博碩士論文 88224002 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:84 、訪客IP:3.93.74.227
姓名 胡小珊( Xiao-Xian Hu)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 探討Alanyl-tRNA synthetase的演化及專一性
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★ 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★ 酵母菌GRS1基因的轉譯起始機制之研究
★ 探討酵母菌ALA1基因的non-AUG轉譯機制★ 酵母菌 alanyl-tRNA synthetase 的細胞內傳輸機制
★ 鑑定酵母菌中具高親和力的tRNA結合蛋白★ 酵母菌ALA1基因的表現調控機制
★ 酵母菌ALA1 基因轉譯起始機制的研究★ 探討一個真核tRNA合成酶的附加區段之轉錄活化活性
★ 一個雙重功能的酵母菌 tRNA 合成酶之研究★ 探討酵母菌中non-AUG起始點的周邊序列對轉譯起始效率的影響
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) 因此我們想要利用AlaRS的N端及一些非專一性的tRNA結合蛋白,來組合一個在活體中具有AlaRS功能的蛋白質。我們的結果顯示,大腸桿菌的AlaRS 位於C端形成多倍體區域在細胞中是必需的。不論是大腸桿菌AlaRS的N端胺基酸1-461或是大腸桿菌AlaRS的N端胺基酸1-699,皆不能補償大腸桿菌AlaRS基因(alaS)刪除株。而融合蛋白質(非專一性tRNA結合蛋白質與大腸桿菌AlaRS的N端胺基酸1-461或是大腸桿菌AlaRS的N端胺基酸1-699的組合)也不能明顯的互補大腸桿菌AlaRS基因(alaS)刪除株。酵母菌的融合蛋白質也不能補償酵母菌AlaRS基因(ALA1)刪除株。但是有趣的是我們發現,將酵母菌細胞質的AlaRS基因構築在具ADH promoter的載體上時,不但可以補償酵母菌AlaRS細胞質刪除株的功能,同時也可以補償酵母菌粒線體刪除株的功能。因此我們推測,在酵母菌中,一個ALA1基因會轉譯出具有細胞質及粒線體雙重功能的AlaRS。
摘要(英) We are motivated to ask whether we could assemble an alanyl-tRNA synthetase that is active in vivo, using the N-terminal domain of AlaRS and nonspecific RNA binding domains. Our results show that the C-terminal oligomerization domain (residue 700-875) of E. coli AlaRS is essential for its in vivo function. Neither N461 (containing residue 1 to 461) nor N699 (containing residue 1 to 699) can complement an alaS (the gene coding for E. coli AlaRS) knockout strain. Fusion of a nonspecific RNA binding domain to either N461 or N699 has no significant effect on its complementing activity. Similar results were obtained using yeast AlaRS as a template for construction of fusion proteins. Interestingly, we found that the putative open reading frame for the yeast cytoplasmic AlaRS, when cloned in a high-copy-number vector under the control of a constitutive ADH promoter, could complement both the cytoplasmic and mitochondrial defects of an ALA1 (the gene coding for yeast AlaRS) disrupted allele, suggesting that a single ALA1 gene codes for both the cytoplasmic and mitochondrial functions of a AlaRS in yeast.
關鍵字(中) 關鍵字(英) ★ Alanyl-tRNA synthetase
★ AlaRS
論文目次 中文摘要 ---------------------------------------------------- 1
英文摘要 ---------------------------------------------------- 3
第一章緒論 ----------------------------------------------- 5
Ⅰ. Aminoacyl-tRNA synthetases(aaRSs)的簡介 ------------- 5
一. aaRS的功能 ---------------------------------------------- 5
二. aaRS 的分類 --------------------------------------------- 7
Ⅱ. Alanyl-tRNA synthetase(AlaRS)的簡介 ----------------- 8
Ⅲ. tRNAAla ---------------------------------------------- 10
Ⅳ. aaRS與tRNA的共同演化 --------------------------------- 12
Ⅴ、非專一性的tRNA 結合蛋白 ------------------------------ 13
一. Arc1p -------------------------------------------------- 13
二. 酵母菌glutaminyl-tRNA synthetase的附加區 (appended domain;AdScGlnRS)------------------------------ 14
第二章研究目的 ------------------------------------------ 15
第三章材料與方法 ---------------------------------------- 16
Ⅰ. 培養基的製備 ----------------------------------------- 16
Ⅱ. 構築野生株及融合酵母菌及大腸桿菌的AlaRS於酵母菌選殖載體
------------------------------------------------------ 19
Ⅲ. 構築野生株及融合大腸桿菌的AlaRS於大腸桿菌表現載體
------------------------------------------------------ 25
Ⅳ. 用西方吸漬法測定AlaRS於酵母菌中的表現
------------------------------------------------------ 27
