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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 吳蕙芬(Whi-Fin Wu) | |
dc.contributor.author | Hsiang-Yun Lien | en |
dc.contributor.author | 連湘芸 | zh_TW |
dc.date.accessioned | 2021-05-20T20:16:34Z | - |
dc.date.available | 2009-07-14 | |
dc.date.available | 2021-05-20T20:16:34Z | - |
dc.date.copyright | 2009-07-14 | |
dc.date.issued | 2009 | |
dc.date.submitted | 2009-07-06 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/9300 | - |
dc.description.abstract | 熱休克蛋白普遍存在於生物體內,作用為幫助生物抵抗環境衝擊。本文針對大腸桿菌熱休克蛋白ClpYQ (HslUV)的基因調控及基質辨識深入研究。遇到環境衝擊時,大腸桿菌會發生熱休克反應,此時折疊錯誤的蛋白促使細胞內熱休克基因表現,這些表現常由調控蛋白sigma factor,如RpoH (或稱σ32) 所調控。本文使用大腸桿菌熱休克基因clpQ+clpY+的啟動子與報導基因lacZ建構轉錄融合基因clpQ+::lacZ (op)及轉譯融合基因clpQ+::lacZ (pr),利用噬菌體λRS45將融合基因clpQ+::lacZ帶入野生株及rpoS -,rpoH -,rpoH -rpoS -突變株後,偵測β-galactosi dase活性以瞭解clpQ+::lacZ的表現。當溫度由30℃升至42℃時,野生株及rpoS -突變株的β-galactosidase活性上升,但rpoH -及rpoH -rpoS -突變株則未見此現象。由β-galactosidase 活性分析與北方點墨法分析結果,可知clpQ+::lacZ轉錄的mRNA訊號強度與β-galactosidase活性成正比,且clpQ+::lacZ與clpQ+clpY+的表現相似。針對clpQ+clpY+啟動子上σ32 (rpoH) 可辨識的保守序列做C→T點突變,此突變使融合基因clpQm(c→t)::lacZ無法被σ32活化,β-galactosidase活性下降。經由遺傳分析結果證實,大腸桿菌clpQ+clpY+的啟動子可被σ32辨識。此外,clpQ+clpY+operon的五端未轉譯區域 (5’-UTR),亦即轉錄起始處 (transcription start site) 與起始密碼 (start codon) 之間,長度為71 bp,此五端未轉譯區域帶有一段inverted repeat sequence (IR序列) 5’ CC CCGTAC TTTTGTACGGGG 3’,此IR序列普遍存在於腸道菌的clpQ+clpY+五端未轉譯區域中。藉由刪除此段IR序列,並與lacZ融合,分析融合基因clpQm2△40bp::lacZ的β-galactosidase 活性,以及此段序列缺失對ClpQ與ClpY間交互作用的影響,顯示IR所形成的stem-loop二級結構在clpQ+clpY+表現時,具有穩定mRNA的效果,本研究為ATP依賴型蛋白酶 (ATP-dependent protease)中,首次發現5’ stem-loop結構具有穩定下游mRNA的功能。在基質辨識的研究部分,ClpYQ以六元環方式組合,其中ClpY負責基質辨識,打開基質結構,並傳送至ClpQ進行分解。ClpY可分為三個作用區(domain),N-terminal domain,I-intermediated domain及C-terminal domain,N domain具有ATPase的功能,C domain則與self-oligomerization及ClpQ的蛋白酶活性相關。本文使用酵母菌雙雜交系統,得知ClpY的I domain負責基質辨識,C domain則可與ClpQ作用,而I domain中的loop L2(175-209 aa)除了與基質結合外,並與後續的基質傳遞及分解相關。另外,本文亦研究大腸桿菌的酵素glutathionylspermidine synthetase之基因gspS,由於病原蟲Trypanosomatida會造成人類昏睡,發炎及死亡,而酵素TryS為此種病原蟲所特有,在人類細胞中不存在,所以TryS的研究對治療Trypanosomatida所造成的疾病極為重要,本文針對大腸桿菌中與TryS構造及功能皆相似的酵素GspS,其基因表現之調控做一初步的探討。大腸桿菌的gspS 長1860 bp,產物為glutathionylspermidine synthetase (GspS),共有619個胺基酸,是一種具有雙重功能的酵素 (bifunctional enzyme),可執行GSH和spermidine之間醯胺鍵的合成與分解。本研究確認gspS為一單獨的轉錄單位而非以操縱子形式存在,且GspS起始密碼上游序列具有啟動子功能,而在in vivo情況下,H2O2與BaeR都可誘導gspS之表現。 | zh_TW |
dc.description.abstract | (1) Heat shock responses are typically observed in E. coli. Upon heat shock, protein misfolding leads to a cascade of intracellular protein synthesis, usually dependent on a sigma factor, i.e., σ32, for their gene expression. In this study, the transcriptional (op) or translational (pr) clpQ+::lacZ fusion gene was constructed, with the clpQ+clpY+ promoter fused to a lacZ reporter gene. The clpQ+::lacZ (op or pr) fusion gene was each crossed into