Ana Camacho & Margarita Salas - COnnecting REpositories · Correspondence: Margarita Salas, Centro de Biolog´ıa Molecular ‘Severo Ochoa’ (CSIC- UAM), Instituto de Biolog´ıa

R E V I E W A R T I C L E

DNAbendingand looping in the transcriptional control ofbacteriophage/29Ana Camacho & Margarita Salas

Centro de Biologıa Molecular ‘Severo Ochoa’ (CSIC-UAM), Instituto de Biologıa Molecular ‘Eladio Vinuela’ (CSIC), Universidad Autonoma de Madrid,

Cantoblanco, Madrid, Spain

Correspondence: Margarita Salas, Centro

de Biologıa Molecular ‘Severo Ochoa’ (CSIC-

UAM), Instituto de Biologıa Molecular ‘Eladio

Vinuela’ (CSIC), Universidad Autonoma de

Madrid, Cantoblanco, 28049 Madrid, Spain.

Tel.:134 91 196 4675; fax: 134 91 196

4420; e-mail: [email protected]

Received 27 January 2010; revised 1 March

2010; accepted 6 March 2010.

Final version published online 20 April 2010.

DOI:10.1111/j.1574-6976.2010.00219.x

Editor: Juan L. Ramos

Keywords

gene regulation; transcriptional regulators;

nucleoprotein complexes;

protein-induced DNA bending;

indirect readout.

Abstract

Recent studies on the regulation of phage f29 gene expression reveal new ways to

accomplish the processes required for the orderly gene expression in prokaryotic

systems. These studies revealed a novel DNA-binding domain in the phage main

transcriptional regulator and the nature and dynamics of the multimeric DNA–

protein complex responsible for the switch from early to late gene expression. This

review describes the features of the regulatory mechanism that leads to the

simultaneous activation and repression of transcription, and discusses it in the

context of the role of the topological modification of the DNA carried out by two

phage-encoded proteins working synergistically with the DNA.

Introduction

During cell infection, viral genes are expressed in a time-

dependent manner for optimization of protein function.

Prokaryotes exert the control of gene expression primarily at

the level of transcription initiation, with RNA polymerase

(RNAP) binding to well-defined DNA promoter sequences,

using diverse mechanisms that include s factors that confer

specificity to the RNAP for a certain set of promoters and

transcription factors or regulators that either help or prevent

RNAP–promoter interactions. In some cases, RNAP–pro-

moter interaction is modified by proteins that, while bind-

ing within the promoter regulatory region, provide a

defined spatial organization resulting in topological altera-

tions of the promoter sequence that are crucial for the

regulation of nearby promoters.

Regulatory proteins were originally classified as activators

or repressors if they enhanced or inhibited transcription,

respectively. More recent studies, however, indicate that

both activators and repressors can exert dual functions,

activating or repressing transcription depending on how

and where they bind to the DNA (Heffernan & Wilcox, 1976;

Aiba, 1983; Choy et al., 1995; Hochschild & Dove, 1998;

Schleif, 2000; Lloyd et al., 2001; Scaffidi & Bianchi, 2001;

Lim et al., 2003; Browning & Busby, 2004; Yang et al., 2004;

van Hijum et al., 2009). Moreover, most transcriptional

regulatory systems rely on the function of more than one

regulatory protein. A fundamental question is how the

functional interaction between proteins regulates differen-

tial gene expression when two or more transcriptional

factors are involved. Studies of promoter regulation by

multiple regulators indicated that both antagonism and

synergism occur between regulators (Hochschild & Ptashne,

1986; Schreiber et al., 2000; Joly et al., 2004).

Many transcription regulators affect the topology of the

DNA locally upon interaction with their binding sequence

and some distort the double helix considerably. DNA

bending is an important feature of their function and

participates in transcription regulation by allowing regula-

tors that bind to distal sites to act synergistically, by

FEMS Microbiol Rev 34 (2010) 828–841c� 2010 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

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permitting correct interactions between regulators and the

transcription machinery, and by providing the appropriate

promoter conformation for its interaction with the RNAP. It

can be anticipated that a protein-induced DNA bend may

synergize or antagonize certain interactions required for

optimal transcription complex formation; this effect is

called structural synergy (Lavigne et al., 1994). In addition,

protein-directed DNA bending may regulate the assembly of

higher order DNA–multiprotein complexes and modulate

gene expression through the lifetime of such complexes

because transcription initiation is a nonequilibrium process

(Grosschedl, 1995; Bourgerie et al., 1997; Perez-Martin & de

Lorenzo, 1997; Kahn & Crothers, 1998; Ross et al., 2000;

Kim et al., 2003; Lewis et al., 2003).

The regulation of gene expression in the development of

the Bacillus subtilis phage f29 has proven to be a very useful

model to analyse new molecular mechanisms of transcrip-

tion regulation. Two phage-encoded early proteins, p4,

which binds to specific DNA sequences, and a nucleoid-like

DNA-binding protein, p6, act synergistically to produce the

switch from early to late gene expression through a novel

mechanism.

Organization of the /29 genome

The f29 viral particle contains a lineal double-stranded

DNA, 19 285 bp long, with a viral-encoded protein cova-

lently linked at the two 50 ends (Fig. 1; Salas, 1991). Viral

gene expression in vegetative cells is regulated in an orderly

sequence; immediately after infection, the host RNAP

transcribes early genes located at the two DNA ends, whereas

transcription of the late genes, located at the middle of the

genome, requires early gene expression (Loskutoff & Pene,

1973; Loskutoff et al., 1973; Sogo et al., 1979). Transcription

initiation and termination sites were mapped in vivo and in

vitro; the corresponding early promoters were named A1,

A2b, A2c, B1, B2, C1 and C2 and the only late promoter A3

(Kawamura & Ito, 1977; Sogo et al., 1979; Murray &

Rabinowitz, 1982; Dobinson & Spiegelman, 1985; Barthel-

emy et al., 1986, 1987; Mellado et al., 1986a, b).

