| 1. |
- Westra, Edze R, et al.
(författare)
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H-NS-mediated repression of CRISPR-based immunity in Escherichia coli K12 can be relieved by the transcription activator LeuO
- 2010
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Ingår i: Molecular Microbiology. - : Wiley. - 0950-382X .- 1365-2958. ; 77:6, s. 1380-1393
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Tidskriftsartikel (refereegranskat)abstract
- The recently discovered prokaryotic CRISPR/Cas defence system provides immunity against viral infections and plasmid conjugation. It has been demonstrated that in Escherichia coli transcription of the Cascade genes (casABCDE) and to some extent the CRISPR array is repressed by heat-stable nucleoid-structuring (H-NS) protein, a global transcriptional repressor. Here we elaborate on the control of the E. coli CRISPR/Cas system, and study the effect on CRISPR-based anti-viral immunity. Transformation of wild-type E. coli K12 with CRISPR spacers that are complementary to phage Lambda does not lead to detectable protection against Lambda infection. However, when an H-NS mutant of E. coli K12 is transformed with the same anti-Lambda CRISPR, this does result in reduced sensitivity to phage infection. In addition, it is demonstrated that LeuO, a LysR-type transcription factor, binds to two sites flanking the casA promoter and the H-NS nucleation site, resulting in derepression of casABCDE12 transcription. Overexpression of LeuO in E. coli K12 containing an anti-Lambda CRISPR leads to an enhanced protection against phage infection. This study demonstrates that in E. coli H-NS and LeuO are antagonistic regulators of CRISPR-based immunity.
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| 2. |
- Amlinger, Lina, et al.
(författare)
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Fluorescent CRISPR Adaptation Reporter for rapid quantification of spacer acquisition
- 2017
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Ingår i: Scientific Reports. - : NATURE PUBLISHING GROUP. - 2045-2322. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- CRISPR-Cas systems are adaptive prokaryotic immune systems protecting against horizontally transferred DNA or RNA such as viruses and other mobile genetic elements. Memory of past invaders is stored as spacers in CRISPR loci in a process called adaptation. Here we developed a novel assay where spacer integration results in fluorescence, enabling detection of memory formation in single cells and quantification of as few as 0.05% cells with expanded CRISPR arrays in a bacterial population. Using this fluorescent CRISPR Adaptation Reporter (f-CAR), we quantified adaptation of the two CRISPR arrays of the type I-E CRISPR-Cas system in Escherichia coli, and confirmed that more integration events are targeted to CRISPR-II than to CRISPR-I. The f-CAR conveniently analyzes and compares many samples, allowing new insights into adaptation. For instance, we show that in an E. coli culture the majority of acquisition events occur in late exponential phase.
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| 3. |
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| 4. |
- Baranowska, Izabella, et al.
(författare)
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Sensory ataxic neuropathy in golden retriever dogs is caused by a deletion in the mitochondrial tRNATyr gene
- 2009
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Ingår i: PLoS Genetics. - : Public Library of Science (PLoS). - 1553-7390 .- 1553-7404. ; 5:5, s. e1000499-
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Tidskriftsartikel (refereegranskat)abstract
- Sensory ataxic neuropathy (SAN) is a recently identified neurological disorder in golden retrievers. Pedigree analysis revealed that all affected dogs belong to one maternal lineage, and a statistical analysis showed that the disorder has a mitochondrial origin. A one base pair deletion in the mitochondrial tRNA(Tyr) gene was identified at position 5304 in affected dogs after re-sequencing the complete mitochondrial genome of seven individuals. The deletion was not found among dogs representing 18 different breeds or in six wolves, ruling out this as a common polymorphism. The mutation could be traced back to a common ancestor of all affected dogs that lived in the 1970s. We used a quantitative oligonucleotide ligation assay to establish the degree of heteroplasmy in blood and tissue samples from affected dogs and controls. Affected dogs and their first to fourth degree relatives had 0-11% wild-type (wt) sequence, while more distant relatives ranged between 5% and 60% wt sequence and all unrelated golden retrievers had 100% wt sequence. Northern blot analysis showed that tRNA(Tyr) had a 10-fold lower steady-state level in affected dogs compared with controls. Four out of five affected dogs showed decreases in mitochondrial ATP production rates and respiratory chain enzyme activities together with morphological alterations in muscle tissue, resembling the changes reported in human mitochondrial pathology. Altogether, these results provide conclusive evidence that the deletion in the mitochondrial tRNA(Tyr) gene is the causative mutation for SAN.
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| 5. |
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| 6. |
- Berghoff, Bork A., et al.
(författare)
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RNA-based regulation in type I toxin-antitoxin systems and its implication for bacterial persistence
- 2017
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Ingår i: Current Genetics. - : Springer Science and Business Media LLC. - 0172-8083 .- 1432-0983. ; 63:6, s. 1011-1016
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Forskningsöversikt (refereegranskat)abstract
- Bacterial dormancy is a valuable survival strategy upon challenging environmental conditions. Dormant cells tolerate the consequences of high stress levels and may re-populate the environment upon return to favorable conditions. Antibiotic-tolerant bacteria-termed persisters-regularly cause relapsing infections, increase the likelihood of antibiotic resistance, and, therefore, earn increasing attention. Their generation often depends on toxins from chromosomal toxin-antitoxin systems. Here, we review recent insights concerning RNA-based control of toxin synthesis, and discuss possible implications for persister generation.
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| 7. |
- Berghoff, Bork A., et al.
(författare)
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RNA-sequence data normalization through in silico prediction of reference genes : the bacterial response to DNA damage as case study
- 2017
-
Ingår i: BioData Mining. - : Springer Science and Business Media LLC. - 1756-0381. ; 10
-
Tidskriftsartikel (refereegranskat)abstract
- Background: Measuring how gene expression changes in the course of an experiment assesses how an organism responds on a molecular level. Sequencing of RNA molecules, and their subsequent quantification, aims to assess global gene expression changes on the RNA level (transcriptome). While advances in high-throughput RNA-sequencing (RNA-seq) technologies allow for inexpensive data generation, accurate post-processing and normalization across samples is required to eliminate any systematic noise introduced by the biochemical and/or technical processes. Existing methods thus either normalize on selected known reference genes that are invariant in expression across the experiment, assume that the majority of genes are invariant, or that the effects of up-and down-regulated genes cancel each other out during the normalization.Results: Here, we present a novel method, moose(2), which predicts invariant genes in silico through a dynamic programming (DP) scheme and applies a quadratic normalization based on this subset. The method allows for specifying a set of known or experimentally validated invariant genes, which guides the DP. We experimentally verified the predictions of this method in the bacterium Escherichia coli, and show how moose(2) is able to (i) estimate the expression value distances between RNA-seq samples, (ii) reduce the variation of expression values across all samples, and (iii) to subsequently reveal new functional groups of genes during the late stages of DNA damage. We further applied the method to three eukaryotic data sets, on which its performance compares favourably to other methods. The software is implemented in C++ and is publicly available from http://grabherr.github.io/moose2/.Conclusions: The proposed RNA-seq normalization method, moose(2), is a valuable alternative to existing methods, with two major advantages: (i) in silico prediction of invariant genes provides a list of potential reference genes for downstream analyses, and (ii) non-linear artefacts in RNA-seq data are handled adequately to minimize variations between replicates.
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| 8. |
- Berghoff, Bork A., et al.
