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研究生:陳瑩綺
研究生(外文):Ying-Chi Chen
論文名稱:E7大腸桿菌素操縱子之溶菌蛋白質基因表現調控
論文名稱(外文):Regulation of the cel gene expression of ColE7 operon
指導教授:翟建富翟建富引用關係
指導教授(外文):Kin-Fu Chak
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2003
畢業學年度:91
語文別:中文
論文頁數:106
中文關鍵詞:大腸桿菌素溶菌蛋白質轉錄終止子基因表現調控
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E7大腸桿菌素操縱子 (ColE7 operon)由cea、cei和cel三個基因所組成,其中cel基因可轉錄、轉譯出溶菌蛋白質 (LysE7),其作用是使細胞膜的通透性增加,以幫助大腸桿菌素複合體分泌至菌體外毒殺敏感細菌,但是過多的溶菌蛋白質表現將會造成菌體自身的溶解死亡,所以菌體如何調控這種對自身而言具有危險性的蛋白質表現是值得深入研究探討的。cel基因是目前在ColE7操縱子中,唯一尚未發現具有啟動子的基因,其基因表現必須藉由上游啟動子的連讀表現 (read through expression),並且可能會受到轉錄終止子T1幹環 (stem-loop)結構影響。本實驗室之前推測當ceiE7-mRNA上發生斷裂後,cei基因在無法完全轉譯的狀況下,位於cei基因與cel基因間的轉錄終止子T1幹環結構會影響cel的核醣體結合,而抑制溶菌蛋白質的合成,也就是藉著ceiE7-mRNA的特異定點切割作用機制來調控cel基因的表現。本論文即針對此推測,建構一系列重組質體以探討調控cel基因表現之機制。
我的實驗結果修訂了原本實驗室對溶菌蛋白質基因celE7調控機制的推測,發現溶菌蛋白質基因的轉譯表現並非完全由ceiE7-mRNA的特異定點切割現象單一因素所主導。目前研究結果顯示,「ceiE7-mRNA的特異定點切割現象」以及「T1 幹環序列的正確性」這兩個因素皆會影響溶菌蛋白質的轉譯表現,且後者的影響較大; 為了進一步確認這個假說,藉由插入核苷酸序列拉開T1 幹環與cel基因的核醣體結合位置之實驗,得知T1幹環結構的確可以調控溶菌蛋白質基因的轉譯表現。這顯示此結構對溶菌蛋白質基因的表現為一重要的調控因素,且環區域 (loop region)序列的改變就能對溶菌蛋白質基因的轉譯表現造成重大之影響。
根據以上實驗結果更進一步推測轉錄終止子T1可能提供一特殊的辨認結構,讓菌體內的RNA結合蛋白質或RNA 解旋酶 (helicase),這類具有鬆解RNA複雜結構活性的分子,與T1幹環的環區域或相關的區域結合,而將其二級結構解開,讓核醣體能順利地結合至核醣體結合位置,進行溶菌蛋白質基因的轉譯表現而達到調控基因表現的目的。為了找尋T1幹環的結合蛋白質,分別在含有或缺乏免疫蛋白質Im7的E. coli中製備S-30 cell free extracts; 同時以Im7為餌,與細胞質進行互動作用後以不同清洗條件所得之pull-down protein fractions,利用EMSA (electrophoretic mobility shift assay)與蛋白質體學技術進行分析。結果發現會與RNA結合產生band shifting的蛋白質可能與Im7沒有相關,並且它們與RNA的結合可能是在非環區域的位置,至於細胞質中何種蛋白質能有解開T1幹環結構的作用?這還有待進一步的以蛋白質體學技術加以分析。
The ColE7 operon comprises the cea, cei, and cel genes. LysE7 protein, the cel gene product, alters the permeability of the outer membrane of Escherichia coli and facilitates colicin-Im7 complex release from the cell. Because of the overexpression of cel is lethal to the host, the regulation of cel expression must be controlled rigidly. celE7 is the third gene of the ColE7 operon and is promoterless, and its regulation of expression of celE7 is still unclear. Our previous studies speculate that the cleavage of the ceiE7-mRNA and the putative T1 stem-loop structure located between the ceiE7 and celE7 genes may inhibit the translational expression of cel gene. Thus, in this work, a serial of pQE70-cei-celHis plasmids was constructed to further investigate the regulation of celE7 expression.
The data revealed that the translational expression of celE7 gene is probably controlled not only by the cleavage of the ceiE7-mRNA but also by the special structure of T1 stem-loop. Both site-directed mutagenesis and insertion mutant elucidated that T1 stem-loop structure plays an important role for regulating the expression of cel gene. Furthermore, we observed that two consecutive GG nucleotides at the topmost region of the putative T1 stem-loop structure are crucial for the expression of cel gene.
Based on these results, we further suggest that the T1 stem-loop structure may offer a recognition and binding site to recruit some unknown RNA binding proteins, like RNA helicase, for unwinding this secondary structure. In order to identify the RNA binding proteins, I prepared S-30 cell free extracts from E. coli and the His-tagged Im7 pull-down protein fractions with mRNA transcribed from cei-cel genes for EMSA (electrophoretic mobility shifting assay). The EMSA results indicated that the proteins bind to the mRNA either with or without Im7 proteins. The results further indicated that the binding of proteins is unrelated to the loop region of the T1 stem-loop structure. It is therefore, the specific proteins for unwinding the T1 stem-loop structure still remains to be resolved.
