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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 詹迺立(Nei-Li Chan) | |
dc.contributor.author | Chia-Lu Chang | en |
dc.contributor.author | 張家璐 | zh_TW |
dc.date.accessioned | 2021-06-16T08:12:01Z | - |
dc.date.available | 2019-02-25 | |
dc.date.copyright | 2014-02-25 | |
dc.date.issued | 2014 | |
dc.date.submitted | 2014-02-17 | |
dc.identifier.citation | Ansari, A.Z., Chael, M.L., and O'Halloran, T.V. (1992). Allosteric underwinding of DNA is a critical step in positive control of transcription by Hg-MerR. Nature 355, 87-89.
Barkay, T., Miller, S.M., and Summers, A.O. (2003). Bacterial mercury resistance from atoms to ecosystems. FEMS microbiology reviews 27, 355-384. Begley, T.P., Walts, A.E., and Walsh, C.T. (1986). Bacterial organomercurial lyase: overproduction, isolation, and characterization. Biochemistry 25, 7186-7192. Brown, N.L., Stoyanov, J.V., Kidd, S.P., and Hobman, J.L. (2003). The MerR family of transcriptional regulators. FEMS microbiology reviews 27, 145-163. Changela, A., Chen, K., Xue, Y., Holschen, J., Outten, C.E., O'Halloran, T.V., and Mondragon, A. (2003). Molecular basis of metal-ion selectivity and zeptomolar sensitivity by CueR. Science 301, 1383-1387. Chen, C.-Y., Hsieh, J.-L., Silver, S., Endo, G., and Huang, C.-C. (2008a). Interactions between two MerR regulators and three operator/promoter regions in the mercury resistance module of Bacillus megaterium. Bioscience, biotechnology, and biochemistry, 0807290996. Chen, C., Hsieh, J., Endo, G., Silver, S., and Huang, C. (2008b). (Bioscience, Biotechnology, and Biochemistry, 72 (9): 2403-2410) Interactions between Two MerR Regulators and Three Operator/Promoter Regions in the Mercury Resistance Module of Bacillus megaterium. Foulkes, E. (2000). Transport of toxic heavy metals across cell membranes. Proceedings of the Society for Experimental Biology and Medicine 223, 234-240. Gochfeld, M. (2003). Cases of mercury exposure, bioavailability, and absorption. Ecotoxicology and Environmental Safety 56, 174-179. Helmann, J.D., Ballard, B.T., and Walsh, C.T. (1990). The MerR metalloregulatory protein binds mercuric ion as a tricoordinate, metal-bridged dimer. Science 247, 946-948. Huang, C.-C., Narita, M., Yamagata, T., and Endo, G. (1999a). Identification of three merB genes and characterization of a broad-spectrum mercury resistance module encoded by a class II transposon of Bacillus megaterium strain MB1. Gene 239, 361-366. Huang, C.-C., Narita, M., Yamagata, T., Itoh, Y., and Endo, G. (1999b). Structure analysis of a class II transposon encoding the mercury resistance of the Gram-positive bacterium Bacillus megaterium MB1, a strain isolated from Minamata Bay, Japan. Gene 234, 361-369. Jarup, L. (2003). Hazards of heavy metal contamination. British medical bulletin 68, 167-182. Lin, T.Y., Kampalath, R.A., Lin, C.