(författare)
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Two regulatory RNA elements affect TisB-dependent depolarization and persister formation
- 2017
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Ingår i: Molecular Microbiology. - : WILEY. - 0950-382X .- 1365-2958. ; 103:6, s. 1020-1033
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Tidskriftsartikel (refereegranskat)abstract
- Bacterial survival strategies involve phenotypic diversity which is generated by regulatory factors and noisy expression of effector proteins. The question of how bacteria exploit regulatory RNAs to make decisions between phenotypes is central to a general understanding of these universal regulators. We investigated the TisB/IstR-1 toxin-antitoxin system of Escherichia coli to appreciate the role of the RNA antitoxin IstR-1 in TisB-dependent depolarization of the inner membrane and persister formation. Persisters are phenotypic variants that have become transiently drug-tolerant by arresting growth. The RNA antitoxin IstR-1 sets a threshold for TisB-dependent depolarization under DNA-damaging conditions, resulting in two sub-populations: polarized and depolarized cells. Furthermore, our data indicate that an inhibitory 5 UTR structure in the tisB mRNA serves as a regulatory RNA element that delays TisB translation to avoid inappropriate depolarization when DNA damage is low. Investigation of the persister sub-population further revealed that both regulatory RNA elements affect persister levels as well as persistence time. This work provides an intriguing example of how bacteria exploit regulatory RNAs to control phenotypic heterogeneity.
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| 9. |
- Darfeuille, Fabien, et al.
(författare)
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An antisense RNA inhibits translation by competing with standby ribosomes
- 2007
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Ingår i: Molecular Cell. - : Elsevier BV. - 1097-2765 .- 1097-4164. ; 26:3, s. 381-392
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Tidskriftsartikel (refereegranskat)abstract
- Most antisense RNAs in bacteria inhibit translation by competing with ribosomes for translation initiation regions (TIRs) on nascent mRNA. We propose a mechanism by which an antisense RNA inhibits translation without binding directly to a TIR. The tisAB locus encodes an SOS-induced toxin, and IstR-1 is the antisense RNA that counteracts toxicity. We show that full-length tisAB mRNA (+1) is translationally inactive and endonucleolytic processing produces an active mRNA (+42). IstR-1 binding inhibits translation of this mRNA, and subsequent RNase III cleavage generates a truncated, inactive mRNA (+106). In vitro translation, toeprinting, and structure mapping suggest that active, but not inactive, tisAB mRNAs contain an upstream ribosome loading or “standby” site. Standby binding is required for initiation at the highly structured tisB TIR. This may involve ribosome sliding to a transiently open tisB TIR. IstR-1 competes with ribosomes by base pairing to the standby site located 100 nucleotides upstream.
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| 10. |
- Di Martino, Maria Letizia, et al.
(författare)
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One Gene and Two Proteins : a Leaderless mRNA Supports the Translation of a Shorter Form of the Shigella VirF Regulator
- 2016
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Ingår i: mBio. - 2161-2129 .- 2150-7511. ; 7:6
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Tidskriftsartikel (refereegranskat)abstract
- VirF, an AraC-like activator, is required to trigger a regulatory cascade that initiates the invasive program of Shigella spp., the etiological agents of bacillary dysentery in humans. VirF expression is activated upon entry into the host and depends on many environmental signals. Here, we show that the virF mRNA is translated into two proteins, the major form, VirF(30) (30 kDa), and the shorter VirF(21) (21 kDa), lacking the N-terminal segment. By site-specific mutagenesis and toeprint analysis, we identified the translation start sites of VirF(30) and VirF(21) and showed that the two different forms of VirF arise from differential translation. Interestingly, in vitro and in vivo translation experiments showed that VirF(21) is also translated from a leaderless mRNA (llmRNA) whose 5' end is at position +309/+310, only 1 or 2 nucleotides upstream of the ATG84 start codon of VirF(21). The llmRNA is transcribed from a gene-internal promoter, which we identified here. Functional analysis revealed that while VirF(30) is responsible for activation of the virulence system, VirF(21) negatively autoregulates virF expression itself. Since VirF(21) modulates the intracellular VirF levels, this suggests that transcription of the llmRNA might occur when the onset of the virulence program is not required. We speculate that environmental cues, like stress conditions, may promote changes in virF mRNA transcription and preferential translation of llmRNA. IMPORTANCE Shigella spp. are a major cause of dysentery in humans. In bacteria of this genus, the activation of the invasive program involves a multitude of signals that act on all layers of the gene regulatory hierarchy. By controlling the essential genes for host cell invasion, VirF is the key regulator of the switch from the noninvasive to the invasive phenotype. Here, we show that the Shigella virF gene encodes two proteins of different sizes, VirF(30) and VirF(21), that are functionally distinct. The major form, VirF(30), activates the genes necessary for virulence, whereas the minor VirF(21), which shares the C-terminal two-thirds of VirF(30), negatively autoregulates virF expression itself. VirF(21) is transcribed from a newly identified gene-internal promoter and, moreover, is translated from an unusual leaderless mRNA. The identification of a new player in regulation adds complexity to the regulation of the Shigella invasive process and may help development of new therapies for shigellosis.
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| 11. |
- Djupedal, Ingela, et al.
(författare)
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Analysis of small RNA in fission yeast; centromeric siRNAs are potentially generated through a structured RNA
- 2009
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Ingår i: EMBO Journal. - : Wiley. - 0261-4189 .- 1460-2075. ; 28:24, s. 3832-3844
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Tidskriftsartikel (refereegranskat)abstract
- The formation of heterochromatin at the centromeres in fission yeast depends on transcription of the outer repeats. These transcripts are processed into siRNAs that target homologous loci for heterochromatin formation. Here, high throughput sequencing of small RNA provides a comprehensive analysis of centromere-derived small RNAs. We found that the centromeric small RNAs are Dcr1 dependent, carry 50-monophosphates and are associated with Ago1. The majority of centromeric small RNAs originate from two remarkably well-conserved sequences that are present in all centromeres. The high degree of similarity suggests that this non-coding sequence in itself may be of importance. Consistent with this, secondary structure-probing experiments indicate that this centromeric RNA is partially double-stranded and is processed by Dicer in vitro. We further demonstrate the existence of small centromeric RNA in rdp1D cells. Our data suggest a pathway for siRNA generation that is distinct from the well-documented model involving RITS/RDRC. We propose that primary transcripts fold into hairpin-like structures that may be processed by Dcr1 into siRNAs, and that these siRNAs may initiate heterochromatin formation independent of RDRC activity. The EMBO Journal (2009) 28, 3832-3844. doi: 10.1038/emboj.2009.351; Published online 26 November 2009
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| 12. |
- Felletti, Michele, et al.
(författare)
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A nascent polypeptide sequence modulates DnaA translation elongation in response to nutrient availability
- 2021
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Ingår i: eLIFE. - : eLife Sciences Publications Ltd. - 2050-084X. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- The ability to regulate DNA replication initiation in response to changing nutrient conditions is an important feature of most cell types. In bacteria, DNA replication is triggered by the initiator protein DnaA, which has long been suggested to respond to nutritional changes; nevertheless, the underlying mechanisms remain poorly understood. Here, we report a novel mechanism that adjusts DnaA synthesis in response to nutrient availability in Caulobacter crescentus. By performing a detailed biochemical and genetic analysis of the dnaA mRNA, we identified a sequence downstream of the dnaA start codon that inhibits DnaA translation elongation upon carbon exhaustion. Our data show that the corresponding peptide sequence, but not the mRNA secondary structure or the codon choice, is critical for this response, suggesting that specific amino acids in the growing DnaA nascent chain tune translational efficiency. Our study provides new insights into DnaA regulation and highlights the importance of translation elongation as a regulatory target. We propose that translation regulation by nascent chain sequences, like the one described, might constitute a general strategy for modulating the synthesis rate of specific proteins under changing conditions.
