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tRNA tracking for direct measurements of protein synthesis kinetics in live cells

Nature Chemical Biologyvolume 14pages618626 (2018) | Download Citation

Abstract

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 tRNAPhe that are in perfect agreement with previous indirect estimates, and once fMet-tRNAfMet 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.

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Acknowledgements

We thank S. Sanyal and M. Ehrenberg (Uppsala University) for sharing components of the reconstituted protein synthesis system; D. Hughes (Uppsala University) for the CH2273 strain; P. Leroy (Uppsala University) for construction of the PL22A9 EF-Tu-mEos2 strain; P. Walter (UCSF) for the pDMF6 plasmid; K. Kipper, A. Boucharin, V. Ćurić and D. Fange for providing technical expertise; E. Amselem for measuring the PSF, and M. Ehrenberg and J. Puglisi for comments on the manuscript. This work was supported by The Swedish Research Council (2015-04111, M.J.), The Wenner-Gren Foundations (M.J., I.L.V.), Carl Tryggers Stiftelse för Vetenskaplig Forskning (CTS 15:243, M.J.), the European Research Council (ERC-2013-CoG 616047 SMILE, J.E.), and Knut and Alice Wallenberg Foundation (J.E.).

Author information

Author notes

  1. These authors contributed equally: Ivan L. Volkov, Martin Lindén.

Affiliations

  1. Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden

    • Ivan L. Volkov
    • , Martin Lindén
    • , Javier Aguirre Rivera
    • , Ka-Weng Ieong
    • , Mikhail Metelev
    • , Johan Elf
    •  & Magnus Johansson

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Contributions

M.J. conceived the project, except for the data analysis and simulation pipelines, which were conceived by M.L. and J.E. M.J. and I.L.V. designed experiments. I.L.V. performed and analyzed in vivo experiments. M.L. generated and analyzed simulated data and wrote analysis code. J.A.R. and M.M. participated in method development and provided reagents. K.-W.I. performed in vitro experiments. M.J., M.L., J.E. and I.L.V. wrote the manuscript.

Competing interests

The authors declare no competing interest.

Corresponding author

Correspondence to Magnus Johansson.

Supplementary information

  1. Supplementary Text and Figures

    Supplementary Tables 1–2, Supplementary Figures 1–6, Supplementary Note

  2. Reporting Summary

  3. Supplementary Video 1

    Experimental and simulated microscopy data of [Cy5]tRNAPhe diffusion in live cells. Top panels show fluorescence microscopy images acquired sequentially with 5 ms camera exposure and 1.5 ms laser illumination (639 nm) per frame.

  4. Supplementary Video 2

    Experimental microscopy data of [Cy5]tRNAfMet diffusion in live cells. The left panel shows raw fluorescence microscopy data acquired at 5 ms camera exposure and 1.5 ms laser illumination (639 nm) per frame.

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DOI

https://doi.org/10.1038/s41589-018-0063-y

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