From the Institut de Génétique et Microbiologie, Unité Mixte de Recherche CNRS no 8621, Université Paris-Sud XI, Bâtiment 409, Centre Universitaire d'Orsay, F-91405 Orsay Cedex, France
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ABSTRACT |
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AlcR is the transcriptional activator in
Aspergillus nidulans, necessary for the induction of the
alc gene cluster. It belongs to the
Zn2Cys6 zinc cluster protein family, but
contains some striking differences compared with other proteins of this
group. In this report, we show that no dimerization element is present in the entire AlcR protein which occurs in solution as a monomer and
binds also to its cognate sites as a monomer. Another important feature
of AlcR is its unique specificity for single sites occurring naturally
as inverted or direct repeats and sharing a common motif, 5'-(T/A)GCGG-3'. Like most other Zn2Cys6
proteins, AlcR contacts directly with the CGG triplet and, in addition,
the upstream adjacent guanine is required for high affinity binding. We
also establish that the flanking regions outside the core play an
essential role in tight binding. From our in vitro
analysis, we propose an optimal AlcR-binding site which is
5'-PuNGCGG-AT rich 3'.
The Aspergillus nidulans protein, AlcR, activates
transcription of genes required for the oxidation of ethanol and other
alc clustered genes whose functions are currently being
studied (1, 2). Transcriptional regulation is mediated by the binding
of AlcR to different cognate sites, encompassing the same consensus core, 5'-(T/A)GCGG-3', organized as direct and inverted repeats, located upstream of these genes (3-6). AlcR is a fungal transcription factor of the zinc binuclear cluster family, containing six cysteines and two zinc atoms (C6 protein class) (7-9). However, AlcR is distinguishable from other proteins in this family by its structural and binding properties. It has an asymmetric structure resulting from
an additional 16 residues between the third and the fourth cysteines
compared with 6-8 residues usually found in the other proteins of this
class (7). Furthermore, in this loop, the proline residue conserved in
all the zinc binuclear cluster domains and which was shown to be
important for zinc binding, as stated for GAL4 (10) and more recently
for PrnA (11), is not present in AlcR. However, these differences have
no effect on the distance between the two zinc atoms and between zinc
and sulfydryl ligands of cysteine (12) which are similar to those found
in the solved structure of GAL4, PPR1, and PUT3 (13-16). Another
important feature of AlcR is the absence of a coiled-coil dimerization
region following the C6 zinc cluster in the other proteins (5). Two
stretches of leucine heptad repeats, downstream of the zinc cluster,
are interrupted by several proline residues known to disrupt a
continuous NMR studies have shown that the isolated AlcR zinc binuclear cluster
domain (residues 1-60) binds to a single copy site as a monomer,
albeit with a low affinity (9). More recently, binding experiments
performed with a longer AlcR protein (1-197), containing the region
downstream of the zinc cluster, have clearly indicated that one AlcR
molecule binds with high affinity to DNA single sites (5 × 10 In this work, we wanted to address several questions. First, it was
important to determine if any functional dimerization element other
than those generally found in the proteins of the C6 class was present
in AlcR, downstream of residue 197. Second, since the AlcR consensus
core includes both the CGG triplet found to interact with the other
well defined zinc binuclear cluster proteins such as GAL4, PPR1, PUT3
(13-16), and the GCG triplet shown to interact with a few other C6
proteins (17), the mode of recognition of AlcR toward its DNA sites was
investigated. Third, it appears that AlcR specificity toward its DNA
target is different from the other proteins of the C6 class, thus the role of the flanking regions outside the consensus core was determined. Since AlcR natural targets are organized as direct or inverted repeats,
the size and the composition of the spacer are also of special interest
in the in vitro study of AlcR binding.
In order to answer these questions, a functional analysis of the
putative dimerization regions of AlcR has been performed as well as a
systematic study by site-directed mutagenesis of AlcR binding to direct
or inverted repeat physiological targets and to an artificial single site.
Strains, Plasmids and Phages--
Escherichia coli
strains CSH50:
The plasmid expressing AlcR(1-197) tagged with 6xHis was constructed
as described previously (5). The AlcR protein expressed was partially
purified on a Ni2+/nitrilotriacetic acid-agarose column at
up to 20% of homogeneity and used for electrophoretic mobility shift assays.
