| 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
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Ingår i: BioData Mining. - : Springer Science and Business Media LLC. - 1756-0381. ; 10
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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. |
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| 13. |
- 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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| 14. |
- Fender, Aurelie, et al.
(författare)
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RNAs actively cycle on the Sm-like protein Hfq
- 2010
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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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| 15. |
- Gerdes, Kenn, et al.
(författare)
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RNA antitoxins
- 2007
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Ingår i: Current Opinion in Microbiology. - : Elsevier BV. - 1369-5274 .- 1879-0364. ; 10:2, s. 117-124
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Forskningsöversikt (refereegranskat)abstract
- Recent genomic analyses revealed a surprisingly large number of toxin–antitoxin loci in free-living prokaryotes. The antitoxins are proteins or antisense RNAs that counteract the toxins. Two antisense RNA-regulated toxin–antitoxin gene families, hok/sok and ldr, are unrelated sequence-wise but have strikingly similar properties at the level of gene and RNA organization. Recently, two SOS-induced toxins were found to be regulated by RNA antitoxins. One such toxin, SymE, exhibits similarity with MazE antitoxin and, surprisingly, inhibits translation. Thus, it is possible that an ancestral antitoxin gene evolved into the present toxin gene (symE) whose translation is repressed by an RNA antitoxin (SymR).
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| 16. |
- Grantcharova, Nina, 1974-
(författare)
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Developmental Control of Cell Division in Streptomyces coelicolor
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cell division in the Gram-positive bacterium Streptomyces coelicolor starts with the assembly of the tubulin homologue FtsZ into a cytokinetic ring (the Z ring) at the site of septation. In stark contrast to the binary fission of most bacteria, the syncytial hyphal cells of S. coelicolor exploit two types of cell division with strikingly different outcomes depending on the developmental stage. The main goal of this study has been to identify developmental mechanisms that modulate this differential performance of the basic cell division machinery.By isolation and characterization of a non-sporulating ftsZ mutant, we demonstrated that the requirements for Z-ring formation differ between the two types of septation. The ftsZ17(Spo) mutation abolished septation without overtly affecting vegetative growth. This mutant was defective in the assembly of FtsZ into regularly spaced Z rings in sporogenic hyphae, suggesting that the assembly of Z rings is developmentally controlled during sporulation.An FtsZ-EGFP translational fusion was constructed and used to visualize the progression of FtsZ ring assembly in vivo. This revealed that polymerization of FtsZ occurred throughout the sporogenic cell, with no evidence for pre-determined nucleation sites, and that the placement of multiple Z rings is a dynamic process and involves remodeling of spiral-shaped FtsZ intermediates into regularly spaced rings. The dynamics of the multiple Z-rings assembly during sporulation was perturbed by the action of the protein CrgA, which is important for coordinating growth and cell division in sporogenic hyphae. CrgA was also found to affect the timing of ftsZ expression and the turnover of the FtsZ protein. S. coelicolor is the main genetic model of the streptomycetes, which are major industrial antibiotic producers. The control of cell division in these organisms differs from that of other bacteria like Escherichia coli. Thus, it is of fundamental importance to clarify how the streptomycetes reproduce themselves.
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| 17. |
- Hammann, Philippe, et al.
(författare)
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A method to map changes in bacterial surface composition induced by regulatory RNAs in Escherichia coli and Staphylococcus aureus
- 2014
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Ingår i: Biochimie. - : Elsevier BV. - 0300-9084 .- 1638-6183. ; 106C, s. 175-179
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Tidskriftsartikel (refereegranskat)abstract
- We have adapted a method to map cell surface proteins and to monitor the effect of specific regulatory RNAs on the surface composition of the bacteria. This method involves direct labeling of surface proteins of living bacteria using fluorescent dyes and a subsequent separation of the crude extract by 2D gel electrophoresis. The strategy yields a substantial enrichment in surface proteins over cytoplasmic proteins. We validated this method by monitoring the effect of the regulatory RNA MicA in Escherichia colt, which regulates the synthesis of several outer membrane proteins, and highlighted the role of Staphylococcus aureus RNAIII for the maintenance of cell wall integrity.
