博碩士論文 101224029 詳細資訊




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姓名 羅可涵(Christine Lwo)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 酵母菌粒線體Gln-tRNAGln的合成機制
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摘要(中) 在蛋白質合成的路徑中,需要tRNA將胺基酸帶到核醣體進行催化反應。而胺基酸接上對應的tRNA形成胺醯化tRNA是由aminoacyl-tRNA synthetases (aaRS) 催化而成。在酵母菌Saccharomyces cerevisiae的細胞質中,Gln-tRNAGln的合成是由GlnRS直接催化而成的,但在其粒線體中,缺乏GlnRS,Gln-tRNAGln是由GluRSc催化Glutamate接上tRNAGln,再經由Glutamyl-tRNAGln amidotransferase (GluAdT),將Glutamate轉變成Glutamine,之前我們曾研究在Escherichia coli GlnRS (EcGlnRS) 的胺基端接上Arc1p可以提供GLN4 剔除株生長所必須的酵素活性,若進一步在此融合蛋白質的胺基端加上一段粒線體標的訊號則此融合蛋白質可以取代粒線體內間接合成Gln-tRNAGln的路徑,這些發現突顯了基因平行轉移的可能性。利用直接合成Gln-tRNAGln 的路徑取代間接合成路徑。我們進一步研究Thermus thermophilus GlnRS (TtGlnRS),發現和EcGlnRS不同的是,若我們在胺基端接上Arc1p無法提供GLN4 剔除株生長所必須的酵素活性,但若進一步在此融合蛋白質的胺基端加上一段粒線體標的訊號(MTS)則此融合蛋白質可以取代粒線體內間接合成Gln-tRNAGln的路徑。根據西方墨點法的實驗結果,發現Arc1p可以增加TtGlnRS在S. cerevisiae的表現量,進一步做蛋白質降解實驗,發現TtGlnRS在粒線體較穩定,體外試驗發現Arc1p-TtGlnRS比TtGlnRS對粒線體tRNAmGln有較高的活性。在Schizosaccharomyces pombe中的GluAdT目前只能找到GatAB兩個次單元,尚未發現GluAdT的第三個次單元,因此我們利用pulldown assay,尋找會和GatB有交互作用的蛋白質,再進一步利用LC/MS/MS進行分析,找到S. pombe中系統編號SPCC777.11,可能是GluAdT的第三次單元體。
摘要(英) Aminoacylation of tRNA is catalyzed by a group of enzymes, called aminoacyl-tRNA synthetases. The resultant aa-tRNA is then delivered to ribosomes for protein translation. In Saccharomyces cerevisiae, cytosolic Gln-tRNAGln is generated by the direct pathway, but mitochondrial Gln-tRNAGln is formed by an indirect pathway involving mischarging by a non-discriminating glutamyl-tRNA synthetase and the subsequent transamidation by a specific Glu-tRNAGln amidotransferase, a heterotrimeric GatFAB. Previous studies showed that fusion of a yeast non-specific tRNA-binding cofactor, Arc1p, to Escherichia coli GlnRS enables the bacterial enzyme to substitute for both the direct and indirect pathways of Gln-tRNAGln synthesis. We showed herein that fusion of Arc1p to Thermus thermophilus GlnRS enabled the bacterial enzyme to substitute for the indirect, but not the direct, pathway of Gln-tRNAGln synthesis. In otherwise, the fission yeast, Schizosaccharomyces pombe use the same mode to synthesis the Gln-tRNAGln by two different pathway. We predict the amidotransferase GatAB ortholog, but cannot figure out the GatC or GatF ortholog. We use the TAP pulldown and LC/MS/MS to demonstrate the third subunit of amidotransferase in S. pombe, and figure out the systematic ID is SPCC777.11.
關鍵字(中) ★ tRNA合成酶
★ 酵母菌
★ 粒線體
關鍵字(英) ★ aminoacyl-tRNA synthetase
★ yeast
★ mitochondria
★ aaRS
論文目次 中文摘要 I
ABSTRACT II
誌謝 III
圖目錄 VII
表目錄 VIII
附錄目錄 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. aaRS的分類 - 2 -
1.2. Glutaminayl-tRNA synthetase (GlnRS)的簡介 - 3 -
1.2.1. GlnRS的生化特性 - 3 -
1.2.2. GlnRS的演化起源 - 3 -
1.2.3. 真核與原核之GlnRS之差異 - 4 -
1.3. Gln-tRNAGln的形成 - 4 -
1.3.1. Gln-tRNAGln的形成的途徑 - 4 -
1.3.2. 間接形成Gln-tRNAGln - 5 -
1.4. 不同酵母菌株 - 6 -
1.4.1. Saccharomyces cerevisiae - 6 -
1.4.2. Schizosaccharomyces pombe - 6 -
1.5. 研究目的 - 7 -
1.5.1. E.Coli GlnRS (EcGlnRS)和T. thermophilus GlnRS (TtGlnRS) 的活性比較 - 7 -
1.5.2. Schizosaccharomyces pombe的Glu-tRNAGln Amidotransferase - 8 -
第二章 材料與方法 - 9 -
2.1. 菌株、載體及培養基 - 9 -
2.1.1. 菌株、基因型及其來源 - 9 -
2.1.2. 載體 - 9 -
2.1.3. 培養基 - 10 -
2.2. 大腸桿菌勝任細胞的製備與轉型作用 - 13 -
2.2.1. 大腸桿菌勝任細胞的製備 - 13 -
2.2.2. 大腸桿菌勝任細胞的轉型作用 (transformation) - 13 -
2.3. 酵母菌勝任細胞的製備與轉型作用 - 14 -
2.3.1. S. cerevisiae勝任細胞的製備 - 14 -
2.3.2. S. cerevisiae勝任細胞的轉型作用 - 14 -
2.3.3. S. pombe勝任細胞的製備 - 15 -
2.3.4. S. pombe勝任細胞的轉型作用 - 16 -
2.4. 質體之選殖 - 16 -
2.5. S. pombe 標記 (TAP-tagging)菌株製備 - 17 -
2.6. 酵母菌菌落PCR - 19 -
2.7. 功能性互補試驗 (Complementation)―測試細胞質功能 - 20 -
2.8. 功能性互補試驗 (Complementation)―測試粒線體功能 - 21 -
2.9. 蛋白質製備 (Protein preparation) - 22 -
2.9.1. 震盪純化法 - 22 -
2.9.2. 三氯醋酸(trichloroacetic acid) TCA變性純化法 - 23 -
2.10. SDS-PAGE 之蛋白質分子量分析 - 24 -
2.11. 西方墨點法 (Western Blotting) - 25 -
2.12. 酵母菌融合蛋白質的表現與純化 - 26 -
2.13. 胺醯化作用分析 (Aminoacylation) - 29 -
第三章 實驗結果 - 30 -
3.2. Arc1p-TtGlnRS和MTS-Arc1p-TtGlnRS的分布位置不同 - 31 -
3.3. Arc1p和MTS可以減少TtGlnRS在酵母菌內的降解 - 32 -
3.4. Arc1p-TtGlnRS比TtGlnRS對粒線體tRNAmGln有較高的活性 - 32 -
第四章 討論 - 34 -
4.1 TtGlnRS和Arc1p-TtGlnRS的差異 - 34 -
4.2 EcGlnRS和TtGlnRS的差異 - 37 -
4.3 Arc1p-TtGlnRS和Ad-EcGlnRS的應用 - 38 -
4.4 S. pombe的GluAdT第三次單元 - 39 -
參考文獻 - 42 -
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指導教授 王健家(Chien-Chia Wang) 審核日期 2015-1-20
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