探討酵母菌Glutaminyl-tRNA synthetase 對於粒腺體功能之影響

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：34

、訪客IP：18.188.20.56

姓名

葉曜榮(Yao-Jung Yeh) 查詢紙本館藏

畢業系所

生命科學系

論文名稱

探討酵母菌Glutaminyl-tRNA synthetase 對於粒腺體功能之影響
(Is glutaminyl-tRNA synthetase essential for themitochondrial function of yeast?)

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]

本電子論文使用權限為同意立即開放。
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。
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)

Aminoacyl-tRNA (簡稱aa-tRNA) 的合成，對於蛋白質的生合成是非常重要的步驟，通常是藉由tRNA合成酶將胺基酸接到相對應的tRNA上。不過在Gln-tRNAGln的合成上卻是一個例外。在之前對於Saccharomyces cerevisiae的研究中指出，細胞質的glutaminyl-tRNA synthetase (簡稱GlnRS )是由GLN4基因所提供的，最近的研究發現細胞質的GlnRS會送到粒腺體中參與Gln-tRNAGln的合成。在本篇論文中我們利用細胞質的valyl-tRNA synthetase作為回報基因進一步找出其粒腺體標的訊號的範圍為包含第524 ~ 564號胺基酸，這段序列接近ATP的結合位 (KMSKS保留區域)，這是少數的例子，其粒腺體標的訊號位於在內部序列當中同時也與主要的催化區重疊，但是對於細胞質的GlnRS是怎樣利用一段非傳統的N端粒腺體標的訊號去作用，其機制還不清楚。在Schizosaccharomyces pombe中並沒有發現有粒腺體標的訊號存在，所以我們認為這樣的標的訊號並不是所有酵母菌都擁有的。藉由功能性互補實驗、回報基因測試，發現GlnRS對於粒腺體功能的維持並不是必要的，因此在粒腺體中可能同時存在兩條途徑去合成Gln-tRNAGln。

摘要(英)

Aminoacyl-tRNA (aa-tRNA) formation, an essential process in protein biosynthesis, is generally achieved by direct attachment of an amino acid to cognate tRNA by the aa-tRNA synthetases. An exception is Gln-tRNAGln synthesis. In Saccharomyces cerevisiae, the cytoplasmic glutaminyl-tRNA synthetase (GlnRS) activity is provided by the translational product of GLN4. Previous reports showed that this cytoplasmic GlnRS is also involved in mitochondrial Gln-tRNAGln synthesis. In this thesis, functional mapping using the cytoplasmic form of valyl-tRNA synthetase as the passenger protein identifies the peptide containing amino acids 524 ~ 564 of the enzyme as the mitochondrial targeting signal (MTS), which is close to the ATP-binding site (KMSKS conserved motif). This is one of the few examples, where MTS is embedded in the internal sequence, and is overlapped with the catalytic core domain of the enzyme. However, the detailed mechanism that enables GlnRS to be imported into mitochondria is not clear. In contrast, no MTS is found in Schizosaccharomyces pombe GlnRS, so this may be not a common feature for all yeast GlnRSs. Complementation tests further suggest that yeast GlnRS is not essential for mitochondrial function, and may serve as a redundant system for Gln-tRNAGln synthesis in mitochondria.

關鍵字(中)

★ tRNA 合成酶
★ 酵母菌

關鍵字(英)

★ GlnRS
★ Aminoacyl-tRNA synthetase
★ AARS

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
1.2原核與真核細胞在轉譯方式上的差異 3
1.3 Glutaminyl-tRNA synthetase (GlnRS)的簡介 4
1.4非專一性的tRNA結合蛋白 6
1.4.1 Arc1p 7
1.4.2 Ad(ScGlnRS) 7
1.5粒腺體標的訊號的特性 8
1.6研究目的 9
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 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 13
2.4質體之建構 14
2.5點突變 (Site-directed Mutagenesis) 14
2.6 GLN4剔除株的建構 16
2.7功能性互補試驗 (Complementation)―測試細胞質功能 17
2.8功能性互補試驗 (Complementation)―測試粒腺體功能 19
2.9蛋白質製備 20
2.10 SDS-PAGE之蛋白質分子量分析 21
2.11西方點墨法 (Western Blotting) 22
2.11酵母菌粒腺體的分離 (Enrichment of mitochondria) 24
第三章結果 27
3.1利用回報基因找出ScGlnRS的粒線體標的訊號 27
3.2鑑定不同物種的GlnRS是否具有粒腺體標的訊號 29
3.3 ScGlnRS對於粒腺體功能不是必要的 31
3.4鑑定PgGlnRS是否同時具有細胞質及粒腺體的異構型 33
3.5利用西方式點墨法證實Arc1p在細胞中的分佈情形 34
第四章討論 36
4.1 ScGlnRS對於粒腺體功能必要性之探討 36
4.2 Arc1p同時會在細胞質及粒腺體中分布 38
第五章參考文獻 40
圖表 44