中文摘要………………………………………………………………………………i
英文摘要……………………………………………………………………………iii
壹、緒論…………………………………………………………………………1 ~ 10
1.1 背景介紹……………………………………………………………………1
1.2 大腸桿菌素E7之介紹………………………………………………………2
1.2.1 大腸桿菌素E7操縱子基因介紹…………………………....……......2
1.2.2 大腸桿菌素之殺菌機制………..………………….……...………....…4
1.3 大腸桿菌素E7操縱子基因表現之調控機制………….……………………6
1.3.1 SOS Response對ColE7 operon表現之調控……………….……….…6
1.3.2 轉錄終止子對ColE7 operon表現之調控…………………..…………7
1.3.3 免疫基因mRNA上特異定點斷裂現象….………………………….. 8
1.4 本論文研究的目的………………………….……...……………...……....…9
貳、實驗材料與方法…………………………………………………………..11 ~ 40
2.1 實驗材料……………………………………………………………………11
2.1.1 細菌菌株………………………………………………………………11
2.1.2 質體及其建構…………………………………………………………11
2.2 實驗方法…………………………………………………………………….16
2.2.1 細菌的培養……………………………………………………………16
2.2.2 大腸桿菌中質體DNA的製備………………………………………..17
2.2.3 聚合酶鏈鎖反應………………………………………………………18
2.2.4 聚合酶鏈鎖反應定位突變法…………………………………………18
2.2.5 DNA片段的純化……………………………………………………..19
2.2.6 限制酶的切割及DNA的連接………………………………………20
2.2.7 細菌的轉型作用………………………………………………………21
2.2.8 選殖菌株之快速篩選…………………………………………………22
2.2.9 蛋白質電泳……………………………………………………………23
2.2.10 西方墨點轉漬分析法…………………………………………………25
2.2.11 活體外轉錄反應………………………………………………………26
2.2.12 S-30 cell free抽出物之製備……...………………………………...27
2.2.13 Electrophoretic mobility shift assay (EMSA) ……………………….28
2.2.14 活體外RNA切割活性分析法……...……………….……………….29
2.2.15 引子延伸作用…………………………………………………………30
2.2.16 電泳……………………………………………………………………31
2.2.17 以Biotin標幟活體外轉錄RNA探討與RNA作用的蛋白質………32
2.2.18 免疫蛋白質Im7相關的蛋白質純化…………………………………33
2.2.19 蛋白質含量之測定……………………………………………………35
2.2.20 二維電白質電泳………………………………………………………36
2.2.21 傳統銀染法……………………………………………………………37
2.2.22 蛋白質電泳膠內切割…………………………………………………38
2.2.23 MALDI-TOF基本原理與流程說明………………………………...39
2.2.24 RNA二級結構分析軟體……………………………………………40
參、實驗結果與討論…………………………………………………………41 ~ 67
3.1 影響溶菌蛋白質基因轉譯表現因素之探討……………………………….41
3.1.1 研究目的與策略………………………………………………………41
3.1.2 實驗結果………………………………………………………………42
I. 以電腦軟體預測轉錄終止子T1之結構及其穩定度…………..42
II. 探討ceiE7-mRNA特異定點切割現象與T1幹環結構對celE7轉譯表現影響之質體建構……………………………………...43
III. 觀察含有pQE70-cei-celHis系列重組質體之E. coli M15菌株之生長情形……………………………………...............................44
IV. 以西方墨點轉漬分析法觀察pQE70-cei-celHis系列重組質體菌株之溶菌蛋白質表現情形………………………………….......45
3.1.3 討論……………………………………………………………………46
3.2 深入探討T1幹環此二級結構對溶菌蛋白質基因轉譯表現之影響……...48
3.2.1 研究目的與策略………………………………………………………48
3.2.2 實驗結果………………………………………………………………49
I. 建構以二十個核苷酸序列插入的pQE70-cei-celHis重組質體.49
II. 含有二十個核苷酸序列插入的pQE70-cei-celHis重組質體之E. coli M15菌株以IPTG誘導基因表現之生長曲線……………..50
III. 以西方墨點轉漬分析法觀察含有二十個核苷酸序列插入的pQE70-cei-celHis重組質體之溶菌蛋白質表現情形…………..51
3.2.3 討論……………………………………………………………………52
3.3 尋找調控T1幹環之蛋白質………………………………………………...54
3.3.1 研究目的與策略………………………………………………………54
3.3.2 實驗結果………………………………………………………………56
3.3.2.1 以pYAN13R與pGEM-3Zf(+) S-30 extracts進行之實驗結果……56
I. 利用活體外RNA切割活性測試以分析ceiE7-mRNA的特異切割現象…………………………………………………………...56
II. 以EMSA分析S-30 cell free extracts與活體外轉錄mRNA的交互作用…………………………………………………………...57
III. 以專一性競爭轉錄物進行EMSA分析S-30 cell free extracts與活體外轉錄mRNA之專一性結合作用……………………….57
IV. 以Biotin標幟活體外轉錄RNA探討與RNA作用的蛋白質…58
3.3.2.2 以免疫蛋白質為釣餌所分離純化的pull-down protein fractions進行之實驗結果………………………………………………………....60
I. 以EMSA分析以不同鹽濃度的清洗條件所得之pull-down protein fractions與活體外轉錄mRNA的交互作用……………60
II. 以EMSA分析以不同imidazole濃度的清洗條件所得之pull-down protein fractions與活體外轉錄mRNA的交互作用..61
III. 將不同清洗條件所得之pull-down protein fractions以二維電白質電泳法分析………………………………………...................63
IV. 以MALDI-TOF質譜儀分析二維電白質電泳膠片上之未知蛋白質………………………………………………………...............64
3.3.3 討論……………………………………………………………………64
3.4 綜合討論…………………………………………………………………….66
肆、圖表……………………………………………………………………....68 ~ 102
伍、參考文獻…………………………………………………………………103 ~ 106
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