Purification and characterization of a repressor for the Bacillus subtilis gnt operon.
The GntR protein is a negative regulator involved in gluconate-inducible expression of the Bacillus subtilis gnt operon which is responsible for gluconate metabolism. The GntR protein has been purified to homogeneity from an overproducing Escherichia coli strain harboring a gntR gene-carrying plasmid. The total amino acid composition and the NH2-terminal amino acid sequence of the purified protein were essentially the same as those deduced from the nucleotide sequence of the gntR gene. Molecular weight determination by gel filtration revealed that the purified protein is in a highly polymerized form, but it likely exists as a dimer when highly diluted. The purified GntR protein was found to be specifically bound to DNA fragments carrying the promoter of the gnt operon in an electrophoretic mobility shift assay. This binding was specifically inhibited by the addition of gluconate or glucono-delta-lactone. The purified protein repressed in vitro transcription from the promoter of the gnt operon. This repression was suppressed by gluconate or glucono-delta-lactone. These results indicate that the GntR protein is a repressor for the gnt operon and that gluconate and glucono-delta-lactone are inducers for this operon.
Miwa
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Y
Department of Biochemistry, Hamamatsu University School of Medicine, Japan.
Fujita
Y
Y
D001412:Bacillus subtilis / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D005942:Gluconates / Q000494:pharmacology; D007783:Lactones; D008970:Molecular Weight; D009876:Operon; D010957:Plasmids; D012097:Repressor Proteins / Q000302:isolation & purification*; D014157:Transcription Factors / Q000302:isolation & purification*; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D005942:Gluconates; D007783:Lactones; D012097:Repressor Proteins; D014157:Transcription Factors; C030691:gluconic acid; C010730:beta-glucono-1,5-lactone