In vitro protein synthesis

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Abstract

This chapter describes in vitro yeast translation system. Molecular characterization of the genes encoding ribosomal RNA, ribosomal proteins, and factors involved in translational initiation, and elongation in yeast make this an exciting system in which to study the biochemistry of translation. Several yeast cellfree–translation systems that are dependent on added messenger RNA (mRNA) are also described. Cell extract can be prepared by gentle glass bead lysis requiring neither prolonged pretreatment before lysis nor ultracentrifugation. The chapter is based on the modification of this method. This method produces active extracts from various haploid and diploid yeast strains which are stable frozen at –70 °. This method is preferred for its reproducibility and ease of preparation. The results obtained with it indicate properties similar to those described for the other systems. Application of this method to cells with genetic alterations in the components of the translational machinery should prove extremely useful for investigation of the mechanism and regulation of protein synthesis. In yeast, both classical and recombinant genetics can be used to determine the structure-function relationship of gene products making up the translational apparatus, unlike the more widely used systems from metazoan eukaryotes. © 1991 © 1991, Elsevier Inc. All rights reserved.

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Leibowitz, M. J., Barbone, F. P., & Georgopoulos, D. E. (1991). In vitro protein synthesis. Methods in Enzymology, 194(C), 536–545. https://doi.org/10.1016/0076-6879(91)94040-J

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