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| 18. |
- Hammarlöf, Disa L., 1980-
(författare)
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EF-Tu and RNase E : Essential and Functionally Connected Proteins
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The rate and accuracy of protein production is the main determinant of bacterial growth. Elongation Factor Tu (EF-Tu) provides the ribosome with aminoacylated tRNAs, and is central for its activity. In Salmonella enterica serovar Typhimurium, EF-Tu is encoded by the genes tufA and tufB. A bacterial cell depending on tufA499-encoded EF-Tu mutant Gln125Arg grows extremely slowly. We found evidence that this is caused by excessive degradation of mRNA, which is suggested to be the result of transcription-translation decoupling because the leading ribosome is ‘starved’ for amino acids and stalls on the nascent mRNA, which is thus exposed to Riboendonuclease RNase E. The slow-growth phenotype can be reversed by mutations in RNase E that reduce the activity of this enzyme. We found that the EF-Tu mutant has increased levels of ppGpp during exponential growth in rich medium. ppGpp is usually produced during starvation, and we propose that Salmonella, depending on mutant EF-Tu, incorrectly senses the resulting situation with ribosomes ‘starving’ for amino acids as a real starvation condition. Thus, RelA produces ppGpp which redirects gene expression from synthesis of ribosomes and favours synthesis of building blocks such as amino acids. When ppGpp levels are reduced, either by over-expression of SpoT or by inactivation of relA, growth of the mutant is improved. We suggest this is because the cell stays in a fast-growth mode. RNase E mutants with a conditionally lethal temperature-sensitive (ts) phenotype were used to address the long-debated question of the essential role of RNase E. Suppressor mutations of the ts phenotype were selected and identified, both in RNase E as well as in extragenic loci. The internal mutations restore the wild-type RNase E function to various degrees, but no single defect was identified that alone could account for the ts phenotype. In contrast, identifying three different classes of extragenic suppressors lead us to suggest that the essential role of RNaseIE is to degrade mRNA. One possibility to explain the importance of this function is that in the absence of mRNA degradation by RNase E, the ribosomes become trapped on defective mRNAs, with detrimental consequences for continued cell growth.
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| 19. |
- He, Lin, et al.
(författare)
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PcnB is required for the rapid degradation of RNAI, the antisense RNA that controls the copy number of ColE1-related plasmids
- 1993
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Ingår i: Molecular Microbiology. - : Wiley. - 0950-382X .- 1365-2958. ; 9:6, s. 1131-1142
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Tidskriftsartikel (refereegranskat)abstract
- The replication of ColE1-related plasmids is controlled by an unstable antisense RNA, RNAI, which can interfere with the successful processing of the RNAII primer of replication. We show here that a host protein, PcnB, supports replication by promoting the decay of RNAI. In bacterial strains deleted for PcnB a stable, active form of RNAI, RNAI*, which appears to be identical to the product of 5'-end processing by RNAase E, accumulates. This leads to a reduction in plasmid copy number. We show, using a GST-PcnB fusion protein, that PcnB does not interfere with RNAI/RNAII binding in vitro. The fusion protein, like PcnB, has polyadenylating activity and is able to polyadenylate RNAI (and also another antisense RNA, CopA) in vitro.
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| 20. |
- Hinas, Andrea, et al.
(författare)
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The small RNA repertoire of Dictyostelium discoideum and its regulation by components of the RNAi pathway
- 2007
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 35:20, s. 6714-6726
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Tidskriftsartikel (refereegranskat)abstract
- Small RNAs play crucial roles in regulation of gene expression in many eukaryotes. Here, we report the cloning and characterization of 1826 nt RNAs in the social amoeba Dictyostelium discoideum. This survey uncovered developmentally regulated microRNA candidates whose biogenesis, at least in one case, is dependent on a Dicer homolog, DrnB. Furthermore, we identified a large number of 21 nt RNAs originating from the DIRS-1 retrotransposon, clusters of which have been suggested to constitute centromeres. Small RNAs from another retrotransposon, Skipper, were significantly up-regulated in strains depleted of the second Dicer-like protein, DrnA, and a putative RNA-dependent RNA polymerase, RrpC. In contrast, the expression of DIRS-1 small RNAs was not altered in any of the analyzed strains. This suggests the presence of multiple RNAi pathways in D. discoideum. In addition, we isolated several small RNAs with antisense complementarity to mRNAs. Three of these mRNAs are developmentally regulated. Interestingly, all three corresponding genes express longer antisense RNAs from which the small RNAs may originate. In at least one case, the longer antisense RNA is complementary to the spliced but not the unspliced pre-mRNA, indicating synthesis by an RNA-dependent RNA polymerase.
