From the Institut de Génétique et
Microbiologie, Université Paris XI, Unité Mixte Recherche
8621, Bâtiment 360, 91405 Orsay Cedex, France, and
Laboratoire de Microscopie Cellulaire et
Moléculaire, Institut Gustave Roussy, Unité Mixte
Recherche 8126, 94805 Villejuif Cedex, France
Received for publication, July 25, 2002, and in revised form, November 18, 2002
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ABSTRACT |
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The Bacillus subtilis LrpC protein
belongs to the Lrp/AsnC family of transcriptional regulators. It binds
the upstream region of the lrpC gene and autoregulates its
expression. In this study, we have dissected the mechanisms that govern
the interaction of LrpC with DNA by electrophoretic mobility shift
assay, electron microscopy, and atomic force microscopy. LrpC is a
structure-specific DNA binding protein that forms stable complexes with
curved sequences containing phased A tracts and wraps DNA to form
spherical, nucleosome-like structures. Formation of such wraps,
initiated by cooperative binding of LrpC to DNA, results from optimal
protein/protein interactions specified by the DNA conformation. In
addition, we have demonstrated that LrpC constrains positive supercoils
by wrapping the DNA in a right-handed superhelix, as visualized by
electron microscopy.
The structural properties of DNA and specific DNA/protein
interactions are crucial for the regulation of fundamental cellular processes such as recombination, replication, and chromosome
organization. In prokaryotes, several small DNA binding proteins
regulate these processes by local changes in DNA conformation, through
the formation of specific nucleoprotein complexes. In Escherichia
coli, these small DNA binding proteins include the CRP, IHF, Fis,
H-NS, Dps, Lrp, and HU proteins (1), and in Bacillus
subtilis, the HU-like protein, HBsu (2). Notably, these proteins
can regulate DNA transcription. For example, CRP, IHF, and Fis
facilitate the association of RNA polymerase with upstream DNA
sequences or with activator proteins, and can enhance the interactions
of activator or repressor proteins at distant sites (3, 4). Moreover,
HU binding to promoter regions modulates the binding of other
transcriptional regulators like CRP (5), LexA (6) and GalR (7). Change of DNA conformation by protein/DNA interaction, however, is not limited
to prokaryotic species. Numerous eukaryotic proteins (e.g. transcription factors, such as TBP or TFIIIA, or structural high mobility group proteins, such as EF-1, SRY (8), HMG1 and HMG2 (9), and
HMG-I(Y) (10)) use similar properties, particularly DNA bending, to
regulate transcription.
The lrpC gene was identified during the B. subtilis genome-sequencing project (11). It encodes a neutral
16.4-kDa protein, LrpC, that forms tetramers in solution. Based on
amino acid sequence identity, it has been assigned to the Lrp/AsnC
family of transcriptional regulators, which in B. subtilis
includes seven Lrp/AsnC-like proteins (12-14). The N-terminal region
of LrpC is predicted to form a typical helix-turn-helix DNA binding
domain, characteristic of numerous transcriptional regulators (15).
Previous experiments have shown that the lrpC gene is
autoregulated (16). In addition, phenotypic analysis of a
lrpC mutant in B. subtilis has revealed a
possible role of LrpC in branched chain amino-acid metabolism, sporulation, and long-term adaptation to stress (16).
To analyze in detail the interactions of LrpC with DNA, the experiments
presented here combine electrophoretic mobility shift assays
(EMSA),1 electron microscopy
(EM), and atomic force microscopy (AFM), using the lrpC
promoter DNA, and curved DNA fragments. These studies show that LrpC
possesses unusual DNA architectural properties not previously assigned
to Lrp-like proteins or other general DNA-structuring proteins and that
DNA curvature and DNA topology (i.e. supra-architecture)
control the order of events in protein/DNA complex formation.
DNAs and Protein--
The 648-bp fragment encompassing the
lrpC promoter region was obtained by a PvuII
digestion of the plasmid pUC18prolrpC (16). Shorter, 331-bp
DNA fragments containing 5'-lrpC region used in EM and AFM
experiments were obtained by PCR and purified by an anion exchange
MonoQ column using a SMART system (Amersham Biosciences);
The
The DNA fragments containing the different curved regions of pBR322
were obtained by PCR amplification (pC4-6, 1773 bp, position 1185-2958; pC7, 1444 bp, position 2576-4020; and pC8, 722 bp, position 3946-307). The 1444-bp fragment containing C7 curved region
was end-labeled at its 3' extremity using a biotinylated primer. The
labeling was revealed by the streptavidin ferritin system (17).
Curvature of the pBR322 was determined using the DNA ReSCue program
(17, 18).
Plasmid pBR322 was used in both EMSA and EM experiments. Supercoiled
pBR322 was from Amersham Biosciences and linear pBR322 DNA was obtained
by a SalI digestion of the plasmid. It was then purified
using the High Pure PCR product purification kit from Roche Molecular
Biochemicals. Relaxed DNA used in relaxation assays was from Lucent
Ltd. Topoisomers of plasmid pTZ18R were prepared by a topoisomerase I
assay in presence of ethidium bromide to obtain negative topoisomers
and in presence of netropsin to obtain positive topoisomers.
The LrpC protein was previously purified (16) and shown to form
tetramers in solution (data not shown; 19). Consequently, all the
concentrations of protein used in this work correspond to LrpC tetramers.
