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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 楊啟伸(Chii-Shen Yang) | |
| dc.contributor.author | Ji-Chau Chang | en |
| dc.contributor.author | 張智超 | zh_TW |
| dc.date.accessioned | 2021-06-08T05:58:07Z | - |
| dc.date.copyright | 2007-09-03 | |
| dc.date.issued | 2007 | |
| dc.date.submitted | 2007-08-29 | |
| dc.identifier.citation | 參考文獻
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/24903 | - |
| dc.description.abstract | 控制基因表現對於細胞存活是很重要的步驟，原核生物與真核生物都具有 RNA polymerase 以調控轉錄作用。轉錄分為起始、延長，及終止三個不同步驟，早期研究則發現轉錄延長階段為 mRNA 合成的關鍵步驟。
真核細胞之細胞核內至少有三種不同 RNA polymerase，其中RNA Pol I 主要負責合成 rRNA，RNA Pol II 負責合成 mRNA，而RNA Pol III 負責合成 tRNA。本論文將研究重點指向酵母菌 RNA Pol II 與 Spt4-Spt5 複合體。早期對 Spt4-Spt5 複合體的研究認為，它在酵母菌轉錄初始階段扮演轉錄因子角色，並調控核染色質結構。然而，近來此複合體已漸漸被證實為細胞內轉錄延長調控因子。 本篇論文中，利用電子顯微鏡的技術輔以單粒子影像分析與三維重建，決定 RNA Pol II 與 Spt4-Spt5 複合體之結構。本論文採取三種策略來增加重建結果的可信度與精確度。首先，以 reference-free 的方式重建 RNA Pol II 與 Spt4-Spt5 複合體結構，克服使用初始模型進行重建所造成的結構失真。接著進行胞外轉錄實驗證實我們所重建的結構具有生理活性，之後將使用奈米金標定的技術與 random conical tilt 的方式，增加定位 Spt4-Spt5 複合體的準確度。 | zh_TW |
| dc.description.abstract | The control of gene expression is essential for cell survival and it is commonly achieved by regulating transcription with different forms of RNA polymerase in both prokaryotes and eukaryotes. Transcription can be mainly divided into initiation, elongation and termination phases and previous studies determined elongation phase as rate-limiting step for proper mRNA production.
At least three different RNA polymerase has been identified in eukaryotic cells, including RNA polymerase I for ribosome, RNA polymerase II (RNA Pol II) for mRNA and RNA polymerase III for transfer-RNA. In this study, we focus on RNA Pol II coupled with Spt4-Spt5 complex in Saccharomyces cerevisiae. Initial studies of Spt4-Spt5 complex in Saccharomyces cerevisiae suggested that they act as transcription factors that modify chromatin structure in transcription initiation. More recently, however, this complex has been generally believed to work as an elongation regulator in vivo. Here, we present the structure of the yeast RNA Pol II with Spt4-Spt5 complex determined by electron microscopy followed by single-particle analysis and three-dimensional reconstruction. Three methods have been adapted to increase the reliability and accuracy of our final structural model: First, we use reference-free methods to reconstruct the structure of RNA Pol II and Spt4-Spt5 complex which overcomes the distortion caused by reference bias using initial model-based method, and secondly, the complex have been assayed by in vitro transcription to show their biological activity. In the future, we will use gold particle labeling and random conical tilt method to increase the accuracy in identifing the position of Spt4-Spt5 in the complex. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T05:58:07Z (GMT). No. of bitstreams: 1 ntu-96-R94B47208-1.pdf: 10166264 bytes, checksum: 02156874933a284ae219c25a3f81e19e (MD5) Previous issue date: 2007 | en |
