Transcription factor (TF) IID, comprised of the TATA-binding protein (TBP) and TBP-associated factors (TAFs), is a general transcription factor required for RNA polymerase II (pol II) transcription on most eukaryotic genes. Recent findings that TAFs may not be globally required for activator-dependent transcription in vivo and in vitro and that both TAF-dependent and TAF-independent promoters are found in yeast suggest that transcriptional activation can occur through at least two different pathways, depending on the presence or absence of TAFs. Using order-of-addition and template challenge assays performed in a human cell-free transcription system reconstituted with recombinant general transcription factors (TFIIB, TBP, TFIIE, TFIIF), a recombinant general cofactor (PC4), and highly purified epitope-tagged multiprotein complexes (TFIID, TFIIH, pol II), we demonstrate that when TBP is used as the TATA-binding factor transcriptional activators such as Gal4-VP16 and human papillomavirus E2 mainly function by facilitating pol II entry to the promoter region. In contrast, when TFIID is used as the TATA-binding factor, promoter recognition by TFIID appears to be the rate-limiting step facilitated by transcriptional activators during preinitiation complex assembly. Using protein-protein pull-down and far-Western analyses, we further show that the presence of TAFs in TFIID facilitates the recruitment of pol II by transcriptional activators, thereby switching the rate-limiting step from pol II entry to promoter recognition. Our findings thus provide distinct molecular mechanisms for TAF-independent and TAF-dependent activation.
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