A novel DNA-binding site for the ferric uptake regulator (Fur) protein from Bradyrhizobium japonicum.
The Fur protein is a global regulator of iron metabolism and other processes in many bacterial species. A key feature of the model of Fur function is the recognition of a DNA element within target promoters with similarity to a 19-bp AT-rich palindromic sequence called a Fur box. The irr gene from Bradyrhizobium japonicum is under the control of Fur. Here, we provide evidence that B. japonicum Fur (BjFur) binds to the irr gene promoter with high affinity despite the absence of DNA sequence similarity to the Fur box consensus. Both Escherichia coli Fur and BjFur bound a synthetic Fur box consensus DNA element in electrophoretic gel mobility shift assays, but only BjFur bound the irr promoter. BjFur maximally protected a 30-bp region in DNase I footprinting analysis that includes three imperfect direct repeat hexamers. BjFur formed a high mobility complex and a low mobility complex with DNA in electrophoretic gel mobility shift assays corresponding to occupancy by a single dimer and two dimers or a tetramer, respectively. A mutation in the downstream direct repeat DNA sequence allowed high mobility complex formation only. In vitro transcription from the wild type irr promoter or from a mutated promoter that allowed only dimer occupancy was repressed by Fur, indicating that the dimer can be a functional repressor unit. Our findings identify a novel DNA-binding element for Fur and suggest that the Fur box consensus may not completely represent the target sequences for bacterial Fur proteins as a whole. In addition, Fur binding to a target promoter is sufficient to repress transcription in vitro.
Friedman
Yali E
YE
Department of Biochemistry and Witebsky Center for Microbial Pathogenesis and Immunology, State University of New York at Buffalo, Buffalo, New York 14214, USA.
O'Brian
Mark R
MR
D001426:Bacterial Proteins / Q000737:chemistry*; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D020369:Bradyrhizobium / Q000378:metabolism*; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012097:Repressor Proteins / Q000737:chemistry*; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D012097:Repressor Proteins; C068477:ferric uptake regulating proteins, bacterial; D004247:DNA; D003850:Deoxyribonuclease I