Bacterial enhancer-binding proteins: unlocking s54-dependent
gene transcription
Mathieu Rappas, Daniel Bose and Xiaodong Zhang
Bacterial transcription relies on the binding of dissociable
sigma (s) factors to RNA polymerase (RNAP) for promoter
specificity. The major variant sigma factor (s54) forms a stable
closed complex with RNAP bound to DNA that rarely
spontaneously isomerises to an open complex. ATP hydrolysis
by bacterial enhancer-binding proteins is used to remodel the
RNAP–s54–DNA closed complex. Recently, a wealth of
structural information on bacterial enhancer-binding proteins
has enabled unprecedented insights into their mechanism.
These data provide a structural basis for nucleotide binding and
hydrolysis, oligomerisation and the conversion of ATPase
activity into remodelling events within the RNAP–s54 closed
complex, and represent advances towards a complete
understanding of the s54-dependent transcription activation
mechanism.
Addresses
Centre for Structural Biology, Division of Molecular Biosciences, Faculty
of Natural Sciences, Imperial College London, London SW7 2AZ, UK
Corresponding author: Zhang, Xiaodong
(xiaodong.zhang@imperial.ac.uk)
Current Opinion in Structural Biology 2007, 17:110–116
This review comes from a themed issue on
Protein–nucleic acid interactions
Edited by James M Berger and Christoph W Müller
Available online 6th December 2006
0959-440X/$ – see front matter
# 2006 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.sbi.2006.11.002
Introduction
Regulated transcription is essential for cellular adapta-
tion, differentiation and growth. In bacteria, transcription
regulation is achieved through the use of dissociable
sigma (s) factors, which bind and direct RNA polymerase
(RNAP) to specific promoter DNA. Predominant s70 and
the alternative s54 form two distinct classes of s factors in
bacteria. The binding of s70–RNAP holoenzyme to pro-
moter DNA is sufficient for promoter melting and tran-
scription initiation — a process collectively known as
open complex formation [1–3]. The alternative s54 class
is functionally distinct, as the binding of a s54–RNAP
holoenzyme to promoter DNA results in a stable closed
promoter complex, unable to initiate transcription [4,5].
This closed complex is stabilised by the interaction of s54
with a repressive fork junction DNA structure near –12,
with respect to the transcription start site (+1); activation
Current Opinion in Structural Biology 2007, 17:110–116
(remodelling) involves losing this repressive structure.
Remodelling the closed complex into a transcriptionally
competent open complex requires energy derived from
ATP hydrolysis catalysed by transcriptional activators.
Activators of closed s54 complexes are members of the
AAA+ (ATPases associated with various cellular activ-
ities) protein family [6–10]. They bind DNA sites located
upstream of the promoter, known as upstream activation
sequences (UASs), and contact the closed complex
through DNA looping, often facilitated by DNA-bending
proteins. This mode of interaction between the activator
and RNAP is similar to that of eukaryotic enhancer-
binding proteins (EBPs), hence s54 activators are also
known as bacterial EBPs (bEBPs) [4,11–13].
bEBPs typically consist of three domains: an N-terminal
regulatory (R) domain, a central AAA+ (C) domain and a
C-terminal DNA-binding (D) domain [14]. Recently,
several high- and low-resolution structures of bEBPs
bound to various nucleotide analogues (AMPPNP,
ATPgS, ADPAlFx, ADP) have become available (see
Table 1), unveiling the details of the s54-dependent
transcription activation mechanism. These include
high-resolution crystal structures of the NtrC1 R and C
domains (nitrogen regulation protein C1, NtrC1R+C)
[15], the PspF C domain (phage shock protein F,
PspFC) [16,17] and the ZraR C and D domains (hydro-
genase regulatory protein G, ZraRC+D) [18]. In addition,
lower resolution electron microscopy (EM) reconstruc-
tions of NtrCR+C+D [19] and PspFC in complex with s54
[17] are now available. Finally, small/wide-angle X-ray
scattering (SAXS/WAXS) models of NtrCR+C+D have
been obtained [19]. ADPAlFx, which mimics the
ATP hydrolysis transition state, allows PspFC to form a
stable oligomer capable of interacting with RNAP–s54–
DNA. Spectroscopic analysis of PspFC–RNAP–s54–DNA
in the presence of ADPAlFx confirms that it is a bona fide
intermediate ‘en route’ to open complex formation [20].
In this review, we summarise recent structural data on
bEBPs and propose a mechanistic model for s54-depen-
dent transcription activation, from initial binding of UAS
DNA to transcription initiation. In particular, we focus on
how the recent structural data have advanced our under-
standing of nucleotide binding and hydrolysis, oligomer-
isation, and the interactions between bEBPs and s54.
Nucleotide binding and hydrolysis
AAA+ proteins are characterised by the highly conserved
Walker A (GxxxxGK[T/S], where x represents any amino
acid) and B (hhhhDE, where h represents hydrophobic
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 Bacterial enhancer-binding proteins Rappas, Bose and Zhang 111

