In vitro reconstitution of translational arrest pathways
In vitro reconstitution of translational arrest pathways
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DOI：
10.1016/j.ymeth.2017.12.018
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发表时间：
2018-03-15
期刊：
影响因子：
4.8
通讯作者：
Shao, Sichen
中科院分区：
文献类型：
--
作者：
Feng, Qing;Shao, Sichen
蛋白质翻译受到严格调控，以确保遗传信息的高保真表达。各种条件导致核糖体在合成新蛋白质时停滞。不同类型的翻译停滞启动特定的mRNA监视、蛋白质质量控制和应激反应途径，这些途径直接影响基因表达和蛋白质稳态。通过在无细胞系统中重建这些过程，我们对这些途径的理解大大增强。体外系统的高度生化可操作性有利于识别关键机制，机械解剖其功能作用，和中间复合物的结构分析。在这里，我们描述的原则和方法，重建和分析在无细胞翻译系统中的翻译停滞途径，使用兔网织红细胞裂解物为例。这些方法可以用来剖析真核生物蛋白质翻译的各种基本、调节和质量控制机制。(C)2017爱思唯尔公司All rights reserved.
Protein translation is tightly regulated to ensure high-fidelity expression of genetic information. Various conditions cause ribosomes to stall while synthesizing new proteins. Different types of translational arrest initiate specific mRNA surveillance, protein quality control, and stress response pathways that directly impact gene expression and protein homeostasis. Our understanding of these pathways is greatly enhanced by reconstituting these processes in cell-free systems. The high degree of biochemical manipulability of in vitro systems facilitates the identification of key machineries, mechanistic dissection of their functional roles, and structural analysis of intermediate complexes. Here, we describe principles and methods for reconstituting and analyzing translational arrest pathways in cell-free translation systems using rabbit reticulocyte lysate as an example. These approaches can be exploited to dissect various fundamental, regulatory, and quality control mechanisms of eukaryotic protein translation. (C) 2017 Elsevier Inc. All rights reserved.