博碩士論文 104821601 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:6 、訪客IP:52.204.98.217
姓名 英德拉(Indra Lasmana Tarigan)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 將mycothiol半胱氨酸連接酶轉化成半胱氨酰-tRNA合成酶
(Converting a mycothiol cysteine ligase into a cysteinyl-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
★ 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★ 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]   至系統瀏覽論文 (2020-1-1以後開放)
摘要(中) Aminoacyl-tRNA synthetase (aaRS)是蛋白質合成必需的酵素,它們的主要作用是將胺基酸接到相對應的tRNA,形成aminoacyl-tRNA,這個aminoacyl-tRNA接著被帶到核醣體進行蛋白質合成,因此aaRS是合成蛋白質的必要酵素,每一個aaRS對應一種胺基酸。過去的研究顯示,有些細菌雖然缺少CysRS,但可以用Serine經過間接路徑合成Cys-tRNA,而大多數細菌則具有一個CysRS來合成Cys-tRNA。我們在此提出證據表明,膿腫分枝桿菌(Mycobacterium abscessus)具有兩個cysteinyl-tRNA synthetase(CysRS)同源基因-CysS1和CysS2 (分別解碼MaCysRS1及MaCysRS2)。這二個同源CysRS蛋白質具有37%的序列identity,80%的序列similarity,且與大腸桿菌的CysRS有(37-42%) identity,進一步序列及親源演化關係分析顯示,MaCysRS2其實是參與細菌體內一個保護性還原劑mycothiol (Msh)合成的一個關鍵酵素MshC,不再是一個參與蛋白質合成的酵素,MshC序列C端缺反密碼結合區域。功能性互補分析顯示,MaCysS1和MaCysS2這二個外源基因可以在酵母菌CysRS剔除株內適度表達蛋白質,可是不能互補酵母菌CysRS的細胞質功能,也就是不能提供酵母菌剔除株生長在5-FOA培養基上所需的CysRS活性;然而,若將一段粒線體標的訊號(MTS)接在MaCysRS1的胺基端,則此融合蛋白質能取代酵母菌CysRS的粒線體功能,也就是能提供酵母菌剔除株生長在YPG培養基上所需的CysRS活性,而MaCysRS2 (或MaMshC)則不能。或許是因為MaMshC只保留CysRS的胺基酸活化區,但缺乏反密碼結合區域(anticodon-binding domain)。最令人驚訝的是,若將Arc1p的tRNA鍵結區段融合到MaCysRS2 (形成MTS-MaCysRS2-Arc1p(M+C)),這個融合蛋白竟然能取代酵母菌CysRS的粒線體功能。這個結果顯示,MaMshC雖具有不同的生物功能及活性,但是能透過融合一個tRNA結合區段讓它轉變成一個具轉譯功能的CysRS

關鍵詞:CysRS,MaCysS,Arc1p
摘要(英) Aminoacyl-tRNA synthetases (aaRSs) belongs to a group of enzymes necessary for protein synthesis. Their main function is to attach an amino acid to its corresponding tRNA to form aminoacyl-tRNA, which is then brought to the ribosome for protein synthesis. One aaRSs corresponds to each amino acid. Previous studies have shown that although some bacteria lack CysRS, they can synthesize Cys-tRNACys through an indirect pathway using serine as a substrate, whereas most bacteria have a CysRS to synthesize Cys-tRNACys. Herein, we present the evidence that that Mycobacterium abscessus possesses two cysteinyl-tRNA synthetase (CysRS) homologues genes that CysS1 and CysS2 (which encode MaCysRS1 and MaCysRS2, respectively). Two homologous CysRSs in M. abscessus have a 37% identity, 80% similarity and 37-42% identity with E. coli CysRS, it is totally different with CysRS in Escherichia coli. Further sequence and phylogenetic analyses showed that MaCysRS2 is actually a mycothiol cysteine-ligase (MshC) which is involved in Mycothiol (MSH) synthesis as a protective thiol. It is not only different protein involved in protein synthesis but also lacks anticodon-binding domain. The result of complementation assay showed that both MaCysS1 and MaCysS2 were moderately expressed in the yeast but failed to complement the cytoplasmic function of the knockout strain, i.e., these two genes cannot provide the required CysRS activity to support the growth of the null allele on 5’-FOA medium. However, if a mitochondrial targeting signal (MTS) was attached to the N-terminal of the MaCysRS1, the fusion protein successfully rescued the growth defect of the knockout strain on YPG, suggesting that this fusion protein can substitute the mitochondrial activity of yeast CysRS. In contrast, MaMshC, even fused to an MTS, could not do so, probably because MaMshC lacks an anticodon-binding domain (ABD). Most surprisingly, fusion of a tRNA-binding domain of Arc1p to MTS-MaMshC, yielding an MTS-MaMshC-Arc1p (M+C), enabled the enzyme to restore the growth of the yeast knockout strain on YPG. This result shows that MaMshC, a bacterial protective thiol-producing enzyme, can be converted to a functional cysteinyl-tRNA synthetase through fusion of a non-specific tRNA-binding domain.

