Purification and characterization of MerR, the regulator of the broad-spectrum mercury resistance genes in Streptomyces lividans 1326.
Streptomyces lividans 1326 carries inducible mercury resistance genes on the chromosome, which are arranged in two divergently transcribed operons. Expression of the genes is negatively regulated by the repressor MerR, which binds in the intercistronic region between the two operons. The merR gene was expressed in E. coli using a T7 RNA polymerase/promoter expression system, and MerR was purified to around 95% homogeneity by ammonium sulfate precipitation, gel filtration and affinity chromatography. Gel filtration showed that the native MerR is a dimer with a molecular mass of 31 kDa. Two DNA binding sites were identified in the intercistronic mer promoter region by footprinting experiments. No evidence for cooperativity in the binding of MerR to the adjacent operator sequences was observed in gel mobility shift assays. The dissociation constants (K(D)) for binding of MerR were: binding site I, 8.5 x 10(-9) M; binding site II, 1.2 x 10(-8) M; and for the complete promoter/operator region 1 x 10(-8) M. The half-life of the MerR-DNA complex was 19.4 min and 18.8 min for binding site I and binding site II, respectively. The K(D) value for binding of mercury(II)chloride to MerR, again determined by mobility shift assay, was 1.1 x 10(-7) M.
Rother
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Institut für Industrielle Genetik, Universität Stuttgart , Germany.
Mattes
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R
Altenbuchner
J
J
D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002413:Cations, Divalent / Q000378:metabolism; D002850:Chromatography, Gel; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D004352:Drug Resistance, Microbial; D005798:Genes, Bacterial; D008628:Mercury / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009875:Operator Regions, Genetic; D011485:Protein Binding; D011994:Recombinant Proteins / Q000302:isolation & purification; D012097:Repressor Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D013302:Streptomyces / Q000187:drug effects* / Q000235:genetics
D001426:Bacterial Proteins; D002413:Cations, Divalent; D004268:DNA-Binding Proteins; C051124:MerR protein, Bacteria; D011994:Recombinant Proteins; D012097:Repressor Proteins; D003850:Deoxyribonuclease I; D008628:Mercury