DNA Looping and Transcription Regulation

Dale EA Lewis, National Institutes of Health, Bethesda, Maryland, USA Sankar Adhya, National Institutes of Health, Bethesda, Maryland, USA
DNA loops, formed by the association of two proteins while bound to spatially separated specific DNA sites, control gene transcription negatively or positively. DNA looping sometimes needs an architectural protein, which acts by bending the DNA segment that loops.

Secondary article
Article Contents
. Introduction . Repression by DNA Looping Requires Multipartite Operators . Bacterial Enhancers: the gln Operon . DNA Looping in the Bacterial Nucleoid . Relating DNA Looping to Transcription Regulation in Eukaryotes

Introduction
When two deoxyribonucleic acid (DNA)-bound proteins are in contact with each other, the intervening DNA forms a loop. Although the term DNA looping was rst proposed to explain the role of transcriptional repressors, whose binding sites were bipartite in nature and spatially separated on DNA (Irani et al., 1983; Dunn et al., 1984), the formation of a DNA loop, although transient, was already surmised to occur in site-specic recombination during the excision of prophage DNA from host chromosomes (Campbell, 1962). DNA looping is now found almost routinely to be a component of DNAmultiprotein complexes involved in transcription regulation, sitespecic recombination, replication initiation, and chromosome condensation and compaction. In most cases, DNA looping aids dispersed DNA-bound proteins to make additional proteinprotein or DNAprotein contacts, which are otherwise prohibiting. As described below, DNA looping serves three biochemical purposes in transcription regulation. First, looping acts as a tether, causing a regulatory protein bound to a site to collide repeatedly with another regulatory protein or ribonucleic acid (RNA) polymerase bound to a dierent site located hundreds of base pairs (bp) away. This arrangement promotes cooperative binding by increasing the local concentration of one protein relative to that of the other. Second, the intervening DNA assumes an altered conformation, which helps or hinders the action of RNA polymerase toward the promoter within the contorted region (DNA allostery). Third, essential regulatory proteins are sequestered by DNA looping and prevented from acting. In this article, we will describe the structure and function of DNA loops in transcription regulation. The premier step of regulation of transcription is at the level of initiation. Regulatory proteins that modulate the initiation step act either as repressors (negative control) or as activators (positive control) of RNA polymerase action at promoters. Although regulatory proteins sometimes achieve their goals indirectly a repressor antagonizing the activity of an activator and vice versa they usually

inuence RNA polymerase by binding to specic DNA sites, which are in cis to the promoters they regulate. The binding site of a repressor is called an operator (o), and that of an activator is usually referred to as an activation site (as). The nomenclature for the regulatory proteins and the DNA sites, however, is of only historical signicance; many regulators act as a repressor in one context and as an activator in another. Because the DNA binding sites of the initially studied repressors and activators were contiguous or even overlapping with their cognate promoter, it was easy to conceive models that explained how a regulator could modulate RNA polymerase activity. For example, a DNA-bound repressor hinders RNA polymerase binding or a DNA-bound activator directly interacts with RNA polymerase and helps transcription. Later discoveries revealed that the DNA controlling elements are sometimes multipartite and are located at distant sites, necessitating the proposal of other molecular mechanisms by which repressors and activators act. Several model systems of repression and activation from distant DNA sites involving DNA looping are described below.

Repression by DNA Looping Requires Multipartite Operators

Two or more spatially separated operator loci are needed to exert tight negative control in several operons. The operators usually encompass the promoter, and one of them may be located adjacent to the promoter it regulates. In these examples, two operator-bound repressors associate, as if they were adjacent, but in the process cause the intervening DNA region to form loops.

The gal operon

Two partially overlapping promoters, P1 and P2, transcribe the gal operon, which contains genes encoding enzymes of d -galactose metabolism (Figure 1a). Transcrip1

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DNA Looping and Transcription Regulation

+1

P1 hbs OI +53.5 galE

OE (a) 60.5 5

P2

+6.5

OE

GalR

(b)

OI

Figure 1 The gal promoter in Escherichia coli. (a) The location of the two overlapping promoters P1 ( 1 1) and P2 ( 2 5), two operators, OE ( 2 60.5) and OI ( 1 53.5), HU-binding site (hbs) at 1 6.5, and the 5 region of the first structural gene, galE. (b) Looping-mediated repression of the gal promoters by GalR GalR interactions (red) in the presence of HU (green). The elements are not drawn to scale.

