In vitro synthesis of 32 translation-factor proteins from a single template reveals impaired ribosomal processivity

Abstract The Protein synthesis Using Recombinant Elements (PURE) system enables transcription and translation of a DNA template from purified components. Therefore, the PURE system-catalyzed generation of RNAs and proteins constituting the PURE system itself represents a major challenge toward a sel...

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Autores principales:	Anne Doerr, David Foschepoth, Anthony C. Forster, Christophe Danelon
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/11e9fe0797a8410691c94ac215af4959
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spelling	oai:doaj.org-article:11e9fe0797a8410691c94ac215af49592021-12-02T14:07:48ZIn vitro synthesis of 32 translation-factor proteins from a single template reveals impaired ribosomal processivity10.1038/s41598-020-80827-82045-2322https://doaj.org/article/11e9fe0797a8410691c94ac215af49592021-01-01T00:00:00Zhttps://doi.org/10.1038/s41598-020-80827-8https://doaj.org/toc/2045-2322Abstract The Protein synthesis Using Recombinant Elements (PURE) system enables transcription and translation of a DNA template from purified components. Therefore, the PURE system-catalyzed generation of RNAs and proteins constituting the PURE system itself represents a major challenge toward a self-replicating minimal cell. In this work, we show that all translation factors (except elongation factor Tu) and 20 aminoacyl-tRNA synthetases can be expressed in the PURE system from a single plasmid encoding 32 proteins in 30 cistrons. Cell-free synthesis of all 32 proteins is confirmed by quantitative mass spectrometry-based proteomic analysis using isotopically labeled amino acids. We find that a significant fraction of the gene products consists of proteins missing their C-terminal ends. The per-codon processivity loss that we measure lies between 1.3 × 10–3 and 13.2 × 10–3, depending on the expression conditions, the version of the PURE system, and the coding sequence. These values are 5 to 50 times higher than those measured in vivo in E. coli. With such an impaired processivity, a considerable fraction of the biosynthesis capacity of the PURE system is wasted, posing an unforeseen challenge toward the development of a self-regenerating PURE system.Anne DoerrDavid FoschepothAnthony C. ForsterChristophe DanelonNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-12 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Anne Doerr David Foschepoth Anthony C. Forster Christophe Danelon In vitro synthesis of 32 translation-factor proteins from a single template reveals impaired ribosomal processivity
description	Abstract The Protein synthesis Using Recombinant Elements (PURE) system enables transcription and translation of a DNA template from purified components. Therefore, the PURE system-catalyzed generation of RNAs and proteins constituting the PURE system itself represents a major challenge toward a self-replicating minimal cell. In this work, we show that all translation factors (except elongation factor Tu) and 20 aminoacyl-tRNA synthetases can be expressed in the PURE system from a single plasmid encoding 32 proteins in 30 cistrons. Cell-free synthesis of all 32 proteins is confirmed by quantitative mass spectrometry-based proteomic analysis using isotopically labeled amino acids. We find that a significant fraction of the gene products consists of proteins missing their C-terminal ends. The per-codon processivity loss that we measure lies between 1.3 × 10–3 and 13.2 × 10–3, depending on the expression conditions, the version of the PURE system, and the coding sequence. These values are 5 to 50 times higher than those measured in vivo in E. coli. With such an impaired processivity, a considerable fraction of the biosynthesis capacity of the PURE system is wasted, posing an unforeseen challenge toward the development of a self-regenerating PURE system.
format	article
author	Anne Doerr David Foschepoth Anthony C. Forster Christophe Danelon
author_facet	Anne Doerr David Foschepoth Anthony C. Forster Christophe Danelon
author_sort	Anne Doerr
title	In vitro synthesis of 32 translation-factor proteins from a single template reveals impaired ribosomal processivity
title_short	In vitro synthesis of 32 translation-factor proteins from a single template reveals impaired ribosomal processivity
title_full	In vitro synthesis of 32 translation-factor proteins from a single template reveals impaired ribosomal processivity
title_fullStr	In vitro synthesis of 32 translation-factor proteins from a single template reveals impaired ribosomal processivity
title_full_unstemmed	In vitro synthesis of 32 translation-factor proteins from a single template reveals impaired ribosomal processivity
title_sort	in vitro synthesis of 32 translation-factor proteins from a single template reveals impaired ribosomal processivity
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/11e9fe0797a8410691c94ac215af4959
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In vitro synthesis of 32 translation-factor proteins from a single template reveals impaired ribosomal processivity

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