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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 吳蕙芬 | |
| dc.contributor.author | Yu-Chun Chen | en |
| dc.contributor.author | 陳郁君 | zh_TW |
| dc.date.accessioned | 2021-06-17T00:18:31Z | - |
| dc.date.available | 2017-07-27 | |
| dc.date.copyright | 2012-07-27 | |
| dc.date.issued | 2012 | |
| dc.date.submitted | 2012-06-28 | |
| dc.identifier.citation | Bi, E. and J. Lutkenhaus. (1990). Analysis of ftsZ mutations that confer resistance to the cell division inhibitor SulA (sfiA). J. Bacteriol. 172: 5602-5609.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/66000 | - |
| dc.description.abstract | 大腸桿菌ClpYQ (HslUV) 蛋白酶為ATP 依賴型蛋白酶的一種，由49 kDa的ClpY 及19 kDa 的ClpQ 組合而成。這類蛋白酶在細胞中可降解不正常堆積的蛋白質或將折疊錯誤的蛋白質恢復正常構形，避免細胞受損。SulA 為一細胞分裂抑制者，當細胞遭遇自由基傷害或是曝露於UV 光下，造成DNA 受損，會誘發SulA 蛋白表現，抑制細胞分裂。目前已知ClpY 是辨識SulA C-端第20 - 30個具高疏水性的胺基酸片段，若將此片段之胺基酸點突變為親水性胺基酸，會降低SulA 與ClpY 之間的交互作用。然而，若將SulA C-端末20 個胺基酸去除，會使SulA 失去活性且成為一聚集之蛋白質。此外，RcsA 亦被認為是ClpYQ 之基質，但RcsA 是如何被ClpY 辨識，目前仍不清楚。
本研究以大腸桿菌表現蛋白質，利用西方墨點法測試ClpY 突變蛋白分解SulA ΔC20、SulA*F143A 的能力，發現ClpY 對於SulA*F143A 突變蛋白及失活且結構已打開之SulAΔC20 突變蛋白的辨識情形與辨識野生型SulA 蛋白相類似。也發現ClpY 第91 個胺基酸Tyrosine 之hydroxyl-group 對於基質的傳送，亦扮演了重要角色。以酵母菌雙雜交系統測試SulA C-端第20 - 30 個胺基酸點突變蛋白與SulA 之間的交互作用，發現這個區域點突變之活性，與能否形成dimer相關。以β-galactosidase 測試RcsA 蛋白於不同溫度下之活性與被ClpYQ 降解的情形，發現RcsA 蛋白的活性隨溫度上升而下降，且β-galactosidase units 值皆因ClpYQ 蛋白酶的出現而下降。之後再利用西方墨點法直接偵測RcsA 蛋白的累積量，在不同溫度下RcsA 的累積量皆相似，誘導ClpYQ 後，累積量皆有下降的趨勢，故RcsA 亦為ClpYQ 之基質。此外也發現，ClpY 對於SulA 及RcsA 之辨識是具相類似的機制。 | zh_TW |
| dc.description.abstract | ClpYQ (HslUV) is an ATP-dependent protease from Escherichia coli, composed of the 49 kDa ClpY and 19 kDa ClpQ. ClpQ peptidase. The degradation of the abnormal proteins or the misfolded proteins is accomplished by this protease to avoid cell damage. When the cells are exposed to UV light or damaged by free radical, the SOS response induced a cell division inhibitor, SulA. ClpY recognizes its C-terminal 129 - 149 hydrophobic amino acids. However, SulA becomes inactive and aggregated while it lacks the last 20 amino acids. Also it was observed that HA-SulA*F143A
was slowly degraded by ClpYQ. In addition, RcsA, as one of its substrates, is not clear for its direct targeted by ClpYQ. In this study, Western blot analysis was used to determine how ClpY is to recognize SulA ΔC20, and SulA*F143A. As results, ClpY recognizes SulAΔC20 and SulA*F143A in a similar way as it did to the wild-type SulA. We showed here that the C129 - 149 region of SulA is related to the monomer or dimer formation. The aromatic ring of ClpY91 (tyrosine,Tyr) is, however, known necessary for the translocation of substrates. In addition, through our study, we also showed that the hydroxyl-group of ClpY91 (Tyr) plays an important role in translocating of the substrates to ClpQ. From an alternative approach, we adopted the assays of β-galactosidase activity of cpsB::lacZ with pTH18kr-rcsA with or without ClpYQ. We found that the activity of RcsA decreases with an increased temperature, and the β-galactosidase levels decrease when ClpYQ is present. Through Western blot analysis, it was shown that the accumulation of RcsA was equal under the different temperature. However, it was reduced when expressing the ClpYQ protease. Therefore, RcsA is one of the substrates for ClpYQ. In addition, we also demonstrated that ClpY recognizes RcsA in a similar way as it did toward SulA. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T00:18:31Z (GMT). No. of bitstreams: 1 ntu-101-R97623024-1.pdf: 2125109 bytes, checksum: 3ac3e0311e9e8a71ac9d7c356e520427 (MD5) Previous issue date: 2012 | en |
