摘要

       先前的研究表明，编码来自大肠杆菌(E.coli) 的精氨酸-tRNA 合成酶 (ArgRS) 的基因 argS 在大肠杆菌转化体 TG1/pUC-argS 中过度表达 1 000 倍，而编码亮氨酰的基因 leuS来自大肠杆菌的 -tRNA 合成酶 (LeuRS) 在同一情况下仅过量生产 35 倍。为了研究这两种氨酰-tRNA 合成酶基因的表达为何如此不同，通过将 leuS 的 5' 侧翼区域替换为 argS 的 5' 侧翼区域构建了融合基因（称为 parg-leuS）。在大肠杆菌转化体TG1/pUC-parg-leuS中，LeuRS的活性仅提高了8.5倍，远低于含有重组质粒pUC18-leuS或pKK-leuS的转化体。然而，通过RNA点杂交，parg-leuS转录产生的mRNA量是野生型leuS的5倍左右，与pKK-leuS相似，表明argS的启动子很强。对三个 mRNA 中起始密码子周围二级结构的分析表明，parg-leuS mRNA 的二级结构是三个 mRNA 中最强的。从结果可以推断融合基因parg-leuS的表达可能在翻译水平受到控制，并且该mRNA的强二级结构可能会阻碍翻译起始并导致低翻译效率。

       Previous studies showed that the gene argS encoding the arginyl-tRNA synthetase(ArgRS) from Escherichia coli(E.coli), was overexpressed 1 000 folds in the E.coli transformant TG1/pUC-argS, while the gene leuS, encoding the leucyl-tRNA synthetase(LeuRS) from E.coli, was only overproduced 35-fold in the same case. To investigate why the expression of these two aminoacyl-tRNA synthetase genes is so different, a fused gene (termed parg-leuS) was constructed by replacement of the 5' flanking region of leuS to 5' flanking region of argS. In the E.coli transformant TG1/pUC-parg-leuS, the activity of LeuRS was only improved 8.5-fold, which was much lower than that of the transformant harboring the recombinant plasmid pUC18-leuS or pKK-leuS. However, by RNA dot hybridization the amount of mRNA produced in the transcription of parg-leuS was about 5 times than that of the wild type leuS, and was similar to that of pKK-leuS, suggesting that the promoter of argS is very strong. Analysis of the secondary structure around the initiation codon among three mRNAs showed that the secondary structure of the mRNA from parg-leuS was the strongest of the three mRNAs. From the results, it could be deduced that expression of the fused gene parg-leuS might be controlled at the translational level and the strong secondary structure of this mRNA may hinder translation initiation and result in a low translation efficiency.

https://europepmc.org/article/med/12058187