CUP2 binds in a bipartite manner to upstream activation sequence c ...

JBIC (1996) 1:451–459 Q SBIC 1996
ORIGINAL ARTICLE
Wendy J. Dixon 7 Carla Inouye 7 Michael Karin
Thomas D. Tullius
CUP2 binds in a bipartite manner to upstream activation sequence c
in the promoter of the yeast copper metallothionein gene
Received: 23 May 1996 / Accepted: 8 July 1996
Abstract Induction of the copper metallothionein
(CUP1) gene of the yeast Saccharomyces cerevisiae is
achieved by CUP2, a transcriptional activator protein
which has a metal-dependent DNA-binding activity. It
is thought that metal binding to CUP2 results in the
formation of several looped regions of the protein
which then are capable of binding to DNA. CUP2
binds to a site near the CUP1 gene called upstream activation
sequence c (UASc), an imperfect inverted repeat.
While CUP2 binds to both half-sites of UASc, the
upstream half-site appears to be more important for
transcriptional activity. A variant of CUP2 called ace1,
in which cysteine-11 is replaced by tyrosine, binds to
DNA but is incapable of activating transcription. We
have used hydroxyl radical footprinting and missing nucleoside
analysis to examine the complexes of wild-type
CUP2 and the ace1 mutant protein with UASc. Our results
indicate that ace1 interacts with a smaller portion
of UASc than does CUP2, providing further evidence
that the DNA-binding domain of CUP2 is complex,
composed of two or more elements that recognize distinct
features of UASc. We also show that CUP2 itself
binds slightly differently to the two half-sites of UASc.
While CUP2 and ace1 bind in a rather similar manner
to the downstream half-site, in the upstream half-site
CUP2 makes more extensive interactions. Our results
W.J. Dixon1 7 T.D. Tullius
Department of Biology, The Johns Hopkins University,
Baltimore, MD 21218, USA
C. Inouye 7 M. Karin
Department of Pharmacology, M-036, School of Medicine,
University of California, San Diego, La Jolla, CA 92093, USA
T.D. Tullius (Y)
Department of Chemistry, The Johns Hopkins University,
3400 North Charles Street, Baltimore, MD 21218, USA
Tel.: c1-410-516-7449; Fax: c1-410-516-8468;
email tom@radical.chm.jhu.edu
1 Present address:
Department of Chemistry, 147–75CH, California Institute
of Technology, Pasadena, CA 91125, USA
suggest that the more crucial role that the upstream
half-site plays in transcriptional activation may be due
to differences in how CUP2 binds to each of the halfsites
of UASc.
Key words Hydroxyl radical footprinting 7 Missing
nucleoside analysis 7 DNA-protein interactions 7
Transcriptional activation 7 Metalloregulatory protein
Introduction
The yeast Saccharomyces cerevisiae synthesizes a copper-inducible
metallothionein [1]. The function of this
protein, encoded by the CUP1 gene, is to maintain a
low level of free copper ion in the cell. The CUP1 protein
consists of 53 amino acids, including 12 cysteines
which bind 8 copper(I) ions [2, 3]. The level of synthesis
of copper metallothionein by yeast is dependent on
the concentration of intracellular copper [4]. Induction
of the CUP1 gene is mediated by CUP2 (also known as
ACE1), a transcriptional activator protein which has a
DNA-binding activity that is dependent on metal ion
binding [5–7].
CUP2 consists of two functional domains. The carboxyl
terminus is rich in negatively-charged amino acid
residues and resembles the acidic activation domains of
the yeast transcription factors GCN4 and GAL4 [8, 9].
In contrast, the amino-terminal domain of CUP2 is rich
in basic amino acid residues and also contains 12 cysteines,
eight of which are arranged in the CysXCys and
CysX2Cys configurations characteristic of metallothionein.
This portion of the protein forms a metal-dependent
DNA-binding domain that binds to the upstream
activation sequences (UAS) of the CUP1 gene [5, 7]. It
was proposed that Cu(I) binds to CUP2 in a cysteinecopper
cluster that is similar to that of the metallothionein
that it regulates [5]. Extended X-ray absorption
fine structure (EXAFS) studies on CUP2 have
since given experimental validity to this hypothesis [10,
11].