參考文獻

Burbaum, J. J., Schimmel, P. (1991) Structural relationships and the classification of aminoacyl-tRNA synthetases. J Biol Chem 266:16965-8.
Cahuzac, B., Berthonneau, E., Birlirakis, N., Guittet, E. and Mirande, M. (2000) A recurrent RNA-binding domain is appended to eukaryotic aminoacyl-tRNA synthetases. EMBO J 19: 445-52.
Carter, C. W. Jr. (1993) Cognition, Mechanism, and Evolutionary Relationships in aminoacyl-tRNA Synthetases. Annu Rev Biochem 62: 715-748
Chang, K. J., and Wang, C. C. (2004) Translation initiation from a naturally occurring non-AUG codon in Saccharomyces cerevisiae. J Biol Chem 279: 13778-13785.
Chatton, B., Walter, P., Ebel, J. P., Lacroute, F., Fasiolo, F. (1988) The yeast VAS1 gene encodes both mitochondrial and cytoplasmic valyl-tRNA synthetases. J Biol Chem 263:52-7
Cusack, S., Berthet-Colominas, C., Härtlein, M., Nassar, N., Leberman, R. (1990) A second class of synthetase structure revealed by X-ray analysis of Escherichia coli seryl-tRNA synthetase at 2.5 A. Nature 347: 249-55
Deinert, K., Fasiolo, F., Hurt, E.C., Simos, G. (2001) Arc1p organizes the yeast aminoacyl-tRNA synthetase complex and stabilizes its interaction with the cognate tRNAs. J Biol Chem 276: 6000-8
Eriani, G., Delarue, M., Poch, O., Gangloff, J., Moras, D. (1990) Partition of tRNA synthetases into two classes based on mutually exclusive sets of sequence motifs. Nature 347: 203-6
Felter, S., Diatewa, M., Schneider, C., and Stahl, A. J. (1981) Yeast mitochondrial and cytoplasmic valyl-tRNA synthetases. Biochem Biophys Res Commun 98: 727-734
Feng, L., Tumbula-Hansen, D., Toogood, H., and Söll, D. (2003) Expanding tRNA recognition of a tRNA synthetase by a single amino acid change. Proc Natl Acad Sci USA 100: 5676–5681.
Gakh O, Cavadini P, Isaya G. (2002) Mitochondrial processing peptidases. Biochim Biophys Acta 1592: 63-77
Galani, K., Grosshans, H., Deinert, K., Simos, G. (2001) The intracellular location of two aminoacyl-tRNA synthetases depends on complex formation with Arc1p. EMBO J 20: 6889-98
Hountondji, C., Dessen, P., and Blanquet, S. (1986) Sequence similarities among the family of aminoacyl-tRNA synthetases. Biochimie 68: 1071-8
Hughes, T.R., Marton, M.J., Jones, A.R., Roberts, C.J., Stoughton, R., Armour, C.D., Bennett, H.A., Coffey, E., Dai, H., He, Y.D., et al. (2000) Functional discovery via a compendium of expression profiles. Cell 102: 109–126.
Ibba, M. and Söll, D. (2004) Aminoacyl-tRNAs: Setting the limits of the genetic code. Genes Dev 18: 731–738.
Ludmerer, S.W., Wright, D.J., and Schimmel, P. (1993) Purification of glutamine tRNA synthetase from Saccharomyces cerevisiae. A monomeric aminoacyl-tRNA synthetase with a large and dispensable NH2-terminal domain. J Biol Chem 268: 5519–5523
Maréchal-Drouard, L., Weil, J. H., and Dietrich, A. (1992) Nuclear-encoded transfer RNAs in plant mitochondria. Annu Rev Cell Biol 8: 115-131
Martinis, S. A., Schimmel, P. (1993) Microhelix aminoacylation by a class I tRNA synthetase. Non-conserved base pairs required for specificity. J Biol Chem 268: 6069-72
Martinis, S. A., Plateau, P., Cavarelli, J., and Florentz, C. (1999) Aminoacyl-tRNA synthetases : A new image for a classical family. Biochimie 81: 683-700
