Abstract
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.
Highlights
Eukaryotic transcriptional activators stimulate the assembly of general transcription factors into a stable preinitiation complex at the start site of class II promoters [1, 2]
transcription factor IID (TFIID) consists of TATA binding protein (TBP) and TBPassociated factors (TAFs), which are essential for the reconsti
Several experimental approaches indicate that binding of TBP to the promoter is rate-limiting in vivo and that activators that interact with TBP stimulate this step in preinitiation complex formation [17,18,19,20,21,22]
Summary
TFIIA-␥ Antibodies (Abs) and Immunoblots—For Ab production, TFIIA-␥ was induced from either the pQE-IIA-␥ or pRSET A-IIA-␥ (see below) constructs in Escherichia coli and purified using Ni-NTA agarose chromatography as described [27]. Plasmid Constructs—The expression construct for human WT TFIIA-␥ (pRSET A-IIA-␥) was created by PCR amplification of the TFIIA-␥ cDNA with a BamHI restriction site immediately preceding the initiation codon and a HindIII restriction site immediately following the termination codon. After digestion, this fragment was cloned into the BamHI and HindIII sites of pRSET A (Invitrogen). In vitro transcription reactions contained 100 ng of DNA template, ϳ100 –200 ng of purified recombinant activator protein, and 40 g of HeLa nuclear extract in a 50-l final reaction volume incubated for 1 h at 30 °C. Purified WT protein expressed from either pQE-IIA-␥ or pRSET A-IIA-␥ has identical in vitro transcription activity (data not shown). Quantitation of the transcription signals was performed on a PhosphorImager 445 SI (Molecular Dynamics) and visualized by autoradiography on X-Omat-AR film (Kodak)
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