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

An Author Correction to this article was published on 05 April 2019

This article has been updated (view changelog)

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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Fig. 1: Tracking of single [Cy5]tRNAPhe in live E. coli cells.
Fig. 2: Internalized [Cy5]tRNAPhe takes an active part in translation.
Fig. 3: [Cy5]tRNAPhe dwell time is longer on slow ribosomes.
Fig. 4: Simulated single-molecule microscopy.
Fig. 5: Analysis of simulated movies.
Fig. 6: In vivo initiation kinetics using Cy5-labeled initiator tRNAfMet.

Change history

  • 05 April 2019

    In the version of this article originally published, the values on the y axis of Fig. 6d were incorrect. They should be 0.00, 0.02, 0.04, 0.06 and 0.08 instead of the previous 0.00, 0.04, 0.08 and 0.12. The error has been corrected in the HTML and PDF versions of this paper.

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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.).

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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.

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Correspondence to Magnus Johansson.

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

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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.

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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Volkov, I.L., Lindén, M., Aguirre Rivera, J. et al. tRNA tracking for direct measurements of protein synthesis kinetics in live cells. Nat Chem Biol 14, 618–626 (2018). https://doi.org/10.1038/s41589-018-0063-y

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