| 1. |
- 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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| 2. |
- 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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| 3. |
- 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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| 4. |
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| 5. |
- Jones, Daniel, et al.
(författare)
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Kinetics of dCas9 target search in Escherichia coli
- 2017
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Ingår i: Science. - : AMER ASSOC ADVANCEMENT SCIENCE. - 0036-8075 .- 1095-9203. ; 357:6358, s. 1420-1423
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Tidskriftsartikel (refereegranskat)abstract
- How fast can a cell locate a specific chromosomal DNA sequence specified by a single-stranded oligonucleotide? To address this question, we investigate the intracellular search processes of the Cas9 protein, which can be programmed by a guide RNA to bind essentially any DNA sequence. This targeting flexibility requires Cas9 to unwind the DNA double helix to test for correct base pairing to the guide RNA. Here we study the search mechanisms of the catalytically inactive Cas9 (dCas9) in living Escherichia coli by combining single-molecule fluorescence microscopy and bulk restriction-protection assays. We find that it takes a single fluorescently labeled dCas9 6 hours to find the correct target sequence, which implies that each potential target is bound for less than 30 milliseconds. Once bound, dCas9 remains associated until replication. To achieve fast targeting, both Cas9 and its guide RNA have to be present at high concentrations.
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| 6. |
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| 7. |
- Persson, Fredrik, et al.
(författare)
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Extracting intracellular diffusive states and transition rates from single-molecule tracking data
- 2013
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Ingår i: Nature Methods. - 1548-7091 .- 1548-7105. ; 10:3, s. 265-269
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Tidskriftsartikel (refereegranskat)abstract
- We provide an analytical tool based on a variational Bayesian treatment of hidden Markov models to combine the information from thousands of short single-molecule trajectories of intracellularly diffusing proteins. The method identifies the number of diffusive states and the state transition rates. Using this method we have created an objective interaction map for Hfq, a protein that mediates interactions between small regulatory RNAs and their mRNA targets.
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| 8. |
- Unoson, Cecilia, et al.
(författare)
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A small SOS-induced toxin is targeted against the inner membrane in Escherichia coli
- 2008
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Ingår i: Molecular Microbiology. - : Wiley. - 0950-382X .- 1365-2958. ; 70:1, s. 258-70
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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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| 9. |
- Unoson, Cecilia, et al.
(författare)
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Dealing with stable structures at ribosome binding sites : bacterial translation and ribosome standby.
- 2007
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Ingår i: RNA Biology. - 1547-6286. ; 4:3, s. 113-117
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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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| 10. |
- Unoson, Cecilia
(författare)
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Small RNA-mediated Regulation of Gene Expression in Escherichia coli
- 2010
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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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