博碩士論文 101224018 詳細資訊




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姓名 陳玥潾(Yueh-lin Chen)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 探討酵母菌粒線體之glutamyl-tRNA synthetase的演化及功能
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摘要(中) 釀酒酵母菌具有兩種GluRS,分別是由不同基因所編碼出來,一種是作用在細胞質的GluRS(GluRSc),由GUS1編碼;另一種則是作用在粒線體的GluRS(GluRSm),由MSE1編碼。GluRSc除了在細胞質胺醯化tRNAnGlu以外,也會跑到粒線體胺醯化tRNAmGln;而GluRSm只會在粒線體胺醯化tRNAmGlu。能辨識非同源tRNAGln的GluRS稱為non-discriminating GluRS(ND-GluRS),只能辨識同源tRNAGlu的GluRS則稱為discriminating GluRS(D-GluRS)。我們在研究中發現,只有細菌的ND-GluRS能取代酵母菌GluRSm,其他真核生物和細菌的D-GluRS則都無法取代酵母菌GluRSm。我們進一步將這些物種的GluRS序列進行比對,發現酵母菌GluRSm上辨識tRNA的重要胺基酸與細菌的ND-GluRS相似。另一方面,我們比對了細胞質的tRNAnGlu和粒線體的tRNAmGlu及tRNAmGln的二級結構序列,發現在accepter stem的第一個核苷酸配對和anticodon loop的第33個和第36個核苷酸有差異性,這可能是這些tRNA被GluRS辨識的位置。因此我們將tRNAmGlu和tRNAmGln這幾個位置上的核苷酸進行互換,進一步做胺醯化活性測試,但發現in vitro transcription的tRNA無法被胺醯化。親源演化的分析結果顯示,真核生物的GluRSm與細菌的D-GluRS較接近,且與古生菌的GluRS較遠,由此可知真核生物粒線體的GluRS可能源自細菌。
論文第二部份,著重於酵母菌Vanderwaltozyma polyspora中glycyl-tRNA synthetase (GlyRS)的研究。在所有已知的酵母菌種中,Saccharomyces cerevisiae 和 Vanderwaltozyma polyspora被發現具有兩個編碼GlyRS的基因。GRS1編碼細胞質和粒線體活性,而GRS2在正常生長情形下是可有可無的。我們在這顯示VpGlyRS2(由V. polyspora GRS2編碼)能有效拯救S. cerevisiae GRS1剔除珠的細胞質功能。雖然VpGlyRS2在正常情況下不表現,但卻能在高溫、高pH值和酒精的環境中被誘導出。此外,在高溫下,VpGlyRS2在細胞內比VpGlyRS1更穩定,且在體外試驗中,VpGlyRS2具有活性。這些結果暗示V. polyspora的GRS2有利於酵母菌在壓力環境下維持生存。
摘要(英) The yeast Saccharomyces cerevisiae possesses two GluRS enzymes that are encoded by two different nuclear genes. Cytoplasmic GluRS (GluRSc) is encoded by GUS1, while mitochondrial GluRS (GluRSm) is encoded by MSE1. GluRSc attaches glutamate to cytoplasmic tRNAnGlu and mitochondrial tRNAmGln. In contrast, GluRSm glutamylates only mitochondrial tRNAmGlu. As GluRSc can attach glutamate to both tRNAnGlu and tRNAmGln, it is called non-discriminating GluRS (ND-GluRS). On the other hand, GluRSm only recognizes tRNAmGlu, it is called discriminating GluRS (D-GluRS).In our study, we found that ND-GluRS of bacteria can rescue a yeast GluRSm knockout strain, but D-GluRS of eukaryotes and other bacteria cannot. Alignment of these GluRSs sequences showed that the amino acids responsible for tRNAGlu recognition are well conserved in yeast GluRSm and the aforementioned ND-GluRSs of bacteria. Comparison of the secondary structure of cytoplasmic tRNAnGlu, mitochondrial tRNAmGlu, and mitochondrial tRNAmGln showed that the first base pair in the accepter stem and nucleosides 33 and 36 in the anticodon loop are highly diverged and may serve as their identity elements. Unfortunately, in vitro transcribed tRNAmGlu and tRNAmGln were inactive for aminoacylation, and further mapping of identity elements in tRNA were thus impossible. Phylogenetic analysis showed that the GluRSm sequences of eukaryotes are clustered in a monophyletic branch that exhibits a higher affinity with GluRS of bacteria than with GluRS of archaea, suggesting that mitochondrial GluRSs of eukaryotes are descended from GluRSs of bacteria.