Ⅴ. AlaRS的活體內分析:互補試驗(Complementation assay)
------------------------------------------------------ 29
Ⅵ. 純化野生株及融合AlaRS -------------------------------- 30
第四章結果 ---------------------------------------------- 32
Ⅰ. 酵母菌的互補(complementation)試驗----------------- 32
Ⅱ. 大腸桿菌的互補(complementation)試驗---------------- 36
Ⅲ. AlaRS於酵母菌中的表現 ------------------------------- 39
Ⅳ. 純化野生株及融合的AlaRS ----------------------------- 40
第五章討論 ---------------------------------------------- 41
第六章參考文獻 ------------------------------------------ 44
第七章附錄 ---------------------------------------------- 52
表目錄 ----------------------------------------------------- iv
圖目錄 ------------------------------------------------------ v
表目錄
表一. aaRS 的分類 ------------------------------------------- 7
表二. ClassⅠ及ClassⅡaaRS的特徵 ---------------------------- 8
圖目錄
圖一. 胺醯化作用 -------------------------------------------- 5
圖二. aaRS在細胞中的功能及角色 ------------------------------ 6
圖三. AlaRS的結構功能單位 ---------------------------------- 10
圖四. tRNA的二級與三級結構 --------------------------------- 11
圖五. 大腸桿菌的tRNAAla ------------------------------------ 11
圖六. aaRS與tRNA演化的圖解 --------------------------------- 12
圖七. 大腸桿菌的glutaminyl-tRNA synthetase(EcGlnRS)與
酵母菌的glutaminyl-tRNA synthetase(ScGlnRS)比對的圖
解---------------------------------------------------- 14
圖八. 構築AlaRS在酵母菌選殖載體的圖示 ---------------------- 24
圖九. 構築AlaRS在大腸桿菌表現載體的圖示 -------------------- 26
圖十. 酵母菌的互補試驗 ------------------------------------- 33
圖十一. 酵母菌AlaRS於5-FOA plate上的生長 ------------------- 34
圖十二. 酵母菌AlaRS於YPG plate上的生長 --------------------- 35
圖十三. 大腸桿菌的互補試驗 --------------------------------- 37
圖十四. 在大腸桿菌中大量表現蛋白質的互補試驗 --------------- 38
圖十五. 選殖載體在酵母菌表現蛋白質的西方吸漬法圖譜
---------------------------------------------------- 39
圖十六. 以Ni-NTA resin純化蛋白質的SDS-PAGE蛋白質圖譜
---------------------------------------------------- 40
參考文獻 Arnez, J. G. and Moras, D. (1997) Structural and functional considerations of the aminoacylation reaction. Trends in Biochemical Sciences 22: 211-216.
Augustine, J. and Francklyn, C. (1997) Design of an active fragment of a ClassⅡ aminoacyl-tRNA synthetases and its significance for synthetase evolution. Biochemistry 36: 3473-3482.
Ausubel, F. M., Brent, R., Kingston, R. D., Moore, D. D., Seidan, J. G., Smith, J. A., and Struhl, K. (1994) Current Protocols in Molecular Biology, copyright by c 1994-2000 by Wiley, J. and Sons, Inc.
Beuning, P. J. and Musier-Forsyth, K. (2000) Hydrolytic editing by a classⅡ-tRMA synthetases. Proceeding of the National Academy of Sciences of the United States America 97: 8916-8920.
Buechter, D. D., and Schimmel, P. (1993) Dissection of a classⅡ tRNA synthetase: determinants for minihelix recognition are tightly associated with domain for amino acid activation. Biochemistry 32: 5267-5272.
Buechter, D. D., and Schimmel, P. (1995) Minor groove recognition of the critical acceptor helix base pair by an appended module of a classⅡtRNA synthetase. Biochemistry 34: 6014-6019.
Burbaum, J. J. and Schimmel, P. (1991) Structional relationships and the classification of aminoacyl-tRNA synthetases. Journal of Biological Chemistry 266: 16965-16968.
Cahuzac, B., Berthonneau, E., Birlirakis, N., Guittet, E., and Mirande, M. (2000) A recurrent RNA-binding domain is appended to eukaryotic aminoacyl-tRNA synthetases. European Molecular Biology Organization Journal 19: 445-452.