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| 13. |
- Fender, Aurelie, et al.
(författare)
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RNAs actively cycle on the Sm-like protein Hfq
- 2010
-
Ingår i: Genes & Development. - : Cold Spring Harbor Laboratory. - 0890-9369 .- 1549-5477. ; 24:23, s. 2621-2626
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Tidskriftsartikel (refereegranskat)abstract
- Hfq, a protein required for small RNA (sRNA)-mediated regulation in bacteria, binds RNA with low-nanomolar K-d values and long half-lives of complexes (>100 min). This cannot be reconciled with the 1-2-min response time of regulation in vivo. We show that RNAs displace each other on Hfq on a short time scale by RNA concentration-driven (active) cycling. Already at submicromolar concentrations of competitor RNA, half-lives of RNA-Hfq complexes are approximate to 1 min. We propose that competitor RNA associates transiently with RNA-Hfq complexes, RNAs exchange binding sites, and one of the RNAs eventually dissociates. This solves the "strong binding-high turn-over" paradox and permits efficient use of the Hfq pool.
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| 14. |
- Hoekzema, Mirthe, et al.
(författare)
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Hfq-dependent mRNA unfolding promotes sRNA-based inhibition of translation
- 2019
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Ingår i: EMBO Journal. - : EMBO Press. - 0261-4189 .- 1460-2075. ; 38:7
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Tidskriftsartikel (refereegranskat)abstract
- Small RNAs post-transcriptionally regulate many processes inbacteria. Base-pairing of sRNAs near ribosome-binding sites inmRNAs inhibits translation, often requiring the RNA chaperoneHfq. In the canonical model, Hfq simultaneously binds sRNAs andmRNA targets to accelerate pairing. Here, we show that theEscher-ichia colisRNAs OmrA and OmrB inhibit translation of the diguany-late cyclase DgcM (previously: YdaM), a player in biofilmregulation. In OmrA/B repression ofdgcM, Hfq is not required as anRNA interaction platform, but rather unfolds an inhibitory RNAstructure that impedes OmrA/B binding. This restructuring involvesdistal face binding of Hfq and is supported by RNA structuremapping. A corresponding mutant protein cannot support inhibi-tionin vitroandin vivo; proximal and rim mutations have negligi-ble effects. Strikingly, OmrA/B-dependent translational inhibitionin vitrois restored, in complete absence of Hfq, by a deoxyoligori-bonucleotide that base-pairs to the biochemically mapped Hfq siteindgcMmRNA. We suggest that Hfq-dependent RNA structureremodeling can promote sRNA access, which represents a mecha-nism distinct from an interaction platform model.
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| 15. |
- Hoekzema, Mirthe
(författare)
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Small RNAs, Big Consequences : Post-transcriptional Regulation and Adaptive Immunity in Bacteria
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- It is nowadays widely accepted that non-coding RNAs play important roles in post-transcriptional regulation of genes in all kingdoms of life. In bacteria, the largest group of RNA regulators are the small RNAs (sRNAs). Almost all sRNAs act through anti-sense base-pairing with target mRNAs, and by doing so regulate their translation and/or stability. As important modulators of gene expression, sRNAs are involved in all aspects of bacterial physiology. My studies aimed to deepen our understanding of the mechanisms behind sRNA-mediated gene regulation. We have shown that translation of the di-guanylate-cyclase YdaM, a major player in the biofilm regulatory cascade, is repressed by the sRNAs OmrA and OmrB. OmrAB require the RNA chaperone protein Hfq for efficient regulation. Interestingly, our results suggest a non-canonical mechanism for Hfq-mediated ydaM-OmrA/B base-pairing. Instead of serving as RNA interaction platform, Hfq restructures the ydaM mRNA to enable sRNA binding. We also addressed the question of how bacteria utilize regulatory RNAs to create phenotypic heterogeneity by studying the role of the tisB/istR-1 type 1 toxin-antitoxin system in SOS-induced persister cell formation in E. coli.In addition, I have investigated the prokaryotic CRISPR-Cas immune system, which has led to the development of two molecular tools. The CRISPR-Cas adaptive immune system consists of a CRISPR array, where palindromic repeats are interspaced by unique spacer sequences derived from foreign genetic elements, and the CRISPR-associated (Cas) proteins. In the adaptation stage, memory is created by insertion of spacer sequences into the CRISPR array. We developed a fluorescent reporter that accurately and sensitively detects spacer integration events (denoted: “acquisition”) in single cells and in real-time. In the effector stage of immunity, crRNAs, consisting of one spacer-repeat unit, associate with the Cas proteins to form a ribonucleoprotein complex that surveys the cell for invader DNA. Target identification occurs by base-pairing between the crRNA and the complementary sequence in the target nucleic acid, which triggers degradation. We repurposed the E. coli type I-E CRISPR-Cas effector complex Cascade for specific reprogrammable transcriptional gene silencing.The studies presented herein thus contributes to our understanding of RNA-based target identification for gene regulation and adaptive immunity.
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| 16. |
- Holmqvist, Erik, 1977-, et al.
(författare)
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A mixed double negative feedback loop between the sRNA MicF and the global regulator Lrp
- 2012
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Ingår i: Molecular Microbiology. - : Wiley-Blackwell. - 0950-382X .- 1365-2958. ; 84:3, s. 414-427
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Tidskriftsartikel (refereegranskat)abstract
- Roughly 10% of all genes in Escherichia coli are controlled by the global transcription factor Lrp, which responds to nutrient availability. Bioinformatically, we identified lrp as one of several putative targets for the sRNA MicF, which is transcriptionally downregulated by Lrp. Deleting micF results in higher Lrp levels, while overexpression of MicF inhibits Lrp synthesis. This effect is by antisense; mutations in the predicted interaction region relieve MicF-dependent repression of Lrp synthesis, and regulation is restored by compensatory mutations. In vitro, MicF sterically interferes with initiation complex formation and inhibits lrp mRNA translation. In vivo, MicF indirectly activates genes in the Lrp regulon by repressing Lrp, and causes severely impaired growth in minimal medium, a phenotype characteristic of lrp deletion strains. The double negative feedback between MicF and Lrp may promote a switch for adequate Lrp-dependent adaptation to nutrient availability. Lrp adds to the growing list of transcription factors that are targeted by sRNAs, thus indicating that perhaps the majority of all bacterial genes may be directly or indirectly controlled by sRNAs.
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| 17. |
- Holmqvist, Erik, 1977-, et al.
