7个水稻线粒体tRNA^(Trp)突变体的克隆和活力测定

Cloning and Characterization of Seven Mutants from Oryza sativa Mitochondrial tRNA^(Trp)

为了研究tRNAs被相应的氨酰酶识别的种属特异性,构建了7个水稻线粒体tRNATrp3个碱基(G73,U72,A68)的单点或多点突变的突变体.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氨酰化的活力分别下降了53.33%～99.79%;被人TrpRS氨酰化的活力则提高了4～330倍,其中以pMPH7的氨酰化活力改变最大.识别位碱基G73,G1/U72,U5/A68对水稻线粒体tRNATrp的种属特异性识别起重要作用.

To reserch the species-specific aminoacylation in tRNAs, seven single or multiple mutations of three bases （G73, U72, A 68） were made in O.sativa mitochondrial tRNA^Trp to the corresponding nucleotides present in human tRNA^Trp. In vitro transcripts of these mutant genes were tryptophanylated by Bacillus subtilis and human tryptophanyl-tRNA synthetases （TrpRS）, and the kinetic parameters were determined. The results showed that the aminoacylation of seven mutant transcripts by B. subtilis TrpRS was 55.33% to 99.79% less efficient than that by wild-type O.sativa mitochondrial tRNA^Trp, but was 4 to 330 times more efficient than that by human TrpRS. The mutant pMPH7 showed a great change of aminoacylation efficiency. Our results indicate that the species-specific identity elements of O.sativa mitochondrial tRNA^Trp are similar to bacterial and eukaryotic （cytoplasm）. They are mainly located at the discriminator base, the first and the fifth pair of bases, the discriminator base G73, two bases in the acceptor stem G1/U72 and U5/A68.