Adenoviruses provide a useful model to study transcriptional regulation and cellular transformation. Our long-term objectives are to understand the biochemical mechanisms involved in transcriptional regulation in higher eukaryotes and how the adenovirus transcriptional regulatory protein, Ela, interacts with the cellular transcription machinery to regulate viral gene expression during permissive infection and contribute to the transformation of non-permissive cells. To pursue these objectives we propose a comprehensive set of genetic and biochemical experiments utilizing in vivo and in vitro approaches. First, we will continue to construct mutations within the Ela protein coding region in order to delineate the protein's functional domains, define the interrelationship between various Ela activities, and provide valuable reagents for biochemical studies. The transcriptional regulatory effects of Ela may result from the interaction of Ela proteins with cellular transcription factors. Normal and mutant Ela proteins will be used to identify and study cellular proteins that are the probable targets of Ela transcriptional regulatory effects. The mechanisms of transcriptional activation by cis-acting enhancer elements and trans-acting Ela proteins will be pursued using as a model the adenovirus E4 promoter. This promoter has extraordinarily high in vitro transcription activity due in large part to an upstream enhancer element, which functions in vitro. The enhancer function involves a sequence-specific interaction with a cellular transcription factor. The interaction of this factor (and other factors) with the E4 promoter will be studied in detail and several strategies are proposed to clone the cellular gene encoding the factor. In addition, the E4 system will be used to directly test specific models of Ela transcriptional activation. Ultimately, we hope to develop in vitro systems that faithfully carry out these Ela-mediated transcriptional regulatory function thus allowing the biochemical mechanisms involved in Ela transcription regulation to be addressed.
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