Chang, K. Y., Varani, G., Bhattacharya, S., Choi, H., and McClain, W. H. (1999) Correlation of deformability at a tRNA recognition site and aminoacylation specificity. Proceeding of the National Academy of Sciences of theUnited States America 96: 11764-11769.
Chihade, J. W. and Schimmel, P. (1999) Assembly of a catalytic unit for RNA microhelix aminoacylation using nonspecific RNA binding domains. Proceeding of the National Academy of Sciences of theUnited States America 96: 12316-12321.
Chihade, J. W., Brown, J. R., Schimmel, P., and Ribas de Pouplana, L. (2000) Origin of mitochondria in relation to evolutionary history of eukaryotic alanyl-tRNA synthetase. Proceeding of the National Academy of Sciences of theUnited States America 97: 12153-12157.
Cilley, C. D. and Williamson, J. R. (1997) Analysis of bacteriophage N protein and peptide binding to boxB RNA using polyacrylamide gel coelectrophosis (PACE). RNA 3: 57-67.
Cusack, S. (1997) Aminoacyl-tRNA synthetases. Current Opinion in Structural Biology 7: 881-889.
Deinert, K., Fasiolo, F., Hert, E. C., and Simos, G. (2001) Arc1p organizes the yeast aminoacyl-tRNA synthetase complex and stabilizes its interaction with the cognate tRNAs. Journal of Biological Chemistry 276: 6000-6008.
Dignam, J. D., Dignam, S. S., and Brumley, L. L. (1991) Alany-tRNA synthetase from Escherichia coli, Bombyx mori and Ratus ratus existence of common structural features. European Journal of Biochemistry 198: 201-210.
Eriane, 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 (London) 347: 203-206.
Fischer, A. E., Beuning, P. J., and Musier-Forsyth, K. (1999) Identification of discriminator base atomic groups that modulate the alanine aminoacylation reaction. Journal of Biological Chemistry 274: 37093-37096.
Hampsey, M. (1997) A review of phenotypes in Saccharomyces cerevisiae. Yeast 13: 1099-1133.
Ho, C., Jasin, M., and Schimmel, P. (1985) Amino acid replacements that compensate for a large polypeptide deletion in an enzyme. Science 229: 189-393.
Hou, Y. M. and Schimmel, P. (1988) A simple structural feature is a major determinant of the identity of a transfer RNA. Nature (London) 133: 140-145.
Hou, Y. M., Zhang, X., Holland, J. A., and Davis, D. R. (2001) An important 2’-OH group for an RNA-protein interaction. Nucleic Acids Research 29: 976-985.
Jasin, M. and Schimmel, P. (1984) Deletion of an essential gene in Escherichia coli by site-aoecific recombination with linear DNA fragment. Journal of Bacteriology 159: 783-789.
Jasin, M., Regan, L., and Schimmel, P. (1983) Modular arrangement of functional domains along the sequence of an aminoacyl tRNA synthetase. Nature (London) 306: 441-447.
Jasin, M., Regan, L., and Schimmel, P. (1984) Dispensable pieces of an aminoacyl tRNA synthetase which activate the catalytic site. Cell 36: 1089-1095.
Jasin, M., Regan, L., and Schimmel, P. (1985) Two mutations in the dispensable part of alanine tRNA synthetase which affect the catalytic activity. Journal of Biological Chemistry 260: 2226-2230.
Kaminska, M., Deniziak, M., Kerjan, P., Barciszewski, J., and Mieande, M. (2000) A recurrent general RNA binding domain appended to plant methionyl-tRNA synthetase acts as a cis-acting cofactor for aminoacylation. European Molecular Biology Organization Journal 19: 6908-6917.
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.
Mathews, C. K. and Van Holde, K. E., (1996) Biochemisty, 2nd ed., The Benjamin/Cummings Publishing Company.
Mirande, M. (1991) Aminoacyl-tRNA synthetase family from prokaryotes and eukaryotes: structural domains and their implications. Progess in Nucleic Acid Research and Molecular Biology 40: 95-142.
Nagan, M. C., Beuming, P., Musier-Forsyth, K., and Cramer, C. J. (2000) Importance of discriminator base stacking interactions: molecular dynamics analysis of A73 microhelix (Ala) variants. Nucleic Acid Research 28: 2527.
Ramos, A. and Varani, G. (1997) Structure of the acceptor stem of Escherichia coli tRNAAla: role of the G3‧U70 base pair in synthetase recognition. Nucleic Acid Research 25: 2083-2090.
Regan, L., Bowie, J., and Schimmel, P. (1987) Polypeptide sequences essential for RNA recognition by an enzyme. Scinece 235:1651-1653.