Keywords: CysRS, MaCysRS, Arc1p
關鍵字(中) ★ CysRS
★ MaCysRS
★ Arc1p
關鍵字(英) ★ CysRS
★ MaCysRS
★ Arc1p
論文目次 Table of Content
Abstract in Chinese i
Abstract in English ii
Acknowledgments iii
Table of Content iv
List of Table vi
List of Figure vii
Abbreviation viii

Chapter I – Introduction 1
1. 1 Cysteine ligase enzymes 1
1. 2 CysRS (Cysteinyl-tRNA synthetase) 2
1. 3 MshC (Mycothiol-synthesizing enzyme C) 2
1. 4 BshC (Bacillithiol-synthesizing enzyme C) 3
1. 5 PPCS (Phosphopantothenoyl-cysteine synthetase) 4
1. 6 GshA (Glutamate-cysteine synthetase) 5
1. 7 Aminoacyl-tRNA synthetase and aminoacylation 5
1. 8 Direct and indirect pathways for cysteinylation 7
1. 9 tRNACys 8

Chapter II – Materials and Methods 9
2. 1 Strain, culture medium, and transformation 9
2. 2 Construction of a yeast CysRS knockout strain 11
2. 3 Plasmid construction 13
2. 4 Cytoplasmic complementation assay 14
2. 5 Mitochondrial complementation assay 14
2. 6 Western blotting assay 14

Chapter III – Results 18
3. 1 S. cerevisiae posseses one dual-functional gene 18
3. 2 CysRS2 in Actinobacteria lacks a tRNA-binding domain 18
3. 3 Converting a bacterial CysRS into a functional yeast cytoplasmic CysRS 19
3. 4 Converting a bacterial CysRS into a functional yeast mitochondrial CysRS 20
3. 5 The C-terminal extension domain of yeast CysRS 20
3. 6 MshC-Arc1p fusion enzymes 21
3. 7 Deletion of ABD of MshC (CysRS2) had affected
losing complementation activity 21
3. 8 MshC(CysRS2)-Sc(Ad)GlnRS fusion enzymes 21
3. 9 Unique features of CysRS and tRNACys 22

