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

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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