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姓名 何宜晏(Yiyan Ho)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 探討酵母菌glycyl-tRNA合成酵素的非傳統生物功能
(Exploring the non-canonical functions of yeast glycyl-tRNA synthetase)
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摘要(中) 在酵母菌中存在二個不同glycyl-tRNA合成酵素(glycyl-tRNA synthetase)的基因, 分別是GRS1 和GRS2。有趣的是GRS1基因同時解碼細胞質及粒腺體的Glycyl-tRNA合成酵素,而GRS2基因則沒有任何已知的功能。近來的研究發現,GRS1基因解碼的glycyl-tRNA合成酵素除了具有傳統胺醯化的功能外,還可能參與轉錄終止作用。我想利用酵母菌雙雜交系統(Yeast two hybrid system)來探究glycyl-tRNA 合成酵素與哪些酵母菌蛋白質有交互作用,希望進一步釐清這個參與蛋白質合成的酵素如何調控轉錄作用,或參與其他的代謝途徑。
我們用酵母菌雙雜交篩選出了三個可能與GRS1 基因產物有交互作用的蛋白質,分別為NFI1(SIZ2)、IFH1、YNL224C。其中NFI1(SIZ2)是一參與SUMO pathway的蛋白質,與蛋白質的後修飾以及蛋白質的活性調控有關;而IFH1是一與FHL1蛋白質有交互作用的蛋白質,可能參與rRNA的修飾作用(rRNA processing);YNL224C是一段胜太鏈,其功能不清楚,但其蛋白質序列中具有一段保留結構區(conserve domain),這段結構區與RNA結合蛋白相類似,推測YNL224C可能也具有RNA結合的功能。
進一步用反向酵母菌雙雜交測試(reverse two hybrid assay),發現了NFI1(SIZ2)與GRS1基因產物的確有交互作用。這些結果顯示: Glycyl-tRNA合成酵素可能參與胺醯化以外的代謝途徑,例如SUMO pathway。
摘要(英) Like most yeast tRNA synthetases, there are two homologous nuclear genes, GRS1 and GRS2, encoding glycyl-tRNA synthetase in the yeast Saccharomyces cerevisiae. However, a recent study argued that GRS1 provides both the cytoplasmic and mitochondrial GlyRS functions, while GRS2 appears to be non-functional. A remarkable feature regarding the biological role of GRS1 was recently identified by a genetic screening, which demonstrated that GlyRS-1 is involved in 3’-end formation of mRNAs. However, the detailed mechanism by which the translational enzyme participates in the process of mRNA termination is largely unknown. In this thesis, we used GRS1 as bait to screen a yeast genomic two-hybrid library, aiming at identifying proteins that interact specifically with GlyRS-1 in vivo. Our results showed that three proteins, NFI1 (SIZ2), IFH1, and YNL224C interact specifically with GlyRS in the two-hybrid screening. Further reverse two-hybrid tests confirmed that GlyRS interact with NFI1, a novel signal transductor that may be involved in SUMO pathway.
