Analysis of aminoacyl- and peptidyl-tRNAs by gel electrophoresis.
Analysis of aminoacyl- and peptidyl-tRNAs by gel electrophoresis.
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DOI：
10.1007/978-1-61779-949-5_19
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发表时间：
2012
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影响因子：
--
通讯作者：
Hayes, Christopher S
中科院分区：
文献类型：
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作者：
Janssen, Brian D;Diner, Elie J;Hayes, Christopher S
在蛋白质合成过程中，核糖体将信使RNA中编码的遗传信息翻译成特定的氨基酸序列。转移RNAs(TRNAs)在这一过程中是关键的适配分子，将氨基酸残基运送到核糖体，并在组装时保持新生的肽链。在这里，我们介绍了从大肠杆菌中分离的氨基酰基和肽基tRNA物种的分析方法。这些方法利用酸性pH下的变性凝胶电泳法来保护连接氨基酸和tRNA的不稳定的酯键。使用tRNA同种受体的探针，通过Northern印迹杂交检测到特定的氨基酰基和肽基-tRNAs。通过用硫酸铜选择性脱酰化氨酰tRNA，可以将小肽tRNA与氨酰tRNA区分开来。此外，可以通过对新生多肽的代谢标记来检测多肽-tRNAs。后一种方法适用于脉冲追逐分析以检测体内多肽-tRNA的周转。我们已经应用这些方法来研究程序性翻译停滞和暂停的核糖体翻转的动力学。
During protein synthesis, the ribosome translates the genetic information encoded within messenger RNAs into defined amino acid sequences. Transfer RNAs (tRNAs) are crucial adaptor molecules in this process, delivering amino acid residues to the ribosome and holding the nascent peptide chain as it is assembled. Here, we present methods for the analysis of aminoacyl- and peptidyl-tRNA species isolated from Escherichia coli. These approaches utilize denaturing gel electrophoresis at acidic pH to preserve the labile ester bonds that link amino acids to tRNA. Specific aminoacyl- and peptidyl-tRNAs are detected by Northern blot hybridization using probes for tRNA isoacceptors. Small peptidyl-tRNAs can be differentiated from aminoacyl-tRNA through selective deacylation of the latter with copper sulfate. Additionally, peptidyl-tRNAs can be detected through metabolic labeling of the nascent peptide. This latter approach is amenable to pulse-chase analysis to examine peptidyl-tRNA turnover in vivo. We have applied these methods to study programmed translational arrests and the kinetics of paused ribosome turnover.