A second part of the thesis is focused on yeast glycyl-tRNA synthetase (GlyRS). In all yeast species, Saccharomyces cerevisiae and Vanderwaltozyma polyspora are the only two yeasts known to contain two GlyRS genes. GRS1 encodes both cytoplasmic and mitochondrial activities, whereas GRS2 is silent and dispensable under normal growth conditions. We show here that VpGlyRS2 (encoded by V. polyspora GRS2) effectively rescued the cytoplasmic defect of a S. cerevisiae GRS1 knockout strain when expressed from a constitutive promoter. Although VpGRS2 was practically silent under normal conditions, but its expression could be induced by high temperature, high pH, and ethanol. In addition, VpGlyRS2 was exclusively localized in the cytoplasm, more stable under heat-shock conditions in vivo, and almost as active as VpGlyRS1 in glycylation of tRNA in vitro. Our study reinforces the hypothesis that GRS2 of V. polyspora is an inducible gene that acts in response to stress to maintain the homeostasis of yeast cells.
關鍵字(中) ★ 胺醯化 關鍵字(英) ★ non-discriminating
★ discriminating
★ glycyl-tRNA synthetase
★ glutamyl-tRNA synthetase
論文目次 中文摘要----------------------------------------------------------------------------------------------------I
英文摘要---------------------------------------------------------------------------------------------------II
致謝--------------------------------------------------------------------------------------------------------III
目錄--------------------------------------------------------------------------------------------------------IV
表目錄-----------------------------------------------------------------------------------------------------IX
圖目錄------------------------------------------------------------------------------------------------------X
附錄-------------------------------------------------------------------------------------------------------XII
物種縮寫檢索表---------------------------------------------------------------------------------------XIII
aaRSs縮寫檢所表--------------------------------------------------------------------------------------XV
第一章 緒論
1.1 Aminoacyl-tRNA synthetases(aaRSs)介紹--------------------------------------------1
1.1.1 aaRSs之生化功能----------------------------------------------------------------------1
1.1.2 aaRSs的分類----------------------------------------------------------------------------1
1.1.3 aaRSs對tRNA 的辨識----------------------------------------------------------------2
1.1.4 原核生物與真核生物之aaRSs的差異----------------------------------------------2
1.1.5 aaRSs的演化----------------------------------------------------------------------------3
1.1.6 aaRSs生理重要性及其他非典型功能----------------------------------------------3
1.2 Glutamyl-tRNA synthetase(GluRS)介紹----------------------------------------------4
1.2.1 GluRS之生理特性----------------------------------------------------------------------4
1.2.2 D-GluRS和ND-GluRS的定義-------------------------------------------------------5
1.2.3 GluRS對tRNAGlu和tRNAGln的辨識------------------------------------------------5
1.2.4 GluRS的演化---------------------------------------------------------------------------6
1.3 Glycyl-tRNA synthetase(GlyRS)介紹-------------------------------------------------6
1.3.1 GlyRS之生理特性-----------------------------------------------------------------------6
1.3.2 Saccharomyces cerevisiae之GRS1和GRS2----------------------------------------6
1.3.3 non-AUG轉譯起始密碼的aaRSs----------------------------------------------------7
1.4 研究目的---------------------------------------------------------------------------------------8
第二章 材料與方法
2.1 質體重組---------------------------------------------------------------------------------------9
2.1.1 GluRS質體建構------------------------------------------------------------------------9
2.1.2 tRNA質體建構-------------------------------------------------------------------------9
2.2 大腸桿菌勝任細胞的製備與轉型---------------------------------------------------------9
2.2.1 大腸桿菌勝任細胞的製備-------------------------------------------------11
2.2.2 大腸桿菌勝任細胞的轉型作用(transformation)-------------------------------11
2.3 酵母菌勝任細胞的製備與轉型-----------------------------------------------------------11
2.3.1 酵母菌勝任細胞的製備---------------------------------------------------------12
2.3.2 酵母菌勝任細胞的轉型作用-------------------------------------------------------12
