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.
Department of Biological Sciences, State University of New York at Buffalo 14260-1300.
P A
PA
Carlson
G B
GB
Koudelka
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