The broad, long-term goal of this project is to understand the functional significance of interactions between promoter specific activators and the general transcription factors in eukaryotes. The Epstein-Barr virus lytic activator, Zta, is a model activator protein that clearly interacts with the general transcription factors TFIIA and TFIID to form a stable preinitiation complex, referred to as Z-D-A. TFIIA is one of several RNA polymerase II accessory proteins essential for activator regulated transcription. However, the fundamental role of TFIIA in activator regulated transcription remains poorly understood. The formation of the Z- D-A complex is dependent on the Zta activation domain, the TAFs in the TFIID fraction, and the number of Zta binding sites in the promoter. These requirements strongly support the functional significance of the Z-D-A complex. However, not all transcriptional activators stimulate a TFIIA- TFIID complex. To investigate the significance of this activator function, we have constructed specific amino acid substitution mutations in the Zta activation domain that fail to stimulate D-A formation, yet are only conditionally defective for transcriptional activation. Preliminary evidence suggests that Z-D-A complex formation is required only under conditions where TFIIA and TFIID binding is rate limiting. The biological significance of this specialized activator function remains unclear. Biologically, Zta functions to disrupt the transcriptional silence of the latent Epstein-Barr virus genome. We hypothesize that Zta has evolved a unique capacity to recruit TFIIA to overcome conditions which maintain transcriptional silence of the latent viral genome. In this proposal, we plan to refine our knowledge of the intermolecular contacts that underpin the Z-D-A complex, and to further investigate the role of TFIIA and the TAFs in mediating activator function. Furthermore, we attempt to identify core promoter elements and cell growth conditions which make TFIIA and TFIID binding rate limiting. These investigations promise to provide general information concerning the role of TFIIA in eukaryotic transcriptional activation mechanisms, to provide molecular details of a unique virus-host interaction, and to correlate the biochemical activity of an activator with a defined biological function.
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