Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, Bern, CH-3012, Switzerland.
Sci Rep. 2019 Jul 25;9(1):10836. doi: 10.1038/s41598-019-47268-4.
Orthogonal aminoacyl-tRNA synthetase/tRNA pairs have emerged as powerful means of site-specifically introducing non-standard amino acids into proteins in vivo. Using amino acids with crosslinking moieties this method allows the identification of transient protein-protein interactions. Here we have introduced a previously characterized evolved tyrosyl-tRNA synthetase/suppressor tRNA pair from E. coli into the parasitic protozoan Trypanosoma brucei. Upon addition of a suitable non-standard amino acid the suppressor tRNA was charged and allowed translation of a green fluorescent protein whose gene contained a nonsense mutation. - T. brucei is unusual in that its mitochondrion lacks tRNA genes indicating that all its organellar tRNAs are imported from the cytosol. Expression of the bacterial tyrosyl-tRNA synthetase in our system is tetracycline-inducible. We have therefore used it to demonstrate that cytosolic aminoacylation of the suppressor tRNA induces its import into the mitochondrion.
正交的氨酰-tRNA 合成酶/tRNA 对已成为在体内将非标准氨基酸特异性引入蛋白质中的有效手段。使用带有交联部分的氨基酸，这种方法可以鉴定瞬时蛋白质-蛋白质相互作用。在这里，我们将先前从大肠杆菌中鉴定出的进化型酪氨酸-tRNA 合成酶/抑制 tRNA 对引入寄生原生动物布氏锥虫中。在添加合适的非标准氨基酸后，抑制 tRNA 被充电，并允许翻译一个绿色荧光蛋白，其基因含有一个无义突变。- 布氏锥虫不同寻常之处在于其线粒体缺乏 tRNA 基因，这表明其所有的细胞器 tRNA 都是从细胞质中导入的。我们系统中细菌酪氨酸-tRNA 合成酶的表达是四环素诱导的。因此，我们用它来证明抑制 tRNA 的细胞质氨酰化诱导其导入线粒体。
