The aminoacyl-tRNA synthetases (aaRSs) catalyze the esterification of amino acidsto their cognate tRNAs. They are specific for both amino acid and tRNA substrates to ensurethe high fidelity required by translation. Leucyl-tRNA synthetase (LeuRS) in one of aaRSsand belongs to class Ⅰ aaRSs. In order to investigate the interaction between LeuRS andtRNALeu, it was necessary that large amount of in vivo tRNALeu were isolated and various mu-tants of tRNALeu was obtained in vitro. In the present work Escherichia coli tRNALeu1 andtRNALeu2 were overproduced in Escherichia coli MT102 and isolated from the transformants,respectively. The kinetic constants of LeuRS for the tRNALeu were the same with previous da-ta. The leucine accepting ability of the unmodified tRNALeu1 and tRNALeu2 transcribed in vitrohad 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 connectivepeptide 1 (CP1) domain has a 3 fold lower specificity for tRNALeu1 than for tRNALeu2, whereasthe native enzyme had the same specificity for their isoacceptors. By in vitro generation of aseries tRNALeu mutants we found that the difference on the first base pair in the acceptor stemsof the two tRNALeu isoacceptors lead to the different rates of leucylation catalyzed by LeuRS-Aand the flexibility of the acceptor stem of tRNALeu might play a key role in its recognition bythe synthetase.%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10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tRNALeu2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10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N-端亲和水解的酰化酶.
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