Key result
Reconstituted yeast in vitro translation system successfully synthesizes long polypeptides and recapitulates ribosome stalling.
Why the study?
Molecular mechanisms modulating eukaryotic translation elongation and termination rates require investigation using a reconstituted in vitro system.
The development of a reconstituted yeast in vitro translation system enables detailed biochemical analyses of eukaryotic translation and potential applications in drug design and novel protein production.
Reconstituted yeast system enables biochemical analyses of translation; leaves open extension to mammalian systems or applications.
The rates of translation elongation or termination in eukaryotes are modulated through cooperative molecular interactions involving mRNA, the ribosome, aminoacyl- and nascent polypeptidyl-tRNAs, and translation factors. To investigate the molecular mechanisms underlying these processes, we developed an in vitro translation system from yeast, reconstituted with purified translation elongation and termination factors, utilizing CrPV IGR IRES-containing mRNA, which functions in the absence of initiation factors. The system is capable of synthesizing not only short oligopeptides but also long reporter proteins such as nanoluciferase. By setting appropriate translation reaction conditions, such as the Mg2+/polyamine concentration, the arrest of translation elongation by known ribosome-stalling sequences (e.g., polyproline and CGA codon repeats) is properly recapitulated in this system. We describe protocols for the preparation of the system components, manipulation of the system, and detection of the translation products. We also mention critical parameters for setting up the translation reaction conditions. This reconstituted translation system not only facilitates biochemical analyses of translation but is also useful for various applications, such as structural and functional studies with the aim of designing drugs that act on eukaryotic ribosomes, and the development of systems for producing novel functional proteins by incorporating unnatural amino acids by eukaryotic ribosomes.
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Nagai et al. (2021) studied this question. In vitro reconstituted yeast translation system was evaluated on Synthesis of long polypeptides and recapitulation of programmed ribosome stalling. An in vitro translation system from yeast was successfully reconstituted using purified translation factors and CrPV IGR IRES-containing mRNA to synthesize long polypeptides and recapitulate ribosome stalling.
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