A Linker Region of the Yeast Zinc Cluster Protein Leu3p Specifies
Binding to Everted Repeat DNA*
Yaël
Mamane
,
Karen
Hellauer
§,
Marie-Hélène
Rochon
, and
Bernard
Turcotte
§¶
From the § Department of Medicine, Royal Victoria
Hospital and
Department of Microbiology and
Immunology, McGill University, Montréal,
Québec, Canada H3A 1A1
 |
ABSTRACT |
Yeast zinc cluster proteins form a major class of
yeast transcriptional regulators. They usually bind as homodimers to
target DNA sequences, with each monomer recognizing a CGG triplet.
Orientation and spacing between the CGG triplet specifies the
recognition sequence for a given zinc cluster protein. For instance,
Gal4p binds to inverted CGG triplets spaced by 11 base pairs whereas Ppr1p recognizes a similar motif but with a spacing of 6 base pairs.
Hap1p, another member of this family, binds to a direct repeat
consisting of two CGG triplets. Other members of this family, such as
Leu3p, also recognize CGG triplets but when oriented in opposite
directions, an everted repeat. This implies that the two zinc clusters
of Leu3p bound to an everted repeat must be oriented in opposite
directions to those of Gal4p or Ppr1p bound to inverted repeats. In
order to map the domain responsible for proper orientation of the zinc
clusters of Leu3p, we constructed chimeric proteins between Leu3p and
Ppr1p and tested their binding to a Leu3p and a Ppr1p site. Our results
show that the linker region, which bridges the zinc cluster to the
dimerization domain, specifies binding of Leu3p to an everted repeat.
We propose that the Leu3p linker projects the two zinc clusters of a
Leu3p homodimer in opposite directions allowing binding to everted
repeats.
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INTRODUCTION |
Sequencing of the yeast (Saccharomyces cerevisiae)
genome has revealed the existence of over 50 proteins that are all
characterized by the presence of 6 cysteine residues (consensus
sequence:
CX2CX6CX5-9CX2CX6-8C) (1, 2). These cysteines are essential for binding to two zinc atoms
involved in proper folding of the cysteine-rich region and are referred
to as zinc cluster proteins (3). Although some zinc cluster proteins
bind as monomers (4, 5), many of them have been shown to bind
specifically to DNA as homodimers with each monomer recognizing a CGG
triplet. The most conserved region of the zinc cluster proteins is the
zinc cluster itself. The zinc cluster is followed by a linker region of
approximately 15 amino acids
(aa)1 in length, which
bridges the zinc cluster to the dimerization domain. The latter has a
coiled-coil structure consisting of heptad repeats with a predominance
of hydrophobic residues at the first and fourth position of the
repeat.
Two strategies aimed at generating a diversity of binding sites for the
zinc cluster proteins have been identified. First, the zinc cluster
proteins usually recognize three different DNA motifs. For instance,
the transcriptional activators of the galactose (Gal4p) and pyrimidine
(Ppr1p) pathways recognize CGG triplets when oriented as inverted
repeats (also called palindromes) (CGG Nx CCG). Another member
of this family, Hap1p, involved in the activation of genes related to
cellular respiration, also recognizes CGG triplets but only when
oriented as a direct repeat (CGG Nx CGG) (6, 7). More recently,
we have identified a third variation of the DNA motif as shown by the
transcriptional activators Leu3p and Pdr3p, which are involved in
controlling expression of genes related to leucine biosynthesis and
multi-drug resistance, respectively. Leu3p and Pdr3p recognize CGG
triplets oriented in opposite directions (CCG Nx CGG, see Figs. 1B and 5), a motif called an everted repeat in analogy to
sites found for some targets of the nuclear receptors like those for retinoic acid (see Ref. 8 and references therein). Alternatively, DNA
targets of Leu3p could be considered to be an inverted repeat with the
sequence CCG. However, a chimeric protein in which the zinc cluster of
Gal4p was replaced by the corresponding region of Leu3p was shown to
recognize a Gal4p site (CGG N11 CCG) but not an inverted
repeat with the sequence CCG N11 CGG (9). Because Leu3p
binds to DNA as a homodimer (10), these observations imply that the two
zinc clusters of Leu3p (and most probably Pdr3) must be oriented in
opposite directions, unlike those of Gal4p or Ppr1p where they have
been shown to face each other (11, 12). Thus, three DNA motifs are used
by the members of the family of zinc cluster proteins: inverted,
direct, and everted repeats.