(författare)
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Impact of bacterial sRNAs in stress responses
- 2017
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Ingår i: Biochemical Society Transactions. - 0300-5127 .- 1470-8752. ; 45, s. 1203-1212
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Forskningsöversikt (refereegranskat)abstract
- Bacterial life is harsh and involves numerous environmental and internal challenges that are perceived as stresses. Consequently, adequate responses to survive, cope with, and counteract stress conditions have evolved. In the last few decades, a class of small, non-coding RNAs (sRNAs) has been shown to be involved as key players in stress responses. This review will discuss - primarily from an enterobacterial perspective - selected stress response pathways that involve antisense-type sRNAs. These include themes of how bacteria deal with severe envelope stress, threats of DNA damage, problems with poisoning due to toxic sugar intermediates, issues of iron homeostasis, and nutrient limitation/starvation. The examples discussed highlight how stress relief can be achieved, and how sRNAs act mechanistically in regulatory circuits. For some cases, we will propose scenarios that may suggest why contributions from post-transcriptional control by sRNAs, rather than transcriptional control alone, appear to be a beneficial and universally selected feature.
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| 18. |
- Holmqvist, Erik, 1977-
(författare)
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Macromolecular Matchmaking : Mechanisms and Biology of Bacterial Small RNAs
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cells sense the properties of the surrounding environment and convert this information into changes in gene expression. Bacteria are, in contrast to many multi-cellular eukaryotes, remarkable in their ability to cope with rapid environmental changes and to endure harsh and extreme milieus. Previously, control of gene expression was thought to be carried out exclusively by proteins. However, it is now clear that small regulatory RNAs (sRNA) also carry out gene regulatory functions. Bacteria such as E. coli harbor a large class of sRNAs that bind to mRNAs to alter translation and/or mRNA stability.By identifying mRNAs that are targeted by sRNAs, my studies have broadened the understanding of the mechanisms that underlie sRNA-dependent gene regulation, and have shed light on the impact that this type of regulation has on bacterial physiology. Control of gene expression often relies on the interplay of many regulators. This interplay is exemplified by our discovery of mutual regulation between the sRNA MicF and the globally acting transcription factor Lrp. Through double negative feedback, these two regulators respond to nutrient availability in the environment which results in reprogramming of downstream gene expression. We have also shown that both the transcription factor CsgD, and the anti-sigma factor FlgM, are repressed by the two sRNAs OmrA and OmrB, suggesting that these sRNAs are important players in the complex regulation that allow bacteria to switch between motility and sessility. Bacterial populations of genetically identical individuals show phenotypic variations when switching to the sessile state due to bistability in gene expression. While bistability has previously been demonstrated to arise from stochastic fluctuations in transcription, our results suggest that bistability possibly may arise from sRNA-dependent regulatory events also on the post-transcriptional level.
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| 19. |
- Holmqvist, Erik, et al.
(författare)
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Massive functional mapping of a 5'-UTR by saturation mutagenesis, phenotypic sorting and deep sequencing
- 2013
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 41:12, s. e122-
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Tidskriftsartikel (refereegranskat)abstract
- We present here a method that enables functional screening of large number of mutations in a single experiment through the combination of random mutagenesis, phenotypic cell sorting and high-throughput sequencing. As a test case, we studied post-transcriptional gene regulation of the bacterial csgD messenger RNA, which is regulated by a small RNA (sRNA). A 109 bp sequence within the csgD 5'-UTR, containing all elements for expression and sRNA-dependent control, was mutagenized close to saturation. We monitored expression from a translational gfp fusion and collected fractions of cells with distinct expression levels by fluorescence-activated cell sorting. Deep sequencing of mutant plasmids from cells in different activity-sorted fractions identified functionally important positions in the messenger RNA that impact on intrinsic (translational activity per se) and extrinsic (sRNA-based) gene regulation. The results obtained corroborate previously published data. In addition to pinpointing nucleotide positions that change expression levels, our approach also reveals mutations that are silent in terms of gene expression and/or regulation. This method provides a simple and informative tool for studies of regulatory sequences in RNA, in particular addressing RNA structure-function relationships (e.g. sRNA-mediated control, riboswitch elements). However, slight protocol modifications also permit mapping of functional DNA elements and functionally important regions in proteins.
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| 20. |
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| 21. |
- Holmqvist, Erik, et al.
(författare)
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The sRNA MicF targets its own regulator Lrp and promotes a positive feedback loop
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- As much as 10% of all genes in Escherichia coli are controlled by the global transcription factor Lrp, whose expression changes depending on the nutritional status of the environment. The output of Lrp regulation can be modulated by cellular leucine, which either enhances or reverses the effect on Lrp-targeted promoters. In a bioinformatic search for sRNA targets, lrp was identified as a putative target for the MicF sRNA, whose expression is negatively regulated by Lrp. A deletion of micF resulted in higher Lrp levels, while overexpression of MicF strongly inhibited Lrp expression. Mutations in the predicted interaction sequence of MicF and lrp relieved MicF-dependent repression of Lrp synthesis, both in vivo and in vitro. The predicted base-pairing interaction was supported by structural probing. Additionally, we show that MicF specifically interferes with initiating ribosomes on the lrp mRNA in vitro. In vivo, MicF-dependent inhibition of Lrp synthesis resulted in increased expression of the livJ gene, a member of the Lrp regulon. Finally, MicF was shown to increase its own expression through Lrp, creating a positive feedback loop. These findings contribute to the understanding of Lrp regulation in particular and the involvement of sRNAs in regulatory networks in general.
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| 22. |
- Ivanova, Natalia, et al.
(författare)
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Structure probing of tmRNA in distinct stages of trans-translation
- 2007
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Ingår i: RNA. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 13:5, s. 713-722
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Tidskriftsartikel (refereegranskat)abstract
- Ribosomes stalled on problematic mRNAs in bacterial cells can be rescued by transfer-messenger RNA (tmRNA), its helperprotein (small protein B, SmpB), and elongation factor Tu (EF-Tu) through a mechanism called trans-translation. In this work weused lead(II) footprinting to probe the interactions of tmRNA with SmpB and other components of the translation machinery atdifferent steps of the trans-translation cycle. Ribosomes with a short nascent peptide stalled on a truncated mRNA were reactedwith Ala-tmRNA EF-Tu GTP, SmpB, and other translation components to initiate and execute trans-translation. Free tmRNA was d dprobed with lead(II) acetate with and without SmpB, and ribosome bound tmRNA was probed in one of four different trans-translation states stabilized by antibiotic addition or selective exclusion of translation components. For comparison, we alsoanalyzed lead(II) cleavage patterns of tmRNA in vivo in a wild-type as well as in an SmpB-deficient Escherichia coli strain. Weobserved some specific cleavages/protections in tmRNA for the individual steps of trans-translation, but the overall tmRNAconformation appeared to be similar in the stages analyzed. Our findings suggest that, in vivo, a dominant fraction of tmRNA isin complex with SmpB and that, in vitro, SmpB remains tmRNA bound at the initial steps of trans-translation.
