The LexA repressor and its isolated amino-terminal domain interact cooperatively with poly[d(A-T)], a contiguous pseudo-operator, but not with random DNA: a circular dichroism study.

The interaction of the entire LexA repressor and its amino-terminal DNA binding domain with poly[d(A-T)] and random DNA has been studied by circular dichroism. Binding of both protein species induces an about 2-fold increase of the positive circular dichroism band at about 270 nm of both polynucleotides, allowing a precise determination of the principal parameters as a function of mono- and divalent salt concentration and pH. Both proteins interact much more strongly (about 2000-fold) with poly[d(A-T)] than with random DNA as expected from the homology with the specific consensus binding site of LexA (CTGTATATATATACAG). For both LexA and its DNA binding domain we find that the interaction with poly[d(A-T)] is cooperative with a cooperativity factor omega of about 50-70 for both proteins over a wide range of solvent conditions, suggesting that the carboxy-terminal domain of LexA is not involved in this type of cooperativity. On the contrary, no cooperativity could be detected for the interaction of the LexA DNA binding domain with a random DNA fragment. The overall binding constant K omega (or simply K in the case of random DNA) depends strongly on the salt concentration as observed for most protein-DNA interactions, but the behavior of LexA is unusual in that the steepness of this salt dependence (delta log K omega/delta log [NaCl]) is much more pronounced at slightly acidic pH values as compared to that at neutral or slightly alkaline pH. The behavior is not easily understood in terms of the current theories on the electrostatic contribution to protein-DNA interactions on the basis of polyelectrolyte theory. A comparison of the overall binding constant K omega of the entire LexA repressor and its DNA binding domain reveals that LexA binds only 20-50-fold stronger under a wide variety of salt and pH conditions. This result tends to demonstrate further that the additional energy due to the dimerization of LexA via the carboxy-terminal domain should be rather weak as expected from the small dimerization constant of LexA (2 X 10(-4) M-1).