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| 21. |
- 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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| 22. |
- 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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| 23. |
- Holmgren, Benjamin T.
(författare)
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Connecting Systemic RNAi to the Endomembrane System in Caenorhabditis elegans
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- RNA interference (RNAi) is a gene regulation mechanism conserved among eukaryotes. To silence gene expression, RNAi relies on a short single-stranded guide RNA to steer the RNA-induced Silencing Complex (RISC) to mRNAs with guide strand-complementary sequences. RNAi is a highly membrane-associated process. The RISC complex is likely loaded at the rough Endoplasmic Reticulum, where it can bind to and degrade mRNAs. Components of the RISC complex also colocalize to late endosomes, and the efficiency of RNAi-mediated silencing is affected by changes in late endosome to lysosome fusion. RNAi can be systemic and inherited, effecting gene silencing in distal tissues and in the offspring.In this thesis, the model organism Caenorhabditis elegans was used to identify and characterize factors connecting systemic and inherited RNAi to the endomembrane system. We identify two SNARE proteins, SEC-22 and SYX-6, that both act as negative regulators of RNAi. SNAREs are necessary for vesicle fusion. Both SEC-22 and SYX-6 localize to late endosomes, and both interact with systemic RNAi protein SID-5 in a yeast two-hybrid (Y2H) screen. We find that in addition to its function in systemic RNAi, SID-5 is required for proper maturation of late endosomes. Furthermore, we identify the putative RNA-binding protein C12D8.1 as a novel regulator of RNAi inheritance. Mutant C12D8.1 animals will have enhanced inheritance of RNAi silencing, which negatively affects the ability of the progeny to silence new targets using RNAi. Finally, we describe a novel, object-based method for estimating significance in colocalization studies. This method helped us describe and quantify spatial relations between fluorophore-labeled proteins in situations where such analyses would otherwise be impossible.In conclusion, the work presented here further elucidates the connection between cellular RNAi, the endomembrane system, and the outside world.
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| 24. |
- Holmqvist, Erik, 1977-, et al.
(författare)
-
A mixed double negative feedback loop between the sRNA MicF and the global regulator Lrp
- 2012
-
Ingår i: Molecular Microbiology. - : Wiley-Blackwell. - 0950-382X .- 1365-2958. ; 84:3, s. 414-427
-
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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| 25. |
- Holmqvist, Erik, 1977-, et al.
(författare)
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Impact of bacterial sRNAs in stress responses
- 2017
-
Ingår i: Biochemical Society Transactions. - 0300-5127 .- 1470-8752. ; 45, s. 1203-1212
-
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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| 26. |
- 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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| 27. |
- 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
-
Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 41:12, s. e122-
-
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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| 28. |
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| 29. |
- Holmqvist, Erik, et al.
(författare)
-
The sRNA MicF targets its own regulator Lrp and promotes a positive feedback loop
-
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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| 30. |
- Holmqvist, Erik, et al.
