Operator sequence context influences amino acid-base-pair interactions in 434 repressor-operator complexes

The 434 repressor binds more tightly to O_R1 than it does to O_R3. The repressor makes several specific contacts with the symmetrically arrayed outer four base-pairs of the 14 base-pair site, and no specific contacts to the central six base-pairs. The sequence of the outer base-pairs of O_R1 and O_R3 differs only by an A→G substitution at position 4 in one half-site of O_R3, while that of central bases is very different. As expected from sequence analysis of wild-type operators, the data show repressor prefers an A·T base-pair at position 4. The magnitude of this preference depends on operator sequence context and solution conditions. Position 4 changes in the context of O_R1 have a greater effect on operator affinity for 434 repressor than do similar changes in O_R3. Although O_R1 and O_R3 display different affinities for 434 repressor, their repressor-operator complexes are similarly insensitive to changes in salt concentration and temperature. By contrast, complexes formed between repressor and position 4 mutant O_R1, bearing an A·T→G·C change, and O_R3, which bears a G·C→A·T change, are affected greatly, and to similar extents, by changes in ionic strength and temperature. Nuclease protection experiments show that 434 repressor protects the DNA phosphate backbone of wild-type operators from cleavage more efficiently than those of mutant operators. These data show that the biochemical and structural properties of a repressor-operator complex, while affected by position 4 base sequence, are independent of the identity of this base. The ability of repressor to recognize the base at position 4 depends on the sequence context at operator positions 5 to 7. Apparently there is an interplay between the bases at operator positions 4 to 7 which has a global effect on the structure of the repressor-operator complex.