A lethal mutant of the catabolite gene activator protein CAP of Escherichia coli

The dimeric catabolite gene activator protein (CAP) of Escherichia coli uses 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 the E. coli strains we used for cloning. We cloned this CAP mutant successfully in cya- strains, where CAP is inactive. Examination of the in vitro binding activities of CAP E181Q, and of in vivo activity 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 lambda consensus operator with G in position-7 and rather weakly to lambda consensus.