用核苷酸转移酶对[3'-32P]标记的tRNA进行测定氨酰化和肽键形成。
[3'-32P]-labeling tRNA with nucleotidyltransferase for assaying aminoacylation and peptide bond formation.
作者信息
Ledoux Sarah, Uhlenbeck Olke C
机构信息
Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL 60208, USA.
出版信息
Methods. 2008 Feb;44(2):74-80. doi: 10.1016/j.ymeth.2007.08.001.
Abstract
The analysis of reactions involving amino acids esterified to tRNAs traditionally uses radiolabeled amino acids. We describe here an alternative assay involving [3'-32P]-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'-32P]-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.
摘要
传统上，对涉及与tRNA酯化的氨基酸的反应进行分析时使用放射性标记的氨基酸。我们在此描述一种替代检测方法，该方法涉及[3'-32P]标记的tRNA，随后进行核酸酶消化和薄层层析分析，它能以高效、定量的方式监测氨酰化反应，同时规避使用放射性标记氨基酸时面临的许多问题。我们还描述了一种类似的检测方法，使用[3'-32P]标记的氨酰-tRNA来确定核糖体上肽键形成的速率。这种类型的检测方法也有可能适用于研究其他涉及与tRNA酯化的氨基酸或肽的反应。
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