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The C repressor protein of phage 16-3, which is required for establishing and maintaining lysogeny, recognizes structurally different operators which differ by 2 bp in the length of the spacer between the conserved palindromic sequences. A 'rotationally flexible protein homodimers' model has been proposed in order to explain the conformational adaptivity of the 16-3 repressor. In this paper, we report on the isolation of a repressor mutant with altered binding specificity which was used to identify a residue-base pair contact and to monitor the spatial relationship of the recognition helix of C repressor to the contacting major groove of DNA within the two kinds of repressor-operator complexes. Our results indicate spatial differences at the interface which may reflect different docking arrangements in recognition of the structurally different operators by the 16-3 repressor.

Prokaryotic repressor-operator systems provide exemplars for the sequence-specific interactions between DNA and protein. The crucial atomic contacts of the two macromolecules are attained in a compact, geometrically defined structure of the DNA-protein complex. The pitch of the DNA interface seems an especially sensitive part of this architecture because changes in its length introduce new spacing and rotational relations in one step. We discovered a natural system that may serve as a model for investigating this problem: the repressor of the 16-3 phage of Rhizobium meliloti (helix-turn-helix class protein) possesses inherent ability to accommodate to various DNA twistings. It binds the cognate operators, which are 5'-ACAA-4 bp-TTGT-3' ([O.sub.L]) and 5'-ACAA-6 bp-TTGT-3' ([O.sub.R]) and thus differ 2 bp in length, and consequently the two half-sites will be rotated with respect to each other by 72[degrees] in the idealized B-DNA (64[degrees] by dinucleotide steps calculations). Furthermore, a synthetic intermediate (DNA sequence) 5'-ACAA-5 bp-TTGT-3' ([O.sub.5]) also binds specifically the repressor. The natural operators and bound repressors can form higher order DNA-protein complexes and perform efficient repression, whereas the synthetic operator-repressor complex cannot do either. The natural operators are bent when complexed with the repressor, whereas the Os operator does not show bending in electrophoretic mobility assay. Possible structures of the complexes are discussed. DNA-protein interactions | transcription regulation | repressor-operator binding | Rhizobium phage