(författare)
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Two antisense RNAs target the transcriptional regulator CsgD to inhibit curli synthesis
- 2010
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Ingår i: EMBO Journal. - : Wiley. - 0261-4189 .- 1460-2075. ; 29:11, s. 1840-1850
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Tidskriftsartikel (refereegranskat)abstract
- Escherichia coli produces proteinaceous surface structures called curli that are involved in adhesion and biofilm formation. CsgD is the transcriptional activator of curli genes. We show here that csgD expression is, in part, controlled post-transcriptionally by two redundant small RNAs (sRNAs), OmrA and OmrB. Their overexpression results in curli deficiency, in accordance with the inhibition of chromosomally encoded, FLAG-tagged CsgD. Downregulation of csgD occurs by a direct antisense interaction within the csgD 5'-UTR, far upstream of the ribosome-binding site (RBS). OmrA/B downregulate plasmid-borne csgD-gfp fusions in vivo, and inhibit CsgD translation in vitro. The RNA chaperone Hfq is required for normal csgD mRNA and OmrA/B levels in the cell, and enhances sRNA-dependent inhibition of csgD translation in vitro. Translational inhibition involves two phylogenetically conserved secondary structure modules that are supported by chemical and enzymatic probing. The 5'-most element is necessary and sufficient for regulation, the one downstream comprises the RBS and affects translational efficiency. OmrA/B are two antisense RNAs that regulate a transcription factor to alter a morphotype and group behaviour.
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| 31. |
- 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
-
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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| 32. |
- 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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| 33. |
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| 34. |
- Lindell, Magnus, 1969-
(författare)
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Lead(II) as a Tool for Probing RNA Structure in vivo
- 2005
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Chemical modification and limited enzymatic hydrolysis are powerful methods to obtain detailed information on the structure and dynamics of RNAs in solution. In the work presented here I have taken advantage of the properties of the divalent metal ion lead(II) to establish it as a new probe for investigating the structure of RNA in vivo. Besides highly specific lead(II)-induced cleavage due to the presence of tight metal ion binding sites, lead(II) is known to cleave RNA within single-stranded regions, loops and bulges. The detailed structural data obtained with three different RNAs: tmRNA, CopT, and the leader region of the ompF mRNA, show that lead(II) has great potential for in vivo studies of RNA structure. In P. fluorescens, the activity and stability of RsmY, a small regulatory RNA, was shown to be strongly dependent on repeated GGA motifs in single-stranded regions. In vivo lead(II) probing essentially confirmed predicted secondary structures and also indicated binding to a protein, RsmA. The potential in using lead(II) for mapping protein binding sites on RNAs was shown for the interaction between E. coli tmRNA and the SmpB protein. In vivo and in vitro data show protections in the tRNA-like domain of tmRNA due to binding to the SmpB protein, indicating that the SmpB protein is associated with the majority of tmRNA in the cell. Furthermore, the overall conformation/ structure of E. coli RNase P was analyzed by probing the native structure of M1 RNA in vivo with lead(II). The observed cleavages suggests that M1 RNA is present in two main conformations in the cell, one being characteristic of free RNase P, and one of an RNase P-tRNA complex. The results also indicate that the C5 protein subunit has only minor effects on the overall structure of the RNA subunit.
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| 35. |
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| 36. |
- Lindell, Magnus, et al.
(författare)
-
Lead(II) cleavage analysis of RNase P RNA in vivo.
- 2005
-
Ingår i: RNA. - 1355-8382. ; 11:9, s. 1348-54
-
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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| 37. |
- Melin, Petter, et al.
(författare)
-
Changes in Aspergillus nidulans gene expression induced by bafilomycin, a Streptomyces-produced antibiotic
- 1999
-
Ingår i: Microbiology. - : Microbiology Society. - 1350-0872 .- 1465-2080. ; 145:5, s. 1115-1122
-
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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| 38. |
- Melin, Petter, et al.
(författare)
-
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
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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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| 39. |
- Melin, Petter, et al.