GalR

HU

tion from P1 (start site of transcription at position marked 1 1) and P2 (start site at 2 5) are regulated by Gal repressor (GalR), which is one of several regulatory proteins that modulate P1 and P2. GalR, the product of an unlinked gene galR, acts by binding to two operators, OE (position 2 60.5) and OI (position 1 53.5), that ank the two promoters. OI is located within the structural gene, galE. The sugar d -galactose, when present, inactivates the repressor, inducing gene transcription. Because of the requirements of dual operators, it was proposed that, in the absence of d -galactose, the two DNA-bound repressor dimers associate to form a 113 bp DNA loop of the intervening segment (Figure 1b). This structure brings about strong repression of both P1 and P2. Incidentally, when a GalR dimer binds to OE without forming a DNA loop, the regulator becomes bifunctional. It enhances transcription from P2 and reduces transcription from P1 (Figure 2, lanes 1 and 2). The bifunctional role of GalR is described in the article on Repression Mechanism. The following genetic analyses support the model of DNA looping-mediated repression. Operator conversion The functional relationship between OE and OI has been studied by converting one of them (by site-directed mutagenesis) to a dierent operator which is the binding site of Lac repressor (LacI), to generate a genotype OGE 2 OLI or OLE 2 OGI (Table 1). OG and OL refer to operators that bind to GalR and LacI, respectively. In both cases the promoters are not repressed, even though both GalR and LacI are present simultaneously in the cell, showing that mere occupation of the two operators is not sucient for repression. In contrast, conversion of both operators to OL restores normal repression in the presence of LacI (Table 1, bottom row). Thus, the gal operon is
2

Figure 2 Effect of Gal repressor (GalR) and the histone like protein HU on the repression of the gal promoters (P1 and P2) in a purified system containing DNA template, RNA polymerase, GalR and HU. Lanes 1 6, DNA template with wild-type operators; lanes 7 8, mutant OI; and lanes 9 10, mutant OE. RNA1 transcripts are from a control promoter. Adapted from Aki and Adhya (1997).

Table 1 Operator conversions in the gal operon Repressor present in cell Operator genotype GalR + LacI R D D R GalR R D D D LacI D D D R None D D D D

R and D represent repression and derepression, respectively, of the gal operon in the presence of GalR, LacI, or both. Adapted from Haber and Adhya (1988).

repressed if both operators are occupied by similar repressors. The most likely explanation is that the repression requires a communication (interaction) between the two DNA-bound repressors; the interaction is not possible between heterologous repressors. Repressor mutants that bind to operators, but do not repress Both GalR and LacI mutants have been described which normally bind to their corresponding operators, OGE 2 OGI for GalR and OLE 2 OLI for LacI, but fail to repress the gal promoters. The behaviour of such mutant

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DNA Looping and Transcription Regulation

repressors is easily explained by the DNA looping model. These mutants bind to DNA, but are defective in making repressorrepressor contacts, which are needed for DNA loop formation. Thus, DNA looping, not mere DNA binding, is needed for repression. For the LacI mutants, it has been shown that they can form dimers that are capable of binding to DNA, but fail to associate to form the tetramers needed for DNA looping. Figure 3 shows direct evidence of DNA looping as observed by electron microscopy. Unlike LacI and AraC (see below), GalRmediated DNA looping has more complex requirements which, together with the nonlooping GalR mutants, are described below. Although the genetic experiments support the existence of DNA looping, GalR binding to OE and OI is not sucient for DNA looping and repression of both P1 and P2 in a puried system; an additional factor, one of the bacterial histone-like proteins named HU, is required (Aki and Adhya, 1997). HU, a heterodimer, acts as a cofactor of GalR in repressing both P1 and P2 (Figure 2, lane 4). The GalR/HU-dependent repression of the two gal promoters, as expected, is sensitive to the inducer, d -galactose (Figure 2, lanes 5 and 6). In addition, GalR/HU-mediated repression requires the binding of GalR to both OE and OI. Mutation of either locus does not bring about repression of both promoters (Figure 2, lanes 710). Consistently, deletion of the genes encoding both subunits of HU derepresses the gal promoter in vivo (Figures 4a,c). HU binds to the gal DNA loop centred at position 1 6.5. HU binding depends on the interaction of GalR to both OE and OI, and thus is sensitive to d -galactose. In addition, HU binding enhances simultaneous GalR binding to OE and OI. This is an example of tripartite cooperativity, although GalR binding to OE and OI in the absence of HU is noncooperative. How does HU binding to region 1 6.5 help DNA looping by GalR? In principle, there are two ways in which HU can act as a cofactor in GalR-mediated DNA loop formation. (1) HU binding promotes DNA looping by bending the DNA and thereby stabilizing a weak interaction between two operator-bound GalRs (Figure 5a). (2) HU helps interaction between two GalRs by HUGalR contacts (Figure 5b). The tripartite cooperativity between HU and two GalRs in DNA binding seems to support this

