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MICROBIAL RESPONSES TO OXIDATIVE STRESS AND
NONCODING RNAS
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Gisela Storz, PhD,Head, Section on Environmental Gene Regulation Aixia Zhang, PhD, Staff Scientist |
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| The Section on Environmental Gene Regulation has two main interests: (1) the characterization of the Escherichia coli and Saccharomyces cerevisiae responses to oxidative stress and transcriptional regulators whose activities are modified by oxidation and reduction; and (2) the identification and characterization of all E. coli noncoding RNAs. Storz, Andrade, Carmel-Harel, Mukopadhyay, Outten, Wood One focus of the group is to study how organisms sense environmental signals and transduce the signals into changes in gene expression and cell physiology. Specifically, we are examining the E. coli and S. cerevisiae responses to oxidative stress. Reactive oxygen species can lead to the damage of almost all cell components (DNA, lipid membranes, and proteins) and have been implicated as causative agents in several degenerative diseases. Most organisms have an adaptive response to defend against oxidants. For example, treatment of both bacterial and yeast cells with low doses of hydrogen peroxide results in the induction of a distinct group of proteins, the decreased expression of other proteins, and resistance to killing by subsequent higher doses of hydrogen peroxide. In bacterial cells, the pivotal regulator of the inducible defenses against hydrogen peroxide 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. Computational and microarray experiments allowed us to identify many of the genes regulated by OxyR. We now are examining the chemical basis of OxyR sensitivity to hydrogen peroxide and the roles of all of OxyR target genes. Compared with the bacterial response to hydrogen peroxide, less is known about the cellular mechanisms used by higher cells to sense and protect against oxidative damage. To initiate studies of the oxidative stress response in eukaryotes, we constructed isogenic S. cerevisiae strains carrying mutations in known signal transduction pathways and compared the oxidant sensitivities and whole genome expression patterns of these mutants. The studies confirmed that the Yap1 transcription factor is critical for resistance to hydrogen peroxide. We have purified the Yap1 protein and have begun biochemical experiments to characterize this redox-sensitive transcription factor. Mass spectrometry analysis revealed that the oxidized form of Yap1p contains two disulfide bonds between C303-C598 and C310-C629. Upon limited proteolysis of oxidized but not reduced Yap1p, we detected a stable domain of about 15 kDa. We purified the Yap1p protease-resistant domain, and mass spectrometry analysis showed that it comprises two separate cysteine-containing peptides of Yap1p. The peptides are separated by 250 amino acids and are joined by the C303-C598 and C310-C629 disulfide bonds. Work to determine the structure of this modular redox domain now is under way. Carmel-Harel O, Wood MJ, Storz G. Regulatory disulfides controlling transcription factor activity in the bacterial and yeast response to oxidative stress. In: Danon A, Gitler C, eds. Cellular Implications of Redox Signaling. London: Imperial College Press, 2003; in press. Outten FW, Wood MJ, Muñoz FM, Storz G. The SufE protein and the SufBCD complex enhance SufS cysteine desulfurase activity as part of a sulfur transfer pathway for Fe-S cluster assembly in E. coli. J Biol Chem 2003;278:45713-45719. Wood MJ, Andrade EC, Storz G. The redox domain of the Yap1p transcription factor contains two disulfide bonds. Biochemistry 2003;42:11982-11991. Zhang A, Wassarman KM, Ortega J, Steven AC, Storz G. The Sm-like Hfq protein increases OxyS RNA interaction with target mRNAs. Mol Cell 2002;9:11-22. Noncoding RNAs Storz, Kawano, Opdyke, Reynolds, Zhang; in collaboration with Gottesman, Rosenow, Tjaden, Wassarman A second focus of the group is to identify untranslated, regulatory RNAs and to elucidate their functions. These noncoding RNAs have been shown to have roles in transcriptional regulation, chromosome replication, RNA processing and modification, mRNA stability and translation, and even protein degradation and translocation. We have been characterizing two previously identified E. coli-regulatory RNAs, OxyS and 6S RNA. The OxyS RNA, whose expression is induced by OxyR in response to oxidative stress, acts as (1) a global regulator that activates and represses the expression of multiple genes and (2) an antimutator that protects cells against DNA damage. Studies of the fhlA and rpoS targets showed that the OxyS RNA represses translation of these genes. We recently found that OxyS RNA action is dependent on the Sm-like Hfq protein and that Hfq 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. Most genome annotation has missed noncoding RNA genes, which are usually poor targets in genetic screens and thus have proven difficult to detect by direct sequence inspection. Accordingly, we have been carrying out systematic screens for additional noncoding RNA genes in E. coli. The screens are all 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 Hfq RNA binding protein allowed us to detect six less well conserved RNAs. A third approach involving size fractionation of total RNA followed by linker ligation and cDNA synthesis has led to the cloning of nine antisense RNAs. Studies to characterize the functions of the newly identified RNAs are ongoing. Storz G. An expanding universe of non-coding RNAs. Science 2002;296:1260-1263. RNA and mRNA targets of Hfq. Mol Microbiol 2003;50:1111-1124. COLLABORATORS Susan Gottesman, PhD, Laboratory of Molecular Biology, NCI, Bethesda MD |
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