Stress-induced molecular damage to ribosomes can impact protein synthesis in cells, but cell-based assays do not provide a clear way to distinguish the effects of ribosome damage from stress responses and damage to other parts of the translation machinery. Here we describe a detailed protocol for the separation of yeast ribosomes from other translational machinery constituents, followed by reconstitution of the translation mixture in vitro. This technique, which we refer to as ribosome separation and reconstitution (RSR), allows chemical modifications of yeast ribosomes without compromising other key translational components. In addition to the characterization of stress-induced ribosome damage, RSR can be applied to a broad range of experimental problems in studies of yeast translation.

中文翻译：

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体外蛋白质翻译核糖体分离和重构系统的开发、验证和应用


应激诱导的核糖体分子损伤会影响细胞中的蛋白质合成，但基于细胞的检测并没有提供明确的方法来区分核糖体损伤与应激反应和翻译机器其他部分损伤的影响。在这里，我们描述了将酵母核糖体与其他翻译机器成分分离的详细方案，然后在体外重建翻译混合物。我们将这种技术称为核糖体分离和重构（RSR），它允许对酵母核糖体进行化学修饰，而不会损害其他关键的翻译成分。除了表征应激诱导的核糖体损伤之外，RSR 还可应用于酵母翻译研究中的广泛实验问题。