In Vitro Reconstitution of Yeast Translation System Capable of Synthesizing Long Polypeptide and Recapitulating Programmed Ribosome Stalling
| Autor: | Yichen Xu, Nono Takeuchi-Tomita, Riku Nagai, Ayaka Shimabukuro, Chang Liu |
|---|---|
| Rok vydání: | 2021 |
| Předmět: |
QH301-705.5
yeast Biochemistry Genetics and Molecular Biology (miscellaneous) Ribosome 03 medical and health sciences 0302 clinical medicine Structural Biology Protocol Initiation factor Biology (General) 030304 developmental biology Polyproline helix chemistry.chemical_classification 0303 health sciences Oligopeptide Messenger RNA Translation (biology) translation termination Yeast Cell biology Amino acid translation elongation in vitro translation chemistry CrPV IGR IRES 030217 neurology & neurosurgery Biotechnology |
| Zdroj: | Methods and Protocols Methods and Protocols, Vol 4, Iss 45, p 45 (2021) |
| ISSN: | 2409-9279 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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