The CTXphi repressor RstR binds DNA cooperatively to form tetrameric repressor-operator complexes.
CTX is a filamentous bacteriophage that encodes cholera toxin and integrates into the Vibrio cholerae genome to form stable lysogens. In CTX lysogens, gene expression originating from the rstA phage promoter is repressed by the phage-encoded repressor RstR. The N-terminal region of RstR contains a helix-turn-helix DNA-binding element similar to the helix-turn-helix of the cI/Cro family of phage repressors, whereas the short C-terminal region is unrelated to the oligomerization domain of cI repressor. Purified His-tagged RstR bound to three extended 50-bp operator sites in the rstA promoter region. Each of the RstR footprints exhibited a characteristic staggered pattern of DNase I-accessible regions that suggested RstR binds DNA as a dimer-of-dimers. In gel permeation chromatography and cross-linking experiments, RstR oligomerized to form dimers and tetramers. RstR was shown to be tetrameric when bound to operator DNA by performing mobility shift experiments with mixtures of RstR and a lengthened active variant of RstR. Binding of RstR to the high affinity O1 site could be fit to a cooperative model of operator binding in which two RstR dimers associate to form tetrameric RstR-operator complexes. The binding of RstR dimers to the left or right halves of O1 operator DNA was not observed in mobility shift assays. These observations support a model in which protein-protein contacts between neighboring RstR dimers contribute to strong operator binding.
Kimsey
Harvey H
HH
Tufts University School of Medicine, Department of Molecular Biology and Microbiology, Boston, Massachusetts 02111, USA. harveykimsey@yahoo.com
Waldor
Matthew K
MK
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D004926:Escherichia coli / Q000821:virology; D017912:Inovirus / Q000235:genetics / Q000378:metabolism*; D009875:Operator Regions, Genetic; D011485:Protein Binding; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D014734:Vibrio cholerae / Q000821:virology; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D012097:Repressor Proteins; C107871:RstR protein, Vibrio cholerae; D014764:Viral Proteins