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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 羅凱尹 | |
| dc.contributor.author | Jing-Ting Shie | en |
| dc.contributor.author | 謝境梃 | zh_TW |
| dc.date.accessioned | 2021-06-17T02:33:11Z | - |
| dc.date.available | 2022-08-24 | |
| dc.date.copyright | 2017-08-24 | |
| dc.date.issued | 2017 | |
| dc.date.submitted | 2017-08-17 | |
| dc.identifier.citation | Allmang, C., P. Mitchell, E. Petfalski, and D. Tollervey. 2000. Degradation of ribosomal RNA precursors by the exosome. Nucleic acids research. 28:1684-1691.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/68742 | - |
| dc.description.abstract | 核醣體生合成的過程中，會有約略200個合成因子參與其中，這些因子主要的功用在幫助核醣體生成、修飾、組裝、以及品質的調控，讓核醣體能夠正確生成。我們實驗室之前發現Bcp1是核醣體蛋白Rpl23的伴護蛋白，具有保護Rpl23結構穩定的功能。我進一步研究發現Bcp1會將剛入核的Rpl23從運輸蛋白(Karyopherins) 上釋放，並和其結合，來確保Rpl23以正確構型結合到核醣體大單元60S上。從前人研究中知道Rkm1是甲基轉移酶，具有將Rpl23上的106與110的離胺酸雙甲基化(dimethylation)的功能，但其甲基化所造成的生理意義尚未被解開。本研究結果發現Rkm1需要Rpl23才會與運輸蛋白結合，一同運輸入核，之後和Bcp1形成三聚體，而Bcp1會活化Rkm1對Rpl23 lysine110的甲基化，在Rpl23甲基化順利完成後，會與Bcp1一同從Rkm1上釋放，接著組裝到60S上。Rkm1缺失時對細胞的生長速度並無明顯影響，但在bcp1突變株中去除Rkm1時，會產生更嚴重的生長缺失，實驗結果發現，Rpl23蛋白質的量於雙突變株中的減少比bcp1突變株更加明顯，證明兩者間有功能上的關聯性，推測為協同保護Rpl23的穩定性。 | zh_TW |
| dc.description.abstract | There are about 200 biogenesis factors participate in the ribosome biosynthesis.These biogenesis factors possess multiple functions such as facilitating the synthesis of ribosome, modifications of rRNAs or ribosomal proteins, assembly of the complex, and quality control of the ribosome. In our previous study, we find that Bcp1 is a chaperone of Rpl23, which can protect and stabilize the structure of Rpl23. In my study, I discover that Bcp1 can release Rpl23 from Karyopherins just after they were imported to nucleus and bind Rpl23 subsequently to ensure incorperation of Rpl23. Rkm1 has been shown as a methyltransferase of Rpl23, adding dimethylation to lysine106 and lysine110. But the physiological significance of lysine methylation stays unclear. Here, I found that Rkm1 co-transport with Rpl23 to nucleus and released by Bcp1. Bcp1, Rkm1 and Rpl23 form hetero-trimer, meanwhile Bcp1 triggers dimethylations on Rpl23 via Rkm1. After accomplishment of dimethylations, Rpl23/Bcp1 complex leaves Rkm1 and delivers Rpl23 to pre-60S. Deletion of RKM1 did not show growth defect, but synthetic growth deficiency was observed in bcp1ts and rkm1Δ double mutant. Moreover, the level of Rpl23 decreased even lower in double mutant. This suggests that both Bcp1 and Rkm1 have functions in protecting the stability of Rpl23. | en |