model. The DNAmultiprotein complex containing the DNA loop is called a repressosome. Repressosome formation The formation of the repressosome requires supercoiled DNA. In the presence of coumermycin, an inhibitor of DNA gyrase, DNA supercoiling is relaxed, resulting in the failure of DNA looping-mediated repression of the gal promoters (Figure 4b). The structure of the repressosome has been analysed by isolating and characterizing galR mutants in which single amino acid substitutions in GalR lead to defects in loop formation but not in DNA binding (Geanacopoulos et al., 1999). All the mutations of GalR that are unable to form repressosome but bind to the DNA occur in a circumscribed surface of the C-terminal domain of the GalR structure, which was modelled after the known structures of the homologous proteins, PurR and LacI. The GalR segment identied by the mutations denes the interface in GalRGalR interactions. Mechanism of repression by DNA looping The critical DNA sequences of P1 and P2 are in the looped segment of the DNA. DNA looping prevents strand separation at the two gal promoters. Deoxyribonuclease (DNAase) I protection analysis also shows that DNA looping prevents open complex formation at the gal promoters. Whatever the mechanism by which DNA looping inhibits open complex formation at the gal promoters, it is clear that the contorted DNA structure makes the promoters inadequate for transcription. When the size of the loop in the gal DNA is increased by engineering from its normal size of 113 bp (11 DNA helical turns) to more than 300 bp, transcription repression does not occur, showing that the inhibitory contortion of DNA goes away in a large DNA loop.

The lac operon

LacI negatively regulates transcription from the lac operon, which encodes enzymes for lactose catabolism in Escherichia coli by binding to three operators, O1, O2 and O3, located at positions 1 11, 1 402 and 2 92, respectively, relative to the transcription start site (Figure 6a). The

Figure 3 Electron micrograph of DNA looping by LacI bound to two lac operators engineered into OE and OI loci of the gal operon of Escherichia coli. The arrows point to the DNA loops (Mandal et al., 1990).
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DNA Looping and Transcription Regulation

300 -Glucuronidase activity (units mL1) 250 +I 200 150 100 50 0 50 0 0.2 0.4 OD 600 nm (a) 0.6 0.8 I

300 250 200 150 100 50 0 50 0 0.2 0.4 OD 600 nm (b) 0.6 0.8 C +C

300 250 200 150 100 50 hupB 0 50 0 0.2 0.4 OD 600 nm (c) 0.6 0.8 hupA

hupA hupB

Figure 4 Repression of galP2 in vivo. The strength of the promoter P2 was monitored by measuring the level of a reporter gene (gusA) product bglucuronidase. (a) In the presence (green) and absence (black) of inducer D-galactose (I). (b) In the presence (red) and absence (black) of coumermycin (C). (c) In the presence and absence of HU; blue, mutated in hupA and hupB genes encoding the a and b subunits of HU, respectively; green, mutated in hupA; red, mutated in hupB. The arrow indicates the point at which D-galactose or coumermycin was added. Adapted from Lewis et al. (1999).

hbs

OE

GalR

GalR

OI

(a)