Protein/DNA Binding--
A typical EMSA mixture
contained 25 mM Tris-HCl, pH 8, 50 mM NaCl,
10% glycerol, 0.1 mM EDTA, 5 mM
MgCl2, 1 mM dithiothreitol, 0.1 mM
phenylmethylsulfonyl fluoride (binding buffer M) with or without 4 mM spermidine, ~0.5 nM
32P-end-labeled DNA probe, and purified LrpC protein in a
volume of 20 µl. For plasmid DNA, unlabeled pBR322 plasmid (2 nM) and binding buffer M without spermidine were used.
After incubation for 10 min at room temperature, the reaction was
loaded onto a 6%
acrylamide/N,N'-methylenebisacrylamide (final
ratio, 80:1) gel containing 10% glycerol or onto a 0.7% agarose gel
in 44.5 mM Tris borate, 2 mM EDTA, pH 8.3 (0.5× Tris borate/EDTA). Electrophoresis was performed at 10 V/cm and
at 4 °C. Radioactive gels were dried, visualized by autoradiography,
and sometimes quantified with a PhosphorImager (Amersham
Biosciences). Nonradioactive gels were stained in 0.5× Tris
borate/EDTA containing 0.2 µg/ml ethidium bromide.
Observation of LrpC/DNA Complexes by Electron
Microscopy and Atomic Force Microscopy--
Complexes were formed as
described for EMSA with the following modifications. Dithiothreitol,
phenylmethylsulfonyl fluoride, and spermidine were removed from binding
buffer M to avoid any interference or artifact that might be caused by
these components. The volume of the assay mixture was 40 µl. After
incubation for 10 min at room temperature, the complexes were purified
by gel filtration (Superose 6B; Amersham Biosciences), with a SMART
system to remove unbound protein and to reduce nonspecific binding. EM observations were performed as described previously (20). 5 µl of
LrpC/DNA complexes, at a concentration of 0.5 µg/ml of DNA, were
deposited onto a 600 mesh copper grid covered with a thin carbon
film activated by a glow discharge in the presence of pentylamine (21).
Grids were washed with aqueous 2% uranyl acetate, dried, and observed
in annular darkfield in a Zeiss 902 electron microscope. Using this
spreading procedure, DNA molecules are rapidly adsorbed onto the carbon
film with no major loss in the tridimensional information (22).
LrpC-DNA complexes were observed at a final magnification of 340,000×
on a TV screen. Images of LrpC-bound DNA molecules were stored and
digitized with a Kontron image processing system as described
previously (17). The data were processed in a PC computer and the
DNA-protein interactions were mapped from 250 complexes. DNA
foreshortening gives an estimation of the length wrapped around the particle.
To analyze LrpC/DNA complexes by AFM, 20 µl of the same solutions
used for EM in presence of 5 mM Mg2+ were
deposited onto freshly cleaved mica and then washed with 0.2% (w/v)
aqueous uranyl acetate (23). The observation was performed in the
tapping mode in air specifically available with nanoscope IIIa (Digital
Instruments/Veeco).
Effect of LrpC on DNA Supercoiling in Vitro--
Different
amounts of LrpC, ranging from 37.5 to 1500 nM (in
tetramers), were incubated with 20 nM of pBR322
(supercoiled or relaxed) at room temperature for 15 min in a total
volume of 10 µl of buffer containing 20 mM Tris-HCl, pH
7.5, 50 mM NaCl, 0.1 mM EDTA, 1 mM
DTT, and 20% glycerol. Wheat germ topoisomerase I (2 unit) was then
added and the incubation continued for 150 min at 37 °C. The DNA was
deproteinized by adding SDS and NaCl to a final concentration of 1%
and 1.7 M respectively, followed by extraction with
phenol/chloroform/isoamyl alcohol (25/24/1 v/v) and the DNA
precipitated with 100% ethanol. The DNA pellet was resuspended in 10 µl of buffer containing 10 mM Tris-HCl, pH 7.5, 1 mM EDTA and loaded onto a 1% agarose gel. One-dimensional electrophoresis was performed for 16 h at 1.5 V/cm in Tris
acetate/EDTA buffer (40 mM Tris-HCl, pH 8.3, 25 mM sodium acetate, and 1 mM EDTA).
Two-dimensional electrophoresis was performed as follows: in the first
dimension, samples were separated in 1% agarose for 6 h at 3 V/cm
in Tris acetate/EDTA buffer. The gel was then equilibrated for 30 min
in Tris acetate/EDTA buffer containing 10 ng/ml ethidium bromide. The
second dimension of electrophoresis was performed for 16 h at 1.3 V/cm in the same buffer. Gels were stained with 0.2 µg/ml ethidium bromide.
The effect of LrpC on lrpC Promoter Architecture--
Previous
experiments have shown that the LrpC protein binds the upstream region
of the lrpC gene in vitro (16, 19). As many
transcriptional regulators are known to modify the geometry of their
target promoters, we wanted to determine whether LrpC displays such a
property. Therefore, the interaction of LrpC with the lrpC
promoter region was visualized by EM. Purified LrpC protein was
incubated with a 648-bp DNA fragment digested from plasmid pUC18prolrpC
that contains the
The simultaneous presence of free DNA molecules and of LrpC/DNA
complexes, which were either partially or completely condensed (Fig.
1a) confirms the cooperative
binding of LrpC to DNA as shown previously (16). Some DNA molecules
displayed thickening that was sometimes associated with bending of the
DNA (Fig. 1; data not shown). Various degrees of organization of the
lrpC promoter were observed, ranging from a local binding of
LrpC along the DNA to DNA wrapping (Fig. 1,
b-e). It is tempting to suggest that these
series of micrographs, as displayed in the order b to
e actually represent the progressive interaction of LrpC
with DNA. Measurements of DNA length in LrpC/DNA complexes indicated
that such interaction corresponds either to less than one turn of the DNA molecule around the protein core (Fig. 1, c and
d) or to the wrapping of more than one turn of the DNA (Fig.
