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 polyme rase II, possibly by contacting one or more of the RNA polymerase II-a ssociated general initiation factors. One candidate target of transact ivators is the TATA-binding protein (TBP), which, when bound to a prom oter, nucleates the formation of a preinitiation complex. Previous stu dies using affinity chromatography techniques have shown that the acti vation domains of certain activators, including the acidic activation domain of E2F-1, can interact with TBP in the absence of DNA Using a s ite directed photoaffinity cross-linking approach, we demonstrate here that the activation domain of the chimeric activator LexA-E2F-1 can b e cross-linked to TBP when both factors are bound to a transcriptional ly responsive RNA polymerase II promoter. Mutations within the activat ion domain of LexA-E2F-1 that impaired its ability to activate transcr iption in vitro were found to reduce cross-linking of LexA-E2F-1 to TB P. The association of initiation factor TFIIB with the TBP-promoter co mplex did not preclude this promoter-dependent cross-linking of LexA-E 2F-1 to TBP. TFIIB itself could also be crosslinked to LexA-E2F-1; how ever, 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 asse mbly of an RNA polymerase II preinitiation complex.