博碩士論文 101224029 詳細資訊




以作者查詢圖書館館藏 以作者查詢臺灣博碩士 以作者查詢全國書目 勘誤回報 、線上人數:83 、訪客IP:3.135.190.135
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
相關論文
★ Kineosphaera limosa 菌株中 phaC 基因之序列分析★ 剪力和組織蛋白去乙醯酶在動靜脈廔管失效扮演的角色
★ Classification of powdery mildews on ornamental plants in northern Taiwan★ 秀麗隱桿線蟲線粒體AlaRS通過非傳統模式識別T型無臂tRNAAla
★ Bacillus thuringiensis contains two prolyl-tRNA synthetases of different origins★ Recognition of tRNA His isoacceptors by human HisRS isoforms
★ Functional replacement of yeast nuclear and mitochondrial RNase P by a protein-only RNase P★ Functional characterization of a noncanonical ProRS in Toxoplasma gondii
★ tRNA aminoacylation by a naturally occurring mini-AlaRS★ Functional Repurposing of C-Ala Domains
★ Recognition of a non-canonical tRNAAla by a non-canonical alanyl-tRNA synthetase★ 探討Alanyl-tRNA synthetase的演化及專一性
★ 酵母菌valyl-tRNA synthetase附加區段的 生物功能之探討★ 探討酵母菌glycyl-tRNA合成酵素的非傳統生物功能
★ 探討酵母菌Valyl-tRNA synthetase的生化活性★ 酵母菌轉譯起始機制的研究
檔案 [Endnote RIS 格式]    [Bibtex 格式]    [相關文章]   [文章引用]   [完整記錄]   [館藏目錄]   [檢視]  [下載]
  1. 本電子論文使用權限為同意立即開放。
  2. 已達開放權限電子全文僅授權使用者為學術研究之目的,進行個人非營利性質之檢索、閱讀、列印。
  3. 請遵守中華民國著作權法之相關規定,切勿任意重製、散佈、改作、轉貼、播送,以免觸法。

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 -
參考文獻 Araiso, Y., Huot, J.L., Sekiguchi, T., Frechin, M., Fischer, F., Enkler, L., Senger, B., Ishitani, R., Becker, H.D., and Nureki, O. (2014). Crystal structure of Saccharomyces cerevisiae mitochondrial GatFAB reveals a novel subunit assembly in tRNA-dependent amidotransferases. Nucleic acids research 42, 6052-6063.
Arnez, J.G., and Moras, D. (1997). Structural and functional considerations of the aminoacylation reaction. Trends in biochemical sciences 22, 211-216.
Asahara, H., and Uhlenbeck, O.C. (2002). The tRNA specificity of Thermus thermophilus EF-Tu. Proceedings of the National Academy of Sciences of the United States of America 99, 3499-3504.
Bahler, J., Wu, J.Q., Longtine, M.S., Shah, N.G., McKenzie, A., 3rd, Steever, A.B., Wach, A., Philippsen, P., and Pringle, J.R. (1998). Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe. Yeast 14, 943-951.
Bhaskaran, H., and Perona, J.J. (2011). Two-step aminoacylation of tRNA without channeling in Archaea. Journal of molecular biology 411, 854-869.
Brown, J.R., and Doolittle, W.F. (1999). Gene descent, duplication, and horizontal transfer in the evolution of glutamyl- and glutaminyl-tRNA synthetases. Journal of molecular evolution 49, 485-495.
Bullock, T.L., Rodriguez-Hernandez, A., Corigliano, E.M., and Perona, J.J. (2008). A rationally engineered misacylating aminoacyl-tRNA synthetase. Proceedings of the National Academy of Sciences of the United States of America 105, 7428-7433.
Burbaum, J.J., and Schimmel, P. (1991). Structural relationships and the classification of aminoacyl-tRNA synthetases. The Journal of biological chemistry 266, 16965-16968.
Carter, C.W., Jr. (1993). Cognition, mechanism, and evolutionary relationships in aminoacyl-tRNA synthetases. Annual review of biochemistry 62, 715-748.
Cathopoulis, T., Chuawong, P., and Hendrickson, T.L. (2007). Novel tRNA aminoacylation mechanisms. Molecular bioSystems 3, 408-418.
Curnow, A.W., Hong, K., Yuan, R., Kim, S., Martins, O., Winkler, W., Henkin, T.M., and Soll, D. (1997). Glu-tRNAGln amidotransferase: a novel heterotrimeric enzyme required for correct decoding of glutamine codons during translation. Proceedings of the National Academy of Sciences of the United States of America 94, 11819-11826.