hbs

Ga lR

anity of LacI for O1 is much greater than that for O2 or O3. Binding of LacI to O1 is an absolute requirement for repression, which is considerably enhanced by the presence of O2 and O3 (Table 2; Figure 6b,c). Unlike GalR, LacI can bind to O1 and O3 or O2 cooperatively without the requirement of another protein, resulting in loop formation of the intervening DNA. This is because LacI is a strong tetramer and can bring together two operators spatially separated by hundreds of base pairs. The rate of ligation of 100200 bp DNA molecules by DNA ligase to small circles depends strongly on the helical phase of the two ligating ends, because misorientation of the helical phase by the insertion of 5 bp (half of a DNA helical turn) sharply decreases the rate of circle formation. Similarly, loop formation by LacI, as expected, is dependent upon the relative angular orientation of the two operators on the DNA helix (Kra mer et al., 1988). Looping occurs more eectively when they are on the same face (i.e. separated by an integral number of DNA helical turns). Direct evidence of DNA looping by LacI has been visualized by electron microscopy. It has been suggested that the binding of LacI to O1 represses transcription initiation from the lac promoter by competing with the binding of RNA polymerase to the promoter, i.e. inhibiting closed complex formation (see article on Repression Mechanism). In this model, O2 and O3 enhance repression by increasing the binding of LacI to O1 as a result of cooperativity.

HU HU

Ga lR

OE

OI
Figure 5 Models of HU in DNA looping. GalR (red), HU (green). (a) HU acts as a DNA bender; (b) HU acts both as a bender and an adaptor. hbs, HUbinding site.

(b)

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DNA Looping and Transcription Regulation

Table 2 Repression of the lac promoter by LacI lac operator present O1 O2 O3 O1 O2 O1 O3 O2 O3 O1 O2 O3 None
Adapted from Oehler et al. (1990)

Relative amount of b-galactosidase synthesis 1 1.7 3 684 72 1300 1300 1300

+1

P lac O1 +11 O2 +402 lacZ

O3 (a) 92

in DNA looping, which blocks access of RNA polymerase to the promoter. When liganded to l -arabinose, the inducer of the ara operon, one monomer of AraC remains bound to I1 and the other monomer switches binding from O2 to I2 ( 2 43), the latter being contiguous to I1 (Figure 7b). Liganded AraC bound to the adjacent I1 2 I2 sites activates the promoter. The activation occurs by a physical contact between the I1 2 I2-bound AraC and RNA polymerase at the promoter. The following evidence supports the model that repression of PBAD by AraC bound to O2 and I1 involve a DNA loop formation. AraC binds to O2 only in the presence of I1, suggesting cooperative binding. DNA helical phasing experiments, as described above, suggested DNA looping in the ara operon. Insertion of 5 bp at a nonessential site between O2 and I1 eliminates repression, but the addition of a further 6 bp to make the insert a full DNA helical turn restores repression. As spacing between O2 and I1 is further increased in a stepwise fashion, repressibility oscillates with a period of 11 bp, the equivalent of a full DNA helical turn (Figure 8).

O2 LacI

O1 LacI

Change in AraC during DNA looping and unlooping Why does an unliganded AraC bind to I1 and O2 to make a loop, whereas a liganded AraC prefers to bind to the tandem I1 I2 sites? Each of the three sites, O2, I1 and I2, constitutes half of a dyad symmetry for the binding of AraC dimer through a helix-turn-helix motif in each monomer. AraC can bind to each adjacent half-site in inverted or direct repeat orientation, or when the half-sites are separated by integral DNA helical turns (Carra and Schleif, 1993). This is because of rotational freedom of the two DNA-binding helix-turn-helix domains from the remainder of the AraC dimer in the absence of the inducer. The relative anities of unliganded and liganded AraC toward O2, I1 and I2 are similar. If the I2 sequence is replaced by O2, providing unliganded AraC an opportunity to bind to the adjacent half-sites (I1 O2), the looping preference is still retained: the AraC dimer, while bound to I1, prefers to bind to a spatially separated site. How does l-arabinose bring about this change? It has been proposed that in the unliganded state of AraC, the Nterminal region, which contains both dimerization and l arabinose-binding domains, interacts with the C-terminal DNA-binding domain to generate a protein conformation that prefers a geometry that is potent for DNA looping. Ligand binding changes the dimerization interfaces, thereby weakening the CN interactions and allowing the Cterminal domain to occupy the adjacent site (Figure 7b). In other words, in the absence of l-arabinose, the N-terminal region sets up the C-terminal domain to favour binding to distal sites, whereas in the presence of ligand the constraint is released to provide the exibility needed for binding to tandem sites.
5

LacI

(b)

O1

(c)

Figure 6 Repression of the lac promoter. (a) The locations of the lac operators, O1 ( 1 11), O2 ( 1 402) and O3 ( 2 92) with respect to the transcription start site of 1 1 for the lac promoter (Plac). The 5 region of the lacZ gene is included. (b) DNA looping by the binding of LacI to O1 and O2. (c) DNA looping by the binding of LacI to O1 and O3.