-C., Zhang, M., Chavarria, K., Lacson, J., and Jay, J.A. (2013). Investigation of mercury methylation pathways in biofilm versus planktonic cultures of Desulfovibrio desulfuricans. Environmental science & technology. Lund, P.A., and Brown, N.L. (1989). Regulation of transcription in Escherichia coli from the mer and merR promoters in the transposon Tn501. Journal of molecular biology 205, 343-353. Mejare, M., and Bulow, L. (2001). Metal-binding proteins and peptides in bioremediation and phytoremediation of heavy metals. TRENDS in Biotechnology 19, 67-73. Omichinski, J.G. (2007). Toward methylmercury bioremediation. Science 317, 205-206. Parkhill, J., Ansari, A.Z., Wright, J.G., Brown, N.L., and O'halloran, T. (1993). Construction and characterization of a mercury-independent MerR activator (MerRAC): transcriptional activation in the absence of Hg (II) is accompanied by DNA distortion. The EMBO journal 12, 413. Ross, W., Park, S., and Summers, A. (1989). Genetic analysis of transcriptional activation and repression in the Tn21 mer operon. Journal of bacteriology 171, 4009-4018. Silver, S., and Phung, L.T. (2005). A bacterial view of the periodic table: genes and proteins for toxic inorganic ions. Journal of Industrial Microbiology and Biotechnology 32, 587-605. Trasande, L., Landrigan, P.J., and Schechter, C. (2005). Public health and economic consequences of methyl mercury toxicity to the developing brain. Environmental health perspectives 113, 590. William Studier, F., Rosenberg, A.H., Dunn, J.J., and Dubendorff, J.W. (1990). [6] Use of T7 RNA polymerase to direct expression of cloned genes. Methods in enzymology 185, 60-89. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/58344 | - |
dc.description.abstract | 汞是自然界中的元素,廣泛存在於空氣、水、土壤之中;近年來,由於快速的工業發展,汞汙染對環境而言是一嚴重的威脅。汞對神經、消化、免疫系統以及肺、腎、皮膚、眼等多種器官均具有毒性,造成多種疾病的發生。微生物修復(biomedeiation)是一種有效且經濟地清除重金屬汙染的方式,一些生存於重金屬環境中的細菌發展出可提供重金屬抗性的操作組。
TnMERI1是最早從革蘭氏陽性菌Bacillus megaterium MB1中被發現的汞抗性操作組。細菌若在含有汞離子(Hg2+)或有機汞的環境下生長,必須利用汞抗性系統mer操作組攜帶之有機汞裂解酶與汞離子還原酶的活性,將有毒性的有機汞還原成沒有毒性且能以昇華方式離開細胞的汞原子。mer操作組之序列啟動子具有雙重對稱的特性(dyad symmetrical sequence),並且在-35和-10兩個區域之間相隔了18~20個鹼基對;由於轉錄聚合酶(RNA polymerase)對 -35與 -10區域相隔17個鹼基對得啟動子呈現較強的轉錄活性,因此mer操作組的基礎轉錄活性較低。MerR1是負責mer操作組調控表達的轉錄因子。當汞離子(Hg2+)不存在時,MerR1雙聚體(dimer)與mer操作組結合並阻止轉錄聚合酶的結合,扮演抑制子(repressor)的角色。反之,在有汞離子(Hg2+)存在的情況下,汞離子會與MerR1雙聚體結合並且引發蛋白構型變化,造成mer操作組DNA扭曲,促使轉錄聚合酶能有效率地結合而起始轉錄作用。MerR1雙聚體可以和兩個汞離子(Hg+2)結合並且當作增強子(activator)來扭曲該段DNA的序列,使轉錄聚合酶(RNA polymerase)能結合上而轉錄作用得以起始。MerR1和汞離子(Hg2+)結合,每個結合位由三個半胱胺酸(cysteine)組成,其中一個單體提供Cys114和Cys123,而另一單體則提供Cys79。這幾個半胱胺酸(cysteine)若發生突變則會導致MerR1雙聚體無法與汞離子(Hg2+)結合,進而抑制mer操作組的表現。 本研究目前已找到適合晶體生長的環境條件,未來期望利用X-射線繞射原理分析,解出在有或無汞離子(Hg2+)存在條件下MerR1和DNA結合的結構,進而更清楚地了解MerR1調控mer操作組的機轉,以及MerR1與汞離子(Hg2+)的結合如何影響DNA之構型改變。 | zh_TW |
dc.description.abstract | Mercury is a naturally occurring element that is found in air, water and soil, and mercury pollution is a severe environmental threat due to rapid industrialization. Exposure to mercury may cause serious health problems. Mercury may have toxic effects on the nervous, digestive and immune systems, and on lungs, kidneys, skin and eyes. Mercury poisoning can result in several diseases. Bioremediation is an efficiently and ecological way to solve the problem. Mercury-resistant bacteria harbor the mer operon in their genome. The mer operon includes certain functional genes along with promoter, regulator, and operator. The mechanism involves the reduction of the highly reactive cationic form of mercury into metallic vapor. The genes, which are responsible for this resistance, are organized in an operon called mer operon. TnMERI1 was found as the first mercury resistance transposon identified from Gram-positive bacteria and was harbored by a Minamata bay sediment isolated bacterial strain which was designated as Bacillus megaterium MB1.
