Metal-selective DNA-binding response of Escherichia coli NikR.
The NikR transcription factor from Escherichia coli is a Ni(II)-dependent repressor that regulates production of the nickel ion transporter encoded by the nik operon. In the previous paper in this issue (Wang, S. C., Dias, A., Bloom, S. L., and Zamble, D. B. (2004) Selectivity of Metal Binding and Metal-Induced Stability of Escherichia coli NikR, Biochemistry 43, 10018-10028) we demonstrated that NikR can bind 1 equiv of Ni(II) or several other divalent transition metals with similar affinities, but that the Ni(II)-loaded protein is less susceptible to thermal or chemical denaturation than other divalent metal complexes. Here, we investigate the metal selectivity of the DNA-binding activity of NikR. Stoichiometric nickel induces binding of nanomolar NikR to the recognition sequence in the nik promoter, but single equivalents of other divalent metals such as Cd(II), Co(II), and Cu(II) also induce a similar DNA-binding affinity. In the presence of excess nickel, DNA-binding experiments indicate that NikR binds to the nik promoter as a tetramer with much higher affinity (20 pM), and it is this response that is selective for nickel. The DNA binding induced by an excess of other divalent metals is weaker, and is enhanced by the addition of stoichiometric nickel. Nickel titrations into a DNA-binding assay reveal a nickel affinity of 30 nM for a second metal-binding site, and in the presence of 30 nM metal only nickel induces detectable DNA binding by Ni(II)-NikR. These experiments support the hypothesis that there are two metal-binding sites and that both contribute to the nickel-selective DNA-binding response. A model for the in vivo activity of NikR is discussed.
Department of Chemistry, University of Toronto, Toronto, Ontario, Canada M5S 3H6.
Stephanie L
SL
Bloom
Deborah B
DB
Zamble
D001665:Binding Sites; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D002413:Cations, Divalent; D003300:Copper / Q000737:chemistry / Q000378:metabolism; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D003850:Deoxyribonuclease I; D024202:Electrophoretic Mobility Shift Assay; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D009532:Nickel / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D012097:Repressor Proteins / Q000737:chemistry* / Q000378:metabolism
D002413:Cations, Divalent; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C402409:NikR protein, E coli; D012097:Repressor Proteins; D003300:Copper; D009532:Nickel; D003850:Deoxyribonuclease I; D002118:Calcium