博碩士論文 104821007 詳細資訊




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姓名 戴愷頫(Kai-Fu Tai)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 嗜酸熱硫化葉菌中去氧核醣核酸結合蛋白Saci_1212之結構性及功能性分析
(Structural and Functional Analysis of the DNA Binding Protein Saci_1212 from the Hyperthermophile Sulfolobus acidocaldarius)
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檔案 [Endnote RIS 格式]    [Bibtex 格式]    至系統瀏覽論文 (2022-8-10以後開放)
摘要(中) 在生物體中,去氧核醣核酸需要被纏繞保存,真核生物以組蛋白作為纏繞核酸的工具,在古生菌中,則有一群分子量7到10 KDa的蛋白質作類似的用途,稱之為「類組蛋白」蛋白質。Sso7c4是硫磺礦硫化葉菌中一個已知的「類組蛋白」蛋白質,本研究的目標蛋白質Saci_1212,則是Sso7c4在嗜酸熱硫化葉菌中的同源蛋白。在先前的研究中已經得知Sso7c4的C端序列(LKEPWK),但正電且結構彈性,會參與去氧核醣核酸的作用。而有趣的是Saci_1212整體結構雖與Sso7c4相似,但C端蛋白質序列(TEEELR)卻帶有3個帶負電荷的麩胺酸殘基,與去氧核醣核酸的電性相排斥。
在本研究中,我們以X光蛋白質晶體繞射的技術得到解析度2.09 埃的Saci_1212蛋白質晶體結構。結構與Sso7c4相似,有著β-loop-β的結構為主體,這樣的折疊構型也與細菌的轉錄調控因子AbrB及 MazE之N端去氧核醣核酸結合區域的結構相似。在蛋白質結構的C端,Sso7c4是一個有彈性的手臂,沒有固定構型;Saci_1212則以三個麩胺酸殘基形成310¬-螺旋的結構,與Sso7c4完全不同。後續以電子顯微鏡拍攝Saci_1212原生型及C端刪除型兩種蛋白質分別以不同的比例與質體PhiX174 RF II DNA作用之影像。從影像分析,得知Saci_1212不同於Sso7c4,不會與去氧核醣核酸有架橋的作用;質體長度並無顯著縮短也顯示它不是一個彎曲者或纏繞者。與C端刪除型相比,原生型完全沒有架橋行為,C端刪除型則有局部架橋,顯示帶負電的C端序列會減少對DNA架橋效應的發生;又根據螢光極化分析的結果,Saci_1212之C端序列刪除後與去氧核醣核酸的結合力上升了20倍,顯示該C端序列降低了整體的結合力,但比較Saci_1212與同源蛋白質Sso7c4的C端刪除型,前者的結合力是後者的24倍,可能有與其他轉錄調控因子競爭去氧核醣核酸結合位的能力,故推測Saci_1212是一個基因轉錄的負調控因子。
摘要(英)
Saci_1212 protein, a Sso7c4 homologue, is commonly believed to be a histone-like protein involved in genomic DNA compaction from Sulfolobus acidocaldarius. Although sequence alignments share high similarity (Saci_1212 vs Sso7c4: 66% identity), their C-termini are quite different in amino acid level. In previous study, we have been demonstrated that basic and flexible C-terminal ends of Sso7c4 (LKEPWK) are involved in binding and bending DNA whereas C-terminus of Saci_1212 (TEEELR) contains three glutamate residues which disfavor to interact with DNA due to the negative charge repulsions.
Here, to obtain a detailed understanding of its architectural properties, we present the crystal structure of wild type Saci_1212 at 2.09 Å resolution. The overall structure of Saci_1212 is similar to Sso7c4 which forms a swapped β-loop-β ′Yin-Yang′ topology. This fold resembles the N-terminal DNA-binding domain of both AbrB and MazE, which are transcriptional regulators in bacteria. The two basic C-termini of Sso7c4 are disordered owing to a lack of corresponding density in the crystal. However, the two C-termini of Saci_1212 adopt the 310-helical conformation by three glutamate residues. Two C-termini of Saci_1212 are quite different from that of Sso7c4 either in amino acid sequence or secondary structure. In fluorescence polarization binding assays, C-terminally truncated proteins exhibited higher binding affinity to 20-bp DNA than the wild-type protein to approximately 20-fold. These FP data further show that the C-terminal ends of Saci_1212 decrease the interaction with DNA. As shown in electron microscopy (EM) images, wild-type Sso7c4 compacts DNA through bridging and bending interactions, whereas the Saci_1212 protein binds DNA without any bridging phenomenon and no compaction of the DNA occurs. A functional role for Saci_1212 as negative transcriptional regulator is suggested.
關鍵字(中) ★ 去氧核醣核酸結合蛋白
★ 嗜酸熱硫化葉菌
★ X射線晶體學
★ 電子顯微鏡
關鍵字(英) ★ DNA binding protein
★ Sulfolobus acidocaldarius
★ X-ray Crystallography
★ Electron Microscope
論文目次
中文摘要...i
Abstract...ii
致謝...iii
目錄...iv
圖目錄...vi
表目錄...vii
一、緒論...1
1-1 菌種...1
1-1-1 古細菌域(Domain Archaea)...1
1-1-2 古生菌硫化葉菌屬...2
1-2 DNA結合蛋白...3
1-2-1 古細菌中的DNA結合蛋白...3
1-2-2 蛋白質Sso7c4和蛋白質Saci_1212...4
1-2-3 DNA結合蛋白質的構築功能...6
1-3 研究動機...7
二、方法與材料...8
2-1建構目標蛋白質基因與載體...8
2-1-1聚合酶鏈鎖反應引子 (PCR primer) 設計...8
2-1-2聚合酶鏈鎖反應擴增目標基因...9
2-1-3利用膠體電泳確認目標基因片段...10
2-1-4純化基因片段...10
2-1-5建構表現質體...11
2-1-6轉型作用 (Transformation)...12
2-1-7基因定序...12
2-2目標蛋白質大量表現...14
2-2-1測試最佳表現條件...14
2-2-2聚丙烯醯胺膠體電泳(SDS Poly-acrylamide-gel-electrophoresis, SDS-PAGE)...14
2-2-3蛋白質大量表現...15
2-2-4純化目標蛋白質...15
2-3晶體形成...17
2-3-1預結晶試驗(PCT, Pre-Crystallization Test)...18
2-3-2蛋白質結晶條件篩選...19
2-4 X光繞射與結構解析...20
2-4-1收集X光繞射數據...20
2-4-2解決相位問題...20
2-4-3結構的建立與優化...21
2-5突變-C端刪除...21
2-6 利用電子顯微鏡(Electron Microscope)觀察蛋白質與DNA結合機制...22
2-7螢光極化分析(Fluorescence polarization Assay)...23
三、結果...24
3-1建構目標蛋白質基因與載體...24
3-2蛋白質表現條件測試...24
3-3測試蛋白質耐熱特性...25
3-4利用陽離子交換樹脂管柱SP column純化蛋白質...26
3-5蛋白質結晶條件篩選...27
3-6蛋白質Saci_1212之結構描述...28
3-7蛋白質與DNA作用機制分析...33
3-8螢光極化分析...34
四、討論...35
4-1 Saci_1212之結構分析...35
4-2電子顯微鏡影像分析...36
4-3螢光極化分析...38
五、參考文獻...39
參考文獻
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指導教授 陳青諭(Chin-Yu Chen) 審核日期 2017-8-14
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