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| 23. |
- Liao, Zhen, 1983-
(författare)
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A small amoeba at the crossroads of the big RNAi world : MicroRNA biogenesis and Argonaute function in Dictyostelium discoideum
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Small non-coding RNA (ncRNA) mediated gene silencing, known as RNAi, is a key regulatory mechanism of gene expression in eukaryotes. MicroRNAs (miRNA), one major type of small ncRNAs, are about 21nt long and bound by Argonaute proteins. This RNA-protein complex, called RISC, silences post-transcriptionally target mRNAs containing partial or full complementary sequence to the miRNA. MiRNAs are generated from step-wise endonucleolytic cleavages of long primary transcripts (pri-miRNAs) by RNase III nucleases. Biogenesis of miRNAs differs between uni- and multicellular eukaryotes, and also between plants and animals. In this thesis, I aimed to understand miRNA maturation in the social amoeba Dictyostelium discoideum, which stands at the crossroads between these phylogenetically distant groups. We showed that Dicer-like protein DrnB is essential for global maturation of D. discoideum miRNAs. The study of two pri-miRNAs revealed the conserved 5’ m7G-cap structures, but different 3’end formation from each other, and also from canonical miRNAs in plants and animals. In agreement with its evolutionary position, D. discoideum miRNA biogenesis showed unique and also shared features with both life groups.D. discoideum grows as a unicellular organism, but can switch to a multicellular development upon starvation. Most miRNAs, many other small ncRNAs, and Argonaute proteins, the core effectors of the RISC, are differentially expressed during development, indicative of a crucial role of RNAi mediated regulation throughout D. discoideum life cycle. Among the five Argonaute homologs in D. discoideum, I investigated the functions of three members, e.g. AgnB, C and E. Judging from their subcellular localization, the phenotypic consequences and transcriptional alteration resulting from single Argonaute gene deletion, our results suggested different roles of AgnB, C and E. Possibly AgnB associates with miRNAs and regulates gene expression post-transcriptionally; while AgnC seems to be involved in nuclear RNAi. Finally, the cytoplasmic AgnE inhibits D. discoideum cell growth and regulates developmental timing via an unknown mechanism.My thesis work expands our knowledge on D. discoideum RNAi with focuses on miRNA biogenesis and potential function of Argonaute proteins and, all together, sheds lights on the evolution of miRNA and RNAi.
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| 24. |
- Lindell, Magnus, et al.
(författare)
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Lead(II) cleavage analysis of RNase P RNA in vivo.
- 2005
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Ingår i: RNA. - 1355-8382. ; 11:9, s. 1348-54
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Tidskriftsartikel (refereegranskat)abstract
- The overall conformation of M1 RNA, the catalytic RNA subunit of RNase P in Escherichia coli, was analyzed in vivo and, in the presence of the C5 protein subunit, in vitro by lead(II) acetate probing. The partial cleavage patterns obtained are congruent with previous structure mapping performed in vitro. Most of the known major and minor cleavages in M1 RNA were supported and could be mapped onto a secondary structure model. The data obtained indicate that C5 has only minor effects on the overall structure of the RNA subunit. The similar cleavage patterns obtained in vitro and in vivo furthermore suggest that the intracellular environment does not greatly alter the overall conformation of M1 RNA within the holoenzyme complex. Moreover, our data indicate that M1 RNA in vivo is present in at least two states-the major fraction is bound to tRNA substrates and a minor fraction is substrate free. Finally, both in this and previous work we found that lead(II) probing data from in vivo experiments conducted on longer RNAs (tmRNA and M1 RNA) generally gives superior resolution compared to parallel in vitro experiments. This may reflect the absence of alternative conformers present in vitro and the more natural state of these RNAs in the cell due to proper, co-transcriptional folding pathways and possibly the presence of RNA chaperones.
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| 25. |
- Melin, Petter, et al.
(författare)
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Changes in Aspergillus nidulans gene expression induced by bafilomycin, a Streptomyces-produced antibiotic
- 1999
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Ingår i: Microbiology. - : Microbiology Society. - 1350-0872 .- 1465-2080. ; 145:5, s. 1115-1122
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Tidskriftsartikel (refereegranskat)abstract
- In natural environments bacteria and filamentous fungi often compete for the same resources. Consequently, production of antibiotic secondary metabolites and defence mechanisms against these compounds have evolved in these organisms. An experimental model has been developed to study the response in fungi exposed to one such antibiotic. The filamentous fungus Aspergillus nidulans was treated with bafilomycin B-1, a Streptomyces-produced antibiotic which reduces radial growth rate and induces morphological changes in fungi. mRNA differential display was used to study changes in fungal gene expression. For five genes, changes in abundance of the corresponding mRNAs, directly or indirectly caused by bafilomycin, were observed. Of these, three were up-regulated and two repressed. With four of these the change in mRNA abundance measured ranged from 10- to 60-fold. However, for one gene the mRNA was only detected after bafilomycin treatment. One of the downregulated mRNAs encodes ASPND1, a glycoprotein that belongs to a known family of antigens identified in aspergilloma patients. One up-regulated mRNA shows sequence similarities, at the amino acid level, with a cell-wall protein of Saccharomyces cerevisiae. The remaining three genes were also cloned and sequenced; their sequences do not correspond to known genes in A. nidulans, and no similarities with published nucleotide or protein sequences in other organisms were found. These results indicate the feasibility of using mRNA differential display to study interactions between bacteria and filamentous fungi.
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| 26. |
- Melin, Petter, et al.
(författare)
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Characterization of phiA, a gene essential for phialide development in Aspergillus nidulans
- 2003
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Ingår i: Fungal Genetics and Biology. - : Academic Press. - 1087-1845 .- 1096-0937. ; 40:3, s. 234-241
-
Tidskriftsartikel (refereegranskat)abstract
- We have previously identified genes and proteins involved in the fungal response to the Streptomyces-produced antibiotics, bafilomycin 131 and concanamycin A, known inhibitors of V-ATPases. Using mRNA differential display we identified an Aspergillus nidulans gene with 30-fold up-regulated expression in the presence of bafilomycin. This gene, here denoted phiA, and its gene product, were further characterized by targeted gene disruption and immunohistochemistry. Phenotypically, the phiA mutation resulted in reduced growth and severely reduced sporulation. The abnormality could be traced to the phialides, which divided several times instead of forming a single flask-shaped cell. The importance of phiA for phialide and conidium development was supported by immunohistochemistry experiments that showed the protein to be mainly present in these two cell types. Attempts to relate phiA to inhibition of V-ATPases did not result in unambiguous conclusions, but suggest the possibility that changed expression of phiA is correlated with growth arrest caused by inhibited V-ATPases.
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| 27. |
- Park, Hyun-Sook, et al.
(författare)
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Novel role for a bacterial nucleoid protein in translation of mRNAs with suboptimal ribosome-binding sites
- 2010
-
Ingår i: Genes & Development. - : Cold Spring Harbor Laboratory. - 0890-9369 .- 1549-5477. ; 24:13, s. 1345-1350
-
Tidskriftsartikel (refereegranskat)abstract
- In Escherichia coli, the major nucleoid protein H-NS limits transcription by acting as a repressor or transcriptional silencer, presumably by its ability to close the looped chromosome domains in the nucleoid through DNA-protein-DNA bridging. Here, we demonstrate the direct involvement of H-NS as a positive factor stimulating translation of the malT mRNA. In vitro studies showed that H-NS facilitates a repositioning of the 30S preinitiation complex on the malT mRNA. H-NS stimulation of translation depended on the AU-rich -35 to -40 region of the mRNA. Several additional examples were found demonstrating a novel function for H-NS in translation of genes with suboptimal ribosome-binding sequences.
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| 28. |
- Rizvanovic, Alisa, et al.