Chapter IV – Discussion and Conclusion 23
4.1 Discussion 23
4.2 Conclusion 28

Reference 29
Appendix 33
參考文獻 1.Burbaum JJ, Schimmel P. 1991. Structural relationships and the classification of aminoacyl-tRNA synthetases. J. Biol. Chem. 266:16965-16968.
2.Carter CW Jr. 1993. Cognition, mechanism, and evolutionary relationships in aminoacyl-tRNA synthetases. Annu. Rev. Biochem. 62:715-748
3.Giege R, Sissler M, Florentz C. 1998. Universal rules and idiosyncratic features in tRNA identity. Nucleic Acids Res. 26:5017-5035
4.Giege R. 2006. The early history of tRNA recognition by aminoacyl-tRNA synthetases. J. Biosciences. 31:477-488
5.Dietrich A, Weil JH, Marechal DL. 1992. Nuclear-encoded transfer RNAs in Plant mitochondria. Annu. Rev. Cell Biol. 8:115-131
6.Sareen D, Steffek M, Newton GL, and Fahey RC. 2002. ATP-Dependent L-Cysteine: 1D-myo¬-Inosityl 2-Amino-2-deoxy-α-D-glucopyranoside Ligase, Mycothiol Biosynthesis Enzyme MshC, Is Related to Class I Cysteinyl-tRNA synthetases. Biochemistry 41:6885-6890
7.Buchmeier NA, Newton GL, Koledin T, and Fahey RC. 2003. Association of mycothiol with protection of Mycobacterium tuberculosis from toxic oxidants and antibiotics. Mol. Microbiol. 47:1723–1732
8.Newton GL, Bewley, CA, Dwyer TJ, Horn R, Aharonowitz Y, Cohen G, Davies J, Faulkner DJ, and Fahey RC. 1995. The structure of U17 isolated from Streptomyces clavuligerus and its properties as an antioxidant thiol Eur. J. Biochem. 230:821-825.
9.Newton GL, Unson M, Anderberg S, Aguilera JA, Oh NN, Cardayre S, Davies J, Av-Gay Y, and Fahey RC. 1999. Characterization of Mycobacterium smegmatis mutant defective in L-D-myo-inosityl-2-amino-2-deoxy-alpha-d-glucopyranoside and mycothiol biosynthesis. Biochem. Biophys. Res. Commun. 255:239- 244
10.Patel M, and Blanchard J. 1999. Synthesis of Des-myo-Inositol Mycothiol and Demonstration of a Mycobacterial Specific Reductase Activity J. Am. Chem. Soc. 120:11538-11539
11.Patel MP, and Blanchard JS. 2001. Mycobacterium tuberculosis Mycothione Reductase:  pH Dependence of the Kinetic Parameters and Kinetic Isotope Effects, Biochemistry 40:3119-3126.
12.Norin A, Van Ophem PW, Piersma SR, Persson B, Duine JA, and Jornvall H. 1997. Mycothiol-dependent formaldehyde dehydrogenase, a prokaryotic medium-chain dehydrogenase/reductase, phylogenetically links different eukaroytic alcohol dehydrogenases primary structure, conformational modelling and functional correlations. Eur. J. Biochem. 248:282-289
13.Musgrave WB, Yi H, Kline D, Cameron JC, Wignes J, Dey S, Pakrasi HB, Jez JM. 2013. Probing the origins of glutathione biosynthesis through biochemical analysis of glutamate-cysteine ligase and glutathione synthetase from a model photosynthetic prokaryote. Biochem J. 15:63-72
14.Meinnel T, Mechulam Y, and Blanquet S. 1995. in tRNA: Structure, Biosynthesis, and Function. ASM Press.Washington DC. pp:251-292,
15.Richards SA. Lipman, and Ya-Ming Hou. 1998. Aminoacylation of tRNA in the evolution of an aminoacyl-tRNA synthetases. Biochemistry. 95:13495–13500.
16.Karina D, Fasiolo F, Eduard C. Hurt, George Simos. 2001. Arc1p Organizes the Yeast Aminoacyl-tRNA Synthetases Complex and Stabilizes Its Interaction with the Cognate tRNAs, J. Bio. Chem. 276:6000-6008
17.Cestari I, Kalidas S, Monnerat S, Anupama A, Phillips MA, and Stuart K. 2013. A Multiple Aminoacyl-tRNA Synthetases Complex That Enhances tRNA-Aminoacylation in African Trypanosomes. 33:4872–4888
18.Gaballa A, Newton GL, Antelmann H, Parsonage D, Upton H, Rawat M, Claiborne A, Fahey RC, and Helmann, J. D. 2010. Biosynthesis and functions of bacillithiol, a major low-molecular-weight thiol in Bacilli. Proc. Natl. Acad. Sci. U.S.A. 107:6482-6486.
19.Andrew J VanDuinen, Kelsey RW, Mary EK, and Paul D Cook. 2015. X ray Crystallographic Structure of BshC, a Unique Enzyme Involved in Bacillithiol Biosynthesis. Biochemistry. 54:100–103.
20.Helmann JD. 2011. Bacillithiol, a new player in bacterial redox homeostasis. Antioxid. Redox Signaling J. 15:123-133
21.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.
22.Liu Y, Nakamura A, Nakazawa Y, Asano N, Ford KA, Hohn MJ, Tanaka I, Yao M, Söll D. 2014. Ancient translation factor is essential for tRNA-dependent cysteine biosynthesis in methanogenic archaea. Proc Natl Acad Sci. 111:10520-10525
