EXPRESSION, PURIFICATION, AND FUNCTIONAL-CHARACTERIZATION OF THE CARBOXYL-TERMINAL DOMAIN FRAGMENT OF BACTERIOPHAGE-434 REPRESSOR

The repressor protein of bacteriophage 434 binds to DNA as a dimer of identical subunits. Its strong dimerization is mediated by the carboxy l-terminal domain. Cooperative interactions between the C-terminal dom ains of two repressor dimers bound at adjacent sites can stabilize pro tein-DNA complexes formed with low-affinity binding sites. We have con structed a plasmid, pCT1, which directs the overproduction of the carb oxyl-terminal domain of 434 repressor. The protein encoded by this pla smid is called CT-1. Cells transformed with pCT1 are unable to be lyso genized by wild-type 434 phage, whereas control cells are lysogenized at an efficiency of 1 to 5%. The CT-1-mediated interference with lysog en formation presumably results from formation of heteromeric complexe s between the phage-encoded repressor and the plasmid-encoded carboxyl -terminal domain fragment. These heteromers are unable to bind DNA and thereby inhibit the repressor's activity in promoting lysogen formati on. Two lines of evidence support this conclusion. First, DNase I foot printing experiments show that at a 2:1 ratio of CT-1 to intact 434 re pressor, purified CT-1 protein prevents the formation of complexes bet ween 434 repressor and its O(R)1 binding site. Second, cross-linking e xperiments reveal that only a specific heterodimeric complex forms bet ween CT-1 and intact 434 repressor. This latter observation indicates that CT-1 interferes with 434 repressor-operator complex formation by preventing dimerization and not by altering the conformation of the DN A-bound repressor dimer. Our other evidence is also consistent with th is suggestion. We have used deletion analysis in an attempt to define the region which mediates the 434 repressor-CT-1 interaction. CT-1 pro teins which have more than the last 14 amino acids removed are unable to interfere with 434 repressor action in vivo.