Characterization of the interaction of the glp repressor of Escherichia coli K-12 with single and tandem glp operator variants.

The glp operons of Escherichia coli are negatively controlled by the glp repressor. Comparison of the repressor-binding affinities for consensus and altered consensus operators in vivo showed that all base substitutions at positions 3, 4, 5, and 8 from the center of the palindromic operator caused a striking decrease in repressor binding. Substitutions at other positions had a severe to no effect on repressor binding, depending on the base substitution. The results obtained indicate that the repressor binds with highest affinity to operators with the half-site WATKYTCGWW, where W is A or T, K is G or T, and Y is C or T. Strong cooperative binding of the repressor to tandem operators was demonstrated in vivo. Cooperativity was maximal when two 20-bp operators were directly repeated or when 2 bp separated the two operators. Cooperativity decreased with the deletion of 2 bp or the addition of 4 bp between the individual operators. Cooperativity was eliminated with a 6-bp insertion between the operators.