A systematically-revised ribosome profiling method for bacteria reveals pauses at single-codon resolution
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.
-
A systematically-revised ribosome profiling method for bacteria reveals pauses at single-codon resolutionNCBI Gene Expression Omnibus, GSE119104.
-
A streamlined ribosome profiling protocol for the characterization of microorganismsNCBI Gene Expression Omnibus, GSE63858.
-
High-resolution view of bacteriophage lambda gene expression by ribosome profilingNCBI Gene Expression Omnibus, GSE47509.
-
Selective ribosome profiling reveals the cotranslational chaperone action of trigger factor in vivoNCBI Gene Expression Omnibus, GSE33671.
-
Global analysis of translation termination in E. coliNCBI Gene Expression Omnibus, GSE88725.
-
The anti-Shine-Dalgarno sequence drives translational pausing and codon choice in bacteriaNCBI Gene Expression Omnibus, GSE35641.
-
An integrated approach reveals regulatory controls on bacterial translation elongationNCBI Gene Expression Omnibus, GSE51052.
-
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.
Metrics
-
- 17,537
- views
-
- 1,783
- downloads
-
- 183
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Further reading
-
- Biochemistry and Chemical Biology
- Microbiology and Infectious Disease
Teichoic acids (TA) are linear phospho-saccharidic polymers and important constituents of the cell envelope of Gram-positive bacteria, either bound to the peptidoglycan as wall teichoic acids (WTA) or to the membrane as lipoteichoic acids (LTA). The composition of TA varies greatly but the presence of both WTA and LTA is highly conserved, hinting at an underlying fundamental function that is distinct from their specific roles in diverse organisms. We report the observation of a periplasmic space in Streptococcus pneumoniae by cryo-electron microscopy of vitreous sections. The thickness and appearance of this region change upon deletion of genes involved in the attachment of TA, supporting their role in the maintenance of a periplasmic space in Gram-positive bacteria as a possible universal function. Consequences of these mutations were further examined by super-resolved microscopy, following metabolic labeling and fluorophore coupling by click chemistry. This novel labeling method also enabled in-gel analysis of cell fractions. With this approach, we were able to titrate the actual amount of TA per cell and to determine the ratio of WTA to LTA. In addition, we followed the change of TA length during growth phases, and discovered that a mutant devoid of LTA accumulates the membrane-bound polymerized TA precursor.
-
- Biochemistry and Chemical Biology
- Computational and Systems Biology
Protein–protein interactions are fundamental to understanding the molecular functions and regulation of proteins. Despite the availability of extensive databases, many interactions remain uncharacterized due to the labor-intensive nature of experimental validation. In this study, we utilized the AlphaFold2 program to predict interactions among proteins localized in the nuage, a germline-specific non-membrane organelle essential for piRNA biogenesis in Drosophila. We screened 20 nuage proteins for 1:1 interactions and predicted dimer structures. Among these, five represented novel interaction candidates. Three pairs, including Spn-E_Squ, were verified by co-immunoprecipitation. Disruption of the salt bridges at the Spn-E_Squ interface confirmed their functional importance, underscoring the predictive model’s accuracy. We extended our analysis to include interactions between three representative nuage components—Vas, Squ, and Tej—and approximately 430 oogenesis-related proteins. Co-immunoprecipitation verified interactions for three pairs: Mei-W68_Squ, CSN3_Squ, and Pka-C1_Tej. Furthermore, we screened the majority of Drosophila proteins (~12,000) for potential interaction with the Piwi protein, a central player in the piRNA pathway, identifying 164 pairs as potential binding partners. This in silico approach not only efficiently identifies potential interaction partners but also significantly bridges the gap by facilitating the integration of bioinformatics and experimental biology.