[3'-32P]-labeling tRNA with nucleotidyltransferase for assaying aminoacylation and peptide bond formation
[3'-32P]-labeling tRNA with nucleotidyltransferase for assaying aminoacylation and peptide bond formation
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DOI：
10.1016/j.ymeth.2007.08.001
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发表时间：
2008-02-01
期刊：
影响因子：
4.8
通讯作者：
Uhlenbeck, Olke C.
中科院分区：
文献类型：
--
作者：
Ledoux, Sarah;Uhlenbeck, Olke C.
分析涉及酯化成tRNA的氨基酸的反应传统上使用放射性标记的氨基酸。在这里，我们描述了一种使用[3‘-P-32]标记的tRNA，然后进行核酸酶消化和TLC分析的替代方法，它允许以有效的、定量的方式监测氨基酰化反应，同时避免了使用放射性标记氨基酸时所面临的许多问题。我们还描述了一种使用[3‘-P-32]标记的AA-tRNAs来确定核糖体上多肽键形成速率的类似方法。这种类型的分析也有可能用于研究与tRNA酯化的氨基酸或多肽有关的其他反应。(C)2007 Elsevier Inc.保留所有权利。
The analysis of reactions involving amino acids esterified to tRNAs traditionally uses radiolabeled amino acids. We describe here an alternative assay involving [3'-P-32]-labeled tRNA followed by nuclease digestion and TLC analysis that permits aminoacylation to be monitored in an efficient, quantitative manner while circumventing many of the problems faced when using radiolabeled amino acids. We also describe a similar assay using [3'-P-32]-labeled aa-tRNAs to determine the rate of peptide bond formation on the ribosome. This type of assay can also potentially be adapted to study other reactions involving an amino acid or peptide esterified to tRNA. (C) 2007 Elsevier Inc. All rights reserved.