| dc.description.tableofcontents | 口試委員會審定書.......................................... i
誌謝..................................................... ii 摘要.................................................... iii Abstract................................................. iv 目錄.......................................................v 表目錄................................................. viii 圖目錄....................................................ix 附圖目錄...................................................x 壹、前言...................................................1 一、ATP 依賴蛋白酶.................................... 1 二、ClpYQ 蛋白酶...................................... 2 三、ClpYQ 蛋白酶基質.................................. 5 四、細胞分裂物SulA.................................... 7 五、蛋白酶與基質SulA.................................. 9 六、胞外莢膜調控蛋白RcsA............................. 13 七、研究動機與目的................................... 15 貳、材料與方法............................................16 一、實驗材料..........................................16 (一) 菌株與質體...................................16 (二) 藥品與試劑...................................17 (三) 器材設備.....................................18 (四) 分析軟體.....................................19 二、實驗方法..........................................19 (一) 一般實驗方法.................................19 (二) SulA (RcsA) 蛋白N 端3x FLAG-tag 標定.........21 (三) 酵母菌雙雜交系統分析 (Yeast two-hybrid system)......................................27 (四) 大腸桿菌選殖基因表現系統.....................30 (五) 西方墨點分析 (Western Blotting) .............33 參、結果..................................................40 一、ClpYQ 蛋白酶降解結構已被打開之基質................40 二、ClpYQ 對於3x FLAG - SulA*F143A 之降解.............41 (一) 3x FLAG-tag 對於SulA*F143A 之影響............41 (二) ClpY 之loop2 及pore1 site 突變對於3x FLAG - SulA*F143A 之結合及辨識......................42 (三) 3x FLAG - tag 對SulA 蛋白之影響..............42 三、SulA 之活性是否受單體或二聚體形式影響.............43 四、RcsA 為ClpYQ 蛋白酶之基質.........................45 (一) RcsA 蛋白於不同溫度之活性與累積量............45 (二)不同溫度下RcsA 被ClpYQ蛋白酶降解之情形........46 (三) ClpY辨識及傳送基質RcsA之區域.................46 肆、討論..................................................48 一、結構已打開之基質被ClpY 辨識.......................48 二、SulA 蛋白被ClpY 辨識區域之點突變及其降解情形......49 三、ClpY 之pore1 點突變對於基質SulA 及其辨識位之點突變之 影響..............................................50 四、SulA 突變蛋白形成Dimer 的與否是否與活性有關.......51 五、ClpYQ 蛋白酶降解RcsA 基質.........................51 (一) 溫度對於RcsA 蛋白的響........................51 (二) ClpY 辨識不同基質之途徑是否相似..............52 伍、結論..................................................53 陸、參考文獻..............................................54 | |
| dc.language.iso | zh-TW | |
| dc.subject | RcsA | zh_TW |
| dc.subject | 蛋白質辨識 | zh_TW |
| dc.subject | ATP依賴蛋白酶 | zh_TW |
| dc.subject | ClpYQ | zh_TW |
| dc.subject | SulA | zh_TW |
| dc.subject | RcsA | en |
| dc.subject | ATP-dependent protease | en |
| dc.subject | Protein recognition | en |
| dc.subject | ClpYQ | en |
| dc.subject | SulA | en |
| dc.title | 大腸桿菌熱休克蛋白酶ClpYQ分解基質之研究 | zh_TW |
| dc.title | The degradation of substrates, SulA and RcsA, by ClpYQ protease in Escherichia coli | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 100-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 黃良得,徐駿森,陳昭瑩,張世宗 | |
| dc.subject.keyword | ATP依賴蛋白酶,ClpYQ,SulA,RcsA,蛋白質辨識, | zh_TW |
| dc.subject.keyword | ATP-dependent protease,ClpYQ,SulA,RcsA,Protein recognition, | en |
| dc.relation.page | 76 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2012-06-28 | |
| 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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