A systematically-revised ribosome profiling method for bacteria reveals pauses at single-codon resolution
- 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.
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A systematically-revised ribosome profiling method for bacteria reveals pauses at single-codon resolutionNCBI Gene Expression Omnibus, GSE119104.
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A streamlined ribosome profiling protocol for the characterization of microorganismsNCBI Gene Expression Omnibus, GSE63858.
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High-resolution view of bacteriophage lambda gene expression by ribosome profilingNCBI Gene Expression Omnibus, GSE47509.
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Selective ribosome profiling reveals the cotranslational chaperone action of trigger factor in vivoNCBI Gene Expression Omnibus, GSE33671.
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Global analysis of translation termination in E. coliNCBI Gene Expression Omnibus, GSE88725.
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The anti-Shine-Dalgarno sequence drives translational pausing and codon choice in bacteriaNCBI Gene Expression Omnibus, GSE35641.
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An integrated approach reveals regulatory controls on bacterial translation elongationNCBI Gene Expression Omnibus, GSE51052.
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Clarifying the Translational Pausing Landscape in Bacteria by Ribosome ProfilingNCBI Gene Expression Omnibus, GSE72899.
Article and author information
Author details
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
© 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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A systematically-revised ribosome profiling method for bacteria reveals pauses at single-codon resolution
eLife 8:e42591.
https://doi.org/10.7554/eLife.42591
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