We have had two main interests: the characterization of 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. Small noncoding RNA genes are of interest, because, although noncoding genes have largely been overlooked until recently, accumulating evidence suggests that many such genes act as important regulators in the cell.
Defenses against oxidative stress
Outten, Storz; in collaboration with Djaman
For some years, we have been investigating how organisms protect against oxidative stress. Genome-wide expression 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. Expression studies and phenotypic characterization of suf mutants revealed that the sufABCDSE operon is specifically regulated in order 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 further enhanced such activity.
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.
Redox regulation of OxyR
Mukhopadhyay,1 Storz; in collaboration with LaRossa, Ryu, Zheng
The key regulator of 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. Measurements of the rate of OxyR activation and the stability of the oxidized conformation have shown that the rapid kinetic reaction path and conformation strain, respectively, drive the oxidation and reduction of OxyR.
Others have suggested that reactive nitrogen species also modulate the activity of OxyR. To evaluate the OxyR contribution to the E. coli response to nitrosative stress, we examined the genome-wide transcriptional responses of cells treated with nitrosylated glutathione or acidified sodium nitrite (NaNO2) during aerobic growth. The assays showed that NorR, a homologue 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. Therefore, our work 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.
Kiley PJ, Storz G. Exploiting thiol modifications. PLoS Biol 2004;2:1714-1717.
Lee C, Lee SM, Mukhopadhyay P, Kim SJ, Lee SC, Ahn W-S, Yu M-H, Storz G, Ryu SE. Redox regulation of OxyR requires specific disulfide bond formation involving a rapid kinetic reaction path. Nat Struct Mol Biol 2004;11:1179-1185.
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
The 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 carried 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; mass-spectrometry analysis showed that the domain 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). The peptides are separated by 250 amino acids and are joined by the C303-C598 and C310-C629 disulfide bonds. We used nuclear magnetic resonance 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. The results revealed the structural basis of redox-dependent Yap1 localization and disclosed a previously unknown mechanism of transcription factor regulation by reversible intramolecular disulfide bond formation.
Wood MJ, Storz G. Oxygen, metabolism and gene expression: the T-Rex connection. Structure 2005;13:2-4.
Wood MJ, Storz G, Tjandra N. Structural basis for redox regulation of Yap1 transcription factor localization. Nature 2004;430:917-921.
Identification of small noncoding RNAs
Kawano, Miranda Rios, Reynolds,2 Storz
Most genome annotation misses noncoding RNA genes; the genes are usually poor targets in genetic screens, and they 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. 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 co-immunoprecipitate with the RNA binding protein Hfq allowed us to detect six less well conserved RNAs. A third approach consisting of size fractionation of total RNA followed by linker ligation and cDNA synthesis has led to the cloning of cis-encoded antisense RNAs.
Kawano M, Reynolds AA, Miranda-Rios J, Storz G. Detection of 5´- and 3´-UTR-derived small RNAs and cis-encoded antisense RNAs in Escherichia coli. Nucleic Acids Res 2005;33:1040-1050.
Characterization of small noncoding RNAs
Fozo, Goodwin, Kawano, Opdyke, Zhang, Storz; in collaboration with Blyn, Chen, Kang
Increasingly, we have been focusing on elucidating the functions of the noncoding RNAs in E. coli. We previously showed that OxyS RNA, whose expression 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. Recently, we elucidated the functions of two other noncoding RNAs that bind to Hfq: the 109 nucleotide MicC RNA and the 105 nucleotide GadY RNA. We found that the MicC RNA represses translation of the OmpC outer membrane porin. Interestingly, under most conditions, the MicC RNA shows the opposite expression to MicF RNA, which represses expression of the OmpF porin. We therefore suggest that the MicF and MicC RNAs act to 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 level of the gadX mRNA and GadX protein. In turn, increased GadX levels result in increased expression of the acid-response genes controlled by the GadX transcription factor. Studies are under way to characterize further the GadY RNA 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, Altuvia S, Wassarman KM. An abundance of RNA regulators. Annu Rev Biochem 2005;74:199-217.
Storz G, Opdyke JA, Zhang A. Controlling mRNA stability and translation with small, noncoding RNAs. Curr Opin Microbiol 2004;7:140-144.
Characterization of small ORFs
Hemm, Miranda Rios, Paul, Soltanzad, Storz
In our genome-wide screens for noncoding RNAs, we found that several short RNAs actually encode small proteins. As noted, genome annotation has largely overlooked small proteins while many biochemical approaches have missed these proteins. However, the few small proteins studied in detail in bacterial and mammalian cells evidence important functions in signaling and cellular defenses. Thus, taking advantage of many of the approaches used to characterize the functions of small noncoding RNAs, we have initiated a project to elucidate the functions of E. coli proteins.
1Partha Mukhopadhyay, PhD, former Postdoctoral Fellow
2April Reynolds, BS, former Predoctoral Fellow
COLLABORATORS
Lawrence B. Blyn, PhD, IBIS Therapeutics, Carlsbad, CA
Shuo Chen, PhD, IBIS Therapeutics, Carlsbad, CA
Ouliana Djaman, MS, University of Illinois, Urbana, IL
Susan Gottesman, PhD, Laboratory of Molecular Biology, NCI, Bethesda, MD
Ju-Gyeong Kang, PhD, Cardiovascular Branch, NHLBI, Bethesda, MD
Robert A. LaRossa, PhD, E.I. DuPont de Nemours and Company, Wilmington, DE
Seong Eon Ryu, PhD, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea
Nico Tjandra, PhD, Laboratory of Biophysical Chemistry, NHLBI, Bethesda, MD
Ming Zheng, PhD, E.I. DuPont de Nemours and Company, Wilmington, DE
For further information, contact storz@helix.nih.gov.