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MICROBIAL RESPONSES TO
OXIDATIVE STRESS
AND FUNCTIONS OF NONCODING RNAS
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Gisela Storz, PhD, Head, Section on Environmental Gene Regulation Aixia
Zhang, PhD, Staff Scientist Matthew
Hemm, PhD, Postdoctoral Fellow Mitsuoki
Kawano, PhD, Postdoctoral Fellow Partha
Mukhopadhyay, PhD, Postdoctoral Fellow Jason
A. Opdyke, PhD, Postdoctoral Fellow F.
Wayne Outten, PhD, Postdoctoral Fellow Matthew
J. Wood, PhD, Postdoctoral Fellow Sarah
Goodwin, BA, Predoctoral Fellow April
Reynolds, BS, Predoctoral Fellow Juan Miranda Rios, PhD, Guest Researcher |
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Our research has two main interests: the
characterization of the Escherichia
coli and Saccharomyces cerevisiae
responses to oxidative stress and the identification and characterization of
all E. coli noncoding RNAs. The
responses to oxidative stress are of interest because the reactive oxygen
species generated during normal aerobic growth can oxidize and damage all
cellular components. Although noncoding RNAs have largely been overlooked
until recently, small, noncoding RNA genes are of interest because
accumulating evidence suggests that many act as important regulators in the
cell. Defenses
against oxidative stress Outten, Wood, Storz; in collaboration
with Djaman We study how organisms sense oxidative stress
and transduce the signals into the induction of defense genes. Microarray
analysis of the E. coli response to
hydrogen peroxide revealed that the sufABCDSE genes, which encode proteins
implicated in iron-sulfur cluster assembly, are among the genes induced by
oxidative stress. Our recent studies have been directed at both examining the
role of the sufABCDSE operon in the
cell and elucidating the functions of the Suf proteins. Expression studies
and phenotypic characterization of suf mutants revealed that the sufABCDSE operon is specifically
regulated to synthesize iron-sulfur clusters when iron metabolism is
disrupted by iron starvation or oxidative stress. Assays of the purified
SufB, C, D, S, and E proteins showed that the SufE protein stimulated the
cysteine desulfurase activity of the SufS protein and that the SufBCD complex
of proteins enhanced the same activity even further. We are undertaking
experiments to elucidate in greater detail the function of the SufBCD
complex. Outten FW, Djaman O, Storz G. A suf operon requirement for Fe-S
cluster assembly during iron starvation in E. coli. Mol Microbiol
2004;52:861-872. Outten FW, Wood MJ, Muñoz FM, Storz G. The
SufE protein and the SufBCD complex enhance SufS cysteine desulferase
activity as part of a sulfur transfer pathway for Fe-S cluster assembly in Escherichia coli. J Biol Chem 2003;278:45713-45719. Redox-regulation
of OxyR Mukopadhyay, Storz; in collaboration with
Bedzyk, LaRossa, Zheng The key regulator of the inducible defenses
against hydrogen peroxide in E. coli
is the OxyR transcription factor. We discovered that OxyR is both the sensor
and transducer of the oxidative stress signal; the oxidized but not the
reduced form of the purified regulator can activate transcription in vitro. OxyR is activated by the
formation of an intramolecular disulfide bond between C199 and C208 and is
deactivated by enzymatic reduction by glutaredoxin 1 together with
glutathione. Structural studies showed that formation of the C199-C208
disulfide bond leads to a large conformational change that alters OxyR
binding to DNA. Others have suggested that the activity of
OxyR is also modulated by reactive nitrogen species. To evaluate the
contribution of OxyR to the E. coli
response to nitrosative stress, we examined the genomewide transcriptional
responses, during aerobic growth, of cells treated with nitrosylated
glutathione or acidified sodium nitrite (NaNO2). Our assays showed
that NorR, a homolog of NO-responsive transcription factors in Ralstonia eutrophus, and Fur, the
global repressor of ferric ion uptake, are major regulators of the response
to reactive nitrogen species. In contrast, SoxR and OxyR, regulators of the E. coli defenses against
superoxide-generating compounds and hydrogen peroxide, respectively, play
minor roles. Moreover, the whole-genome expression patterns showed that
additional regulators of the E. coli response
to reactive nitrogen species remain to be identified. This study led us to
propose that the E. coli
transcriptional response to reactive nitrogen species is a composite response
mediated by the modification of several transcription factors containing iron
or redox-active cysteines, some specifically designed to sense NO and its
derivatives and others collaterally activated by the reactive nitrogen
species. Mukhopadhyay P, Zheng M, Bedzyk LA, LaRossa
RA, Storz, G. Prominent roles of the NorR and Fur regulators in the Escherichia coli transcriptional
response to reactive nitrogen species. Proc
Natl Acad Sci USA 2004;101:745-750. Redox-regulation
of Yap1 Wood, Storz; in collaboration with
Tjandra A central regulator of the response to
oxidative stress in S. cerevisiae
is the Yap1 transcription factor. Upon activation by increased levels of
reactive oxygen species, Yap1 rapidly redistributes to the nucleus, where it
regulates the expression of up to 70 genes. We purified the Yap1 protein and
have been carrying out biochemical experiments to characterize this
redox-sensitive transcription factor. Mass-spectrometric analysis revealed
that the oxidized form of Yap1p contains two disulfide bonds between
C303-C598 and C310-C629. We detected a stable domain of about 15 kDa upon limited
proteolysis of oxidized but not reduced Yap1p. We purified the Yap1p protease–resistant
domain and used mass spectrometry analysis to show that it comprised two
separate cysteine-containing peptides of Yap1p: the amino-terminal
cysteine-rich domain (n-CRD) and the carboxy-terminal cysteine-rich domain
(c-CRD). These peptides are separated by 250 amino acids and are joined by
the C303-C598 and C310-C629 disulfide bonds. We used NMR spectroscopy to
determine the high-resolution solution structure of the redox-domain. In the
active oxidized form, a nuclear export signal (NES) in the c-CRD is masked by
disulfide bond–mediated interactions with a conserved alpha helix in the
n-CRD. Point mutations that weaken the hydrophobic interactions between the n-CRD
alpha-helix and the c-CRD abolished redox-regulated changes in subcellular
localization of Yap1. Upon reduction of the disulfide bonds, Yap1 undergoes a
change to an unstructured conformation that exposes the NES and allows
redistribution to the cytoplasm. Our results revealed the structural basis of
redox-dependent Yap1 localization and provided a previously unknown mechanism
of transcription factor regulation by reversible intramolecular disulfide
bond formation. Carmel-Harel O, Wood MJ, Storz G. Regulatory
disulfides controlling transcription factor activity in the bacterial and
yeast responses to oxidative stress. In: Gitler C, Danon A, eds. Cellular Implications of Redox Signaling.
