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Author:
Date:
2020-12-05
[Abstract] Transfer RNA (tRNA) is an essential link between the genetic code and proteins. During the process of translation, tRNA is charged with its cognate amino acid and delivers it to the ribosome, thus serving as a substrate of protein synthesis. To analyze the charging state of a particular tRNA, total RNA is purified and analyzed on an acid-urea gel. Separated RNA is then transferred to a membrane and detected with a probe for the tRNA of interest. Here, we present an improved protocol to analyze the tRNA charging state in the α-proteobacterium Rhodopseudomonas palustris. Compared to the classical method, the RNA isolation step is optimized to suit this organism. Additionally, a non-radioactive platform is used for electrophoresis and Northern blots. This significantly reduces ...
[摘要] [摘要]转移RNA（tRNA）是遗传密码与蛋白质之间的重要纽带。在翻译过程中，tRNA带有其同源氨基酸，并将其传递至核糖体，因此可作为蛋白质合成的底物。为了分析特定tRNA的电荷状态，纯化总RNA并在酸性尿素凝胶上进行分析。然后将分离的RNA转移到膜上并用目标tRNA的探针进行检测。在这里，我们提出了一种改进的协议来分析α-变形杆菌Rhodopseudomonas palustris中的tRNA充电状态 。与传统方法相比，优化了RNA分离步骤以适合这种生物。另外，非放射性平台用于电泳和RNA印迹。这显着减少了此协议所需的时间和精力。
[背景] tRNA的主要功能是，与其他翻译因素的帮助，以确保mRNA的蛋白质的准确的翻译。氨基酰基-tRNA（带电）将氨基酸带到核糖体中以延长肽段，然后释放不带电荷的tRNA。tRNA的充电状态主要取决于可用资源（即氨基酸）及其被核糖体的消耗量。为了分析细胞tRNA的充电状态，已经开发了使用酸性脲凝胶分离总RNA并通过Northern印迹检测感兴趣的tRNA的方法（Janssen等人，2012； ...
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