The yeast general transcription factor TFIIA is composed of two polypeptide subunits.
The general transcription factor TFIIA was purified from yeast. A key step in the purification was affinity chromatography using a column containing the adenovirus major late promoter with bound recombinant TFIID to which TFIIA binds with high affinity. TFIIA activity copurifies with two polypeptides of molecular mass 32 and 13.5 kDa. Elution and renaturation of these two polypeptides from sodium dodecyl sulfate-polyacrylamide gels showed that both polypeptides were required for TFIIA activity. TFIIA activity was measured by both a native gel shift assay and by in vitro complementation of transcription using yeast nuclear extracts depleted of TFIIA. The purified renatured yeast TFIIA also complements basal level transcription using a mammalian transcription system depleted of TFIIA. Native TFIIA has an apparent molecular mass of approximately 90 kDa measured by gel filtration chromatography. TFIIA binds to a TFIID.TATA element.DNA complex with an apparent equilibrium dissociation constant (KD) of 20 pM. This affinity is about 100-fold greater than the affinity of TFIID for TATA elements and much greater than the affinity of TFIIA for TFIID not bound to DNA.
Fred Hutchinson Cancer Research Center, Seattle, Washington 98104.
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D002467:Cell Nucleus / Q000378:metabolism; D002850:Chromatography, Gel; D004271:DNA, Fungal / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D010455:Peptides / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D035561:Transcription Factor TFIIA; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic
D004271:DNA, Fungal; D010455:Peptides; D035561:Transcription Factor TFIIA; D014157:Transcription Factors