Purification and characterization of a poly(dA-dT) lux-specific DNA-binding protein from Vibrio harveyi and identification as LuxR.
A lux-specific DNA-binding protein was purified to homogeneity from Vibrio harveyi by chromatography on DEAE-Sepharose, DNA-cellulose, Superose 12, and Mono Q. A single polypeptide of M(r) = 23,000 was found on SDS-polyacrylamide gel electrophoresis with an amino-terminal sequence corresponding to that predicted for luxR, a gene that causes a shift in the transcriptional start site from position -123 to -26 base pairs upstream of the initiation codon of luxC in the V. harveyi lux operon and is required for high expression of lux mRNA in recombinant Escherichia coli. Identification of the DNA-binding protein as LuxR was confirmed by showing its absence in V. harveyi luxR-mutants and its synthesis in recombinant E. coli containing V. harveyi luxR. The LuxR protein was shown to bind to two specific (A + T)-rich regions of DNA upstream of the V. harveyi luxC gene: region A, -290 to -253 base pairs, and region B, -170 to -116 base pairs. Synthetic poly(dA-dT) but not poly(dA)-poly(dT) competed with the lux DNA for binding to LuxR suggesting that this protein may be a novel poly(dA-dT)-binding protein in prokaryotes. The LuxR protein inhibited transcription from the -123 promoter in vitro; however, transcription from the -26 promoter was not reconstituted suggesting the possible requirement for other factors in lux gene regulation. LuxR shared sequence identity with two proteins linked to the regulation of enzymes involved in electron transport indicating that it may be a member of a family of regulators of metabolic functions responsible for diverting electrons from the respiratory chain.
Swartzman
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Department of Biochemistry, McGill University, Montreal, Quebec, Canada.
Meighen
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EA
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D002850:Chromatography, Gel; D004269:DNA, Bacterial / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D003850:Deoxyribonuclease I; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D011067:Poly dA-dT / Q000378:metabolism*; D011485:Protein Binding; D012097:Repressor Proteins; D017386:Sequence Homology, Amino Acid; D015534:Trans-Activators; D014733:Vibrio / Q000378:metabolism*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D015534:Trans-Activators; C064778:LuxR autoinducer binding proteins; D011067:Poly dA-dT; D003850:Deoxyribonuclease I