探討酵母菌Valyl-tRNA synthetase的生化活性

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：111

、訪客IP：3.145.111.115

姓名

林于慧(Yu-Hui Lin) 查詢紙本館藏

畢業系所

生命科學系

論文名稱

探討酵母菌Valyl-tRNA synthetase的生化活性
(Exploring the biological fumction of yeast Valyl-tRNA synthetase)

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
★ 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

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)

不同於大部分的tRNA合成酵素，酵母菌中的細胞質與粒腺體Valine-tRNA合成酵素（ValRS）是由同一個細胞核基因所解碼出來;這兩個異構型唯一的不同處是在於新合成的粒腺體合成酵素多了一段由46個胺基酸組成的"粒腺體標的訊號"。雖然在活體外這兩個合成酵素有幾乎相同的活性，但是在活體內這兩個合成酵素因為存在不同的胞器內，所以無法互相取代。我們發現此合成酵素的胺基端具有一段富含Lysine的附加區段（胺基酸1~97），但是此附加區段並不直接參與胺醯化作用，也不是細胞內蛋白質合成功能之所需;利用two-hybrid系統我們發現ValRS的附加區段能夠活化細胞核內回報基因的轉錄作用，而這種轉錄活化功能並未發現於其他已知的aaRS附加區段，且其序列亦與已知的轉錄活化蛋白質不同，因此推斷可能是一個新的轉錄活化因子。

摘要(英)

In yeast, there are typically two distinct nuclear-encoded tRNA synthetases for each amino acids; one function in the cytoplasm and the other in the mitochondria. Previous studies showed that VAS1 is the only gene coding for valyl-tRNA synthetase （ValRS） in the yeast genome. This gene encodes both the cytoplasmic and mitochondrial forms of valyl-tRNA synthetase. These two isoforms have essentially identical polypeptide sequences except for a 46-amino acid leader at the N-terminus of the mitochondrial precursor, which functions as a mitochondrial targeting signal. In vitro, the two isoforms of ValRS cannot distinguish between tRNA species from the cytoplasm and mitochondria, but the enzymes cannot substitute for each other in vivo due to differential partitioning. Also, like many other eukaryotic tRNA synthetase, the cytoplasmic form of yeast ValRS has a lysine-rich polypeptide extension of 97 amino acids appended to its N-terminus. I found that the appended domain did not participate in aminoacylation and was largely dispensible for function in vivo. Surprisingly, our results showed that this appended domain exhibited a strong transcriptional activation activity that was never found in any other tRNA synthetase tested. This suggests that this appended domain may be a novel transcriptional activator.

關鍵字(中)

★ 酵母菌

關鍵字(英)

★ yeast
★ Valyl-tRNA synthetase

論文目次

第一章緒論
I. 何謂aaRS（Aminoacyl-tRNA synthetase;tRNA合成酵素） -------------------------------------------------------------------------1
A. aaRS的生化功能----------------------------------------------- 1
B. ClassI和ClassII aaRS----------------------------------------- 2
II. Prokaryote及Eukaryote中的aaRS ----------------------------3
A. 分佈情形---------------------------------------------------------3
B. aaRS形成complex -------------------------------------------- 3
III. aaRS除了胺醯化作用之外具有非傳統的功能-------------4
IV. 酵母菌中的aaRS-------------------------------------------------5
A. aaRS 的合成--------------------------------------------------5
B. 選擇性轉錄及轉譯作用 ------------------------------------5
C. Valyl-tRNA synthetase（ValRS）----------------------- 7
1. ValRS的生化特性---------------------------------------7
2. 酵母菌中的ValRS---------------------------------------7
第二章材料與方法------------------------------------------------ 10
I. 實驗材料-----------------------------------------------------------10
II. 實驗方法----------------------------------------------------------- 11
A. 核酸的製備---------------------------------------------------11
1. 質體DNA的小量純化---------------------------------11
2. 啟動子DNA的製備------------------------------------ 12
B. 大腸桿菌勝任細胞的製備與轉型作用------------------13
1. 大腸桿菌勝任細胞的製備-----------------------------13
2. 大腸桿菌勝任細胞的轉型作用-----------------------14
C. 酵母菌勝任細胞的製備與轉型作用---------------------15
1. 酵母菌勝任細胞的製備--------------------------------15
2. 酵母菌勝任細胞的轉型作用--------------------------15
D. 大腸桿菌融合蛋白質的表現、純化與濃縮------------16
1. 大腸桿菌融合蛋白質的表現--------------------------16
2. 大腸桿菌融合蛋白質的純化--------------------------17
3. 大腸桿菌融合蛋白質的濃縮--------------------------19
E. 酵母菌融合蛋白質的表現、純化與濃縮----------------20
F. 西方墨點法（Western blot）------------------------------20
1. 酵母菌粗萃物的製備 ----------------------------------20
2. 正十二烷硫酸鈉-聚丙醯胺凝膠電泳（Sodium dodecyl sulfate polyacrylamide gel electrophoresis，SDS-PAGE）------------------------21
3. 蛋白質的轉漬--------------------------------------------21
G. 胺醯化作用分析---------------------------------------------22
1. 蛋白質的濃度測定與稀釋-----------------------------22
2. 胺醯化作用-----------------------------------------------22
H. 聚丙烯醯胺親和力共電泳（Polyacrylamide Affinity Coelectrophoresis，PACE）製膠與電泳--------------24
第三章結果---------------------------------------------------------------25
I. 附加區段對於ValRS胺醯化的影響---------------------------25
II. ValRS是否具有調控自己基因表現的能力？---------------27
1. 酵母菌ValRS的附加區段不會鍵結DNA-----------29
2. 酵母菌ValRS不會鍵結DNA，也不具有專一性---30
第四章討論---------------------------------------------------------------32
參考文獻--------------------------------------------------------------------36
表------------------------------------------------------------------------------39
圖------------------------------------------------------------------------------42