(författare)
-
Disruption of the gene encoding the V-ATPase subunit A results in inhibition of normal growth and abolished sporulation in Aspergillus nidulans
- 2003
-
Ingår i: Microbiology. - : Microbiology Society. - 1350-0872 .- 1465-2080. ; 150:3, s. 743-748
-
Tidskriftsartikel (refereegranskat)abstract
- The authors have previously reported on molecular responses of Aspergillus nidulans to bacterial antifungal metabolites, e.g. bafilomycins and the related concanamycins. These compounds are known inhibitors of V-ATPases and cause dramatic effects on mycelial growth and morphology. In Neurospora crassa, studies have shown that disruption of the gene encoding subunit A of the V-ATPase results in morphological changes and reduced growth similar to those observed after addition of concanamycin. This phenotype, and the fact that this mutation confers resistance to concanamycin, suggests that V-ATPase is the main (or only) target for the antibiotics. However, growth inhibition and morphology changes in, for example, A. nidulans and Penicillium roqueforti are more severe, and thus other targets are possible. In this study, the vmaA gene of A. nidulans, encoding the subunit A of V-ATPase, was disrupted by homologous recombination. The resulting vmaA1 mutant strain displayed extremely slow growth and failed to produce asexual spores. Furthermore, an altered morphology similar to that caused by addition of V-ATPase inhibitors, i.e. bafilomycin or concanamycin, was observed, indicating that V-ATPase is the main target for the antibiotics also in A. nidulans. The vmaA1 mutant was not viable at pH values above 7 and was highly sensitive to high Zn2+ concentrations, in agreement with previous results from studies of Saccharomyces cerevisiae and N. crassa.
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| 40. |
- 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
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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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| 41. |
- Quax, Tessa E. F., et al.
(författare)
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Differential Translation Tunes Uneven Production of Operon-Encoded Proteins
- 2013
-
Ingår i: Cell Reports. - : Elsevier BV. - 2211-1247. ; 4:5, s. 938-944
-
Tidskriftsartikel (refereegranskat)abstract
- Clustering of functionally related genes in operons allows for coregulated gene expression in prokaryotes. This is advantageous when equal amounts of gene products are required. Production of protein complexes with an uneven stoichiometry, however, requires tuning mechanisms to generate subunits in appropriate relative quantities. Using comparative genomic analysis, we show that differential translation is a key determinant of modulated expression of genes clustered in operons and that codon bias generally is the best in silico indicator of unequal protein production. Variable ribosome density profiles of polycistronic transcripts correlate strongly with differential translation patterns. In addition, we provide experimental evidence that de novo initiation of translation can occur at intercistronic sites, allowing for differential translation of any gene irrespective of its position on a polycistronic messenger. Thus, modulation of translation efficiency appears to be a universal mode of control in bacteria and archaea that allows for differential production of operon-encoded proteins.
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| 42. |
- Reimegård, Johan, et al.
(författare)
-
AntisenseRNA: fast, specific target prediction for bacterial sRNAs through models of interaction and evolutionary conservation
-
Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Small regulatory RNAs (sRNAs) are ubiquitously present in many different bacteria and are often conserved in closely related species. The majority of the sRNAs acts as antisense RNAs via basepairing to target mRNAs and mediates up- or down-regulation. This involves effects on translation and/or mRNA stability. Since antisense-target RNA interaction sites are often short and non-contiguous, prediction of targets is a non-trivial task. Thus, most sRNA have not yet been assigned to specific target genes, and this motivates a need for computational target prediction programs. Available programs use criteria such as, for instance, a "seed" antisense-target interaction and the structural accessibility of the interaction site to indentify sRNA targets. So far, the realization that RNA/RNA interaction is a hierarchical multi-step process has not been incorporated into algorithms, and neither has conservation of interactions between related species. We have designed a target prediction program, AntisenseRNA, which considers the interaction as a multistep process, in which the intramolecular structure of the two RNAs and the phylogenetic conservation of basepairs are taken into account. AntisenseRNA successfully identifies most of the experimentally validated sRNA targets in Escherichia coli and predicts their interaction sites with high specificity and sensitivity. AntisenseRNA is about ten times faster than its best competitor programs, suggesting it to be a good choice for identification of new targets for sRNAs.
