Binding of ArsR, the repressor of the Staphylococcus xylosus (pSX267) arsenic resistance operon to a sequence with dyad symmetry within the ars promoter.
arsR, the first gene of the Staphylococcus xylosus (pSX267) arsenic/antimonite resistance (ars) operon encodes a negative regulatory protein, ArsR, which mediates inducibility of the resistances by arsenic and antimony compounds. ArsR, which has no obvious DNA-binding motif in its primary structure, was purified from an ArsR-overproducing Escherichia coli strain and identified as a DNA-binding protein by its behaviour in gel mobility shift assays. ArsR had a specific affinity for a 312 bp DNA restriction fragment carrying the ars promoter; the minimum sequence complexed by ArsR was a 75 bp polymerase chain reaction (PCR) fragment, which mainly comprised the -35 and -10 regions of the promoter. The effect of inducers on the DNA-binding activity of ArsR was examined by in vitro induction assays; only arsenite inhibited DNA-binding of the repressor. DNase I footprinting revealed two protected regions within the promoter region, spanning 23 and 9 nucleotides, respectively. Furthermore, a new cleavage site for DNase I between the protected regions was made accessible by binding of the repressor. The footprints cover a region of three inverted repeats located between the -35 and -10 motifs of the ars promoter. By high resolution footprinting with the hydroxy radical, five sites of close contact between the protein and DNA were identified.
Rosenstein
R
R
Lehrstuhl für Mikrobielle Genetik, Universität Tübingen, Germany.
Nikoleit
K
K
Götz
F
F
D000965:Antimony / Q000494:pharmacology; D001151:Arsenic / Q000494:pharmacology*; D001483:Base Sequence; D004352:Drug Resistance, Microbial; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D009876:Operon; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012097:Repressor Proteins / Q000378:metabolism*; D013210:Staphylococcus / Q000235:genetics*
D012097:Repressor Proteins; D000965:Antimony; D001151:Arsenic