Regulation of gene expression at the level of transcription is one of the major means of regulating cell growth and differentiation. Abnormal transcriptional regulation plays an important role in neoplastic transformation. Despite a great amount of research activity on the mechanisms of transcriptional regulation, we are only beginning to get a picture of the multiple transcription factors and how they promote, activate, and repress specific transcription. Very little is presently known about the details of the subunit interactions within RNA polymerase II itself of how these subunits interact with and are modulated by the numerous transcription factors. We will concentrate on the structure, function and regulation of the transcription machinery, with an emphasis on RNA polymerase II. We will study the RNA polymerase II subunit organization: by identifying stable subassemblies, by protein-protein crosslinking, and by collaborating on the 3-D structure determination of the yeast enzyme. We will determine which RNA polymerase II subunits are involved in interactions with the general transcription factors both in factor-polymerase complexes in solution and in reinitiation complexes formed on a minimal promoter system (involving the human IgH promoter, RNA polymerase II and the most essential general transcription factors TBP, TFIIB, and RAP30). We will extend our recent results on the importance of the C-terminal domain of the largest subunit of RNA polymerase II and determine the factors that interact with this domain. While we are ultimately interested in human transcriptional regulation, we will also work extensively with yeast because of this superior genetics and its abundance and ability to substitute for human polymerase during transcription in vitro in extracts and purified systems. This work will build on our strengths, experience, and previous work on the identification, purification, and characterization of RNA polymerases and transcription factors. We will rely heavily on protein-protein and protein-DNA crosslinking; on the use of monoclonal antibodies to detect, inhibit, and immunopurify parts of the transcription apparatus; and on improved methods for efficient renaturation of proteins eluted from gels, blotted onto membranes, or from solubilized inclusion bodies. We are convinced that a basic, thorough study of RNA polymerase II structure and key interactions with transcription factors and DNA will provide major new insights into the mechanism of specific transcription and how that transcription is controlled. A long-term goal of this research is to use our detailed knowledge of specific polymerase- transcription factor interactions to design agents that interfere wit abnormal regulatory interactions crucial to maintaining the neoplastic state.
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