Coactivators for a proline-rich activator purified from the multisubunit human TFIID complex.
The mechanisms of transcriptional activation directed by sequence-specific regulators is central to understanding gene regulation. Here, we report the isolation of coactivators responsible for mediating transcriptional activation by Gal4-Pro, a hybrid regulator containing the proline-rich activation domain of human CTF/NFI. Chromatographic studies indicate that endogenous human TFIID consists of a multisubunit complex containing the TATA-binding protein (TBP), coactivators, and other associated factors. A fraction containing the coactivator activity was separated from the endogenous TBP after disrupting the tightly associated complex with urea. The urea-purified TBP was active for basal level transcription but no longer could support activation by Gal4-Pro. However, when the two separated components were added together, activated levels of transcription were restored in the presence of Gal4-Pro. Immunoaffinity purification of the TFIID complex identifies several polypeptides specifically associated with the endogenous TBP, some or all of which function as coactivators when reconstituted with Gal4-Pro. The isolated coactivators also mediate activation by a chimeric glutamine-rich activator derived from Sp1 but not the Gal4-VP16 activator, suggesting distinct factor requirements for different types of transcriptional regulators.
Tanese
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N
Howard Hughes Medical Institute, Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Pugh
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BF
Tjian
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R
D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D011233:Precipitin Tests; D055232:Proline-Rich Protein Domains; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D016385:TATA Box; D035362:Transcription Factor TFIID; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D010455:Peptides; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D035362:Transcription Factor TFIID; D014157:Transcription Factors