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为了研究原核生物和真核生物(细胞质)色氨酰tRNA合成酶(TrpRS)对线粒体tRNATrp识别的种属特异性,构建了7个水稻线粒体tRNATrp三位点(G73,U72,A68)的单点或多点突变的突变体.这些突变基因,经体外转录后分别用枯草杆菌(B.subtilis)和人色氨酰tRNA合成酶(TrpRS)进行氨酰化反应,并测定它们的动力学常数.结果表明,与野生型水稻线粒体tRNATrp相比,7个突变体的转录产物被B.subtilisTrpRS氨酰化的活力分别降低了53.33%~99.79%,被人TrpRS氨酰化的活力却分别提高了4~330倍,其中以MPH7(水稻线粒体tRNATrp的三碱基(G73,U72和C68)全部突变为人tRNATrp的三碱基序列)的氨酰化活力改变最大.实验结果证明,与真核生物和原核生物细胞质tRNATrp的种族特异性类似,水稻线粒体tRNATrp的种属特异性元件也主要处于氨基酸接受茎的识别位碱基、第一和第五对碱基对,亦即识别位碱基G73,氨基酸接受茎上的两个碱基对G1/U72和U5/A68.本研究为线粒体tRNATrp起源于真细菌的推论提供了实验依据.
In order to investigate the species-specific recognition of mitochondrial tRNATrp by pro-nuclear and eukaryotic (TrpRS) trichosporon, a single point of seven tRNATrp three-locus (G73, U72, A68) Or mutants with multiple mutations.These mutant genes were subjected to aminoacylation with B. subtilis and human tryptophan tRNA synthetase (TrpRS) respectively after in vitro transcription and their kinetic constants were determined. The results showed that compared with mitochondria tRNATrp in wild type rice, the transcripts of the seven mutants were reduced by 53.33% -99.79% in their aminoacylation by B. subtilis TrpRS, but their aminoacylation by human TrpRS was increased by 4 ~ 330-fold, of which the aminoacylation activity of MPH7 (the three bases of tRNATrp in rice was completely mutated to human tRNATrp) showed the greatest change in aminoacylation activity.The results of experiments showed that the aminoacylation of MPH7 The racial specificity of the biological cytoplasm tRNATrp is similar. The species-specific elements of the mitochondrial tRNATrp in rice are also mainly located at the amino acid bases of the recognition base of the stem, the first and fifth pairs of bases, ie, the recognition of the base G73, the amino acid acceptor Two bases on the stem G1 / U72 and U5 / A68. In this study, the origin of mitochondria tRNATrp to provide an experimental basis for the inference eubacteria.