摘要

       编码大肠杆菌 (E .coli)精氨酰 tRNA合成酶 (ArgRS)的基因 (argS)和编码亮氨酰 tRNA合成酶 (LeuRS)的基因(LeuS)分别插入 pUC1 8后 ,各自在E .coliTG1转化子中的表达有很大的差异 (高表达倍数分别为 1 0 0 0和 3 5倍 )。为了调查造成其表达差异的原因 ,用argS的 5′上游非编码区取代leuS的 5′上游非编码区 ,构建了融合基因 parg leuS ;将它插入质粒 pUC1 8后转化E .coliTG1 ,发现仅使LeuRS活力提高了约 8.5倍 ,这远低于野生型leuS或带有强启动子trp lac的pKK leuS的。但是 ,RNA斑点杂交却发现从 parg leuS转录出来的leuSmRNA是野生型leuS转录的 5倍 ,几乎与从 pKK leuS中转录出来的量差不多 ,说明argS的启动子很强 ,接近于trp lac。分析三种leuSmRNA在起始密码子附近的二级结构 ,发现从 parg leuS转录的mRNA的二级结构最强。一般认为 ,这种二级结构将会妨碍核糖体的结合 ,降低翻译效率。从这些结果可以推测 ,parg leuS可能就是因为其mRNA在起始密码子附近的强二级结构而使表达受限于翻译水平。

       The gene (argS) encoding the arginyl tRNA synthetase (ArgRS) of Escherichia coli (E.coli) and the gene (LeuS) encoding the leucyl tRNA synthetase (LeuRS) were inserted into pUC18, and they were transformed in E.coliTG1. There is a big difference in the expression in the child (high expression folds are 100 and 35 times respectively). In order to investigate the cause of the difference in expression, the 5'upstream non-coding region of argS was replaced with the 5'upstream non-coding region of leuS, and the fusion gene parg leuS was constructed; it was inserted into the plasmid pUC1 8 and transformed into E. The activity of LeuRS increased by about 8.5 times, which was much lower than that of wild-type leuS or pKK leuS with a strong promoter trp lac. However, RNA dot blot hybridization found that the leuS mRNA transcribed from parg leuS was 5 times that of wild-type leuS, which was almost the same as the amount transcribed from pKK leuS, indicating that the promoter of argS is very strong, close to trp lac. Analyzing the secondary structure of the three types of leuS mRNA near the start codon, it was found that the mRNA transcribed from parg leuS had the strongest secondary structure. It is generally believed that this secondary structure will hinder the binding of ribosomes and reduce translation efficiency. From these results, it can be inferred that parg leuS may be restricted to the level of translation due to the strong secondary structure of its mRNA near the start codon.

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