Ribas de Pouplana, L. and Schimmel, P. (1997) Reconstruction of quaternary structures of ClassⅡ tRNA synthetases by rational mutagenesis of a conserved domain. Biochemistry 36: 15041-15048.
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.
Ripmaster, T. L., Shiba, K., and Schimmel, P. (1995) Wide cross-species aminoacyl-tRNA synthetase replacement in vivo: Yeast cytoplasmic alanine enzyme replaced by human polymyositis serum antigen. Proceeding of the National Academy of Sciences of theUnited States America 92: 4932-4936.
Sambrook, J., Fritsch, E. F., and Maniatis, T., (1989) Molecular cloning:a laboratory manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Schimmel, P. (1991) Classes of aminoacyl-tRNA synthetases and the establishment of the genetic code. Trends in Biochemical Sciences 16: 1-3.
Schimmel, P. and Henderson, B. (1994) Possible role of aminoacyl-RNA complexes in noncoded peptide synthesis and origin of coded synthesis. Proceeding of the National Academy of Sciences of theUnited States America 91: 11283-11286.
Schimmel, P. and Ribas de Pouplana, L. (1995) Transfer RNA: from minihelix to genetic code. Cell 81: 983-986.
Schimmel, P. and Ribas de Pouplana, L. (2000) Footprints of aminoacyl-tRNA synthetases are everywhere. Trends in Biochemical Sciences 25: 207-209.
Schimmel, P. and Ripmaster, T. (1995) Modular design of components of the operational RNA code for alanine in evolution. Trends in Biochemical Sciences 20: 333-334.
Schimmel, P. and Schmidt, E. (1995) Making connections: RNA-dependent amino acid recognition. Trends in Biochemical Sciences 20: 1-2.
Schimmel, P., Gieget, R., Morast, D., and Yokoyama, S. (1993) An operational RNA code for amino acids and possible relationship to genetic code. Proceeding of the National Academy of Sciences of theUnited States America 90: 8763-8768.
Shiba, K., Motegi, H., and Schimmel, P. (1997) Maintaining genetic code through adaptations of tRNA synthetases to taxonamic domains. Trends in Biochemical Sciences 22: 453-457.
Shiba, K., Ripmaster, T., Suzuki, N., Nichols, R., Plotz, P., Noda, T., and Schimmel P. (1995) Human alanyl-tRNA synthetases: conservation in evolution of catalytic core and microhelix recognition. Biochemistry 34: 10340-10349.
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. European Molecular Biology Organization Journal 15: 5437-5448.
Sood, S. M., Slattery, C. W., Filley, S. J., Wu, M. X., and Hill, K. A. W. (1996) Further characterization of Escherichia coli alanyl-tRNA synthetase. Archives of Biochemistry and Biophysics 328: 295-301.
Sprinzl, M., Steegborn, C., Hubel, F., and Steinberg, S. (1996) Compilation of tRNA sequences and sequences of tRNA genes. Nucleic Acid Research 24: 68-72.
Stathopoulos, C., Li, T., Longman, R., Vothknecht, U. C., Becker, H. D., lbba, M., and Soll, D. (2000) One polypeptide with two aminoacyl-tRNA synthetases activities. Science 287: 479-482.
Strazewski, P., Biala, E., Gabriel, K., and McClain, W. H. (1999) The relationship of thermodynamic stability at a G×U recognition site to tRNA aminoacylation specificity. RNA 5: 1490-1494.
Wang, C. C. and Schimmel, P. (1999) Species barrier to RNA recognition overcome with nonspecific RNA binding domains. Journal of Biological Chemistry 274: 16508-16512.
Wang, C. C., Morales, A. J., and Schimmel, P. (2000) Functional redundancy in the nonspecific RNA binding domain of a ClassⅠtRNA synthetases. Journal of Biological Chemistry 275: 17180-17186.
Whelihan, E. F. and Schimmel, P. (1997) Rescuing an essential enzyme-RNA complex with a non-essential appended domain. European Molecular Biology Organization Journal 16: 2968-2974.
Wilhelm, M. L., Reinbolt, J., Gangloff, J., Dirheimer, G., and Wilhelm, F. X. (1994) Transfer RNA binding protein in the nucleus of Saccharomyces cerevisiae. Federation of European Biochemical Societies letters 349: 260-264.
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 64: 202-236.
Wolin, S. L. and Matera, A. G. (1999) The trials and travels of tRNA. Genes & Development 13: 1-10.
指導教授 王健家(chien-chia Wang) 審核日期 2001-7-10
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