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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 楊健志(Chien-Chih Yang) | |
| dc.contributor.author | Chien-Hung Chung | en |
| dc.contributor.author | 鍾建弘 | zh_TW |
| dc.date.accessioned | 2021-06-17T08:11:24Z | - |
| dc.date.available | 2024-08-20 | |
| dc.date.copyright | 2019-08-20 | |
| dc.date.issued | 2019 | |
| dc.date.submitted | 2019-08-15 | |
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Hirtreiter, A., et al., Spt4/5 stimulates transcription elongation through the RNA polymerase clamp coiled-coil motif. Nucleic Acids Research, 2010. 38(12): p. 4040-4051. 42. Li, W., C. Giles, and S. Li, Insights into how Spt5 functions in transcription elongation and repressing transcription coupled DNA repair. Nucleic Acids Research, 2014. 42(11): p. 7069-7083. 43. Kim, D.K., et al., Structure-function analysis of human Spt4: evidence that hSpt4 and hSpt5 exert their roles in transcriptional elongation as parts of the DSIF complex. Genes Cells, 2003. 8(4): p. 371-8. 44. Mosley, A.L., et al., Quantitative proteomics demonstrates that the RNA polymerase II subunits Rpb4 and Rpb7 dissociate during transcriptional elongation. Mol Cell Proteomics, 2013. 12(6): p. 1530-8. 45. Allepuz-Fuster, P., et al., Rpb4/7 facilitates RNA polymerase II CTD dephosphorylation. Nucleic Acids Res, 2014. 42(22): p. 13674-88. 46. Bernecky, C., J.M. Plitzko, and P. Cramer, Structure of a transcribing RNA polymerase II–DSIF complex reveals a multidentate DNA–RNA clamp. Nature Structural &Amp; Molecular Biology, 2017. 24: p. 809. 47. Liu, Y., et al., Phosphorylation of the transcription elongation factor Spt5 by yeast Bur1 kinase stimulates recruitment of the PAF complex. Mol Cell Biol, 2009. 29(17): p. 4852-63. 48. Kecman, T., et al., Elongation/Termination Factor Exchange Mediated by PP1 Phosphatase Orchestrates Transcription Termination. Cell Reports, 2018. 25(1): p. 259-269.e5. 49. Mitsuzawa, H., E. Kanda, and A. Ishihama, Rpb7 subunit of RNA polymerase II interacts with an RNA-binding protein involved in processing of transcripts. Nucleic acids research, 2003. 31(16): p. 4696-4701. 50. Runner, V.M., V. Podolny, and S. Buratowski, The Rpb4 subunit of RNA polymerase II contributes to cotranscriptional recruitment of 3' processing factors. Mol Cell Biol, 2008. 28(6): p. 1883-91. 51. Wittmann, S., et al., The conserved protein Seb1 drives transcription termination by binding RNA polymerase II and nascent RNA. Nature Communications, 2017. 8: p. 14861. 52. Vilborg, A., et al., Widespread Inducible Transcription Downstream of Human Genes. Molecular Cell, 2015. 59(3): p. 449-461. 53. Elbashir, S.M., et al., Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature, 2001. 411(6836): p. 494-8. 54. Ohtsuka, M., et al., MicroRNA Processing and Human Cancer. J Clin Med, 2015. 4(8): p. 1651-67. 55. Goodrich, J.A. and J.F. Kugel, Non-coding-RNA regulators of RNA polymerase II transcription. Nature Reviews Molecular Cell Biology, 2006. 7(8): p. 612-616. 56. Guttman, M., et al., Chromatin signature reveals over a thousand highly conserved large non-coding RNAs in mammals. Nature, 2009. 458(7235): p. 223-227. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/73833 | - |
| dc.description.abstract | 在真核生物中,核糖核酸聚合酶二型是由12個次單元體構築的巨型蛋白質複合體,負責轉錄信使RNA以及小核RNA、小分子RNA,而轉錄因子Spt5對於核糖核酸聚合酶二型轉錄的初始、延長、以及終止階段都扮演了角色。
本篇論文,以出芽酵母過量表達可被半乳糖誘導表現的Spt5,以體外的轉錄實驗驗證了蛋白質的功能性,並利用冷凍電顯技術對於Spt5與核糖核酸聚合酶二型的複合體進行三維結構的組建。在結構影像中我們有意外的發現,本篇首次揭露Spt5與核糖核酸聚合酶二型的結合,會造成其柄狀構型的Rpb4/7自核糖核酸聚合酶二型分離,並且這個現象也在生理狀況被觀測到。 總結來說,本篇成功過量表現有功能性的蛋白質Spt5,其與核糖核酸聚合酶二型的結合造成Rpb4/7的分離,實驗結果可能揭露了一個新的Spt5對於轉錄調控的機制。 | zh_TW |
| dc.description.abstract | In the eukaryotes, RNA polymerase Ⅱ is a large protein complex consist of 12 subunits·It catalyzes the transcription of DNA to pre-mRNA、Small nuclear RNA and microRNA. Transcription Factor Spt5 play roles in RNA polymerase Ⅱ mediated transcription, from initiation to termination.