1, a, arrow, and e), which thus seems
shorter.
LrpC Wraps DNA to Form Nucleosome-like Structures--
The
conformation of the 648-bp DNA fragment containing the lrpC
promoter was significantly altered when bound by the LrpC protein. To
further investigate this change in DNA conformation, we analyzed the
interactions of LrpC with the
We also used AFM under air-dried conditions to obtain topographic
information about the LrpC/DNA complexes. The main results shown in
Fig. 2A, b, confirmed that the thickenings
observed by EM (Figs. 1 and 7) were really caused by the presence of
the protein. LrpC covered various lengths of DNA, but such complexes
were not always associated with DNA bending. LrpC binding therefore
seems to progress until stable wrappings are formed (Fig.
2A, a) resulting also in spherical structures.
Localization of the LrpC Binding Site on the lrpC
Promoter--
To map the location of LrpC binding to the
lrpC promoter DNA fragment, we have used the Binding of LrpC to Linear versus Supercoiled Plasmid DNA--
LrpC
DNA binding properties described above are not restricted to the
lrpC promoter region. This was first demonstrated in an EMSA
by using pBR322 DNA as a competitor for the previously shown binding of
LrpC to a 32P-labeled lrpC promoter DNA fragment
(Fig. 3, A and C)
(16). Increasing concentrations of plasmid DNA were able to disrupt the
highly retarded radioactive complex LrpC/lrpC promoter (Fig. 3, A and C). Moreover, a remarkable difference
was observed in the competing ability of the linear and of the
supercoiled pBR322 monitored by EMSA. Up to 0.2 nM the
linear form is more able than the supercoiled form to compete with the
labeled lrpC promoter region for the LrpC protein.
Consequently, a larger fraction of the LrpC bound DNA is released from
the LrpC protein and can move further in the electric field (Fig.
3A). At higher pBR322 concentrations, the supercoiled form
is more effective to bind the LrpC protein and the totality of the
labeled DNA is even free from the LrpC protein (Fig.
3C).
The binding between LrpC and pBR322 DNA was also confirmed directly
(Fig. 3, B and D). At low LrpC concentrations, a
small proportion of linear pBR322 was shifted and led to clearly
defined retarded complexes (Fig. 3B, arrows)
whereas at higher concentrations of LrpC, the totality of the DNA
remained in the wells. Curiously, the binding of LrpC to supercoiled
pBR322 was somewhat different (Fig. 3D). Whereas linear
pBR322 caused gel retardation as expected on the basis of the
competition experiment, the supercoiled pBR322 showed very weak
retardation of the totality of the DNA at LrpC concentrations of 37.5 and 75 nM (Fig. 3D). At higher concentrations of
LrpC (150 and 225 nM), the protein even slightly increased the mobility of most of the supercoiled DNA. At 300 nM
LrpC, a large proportion of the DNA was stuck in the wells. In
comparison, the open circular form of plasmid pBR322 was not bound at
LrpC concentrations below 150 nM, suggesting a lower
affinity of LrpC for open circular DNA. Altogether, these results show
that LrpC interacts quite differently with the various topological
forms of the same DNA molecule.
Selective Recognition of LrpC within the Different Curved Regions
of pBR322--
LrpC shows selectivity in forming complexes with linear
pBR322 DNA. The presence of preferential LrpC binding sites was
investigated by EMSA. The 1444-bp TaqI-TaqI
restriction fragment (see Fig. 5B for details) was
preferentially bound by LrpC (data not shown). It contains a curved
region described previously as C7 (Fig.
4A; Ref. 17). To identify more
precisely the region(s) recognized by LrpC within this fragment, it was
cleaved by restriction enzymes into three different sets of DNA
fragments (Fig. 4B). Interestingly, a 517-bp fragment that
encompasses the C7 curved sequence was preferentially bound by LrpC
(Fig. 4C, 1). When the curvature or its position
within the fragment was altered, the preferential binding was lost
(Fig. 4C, 2; the 517-bp fragment is cut into 349- and 168-bp fragments). Finally, a 361-bp fragment containing only the
C7 region was specifically bound by LrpC (Fig. 4C,
3). A precise localization of the LrpC binding site was
performed using a 1444-bp biotinylated fragment and EM observation (see "Experimental Procedures"; Fig. 5A, c and
d). The complexes were visualized between positions 490 and
720, covering about 80 bp (± 20 bp) (data not shown). This corresponds
exactly to the position of the curved region detected in C7 (Fig.
4A).
Because pBR322 contains three other major curved regions
(i.e. C4, C6, and C8 (17)) (Fig.
5B), we sought to investigate the differential affinity of LrpC for these curved regions. To this
effect, three DNA fragments were amplified from pBR322 by PCR. These
contained the C4-C6 region (pc4-6), the C7 region (pc7), and the C8
region (pc8). The three fragments were mixed at equimolar concentration, incubated at an LrpC/DNA molar ratio of 12.5, and 200 complexes were analyzed by EM (Fig. 5A, a). No
LrpC/pc4-6 complexes were observed, whereas 44% of the pc8 fragments
were complexed with LrpC. Consistent with the results presented above, 78% of the pc7 fragments were found to be associated with LrpC. LrpC
binding to the C7 and C8 regions led to the formation of stable
wraps/loops as observed with the lrpC promoter region (Fig. 5A, a-d). Therefore, the presence of curvature
favored the wrapping of DNA around LrpC. Interestingly, the sequence
analysis of pc8 revealed that it has two series of oligoA tracts in
phase (i.e. on the same side of the DNA double helix) that
create two successive, sharply curved domains that could be potential
targets for LrpC. Indeed, double wrappings were frequently observed
within LrpC/pc8 complexes, as is clearly visible in Fig. 5A,
b.
