Nucleic Acids Research, Vol 24, Issue 8 1453-1459, Copyright © 1996 by Oxford University Press
N Ha, K Hellauer and B Turcotte
The yeast zinc cluster protein HAP1, a member of the GAL4 family, is a
transcriptional activator that binds as a homodimer to target DNA
sequences. These targets include the upstream activating sequences of the
CYC1 and CYC7 genes, which have no obvious sequence similarity. Even though
both sites have the same affinity for HAP1, activation differs at these two
sites, even when the sequences are placed in an identical promoter context.
In addition, mutants of HAP1 that can bind to both sites but are
specifically transcriptionally inactive at CYC7 have been previously
isolated. In order to identify nucleotides that are responsible for this
differential activity, we have performed random and site-directed
mutagenesis of these target sites and assayed their binding to HAP1 in
vitro and their activity in vivo in reporter plasmids. Our results show
that HAP1 binding sites are degenerate forms of the direct repeat CGG N3 TA
N CGG N3 TA. Moreover, we show that activity of HAP1 mutants defective for
activation of the CYC7gene is restored by specific mutations in the CYC7
binding site. Conversely, other mutations of the target sites prevent
activation by HAP1, without interfering with DNA binding. The results
suggest that the sequence of the target sites influences the conformation
and, hence, the activity of DNA-bound HAP1.
ARTICLES
Mutations in target DNA elements of yeast HAP1 modulate its transcriptional activity without affecting DNA binding
Department of Medicine, McGill University, Royal Victoria Hospital, Montreal Quebec, Canada.
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