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研究生:施如珊
研究生(外文):Ru-Shan Shy
論文名稱:篩選大腸桿菌熱休克蛋白ClpYIdomain之突變蛋白與其辨識專一性基質之研究
論文名稱(外文):The mutantion studies of ClpY I domain and idenitification of its function for specific substrates recognition
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:2004
畢業學年度:92
語文別:中文
論文頁數:76
中文關鍵詞:辨識區域熱休克蛋白大腸桿菌
外文關鍵詞:RpoHClpYloopRcsAI domainHslUSulAmutation
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ATP-dependent protease普遍存在於細菌、古細菌及真核細胞中。這一類型的蛋白酶在細胞中主要弁酮偕�持調節蛋白在細胞當中的量,分解細胞內不正常堆積之蛋白質或將摺疊錯誤的蛋白質重新調整回正常構形。ClpQY蛋白酶為ATP-dependent protease 中的一種,由ClpQ(19kDa)及ClpY(49kDa)構成。其中ClpY有著辨識基質、將基質打開及送往ClpQ的弁遄A其具有水解ATP的能力。關於ClpY是如何辨識基質及與其如何作用,機制目前未知。根據先前的研究指出,ClpY與基質的辨識結合區有兩種不同的說法,一為根據Clp家族蛋白酶的序列比對,認為基質辨識區位於SSD( Sensor and Substrate Discrimination Domain )上;另一則是根據ClpQY的結晶結構,推測基質辨識區位於I domain( Intermideate Domain )上。近來有研究指出ClpY之C端的弁酮飢峖角誘衙暺P活化ClpQ。
於本研究中,利用酵母菌雙雜交系統挑選出能與ClpY基質作用之ClpY突變蛋白,並進一步分析在酵母菌及大腸桿菌系統中,先前所挑選出的突變蛋白與專一性基質SulA、RcsA的作用。在酵母菌系統中,利用lacZ gene與leu2 gene expression測試ClpY突變蛋白與基質的辨識、結合作用;在大腸桿菌系統中,利用MMS test及cps-lacZ expression test進一步了解突變蛋白辨識、分解基質及影響細胞生長的能力。本論文在大腸桿菌AC3112系統進行MMS test及cps-lacZ expression test,發現:突變蛋白ClpY ∆I domain及∆I domain loop(175-209)會使ClpY無法與基質作用,此意味著ClpY I domain及 I domain loop(175-209)對於ClpY辨識專一性基質是相當重要的。另外,經由挑選出的ClpY突變蛋白發現 I domain loop(175-209)上的突變M187I、A188S、S197R、L199Q及N205K對於ClpY與專一性基質作用較其他突變蛋白影響為大,證明I domain loop對ClpY對基質辨識作用的重要性。
Recognition of the appropriate molecular targets is critical for most biological activity in the cells. For irreversible processes such as protein degradation, recognizing of the correct substrates are important because cleavage of the wrong targets might be damaging to the cells or even lethal. It is required for cells to degrade proper targets like misfolded or abnormal proteins and to control the levels of critical short-lived regulatory proteins. The ATP-dependent proteases play a role in phenomena described above. In bacteria and other organisms, many of intracellular proteases have to hydrolyze ATP to degrade complex substrates;including peptide enzymes, such as Lon, and two-component protease, such as ClpXP, ClpQY, ClpAP. In ClpQY, the small subunit ClpQ (19kDa) is a peptidase, and the larger subunit ClpY (49kDa) exhibits both ATPase and chaperone activities. ClpY can recognize, unfold, and translocate the specific substrates. It is unclear about the mechanisms of how the ClpY recognizes, binds and translocates the specific substrates to ClpQ and the ClpQY degrades the substrates. In this study, the yeast two-hybrid system was used to screen the ClpY mutants either interact or not with the specific substrates. To test the abilities of interaction of ClpY mutants with substrates, lacZ and leu2 expressions was used in yeast system;cps-lacZ expression was used to detect the RcsA degraded by the ClpY mutants in the presence of ClpQ, MMS test was used to detect the SulA degradation.In addition, the ClpYmutants were tested of their influences on cell growth while overproducted.We show that the loop of I domain(175-209) is necessary for substrate recognition and the altered specific amino acid residues on the loop have an influence on ClpY cellular activities.
Abstract 3
中文摘要 4
壹、 前言 5
一、ATP-dependent protease (ATP依賴蛋白酶) 5
二、熱休克蛋白酶HslVU(ClpQY) 6
三、Two component protease ClpQY 6
四、ClpYQ蛋白酶結構及其與基質辨識相關之研究 7
(一) ClpYQ蛋白酶結構 7
五、ClpQY之基質 9
六、研究目的 10
貳、 研究方法 11
一、實驗材料 11
1、菌株及質體 11
2、培養基 12
3、相關酵素 12
4、實驗相關核酸引子 12
二、實驗方法 12
1. Mini-preparation of plasmid DNA from bacteria 12
2. Midi-preparation of plasmid DNA from bacteria 13
3. Preparation of plasmid DNA from yeast 13
4. preparation of compentent cells 13
5. Transformation 14
6. Mutagenize I domain of clpY & whole clpY (Error prone PCR) 16
7. Site directed mutagenesis 17
8. Screening of I domain & ClpY mutants in yeast two hybrid system 18
9. Analysis of reporter gene in yeast 18
10. cps-lacZ expression: β-galactosidase assay in bacteria 20
11. SulA degradation test: MMS assay 20
12. SDS蛋白質膠體電泳 21
13. Western Blotting 22
參、實驗結果 25
一、 Screening ClpY mutants in yeast two-hybrid system 25
二、 lacZ expression and leu2 expression test 26
三、 MMS test in pBAD24 system 29
四、 cps-lacZ expression test 32
肆、討論 34
伍、參考文獻 38
陸、表 46
柒、圖 50
伍、參考文獻
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