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

Nature Chemical Biologyvolume 14pages618626 (2018) | Download Citation


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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  1. 1.

    Sanamrad, A. et al. Single-particle tracking reveals that free ribosomal subunits are not excluded from the Escherichia coli nucleoid. Proc. Natl. Acad. Sci. USA 111, 11413–11418 (2014).

  2. 2.

    Uphoff, S., Reyes-Lamothe, R., Garza de Leon, F., Sherratt, D. J. & Kapanidis, A. N. Single-molecule DNA repair in live bacteria. Proc. Natl. Acad. Sci. USA 110, 8063–8068 (2013).

  3. 3.

    Persson, F., Lindén, M., Unoson, C. & Elf, J. Extracting intracellular diffusive states and transition rates from single-molecule tracking data. Nat. Methods 10, 265–269 (2013).

  4. 4.

    Crawford, R. et al. Long-lived intracellular single-molecule fluorescence using electroporated molecules. Biophys. J. 105, 2439–2450 (2013).

  5. 5.

    Plochowietz, A., Farrell, I., Smilansky, Z., Cooperman, B. S. & Kapanidis, A. N. In vivo single-RNA tracking shows that most tRNA diffuses freely in live bacteria. Nucleic Acids Res. 45, 926–937 (2017).

  6. 6.

    Cochella, L. & Green, R. An active role for tRNA in decoding beyond codon:anticodon pairing. Science 308, 1178–1180 (2005).

  7. 7.

    Johansson, M., Bouakaz, E., Lovmar, M. & Ehrenberg, M. The kinetics of ribosomal peptidyl transfer revisited. Mol. Cell 30, 589–598 (2008).

  8. 8.

    Sievers, A., Beringer, M., Rodnina, M. V. & Wolfenden, R. The ribosome as an entropy trap. Proc. Natl. Acad. Sci. USA 101, 7897–7901 (2004).

  9. 9.

    Selmer, M. et al. Structure of the 70S ribosome complexed with mRNA and tRNA. Science 313, 1935–1942 (2006).

  10. 10.

    Valle, M. et al. Incorporation of aminoacyl-tRNA into the ribosome as seen by cryo-electron microscopy. Nat. Struct. Biol. 10, 899–906 (2003).

  11. 11.

    Demeshkina, N., Jenner, L., Westhof, E., Yusupov, M. & Yusupova, G. A new understanding of the decoding principle on the ribosome. Nature 484, 256–259 (2012).

  12. 12.

    Blanchard, S. C., Kim, H. D., Gonzalez, R. L. Jr., Puglisi, J. D. & Chu, S. tRNA dynamics on the ribosome during translation. Proc. Natl. Acad. Sci. USA 101, 12893–12898 (2004).

  13. 13.

    Chen, J. et al. Dynamic pathways of -1 translational frameshifting. Nature 512, 328–332 (2014).

  14. 14.

    Rodnina, M. V. The ribosome in action: Tuning of translational efficiency and protein folding. Protein Sci. 25, 1390–1406 (2016).

  15. 15.

    Antoun, A., Pavlov, M. Y., Lovmar, M. & Ehrenberg, M. How initiation factors maximize the accuracy of tRNA selection in initiation of bacterial protein synthesis. Mol. Cell 23, 183–193 (2006).

  16. 16.

    Antoun, A., Pavlov, M. Y., Lovmar, M. & Ehrenberg, M. How initiation factors tune the rate of initiation of protein synthesis in bacteria. EMBO J. 25, 2539–2550 (2006).

  17. 17.

    Loy, G. & Zelinsky, A. Fast radial symmetry for detecting points of interest. IEEE Trans. Pattern Anal. Mach. Intell. 25, 959–973 (2003).

  18. 18.

    Lindén, M., Ćurić, V., Amselem, E. & Elf, J. Pointwise error estimates in localization microscopy. Nat. Commun. 8, 15115 (2017).

  19. 19.

    Jaqaman, K. et al. Robust single-particle tracking in live-cell time-lapse sequences. Nat. Methods 5, 695–702 (2008).

  20. 20.

    Bakshi, S., Siryaporn, A., Goulian, M. & Weisshaar, J. C. Superresolution imaging of ribosomes and RNA polymerase in live Escherichia coli cells. Mol. Microbiol. 85, 21–38 (2012).

  21. 21.

    Burnham, K. P. & Anderson, D. R. Model Selection and Multimodel Inference: a Practical Information-theoretic Approach. 2nd edn. (Springer, New York, 2002).

  22. 22.

    Furano, A. V. Content of elongation factor Tu in Escherichia coli. Proc. Natl. Acad. Sci. USA 72, 4780–4784 (1975).

  23. 23.

    Avcilar-Kucukgoze, I. et al. Discharging tRNAs: a tug of war between translation and detoxification in Escherichia coli. Nucleic Acids Res. 44, 8324–8334 (2016).

  24. 24.

    Dittmar, K. A., Sørensen, M. A., Elf, J., Ehrenberg, M. & Pan, T. Selective charging of tRNA isoacceptors induced by amino-acid starvation. EMBO Rep. 6, 151–157 (2005).

  25. 25.

    Balzarotti, F. et al. Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes. Science 355, 606–612 (2017).

  26. 26.

    Nakahigashi, K. et al. Comprehensive identification of translation start sites by tetracycline-inhibited ribosome profiling. DNA Res. 23, 193–201 (2016).

  27. 27.

    Chen, J., Petrov, A., Tsai, A., O’Leary, S. E. & Puglisi, J. D. Coordinated conformational and compositional dynamics drive ribosome translocation. Nat. Struct. Mol. Biol. 20, 718–727 (2013).

