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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 鄭貽生(Yi-Sheng Cheng) | |
dc.contributor.author | Chia-Yu Chien | en |
dc.contributor.author | 簡嘉佑 | zh_TW |
dc.date.accessioned | 2021-06-16T05:28:49Z | - |
dc.date.available | 2014-08-21 | |
dc.date.copyright | 2014-08-21 | |
dc.date.issued | 2014 | |
dc.date.submitted | 2014-08-14 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/56442 | - |
dc.description.abstract | 在光敏素調控的訊息傳導路徑中,PHYTOCHROME INTERACTING FACTOR3 (PIF3)被認為是負調控光訊息反應的一個重要因子。先前的研究也指出PIF3是隸屬於可以結合至G-box序列的bHLH轉錄因子巨家族的成員。在本研究中,旨在探討PIF3與G-box DNA專一性結合之機制。
藉由膠體過濾層析法(gel filtration chromatography)以及分析型超高速離心(analyticalultracentrifugation; AUC)的結果顯示,bHLH是以二聚體形式與G-box(CACGTG)進行結合。經蛋白質序列比對及結構模擬等生物資訊分析方式,發現數個重要的疏水性胺基酸:Met60、Val57、Gln56、Leu53、Leu50、Tyr49、Ile47、Leu27以及Met24,其之間形成六層疏水性作用力介面以穩固蛋白質二聚體的形成。另以螢光基礎電泳遷移滯實驗(fluorescein-based electrophoretic mobility shift assay; fEMSA)測量bHLH與G-box專一性結合的能力,結果顯示bHLH除了可以與G-box結合外,也可以與其他不同E-box(CANNTG)進行結合。從蛋白質結構模擬分析,bHLH中與DNA結合的重要保守性殘基為His9、Glu13以及Arg17,Arg17負責與G-box中第四號鹼基形成氫鍵,由於在fEMSA實驗中發現bHLH在第四號鹼基位置分別置換四種鹼基後皆具結合能力,因此推估Arg17並非扮演DNA辨認的角色,另外也發現E-box上的第三號鹼基突變為空間較大的G後,整體結合能力較G-box下降許多。總結以上研究結果,可以推論一個bHLH與G-box之間可能的結合機制:藉疏水性胺基酸交互作用之二聚體bHLH先以帶正電結合溝槽與雙股DNA進行初步結合,辨認到core region之正股六號鹼基(G)以及反股之二號鹼基(A),進行氫鍵結合後,進一步靠著中心的正股四號鹼基(G)進行氫鍵結合,形成一氫鍵網絡以穩固蛋白質在DNA上的構型。 | zh_TW |
dc.description.abstract | It is crucial for plants to sense light signals by phytochromes in development and growth. Among phytochrome signaling pathway, PHYTOCHROME INTERACTING FACTOR3 (PIF3) is believed to act as a key component that negatively regulates several light responses in plants. Previous studies have identified PIF3 as a member of basic helix-loop-helix (bHLH) transcription factor superfamily that can directly bind to a typical G-box (CACGTG) DNA. In this study, we aimed to reveal the binding mechanism between PIF3 and G-box DNA from structural view point.