Electrophoretic Mobility Shift Assays--
Oligonucleotide
probes containing either direct repeat targets a or
c or inverted repeat target b from the
alcA promoter or an artificial single copy site
sc and its mutated derivatives were used in most gel shift
experiments. Sequences of these probes, as present on the top strand of
the alcA promoter sequence, are given below: a,
5'-CCCACTTGTCCGTCCGCATCGGCATCCGCAGCTCGGG-3'; b, 5'-GATGCATGCGGAACCGCACGAGGGC-3';
c, 5'-CTTTCTGGTACTGTCCGCACGGGATGTCCGCACGGAGA-3'. The conserved repeats are marked in boldface type. The sequence of a single
copy site probe sc and its mutated variants is indicated in
Table II. Probes with a modified spacer region (underlined below) have
the following sequence: inverted repeats: N2,
5'-ATGCATGCGGGCCCGCACGAGGGC-3'; N3,
5'-GATGCATGCGGAAACCGCACGAGGGC-3';
N12,
5'-GATGCATGCGGTTCAAATAAGATCCGCACGAGGGC-3'; N17,
5'-GATGCATGCGGTTCAAATCTATAAAGATCCGCACGAGGGC-3'; direct repeat, N10,
5'-CTTTCTGGTACTGTCCGCACGGGATGTCCGCACGGAGA-3'; N13,
5'-CTTTCTGGTACTGTCCGCACGGAGAGATGTCCGCACGGAGA-3', where N corresponds to the number of nucleotides separating the CGG
triplets in the consensus core. Sequences of the other probes used in
this work are shown in the figures. In all cases, only the top strand
of the oligonucleotides is shown. The binding reactions were analyzed
by gel electrophoretic mobility shift assays as described
previously (5) and were quantified with a PhosphorImager (Molecular
Dynamics). In experiments determining the relative binding efficiencies
of oligonucleotides containing sc or mutated sequences, the
protein concentration of AlcR(1-197) was calibrated to yield
approximately 30-50% of bound probe. Binding affinities were relative
to the binding of AlcR(1-197) to an artificial probe sc
normalized to 1 or 100.
Footprint Analysis--
Methylation interference and
depurination interference footprinting assays were carried out as
described earlier (4). Generally, 105 cpm of end-labeled
single strand DNA probe were annealed with the complementary,
non-labeled strand and then subjected to chemical modifications either
with dimethyl sulfate, for partial methylation of guanines, or
piperidine formate, for partial depurination, according to the
procedure of Maxam and Gilbert (19). Briefly, the modified DNA probes
were incubated with sufficient partially purified AlcR(1-197) protein
(300-500 ng) to bind approximately 30-50% of the probe. The protein
bound and free DNAs were separated by preparative gel mobility shift
technique and electroeluted from gel slices. Recovered DNAs were
treated with 1 M piperidine and resolved on a 16%
polyacrylamide gel containing 8 M urea.
In Vitro Transcription-Translation--
A full-length AlcR
protein (residues 1 to 821) and a truncated NirA protein (residues 1 to
222) were expressed in vitro using the TNT T7-coupled
reticulocyte lysate system (Promega) according to the recommendations
of the supplier. For this purpose the AlcR coding sequence was cloned
into the T7 expression vector pET-22b. The pNirA(1-222) plasmid
expressing NirA (1-222) was kindly provided by Dr. M. I. Muro-Pastor. 3-20 µl of translation products were incubated for
1 h with 0.005% glutaraldehyde in 50 mM potassium phosphate buffer, pH 7.5, and afterward analyzed either by 8 or 10%
SDS-polyacrylamide gel electrophoresis followed by fluorography.
Evidence for AlcR Monomers: AlcR Is a Monomeric Protein--
Our
previous studies performed with a truncated protein predicted that the
active form of AlcR binds DNA in vitro as a monomer (5).
This result was further confirmed by calibrated gel filtration chromatography. The AlcR(1-197) protein migrates with an apparent molecular mass of 18 kDa (data not shown), a value close to that expected for a monomer (22 kDa). Nevertheless, the truncated protein could be lacking a downstream dimerization domain. In order to determine if AlcR regions other than those usually found in the C6
class proteins could drive dimerization of AlcR, another method based
on the property of the bacteriophage
One could argue that the lack of dimerization could be the result of a
possible insolubility or misfolding of the chimeric
To obtain additional evidence that AlcR does not form dimers, at least
in solution, we compared the dimerization of AlcR and NirA by
cross-linking each protein in solution. NirA has been recently shown to
form stable dimers (23). A full-length AlcR protein and a truncated
NirA(1-222) version containing its dimerization interface were
translated in vitro and after treatment with glutaraldehyde were analyzed by SDS-gel electrophoresis. As shown in Fig.
1, the majority of NirA(1-222) dimerizes
whereas AlcR(1-821) stays monomeric in solution even in conditions
when the translated product is increased 7-fold in the reaction.
Mode of AlcR Interaction with Its Cognate Targets--
Our
previous in vitro DNA binding studies using alcA
physiological repeated sites have shown the formation of two complexes (a fast migrating complex CI and a slow migrating complex CII) with
symmetric sites, which correspond to the binding of one and two AlcR
molecules to both sites, respectively (Refs. 5 and 6 and Fig.
4A). The apparent Kd for both complexes was not significantly different suggesting a lack of cooperativity (5).
However, one retarded complex CI is preferentially observed with all
the direct repeat targets tested. Furthermore, we have demonstrated
that AlcR is able to recognize a single copy site (5, 9) with the same
range of affinity. Thus, it seemed necessary to examine for all these
types of DNA targets whether AlcR uses a general mode of recognition or
a pattern of DNA-protein interaction that is dependent upon the target
configuration (direct or inverted repeat or a single copy site). Two
different footprint interference techniques, namely methylation and
depurination interference, were applied to establish the base-specific
contacts within direct repeat targets a and c and
inverted repeat site b in the alcA promoter (see
Fig. 2). All these targets have been
shown to be functional in the alcA promoter region (6). These experiments were performed with AlcR(1:197), His-tagged protein,
which contains a region downstream of the zinc binuclear cluster
necessary for high affinity binding (5).
