TATA-binding protein-associated factors enhance the recruitment of RNA polymerase II by transcriptional activators.
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.
Wu
S Y
SY
Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4935, USA.
Chiang
C M
CM
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002474:Cell-Free System; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004789:Enzyme Activation; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007313:Insecta; D008954:Models, Biological; D008957:Models, Genetic; D027383:Papillomaviridae / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012319:RNA Polymerase II / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D035181:TATA-Box Binding Protein; D035362:Transcription Factor TFIID; D014157:Transcription Factors / Q000378:metabolism*; D019473:Transcription Factors, TFII / Q000378:metabolism; D015533:Transcriptional Activation
D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D035181:TATA-Box Binding Protein; D035362:Transcription Factor TFIID; D014157:Transcription Factors; D019473:Transcription Factors, TFII; D005982:Glutathione Transferase; D012319:RNA Polymerase II