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Mol Cell. 2019 Mar 7;73(5):959-970.e5. doi: 10.1016/j.molcel.2018.12.009. Epub 2019 Jan 24.

High-Resolution Ribosome Profiling Defines Discrete Ribosome Elongation States and Translational Regulation during Cellular Stress.

Author information

1
Howard Hughes Medical Institute (HHMI); Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
2
Howard Hughes Medical Institute (HHMI); Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. Electronic address: ragreen@jhmi.edu.

Abstract

Ribosomes undergo substantial conformational changes during translation elongation to accommodate incoming aminoacyl-tRNAs and translocate along the mRNA template. We used multiple elongation inhibitors and chemical probing to define ribosome conformational states corresponding to differently sized ribosome-protected mRNA fragments (RPFs) generated by ribosome profiling. We show, using various genetic and environmental perturbations, that short 20-22 or classical 27-29 nucleotide RPFs correspond to ribosomes with open or occupied ribosomal A sites, respectively. These distinct states of translation elongation are readily discerned by ribosome profiling in all eukaryotes we tested, including fungi, worms, and mammals. This high-resolution ribosome profiling approach reveals mechanisms of translation-elongation arrest during distinct stress conditions. Hyperosmotic stress inhibits translocation through Rck2-dependent eEF2 phosphorylation, whereas oxidative stress traps ribosomes in a pre-translocation state, independent of Rck2-driven eEF2 phosphorylation. These results provide insights and approaches for defining the molecular events that impact translation elongation throughout biology.

KEYWORDS:

eEF2 phosphorylation; ribosome functional states; ribosome profiling; translation elongation

PMID:
30686592
PMCID:
PMC6411040
[Available on 2020-03-07]
DOI:
10.1016/j.molcel.2018.12.009
[Indexed for MEDLINE]

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