Transcription factor IIA mutations show activator-specific defects and reveal a IIA function distinct from stimulation of TBP-DNA binding.
The general transcription factor IIA (TFIIA) binds to the TATA binding protein (TBP) and mediates transcriptional activation by distinct classes of activators. To elucidate the function of TFIIA in transcriptional activation, point mutants were created in the human TFIIA-gamma subunit at positions conserved with the yeast homologue. We have identified a class of TFIIA mutants that stimulate TBP-DNA binding (T-A complex) but fail to support transcriptional activation by several different activators, suggesting that these mutants are defective in their ability to facilitate an activation step subsequent to TBP promoter binding. Point mutations of the hydrophobic core of conserved residues from 65 to 74 resulted in various activation-defective phenotypes. These residues were found to be important for TFIIA gamma-gamma interactions, suggesting that gamma-gamma interactions are critical for TFIIA function as a coactivator. A subset of these TFIIA-gamma mutations disrupted transcriptional activation by all activators tested, except for the Epstein-Barr virus-encoded Zta protein. The gamma Y65F, gamma W72A, and gamma W72F mutants mediate Zta activation, but not GAL4-AH, AP-1, GAL4-CTF, or GAL4-VP16 activation. The gamma W72A mutant failed to stimulate TFIID-DNA binding (D-A complex) but was able to form a complex with TFIID and DNA in the presence of Zta (Z-D-A complex). Thus, the ability of Zta to activate transcription with gamma W72A appears to result from a unique ability to form the stable Z-D-A complex with this mutant. Our results show that different activators utilize the general factor TFIIA in unique ways and that TFIIA contributes transcription activation functions in addition to the facilitation of TBP-DNA binding.
Ozer
J
J
Wistar Institute, Philadelphia Pennsylvania 19104, USA.
Bolden
A H
AH
Lieberman
P M
PM
D000409:Alanine; D000595:Amino Acid Sequence; D017124:Conserved Sequence; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000378:metabolism*; D005982:Glutathione Transferase / Q000096:biosynthesis; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D016385:TATA Box; D035181:TATA-Box Binding Protein; D035561:Transcription Factor TFIIA; D035362:Transcription Factor TFIID; D014157:Transcription Factors / Q000096:biosynthesis / Q000378:metabolism*; D015533:Transcriptional Activation
D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D035181:TATA-Box Binding Protein; D035561:Transcription Factor TFIIA; D035362:Transcription Factor TFIID; D014157:Transcription Factors; D005982:Glutathione Transferase; D000409:Alanine