臺灣博碩士論文加值系統

RNA聚合酶II具有合成mRNA的功能，在真核細胞中扮演重要的角色。RNA聚合酶II之轉錄延長複合體的X-ray晶體繞射結構已達3.3Å解析度，但由於mRNA具有彈性易擺動，因此轉錄出的mRNA離開RNA聚合酶II之轉錄延長複合體的正確出口途徑，至今仍不是很清楚。
在本文的研究中，利用一種非傳統螢光標定方法來標記RNA聚合酶II次單元，並設計兩種不同長度的RNA，在RNA 5’標記上配對螢光染劑，如此便可測量RNA與RNA聚合酶II次單元之間的距離，推導出轉錄後mRNA的出口路徑。從膠內螢光共振能量轉移技術的結果確定RNA聚合酶II轉錄延長複合體的存在，並成為支持單分子螢光技術實驗結果的另一項證據。
尤有進者，我們成功地建立本實驗室之單分子螢光技術，並利用其可以測量到10-10m解析度之特質，發現Rpb3與不同長度RNA測量螢光轉移共振能量，符合膠內螢光共振能量轉移的結果。此實驗結果在研究RNA的出口途徑之問題上，有十分顯著之意義。

Together with six general transcription factors and promoter DNA, yeast RNA polymerase II (RNA pol II) forms the core enzyme responsible for the synthesis of messenger RNA from genome DNA, a crucial role in eukaryote. The atomic structures of RNA polymerase II elongation complex have been resolved by using X-ray crystallography technology, but the nascent RNA exit route on the complex remains unknown.
Fluorescence resonance energy transfer (FRET) is a physical tool that can measure intramolecular or intermolecular distance to nanometer precision. In this work, we use a novel strategy that enables us to measure the distance between 5’ end of designed RNA primers and any subunits in the RNA pol II elongation complex. We have successfully determined the position of 5’ end of the synthesized 10mer and 17mer RNA primers and thus mapped the exiting route of RNA from RNA pol II. Furthermore, we successfully used the in gel fluorescence resonance energy transfer (gelFRET) assay to demonstrate the formation of RNA pol II elongation complex. We further use smFRET to measure the distance between 5’ RNA and c-terminal of Rpb3.
Our finding that 10mer RNA assumes higher FRET with the c-terminal of Rpb3 than 17mer RNA suggests the hypothesis RNA exits through a channel toward Rpb7 instead of towards Rpb8.

目 錄……………………………………………………………………………..… I
縮寫表…………………………………………………………………………….… V
中文摘要……………………………………………………………………………VII
Abstract…………………………………………………………………………… VIII

