title: Characterization of a second MetR-binding site in the metE metR regulatory region of Salmonella typhimurium.
abstract: Transcription of the metE gene in Salmonella typhimurium and Escherichia coli is positively regulated by the MetR protein, with homocysteine serving as a coactivator. It was shown previously that MetR binds to and protects from DNase I digestion a 24-bp sequence in the metE metR regulatory region from nucleotides -48 to -71 relative to the metE transcription initiation site (designated as site 1). In this study, we show that purified MetR protein also binds to and protects a second 24-bp sequence adjacent to the original site, from nucleotides -24 to -47 relative to the metE transcription initiation site (designated as site 2). Single and multiple base changes were introduced into sites 1 and 2 in a metE-lacZ fusion. Base pair changes in site 1 or site 2 away from the MetR consensus binding sequence resulted in decreased metE-lacZ expression, suggesting that both sites are necessary for expression. DNase I footprint analysis showed that MetR bound at the high-affinity site 1 enhances MetR binding at the low-affinity site 2. A 2-bp change in site 2 toward the MetR consensus binding sequence resulted in high metE-lacZ expression; the increased expression was MetR dependent but homocysteine independent.
authors.affiliation: Department of Microbiology, University of Iowa, Iowa City 52242.
authors.forename: W F
authors.initials: WF
authors.lastname: Wu
authors.forename: M L
authors.initials: ML
authors.lastname: Urbanowski
authors.forename: G V
authors.initials: GV
authors.lastname: Stauffer
mesh_terms: D013766:5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D005809:Genes, Regulator; D007763:Lac Operon; D008780:Methyltransferases; D008969:Molecular Sequence Data; D009154:Mutation; D012486:Salmonella typhimurium / Q000235:genetics*; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C058169:MetR protein, Bacteria; D015534:Trans-Activators; D008780:Methyltransferases; D013766:5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase; C058170:metE protein, E coli