Early transcription, starting at the main early promoters

A2b and A2c, gives rise to a polycistronic mRNA-encoding

proteins p6 (double-strand DNA-binding protein), p5 (sin-

gle-strand DNA-binding protein), p4 (transcriptional reg-

ulator), p3 (terminal protein), p2 (DNA polymerase), p1

(membrane protein involved in DNA replication; Serrano-

Heras et al., 2003), p56 (inhibitor of the host uracil-DNA

glycosylase; Serrano-Heras et al., 2006), ORFs 58 and 47 and

a small RNA required for packaging of the genome into the

viral proheads (Guo et al., 1987), although the major

production of this small RNA derives from promoter A1.

Transcription from promoters A2b and A2c terminates

mostly at the left end of the genome; however, a transcrip-

tional terminator (TA1) is located within gene 4, and there-

fore, there are transcripts coding only for proteins p6 and

p5, two proteins that are known to be synthesized in large

amounts in infected cells (Barthelemy et al., 1987). Early

promoters C2 and C1 are located at the right end of the

genome and its mRNA terminates at the bidirectional

terminator TD1. Transcripts starting from promoter C2

encode protein p17 required for the ejection of the genome

(Carrascosa et al., 1976; Crucitti et al., 2003; Gonzalez-Huici

et al., 2006), protein p16.7 involved in the replication of the

DNA (Serna-Rico et al., 2002) and several small ORFs of

unknown function (Meijer et al., 2001). No function has

been proposed for the transcripts derived from promoter

C1. All the late genes are expressed from a single promoter,

A3, and its transcript terminates at terminator TD1. Late

genes 7–13 and 16 are required for the morphogenesis of the

viral particle, and genes 14 and 15 are involved in the lysis of

the host. Two small noncoding RNAs, synthesized from

promoters B1 and B2 and terminating at terminators TB1

and TB2, are antisense to the late genes mRNA sequence.

The RNA derived from promoter B2 includes a sequence

complementary to gene 14 mRNA encoding the holin

protein (Barthelemy et al., 1987). This mRNA may control

holin synthesis and, therefore, provide the biological clock

for f29-induced host cell lysis.

Transcription machinery

Prokaryotic RNAPs constitute two families of enzymes; the

most abundant one includes multisubunit enzymes and the

other contains single-subunit enzymes such as the well-

characterized RNAPs of phages T7 (McAllister & Raskin,

1993) and N4 (Falco et al., 1980). f29 DNA transcription

Fig. 1. Genetic and transcription map of the bacteriophage f29 genome. The locations of promoters A1, A2c, A2b, A3, B1, B2, C1 and C2 are

indicated by vertical bars. Arrows indicate the direction of transcription, with the arrowheads at the termination sites (TA1, TB1, TB2 and TD1). The

genetic map is depicted. Phage terminal protein (TP) is shown as a black circle.

FEMS Microbiol Rev 34 (2010) 828–841 c� 2010 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

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requires the multisubunit DNA-dependent RNAP holoen-

zyme of B. subtilis, sA-RNAP (Sogo et al., 1979). The core

enzyme has a subunit composition of b, b0, a2 and o,

homologous to the Escherichia coli enzyme subunits. The

B. subtilis vegetative sA-subunit is homologous to E. coli s70;

both recognize the same consensus sequences at the � 35

and � 10 hexamers (Chang et al., 1992; Helmann, 1995;

Voskuil et al., 1995; Minakhin et al., 2001; Campbell et al.,

2002; Ebright, 2002; Gruber & Gross, 2003; Mathew &

Chatterji, 2006; Johnston et al., 2009). The RNAP is a

remarkable molecular machine. During transcription initia-

tion, it must recognize promoter DNA from a vast excess of

nonpromoter DNA sequences, separate the duplex chain,

expose the template strand to initiate RNA synthesis and it

must change from being a sequence-specific to a non-

sequence-specific DNA-binding protein that moves forward

along the DNA before beginning processive elongation.

The crystallographic studies of a core enzyme (Zhang et al.,

1999; Darst, 2001), of the prokaryotic initiation factor s(Malhotra et al., 1996; Campbell et al., 2002; Schwartz et al.,

2008), and of the initiation form of the prokaryotic RNAP

with and without promoter DNA (Murakami et al.,

2002a, b; Vassylyev et al., 2002), provided valuable structural

information on transcription initiation (Young et al., 2002).

RNAPs are highly processive and faithful, synthesizing RNA

molecules thousands of nucleotides long without a single

mismatch. Recent data reveal the importance of a mobile

structural element, the ‘trigger loop’, in elongation control

and fidelity (Bar-Nahum et al., 2005; Toulokhonov et al.,

2007; Nudler, 2009; Svetlov & Nudler, 2009; Wang et al.,

2009).

The interaction of the holoenzyme (RNAP) with promo-

ter DNA (P) initiates a series of structural transitions from

the initial closed promoter complex (RPc) to the transcrip-

tion-competent open complex (RPo). Double-stranded

DNA is melted over a region spanning the transcription

start site 11, in the following process:

RNAPþ P2RPc2I2Rpo

where ‘I’ represents several intermediate states. RPo is in a

rapid equilibrium with I, and this equilibrium depends on

many factors (i.e. temperature, Mg21, promoter sequence).