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| dc.description.tableofcontents | 目錄
誌謝 i 摘要 ii Abstract iii 目錄 iv 表目錄 vii 圖目錄 viii 一、文獻回顧與探討 1 1.1核醣體 1 1.2核醣體的生合成 1 1.2.1核醣體成熟 1 a. pre-60S的成熟 2 b. pre-40S的成熟 3 c.出核機制 4 1. 2.2核醣體功能的校對 5 1.3核醣體蛋白的伴護蛋白 (Chaperon) 6 1.4研究目標蛋白 7 1.4.1 Bcp1 7 1.4.2 Rpl23 8 1.4.3 Rkm1 8 1.5 Ribosomopathy 9 二、研究動機 10 三、研究材料與方法 11 3.1 菌株與質體 11 附表一、二 11 3.2質體製備 11 3.2.1 PCR 11 3.2.2 電泳 11 3.2.3 DNA電泳膠體純化 11 3.2.4限制酶切割 11 3.2.5 接合作用 12 3.3轉型作用 12 3.3.1大腸桿菌轉型作用 12 3.2.2快速啤酒酵母轉殖作用(Quick yeast transformation) 12 3.4生長測試(Growth assay) 13 3.5多核醣體圖譜分析法（Polysome profile） 13 3.6 免疫沉澱 13 3.7 In vitro interaction 14 3.7.1 RanGTP的純化與製備 14 3.7.2Bcp1&Rpl23 14 a. Bcp1-HIS6 ( PKL502 )表現與純化 15 b. Rpl23 no tag ( PKL346 )表現 15 c. Bcp1&Rpl23複合體純化 15 3.7.3 Rkm1 16 a. GST-Rkm1 ( PKL519)表現 16 b. HIS-Rkm1 ( PKL519)表現 16 3.8 蛋白質染色 (Coomassie blue) 16 3.9 Native PAGE 17 3.10 Cross-link 17 3.11膠體過濾法(Gel filtration Chromatography) 17 3.12 in vitro methylation分析 18 3.13單點突變株的建構 19 四、結果 20 4.1 Bcp1為Rpl23的伴護蛋白且為釋放子(escortin) 20 4.2 Bcp1、Rpl23與Rkm1會形成三聚複合體 22 4.3 Rkm1與Rpl23協同入核 23 4.4 Bcp1的N端結構缺失會造成細胞的生長缺失 25 4.5 Bcp1透過N端與Rkm1結合 26 4.6 Bcp1的N端結構與入核運輸相關 27 4.7 Rpl23透過胺基酸41-52的Loop結構與Bcp1結合 28 4.8 Bcp1會影響Rkm1對Rpl23的甲基化程度 29 4.9 Rpl23的甲基化會影響與Rkm1的親和能力 30 4.10 Bcp1與Rkm1可以協同幫助Rpl23的穩定 32 4.11 Bcp1與Rkm1可以協同幫助Rpl23的穩定 33 五、討論 34 5.1 Bcp1為Rpl23的釋放子 34 5.2 Rpl23與Rkm1複合體 35 5.3 Bcp1的結構在生理上的意義 37 5.4 Rpl23的甲基化會影響結構正確性 39 5.5 Bcp1可能藉由活化Rkm1對Rpl23的甲基化來調控Rpl23蛋白質的表現量 40 六、參考文獻 42 | |
| dc.language.iso | zh-TW | |
| dc.title | Bcp1和甲基轉移酶Rkm1協同調控核醣體蛋白Rpl23的穩定性 | zh_TW |
| dc.title | Bcp1 and methyltransferase Rkm1 regulate the stability of ribosomal protein Rpl23 coordinately | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 105-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 黃偉邦,冀宏源,吳惠芬,徐駿森 | |
| dc.subject.keyword | 核醣體,Bcp1,Rkm1,Rpl23,核醣體蛋白,甲基化,伴護子, | zh_TW |
| dc.subject.keyword | Ribsome,Bcp1,Rpl23,Rkm1,ribosomal protein,methylation,chaperone, | en |
| dc.relation.page | 75 | |
| dc.identifier.doi | 10.6342/NTU201703627 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2017-08-18 | |
| 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 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-106-1.pdf 目前未授權公開取用 | 3.33 MB | Adobe PDF |
系統中的文件，除了特別指名其著作權條款之外，均受到著作權保護，並且保留所有的權利。