  28. 28.

    Liang, S. T., Xu, Y. C., Dennis, P. & Bremer, H. mRNA composition and control of bacterial gene expression. J. Bacteriol. 182, 3037–3044 (2000).

  29. 29.

    Borg, A. & Ehrenberg, M. Determinants of the rate of mRNA translocation in bacterial protein synthesis. J. Mol. Biol. 427, 1835–1847 (2015).

  30. 30.

    Bilgin, N., Claesens, F., Pahverk, H. & Ehrenberg, M. Kinetic properties of Escherichia coli ribosomes with altered forms of S12. J. Mol. Biol. 224, 1011–1027 (1992).

  31. 31.

    Ruusala, T., Andersson, D., Ehrenberg, M. & Kurland, C. G. Hyper-accurate ribosomes inhibit growth. EMBO J. 3, 2575–2580 (1984).

  32. 32.

    Fange, D., Mahmutovic, A. & Elf, J. MesoRD 1.0: Stochastic reaction-diffusion simulations in the microscopic limit. Bioinformatics 28, 3155–3157 (2012).

  33. 33.

    Lindén, M., Ćurić, V., Boucharin, A., Fange, D. & Elf, J. Simulated single molecule microscopy with SMeagol. Bioinformatics 32, 2394–2395 (2016).

  34. 34.

    Subramaniam, A. R., Zid, B. M. & O’Shea, E. K. An integrated approach reveals regulatory controls on bacterial translation elongation. Cell 159, 1200–1211 (2014).

  35. 35.

    Goyal, A., Belardinelli, R., Maracci, C., Milón, P. & Rodnina, M. V. Directional transition from initiation to elongation in bacterial translation. Nucleic Acids Res. 43, 10700–10712 (2015).

  36. 36.

    Milon, P. et al. The ribosome-bound initiation factor 2 recruits initiator tRNA to the 30S initiation complex. EMBO Rep. 11, 312–316 (2010).

  37. 37.

    Pan, D., Qin, H. & Cooperman, B. S. Synthesis and functional activity of tRNAs labeled with fluorescent hydrazides in the D-loop. RNA 15, 346–354 (2009).

  38. 38.

    Smith, A., Naik, P. A. & Tsai, C. L. Markov-switching model selection using Kullback-Leibler divergence. J. Econom. 134, 553–577 (2006).

  39. 39.

    Mondal, J., Bratton, B. P., Li, Y., Yethiraj, A. & Weisshaar, J. C. Entropy-based mechanism of ribosome-nucleoid segregation in E. coli cells. Biophys. J. 100, 2605–2613 (2011).

  40. 40.

    Johansson, M. et al. pH-sensitivity of the ribosomal peptidyl transfer reaction dependent on the identity of the A-site aminoacyl-tRNA. Proc. Natl. Acad. Sci. USA 108, 79–84 (2011).

  41. 41.

    Zhang, J., Ieong, K. W., Johansson, M. & Ehrenberg, M. Accuracy of initial codon selection by aminoacyl-tRNAs on the mRNA-programmed bacterial ribosome. Proc. Natl. Acad. Sci. USA 112, 9602–9607 (2015).

  42. 42.

    Zhang, J., Ieong, K. W., Mellenius, H. & Ehrenberg, M. Proofreading neutralizes potential error hotspots in genetic code translation by transfer RNAs. RNA 22, 896–904 (2016).

  43. 43.

    Dong, H., Nilsson, L. & Kurland, C. G. Co-variation of tRNA abundance and codon usage in Escherichia coli at different growth rates. J. Mol. Biol. 260, 649–663 (1996).

  44. 44.

    Gromadski, K. B., Daviter, T. & Rodnina, M. V. A uniform response to mismatches in codon-anticodon complexes ensures ribosomal fidelity. Mol. Cell 21, 369–377 (2006).

  45. 45.

    Kurland, C.G., Hughes, D. & Ehrenberg, M. in Escherichia coli and Salmonella: Cellular and Molecular Biology (ed. Neidhardt, F.C.) (ASM Press, Washington, 1996).

  46. 46.

    Schmidt, C. M., Shis, D. L., Nguyen-Huu, T. D. & Bennett, M. R. Stable maintenance of multiple plasmids in E. coli using a single selective marker. ACS Synth. Biol. 1, 445–450 (2012).

  47. 47.

    Tenson, T., Herrera, J. V., Kloss, P., Guarneros, G. & Mankin, A. S. Inhibition of translation and cell growth by minigene expression. J. Bacteriol. 181, 1617–1622 (1999).

  48. 48.

    Hanahan, D., Jessee, J. & Bloom, F. R. Plasmid transformation of Escherichia coli and other bacteria. Methods Enzymol. 204, 63–113 (1991).

  49. 49.

    Parthasarathy, R. Rapid, accurate particle tracking by calculation of radial symmetry centers. Nat. Methods 9, 724–726 (2012).

  50. 50.

    Harpsøe, K. B. W., Andersen, M. I. & Kjægaard, P. Bayesian photon counting with electron-multiplying charge coupled devices (EMCCDs). Astron. Astrophys. 537, A50 (2012).

  51. 51.

    Ranefall, P., Sadanandan, S.K. & Wählby, C. in 2016 IEEE 13th International Symposium on Biomedical Imaging (ISBI). 205–208 (IEEE, 2016).

  52. 52.

    Vestergaard, C. L., Blainey, P. C. & Flyvbjerg, H. Optimal estimation of diffusion coefficients from single-particle trajectories. Phys. Rev. E 89, 022726 (2014).

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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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Author notes

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


  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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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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The authors declare no competing interest.

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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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