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, the investigator is only beginning to get a picture of the multiple transcription factors and how they activate and repress specific transcription. Very little is presently known about the details of the subunit interactions within RNA polymerase II (RNAP II) itself or how these subunits interact with and are modulated by the numerous transcription factors. The goal is to understand the structure, function, and regulation of the eukaryotic transcription machinery, with an emphasis on RNAP II. Dr. Burgess will study the RNAP II subunit organization and architecture and locate sites of interaction with general transcription factors both in solution and in pre-initiation complexes formed on a minimal promoter system. These studies build on experience and progress in RNA polymerase and transcription factor purification, and in the preparation and use of MAbs to quantify and purify proteins and to inhibit specific functional interactions. New techniques, recently developed in the principal investigator's laboratory, will allow him to rapidly map interaction domains on RNAP II subunits and factors. He has recently developed a versatile system for expressing yeast RNAP II subunits in yeast, that now allows to ask detailed questions about RNAP II assembly and function in vivo. Excellent progress in his collaboration with Dr. Roger Kornberg on the 3-D crystal structure of yeast RNAP II provides with a strong framework upon which to place the subunit and factor interaction mapping results. Dr. Burgess proposes to continue the most promising work from his previous grant period and to undertake a new project in collaboration with Dr. Peggy Farnham, who shares the floor of McArdle, to study the interaction of the activation domain (AD) of hun1an transcription factor E2F 1 with the rest of the transcription machinery and, using single chain antibodies to E2Fl AD, to interfere with its function in vitro and in vivo. A long-term goal of this research is to use the detailed knowledge of specific polymerase/transcription factor interactions to design agents that interfere with abnormal regulatory interactions crucial to maintaining the neoplastic state.
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