An RNAP–promoter complex capable of transcription

initiation (RPo) is formed through interactions that involve

several regions of the RNAP and that span 70–80 bp of

promoter DNA (�� 60 to 120 with respect to the tran-

scription start site 11) (Mekler et al., 2002; Naryshkin et al.,

2009). Each RNAP subunit, with the exception of o(Malhotra et al., 1996; Mathew & Chatterji, 2006), partici-

pates in these interactions, although a majority of the

sequence-specific interactions occur with the s-subunit.

Recognition of the promoter sequence involves the interac-

tions of s with the two elements of conserved sequence

located at � 10 and � 35 bp upstream of the promoter start

site, and with the � 10 extended element, the TG dinucleo-

tide at position � 14/� 15. At some promoters, the

C-terminal domain of the a subunits (aCTD) interacts

specifically with an AT-rich sequence, the UP element,

located upstream of the � 35 element (Fig. 2a; Ross et al.,

1993; Barne et al., 1997; Gourse et al., 2000; Cellai et al.,

2007; Haugen et al., 2008; Ross & Gourse, 2009).

Recognition of the /29 promoters

Promoters recognized by the sA-RNAP vary widely in

overall strength and in the extent of similarity to the

consensus sequences of its recognition elements. Some B.

subtilis genes are highly transcribed, while others are barely

transcribed. This is due in part to the promoter sequence

Fig. 2. Elements of bacteriophage f29 main

promoters. (a) Prokaryotic promoter with the

basic elements for RNAP recognition: UP, � 10

and �35 elements. (b) f29 promoters are

aligned to their � 10 element. The TG –10

extended motif is indicated in blue letters.

Positions of the RNAP s and a subunits

interaction in the promoter sequence are de-

noted by arrows. Sequences for UP elements are

in red, with the nucleotides at promoters

A2c and A2b contacted by the aCTD domain

underlined.


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itself and to the fact that transcriptional regulation takes

place mainly during the binding of RNAP to the DNA.

The main f29 promoters, early promoters A1, A2b, A2c

and C2 and late promoter A3 may be considered as examples

of evolutionary selection by the number of conserved

elements present in their sequences (see Fig. 2b) that would

bring the RNAP selectively to their sequences among the

large excess of bacterial host promoters. They are highly

homologous to the consensus hexamers at positions � 10

and � 35, except at the � 35 position of the late promoter

A3. The promoter spacers, or number of base pairs between

the � 10 and the � 35 elements, are 17–18 bp, the appro-

priate distance for simultaneous interaction of s with both

elements in order to anchor the RNAP to the promoter

(Hook-Barnard & Hinton, 2009). In addition, all the

promoters have the dinucleotide TG at position � 14/� 15

(� 10 extended motif), which is an additional anchor site

for sA-RNAP during closed-complex formation (Camacho

& Salas, 1999). Based on the amino acid homology between

sA and s70, the dinucleotide TG is most probably recog-

nized by sA-RNAP through the interaction with sA region

s3 (Sanderson et al., 2003).

In addition, promoters A1, A2b, A2c and C2 have an

AT-rich sequence or UP element immediately upstream of

the � 35 hexamer (Camacho & Salas, 2004; Meijer & Salas,

2004). The function of the UP element and the interaction

of the aCTD domain of the sA-RNAP with this element

were studied assaying transcription from promoters A1, A2c

and A2b with either wild-type RNAP or a truncated RNAP

devoid of the aCTD domains. Deletion of the aCTDs

domains drastically decreased transcription from promoters

A2c and A2b, but not that of promoter A1, and chemical

footprinting allowed identification of the aCTDs-binding

sites. At promoter A2c, the aCTDs bind to the promoter

sequence at positions centred at � 41 and � 51 from

the promoter start site; at promoter A2b, only one aCTD

contact was detected at a site centred at position � 51

(Fig. 2b; Camacho & Salas, 2004).

Transcriptional control of the viral cycle

The expression of the viral genes is regulated along the

infection so that the proteins are synthesized at the required

time. In vivo, using conditional lethal mutants of phage f29,

two proteins have been shown to be required for this timely

expression of the viral genes. Mutants in early genes 4 and 6

have impaired transcription when they infect nonsuppressor

bacteria (Monsalve et al., 1995; Camacho & Salas, 2000).

Early transcription driven from promoters C2, A2b and A2c

starts as soon as the genome enters the bacterium and

interacts with sA-RNAP, resulting in the synthesis of the

early proteins, among them proteins p4 and p6. Transcripts

from promoter C2 reach a maximum at 10 min of infection

and those derived from promoters A2b and A2c reach a

maximum by 20 min, decreasing afterwards. The synthesis of

p6 results in the activation of the initiation of viral DNA

synthesis and in the repression of early promoters A2b,

A2c and C2. Protein p6 binds in a nonsequence specific

manner, but preferentially to bendable sequences of the phage

DNA, forming extended multimeric complexes (Serrano et al.,

1989; Gonzalez-Huici et al., 2004). The multimeric complex

formed at the right end of the genome represses promoter C2

(Barthelemy et al., 1989; Camacho & Salas, 2001a).

Protein p4 plays a major role in the control of early

promoters A2b, A2c and late promoter A3 (Rojo et al.,

1998). In vivo, the synthesis of p4 is required for the

activation of late promoter A3 and for the repression of

early promoters A2b and A2c (Monsalve et al., 1995).

In vitro, protein p4 activates transcription from the late

promoter A3 by stabilizing sA-RNAP at the promoter

(Barthelemy & Salas, 1989; Nuez et al., 1992). Activation

requires an interaction between the C-terminal domain of

the RNAP a-subunit and the protein p4 residue Arg120; this

interaction also represses the early A2c promoter in vitro in

the absence of protein p6 (Mencıa et al., 1996a, b; Monsalve

et al., 1996; Calles et al., 2002). Protein p4 represses the A2b

promoter by interfering with RNAP binding.

