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

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-