大肠杆菌tRNA^(Leu)的提取、纯化及性质研究  

Preparation,Purification and Properties of Escherichia coli tRNA Leu

作　　者：涂华民[1] 孟建新[1] 杨燕生[1] 李勇[2] 刘文[2] 王恩多[2] 

机构地区：[1]中山大学化学系 [2]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

出　　处：《中国生物化学与分子生物学报》1998年第6期697-703,共7页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家教委博士点基金;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基金

摘　　要：采用高表达大肠杆菌ｔＲＮＡＬｅｕ菌株提取、纯化了亮氨酸等受体转移核糖核酸ｔＲＮＡＬｅｕ１和ｔＲＮＡＬｅｕ２．利用稳态动力学手段研究了ｔＲＮＡＬｅｕ１及脱镁ｔＲＮＡＬｅｕ１在不同稀土离子作用下与纯化亮氨酰－ｔＲＮＡ合成酶的氨酰化作用．ｔＲＮＡＬｅｕ１与亮氨酰－ｔＲＮＡ合成酶的结合及催化效率均受参与稀土离子的影响，表观Ｋｍ值有较明显的变化．结果表明，亮氨酰－ｔＲＮＡ合成酶催化的ｔＲＮＡＬｅｕ１氨酰化反应所需Ｍｇ２＋能够被稀土离子取代，但亲合性能不同．The biological activity of a nucleic acid is instinctively associated with its three dimensional structure.Metal ions affect the secondary and tertiary structures,stabilities,and conformational states of RNA and DNA.The interaction of rare earth ions (RE 3+ ) with tRNA had been studied by the spectroscopy methods.The results showed that the structural role of Mg 2+ could be substituted by RE 3+ ,but this substitution induced a conformational change of tRNA that might affect the biological function of tRNA.In order to study the biological effect of RE 3+ ,leucine isoacceptor transfer ribonucleic acid of tRNA Leu 1 and tRNA Leu 2 from E.coli was prepared and purified using an overexpressed strain.A detailed steady state kinetic study of the aminoacylation of the tRNA Leu 1 and Mg 2+ free tRNA Leu 1 from E.coli with different RE 3+ by the purified E.coli Leucyl tRNA synthetase (LeuRS) was carried out.The binding of tRNA Leu 1 to the LeuRS and the catalytic efficiency was affected by the participation of the RE 3+ .The apparent K m value for native and Mg 2+ free tRNA Leu 1 was markedly changed.These results indicated that Mg 2+ requirement of the aminoacylation reaction catalyzed by LeuRS from E.coli could be replaced by RE 3+ ,but the affinities for tRNA Leu 1 with LeuRS was different.

关 键 词：氨酰化反应 稀土离子 TRNA aaRS 大肠杆菌 

分 类 号：Q751[生物学—分子生物学] Q933