In this study, we examined the oligomeric states of apo form bHLH and bHLH-DNA by gel filtration chromatography and analytical ultracentrifugation. The results indicated that bHLH might form a homodimer to associate with G-box DNA. We also found some hydrophobic residues, including Met60, Val57, Gln56, Leu53, Leu50, Tyr49, Ile47, Leu27 and Met24 by sequence alignment and modellng. These residues formed a six-layer hydrophobic interaction interface that might be critical for bHLH dimer formation. From the fluorescein-based electrophoretic mobility shift assay (fEMSA) results, we found that not only G-box but also E-box (CANNTG) could bind to bHLH protein. In addition, we found some conserved residues His9, Glu13 and Arg17 that might play critical roles in G-box recognition. Combined with fEMSA and modelling results, the Arg17 that formed hydrogen bonds with the 4th base of G-box may not act as a DNA recognition residue. Although the 3th base of G-box did not participate in hydrogen bond formation, it might have steric hindrance that affected the hydrogen bond formation between Arg17 and the 4th base of G-box. In summary, we concluded a possible mechanism of bHLH binding to G-box sequence: The bHLH formed homodimers by the hydrophobic residues first. The positive-charged binding groove of bHLH could stabilize DNA at the groove. Then, the His9 and Glu13 could recognize the 6th base of G-box and the the 2th base of anti-sense G-box. The Arg17 then formed hydrogen bonds with the 4th base of G-box. Finally, they could form a hydrogen bond network to stabilize the protein-DNA structure. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T05:28:49Z (GMT). No. of bitstreams: 1 ntu-103-R01b42004-1.pdf: 2416722 bytes, checksum: 9b5ded962c2e672a41b23d84a2024ac1 (MD5) Previous issue date: 2014 | en |
dc.description.tableofcontents | 中文摘要 v
Abstract vi 縮寫對照表 viii 第一章 前言 - 1 - 一、光敏素交互作用因子(phytochrome interacting factors; PIFs)家族 - 1 - 二、PIFs家族與植物荷爾蒙和其他環境因子之間的訊息調控 - 3 - 三、PHYTOCHROME INTERACTING FACTOR3簡介 - 7 - 四、Basic helix-loop-helix (bHLH)之蛋白質結構 - 9 - 五、研究目標 - 11 - 第二章 材料與方法 - 12 - 1. 材料 - 12 - 2. 方法 - 12 - (一) bHLH功能域基因表現載體之構築 - 12 - (二) bHLH蛋白質之表現與純化 - 12 - 1. 大腸桿菌勝任細胞(Competent cell)之製備 - 12 - 2. 大腸桿菌之轉形作用(Transformation) - 13 - 3. 重組蛋白質之大量表現及純化(Over-Expression and purification) - 13 - 4. 蛋白質濃縮以及蛋白質緩衝液置換 - 14 - 5. 蛋白質之定量分析 - 14 - 6. SDS-聚丙烯醯胺膠體電泳(Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis, SDS-PAGE) - 15 - 7. 西方墨點法(western blot)分析 - 15 - (三) PIF3之bHLH domain對不同E-box解離常數(Dissociation constant, KD)之分析 - 16 - 1. 螢光電泳遷移率實驗(fluorescein-based electrophoretic mobility shift assay, fEMSA) - 16 - 1.1 雙股DNA的製備 - 16 - 1.2 樣品製備 - 17 - 1.3 原態聚丙烯醯胺膠體電泳(native-PAGE) - 17 - 2. 