Simian virus 40 large T antigen is a promiscuous activator of many viral and cellular promoters. A very simple promoter structure is required for transcriptional activation: a TATA or initiator element with an upstream site for a sequence-specific promoter binding factor. Our data suggest that T antigen-mediated transcriptional activation of these simple promoters occurs through direct protein-protein interactions with athe transcription complex on the promoter. Large T antigen can interact with both sequence-specific promoter binding factors and the basal transcription complex, most notably with the TATA binding protein (TBP) and other components of TFIID. However, this interaction with TFIID alone does not cause activation; large T antigen cannot activate a promoter containing only a TATA element, the presence of the upstream bound factor is essential. T antigen appears to facilitate interactions between the upstream bound sequence-specific promoter factor and TFIID. This model suggests that T antigen functions in a manner similar to the TBP associated factors (TAFs), cellular proteins which associate with TBP to form TFIID and provide the communication between sequence-specific promoter factors and the basal transcriptional complex to activate transcription. In addition, our recent experiments suggest that a transactivating protein of the human cytomegalovirus (HCMV), the 86 kDa immediate early protein (IEP559aa: ppUL122a), functions similarly to T antigen in transcriptional activation. We have initiated experiments using the HCMV immediate early proteins in parallel studies with T antigen.
The specific aims of this proposal are to understand athe transcriptional activation mechanisms of these viral activator proteins.
in aim 1 the nature of interactions between the viral activator proteins and components of the basal transcription apparatus will be characterized in vitro and in vivo. These data will be correlated with the experiments of aim 2 which will examine the effects of the interactions of the viral activators on TFIID function.
In aim 3 the ability of purified recombinant viral activator proteins to mediate activated in vitro transcription will be examined using partial TFIID complexes constructed from purified recombinant TAFs and TBP. We will determine the minimal complex required and whether viral activator proteins can substitute for specific TAFs.
In aim 4 the interaction between the viral activators and sequence-specific promoter factors will be characterized to determine how this may correlate with basal transcription complex interactions and activation.
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