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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 徐駿森(Chun-Hua Hsu) | |
| dc.contributor.author | CHIA-YEE CHUN | en |
| dc.contributor.author | 莊佳儀 | zh_TW |
| dc.date.accessioned | 2021-06-08T01:46:05Z | - |
| dc.date.copyright | 2016-08-31 | |
| dc.date.issued | 2016 | |
| dc.date.submitted | 2016-08-11 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/19131 | - |
| dc.description.abstract | CupR 是來自耐金屬貪銅菌 (Cupriavidus metallidurans CH34) 之金屬調節轉錄因子 (metalloregulatory transcription factor)，能夠調控其操縱子之基因表現與否。CupR 二聚體受到金屬離子所引發的構型變化，將導致操縱子轉錄調控區塊 (operator/promoter site) RNA 聚合酶結合位 -35 和 -10 區塊旋轉至同一平面上，因而活化轉錄作用。然而至目前為止，CupR 與其專一性啟動子區域結合的細節尚未被釐清。
為了瞭解 CupR 對其特異性 (specific) DNA 序列之辨識以及二者結合對蛋白質二級結構和 DNA 構型的變化，本研究利用電泳遷移率實驗 (Electrophoretic Mobility Shift Assay, EMSA)、恆溫滴定微量熱實驗 (Isothermal Titration Calorimetry, ITC)、圓二色光譜分析實驗 (Ciruclar Dichroism, CD) 對 CupR 與不同序列 DNA 之結合特性做比較探討，再藉由結構模擬以及分子對接技術 (Molecular Docking) 分析 CupR 對相似序列之辨識差異。CupR 與 DNA 之結合能力藉由電泳遷移率實驗分析，顯示 CupR 與其轉錄調控區塊 (PcupA) 的結合能力相較於非特異性核酸序列明顯較強，且亞金離子存在之環境下使二者結合能力提升。CupR 與 DNA 結合過程之熱力學參數，指出 CupR 與 PcupA 的結合過程中所量測得到之熱焓與亂度皆為正值，說明 CupR 主要是以氫鍵穩定與 PcupA 結合。經由 CupR 二聚體模擬結構與其轉錄調控區塊 PcupA 所建構的複合體 (complex) 與文獻所提供之同源蛋白質 CueR-DNA 結構作比較，結果顯示 CupR-DNA 其中三個參與交互作用的鹼基可能對於 CupR 辨識並結合專一性序列重要。 | zh_TW |
| dc.description.abstract | CupR, the metalloregulatory transcription factor from Cupriavidus metallidurans strain 34, regulates gene expression of metal-resistance operon. When the dimeric CupR coordinates to a metal ion, the metal-CupR complex causes an allosteric underwinding of the DNA at the operator/promoter site, which realigns the -35 and -10 sequences of the promoter. Subsequently, RNA polymerase can contact the promoter sequences leading to transcription activation. So far the detail recognition of CupR with its specific DNA-binding sequences remains unknown.
In this research, we study the binding properties of CupR toward specific or non-specific DNA sequences through EMSA (Electrophoretic Mobility Shift Assay), ITC (Isothermal Titration Calorimetry) and CD (Circular Dichroism Spectroscopy). Furthermore, homology modeling and docking methods were used to investigate the recognition modes between CupR and various DNA sequences. Data from EMSA reveals that CupR posesses best binding ability with promoter sequences (PcupA) compared to others. Moreover, chelation of CupR with Au+ ion makes CupR binding to PcupA stronger. Additionally, the ITC thermodynamic parameters of CupR bound to promoter sequences (PcupA) shows a very negative enthalpy and a negative entropy effect which indicating that formation of CupR and PcupA is mainly via hydrogen-bonding. Molecular models of CupR in complexes with various DNAs are analyzed by NucPlot and reveal that three bases may be important for the recognition between CupR and promoter sequence. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T01:46:05Z (GMT). No. of bitstreams: 1 ntu-105-R03623029-1.pdf: 7740252 bytes, checksum: c6009d37a6575126e31ed9165a73468e (MD5) Previous issue date: 2016 | en |
