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: | , , , |
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| Formato: | article |
| Lenguaje: | EN |
| Publicado: |
Nature Portfolio
2021
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| Materias: | |
| Acceso en línea: | https://doaj.org/article/11e9fe0797a8410691c94ac215af4959 |
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| Sumario: | 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. |
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