探討一個tRNAHis修飾酵素的生理功能

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姓名

羅雅庭(Ya-Ting Lo) 查詢紙本館藏

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生命科學系

論文名稱

探討一個tRNAHis修飾酵素的生理功能
(Exploring the physiological functions of a tRNAHis-modifying enzyme)

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摘要(中)

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。

摘要(英)

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.

關鍵字(中)

★ 修飾酵素

關鍵字(英)

★ HisRS
★ Thg1
★ tRNAHis

論文目次

中文摘要 I
英文摘要 II
誌謝 III
目錄 IV
圖目錄 VII
表目錄 VIII
縮寫檢索表 IX
第一章緒論 1
1.1 Aminoacyl-tRNA synthetases (aaRSs)的簡介 1
1.1.1 aaRS的功能 1
1.1.2 aaRSs 的分類 2
1.1.3 aaRSs 的分子演化 2
1.2 Histidyl-tRNA synthetase (HisRS)的簡介 3
1.2.1 HisRS的生化特性 3
1.2.2 HisRS辨識tRNAHis的G-1 3
1.2.3 HisRS辨識tRNAHis的N73 4
1.3 tRNAHis guanylyltransferase (Thg1)的簡介 4
1.3.1 Thg1的生理功能 4
1.3.2 Thg1的G-1加入反應 5
1.4 Thg1-like proteins (TLPs)的簡介 5
1.4.1 TLP的生理功能 5
1.4.2 TLP的生化活性 6
1.5 Acr1p的簡介 6
1.6 研究目的 7
第二章材料與方法 9
2.1 菌株、載體及培養基 9
2.2 大腸桿菌勝任細胞的製備與轉型作用 10
2.2.1大腸桿菌勝任細胞的製備 11
2.2.2大腸桿菌勝任細胞的轉型作用 (transformation) 11
2.3 酵母菌勝任細胞的製備與轉型作用 12
2.3.1酵母菌勝任細胞的製備 12
2.3.2酵母菌勝任細胞的轉型作用 12
2.4 質體之選殖 13
2.5 功能性互補試驗 (Complementation)―測試細胞質功能 13
2.6 功能性互補試驗 (Complementation)―測試粒線體功能 14
2.7 蛋白質純化 (Protein purification) 15
2.8 SDS-PAGE之蛋白質分離 16
2.9 西方點墨法 (Western Blotting) 17
2.10 酵母菌融合蛋白質的表現和純化 18
2.11 aaRS胺醯化反應 (aminoacylation assay) 21
第三章結果 23
3.1 具有Thg1的物種 23
3.1.1 不同物種Thg1的差異性 23
3.1.2人類Thg1和果蠅Thg1的粒線體標的訊號 23
3.1.3不同物種Thg1的功能 24
3.1.4酵母菌Thg1送入粒線體的影響 24
3.1.5阿拉伯芥THG1基因的功能 25
3.2 Thg1酵素之應用 26
3.2.1 Thg1和TLP的功能應用 26
3.2.2 探討Thg1-TLP融合蛋白的功能 26
3.2.3 Thg1-Arc1p融合蛋白的生物毒性 27
3.3 不具Thg1以及TLP的物種 28
3.3.1 線蟲tRNAHis的特別之處 28
3.3.2 G-1對線蟲HisRS辨識的重要性 28
3.3.3 線蟲HisRS辨識tRNAHis的生化活性 29
第四章討論 31
4.1 Thg1演化上的意義 31
4.2 Thg1融合蛋白的應用性 32
4.3線蟲HisRS 的生化活性 33
參考文獻 34
圖表 38
MEI引子及質體清單 56

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指導教授

王健家(Chien-Chia Wang)

審核日期

2016-7-26

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