探討酵母菌glycyl-tRNA合成酵素的非傳統生物功能

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姓名

何宜晏(Yiyan Ho) 查詢紙本館藏

畢業系所

生命科學系

論文名稱

探討酵母菌glycyl-tRNA合成酵素的非傳統生物功能
(Exploring the non-canonical functions of yeast glycyl-tRNA synthetase)

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摘要(中)

在酵母菌中存在二個不同glycyl-tRNA合成酵素(glycyl-tRNA synthetase)的基因, 分別是GRS1 和GRS2。有趣的是GRS1基因同時解碼細胞質及粒腺體的Glycyl-tRNA合成酵素，而GRS2基因則沒有任何已知的功能。近來的研究發現，GRS1基因解碼的glycyl-tRNA合成酵素除了具有傳統胺醯化的功能外，還可能參與轉錄終止作用。我想利用酵母菌雙雜交系統(Yeast two hybrid system)來探究glycyl-tRNA 合成酵素與哪些酵母菌蛋白質有交互作用，希望進一步釐清這個參與蛋白質合成的酵素如何調控轉錄作用，或參與其他的代謝途徑。
我們用酵母菌雙雜交篩選出了三個可能與GRS1 基因產物有交互作用的蛋白質，分別為NFI1(SIZ2)、IFH1、YNL224C。其中NFI1(SIZ2)是一參與SUMO pathway的蛋白質，與蛋白質的後修飾以及蛋白質的活性調控有關；而IFH1是一與FHL1蛋白質有交互作用的蛋白質，可能參與rRNA的修飾作用(rRNA processing)；YNL224C是一段胜太鏈，其功能不清楚，但其蛋白質序列中具有一段保留結構區(conserve domain)，這段結構區與RNA結合蛋白相類似，推測YNL224C可能也具有RNA結合的功能。
進一步用反向酵母菌雙雜交測試(reverse two hybrid assay)，發現了NFI1(SIZ2)與GRS1基因產物的確有交互作用。這些結果顯示： Glycyl-tRNA合成酵素可能參與胺醯化以外的代謝途徑，例如SUMO pathway。

摘要(英)

Like most yeast tRNA synthetases, there are two homologous nuclear genes, GRS1 and GRS2, encoding glycyl-tRNA synthetase in the yeast Saccharomyces cerevisiae. However, a recent study argued that GRS1 provides both the cytoplasmic and mitochondrial GlyRS functions, while GRS2 appears to be non-functional. A remarkable feature regarding the biological role of GRS1 was recently identified by a genetic screening, which demonstrated that GlyRS-1 is involved in 3’-end formation of mRNAs. However, the detailed mechanism by which the translational enzyme participates in the process of mRNA termination is largely unknown. In this thesis, we used GRS1 as bait to screen a yeast genomic two-hybrid library, aiming at identifying proteins that interact specifically with GlyRS-1 in vivo. Our results showed that three proteins, NFI1 (SIZ2), IFH1, and YNL224C interact specifically with GlyRS in the two-hybrid screening. Further reverse two-hybrid tests confirmed that GlyRS interact with NFI1, a novel signal transductor that may be involved in SUMO pathway.

關鍵字(中)

★ 酵母菌雙雜交系統

關鍵字(英)

★ yeast two hybrid system

論文目次

目錄
目錄----------------------------------------------------------------------I
表目錄-----------------------------------------------------------------IV
圖目錄------------------------------------------------------------------V
縮字表-----------------------------------------------------------------VI
第一章前言-----------------------------------------------------------1
壹、 aminoacyl-tRNA合成酵素----------------------------------1
一、aminoacyl-tRNA合成酵素之功能
二、aminoacyl-tRNA合成酵素的分類
三、aminoacyl-tRNA合成酵素的其他生物功能
貳、酵母菌雙雜交系統(yeast two hybrid system)簡介------3
一、簡介
二、LexA 酵母菌雙雜交系統
參、 Glycyl-tRNA合成酵素----------------------------------------4
肆、研究目的---------------------------------------------------------6
第二章材料與方法--------------------------------------------------7
壹、實驗材料---------------------------------------------------------7
一、大腸桿菌株
二、酵母菌株
三、載體
四、核酸引子
貳、實驗方法----------------------------------------------------------9
一、核酸的製備
二、大腸桿菌勝任細胞的製備與轉殖作用
三、酵母菌Genomic DNA 的抽取與純化
四、酵母菌雙雜交系統
1. 酵母菌GlyRS基因的增幅與選殖
2. 酵母菌勝任細胞的製備與轉殖作用
3. 利用報到基因進行酵母菌雙雜交系統的篩選
4. 酵母菌質體DNA的抽取
5. 利用限制酵素將酵母菌質體DNA分類
五、酵母菌融合蛋白質的表現與西方墨點法
第三章結果----------------------------------------------------------25
壹、GRS1基因的建構---------------------------------------------25
貳、接合蛋白的表達偵測-----------------------------------------25
參、融合蛋白本身是否啟動報導基因--------------------------26
肆、相互作用的偵測-----------------------------------------------27
伍、用反向雙雜交偵測法(reverse two hybrid assay)確定交互作用--------------------------------------------------------------------28
第四章討論----------------------------------------------------------30
壹、結果的討論-----------------------------------------------------30
貳、實驗方法的討論-----------------------------------------------32
參考文獻--------------------------------------------------------------35
表-----------------------------------------------------------------------43
圖-----------------------------------------------------------------------46
附錄--------------------------------------------------------------------58
表目錄
表一、aminoacyl-tRNA合成酵素的分---------------------------43
表二、aminoacyl-tRNA合成酵素族群的差異------------------44
表三、與Glycyl-tRNA 合成酵素交互作用的蛋白質簡表---45
圖目錄
圖一、tRNA的胺醯化作用-----------------------------------------46
圖二、aminoacyl-tRNA合成酵素參與多種生物功能---------47
圖三、LexA 酵母菌雙雜交系統示意圖-------------------------48
圖四、GRS1基因轉殖質體的建構-------------------------------49
圖五、LexA-GlyRS 融合蛋白的表現----------------------------50
圖六、LexA-GlyRS 融合蛋白的報導基因測試----------------51
圖七、酵母菌雙雜交系統基因庫篩選示意圖------------------52
圖八、GlyRS與酵母菌基因庫篩選分類------------------------53
圖九、GlyRS 與候選者的報導基因測試------------------------54
圖十、SIZ2與GlyRS 交互作用的蛋白質序列----------------55
圖十一、LexA-SIZ2融合蛋白的表現----------------------------56
圖十二、GlyRS 與 SIZ2 的反向酵母菌雙雜交偵測--------57

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指導教授

王健家(Chien-Chia Wang)

審核日期

2003-1-17

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