The operator/promoter region of mer operon is composed of pseudopalindromic promoter binding site and unnormal spaces, 18~20 bps, between -35 and -10 box. RNA polymerase only recognize 17 bp spaces. It need regulator MerR to distort DNA and then shorten the space. When Hg2+ is absent, MerR1 dimer bound to mer operon function as repressor. In the present of Hg2+, MerR1 dimer bound Hg2+ becomes activator to distort and bend DNA, then RNA polymerase binds to -10 and -35 box to start transcription of mer operon. The binding sites of Hg2+ is consisted of three cysteines, one monomer of MerR offers two cysteines ( cys114 and cys123) and the other one offers one cysteine (cys79). If one of those cysteines was mutated, MerR dimer can not bind to Hg2+, resulting to inhibit mer operon expression. To understand the structural basis of MerR1-mediated transcriptional regulation and how mercury ion bind to MerR1, we try to solve the crystal structure of apo-MerR1 and Hg-MerR1 bound to DNA from Gram-positive bacteria Bacillus megaterium MB1. From our results, we get several conditions of crystallization, and we optimize the condition to get better quality of crystal for structural determination. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T08:12:01Z (GMT). No. of bitstreams: 1 ntu-103-R98442028-1.pdf: 2556373 bytes, checksum: e8dde5766f3ddc489ceb2eddafb94a81 (MD5) Previous issue date: 2014 | en |
dc.description.tableofcontents | 目錄
目錄............................................................................................................II 圖目錄.............................................................................................................V 表目錄....................................................................................................................VII 縮寫表...................................................................................................................VIII 中文摘要.........................................................................................................................IX 英文摘要.........................................................................................................................XI 一、 前言..........................................................................................................................1 1-1重金屬污染........................................................................................................1 1-2重金屬對生物體的危害....................................................................................2 1-3細菌抗汞機制及其重要性................................................................................2 1-4 細菌抗汞機制的介紹.......................................................................................4 1-5 Bacillus megaterium MB1轉位子TnMERI1汞抗性操作組.........................4 1-5-1 Bacillus megaterium MB1的汞抗性操作組的介紹.................................4 1-5-2 汞抗性操作組調控因子 (MerR1) ..........................................................5 1-6 研究動機及目的...............................................................................................6 二、 材料與方法..............................................................................................................7 2-1 蛋白質表現質體之構築...................................................................................7 2-1-1 pET21b-merR1.........................................................................................7 2-1-2 PMAL-C2F-MBP....................................................................................7. 2-1-3 pET21b-merR1-MBP...............................................................................8 2-2 蛋白表現量的測試. ........................................................................................10 2-2-1 MerR1蛋白之表現...................................................................................10 2-2-2 MerR1-MBP蛋白之表現.........................................................................11 2-3 蛋白純化..........................................................................................................12 2-3-1 MerR1蛋白之純化...................................................................................12 2-3-2 MerR1-MBP的純化..................................................................................14 2-4 蛋白質濃縮與定量..........................................................................................19 2-5 蛋白質均質性測定..........................................................................................19 2-6 蛋白晶體培養..................................................................................................20 2-6-1 PCT (Pre-Crystallization Test) ..................................................................20 2-6-2 MerR1-DNA及MerR-DNA-Hg2+複合體..............................................20 2-6-3 晶體生長條件測試...................................................................................20 2-6-4 微調養晶條件...........................................................................................21 2-6-5 添加物試驗...............................................................................................21 2-7 蛋白質晶體之X-ray繞射數據的分析與收集.................................................22 2-7-1 蛋白質晶體冷凍保護 (cryo-protection) ................................................22 2-7-2 單晶繞射實驗...........................................................................................23 2-8 TopoI-Readout Assay.........................................................................................23 三、 結果.........................................................................................................................25 3-1 構築MerR1-MBP 