Protein p6 cooperates with p4 in transcription regulation

(Elıas-Arnanz & Salas, 1999). Both proteins bind synergisti-

cally to the sequence containing early promoters A2c, A2b

and late promoter A3, resulting in a multimeric complex

that induces the switch from early to late transcription by

repressing early promoters A2c and A2b and simultaneously

activating late promoter A3 (Camacho & Salas, 2001b).

Protein p4, the /29 transcriptionalregulator

Structure of protein p4

Protein p4 is a dimer of two identical subunits of 124 amino

acids; it has no sequence homology with other proteins,

except for the corresponding proteins of the bacteriophage

f29 family. In the 3D structure (Fig. 3a), each monomer

(45� 20� 20 A3) has central five-stranded antiparallel b-

sheets, four a helices and one 310-helix (Badia et al., 2006).

Besides this, there is a peculiar structural element, referred

to as the N-hook, that is present at the N-terminus; the

polypeptide chain from Pro2 to Gln5 runs antiparallel to the

stretch from Arg6 to Asp11, the motif being stabilized by

both H-bonding and hydrophobic interactions. The struc-

ture of the p4 dimer presents an elongated S-shape, 80 A

long and 25 A wide, where both promoters establish exten-

sive protein–protein contacts, through an interface of 820 A2

with good surface complementarity.





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Protein p4--DNA complex

Protein p4 is a DNA-binding protein (Barthelemy & Salas,

1989; Nuez et al., 1992, 1994) that binds as a dimer

specifically to two regions of the phage genome (named

region 1 and region 2; Fig. 4) spanning from nucleotides

� 25 to � 84 and � 27 to � 87 relative to promoters

A2c and A2b transcription start sites, respectively (Perez-

Lago et al., 2005a). Each region contains two imperfect

inverted repeats and each inverted repeat is an independent

p4-binding site with the consensus sequence: 50-CTTT

TT-15 bp-AAAATGTT-3 0. The binding sites of region 1 are

named sites 1 and 2 and those in region 2 are named sites 3

and 4 (Fig. 4). Protein p4 binds twofold more efficiently to

site 3 than to site 1, and fivefold better to site 1 than to site 2;

site 4 is the lower affinity-binding site.

In the p4-site 3 crystal, the p4 dimer contacts three separate

A/T-rich areas on the same face of the DNA with three

positively charged patches (Fig. 3). Two of these areas are

contacted by residues of the N-hook motif because each hook

intrudes into the DNA major groove, where protein p4 Arg6

establishes hydrogen bonds with the DNA bases G� 13, and

Thr4 contacts the phosphate of the bases at position � 12. In

addition, the kinked helix a1 residue Tyr33 contacts DNA

phosphates T� 8, and residues Lys51 and Arg54 contact

phosphates of the central minor groove (Badia et al., 2006).

Direct and indirect readout in the recognition ofthe p4-binding site

The analysis of p4 mutant proteins as well as the p4

interaction with mutant-binding sites provided important

Fig. 3. Structure of the p4–DNA complex

and sequence-specific recognition.

(a) Representation of the p4 dimer (ribbons) in

complex with a DNA encompassing the site 3

sequence. Adapted from Badia et al. (2006). (b)

Site 3 sequence with the two external A-tracts in

boxes and the interactions of Arg6 with G � 13,

and Thr4 and Tyr33 with phosphates �13 and

�8, respectively, marked with arrows.

Fig. 4. Protein p4-binding sites. Detail of the f29 genome intergenic region between the early promoters A2c and A2b, and the late promoter A3 with

the � 10 and �35 elements indicated. In this intergenic region, there are four p4-binding sites. Protein p4 monomers are drawn as elliptical objects with

their N-hook DNA recognition motifs. Monomers of the p4 dimer are represented in green and purple and each monomer is distinguished as A and B,

respectively. Sequence of the p4-binding sites 1–4 with the external symmetry indicated by arrows. The guanines recognized by Arg6 residues are in red.





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insights into the determinants of p4–DNA complex forma-

tion. Alanine substitution of Arg6, the only amino acid

responsible for base interaction, and of Thr4 and of Tyr33,

which reside near the recognition N-hook and interact with

phosphates, resulted in proteins with impaired DNA bind-

ing; in contrast, mutations at the dimerization region

(Lys51Ala and Arg54Ala) resulted in a slight loss of binding

ability. Hence, the N-hook motif is a newly defined protein

substructure that establishes proper recognition of the DNA

sequence by penetrating the DNA major groove (Badia et al.,

2006).

Although most proteins recognize their target DNA

sequence through interactions between their amino acids

and groups of the bases in the DNA (direct readout),

protein–DNA complex formation often requires additional

coupling in which bases not contacted by the protein or

apparent unspecific interactions such as those with phos-

phates confer specificity to nucleoprotein complexes. This

mechanism has been called ‘indirect readout’ (Kalodimos

et al., 2004a, b; Napoli et al., 2006). The affinity of a protein

for its DNA target by indirect readout relies on the fact that

B-DNA exhibits a high degree of sequence-dependent

structural variation that includes the recognition of aspects

of DNA structure such as intrinsic curvature, the topology

of major or minor grooves, the local geometry of backbone

phosphates, flexibility or deformability and water-mediated

hydrogen bonds (Hogan & Austin, 1987; von Hippel, 1994).