關鍵字(中) ★ 酵母菌雙雜交系統 關鍵字(英) ★ yeast two hybrid system
論文目次 目錄
目錄----------------------------------------------------------------------I
表目錄-----------------------------------------------------------------IV
圖目錄------------------------------------------------------------------V
縮字表-----------------------------------------------------------------VI
第一章 前言-----------------------------------------------------------1
壹、 aminoacyl-tRNA合成酵素----------------------------------1
一、aminoacyl-tRNA合成酵素之功能
二、aminoacyl-tRNA合成酵素的分類
三、aminoacyl-tRNA合成酵素的其他生物功能
貳、 酵母菌雙雜交系統(yeast two hybrid system)簡介------3
一、簡介
二、LexA 酵母菌雙雜交系統
參、 Glycyl-tRNA合成酵素----------------------------------------4
肆、 研究目的---------------------------------------------------------6
第二章 材料與方法--------------------------------------------------7
壹、 實驗材料---------------------------------------------------------7
一、大腸桿菌株
二、酵母菌株
三、載體
四、核酸引子
貳、實驗方法----------------------------------------------------------9
一、核酸的製備
二、大腸桿菌勝任細胞的製備與轉殖作用
三、酵母菌Genomic DNA 的抽取與純化
四、酵母菌雙雜交系統
1. 酵母菌GlyRS基因的增幅與選殖
2. 酵母菌勝任細胞的製備與轉殖作用
3. 利用報到基因進行酵母菌雙雜交系統的篩選
4. 酵母菌質體DNA的抽取
5. 利用限制酵素將酵母菌質體DNA分類
五、酵母菌融合蛋白質的表現與西方墨點法
第三章 結果----------------------------------------------------------25
壹、GRS1基因的建構---------------------------------------------25
貳、接合蛋白的表達偵測-----------------------------------------25
參、融合蛋白本身是否啟動報導基因--------------------------26
肆、相互作用的偵測-----------------------------------------------27
伍、用反向雙雜交偵測法(reverse two hybrid assay)確定交互作用--------------------------------------------------------------------28
第四章 討論----------------------------------------------------------30
壹、結果的討論-----------------------------------------------------30
貳、實驗方法的討論-----------------------------------------------32
參考文獻--------------------------------------------------------------35
表-----------------------------------------------------------------------43
圖-----------------------------------------------------------------------46
附錄--------------------------------------------------------------------58
表目錄
表一、aminoacyl-tRNA合成酵素的分---------------------------43
表二、aminoacyl-tRNA合成酵素族群的差異------------------44
表三、與Glycyl-tRNA 合成酵素交互作用的蛋白質簡表---45
圖目錄
圖一、tRNA的胺醯化作用-----------------------------------------46
圖二、aminoacyl-tRNA合成酵素參與多種生物功能---------47
圖三、LexA 酵母菌雙雜交系統示意圖-------------------------48
圖四、GRS1基因轉殖質體的建構-------------------------------49
圖五、LexA-GlyRS 融合蛋白的表現----------------------------50
圖六、LexA-GlyRS 融合蛋白的報導基因測試----------------51
圖七、酵母菌雙雜交系統基因庫篩選示意圖------------------52
圖八、GlyRS與酵母菌基因庫篩選分類------------------------53
圖九、GlyRS 與候選者的報導基因測試------------------------54
圖十、SIZ2與GlyRS 交互作用的蛋白質序列----------------55
圖十一、LexA-SIZ2融合蛋白的表現----------------------------56
圖十二、GlyRS 與 SIZ2 的反向酵母菌雙雜交偵測--------57
參考文獻 參考文獻
Arnez, J. G., Dock-Bregeon, A. C. and Moras, D. (1999) Glycyl-tRNA synthetase uses a negatively charged pit for specific recognition and activation of glycine.J. Mol. Biol. 286:1449-1459.
Azad, A. K., Stanford, D. R., Sarkar, S. and Hopper, A. K. (2001) Role of nuclear pools of aminoacyl-tRNA synthetases in tRNA nuclear export. Mol. Biol. Cell.12: 1381-1392.
Bialkowska, A. and Kurlandzka, A. (2002) Proteins interacting with Lin1p, a putative link between chromosome segregation, mRNA splicing and DNA replication in Saccharomyces cerevisiae. Yeast.19: 1323-1333
Briand, J. F., Navarro, F., Gadal, O. and Thuriaux, P. (2001) Cross talk between tRNA and rRNA synthesis in Saccharomyces cerevisiae. Mol. Cell. Biol.21: 189-195.
Cherel, I. and Thuriaux, P. (1995) The IFH1 gene product interacts with a fork head protein in Saccharomyces cerevisiae. Yeast.11: 261-270.
CLONTECH Laboratories, Inc., (1996) MATCHMAKER LexA Two-Hybrid System and MATCHMAKER LexA Libraries User Manual
Dagkessamanskaia, A., Martin-Yken, H., Basmaji, F., Briza, P. and Francois, J. (2001) Interaction of Knr4 protein, a protein involved in cell wall synthesis, with tyrosine tRNA synthetase encoded by TYS1 in Saccharomyces cerevisiae. FEMS Microbiol. Lett. 200:53-58.
