Aminoacyl–tRNA synthetases (aaRSs) 是一群古老的酵素，它們的主要功能是 將一個胺基酸接到相對應的tRNA，接著aa-tRNA會被送去核醣體參與蛋白質合成。 在真核細胞內，蛋白質合成發生在細胞質及胞器(例如粒線體)，因此細胞會合成二 套aaRSs，其中一套作用在細胞質，另一套作用在粒腺體。一般而言，對應一個特 定胺基酸的細胞質及粒線體aaRSs是由二個不同的細胞核基因解碼，但是也有少數 例外。在本研究中我們發現弓形蟲(Toxoplasma gondii) 只有一個 prolyl-tRNA synthetase (TgProRS)基因，但是有二個不同的tRNAPro (一個是E-type，另一個是Ptype)， 細胞核基因解碼的tRNAPro作用在細胞質及粒線體，它的序列屬於E-type； 頂質體基因解碼的tRNAPro作用在頂質體, 它的序列屬於P-type。序列比對及親緣演 化關係顯示TgProRS屬於 E-type ProRS，胺醯化活性測試也顯示這個酵素能有效地 胺醯化E-type tRNAPro，但是胺醯化 P-type tRNAPro的效率則明顯偏低。可是，互補 試驗顯示這個酵素能有效取代酵母菌粒線體ProRS (P-type ProRS)，這也顯示這個酵 素能胺醯化P-type tRNAPro到某一個程度。總而言之，我們的研究結果顯示TgProRS 是一個非典型的ProRS，能同時胺醯化P-type及E-type tRNAsPr;Aminoacyl–tRNA synthetases (aaRSs) belong to a group of ancient enzymes that plays a key role in protein synthesis by attaching a specific amino acid to its cognate tRNA. In eukaryotes, protein synthesis occurs not only in the cytoplasm but also in organelles. Thus, two distinct sets of aaRSs are required, one for the cytoplasm and the other for organelles. In most cases, the cytoplasmic and organellar isoforms of a given aaRS are encoded by two different nuclear genes, each recognizing its own tRNA isoacceptors. In this research we found that Toxoplasma gondii contains only one nuclear prolyl-tRNA synthetase (TgProRS) gene, but it contains two different tRNAPro isoacceptors, a nuclear-encoded E-type tRNAPro (tRNAn Pro), which functions in the cytoplasm and mitochondria, and an apicoplast-encoded P-type tRNAPro (tRNAa Pro), which functions in apicoplast. Sequence alignment and phylogenetic analysisindicated that TgProRS possesses the unique C-terminal appended domain that is unique to an E-type ProRS. Amininoacylation assays showed that TgProRS can efficiently charge yeast tRNAPro, which represents an E-type tRNAPro, but it can hardly charge E. coli tRNAPro and B. thuringiensis tRNAPro, both of which represent a P-type tRNAPro. However, contrary to our expectations, TgProRS robustly rescued a yeast mitochondrial ProRS knockout strain when it was overexpressed, suggesting that TgProRS can charge the yeast mitochondrial P-type tRNAPro to a level sufficient to maintain normal mitochondrial function. Our results suggest that TgProRS is a noncanonical ProRS that charges both the P- and E-types of tRNAPro