This mechanism has been used to explain some aspects of

the affinity of several prokaryotic transcriptional regulators

for its target sequences. The E. coli catabolite activator

protein (CAP; also known as the cAMP receptor protein,

CRP) is selective for a pyrimidine–purine step that involves

sequence effects on the energy of kink formation (Garten-

berg & Crothers, 1988). Bacteriophage 434 repressor recog-

nizes structural features on the central base pair of its target

sequence (Mauro et al., 2003), and water-mediated contacts

are known to play an important recognition role in the trp

repressor–operator system (Bareket-Samish et al., 1998).

B-DNA sequences have a dissimilar distribution of hydro-

gen-bond donor and acceptor groups on their bases, but

similar sugar-phosphate backbones. In the case of p4, Thr4-

and Tyr33-phosphate backbone contacts are insufficient to

explain the affinity of p4 and its sequence specificity, and the

recognition of other aspects of the sequence topology

through an indirect readout mechanism should also account

for p4 sequence specificity.

The site 3 sequence includes three A-tracts, and similar

A-tracts are present in the other three p4-binding sites (Fig.

4; Perez-Lago et al., 2005a). Residue Thr4 contacts the DNA

backbone at one edge of the two external A-tracts, with

Tyr33 contacting the opposite edge of those A-tracts. Sub-

stitution of the AT base pairs at positions � 10 or � 11 for

less deformable base pairs, which results in an increase in the

energy required to distort the DNA, strongly diminished the

affinity of p4 for DNA (Mendieta et al., 2007). Therefore, the

effect of base substitutions at those A-tracts on p4 binding

support sequence recognition by both an indirect readout

mechanism and the overwinding of the minor groove at the

A-tracks mediated by p4 binding.

The structural stability of the protein and DNA in the

p4–DNA complex and the relative roles played by direct and

indirect readout in sequence-specific recognition by protein

p4 were studied by Molecular Dynamics Simulations (Men-

dieta et al., 2007). In a Molecular Dynamic Simulation, the

distances Arg6-G � 13, Thr4-phosphate � 13 and Tyr33-

phosphate � 8 were maintained at about 3 A in both p4

monomers; however, the monomers differed in its interac-

tion with the DNA. In monomer A (Fig. 3), the contacts

with DNA remained constant through the simulation while

the contacts of monomer B presented fluctuations. Because

both p4 monomers are identical, but its binding site is an

imperfect inverted repeat, this slight asymmetry of the

sequence could give rise to monomer–DNA nonhomolo-

gous interactions within the p4–DNA complex. In fact, p4

binding to inverted symmetric sites bearing the sequence

50-AAAATG-30 (the sequence contacted by monomer B) at

both ends yielded fourfold higher binding affinity than for a

site with the inverted symmetric sequence 50-CTTTTT-30

(the sequence contacted by monomer A). In agreement with

this result, the effect of individual substitution of the

guanine at positions 1 or � 13 by adenine was more

deleterious when the site lacked the guanine on the mono-

mer B sequence than when the guanine was mutated on the

monomer A sequence. Hence, the protein monomers dis-

play different binding entropies due to the slight asymmetry

of the inverted repeats; since the pyrimidine–purine T/G

step is more susceptible to deformation than the A/G step

because it has a smaller amount of base overlap, the T/G

bases of monomer B may present a better orientation of the

G for its interaction with Arg6. Therefore, the sequence-

dependent characteristics of the external A-tracts provide an

indirect readout by affecting the optimal complementarity

both for amino acid–base hydrogen bonding and for pre-

cisely positioned interactions between amino acids and

DNA phosphates.

DNA bending mediated by protein p4

DNA bending and looping are involved in the control of

transcription, enabling distal DNA regions and proteins to

interact with each other to coordinate gene activity, as

demonstrated for the E. coli araCBAD, gal, lac and deo

operon systems (Dunn et al., 1984; Adhya, 1989; Matthews,

1992; Schleif, 2000), in eukaryotic enhancers and promoters

(Milani et al., 2007) and in the lysogenic-to-lytic switch of

phage l (Ptashne, 2006).





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In the p4–DNA crystal structure, with a dimer of protein

bound to site 3, the DNA is curved (Fig. 3a; Badia et al.,

2006). This DNA conformation is an energetically strained

form stabilized only by interaction with p4, because the

measurement of the Ca atoms root-mean-square-deviation

values vs. simulation time of DNA and p4 in a Molecular

Dynamic Simulation reflected the dynamic conformations

of the DNA and the invariable conformation for p4 (Men-

dieta et al., 2007). The DNA adapts to fit the protein,

which confers the conformation to the DNA through local

modification of the minor groove width at the three A-tract

areas of amino acid-phosphate contacts (Fig. 3). There, the

minor groove facing p4 narrowed from the 11.5 A of a

regular B-DNA width to 9 A, while it widened up to 15 A

on the opposite face of the minor groove, resulting in

bending of the DNA that evolves rapidly from a low

curvature (with a mean value of 28.1� 8.11) to reach a

curvature with a mean value of 68� 7.51. This is in agree-

ment with the results obtained by Rojo et al. (1990) on the

p4-mediated bending of the DNA.

