大肠杆菌亮氨酰-tRNA合成酶与tRNA^(Leu)的相互作用 头孢菌素酰化酶的表达、翻译后加工和亚基重组(英文)  

Study on the Interaction Between Leucyl-tRNA Synthetase and tRNA^(Leu) from Escherichia ColiGene Expression,Post-Translational Processing and Subunits Reconstitution in Vivo of Cephalosporin Acylase from Pseudomonas

作　　者：李勇[1] 王恩多[1] 

机构地区：[1]中国科学院上海生命科学研究院生物化学与细胞生物学研究所分子生物学国家重点实验室,上海200031

出　　处：《中国科学院研究生院学报》2003年第1期117-122,共6页Journal of the Graduate School of the Chinese Academy of Sciences

基　　金：supportedbytheNationalNaturalScienceFoundationofChina(3 973 0 12 0)andtheChineseAcademyofSciences(GrantKJ95 1 B1 610) supportedbythe 863projectinChina(10 3 13 0 2 0 1)

摘　　要：第一部分:为得到大量的大肠杆菌tRNALeu1 和tRNALeu2 ,研究tRNA与亮氨酰 tRNA合成酶(LeuRS)的相互作用,用基因克隆的方法高表达并纯化了它们;测定了接受活力和被LeuRS催化的动力学常数;改进了RNA体外转录必需的T7RNA聚合酶的纯化方法,优化了转录条件,转录序列比文献值高 1 0倍.用体外转录方法得到了各种在接受茎突变的tRNALeu变种,研究了一个在CP1结构域插入 40个氨基酸残基的变种LeuRS A对tRNALeu2种等受体的识别,证明了tRNA Leu1 和tRNA Leu2 接受茎上的第一对硷基对是否摆动是LeuRS A识别的关键.　　第二部分:在大肠杆菌中高表达头孢菌素酰化酶基因 1 0倍以上.研究了它的α 亚基的N 端变化(LAEP →MGIP )对酶学动力学常数的影响.构建了带有不同抗菌素抗性和启动子的高表达质粒,研究了它们表达的特点,分析了这些质粒的不同用途.用含有编码该酶两个亚基的基因的相容质粒共转化的方法.研究了它们在体内的重组和前体的加工.研究结果表明,该酶可能属于一类新的NThe aminoacyl tRNA synthetases (aaRSs) catalyze the esterification of amino acids to their cognate tRNAs. They are specific for both amino acid and tRNA substrates to ensure the high fidelity required by translation. Leucyl tRNA synthetase (LeuRS) in one of aaRSs and belongs to class I aaRSs. In order to investigate the interaction between LeuRS and tRNA Leu , it was necessary that large amount of in vivo tRNA Leu were isolated and various mutants of tRNA Leu was obtained in vitro . In the present work Escherichia coli tRNA Leu 1 and tRNA Leu 2 were overproduced in Escherichia coli MT102 and isolated from the transformants, respectively.The kinetic constants of LeuRS for the tRNA Leu were the same with previous data. The leucine accepting ability of the unmodified tRNA Leu 1 and tRNA Leu 2 transcribed in vitro had little difference, but only one fourth that of the modified ones. It was found avariant (LeuRS A) of Escherichia coli LeuRS carrying a 40 residue long duplication in its connective peptide 1 (CP1) domain has a 3 fold lower specificity for tRNA Leu 1 than for tRNA Leu 2,whereas the native enzyme had the same specificity for their isoacceptors. By in vitro generation of a series tRNA Leu mutants we found that the difference on the first base pair in the acceptor stems of the two tRNA Leu isoacceptors lead to the different rates of leucylation catalyzed by LeuRS A and the flexibility of the acceptor stem of tRNA Leu might play a key role in its recognition by the synthetase. Cephalosporin acylases are a group of enzymes that hydrolyze cephalosporin C (CPC) and/or glutaryl 7 amino cephalosporanic acid (GL 7ACA) to produce 7 amino cephalosporanic acid (7 ACA) \ . The acylase from Pseudomonas sp . 130 (CA 130) shows high activity on GL 7ACA and glutaryl 7 aminodesacetoxycephalosporanic acid (GL 7ADCA) but much lower activity on CPC and penicillin G. The gene encoding the enzyme i

关 键 词：大肠杆菌 tRNA^Leu 亮氨酰-TRNA合成酶 相互作用 头孢菌素酰化酶 亚基重组 基因表达 基因克隆 

分 类 号：Q814[生物学—生物工程] Q78