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Direct measurements of mRNA translation kinetics in living cells

Author

Listed:
  • Mikhail Metelev

    (Uppsala University)

  • Erik Lundin

    (Uppsala University)

  • Ivan L. Volkov

    (Uppsala University)

  • Arvid H. Gynnå

    (Uppsala University)

  • Johan Elf

    (Uppsala University)

  • Magnus Johansson

    (Uppsala University)

Abstract

Ribosome mediated mRNA translation is central to life. The cycle of translation, however, has been characterized mostly using reconstituted systems, with only few techniques applicable for studies in the living cell. Here we describe a live-cell ribosome-labeling method, which allows us to characterize the whole processes of finding and translating an mRNA, using single-molecule tracking techniques. We find that more than 90% of both bacterial ribosomal subunits are engaged in translation at any particular time, and that the 30S and 50S ribosomal subunits spend the same average time bound to an mRNA, revealing that 30S re-initiation on poly-cistronic mRNAs is not prevalent in E. coli. Instead, our results are best explained by substantial 70S re-initiation of translation of poly-cistronic mRNAs, which is further corroborated by experiments with translation initiation inhibitors. Finally, we find that a variety of previously described orthogonal ribosomes, with altered anti-Shine-Dalgarno sequences, show significant binding to endogenous mRNAs.

Suggested Citation

  • Mikhail Metelev & Erik Lundin & Ivan L. Volkov & Arvid H. Gynnå & Johan Elf & Magnus Johansson, 2022. "Direct measurements of mRNA translation kinetics in living cells," Nature Communications, Nature, vol. 13(1), pages 1-13, December.
    • Handle: RePEc:nat:natcom:v:13:y:2022:i:1:d:10.1038_s41467-022-29515-x
    DOI: 10.1038/s41467-022-29515-x
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    References listed on IDEAS

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    1. Sandip Kaledhonkar & Ziao Fu & Kelvin Caban & Wen Li & Bo Chen & Ming Sun & Ruben L. Gonzalez & Joachim Frank, 2019. "Late steps in bacterial translation initiation visualized using time-resolved cryo-EM," Nature, Nature, vol. 570(7761), pages 400-404, June.
    2. Martin Lindén & Vladimir Ćurić & Elias Amselem & Johan Elf, 2017. "Pointwise error estimates in localization microscopy," Nature Communications, Nature, vol. 8(1), pages 1-9, August.
    3. Bo Yan & Matthew Boitano & Tyson A. Clark & Laurence Ettwiller, 2018. "SMRT-Cappable-seq reveals complex operon variants in bacteria," Nature Communications, Nature, vol. 9(1), pages 1-11, December.
    4. Madeleine Huber & Guilhem Faure & Sebastian Laass & Esther Kolbe & Kristina Seitz & Christina Wehrheim & Yuri I. Wolf & Eugene V. Koonin & Jörg Soppa, 2019. "Translational coupling via termination-reinitiation in archaea and bacteria," Nature Communications, Nature, vol. 10(1), pages 1-11, December.
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    Cited by:

    1. Elias Amselem & Bo Broadwater & Tora Hävermark & Magnus Johansson & Johan Elf, 2023. "Real-time single-molecule 3D tracking in E. coli based on cross-entropy minimization," Nature Communications, Nature, vol. 14(1), pages 1-11, December.

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