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標題: | 阿拉伯芥PIF3與G-box序列結合的分子機制之研究 Study on the molecular mechanism of Arabidopsis PIF3 binding to G-box cis-elements |
作者: | Chia-Yu Chien 簡嘉佑 |
指導教授: | 鄭貽生(Yi-Sheng Cheng) |
關鍵字: | 光敏素交互作用因子,光訊息傳遞路徑, PIF3,bHLH,G-box,fEMSA, |
出版年 : | 2014 |
學位: | 碩士 |
摘要: | 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。 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. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/56442 |
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顯示於系所單位: | 植物科學研究所 |
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