A Lethal Mutant of the Catabolite Gene Activator Protein CAP ofEscherichia coli

The dimeric catabolite gene activator protein (CAP) ofEscherichia coliuses its recognition helix to bind with each subunit the DNA sequence motif 5′ G_-7T_-6G_-5A_-4 3′. It makes a direct amino acid-base contact with E181 and cytosine in position -5′ on the reverse strand. While testing mutants of CAP in position 181 for specificity changes, we found that CAP E181Q is lethal in high amounts for theE. colistrains we used for cloning. We cloned this CAP mutant successfully incya^-strains, where CAP is inactive. Examination of thein vitrobinding activities of CAP E181Q, and ofin vivoactivity when present in low, non-lethal amounts, revealed loss of specificity but not of binding capacity for its DNA targets. It binds well to CAP consensus with G or T in position -5, better to CAP consensus with A, C in position -5, quite well to λ consensus operator with G in position -7 and rather weakly to λ consensus.