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Unoson, Cecilia
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Jones, D., Leroy, P., Unoson, C., Fange, D., Curic, V., Lawson, M. J. & Elf, J. (2017). Kinetics of dCas9 target search in Escherichia coli. Science, 357(6358), 1420-1423
Öppna denna publikation i ny flik eller fönster >>Kinetics of dCas9 target search in Escherichia coli
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2017 (Engelska)Ingår i: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 357, nr 6358, s. 1420-1423Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

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.

Ort, förlag, år, upplaga, sidor
AMER ASSOC ADVANCEMENT SCIENCE, 2017
Nationell ämneskategori
Biologiska vetenskaper
Identifikatorer
urn:nbn:se:uu:diva-337092 (URN)10.1126/science.aah7084 (DOI)000411880800052 ()28963258 (PubMedID)
Forskningsfinansiär
EU, Europeiska forskningsrådetVetenskapsrådetKnut och Alice Wallenbergs Stiftelse
Tillgänglig från: 2018-01-25 Skapad: 2018-01-25 Senast uppdaterad: 2018-01-25Bibliografiskt granskad
Persson, F., Linden, M., Unoson, C. & Elf, J. (2013). A Bayesian Approach to Single Particle Tracking Analysis. Paper presented at 57th Annual Meeting of the Biophysical-Society, FEB 02-06, 2013, Philadelphia, PA. Biophysical Journal, 104(2), 177A-177A
Öppna denna publikation i ny flik eller fönster >>A Bayesian Approach to Single Particle Tracking Analysis
2013 (Engelska)Ingår i: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 104, nr 2, s. 177A-177AArtikel i tidskrift, Meeting abstract (Övrigt vetenskapligt) Published
Nationell ämneskategori
Naturvetenskap
Identifikatorer
urn:nbn:se:uu:diva-199047 (URN)10.1016/j.bpj.2012.11.998 (DOI)000316074301407 ()
Konferens
57th Annual Meeting of the Biophysical-Society, FEB 02-06, 2013, Philadelphia, PA
Tillgänglig från: 2013-05-03 Skapad: 2013-05-02 Senast uppdaterad: 2017-12-06Bibliografiskt granskad
Persson, F., Linden, M., Unoson, C. & Elf, J. (2013). Extracting intracellular diffusive states and transition rates from single-molecule tracking data. Nature Methods, 10(3), 265-269
Öppna denna publikation i ny flik eller fönster >>Extracting intracellular diffusive states and transition rates from single-molecule tracking data
2013 (Engelska)Ingår i: Nature Methods, ISSN 1548-7091, E-ISSN 1548-7105, Vol. 10, nr 3, s. 265-269Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

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.

Nationell ämneskategori
Naturvetenskap
Identifikatorer
urn:nbn:se:uu:diva-199738 (URN)10.1038/NMETH.2367 (DOI)000316650800026 ()
Tillgänglig från: 2013-05-13 Skapad: 2013-05-13 Senast uppdaterad: 2017-12-06Bibliografiskt granskad
Holmqvist, E., Unoson, C., Reimegård, J. & Wagner, G. E. H. (2012). A mixed double negative feedback loop between the sRNA MicF and the global regulator Lrp. Molecular Microbiology, 84(3), 414-427
Öppna denna publikation i ny flik eller fönster >>A mixed double negative feedback loop between the sRNA MicF and the global regulator Lrp
2012 (Engelska)Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 84, nr 3, s. 414-427Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

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.

Ort, förlag, år, upplaga, sidor
Wiley-Blackwell, 2012
Nyckelord
MicF, Lrp, sRNA, transcription factor, feedback regulation
Nationell ämneskategori
Mikrobiologi
Forskningsämne
Biologi med inriktning mot mikrobiologi
Identifikatorer
urn:nbn:se:uu:diva-171639 (URN)10.1111/j.1365-2958.2012.07994.x (DOI)000303050900003 ()22324810 (PubMedID)
Tillgänglig från: 2012-03-25 Skapad: 2012-03-25 Senast uppdaterad: 2017-12-07Bibliografiskt granskad
Wagner, G. E. H. & Unoson, C. (2012). The toxin-antitoxin system tisB-istR1 Expression, regulation and biological role in persister phenotypes. RNA Biology, 9(12), 1513-1519
Öppna denna publikation i ny flik eller fönster >>The toxin-antitoxin system tisB-istR1 Expression, regulation and biological role in persister phenotypes
2012 (Engelska)Ingår i: RNA Biology, ISSN 1547-6286, E-ISSN 1555-8584, Vol. 9, nr 12, s. 1513-1519Artikel, forskningsöversikt (Refereegranskat) Published
Abstract [en]

Chromosomally encoded toxin-antitoxin (TA) systems are abundantly present in bacteria and archaea. They have become a hot topic in recent years, because-after many frustrating years of searching for biological functions-some are now known to play roles in persister formation. Persisterscells represent a subset of a bacterial population that enters a dormant state and thus becomes refractory to the action of antibiotics. TA modules come in several different flavors, regarding the nature of their gene products, their molecular mechanisms of regulation, their cellular targets, and probably their role in physiology. This review will primarily focus on the SOS-associated tisB/istR1 system in Escherichia coli and discuss its nuts and bolts as well as its effect in promoting a subpopulation phenotype that likely benefits long-term survival of a stressed population.

Nyckelord
Toxin-antitoxin system, persisters, antisense RNA, sRNA, translational control
Nationell ämneskategori
Medicin och hälsovetenskap
Identifikatorer
urn:nbn:se:uu:diva-197619 (URN)10.4161/rna.22578 (DOI)000314451400016 ()
Tillgänglig från: 2013-04-01 Skapad: 2013-04-01 Senast uppdaterad: 2017-12-06Bibliografiskt granskad
Holmqvist, E., Unoson, C., Reimegård, J. & Wagner, G. E. H.The sRNA MicF targets its own regulator Lrp and promotes a positive feedback loop.
Öppna denna publikation i ny flik eller fönster >>The sRNA MicF targets its own regulator Lrp and promotes a positive feedback loop
(Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
Abstract [en]

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.

Nyckelord
MicF, Lrp, antisense RNA, transcription factor, positive feedback loop
Nationell ämneskategori
Naturvetenskap
Identifikatorer
urn:nbn:se:uu:diva-130884 (URN)
Tillgänglig från: 2010-09-15 Skapad: 2010-09-15 Senast uppdaterad: 2014-05-28Bibliografiskt granskad
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