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Deletion studies have shown that UASc is required
for copper-induced transcription of the CUP1 gene [12,
13]. Initially, specific CUP2-DNA interactions were examined
by mutagenesis of UASc. Individual transition
mutants within this region of the CUP1 promoter (positions
–105 to –148 relative to the transcription start site)
were constructed and assayed for transcriptional activity
[5]. All mutants that were completely noninducible
were found in a 16-bp region in the upstream half of
UASc. In the downstream half of UASc, no mutants
were completely noninducible, although some mutants
were substantially less inducible than the wild-type construct.
These results led Hamer and coworkers [5] to
propose that the 16-bp region in the upstream half of
UASc is a potential binding site for CUP2.
Further biochemical analysis of the mode of binding
of CUP2 has revealed that CUP2 indeed binds to both
halves of UASc. A variant CUP2 protein, called ace1,
in which a single cysteine residue is substituted by tyrosine,
binds to DNA but is incapable of inducing transcription.
Examination of the specific interactions made
by CUP2 and ace1 with DNA indicated that the DNAbinding
domain of CUP2 is complex, being composed
of two or more elements that recognize distinct features
of UASc [14].
We report here the results of hydroxyl radical footprinting
[15] and missing nucleoside experiments [16]
for CUP2 or ace1 bound to a restriction fragment containing
UASc. Using these methods we have determined
which nucleotides are protected or contacted by
the two proteins. We show that ace1 contacts a smaller
region of the upstream half-site of UASc, consistent
with previously published experiments [14, 17]. Our results
provide further evidence that the DNA-binding
domain of CUP2 is complex, composed of multiple elements
that recognize distinct features of UASc. In addition,
our studies suggest that the disparity in the effect
on transcription of mutations in the two half-sites of
UASc is likely to be due to differences in the interactions
of CUP2 with each half-site.
Materials and methods
Plasmids, DNA molecules and proteins
Plasmid pUASc, containing the 5b-noncoding region of the CUP1
gene from positions –145 to –105, was the source of the DNA
used in these experiments. DNA restriction fragments were radiolabeled
at either the 5b or 3b end of the HindIII site, digested
with EcoRI, and purified by gel electrophoresis. The proteins
used were expressed in and purified from Escherichia coli transformed
with plasmid pET7CUP2, pET7CUP2TR, or pET7ace1,
as described previously [14].
Binding conditions for the CUP2-DNA complex
Binding solutions contained 0.05–0.10 ng singly end-labeled 94-bp
restriction fragment that contained UASc from the CUP1 promoter
(100000–200000 dpm), 1 mg BSA, 1% polyvinyl alcohol,
20 ng poly(dI7dC), 12.5 mM Hepes-NaOH (pH 8), 50 mM KCl,
6.25 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, and 0–500 ng
CUP2 protein, in a total volume of 20 ml. The solution was incubated
on ice for 15 min.
Hydroxyl radical foot printing
The hydroxyl radical cleavage reaction was performed as previously
described [15, 18]. The sample containing the protein-
DNA complex was warmed to room temperature for 1 min, and
then the cutting reagents were added. The final concentrations of
cutting reagents were 1 mM Fe(II), 2 mM EDTA, 0.003% H 2O 2,
and 20 mM sodium ascorbate. After 2 min the reaction was stopped
by addition of thiourea to a final concentration of 24 mM.
DNA was precipitated twice by addition of ethanol, rinsed, dried
under vacuum, resuspended in 3 ml of formamide-dye mixture,
heated to 90 7C for 5 min, and electrophoresed on a denaturing
gel (12% polyacrylamide, 8.3 M urea). The gel was dried, autoradiographed,
and scanned with a Joyce-Loebl Chromoscan 3 densitometer.
Missing nucleoside experiment
Missing nucleoside experiments were performed as previously described
[16]. The DNA molecule used was a 94-bp restriction
fragment containing UASc from the CUP1 promoter. Gapped
DNA containing UASc was produced by adding 12 ml of each of
the hydroxyl radical-generating reagents to a sample containing
approximately 1.5 ng singly end-labeled DNA (3000000 dpm) in
60 ml of 10 mM Tris-HCl buffer (pH 8.0), 1 mM EDTA. Final concentrations
of the cleavage reagents were 1 mM Fe(II), 2 mM
EDTA, 0.003% H2O2, and 20 mM sodium ascorbate. The reaction
was allowed to proceed for 2 min at room temperature and then
stopped by addition of 30 ml of 0.1 M thiourea. The DNA was
precipitated with ethanol, rinsed, and dried.
