A systematically-revised ribosome profiling method for bacteria reveals pauses at single-codon resolution

  1. Fuad Mohammad
  2. Rachel Green
  3. Allen R Buskirk  Is a corresponding author
  1. Johns Hopkins University School of Medicine, United States

Abstract

In eukaryotes, ribosome profiling provides insight into the mechanism of protein synthesis at the codon level. In bacteria, however, the method has been more problematic and no consensus has emerged for how to best prepare profiling samples. Here, we identify the sources of these problems and describe new solutions for arresting translation and harvesting cells in order to overcome them. These improvements remove confounding artifacts and improve the resolution to allow analyses of ribosome behavior at the codon level. With a clearer view of the translational landscape in vivo, we observe that filtering cultures leads to translational pauses at serine and glycine codons through the reduction of tRNA aminoacylation levels. This observation illustrates how bacterial ribosome profiling studies can yield insight into the mechanism of protein synthesis at the codon level and how these mechanisms are regulated in response to changes in the physiology of the cell.

Data availability

Sequencing data have been deposited in GEO under accession code GSE119104.

The following data sets were generated
The following previously published data sets were used

Article and author information

Author details

  1. Fuad Mohammad

    Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, United States
    Competing interests
    No competing interests declared.
  2. Rachel Green

    Department of Molecular Biology and Genetics, Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, United States
    Competing interests
    Rachel Green, Reviewing editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9337-2003
  3. Allen R Buskirk

    Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, United States
    For correspondence
    buskirk@jhmi.edu
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2720-6896

Funding

National Institute of General Medical Sciences (GM110113)

  • Allen R Buskirk

Howard Hughes Medical Institute

  • Rachel Green

National Institute of General Medical Sciences (GM105816)

  • Allen R Buskirk

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Alan G Hinnebusch, Eunice Kennedy Shriver National Institute of Child Health and Human Development, United States

Version history

  1. Received: October 4, 2018
  2. Accepted: February 5, 2019
  3. Accepted Manuscript published: February 6, 2019 (version 1)
  4. Version of Record published: February 15, 2019 (version 2)

Copyright

© 2019, Mohammad et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Fuad Mohammad
  2. Rachel Green
  3. Allen R Buskirk
(2019)
A systematically-revised ribosome profiling method for bacteria reveals pauses at single-codon resolution
eLife 8:e42591.
https://doi.org/10.7554/eLife.42591

Share this article

https://doi.org/10.7554/eLife.42591

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