The broad, long range goal of this proposal is to learn how genes are controlled by remotely bound activator proteins. This is a problem of great significance since the transcription of most genes in human cells is controlled in this manner. These genes include oncogenes and those controlling the production of other proteins whose functions are critical for the health of the cells and the organism. This process is of such complexity and interest that an enormous scientific effort is currently being applied to its solution. However, the recent discovery of an analogous process in bacterial cells provides entry into studying the problem in a much simpler and more accessible system. A rare bacterial protein, sigma 54, resembles closely analogous proteins in mammalian cells and is required to mediate the control from distant DNA sites in E. Coli. The mechanism of action of sigma 54 will be determined in this proposal. the entire length of the protein will be covered by mutations and the mutant proteins expressed from plasmids in cells lacking the wild-type protein. In vivo probing, genetic screens, and selected in vitro experiments will be applied to learn the roles of each part of the protein in mediating this long range control. The way in which these domains interact with each other and with the DNA and the other components of the transcription apparatus will be determined. New methods of in vivo probing will be developed in order to speed and extend this work. It is expected that the analysis of this simple system will precede analysis of more complex systems and thus help guide the tremendous and important ongoing effort to learn the role of oncogene control proteins and other analogous factors in mammalian cells.
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