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Gisela Storz

Publications and source records attributed to Gisela Storz.

At least 19 recordsLinked to original sources
PubMed2006

Mutational analysis to define an activating region on the redox-sensitive transcriptional regulator OxyR.

The OxyR transcription factor is a key regulator of the Escherichia coli response to oxidative stress. Previous studies showed that OxyR binding to a target promoter enhances RNA polymerase binding and vice versa, suggesting a direct interaction between OxyR and RNA polymerase. To identify the region of OxyR that might contact RNA polymerase, we carried out alanine scanning and random mutagenesis of oxyR. The combination of these approaches led to the identification of several mutants defective in the activation of an OxyR target gene. A subset of the mutations map to the DNA-binding domain, other mutations appear to affect dimerization of the regulatory domain, while another group is suggested to affect disulfide bond formation. The two mutations, D142A and R273H, giving the most dramatic phenotype are located in a patch on the surface of the oxidized OxyR protein and possibly define an activating region on OxyR.

Amino Acid Sequence↗
PubMed2006

Modulating the outer membrane with small RNAs.

MicF, one of the first chromosomally encoded regulatory small RNAs (sRNAs) to be discovered, was found to modulate the expression of OmpF, an abundant outer membrane protein. Several recent papers have now shown that this is not an isolated case. At least five other sRNAs also regulate the synthesis of outer membrane porins, and additional sRNAs modulate the expression of other outer membrane proteins. Here we review what is known about these sRNAs and discuss the implications of this regulation.

Bacterial Outer Membrane Proteins↗
PubMed2006

Target prediction for small, noncoding RNAs in bacteria.

Many small, noncoding RNAs in bacteria act as post-transcriptional regulators by basepairing with target mRNAs. While the number of characterized small RNAs (sRNAs) has steadily increased, only a limited number of the corresponding mRNA targets have been identified. Here we present a program, TargetRNA, that predicts the targets of these bacterial RNA regulators. The program was evaluated by assessing whether previously known targets could be identified. The program was then used to predict targets for the Escherichia coli RNAs RyhB, OmrA, OmrB and OxyS, and the predictions were compared with changes in whole genome expression patterns observed upon expression of the sRNAs. Our results show that TargetRNA is a useful tool for finding mRNA targets of sRNAs, although its rate of success varies between sRNAs.

Base Pairing↗
PubMed2006

An antibody-based microarray assay for small RNA detection.

Detection of RNAs on microarrays is rapidly becoming a standard approach for molecular biologists. However, current methods frequently discriminate against structured and/or small RNA species. Here we present an approach that bypasses these problems. Unmodified RNA is hybridized directly to DNA microarrays and detected with the high-affinity, nucleotide sequence-independent, DNA/RNA hybrid-specific mouse monoclonal antibody S9.6. Subsequent reactions with a fluorescently-labeled anti-mouse IgG antibody or biotin-labeled anti-mouse IgG together with fluorescently labeled streptavidin produces a signal that can be measured in a standard microarray scanner. The antibody-based method was able to detect low abundance small RNAs of Escherichia coli much more efficiently than the commonly-used cDNA-based method. A specific small RNA was detected in amounts of 0.25 fmol (i.e. concentration of 10 pM in a 25 microl reaction). The method is an efficient, robust and inexpensive technique that allows quantitative analysis of gene expression and does not discriminate against short or structured RNAs.

Antibodies, Monoclonal↗
PubMed2005

Detection of low-level promoter activity within open reading frame sequences of Escherichia coli.

The search for promoters has largely been confined to sequences upstream of open reading frames (ORFs) or stable RNA genes. Here we used a cloning approach to discover other potential promoters in Escherichia coli. Chromosomal fragments of approximately 160 bp were fused to a promoterless lacZ reporter gene on a multi-copy plasmid. Eight clones were deliberately selected for high activity and 105 clones were selected at random. All eight of the high-activity clones carried promoters that were located upstream of an ORF. Among the randomly-selected clones, 56 had significantly elevated activity. Of these, 7 had inserts which also mapped upstream of an ORF, while 49 mapped within or downstream of ORFs. Surprisingly, the eight promoters selected for high activity matched the canonical sigma70 -35 and -10 sequences no better than sequences from the randomly-selected clones. For six of the nine most active sequences with orientations opposite to that of the ORF, chromosomal expression was detected by RT-PCR, but defined transcripts were not detected by northern analysis. Our results indicate that the E.coli chromosome carries numerous -35 and -10 sequences with weak promoter activity but that most are not productively expressed because other features needed to enhance promoter activity and transcript stability are absent.

