MOLECULAR BASIS OF CONTROL IN THE L-ARABINOSE OPERON

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Genetic and biochemical experiments are described which will analyze the molecular interactions that control expression of the L-arabinose operon in Escherichia coli. The araBAD operon and araC regulatory gene promoters will be saturated with mutants using in vitro mutagenesis. The methods used will include (1) synthetic oligonucleotide-directed mutagenesis, (2) heavy mutagenesis with hydroxylamine, and (3) site-directed chemical modification. Mutations created in vitro will be transferred to the chromosome for analysis of their effect on expression of the ara genes. The possible functional significance of a putative polypeptide coded for by araC leader mRNA will be assessed. A comparison will be made at the DNA sequence level of ara promoters and the araC gene from other Enterobacteriaceae. The interaction of small molecules with purified araC regulatory protein will be studied by spectrofluorimetric methods. Finally, an attempt will be made to determine the 3-dimensional structure of the araC regulatory protein with and without L-arabinose and araC protein complexed with araI and araO DNA.