Here, we use Saccharomyces cerevisiae system to overexpress galactose inducible Spt5, and characterized its function and interaction with RNA polymerase Ⅱ in a in vitro transcription system. We use Cryo-EM and 3D-reconstruction to demonstrate structure of Spt5/ RNA polymerase Ⅱ complex. Surprisingly, our structure image rollout Spt5 binding with RNA polymerase Ⅱ will lead stalk-like Rpb4/7 heterodimer dissociate from RNA polymerase Ⅱ, and this event will occur during in vivo transcription. In summary, we successfully overexpress functional protein Spt5.Spt5 bind to RNA polymerase Ⅱ will lead Rpb4/7 dissociate from RNA polymerase Ⅱ, this result may reveal a new mechanism of Spt5 to regulate transcription process 。 | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T08:11:24Z (GMT). No. of bitstreams: 1 ntu-108-R04b22057-1.pdf: 10211587 bytes, checksum: a83999f71f5db0f077dad8769c7846bf (MD5) Previous issue date: 2019 | en |
| dc.description.tableofcontents | 目錄
口試委員會審定書 Ⅰ 中文摘要 Ⅱ Abstract Ⅲ 目錄 Ⅳ 圖目錄 Ⅶ 表目錄 Ⅷ 縮寫表 IV 第一章 緒論 1 1.1酵母菌的核糖核酸聚合酶 II (RNA polymerase II,RNAP II) 與次單元體 RPB4,7 1 1.2 轉錄因子Spt5 6 1.3 串聯式親和性純化 9 1.4 冷凍電子顯微鏡 11 1.5 研究目的以及實驗設計 12 第二章 材料與方法 17 2.1 實驗材料 17 2.1.1菌種 17 2.1.2質體 17 2.1.3實驗試劑 17 2.1.4培養基 21 2.1.5 緩衝液、溶液 22 2.2實驗器材 23 2.3 實驗方法 24 2.3.1聚合酶連鎖反應(Polymerase chain reaction/PCR) 24 2.3.2無縫克隆 26 2.4.3限制酶切割反應 27 2.3.4 DNA連接反應 27 2.3.5培養基之製備 28 2.3.6大腸桿菌的轉形作用 28 2.3.7大腸桿菌Colony PCR 29 2.3.8DNA 瓊脂醣凝膠電泳（DNA agarose gel electrophoresis） 30 2.3.9酵母菌勝任細胞製備 31 2.3.10酵母菌轉形作用 32 2.3.11酵母菌的質體萃取 33 2.3.12 酵母菌 Colony PCR 34 2.3.13 RNAP II的純化 35 2.3.14 Spt5純化 37 2.3.14.1 Spt5小量純化及分析 37 2.3.14.2 Spt5大量純化 38 2.3.15 Bradford protein assay 40 2.3.16 SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) 40 2.3.17西方墨點法(Western Blot) 41 2.3.18膠體內水解(In