Regulation of high affinity nickel uptake in bacteria. Ni2+-Dependent interaction of NikR with wild-type and mutant operator sites.
Escherichia coli actively imports nickel via the ATP-dependent NikABCDE permease. NikR, a protein of the ribbon-helix-helix family of transcription factors, represses expression of the nikABCDE operon in the presence of excessive concentrations of intracellular nickel. Here, the NikR operator site is identified within the nikABCDE promoter by footprinting and mutational analyses. The operator consists of two dyad-symmetric 5'-GTATGA-3' recognition sequences separated by 16 base pairs. Mutations in the GTATGA sequences reduce NikR binding affinity in vitro and reduce repression of a P(nik)-lacZ fusion in vivo. Moreover, NikR is shown to be a direct sensor of nickel ions. Strong operator binding requires the continual presence of 20-50 micrometer nickel, indicating the presence of a low affinity nickel-binding site, and NikR dimers also contain two high affinity nickel-binding sites. In addition to both GTATGA sites and nickel, high affinity operator binding also requires the C-terminal domain of NikR.
Chivers
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PT
Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Sauer
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RT
D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D004247:DNA / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D017930:Genes, Reporter; D007477:Ions; D008745:Methylation; D008969:Molecular Sequence Data; D009154:Mutation; D009532:Nickel / Q000493:pharmacokinetics*; D009875:Operator Regions, Genetic; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012689:Sequence Homology, Nucleic Acid; D013053:Spectrophotometry; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000378:metabolism
D029968:Escherichia coli Proteins; D007477:Ions; C402409:NikR protein, E coli; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D009532:Nickel; D004247:DNA; D003850:Deoxyribonuclease I; D001616:beta-Galactosidase