In vivo and in vitro analysis of poly(A) length effects on mRNA translation.
In vivo and in vitro analysis of poly(A) length effects on mRNA translation.
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Poly(A) 长度对 mRNA 翻译影响的体内和体外分析。
DOI：
10.1007/978-1-59745-033-1_15
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发表时间：
2008
期刊：
影响因子：
--
通讯作者：
Schoenberg,DanielR
中科院分区：
文献类型：
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作者：
Peng,Jing;Murray,ElizabethL;Schoenberg,DanielR
在翻译水平上调节基因表达控制着多种生物学事件，如发育、长期记忆、应激反应、某些营养物质的运输和储存以及病毒感染。稳定状态下的蛋白质合成可以直接用蛋白质印迹或使用易于检测的报告，如荧光素酶来测量。然而，这些方法并没有测量mRNA与核糖体的关联，这对于理解翻译的机制和动力学更有意义。本章描述了如何使用蔗糖密度梯度来分析多糖体图谱。从梯度组分中提取的RNA或蛋白质样品通常用于进一步分析它们与翻译核糖体的关联。我们还描述了一个由HeLa S3细胞胞浆提取物制备的体外翻译系统，该系统显示出对RNA帽和聚(A)长度尾巴的长度的依赖性，这两个特征都是体内翻译的特征。这对于研究翻译控制中的顺式和反式作用因素特别有用。最后，我们描述了一种用体外制备的RNA转染细胞的方法，以研究聚(A)长度对翻译的影响。这种方法对于表征与聚(A)共同调节翻译的顺式作用元件特别有用。
Regulating gene expression at the translational level controls a wide variety of biological events such as development, long-term memory, stress response, transport and storage of certain nutrients, and viral infection. Protein synthesis at steady-state level can be directly measured with Western blot or using an easy-to-detect reporter such as luciferase. However, these methods do not measure the association of mRNA with ribosomes, which is more meaningful in understanding the mechanism and dynamics of translation. This chapter describes the use of sucrose density gradients for analysis of polysome profiles. RNA or protein samples extracted from gradient fractions are commonly used for further analysis of their association with translating ribosomes. We also describe an in vitro translation system prepared from HeLa S3 cell cytoplasmic extract that shows dependency on the mRNA cap and length of the poly(A) length tail, both features of translation in vivo. This is particularly useful to study thecis- andtrans-acting factors involved in translational control. Lastly, we describe a method for transfecting cells with an in vitro prepared RNA to study the impact of poly(A) length on translation. This approach is particularly useful for characterizingcis-acting elements that work in conjunction with poly(A) in regulating translation.