For both asymmetric targets a and c, only a
slight reduction of signal at positions encompassing the consensus motif 5'-GCGG-3' and the adjacent A (position 6) was detected (Fig. 2,
A and B). The equal reduction of band intensities
for both repeats is indicative of a random occupancy of binding sites which results in formation of one retarded complex, CI. Our present results might seem contradictory to the previous footprinting data
which demonstrated that only one of the two binding sites, namely
c2, can be occupied by AlcR. However, the
oligonucleotide probe utilized earlier was 6 bp shorter at the 5' end
than the one we used in the current studies. Apparently, the limited
size of flanking regions impaired the AlcR binding to the adjacent site
c1.
Similarly, AlcR exhibits the same manner of site recognition, when
fixed on the symmetric target b, giving rise to the fast
migrating complex, CI. Only a slight interference is noticable by both
methylation and missing-contact footprint analyses (Fig.
2D). Thus, no site preference for binding of the first AlcR molecule to either direct or inverted repeat sites in the
alcA promoter was observed. A depurination footprint pattern
obtained with complex CII revealed a strong interference of all the G
residues within the consensus motif in both strands. In addition, two
central A residues separating inverted repeats exhibited strong
interference (Fig. 2D). Methylation of two G residues at
positions 2 and 4 in both strands interfered strongly with AlcR
binding, while methylation of the adjacent G (position 5) had weaker
effects. These results indicate that AlcR interacts mainly in the major
groove, although minor groove contacts exist. For example, contacts
with N3 of A in the spacer sequence in the symmetric repeat
b, as shown by methylation footprinting data, occur in the
minor groove (Fig. 2D). Within each recognition motif AlcR
makes direct, sequence-specific contacts with bases on the same strand,
since methylation of the N-7 position of G (position 3) in the C-rich
strand of the consensus core resulted in weak, if any, interference.
Footprint experiments performed with an artificial single copy site
(sc), designed after disruption of one of the repeats in the
symmetric target b, suggest that the AlcR molecule establishes direct base-specific contacts within the conserved repeat
and does not interact with neighboring nucleotides (Fig. 2C). The pattern of depurination interference appeared to be
similar for all types of targets (inverted or direct repeat or a single copy site), indicating that AlcR uses a unique strategy for DNA recognition.
AlcR Interacts Directly with the CGG Triplet--
Most of the zinc
binuclear cluster proteins have been shown to interact directly with
CGG triplets present at the ends of their cognate targets, whereas some
activators of this class are able to recognize the CGC triplet (25).
According to footprinting data, the AlcR-binding site represents a
combination of both triplets found on opposite strands. To distinguish
among these various putative sites as well as to examine the
contribution of each interacting base, we tested the effect of base
substitutions within the consensus motif using saturation mutagenesis.
AlcR binding affinities for all mutant-binding sites were estimated
from gel band shift experiments and compared with that of the single
copy probe sc. Changes of any base within the CGG triplet
severely reduced the affinity for AlcR. Of these bases, the G residues at positions 4 and 5 were the most sensitive to change, since substitution with any of the other three bases resulted in a complete loss of binding (Table II). Mutation of C
in position 3 resulted also in a drastic decrease of AlcR binding
(50-100). A large decrease (10-20 fold) in binding activity was
observed by mutations in position 2. Although this reduction is smaller
than those found when mutations are introduced in the terminal CGG
triplet, it is substantial compared with the effect of substitutions at
position 1. The highly conserved T at this position makes a negligible contribution to AlcR binding affinity, since any nucleotide gives reasonably strong binding. Consistent with the footprinting data, these
results confirm the absence of essential base-specific contacts at this
position. On the whole, our results suggest that, like other proteins
of the zinc binuclear cluster family, AlcR recognizes the CGG triplet
by establishing crucial contacts, presumably, in the major groove.
However, unlike the other proteins of this C6 class, position 2 occupied by G is also essential for AlcR high affinity binding. The
first nucleotide seems to be important for tight binding rather than
for a specific interaction with the DNA.
The Distance between Two Sites Specifies Binding of Two AlcR
Molecules--
All the naturally occurring AlcR inverted repeat
targets identified in the alc promoters display conserved
central bases, APu (2 bp)2
separating two inverted motifs, whereas within direct repeats the
spacing sequence varies greatly (3-8 bp) (Fig.
3). Moreover, in symmetric sites, these
bases were shown to be involved in direct contacts with AlcR (see Fig.
2). We wanted to know if the composition and spacing length are
critical for AlcR binding. Since removal of these bases by the
missing-contact technique interferes with AlcR binding, it seemed
important to estimate the contribution of the inner 2-bp spacer in
symmetric sites, for protein binding. Substitution of the spacer
sequence in the alcA symmetric site from AA to GC bases
which is never encountered in natural palindromic alc sites,
reduced AlcR binding by 3-5-fold (Fig.
