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Published on: August 22, 2017
A method to enrich polypeptidyl-tRNAs to capture snapshots of translation in the cell
Ayako Yamakawa1, Tatsuya Niwa1,2, Yuhei Chadani2
1School of Life Science and Technology, Tokyo Institute of Technology, S2-19, Nagatsuta 4259, Midori-ku, Yokohama 226-8503, Japan.
Insights
Researchers developed a new method to capture and identify nascent proteins (pep-tRNAs) during translation. This technique reveals insights into protein synthesis regulation and cellular responses to stress.
Area of Science:
- Molecular Biology
- Proteomics
- Gene Expression
Background:
- Proteins are essential for life, synthesized as nascent chains attached to tRNA within ribosomes.
- Nascent chains, or peptidyl-tRNAs (pep-tRNAs), are transient intermediates but play roles in cellular functions like gene expression.
- Understanding translational regulation requires methods to analyze pep-tRNAs on a large scale.
Purpose of the Study:
- To develop a general method for large-scale handling and identification of pep-tRNAs.
- To investigate the role of pep-tRNAs in cellular processes and responses.
Main Methods:
- Developed a novel method named peptidyl-tRNA enrichment using organic extraction and silica adsorption (PETEOS).
- Identified polypeptide moieties of captured pep-tRNAs using mass spectrometry.
- Applied PETEOS to Escherichia coli as a proof-of-concept.
Main Results:
- Identified approximately 800 proteins derived from pep-tRNAs in E. coli.
- Observed an N-terminal bias in identified proteins, indicating capture of translation intermediates.
- Detected changes in the pep-tRNA profile under heat shock and antibiotic stress conditions.
Conclusions:
- PETEOS is an effective method for enriching and identifying pep-tRNAs.
- This technique complements existing methods for studying nascent protein chains.
- PETEOS provides insights into translational regulation and cellular responses to environmental changes.
Abstract:
Life depends on proteins, which all exist in nascent states when the growing polypeptide chain is covalently attached to a tRNA within the ribosome. Although the nascent chains, i.e. polypeptidyl-tRNAs (pep-tRNAs), are considered as merely transient intermediates during protein synthesis, recent advances have revealed that they are directly involved in a variety of cell functions, such as gene expression control. An increasing appreciation for fine-tuning at translational levels demands a general method to handle the pep-tRNAs on a large scale. Here, we developed a method termed peptidyl-tRNA enrichment using organic extraction and silica adsorption (PETEOS), and then identify their polypeptide moieties by mass spectrometry. As a proof-of-concept experiment using Escherichia coli, we identified ∼800 proteins derived from the pep-tRNAs, which were markedly biased towards the N-termini in the proteins, reflecting that PETEOS captured the intermediate pep-tRNA population during translation. Furthermore, we observed the changes in the pep-tRNA set in response to heat shock or antibiotic treatments. In summary, PETEOS will complement conventional methods to investigate nascent chains in the cell.
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