The ara operon

A more interesting example of DNA looping-mediated repression without the need for another factor is the regulation of the ara operon, which encodes enzymes of l arabinose catabolism, in E. coli. Three loci called O2, I1 and I2, are involved in the regulation of the ara operon by a regulator, called AraC (Figure 7a). The AraC protein represses the ara promoter by binding to O2 ( 2 275) and I1 ( 2 64), separated by 211 bp and forming a loop of the intervening DNA segment. Mutations in DNA sites that reduce repression of the promoter in the absence of l arabinose map only in O2 and I1. Unlike GalR and LacI, which form DNA loops as tetramers, AraC makes a DNA loop as dimers. Each subunit of AraC dimer is capable of binding to a DNA site. During repression of the arabinose promoter, an AraC dimer simultaneously binds to O2 and I1, resulting

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LacI O3

DNA Looping and Transcription Regulation

275

64

43

+1

P BAD araB

O2 (a)

I1

I2

O2 AraC AraC (+) L-Arabinose +1 P BAD () L-Arabinose araB Activation of PBAD O2 AraC AraC I1 I2 P BAD araB

I1 (b)

I2

Looping repression of PBAD

Figure 7 DNA looping at the ara promoter (PBAD). (a) The location of half-sites O2 ( 2 275), I1 ( 2 64) and I2 ( 2 43) with respect to the transcription start site of promoter. The 5 region of the araB structural gene is included. (b) The binding of AraC (red) to O2 and I1 in the absence of inducer (L-arabinose) forms a DNA loop. The binding of AraC (green) to I1 and I2 in the presence of inducer destroys DNA looping and activates PBAD.

600

300

0 100

120

140

160

180

200

220

240

Distance between O2 and I2 (base pairs)

Figure 8 Periodicity in the repression of the promoter at the ara operon. The periodicity oscillates between repression and derepression depending on the distance between O2 and I1 Adapted from Lee and Schleif (1989).

Bacterial Enhancers: the gln Operon

The intrinsic activity of many promoters is very low: they are naturally defective and resurrected by activators in response to cellular demands. As mentioned above, the activators act by binding to cognate DNA sites, which are usually found in bacteria close to or partially overlapping with the promoter. Occasionally, the activator binding sites are located hundreds of base pairs upstream of the promoter. An activation site located far from the promoter, commonly found in eukaryotic chromosomes, is called an enhancer element. A unique class of activators that bind to enhancers is found in Eubacteria. These activators function in conjunction with promoters recognized only by the RNA polymerase holoenzyme, which contains a specialized sigma factor, s54. s54 holoenzyme acts on promoter elements located at 2 12 and 2 24 relative to 1 1, instead of 2 10 and 2 35 promoter regions, which are recognized by the major s70 holoenzyme.
6

The best studied activator in this group is the nitrogen regulatory protein, NtrC (also known as NRI). This protein activates transcription from the major promoter, p2, of the glnALG operon, which encodes enzymes of glutamine biosynthesis in E. coli and Salmonella typhimurium. NtrC acts by binding to two DNA sites, which compose the enhancer, and are located at 2 110 and 2 140 upstream of the transcription start site (Figure 9a). For a high level of activation, both enhancer sites are needed. NtrC is an apoactivator which is phosphorylated by NtrB (also called NRII) during nitrogen limitation to become an activator (NtrC  P) (Ninfa and Magasanik, 1986). When ammonia is in excess, NtrB, PII (the product of glnB gene) and adenosine triphosphate (ATP), acting together, dephosphorylate NtrC  P. Therefore, NtrB regulates the activity of NtrC. NtrC  P has a half-life of only 3.6 5.0 min. Biochemically, s54 holoenzyme binds to the promoter on its own to form a closed complex. NtrC  P acts by catalysing the isomerization of the closed complex to an open form at the p2 promoter in the presence of ATP. This clearly demonstrates that an enhancer is not necessary for recruiting RNA polymerase, but helps at a post-RNA polymerase binding step of transcription initiation.