4A). This is in agreement with
results of the footprinting analysis (Fig. 2D) showing that
contacts established within the spacing region contribute to some
extent to specific binding and, hence, interaction extends beyond the
consensus core.
To test whether the natural spacing between inverted repeats is a
prerequisite for binding, we assayed AlcR binding to mutant probes with
increasing spacer lengths (Fig. 4). As seen in the model summarized in
Fig. 5, the 2-bp spacing between inverted repeats in the alcA promoter resulted in positioning of the
cores on the opposite sides of the DNA helix. In this case, two AlcR molecules are presumed to be in a head to head orientation.
Surprisingly, increasing the spacer length just by one nucleotide
(spacer N3) completely abolished the formation of the slow migrating
complex CII (containing two AlcR molecules), suggesting that one AlcR molecule is bound with an affinity similar to that of a single site
(Fig. 4A). Footprinting analysis performed with the CI
complex (containing one AlcR molecule) showed a random occupancy of the two inverted repeats (results not shown). A similar pattern of binding
was also observed when the spacer region was gradually increased to 7 bp, thereby placing the two interacting CGG triplets on the same face
of the double helix. The absence of simultaneous binding of two AlcR
molecules to such modified palindromes might be due to the fact that
fixation of the first AlcR molecule on the DNA induces a conformational
change in the DNA, preventing the binding of the second AlcR molecule.
Another hypothesis, implying the interaction between two AlcR
molecules, seems less convincing. If such an interaction did exist, a
strong cooperativity of AlcR binding would have been observed, which
was not the case (5, 24). More likely, the phasing of the sites is
important for binding of two AlcR molecules. Increasing the spacer
length to 12 bp, corresponding to one additional turn of the DNA helix, did not restore normal binding of AlcR. We found that the protein did
indeed bind to the second site but with an affinity much lower than
with the wild type probe (Fig. 4A, spacer N12). This
decrease in affinity could be due to changes in spacing rather than in adjacent sequences to the consensus core, which are unchanged. The same
decrease was already observed when changing the two nucleotides of the
spacer (Fig. 4A, spacer GC). These results are in agreement with data
presented below, showing a general role of flanking regions in high
binding affinity. In contrast, a further increase in the distance
between triplets by one-half turn of helix (spacer N-17), results in
the formation of the slow migrating complex CII with a similar range of
affinity characteristic of the wild type probe (3-fold lower). In that
case, the two repeated sites are placed on the same face of the double
helix but one turn apart.
Natural spacing between physiologically functional direct repeats in
the alcA promoter varies from 7 to 8 bp (Fig. 3), which corresponds to a distance between the CGG triplets of 9 and 10 bp,
respectively. Hence, both sites partly lie on the same face on the DNA
helix (Fig. 5). Assuming a head to tail orientation of AlcR for this
case, such a positioning of direct repeated sites on DNA obviously does
not favor simultaneous occupancy of both sites by AlcR, as observed
previously (5, 6) and in Fig. 4B (spacer N-10). Introducing
an additional 3 bp into the spacer region of target c
removes the second CGG triplet on the opposite side of the double helix
(spacer N-13). Indeed, the second site is then accessible for AlcR high
affinity binding resulting in the formation of the complex CII.
Therefore, with both inverted or direct repeats, the intervening
sequence is involved in the binding of two AlcR molecules.
Role of the Flanking Regions Outside the Consensus
Core--
Sequence alignment of AlcR symmetric sites found in the AlcR
regulated promoters, allowed the identification of conserved base pairs
outside the consensus motif (Fig.
6A). Thus, the 5'-proximal position is characterized by the presence of a purine preceded by C or
A. It appears that the 5'-proximal purine is more important for high
affinity binding, since its replacement results in a 10-fold decrease
in binding (Fig. 6B). This indicates that, in addition to
the conserved A immediately downstream of the consensus motif, shown to
interact directly with AlcR, the 5'-proximal bp contributes
significantly to binding. This result is consistent with the
observation that AlcR high affinity binding sites in direct repeat
targets of the alcA promoter are often flanked by the same
conserved nucleotides (Fig. 6C). In contrast, sites with low
binding affinity are followed by a G-rich stretch (Fig. 6C). In order to simplify the analysis, nonphysiological artificial single
copy sites were further investigated. Mutagenesis in the 5'- and
3'-flanking regions was carried out. In Fig. 6D only the mutated oligonucleotides relevant to AlcR binding are shown. In fact,
the purine 5'-proximal to the binding site plays an active role in DNA
recognition by AlcR. Substitution of A by T reduced the affinity
5-fold, whereas mutating the preceding position The activator AlcR belongs to the zinc binuclear cluster family
that includes a number of other fungal transcription factors. These
factors share common regions essential for DNA binding (13, 17, 25,
26). They bind as homodimers to their DNA cognate targets, organized
either as inverted or direct repeats containing the interacting CGG
triplets at both ends. Their DNA-binding module, usually found at the
NH2 terminus, comprises a highly conserved zinc binuclear
cluster domain, followed by a less conserved linker and then a
coiled-coil dimerization motif. AlcR also contains, at its amino
terminus, a DNA-binding domain with two zinc atoms linked to six
cysteine residues arranged as a compact structure (7, 8). This is
similar to that of GAL4, PPR1, and PUT3, whose three-dimensional
structures have been resolved (13-16). Mutations in any cysteine of
the AlcR zinc cluster result in a loss of DNA binding activity (12).
