在酵母菌體內具有兩套aminoacyl-tRNA synthetases (aaRSs)，一套作用在細胞質，另一套作用在粒線體。然而細胞質和粒線體的HisRS卻是由同一個基因HTS1經由兩個不同的AUG轉譯起始點做出來的。過去的研究指出大腸桿菌和酵母菌的tRNAHis 在-1:73位置是重要的aaRS辨識核?酸，而在酵母菌細胞質和粒線體tRNAHis相對位置分別是G-1:A73和G-1:C73，在大腸桿菌中則是G-1:C73，且大腸桿菌的histidyl-tRNA synthetase (HisRS) 無法取代酵母菌細胞質HisRS的功能，若將其送入粒線體則可取代。我們也進一步在體外實驗中證實，大腸桿菌HisRS無法辨識G-1:A73的酵母菌細胞質tRNAHis。由此推論G-1:A73和G-1:C73分別是真核生物細胞質和粒線體的保守辨識位置，我們的實驗結果也顯示人類細胞含有HisRS分為粒線體與細胞質異構型HisRS，它們可以分別取代酵母菌粒線體和細胞質的HisRS功能。值得一提的，我們的實驗結果發現果蠅和線蟲的HisRS也都是雙重功能基因，可以同時作用在細胞質和粒線體，且果蠅HisRS應該是由非AUG起始密碼做出帶有粒線體標的訊號的異構型。除此之外，人類和酵母菌HisRS經過結構域互換的結果顯示對HisRS 而言N端結構域才是辨識tRNAHis 的重要區域。因此我們更細部探討，發現在體內實驗中酵母菌HisRS的motif 2 loop突變會影響它在粒線體中的活性，但不影響在細胞質。由體外胺醯化反應結果可以發現motif 2 loop突變使其催化細胞質tRNAHis效率約下降30倍，而且幾乎無法辨識粒線體tRNAHis。由此發現我們認為酵母菌HisRS之motif 2 loop對於辨識-1:73位置相當重要，尤其是G-1:C73。這對於雙功能HisRS基因在演化上的策略提供了新的線索。 There are two distinct sets of aminoacyl-tRNA synthetases (aaRSs) in yeast, one localized in the cytoplasm and the other in mitochondria. Paradoxically, there is only one histidyl-tRNA synthetase (HisRS) gene, HTS1, in the yeast nuclear genome. Recent studies indicate that this gene encodes both cytoplasmic and mitochondrial forms of HisRS through alternative initiation of translation from two in-frame AUG initiator codons. We report herein that a similar, dual-functional phenotype is conserved in the HTS1 genes of many eukaryotes. While E. coli tRNAHis contains a G-1:C73 base pair as the major determinant, yeast cytosolic and mitochondrial tRNAHis isoacceptors respectively contain G-1:A73 and G-1:C73 at the same positions. We found that human cytoplasmic and mitochondrial HisRS can respectively substitute for the cytoplasmic and mitochondrial activities of yeast HTS1. Furthermore, The HisRS gene of Drosophila melanogaster and Caenorhabditis elegans are also dual functional in yeast. Domain swapping between yeast HisRS and human HisRS suggested that the N-terminal domains is important for tRNAHis recognition. Moreover, mutations in motif 2 loop largely impaired the mitochondrial but slightly cytoplasmic activity. This finding suggests that the motif 2 loop of yeast HisRS is important for recognition of G-1:C73. This may provide a clue of evolution strategy of the dual-functional HTS1 gene.
