Eukaryotic polyribosome profile analysis.
Eukaryotic polyribosome profile analysis.
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DOI：
10.3791/1948
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发表时间：
2010-06-15
期刊：
影响因子：
--
通讯作者：
Kinzy, Terri G
中科院分区：
文献类型：
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作者：
Esposito, Anthony M;Mateyak, Maria;Kinzy, Terri G
蛋白质合成是一个复杂的细胞过程，在许多水平上受到调节。例如，在起始阶段或延伸阶段，可以通过多种细胞应激（例如氨基酸饥饿或生长因子撤回）来抑制整体翻译。或者，单个mRNA的翻译可以通过mRNA定位或同源microRNA的存在来调节。蛋白质合成的研究经常利用多聚核糖体分析来阐明蛋白质合成中的翻译调节或缺陷的机制。在该测定中，从真核细胞中分离mRNA/核糖体复合物。蔗糖密度梯度将结合到多个核糖体（称为多聚核糖体）的mRNA与结合到单个核糖体或单核糖体的mRNA分开。梯度的分级允许分离和定量不同的核糖体群体及其相关的mRNA或蛋白质。在限定条件下多聚核糖体与单体的比率的差异可以指示翻译起始或延伸/终止中的缺陷。检查多核糖体组分中存在的mRNA可以揭示被翻译的单个mRNA的队列是否随实验条件而变化。此外，可以通过分析也被梯度分离的核糖体大亚基峰和小亚基峰来监测核糖体组装。在这个视频中，我们提出了一种从酵母细胞中制备粗核糖体提取物的方法，通过蔗糖梯度分离提取物并解释结果。这一程序很容易适用于哺乳动物细胞。
Protein synthesis is a complex cellular process that is regulated at many levels. For example, global translation can be inhibited at the initiation phase or the elongation phase by a variety of cellular stresses such as amino acid starvation or growth factor withdrawal. Alternatively, translation of individual mRNAs can be regulated by mRNA localization or the presence of cognate microRNAs. Studies of protein synthesis frequently utilize polyribosome analysis to shed light on the mechanisms of translation regulation or defects in protein synthesis. In this assay, mRNA/ribosome complexes are isolated from eukaryotic cells. A sucrose density gradient separates mRNAs bound to multiple ribosomes known as polyribosomes from mRNAs bound to a single ribosome or monosome. Fractionation of the gradients allows isolation and quantification of the different ribosomal populations and their associated mRNAs or proteins. Differences in the ratio of polyribosomes to monosomes under defined conditions can be indicative of defects in either translation initiation or elongation/termination. Examination of the mRNAs present in the polyribosome fractions can reveal whether the cohort of individual mRNAs being translated changes with experimental conditions. In addition, ribosome assembly can be monitored by analysis of the small and large ribosomal subunit peaks which are also separated by the gradient. In this video, we present a method for the preparation of crude ribosomal extracts from yeast cells, separation of the extract by sucrose gradient and interpretation of the results. This procedure is readily adaptable to mammalian cells.