Carboxyl-terminal domain dimer interface mutant 434 repressors have altered dimerization and DNA binding specificities.

Strong dimerization of the repressor, mediated by the carboxyl (C)-terminal domain, is a prerequisite for forming a specific complex with DNA and cooperative DNA binding to form tetramers. We have generated a computer model of the C-terminal domain of the 434 repressor based on the crystal structure of the homologous UmuD' protein. This model predicts that residues in the primary sequence between 93 and 168 contribute to the dimer interface. We changed several amino acid residues located in this region. Gel filtration and crosslinking assays were used to characterize the strength and specificity of dimerization of the purified repressor C-terminal domain dimer interface mutants. These results indicate that amino acid residues K121, H139, D161 and N163 contribute to the strength and/or specificity of dimerization. The relative affinity of the bacteriophage 434 repressor for 434 operators is determined, in part, by the repressor's ability to detect sequence-dependent structural alterations in the non-contacted region at the center of an operator site. We find that the relative ability of C-terminal domain dimer interface mutant repressors to dimerize does not necessarily predict their relative abilities to bind DNA, and that these proteins are deficient in detecting non-contacted base-dependent differences in operator strength. Our results show that the structure of the DNA in complex with these mutant proteins differs from that found in wild-type repressor-operator complexes, even though the sites of these mutations lie in a separate domain from that which contacts the DNA. These observations demonstrate that the structural integrity of the C-terminal domain dimer interface is required to appropriately orient the DNA binding information contained within the DNA-contacting N-terminal domain.