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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: msalas@cbm.uam.es
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
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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.
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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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