In the interaction of p4 with the entire f29 genome, two

tetramers of p4 bind to the two DNA regions; one of the

tetramers interacts with region 1 located between promoters

A2c and A2b, and the other to region 2 between promoters

A2b and A3 (Fig. 4). The topology of the sequence encom-

passing region 1 and region 2 was analysed both by circular

permutation and by the analysis of the mobility of DNA

during low-temperature polyacrylamide gel electrophoresis

(Rojo et al., 1990; Perez-Lago et al., 2005a). Region 2 has a

static DNA bend enhanced upon p4 binding while region 1

is straight. Because each p4 tetramer binds similarly to

regions 1 and 2, and binding results in a similar �851 DNA

bend, a sequence-directed curvature is not an essential

requirement for p4 binding and bending (Perez-Lago et al.,

2005a). The architecture imposed on the DNA when p4

is bound to the four binding sites, analysed by atomic

force microscopy (AFM), showed that the DNA is bent

around p4 (Gutierrez del Arroyo et al., 2009). The

angle built by the two DNA strands around p4 showed a

broad variation, with hji = 991 for region 2 to 1061 for

region 1, and in addition, when both region 1 and region 2

were simultaneously occupied, there was a configuration

where both angles contributed to build the closing of a

hairpin.

Model for the mechanism of p4--DNA binding

In the p4–DNA complex, stability is a consequence of p4-

induced conformational modification of the DNA from the

canonical B form through an indirect readout mechanism,

whereas the primary function of the DNA is its ability to

acquire a conformation capable of enhancing positive inter-

actions with its cognate protein. Taking into account that

sequence asymmetry is functionally required for p4–DNA

interaction, that p4 curves the DNA, and that the distance

from G � 13 to G 113 is about 90 A, while the 75 A distance

from the Arg6 of one of the monomers to the Arg6 of the

other monomer is too short for simultaneous interaction of

both monomers at the inverted repeats of the p4-binding

site, we propose a zipper-binding model (Fig. 5), where one

of the p4 monomers interacts first with the higher entropic

stability inverted repeat sequence, 50-AAAATG-30, introdu-

cing its N-hook into the major groove and providing an

Arg6-G 113-specific interaction. Subsequent local minor

groove narrowing will allow interactions between basic

residues of p4 with the DNA phosphates around positions

� 1, and the progressive bend of the DNA would approx-

imate the 50-AAAAAG-30 inverted repeat to the hook of the

second monomer.

Fig. 5. Protein p4–DNA-binding mechanism:

Zipper model. The distance from site 3 G � 13

to G 113 is about 90 A, while the distance from

the p4 Arg6 of one of the monomers to Arg6 of

the other monomer is only 75 A; the initial

interaction of one of the p4 monomers with DNA

is indicated (a); subsequent interactions between

basic residues of both monomers with the central

minor groove (b); the progressive bend of the

DNA would approximate the 50-AAAAAGTT-30

end to the hook of the second monomer (c).





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The second /29 transcriptional regulator:protein p6

Protein p6 is a 103 amino acid DNA-binding protein, able to

bind to the phage genome. Protein binding results in a broad

nucleoprotein complex, where the DNA, remarkably curved

by approximately 661 every 12 bp, adopts a right-handed

toroidal conformation (Prieto et al., 1988; Serrano et al.,

1990, 1993b). Protein p6 binds preferentially to the terminal

left and right sequences of the phage genome, but with a low

affinity (affinity constant keff = 105 M�1; Gutierrez et al.,

1994). The in vitro formation of p6–DNA complexes, scat-

tered through virtually the entire genome, led to the proposal

that protein p6 plays a structural role in the organization of

the viral genome into a highly dynamic nucleoprotein

complex. The p6–DNA complex is involved in the activation

of the initiation of DNA replication (Blanco et al., 1986;

Serrano et al., 1993a), in the repression of the early promoter

C2 (Whiteley et al., 1986; Barthelemy et al., 1989; Camacho

& Salas, 2000, 2001a) and in the regulation of the central

promoter region cooperating with the transcriptional reg-

ulator protein p4 (Elıas-Arnanz & Salas, 1999).

Repression of early promoter C2 mediated byprotein p6

Prokaryotic transcription repression generally involves se-

quence-specific DNA-binding proteins whose binding site is

frequently near or within the promoter sequence; intimate

contacts occur between the repressor and RNAP in many

cases. Protein p6 belongs to the so-called prokaryotic nucleoid

proteins, such as the E. coli proteins H-NS and HU (Drlica &

Rouviere-Yaniv, 1987), which are involved in repression of

transcription (Adhya et al., 1998). Upon p6 synthesis, the

protein binds to the phage DNA in an apparent nonsequence

specific manner, but with a preference for certain sequences,

called ‘nucleation sites’, located at the genome ends (Serrano

et al., 1989). The complex formed at the right genome end

represses promoter C2 located 160 bp from the end; however,

p6 does not affect the transcription from promoter A1 located

at 321 bp from the left end of the genome. This different effect

on the C2 and A1 promoters may depend on the orientation

of the transcription unit relative to the right-handed toroidal

conformation of the DNA in the p6–DNA complex and/or the

location of the � 35 promoter element with respect to the p6

‘nucleation sites’. Transcription from promoter C2 is codirec-

tional to the growth of the p6–DNA complex and its � 35

element is adjacent to the right-end nucleation site; in

contrast, the direction of transcription from promoter A1 is

opposite to the direction of p6–DNA complex formation and

the A1 � 35 element is distal from the left-end nucleation site.

DNAse I-footprint assays demonstrated than the

p6–DNA complex does not occlude RNAP binding to

the C2 promoter sequence, but affects the stability of the

transcriptional closed complex because p6 and RNAP could

be detected on the same DNA molecule when the ternary

complex RNAP–p6–DNA is allowed to be formed (Cama-

cho & Salas, 2001a). This suggests that the recognition of

promoter C2 by RNAP occurs as in the case of the lac UV5

promoter (Buckle et al., 1999), where RNAP contacts with

the promoter to form a stressed intermediate that is relaxed

by transferring the torsional stress to the DNA during

closed-complex formation. Protein p6 could repress C2

transcription by impeding this stress transference when the

promoter is occupied by the repressor.