Cusack, S., Hartlein, M., and Leberman, R. (1991). Sequence, structural and evolutionary relationships between class 2 aminoacyl-tRNA synthetases. Nucleic acids research 19, 3489-3498.
Delarue, M. (1995). Aminoacyl-tRNA synthetases. Current opinion in structural biology 5, 48-55.
Eriani, G., Delarue, M., Poch, O., Gangloff, J., and Moras, D. (1990). Partition of tRNA synthetases into two classes based on mutually exclusive sets of sequence motifs. Nature 347, 203-206.
Frechin, M., Senger, B., Braye, M., Kern, D., Martin, R.P., and Becker, H.D. (2009). Yeast mitochondrial Gln-tRNA(Gln) is generated by a GatFAB-mediated transamidation pathway involving Arc1p-controlled subcellular sorting of cytosolic GluRS. Genes & development 23, 1119-1130.
Galibert, F., Alexandraki, D., Baur, A., Boles, E., Chalwatzis, N., Chuat, J.C., Coster, F., Cziepluch, C., De Haan, M., Domdey, H., et al. (1996). Complete nucleotide sequence of Saccharomyces cerevisiae chromosome X. The EMBO journal 15, 2031-2049.
Guo, M., and Schimmel, P. (2013). Essential nontranslational functions of tRNA synthetases. Nature chemical biology 9, 145-153.
Hayase, Y., Jahn, M., Rogers, M.J., Sylvers, L.A., Koizumi, M., Inoue, H., Ohtsuka, E., and Soll, D. (1992). Recognition of bases in Escherichia coli tRNA(Gln) by glutaminyl-tRNA synthetase: a complete identity set. The EMBO journal 11, 4159-4165.
Hiraoka, Y., Kawamata, K., Haraguchi, T., and Chikashige, Y. (2009). Codon usage bias is correlated with gene expression levels in the fission yeast Schizosaccharomyces pombe. Genes to cells : devoted to molecular & cellular mechanisms 14, 499-509.
Horiuchi, K.Y., Harpel, M.R., Shen, L., Luo, Y., Rogers, K.C., and Copeland, R.A. (2001). Mechanistic studies of reaction coupling in Glu-tRNAGln amidotransferase. Biochemistry 40, 6450-6457.
Ibba, M., Becker, H.D., Stathopoulos, C., Tumbula, D.L., and Soll, D. (2000). The adaptor hypothesis revisited. Trends in biochemical sciences 25, 311-316.
Ibba, M., and Soll, D. (2000). Aminoacyl-tRNA synthesis. Annual review of biochemistry 69, 617-650.
Kim, D.U., Hayles, J., Kim, D., Wood, V., Park, H.O., Won, M., Yoo, H.S., Duhig, T., Nam, M., Palmer, G., et al. (2010). Analysis of a genome-wide set of gene deletions in the fission yeast Schizosaccharomyces pombe. Nature biotechnology 28, 617-623.
Kurtzman, C.P., and Fell, J.W. (2000). The yeasts : a taxonomic study, 4th edn (Amsterdam ; New York: Elsevier).
Lamour, V., Quevillon, S., Diriong, S., N′Guyen, V.C., Lipinski, M., and Mirande, M. (1994). Evolution of the Glx-tRNA synthetase family: the glutaminyl enzyme as a case of horizontal gene transfer. Proceedings of the National Academy of Sciences of the United States of America 91, 8670-8674.
Lang, B.F., Cedergren, R., and Gray, M.W. (1987). The mitochondrial genome of the fission yeast, Schizosaccharomyces pombe. Sequence of the large-subunit ribosomal RNA gene, comparison of potential secondary structure in fungal mitochondrial large-subunit rRNAs and evolutionary considerations. European journal of biochemistry / FEBS 169, 527-537.
Lapointe, J., Duplain, L., and Proulx, M. (1986). A single glutamyl-tRNA synthetase aminoacylates tRNAGlu and tRNAGln in Bacillus subtilis and efficiently misacylates Escherichia coli tRNAGln1 in vitro. Journal of bacteriology 165, 88-93.
Liao, C.C., Lin, C.H., Chen, S.J., and Wang, C.C. (2012). Trans-kingdom rescue of Gln-tRNAGln synthesis in yeast cytoplasm and mitochondria. Nucleic acids research 40, 9171-9181.
MacNeill, S.A. (2002). Genome sequencing: and then there were six. Current biology : CB 12, R294-296.