2.4 功能性互補試驗(complementation)--------------------------------------------------13
2.4.1 粒線體功能測試----------------------------------------------------------------------14
2.5 蛋白質表現測定-----------------------------------------------------------------------------14
2.5.1 蛋白質製備----------------------------------------------------------------------------15
2.5.2 Sodium Dodecyl Sulfate-Polyacrylamide Gel(SDS-PAGE)----------------15
2.5.3 Coomassie Brilliant Blue------------------------------------------------------------16
2.5.4 西方墨點法----------------------------------------------------------------------------16
2.6 酵母菌融合蛋白表現與純化--------------------------------------------------------------18
2.6.1 細胞破碎儀破菌-----------------------------------------------------------------------19
2.6.2 Ni-NTA純化---------------------------------------------------------------------------19
2.7 Fluorescence location assay----------------------------------------------------------------20
2.8 點突變(Site-directed mutagenesis)----------------------------------------------------20
2.9 純化mitochondrial tRNA ------------------------------------------------------------------21
2.9.1 細胞破碎儀破菌-----------------------------------------------------------------------22
2.9.2 純化toatl RNA-----------------------------------------------------------------------22
2.9.3 萃取total tRNA-----------------------------------------------------------------------22
2.9.4 純化特定tRNA-----------------------------------------------------------------------23
2.10 製備in vitro transcription tRNA ---------------------------------------------------------23
2.11 Aminoacylation assay-----------------------------------------------------------------------24
2.12 Cycloheximide-chase assay ----------------------------------------------------------------25
2.13 Circular dichroism(CD)spectroscopy ------------------------------------------------26
2.14 Fast protein liquid chromatography (FPLC)-----------------------------------------26
第三章 結果
3.1 酵母菌細胞質和粒線體之GluRS差異--------------------------------------------------27
3.2 不同真核生物之GluRSm對酵母菌粒線體互補性測試-------------------------------27
3.3 不同原核生物之GluRS對酵母菌粒線體互補性測試--------------------------------28
3.4 Synechocystis sp.之GluRS在酵母菌內的表現位置------------------------------------28
3.5 ScGluRSc和ScGluRSm之活性測試-------------------------------------------------------29
3.6 古生菌與原核生物GluRS和真核生物GluRSm序列比對-----------------------------30
3.7 酵母菌GRS1和GRS2之細胞質和粒線體的互補性測試--------------------------30
3.8 酵母菌V. polyspora之GlyRS的蛋白質穩定性--------------------------------------31
3.9 酵母菌V. polyspora之GRS1和GRS2之mRNAs的表現-------------------------32
3.10 酵母菌V. polyspora之GlyRS2在細胞內的表現情形------------------------------33
3.11 酵母菌V. polyspora之GlyRS對不同溫度的活性測試----------------------------33
3.12 不同溫度下VpGlyRS的二級結構的穩定性------------------------------------------33
3.13 VpGlyRS1和VpGlyRS2的蛋白質分子型---------------------------------------------34
第四章 討論
4.1 不同物種對酵母菌GluRSm的功能性取代--------------------------------------------35
4.2 預測D-GluRS和ND-GluRS辨識tRNA的部位----------------------------------------35
4.3 真核生物粒線體tRNA的後修飾--------------------------------------------------------36
4.4 真核生物GluRSm辨識tRNA的胺基酸-------------------------------------------------36
4.5 D-GluRS和ND-GluRS之演化假說-------------------------------------------------------37
4.6 酵母菌V. polyspora之GlyRS的功能性取代-----------------------------------------38
4.7 酵母菌GRS2為誘導型基因--------------------------------------------------------------39
4.8 在細胞內酵母菌GlyRS2對高溫具有穩定性------------------------------------------39
4.9 In vitro的酵母菌GlyRS2對高溫度穩定性差------------------------------------------39
4.10 酵母菌VpGlyRS的蛋白質分子型態-----------------------------------------------------40
4.11 酵母菌GlyRS2之生理功能假說---------------------------------------------------------40
參考文獻--------------------------------------------------------------------------------------------------41
圖表--------------------------------------------------------------------------------------------------------46
附錄--------------------------------------------------------------------------------------------------------66
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指導教授 王健家(Chien-chia Wang) 審核日期 2015-3-2
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