Robakis N, Cenatiempo Y, Meza-Basso L, Brot N, Weissbach H
Methods Enzymol. 1983;101:690-706. doi: 10.1016/0076-6879(83)01048-4.
In this report, a simplified coupled DNA-directed in vitro system has been described that is based on the formation of the first di- or tripeptide of the gene product. This system is gene specific and quantitative, and the assay (especially the extraction procedure) is very rapid. The fact that both transcription and translation initiation occur in this system makes it ideally suited for studies on the regulation of prokaryotic gene expression. The ideal templates are plasmids, DNA fragments or purified mRNAs that direct the synthesis of a limited number of products with different second amino acids. An essential requirement is that the initial sequence of the protein products be known, although this system could be used to determine the second amino acid in cases where there is some doubt from the DNA sequence as to where a particular protein initiates. A difficulty arises when a plasmid contains more than one gene whose protein products have the same initial dipeptide. One solution to the problem is to measure tripeptide formation if the third amino acid is different. A second procedure, if the code word for the second amino acid differs between the genes, is to use purified isoacceptor tRNA species to distinguish the products. Another important application of tripeptide synthesis is that it can be used as a measure of the amount of active mRNA present in a mixture of mRNAs. The use of a ribosomal high-salt wash instead of the purified initiation and elongation factors greatly simplifies this system and should make it suitable for routine analysis in most laboratories.
在本报告中，描述了一种简化的耦合DNA定向体外系统，该系统基于基因产物的首个二肽或三肽的形成。该系统具有基因特异性和定量性，并且检测方法（尤其是提取过程）非常快速。该系统中同时发生转录和翻译起始这一事实使其非常适合用于原核基因表达调控的研究。理想的模板是质粒、DNA片段或纯化的mRNA，它们可指导合成数量有限的、带有不同第二个氨基酸的产物。一个基本要求是蛋白质产物的初始序列是已知的，尽管在根据DNA序列对特定蛋白质的起始位置存在一些疑问的情况下，该系统可用于确定第二个氨基酸。当一个质粒包含多个其蛋白质产物具有相同初始二肽的基因时，就会出现困难。解决该问题的一种方法是，如果第三个氨基酸不同，则测量三肽的形成。如果不同基因中第二个氨基酸的密码子不同，另一种方法是使用纯化的同工受体tRNA种类来区分产物。三肽合成的另一个重要应用是，它可用于测量mRNA混合物中存在的活性mRNA的量。使用核糖体高盐洗脱液而非纯化的起始因子和延伸因子极大地简化了该系统，使其适用于大多数实验室的常规分析。
