Analysis of TFIIA Function In Vivo: Evidence for a Role in TATA-Binding Protein Recruitment and Gene-Specific Activation

Abstract

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.

ACKNOWLEDGMENTS

We thank the following individuals for the gifts of plasmids and strains: Cori Detweiler and Joachim Li, Steve Hahn, Steve Buratowski, Greg Prelich, Brendan Cormack and Kevin Struhl, Robert Nash, Michael Grunstein, and Fred Winston. We are very grateful to Jim Geiger for crystal structure coordinates and valuable discussions, to Steve Hahn for antisera against TFIIA, to Jonathan Warner for antisera against L3, and to Tony Imbalzano and Bob Kingston for recombinant yeast TFIIA. We thank Jeff Brodsky and members of his laboratory for technical assistance and Greg Prelich, Martin Schmidt, Peggy Shirra, and Fred Winston for helpful discussions and critical reading of the manuscript.

This work was supported by NIH grant GM52593 and by an NSF Career Development Award (MCB-9600955) to K.M.A. S.E.G. was supported by an NSF REU Award.