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tRNA tracking for direct measurements of protein synthesis kinetics in live cells
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.ORCID iD: 0000-0003-4200-0191
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
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2018 (English)In: Nature Chemical Biology, ISSN 1552-4450, E-ISSN 1552-4469, Vol. 14, no 6, p. 618-626Article in journal (Refereed) Published
Abstract [en]

Our ability to directly relate results from test-tube biochemical experiments to the kinetics in living cells is very limited. Here we present experimental and analytical tools to directly study the kinetics of fast biochemical reactions in live cells. Dye-labeled molecules are electroporated into bacterial cells and tracked using super-resolved single-molecule microscopy.Trajectories are analyzed by machine-learning algorithms to directly monitor transitions between bound and free states. In particular, we measure the dwell time of tRNAs on ribosomes, and hence achieve direct measurements of translation rates inside living cells at codon resolution. We find elongation rates with tRNA(Phe) that are in perfect agreement with previous indirect estimates, and once fMet-tRNA(fMet) has bound to the 30S ribosomal subunit, initiation of translation is surprisingly fast and does not limit the overall rate of protein synthesis. The experimental and analytical tools for direct kinetics measurements in live cells have applications far beyond bacterial protein synthesis.

Place, publisher, year, edition, pages
NATURE PUBLISHING GROUP , 2018. Vol. 14, no 6, p. 618-626
National Category
Biochemistry and Molecular Biology Cell and Molecular Biology
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URN: urn:nbn:se:uu:diva-359663DOI: 10.1038/s41589-018-0063-yISI: 000435445100019PubMedID: 29769736OAI: oai:DiVA.org:uu-359663DiVA, id: diva2:1245587
Funder
Swedish Research Council, 2015-04111EU, European Research Council, ERC-2013-CoG 616047 SMILEKnut and Alice Wallenberg FoundationWenner-Gren FoundationsCarl Tryggers foundation , CTS 15:243Available from: 2018-09-05 Created: 2018-09-05 Last updated: 2018-09-05Bibliographically approved

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Volkov, IvanLindén, MartinAguirre, JavierIeong, Ka-WengMetelev, MikhailElf, JohanJohansson, Magnus

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