LrpC Positively Supercoils DNA--
After showing the influence of
DNA curvature on the formation of the LrpC/DNA complexes, we
investigated the effect of LrpC on DNA topology. Increasing amounts of
purified LrpC protein were incubated with relaxed closed circular
pBR322 DNA. Subsequently, wheat germ topoisomerase I was added to relax
any formation of compensatory supercoils elsewhere in the free DNA. The
pBR322 DNA was deproteinized and analyzed by agarose gel
electrophoresis to resolve topoisomers (Fig.
6A). Incubation of pBR322 DNA
with increasing concentrations of LrpC in combination with the action of the topoisomerase I resulted in extended supercoiling and generated a large distribution of distinct topoisomers. Indeed, at a LrpC/DNA molar ratio of 75:1 (one tetramer of LrpC per 60 bp), 11 topoisomers could be resolved (Fig. 6A, lane b). These
experiments clearly demonstrate that LrpC interaction with pBR322 in
presence of topoisomerase I introduces supercoils into a closed
circular DNA, consistent with Ref. 19.
To ascertain whether the supercoils constrained by LrpC were negative
or positive, pBR322 samples that were incubated without LrpC (Fig.
6A, a) or with 1500 nM LrpC (Fig.
6A, b) were separated by two-dimensional agarose
gel electrophoresis (Fig. 6B). A mixture of negatively and
positively supercoiled topoisomers migrates as a biphasic arched
pattern of bands. In the presence of LrpC, the arch of topoisomers
corresponded exclusively to positively supercoiled topoisomers (Fig.
6B, b), whereas, without LrpC, the distribution of DNA
topoisomers corresponded to the relaxed state (Fig. 6B,
a). Therefore, it can be concluded that most of the DNA
bound to LrpC protein is positively supercoiled.
LrpC Wraps DNA in a Right-handed Superhelix--
We have shown
that LrpC binds to negatively supercoiled pBR322 DNA (Fig.
3B); however, LrpC constrains positive supercoils in closed
circular DNA (Fig. 6). Therefore, binding of LrpC to different forms of
pBR322 plasmid DNA was further analyzed by EM. LrpC was incubated with
an equimolar concentration of linear and supercoiled plasmid DNA at a
LrpC/DNA ratio of 37/1 (corresponding to one LrpC tetramer per 118 bp).
Only 5% of the open form of the plasmid (linear and traces of open
circular DNA) were complexed with LrpC opposed to almost 100% for the
supercoiled DNA. In the cases where LrpC associated with the linear or
open circular plasmids, only one or two wrappings, appearing as small
loops, were formed (Fig. 7d;
data not shown). In contrast, the assembly of LrpC with negatively
supercoiled DNA led to formation of five to six homogeneously structured loops (Fig. 7, b-d). This confirmed
the selective affinity of LrpC for supercoiled DNA compared with linear
DNA at high LrpC/DNA ratios. Moreover, these LrpC/DNA wrappings were
frequently near each other in a restricted part of the molecule (Fig.
7b). The resulting topological constraints induced by loop
formation seemed to be compensated by tight winding in other parts of
the DNA, compared with free DNA molecules (compare Figs. 7b
and 7a). Such a partition of DNA structural domains is
clearly caused by an increase in the free negative supercoiling to
compensate for the LrpC-restrained positive supercoils. This clearly
demonstrates that the DNA is wrapped around LrpC as a right-handed
superhelix, because left-handed wrapping of negatively supercoiled DNA
would result in an apparent relaxation of the molecule. Furthermore, compared with free DNA (Fig. 7a), the part of the DNA
exhibiting tight winding displayed thickening that could be caused by a
local polymerization of LrpC on the DNA (Fig. 7, b and
c). We also observed that within the same samples several
supercoiled DNA molecules were highly compacted by the LrpC protein
(Fig. 7d).
Considering the properties of LrpC, it was important to
monitor its binding to positively supercoiled DNA. To this effect, we
used a pBR322-derivative plasmid, pTZ18R, also containing the C7 and C8
regions. With the native negatively supercoiled form of pTZ18R
( We previously identified LrpC as the seventh member of the
Lrp/AsnC family of proteins in B. subtilis, and we have
shown that LrpC positively autoregulates its own gene. In this study,
we have analyzed in detail the interactions of LrpC with DNA, with respect to DNA conformation, curvature, and topology, using EMSA, EM,
and AFM. We showed that LrpC progresses unspecifically along DNA,
preferentially recognizes a specific type of DNA curvature, and wraps
DNA in a right-handed superhelix to form looped structures. In
addition, we propose that its oligomerization on DNA is not random but
is orientated by DNA conformation, mainly its bendability and its
topological state. Moreover, LrpC is an unusual bacterial DNA
architectural protein because of its capacity to constrain positive
supercoiling. We propose a model for dynamic interactions between LrpC
and DNA.
