The binding of the cyclic AMP receptor protein to synthetic DNA sites containing permutations in the consensus sequence TGTGA

Abstract

The binding of the cyclic AMP receptor protein (CRP) to symmetrical synthetic DNA-binding sites was investigated with a gel-retardation assay. A set of ten different sequences was employed, comprising all base permutations at positions 2, 4, and 5 of the consensus sequence 5′(TGTGA)3′. We show that: (i) CRP has a higher affinity for the completely symmetrical site than towards the lac wild-type site; (ii) base substitutions at position 2 lead to either a complete loss of specific CRP binding (G—C), a reduction in specific CRP binding (G—A) or only marginal effects on specific CRP binding (G—T); (iii) changes at position 4 abolish (G—C; G—A) or reduce (G—T) specific CRP binding; and (iv) base permutations at position 5 reduce specific CRP binding, but never completely abolish it. Thus position 4, and to a lesser extent position 2, in the DNA consensus sequence are the most crucial ones for specific binding by CRP.