Dou, X., Limmer, S. and Kreutzer, R. (2001) DNA-binding of phenylalanyl-tRNA synthetase is accompanied by loop formation of the double-stranded DNA. J. Mol. Biol.305: 451-458.
Duchene, A., M., Peeters, N., Dietrich, A., Cosset, A., Small, I. D. and Wintz, H. (2001) Overlapping destinations for two dual targeted glycyl-tRNA synthetases in Arabidopsis thaliana and Phaseolus vulgaris. J. Biol. Chem.276: 15275-15283.
Flores, A., Briand, J. F., Gadal, O., Andrau, J. C., Rubbi, L., Van Mullem, V., Boschiero, C., Goussot, M., Marck, C., Carles, C., Thuriaux, P., Sentenac, A. and Werner, M. (1999) A protein-protein interaction map of yeast RNA polymerase III. Proc. Natl. Acad. Sci. U. S. A. 96: 7815-7820.
Freist, W., Logan, D. T. and Gauss, D. H. (1996) Glycyl-tRNA synthetase. Biol. Chem. Hoppe. Seyler. 377: 343-356. Review
Golemis, E. A., Gyuris, J. and Brent, R. (1994) Interaction trap/two-hybrid systems to identify interacting proteins. In Current Protocols in Molecular Biology (John Wiley & Sons, Inc.), Ch. 13.14.
Golemis, E. A., Gyuris, J. and Brent, R. (1996) Analysis of protein interactions; and Interaction trap/two-hybrid systems to identify interacting proteins. In Current Protocols in Molecular Biology (John
Wiley & Sons, Inc.), Ch. 20.0 and 20.1.
Hochstrasser, M. (2001) SP-RING for SUMO: new functions bloom for a ubiquitin-like protein. Cell. 107: 5-8. Review.
Houman, F., Rho, S. B., Zhang, J., Shen, X., Wang, C. C., Schimmel, P. and Martinis, S. A. (2000) A prokaryote and human tRNA synthetase provide an essential RNA splicing function in yeast mitochondria. Proc. Natl. Acad. Sci. U. S .A. 97: 13743-13748.
Ito, T., Chiba, T., Ozawa, R., Yoshida, M., Hattori, M. and Sakaki, Y. (2001) A comprehensive two-hybrid analysis to explore the yeast protein interactome. Proc. Natl. Acad. Sci. U. S .A. 98: 4569-4574.
Ito, T., Ota, K., Kubota, H., Yamaguchi, Y., Chiba, T., Sakuraba, K., and Yoshida, M. (2002) Roles for the two-hybrid system in exploration of the yeast protein interactome. Mol. Cell. Proteomics. 1:561-566.
Johnson E. S. and Gupta, A. A. (2001) An E3-like factor that promotes SUMO conjugation to the yeast septins. Cell. 106: 735-744.
Kowal, A. K., Kohrer, C. and RajBhandary, U. L. (2001) Twenty-first aminoacyl-tRNA synthetase-suppressor tRNA pairs for possible use in site-specific incorporation of amino acid analogues into proteins in eukaryotes and in eubacteria. Proc. Natl. Acad. Sci. U. S .A.98: 2268-2273
Logan, D. T., Cura, V., Touzel, J. P., Kern, D. and Moras, D. (1994) Crystallisation of the glycyl-tRNA synthetase from Thermus thermophilus and initial crystallographic data. J. Mol. Biol. 241: 732-735.
Magrath, C. and Hyman, L. E. (1999) A mutation in GRS1, a glycyl-tRNA synthetase, affects 3'-end formation in Saccharomyces cerevisiae. Genetics.152: 129-141.
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. Review
Martinis, S. A., Plateau, P., Cavarelli, J. and Florentz, C. (1999) Aminoacyl-tRNA synthetases: a family of expanding functions. Mittelwihr, France, October 10-15, 1999.EMBO. J. 18: 4591-4596
Mazauric, M. H., Roy, H. and Kern, D. (1999) tRNA glycylation system from Thermus thermophilus. tRNAGly identity and functional interrelation with the glycylation systems from other phylae. Biochemistry 38: 13094-13105.