Table 1

Recent structures of bacterial enhancer-binding proteins (determined since 2003).

Structure Organism Technique Resolution (Å) PDB/EMD code References

NtrC R domain (BeF complex) Salmonella typhimurium NMR – 1J56, 1KRX, 1KRW [25]
NtrC1 R and C domains Aquifex aeolicus X-ray 2.4 1NY5 [15]
NtrC1 C domain Aquifex aeolicus X-ray 3.1 1NY6 [15]
PspF1–275 C domain (apo, apo Escherichia coli X-ray 1.75, 1.7 2BJW, 2BJV [17]
R168A mutant)
NtrC1 R domain Aquifex aeolicus X-ray 3.03 1ZY2 [33]
PspF1–275 C domain (AMPPNP, Escherichia coli X-ray 1.9, 1.8, 1.9, 2.1 2C99, 2C96, 2C98, [16]
ATP, ADP, MgATP R227A mutant 2C9C
complexes)
ZraR C and D domains Salmonella typhimurium X-ray 3.0 1OJL [18]
s54–PspF C domain (ADPAlFx Sinorhizobium meliloti and Cryo-EM 20 EMD-1109 [17]
complex) Escherichia coli
NtrC R and C and D domains Salmonella typhimurium SAXS/WAXS – – [19]
(BeF and ADP complex)
NtrC R and C domains (ADP, Salmonella typhimurium Negative-stain EM 25, 28 EMD-1218 [19]
ADPAlFx complexes)

