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 carboxyl-terminal domain. Cooperative interactions between the C-terminal domains of two repressor dimers bound at adjacent sites can stabilize protein-DNA complexes formed with low-affinity binding sites. We have constructed a plasmid, pCT1, which directs the overproduction of the carboxyl-terminal domain of 434 repressor. The protein encoded by this plasmid is called CT-1. Cells transformed with pCT1 are unable to be lysogenized by wild-type 434 phage, whereas control cells are lysogenized at an efficiency of 1 to 5%. The CT-1-mediated interference with lysogen formation presumably results from formation of heteromeric complexes 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 formation. Two lines of evidence support this conclusion. First, DNase I footprinting experiments show that at a 2:1 ratio of CT-1 to intact 434 repressor, purified CT-1 protein prevents the formation of complexes between 434 repressor and its OR1 binding site. Second, cross-linking experiments reveal that only a specific heterodimeric complex forms between 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 DNA-bound repressor dimer. Our other evidence is also consistent with this suggestion. We have used deletion analysis in an attempt to define the region which mediates the 434 repressor-CT-1 interaction. CT-1 proteins which have more than the last 14 amino acids removed are unable to interfere with 434 repressor action in vivo.
Carlson
P A
PA
Department of Biological Sciences, State University of New York at Buffalo 14260-1300.
Koudelka
G B
GB
D000595:Amino Acid Sequence; D003090:Coliphages; D004252:DNA Mutational Analysis; D004926:Escherichia coli; D008242:Lysogeny / Q000235:genetics; D008969:Molecular Sequence Data; D009875:Operator Regions, Genetic; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000235:genetics; D010957:Plasmids / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D012097:Repressor Proteins / Q000096:biosynthesis* / Q000235:genetics; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D014170:Transformation, Genetic; D014764:Viral Proteins
C063367:434-repressor protein, Bacteriophage 434; D010446:Peptide Fragments; D012097:Repressor Proteins; D014764:Viral Proteins