Use of a genetically introduced cross-linker to identify interaction sites of acidic activators within native transcription factor IID and SAGA.
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.
Klein
Joachim
J
Department of Microbiology, Goethe University, 60439 Frankfurt am Main, Germany.
Nolden
Mark
M
Sanders
Steven L
SL
Kirchner
Jay
J
Weil
P Anthony
PA
Melcher
Karsten
K
D000123:Acetyltransferases / Q000737:chemistry / Q000378:metabolism*; D020816:Amino Acid Motifs; D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D010450:Endopeptidases / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D051548:Histone Acetyltransferases; D006657:Histones; D008954:Models, Biological; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D035381:TATA-Binding Protein Associated Factors / Q000378:metabolism; D035362:Transcription Factor TFIID / Q000737:chemistry / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D015003:Yeasts
C090575:ADA2 protein, S cerevisiae; D003432:Cross-Linking Reagents; D006657:Histones; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C116141:TAF9 protein, S cerevisiae; D035381:TATA-Binding Protein Associated Factors; D035362:Transcription Factor TFIID; D014157:Transcription Factors; D006639:Histidine; D000123:Acetyltransferases; D051548:Histone Acetyltransferases; D010450:Endopeptidases; C099488:TEV protease