第一章 研究背景……………………………………………………….……….…..1
1.1 真核細胞轉錄……………………………………………………………...2
1.1.1 真核細胞轉錄循環 2
1.1.2 轉錄延長複合體 (Transcription elongation complex, TEC) 2
1.1.3 轉錄後mRNA離開路徑 3
1.1.4 缺乏啟動子序列之胞外轉錄活性測試 4
1.2 酵母菌TAP-tag親和純化…………………………………………....……5
1.3 攜鈣素 (Calmodulin) ………………………………………...…….….…..5
1.4 在膠內電泳觀察螢光共振能量轉移………………………….…….….…6
1.5 單分子螢光共振能量轉移技術………………………………...……..…..7
1.6 全反射顯微鏡系統……………………………………………….….….…8
1.7 論文研究方向及目的…………………………………….……………..…8
1.7.1 如何使RNA pol II特定次單元被標定上探針 9
1.7.2 利用FRET技術觀察TEC中mRNA 出口路徑 9
第二章 材料與方法…………………………………………………………….......13
2.1 實驗材料與藥品………………………………….…………………….…13
2.2 菌種………………………………………………………………..……....13
2.2.1 酵母菌株Saccharomyces cerevisia 13
2.2.2 質體 13
2.3 實驗儀器設備……………………………………………………..…...…14
2.4 酵母菌純化…………………………………………………………..…...14
2.4.1 重組RNA聚合酶 (RNA pol) 於酵母菌中表現 14
2.4.2 打破酵母菌細胞 15
2.4.3 RNA pol II 純化 15
2.4.3.1 RNA pol II-TAP / TFIIF complex 純化 15
2.4.3.1.1 TEV切除IgG 16
2.4.3.2 Pure RNA pol II complex純化 16
2.5 缺乏啟動子轉錄活性測試 ……………………………………………...17
2.5.1 製備 RNA pol II 轉錄模版 17
2.5.2 製備 T7 pol 轉錄模版 17
2.5.3 螢光胞外轉錄反應活性測定 17
2.5.3.1 酵母菌 RNA pol 胞外轉錄反應 17
2.5.3.2 T7 RNA pol 胞外轉錄反應 18
2.5.4 放射線胞外轉錄反應活性測定 18
2.5.4.1酵母菌 RNA pol 胞外轉錄反應 18
2.5.4.2 T7 RNA pol 胞外轉錄反應 19
2.6 大腸桿菌之 CaMD3C 攜鈣素表現系統…………………………..…..…19
2.6.1 大腸桿菌之 CaMD3C 攜鈣素純化 19
2.6.2 利用Lucifer yellow vinyl sulfone (LY-VS) 標定CaMD3C 21
2.6.3 CaMD3C標記螢光染劑 22
2.7 轉錄延長複合體………...……………………………………................….25
2.7.1 DNA與RNA核酸序列設計 25
2.7.2 形成RNA轉錄複合體 26
2.7.2.1 形成TEC設計1 26
2.7.2.2 形成TEC設計2 27
2.7.3 TEC活性測試 27
2.8 膠體內水解 (In gel digestion )………………………….………...…....…28
2.9 溶液內螢光共振能量轉移測定…………………………………..…........29
2.10 膠體內螢光轉移共振能量測定………………………………………....30
2.10.1 膠體內螢光共振能量轉移流程 30
2.10.2 如何計算螢光共振能量轉移 30
2.11 單分子螢光顯微鏡實驗………………………………………................31
2.11.1 清洗石英 (Quatz) 與玻璃 (Glass) 蓋玻片 31
2.11.2 製備單分子螢光顯微鏡樣品 32
2.11.3 單分子螢光共振轉移取樣步驟 33
2.11.4 單分子螢光共振結果分析 33
第三章　結果與討論……………………………………………………………..…35
3.1 攜鈣素突變株 (CaMD3C) 的表現與純化…………………................….35　　
3.2 攜鈣素突變株 (CaMD3C) 的硫醇基標定測試……..………………...…35
　　3.3 攜鈣素突變株 (CaMD3C) 標定螢光染劑…………………………….…35
3.4 酵母菌RNA pol II表現純化......................................................................36
3.4.1 重組蛋白質RNA pol II (Rpb3-TAP) 表現與純化 36
3.4.2 重組蛋白質RNA pol II (Rpb4-TAP) 表現與純化 37
　　3.5 RNA聚合酶II胞外轉錄活性測試系統建立……………………….…....38
3.5.1 螢光胞外轉錄系統建立 38
3.5.2 放射線胞外轉錄系統建立 39
3.5.3 不同RNA polⅡ活性測試 39
3.5.4 二價離子對於RNA polⅡ轉錄活性的影響 40
　　3.6 形成轉錄延長複合體 (TEC) ………………………….….………............40
3.7 TEC活性測試………………………………………………………….... 42
3.8 溶液內螢光共振能量轉移測定…………………………..…………..….42
3.9 原態電泳膠片螢光共振能轉移效率…………………………………....43
3.9.1 利用膠內測定標準樣品螢光能量轉移效率 43
3.9.2 利用膠內測定TEC螢光能量轉移效率 44
3.9.2.1 TEC 1 結果 44
3.9.2.2 TEC 2結果 44
　　3.10 單分子螢光共振轉移效率測定…………………………………….........45
第四章 總結與未來展望………………………………………………….….....…...48
4.1 單分子螢光觀測轉錄延長複合體 (TEC) 48
4.2 轉錄後mRNA出口路徑 48
參考文獻…………………………………………………………………….…....…..49
結果圖與表………………………………………………………………….………..54
附件………………………………………………………………………...………….81

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