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| 43. |
- Reimegård, Johan
(författare)
-
Making Sense of Antisense
- 2010
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- RNA is a highly versatile molecule with functions that span from being a messenger in the transfer from DNA to protein, a catalytic molecule important for key processes in the cell to a regulator of gene expression. The post-genomic era and the use of new techniques to sequence RNAs have dramatically increased the number of regulatory RNAs during the last decade. Many of these are antisense RNAs, as for example the miRNA in eukaryotes and most sRNAs in bacteria. Antisense RNAs bind to specific targets by basepairing and thereby regulate their expression. A major step towards an understanding of the biological role of a miRNA or an sRNA is taken when one identifies which target it regulates. We have used RNA libraries to study the RNA interference pathway during development in the unicellular model organism Dictyostelium discoideum. We have also, by combining computational and experimental methods, discovered the first miRNAs in this organism and shown that they have different expression profiles during development. In parallel, we have developed a novel approach to predict targets for sRNAs in bacteria and used it to discover sRNA/target RNA interactions in the model organism Escherichia coli. We have found evidence for, and further characterized, three of these predicted sRNA/target interactions. For instance, the sRNA MicA is important for regulation of the outer membrane protein OmpA, the sRNAs OmrA and OmrB regulate the transcription factor CsgD, which is important in the sessile lifestyle of E. coli, and MicF regulates its own expression in a feed forward loop via the regulatory protein Lrp. In conclusion, we have discovered novel antisense RNAs, e.g. miRNAs in D. discoideum, developed an approach to identify targets for antisense RNAs, i.e. a target prediction program for sRNAs in bacteria, and verified and characterized some of the predicted antisense RNA interactions.
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| 44. |
- 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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| 45. |
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| 46. |
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| 47. |
- Romilly, Cedric, et al.
(författare)
-
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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| 48. |
- 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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| 49. |
- 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
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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
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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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- Sjögren, Ann-Sofie, 1961-
(författare)
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Structure and function studies on nrdB group I intron from bacteriophage T4
- 1997
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The nrdB gene of bacteriophage T4 contains a group I intron which is autocatalytic in water solutions containing salts. The group I intron self-splicing reaction consists of two consecutive transesterfication steps. The first step is the attack of an external guanosine on phosphor at the 5'-splice site. The external guanosine becomes covalently attached via its 3'-hydroxyl to the phosphor of the 5'-splice site. The second step follows where the hydroxyl of the 3'-end of the upstream exon attacks the phosphor at the 3'-end of the intron. The intron is liberated with a covalently attached guanosine and a functional messenger RNA is produced.The core of the nrdB group I intron was used in computer modelling. The 3D arrangement was according to a model by Kim and Cech originally proposed for the Tetrahymena group I intron. Our model consisted of three helices where a binding site for the cosubstrate guanosine and inhibitor arginine was located between the helix P1 and helix P7. Molecular dynamics simulation forced the cosubstrate guanosine closer to the splice site at the 5'-end of the intron.2'-Amino-2'-deoxyguanosine was found to be a cosubstrate of the nrdB group I intron self-splicing reaction. The catalytic efficiency for 2'-amino-2'-deoxyguanosine was 22-fold lower compared to guanosine catalysed self-splicing in the reaction of the wild type premessenger RNA of the nrdB gene. The wild type premessenger RNA was reconstructed and the splicing of the new shortened nrdB pre-mRNA was investigated by analysing splicing products, and the pH dependence for guanosine and 2'-amino-2'-deoxyguanosine. The study supported that the self-splicing reaction of the shortened nrdB pre-mRNA was amenable to kinetic analysis and that the chemical cleavage step was monitored under certain reaction conditions.The 2'-amino-2'-deoxyguanosine was susbsequently used to probe metal ion interaction with the cosubstrate in group I intron self-splicing. The rate of the cleavage step, catalysed by 2'-amino-2'-deoxyguanosine or guanosine, was examined in presence of different divalent metal ions or mixtures thereof. It could be demonstrated that 2'-amino-2'-deoxyguanosine becomes a better cosubstrate in presence of Mn2+ or Mg2+ and Zn2+ compared to Mg2+ alone. The increase in rate for 2'-amino-2'-deoxyguanosine was 35-fold for mixtures of Mg2+ and Zn2+ while the rate for guanosine was not affected. The results are the first experimental evidence that support the proposed two-metal-ion mechanism for group I introns.
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