The structure of RNA polymerase is very similar from bacteria to man. This enzyme is responsible for the bulk of differential gene expression in all organisms. The properties of the large enzyme are modified by interactions with proteins and small molecule effectors. In this proposal a bacterial system is used to learn how the common core can have its properties changed. 3 sigma factors are used, the main housekeeping sigma70, sigma38 and sigma54, each of which causes the core to assume a different mechanism of action. Sigma38 causes the core to respond uniquely to several small molecule effectors. Sigma54 causes the core to respond to enhancer binding proteins. Sigma70 induces neither of these responses. A combination of genetic screens, band shift assays and footprinting analysis will be used to investigate this problem. Screens are devised to identify the parts of sigmas that are involved in these responses. The biochemical assays are used on the mutants obtained to learn why they are no longer able to alter the behavior of the core RNA polymerase. These mutants will be mapped on the known high resolution structures. The outcome is expected to be a description of the 3-dimensional domains on sigma that are critical for changing how the core polymerase works and new knowledge about how this happens. Because the core polymerase core is so conserved we expect that its manner of response will also be conserved, enlarging the possibilities for understanding the basis for human diseases of gene expression. ? ?
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