(författare)
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The RNA-binding protein ProQ promotes antibiotic persistence in Salmonella
- 2022
-
Ingår i: Molecular and Cellular Biology. - : American Society for Microbiology. - 0270-7306 .- 1098-5549. ; 13:6
-
Tidskriftsartikel (refereegranskat)abstract
- Bacterial populations can survive the exposure to antibiotics through transient phenotypic and gene expression changes. These changes can be attributed to a small subpopulation of bacteria, giving rise to antibiotic persistence. Even though this phenomenon has been known for decades, much is still to be learnt about the mechanisms that drive persister formation. The RNA-binding protein ProQ has recently emerged as a global regulator of gene expression. Here, we show that ProQ impacts persister formation in Salmonella. ProQ contributes to growth-arrest in single cells, which are able to survive treatment with high concentrations of different antibiotics. The underlying mechanism for ProQ-dependent persister formation involves activation of the flagellar pathway. Importantly, we show that the ProQ-dependent phenotype is relevant during macrophage infection and allows Salmonella to survive the combined action of host immune defences and antibiotics. Together, our data highlights the importance of ProQ in Salmonella persistence and pathogenesis.
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| 29. |
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| 30. |
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| 31. |
- Romilly, Cedric, et al.
(författare)
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An RNA pseudoknot is essential for standby-mediated translation of the tisB toxin mRNA in Escherichia coli
- 2020
-
Ingår i: Nucleic Acids Research. - : OXFORD UNIV PRESS. - 0305-1048 .- 1362-4962. ; 48:21, s. 12336-12347
-
Tidskriftsartikel (refereegranskat)abstract
- In response to DNA damage, Escherichia coli cells activate the expression of the toxin gene tisB of the toxin-antitoxin system tisB-istR1. Of three isoforms, only the processed, highly structured +42 tisB mRNA is active. Translation requires a standby site, composed of two essential elements: a single-stranded region located 100 nucleotides upstream of the sequestered RBS, and a structure near the 5'-end of the active mRNA. Here, we propose that this 5'-structure is an RNA pseudoknot which is required for 30S and protein S1-alone binding to the mRNA. Point mutations that prevent formation of this pseudoknot inhibit formation of translation initiation complexes, impair S1 and 30S binding to the mRNA, and render the tisB mRNA non-toxic in vivo. A set of mutations created in either the left or right arm of stem 2 of the pseudoknot entailed loss of toxicity upon overexpression of the corresponding mRNA variants. Combining the matching right-left arm mutations entirely restored toxicity levels to that of the wild-type, active mRNA. Finally, since many pseudoknots have high affinity for S1, we predicted similar pseudoknots in non-homologous type I toxin-antitoxin systems that exhibit features similar to that of tisB-IstR1, suggesting a shared requirement for standby acting at great distances.
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| 32. |
- Romilly, Cedric, et al.
(författare)
-
Small RNAs OmrA and OmrB promote class III flagellar gene expression by inhibiting the synthesis of anti-Sigma factor FlgM
- 2020
-
Ingår i: RNA Biology. - : Informa UK Limited. - 1547-6286 .- 1555-8584. ; 17:6, s. 872-880
-
Tidskriftsartikel (refereegranskat)abstract
- Bacteria can move by a variety of mechanisms, the best understood being flagella-mediated motility. Flagellar genes are organized in a three-tiered cascade allowing for temporally regulated expression that involves both transcriptional and post-transcriptional control. The class I operon encodes the master regulator FlhDC that drives class II gene transcription. Class II genes include fliA and flgM, which encode the Sigma factor sigma(28), required for class III transcription, and the anti-Sigma factor FlgM, which inhibits sigma(28) activity, respectively. The flhDC mRNA is regulated by several small regulatory RNAs (sRNAs). Two of these, the sequence-related OmrA and OmrB RNAs, inhibit FlhD synthesis. Here, we report on a second layer of sRNA-mediated control downstream of FhlDC in the flagella pathway. By mutational analysis, we confirm that a predicted interaction between the conserved 5MODIFIER LETTER PRIME seed sequences of OmrA/B and the early coding sequence in flgM mRNA reduces FlgM expression. Regulation is dependent on the global RNA-binding protein Hfq. In vitro experiments support a canonical mechanism: binding of OmrA/B prevents ribosome loading and decreases FlgM protein synthesis. Simultaneous inhibition of both FlhD and FlgM synthesis by OmrA/B complicated an assessment of how regulation of FlgM alone impacts class III gene transcription. Using a combinatorial mutation strategy, we were able to uncouple these two targets and demonstrate that OmrA/B-dependent inhibition of FlgM synthesis liberates sigma(28) to ultimately promote higher expression of the class III flagellin gene fliC.
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| 33. |
- Romilly, Cedric, et al.
(författare)
-
The ribosomal protein S1-dependent standby site in tisB mRNA consists of a single-stranded region and a 5 ' structure element
- 2019
-
Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : NATL ACAD SCIENCES. - 0027-8424 .- 1091-6490. ; 116:32, s. 15901-15906
-
Tidskriftsartikel (refereegranskat)abstract
- In bacteria, stable RNA structures that sequester ribosome-binding sites (RBS) impair translation initiation, and thus protein output. In some cases, ribosome standby can overcome inhibition by structure: 30S subunits bind sequence-nonspecifically to a single-stranded region and, on breathing of the inhibitory structure, relocate to the RBS for initiation. Standby can occur over long distances, as in the active, +42 tisB mRNA, encoding a toxin. This mRNA is translationally silenced by an antitoxin sRNA, IstR-1, that base pairs to the standby site. In tisB and other cases, a direct interaction between 30S subunits and a standby site has remained elusive. Based on fluorescence anisotropy experiments, ribosome toeprinting results, in vitro translation assays, and cross-linking-immunoprecipitation (CLIP) in vitro, carried out on standby-proficient and standby-deficient tisB mRNAs, we provide a thorough characterization of the tisB standby site. 30S subunits and ribosomal protein S1 alone display high-affinity binding to standby-competent fluorescein-labeled +42 mRNA, but not to mRNAs that lack functional standby sites. Ribosomal protein S1 is essential for standby, as 30 Delta S1 subunits do not support standby-dependent toeprints and TisB translation in vitro. S1 alone- and 30S-CLIP followed by RNA-seq mapping shows that the functional tisB standby site consists of the expected single-stranded region, but surprisingly, also a 5'-end stem-loop structure. Removal of the latter by 5'-truncations, or disruption of the stem, abolishes 30S binding and standby activity. Based on the CLIP-read mapping, the long-distance standby effect in +42 tisB mRNA (similar to 100 nt) is tentatively explained by S1-dependent directional unfolding toward the downstream RBS.
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| 34. |
- Slagter-Jäger, Jacoba G., et al.
(författare)
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Loop swapping in an antisense RNA/target RNA pair changes directionality of helix progression
- 2003
-
Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 278:37, s. 35558-35563
-
Tidskriftsartikel (refereegranskat)abstract
- The binding pathway of the natural antisense RNA CopA to its target CopT proceeds through a hierarchical order of steps. It initiates by reversible loop-loop contacts followed by unidirectional helix progression into the upper stems. This involves extensive breakage of intramolecular base pairs and the subsequent formation of two intermolecular helices, B and B'. Based on the known tRNA anticodon loop structure and on results from the Sok/Hok antisense/target RNA system, it had been suggested that a U-turn (or pi-turn) in the loop of CopT might determine the directionality of helix progression. Data presented here show that the putative U-turn is one of the structural elements of antisense/target RNA pairs required to achieve fast binding kinetics. Swapping of the hypothetical U-turn structure from the target RNA to the antisense RNA retained regulatory performance in vivo and binding rates in vitro but altered the binding pathway by changing the direction in which the initiating helix was extended. In addition, our data indicate that a helical stem immediately adjacent to the target loop sequence is required to provide a scaffold for the U-turn.
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| 35. |
- Sterk, Maaike M.
