探索修饰核糖体功能的人兔杂交翻译系统。

Eriko Matsuura-Suzuki, Hirotaka Toh, Shintaro Iwasaki
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引用次数: 0

摘要

体外翻译系统是研究翻译调控的一个有用的生化工具。尽管从哺乳动物中制备具有翻译能力的细胞提取物一直是一个挑战,但市售的兔网织细胞裂解液(RRL)是一个例外。然而,它的有效使用,研究翻译机器的机制,如核糖体在RRL,提出了一个分析障碍。为了克服这一问题,基于将纯化的人类核糖体补充到核糖体缺失的RRL的杂交翻译系统已经开发出来。在这里,我们描述了该系统的一步一步的协议,以研究由缺乏翻译后修饰的核糖体蛋白驱动的翻译。此外,我们将这种方法与先前开发的报告mRNA结合起来评估翻译延伸的进程性。该方法可用于研究异源核糖体的效力。
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Human-rabbit Hybrid Translation System to Explore the Function of Modified Ribosomes.

In vitro translation systems are a useful biochemical tool to research translational regulation. Although the preparation of translation-competent cell extracts from mammals has often been a challenge, the commercially available rabbit reticulocyte lysate (RRL) is an exception. However, its valid use, investigating the mechanism of translation machinery such as ribosomes in RRL, presents an analytic hurdle. To overcome this issue, the hybrid translation system, which is based on the supplementation of purified human ribosomes into ribosome-depleted RRL, has been developed. Here, we describe the step-by-step protocol of this system to study translation driven by ribosomes lacking post-translational modifications of the ribosomal protein. Moreover, we combined this approach with a previously developed reporter mRNA to assess the processivity of translation elongation. This protocol could be used to study the potency of heterologous ribosomes.

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