Repressor assembly at trp binding sites is dependent on the identity of the intervening dinucleotide between the binding half sites.

The interaction of trp repressor with its DNA targets is unusual in that specific recognition in this system does not rely exclusively on direct hydrogen bonds to the DNA bases that are crucial for sequence-specific recognition. It has been suggested that trp operators are mainly recognized by water-mediated interactions and by structural recognition of DNA deformability. Here we study the effect of the central dinucleotide on the mode of interaction of the trp repressor with its binding sites. The study was carried out on two consensus sequences: (1) trpTA, the consensus of naturally occurring trp binding sites, containing a T-A step between the two hexameric half-site sequences, ACTAGT; (2) trpAC, a consensus sequence derived from a functional selection study, containing a central A-C step. We show that the identity of the central dinucleotide does not affect the interaction of the first trp repressor molecule with the primary DNA target site, however, it influences the assembly of additional repressor molecules at adjacent sites. Central A-C steps stabilize tandem binding, whereas T-A steps destabilize it. It has been previously suggested that in vivo regulation of trp operators is due to their differential ability to bind multiple repressor molecules. The observations presented here support this model. We ascribe this ability to two sequence-dependent factors which act together: the identity and number of half-site sequences, recognized by water-mediated hydrogen bonds, and the ability of the intervening dinucleotides to form direct bidentate hydrogen bonds to the repressor. Furthermore, we measured the intrinsic and the induced bending of trp operators by the repressor. We find that the operators are straight in their free form, bent by 23 degrees when bound by a single trp repressor molecule, and bent by 30 degrees when bound by two repressor molecules.