| dc.description.tableofcontents | 壹、前言 1
一、金屬調節轉錄因子 1 二、MerR 家族之金屬調節轉錄因子 2 三、CupR 以及 cup 操縱子 4 四、一價金屬離子調控轉錄因子 5 五、蛋白質與 DNA 之結合 6 六、研究目標 7 貳、材料與方法 8 一、實驗材料 8 1. 細菌染色體 DNA 8 2. 蛋白質表現載體之構築 8 3. 菌株及培養系統 8 4. 雙股去氧核酸序列設計 8 5. 金屬種類 9 二、實驗方法 10 1. 細菌染色體 DNA 的抽取 10 2. 大腸桿菌勝任細胞 (Competent cell) 之製備 10 3. 轉型作用 11 4. 質體 DNA 抽取 11 5. DNA 定序比對 11 6. SDS 蛋白質膠體電泳分析 11 7. 重組蛋白質之大量表現 12 8. 重組蛋白質之純化 12 9. 蛋白質透析與濃縮 14 10. 膠體過濾層析法 14 11. 蛋白質之定量分析 15 12. 胺基酸序列比對 15 13. 親緣關係圖將胺基酸序列 16 14. 蛋白質與 DNA 之結構模型建立 16 15. 分子對接 (Molecular docking) 與分析 17 16. 雙股去氧核糖核酸序列製備 18 17. 圓二光譜儀測試蛋白質與 DNA 結合的穩定性 18 18. 恆溫滴定微量熱實驗 20 19. 電泳遷移率實驗 21 20. 蛋白質晶體之培養 22 參、實驗結果 26 一、構築 pET-21a(+)-CupR 載體 26 二、CupR蛋白質小量表現 26 三、CupR 蛋白質純化 26 四、膠體過濾層析法分析 CupR 的分子量 27 五、MerR family 蛋白質胺基酸序列比對 27 六、CupR 蛋白質二聚體結構模擬 28 1. CupR 蛋白質二聚體模擬結構之建立 28 2. CupR 結構分析 29 七、CupR 的二級結構和基本性質 29 1. 蛋白質之熱穩定性 29 2. 蛋白質在酸鹼環境的耐受性 30 八、電泳遷移率實驗結合能力測試 30 1. 牛血清蛋白對照組之結合能力測試 30 2. CupR 與四種雙股 DNA 之結合能力測試 30 3. CupR 與四種雙股 DNA 之非特異性結合 31 九、以測定熱量變化觀察 CupR與雙股 DNA 之結合 31 十、於金屬離子存在下CupR 與四種雙股 DNA 之結合能力測試 32 1. CupR 與雙股 DNAspe 之結合 32 2. CupR 與非特異性雙股 DNA 之結合 32 十一、CupR 與雙股 DNA 的結合對二級結構之影響 33 1. 蛋白質構型變化 33 2. 雙股 DNA 構型變化 34 3. Au+ 添加順序對與 CupR 結合之雙股 DNA 的影響 35 十二、與雙股DNA、金屬離子結合之 CupR 熱穩定性 35 十三、CupR 晶體條件篩選 36 十四、CupR 與 DNA 之對接 (docking) 與分析 37 1. CueR-DNA 控制組 37 2. CupR 與雙股 DNA 之對接 37 3. 雙股 DNA 序列之置換 38 十五、CupR 與雙股 DNACueR(TG) 之結合能力 39 肆、討論 40 一、CupR 蛋白質的表現純化與熱變性分析 40 二、CupR 二聚體模擬結構之建立 40 三、CupR 與 DNA 結合之構型變化探討 41 四、CupR 與 DNA 結合之熱量變化探討 41 五、CupR 晶體培養 42 六、CupR 對轉錄調控區塊之辨識 43 伍、圖表 45 陸、參考文獻 83 柒、附錄 89 | |
| dc.language.iso | zh-TW | |
| dc.subject | 專一性 | zh_TW |
| dc.subject | 耐金屬貪銅菌 | zh_TW |
| dc.subject | 金屬調節轉錄因子 | zh_TW |
| dc.subject | 轉錄調控區塊 | zh_TW |
| dc.subject | 蛋白質與核酸結合 | zh_TW |
| dc.subject | Transcriptional regulation operon | en |
| dc.subject | Protein-DNA binding | en |
| dc.subject | Cupriavidus metallidurans strain 34 | en |
| dc.subject | Specificity | en |
| dc.subject | Metalloregulatory transcription factor CupR | en |
| dc.title | 從結構和能量觀點探討耐金屬貪銅菌之金屬調節轉錄因子 CupR 與 DNA 結合的特異性 | zh_TW |
| dc.title | Structural and Energetic Basis of DNA Specificity by Metalloregulatory Transcription Factor CupR in Cupriavidus metallidurans | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 104-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 詹迺立(Nei-Li Chan),劉沛棻(Pei-Fen Liu),葉怡均(Yi-Chun Yeh) | |
| dc.subject.keyword | 耐金屬貪銅菌,金屬調節轉錄因子,轉錄調控區塊,蛋白質與核酸結合,專一性, | zh_TW |
| dc.subject.keyword | Cupriavidus metallidurans strain 34,Metalloregulatory transcription factor CupR,Transcriptional regulation operon,Protein-DNA binding,Specificity, | en |
| dc.relation.page | 99 | |
| dc.identifier.doi | 10.6342/NTU201602219 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2016-08-11 | |
| dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
| dc.contributor.author-dept | 農業化學研究所 | zh_TW |
| 顯示於系所單位： | 農業化學系 | |
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