Demonstration of two operator elements in gal: in vitro repressor binding studies.

Abstract

Genetic and DNA base sequence analyses of cis-dominant mutations that derepress the gal operon of Escherichia coli suggested the existence of 2 operator loci needed for gal repression. One (OE) is located immediately upstream to the 2 overlapping gal promoters and the other (OI) is inside the first structural gene. The ability of wild-type and mutant OE and OI DNA sequences to bind to gal repressor was studied. The repressor was purified from cells containing a multicopy plasmid in which the repressor gene is brought under the control of phage .lambda.PL promoter. The DNA-repressor interactions are detected by the change in electrophoretic mobility of labeled DNA that accompanies its complex formation with repressor protein. The purified repressor shows concentration-dependent binding to both .**GRAPHIC**. and .**GRAPHIC**. but not to .**GRAPHIC**. and .**GRAPHIC**. sequences. These results authenticate the proposed operator role of the 2 homologous gal DNA control elements and thereby establish that the negative control of the gal operon requires repressor binding at both OE and OI, which are separated by > 90 base pairs.