An Octameric Model for LrpC/DNA Interactions--
We
have provided evidence that LrpC wraps DNA and forms stable complexes
resembling nucleosomes with various DNA fragments, including the
lrpC promoter region, where its binding coincides with the
P1 promoter. Formation of stable complexes between LrpC and
DNA results from protein-protein assembly. DNA flexibility or intrinsic
curvature favors protein-protein interactions within one DNA fragment
to form a stable protein core. This results in a progressive bending of
the DNA that leads to loop formation through a complete wrapping of the
DNA around the protein (Fig. 8B). LrpC could interact with
DNA through its N-terminal helix-turn-helix motif and oligomerize
through its C-terminal domain (Fig. 8A). The radius of
curvature measured in the LrpC/DNA complexes correlates perfectly with
the sizes of the octameric model presented for the recently
crystallized LrpA protein of Pyrococcus furiosus, in which
the four dimerized N-terminal DNA binding domains are diametrically
opposed (25). Moreover, as with LrpA, LrpC has been shown to form
dimers and multimers of dimers, mainly tetramers in solution (Ref. 19;
data not shown).
DNA Conformation and Formation of Stable LrpC/DNA
Complexes--
DNA bendability determines the path of the double helix
axis and contributes to the thermodynamic stabilization of the
DNA/protein complexes. DNA bendability results from an increased local
flexibility (26) and/or from a static intrinsic curvature, such as
kinks or smooth continuous curvature, with either planar or torsional bending (27, 28). We demonstrated here that among different types of
curvature, LrpC forms stable complexes with curved regions containing
phased A tracts in pBR322 (C7 and C8; Fig. 5); in the lrpC
promoter region (between the DNA Topology and LrpC/DNA Interactions--
We have
shown that LrpC/DNA complex formation is influenced by DNA topology
and, moreover, that LrpC constrains positive supercoiling. We propose
that the formation of a first complex in supercoiled molecules is
promoted by one curved region localized at one of the apices, as
observed previously for the transcription activator NR1 (32) and the
Tth 111 glutamine synthetase (33) (Fig. 7 and 8D,
1). Because LrpC induces the formation of a positively supercoiled loop, a new negatively supercoiled loop is then created in
the vicinity of the first complex to maintain a constant linking number. The formation of a second complex is favored by cooperative effects, which promote LrpC recruitment to the flanking DNA regions (Fig. 8D, 2). This will induce the formation of a
new positive supercoil and subsequently of a compensatory negative
supercoil that will be again targeted by an LrpC oligomer (Fig.
8D, 3). This model explains the formation of
successive wrappings in a very close proximity by an invasive
mechanism, which induces partition of topological domains between
LrpC-restrained positive supercoils and free negative ones (Fig.
8D, 4). To our knowledge, we present the first
visualization of such a partition in negative and positive supercoiled
domains within a single DNA molecule and therefore demonstrate that
positive supercoiling mediated by LrpC is caused by a right-handed DNA
wrapping (Fig. 7, b and c). The presence of a
right-handed DNA superhelix wrapped around a protein core in a
negatively supercoiled environment represents a new topological paradox
that could be explained by the following considerations.
The affinity of LrpC for DNA increases with supercoiling, either
positive or negative, because supercoiling favors loop formation. Whatever their chirality, these loops promote protein/protein assembly,
and the stabilization of the complexes leads to the formation of a
right-handed DNA helix. Such DNA transition triggered by LrpC should
require minimal energy, as shown for interaction of
(H3-H4)2 tetramer with supercoiled DNA (34).
Such a topological partition in the plasmid induces an accumulation of
negative topological constraints in the free DNA, which reduces its
flexibility. Consequently, right-handed DNA wrapping around the LrpC
protein core is no longer favored, and an alternative mode of
protein-protein interaction is adopted; any additional LrpC protein
polymerizes along the hypernegatively supercoiled DNA (Figs. 7 and
8).
This bimodal assembly of LrpC within nucleoprotein complexes is related
to two types of DNA condensation, determined by the topological state
of DNA. The first one results from successive DNA wrappings mainly
observed with linear fragments and in regions of negatively supercoiled
DNA (Figs. 1, 2, and 7). The second one results from polymerization of
LrpC along DNA and bridging of DNA segments within a circular plasmid
to finally induce its folding. This could be observed with open
circular plasmids (data not shown) or with slightly positive
supercoiled plasmids, where right-handed DNA wrapping by LrpC led to a
relaxation of the free DNA (Fig. 7), followed by additional
polymerization of the protein along DNA. The same type of interactions
(i.e. polymerization and bridging) is observed with
"straight" uncurved DNA (data not shown; Fig. 8C).
LrpC, an Unusual DNA Architecture Protein--
The capacities of
LrpC to drastically modify DNA structure by DNA bending and wrapping,
and the fact that it probably uses these properties to modulate the
geometry of promoters, confirms that LrpC belongs to the DNA
architectural protein family. However, it seems that LrpC possesses
unusual properties among eubacterial DNA structuring proteins,
including the Lrp-like family. Whereas E. coli Lrp has been
proposed to wrap DNA (35), LrpC, along with the Smj12 protein from
Sulfolobus solfataricus (which also overwinds DNA) and the
PutR protein from Agrobacterium tumefaciens, are the only
members of the Lrp-like family for which DNA wrapping has been firmly
demonstrated (36, 37). Looped structures formed by LrpC where 80 bp of DNA is wrapped resemble eukaryotic dimers of
(H3-H4) and archaeal HMf or HTz tetrasomes
(Fig. 2; 38, 39). Eukaryotic dimers of (H3-H4)
(35) and HMf tetramers (40) are able, under certain conditions, to
constrain positive supercoils, as observed for LrpC. Other proteins
that activate transcription, such as the eukaryotic transcription
factors UBF or SWI/SNF or the B. subtilis PurR regulator,
are known to bind upstream of the promoters they regulate and to
introduce one positive supercoil (41-44). Therefore, it is likely that
the capacity of LrpC to induce right-handed supercoiling is involved in
its regulatory activity. In addition, like the HMf proteins, eukaryotic
histones and the high mobility group proteins, LrpC highly compacts
DNA. Such ability has not been described thus far for other members of
the Lrp-like family. This work shows that the B. subtilis
LrpC protein displays a mosaic of properties present in archaeal and
eukaryotic histones, Lrp-like proteins, transcription factors, and
eubacterial DNA-structuring proteins. Consequently, LrpC is a unique
member of the DNA architectural family of proteins.