Base Sequence↗
PubMed2005

Detection of 5'- and 3'-UTR-derived small RNAs and cis-encoded antisense RNAs in Escherichia coli.

Evidence is accumulating that small, noncoding RNAs are important regulatory molecules. Computational and experimental searches have led to the identification of approximately 60 small RNA genes in Escherichia coli. However, most of these studies focused on the intergenic regions and assumed that small RNAs were >50 nt. Thus, the previous screens missed small RNAs encoded on the antisense strand of protein-coding genes and small RNAs of <50 nt. To identify additional small RNAs, we carried out a cloning-based screen focused on RNAs of 30-65 nt. In this screen, we identified RNA species corresponding to fragments of rRNAs, tRNAs and known small RNAs. Several of the small RNAs also corresponded to 5'- and 3'-untranslated regions (UTRs) and internal fragments of mRNAs. Four of the 3'-UTR-derived RNAs were highly abundant and two showed expression patterns that differed from the corresponding mRNAs, suggesting independent functions for the 3'-UTR-derived small RNAs. We also detected three previously unidentified RNAs encoded in intergenic regions and RNAs from the long direct repeat and hok/sok elements. In addition, we identified a few small RNAs that are expressed opposite protein-coding genes and could base pair with 5' or 3' ends of the mRNAs with perfect complementarity.

3' Untranslated Regions↗
PubMed2005

An abundance of RNA regulators.

The importance of small, noncoding RNAs that act as regulators of transcription, of RNA modification or stability, and of mRNA translation is becoming increasingly apparent. Here we discuss current knowledge of regulatory RNA function and review how the RNAs have been identified in a variety of organisms. Many of the regulatory RNAs act through base-pairing interactions with target RNAs. The base-pairing RNAs can be grouped into two general classes: those that are encoded on the opposite strand of their target RNAs such that they contain perfect complementarity with their targets, and those that are encoded at separate locations on the chromosome and have imperfect base-pairing potential with their targets. Other regulatory RNAs act by modifying protein activity, in some cases by mimicking the structures of other RNA or DNA molecules.

Animals↗
PubMed2004

Redox regulation of OxyR requires specific disulfide bond formation involving a rapid kinetic reaction path.

The Escherichia coli OxyR transcription factor is activated by cellular hydrogen peroxide through the oxidation of reactive cysteines. Although there is substantial evidence for specific disulfide bond formation in the oxidative activation of OxyR, the presence of the disulfide bond has remained controversial. By mass spectrometry analyses and in vivo labeling assays we found that oxidation of OxyR in the formation of a specific disulfide bond between Cys199 and Cys208 in the wild-type protein. In addition, using time-resolved kinetic analyses, we determined that OxyR activation occurs at a rate of 9.7 s(-1). The disulfide bond-mediated conformation switch results in a metastable form that is locally strained by approximately 3 kcal mol(-1). On the basis of these observations we conclude that OxyR activation requires specific disulfide bond formation and that the rapid kinetic reaction path and conformation strain, respectively, drive the oxidation and reduction of OxyR.

DNA-Binding Proteins↗
PubMed2004

Structural basis for redox regulation of Yap1 transcription factor localization.

The ability of organisms to alter their gene expression patterns in response to environmental changes is essential for viability. A central regulator of the response to oxidative stress in Saccharomyces 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. Here we identify a redox-regulated domain of Yap1 and determine its high-resolution solution structure. In the active oxidized form, a nuclear export signal (NES) in the carboxy-terminal cysteine-rich domain is masked by disulphide-bond-mediated interactions with a conserved amino-terminal alpha-helix. Point mutations that weaken the hydrophobic interactions between the N-terminal alpha-helix and the C-terminal NES-containing domain abolished redox-regulated changes in subcellular localization of Yap1. Upon reduction of the disulphide bonds, Yap1 undergoes a change to an unstructured conformation that exposes the NES and allows redistribution to the cytoplasm. These results reveal the structural basis of redox-dependent Yap1 localization and provide a previously unknown mechanism of transcription factor regulation by reversible intramolecular disulphide bond formation.

