The promoter and regulatory region of the ilvlH operon of Escherichia coli K-12 has three interesting features: dependence of expression upon relatively distant upstream sequences, high- affinity binding to a rare E. coli protein, and anomalous migration during electrophoresis. Preliminary experiments indicate that some unusual structural feature underlies the phenomenon of anomalous migration. The goals of this research are to investigate these features and possible relationships between them. At present, there are few examples of DNA regions having an unusual structural feature of the type manifested by the ilvlH regulatory region and almost nothing is known about the underlying basis for such unusual structural features and whether gene expression is influenced by them. Protection and binding interference experiments will be carried out to define sites at which binding occurs. In addition, the size and subunit composition of the binding protein will be determined and the binding constant and stoichiometry of binding will be measured. Mutations will be made within the ilvlH promoter and regulatory region using site-directed procedures and the resulting mutations will be analyzed for their effects upon in vivo expression of the operon, in vitro transcription, binding, and anomalous migration. In addition, mutations within the locus that codes for the binding protein will be isolated to determine the effect that null alleles have upon expression of the ilvlH operon. Finally, experiments will be carried out to investigate the basis for anomalous migration. The overall goals of this research are to understand how DNA structure affects gene expression. Advances in medicine depend upon such basic information.
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