However, AlcR is distinguished by a number of major features from the
other proteins of this class as will be discussed later. These features
most probably account for the unique AlcR specificity which is
characterized by in vitro binding to single sites (5, 6, 9),
containing the consensus core 5'-(T/A)GCGG-3' and, in vivo
by the functional relevance of these sites, organized in repeated
elements, which are defined as UASalc (3, 4, 6, 27).
One striking structural difference between AlcR and the zinc cluster
proteins is the absence of a predicted dimerization domain, consisting
of a coiled-coil downstream of the DNA-binding domain (17, 25). This
observation allowed us to suggest that, in contrast to most other
proteins of this class, AlcR could function as a monomer. In fact,
several lines of evidence support the idea that AlcR is a monomer and
binds to DNA as a monomer. These include the data presented in this
paper as well as in vitro and in vivo data
published previously. First, as follows from our previous in
vitro studies, the truncated AlcR(1-197) protein which despite containing putative leucine heptads, was unable to form dimers, either
in solution or upon binding to DNA (5). According to calibrated gel
filtration chromatography the AlcR(1-197) protein has the molecular
weight expected for a monomer (results not shown). Furthermore,
glutaraldehyde cross-linking data show that the full-length AlcR
protein does not form stable dimers in solution. Second, the in
vivo test with the CI The AlcR activator does not possess the linker region present in most
proteins of the Cys6Zn2 class, which directs
each protein to its preferred DNA repeated sites depending on the
length and composition of the spacer between the half-sites (28). This observation correlates with the finding that spacing between the AlcR
repeated binding sites does not seem to affect specific recognition, despite the fact that its length is important in natural palindromic targets. In contrast to most other proteins of this class which bind as
dimers, the in vitro binding affinity of AlcR was not changed significantly upon alterations of the spacing length. Rather,
variations in the number of intervening bases specify the number of
AlcR molecules fixed simultaneously on repeated DNA sites. Separated by
2 bp, the interacting cores lie exactly on the opposite sides of the
double helix. Such a relative disposition of sites on the DNA makes
both of them accessible for contacts with AlcR present in a head to
head orientation (see Fig. 5). Placing both sites, whatever their
orientation on the same face of the DNA helix, results in binding of
only one AlcR molecule to repeated targets. Either the first AlcR
molecule when bound to its site sterically blocks the binding of a
second molecule or it distorts the DNA, thereby preventing further
binding to the adjacent site. Unfortunately, our results do not allow
us to discriminate between these hypotheses. In any case, the
disappearance of the second complex CII is unlikely explained by the
loss of protein-protein interaction. First, increasing the spacing
distance restores the formation of the slowly migrating complex CII
containing two AlcR molecules; second, cooperativity of binding has
never been observed, as expected if the binding of two AlcR molecules is independent. The total number of AlcR molecules bound to the promoter region might, in turn, be important for synergistic
transcriptional activation of the alc genes as has been
shown for the alcA gene (6).
All the AlcR sites ((T/A)GCGG) in alc promoters encompass
the same subsite CGG which is recognized by the other proteins of the
zinc cluster family (17, 25). Like most other proteins of this class,
AlcR is shown to recognize the same triplet via establishing direct
contacts in the major groove of DNA. The zinc cluster proteins all
share, between the second and third cysteines, a constant number of
highly conserved basic residues. X-ray and NMR studies on GAL4, PPR1,
and PUT3 (13-16) have shown that this sequence is the recognition
module in which one conserved basic residue at position 4, relative to
the third cysteine, makes specific contacts through the N-7 of the two
G residues of the CGG triplet in the DNA major groove. Interestingly,
in AlcR, lysine 19 is at the same position as Lys18 in GAL4
and Lys41 in PPR1 (7, 29). It would seem reasonable to
expect that this residue makes contacts with G residues in the CGG
triplet. However, assuming the monomeric structure of AlcR, this
triplet alone is obviously not sufficient for binding selectivity. In fact, we show here that bases proximal to the consensus core play an
important role in high affinity binding. Thus, the 5' adjacent G and 3'
A are involved in direct base-specific contacts and, therefore,
contribute greatly to binding. This also distinguishes AlcR from GAL4
and PPR1 for which specific nucleotides flanking the recognition
triplet, are not necessary for binding (30). In the AlcR consensus
sequence, the three G residues appeared to be extremely important for
binding affinity, which allowed us to extend the recognition unit to
the sequence 5'-GCGG-3'. Furthermore, the presence of an AT-rich region
3' and a purine 5' to the consensus sequence favors high affinity
binding by AlcR. Another regulator, MIG1 which is a Saccharomyces
cerevisiae zinc finger protein also requires an adjacent AT-rich
region for optimal binding (31). It was speculated that this region
facilitates DNA bending and thereby enhances MIG1 affinity for its site
as suggested here for AlcR. Another possible explanation for the importance of the flanking regions would be that AlcR establishes specific contacts with DNA outside the core. Although AlcR is indeed
able to establish direct contacts, at least with the first 3' proximal
base, the fact that no single base in the 3'-flanking region is
essential for binding indicates that a certain DNA structure rather
than an unique sequence is important. Our data allow us to propose a
new AlcR-binding site: 5'-PuNGCGG-AT rich-3'.
