A gene of interest was identified by SDS-PAGE of cell lysates from wild-type and fur-mutant cells, which indicated a protein (identified as pfr) was highly up-regulated in the fur mutant. Primer extension identified the transcription start site and demonstrated 30x pfr transcription in the fur mutant compared to the wild type, suggesting fur represses pfr. Further, in iron-variable assays, increased iron was shown to increase pfr transcript, and decreased iron reduced pfr transcript, though the fur mutant showed no response to changes in iron concentration. DNAse I protection demonstrated specific binding in the presence of a non-specific competitor, and revealed three putative binding sites displaying a “unique repeated pattern of dual binding sites flanking a hypersensitive site.” Additional protection assays of iron-variable cells showed fur-Fe2+ complex has reduced affinity for the pfr binding sites. The sites were aligned with each other and the Fur box of E. coli, and showed limited conservation.
Regulated genes for each binding site are displayed below. Gene regulation diagrams show binding sites, positively-regulated genes, negatively-regulated genes, both positively and negatively regulated genes, genes with unspecified type of regulation. For each indvidual site, experimental techniques used to determine the site are also given.
|Site sequence||Regulated genes||Gene diagram||Experimental techniques||TF function||TF type|