Aminoacyl-tRNA synthetases (aaRSs)是一群必要的轉譯酵素，它們的主要功能是將胺基酸接到相對應的 tRNA上，因此aaRSs必須精確地辨認其相對應tRNA，蛋白質轉譯才不會出錯，Histidyl-tRNA synthetase (HisRS)主要辨識histidine tRNA (tRNAHis)上的G-1，G-1只存在於tRNAHis，不存在於其他種類tRNA。在酵母菌粒線體中，G-1是由粒線體的基因體直接轉錄而來；而細胞質中G-1則是藉由tRNAHis guanylyltransferase (Thg1)以後修飾作用的方式加上去的，Thg1辨識tRNAHis的反密碼(anticodon)，並且以其3′–5′聚合酶的活性將Ｇ-1加在tRNAHis的5’端。然而我的實驗結果顯示人類和果蠅的Thg1都帶有一段粒線體標的訊號(mitochondria targeting signal；MTS)，這個訊號可以幫助Thg1進入粒線體。此外，我也發現阿拉伯芥具有兩個THG1基因，這二個基因的序列比酵母菌的THG1長許多，因此我想進一步探討哪一個才是具功能的阿拉伯芥Thg1。黏菌具有多個Thg1-Like Proteins (TLPs)，可以用來編輯mitochondrial tRNA (mt-tRNA)，我希望利用此特性建構一個3’→5’聚合酶酵素，增加其應用性。雖然線蟲不具有THG1基因，但它本身的細胞質tRNAHis 卻保有G-1，我的實驗結果顯示:線蟲的HisRSs偏好具有G-1的tRNAHis，該酵素如何胺醯化不具G-1的粒線體tRNAHis仍是未知，我希望透過研究各物種間的Thg1及HisRS特性，進一步解開HisRS的的作用機制。;Aminoacyl-tRNA synthetases (aaRSs) are a family of essential translation enzymes, each of which catalyzes the coupling of a specific amino acid to its cognate tRNAs. Histidine tRNA (tRNAHis) is unique among tRNA species as it carries an additional nucleotide at its 5′ terminus. This unusual G-1 residue is the major identity element of tRNAHis, and is essential for recognition by histidyl-tRNA synthetase (HisRS). In yeast, G-1 of mitochondrial tRNAHis (denoted as tRNAmHis) is genome-encoded, while G-1 of cytoplasmic tRNAHis (denoted as tRNAnHis) is added post-transcriptionally by tRNAHis guanylyltransferase (Thg1). Thg1 possesses efficient 3′–5′ polymerase activity that specifically adds the G-1 residue by recognizing the anticodon of tRNAHis. We reported herein that the Thg1 homologues of Homo sapiens and Drosophila melanogaster possess a mitochondrial targeting signal (MTS) that can deliver the protein into mitochondria for functioning. Moreover, we found that Arabidopsis thaliana contains two THG1 genes, the sequences of which consist of two similar repeats. The specific aims of this project are to elucidate the mechanism by which G-1 can be specifically added to tRNAHis and to decipher how certain HisRSs can recognize tRNAHis without G-1. In addition, we would like to characterize the four Thg1-like proteins (TLPs) of Dictyostelium discoideum. Despite the fact that Caenorhabditis elegans lacks a THG1 gene, its HisRS can recognize both cytoplasmic and mitochondrial tRNAHis isoacceptors. Our results showed that C. elegans HisRS preferred tRNAHis with G-1. It is still unclear how this enzyme can recognize the mitochondrial tRNAHis without G-1.
