Distinct domains of hTAFII100 are required for functional interaction with transcription factor TFIIF beta (RAP30) and incorporation into the TFIID complex.
TFIID is the DNA binding component of the RNA polymerase II transcriptional machinery and is composed of the TATA binding protein (TBP) and TBP-associated factors (TAFIIs). Here we report the characterization of a new human TAF, hTAFII100, which is the human homologue of Drosophila TAFII80 and yeast TAFII90. hTAFII100 interacts strongly with hTAFII250, hTAFII55 and hTAFII28, less with hTAFII20 and hTAFII18, weakly with TBP and not at all with delta NTAFII135 and hTAFII30. Deletion analysis revealed that the C-terminal half of hTAFII100, which contains six WD-40 repeats, is not required for incorporation into the TFIID complex. Our results suggest that hTAFII100 can be divided into two domains, the N-terminal region responsible for interactions within the TFIID complex and the C-terminal WD repeat-containing half responsible for interactions between hTAFII100 and other factors. An anti-hTAFII100 antibody, raised against a C-terminal epitope, selectively inhibited basal TFIID-dependent in vitro transcription and the specific interaction between hTAFII100 and the 30 kDa subunit of TFIIF (RAP30). We demonstrate that the hTAFII100-TFIIF interaction supports pre-initiation complex formation in the presence of TFIID. Thus, this is the first demonstration that a TAFII functionally interacts with a basal transcription factor in vitro.
Dubrovskaya
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Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), CNRS/INSERM/ULP, Illkirch, Strasbourg, France.
Lavigne
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AC
Davidson
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Acker
J
J
Staub
A
A
Tora
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L
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D018411:Spodoptera / Q000166:cytology; D035381:TATA-Binding Protein Associated Factors; D035362:Transcription Factor TFIID; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019473:Transcription Factors, TFII
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D004268:DNA-Binding Proteins; C100838:TAF5 protein, human; D035381:TATA-Binding Protein Associated Factors; D035362:Transcription Factor TFIID; D014157:Transcription Factors; D019473:Transcription Factors, TFII; C056604:transcription factor TFIIF