amino acids) motifs, responsible for nucleotide binding
and hydrolysis, respectively, and the sensor I and sensor II
motifs, responsible for sensing nucleotide states [6–10].
Several medium- and high-resolution structures of bEBPs
confirm that, in common with all other AAA+ proteins, the
C domains of bEBPs fold into an a/b subdomain followed
by an a-helical subdomain. Nucleotide binds in the cleft
between these two subdomains (Figure 1) and between
two adjacent protomers (Figure 2). The C domain of
bEBPs is distinguished from other AAA+ family members
by the signature motif GAFTGA, located within surface
loop L1 of the a/b subdomain [4,15,17,18]. Another
surface loop, called L2, is also found in some AAA+ DNA
helicases, including RuvB [17,21].
Crystal structures of PspFC in complex with different
nucleotides have shed light on the conformational
changes that occur in the nucleotide-binding pocket
during the hydrolysis cycle. Comparison of the different
nucleotide-bound structures identified regions and resi-
dues important for nucleotide binding and hydrolysis, as
illustrated in Figure 1 [16]. Most interestingly, during
the early stages of ATP hydrolysis, a newly formed
interaction between a catalytic residue (E108 of Walker
B) and a highly conserved structural residue (N64) acts as
an ‘atomic switch’, enabling the bEBP to sense changes
within the catalytic site and transmit them to the L1 and
L2 loops (Figure 1).
Oligomerisation of the bEBP
bEBPs are typically dimeric in their inactive state, with
the major dimerisation determinants existing either in the
R domain, as in DctD (C4-dicarboxylic acid transport
protein D) and NtrC1 [13,22,23], or in the D domain,
as in NtrC [15,24,25]. However, in response to stimu-
latory signals, bEBPs form higher order oligomers via
their C AAA+ domain, which in turn stimulates their
ATPase activity and transcription activation. The exact
functional oligomeric state of bEBPs has been a matter of
debate. Whereas the first X-ray structure of an oligomeric
AAA+ domain (NtrC1C) was heptameric [15], the crystal
structure of ZraRC+D revealed a hexameric structure [18]
(Figure 2), and nano-ESI (electrospray ionisation) mass
spectrometry and three-dimensional cryo-EM reconstruc-
tion revealed that PspFC is a hexamer when in complex
with s54 at the point of ATP hydrolysis [17]. Moreover,
the studies of ZraR and the three-dimensional cryo-EM
reconstruction of the PspF–s54 complex suggest that
bEBPs form non-planar asymmetric hexamers, indicating
that they may follow a non-concerted nucleotide binding/
hydrolysis mechanism. This is consistent with recent
biochemical studies [26], and is similar to that exhibited
by other AAA+ proteins, including ClpX [27], papilloma-
virus E1 replicative helicase [28] and FtsH [29].
Recent work on NorR (nitric oxide regulatory protein R)
suggests that three inactive NorR dimers bind to three
adjacent UAS sites, resulting in an increased local con-
centration of NorR and facilitating oligomerisation upon
activation of the regulatory GAF domain [30–32]. Oligo-
merisation of NorR would also promote DNA bending
and DNA looping, essential for closed complex interac-
tion. Binding to DNA as dimers is supported by the
dimeric DNA-binding domains observed in the structure
of ZraR and the recently published negative-stain EM
reconstruction of NtrC, which reveals a symmetry mis-
match between the hexameric arrangement of both the C
and R domains and the ‘trimer of dimers’ arrangement of
the D domains [18,19] (Figure 2).
The R domains sense specific environmental cues and
adjust accordingly the activity of the AAA+ domain [10].
One way of modulating AAA+ domain activity is to
regulate oligomerisation, either negatively (as in NtrC1
and DctD) or positively (as in NtrC and ZraR). The
inactive NtrC1 dimer is maintained by a tight interaction

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112 Protein–nucleic acid interactions

Figure 1

Nucleotide binding and hydrolysis. (a) High-resolution crystal structures of PspFC in complex with ATP, ADP and, shown, MgATP (using
hydrolysis-deficient mutant R227A) reveal distinct changes during the ATP hydrolysis cycle. Nucleotide exchange induces changes in the
Walker A motif, loops L1 and L2, sensor II and the linker region between the two subdomains (blue). Nucleotide hydrolysis induces changes in
linker 1 and linker 2, L1 and L2, Walker B, sensor I and the linker region between the two subdomains (red). Regions that are affected by both
nucleotide exchange and hydrolysis are highlighted in magenta. (b) Enlarged view of the nucleotide-binding pocket. Residues involved in
nucleotide binding and hydrolysis are labelled.

Figure 2

Functional oligomerisation. (a) NtrC1R+C inactive dimer. (b) Schematic oligomeric arrangement of bEBPs. (c) Side and top views of an X-ray
crystallographic structure of the ZraRC+D hexamer. Side and top views of (d) SAXS/WAXS and (e) 3D EM models of activated (ADPAlFx-bound)
NtrCR+C+D, showing R domains on the periphery and D domains beneath the plane of the hexameric C domain.

