ArticlePloS one2016
Site-Specific Cleavage of Ribosomal RNA in Escherichia coli-Based Cell-Free Protein Synthesis Systems.
Article in PloS one, 2016. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 14 papers.
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Who cites it
14 citing papers in PubMed, 23 citations in OpenAlex.
- Carrot and stick: how RNase R contributes to function and destruction of the translation machinery.RNA biology · 2025Review
- Role of VapBC4 toxin-antitoxin system ofmBio · 2024Article
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- A cell-free strategy for host-specific profiling of intracellular antibiotic sensitivity and resistance.npj antimicrobials and resistance · 2023Article
- Designing of an extract production protocol for industrial application of cell-free protein synthesis technology: Building from a current best practice to a quality by design approach.Engineering biology · 2023Review
- Structural Insights into the Distortion of the Ribosomal Small Subunit at Different Magnesium Concentrations.Biomolecules · 2023Article
- Advancing synthetic biology through cell-free protein synthesis.Computational and structural biotechnology journal · 2023Review
- COmBio · 2020Article
- Models for Cell-Free Synthetic Biology: Make Prototyping Easier, Better, and Faster.Frontiers in bioengineering and biotechnology · 2018Review
- Cell-Free Protein Synthesis From Fast-GrowingFrontiers in microbiology · 2018Article
- Cell-free protein synthesis from non-growing, stressed Escherichia coli.Scientific reports · 2017Article
- A reproducible and scalable procedure for preparing bacterial extracts for cell-free protein synthesis.Journal of biochemistry · 2017Article
- Bacterial cell-free expression technology toSynthetic and systems biotechnology · 2017Review
- Translation inhibition and resource balance in the TX-TL cell-free gene expression system.Synthetic biology (Oxford, England) · 2017Article
Corrections and comments
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Authors and funding
5 authors at 2 institutions in 2 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Cell-free protein synthesis, which mimics the biological protein production system, allows rapid expression of proteins without the need to maintain a viable cell. Nevertheless, cell-free protein expression relies on active in vivo translation machinery including ribosomes and translation factors. Here, we examined the integrity of the protein synthesis machinery, namely the functionality of ribosomes, during (i) the cell-free extract preparation and (ii) the performance of in vitro protein synthesis by analyzing crucial components involved in translation. Monitoring the 16S rRNA, 23S rRNA, elongation factors and ribosomal protein S1, we show that processing of a cell-free extract results in no substantial alteration of the translation machinery. Moreover, we reveal that the 16S rRNA is specifically cleaved at helix 44 during in vitro translation reactions, resulting in the removal of the anti-Shine-Dalgarno sequence. These defective ribosomes accumulate in the cell-free system. We demonstrate that the specific cleavage of the 16S rRNA is triggered by the decreased concentrations of Mg2+. In addition, we provide evidence that helix 44 of the 30S ribosomal subunit serves as a point-of-entry for ribosome degradation in Escherichia coli. Our results suggest that Mg2+ homeostasis is fundamental to preserving functional ribosomes in cell-free protein synthesis systems, which is of major importance for cell-free protein synthesis at preparative scale, in order to create highly efficient technical in vitro systems.
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