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

Klein J, Nolden M, Sanders SL, Kirchner J, Weil PA, Melcher K
J Biol Chem. 2003 278 (9): 6779-86

PMID: 12501245 · DOI:10.1074/jbc.M212514200

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.

MeSH Terms (20)

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

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