Mobility shift assay
CUP2 or ace1 was bound to UASc by adding protein to a solution
containing approximately 0.25–0.5 ng of radiolabeled gapped
DNA (500000–1 000000 dpm), 1 mg BSA, 1% polyvinyl alcohol,
20 ng poly(dI!dC), 12.5 mM Hepes-NaOH (pH 8), 50 mM KCl,
6.25 mM MgCl2, 0.05 mM EDTA, and 0.5 mM DTT, in a total volume
of 10 ml. The mixture was incubated on ice for 15 min,
warmed to room temperature, and then 2 ml of 30% glycerol was
added. To separate protein-bound DNA from free DNA, samples
were loaded on an 8% nondenaturing gel (80:1 acrylamide:bis).
Electrophoresis was performed at room temperature at 125 V for
3 h in a buffer consisting of 25 mM Tris-borate-HCl (pH 8.3), and
2.5 mM EDTA. The wet gel was autoradiographed for 1 h. The
desired bands were excised from the gel and crushed. DNA was
eluted by soaking the crushed gel slice in buffer [50 mM ammonium
acetate, 1 mM EDTA (pH 6.5)] at 37 7C overnight. DNA was
precipitated with ethanol twice, rinsed, dried, resuspended in 3 ml
of formamide-dye mixture, heated to 90 7C for 5 min, and electrophoresed
on a denaturing gel (10% polyacrylamide, 8.3 M urea).
Results
Hydroxyl radical footprinting
The hydroxyl radical footprints of the ace1 mutant protein,
in which Cys11 is substituted by Tyr, are presented
in Fig. 1. These hydroxyl radical footprints are virtually
identical to those of the wild-type CUP2 protein bound

Fig. 1A–D Densitometer scans of hydroxyl radical footprints of
ace1 bound to UASc. Shown are scans of autoradiographs of hydroxyl
radical cleavage of A the top strand of UASc in the absence
of protein, B the top strand with bound ace1, C the bottom
strand with bound ace1, and D the bottom strand in the absence
of protein. Nucleotides are numbered relative to the start site of
transcription. The vertical line marks the pseudodyad symmetry
axis located at position P124. The horizontal line at the bottom
delineates UASc
to UASc (data not shown). We also obtained similar
footprints for a truncated version of CUP2 (called
CUP2TR) which contains only the amino terminus of
the protein (data not shown), indicating that only the
amino-terminal domain interacts with DNA, as suggested
previously by mobility shift gel electrophoresis
assays [14].
All three proteins protect two regions on each
strand, with the strongest protections centered at positions
–132 and –112 (top strand) and –137 and –115
(bottom strand). At lower concentrations of ace1 only a
single region of protection, at –112 (top strand) and
–115 (bottom strand), is seen. The bottom strand also
has a weaker site of protection between positions –120
and –115. A very weak protection is seen in the central
region of UASc, covering two or three nucleotides centered
at –122 (top strand) and –126 (bottom strand).
The strong hydroxyl radical protections are identical
to those determined previously for CUP2 binding to
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UASc [14]. The two strong protections at the extremities
of the binding site are offset from each other by
three or five nucleotides in the 3b direction from one
strand to the other, indicating that CUP2 crosses the
minor groove of the DNA at those points [15, 18].
In the central region of the binding site we find that
the hydroxyl radical protections are weaker and appear
to be offset in the 3b direction. In previous work we
reported an offset in the 5b direction for the weak footprint
of CUP2 at the center of UASc [14]. There are
two differences between the earlier footprinting experiments
and those reported here that might be responsible
for the difference in the footprint at the center of
the binding site. One difference is the presence of an
additional upstream activation site, UASd, within the
restriction fragment used in the earlier work. The other
difference was the use in the previous experiments of a
mobility shift gel to separate protein-DNA complexes
from unbound DNA after performing hydroxyl radical
footprinting. More recent hydroxyl radical footprints of
CUP2 bound to DNA containing both UASc and
UASd, performed in the conventional manner without
separation of protein-DNA complexes from unbound
DNA, show a protected region in the center of UASc
of moderate intensity and offset in the 3b direction
(Dixon et al., in preparation). This result suggests that
the protections at the center of UASc seen in the previously
published hydroxyl radical footprint of CUP2
[14] are an artifactual result due either to the low resolution
between free DNA and protein-DNA complexes
in the mobility shift gel or to instability of the complex
over the time between footprinting and gel loading.
