The cheA locus of Escherichia coli consists of two overlapping genes, one of the few examples of overlapping genes in a non-viral system. One goal of this work is to learn more about the role of overlapping genes in bacteria - how they evolved and how their expression is regulated. In the case of cheA, which is involved in chemotactic behavior, the two genes are known to be in the same reading frame, but initiate translation at different start points. Mutations affecting initiation efficiency at either of these start sites will be isolated with the aid of cheA-lacZ gene fusions in which a functional Beta-galactosidase gene is translationally joined to the carboxyl coding region of the cheA locus. These fusions synthesize two large hybrid proteins with Beta-galactosidase activity and will be used to select mutants with increased or decreased enzyme levels due to start site alterations. Mutants altered at the internal start site, which should be incapable of synthesizing the smaller cheA product, will be examined for chemotactic ability to see whether the smaller protein is really needed in wild type E. coli. Other work dealing with the functional roles of the two cheA proteins will involve nucleotide sequence analysis of the cheA locus to determine the primary structures of both proteins, reversion analyses to identify other cell components with which the cheA products interact, and cell fractionation studies to see whether the two proteins are localized in different parts of the cell. The second major goal of this study is to exploit the unique features of the cheA system to learn more about the process of translation initiation. The initiation properties of start site mutants will be compared to their sequence alterations to identify the factors most critical to initiation.
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