Given that AlcR differs by its structural organization from most other
proteins of the same class, one could ask which determinants other than
the zinc cluster might define in vivo its binding
specificity. One candidate for this role could be the
NH2-terminal region adjacent to the zinc cluster. Recent
observations have shown both in vitro and in vivo
that, unexpectedly outside the AlcR zinc cluster domain, the
NH2-terminal region plays a major role in site-specific
recognition (32), whereas the downstream region is necessary for high
affinity binding (5). An additional, so-called middle homology region may also assist in in vivo binding selectivity. This region
was identified by sequence alignment in most of the known zinc cluster proteins and was suggested to participate in DNA target discrimination (17, 26). Preliminary analysis allowed us to localize similar stretches
of moderate homology within the AlcR
sequence.4 We
cannot exclude, however, an alternative explanation involving an
accessory protein required for the transcriptional activation of the
alc genes.
In conclusion, although it has been proposed previously that some zinc
cluster proteins may bind their targets as monomers, AlcR appears to be
the first example thus far described, for which detailed evidence,
albeit indirect is now available. This gives new insights into the
general picture of the zinc binuclear cluster protein family.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
-helix.
8 M) (5, 6). Furthermore, using a
transcription-translation assay, we have shown that no dimerization
sequence is present up to residue 197 which comprises one-fourth of the
protein length (5).
EXPERIMENTAL PROCEDURES
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
(proLac) F
(proABLacIqZ)
M15,
traD36 was used in phage immunity tests. Wild type phage
was employed for immunity tests. The pCIR1-7 plasmids were obtained from the pC132 vector (18) which harbors a CI rop fusion by replacing the rop gene with the alcR regions of
interest. The different AlcR domains were amplified by polymerase chain
reaction using specific oligonucleotides containing SalI and
BamHI sites at their 5' and 3' termini, respectively. The
polymerase chain reaction products were cut by SalI and
BamHI and cloned in the pC132 plasmid treated with the same
enzymes to excise the rop sequence. To generate plasmids
pCIR8 and pCIR9 containing the alcR fusion with the
cI repressor and the rop genes, the
alcR fragment was amplified by polymerase chain reaction
primed from specific oligonucleotides containing SalI sites
at both ends and cloned into the pC132 plasmid cut by SalI.
In all these constructs the AlcR domains were fused in-frame with the
phage CI repressor NH2-terminal domain at the 5' end
and the
-galactosidase
-peptide gene at the 3' end. The AlcR
coding sequences were separated from the
-peptide by an amber codon
introduced during amplification to allow for synthesis of a bipartite
(CI:AlcR), tripartite (CI:AlcR:
-peptide), or tetrapartite
(CI:AlcR:Rop:
-peptide) fusion protein depending on the suppressor
background of the host. The supE (71.18) strain bearing fusion plasmids
forms blue colonies on
5-bromo-4-chloro-3-indoyl-
-D-galactoside indicator
plates. Bacterial cells transformed by plasmids were assayed by spot
tests for sensitivity to
phages at different concentrations on
lawns of transformed bacteria.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
CI repressor to function as a
dimer was used (20). When a dimerization element is fused to the
NH2-terminal DNA-binding domain of the
CI repressor, E. coli cells are immune to superinfection by
phage,
while cells that contain the CI DNA-binding domain alone are sensitive.
This system was used previously to analyze the residues involved in the
leucine zipper from the yeast activator GCN4 (20), in TAT homodimerization (21), Myc/Max heterodimer formation (22), and
dimerization of the NirA zinc cluster protein (23). Gene fusions
between the NH2-terminal domain of
CI repressor and overlapping AlcR sequences covering the whole protein (1-821), including the zinc cluster domain (1-59), were performed (as described under "Experimental Procedures"). None of the AlcR sequences tested conferred immunity to
infection when transformed into E. coli, implying that no dimerization element was present (Table
I).
In vivo assays of different AlcR regions for putative dimerization
elements
CI-AlcR
protein. This hypothesis was ruled out by assaying the
-galactosidase activity driven by all the fusion constructs (see "Experimental Procedures"). In order to ensure that AlcR sequences did not prevent per se, dimerization of
CI repressor, a
control dimerization element, originating from the Rop protein, was
introduced at the carboxyl terminus of AlcR sequences (pCIR8, pCIR9).