Missing nucleoside experiments
To examine more closely the interactions required for
formation of the CUP2 or ace1 complex with UASc,
missing nucleoside experiments [16] were performed.
DNA containing UASc was treated with the hydroxyl
radical to randomly remove nucleosides. The collection
of gapped DNA molecules was then fractionated by
mobility shift gel electrophoresis based on the ability to
form specific protein-DNA complexes.
When either CUP2 or ace1 is incubated with an intact
or with a gapped DNA molecule containing UASc
and then electrophoresed on a mobility shift gel, two
slower-moving bands appear in addition to the band
containing free DNA [14]. The faster of the two protein-UASc
complexes is termed complex I and the
slower is termed complex II (Fig. 2). We presume that
these complexes represent singly and doubly occupied
UASc, respectively. For the missing nucleoside experiment
we isolated DNA from these three fractions (unbound,
complex I, and complex II). The DNA was then
electrophoresed on a denaturing gel. Densitometer
scans of autoradiographs of the DNA cleavage products
are shown in Fig. 3 and Fig. 4 for CUP2 and ace1,
respectively.

454
Fig. 2A, B Mobility shift gel
electrophoresis of CUP2 and
ace1 bound to UASc. A The
autoradiograph of a native gel
on which was run the CUP2-
UASc complex. In the lanes
on the left the top strand of
UASc is radiolabeled, and in
the lanes on the right the bottom
strand is radiolabeled.
The outer lanes contain only
ungapped DNA. The other
lanes contain gapped DNA
and CUP2. The bands labeled
unbound contain only DNA.
The faster-migrating protein-
UASc complex is labeled
complex I and the slower is labeled
complex II. B The autoradiograph
of a mobility shift
electrophoresis gel on which
was run the ace1-UASc complex.
In the lanes on the left
the top strand of UASc is radiolabeled,
and in the lanes
on the right the bottom strand
is radiolabeled. The outer
lanes contain ungapped DNA.
The lanes next to the outer
lanes contain gapped DNA.
The remaining lanes contain
gapped DNA and ace1. The
bands labeled unbound contain
only DNA. The faster-migrating
ace1-UASc complex is
labeled complex I and the
slower one is labeled complex
II
The results of the missing nucleoside experiment are
interpreted in the following way. An increase in the
amount of DNA in a band in the unbound fraction indicates
that the loss of this nucleoside inhibits formation
of the protein-DNA complex. Correspondingly, a
reduced amount of gapped DNA at a particular sequence
position in the complex I and complex II fractions
indicates that loss of this nucleoside reduces the
formation of these complexes.
Missing nucleoside analysis of CUP2
The results of our missing nucleoside experiments show
that formation of complex II requires that CUP2 occupy
both half-sites of UASc (Fig. 3). In each half of
UASc we find a series of bands of reduced intensity,
indicating that the loss of any of these nucleosides inhibits
formation of complex II. For both strands, a region
of reduced band intensity is observed between nucleotides
–119 and –112 in the downstream half-site. In the
upstream half-site, a longer stretch of DNA, from positions
–129 to –142, is required for formation of complex
II.
On the top strand, the nucleosides required for formation
of complex II are found in two regions: strong
reductions in band intensity in the bound fraction are
observed at nucleosides –135 to –129, while moderate
reductions in band intensity are seen at nucleosides
–142 to –140. On the bottom strand, 11 nucleosides between
positions –141 to –129 are seen to be required for
formation of complex II.