Diversity of target sites is further increased by changing the spacing
between the CGG triplets. Gal4p binds with high affinity to an inverted
repeat only when the spacing between CGG triplets is 11 base pairs (bp)
(13, 14). Other members of this family recognize inverted repeats with
distinct spacings: 10 bp for the transcriptional activator of the
proline pathway, Put3p (15) or 6 bp for Ppr1p (13, 16). The binding of
Hap1p is restricted to direct repeats with a spacing of 6 bp (6, 7).
Similarly, binding of Leu3p or Pdr3p to everted repeats is strictly
dependent on spacing between the CGG triplets of 4 bp and 0 bp,
respectively (9). Thus, the requirement for a proper spacing is
important in generating a diversity of target sites. Binding of a given zinc cluster protein to an alternate DNA motif does not appear to
occur. For example, all the known target sites for Gal4p or the
Kluyveromyces lactis homologue Lac9p are inverted repeats (see Refs. 14, 17, and 18 for a compilation of the DNA targets). Only
direct repeats were recovered from a random site selection performed
with Hap1p (7), in agreement with the fact that all known targets of
Hap1p are imperfect direct repeats (6, 19-24).
The crystal structure of the Gal4p DNA binding domain bound to a
consensus DNA site revealed a symmetrical structure of the two
monomers, each interacting with a CGG triplet (11). The linker has an
extended structure and the dimerization domain lies perpendicular to
the minor groove, in the middle of the upstream activating sequence
(UAS). Other studies have revealed a remarkable similarity in the zinc
cluster structures of Ppr1p, Put3p, and Gal4p (12, 25) (reviewed in
Ref. 26). However, the linker region of Ppr1p has a folded structure
and the Ppr1p linker and the coiled-coil of each monomer are arranged
asymmetrically. Folding of the linker renders Ppr1p dimers more
compact, leading to recognition of CGG inverted repeats with a reduced
spacing of 6 bp. These results are in agreement with studies of Gal4p,
Ppr1p, and Put3p chimeras, which showed that the linker region and the
beginning of the dimerization domain specify binding to an inverted
repeat of a given spacing (27). However, use of chimeric proteins
between Hap1p and Ppr1p has demonstrated that, in contrast to Gal4p and Ppr1p, the zinc cluster of Hap1p is solely responsible for positioning the two monomers to a direct repeat (28).
We wished to determine the region of the zinc cluster proteins
responsible for binding to an inverted motif or an everted repeat. We
constructed chimeric proteins derived from Leu3p and Ppr1p and tested
their binding in an electrophoretic mobility shift assay (EMSA). Our
results show that the linker region of Leu3p specifies binding to an
everted repeat.
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EXPERIMENTAL PROCEDURES |
GST Expression Vectors--
A portion of the PPR1
gene (29) that encodes its DNA binding domain (aa 1-144) was
PCR amplified with Vent polymerase (New England Biolabs) using the
primers 5'-CGGGATCCATGAAGCAGAAAAAATTTAA-3' (initiator codon
in bold) and 5'-GGAATTCTATTTCTGAACCAAA-3' and yeast genomic DNA
isolated from the strain YPH499 (30) as a template. The PCR product was
cut by BamHI and EcoRI, purified on a column (PCR
purification kit, Promega), and subcloned into pGEX-f (9) cut with the
same enzymes to give pPPR1(1-144). This plasmid has the
PPR1 coding sequences in frame with the GST sequences and a
stop codon, introduced during PCR amplification, after aa 144. Sequencing of the coding region of PPR1 showed that no
mutations were introduced during PCR amplification. A similar expression vector for Leu3p, pLEU3(1-147), has been described previously (9). Two strategies were used to generate expression vectors
for chimeric proteins: site-directed mutagenesis (31) and PCR using the
megaprimer technique (32). All the constructs were sequenced to confirm
their integrity.
