探討酵母菌Histidyl-tRNA synthetase的特性

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姓名

鄭鈺儒(Yu-ju Cheng) 查詢紙本館藏

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生命科學系

論文名稱

探討酵母菌Histidyl-tRNA synthetase的特性
(Characterization of yeast Histidyl-tRNA synthetase.)

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摘要(中)

在酵母菌中具有兩套aminoacyl-tRNA synthetases (aaRSs)，一套在細胞質作用，另一套在粒線體作用。然而酵母菌中，只有一個histidyl-tRNA synthetase (HisRS) 基因HTS1，利用兩個不同的起始密碼轉譯出兩個酵素異構型。在本論文中我們研究不同種的酵母菌HTS1基因，結果顯示所有酵母菌都是使用相似的轉譯機制，利用一個基因轉譯出在細胞質和粒線體作用的HisRS。在E. coli tRNAHis 上G-1:C73是一個重要的決定基，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。實驗結果顯示E. coli HisRS無法取代酵母菌細胞質HisRS的活性，可是如果將E. coli HisRS送入粒線體之後，就可以互補酵母菌 HTS1剔除株粒線體的功能。此外，我們發現酵母菌 S. cerevisiae HisRS的胺基端具有一段附加區段，L. elongisporous HisRS中間則有一段插入區段，這兩個區段主要影響酵素對tRNA的KM，對於酵素的kcat則只有輕微的影響，因此，我們猜測此附加區段及插入區段可能用來增加整個酵素對tRNA的親和力。

摘要(英)

In yeast, there are two distinct sets of aminoacyl-tRNA synthetases (aaRSs), 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. As it turns out, this gene encodes both cytoplasmic and mitochondrial forms of HisRS through alternative initiation of translation from two in-frame initiator codons. We herein report that a dual-functional phenotype was conserved in the HTS1 genes of other yeast species. While E. coli tRNAHis species contained a G-1:C73 base pair as the major determinant, yeast cytosolic and mitochondrial tRNAHis isoacceptors respectively contained G-1:A73 and G-1:C73 at the same position. Even though E. coli HisRS failed to substitute for the cytoplasmic activity of yeast HTS1, the bacterial enzyme, upon being imported into mitochondria, could rescue growth defect of a S. cerevisiae HTS1 knockout strain. In addition, we found that the N-terminal appended domain of S. cerevisiae HisRS and an insertion domain of L. elogisporous HisRS significantly contributed to KM of the enzyme for tRNAHis, but had relatively minor effect on the kcat. Our study suggests that the appended domain or insertion domain of yeast HisRS acts as an auxiliary tRNA-binding domain to improve the overall tRNA-binding affinity of the enzyme.

關鍵字(中)

★ 酵母菌
★ 蛋白質合成
★ 胺醯化

關鍵字(英)

★ histidyl-tRNA synthetase
★ aminoacylation

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 IX
縮寫檢索表 X
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轉譯例外 - 3 -
1.3 Histidyl-tRNA synthetase (HisRS)的簡介 - 4 -
1.3.1 HisRS的生化特性 - 4 -
1.3.2 HTS1基因在酵母菌中的表現 - 5 -
1.4 tRNAHis的辨認位置 - 6 -
1.5 酵母菌的附加區段 - 7 -
1.6 研究目的 - 8 -
第二章材料與方法 - 10 -
2.1 菌株、載體及培養基 - 10 -
2.2 大腸桿菌勝任細胞的製備與轉型作用 - 11 -
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) - 12 -
2.3 酵母菌勝任細胞的製備與轉型作用 - 13 -
2.3.1酵母菌勝任細胞的製備 - 13 -
2.3.2酵母菌勝任細胞的轉型作用 - 13 -
2.4 質體之選殖 - 14 -
2.5 點突變 (Site-directed Mutagenesis) - 15 -
2.6 功能性互補試驗 (Complementation)─測試細胞質功能 - 16 -
2.7 功能性互補試驗 (Complementation)─測試粒腺體功能 - 17 -
2.8 蛋白質製備 (Protein preparation) - 19 -
2.9 SDS-PAGE之蛋白質分子量分析 - 20 -
2.10 西方點墨法 (Western Blotting) - 21 -
2.11 Cycleheximide-chase assay之蛋白質穩定度測試 - 22 -
2.12 免疫螢光分析 (immunofluorescence analysis) - 23 -
2.13 酵母菌融合蛋白質的表現和純化 - 23 -
2.14 aaRS胺醯化反應 (aminoacylation assay) - 27 -
第三章結果 - 29 -
3.1 不同酵母菌之間HisRS的相互關係。 - 29 -
3.2 不同物種HTS1的轉譯機制 - 30 -
3.3 原核生物和真核生物之間HisRS的相互關係。 - 34 -
3.4 原核生物和真核生物之間HisRS生化活性差異。 - 37 -
3.5 HisRS附加區段對酵母菌的重要性。 - 38 -
3.6 HisRS附加區段對蛋白質穩定度的影響。 - 41 -
3.7 HisRS附加區段對酵素生化活性的影響。 - 42 -
第四章討論 - 44 -
4.1 酵母菌HTS1基因的表現 - 44 -
4.2 tRNAHis和HisRS的相互關係 - 46 -
4.3 酵母菌HisRS附加區段的特性 - 48 -
參考文獻 - 52 -
圖表 - 58 -

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指導教授

王健家(Chien-Chia Wang)

審核日期

2012-7-6

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