In the DNA fraction obtained from complex I, we
observe a fairly even cutting pattern that contains regions
having a small reduction in band intensity within
each half-site. Bands are slightly lower in intensity in
the upstream half-site. Thus, a gap at any single nucleoside
within UASc is not sufficient to prevent formation

Fig. 3 Densitometer scans of the missing nucleoside experiment
for CUP2. In the experiment that produced the upper four scans,
the top strand of UASc was radiolabeled; to produce the lower
four scans, the bottom strand was radiolabeled. To the left of each
scan, the DNA fraction from the mobility shift gel (see Fig. 2) is
indicated. The horizontal line in the center of the figure represents
the position of UASc. The numbers indicate the positions of
nucleotides relative to the start site of transcription
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Fig. 4 Densitometer scans of the missing nucleoside experiment
for ace1. In the experiment that produced the upper three scans,
the top strand of UASc was radiolabeled; to produce the lower
three scans, the bottom strand was radiolabeled. To the left of
each scan, the DNA fraction from the mobility shift gel (see
Fig. 2) is indicated. The horizontal line in the center of the figure
represents the position of UASc. The numbers indicate the positions
of nucleotides relative to the start site of transcription
of complex I. In other words, if there is a gap in one
half-site, the protein is still able to bind to the other
half-site and form a singly occupied UASc.
In general, sequence positions at which we see enhanced
band intensity in the unbound lane are the
same sequence positions at which we observe reduced
intensity in complex II. However, there are two exceptions.
On the bottom strand in the upstream half-site at
positions –129 to –141 and on the top strand in the
downstream half-site at positions –119 to –112, no enhancement
in band intensity in the unbound lane is observed,
corresponding to the reduced intensity seen at
these positions in the complex II sample. Similar miss-

456
ing nucleoside results were observed with the CUP2TR
protein (data not shown).
Missing nucleoside analysis of ace1
The missing nucleoside pattern we find for the ace1
complex II fraction reveals that ace1 makes important
interactions with a stretch of nine base pairs within
each half-site of UASc, with both nucleosides of each
base pair contributing to binding (Fig. 4). These two regions,
nucleotides –120 to –112 in the downstream halfsite
and –136 to –127 in the upstream half-site, are symmetrical
about the pseudodyad at base pair –124.
The cleavage pattern of DNA isolated from complex
I indicates that only nucleosides –120 to –112 in the
downstream half-site of UASc are essential for binding
of ace1. In the unbound fraction a complementary enhancement
of band intensities is observed. Our results
for the unbound fraction and for complex I, along with
previously reported methylation interference data [14],
indicate that ace1 has a strong preference for binding to
the downstream half-site. Thus, under the conditions of
our experiment, the affinity of ace1 for the upstream
half-site is so low relative to its affinity for the downstream
site that there is little binding of ace1 to the upstream
site even when the downstream site has suffered
the loss of a critical nucleoside.
Discussion
CUP2 binding to UASc
The results of hydroxyl radical footprinting and missing
nucleoside experiments indicate that CUP2 interacts
with both half-sites of UASc, spanning sequence positions
–142 to –109 (Fig. 5). In each half-site, these interactions
extend one and one half turns from the dyad
center. Within each half-site there is one strong hydroxyl
radical footprint which is offset in the 3b direction
from one strand to the other, indicative of protection
across the minor groove [15]. Previous methylation interference
studies indicated that CUP2 interacts with
the major groove of the DNA directly flanking the regions
protected from hydroxyl radical cleavage [14].
We observe missing nucleoside signals throughout the
regions where methylation interference signals occur
and where hydroxyl radical footprints are seen.
Collectively, these data lead to a model for the complex
in which CUP2 makes contacts in the major
groove one half turn and one and one half turns to
either side of the center of UASc, with the protein
crossing over the minor groove between these major
groove interactions (Fig. 5). Although UASc can be
considered to be an inverted repeat around a dyad axis
of symmetry centered at position –124, if the G7C base
pair at position –120 is eliminated [14], the interactions
of CUP2 with the two half-sites are not completely
Fig. 5 Compilation of DNA binding data for CUP2 mapped on a
10.5 bp per turn double helical representation of UASc. The sequence
of UASc is shown below the DNA helix. Dots are placed
every 10 bp, from position –140 at the left to –110 at the right.
