Use of a genetically introduced cross-linker to identify interaction sites of acidic activators within native transcription factor IID and SAGA

J Biol Chem. 2003 Feb 28;278(9):6779-86. doi: 10.1074/jbc.M212514200. Epub 2002 Dec 24.


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

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Acetyltransferases / chemistry
  • Acetyltransferases / metabolism*
  • Amino Acid Motifs
  • Binding Sites
  • Cross-Linking Reagents / pharmacology*
  • Endopeptidases / metabolism
  • Histidine / chemistry
  • Histone Acetyltransferases
  • Histones
  • Models, Biological
  • Protein Binding
  • Protein Folding
  • Protein Structure, Tertiary
  • Recombinant Fusion Proteins / metabolism
  • Recombinant Proteins / metabolism
  • Saccharomyces cerevisiae Proteins / chemistry
  • Saccharomyces cerevisiae Proteins / metabolism*
  • TATA-Binding Protein Associated Factors / metabolism
  • Transcription Factor TFIID / chemistry
  • Transcription Factor TFIID / metabolism*
  • Transcription Factors / metabolism
  • Transcription, Genetic
  • Yeasts


  • ADA2 protein, S cerevisiae
  • Cross-Linking Reagents
  • Histones
  • Recombinant Fusion Proteins
  • Recombinant Proteins
  • Saccharomyces cerevisiae Proteins
  • TAF9 protein, S cerevisiae
  • TATA-Binding Protein Associated Factors
  • Transcription Factor TFIID
  • Transcription Factors
  • Histidine
  • Acetyltransferases
  • Histone Acetyltransferases
  • Endopeptidases
  • TEV protease