Active Transport, Cell Nucleus↗
PubMed2004

Prominent roles of the NorR and Fur regulators in the Escherichia coli transcriptional response to reactive nitrogen species.

We examined the genomewide transcriptional responses of Escherichia coli treated with nitrosylated glutathione or the nitric oxide (NO)-generator acidified sodium nitrite (NaNO(2)) during aerobic growth. These 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, have minor roles. Moreover, additional regulators of the E. coli response to reactive nitrogen species remain to be identified because several of the induced genes were regulated normally in norR, fur, soxRS, and oxyR mutant strains. We propose that the E. coli transcriptional response to reactive nitrogen species is a composite response mediated by the modification of multiple transcription factors containing iron or redox-active cysteines, some specifically designed to sense NO and its derivatives and others that are collaterally activated by the reactive nitrogen species.

Bacterial Proteins↗
PubMed2004

Controlling mRNA stability and translation with small, noncoding RNAs.

Recent studies have led to the identification of more than 50 small regulatory RNAs in Escherichia coli. Only a subset of these RNAs has been characterized. However, it is clear that many of the RNAs, such as the MicF, OxyS, DsrA, Spot42 and RyhB RNAs, act by basepairing to activate or repress translation or to destabilize mRNAs. Basepairing between these regulatory RNAs and their target mRNAs requires the Sm-like Hfq protein which most likely functions as an RNA chaperone to increase RNA unfolding or local target RNA concentration. Here we summarize the physiological roles of the basepairing RNAs, examine their prevalence in bacteria and discuss unresolved questions regarding their mechanisms of action.

Escherichia coli↗
PubMed2004

A suf operon requirement for Fe-S cluster assembly during iron starvation in Escherichia coli.

The suf and isc operons of Escherichia coli have been implicated in Fe-S cluster assembly. However, it has been unclear why E. coli has two systems for Fe-S cluster biosynthesis. We have examined the regulatory characteristics and mutant phenotypes of both operons to discern if the two operons have redundant functions or if their cellular roles are divergent. Both operons are similarly induced by hydrogen peroxide and the iron chelator 2,2'-dipyridyl, although by different mechanisms. Regulation of the isc operon is mediated by IscR, whereas the suf operon requires OxyR and IHF for the response to oxidative stress and Fur for induction by iron starvation. Simultaneous deletion of iscS and most suf genes is synthetically lethal. However, although the suf and isc operons have overlapping functions, they act as distinct complexes because the SufS desulphurase alone cannot substitute for the IscS enzyme. In addition, suf deletion mutants are more sensitive to iron starvation than isc mutants, and the activity of the Fe-S enzyme gluconate dehydratase is diminished in the suf mutant during iron starvation. These findings are consistent with the model that the isc operon encodes the housekeeping Fe-S cluster assembly system in E. coli, whereas the suf operon is specifically adapted to synthesize Fe-S clusters when iron or sulphur metabolism is disrupted by iron starvation or oxidative stress.

Base Sequence↗
PubMed2004

MicC, a second small-RNA regulator of Omp protein expression in Escherichia coli.

In a previous bioinformatics-based search for novel small-RNA genes encoded by the Escherichia coli genome, we identified a region, IS063, located between the ompN and ydbK genes, that encodes an approximately 100-nucleotide small-RNA transcript. Here we show that the expression of this small RNA is increased at a low temperature and in minimal medium. Twenty-two nucleotides at the 5' end of this transcript have the potential to form base pairs with the leader sequence of the mRNA encoding the outer membrane protein OmpC. The deletion of IS063 increased the expression of an ompC-luc translational fusion 1.5- to 2-fold, and a 10-fold overexpression of the small RNA led to a 2- to 3-fold repression of the fusion. Deletion and overexpression of the IS063 RNA also resulted in increases and decreases, respectively, in OmpC protein levels. Taken together, these results suggest that IS063 is a regulator of OmpC expression; thus, the small RNA has been renamed MicC. The antisense regulation was further demonstrated by the finding that micC mutations were suppressed by compensatory mutations in the ompC mRNA. MicC was also shown to inhibit ribosome binding to the ompC mRNA leader in vitro and to require the Hfq RNA chaperone for its function. We suggest that the MicF and MicC RNAs act in conjunction with the EnvZ-OmpR two-component system to control the OmpF/OmpC protein ratio in response to a variety of environmental stimuli.