Results presented in Table I show clearly that this Rop sequence is
able to drive
CI dimerization when fused at the carboxyl-terminal region of AlcR, indicating that AlcR sequences did not prevent dimerization. Another hypothesis could be that the AlcR dimerization element consists of separate domains, comprising interacting
helical regions. The only possibility, according to computer prediction analysis of AlcR (24) and preliminary results of AlcR NMR
studies,1 would
be the interaction of two regions, one of 16 residues between the third
and fourth cysteines and the second region downstream of the zinc
cluster at residues 310 to 343. Results presented in Table I (pCIR4)
argue against this assumption. In fact, the AlcR region from residues 1 to 367 does not confer immunity to
infection whereas the AlcR-Rop
fusion does. Therefore, the absence of immunity, whatever the
alcR sequence fused to
CI repressor, indicates that
under these conditions the AlcR dimerization sequence, if any exists,
is either not functional or not strong enough to promote in
vivo formation of dimers of CI repressor.
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Fig. 1.
Absence of AlcR dimerization in
solution. A full-length AlcR(1-821) protein was translated
in vitro and 3-20 µl of translation mixtures were
subjected to glutaraldehyde cross-linking for 0 ( ) and 60 (+) min,
respectively, as described under "Experimental Procedures." As a
control, a truncated NirA(1-222) protein, which forms dimers (23), was
treated in a similar way. The products were detected by 10%
SDS-polyacrylamide gel electrophoresis for NirA or 8%
SDS-polyacrylamide gel electrophoresis for AlcR followed by
fluorography. Arrows indicate monomeric and dimeric species.
The migration of molecular weight markers is indicated.
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Fig. 2.
Depurination (G + A) and methylation (G)
interference of contacts between AlcR(1-197) and its DNA-binding sites
in the alcA promoter. A, direct repeat
target a; B, direct repeat target c;
C, single copy site sc; D, inverted repeat target
b. Lanes: CI and CII, bound DNA
isolated from fast and slow migrating complexes, respectively;
P, free probe. Probes were labeled on the top and bottom
strand, respectively. For experimental details, see "Experimental
Procedures." Arrows indicate the orientation of the
conserved sequence 5'-TGCGG-3'. Positions numbered above the
sequence correspond to 5' to 3' orientation of this motif.
Circles represent purine contacts identified by base-missing
interference and squares symbolize G contacts identified by
methylation interference in the slow migrating complex CII. In all
cases, closed symbols denote strong and open
symbols weak base contacts. A summary of the interference assays
is presented at the bottom of each panel.
Summary of the affinities for AlcR(1-197) for DNA-binding sites
containing all possible mutations in the conserved motif
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Fig. 3.
Localization of the AlcR-binding sites in the
alcA and alcR promoters.
Arrows indicate the orientation of the conserved repeat
5'-(T/A)GCGG-3'. Numbers between the
arrows correspond to numbers of intervening bp between
repeats. Positions of the binding sites are shown below,
relative to the start of translation +1. tsp, transcription
start point.
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Fig. 4.
Effect of altering the composition and length
of the spacer region between inverted (A) and direct
(B) repeat targets b and c in the alcA
promoter on binding of AlcR(1-197). Electrophoretic
mobility shift assay was performed in the presence of increasing
amounts of the partially purified AlcR(1-197) protein as noted
above the gel, with 50 fmol of each radiolabeled probe.
Arrows indicate the orientation of the consensus motif
TGCGG. Sequences of the probes are identical to the alcA WT
probes b and c, except for the composition of the
spacer region which is presented below each panel. N
corresponds to the number of nucleotides in the spacer sequence
separating the CGG triplets in the consensus.
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Fig. 5.
A schematic model of AlcR bound to its
cognate targets. The model illustrates how two AlcR molecules
interact with inverted repeat target b, whereas only one
molecule is able to bind to direct repeat target c. The
interacting bases within the consensus core GCGG on both strands are
displayed on a split projection of B-form DNA. Only the top strand of
each target is shown. Full circles indicate G residues in
both strands which are involved in direct contacts with AlcR molecules
(as shown shaded). Putative interacting G residues of a
second site within direct repeat c are denoted by open
circles. A putative position of another AlcR molecule is shown by
a dashed line. A head to head and head to tail orientation
of AlcR molecules correspond to symmetric and asymmetric organization
of the target, respectively, presented below.
2 (C to G) had little
effect. Apart from the highly conserved A 3' to the consensus core
which, as expected from footprinting analysis, contributes to binding,
mutagenesis of any other base in the 3'-flanking region did not lead to
any significant effect (results not shown). However, while no single
mutation in this region is essential, binding of AlcR is dependent on
the overall composition of the sequence 3'-proximal to the site.
Changing the box composition of nucleotides from AT-rich to G-rich
results in a noticable decrease (3-9-fold) of AlcR binding (Fig.
6D). Based on these data, it can now be explained why some
of the AlcR-binding sites, as for example, the direct repeated sites
d in the alcR promoter (Fig. 3) are not active
in vivo.3
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Fig. 6.
