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Single-particle tracking reveals that free ribosomal subunits are not excluded from the Escherichia coli nucleoid
Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Beräknings- och systembiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Beräknings- och systembiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Beräknings- och systembiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Beräknings- och systembiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
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2014 (engelsk)Inngår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 111, nr 31, s. 11413-11418Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

Biochemical and genetic data show that ribosomes closely follow RNA polymerases that are transcribing protein-coding genes in bacteria. At the same time, electron and fluorescence microscopy have revealed that ribosomes are excluded from the Escherichia coli nucleoid, which seems to be inconsistent with fast translation initiation on nascent mRNA transcripts. The apparent paradox can be reconciled if translation of nascent mRNAs can start throughout the nucleoid before they relocate to the periphery. However, this mechanism requires that free ribosomal subunits are not excluded from the nucleoid. Here, we use single-particle tracking in living E. coli cells to determine the fractions of free ribosomal subunits, classify individual subunits as free or mRNA-bound, and quantify the degree of exclusion of bound and free subunits separately. We show that free subunits are not excluded from the nucleoid. This finding strongly suggests that translation of nascent mRNAs can start throughout the nucleoid, which reconciles the spatial separation of DNA and ribosomes with cotranscriptional translation. We also show that, after translation inhibition, free subunit precursors are partially excluded from the compacted nucleoid. This finding indicates that it is active translation that normally allows ribosomal subunits to assemble on nascent mRNAs throughout the nucleoid and that the effects of translation inhibitors are enhanced by the limited access of ribosomal subunits to nascent mRNAs in the compacted nucleoid.

sted, utgiver, år, opplag, sider
2014. Vol. 111, nr 31, s. 11413-11418
Emneord [en]
nucleoid exclusion, transcription-translation coupling, antibiotics, single-molecule tracking, single-molecule imaging
HSV kategori
Identifikatorer
URN: urn:nbn:se:uu:diva-229101DOI: 10.1073/pnas.1411558111ISI: 000339807200043PubMedID: 25056965OAI: oai:DiVA.org:uu-229101DiVA, id: diva2:735704
Tilgjengelig fra: 2014-07-30 Laget: 2014-07-30 Sist oppdatert: 2017-12-05bibliografisk kontrollert
Inngår i avhandling
1. Biological Insights from Single-Particle Tracking in Living Cells
Åpne denne publikasjonen i ny fane eller vindu >>Biological Insights from Single-Particle Tracking in Living Cells
2014 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
Abstract [en]

Single-particle tracking is a technique that allows for quantitative analysis of the localization and movement of particles. In this technique, trajectories are constructed by determining and connecting the positions of individual particles from consecutive images. Recent advances have made it possible to track hundreds of particles in an individual cell by labeling the particles of interest with photoactivatable or photoconvertible fluorescent proteins and tracking one or a few at a time.

Single-particle tracking can be used to study the diffusion of particles. Here, we use intracellular single-particle tracking and trajectory simulations to study the diffusion of the fluorescent protein mEos2 in living Escherichia coli cells. Our data are consistent with a simple model in which mEos2 diffuses normally at 13 µm2 s−1 in the E. coli cytoplasm. Our approach can be used to study the diffusion of intracellular particles that can be labeled with mEos2 and are present at high copy numbers.

Single-particle tracking can also be used to determine whether an individual particle is bound or free if the free particle diffuses significantly faster than its binding targets and remains bound or free for a long time. Here, we use single-particle tracking in living E. coli cells to determine the fractions of free ribosomal subunits, classify individual subunits as free or mRNA-bound, and quantify the degree of exclusion of bound and free subunits separately. We show that, unlike bound subunits, free subunits are not excluded from the nucleoid. This finding strongly suggests that translation of nascent mRNAs can start throughout the nucleoid, which reconciles the spatial separation of DNA and ribosomes with co-transcriptional translation. We also show that, after translation inhibition, free subunit precursors are partially excluded from the compacted nucleoid. This finding indicates that it is active translation that normally allows ribosomal subunits to assemble on nascent mRNAs throughout the nucleoid and that the effects of translation inhibitors are enhanced by the limited access of ribosomal subunits to nascent mRNAs in the compacted nucleoid.

sted, utgiver, år, opplag, sider
Uppsala: Acta Universitatis Upsaliensis, 2014. s. 65
Serie
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1159
Emneord
single-particle tracking, intracellular diffusion, nucleoid exclusion, transcription-translation coupling, antibiotics
HSV kategori
Forskningsprogram
Biologi med inriktning mot molekylär bioteknik
Identifikatorer
urn:nbn:se:uu:diva-229342 (URN)978-91-554-8991-5 (ISBN)
Disputas
2014-09-05, lecture hall B42, Uppsala Biomedical Centre, Husargatan 3, Uppsala, 13:00 (engelsk)
Opponent
Veileder
Tilgjengelig fra: 2014-08-15 Laget: 2014-08-06 Sist oppdatert: 2014-09-08bibliografisk kontrollert

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