(författare)
-
Ribosome standby sites and other structural aspects of translation initiation regions in Escherichia coli
- 2018
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Translation initiation, which is rate-limiting in protein synthesis, is often the step at which regulation occurs. Here, we investigated several mechanisms of translation initiation in Escherichia coli, including their control. First, we showed that translation of the transcriptional regulator CsgD is inhibited by two sRNAs through a direct antisense mechanism.In some bacterial mRNAs, the ribosome binding site (RBS) is sequestered in a stable structure, which generally generates very low protein output. Yet, these mRNAs are often efficiently translated, which suggested the requirement for “ribosome standby sites”. Here, we investigated the structure and sequence features of an effective standby site using plasmid-borne GFP reporter constructs, and showed that relatively short, single-stranded regions near a structurally sequestered RBS can profoundly increase translation rates. Both the length and the sequence of these single-stranded regions are important for standby site efficiency, and the standby site needs to be single-stranded. This work serves as a proof-of-principle study of the ribosome standby model.To investigate the sequence-dependency of standby sites further, we used an unbiased approach, creating plasmid libraries containing millions of different standby sites in the same reporter plasmid as before. Cells were sorted by fluorescence according to translational levels, and standby sites analyzed by deep sequencing. This analysis showed that efficient standby sites have a low GC-content and rarely contain Shine-Dalgarno sequences. Additionally, nucleotides near the 3’-border of the standby region affect translation efficiency more than those closer to the 5’-end. Mutational and structure-probing experiments are planned to verify these findings.
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| 36. |
- Sterk, Maaike, et al.
(författare)
-
Unstructured 5'-tails act through ribosome standby to override inhibitory structure at ribosome binding sites.
- 2018
-
Ingår i: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 46:8, s. 4188-4199
-
Tidskriftsartikel (refereegranskat)abstract
- Initiation is the rate-limiting step in translation. It is well-known that stable structure at a ribosome binding site (RBS) impedes initiation. The ribosome standby model of de Smit and van Duin, based on studies of the MS2 phage coat cistron, proposed how high translation rates can be reconciled with stable, inhibitory structures at an RBS. Here, we revisited the coat protein system and assessed the translation efficiency from its sequestered RBS by introducing standby mutations. Further experiments with gfp reporter constructs assessed the effects of 5-tails-as standby sites-with respect to length and sequence contributions. In particular, combining in vivo and in vitro assays, we can show that tails of CA-dinucleotide repeats-and to a lesser extent, AU-repeats-dramatically increase translation rates. Tails of increasing length reach maximal rate-enhancing effects at 16-18 nucleotides. These standby tails are single-stranded and do not exert their effect by structure changes in the neighboring RBS stem-loop. In vitro translation and toeprinting assays furthermore demonstrate that standby effects are exerted at the level of translation initiation. Finally, as expected, destabilizing mutations within the coat RBS indicate an interplay with the effects of standby tails.
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| 37. |
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| 38. |
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| 39. |
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| 40. |
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| 41. |
- Udekwu, Klas I, et al.
(författare)
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Hfq-dependent regulation of OmpA synthesis is mediated by an an-tisense RNA.
- 2005
-
Ingår i: Genes & Development. - : Cold Spring Harbor Laboratory. - 0890-9369 .- 1549-5477. ; 19:19, s. 2355-2366
-
Tidskriftsartikel (refereegranskat)abstract
- This paper shows that the small RNA MicA (previously SraD) is an antisense regulator of ompA in Escherichia coli. MicA accumulates upon entry into stationary phase and down-regulates the level of ompA mRNA. Regulation of ompA (outer membrane protein A), previously attributed to Hfq/mRNA binding, is lost upon deletion of the micA gene, whereas overexpression of MicA inhibits the synthesis of OmpA. In vitro, MicA binds to the ompA mRNA leader. Enzymatic and chemical probing was used to map the structures of MicA, the ompA mRNA leader, and the complex formed upon binding. MicA binding generates a footprint across the ompA Shine-Dalgarno sequence, consistent with a 12 + 4 base-pair interaction, which is additionally supported by the effect of mutations in vivo and by bioinformatics analysis of enterobacterial micA/ompA homolog sequences. MicA is conserved in many enterobacteria, as is its ompA target site. In vitro toeprinting confirmed that binding of MicA specifically interferes with ribosome binding. We propose that MicA, when present at high levels, blocks ribosome binding at the ompA translation start site, which—in line with previous work—secondarily facilitates RNase E cleavage and subsequent mRNA decay. MicA requires the presence of the Hfq protein, although the mechanistic basis for this remains unclear.
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| 42. |
- Udekwu, Klas I., et al.
(författare)
-
Sigma E controls biogenesis of the antisense RNA MicA
- 2007
-
Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 35:4, s. 1279-1288
-
Tidskriftsartikel (refereegranskat)abstract
- Adaptation stress responses in the Gram-negative bacterium Escherichia coli and its relatives involve a growing list of small regulatory RNAs (sRNAs). Previous work by us and others showed that the antisense RNA MicA downregulates the synthesis of the outer membrane protein OmpA upon entry into stationary phase. This regulation is Hfq-dependent and occurs by MicA-dependent translational inhibition which facilitates mRNA decay. In this article, we investigate the transcriptional regulation of the micA gene. Induction of MicA is dependent on the alarmone ppGpp, suggestive of alternative σ factor involvement, yet MicA accumulates in the absence of the general stress/stationary phase σS. We identified stress conditions that induce high MicA levels even during exponential growth - a phase in which MicA levels are low (ethanol, hyperosmolarity and heat shock). Such treatments are sensed as envelope stress, upon which the extracytoplasmic sigma factor σE is activated. The strict dependence of micA transcription on σE is supported by three observations. Induced overexpression of σE increases micA transcription, an ΔrpoE mutant displays undetectable MicA levels and the micA promoter has the consensus σE signature. Thus, MicA is part of the σE regulon and downregulates its target gene, ompA, probably to alleviate membrane stress.
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| 43. |
- Unoson, Cecilia, et al.
(författare)
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A small SOS-induced toxin is targeted against the inner membrane in Escherichia coli
- 2008
-
Ingår i: Molecular Microbiology. - : Wiley. - 0950-382X .- 1365-2958. ; 70:1, s. 258-70
-
Tidskriftsartikel (refereegranskat)abstract
- We previously reported on an SOS-induced toxin, TisB, in Escherichia coli and its regulation by the RNA antitoxin IstR-1. Here, we addressed the mode of action of TisB. By placing the tisB reading frame downstream of a controllable promoter on a plasmid, toxicity could be analysed in the absence of the global SOS response. Upon induction of TisB, cell growth was inhibited and plating efficiency decreased rapidly. The onset of toxicity correlated with a drastic decrease in transcription, translation and replication rates. Cellular RNA was degraded, but in vitro experiments showed that TisB did not affect translation or transcription directly. Thus, these effects are downstream consequences of membrane damage: TisB is predicted to be hydrophobic and membrane spanning, and Western analyses demonstrated that this peptide was strictly localized to the cytoplasmic membrane fraction. Membrane damage and cell killing under tisB multicopy expression are also seen by live/death staining and the formation of ghost cells. This is reminiscent of another toxin, Hok of plasmid R1, which also targets the membrane. The biological significance of the istR/tisB locus is still elusive; deletion of the entire locus gave no fitness phenotype in competition experiments.
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| 44. |
- Unoson, Cecilia, et al.
