A comprehensive analysis of Gcn4p coactivator requirements reveals
novel genetic and physical interactions.
Mark J. Swanson, Laarni Sumibcay, Anna Krueger, Soon-ja Kim, Fan
Zhang, Hongfang Qiu, Alan G. Hinnebusch
LGRD/NICHD, National Institutes of Health, 9000 Rockville Pike,
Bethesda, MD 20892, USA
Eukaryotic transcriptional regulation requires sequence specific DNA
binding proteins to activate transcription from target genes organized
in repressive chromatin. Activators recruit multisubunit coactivator
complexes harboring activities to alter chromatin and recruit general
transcription factors and RNA polII. Budding yeast cells respond to
amino acid starvation by inducing Gcn4p, an activator of amino acid
biosynthetic genes. Our previous studies showed that SAGA, SWI/SNF and
SRB/MED coactivators can interact with Gcn4p in vitro, and mutations in
subunits of these complexes decrease activation by Gcn4p in vivo. We are
conducting a comprehensive analysis of Gcn4p coactivator requirements by
testing all viable mutants from the Saccharomyces Deletion
Project for defects in activation by Gcn4p in vivo. Our data confirm
that Gcn4p requires SAGA, SWI/SNF and SRB/MED and identify key
nonessential subunits in these complexes required for activation in
vivo. We uncovered a strong dependence on CCR4/NOT and significant
requirements for RSC, PAF1/mediator and TFIID. In vitro binding
experiments suggest that Gcn4p interacts specifically with CCR4/NOT and
RSC but not with TFIID or PAF1/mediator. Interaction assays reveal that
deletions of certain SAGA and SRB/MED subunits with strong activation
defects in vivo disrupt in vitro binding of Gcn4p to the complex; other
deletions do not reduce in vitro binding to Gcn4p and may impair a
biochemical function of the coactivator in vivo.
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