探討酵母菌 Histidine tRNA 之辨識

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李宜學(Yi-Siao Li) 查詢紙本館藏

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生命科學系

論文名稱

探討酵母菌 Histidine tRNA 之辨識

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摘要(中)

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辨識核苷酸，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。

摘要(英)

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.

關鍵字(中)

★ 酵母菌
★ 胺醯-tRNA合成酶

關鍵字(英)

★ aaRS
★ tRNAHis
★ G-1
★ yeast

論文目次

目錄
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
1.2 原核生物與真核生物蛋白質轉譯方式 2
1.2.1原核生物與真核生物蛋白質合成 2
1.2.2 真核細胞aaRS雙重功能基因 3
1.3 Histidyl-tRNA synthetase (HisRS)的簡介 4
1.3.1 HisRS的生化特性 4
1.3.2酵母菌的HTS1基因 4
1.4 tRNAHis的辨認位置 5
1.4.1 tRNAHis特別且保守的G-1核苷酸 5
1.4.2 真核和原核生物tRNAHis的N73鹼基 6
第二章材料與方法 7
2.1 菌株、載體及培養基 7
2.2 大腸桿菌勝任細胞的製備與轉型作用 8
2.2.1大腸桿菌勝任細胞的製備 8
2.2.2大腸桿菌勝任細胞的轉型作用 (transformation) 9
2.3 酵母菌勝任細胞的製備與轉型作用 9
2.3.1酵母菌勝任細胞的製備 9
2.3.2酵母菌勝任細胞的轉型作用 10
2.4 質體之選殖 10
2.5 點突變 (Site-directed Mutagenesis) 11
2.6 功能性互補試驗 (Complementation)―測試細胞質功能 12
2.7 功能性互補試驗 (Complementation)―測試粒線體功能 13
2.8 蛋白質製備 (Protein preparation) 14
2.9 SDS-PAGE之蛋白質分子量分析 15
2.10 西方點墨法 (Western Blotting) 15
2.11 酵母菌融合蛋白質的表現和純化 17
2.12 aaRS胺醯化反應 (aminoacylation assay) 20
第三章結果 22
3.1 酵母菌HisRS 的生化活性 22
3.2 酵母菌tRNAHis 其它識別元素 23
3.3 高等真核生物HisRS專一性與功能 24
3.4 G-1對真核生物HisRS的重要性 26
3.5 酵母菌和人類HisRS 之N端與C端結構域功能 27
3.6 酵母菌HisRS Motif 2 loop的重要性 28
第四章討論 29
參考文獻 33
圖表 36

參考文獻

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指導教授

王健家(Chien-Chia Wang)

審核日期

2013-7-25

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