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

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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