A method to enrich polypeptidyl-tRNAs to capture snapshots of translation in the cell.
Journal
Nucleic acids research
ISSN: 1362-4962
Titre abrégé: Nucleic Acids Res
Pays: England
ID NLM: 0411011
Informations de publication
Date de publication:
21 03 2023
21 03 2023
Historique:
accepted:
25
12
2022
revised:
02
12
2022
received:
19
10
2022
pubmed:
31
1
2023
medline:
21
3
2023
entrez:
30
1
2023
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 36715318
pii: 7009116
doi: 10.1093/nar/gkac1276
pmc: PMC10018338
doi:
Substances chimiques
RNA, Transfer
9014-25-9
Peptides
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
e30Informations de copyright
© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.
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