競爭式螢光電泳遷移率實驗 (competitive fEMSA) - 17 - 3. 數據分析方式 - 17 - (四) bHLH蛋白質之多聚體(oligomer)組成與cis-element結合形式之分析 - 18 - 1. 膠體過濾層析法(gel filtration chromatography) - 18 - 2. 分析型超高速離心實驗(analytical ultracentrifugation, AUC) - 18 - (五) 蛋白質結晶實驗 - 19 - 1. Pre-crystallization test (PCT測試) - 19 - 2. bHLH蛋白質與雙股DNA複合體之結晶條件篩選 - 20 - 3. 蛋白質篩選溶液之微調 - 20 - (六) bHLH蛋白質之二聚體結構建立 - 21 - 1. PIF3之bHLH domain與已解出結構之bHLH家族成員的胺基酸序列比對 - 21 - 2. bHLH蛋白質之二聚體模型建立 - 21 - 第三章 結果 - 22 - 一、 PIF3 bHLH domain的表現與純化 - 22 - (1) 利用親合性層析管柱Ni-NTA得到具相對純度以及大量的bHLH-His6 - 22 - (2) 利用膠體過濾層析法得到極高純度的bHLH-His6 - 22 - 二、bHLH domain以及bHLH和雙股DNA在溶液中多聚體之組成分析 - 23 - (1)膠體過濾層析法分析bHLH在溶液中多聚體的形式 - 23 - (2) 分析型超高速離心法分析bHLH和DNA在溶液中多聚體之組成分析 - 23 - 三、bHLH蛋白質之晶體篩選 - 24 - 四、bHLH蛋白質二聚體結構模擬 - 25 - (1) bHLH蛋白質二聚體模擬結構之建立 - 25 - (2) bHLH模擬結構與實際人類BMAL1-CLOCK-E-box結構之間的比對 - 26 - 五、bHLH蛋白質與不同E-box之結合能力分析 - 27 - 第四章 討論 - 29 - 一、 PIF3 蛋白質的表現與純化 - 29 - 二、 bHLH之多聚體組成探討 - 30 - 三、 PIF3之bHLH domain蛋白質結構模擬探討 - 30 - 四、 bHLH與不同E-box序列結合能力之探討 - 31 - 第五章 結論 - 33 - 參考文獻 - 35 - 圖表 - 46 - 圖一、bHLH蛋白質大量表現及純化結果 - 46 - 圖二、以西方墨點法(Western blot)檢測蛋白質表現及純化各階段之結果 - 47 - 圖三、膠體過濾層析實驗分析標準分子量之蛋白質 - 48 - 圖四、bHLH蛋白質之多聚體組成分析 - 49 - 圖五、bHLH蛋白質與雙股DNA結合形式之AUC分析 - 50 - 圖六、篩晶後得到之bHLH與雙股DNA複合體結晶 - 51 - 圖七、以fEMSA實驗驗證bHLH蛋白質與雙股DNA結合的能力 - 52 - 圖八-1、利用fEMSA檢測各個E-box和bHLH之間的親和性 - 53 - 圖八-2、利用fEMSA檢測各個E-box和bHLH之間的親和性 - 54 - 圖八-3、利用fEMSA檢測各個E-box和bHLH之間的親和性 - 55 - 圖八-4、利用fEMSA檢測各個E-box和bHLH之間的親和性 - 56 - 圖九、阿拉伯芥PIF3之bHLH domain三級模擬結構 - 57 - 圖十、以模擬結構探討bHLH形成二聚體之重要胺基酸位點 - 59 - 圖十一、bHLH與G-box DNA結合的細部機制 - 60 - 表一、PIF3之bHLH domain與其他已解出結構之bHLH序列比對後結果 - 61 - 表二、利用Z-Dock進行二聚體模型建立之分數最高前五名模型 - 62 - 表三、不同E-box序列對於bHLH之解離常數KD分析整理 - 63 - 表四、蛋白質養晶母液成分之微調整理 - 64 - 附錄 - 65 - 附錄一、聚合酶連鎖反應參數設定 - 65 - 附錄二、PIF3之bHLH domain重組蛋白純化之試劑 - 65 - 附錄三、SDS 膠體電泳之試劑 - 66 - 附錄四、西方墨點法(western blot)之試劑 - 68 - 附錄五、fEMSA之試劑 - 69 - 附錄六、蛋白質結晶濃度之PCT試劑 - 70 - 附圖一、阿拉伯芥PIFs家族成員 - 71 - 附圖二、PIFs家族參與在phytochrome的光傳遞路徑中 - 72 - 附圖三、動物界中bHLH蛋白質之六種分類 - 73 - 附圖四、坐式蒸氣擴散法以及懸吊式蒸氣擴散法結晶方法示意圖 - 74 - 附圖五、蛋白質表現載體pET21b之圖譜 - 75 - 附圖六、阿拉伯芥PIF3 蛋白質domain分布圖 - 76 - | |
dc.language.iso | zh-TW | |
dc.title | 阿拉伯芥PIF3與G-box序列結合的分子機制之研究 | zh_TW |
dc.title | Study on the molecular mechanism of Arabidopsis PIF3 binding to G-box cis-elements | en |
dc.type | Thesis | |
dc.date.schoolyear | 102-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 吳克強(Keqiang Wu),徐駿森(Chun-Hua Hsu),張世宗(Shih-Chung Chang) | |
dc.subject.keyword | 光敏素交互作用因子,光訊息傳遞路徑, | zh_TW |
dc.subject.keyword | PIF3,bHLH,G-box,fEMSA, | en |
dc.relation.page | 76 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2014-08-14 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 植物科學研究所 | zh_TW |
顯示於系所單位: | 植物科學研究所 |
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