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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
Identifiers
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: 2020-08-21Bibliographically approved
In thesis
1. Tracking single molecules in uncharted territory: A single-molecule method to study kinetics in live bacteria
Open this publication in new window or tab >>Tracking single molecules in uncharted territory: A single-molecule method to study kinetics in live bacteria
2020 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The synthesis of proteins, also known as translation, is a fundamental process in every living organism. The steps in the translation of genetic information to functional proteins have been meticulously studied, mostly using in vitro techniques, yielding a detailed model of their mechanisms. However, the use of minimal cell-free systems allows for the possibility to miss interactions from absent components or that reactions are affected by the buffer composition. The work presented in this thesis opens a way to study the kinetics of complex molecular processes, like protein synthesis, directly inside live bacterial cells in real time. We developed and optimized a method to deliver dye-labeled macromolecules inside live cells and generate a kinetic model of the particle’s interactions based on its diffusion inside the cell.

This method facilitated the study of translation elongation and initiation directly in live cells. Our measurements of reaction times of tRNA in the ribosome, agree with previous reports from in vitro techniques. We further applied the method to examine the effects of three aminoglycoside antibiotics and erythromycin directly in live cells. The aminoglycoside antibiotics slowed-down protein synthesis 2- to 4-fold, while the number of elongation cycles per initiation event decreased significantly. In the case of erythromycin, cells showed a 4-fold slower protein synthesis. Additionally, we measured the kinetics of sequence-specific effects of erythromycin: translational arrest, and peptidyl-tRNA drop-off; these in vivo measurements revealed a complex mechanism of action of the drug, in agreement with models suggested by previous experiments. Additionally, we applied the method to measure the effects, on the kinetics of protein synthesis, caused by modifications in the C-terminal tail of the S13 ribosomal protein. Our measurements showed that specific mutations led to different changes in the occupancy and dwell-time of labeled-tRNA in the ribosome.

To summarize, the present work will guide the reader through the development of a method to study the kinetics of protein synthesis directly in live bacterial cells, as well as its application to characterize the effects of different antibiotics within the complex environment of a living organism.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2020. p. 60
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1957
Keywords
single-molecule, protein synthesis, fluorescence microscopy, antibiotics, aminoglycosides, macrolides, apramycin, gentamicin, paromomycin, S13, translation, bacteria
National Category
Natural Sciences Biochemistry and Molecular Biology
Research subject
Molecular Life Sciences
Identifiers
urn:nbn:se:uu:diva-417642 (URN)978-91-513-0992-7 (ISBN)
Public defence
2020-10-09, Room A1:111a, BMC, Husargatan 3, Uppsala, 14:15 (English)
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Supervisors
Available from: 2020-09-16 Created: 2020-08-21 Last updated: 2020-10-06

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