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Elimination of Ribosome Inactivating Factors Improves the Efficiency of Bacillus subtilis and Saccharomyces cerevisiae Cell-Free Translation Systems.

ABSTRACT: Cell-free translation systems based on cellular lysates optimized for in vitro protein synthesis have multiple applications both in basic and applied science, ranging from studies of translational regulation to cell-free production of proteins and ribosome-nascent chain complexes. In order to achieve both high activity and reproducibility in a translation system, it is essential that the ribosomes in the cellular lysate are enzymatically active. Here we demonstrate that genomic disruption of genes encoding ribosome inactivating factors - HPF in Bacillus subtilis and Stm1 in Saccharomyces cerevisiae - robustly improve the activities of bacterial and yeast translation systems. Importantly, the elimination of B. subtilis HPF results in a complete loss of 100S ribosomes, which otherwise interfere with disome-based approaches for preparation of stalled ribosomal complexes for cryo-electron microscopy studies.

SUBMITTER: Brodiazhenko T 

PROVIDER: S-EPMC6305275 | biostudies-other | 2018

REPOSITORIES: biostudies-other

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Elimination of Ribosome Inactivating Factors Improves the Efficiency of <i>Bacillus subtilis</i> and <i>Saccharomyces cerevisiae</i> Cell-Free Translation Systems.

Brodiazhenko Tetiana T   Johansson Marcus J O MJO   Takada Hiraku H   Nissan Tracy T   Hauryliuk Vasili V   Murina Victoriia V  

Frontiers in microbiology 20181218

Cell-free translation systems based on cellular lysates optimized for <i>in vitro</i> protein synthesis have multiple applications both in basic and applied science, ranging from studies of translational regulation to cell-free production of proteins and ribosome-nascent chain complexes. In order to achieve both high activity and reproducibility in a translation system, it is essential that the ribosomes in the cellular lysate are enzymatically active. Here we demonstrate that genomic disruption  ...[more]