A fascinating hypothesis is that micro-organisms have developed a DNA
overwinding activity to compensate for the DNA underwinding activity
displayed by more common nucleoid-associated proteins such as H-NS, HU,
IHF, or Fis. The LrpC protein could be a prototypical member of a new
family of proteins that overwinds DNA and, together with
topoisomerases, modulates the global supercoiling density or, more
likely, the local DNA topology during certain DNA transactions. Indeed,
at 10 to 80 tetramers per cell, LrpC is not particularly abundant in
the cell under normal growth conditions (16); this could limit its role
as global chromosome organizer and suggests a role in sensing locally
DNA architecture. In light of what we have learned concerning LrpC, it
would be interesting to evaluate the DNA binding and DNA structuring
properties of the six other B. subtilis Lrp/AsnC proteins to
gain a fuller appreciation of the role for this family in bacterial physiology.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
fragments correspond, respectively, to
225 to +106 and
270
to +61 with respect to the P1 transcription start site.
fragment was biotinylated at its 5'- or 3'-extremity and
dimerized using streptavidin. After dimerization of the
fragment the promoters P1/P2 are localized near the
extremities when using the 5'-biotinylated fragment (5'
-dimers) or
near the center using the 3'-biotinylated fragment (3'
-dimers).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
fragment in Fig. 2A, this is 331-bp of
the 5'-lrpC region (16). Protein/DNA complexes were visualized by EM using an annular darkfield mode (24).
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Fig. 1.
Visualization of LrpC binding to the
lrpC promoter region. A 648-bp fragment (1 nM) containing 331 bp of the 5'-lrpC region
( 225 to +106 with respect to P1) and flanked by 120 and
197 bp of pUC18 plasmid DNA was mixed with purified LrpC at a
protein/DNA molar ratio of 6:1 (one tetramer/100 bp). LrpC/DNA
complexes were visualized by EM. a, different types of
protein/DNA complexes that formed compared with uncomplexed DNA
(molecule at bottom right). The presence of the protein
correlates with thickening of some parts of the DNA. The LrpC/DNA
complex indicated by an arrow shows highly condensed DNA
caused by successive wrappings. b-e,
representative LrpC/DNA complexes indicating the different steps in the
wrapping mechanism. In e, a tight wrapping of more than one
superhelical turn of DNA is shown. Scale bar, 50 nm.
fragment itself, which encompasses
only the 5'-lrpC region (Fig.
2A,
fragment,
225 to
+106 with respect to the P1 transcription start site). Protein/DNA complexes were allowed to form for different lengths of
time using a LrpC/DNA molar ratio of 4:1 (LrpC protein concentration in
tetramers, its preferred quaternary structure in solution). The
complexes were subsequently visualized by EM at high magnification (140,000×). After 1-10 min, the complexes seemed to be localized at
various positions of the
fragment, although a preference at or near
the extremities was observed (data not shown). After a longer
incubation time (15 min), LrpC seemed to be nearer to the center of the
fragment, with the DNA molecule clearly tightly wrapped around it
(Fig. 2A). This LrpC-mediated DNA wrapping creates spherical
structures resembling nucleosomes (Fig. 2A, a).
The contour length of the DNA wrapped around LrpC (averaged from
measuring 50 LrpC/DNA complexes; see "Experimental Procedures") was
28 ± 4 nm, which corresponds to 80 ± 12 bp, and the radius
of curvature was 4.5 ± 0.2 nm. The presence of intrinsic
curvature in the lrpC promoter region presumably promotes
the DNA wrapping around LrpC (16). As shown in Fig. 1, several LrpC/DNA
complexes resulted from multiple wrappings of the DNA that induced a
highly ordered condensation (data not shown).
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Fig. 2.
Structure and mapping of the LrpC/DNA
complex. A, EM and AFM visualization of LrpC/DNA
nucleosome-like complexes. Purified LrpC was incubated with the DNA fragment (
225 to +106 with respect to the lrpC P1promoter)
and the wrapping of DNA around LrpC was analyzed by EM (a)
(140,000×). b and c, representative LrpC/
DNA
fragment visualized by AFM without (b) or with
(c) DNA wrapping. B, mapping the LrpC binding
site within the lrpC promoter region. The
fragment
(
270 to +61 with respect to P1) was biotinylated at its
5'- or 3'-extremity and dimerized using streptavidin (5'
- and
3'
-dimers, respectively). Promoters P1/P2 are
localized near the extremities of 5'
-dimers (a and
b) or near the center of 3'
-dimers (c).
