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.
Keywords: in vitro translation; CrPV IGR IRES; translation elongation; translation termination; yeast.
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