gel digestion) 43 2.3.19 Spt5功能性試驗 44 2.3.19.1抗轉錄中止試驗 44 2.3.19.2轉錄效能試驗 45 2.3.20 Spt5與RNAP II之結合 46 2.3.21 Ruby Protein Gel Stain 47 第三章 結果 48 3.1 表現質體的建構 48 3.1.1質體GAL1/10 KanR SPT5-CBP-TEV-ProteinA 48 3.1.2質體GAL1/10 KanR SPT5 632-cpEGFP-CBP-TEV-ProteinA 48 3.1.3質體GAL1-SPT5 632-cpEGFP- CBP-TEV-ProteinA GAL-10 -SPT4 48 3.1.4質體GAL1-SPT5-CBP-TEV-ProteinA GAL10-SPT4-SBP 49 3.1.5質體GAL1 SPT5 TEV-10H-SBP-HRV3C-ProteinA GAL10 SPT4-SBP 49 3.1.6質體GAL1-SPT5 632-cpEGFP- TEV-10H -SBP-HRV3C-ProteinAGAL-10-Spt4 -SBP 49 3.2酵母菌 RNAPⅡ之純化 50 3.3Spt5、Spt5-cpEGFP純化,蛋白質的分析及鑑定 52 3.3.1.1 Spt5/Spt5-632-cpEGFP小量表現 52 3.3.1.2 Spt5/Spt5-632-cpEGFP SDS PAGE 52 3.3.1.3 Spt5/Spt5-632-cpEGFP Western blot 52 3.3.1.4 Spt5-632-cpEGFP之酵母菌的細胞螢光影像 53 3.3.1.5 Spt4/5;Spt4/5-632-cpEGFP液相層析電灑法串聯式質譜(LC/MS/MS)分析結果 53 3.3.2蛋白質隨誘導時間的表現量變化 53 3.4 Spt5-632-cpEGFP的功能性測試 63 3.4.1 Spt5-632-cpEGFP 抗轉錄中止試驗 63 3.4.2 Spt5-632-cpEGFP 轉錄效能試驗 64 3.5 Spt5/RNAPⅡ、Spt5-632-cpEGFP/RNAPⅡ 複合體 67 3.6 Spt5/RNAPⅡ、Spt5-632-cpEGFP/RNAPⅡ 複合體 之負染影像 68 3.7 Spt5/RNAPⅡ、Spt5-632-cpEGFP/RNAPⅡ 複合體 之電顯影像暨2D、3D結構 70 3.8 Spt5/RNAPⅡ 複合體 SDS PAGE 79 第四章 討論 81 4.1蛋白質的表現與鑒定 81 4-2 Spt5、Spt5-632-cpEGFP與RNAPⅡ的結合造成Rpb4/7的脫離 82 4-3未來的研究方向 87 參考文獻 94 附錄 99 圖目錄 圖一、三域間核醣核酸聚合酶之結構差異 3 圖二、RNAPⅡ之結構與各次單元體之分佈 3 圖三、Rpb1與Rpb1 CTD之重複序列 4 圖四、Rpb1 CTD磷酸化與轉錄進程的關係示意圖 4 圖五、不同CTD位點的磷酸化對於蛋白質作用力的影響 5 圖六、Rpb4/7異源複合體及其柄狀構型 5 圖七、酵母菌Spt5種間(species)之蛋白質序列分析 7 圖八、Spt5於三域之間的差異 8 圖九、串聯式親和性純化之示意以及設計之變動 10 圖十、GAL1/10雙向表達Spt4/5及菌株BCY123的誘導強化機制 14 圖十一、RNAPII/DSIF複合體相關聯的的結構影像回顧 14 圖十二、嗜甲醇酵母RNAP-DSIF-TFⅡF結構圖 15 圖十三、人類DSIF、TFⅡF與野豬RNAPⅡ結構圖 15 圖十四、出芽酵母Spt4-Spt5 NGN 融合蛋白質之晶體結構 16 圖十五、Spt5純化流程之示意圖 39 圖十六.