Role of the flanking regions outside the
consensus motif for AlcR binding. A, sequence
comparison of AlcR symmetric DNA-binding sites found in various
alc gene promoters (Refs. 3-5 and 33, and see Fig. 3). For
the sake of consistency, the sequence of the AlcR-binding sites in the
aldA promoter was inverted with respect to its natural
orientation within the promoter region. Positions of the targets with
respect to their translation start sites are indicated on the
right. For simplicity, only the upper strand is shown.
Arrows indicate the orientation of the repeated sequence.
Conserved nucleotides outside the consensus repeat are
boxed. B, effect of mutations in the conserved
positions outside the inverted repeat target b in the
alcA promoter. Each mutant probe is named after the mutant
nucleotide in the corresponding position. Numbers are
indicated above the sequence. Mutated nucleotides are marked
in boldface type. The complete sequence of the wild type
probe b is presented under "Experimental Procedures."
Relative binding affinity was determined in gel shift experiments as
the affinity of the AlcR(1-197) protein for a mutant site compared
with that for the target b (normalized to 1). C,
sequence comparison of AlcR DNA-binding sites within direct repeat
sites in the alcA and alcR promoters (see Fig.
3). All the binding sites were orientated in the 5' to 3' direction of
the TGCGG conserved repeat indicated by an arrow. Site
positions relative to their starts of translation are indicated on the
right. Conserved nucleotides flanking the consensus repeat
are boxed. Binding affinities were estimated as described
earlier and compared with that for the single site probe sc.
D, mutational analysis of the flanking regions of the
artificial AlcR single copy site sc. Each mutant probe is
named after the corresponding mutation outside the conserved motif.
Numbers correspond to the mutated base positions and
letters correspond to nucleotides introduced at these
positions. Mutated nucleotides are marked in boldface type.
The complete sequence of the probe sc is present in Table
II. The other probes are identical except for the mutations, as
indicated. Electrophoretic mobility shift assay were performed in the
presence of increasing amounts of AlcR(1-197) with labeled probes (50 fmol) as described under "Experimental Procedures." Binding
affinities were relative to AlcR(1-197) binding to the probe
sc.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
phage repressor clearly indicate that
within the entire AlcR sequence no region is able to promote functional
dimerization of the CI repressor. Third, in vitro binding experiments and analysis of interacting bases also show that a single
AlcR molecule is able to bind to a single copy site with the same
affinity and the same pattern of base interaction. NMR studies have
also shown that one AlcR molecule is bound to a single site (9). The
absence of cooperativity upon binding of two AlcR molecules to inverted
repeat targets (5, 6) is consistent with AlcR binding DNA as a monomer.
Finally, physiological studies by site-directed mutagenesis of AlcR
targets on the alcA promoter also favor the idea that AlcR
acts in vivo as a monomer. AlcR was shown to possess a
unique specificity, being able to recognize in vitro single
sites organized as inverted or direct repeats in AlcR controlled
promoters. Both types of targets are functional in vivo in
the alcA promoter (6). However, in vivo there are no strict
requirements for spacing between the direct repeats in the
alcA promoter and furthermore, the spacing could be
increased in such a manner that it would be very difficult to imagine
that AlcR binds as a dimer (6). Moreover, disruption of any of the three individual AlcR-binding sites within the target c leaving two other sites intact, lead to an active alcA
promoter, a result which would not be expected if AlcR is a dimeric
protein. Our attempts to directly isolate in A. nidulans and
in E. coli the full-length AlcR protein were unsuccessful.
Therefore, we must stress that at this stage only indirect lines of
evidence can be presented, which nevertheless when taken together all
strongly indicate that AlcR is a monomer.
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ACKNOWLEDGEMENTS |
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We thank Prof. B. Holland for the English version of the manuscript, and M. Mathieu for assistance in the construction and test of the rop plasmids.
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FOOTNOTES |
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* This work was supported in part by Centre National de la Recherche Scientifique Grant URA D 2225, by the Université Paris-Sud XI, and by European Communities Contract BIO4-CT96-0535.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.
Supported by grants from NATO and as an Invited Professor.
§ Recipient of doctoral fellowship from the Ministère de l'Education Nationale, de l'Enseignement Supérieur et de la Recherche of the French Government. Present address: Molecular Genetics of Industrial Microorganisms, Wageningen Agricultural University, Dreijenlaan 2, 6703 HA Wageningen, The Netherlands.
¶ To whom correspondence should be addressed. Tel.: 33-1-69-15-63-28; Fax: 33-1-69-15-78-08; E-mail: felenbok{at}igmors.u-psud.fr.
1 R. Cerdan, F. Penin, F. Lenouvel, B. Felenbok, and E. Guittet, unpublished results.
3 M. Mathieu and B. Felenbok, unpublished results.
4 I. Nikolaev and B. Felenbok, unpublished results.
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ABBREVIATIONS |
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The abbreviation used is: bp, base pair(s).
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REFERENCES |
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