lambda phage. The λclpQ+::lac (op), a transcriptional fusion gene, was used to form lysogens in the wild-type, rpoH - or/and rpoS - mutants. Upon shifting the temperature up from 30 ℃ to 42 ℃, the wild-type λclpQ+::lacZ(op) demonstrates an increased β-galactosidase activity. However, the β-galactosidase activity of clpQ+::lacZ(op) was decreased in the rpoH - and rpoH -rpoS - mutants but not in the rpoS - mutant. The levels of clpQ+::lacZ mRNA transcripts correlated well to their β-galactosidase activity. Similarly, the expression of the clpQ+::lacZ gene fusion was nearly identical to the clpQ+clpY+ transcript under the in vivo condition. The clpQm(c→t)::lacZ, containing a C to T point mutation in the -10 promoter region for RpoH binding, showed decreased β-galactosidase activity, independent of activation by RpoH. Thus, through a genetic analysis, the clpQ+clpY+ promoter is in vivo recognized by σ32. The transcriptional start point of the clpQ+clpY+ gene lies 71 bases upstream from the clpQ+ start codon. An untranslated region (UTR) upstream of this mRNA contains a 20 bp inverted repeat (IR) sequence 5’CCCCGTACTTTTGTAC GGGG3’, which is unique for the clpQ+clpY+ operon. In addition, from the wild bacterial genome, the 5’UTR of clpQ+clpY+ also exists in other bacterial species. The clpQ+clpY+ message carries a conserved 71 bp at the 5’ untranslated region (5’UTR) that is predicted to form the stem-loop structure by analysis of its RNA secondary structure. The clpQm2△40bp::lacZ, with a 40 bp deletion in the 5’UTR, showed a decreased β-galactosidase activity. In addition, from our results, it is suggested that this stem-loop structure is necessary for the stability of the clpQ+clpY+ message. It is noteworthy that this is the first example in the ATP dependent protease to demonstrate that the 5’ stem-loop structure itself participates in the stability of its downstream mRNA. (2) Regarding ClpY substrate recognition study, in the presence of ATP, the ClpYQ complex forms an active protease with an Y6Q6Q6Y6 configuration. ClpY binds, unfolds, and transfers the substrates outside the cylinder into a catalytic core where ClpQ degrades the substrates. The ClpY molecule is divided into three domains: the N-terminal domain, I-intermediate domain and C-terminal domain. The N domain has an ATPase activity, and the C domain is responsible for self-oligomerization of ClpY. Using the yeast two-hybrid system, we show that domain I of ClpY is responsible for recognition of its natural substrates while domain C is necessary for association with ClpQ. The loop 175-209 aa plays a role in substrate tethering. (3) In addition to clpQ+clpY+, gspS+ in Escherichia coli is included in this study. Parasitic Trypanosoma species cause serious tropical diseases such as kala-azar, African sleeping