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Nascentome Analysis Uncovers Futile Protein Synthesis in Escherichia coli

Although co-translational biological processes attract much attention, no general and easy method has been available to detect cellular nascent polypeptide chains, which we propose to call collectively a “nascentome.” We developed a method to selectively detect polypeptide portions of cellular polyp...

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Autores principales: Ito, Koreaki, Chadani, Yuhei, Nakamori, Kenta, Chiba, Shinobu, Akiyama, Yoshinori, Abo, Tatsuhiko
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2011
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3230602/
https://www.ncbi.nlm.nih.gov/pubmed/22162769
http://dx.doi.org/10.1371/journal.pone.0028413
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author Ito, Koreaki
Chadani, Yuhei
Nakamori, Kenta
Chiba, Shinobu
Akiyama, Yoshinori
Abo, Tatsuhiko
author_facet Ito, Koreaki
Chadani, Yuhei
Nakamori, Kenta
Chiba, Shinobu
Akiyama, Yoshinori
Abo, Tatsuhiko
author_sort Ito, Koreaki
collection PubMed
description Although co-translational biological processes attract much attention, no general and easy method has been available to detect cellular nascent polypeptide chains, which we propose to call collectively a “nascentome.” We developed a method to selectively detect polypeptide portions of cellular polypeptidyl-tRNAs and used it to study the generality of the quality control reactions that rescue dead-end translation complexes. To detect nascent polypeptides, having their growing ends covalently attached to a tRNA, cellular extracts are separated by SDS-PAGE in two dimensions, first with the peptidyl-tRNA ester bonds preserved and subsequently after their in-gel cleavage. Pulse-labeled nascent polypeptides of Escherichia coli form a characteristic line below the main diagonal line, because each of them had contained a tRNA of nearly uniform size in the first-dimension electrophoresis but not in the second-dimension. The detection of nascent polypeptides, separately from any translation-completed polypeptides or degradation products thereof, allows us to follow their fates to gain deeper insights into protein biogenesis and quality control pathways. It was revealed that polypeptidyl-tRNAs were significantly stabilized in E. coli upon dysfunction of the tmRNA-ArfA ribosome-rescuing system, whose function had only been studied previously using model constructs. Our results suggest that E. coli cells are intrinsically producing aberrant translation products, which are normally eliminated by the ribosome-rescuing mechanisms.
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spelling pubmed-32306022011-12-08 Nascentome Analysis Uncovers Futile Protein Synthesis in Escherichia coli Ito, Koreaki Chadani, Yuhei Nakamori, Kenta Chiba, Shinobu Akiyama, Yoshinori Abo, Tatsuhiko PLoS One Research Article Although co-translational biological processes attract much attention, no general and easy method has been available to detect cellular nascent polypeptide chains, which we propose to call collectively a “nascentome.” We developed a method to selectively detect polypeptide portions of cellular polypeptidyl-tRNAs and used it to study the generality of the quality control reactions that rescue dead-end translation complexes. To detect nascent polypeptides, having their growing ends covalently attached to a tRNA, cellular extracts are separated by SDS-PAGE in two dimensions, first with the peptidyl-tRNA ester bonds preserved and subsequently after their in-gel cleavage. Pulse-labeled nascent polypeptides of Escherichia coli form a characteristic line below the main diagonal line, because each of them had contained a tRNA of nearly uniform size in the first-dimension electrophoresis but not in the second-dimension. The detection of nascent polypeptides, separately from any translation-completed polypeptides or degradation products thereof, allows us to follow their fates to gain deeper insights into protein biogenesis and quality control pathways. It was revealed that polypeptidyl-tRNAs were significantly stabilized in E. coli upon dysfunction of the tmRNA-ArfA ribosome-rescuing system, whose function had only been studied previously using model constructs. Our results suggest that E. coli cells are intrinsically producing aberrant translation products, which are normally eliminated by the ribosome-rescuing mechanisms. Public Library of Science 2011-12-05 /pmc/articles/PMC3230602/ /pubmed/22162769 http://dx.doi.org/10.1371/journal.pone.0028413 Text en Ito et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Ito, Koreaki
Chadani, Yuhei
Nakamori, Kenta
Chiba, Shinobu
Akiyama, Yoshinori
Abo, Tatsuhiko
Nascentome Analysis Uncovers Futile Protein Synthesis in Escherichia coli
title Nascentome Analysis Uncovers Futile Protein Synthesis in Escherichia coli
title_full Nascentome Analysis Uncovers Futile Protein Synthesis in Escherichia coli
title_fullStr Nascentome Analysis Uncovers Futile Protein Synthesis in Escherichia coli
title_full_unstemmed Nascentome Analysis Uncovers Futile Protein Synthesis in Escherichia coli
title_short Nascentome Analysis Uncovers Futile Protein Synthesis in Escherichia coli
title_sort nascentome analysis uncovers futile protein synthesis in escherichia coli
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3230602/
https://www.ncbi.nlm.nih.gov/pubmed/22162769
http://dx.doi.org/10.1371/journal.pone.0028413
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