To facilitate construction of expression vectors for chimeric proteins,
we introduced, in the same reading frame, a unique site
(SphI or BglII) within specific regions of the
LEU3 and PPR1. SphI sites were
introduced at PPR1 sequences corresponding to the
first or the last cysteine as described for LEU3 (9) using the
oligonucleotides 5'-TCTAGAACTGCATGCAAACGATGTCGATT-3' and
5'-CAAAATTAGAGGTAGCATGCGTTTCTTTGGACCC-3'. A BglII site was
also introduced in LEU3 at the border between the linker and
the dimerization domain using the oligonucleotide 5'-GAACTTATAAAAGAAGATCTAACGAAGCCATTGA-3'. PPR1 possesses a
natural BglII at this position. To construct chimeras,
PPR1 and LEU3 DNAs were digested with
SphI or BglII and the various fragments derived from LEU3 or PPR1 ligated to give LP-A, LP-D,
LP-F, LP-H, LP-5 (encoding the N terminus and the zinc cluster of
LEU3 followed by the linker and the dimerization domain of
PPR1), and LP-6 (encoding the N terminus and the zinc
cluster of PPR1 followed by the linker and the dimerization
domain of LEU3). LP-E, LP-G, LP-B, and Leu3-1 were
constructed by using PCR with Vent polymerase (New England BioLabs).
LP-E (extended version of LP-D) was constructed by amplifying a portion
of pLEU3(1-147) using the primers
5'-AGGACGTTCCAAGATCTTACGTCTTTTTTCTGGAAGATAGATTCAAGGAACTCACCAGAA-3' and
3'-GST 5'-CCGGGAGCTGCATGTGTCAGAGG-3'. The PCR product was digested with
BglII and EcoRI, purified on a column (PCR
purification kit; Promega), and subcloned into pLP-D cut with
BglII and EcoRI. LP-I (an extended version of
LP-H) was constructed by using the megaprimer technique. A region of
LP-H was amplified with Vent (New England BioLabs) PCR using the
primers
5'-AGAAGAACTTATAAAAGAGCAAGGAACGAAGCCATTGAAAAAAGATTGGCTGTCATGATG-3' and
5'-GGAATTCAAAGTGTTTTGTATG-3'. The PCR product was purified on a
column (PCR purification kit; Promega). This product containing the
desired mutation and the oligonucleotide 5'-AGCACCGGAGCCATGCACTA-3' were used as primers with LP-H as a template. The PCR product was
digested with NsiI and EcoRI and subcloned into
LP-H cut with the same restriction enzymes.
Alanine scan mutants of LEU3 were obtained using the following
oligonucleotides: 78A, 5'-AGAAGAACTTATGCAAGAGCAAGGAAC-3'; 79A, 5'-AGAACTTATAAAGCAGCAAGGAACGAA-3'; 81A,
5'-TATAAAAGAGCAGCGAACGAAGCCATT-3'; 82A,
5'-AAAAGAGCAAGGGCCGAAGCCATTGAA-3'; 83A,
5'-AGAGCAAGGAACGCAGCCATTGAAAAA-3'; 85A,
5'-AGGAACGAAGCCGCTGAAAAAAGATTC-3'; 86A,
5'-AACGAAGCCATTGCAAAAAGATTCAAG-3'; 87A,
5'-GAAGCCATTGAAGCAAGATTCAAGGAA-3'; 88A,
5'-GCCATTGAAAAAGCATTCAAGGAACTC-3'; 89A,
5'-ATTGAAAAAAGAGCCAAGGAACTCACC-3'; 90A,
5'-GAAAAAAGATTCGCGGAACTCACCAGA-3'; 91A,
5'-AAAAGATTCAAGGCACTCACCAGAACT-3'; 92A,
5'-AGATTCAAGGAAGCCACCAGAACTTTG-3'.