載體..................................................................................25 3-2蛋白表現量的測試. .........................................................................................25 3-2-1 MerR1蛋白的表現....................................................................................25 3-2-2 MerR1-MBP融合蛋白的表現..................................................................25 3-3 蛋白質純化......................................................................................................26 3-3-1 MerR1 蛋白純化......................................................................................26 3-3-2 MerR1-MBP 融合蛋白純化....................................................................26 3-4 蛋白質均質性測定..........................................................................................27 3-5 蛋白質晶體培養..............................................................................................28 3-5-1 MerR1蛋白質及B3 DNA晶體培養......................................................28 3-5-2 MerR1-MBP融合蛋白晶體培養.............................................................28 3-6 TopoI readout assay.........................................................................................28 四、 討論.........................................................................................................................30 4-1 選擇MBP蛋白之分析...................................................................................30 4-2 晶體培養之探討..............................................................................................30 4-3 TopoI-Readout Assay........................................................................................31 4-4 TnMERI1汞抗性操作組調控因子MerR1和汞離子結合後 所發生的構型變化...........................................................................................32 圖.....................................................................................................................................33 表.....................................................................................................................................72 參考文獻.........................................................................................................................78 圖目錄 圖1-1汞循環圖..............................................................................................................33 圖1-2 革蘭氏陰性菌抵抗重金屬危害之示意圖.........................................................34 圖1-3 MerR 家族蛋白...................................................................................................35 圖1-4 革蘭氏陰性菌的汞抗性系統.............................................................................36 圖1-5 Bacillus megaterium MB1轉位子TnMERI1汞抗性操縱組............................37 圖1-6革蘭氏陽性菌TnMERI1汞抗性操作組的調控蛋白與其操作子/啟動子區域 (O/P)的結合能力比較.......................................................................................38 圖1-7 MerR家族蛋白之序列比對 (一) ..................................................................39 圖1-8 MerR家族蛋白之序列比對 (二) ..................................................................40 圖1-9 TnMERI1的三個啟動子/操作子 區域之序列..............................................41 圖1-10 汞抗性操作組調控因子MerR作用機制.......................................................42 圖2-1構築MerR1 表現載體:pET-21b(+)................................................................43 圖2-2 構築MBP 表現載體:pMAL-c2F..................................................................44 圖2-3蛋白質溶液過飽和曲線.....................................................................................45 圖2-4蒸氣擴散法 (Vapor diffusion) ..........................................................................46 圖2-5 TopoI-Readout Assay原理示意圖...................................................................47 圖2-6 TopoI-Readout Assay實驗之DNA序列........................................................48 圖 3-1 Colony PCR確認結果.......................................................................................49 圖 3-2 以限制酶 EcoRI 及 XhoI 進行 double digestion 之結果...........................50 圖 3-3 以限制酶 NdeI 及 XhoI 進行 double digestion 之結果.............................51 圖3-4 MerR1-MBP 融合蛋白小量表現...................................................................52 圖3-5 MerR1經親和性管柱 (Heparin-affinity column) 之層析結果....................53 圖3-6 MerR1經疏水性管柱 (Phenyl column) 之層析結果...................................54 圖3-7 MerR1經分子篩 (gel filtration) 之層析結果...............................................55 圖3-8 MerR1-MBP融合蛋白經親和性管柱 (Ni-affinity column)之層析結果......56 圖3-9 MerR1-MBP融合蛋白經親和性管柱 (MBP column)之層析結果...............57 圖3-10 MerR1-MBP融合蛋白經分子篩 (gel filtration)之層析結果......................58 圖3-11 MerR1-MBP融合蛋白經親和性管柱(Heparin-affinity column)層析結果..59 圖3-12 MerR1-MBP融合蛋白經親和性管柱 (MBP column)之層析結果……......60 圖3-13 MerR1-MBP融合蛋白經分子篩 (gel filtration)之層析結果……………...61 圖3-14 利用DLS測得MerR1-MBP 融合蛋白的均質性與粒徑大小 (一)………62 圖3-15 利用DLS測得MerR1-MBP 融合蛋白的均質性與粒徑大小 (二)……….63 圖3-16 利用DLS測得MerR1蛋白的均質性與粒徑大小…………………………64 圖3-17 4 | |
dc.language.iso | zh-TW | |
dc.title | TnMERI1汞抗性操作組轉錄調控因子MerR1之結構與功能解析 | zh_TW |
dc.title | Structural studies of transcription regulator MerR1 from mercury resistance transposon TnMERI1 | en |
dc.type | Thesis | |
dc.date.schoolyear | 102-1 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 徐駿森(Jun-Sen Hsu),黃介辰(Jie-Chen Huang) | |
dc.subject.keyword | 汞,TnMERI1,MerR1,mer操作組,X-射線繞射原理分析, | zh_TW |
dc.subject.keyword | mercury,TnMERI1,MerR1,mer operon,X-ray crystallography, | en |
dc.relation.page | 80 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2014-02-17 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 生物化學暨分子生物學研究所 | zh_TW |
顯示於系所單位: | 生物化學暨分子生物學科研究所 |
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