Mudge, S. J., Williams, J. H., Eyre, H. J., Sutherland, G. R., Cowan, P. J. and Power, D. A. (1998) Complex organisation of the 5'-end of the human glycine tRNA synthetase gene. Gene.209: 45-50.
Nameki, N., Tamura, K., Asahara, H. and Hasegawa, T. (1997) Recognition of tRNA(Gly) by three widely diverged glycyl-tRNA synthetases. J. Mol. Biol. 268: 640-647.
Rho, S. B. and Martinis, S. A. (2000) The bI4 group I intron binds directly to both its protein splicing partners, a tRNA synthetase and maturase, to facilitate RNA splicing activity. RNA. 6: 1882-1894.
Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY)
Scandurro, A. B., Weldon, C. W., Figueroa, Y. G., Alam, J. and Beckman, B. S. (2001) Gene microarray analysis reveals a novel hypoxia signal transduction pathway in human hepatocellular carcinoma cells. Int. J. Oncol. 19: 129-135.
Strunnikov, A. V., Aravind, L. and Koonin, E. V. (2001) Saccharomyces cerevisiae SMT4 encodes an evolutionarily conserved protease with a role in chromosome condensation regulation. Genetics.158: 95-107.
Stephens D. J. and Banting, G. (2000) The use of yeast two-hybrid screens in studies of protein:protein interactions involved in trafficking. Traffic. 1: 763-768. Review
Szymanski, M., Deniziak, M. and Barciszewski, J. (2000) The new aspects of aminoacyl-tRNA synthetases. Acta. Biochim. Pol. 47: 821-834. Review.
Tolkunova, E., Park, H., Xia, J., King, M. P. and Davidson, E. (2000) The human lysyl-tRNA synthetase gene encodes both the cytoplasmic and mitochondrial enzymes by means of an unusual alternative splicing of the primary transcript. J. Biol. Chem. 275: 35063-35069.
Turner, R. J., Lovato, M. and Schimmel, P. (2000) One of two genes encoding glycyl-tRNA synthetase in Saccharomyces cerevisiae provides mitochondrial and cytoplasmic functions. J. Biol. Chem. 275: 27681-27688.
Tussie-Luna, M. I., Michel, B., Hakre, S. and Roy, A. L. (2002) The SUMO ubiquitin-protein isopeptide ligase family member Miz1/PIASxbeta /Siz2 is a transcriptional cofactor for TFII-I. J. Biol. Chem. 277: 43185-43193.
Uetz, P., Giot, L., Cagney, G., Mansfield, T. A., Judson, R. S., Knight, J. R., Lockshon, D., Narayan, V., Srinivasan, M., Pochart, P., Qureshi-Emili, A., Li, Y., Godwin, B., Conover, D., Kalbfleisch, T., Vijayadamodar, G., Yang, M., Johnston, M., Fields, S. and Rothberg, J. M. (2000) A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae. Nature. 403 :623-627.
Uwer, U., Willmitzer, L. and Altmann, T. (1998) Inactivation of a glycyl-tRNA synthetase leads to an arrest in plant embryo development. Plant Cell. 10: 1277-1294.
Vidal, M., Brachmann, R. K., Fattaey, A., Harlow, E. and Boeke, J. D. (1996) Reverse two-hybrid and one-hybrid systems to detect dissociation of protein-protein and DNA-protein interactions. Proc. Natl. Acad. Sci. U. S. A. 93:10315-10320
Vidal, M. and Legrain, P. (1999) Yeast forward and reverse 'n'-hybrid systems. Nucleic Acids Res.27: 919-929. Review.
White, M. A. (1996)The yeast two-hybrid system: forward and reverse. Proc. Natl. Acad. Sci. U. S. A. 93: 10001-10003. Review
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. Res. 9: 689-710.
Wrobel, C., Schmidt, E. V. and Polymenis, M. (1999) CDC64 encodes cytoplasmic alanyl-tRNA synthetase, Ala1p, of Saccharomyces cerevisiae. J. Bacteriol. 181: 7618-7620.
指導教授 王健家(Chien-Chia Wang) 審核日期 2003-1-17
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