大肠杆菌tRNA~(Leu)的提取、纯化及性质研究

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．

Preparation,Purification and Properties of Escherichia coli tRNA Leu

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.