Fluorescence-based analysis of aminoacyl- and peptidyl-tRNA by low-pH sodium dodecyl sulfate-polyacrylamide gel electrophoresis
Fluorescence-based analysis of aminoacyl- and peptidyl-tRNA by low-pH sodium dodecyl sulfate-polyacrylamide gel electrophoresis
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DOI：
10.1016/j.ab.2007.01.019
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发表时间：
2007-05-01
影响因子：
2.9
通讯作者：
Cavagnero, Silvia
中科院分区：
文献类型：
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作者：
Kirchdoerfer, Robert N.;Huang, Joseph J-T.;Cavagnero, Silvia
蛋白质生物合成的关键组分氨酰和肽酰tRNA的正确表征在翻译的多方面研究中至关重要。核糖体相关氨酰和肽酰-tRNA的分析通常通过蔗糖梯度和闪烁计数进行[1]。这种方法适合于获得关于整个核糖体复合物的信息，但它不能提供关于氨酰-和肽酰-tRNA的分子大小的直接知识。先前对肽基-tRNA进行直接分析的尝试是基于天然凝胶电泳[2]。在制备规模上，已经通过离子交换[3，4]、反相、尺寸排阻或亲和色谱法分离氨酰基-和肽基-tRNA。然而，后者的方法不像凝胶电泳那么方便，可能需要大量的样品。小规模凝胶电泳是一种有效的工具，用于分析表征生物分子。虽然通常通过含脲凝胶检测高度带负电荷的RNA，但肽和蛋白质需要SDS的存在，SDS用作能够用负电荷涂覆多肽表面的变性剂。上述策略使蛋白质或RNA既能展开，又能被表面负电荷覆盖，从而使分子区分仅基于分子大小。然而，肽基-tRNA的电泳分析提出了特殊的挑战，因为这种分子包含多肽和RNA的特性。此外，连接两个部分的酯键是容易水解的。在这里，我们报告了一种通用的方法，通过3层十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）1在三乙酸（TA）的存在下，在pH 5.7的可变链长的氨酰和肽基-tRNA的直接分析。该凝胶系统特别强大，因为（a）它与肽和RNA组分相容，（B）它使连接3′ tRNA末端和C-末端氨基酸羧基的酯键的水解最小化，以及（c）它适用于在存在和不存在共价结合的tRNA的情况下检测各种肽大小。本文提出将荧光检测与TA凝胶结合[5]。
Proper characterization of aminoacyl-and peptidyl-tRNA, key components of protein biosynthesis, is of crucial importance in the study of the multifaceted aspects of translation. Analysis of ribosome-associated aminoacyl-and peptidyl-tRNAs has often been approached via sucrose gradients followed by scintillation counting [1]. This method is suitable to gain information about the entire ribosomal complex but it fails to provide direct knowledge on the molecular size of aminoacyl-and peptidyl-tRNA. Previous attempts to perform direct analysis on peptidyl-tRNA were based on native gel electrophoresis [2]. On the preparative scale, aminoacyl-and peptidyl-tRNAs have been isolated by ion exchange [3, 4], reverse phase, size exclusion or affinity chromatography. However, the latter approaches are not as convenient as gel electrophoresis and may require larger amount of samples.Small scale gel electrophoresis is an effective tool for the analytical characterization of biomolecules. While the highly negatively charged RNA is typically detected via ureacontaining gels, peptides and proteins require the presence of SDS, which serves as a denaturing agent able to coat the polypeptide surface with negative charges. The above strategies enable proteins or RNA to be both unfolded and coated with surface negative charges, allowing molecular discrimination to be based solely on molecular size. Electrophoretic analysis of peptidyl-tRNA, however, poses special challenges because this molecule embraces both polypeptide and RNA character. Furthermore, the ester bond linking the two moieties is easily hydrolysable. Here, we report a versatile method for the direct analysis of both aminoacyl-and peptidyl-tRNAs of variable chain length via 3-layer sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) 1 in the presence of tris-acetate (TA) at pH 5.7. This gel system is particularly powerful in that (a) it is compatible with both the peptide and RNA components,(b) it minimizes hydrolysis of the ester bond linking the 3′ tRNA end to the C-terminal amino acid carboxyl group, and (c) it is suitable for the detection of a wide range of peptide sizes both in the presence and absence of covalently bound tRNA. Fluorescence detection is proposed here in conjunction with the TA gels [5].