將mycothiol半胱氨酸連接酶轉化成半胱氨酰-tRNA合成酶

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英德拉(Indra Lasmana Tarigan) 查詢紙本館藏

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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)

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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

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指導教授

王健家(Chien Chia Wang)

審核日期

2018-1-9

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