Interaction of Bacillus subtilis Fur (ferric uptake repressor) with the dhb operator in vitro and in vivo.
Bacillus subtilis contains three metalloregulatory proteins belonging to the ferric uptake repressor (Fur) family: Fur, Zur, and PerR. We have overproduced and purified Fur protein and analyzed its interaction with the operator region controlling the expression of the dihydroxybenzoate siderophore biosynthesis (dhb) operon. The purified protein binds with high affinity and selectivity to the dhb regulatory region. DNA binding does not require added iron, nor is binding reduced by dialysis of Fur against EDTA or treatment with Chelex. Fur selectively inhibits transcription from the dhb promoter by sigmaA RNA polymerase, even if Fur is added after RNA polymerase holoenzyme. Since neither DNA binding nor inhibition of transcription requires the addition of ferrous ion in vitro, the mechanism by which iron regulates Fur function in vivo is not obvious. Mutagenesis of the fur gene reveals that in vivo repression of the dhb operon by iron requires His97, a residue thought to be involved in iron sensing in other Fur homologs. Moreover, we identify His96 as a second likely iron ligand, since a His96Ala mutant mediates repression at 50 microM but not at 5 microM iron. Our data lead us to suggest that Fur is able to bind DNA independently of bound iron and that the in vivo role of iron is to counteract the effect of an inhibitory factor, perhaps another metal ion, that antagonizes this DNA-binding activity.
Section of Microbiology, Cornell University, Ithaca, New York 14853-8101, USA.
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Helmann
D001412:Bacillus subtilis / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D062385:Hydroxybenzoates / Q000378:metabolism*; D007501:Iron / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009875:Operator Regions, Genetic; D009876:Operon; D016133:Polymerase Chain Reaction; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D017262:Siderophores / Q000096:biosynthesis*; D012808:Sigma Factor / Q000378:metabolism; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D062385:Hydroxybenzoates; D012097:Repressor Proteins; D017262:Siderophores; D012808:Sigma Factor; C068477:ferric uptake regulating proteins, bacterial; D007501:Iron; D012321:DNA-Directed RNA Polymerases