Promoter-dependent photocross-linking of the acidic transcriptional activator E2F-1 to the TATA-binding protein.
Sequence-specific transcriptional activators, such as the human factor E2F-1, increase the rate of initiation of transcription by RNA polymerase II, possibly by contacting one or more of the RNA polymerase II-associated general initiation factors. One candidate target of transactivators is the TATA-binding protein (TBP), which, when bound to a promoter, nucleates the formation of a preinitiation complex. Previous studies using affinity chromatography techniques have shown that the activation domains of certain activators, including the acidic activation domain of E2F-1, can interact with TBP in the absence of DNA. Using a site-directed photoaffinity cross-linking approach, we demonstrate here that the activation domain of the chimeric activator LexA-E2F-1 can be cross-linked to TBP when both factors are bound to a transcriptionally responsive RNA polymerase II promoter. Mutations within the activation domain of LexA-E2F-1 that impaired its ability to activate transcription in vitro were found to reduce cross-linking of LexA-E2F-1 to TBP. The association of initiation factor TFIIB with the TBP-promoter complex did not preclude this promoter-dependent cross-linking to LexA-E2F-1; however, this cross-linking was promoter-independent. In contrast, TFIIA strongly inhibited the promoter-dependent cross-linking of LexA-E2F-1 to TBP. These results directly demonstrate that acidic activators such as E2F-1 can interact with TBP during the earliest stages in the assembly of an RNA polymerase II preinitiation complex.
Emili
A
A
Banting and Best Department of Medical Research, University of Toronto, Ontario, Canada.
Ingles
C J
CJ
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins / Q000378:metabolism*; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D008027:Light; D011401:Promoter Regions, Genetic; D056465:Retinoblastoma-Binding Protein 1; D016385:TATA Box; D035181:TATA-Box Binding Protein; D050680:Transcription Factor DP1; D035561:Transcription Factor TFIIA; D035581:Transcription Factor TFIIB; D014157:Transcription Factors / Q000378:metabolism* / Q000494:pharmacology; D015533:Transcriptional Activation
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D056465:Retinoblastoma-Binding Protein 1; D035181:TATA-Box Binding Protein; D050680:Transcription Factor DP1; D035561:Transcription Factor TFIIA; D035581:Transcription Factor TFIIB; D014157:Transcription Factors