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研究生:陳盟靜
研究生(外文):Chen Mengching
論文名稱:以酵母菌雙雜交系統分析大腸桿菌ClpYQ蛋白酶中ClpQ/ClpQ次單元體的互相作用並尋找和ClpY互相作用的分子
論文名稱(外文):An analysis of subunit interaction of ClpQ/ClpQ molecules and searching for interactive molecules of ClpYQ protease in E.coliby using yeast two-hybrid system
指導教授:吳蕙芬
指導教授(外文):Whi-Fin Wu
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:農業化學研究所
學門:農業科學學門
學類:農業化學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:100
中文關鍵詞:ClpYQ蛋白酶酵母菌雙雜交系統大腸桿菌
外文關鍵詞:ClpYQ proteaseyeast two-hybrid systemE.coli
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大腸桿菌(Eschericia coli)的ClpYQ蛋白酶屬於熱休克蛋白(heat shock protein)的一種,ClpYQ蛋白酶是由ClpY與ClpQ兩種次單元體所組成,分子量分別為49kDa與19kDa。
由前人的研究已知ClpY與ClpQ在菌體內會各自形成六元環(hexamer ring),再組合成完整的作用結構,本實驗以酵母菌雙雜交系統(Yeast two hybrid system)分析ClpQ單體與ClpQ單體互相作用的情形,發現第133個胺基酸Alanine突變成Threonine,造成ClpQ單體間的作用下降,進一步利用ClpQ的缺失變異(deletion),確認ClpQ單體間的作用區(domain)在羧基端。
ClpYQ蛋白酶分解基質時,是由ClpY次單元體負責基質的辨認與結合,ClpQ再將基質分解。我建構大腸桿菌的基因庫(genomic library),利用ClpY當作餌,篩選會和ClpY互相作用的分子,篩選到編號lib 1-36,轉譯出77胺基酸的片段可以和ClpY互相作用,但仍須做進一步研究其在菌體內的open reading frame。
為了確認ClpY與基質互相作用的功能區(domain),以利篩選,使用ClpY的缺失變異與次要基質RcsA的作用,發現ClpY與基質的作用在PDZ及I domain。

The E.coli ClpYQ protease are consisted of ClpY and ClpQ, and are heat shock proteins. The large subunit, ClpY, is 49 kDa; and the small subunit, ClpQ is 19 kDa.
The ClpY and ClpQ individually form identical homo-ring structures in vitro and assemble into a four ring ClpYQ complex. I used the yeast two-hybrid methods to analyze an interaction domain within ClpQ molecules. The ClpQ 133th amino acid alanine mutated to threonine decreases the interaction. The results from assays of the interactions between the ClpQ deletion mutant and itself, suggest that the C domain is responsible for the interaction.
RcsA is one substrate of the ClpYQ proteas. The ClpY subunit is responsible for substrate recognition and binding. Use the ClpY as a bait to screen E.coli genomic library. It came out the library no. 1-36 which is translated 77 amino acid and show an interaction with ClpY strongly. More study is needed to identify that there is an ORF of lib 1-36 in the cell.
To confirm an interaction domain of ClpY with RcsA, I tested an interaction between the ClpY deletion mutants and RcsA. The data showed the PDZ and I domains of ClpY are involved in substrates recognitions.

壹、前言......................... 1
一、 ClpYQ的簡介 ................... 1
(一) 大腸桿菌clpQclpY操作子 (operon) 的發現......1
(二) ClpYQ的功能.................... 2
1. ClpYQ是蛋白酶................. 2
2. ClpYQ屬於two-component protease........ 2
(三) ClpYQ蛋白酶的結構與功能.............. 4
1. ClpYQ complex................. 4
2. ATP對於ClpYQ複合體的影響........... 6
a.蛋白分解活性的表現.............. 6
b.ClpY聚合蛋白 (oligomer) 的形成........ 7
3. ClpY辨認基質區域的研究.............7
(四) ClpYQ蛋白酶的作用基質............... 8
二、酵母菌雙雜交系統的原理簡介 ............ 9
(一) 酵母菌雙雜交系統 (yeast two-hybrid system) 的原理. 9
(二) 酵母菌雙雜交系統 (yeast two-hybrid system) 的發展. 11
1. GAL4酵母菌雙雜交系統............11
2. LexA酵母菌雙雜交系統............11
三、研究目的 ..................... 13
貳、材料與方法 .................... 14
一、研究材料 ..................... 14
1.大腸桿菌................... 14
2.LexA酵母菌雙雜交系統............. 14
二、 研究方法 ..................... 16
1.基因選殖...................... 16
(1)聚合酶連鎖反應................. 16
(2)限制酶作用................... 17
(3)DNA分子的接合作用................ 17
(4)轉形作用.................... 17
(5)選殖質體的確認................. 18
2.質體的萃取..................... 18
從大腸桿菌中抽取質體
(1) 小量製備................... 18
(2) 大量製備................... 19
從酵母菌中抽取質體
Lysis/glass beads................. 19
3.轉形法........................ 20
大腸桿菌轉形法
(1)熱處理轉形法................. 20
(2)電穿孔轉形法................. 21
(3)TSS法..................... 22
酵母菌轉形法
(1)高效率轉形法................ 22
(2)大規模熱休克轉形法.............. 22
4.建構clpQ突變株................... 23 (1)Hydroxylamine Mutagenesis of plasmid DNA..23
(2)Error-prone PCR............... 24
5.報導基因的偵測.................... 24
(1) LEU2表現的測試............... 24
(2) LacZ表現的偵測............... 24
6.西方式免疫轉印法.................. 26
(1)抽取酵母菌蛋白質樣本............ 26
(2)以SDS-PAGE分離蛋白質............ 26
(3)蛋白質的轉印................ 26
(4)免疫呈色反應................ 27
7.北方式免疫轉印法.................. 28
(1) 抽取大腸桿菌total RNA .......... 28
(2) RNA電泳.................. 29
(3) 北方轉漬法 ................ 29
參、結果 ......................... 32
一、ClpQ與ClpQ單體間互相作用的區段
(一) ClpQ突變株篩選的結果............... 32
(二) 由ClpQ的結構預測ClpQ/ClpQ互相作用的區段..... 33
(三) 選殖質體的建構.................. 33
(四) ClpQ/ClpQ互相作用區段的確認............ 34
1.篩選所得的突變株(5Q, 3W) Q/Q間互相作用
的報導基因活性表現................. 34
2.在ClpQ缺失變異株(deletion mutation)
Q/Q間互相作用時,報導基因的活性表現....... 34
二、E.coli ClpQ與 B. subtilis ClpQ辨識作用區的確認 
1.E. coli ClpQ和 B. subtilis ClpQ的相互作用..... 35
2.20種微生物和ClpQ相似序列的比對 ......... 35
3.ClpQ PO和野生型ClpQ之間相互作用時,
報導基因活性的測試....... 36
4.B. subtilis Q/Q互相作用的區段及融合蛋白的相互作用. 36
三、以ClpY篩選大腸桿菌基因庫的結果 ......... 37
四、ClpY次單元體與基質RcsA作用區段的確認 ..... 38
肆、討論 ........................ 39
一、ClpQ/ClpQ單體間相互作用之結構模式 ....... 39
二、E.coli ClpQ
與B. subtilis ClpQ辨識作用之結構模式 ..... 40
三、lib 1-36 的Open reading frame ......... 40
四、ClpY辨認基質的作用區 ............... 41
五、實驗系統的討論 .................. 42
伍、參考文獻 ..................... 43
附錄表 ..........................I
表.............................II
附錄圖 .......................... i
圖 ............................. ix

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