Development, validation, and application of the ribosome separation and reconstitution system for protein translation in vitro.
Development, validation, and application of the ribosome separation and reconstitution system for protein translation in vitro.
复制标题
DOI：
10.1261/rna.078852.121
复制
发表时间：
2021-12
期刊：
影响因子：
--
通讯作者：
Shcherbik N
中科院分区：
文献类型：
--
作者：
Trainor BM;Pestov DG;Shcherbik N
应激诱导的对核糖体的分子损伤可以影响细胞中的蛋白质合成，但是基于细胞的测定没有提供明确的方法来区分核糖体损伤与应激反应的影响以及对翻译机器的其他部分的损伤。在这里，我们描述了一个详细的协议，从其他翻译机器成分的酵母核糖体的分离，然后在体外重建的翻译混合物。这种技术，我们称之为核糖体分离和重建（RSR），允许酵母核糖体的化学修饰，而不损害其他关键的翻译组件。除了表征应激诱导的核糖体损伤外，RSR还可以应用于酵母翻译研究中的广泛实验问题。
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.