一种用于tRNA氨基酰化动力学的新检测方法。
A new assay for tRNA aminoacylation kinetics.
作者信息
Wolfson A D, Pleiss J A, Uhlenbeck O C
机构信息
Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215, USA.
出版信息
RNA. 1998 Aug;4(8):1019-23. doi: 10.1017/s1355838298980700.
Abstract
An improved quantitative assay for tRNA aminoacylation is presented based on charging of a nicked tRNA followed by separation of an aminoacylated 3'-fragment on an acidic denaturing polyacrylamide gel. Kinetic parameters of tRNA aminoacylation by Escherichia coli AlaRS obtained by the new method are in excellent agreement with those measured by the conventional method. This assay provides several advantages over the traditional methods of measuring tRNA aminoacylation: (1) the fraction of aminoacyl-tRNA is measured directly; (2) data can be obtained at saturating amino acid concentrations; and (3) the assay is significantly more sensitive.
摘要
本文介绍了一种改进的tRNA氨基酰化定量测定方法，该方法基于对带切口的tRNA进行氨基酰化，然后在酸性变性聚丙烯酰胺凝胶上分离氨基酰化的3'-片段。通过新方法获得的大肠杆菌丙氨酸-tRNA合成酶（AlaRS）对tRNA进行氨基酰化的动力学参数与传统方法测得的参数高度一致。与传统的tRNA氨基酰化测量方法相比，该测定方法具有以下几个优点：（1）直接测量氨基酰-tRNA的比例；（2）可以在氨基酸浓度饱和时获得数据；（3）该测定方法的灵敏度显著更高。
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