DIMERIZATION SPECIFICITY OF P22 AND 434-REPRESSORS IS DETERMINED BY MULTIPLE POLYPEPTIDE SEGMENTS

The repressor protein of bacteriophage P22 binds to DNA as a homodimer . This dimerization is absolutely required for DNA binding, Dimerizati on is mediated by interactions between amino acids in the carboxyl (C) -terminal domain. We have constructed a plasmid, p22CT-1, which direct s the overproduction of just the C-terminal domain of the P22 represso r (P22CT-1), Addition of P22CT-1 to DNA-bound P22 repressor causes the dissociation of the complex, Cross-linking experiments show that P22C T-1 forms specific heterodimers with the intact P22 repressor protein, indicating that inhibition of P22 repressor DNA binding by P22CT-1 is mediated by the formation of DNA binding-inactive P22 repressor:P22CT -1 heterodimers. We have taken advantage of the highly conserved amino acid sequences within the C-terminal domains of the P22 and 434 repre ssors and have created chimeric proteins to help identify amino acid r egions required for dimerization specificity. Our results indicate tha t the dimerization specificity region of these proteins is concentrate d in three segments of amino acid sequence that are spread across the C-terminal domain of each of the two phage repressors, We also show th at the set of amino acids that forms the cooperativity interface of th e P22 repressor may be distinct from those that form its dimer interfa ce. Furthermore, cooperativity studies of the wild-type and chimeric p roteins suggest that the location of cooperativity interface in the 43 4 repressor may also be distinct from that of its dimerization interfa ce. Interestingly, changes in the dimer interface decreases the abilit y of the 434 repressor to discriminate between its wild-type binding s ites, O(R)1, 0(R)2, and 0(R)3. Since 434 repressor discrimination betw een these sites depends in large part on the ability of this protein t o recognize sequence-specific differences in DNA structure and flexibi lity, this result indicates that the C-terminal domain is intimately i nvolved in the recognition of sequence-dependent differences in DNA st ructure and flexibility.