(författare)
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Dealing with stable structures at ribosome binding sites : bacterial translation and ribosome standby.
- 2007
-
Ingår i: RNA Biology. - 1547-6286. ; 4:3, s. 113-117
-
Tidskriftsartikel (refereegranskat)abstract
- Bacterial ribosomes have great difficulties to initiate translation on stable structures within mRNAs. Translational coupling and induced structure changes are strategies to open up inhibitory RNA structures encompassing ribosome binding sites (RBS). There are, however, mRNAs in which stable structures are not unfolded, but that are nevertheless efficiently initiated at high rates. de Smit and van Duin(1) proposed a "ribosome standby" model to theoretically solve this paradox: the 30S ribosome binds nonspecifically to an accessible site on the mRNA (standby site), waiting for a transient opening of a stable RBS hairpin. Upon unfolding, the 30S subunit relocates to form a productive initiation complex. Recent reports have provided experimental support for this model. This review will describe and compare two different flavors of standby sites, their properties, and their likely implications. We also discuss the possibility that ribosome standby may be a more general strategy to obtain high translation rates.
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| 45. |
- Unoson, Cecilia
(författare)
-
Small RNA-mediated Regulation of Gene Expression in Escherichia coli
- 2010
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Non-coding RNAs are highly abundant regulators of gene expression in all kingdoms of life that often play important roles in vital cellular functions. In bacteria, small regulatory RNAs (sRNAs) usually act post-transcriptionally by regulating mRNAs through base pairing within ribosome binding sites (RBS), thereby inhibiting translation initiation. tisB encodes a toxin, TisB, whose synthesis is controlled by the sRNA IstR-1. Intriguingly, IstR-1 base pairs far upstream of the RBS but nevertheless inhibits translation initiation. The tisB mRNA is unusual in that ribosomes cannot access the RBS directly, but instead need an unstructured upstream region. This is precisely where IstR-1 exerts its inhibitory effect. We propose this region to serve as a ribosome loading site (standby site) which permits ribosomes to overcome the obstacle of inhibitory RBS-containing structures. Sequence-independent ribosome binding to the standby site allows for efficient relocation to the RBS structure when it is transiently open. Thus, standby sites are translation enhancer elements. I also characterized TisB-mediated toxicity. The hydrophobic protein TisB is targeted to the inner membrane and causes damage. This decreases the intracellular ATP concentration and entails decreased replication, transcription and translation rates. It is likely that this toxin is involved in multidrug tolerance under certain conditions. We identified the sRNA MicF as a negative regulator of lrp expression. Lrp is a global transcription factor that controls genes involved in amino acid metabolism and transport of small molecules. Interestingly, Lrp also downregulates MicF. Thus, this study established that the mutual downregulation of MicF/Lrp creates a positive feedback loop which gives a switch-like behavior important for fast adaptations.
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| 46. |
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| 47. |
- Vogel, Jörg, et al.
(författare)
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RNomics in Escherichia coli detects new sRNA species and indicates parallel transcriptional output in bacteria
- 2003
-
Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 31:22, s. 6435-6443
-
Tidskriftsartikel (refereegranskat)abstract
- Recent bioinformatics‐aided searches have identified many new small RNAs (sRNAs) in the intergenic regions of the bacterium Escherichia coli. Here, a shot‐gun cloning approach (RNomics) was used to generate cDNA libraries of small sized RNAs. Besides many of the known sRNAs, we found new species that were not predicted previously. The present work brings the number of sRNAs in E.coli to 62. Experimental transcription start site mapping showed that some sRNAs were encoded from independent genes, while others were processed from mRNA leaders or trailers, indicative of a parallel transcriptional output generating sRNAs co‐expressed with mRNAs. Two of these RNAs (SroA and SroG) consist of known (THI and RFN) riboswitch elements. We also show that two recently identified sRNAs (RyeB and SraC/RyeA) interact, resulting in RNase III‐dependent cleavage. To the best of our knowledge, this represents the first case of two non‐coding RNAs interacting by a putative antisense mechanism. In addition, intracellular metabolic stabilities of sRNAs were determined, including ones from previous screens. The wide range of half‐lives (<2 to >32 min) indicates that sRNAs cannot generally be assumed to be metabolically stable. The experimental characterization of sRNAs analyzed here suggests that the definition of an sRNA is more complex than previously assumed.
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| 48. |
- Vogel, Jörg, et al.
(författare)
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Target identification of small noncoding RNAs in bacteria
- 2007
-
Ingår i: Current Opinion in Microbiology. - : Elsevier BV. - 1369-5274 .- 1879-0364. ; 10:3, s. 262-270
-
Forskningsöversikt (refereegranskat)abstract
- Small noncoding RNAs have been discovered at a staggering rate in Escherichia coli and many other bacteria. Most of the sRNAs of known function regulate gene expression by binding to specific mRNAs or proteins. Given the scores of sRNAs of unknown function, the identification of their cellular targets has become urgent. Here, we review the diverse strategies that have been used to identify and validate bacterial sRNA targets. These include the pulse-expression of sRNAs followed by global transcriptome analysis (microarrays), new biocomputational prediction algorithms, and novel gfp reporter gene fusions to validate candidate target gene regulation.
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| 49. |
- Vogel, Jörg, et al.
(författare)
-
The small RNA IstR inhibits synthesis of an SOS-induced toxic peptide
- 2004
-
Ingår i: Current Biology. - : Elsevier BV. - 0960-9822 .- 1879-0445. ; 14:24, s. 2271-2276
-
Tidskriftsartikel (refereegranskat)abstract
- More than 60 small RNAs (sRNA) have been identified in E. coli 1, 2, 3, 4, 5, 6 and 7. The functions of the majority of these sRNAs are still unclear. For the few sRNAs characterized, expression and functional studies indicate that they act under stress conditions 8, 9, 10, 11, 12, 13 and 14. Here, we describe a novel E. coli chromosome locus that is part of the SOS response to DNA damage. This locus encodes two sRNAs, IstR-1 and IstR-2, and a toxic peptide, TisB, encoded by tisAB mRNA. Transcription of tisAB and istR-2 is SOS regulated, whereas IstR-1 is present throughout growth. IstR-1 inhibits toxicity by base-pairing to a short region in the tisAB mRNA. This antisense interaction entails RNase III-dependent cleavage, thereby inactivating the mRNA for translation. In the absence of the SOS response, IstR-1 is present in high excess over its target. However, SOS induction leads to depletion of the IstR-1 pool, concomitant with accumulation of tisAB mRNA. Under such conditions, TisB exerts its toxic effect, slowing down growth. We propose that the inhibitory sRNA prevents inadvertent TisB synthesis during normal growth and, possibly, also limits SOS-induced toxicity. Our study adds the SOS regulon to the growing list of global regulatory circuits controlled by sRNA genes.
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| 50. |
- Wagner, E. Gerhart H., et al.
(författare)
-
The Length of a DNA T-Tract Modulates Expression of a Virulence-Regulating sRNA
- 2020
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Ingår i: Molecular Cell. - : Elsevier BV. - 1097-2765 .- 1097-4164. ; 80:2, s. 175-177
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Eisenbart et al. (2020) find an SSR-associated sRNA, NikS, that is subject to variable repeat-controlled expression. NikS regulates H. pylori virulence by post-transcriptionally repressing pathogenicity factors, including CagA and VacA, via base-pairing to their mRNAs.
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