编码大肠杆菌精氨酰tRNA合成酶的基因(argS)拥有一个强启动子(英文)  

A Strong Promoter Provided with the Gene Encoding Arginyl-tRNA Synthetase(argS) from Escherichia coli

作　　者：刘默芳[1] 李彤[1] 尹兆宝[2] 徐敏刚 王恩多[1] 王应睐 

机构地区：[1]中国科学院上海生物化学研究所,分子生物学国家重点实验室,上海200031 [2]上海中医药大学老年病研究所,上海200032

出　　处：《生物化学与生物物理学报》2000年第5期435-440,共6页

基　　金：国家自然科学基金资助项目 !No .39670 4 1 2&&

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。Previous studies showed that the gene argS encoding the arginyl-tRNA synthe tase(ArgRS) from Escherichia coli(E. coli), was overexpressed 1 000-fold in the E. coli transformant TG1/pUC-argS, while the gene leuS, encodi ng 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 aminoa cyl-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′ fl anking 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 tha n 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 that of the wild type le uS, and was similar to that of pKK-leuS, suggesting that the promoter of argS is very strong. Analysis of the secondary structure around the initiati on 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 contr olled at the translational level and the strong secondary structure of this mRNA may hinder translation initiation and result in a low translation efficiency.

关 键 词：精氨酰-TRNA合成酶 亮氨酰-TRNA合成酶 强启动子 

分 类 号：Q756[生物学—分子生物学]