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J Mol Biol. 1993 Dec 5;234(3):542-53.

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

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Department of Biological Sciences, State University of New York at Buffalo 14260.


The 434 repressor binds more tightly to OR1 than it does to OR3. 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 OR1 and OR3 differs only by an A-->G substitution at position 4 in one half-site of OR3, 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 OR1 have a greater effect on operator affinity for 434 repressor than do similar changes in OR3. Although OR1 and OR3 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 OR1, bearing an A.T-->G.C change, and OR3, 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.

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