Protein Purification--
Expression and purification of the
proteins were performed as described (9) except that cells were
freeze/thawed three times and sonicated for 10 s. For all
proteins, the GST moiety was removed by cleavage with thrombin. The
polypeptides were 50-90% pure as judged by SDS-polyacrylamide gel
analysis followed by Coomassie staining. Ppr1p has a significantly
faster mobility than Leu3p on a denaturing gel even though they have a
very similar molecular size (Leu3p, 17 kDa; Ppr1p, 16 kDa). This may be
due to abnormal migration because of the high content of glutamic acids
in the DNA binding domain of Leu3p as observed for the chicken progesterone receptor (40). Chimeric proteins migrated at intermediary positions relative to Leu3p and Ppr1p (data not shown).
Electrophoretic Mobility Shift Assays--
DNA sequences
of the probes used in EMSA are TCGACCTGCCGGTACCGGCTTGGTCGA (Leu3p site:
UAS of ILV2; Ref. 33) and TCTTCGGCAATTGCCGAAGA (Ppr1p
site, Ref. 11). EMSAs were performed using salmon sperm DNA as
nonspecific competitor as described (9) with increasing concentrations
(1-, 2-, 4-, 8-, and 16-fold) of recombinant proteins. The gels were
run at 4 °C. Dissociation constants (Kd values)
were estimated (34) by performing EMSAs in the absence of competitor
DNA with a fixed amount of each protein and decreasing concentrations
of the appropriate probe. The ratio of bound to free DNA was plotted
against [bound DNA] and the Kd
estimated from the slope of the graph. Percentage of binding
was measured using a Phosphorimager (Fuji).
 |
RESULTS |
We were interested in determining which region of the DNA binding
domain of Leu3p and Ppr1p controlled recognition of inverted and
everted CGG triplets. We constructed chimeric proteins between Ppr1p
and Leu3p. Thus, the DNA binding domains of Ppr1p (aa 1-144), Leu3p
(aa 1-147) (hereafter referred to as Ppr1p and Leu3p, respectively) or
chimeras were expressed in bacteria as GST fusion proteins. The
polypeptides were then purified and the GST moiety removed by cleavage
with thrombin. The purified polypeptides were then used in an EMSA
using the UAS of ILV2 (Leu3p site) (or mutants) and a
consensus site for Ppr1p (12) (see Fig.
1B).

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Fig. 1.
A, amino acid sequence comparison of the
core of the Leu3p and Ppr1p DNA binding domains. Cysteine residues that
coordinate zinc atoms are shown underlined and in bold
characters. Hydrophobic residues found at positions a and d of the
heptad repeats (dimerization domain) are in bold characters.
Borders between the zinc cluster domain, the linker region, and the
dimerization domains were defined according to previous studies (11,
12, 27, 28) and this work. B, probes used in electrophoretic
mobility shift assays. Arrows correspond to the CGG triplets
recognized by Leu3p or Ppr1p. The Leu3p probe is derived from the UAS
of ILV2 (33) flanked by the sequence TCGA. Mutant probes
have been described previously (9), and mutations are
underlined. The Ppr1p probe is a consensus site for that
protein (12).
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The Linker or the Dimerization Domains of Leu3p Specify Binding to
Everted Repeats--
A first set of chimeric proteins containing
N-terminal sequences of Ppr1p of increasing length were constructed as
shown in Fig. 2A. Control
experiments demonstrated that Leu3p bound to its target site but not to
a Ppr1p site and vice versa (Fig. 2B). Deletion
of the first 28 aa of Leu3p resulted in a complex of faster mobility
(Fig. 2B) but did not alter the specificity or the affinity
of binding of the truncated protein (construct Leu3-1, Fig. 2,
A and B). A chimeric protein, where the N
terminus of Leu3p was substituted with that of Ppr1p (construct LP-A),
gave two major complexes with a probe corresponding to a Leu3p site. In
order to test if the fast migrating complex corresponds to a truncated
protein or a monomer, we used a mutant probe carrying a mutation in one
everted CGG triplet. If the fast migrating complex corresponds to a
monomer, significant binding should be observed with the probe because
one CGG triplet is left intact. However, this mutant probe (probe 8A,
Fig. 1B), known to greatly impair binding of Leu3p in
vitro (9), also drastically reduced binding of LP-A suggesting
that the fast migrating complexes correspond to a truncated form of
LP-A. No complex was detected in the presence of LP-A when a Ppr1p site
was used (Fig. 2B), suggesting that the N terminus of Leu3p
is not involved in discriminating between a Leu3p and a Ppr1p site.

