Reconstitution of yeast translation elongation and termination in vitro utilizing CrPV IRES-containing mRNA

Abstract We developed an in vitro translation system from yeast, reconstituted with purified translation elongation and termination factors and programmed by CrPV IGR IRES-containing mRNA, which functions in the absence of initiation factors. The system is capable of synthesizing the active reporter...

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Bibliographic Details
Published in:Journal of biochemistry (Tokyo) 2020-05, Vol.167 (5), p.441-450
Main Authors: Abe, Taisho, Nagai, Riku, Imataka, Hiroaki, Takeuchi-Tomita, Nono
Format: Article
Language:English
Subjects:
Peptide Chain Elongation, Translational
Peptide Chain Termination, Translational
Peptides - metabolism
RNA, Messenger - metabolism
Saccharomyces cerevisiae - genetics
Saccharomyces cerevisiae - metabolism
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Summary:Abstract We developed an in vitro translation system from yeast, reconstituted with purified translation elongation and termination factors and programmed by CrPV IGR IRES-containing mRNA, which functions in the absence of initiation factors. The system is capable of synthesizing the active reporter protein, nanoLuciferase, with a molecular weight of 19 kDa. The protein synthesis by the system is appropriately regulated by controlling its composition, including translation factors, amino acids and antibiotics. We found that a high eEF1A concentration relative to the ribosome concentration is critically required for efficient IRES-mediated translation initiation, to ensure its dominance over IRES-independent random internal translation initiation.
ISSN:0021-924X
1756-2651
DOI:10.1093/jb/mvaa021