23.Nicely NI, Parsonage D, Paige C, Newton GL, Fahey RC, Leonardi R, Jackowski S, Mallett TC, Claiborne A. 2007. Structure of the Type III Pantothenate Kinase from Bacillus anthracis at 2.0 Å Resolution: Implications for Coenzyme A-Dependent Redox Biology. J. Biochem. 46:3234-3245
24.Chen SJ, Lee CY, Lin ST, Wang CC. 2011. Rescuing a Dysfunctional Homologues of a Yeast Glycyl-tRNA Synthetase Gene. ACS Chem Biol.6:1182-1187
25.Vilchèze C, Av-Gay Y, Attarian R, Liu Z, Hazbón MH, Colangeli R, Chen B, Liu W, Alland D, Sacchettini JC, Jacobs WR Jr. 2008. Mycothiol biosynthesis is essential for ethionamide susceptibility in Mycobacterium tuberculosis. Mol Microbiol. 69:1316-1329
26.Tremblay LW, Fan F, Vetting MW, and Blanchard JS. 2008. The 1.6Å Crystal Structure of Mycobacterium smegmatis MshC: The Penultimate Enzyme in the Mycothiol Biosynthetic Pathway. Biochemistry. 47:13326-13335.
27.Fan F, Luxenburger A, Painter GF, Blanchard JS. 2007. Steady-state and pre-steady-state kinetic analysis of Mycobacterium smegmatis cysteine ligase (MshC). Biochemistry. 2007 46:11421-11429
28.Martinez DL, Tsuchiya Y, Gout I. 2014. Coenzyme A biosynthetic machinery in mammalian cells. Biochem Soc Trans. 42:1112-1117
29.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. 347:203–206
30.Jiangwei Y, James D. Patrone, and Garry DD. 2009. Characterization and Kinetics of Phosphopantothenoylcysteine Synthetase from Enterococcus faecalis. J. Biochem. 48:2799-2806
31.Burbaum JJ, Schimmel P. 1991.Structural Relationships and the classification of Aminoacyl-tRNA synthetases. J. Biol. Chem. 266:16965–16968
32.Arnez JG, Moras D. 1997. Structural and functional considerations of the aminoacylation reaction. Trends Biochem. Sci. 22:211–216
33.Ekaterina I, Biterova and Joseph J. Baryck. 2009. Mechanistic details of glutathione biosynthesis revealed by crystal structures of Saccharomyces cerevisiae glutamate cysteine ligase. Ligase. J. Biol. Chem 47:32700–32708
34.Hibi T, Nii H, Nakatsu T, Kimura A, Kato H, Hiratake J, Oda J. 2004. Crystal structure of -glutamylcysteine synthetase: Insights into the mechanism of catalysis by a key enzyme for glutathione homeostasis. Proc Natl Acad Sci. 101:15052–15057
35.Chun-Mei Zhang, Cuiping Liu, Simon Slater, Ya-Ming Hou. 2008. Aminoacylation of tRNA with phosphoserine for synthesis of cysteinyl-tRNACys. Nat. Structure Mol. Biol. 15:507-514
36.George AK and John A. 1993. Recognition of tRNACys by Escherichia coli Cysteinyl-tRNA Synthetase. J. Biochemistry. 32:7435-7444.
37.Xiaotian Ming, Kristina Smith, Hiroaki Suga, Ya-Ming Hou. 2002. Recognition of tRNA Backbone for Aminoacylation with Cysteine: Evolution from Escherichia coli to Human, 318:1207-1220
38.Kate J Newberry, Ya-Ming Hou, and John J Perona. 2002. Structural Origins of amino acid selection without editing by cysteinyl-tRNA synthetase. EMBO J. 21:2778-2787
39.Dipti S, Newton GL, Fahey RC, Buchmeier NA. 2003. Mycothiol Is Essential for Growth of Mycobacterium tuberculosis Erdman. J. Bacteriology. 185:6736-6740
40.Hausmann CD, and Ibba M. 2008. Aminacyl-tRNA synthetase complex: molecular multitasking revealed. FEMS Microbiol. Rev. 32:705-721.
41.Scott I, Hauenstein, and John, JP. 2008. Redundant Synthesis of Cysteinyl-tRNACys in Methanosarcina mazei. J Biol Chem. 283:22007–22017.
42.Cuiping Liu, Howard Gamper, Hanqing Liu, Barry S, Cooperman, and Ya-Ming Hou. 2011. Potential for interdependent development of tRNA determinants for aminoacylation and ribosome decoding. Nature Communication. 2:329-341
43.Joseph M. Jez, Rebecca E, Cahoon, and Sixue Chen. 2004. Arabidopsis Thaliana Glutamate-Cysteine Ligase Functional Properties, Kinetic Mechanism, and Regulation of Activity. J Biol Chem. 279:33463–33470
44.Griffith OW, and Mulcahy RT. 1999. The enzymes of glutathione synthesis: gamma-glutamylcysteine synthetase. Adv. Enzymol. Relat. Areas Mol. Biol. 73:209–267
45.Lu SC. 2009. Regulation of glutathione synthesis. Mol. Aspects Med. 30:42–59

指導教授 王健家(Chien Chia Wang) 審核日期 2018-1-9
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