Monomers of
fragments are also present in the preparation and are
complexed at their extremities by LrpC. The precise LrpC binding sites
of 250 LrpC/5'
dimers complexes were mapped, and the data presented
in the histogram show the total percentage of interactions within 20-bp
windows. m and d represent monomers and dimers of
fragments, respectively. A and B, scale
bars, 50 nm. The
and
fragments used above are represented
with the wedge-curved sequence (black box) localized between
P2 and P1
35 box, whereas the junction-curved
DNA is localized between P1
35 box and the lrpC
ATG.
fragment
(Fig. 2B,
270 to +61 with respect to the P1
transcription start site). The
fragment has the same length as the
fragment, but the P1 promoter region is much closer to
the extremity of the DNA molecule. To distinguish the extremity of the
fragment that contains the P1 promoter,
fragments were
bridged by their 5' (5'
dimers) or 3' (3'
dimers) extremities
(see "Experimental Procedures"). In the 5'
dimers, the
P1 promoter regions localized at the extremities of the
dimers. In the 3'
dimers, the P1 promoter regions are
gathered at the center of the dimers. When LrpC was incubated with
either the 5'
dimers (Fig. 2B, a and
b) or the 3'
dimers (Fig. 2B, c), its binding coincided with the position of the P1 promoter
as visualized by EM. Some unbridged
monomers present in the
preparation were complexed with LrpC at their extremities. 250 LrpC/5'
complexes were mapped to the precise location of LrpC. 78%
of the LrpC/5'
dimer complexes had LrpC bound at the P1
promoter region. Only 10-20% of the complexes had LrpC localized at
the P2 promoter region. The average length of DNA complexed
with LrpC was 90 bp with an S.D. of 42.5 bp. Multiple DNA wrappings
around LrpC were also observed here (data not shown).
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Fig. 3.
LrpC binding has different effects on linear
and on supercoiled plasmid DNA. In competitive EMSA,
32P-labeled 5'-lrpC fragment (0.5 nM) was incubated with an excess of purified LrpC (12 nM) and increasing amounts (0 to 2 nM) of
unlabeled linear pBR322 (pBRlin, A) or supercoiled pBR322
(pBRsc, C) as competitor DNA. Complexes were resolved by
migration through a 6% polyacrylamide gel. In direct plasmid EMSA, 2 nM of linear pBR322 DNA (B) or negatively
supercoiled pBR322 DNA (D) was incubated with an increasing
concentration of LrpC (0 to 300 nM). Complexes were
resolved by migration through a 0.7% agarose gel. Protein/DNA
complexes were visualized by staining with ethidium bromide. 400 ng of
bovine serum albumin (BSA) was incubated with the plasmids
as a negative control. Linear plasmid (lin) corresponds to
SalI-digested supercoiled pBR322 (sc). LrpC/lin
complexes are indicated by arrows. Purified supercoiled
pBR322 contains traces of open circular plasmid as indicated
(oc).
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Fig. 4.
Identification of a curved region of pBR322
to which LrpC preferentially binds. A, analysis of DNA
curvature. A 1444-bp TaqI-TaqI fragment of pBR322
(position 2576 to 4020) that is preferentially bound by LrpC was
subjected to curvature detection analysis using the DNA ReSCue program
(18). The propensity plots indicating curvature (degree per base pair)
according to Refs. 45 (gray line) and 46 (black
line), plotted against the position in the sequence, are
presented. The DNA corresponding to the C7 curved region is indicated
(410 to 750 bp in the 1444-bp TaqI-TaqI fragment
that corresponds to 2986 to 3301 bp in pBR322). The C7 DNA contains two
curved regions, the first only slightly curved and located between 410 and 600 bp with a maximum curvature at 550 bp and a second one extended
from 600 to 750 bp with a maximum curvature at 650 bp. B,
schematic representation of the 1444-bp fragment with the C7 curved
region (thick line). The maximum of the major curvature (650 bp) is indicated by the thick arrow, whereas the maximum of
the minor curvature (550 bp) is indicated by the thin arrow.
The positions of relevant restriction endonuclease cleavage sites are
shown. Three sets of DNA fragments encompassing different regions of
the 1444-bp fragment were generated by using different enzyme
combinations. *, restriction fragments that include a part of or the
entire C7 curved region. C, EMSA of LrpC interactions with
restricted segments of the 1444-bp TaqI-TaqI
fragment of pBR322. The 1444-bp TaqI-TaqI
fragment (10 nM) was digested with
AvaII-HinfI (1),
HinfI-BspHI (2), or
AvaII-HinfI-Eco57I (3) and
incubated with increasing concentrations of LrpC (0 to 300 nM). Complexes were resolved through a 6% acrylamide gel
in 0.5× Tris borate/EDTA at 4 °C. M, Promega 100-bp DNA
ladder. *, restriction fragments that include a part of or the entire
C7 curved region.
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Fig. 5.
LrpC binds preferentially to the C7 and C8
regions of pBR322. A, EM visualization of LrpC
association with curved DNA. Three PCR fragments were generated that
contain the C4-C6 (1773 bp, pc4-6), C7 (1444 bp, pc7), and C8 (722 bp,
pc8) pBR322 curved regions. These fragments (5 nM) were co-incubated with 100 nM of LrpC.
Complexes formed were observed by EM (a and b).
To localize the binding of LrpC to the C7 curved region, the pc7
fragment was labeled by biotin-streptavidin-ferritin at its
3'-extremity and complexes formed with LrpC were visualized by EM
(c and d). The bar represents 100 nm.
B, schematic representation of the curved regions of pBR322.
This diagram is adapted from Ref. 17. Minor curved regions including
C1, C2, C3, and C5 are hatched. Major curved regions C4, C6,
C7, and C8 are indicated by black boxes. PCR fragments
containing C4-C6, C7, or C8 regions are represented by
arrows.
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Fig. 6.