sickness, and Chagas diseases. Trypanothione synthetase (TryS) is a protein unique to Trypanosoma. However, Escherichia coli produce only the metabolic intermediate GspdSH by enzyme GspS, but not trypanothione. Evolutionary, TryS and GspS share the similarly functional domains. The gspS of E. coli, encoding a bifunctional enzyme GspS of 619 amino acids, is a gene with 1860 bp. GspS is responsible for the activities of amidase and synthetase between GSH and spermidine.In this study, we showed that gspS in E. coli is an unique transcriptional unit, and the singular promoter was present in the upstream region of the GspS structural gene. In addition, the gspS promoter is in vivo induced by H2O2 and BaeR. | en |
dc.description.provenance | Made available in DSpace on 2021-05-20T20:16:34Z (GMT). No. of bitstreams: 1 ntu-98-D92623005-1.pdf: 1670646 bytes, checksum: 7497818137ceaa8971ba2086895226bc (MD5) Previous issue date: 2009 | en |
dc.description.tableofcontents | 目錄
誌謝 i 摘要 ii Abstract iv 目錄 vii 表目錄 x 圖目錄 xi 已發表文章 xii 第一章 前言 1 1.1 clpQ+clpY+研究源起 1 1.2 熱休克蛋白 (heat shock proteins) 與ATP依賴型蛋白酶 (ATP-dependent protease) 簡介 1 1.3 ClpYQ蛋白酶之簡介 2 1.3.1 clpQ+clpY+操縱子 (operon) 的發現及命名 2 1.3.2 ClpYQ為熱休克蛋白的證據 3 1.4 ClpYQ為ATP依賴型蛋白酶之研究 3 1.4.1 ClpY為ATPase之研究 3 1.4.2 ClpY具chaperone功能之研究 4 1.4.3 ClpYQ蛋白酶為Threonine蛋白酶 5 1.4.4 ClpYQ蛋白酶為ATP依賴型蛋白酶 6 1.5 ClpYQ蛋白複合體的結構 7 1.5.1 ClpQ及ClpY可形成複合體 7 1.5.2 由蛋白分子量推測ClpYQ蛋白複合體結構 7 1.5.3 由電顯影像分析ClpYQ蛋白複合體結構 8 1.6 ClpYQ蛋白酶基質辨識區域之相關研究 9 1.7 ClpYQ蛋白酶基質之研究 10 1.7.1 ClpYQ蛋白酶可分解異蛋白 (abnormal proteins) 10 1.7.2 ClpYQ蛋白酶可分解SulA 10 1.7.3 ClpYQ蛋白酶可分解RcsA 12 1.8 clpQ+clpY+實驗目的 12 1.9 gspS+研究源起 13 1.10 GspdSH簡介 14 1.10.1 GspdSH組成物質: GSH及spermidine 14 1.10.2 大腸桿菌中GspdSH及其合成酵素GspS相關之研究 15 1.11 雙成份控制系統BaeSR之研究 17 1.12 gspS+實驗目的 19 第二章 調控蛋白sigma factors對clpQ+clpY+表現的影響 21 2.1 摘要 21 2.2 材料與方法 21 2.2.1 建立clpQ+clpY+上游啟動子區域 ( promoter region ) 表現系統 21 2.2.2 β-galactosidase活性分析 24 2.2.3 北方點墨法 (Northern blotting) 25 2.2.4 引子延伸實驗 (primer extension) 26 2.3 結果 28 2.3.1 clpQ+::lacZ轉錄及轉譯融合基因的表現 28 2.3.2 RpoH對clpQ+::lacZ基因表現的影響 28 2.3.3 clpQm1::lacZ啟動子上的點突變對clpQm1::lacZ基因表現的影響 31 2.3.4 clpQm(c→t)::lacZ及clpQm1::lacZ啟動子上的點突變不影響轉錄起始點 32 2.3.5 clpQ+clpY+ 操縱子 (operon) 的mRNA表現與clpQ+::lacZ的β-galactosidase活性 32 2.3.6 不同蛋白酶缺失對clpQ+::lacZ的影響 33 2.4 討論 34 第三章 clpQ+clpY+ 啟動子五端未轉譯區域 (5’-UTR) 對ClpQ表現的影響 36 3.1 摘要 36 3.2 材料與方法 36 3.2.1 建立AC3112(cpsB::lacZ)/pBAD33-clpQ/pBAD24-clpY表現系統 36 3.2.2 AC3112/pBAD33-clpQ/pBAD24-clpY中,ClpYQ對兩種基質RcsA及SulA的分解能力測試 38 3.2.3 β-galactosidase活性分析 39 3.2.4 北方點墨法 (Northern blotting) 39 3.2.5 mRNA穩定性測試 41 3.2.6 西方點墨法 (Western blotting) 41 3.2.7 預測RNA二級結構所使用的網站 43 3.3 結果 43 3.3.1 clpQ+clpY+ 啟動子的五端未轉譯區域 (5’-UTR) 對clpQ+::lacZ表現的影響 43 3.3.2 五端未轉譯區域 (5’-UTR) 中的IR序列對clpQ+ clpY+表現的影響 44 3.3.3 IR序列所形成的stem-loop結構對clpQ+ clpY+表現的影響 46 3.4 討論 47 第四章 ClpY基質辨識位置之探討 49 4.1 摘要 49 4.2 