形成Spt5、 RNAPⅡ複合體之流程示意圖 47 圖十七、RNAPⅡ(Rpb4-10H) 純化暨分析 SDS-PAGE 51 圖十八、Spt5、Spt5-632-cpEGFP 純化暨分析 55 圖十九、酵母菌誘導表現Spt5-632-cpEGFP融合蛋白質之細胞螢光影像 56 圖二十之一、進行膠體內水解(In gel digest)之膠片 57 圖二十之二、液相層析電灑法串聯式質譜(LC/MS/MS)分析結果a 58 圖二十之三、液相層析電灑法串聯式質譜(LC/MS/MS)分析結果b 59 圖二十之四、液相層析電灑法串聯式質譜(LC/MS/MS)分析結果e 60 圖二十之五、液相層析電灑法串聯式質譜(LC/MS/MS)分析結果f 61 圖二十一、蛋白質隨誘導時間的表現量變化 62 圖二十二.模板DNA 與RNA引物的結合示意圖 64 圖二十三、Spt5-632-cpEGFP的功能性測試 65 圖二十四.DSIF對於轉錄效率之影響 66 圖二十五.形成Spt5、 RNAPⅡ複合體之流程示意圖 67 圖二十六.(JEM-1400 )Spt5、Spt5-632-cpEGFP/RNAP Ⅱ複合體之負染影像 69 圖二十七之一.(Titan-Krios)Spt5/RNAPⅡ複合體之冷凍電顯下之影像 71 圖二十七之二. Spt5-632-cpEGFP/RNAPⅡ複合體之冷凍電顯影像的二維分類 72 圖二十七之三. Spt5-632-cpEGFP/RNAPⅡ複合體之冷凍電顯下之影像的三維結構 73 圖二十七之四. Spt5-632-cpEGFP/RNAPⅡ複合體三維結構之黃金標準傅立葉殼層關聯函數(Gold Standard Fourier shell correlation，FSC)曲線 74 圖二十八之一.(Titan-Krios)Spt5/RNAPⅡ複合體之冷凍電顯下之影像 75 圖二十八之二.Spt5/RNAPⅡ複合體之冷凍電顯影像的二維分類 76 圖二十八之三. Spt5/RNAPⅡ複合體之冷凍低溫電顯下之影像的三維結構 77 圖二十八之四. Spt5/RNAPⅡ複合體三維結構之黃金標準傅立葉殼層關聯函數曲線 78 圖二十九.Spt5/RNAPⅡ複合體之SDS PAGE(Ruby staining) 80 圖三十.多個轉錄相關蛋白結合缺乏Rpb4/7之RNAPⅡ 86 圖三十一.Spt4/5對於Rpb4/7分離機制的調控假設 89 表目錄 表一、 三域系統中RNA 聚合酶之各次單元構成 90 表二、 表現蛋白質分子量對照表 91 表三、 引子及模板列表 92 | |
| dc.language.iso | zh-TW | |
| dc.title | 出芽酵母Spt5在RNA polymeraseⅡ上的功能與結構的觀察 | zh_TW |
| dc.title | The observation on function and structure of Saccharomyces cerevisiaes Spt5 associates with RNA polymeraseⅡ | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 107-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.coadvisor | 章為皓(Wei-Hau Chang) | |
| dc.contributor.oralexamcommittee | 陳佩燁(PEI-YEH CHEN) | |
| dc.subject.keyword | 核糖核酸聚合?二型,Spt5,冷凍電顯,Rpb4/7, | zh_TW |
| dc.subject.keyword | RNA polymerase Ⅱ,Spt5,Cryo-EM,Rpb4/7, | en |
| dc.relation.page | 115 | |
| dc.identifier.doi | 10.6342/NTU201903614 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2019-08-16 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科技學系 | zh_TW |
| 顯示於系所單位： | 生化科技學系 | |
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