PMID:8631739
| Citation |
Hidalgo, E and Demple, B (1996) Activation of SoxR-dependent transcription in vitro by noncatalytic or NifS-mediated assembly of [2Fe-2S] clusters into apo-SoxR. J. Biol. Chem. 271:7269-72 |
|---|---|
| Abstract |
SoxR is a transcriptional activator that senses superoxide and nitric oxide stress in Escherichia coli. The active protein isolated from E. coli contains a pair of [2Fe-2S] clusters per SoxR dimer. We previously demonstrated that the iron-free protein (apo-SoxR), isolated during purification in thiol-containing buffers, binds soxS promoter DNA with an affinity equal to that of the metalloprotein (Fe-SoxR), but lacks significant ability to activate transcription in vitro. Here we demonstrate the reversibility of this process: the full transcriptional activity of SoxR can be restored by in vitro assembly of iron-sulfur clusters into the apoprotein. Two methods were used to synthesize the metallocenters of SoxR: (i) nonenzymatic, in which apo-SoxR, incubated in the presence of iron, inorganic sulfide, and a reducing agent, regained full transcriptional activity in 5-6 h; (ii) enzymatic, in which NifS protein of Azotobacter vinelandii regenerated active Fe-SoxR in as little as 2 min. Analysis by electron paramagnetic resonance spectroscopy indicated that binuclear [2Fe-2S] clusters were restored by both the enzymatic and nonenzymatic reconstitutions. A mutant SoxR protein missing one of its four cysteine residues failed to undergo either transcriptional activation or the formation of [2Fe-2S] centers, even in the presence of NifS. Thus, only the presence of an iron-sulfur center is required to restore transcriptional activity to apo-SoxR. Moreover, the catalytic generation of [2Fe-2S] centers extends the known specificity of this enzyme beyond that already shown for [4Fe-4S] centers. Catalytic generation of [2Fe-2S]-containing SoxR could allow for rapid activation of this transcription factor in vivo. |
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| Keywords |
Apoproteins/metabolism; Azotobacter vinelandii/metabolism; Bacterial Proteins/biosynthesis; Bacterial Proteins/isolation & purification; Bacterial Proteins/metabolism; Base Sequence; DNA Primers; Electron Spin Resonance Spectroscopy; Escherichia coli/metabolism; Escherichia coli Proteins; Gene Expression Regulation; Genes, Bacterial; Iron/analysis; Iron-Sulfur Proteins/biosynthesis; Iron-Sulfur Proteins/metabolism; Molecular Sequence Data; Nitrogen Fixation; Promoter Regions, Genetic; Sulfur/analysis; Trans-Activators/metabolism; Transcription Factors/biosynthesis; Transcription Factors/isolation & purification; Transcription Factors/metabolism; Transcription, Genetic |
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