Horizontal arrows demarcate the region of almost perfect dyad
symmetry. The ellipse in the center of the DNA helix is placed at
position –124, the site of the pseudodyad. A rectangle encloses the
extra G7C base pair at position –120 that interrupts dyad symmetry.
The results of several kinds of experiment are mapped on the
DNA helix. Bases at which missing nucleoside signals are observed
are marked by filled squares. Nucleotides protected from
cleavage by the hydroxyl radical are indicated by circles on the
sugar-phosphate backbone. Bases at which methylation interference
was observed [14] are marked by a carat near the DNA helix.
The results of analysis of point mutations [5] are indicated by
rectangles below the DNA sequence: solid noninducible, moderately
shaded ~25% inducible, lightly shaded ~75% inducible.
The bracket above the DNA helix spanning positions –142 to
–139 highlights the additional asymmetric contacts made by
CUP2 in the upstream half-site of UASc (see text for discussion).
No corresponding contacts symmetrically disposed across the
dyad at sequence positions –108 to –105 are observed
symmetrical. CUP2 makes energetically important contacts
with an additional 4 base pairs in the upstream
half-site. The location of these additional contacts is indicated
by the bracket above nucleotides –142 to –139
in Fig. 5, outside the UASc inverted repeat.
In general these results agree with previous DNase I
footprinting and methylation interference experiments
[14]. In addition, the nucleotide contacts we observe in
the upstream half of UASc are nearly identical to those
recently found by the related missing contact technique
for wild-type CUP2 bound to a DNA construct containing
only the upstream half-site [17].
Comparison with point mutation analysis
The missing nucleoside experiment reveals the nucleosides
that make energetically important contributions
to the formation of a protein-DNA complex. It might
be expected that the essential base pairs revealed by
point mutation studies would be a subset of the contacts
found in missing nucleoside experiments.
Hamer and coworkers [5] found 21 transition mutations
in UASc that decrease the level of copper-induced
transcription. Their results are summarized in
Fig. 5. Twelve of these mutants occur in the upstream
half of UASc, 11 of which are completely noninducible.
These results led these workers to propose as the CUP2
binding site a 16-bp region in the upstream half-site of
UASc [5]. From the results presented here, and from

previous work [14], the binding region for CUP2 clearly
encompasses 32 bp. Quantitative binding experiments
[19] show that the affinity of CUP2 for the two halfsites
differs only sixfold. The much greater disparity in
the effect of mutations in the two half-sites suggests
that transcriptional activation is more critically dependent
on the CUP2 molecule that is bound to the upstream
half-site.
A comparison of the 11 completely noninducible
mutations with the missing nucleoside signals in the upstream
half-site reveals that in general the mutations
constitute a subset of the missing nucleoside signals.
Seven of the mutations occur in or near the two regions
where the protein appears to interact directly with
bases in the major groove. In the region where the protein
crosses the minor groove, three T7A base pairs in
the TTTT sequence inhibit inducible transcription
when mutated, suggesting either that the protein makes
direct contacts with the minor groove, or that the structural
integrity of the A tract must be maintained for
protein binding.
ace1 binding to UASc
The ace1 protein binds with markedly higher affinity to
the downstream half of UASc [14], forming a stable
singly occupied complex. In the fully occupied complex,
ace1 makes symmetrical interactions with the two halfsites.
The hydroxyl radical footprint of ace1 is identical
for each half-site, unlike the DNase I footprint, in
which protection is greater for the downstream half-site
[14]. Analogous footprinting behavior has been noted
for another DNA-binding metalloprotein, TFIIIA [20].
In footprinting experiments on a series of TFIIIA mutants
in which successive zinc fingers were deleted, it
was found that for certain finger deletion mutants the
hydroxyl radical footprint was larger than the DNase I
footprint. This phenomenon probably reflects competition
between DNase I and a weakly interacting zinc finger
for binding to DNA. The hydroxyl radical, in contrast,
cleaves DNA without prior complexation, so a
weakly bound protein domain would still give a footprint.
Perhaps a similar effect is operative for ace1.
