The ferric uptake regulator (Fur) protein from Bradyrhizobium japonicum is an iron-responsive transcriptional repressor in vitro.
The Fur protein represses transcription of iron-responsive genes in bacteria. The discovery that Fur is a zinc metalloprotein and the use of surrogate metals for Fe(2+) for in vitro studies question whether Fur is a direct iron sensor. In the present study, we show that the affinity of Fur from Bradyrhizobium japonicum (BjFur) for its target DNA increases 30-fold in the presence of metal, with a K(d) value of about 2 nM. DNase I footprinting experiments showed that BjFur protected its binding site within the irr gene promoter in the presence of Fe(2+) but not in the absence of metal, showing that DNA binding is Fe(2+)-dependent. BjFur did not inhibit in vitro transcription from the irr promoter using purified components in the absence of metal, but BjFur repressed transcription in the presence of Fe(2+). Thus, BjFur is an iron-responsive transcriptional repressor in vitro. A regulatory Fe(2+)-binding site (site 1) and a structural Zn(2+)-binding site (site 2) inferred from the recent crystal structure of Fur from Pseudomonas aeruginosa are composed of amino acids highly conserved in many Fur proteins, including BjFur. BjFur mutants containing substitutions in site 1 (BjFurS1) or site 2 (BjFurS2) bound DNA with high affinity and repressed transcription in vitro in an Fe(2+)-dependent manner. Interestingly, only a single dimer of BjFurS2 occupied the irr promoter, whereas the wild type and BjFurS1 displayed one- or two-dimer occupancy. We suggest that the putative functions for metal-binding sites deduced from the structure of P. aeruginosa Fur cannot be extrapolated to bacterial Fur proteins as a whole.
Friedman
Yali E
YE
Department of Biochemistry and Witebsky Center for Microbial Pathogenesis and Immunology, State University of New York, Buffalo, 14214, USA.
O'Brian
Mark R
MR
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism / Q000502:physiology*; D001665:Binding Sites; D020369:Bradyrhizobium / Q000378:metabolism*; D018360:Crystallography, X-Ray; D004247:DNA / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000737:chemistry / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D007501:Iron / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008670:Metals / Q000737:chemistry; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011550:Pseudomonas aeruginosa / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism / Q000502:physiology*; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic; D015032:Zinc / Q000737:chemistry
D001426:Bacterial Proteins; D008670:Metals; D012097:Repressor Proteins; C068477:ferric uptake regulating proteins, bacterial; D004247:DNA; D007501:Iron; D003850:Deoxyribonuclease I; D015032:Zinc