Ternary p4--p6--DNA complex

Proteins p4 and p6 bind to the sequence containing promo-

ters A2c, A2b and A3. The resulting complex induces the

switch from early to late transcription by activating the late

promoter A3 and simultaneously repressing early promoters

A2c and A2b. Binding of both p4 and p6 to DNA encom-

passing promoters A2c, A2b and A3, studied by hydroxyl

radical footprinting, showed that p4 maintains its contacts

at sites 1, 3 and 4; two or three dimers of p6 polymerize from

the end of site 1 to the beginning of site 3, synergizing the

binding of p4 most likely by anchoring the p4 A monomers

to the lower affinity sequence of sites 1 and 3, respectively

(Fig. 6a). Increasing the concentrations of p6 results in

progressive polymerization of p6 beyond sites 4 and 1

(Camacho & Salas, 2001b, 2004).

In the ternary p4–p6–DNA complex, a number of DNA

positions are DNAse I hypersensitive, indicating that the

DNA helix is highly distorted. This observation provides an

insight into a possible mechanism for controlling the

transcription through topological modification of the pro-

moters region. Topological modification induced on the

promoters sequence has been studied by performing direct

visualization of the protein–DNA complexes at the single

molecule level by AFM (Beloin et al., 2003; Moreno-Herrero

et al., 2003; Sorel et al., 2006). When we applied AFM in a

liquid environment to examine the topology of the ternary

complex formed by the f29 genome containing promoters

A2c, A2b and A3, and the two transcriptional regulatory

proteins, p4 and p6, the most recurrent structure was a

hairpin turn conformation of the DNA chain with a length

ranging from 10 to 43 nm, where the most frequent hairpin

had a length comparable to that of the p4–DNA complexes

(average length of 13� 4 nm; Gutierrez del Arroyo et al.,

2009). Moreover, the presence of p6 produced a much

higher number of hairpins within the complexes, in agree-

ment with the fact that p4 is stabilized by binding of p6.

Further enlargement of the hairpin structure could be due to

additional binding of p6 beyond p4 bound at sites 1 and 3.

DNA looping is a general phenomenon for the regulation

of transcription initiation, replication, recombination and





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genome condensation and generally results from holding

two noncontiguous sites of the DNA together by sequence-

specific DNA-binding proteins, the stability of the loops

being important for the proper function (Echols, 1990;

Schleif, 1992). The distances between the two end points of

the loop can range from a few base pairs to several kilobase

pairs. The feasibility of DNA loop formation over short

distances depends on the flexibility of the intervening DNA.

Double-stranded DNA is a semi-flexible polymer with a

persistence length of 50 nm (about 150 bp) (Shore et al.,

1981). Segments of DNA that are longer than the persistence

length are relatively easy to bend, whereas segments shorter

than the persistence length behave as stiff rods and are not

easily bendable. Several factors such as supercoiling, intrin-

sically distorted (kinked) structures and auxiliary proteins

(as architectural proteins) that create or maintain distorted

or curved structures, can reduce DNA stiffness and lower the

energy required for loop formation. The importance of such

factors becomes more pronounced when the intervening

DNA length is o 200 bp (Kramer et al., 1987; Lee & Schleif,

1989; Lewis & Adhya, 2002; Semsey et al., 2005). The short

distance (120 bp) between site 1 and site 3 may impede

stable p4-hairpin and require p6 binding to lower the energy

required to maintain the hairpin.

Within the f29-like genus of phages, bacteriophage Nf

encodes proteins homologous to p4 and p6 (named p4N and

p6N) and has promoters functionally homologous to the

f29 promoters A2b, A2c and A3, which are also coordi-

nately controlled by p4N and p6N. As in f29, p4N is required

for the activation of promoter A3, and the additional

presence of protein p6N results in the repression of the early

promoters A2c and A2b (Perez-Lago et al., 2005b). Binding

of p4N and p6N to the promoters region results in nucleo-

protein-hairpin structures covering from downstream of

promoter A2c to downstream of promoter A3, similar to

those described for f29 (Gutierrez del Arroyo et al., 2009).

Hence, phage f29 and Nf follow a common mechanism for

the switch from early to late transcription, supporting the

hypothesis that a nucleoprotein-hairpin may play a key

structural role in f29 and its related phage family.

Regulation of the switch from early to latetranscription by the p4--p6--DNAnucleoprotein complex

The majority of the regulators appear to function by

establishing a direct interaction with the initiating RNAP–

promoter transcription complex such as bacteriophage l cI

protein and E. coli cyclic AMP receptor protein (CRP)

(Ptashne & Gann, 1997; Barnard et al., 2004), and a small

number of regulators function by altering the conformation

of the promoter DNA such that it is better recognized by the

RNAP (Brown et al., 2003).

Upon f29 infection of B. subtilis, the host RNAP recog-

nizes and starts transcription from, among others, early

promoters A2b and A2c. RNAP recognizes promoter A2c

through interaction of its s-subunit with the � 10, � 35

and the � 10 extended motif elements and the interaction of

the aCTDs at positions centred at � 41 and � 51 bp from

the promoter start site. At promoter A2b, the s-subunit is

Fig. 6. Synchronized regulation of promoters A2c, A2b and A3. (a) Schematic representation of contact positions of the nucleoprotein complex

p4–p6–DNA obtained by hydroxyl radical footprinting at the DNA sequence including promoters A2c, A2b and A3 (protein p4 dimer in violet–green and

protein p6, in yellow). The �10 and � 35 elements of each promoter are indicated. (b) AFM of the RNAP–p4–p6–DNA quaternary complex with the

DNA helix and nucleoprotein-hairpin remarked. (c) Model representing the regulation of promoters A2c, A2b and A3 in the presence of proteins p4 and

p6. The RNAP is represented stably bound to promoter A3, but in an unstable interaction with promoter A2c. Proteins p4 (violet–green) and p6 (yellow)

binding to the sequence between promoters A2c and A3 results in the formation of the nucleoprotein-hairpin with the apex at promoter A2b. (b) and

(c) are taken from Gutierrez del Arroyo et al., 2009.