Base Sequence↗
PubMed2004

GadY, a small-RNA regulator of acid response genes in Escherichia coli.

A previous bioinformatics-based search for small RNAs in Escherichia coli identified a novel RNA named IS183. The gene encoding this small RNA is located between and on the opposite strand of genes encoding two transcriptional regulators of the acid response, gadX (yhiX) and gadW (yhiW). Given that IS183 is encoded in the gad gene cluster and because of its role in regulating acid response genes reported here, this RNA has been renamed GadY. We show that GadY exists in three forms, a long form consisting of 105 nucleotides and two processed forms, consisting of 90 and 59 nucleotides. The expression of this small RNA is highly induced during stationary phase in a manner that is dependent on the alternative sigma factor sigmaS. Overexpression of the three GadY RNA forms resulted in increased levels of the mRNA encoding the GadX transcriptional activator, which in turn caused increased levels of the GadA and GadB glutamate decarboxylases. A promoter mutation which abolished gadY expression resulted in a reduction in the amount of gadX mRNA during stationary phase. The gadY gene was shown to overlap the 3' end of the gadX gene, and this overlap region was found to be necessary for the GadY-dependent accumulation of gadX mRNA. We suggest that during stationary phase, GadY forms base pairs with the 3'-untranslated region of the gadX mRNA and confers increased stability, allowing for gadX mRNA accumulation and the increased expression of downstream acid resistance genes.

AraC Transcription Factor↗
PubMed2003

The redox domain of the Yap1p transcription factor contains two disulfide bonds.

The subcellular localization of the Saccharomyces cerevisiae transcription factor Yap1p is regulated by oxidation and reduction. We purified Yap1p from yeast and characterized its properties in vitro. Electrophoretic mobility shift assays showed that the purified protein can specifically bind the TRX2 target promoter. Yap1p was purified under reducing conditions, but removal of reducing agents resulted in the formation of an oxidized Yap1p species with properties similar to in vivo oxidized Yap1p. MALDI-TOF mass spectrometry analysis revealed that the oxidized form of Yap1p contains two disulfide bonds between C303-C598 and C310-C629. A stable domain of approximately 15 kDa was detected upon limited proteolysis of oxidized but not reduced Yap1p. This Yap1p protease resistant domain was purified, and MALDI-TOF mass spectrometry analysis showed that it was comprised of two separate cysteine-containing peptides of Yap1p. These peptides are separated by 250 amino acids and are joined by the C303-C598 and C310-C629 disulfide bonds. Taken together, these data suggest that the domain that controls Yap1p subcellular localization is modular and contains a redox center comprised of four cysteine residues.

Amino Acid Sequence↗
PubMed2003

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 Escherichia coli.

The sufABCDSE operon of the Gram-negative bacterium Escherichia coli is induced by oxidative stress and iron deprivation. To examine the biochemical roles of the Suf proteins, we purified all of the proteins and assayed their effect on SufS cysteine desulfurase activity. Here we report that the SufE protein can stimulate the cysteine desulfurase activity of the SufS enzyme up to 8-fold and accepts sulfane sulfur from SufS. This sulfur transfer process from SufS to SufE is sheltered from the environment based on its resistance to added reductants and on the analysis of available crystal structures of the proteins. We also found that the SufB, SufC, and SufD proteins associate in a stable complex and that, in the presence of SufE, the SufBCD complex further stimulates SufS activity up to 32-fold. Thus, the SufE protein and the SufBCD complex act synergistically to modulate the cysteine desulfurase activity of SufS. We propose that this sulfur transfer mechanism may be important for limiting sulfide release during oxidative stress conditions in vivo.

Amino Acid Sequence↗