| dc.description.tableofcontents | 目 錄
目錄 I 縮寫表 IV 中文摘要 VI Abstract VII 第一章 緒論 1 1.1 真核生物轉錄初始機制 1 1.2 真核生物轉錄延長階段與相關因子討論 8 1.3 Spt4-Spt5 complex 生理背景與論文研究目標 9 1.4 冷凍電子顯微鏡 11 1.5 奈米金標定技術 13 1.6 Tandem Affinity Purification (TAP) method 15 第二章 材料與方法 18 2.1 固態培養與菌種保存 18 2.1.1 液態培養 18 2.2 實驗藥品 18 2.3 實驗儀器 19 2.4 實驗方法 22 2.4.1 酵母菌培養與 RNA Pol II/Spt4-Spt5 複合體純化 22 2.4.1.1 酵母菌培養 22 2.4.1.2 玻璃珠研磨法破菌 23 2.4.1.3 RNA Pol II/Spt4-Spt5 IgG 親和性管柱一次純化 25 2.4.1.4 Bradford protein–binding assay 定量 26 2.4.1.5 SDS 膠體電泳 26 2.4.1.6 蛋白質染色 26 2.4.1.7 膠體內水解 (In-Gel Digestion) 26 2.4.1.8 RNA Pol II/Spt4-Spt5 (Spt5-CBP) 攜鈣素親和性 管柱二次純化 28 2.4.2 酵母菌 Spt4-Spt5 複合體純化 29 2.4.2.1 酵母菌 RNA Pol II/Spt4-Spt5 (Spt4-TAP) IgG 親和性管柱一次純化 29 2.4.2.2 Spt4-Spt5 複合體攜鈣素親和性管柱二次純化 30 2.4.3 電子顯微鏡樣品製備 31 2.4.3.1 製備覆蓋碳膜之銅網格 31 2.4.3.2 電子顯微鏡負染樣品製備 32 2.4.3.3 電子顯微鏡冷凍樣品製備 33 2.4.3.4 冷凍負染樣品製備 34 2.4.4 穿透式電子顯微鏡造影 36 2.4.5 三維重建 (Three-Dimensional Reconstruction) 36 2.4.5.1 Reference-Free Methods 36 2.4.5.2 Refinement 37 第三章 結果與討論 39 3.1 酵母菌 RNA Pol II/Spt4-Spt5 複合體純化結果與討論 39 3.1.1 酵母菌 RNA Pol II/Spt4-Spt5 (Spt4-TAP) 39 3.1.1.1 RNA Pol II/Spt4-TAP tag 純化結果 39 3.1.1.2 RNA Pol II/Spt4-TAP tag 純化討論 42 3.1.2.1 RNA Pol II/Spt5-TAP tag 純化結果 45 3.1.2.2 RNA Pol II/Spt5-TAP tag 純化討論 48 3.1.3.1 RNA Pol II/Spt4-Spt5 from Cornell University 51 3.1.3.2 RNA Pol II/Spt4-Spt5 討論 52 3.2 穿透式電子顯微鏡影像分析與三維重建 58 3.2.1 RNA Pol II/Spt4-Spt5 (Spt4-TAP) 造影結果與三維重 建 58 3.2.1.1 RNA Pol II/Spt4-Spt5 (Spt4-TAP) 重建結果討 論 58 3.2.2 RNA Pol II/Spt4-TAP Reference-Free 三維重建 58 3.2.2.1 RNA Pol II/Spt4-TAP Reference-Free 重建結構 討論 59 3.2.3 RNA Pol II/Spt5-TAP造影結果與三維重建 59 3.2.3.1 RNA Pol II/Spt5-TAP造影結果討論 59 3.2.3.2 RNA Pol II/Spt5-TAP Reference-Free重建結構 討論 60 3.2.4 RNA Pol II/Spt5-TAP Reference-Based 重建結果 61 3.2.4.1 RNA Pol II/Spt5-TAP Reference-Based 重建討 論 62 3.2.5 RNA Pol II/Spt4-Spt5 (Spt4-TAP) 與攜鈣素奈米金標 定 63 3.2.5.1 電子顯微鏡負染色造影結果 63 3.2.5.2 奈米金負染色造影結果討論 63 3.2.6 低溫電子顯微鏡造影討論 63 第四章 結論與未來展望 64 結果圖 65 參考文獻 85 附錄 91 | |
| dc.language.iso | zh-TW | |
| dc.title | 以電子顯微鏡解析酵母菌 RNA 聚合酶 II 與 Spt4-Spt5 複合體之三維結構 | zh_TW |
| dc.title | Three Dimensional Electron Microscopy Reconstruction of Yeast RNA Polymerase II with Spt4-Spt5 complex | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 95-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.coadvisor | 章為皓(Wei-Hau Chang) | |
| dc.contributor.oralexamcommittee | 李平篤(Ping-Du Lee),王愛玉(Ai-Yu Wang) | |
| dc.subject.keyword | 電子顯微鏡,酵母菌,聚合酶,三維重建, | zh_TW |
| dc.subject.keyword | Electron Microscopy,Yeast,Polymerase,Three-Dimensional Reconstruction, | en |
| dc.relation.page | 92 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2007-08-29 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 微生物與生化學研究所 | zh_TW |
| 顯示於系所單位： | 微生物學科所 | |
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