Marguerat, S., Schmidt, A., Codlin, S., Chen, W., Aebersold, R., and Bahler, J. (2012). Quantitative analysis of fission yeast transcriptomes and proteomes in proliferating and quiescent cells. Cell 151, 671-683.
Mirande, M. (1991). Aminoacyl-tRNA synthetases and DNA replication. Molecular mimicry between RNAII and tRNA(Lys). FEBS letters 283, 1-3.
Mitchison, J.M. (1957). The growth of single cells. I. Schizosaccharomyces pombe. Experimental cell research 13, 244-262.
Park, S.G., Schimmel, P., and Kim, S. (2008). Aminoacyl tRNA synthetases and their connections to disease. Proceedings of the National Academy of Sciences of the United States of America 105, 11043-11049.
Perona, J.J., Rould, M.A., and Steitz, T.A. (1993). Structural basis for transfer RNA aminoacylation by Escherichia coli glutaminyl-tRNA synthetase. Biochemistry 32, 8758-8771.
Rigaut, G., Shevchenko, A., Rutz, B., Wilm, M., Mann, M., and Seraphin, B. (1999). A generic protein purification method for protein complex characterization and proteome exploration. Nature biotechnology 17, 1030-1032.
Rinehart, J., Krett, B., Rubio, M.A., Alfonzo, J.D., and Soll, D. (2005). Saccharomyces cerevisiae imports the cytosolic pathway for Gln-tRNA synthesis into the mitochondrion. Genes & development 19, 583-592.
Saha, R., Dasgupta, S., Basu, G., and Roy, S. (2009). A chimaeric glutamyl:glutaminyl-tRNA synthetase: implications for evolution. The Biochemical journal 417, 449-455.
Sheppard, K., Yuan, J., Hohn, M.J., Jester, B., Devine, K.M., and Soll, D. (2008). From one amino acid to another: tRNA-dependent amino acid biosynthesis. Nucleic acids research 36, 1813-1825.
Simos, G., Segref, A., Fasiolo, F., Hellmuth, K., Shevchenko, A., Mann, M., and Hurt, E.C. (1996). The yeast protein Arc1p binds to tRNA and functions as a cofactor for the methionyl- and glutamyl-tRNA synthetases. The EMBO journal 15, 5437-5448.
Smith, C.L., Matsumoto, T., Niwa, O., Klco, S., Fan, J.B., Yanagida, M., and Cantor, C.R. (1987). An electrophoretic karyotype for Schizosaccharomyces pombe by pulsed field gel electrophoresis. Nucleic acids research 15, 4481-4489.
Waadt, R., Schlucking, K., Schroeder, J.I., and Kudla, J. (2014). Protein fragment bimolecular fluorescence complementation analyses for the in vivo study of protein-protein interactions and cellular protein complex localizations. Methods in molecular biology 1062, 629-658.
Wang, C.C., and Schimmel, P. (1999). Species barrier to RNA recognition overcome with nonspecific RNA binding domains. The Journal of biological chemistry 274, 16508-16512.
Woese, C.R., Olsen, G.J., Ibba, M., and Soll, D. (2000). Aminoacyl-tRNA synthetases, the genetic code, and the evolutionary process. Microbiology and molecular biology reviews : MMBR 64, 202-236.
Wolf, Y.I., Aravind, L., Grishin, N.V., and Koonin, E.V. (1999). Evolution of aminoacyl-tRNA synthetases--analysis of unique domain architectures and phylogenetic trees reveals a complex history of horizontal gene transfer events. Genome research 9, 689-710.
Wood, V., Gwilliam, R., Rajandream, M.A., Lyne, M., Lyne, R., Stewart, A., Sgouros, J., Peat, N., Hayles, J., Baker, S., et al. (2002). The genome sequence of Schizosaccharomyces pombe. Nature 415, 871-880.
唐蕙苓 (2002). 酵母菌轉譯起始機制的研究;Elucidating a novel translation initiation mechanism for a eukaryotic gene. In 生命科學系 (台灣: 國立中央大學).
指導教授 王健家(Chien-Chia Wang) 審核日期 2015-1-20
推文 facebook   plurk   twitter   funp   google   live   udn   HD   myshare   reddit   netvibes   friend   youpush   delicious   baidu   
網路書籤 Google bookmarks   del.icio.us   hemidemi   myshare   

若有論文相關問題,請聯絡國立中央大學圖書館推廣服務組 TEL:(03)422-7151轉57407,或E-mail聯絡  - 隱私權政策聲明