Mirande, M. (1991) Aminoacyl-tRNA synthetase family from prokaryotes and eukaryotes: structural domains and their implications. Prog Nucleic Acid Res Mol Biol 40:95-142.
Mulero, J.J., Rosenthal, J.K., and Fox, T.D. (1994) PET112, a Saccharomyces cerevisiae nuclear gene required to maintain rho+ mitochondrial DNA. Curr Genet 25: 299–304.
Neupert, W. (1997) Protein import into mitochondria. Annu Rev Biochem 66: 863-917.
Pelchat, M. and Lapointe, J. (1999) Aminoacyl-tRNA synthetase genes of Bacillus subtilis: organization and regulation. Biochem Cel Biol 77: 343-347
Ribas de Pouplana, L. and Schimmel, P. (2001) Two classes of tRNA synthetases suggested by sterically Compatible dockings on tRNA acceptor stem. Cell 104: 191-193.
Saraste, M., Walker, J.E. (1982) Internal sequence repeats and the path of polypeptide in mitochondrial ADP/ATP translocase. FEBS J 144: 250-4
Schimmel, P., R. and Soll, D. (1979) Aminoacyl-tRNA synthetases: general features and recognition of transfer RNAs. Annu Rev Biochem 48:601-48.
Schön, A., Kannangara, C.G., Gough, S., and Söll, D. 1988. Protein biosynthesis in rganelles requires misaminoacylation of tRNA. Nature 331: 187–190.
Shen, W.-C., Selvakumar, D., Standford, D. R., and Hopper, A., K. (1993) The Saccharomyces cerevisiae LOS1 gene involved in pre-tRNA splicing encodes a nuclear protein that behaves as a component of the nuclear matrix. J Biol Chem 268: 19436–19444
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. EMBO J 15: 5437–5448
Simos, G., Sauer, A., Fasiolo, F., and Hurt, E. C. (1998) A conserved domain within Arc1p delivers tRNA to aminoacyl-tRNA synthetases. Mol Cell 1: 235–242
Tang, H. L., Yeh, L. S., Chen, N. K., Ripmaster, T., Schimmel, P., Wang, C. C. (2004) Translation of a yeast mitochondrial tRNA synthetase initiated at redundant non-AUG codons. J Biol Chem 279: 49656-63
Rinehart, J., Krett, B., Juan, D., and Söll, D. (2007) Saccharomyces cerevisiae imports the cytosolic pathway for Gln-tRNA synthesis into the mitochondrion. Genes Dev 19: 583-592
Whelihan, E.F., Schimmel, P. (1997) Rescuing an essential enzyme-RNA complex with a non-essential appended domain. EMBO J 16: 2968-74:
Wang, C. C. and Schimmel, P. (1999) Species barrier to RNA recognition overcome with nonspecific RNA binding domains. J Biol Chem 274: 16508-16512.
Wilhelm, M. L., Reinbolt, J., Gangloff, J., Dirheimer, G., and Wilhelm, F. X. (1994) Transfer RNA binding protein in the nucleus of Saccharomyces cerevisiae. FEBS J 349: 260-264
Woese, C.R., Olsen, G.J., Ibba, M., and Söll, D. (2000) Aminoacyl-tRNA synthetases, the genetic code, and the evolutionary process. Microbiol Mol Biol Rev 64: 202–236.
Xin, Y., Weidong, L., and Eric, A. F. (2000) The ‘KMSKS’ motif in Tyrosyl-tRNA synthetase participates in initial binding of tRNATyr. Biochimie 39: 340-347
Yang, D., Oyaizu, Y., Oyaizu, H., Olsen, G.J., and Woese, C.R. (1985) Mitochondrial origins. Proc Natl Acad Sci USA 82: 4443–4447.
黃曉芸 (2005)酵母菌ALA1基因轉譯起始機制的研究。中央大學碩士論文
桂妤 (2006)一個雙重功能的酵母菌tRNA合成酶之研究。中央大學碩士
論文
張嘉珮 (2007)酵母菌使用罕見轉譯起始密碼的可能性探討。中央大學碩士
論文

指導教授

王健家(Chien-Chia Wang)

審核日期

2008-7-1

推文