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Fig. 2.
Binding of Leu3p-Ppr1p chimeras to a Leu3p or
a Ppr1p site. A, schematic view of the chimeras.
Subdomains of the DNA binding domains of Leu3p or Ppr1p are shown on
top. Numbers correspond to the amino acids of
Leu3p and Ppr1p present in a given chimera. Kd
values are listed at right. No binding is
indicated by . B, EMSA was performed with a Leu3p site, a
Leu3p mutant site (mutant 8A, Fig. 1B) or a Ppr1p site as
indicated on the bottom using wild type Leu3p, Ppr1p, or
chimeras as indicated on top of the figure.
Triangles correspond to increasing protein concentrations
(1-, 2-, 4-, 8-, and 16-fold). The lowest protein concentrations used
were 45 nM for Leu3 and Leu3-1, 90 nM for
LP-A, 160 nM for Ppr1. and 60 nM for LP-B. For
the sequence of the probes, refer to Fig. 1B.
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Moreover, the chimera LP-B, which contains the N terminus and the zinc
cluster of Ppr1p followed by the linker and dimerization domains of
Leu3p, bound to a Leu3p site with high affinity. Because the Leu3p site
also contains an inverted CGG triplet spaced by 2 bp, we also tested
mutant probes to verify that the chimeric proteins LP-A, LP-B (Fig.
2A) and LP-I (Fig.
3A) recognize the everted but
not the inverted motif. Binding was abolished with a mutation that
targets the everted repeat (mutant probe 8A, Fig. 1B) but
not with a mutation targeting the inverted repeat (probe 5A, Fig.
1B) (data not shown). These results strongly suggest that
the chimeric proteins LP-A and LP-B have the same binding specificity
as wild type Leu3p. Interestingly, when the junction between the Ppr1p
and the Leu3p chimera was shifted so that the linker region contains 4 additional aa derived from Ppr1p (chimera LP-C, Fig. 2A), no
binding was detected at a Leu3p or a Ppr1p site (data not shown). Thus,
the data define aa 70 as the N-terminal boundary of Leu3p sequences
required for binding to an everted repeat. These results indicate that
the N-terminal region and the zinc cluster of Leu3p are not involved in
specifying binding to an everted repeat.

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Fig. 3.
Binding of Leu3p-Ppr1p chimeras to a Leu3p or
a Ppr1p site. A, schematic view of the chimeras. Subdomains
of the DNA binding domains of Leu3p or Ppr1p are shown on
top. Numbers correspond to the amino acids of
Leu3p and Ppr1p present in a given chimera. Kd
values are listed at right. No binding is indicated by .
NT, Kd not tested. B, EMSA was
performed with a Leu3p site, a Leu3p mutant site (mutant 8A, Fig.
1B) or a Ppr1p site as indicated on the bottom
using Leu3p-Ppr1p chimeras as indicated on top of the
figure. Triangles correspond to increasing protein
concentrations (1-, 2-, 4-, 8-, and 16-fold). The lowest protein
concentrations used were 250 nM for LP-F and LP-G and 60 nM for LP-I. For the sequence of the probes, refer to Fig.
1B.
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Other chimeric proteins, LP-D, and LP-E (Fig. 2A), which
contain Ppr1p sequences up to the sixth aa of the dimerization domain, did not form a complex with a mobility comparable to wild type proteins
on either probe (data not shown). In summary, these experiments define
the region of Leu3p critical for recognition of an everted repeat at
the C terminus of its DNA binding domain and include the linker and the
dimerization domain (aa 70-147).
