificity to elicit a

160 degree bend in the



Dps binds DNA uniformly with no

apparent sequence specificity,

driving the chromosome of

starved cells into a crystalline


Fig. 18.1  The principal nucleoid-associated proteins of E. coli. (a) The figure depicts the fluctuations

in the levels of the major nucleoid-associated proteins that occur with changes in growth phase (data

taken from Talukder et al. 1999). (b) Panels i, ii, iii, iv and v illustrate the main impact of HU, H-NS,

IHF, FIS and Dps proteins respectively on chromosome structure. The various nucleoid-associated

proteins are shown as spheres and DNA is shown as a solid line


D.F. Browning et al.

18.3 Repression and Antirepression at the Escherichia coli nir

Operon Promoter

Nucleoid-associated proteins play quite subtle roles at some complex promoters

and perhaps the best example of this is found at the E. coli nir operon regulatory

region. The nir operon encodes a cytoplasmic nitrite reductase that catalyses the

NADH-dependent reduction of nitrite ions to ammonia (Harborne et  al. 1992).

Transcription from a single startpoint is controlled by a promoter upstream of the

nirB gene (Jayaraman et al. 1988). At this promoter, H-NS is an overall repressor,

whereas FIS and IHF function in concert to confer codependence on two activators

(Browning et al. 2000).

Early studies had shown that the nir promoter is optimally active when cells are

grown in anaerobic conditions in the presence of nitrite or nitrate ions, and also that

higher activities are found in rich media (Bell et  al. 1990; Page et  al. 1990).

Induction in anaerobic conditions is due to the activity of FNR, a global transcription activator responsible for the induction of over 100 different transcription units

in E. coli in response to low oxygen levels (Browning et al. 2002a). FNR dimerisation, and hence specific binding at target promoters, requires the formation of an

iron-sulphur cluster that is destroyed by oxygen. However as oxygen levels

decrease, the iron-sulphur cluster forms, FNR dimerises and binds to target sites,

and is then able to activate transcription. In most cases, including the nir promoter,

the FNR dimer binds to a target near position −41 and functions as a simple activator

of transcription initiation (Wing et al. 1995; Fig. 18.2).

In addition to being dependent on FNR, expression from the nir promoter is

dependent on activation by NarL (or its homologue, NarP), that are response regulators, which are both activated by nitrate or nitrite ions via the membrane bound

NarX and NarQ sensor kinases. It is this dependence that ensures that nir operon

induction is coupled to the presence of nitrate or nitrite ions in the environment, as

well as the lack of oxygen (Tyson et al. 1993, 1994). NarL binds as a dimer to a DNA

site just upstream of the DNA site for FNR at the nir promoter (Fig. 18.2) and has

no effect on FNR binding. Since FNR is perfectly able to function as an activator

alone, this raises the question of why NarL is needed. The explanation for this follows from the observation that the sequences upstream of the DNA site for FNR

carry targets for FIS at position −142 and for IHF at positions −88 and −115

(Browning et al. 2000, 2004a). Genetic experiments show that FNR-dependent activation is suppressed by the binding together of FIS at position -142 and IHF at position −88. In vitro experiments with purified components demonstrate that FNR is

able to activate open complex formation at the nir promoter and that the complex is

destabilized by binding of FIS and IHF. The suppression mediated by FIS and IHF

is relieved upon binding of NarL, which displaces IHF from position −88 (Fig. 18.2).

Thus here, the role of the second activator, NarL, is not to activate directly, but rather

to prevent FIS and IHF from interfering with the function of the primary activator

(FNR). Hence, point mutations that destroy the DNA sites for FIS and IHF at positions -142 and -88 respectively, release the requirement for NarL, and convert the

nir promoter into a simple FNR-dependent promoter (Wu et al. 1998).

18  Transcriptional Regulation by Nucleoid-Associated Proteins


a Anaerobic.

















b Anaerobic plus NO2− / NO3−.






NarL /P

−88 H-NS −69.5





−15.5 +1


Fig. 18.2  Transcription regulation at the E. coli nir operon promoter. (a) Anaerobic conditions.

Transcription initiation is dependent on FNR. The binding of FIS to FIS I and IHF to IHF I inhibits

FNR-activated transcription (−ve), whilst IHF binding to the lower affinity IHF II site stimulates

transcription (+ve). The mechanisms by which upstream bound FIS and IHF modulate FNRdependent transcription initiation at the nir promoter are not understood. In addition, the binding of

H-NS and FruR can down-regulate transcription initiation at the nir promoter. (b) Anaerobic conditions plus nitrite or nitrate. The binding of NarL (or its homologue, NarP) displaces IHF from IHF

I, thus counteracting the repression mediated by IHF and FIS, and enabling maximal FNRdependent transcription. In contrast, NarL and NarP have little effect on repression by H-NS and

FruR, whose effects are modulated by temperature and nutrient richness respectively

Further analysis has revealed a complication: IHF binding to a weaker second

site at position −115 promotes rather than suppresses FNR-dependent transcription

activation (Browning et  al. 2004a). Thus, IHF bound at two adjacent sites (at

positions −88 and −115) have opposite effects on nir promoter activity (Fig. 18.2).

Interestingly, the relative IHF binding affinities at the two sites differ in diverse

enteric bacteria and this sets the basal level of FNR-dependent activation in the

absence of NarL (Browning et  al. 2008). Hence the basal NarL-independent

activity of the nir promoter is increased by mutations that improve IHF binding at

position −115 and decreased by mutations that destroy binding (Browning et  al.

2004a). A second complication is that H-NS globally down-regulates nir promoter

activity and, thus, three different nucleoid-associated proteins participate directly in

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