Abstract
An important goal is to identify the direct activation domain (AD)-interacting components of the transcriptional machinery within the context of native complexes. Toward this end, we first demonstrate that the multisubunit TFIID, SAGA, mediator, and Swi/Snf coactivator complexes from transcriptionally competent whole-cell yeast extracts were all capable of specifically interacting with the prototypic acidic ADs of Gal4 and VP16. We then used hexahistidine tags as genetically introduced activation domain-localized cross-linking receptors. In combination with immunological reagents against all subunits of TFIID and SAGA, we systematically identified the direct AD-interacting subunits within the AD-TFIID and AD-SAGA coactivator complexes enriched from whole-cell extracts and confirmed these results using purified TFIID and partially purified SAGA. Both ADs directly cross-linked to TBP and to a subset of TFIID and SAGA subunits that carry histone-fold motifs.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Acetyltransferases / chemistry
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Acetyltransferases / metabolism*
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Amino Acid Motifs
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Binding Sites
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Cross-Linking Reagents / pharmacology*
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Endopeptidases / metabolism
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Histidine / chemistry
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Histone Acetyltransferases
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Histones
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Models, Biological
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Protein Binding
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Protein Folding
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Protein Structure, Tertiary
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Recombinant Fusion Proteins / metabolism
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Recombinant Proteins / metabolism
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Saccharomyces cerevisiae Proteins / chemistry
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Saccharomyces cerevisiae Proteins / metabolism*
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TATA-Binding Protein Associated Factors / metabolism
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Transcription Factor TFIID / chemistry
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Transcription Factor TFIID / metabolism*
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Transcription Factors / metabolism
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Transcription, Genetic
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Yeasts
Substances
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ADA2 protein, S cerevisiae
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Cross-Linking Reagents
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Histones
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Recombinant Fusion Proteins
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Recombinant Proteins
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Saccharomyces cerevisiae Proteins
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TAF9 protein, S cerevisiae
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TATA-Binding Protein Associated Factors
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Transcription Factor TFIID
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Transcription Factors
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Histidine
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Acetyltransferases
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Histone Acetyltransferases
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Endopeptidases
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TEV protease