Effect of non-contacted bases on the affinity of 434 operator for 434 repressor and Cro

The repressor of phage 434 binds to six operator sites on the phage chromosome. A comparison of the sequences of these 14-base-pair (bp) operator sites reveals a striking pattern: at five of the six sites, the symmetrically arrayed outer eight base pairs (four in each half-site) are identical and the remaining site differs at only one position (Fig. 1b). In contrast, the sequences of the inner four base pairs are highly variable. Crystallographic analysis of the repressor-operator complex shows that at each half-site, the 'recognition alpha-helix' of the repressor is positioned in the major groove such that it could contact the outermost five base pairs, but not the innermost two (Fig. 1a). We show in this paper that the sequence of the central base pairs of the operator (two in each half-site) have a significant role in determining operator affinity for repressor, despite the evidence presented here and in the accompanying paper that these base pairs are not contacted by repressor. We also show that these central base pairs influence operator affinity for Cro, a second gene regulatory protein encoded by phage 434. We discuss the likely structural basis for this evidently indirect, but sequence-dependent, effect of the central base pairs of the operator on its affinity for the two regulatory proteins.