In agreement with the results of earlier methylation
interference experiments, we find that the majority of
ace1 contacts occur in the major groove one half turn
from the pseudodyad (Fig. 6). Additionally, missing nucleoside
signals occur further upstream or downstream
at nucleosides that are found to be protected in hydroxyl
radical footprinting experiments. In previous work it
was found that methylation of guanines in this region
(positions –133 and –132) does not interfere with ace1
binding [14], indicating that the missing nucleoside signals
result either because ace1 contacts the sugar-phosphate
backbone or because the missing nucleoside gaps
cause structural changes in the DNA that inhibit ace1
binding [21]. Since hydroxyl radical footprints are seen
at these guanines, it seems most plausible that ace1
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Fig. 6 Compilation of DNA binding data for ace1 mapped on a
10.5 bp per turn double helical representation of UASc. The sequence
of UASc is shown below the DNA helix. Dots are placed
every 10 bp, from –140 at the left to –110 at the right. Horizontal
arrows demarcate the region of almost perfect dyad symmetry.
The ellipse in the center of the DNA helix is placed at position
–124, the site of the pseudodyad. A rectangle encloses the extra
G7C base pair at position –120 that interrupts dyad symmetry.
The results of several kinds of experiment are mapped on the
DNA helix. Bases at which missing nucleoside signals are observed
are marked by squares: stippled signals observed for complex
I, stippled plus solid signals observed for complex II. Nucleotides
protected from cleavage by the hydroxyl radical are indicated
by circles on the sugar-phosphate backbone. Bases at which
methylation interference was observed [14] are marked by a carat
near the DNA helix
contacts the sugar-phosphate backbone at these nucleotides.
Our results indicate that ace1 binds in the major
groove one half turn from the dyad and protects the
minor groove one turn from the dyad (Fig. 6).
Comparison of the CUP2- and ace1-DNA complexes
Although the hydroxyl radical footprint of ace1 is identical
to the footprint of CUP2, missing nucleoside experiments
identify a smaller region of contact between
ace1 and DNA. The major groove contacts made by
CUP2 at the outer edge of the binding site are not
found for ace1. A similar result was seen in previous
methylation interference experiments [14]. Our missing
nucleoside data now provide us with a precise assessment
of the differences in nucleoside contacts made by
CUP2 and ace1.
In the downstream half-site, missing nucleoside experiments
show that CUP2 and ace1 interact with
UASc in almost an identical manner (Fig. 7). One methylation
interference signal at the outer edge of the
downstream half-site is present for CUP2 but absent
for ace1 (compare Fig. 5 and Fig. 6), indicating that
CUP2 contacts the guanine in the major groove near
the downstream end of this half-site while ace1 does
not. In this half-site, the positions of nucleosides giving
missing nucleoside signals are identical for the two proteins,
and the strongest signals are found at the same
nucleotide positions.
In the upstream half-site, differences in the interactions
of the two proteins with DNA are more pronounced.
The missing nucleoside patterns clearly are
not identical (Fig. 7). On the top strand ace1 gives
strong signals at positions –128 to –130, while for CUP2
there is little or no signal apparent at these positions.

458
This difference is most clearly appreciated when the intensities
of bands at positions –128 and –127 are compared
(Fig. 7B): for ace1, the intensity of the band at
position –128 is substantially reduced compared to the
band at –127, while for CUP2 the bands at –128 and
–127 have almost equal intensity. Further upstream,
CUP2 exhibits missing nucleoside signals at position
–140 to –142, while ace1 does not. Although on the top
strand these differences are small, the comparison
shown in Fig. 7A demonstrates that while at position
–139 the two proteins have similar signals, at position
–140 the band intensity for CUP2 is significantly lower
than that for ace1. Additional evidence for these contacts
is provided by the scan of the cleavage pattern of
the unbound fraction for CUP2 (see Fig. 3), in which a
peak of enhanced band intensity is observed at position
–141.
The differences in missing nucleoside patterns are
most pronounced on the bottom strand. The most striking
difference occurs at positions –139 to –141, where
significant missing nucleoside signals are found for
CUP2 while ace1 shows none (Fig. 7B).