參考文獻

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 5, 1381-1392
Burbaum J. J., and Schimmel P. (1991) Structural relationships and the classification of aminoacyl-tRNA synthetases. J. Biol. Chem. 266, 16965-16968
Chatton B., Walter P., Ebel J. P., Lacroute F., and Fasiolo F. (1988) The yeast VAS1gene encodes both mitochondrial and cytoplasmic valyl-tRNA synthetase. J. Biol. Chem. 263, 52-57
Cerini C., Kerjan P., Astier M., Gratecos D., Mirande M., and Semeriva M. (1991) A component of the multisynthetase complex is a multifunctional aminoacyl-tRNA synthetase. EMBO 10, 4267-4277
Cusack, S. (1997) Aminoacyl-tRNA synthetases. Curr. Opin. Struct. Biol. 6,
881-889
Crepin T., Schmitt E., Blanquet S., and Mechulam Y. (2002) Structure and function of the C-terminal domain of methionyl-tRNA synthetase. Biochemistry 43, 13003-13011
Dou X., Limmer S., 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
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
Felter S., Diatewa M., Schneider C., and Stahl A. J. (1981) Yeast mitochondrial and cytoplasmic valyl-tRNA synthetase. Biochem. Biophys. Res. Commun. 98, 727-734
Fukai S., Nureki O., Sekine S., Shimada A., Tao J., Vassylyev D. G.,and Yokoyama S. (2000) Structural basis for double-sieve discrimination of L-valine from L-isoleucine and L-threonine by the complex of tRNA(Val) and valyl-tRNA synthetase. Cell 103, 793-803
Francin M., and Mirande M. (2003) Functional dissection of the eukaryotic- specific tRNA-interacting factor of lysyl-tRNA synthetase. J Biol Chem 3, 1472-1479
Hountondji C., Dessen P., and Blanquet S. (1986) Sequence similarities among the family of aminoacyl-tRNA synthetases. Biochimie. 68, 1071-1078
Hooper, A.K. (1998) Nuclei functions charge ahead. Science 282, 2003–2004
Lluís R.P., Douglas B., Niranjan Y.S., and Schimmel P. (1998) Functional analysis of peptide motif for RNA microhelix binding suggests new family of RNA-binding domains. EMBO 17, 5449-5457
Minella O., Mulner-Lorillon O., Bec G., Cormier P., and Belle R. (1998) Multiple phosphorylation sites and quaternary organization of guanine-nucleotide exchange complex of elongation factor-1 (EF-1betagammadelta/ValRS) control the various functions of EF-1alpha. Biosci Rep.18, 119-127
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
Natsoulis G., Hilger F., and Fink G. R. (1986) The HTS1 gene encodes both the cytoplasmic and mitochondrial histidine tRNA synthetases of S. cerevisiae. Cell 46, 235-243
Schimmel, P.R, ans Soll, D. (1979) Aminoacyl-tRNA synthetases: general features and recognition of transfer RNAs. Annu. Rev. Biochem. 48, 601-648
Scott D. P., and Schimmel P. (1981) An aminoacyl tRNA synthetase binds to a specific DNA sequence and regulates its gene transcription. Nature 291, 632-635
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 19, 5437-5448
Schimmel P., and Wang C. C. (1999) Getting tRNA synthetases into the nucleus. TIBS 24, 127-128
Souciet G., Menand B., Ovesna J., Cosset A., Dietrich A., and Wintz H. (1999) Characterization of two bifunctional arabdopsis thaliana genes coding for mitochondrial and cytosolic forms of valyl-tRNA synthetase and threonyl-tRNA synthetase by alternative use of two in-frame AUGs. Eur. J. Biochem. 266, 848-854
Weber L. F., Marechal D. L., Folkerts O., Hanson M., and Grienenberger J. M. (1993) Localization of tRNA genes on the Petunia hybrida 3704 mitochondrial
genome. Plant Mol Biol.21, 403-407
Wang C. C. and Schimmel P. (1999) Species barrier to RNA recognition overcome with nonspecific RNA binding domains. J. Biol. Chem. 274, 16508-16512
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.97, 13743-13748
Stathopoulos C., Li T., Longman R., Vothknecht U. C., Becker H. D., Ibba M., and Soll D.(2000) One polypeptide with two aminoacyl-tRNA synthetase activities. Science 287, 5438-5448
Wang C. C., Morales A. J., and Schimmel P. (2000) Functional redundancy in the
nonspecific RNA binding domain of a class I tRNA synthetase. J. Biol. Chem. 22, 17180-17186
謝佳容，酵母菌Valyl-tRNA synthetase 附加區段的生物功能之探討，民國91年，國立中央大學生命科學研究所

指導教授

王健家(Chien-Chia Wang)

審核日期

2003-7-18

推文