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 Bacterial enhancer-binding proteins Rappas, Bose and Zhang 113

between R domains that locks the AAA+ domains in a
‘front to front’ orientation, thus preventing oligomerisa-
tion (Figure 2). Phosphorylation of the R domain alters its
conformation, causing greater conformational flexibility
of the C domains and allowing repacking in the ‘front to
back’ orientation necessary for oligomerisation [15,33].
In NtrC, phosphorylation exposes a hydrophobic patch in
the R domain, which allows the R domain of one protomer
to bind to the outside edge of the adjacent protomer,
thereby actively facilitating oligomerisation [19]. The
resulting conformation, with the R domains positioned on
the periphery of the hexameric ring, would prevent steric
clashes when contacting the s54 holoenzyme [19].
Stable hexamers have been observed for PspF, NtrC,
NtrC1 and ZraR in the presence of different nucleotides
[15,18,19,26,34], implying that nucleotides play sig-
nificant roles in oligomerisation. This is consistent with
crystal structures in which the nucleotide binds between
protomers. In AAA+ proteins, R fingers are proposed to be
involved in intersubunit catalysis and nucleotide sensing
[6–9]. In bEBPs, two highly conserved R fingers and
sensor II residues are indeed located at the protomer
interface, ideally situated to sense changes in the nucleo-
tide-binding pocket and correspondingly affect the oli-
gomeric organisation.
Interactions with s54
To remodel the closed promoter complex, the UAS-
bound bEBP must loop out the intervening DNA and
make contact with the complex at the promoter. Studies
using variable operator lengths have revealed the impor-
tance of the geometry of this process, showing that bEBPs
must bind to the opposite face of the promoter DNA to

Figure 3

Mechanistic model of s54-dependent transcription activation. (a) Inactive dimers bind to the UAS on the opposite DNA face to the RNAP–s54
complex, promoting oligomerisation. (b) Upon stimuli and through DNA looping, hexameric bEBP contacts RNAP–s54. (c) Role of the proposed
atomic switch pair during the ATP hydrolysis cycle. (i) Locked state of GAFTGA (ADP state); there is no interaction between N64 and E108.
(ii) Released state of GAFTGA (apo state). (iii) ‘Open’ state of GAFTGA (ATP state); interaction between N64 and E108 is established. (iv) ‘Engaged’
state of GAFTGA. Upon nucleotide binding and initial hydrolysis (ii and iii), changes in the nucleotide-binding pocket are sensed by the atomic
switch and transmitted through a conformational signalling pathway to allow a productive interaction between the L1 and L2 loops of bEBP and
s54 (iv). g-Phosphate release changes L1 and L2 into the locked conformation (i). (d) Combining available structural information, the transcriptional
intermediate complex shown induces changes in RNAP–s54–DNA that result in open complex formation.

