In vitro characterization of constitutive CysB proteins from Salmonella typhimurium.
Expression of the cysteine regulon in Salmonella typhimurium and Escherichia coli is controlled by the LysR-type transcriptional activator CysB and by the inducer N-acetyl-L-serine. Sulphide and thiosulphate are anti-inducers. Two highly purified constitutive CysB proteins, CysB(T149M) and CysB(T149P), were found to bind to the cysJIH, cysK and cysP promoters, to activate transcription from the cysJIH and cysK promoters in the absence of N-acetyl-L-serine, and to be insensitive to the effects of anti-inducers. At 10 mM MgCl2, the in vitro transcription activity of CysB(T149M) was maximal without N-acetyl-L-serine, but that of CysB(T149P) was increased by inducer. At 2 mM MgCl2, both proteins were fully active without inducer. A third mutant protein, CysB(W166R), was totally inactive at 10 mM MgCl2, but gave constitutive expression of the cysK and cysJIH promoters at 2 mM MgCl2. Surprisingly, wild-type CysB was also constitutive for the cysK promoter at 2 mM mgCl2 but not at 10 mM MgCl2; it required inducer for cysJIH promoter activation at both concentrations. Mutagenic studies indicated that this difference between promoters is due to the distance between activation site half-sites, which are separated by 1 bp in the cysJIH promoter and by 2 bp in the cysK promoter. We speculate that inducer acts to decrease the distance between the binding domains of two CysB subunits that interact with an activation site. In vitro activities of wild-type and mutant CysB proteins correlated much better with in vivo behaviour at 2 mM than at 10 mM MgCl2, suggesting that the former is the more physiological concentration.
Colyer
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Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA.
Kredich
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D000483:Alleles; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003545:Cysteine / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D009690:Nucleic Acid Conformation; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D018085:Regulon / Q000187:drug effects; D012486:Salmonella typhimurium / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D012694:Serine / Q000031:analogs & derivatives / Q000494:pharmacology; D013885:Thiosulfates / Q000494:pharmacology; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation
D001426:Bacterial Proteins; C062886:CysB protein, Bacteria; C489265:CysB protein, Salmonella typhimurium; D004269:DNA, Bacterial; D013885:Thiosulfates; D015534:Trans-Activators; D012694:Serine; D003545:Cysteine; C012162:N-acetylserine