Nucleic Acids Research, Vol 25, Issue 8 1469-1475, Copyright © 1997 by Oxford University Press
TM Bradley, E Hidalgo, V Leautaud, H Ding and B Demple
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.
ARTICLES
Cysteine-to-alanine replacements in the Escherichia coli SoxR protein and the role of the [2Fe-2S] centers in transcriptional activation
Department of Molecular and Cellular Toxicology, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 02115, USA.
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