Reconstitution of human TFIIA activity from recombinant polypeptides: a role in TFIID-mediated transcription.
Human TFIIA activity is composed of three subunits (alpha, beta, gamma). Here we report the isolation of a human cDNA clone encoding the gamma-subunit and the reconstitution of TFIIA activity from recombinant polypeptides (holo-TFIIA). Protein-protein interaction analysis established that the beta and gamma subunits of TFIIA interact with the TBP component of TFIID. The alpha-subunit is recruited into the complex by association with the gamma-subunit. Functional studies indicate that recombinant TFIIA stimulates basal TFIID-dependent transcription but is without effect on TBP-dependent transcription. Our studies indicate that TFIIA not only functions by physically removing negative components present in TFIID (antirepression), as demonstrated previously, but that it can stimulate basal transcription through components of the TFIID complex. Holo-TFIIA also stimulated activation of transcription in vitro as well as in vivo in transfected HeLa cells.
Howard Hughes Medical Institute, Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway 08854-5635.
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D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D035181:TATA-Box Binding Protein; D015534:Trans-Activators / Q000378:metabolism; D035561:Transcription Factor TFIIA; D035362:Transcription Factor TFIID; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C070633:Gal-VP16; D011994:Recombinant Proteins; D035181:TATA-Box Binding Protein; D015534:Trans-Activators; D035561:Transcription Factor TFIIA; D035362:Transcription Factor TFIID; D014157:Transcription Factors