Tan Timothy H P, Bochud-Allemann Natacha, Horn Elke K, Schneider Andre
Department of Biology/Zoology, University of Fribourg, Chemin du Musee 10, CH-1700 Fribourg, Switzerland.
Proc Natl Acad Sci U S A. 2002 Feb 5;99(3):1152-7. doi: 10.1073/pnas.022522299. Epub 2002 Jan 15.
The mitochondrion of Trypanosoma brucei lacks tRNA genes. Its translation system therefore depends on the import of cytosolic, nucleus-encoded tRNAs. Thus, most trypanosomal tRNAs function in both the cytosol and the mitochondrion, and all are of the eukaryotic type. This is also the case for the elongator tRNA(Met), whereas the only other trypanosomal tRNA(Met), the eukaryotic initiator, is found exclusively in the cytosol. Unlike their cytosolic counterparts, organellar initiator tRNAs(Met) carry a formylated methionine. This raises the question of how initiation of translation works in trypanosomal mitochondria, where only elongator tRNA(Met) is found. Using in organello charging and formylation assays, we show that unexpectedly a fraction of elongator tRNA(Met) becomes formylated after import into mitochondria. Furthermore, in vitro experiments with mitochondrial extracts demonstrate that only the trypanosomal elongator and not the initiator tRNA(Met) is recognized by the formylation activity. Finally, RNA interference assays identify the gene encoding the trypanosomal formylase activity. Whereas the predicted protein is homologous to prokaryotic and mitochondrial methionyl-tRNA(Met) formyltransferases, it has about twice the mass of any of these proteins.
布氏锥虫的线粒体缺乏tRNA基因。因此，其翻译系统依赖于从细胞质中导入由细胞核编码的tRNA。所以，大多数锥虫tRNA在细胞质和线粒体中都发挥作用，并且都是真核类型。延长因子tRNA(Met)也是如此，而锥虫中另一种tRNA(Met)，即真核起始因子，仅存在于细胞质中。与细胞质中的对应物不同，细胞器起始因子tRNA(Met)携带甲酰化甲硫氨酸。这就引出了一个问题，在仅发现延长因子tRNA(Met)的锥虫线粒体中，翻译起始是如何进行的。通过细胞器内的充电和甲酰化分析，我们发现，意外的是，一部分延长因子tRNA(Met)在导入线粒体后会被甲酰化。此外，用线粒体提取物进行的体外实验表明，甲酰化活性只能识别锥虫延长因子tRNA(Met)，而不能识别起始因子tRNA(Met)。最后，RNA干扰分析确定了编码锥虫甲酰化酶活性的基因。尽管预测的蛋白质与原核和线粒体甲硫氨酰-tRNA(Met)甲酰转移酶同源，但它的质量约为这些蛋白质中任何一种的两倍。
