Abstract
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.
Highlights
The reconstitution of biological processes is critical for the investigation of the underlying molecular mechanisms
To prepare aminoacyl-tRNAs, a yeast tRNA mixture was charged by yeast S100 extract, which we found to be capable of template-dependent synthesis of long polypeptides
Other applications of this system include the template-dependent synthesis of proteins containing unnatural amino acids by eukaryotic ribosomes, using synthetic tRNAs that have been appropriately charged for example, with ribozymes
Summary
The reconstitution of biological processes is critical for the investigation of the underlying molecular mechanisms. We have recently developed an in vitro reconstituted translation system that is capable of synthesizing long peptides [18] In this system, in order to bypass the complex initiation process, we exploited CrPV IGR IRES (intergenic region internal ribosome entry site sequence from cricket paralysis virus)-containing mRNA, whereby yeast 80S ribosomes initiate in the absence of initiator tRNA or any eukaryotic translation initiation factors [19,20]. As the system allows for research into the effect of mRNA- or nascent peptide-context on translation termination, it could be applied for the development of readthrough-inducing agents for treating genetic diseases caused by nonsense mutations Other applications of this system include the template-dependent synthesis of proteins containing unnatural amino acids by eukaryotic ribosomes, using synthetic tRNAs that have been appropriately charged for example, with ribozymes. In combination with ribosome- or mRNA-display systems, this system could serve as a powerful in vitro screening system for novel functional proteins, such as antimicrobial peptides with cytotoxicity
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