LrpC constrains positive supercoils
in relaxed pBR322. Topoisomerase I relaxation assay. A,
relaxed pBR322 (20 nM) was incubated with increasing
amounts of LrpC (37.5 to 1500 nM) for 15 min at room
temperature. TopoI was then added and incubation continued for 150 min
at 37 °C. Deproteinized samples were separated on a one-dimensional
agarose gel. B, pBR322 DNA without LrpC (a), and
with 1500 nm LrpC (b), as indicated, were separated by
two-dimensional agarose gel electrophoresis with ethidium bromide in
the second dimension. The positive topoisomers constrained by LrpC are
clearly visible in sample b.
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Fig. 7.
EM visualization of complexes between LrpC
and supercoiled plasmid DNA. a, a molecule of naked
supercoiled pBR322. b-d, LrpC was incubated with
an equimolar concentration of supercoiled pBR322 and linear plasmid DNA
(protein/DNA molar ratio of 37:1 corresponding to one LrpC tetramer per
118 bp). A typical LrpC/supercoiled pBR322 complex conformation that
was predominantly observed is shown in b and c.
LrpC was rarely associated with linear DNA or open circular DNA
molecules. However, a complex of LrpC with open circular pBR322 is
shown on the left side of d. The ability of LrpC to highly
condense supercoiled DNA is shown at the bottom right of
d-g, corresponding to complexes of LrpC with
positively supercoiled pTZ18R plasmid ( LZ = +4) at a LrpC/DNA
molar ratio of 8 (e) and 27 (f and g).
g, top left, free pTZ18R DNA molecule.
Scale bar, 50 nm.
Lk =
15), we obtained the same pictures as with
pBR322 (LrpC/DNA ratio of 28/1 corresponding to 1 LrpC tetramer per 100 bp, data not shown). However, when pTZ18R was artificially positively supercoiled (
Lk = +4), only two loop complexes were
observed (Fig. 7e, LrpC/DNA ratio of 8:1). Because positive
supercoils are introduced by LrpC binding, the unbound DNA region is
relaxed. When the LrpC/DNA ratio was increased to 27:1, a mixture of
two types of complexes was observed: the two loops complexes already observed at a lower LrpC/DNA ratio and new complexes showing a very
organized folding of the pTZ18R (+4) on itself (Fig. 7, f and g). As observed with native pBR322, LrpC was able to
massively cover the DNA through cooperative mechanisms, thus
promoting intramolecular condensation of DNA through LrpC/LrpC interactions.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 8.
A model for LrpC-DNA interaction.
A, octameric model of quaternary structure of LrpC
interacting with DNA adapted from comparison with the P. furiosus LrpA protein octameric model (25). N-terminal
helix-turn-helix DNA-binding domain and C-terminal oligomerization
domain are represented by open squares and open
circles, respectively. B, interaction of LrpC with
flexible/curved DNA induces a complete wrapping of the DNA around the
protein to form a nucleosome-like structure. C, interaction
of LrpC with straight DNA (i.e. uncurved or
hyperconstrained) leads to polymerization of LrpC and bridging of DNA
fragments through protein-protein interactions. D,
interaction of LrpC with a negatively supercoiled plasmid DNA. LrpC
creates a positively supercoiled loop that is compensated by a new
negatively supercoiled loop. The latter is a target for LrpC. This
model explains the formation of successive wrappings in a very close
proximity by an invasive mechanism that induces partition of
topological domains between LrpC-restrained positive supercoils and
free negative ones.
35 box of P1 and the ATG,
Fig. 2B); and in synthetic curved DNA molecules (data not
shown). These A tract motifs are related to the junction model, in
which the deflection of the helix axis is localized at junctions
between B' form structure of A tracts and B form (29). Furthermore, all
regions stably bound by LrpC contained phased A tracts preceded by a C
(i.e. C(A)n motifs). In contrast, LrpC does not
form any complexes with the pBR322 curved C4-C6 region (Fig. 5) or
with the highly curved region located upstream of the
35 box of the P1 lrpC promoter (Fig. 2B; Ref. 16). In these
fragments, curvature is more related to the wedge model, which
attributes small deflections of the helix axis at every base-pair step,
with a predominant contribution of the AA dinucleotide (30). Although
junction and wedge models are comparable in their general predictions
of DNA curvature for fragments, including phased A tracts, they differ for curved fragments without A tract motifs. Moreover, the wedge model
does not take into account cooperativity effects in the stacking of AT
base pairs within A tracts (31). Our results clearly show that LrpC
discriminates between different types of curvature to form stable
complexes within C(A)n phased motifs.
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ACKNOWLEDGEMENTS |
---|
We thank R. Exley for help with the English language, P. Deighan, F. Confalonieri, and W. F. Stevens for helpful discussions, and S. Lyonnais for help with figures.
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FOOTNOTES |
---|
* This work was supported in part by grants from CNRS/Université Paris XI (UMR C8621 and ARC 9490 to F. L. H. and CNRS UMR 8126 to E. L. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: Groupe de Génétique des Biofilms, Bâtiment Fernbach, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris cedex 15, France.
¶ Supported by fellowships from the French Ministère de l'Éducation Nationale, de l'Enseignement Supérieur et de la Recherche, and Fondation pour la Recherche Médicale.
** To whom correspondence should be addressed. Tel.: 33-1-69-15-63-62; Fax: 33-1-69-15-63-34; E-mail: francoise.le-hegarat@igmors.u-psud.fr.
Published, JBC Papers in Press, November 27, 2002, DOI 10.1074/jbc.M207489200
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ABBREVIATIONS |
---|
The abbreviations used are: EMSA, electrophoretic mobility shift assay; EM, electron microscopy; AFM, atomic force microscopy.
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