The Linker of Leu3p Specifies Binding to Everted
Repeats--
In order to map more precisely the region of Leu3p
responsible for binding to an everted repeat, we tested a series of
converse chimeras that contain increasing portions of the Leu3p
sequences at the N terminus of Ppr1p. Swapping the N terminus of Ppr1p
with the corresponding region of Leu3p (chimera LP-F) did not prevent binding to a Ppr1p site. In addition, LP-G, which contains both the
N-terminal and the zinc cluster regions of Leu3p (followed by Ppr1p
sequences), bound to a Ppr1p site (with reduced affinity) but not a
Leu3p site (Fig. 3B). However, addition of the linker of
Leu3p (chimera LP-I) switched the specificity of binding to a Leu3p
site. Two complexes are detected when using a Leu3p site. The slow
complex migrates at a position similar to Leu3p and is not detected
with a mutant probe (Fig. 3B), strongly suggesting that it
corresponds to a dimer. In contrast, the fast migrating complex is also
seen with a mutant of a Leu3p site as well as with a Ppr1p site. Thus,
the fast migrating complex probably corresponds to a monomer. Similar
observations were made for some Hap1p-Ppr1p chimeras (28). Because LP-I
contains most of the dimerization domain of Ppr1p and recognizes a
Leu3p site, this strongly suggests that the dimerization domain of
Leu3p (with the exception of the first 6 aa) is not responsible for the
specific orientation of the zinc clusters of a Leu3p homodimer. A
chimera (LP-H) carrying a shorter segment of the Leu3p linker did not
bind to either probe tested (Fig. 3A). Taken together, our
results show that the critical region of Leu3p that specifies binding
to everted repeats maps to the linker region and the beginning of the
dimerization domain between aa 70 and 87.
Alanine Scan Mutagenesis of the Linker-Dimerization Junction of
Leu3p--
We then focused our analysis on the Leu3p region that
encompasses the linker and the dimerization domain. Each of these
residues (aa 78-92) was mutated to alanine and the binding of mutant
proteins was analyzed by EMSA as shown in Fig.
4. Three mutants (I85A, F89A, and L92A)
gave rise to higher mobility DNA-protein complexes (Fig. 4). The
hydrophobic residues isoleucine 85, phenylalanine 89, and leucine 92 correspond to positions "a" or "d" of a heptad repeat (Fig. 4),
which was shown to be involved in dimerization of Gal4p, Ppr1p, and
Put3p (11, 12, 25). We suggest that these mutants are defective in
dimerization and, as a result, bind to DNA as monomers as observed for
Hap1p with mutant sites (28). Thus, the beginning of the dimerization
domain would map to aa 82. Moreover, our alanine scan identified one aa
of the linker region as a critical residue. Indeed, binding was
abolished with a change from arginine to alanine at position 81, just
before the dimerization domain. This is in agreement with the absence of binding of the chimera LP-H that lacks aa 80-147 of Leu3p (Fig. 3A). Substitution of aa 86 and 87 to alanine reduced DNA
binding. Many other changes (positions 78, 79, 86, 90, and 91) had no
or minor effects on binding of Leu3p or resulted in increased binding (positions 82, 83, and 88).

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Fig. 4.
Alanine scan mutagenesis of the
linker-dimerization junction of Leu3p. The amino acid sequence of
the linker and part of the dimerization domain of Leu3p is given on
top of the figure. The last cysteine of the zinc cluster is
circled. Arrows correspond to the heptad repeats
present in the dimerization domain of Leu3p. EMSA is shown at the
bottom with each amino acid tested changed to alanine and
numbered according to the initiation codon. WT,
wild type Leu3p. The probe used in the EMSA is the UAS of
ILV2 (see Fig. 1B). Percentage of homodimer
binding is given at the bottom of the figure.