Wood MJ, Andrade EC, Storz G. The redox domain
of the Yap1p transcription factor contains two disulfide bonds. Biochemistry 2003;42:11982-11991. Wood MJ, Storz G, Tjandra N. Structural basis
for redox regulation of Yap1 transcription factor localization. Nature 2004;430:917-921. Identification
of noncoding RNAs Kawano, Miranda Rios, Reynolds, Zhang,
Storz; in collaboration with Gottesman, Rosenow, Tjaden, Wassarman Most genome annotation has missed noncoding
RNA genes; the genes are usually poor targets in genetic screens and have
been difficult to detect by direct sequence inspection. Thus, we have been
carrying out systematic screens for additional noncoding RNA genes in E. coli, screens that are applicable
to other organisms. One approach, based on computer searches of intergenic
regions for extended regions of conservation among closely related species,
has led to the identification of 17 conserved noncoding RNAs. Another screen
for noncoding RNAs that coimmunoprecipitate with the RNA binding protein Hfq
allowed us to detect six less well conserved RNAs. In yet another approach,
size fractionation of total RNA followed by linker ligation and cDNA
synthesis has led to the cloning of cis-encoded
antisense RNAs. Zhang A, Wassarman KM, Rosenow C, Characterization
of noncoding RNAs Goodwin, Kawano,
Miranda Rios, Opdyke, Zhang, Storz; in collaboration with Blyn, Chen, Kang We have succeeded in elucidating the functions
of the noncoding RNAs in E. coli.
We previously showed that OxyS RNA, the expression of which is induced by
OxyR in response to oxidative stress, acts to repress translation by base pairing
with target mRNAs. OxyS RNA action is dependent on the Sm-like Hfq protein,
which functions as a chaperone to facilitate OxyS RNA base pairing with its
target mRNAs. We also discovered that the abundant 6S RNA binds to and
modifies RNA polymerase. In the past year, we elucidated the functions of two
recently discovered noncoding RNAs that bind to Hfq: the 109-nucleotide MicC
RNA and the 105-nucleotide GadY RNA. We found that MicC RNA represses
translation of the OmpC outer membrane porin. Interestingly, under most
conditions, MicC RNA shows the opposite expression to MicF RNA, which
represses expression of the OmpF porin. Thus, we suggest that the MicF and
MicC RNAs control the OmpF:OmpC protein ratio in response to a variety of
environmental stimuli. In contrast, base pairing between the GadY RNA and the
3´-untranslated region (3´ UTR) of the gadX
mRNA encoded opposite gadY leads to
increased stability of the gadX
mRNA. Enhanced gadX mRNA stability
results in increased GadX levels and increased expression of the
acid-response genes controlled by the GadX transcription factor. We are
continuing to characterize further the GadY RNA activity and the roles of
other newly discovered noncoding RNAs. Chen S, Zhang A, Blyn LB, Storz G. MicC, a
second small RNA regulator of Omp protein expression in Escherichia coli. J
Bacteriol 2004;186:6689-6697. Opdyke JA, Kang J-G, Storz G. GadY, a small
RNA regulator of acid response genes in Escherichia
coli. J Bacteriol
2004;186:6698-6705. Storz G, Opdyke JA, Zhang A. Controlling mRNA
stability and translation with small, noncoding RNAs. Curr Opin Microbiol 2004;7:140-144. COLLABORATORS Laura A. Bedzyk, MS, Central Research and Development E.I.
DuPont de Nemours and Company, Shuo Chen, PhD, IBIS Therapeutics, Susan Gottesman, PhD, Laboratory of Molecular Biology, NCI, Ju-Gyeong Kang, PhD, Cardiovascular Branch, NHLBI, Robert A. LaRossa, PhD, Central Research and Development, E.I.
DuPont de Nemours and Company, Carsten Rosenow, PhD, Affymetrix, Brian C. Tjaden, PhD, Computer Science Department, Nico Tjandra, PhD, Laboratory of Biophysical Chemistry,
NHLBI, Karen M. Wassarman, PhD, Department of Bacteriology, Ming
Zheng, PhD, Central Research and
Development, E.I. DuPont de Nemours and Company, For
further information, contact storz@helix.nih.gov |
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