水稻线粒体tRNA^(Trp)种属特异性元件研究

Species-specific element of Oryza sativa mitochondria tRNA^(Trp)

为了研究水稻线粒体tRNATrp的种属特异性元件,在野生型水稻线粒体tRNATrp的基础上,设计并完成了3种向人tRNATrp的突变,体外转录并用枯草杆菌和人这两种不同种属来源的色氨酰-tRNA合成酶(TrpRS)测定了这些tRNATrp分子的氨酰化活力(Kcat/KM).结果表明,与野生型水稻线粒体tRNATrp相比,3个突变体被人TrpRS氨酰化的活力分别提高了354、407和803倍,其中以PMPH3(水稻线粒体tRNATrp的氨基酸接受茎的C2-G71和G3-C70都突变为人tRNATrp的氨基酸接受茎的相应部位)的氨酰化活力改变最大.而3个突变体对B.subtilisTrpRS氨酰化活力有进一步负影响,氨酰化活力微弱.说明水稻线粒体tRNATrp氨基酸接受茎上的第2个碱基对C2-G71和第3个碱基对G3-C70在人色氨酰-tRNA合成酶识别过程中有着极为重要的作用,是水稻线粒体tRNATrp的种属特异性元件.

To discover the species-specific element of Oryza sativa mitochondria tRNATrp , three mutants from Oryza sativa mitochondria tRNA^Trp to human tRNA^Trp（PMPH1 ： 2-71 （CA→GU） ; PMPH2 ： 3-70 （GC→CG） ; PMPH3 ： 2 71（CA→GU） and 3-70 （GC→CG） were constructed and transcribed in vitro with T7 RNA polymerase. The ki- netic parameters （Kcat/KM）of B. subtilis tryptophanyl-tRNA synthetase （TrpRS）and human TrpRS were determined with three mutant-type tRNA^Trp. The results showed that the aminoacryl activities of these three mutants were 354, 407, and 803 times higher than the wild-type Oryza sativa mitochondria tRNA^Trp. The PMPH3 based on Oryza sativa mitochondrial tRNA^Trp with its C2/G71and G3/C70 bases were transplanted by Human counterparts showed a large increase in aminoacylation efficiency by human TrpRS. The three mutants all showed a weak aminoacylation efficiency by B. subtilis TrpRS as compared with the wild-type Oryza sativa Mitochondrial tRNAwrp. It was concluded that the bases of C2-G71 and G3-C70 play a very important role in recognition by human TrpRS and is the species-specific elements in Oryza sativa mitochondria tRNATrp.