TAFII-independent activation mediated by human TBP in the presence of the positive cofactor PC4.
TFIID is a multiprotein complex comprised of the TATA-binding protein (TBP) and an array of TBP-associated factors (TAFIIs). Whereas TBP is sufficient for basal transcription in conjunction with other general transcription factors and RNA polymerase II, TAFIIs are additionally required for activator-dependent transcription in mammalian cell-free transcription systems. However, recent in vivo studies carried out in yeast suggest that TAFIIs are not globally required for activator function. The discrepancy between in vivo yeast studies and in vitro mammalian cell-free systems remains to be resolved. In this study, we describe a mammalian cell-free transcription system reconstituted with only recombinant proteins and epitope-tagged multiprotein complexes. Transcriptional activation can be recapitulated in this highly purified in vitro transcription system in the absence of TAFIIs. This TBP-mediated activation is not induced by human mediator, another transcriptional coactivator complex potentially implicated in activator response. In contrast, general transcription factors TFIIH and TFIIA play a significant role in TBP-mediated activation, which can be detected in vitro with Gal4 fusion proteins containing various transcriptional activation domains. Our data, therefore, suggest that TFIIH and TFIIA can mediate activator function in the absence of TAFIIs.
Department of Biochemistry, University of Illinois, Urbana 61801, USA.
S Y
SY
Wu
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Kershnar
C M
CM
Chiang
D002458:Cell Fractionation; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D004357:Drug Synergism; D005656:Fungal Proteins / Q000502:physiology; D006367:HeLa Cells; D017405:Herpes Simplex Virus Protein Vmw65 / Q000502:physiology; D006801:Humans; D008565:Membrane Proteins / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000502:physiology; D012045:Regulatory Sequences, Nucleic Acid; D016385:TATA Box / Q000502:physiology*; D035181:TATA-Box Binding Protein; D015534:Trans-Activators / Q000502:physiology; D035561:Transcription Factor TFIIA; D035362:Transcription Factor TFIID; D051758:Transcription Factor TFIIH; D014157:Transcription Factors / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology*; D019473:Transcription Factors, TFII / Q000378:metabolism / Q000502:physiology; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C070633:Gal-VP16; D017405:Herpes Simplex Virus Protein Vmw65; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; C509201:SUB1 protein, human; D035181:TATA-Box Binding Protein; D015534:Trans-Activators; D035561:Transcription Factor TFIIA; D035362:Transcription Factor TFIID; D014157:Transcription Factors; D019473:Transcription Factors, TFII; C066644:transcription factor TFIIE; D051758:Transcription Factor TFIIH; C056604:transcription factor TFIIF