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Elife. 2019 Feb 6;8. pii: e42591. doi: 10.7554/eLife.42591.

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

Author information

1
Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, United States.
2
Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, 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.

KEYWORDS:

E. coli; bacteria; biochemistry; chemical biology; chromosomes; gene expression; pausing; ribosome profiling; serine

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