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Molecular and Cellular Biology, December 1999, p. 8673-8685, Vol. 19, No. 12
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Analysis of TFIIA Function In Vivo: Evidence for a
Role in TATA-Binding Protein Recruitment and Gene-Specific
Activation
Qing
Liu,
Scott E.
Gabriel,
Kelli L.
Roinick,
Robert
D.
Ward, and
Karen M.
Arndt*
Department of Biological Sciences, University
of Pittsburgh, Pittsburgh, Pennsylvania 15260
Received 11 June 1999/Returned for modification 16 July
1999/Accepted 7 September 1999
Activation of transcription can occur by the facilitated
recruitment of TFIID to promoters by gene-specific activators. To investigate the role of TFIIA in TFIID recruitment in vivo, we exploited a class of yeast TATA-binding protein (TBP) mutants that is
activation and DNA binding defective. We found that co-overexpression of TOA1 and TOA2, the genes that encode yeast
TFIIA, overcomes the activation defects caused by the TBP mutants.
Using a genetic screen, we isolated a new class of TFIIA mutants and
identified three regions on TFIIA that are likely to be involved in TBP
recruitment or stabilization of the TBP-TATA complex in vivo. Amino
acid replacements in only one of these regions enhance TFIIA-TBP-DNA
complex formation in vitro, suggesting that the other regions are
involved in regulatory interactions. To determine the relative
importance of TFIIA in the regulation of different genes, we
constructed yeast strains to conditionally deplete TFIIA levels prior
to gene activation. While the activation of certain genes, such as
INO1, was dramatically impaired by TFIIA depletion,
activation of other genes, such as CUP1, was unaffected.
These data suggest that TFIIA facilitates DNA binding by TBP in vivo,
that TFIIA may be regulated by factors that target distinct regions of
the protein, and that promoters vary significantly in the degree to
which they require TFIIA for activation.
*
Corresponding author. Mailing address: Department of
Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260. Phone: (412) 624-6963. Fax: (412) 624-4759. E-mail:
arndt{at}vms.cis.pitt.edu.
Molecular and Cellular Biology, December 1999, p. 8673-8685, Vol. 19, No. 12
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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