Dimerization is essential for DNA binding and repression by the ArsR metalloregulatory protein of Escherichia coli.
Arsenical resistance (ars) operons produce resistance to trivalent and pentavalent salts of the metalloids arsenic and antimony in cells of Escherichia coli. The first gene in the operon, arsR, was previously shown to encode a homodimeric trans-acting metalloregulatory repressor protein. Dimerization of ArsR was investigated using the yeast two-hybrid system in which the ArsR protein was fused to the Saccharomyces cerevisiae GAL4 DNA-binding domain and GAL4 activation domain to produce chimeric proteins. Transcriptional activation of lacZ reporter indicated that dimerization of the ArsR is stable in yeast. The results indicated that residues 1-8 and 90-117 are not required for ArsR dimerization. The genes for a series of truncated ArsR proteins containing six histidine tags were constructed and the proteins purified. The mass of each recombinant protein, as determined by size exclusion chromatography, was consistent with the results from two-hybrid analysis. The results of beta-galactosidase assays in vivo and gel mobility shift assays in vitro showed that dimers retained the ability to bind to the ars promoter and to respond to inducer, whereas monomeric ArsRs did neither. These results suggest that a core sequence of about 80 residues has all of the information necessary for dimerization, repression, and metal recognition.
Xu
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Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Rosen
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BP
D000595:Amino Acid Sequence; D001152:Arsenicals / Q000378:metabolism; D001426:Bacterial Proteins; D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D019281:Dimerization; D004791:Enzyme Inhibitors / Q000378:metabolism; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D017017:Methanococcus; D008969:Molecular Sequence Data; D009876:Operon; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012097:Repressor Proteins / Q000378:metabolism; D017384:Sequence Deletion; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*
C489527:ArsR protein, E coli; D001152:Arsenicals; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D012097:Repressor Proteins; D015534:Trans-Activators; C029341:oxophenylarsine; D004247:DNA