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 |
DISCUSSION |
Zinc cluster proteins usually bind as homodimers to specific DNA
sequences. A major determinant of binding is the recognition of a CGG
triplet by each of the two monomers. Two mechanisms that allow zinc
cluster proteins to bind to diverse DNA sequences have been identified:
1) variation of the relative orientation of the CGG triplets (inverted,
everted, and direct repeats) and 2) alternate spacing between the CGG
triplets. Previous studies have shown that the linker region, located
between the zinc cluster and the dimerization domain, specifies binding
to sites with a given spacing, as shown for Gal4p, Put3p, and Ppr1p
that bind to inverted sites with a spacing of 11, 10, and 6 bp,
respectively (27).
We show here that the linker of Leu3p specifies the relative
orientation of the zinc clusters. The chimera LP-G (Fig.
3A), which contains the N terminus and the zinc cluster of
Leu3p followed by the linker and the dimerization domain of Ppr1p,
bound to a Ppr1p site. Specificity of binding was switched to a Leu3p
site when the fusion protein contained the linker region of Leu3p
(LP-I, Fig. 3A). Similar results were obtained with converse
fusions. For instance, a fusion protein (LP-B), consisting of the N
terminus and the zinc cluster of Ppr1p followed by Leu3p sequences,
recognized a Leu3p site. However, chimeric proteins LP-D, and LP-E
(Fig. 2A) containing Ppr1p sequences (including the linker)
followed by the dimerization domain of Leu3p failed to bind to DNA. It has been shown that Ppr1p is almost insensitive to mutations outside the CGG triplets (13). In contrast, we have shown that, for Leu3p,
nucleotides located between the two everted CGG triplets are important
for binding in vitro and activity in vivo (41). For example, there is a requirement for a C 5' to the CGG triplet (CCGG) for binding in vitro of Leu3p. The Ppr1p site has the
sequence TCGG, which may prevent binding of chimeras LP-D and LP-E even if their zinc clusters are properly oriented to recognize an inverted repeat. The Leu3p linker region may also specify the spacing between the everted CGG triplets preventing binding to sites with alternate spacing.
Role of the Linker and Dimerization Domains--
Alanine scan
mutagenesis of the linker-dimerization region of Leu3p has revealed aa
important for DNA binding. For instance, an arginine (position 81),
located in the linker region, is essential for binding of Leu3p. Other
key aa are located in the dimerization domain, which contains the
heptad repeat region. Mutagenesis of isoleucine 85, phenylalanine 89, and leucine 92 disrupts Leu3p dimerization. However, the Leu3p
dimerization domain does not play a role in specifying binding to
everted repeats. Substitution of the dimerization domain of Leu3p by
the corresponding region of Ppr1p does not disrupt binding to a Leu3p
site (chimera LP-I, Fig. 3A). Similarly, most of the
dimerization domain of Gal4p or Hap1p can be replaced by that of Ppr1p
without affecting binding specificity (7, 27). Many characterized zinc
cluster proteins bind to DNA as homodimers. However, there is good
evidence that the yeast zinc cluster proteins Oaf1p and Oaf2p
(35) can form heterodimers. One possibility is that some dimerization
domains of zinc cluster proteins direct formation of specific
heterodimers resulting in increased diversity of binding sites as
observed for other transcriptional regulators like the nuclear
receptors and leucine zipper proteins (36, 37).
Role of the Zinc Cluster Region--
Similar to the dimerization
domain, the zinc clusters of Leu3p and Ppr1p can be exchanged without
affecting binding specificity. These results are supported by our
previous data which showed that the replacement of the zinc cluster of
Gal4p by the one of Leu3p does not affect binding to a Gal4p site (9).