Derepression of promoter

Activation by DNA looping

How does bound NtrC located at a distance from bound RNA polymerase stimulate transcription of p2 promoter? The ability to act from a distance is one of the features of a protein bound at an enhancer site. Transcription activation from the p2 promoter is not aected when the enhancer site for NtrC is moved upstream or downstream by more than 1000 bp. The enhancer-bound NtrC  P physically contacts the promoter-bound RNA polymerase

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DNA Looping and Transcription Regulation

+1 140 110 NtrC-binding sites (a) NtrC~P +1 (b) 132 NifA-binding site (c) IHF (d) NifA
54 54

p2 glnA

p2 glnA +1

RNAP

56 IHF-binding site

p nifH

+1

p nifH p2 +1 glnH

RNAP

121 108 NtrC-binding sites (e) IHF (f)

42 IHF-binding site

NtrC~P +1
54

p2 glnH

RNAP

Figure 9 DNA looping at the promoters of glnA, glnH and nifH. (a) The enhancer binding sites of the nitrogen regulatory protein NtrC are located at 2 140 and 2 110 upstream of the p2 promoter of the glnALG operon of Escherichia coli. (b) DNA looping for activation of p2 by the interaction of NtrC  P (green) bound to the enhancer sites and s54 RNA polymerase (RNAP) (red) bound to the promoter. (c) The spatial relationship of the enhancer site ( 2 132) and the integration host factor (IHF) site ( 2 56) with respect to the promoter (p) of the nifHDK operon. (d) DNA looping at the nifHDK operon by the binding of IHF (blue), NifA (green) and s54 RNA polymerase (red) to their respective binding sites. (e) The spatial relationship of enhancer sites ( 2 121 and 2 108) and the IHF site ( 2 42) with respect to the promoter (p2) of the glnHPQ operon. (f) DNA looping at the glnHPQ operon by the binding of IHF (blue), NtrC  P (green) and s54 RNA polymerase (red) to their respective binding sites.

Klebsiella pneumoniae and of the glutamine permease operon, glnHPQ, in E. coli (Hoover et al., 1990). In the nifHDK operon, the NifA activator binds to a distally located enhancer and activates transcription at the promoter in the presence of IHF. In vitro, NifA together with IHF stimulates transcription. IHF binds to a site located at position 2 56, between the NifA-binding site and the promoter. IHF bends the DNA and brings the enhancer-bound NifA in contact with the promoter-bound RNA polymerase by DNA looping for transcription activation (Figure 9c,d). The DNA bending by IHF has been observed by electron microscopy. In the absence of IHF, the probability of bound RNA polymerase and bound NifA contacting each other is very low. Although NtrC and NifA are similar in their function and structure, NifA does not need to be phosphorylated by an NtrB-like protein for transcription activation. In the p2 promoter of the glnHPQ operon, the position of NtrC and RNA polymerase binding sites relative to the IHF-binding site is important for the formation of proteinprotein contacts between the activator and RNA polymerase (Figure 9e,f). When IHF, NtrC and RNA polymerase binding sites are all on the same face of the DNA, transcription is enhanced. When an extra 5-bp DNA segment is inserted either between the enhancer and IHF-binding sites or IHF and RNA polymerase-binding sites to change their relative angular orientations on the DNA, transcription enhancement fails. Thus, IHF plays an architectural role in DNA looping by aligning the DNA-bound regulator and RNA polymerase to facilitate proteinprotein contact.

DNA Looping in the Bacterial Nucleoid

The E. coli chromosomal DNA, a single circular molecule of 4.7 million base pairs, is compacted to a density of 30 mg mL 2 1, which is three times greater than the density that would exist if the DNA molecule were evenly distributed throughout the cell (10 mg mL 2 1). The condensation is mediated by DNA supercoiling and the binding of several small proteins, including HU and other histone like proteins called IHF, FIS and HNS (Murphy and Zimmerman, 1997). The formation of repressosomelike structures, as described above, involving both sequence nonspecic and sequence-specic DNA-binding proteins also contributes to chromosome condensation. The compaction of bacterial DNA by the HU groups of proteins occurs in such a way that enables the bacterial chromosome, called a nucleoid, to be distinguished from eukaryotic chromatin (Figure 10). The nucleoid structure, unlike chromatin, is not very stable; lysis of bacterial cells under gentle conditions destabilizes the overall structure. This is expected, because most regions of the bacterial DNA, unlike eukaryotic DNA, must be instantaneously available to participate in DNA metabolic reactions in
7

by forming DNA loops, as observed by electron or scanning force microscopy (Figure 9b). The following experiments suggest that a cis conguration of both sites (on the same DNA) as well as the DNA sequence between them are not critical to achieve transcription from p2. When the enhancer and the promoter are placed in a trans conguration (i.e. on dierent DNA circles linked by a catenate structure), NtrC still functions and activates transcription from p2 (Wedel et al., 1990). This result shows that a proteinprotein contact between enhancerbound activator and promoter-bound RNA polymerase is sucient, whereas the intervening DNA sequence is not important for transcription activation. Unlike the glnALG operon, another DNA-binding protein, integration host factor (IHF), is required for activation of the nitrogen xation operon, nifHDK, in