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also bound to the � 10, � 35 and � 10 extended motif

elements, but here, only one aCTD binds to the promoter at

a position centred at � 51 bp from the promoter start site.

Efficient stabilization of the closed complex at late promoter

A3 requires protein p4, because the match to the consensus

� 35 element is poor (Nuez et al., 1992). The synthesis of

early mRNA from promoters A2c and A2b gives rise to the

synthesis of proteins p4 and p6.

The p4-binding sites are placed between promoters A2c

and A3; sites 1 and 3 overlap the � 35 elements and the

binding sites of the aCTDs at promoters A2c and A2b (see

Fig. 4). In the cocrystal p4–DNA, the DNA is curved and

shows a twist of the DNA axis of 27.71; as a consequence,

each tetramer of p4 on two consecutive binding sites might

generate a left-handed loose superhelix, where the p4-

tetramer of region 1 might cooperate with the region 2

tetramer to induce a twisting of the helix at the middle point

that contains the � 10 element of promoter A2b, an AT-rich

region characterized by its capacity to be distorted (Gu-

tierrez del Arroyo et al., 2009). Binding of p4 to its four sites

results in a 13 nm hairpin structure that might be the

triggering factor for the preferential binding of p6 between

p4 sites 1 and 3 that leads to the stabilization of the

nucleoprotein-hairpin with a change in the activity of

promoters A2c, A2b and A3. Under these conditions, RNAP

recognizes promoter A2c, giving rise to a transcriptional

closed complex where isomerization to an open complex is

impeded (Camacho & Salas, 2004); no transcription com-

plex is detected at promoter A2b most probably due to its

dependence on aCTD binding that is competed by p4, and

due to the topological modification of the promoter se-

quence located at the apex of the nucleoprotein-hairpin (see

Fig. 6c). On the other hand, protein p4 bound at site 3

interacts with RNAP, overcoming the rate-limiting step,

closed-complex formation of promoter A3 (Mencıa et al.,

1996b). Activation of promoter A3 may be enhanced by the

hairpin through a more appropriate p4–RNAP interaction,

and because the hairpin brings promoters A2c and A3 in

close proximity, it would increase the local concentration of

the enzyme at promoter A3.

Concluding remarks

Transcription of the f29 genome is tightly regulated to

ensure appropriate gene expression. On the one hand, the

expressions of those early genes expressed from promoter

C2 are highly repressed by protein p6, a low-specificity

DNA-binding protein, while the main block of early tran-

scription from promoters A2c and A2b that involves those

essential genes required for the synthesis of the viral DNA

and the activation of the late promoter A3 are simulta-

neously regulated in a sophisticated manner by proteins p4

and p6, where protein p4 plays a leading role in the process.

The study of regulatory protein p4 revealed novel para-

digms: (1) its structure has provided a new DNA-binding

motif, the N-hook, to the repertoire of the DNA-binding

proteins; (2) the protein recognizes its binding sites through

minimal contacts that include both direct and indirect

readout mechanisms; and (3) p4 is capable of bending the

120 bp of the sequence between the promoters to the

structure of a nucleoprotein-hairpin. This nucleoprotein-

hairpin, stabilized by the incorporation of p6 into the

nucleoprotein complex, allows the close proximity of late

promoter A3 to the early promoter A2c.

We envision the switch from early to late transcription as

the interplay between the RNAP and the p4–p6 complex for

binding to the sequences containing promoters A2c, A2b

and A3. Although RNAP has a higher binding affinity than

p4 and p6 for promoters A2c and A2b, efficient complex

formation and transcription initiation requires appropriate

positioning of the proteins. At the onset of infection, there

are few molecules of p4 and p6; therefore, RNAP recognizes

and transcribes promoters A2c and A2b, but not promoter

A3. As the concentration of proteins p4 and p6 increases, the

p4–p6 nucleoprotein complex extends over the region

impairing the correct interaction of the RNAP at promoters

A2c and A2b and consequently decreasing the efficiency of

transcription. However, if promoters A2c and A2b were

totally repressed at a time when exponential DNA synthesis

occurs, the synthesis of the early proteins expressed from

these promoters, and required for replication, could be

compromised. The phage could overcome this situation by

maintaining a controlled synthesis of early proteins by a

feedback regulatory mechanism. Activation of the late

promoter A3 takes place mainly by the stabilization of the

RNAP through its interaction with the p4–p6 nucleoprotein

complex.

Acknowledgements

We are grateful to the very many colleagues who have

discussed their ideas with us and we apologize to those

whose work we could not cite due to space limitations. Work

was funded by Grants BFU2008-00215/BMC to M.S. and

BFU2008-01216/BMC to A.C., from the Ministerio de

Ciencia e Innovacion of Spain, by Grant S-0505/MAT/

0283-05 to M.S. from the Madrid Autonomous Commu-

nity, by the Intramural-CSIC Grant 2007 20I007 to A.C. and

by an Institutional Grant from the Fundacion Ramon Areces

to the Centro de Biologıa Molecular Severo Ochoa.

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Ana Camacho & Margarita Salas - COnnecting REpositories · Correspondence: Margarita Salas, Centro de Biolog´ıa Molecular ‘Severo Ochoa’ (CSIC- UAM), Instituto de Biolog´ıa