Many residues known to interact with the CGG triplet, like a lysine at
the fourth aa after the second cysteine of the zinc cluster, are
conserved in Gal4p, Ppr1p, and Leu3p. This conservation extends to
Hap1p (1) but, in contrast to Gal4p, Ppr1p, and Leu3p, this region of
Hap1p has also been shown to be responsible for orienting the zinc
clusters (28). It has been proposed (28) that the zinc clusters of
Hap1p interact with each other allowing binding to asymmetric sites
unlike Leu3p and Ppr1p that recognize symmetric targets as depicted in
Fig. 5. In addition, zinc cluster
proteins Put3p, Tea1p, and Cha4p recognize a similar site: inverted CGG triplets spaced by 10 bp (15, 38, 39), while Leu3p and Uga3p recognize
an everted repeat spaced by 4 bp (41). However, Tea1p does not bind to
a Put3p site (38) and Leu3p does not recognize a Uga3p site (41). It is
possible that an additional role of the zinc cluster (or maybe the
dimerization domain) is to discriminate between highly related sites by
contacting nucleotides that flank the CGG triplets.

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Fig. 5.
Model for binding of zinc cluster proteins to
inverted, direct, or everted repeats. Drawings are based on the
crystal structure of the DNA binding domains of Gal4p (11) and Ppr1p
(12), the analysis of Hap1p-Ppr1p chimeras (28), and Leu3p-Ppr1p
chimeras (this study). Shaded bars indicate interactions
between the zinc clusters of Hap1p.
|
|
A Model for Binding of Zinc Cluster Proteins to DNA--
From the
data presented in this study and others, we propose a model for binding
of the zinc cluster proteins to three different DNA motifs as shown in
Fig. 5. We propose that the C-terminal region of the linker, at the
border of the dimerization domain, projects the two zinc clusters of
the Leu3p homodimer so that they are oriented in opposite directions
allowing binding to an everted repeat. Arginine at position 81, which
was shown to be critical for binding of Leu3p (Fig. 4), may be involved
in projecting the zinc cluster to the right orientation. Because Leu3p
does not bind to mutants of the UAS of LEU2 carrying a
shorter (3 bp) or a longer (5 bp) spacing between the CGG triplets (9),
the linker region must have a rigid structure that prevents binding to
sites with different spacings and, most probably, to sites with
alternate DNA motifs. Similarly, the zinc clusters of Gal4p or Ppr1p
are symmetrically arranged with the linker that controls the
head-to-head orientation of the two zinc clusters (Fig. 5). In
addition, the linkers of Gal4p, Ppr1p, and Leu3p determine the distance
between the zinc clusters and, consequently, restrict the recognition
site to CGG triplets with specific spacings. Comparison of the linker
regions of members of the zinc cluster family shows no obvious homology
(1). Therefore, it is not possible to predict the motif recognized by a
given zinc cluster protein by aa comparison.
In conclusion, recognition of CGG triplets is achieved by the
homologous zinc cluster region of members of the Gal4p/Ppr1p/Leu3p family while the adjacent linker region controls the relative orientation of the zinc clusters thus allowing recognition of inverted
repeats or everted repeats. It will be interesting to correlate the
analysis of Leu3p-Ppr1p chimeras with the crystal structure of the
Leu3p DNA binding domain.
 |
ACKNOWLEDGEMENTS |
We are grateful to Dr. J. White for critical
review of the manuscript and to Dr. A. Nepveu and members of the
Laboratory of Molecular Endocrinology for very helpful discussions. We
are grateful to Dr. H. Zingg and K. Chu for advice.
 |
FOOTNOTES |
*
This work was supported in part by grants from the National
Science and Engineering Research Council of Canada, the Canadian Genome
Analysis and Technology program, and the Medical Research Council of
Canada.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.
¶
Scholar of the Medical Research Council of Canada. To whom
correspondence should be addressed: Dept. of Medicine, Royal Victoria Hospital, McGill University, 687 Pine Ave. West, Montréal,
Québec, Canada H3A 1A1. Tel.: 514-842-1231 (ext. 5046); Fax:
514-982-0893; E-mail: turcotte{at}lan1.molonc.mcgill.ca.
1
The abbreviations used are: aa, amino
acid(s); EMSA, electrophoretic mobility shift assay; UAS, upstream
activating sequence; bp, base pair(s); GST, glutathione
S-transferase; PCR, polymerase chain reaction; aa, amino
acid(s).
 |
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