Detection of tetramerization domains in vivo by cooperative DNA binding to tandem lambda operator sites.
Chimeric proteins comprising the N-terminal DNA binding domain of lambda repressor fused to a fragment of a foreign protein have been used to detect oligomerization of the latter. Fusions containing dimeric and tetrameric leucine zipper domains can be distinguished based on their in vivo repressor activities on a pair of cat-lacZ reporter strains. Repressor fusions are unable to efficiently repress transcription from a synthetic promoter that overlaps a weak operator site; repression by tetrameric, but not dimeric, fusion proteins is increased by the presence of a strong, upstream operator site. To construct reporters we developed a shuttle system that allows rapid construction of single-copy operon fusions in E. coli, with both cat and lacZ as reporters.
Zeng
X
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Department of Biochemistry and Biophysics, Texas A&M University, College Station 77843-2128, USA.
Hu
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JC
D010582:Bacteriophage lambda / Q000276:immunology / Q000472:pathogenicity; D001483:Base Sequence; D001665:Binding Sites; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000235:genetics; D016350:Leucine Zippers / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000235:genetics / Q000276:immunology; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012097:Repressor Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase; D001616:beta-Galactosidase