材料與方法 49 4.2.1 酵母菌雙雜交系統的菌株、載體及培養基 49 4.2.2 leu2 expression : 生長測試 50 4.2.3 lacZ expression:X-gal測試 50 4.2.4 lacZ expression:b-galactosidase活性分析 51 4.2.5 ClpY及其突變分解基質之偵測 52 4.3 結果 52 4.3.1 ClpY基質辨識:ClpY△I+7Gly及ClpY△L1, △L2與SulA之間沒有交互作用 52 4.3.2 ClpY基質辨識: loopL2上的點突變ClpYL199Q造成ClpYL199Q/ClpQ無法分解基質 53 4.3.3 ClpY基質辨識:ClpY的基質辨識與loopL2上的疏水性胺基酸密切相關 53 4.3.4 ClpY與ClpQ之交互作用: ClpY△L1,ClpY△L2,ClpY△L1, △L2及ClpY△I+7Gly能與ClpQE61C結合,ClpY△c,ClpYX則否 54 4.4 討論 55 第五章 大腸桿菌gspS基因表現之調控 56 5.1 摘要 56 5.2 材料與方法 56 5.2.1 建立gspS上游啟動子區域表現系統 56 5.2.2 β-galactosidase活性分析 58 5.2.3 預測啟動子、密碼子及操縱子所使用的網站 58 5.3 結果 59 5.3.1 gspS 上游序列之探討 59 5.3.2 影響gspS表現的因素 64 5.4 討論 67 參考文獻 69 表目錄 表一、使用於clpQ+clpY+之研究的菌株 76 表二、使用於clpQ+clpY+之研究的載體與噬菌體 77 表三、使用於clpQ+clpY+之研究的引子 78 表四、使用於gspS之研究的菌株 79 表五、使用於gspS之研究的載體與噬菌體 80 表六、使用於gspS之研究的引子 81 表七、操縱子預測結果 82 表八、SG20250/pRS415-gspS-lacZ(op)與SG20250/λRS45-gspS-lacZ的β-galactosidase活性分析 83 表九、BaeR對gspS-lacZ表現之影響 84 表十、BaeR與H2O2對gspS-lacZ表現的影響 85 圖目錄 圖一、clpQ+ clpY+操縱子的啟動子區域之序列圖示 86 圖二、不同長度的轉錄及轉譯融合基因clpQ+clpY+::lacZ之活性 87 圖三、wt, rpoS -, rpoH -, rpoS –rpoH –四種菌株內,42 ℃對clpQ+::lacZ的熱誘導 88 圖四、42 ℃對clpQ+::lacZ熱誘導受啟動子點突變(C→T)的影響 89 圖五、啟動子點突變 (A→C) 對clpQm1::lacZ表現的影響 90 圖六、clpQ+啟動子點突變對轉錄起始點(+1)沒有影響 91 圖七、clpQ+::lacZ 與clpQ+clpY+在30℃時的mRNA表現 92 圖八、蛋白酶缺失對clpQ+::lacZ表現的影響 93 圖九、去除5’-UTR對clpQm2△40bp::lacZ表現的影響 94 圖十、五端未轉譯區域 (5’-UTR) 中的IR序列對clpQ+ clpY+表現的影響 95 圖十一、IR序列對ClpQ/ClpY 交互作用之影響 96 圖十二、IR 序列所形成的stem-loop結構對clpQ+ clpY+表現的影響 97 圖十三、不同菌種間clpQ+ clpY+五端未轉譯區域(5’-UTR)保守性序列之比對 98 圖十四、ClpY三個主要作用區示意圖 99 圖十五、ClpY的loop L1與loop L2與SulA間交互作用之結果。 100 圖十六、四組ClpY點突變M187I,A188S,L199Q,N205K與基質結合及分解基質能力 101 圖十七、三組ClpY突變I186N,E193L,E194L,Q198L,Q200L與基質結合及分解基質能力 102 圖十八、帶有ClpQE61C/ClpY融合蛋白的EGY48/p8op-lacZ,其Leu2的表現 103 圖十九、gspS 上游區域之啟動子預測 104 圖二十、gspS上游區域的編碼區預測 105 圖二十一、啟動子與編碼區預測結果比較 106 圖二十二、編碼區與操縱子預測結果比較 107 圖二十三、gspS-lacZ融合基因之建構 108 圖二十四、H2O2對gspS-lacZ表現的影響 109 圖二十五、BaeR與H2O2對HY1002(gspS-lacZ)的生長狀況之影響 110 圖二十六、H2O2對HY1002(gspS-lacZ)的生長狀況之影響 111 附圖一、大腸桿菌 ClpYQ 結構圖 112 | |
dc.language.iso | zh-TW | |
dc.title | 大腸桿菌clpQ+clpY+及gspS+基因之研究:基因之調控及其基質辨識 | zh_TW |
dc.title | Investigation of clpQ+clpY+ and gspS+ in Escherichia coli: gene regulation and substrate recognition | en |
dc.type | Thesis | |
dc.date.schoolyear | 97-2 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 楊盛行,陳昭瑩,杜宜殷,張傳雄,張世宗 | |
dc.subject.keyword | 大腸桿菌,熱休克蛋白,ATP依賴型蛋白酶,ClpYQ(HslUV),啟動子活性,基因調控,RNA二級結構,mRNA穩定性,基質辨識,麩氨基硫─精胺質合成酶,。, | zh_TW |
dc.subject.keyword | Escherichia coli,heat shock,ATP dependent protease ClpYQ(HslUV),promoter activity,gene regulation,mRNA stability,substrate recognition,glutathionylspermidine synthetase (GspS), | en |
dc.relation.page | 112 | |
dc.rights.note | 同意授權(全球公開) | |
dc.date.accepted | 2009-07-06 | |
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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