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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 梁國淦(Kuo-kan Liang) | |
| dc.contributor.author | Yang-Chih Liu | en |
| dc.contributor.author | 劉洋志 | zh_TW |
| dc.date.accessioned | 2021-06-17T03:45:36Z | - |
| dc.date.available | 2020-08-24 | |
| dc.date.copyright | 2020-08-24 | |
| dc.date.issued | 2020 | |
| dc.date.submitted | 2020-08-19 | |
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Master thesis, Department of chemical engineering and biotechnology, National Taipei University of Technology. (2008) Wu, Y.-M. et al., Regulation of mammalian transcription by Gdown1 through a novel steric crosstalk revealed by cryo-EM, The EMBO Journal (2012) 31, 3575–3587 Zhou, J. et al., Histone deacetylase Rpd3 antagonizes Sir2-dependent silent chromatin propagation. Nucleic Acids Res (2009) 37(11):3699-713 | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/70138 | - |
| dc.description.abstract | 在真核生物內，轉錄延長因子Spt5對於基因表達扮演重要角色。此外，它也是個多功能因子，它不僅促進了RNA polymerase II（RNAPII or Pol II）從初始到終止階段之間的轉錄作用。也對於人類神經退化性疾病與愛滋病造成影響。於是Spt5-Pol II的研究就非常重要。本實驗室同仁先前對於Spt5-Pol II複合體，使用冷凍電子顯微鏡(cryo-EM)結構，說明Spt5可能會改變Pol II與Spt5-Pol II轉錄複合體的立體構型。因此，我們利用啤酒酵母菌的Spt5與RNA polymerase II，研究Spt5對於Pol II的結構影響。為了克服Spt5產量的要求，我們針對Spt5建立了一個啤酒酵母過量表達系統。首先，我優化了此過量表達系統與開發了一個技術。本技術利用免疫沈澱固定Pol II。另外，我利用size exclusion chromatography做競爭性分析試驗，藉此評估Spt5與Pol II的互動關係。最後，我發現了在缺乏DNA-RNA scaffold結構存在，Spt5會把次複合體Rpb4/7從RNA polymerase II上剔除。最重要的是，此發現對於近一步探討Spt5與RNA polymerase II的互動情形，開啟了新的可能。 | zh_TW |
| dc.description.abstract | In the eukaryotes, transcription elongation factor Spt5 plays important role in the gene expression. Also, Spt5 is a multi-functional factor. It not only facilitates the RNA polymerase II (RNAPII or Pol II) mediated transcription, from initiation to termination, but also has effects on human neurodegenerative disease and AIDS. Thus, the study on Spt5 and Pol II is very important. Our previous cryo-EM structure study of Spt5-Pol II complex suggested that Spt5 could alter the architecture of Pol II transcribing complex. Therefore, we studied the Spt5 effect on the architecture of Pol II using the S. cerevisiae’s Spt5 and Pol II. To overcome the demand on the quantity of Spt5, we construct an over-expressing system of S. cerevisiae to produce Spt5. Here, I optimized the S. cerevisiae over-expressing system for Spt5 and developed an immobilized Pol II assay with immuno-precipitation (IP) and Spt5-Pol II competition assay with size exclusion chromatography allowing for evaluating the interactions between Spt5 and Pol II both from S. cerevisiae. Finally, I discovered that Spt5 may dissociate subcomplex Rpb4/7 from core structure of RNA polymerase II without DNA-RNA scaffold. Last but not least, the findings initiate new possibility | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T03:45:36Z (GMT). No. of bitstreams: 1 U0001-1708202018301600.pdf: 73517363 bytes, checksum: 79a64895246790b76e9344ef40c39989 (MD5) Previous issue date: 2020 | en |
| dc.description.tableofcontents | 中文摘要 III ABSTRACT IV 目錄 V 圖目錄 VII 表目錄 IX TABLE OF ABBREVIATIONS X CHAPTER 1 INTRODUCTION 1 1.1 THE EFFECT OF SPT5 ON BUDDING YEAST 5 1.2 THE FUNCTION OF THE C-TERMINAL DOMAIN OF SPT5WT 12 1.3 THE ROLE OF RNA POLYMERASE 2 12 1.4 THE SYSTEM FOR OVEREXPRESSING WILD TYPE SPT5 14 1.5 THE TANDEM AFFINITY PURIFICATION (TAP) TECHNOLOGY 16 1.6 EXPERIMENT DESIGN AND PURPOSE 18 CHAPTER 2 MATERIAL AND METHODS 21 2.1 TAP TAGGING RNAPII 21 2.2 OVEREXPRESSION AND PURIFICATION OF A RECOMBINANT SPT5 IN S. CEREVISIAE 21 2.3 THE IMMOBILIZED-PROTEIN ASSAY 24 2.4 COMPETITION ASSAY 29 2.5 PURIFICATION OF WILD TYPE SPT5 31 2.6 WESTERN BLOTTING 32 2.7 COLONY POLYMERASE CHAIN REACTION (PCR) 33 2.8 QUICK-FUSION CLONING TECHNOLOGY 36 2.9 CHROMOSOME AND PLASMIDS EXTRACT FROM S. CEREVISIAE. 38 2.10 MATERIALS 40 CHAPTER 3 DISCUSSION AND RESULTS 43 3.1 TRANSFORMATION OF S. CEREVISIAE. 43 3.2 EXPRESSION OF RECOMBINANT PROTEIN: RNAPII AND SPT5 44 3.3 PURIFICATION OF RECOMBINANT PROTEIN: RNAPII AND SPT5 49 3.4 THE IMMOBILIZED PROTEIN ASSAYS 54 3.5 COMPETITION ASSAY 61 CHAPTER 4 CONCLUSION AND FUTURE WORKS 64 REFERENCES 66 SUPPLEMENTARY DATA 72 THE IN VITRO TRANSCRIPTION ASSAYS 72 THE IMMUNE BLOTTING FOR THE FLOW-THROUGH SOLUTION 73 IMMOBILIZED-POL II ASSAY BY SDS-PAGE ANALYSIS 75 | |
| dc.language.iso | en | |
| dc.subject | 第二型RNA聚合酶 | zh_TW |
| dc.subject | Spt5 | zh_TW |
| dc.subject | 冷凍電子顯微鏡 | zh_TW |
| dc.subject | Rpb4/7 | zh_TW |
| dc.subject | 啤酒酵母 | zh_TW |
| dc.subject | RNA polymerase II | en |
| dc.subject | Spt5 | en |
| dc.subject | S.cerevisiae | en |
| dc.subject | Rpb4/7 | en |
| dc.subject | cryo-EM | en |
| dc.title | 啤酒酵母的Spt5 對於第二型RNA聚合酶複合體之結構的影響 | zh_TW |
| dc.title | Effect of Spt5 on RNAPII complex architecture in Saccharomyces cerevisiae | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 108-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.coadvisor | 章為皓(Wei-hau Chang) | |
| dc.contributor.oralexamcommittee | 陳佩燁(Rita P.-Y. Chen),楊健志(Chien-Chih Yang) | |
| dc.subject.keyword | Spt5,第二型RNA聚合酶,冷凍電子顯微鏡,Rpb4/7,啤酒酵母, | zh_TW |
| dc.subject.keyword | Spt5,RNA polymerase II,cryo-EM,Rpb4/7,S.cerevisiae, | en |
| dc.relation.page | 78 | |
| dc.identifier.doi | 10.6342/NTU202003835 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2020-08-20 | |
| 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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