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114 Protein–nucleic acid interactions
that bound by the s54 holoenzyme to efficiently initiate
transcription [35]. The bEBP GAFTGA signature motif
in the AAA+ domain forms the main contact between
bEBPs and s54; mutations within this motif generally
abolish s54 interaction and transcription initiation [36,37].
As revealed by the crystal structures of NtrC1 and ZraR,
GAFTGA is located at the tip of the L1 loop, which is
inserted into helix 3 located at the surface of the a/b
subdomain and near the hexameric pore.
From these structures, it is not obvious how the
GAFTGA motif could interact with s54. Hydrophobic
interactions involving the phenylalanine of GAFTGA, a
valine from the adjacent L2 loop (Figure 1) and residues
from helix 3 lock GAFTGA in a conformation pointing
towards the central pore of the ring [15]. This con-
formation is unfavourable for stable s54 interaction, as
the GAFTGA motif is buried and not readily accessible.
This is supported by biochemical studies indicating
that the s54-binding site may be masked before ATP
hydrolysis and that the threonine of GAFTGA might
make a direct interaction with s54 [36]. Fitting the crystal
structure of PspFC into the low-resolution three-dimen-
sional cryo-EM map of the PspFC–s54 complex revealed
that the L1 loop (with GAFTGA at its tip) has to project
upwards from the plane of the hexameric PspF ring
to contact s54 [17]. A raised ‘rim’ corresponding to
the L1 and L2 loops also appears in NtrC in the presence
of the ATP hydrolysis transition state analogue
ADPAlFx and is absent in the presence of ADP
[19]. The raised conformation of L1 and L2 is thought
to happen at the point of ATP hydrolysis and to be part
of a coordinated series of movements that occurs in
response to changing nucleotide states during the hydro-
lysis cycle.
The chemical energy released by ATP hydrolysis in the
active site is converted into conformational changes that
ultimately lead to the relocation of the L1 and L2 loops.
Recent studies on PspFc in complex with different
nucleotides provided a structural basis for this conversion
[16]. The changes in the nucleotide-binding pocket are
sensed by the atomic switch pair (E108–N64) and com-
municated through a conformational signalling pathway,
resulting in the rotation of helix 3, where L1 is inserted
(Figure 3). This disrupts the above-mentioned hydro-
phobic interactions that lock L1 and the GAFTGA motif,
and moves L1 into the raised conformation. The inter-
action between a residue in L1 (E81) and a residue in L2
(R131) also ensures the coordinated movement of these
two loops.
Conclusions: from binding to initiation
Recent structural studies have provided a detailed under-
standing of bEBP nucleotide binding and hydrolysis,
functional oligomerisation and interactions with s54.
Combining the structural information discussed above,
Current Opinion in Structural Biology 2007, 17:110–116
we present the current mechanistic model of bEBP-
dependent transcription activation (Figure 3).
Dimeric bEBPs bind to the UAS DNA distantly and to
the opposite face of DNA to that bound by RNAP–s54,
thereby increasing the local concentration of bEBPs.
Upon receipt of an appropriate stimulus, the R domain
facilitates formation of an unstable hexamer, capable of
binding and hydrolysing ATP. The oligomerisation of
the bEBPs and other DNA-bending proteins would
then facilitate DNA looping between the UAS and
the promoter, bringing the bEBP into close proximity
with RNAP–s54. Nucleotide binding stabilises the hex-
amer and initial hydrolysis within certain subunits is
sensed by the atomic switch pair (PspF E108–N64)
and transmitted through the conformational signalling
pathway to release the locked L1 and L2 loops. Exposure
of one or more GAFTGA motifs within the hexamer
leads to a stable interaction with s54. Completion of
hydrolysis and g-phosphate release (from ATP to
ADP) disrupts the atomic switch pair interaction, releas-
ing the conformational strain that caused the exposure of
the L1 and L2 loops and allowing the L1-locking
mechanism to be re-engaged [15,16,17]. Changes
in L1 conformation will cause changes in RNAP–s54
that result in the release of fork junction DNA repression,
leading to open complex formation and transcription
initiation.
Our current data provide evidence of the origin of con-
formational changes in the activator (Figure 3) and a
structural model for the transcriptional intermediate com-
plex, as shown in Figure 3. However, we still lack a
detailed understanding of how the energy released from
nucleotide hydrolysis in the activator is used to remodel
the RNAP–s54–DNA closed complex, resulting in DNA
melting and open complex formation. This requires a
mechanistic understanding of how the nucleotide activity
of one subunit influences the activity and functionality of
other subunits within the hexameric bEBP. In addition,
structures and dynamics of bEBP in complex with
RNAP–s54–DNA are necessary for a complete under-
standing of the s54-dependent transcription activation
process.
Acknowledgements
We are indebted to many useful discussions with Martin Buck, Paul
Freemont, Tillmann Pape, Ramesh Wignerawaraj, Nicolas Joly and Jorg
Schumacher. This work is supported by the Wellcome Trust and the
Biotechnology and Biological Sciences Research Council.
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