These missing nucleoside results indicate that CUP2
contacts four additional base pairs in the upstream halfsite,
with greater interaction with the bottom strand
than is the case for ace1. This conclusion is consistent
with previous methylation interference results [14],
which showed that three of the guanines within this region
interfere with the formation of the CUP2-DNA
complex when methylated, while formation of the ace1-
DNA complex is unaffected. A variant of the CUP2
protein, in which the three most amino-terminal cysteines
(Cys11, Cys14 and Cys23) were carboxymethylated,
was found to contact a nearly identical set of nucleotides
as we find for ace1 [17]. These results indicate
that in both half-sites CUP2 contacts the outermost major
groove while ace1 does not, and that the contacts
made by CUP2 with the major groove are more extensive
in the upstream half-site.
Biological implications
In this paper we have examined the binding of CUP2
and the related protein ace1 to UASc. These studies
have shown that ace1 interacts with a smaller portion of
UASc than does CUP2. Our results are consistent with
previous suggestions that CUP2 possesses a bipartite
DNA-binding domain. The loss of a metal coordination
site due to substitution of Tyr for Cys11 [22] apparently
results in the loss of a DNA-binding element which
normally contacts the major groove of the DNA at the
extremities of UASc. The fewer DNA contacts made
by ace1 explains its tenfold lower binding affinity for
UASc compared to CUP2 [14]. It is plausible that the
mutation in ace1, Cys11Tyr, leads to disruption of a
DNA-binding domain. Six basic amino acids, four of
which are clustered together, occur between Cys11 and
the next cysteine in the sequence, Cys43.
Fig. 7A, B Comparison of missing nucleoside patterns for CUP2
and ace1. A Overlay of densitometer scans of missing nucleoside
patterns obtained from complex II (dotted line CUP2, solid line
ace1). B The densitometer scan of the missing nucleoside pattern
obtained from complex II (dotted line) is superimposed upon the
densitometer scan of the hydroxyl radical cleavage pattern of free
(control) DNA (solid line). Top two scans top strand, bottom two
scans bottom strand
Although UASc can be considered to be pseudopalindromic,
mutational analysis has indicated that the upstream
half-site is particularly crucial for transcriptional
activity [5]. Either this half-site is at the appropriate
distance and orientation relative to the TATA box of
the CUP1 gene or there is some difference in the way
that CUP2 binds to the two half-sites. The missing nucleoside
results presented here reveal that the interac-

tions made by CUP2 at each half-site are not completely
symmetrical. CUP2 contacts 4 base pairs more in the
upstream than in the downstream half-site.
In comparing the binding of ace1 and CUP2 to each
half-site, we observed that in the downstream half-site
the two proteins produce almost identical footprinting
and missing nucleoside signals. We therefore conclude
that CUP2 and ace1 bind in a nearly identical manner
to the downstream half-site. Since the mutational data
indicate that this half-site is biologically nonfunctional,
it appears that the mode of binding of CUP2 to the
downstream site results in an inability to activate transcription.
In the upstream half-site, CUP2 contacts more nucleotides
than does ace1. Since this is the half-site implicated
by mutational studies to be necessary for
CUP2-mediated transcriptional activation, the different
structure of the CUP2-DNA complex in this half-site
compared to the downstream site might be related to
the ability of the protein to activate transcription. Possible
models include (1) a change in DNA conformation
induced by binding of CUP2, which would facilitate
the binding of the transcriptional machinery to the
TATA box, or (2) a change in the conformation of
CUP2 upon binding to the upstream site, which would
in turn position the activation domain of the protein to
interact productively with the transcriptional machinery.
The structural change caused by a single-amino-acid
difference leads to the lack of function for one of the
DNA-binding elements of ace1. For CUP2, this DNAbinding
element is functional, but in the downstream
half-site of UASc, apparently it does not interact with
DNA or at least not in the same way as in the upstream
half-site. Alternatively, the DNA-binding element may
not be positioned correctly to make specific contacts
with the DNA. Crystallographic studies of the glucocorticoid
receptor bound to DNA demonstrate that a
1-bp translocation of the protein relative to the DNAbinding
sequence leads to nonspecific instead of specific
binding [23]. It is possible that the additional base
pair within the palindromic sequence of the downstream
half-site at sequence position –120 alters the po-
459
sitioning of CUP2 relative to DNA, thereby making
one DNA-binding unit of the protein unable to make
specific contacts with the DNA.
Acknowledgements This work was supported by PHS grants
GM 41930 (TDT) and ES 04151 (MK).
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