Cysteine-to-alanine replacements in the Escherichia coli SoxR protein and the role of the [2Fe-2S] centers in transcriptional activation.
The Escherichia coli soxRS regulon activates oxidative stress and antibiotic resistance genes in two transcriptional stages. SoxR protein becomes activated in cells exposed to excess superoxide or nitric oxide and then stimulates transcription of the soxS gene, whose product in turn activates>/=10 regulon promoters. Purified SoxR protein is a homodimer containing a pair of [2Fe-2S] centers essential for soxS transcription in vitro . The [2Fe-2S] centers are thought to be anchored by a C-terminal cluster of four cysteine residues in SoxR. Here we analyze mutant SoxR derivatives with individual cysteines replaced by alanine residues (Cys-->Ala). The mutant proteins in cell-free extracts bound the soxS promoter with wild-type affinity, but upon purification lacked Fe or detectable transcriptional activity for soxS in vitro . Electron paramagnetic resonance measurements in vivo indicated that the Cys-->Ala proteins lacked the [2Fe-2S] centers seen for wild-type SoxR. The Cys-->Ala mutant proteins failed to activate soxS expression in vivo in response to paraquat, a superoxide- generating agent. However, when expressed to approximately 5% of the cell protein, the Cys-->Ala derivatives increased basal soxS transcription 2-4-fold. Overexpression of the Cys119-->Ala mutant protein strongly interfered with soxS activation by wild-type SoxR in response to paraquat. These studies demonstrate the essential role of the [2Fe-2S] centers for SoxR activation in vivo ; the data may also indicate oxidant-independent mechanisms of transcriptional activation by SoxR.
Bradley
T M
TM
Department of Molecular and Cellular Toxicology, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 02115, USA.
Hidalgo
E
E
Leautaud
V
V
Ding
H
H
Demple
B
B
D000409:Alanine; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D003545:Cysteine; D017931:DNA Primers; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D001616:beta-Galactosidase / Q000378:metabolism
D001426:Bacterial Proteins; D017931:DNA Primers; D007506:Iron-Sulfur Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C070724:SoxR protein, Bacteria; D001616:beta-Galactosidase; D003545:Cysteine; D000409:Alanine