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

Abstract
Data availability
Article and author information
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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

(2018) A systematically-revised ribosome profiling method for bacteria reveals pauses at single-codon resolution
NCBI Gene Expression Omnibus, GSE119104.

https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE119104

The following previously published data sets were used

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Article and author information

Author details

Fuad Mohammad

Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, United States

Competing interests
No competing interests declared.
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.

"This ORCID iD identifies the author of this article:" 0000-0001-9337-2003
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.

"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.

Copyright

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.