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DNA Looping and Transcription Regulation

Relating DNA Looping to Transcription Regulation in Eukaryotes

DNA regulatory elements located thousands of base pairs away from promoters regulate transcription initiation in eukaryotic organisms both positively and negatively. In positive controls, activators bind to enhancer sites and increase transcription from the promoters. In negative controls, repressors interact with the distant DNA elements (called silencers) and quench transcription. Although other mechanisms have been suggested, both enhancers and silencers have been shown to act by DNA looping in several systems. For enhancers, the DNAbound activators communicate with general transcription factors at the promoter by DNA looping. The communication between the enhancer and the promoter involves recruitment of other proteins (coactivators and mediators) to act as a bridge. Similarly, silencer-bound repressors recruit other factors, which in turn interact with RNA polymerase by DNA looping, but in the process downregulate transcription initiation. However, DNA looping itself may not be critical for transcription regulation by proteins bound far away from the promoter, as has been shown in the NtrC system in bacteria. For example, DNA catenane structures have been used to show that an enhancer, present on one DNA circle, can activate a cognate promoter located in trans (i.e. on another DNA circle) in Xenopus laevis (Dunaway and Dro ge, 1989). In addition, when a b-globin gene DNA is attached noncovalently by streptavidin to an enhancer DNA of simian virus 40 or cytomegalovirus, transcription is enhanced, suggesting that increasing the local concentration of activators is a key factor in transcriptional regulation by DNA looping.

Figure 10 Schematic diagram of the Escherichia coli chromosome showing loop domain structures during DNA compaction. Adapted from Trun and Marko (1998).

response to extracellular or intracellular signals. In packaging DNA, the HU groups of proteins bend DNA and serve the purpose of reducing the end-to-end distance of the two arms of the bent locus. These proteins, which are usually present in large numbers in a cell, bind to many DNA sites because their DNA-binding activities have semisequence specicity or no specicity at all. Although DNA bending by DNA-binding proteins plays a signicant role in DNA condensation, the major factor in nucleoid formation is DNA looping. The E. coli chromosomal DNA is negatively supercoiled. It is advantageous to partition the chromosome into topologically independent segments (domains). The formation of an independent topological domain prevents rotation of the DNA strands at the ends of the domain. Each domain has dierent degrees of supercoiling, allowing promoters to be transcribed at dierent superhelical density of DNA. DNA supercoiling is known to increase or decrease transcription from dierent promoters. Independent domains also permit the introduction of transient nicks into DNA for DNA repair purposes, without loss of superhelicity of the entire chromosome. Chromosomes isolated by special methods, when observed by electron microscopy, show about 45 independent topological domains, which in reality are DNA loops (Sinden and Pettijohn, 1981). The nature of the protein(s) involved in the formation of such loops, whose size ranges from 50 to 100 kbp, is not known. A suspected candidate is DNA gyrase, which may be anchored to the bacterial membrane to prevent rotation.

References
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DNA Looping and Transcription Regulation

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Further Reading
Adhya S, Geanacopoulos M, Lewis DEA, Roy S and Aki T (1998) Transcription regulation by repressosome and RNA polymerase contact. Cold Spring Harbor Symposia on Quantitative Biology 63: 19. Collado-Vides J, Magasanik B and Gralla JD (1991) Control site location and transcriptional regulation in Escherichia coli. Microbiological Reviews 55: 371394. Kustu S, Santero E, Keener J, Popham D and Weiss D (1989) Expression of s54 (ntrA)-dependent genes is probably united by a common mechanism. Microbiological Reviews 53: 367376. Martin K, Huo L and Schleif RF (1986) The DNA loop model for ara repression: AraC protein occupies the proposed loop sites in vivo and repression-negative mutations lie in the same sites. Proceedings of the National Academy of Sciences of the USA 83: 36543658.

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net