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

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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