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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/53987完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | ?進德(Jin-Der Wen) | |
| dc.contributor.author | Shu-Ya Chang | en |
| dc.contributor.author | 張舒雅 | zh_TW |
| dc.date.accessioned | 2021-06-16T02:35:38Z | - |
| dc.date.available | 2018-08-07 | |
| dc.date.copyright | 2015-08-07 | |
| dc.date.issued | 2015 | |
| dc.date.submitted | 2015-07-27 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/53987 | - |
| dc.description.abstract | 許多mRNA在一般情況下會形成二級結構，然而，當轉譯作用進行時，其二級結構必須被解開使得密碼子能夠被呈現出來。先前研究指出核醣體自己本身在轉譯作用進行時即具有解旋酶(helicase)的活性，可以解開mRNA所形成的二級結構。大腸桿菌中rpsO基因轉錄本的5’未轉譯區(5’UTR)能夠藉由形成假結或雙髮夾結構來調控其本身的轉譯作用，但因為雙髮夾結構的SD(Shine-Dalgarno)序列無法被呈現，所以核醣體只能與假結結構結合並開始轉譯作用的進行。除此之外，在假結結構中只有SD序列是暴露出來的，而起始密碼子下游卻緊鄰二級結構，所以核醣體必須解開下游的二級結構才能完成轉譯起始作用。然而，目前對於假結結構在起始作用中哪個階段被核醣體解開的仍然不清楚。
本篇研究透過單分子螢光共振能量轉移技術來探討當核醣體小次單元及起始tRNA存在時，rpsO基因轉錄本之5’未轉錄區的結構會有何變化。研究結果發現，當核醣體小次單元存在並與mRNA結合時，mRNA形成假結結構的比例會增加，與先前研究核醣體小次單元只能和假結結構結合的結果相符。不只如此，當30S結合到假結結構的SD序列上時，核醣體小次單元能夠藉由解開部分mRNA的二級結構來找到起始密碼子AUG。而當同時有30S及起始tRNA出現時，起始tRNA能夠幫助核醣體小次單元將下游的二級結構全部解開，使得前轉譯起始複合體可以完成。以上研究結果顯示，在轉譯起始階段，核醣體小次單元自己本身即可表現解旋酶活性，將具有二級結構的mRNA解開，讓轉譯作用可以順利進行。 | zh_TW |
| dc.description.abstract | Many mRNAs fold into secondary structures; however, their codons must be in single-stranded form to be translated. Previous research has revealed that the ribosome itself has helicase activity during the translation process. The rpsO gene transcript of Escherichia coli regulates its own translation through the 5’ untranslated region (5’ UTR), which can fold into a pseudoknot or a double-hairpin conformation. The two structures can be interchanged spontaneously, but the ribosome can only bind to the pseudoknot to initiate translation. On the pseudoknot, only the Shine-Dalgarno (SD) sequence but not the AUG start codon is fully exposed, so the ribosome has to unwind part of the secondary structure to complete the initiation. However, it remains unclear in which
stage the conformation is opened by the ribosome. In this study, we characterize the conformational change of the rpsO 5’ UTR in the presence of the 30S ribosomal subunit and initiator tRNA (charged formyl-methionyl tRNA) by using single-molecule fluorescence resonance energy transfer (smFRET). Our results show that the population of the pseudoknot form is increased when 30S binds to the RNA. 30S would begin to search for the AUG start codon after binding to the SD sequence of the pseudoknot by partially unwinding the local structures. In the presence of the initiator tRNA, 30S may completely unwind a stem of the pseudoknot and form the pre-initiation complex. These results demonstrate that the 30S ribosomal subunit alone can perform its helicase activity during the initiation stage. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T02:35:38Z (GMT). No. of bitstreams: 1 ntu-104-R02b43027-1.pdf: 8401237 bytes, checksum: b2a3d39d88d1762b78a239059fa5a84f (MD5) Previous issue date: 2015 | en |
| dc.description.tableofcontents | 目錄
口試委員審定書 ii 致謝 iii 中文摘要 iv ABSTRACT v 目錄 vi 圖目錄 ix 表目錄 x 第一章 導論 1 1.1 核醣體 1 1.1.1 功能 1 1.1.2 結構 1 1.1.3 解旋作用 2 1.2 轉譯作用 3 1.2.1 起始階段 3 1.2.2 延長階段 4 1.2.3 終止階段 4 1.3 rpsO基因 5 1.4 單分子技術 6 1.4.1 簡介 6 1.4.2 螢光共振能量轉移 (fluorescence resonance energy transfer ) 6 1.5 研究動機 7 第二章 材料與方法 8 2.1 材料 8 2.1.1 勝任細胞品系 8 2.1.2 質體 8 2.1.3 試劑 8 2.1.4 藥品 9 2.1.5 酵素 10 2.1.6 載體構築序列及引子設計 10 2.1.7 溶液 12 2.2 方法 14 2.2.1 載體構築及純化 14 2.2.2 聚合酶連鎖反應 (Polymerase Chain Reaction, PCR) 14 2.2.3 細胞外轉錄作用 ( in vitro transcription ) 15 2.2.4 細胞外轉譯作用 ( in vitro translation ) 15 2.2.5 DNA引子及RNA黏合反應 16 2.2.6 核醣體30S小次單元的純化 16 2.2.7 單分子螢光共振能量轉移實驗 18 第三章 結果 21 3.1 RNA樣本的製備 21 3.2 確認rpsO基因結構的FRET效率 21 3.2.1 雙髮夾結構的FRET效率 21 3.2.2 假結結構的FRET效率 22 3.2.3 rpsO基因轉錄本5’UTR的FRET效率 22 3.3 核醣體小次單元30S對rpsO基因轉錄本的影響 23 3.4 核醣體30S小次單元解開假結結構時的FRET效率 24 3.5 起始tRNA能協助30S小次單元完整解開二級解構 25 3.6 進一步確認30S和起始tRNA與rpsO基因轉錄本結構變化的關係 26 3.6.1 加強SD序列可穩定30S與RNA的結合 26 3.6.2 去除起始密碼子AUG對轉譯起始作用的影響 27 第四章 討論 29 4.1 解開二級結構的能量來源 29 4.2 核醣體30S小次單元具有解旋能力的部位 29 4.3 核醣體蛋白S15對rpsO基因轉錄本在轉譯起始作用的影響 30 4.4 rpsO基因轉錄本的5’ UTR形成雙髮夾結構的功能 31 4.5 起始tRNA協助30S解開二級結構的作用機制 32 4.6 核醣體30S小次單元的解旋能力與解旋酶的異同 33 4.7 未來展望 34 參考文獻 35 圖目錄 圖1. pS15WT-s載體構築示意圖及部分序列示意圖 41 圖2. 核醣體純化之結果 42 圖3. 單分子螢光共振能量轉移實驗使用之RNA轉錄本 43 圖4. 單分子螢光共振能量轉移實驗設置 44 圖5. rpsO基因轉錄本5’UTR結構與DNA探針之相對位置示意圖 45 圖6. 雙髮夾結構(mHPGC)序列及FRET與分子螢光強度關係圖 47 圖7. 雙髮夾結構(mHPGC)的FRET效率及時間軌跡圖 49 圖8. 假結結構(dAC)序列及FRET與分子螢光強度關係圖 51 圖9. 假結結構(dAC)的FRET效率及時間軌跡圖 53 圖10. rpsO 5’UTR的FRET與分子螢光總強度及FRET效率示意圖 55 圖11. rpsO基因轉錄本5’UTR的時間軌跡圖 57 圖12. 30S存在時5’UTR的FRET與分子螢光總強度及轉換效率示意圖 58 圖13. 核醣體30S存在時rpsO 5’UTR的時間軌跡圖 60 圖14. mHP1序列及FRET與分子螢光強度關係圖 61 圖15. mHP1的FRET效率及時間軌跡圖 62 圖16. 起始tRNA與30S同時存在時5’UTR的FRET與分子螢光總強度及轉換效率示意圖 64 圖17. 起始tRNA與30S同時存在時rpsO 5’UTR的時間軌跡圖 66 圖18. 強SD結構(mPKsSD)序列及FRET與分子螢光強度關係圖 67 圖19. 強SD結構(mPKsSD)的FRET效率及時間軌跡圖 68 圖20. 去除AUG結構(mdAUG)序列及FRET與分子螢光強度關係圖 69 圖21. 去除AUG結構(mdAUG)的FRET效率及時間軌跡圖 70 圖22. 細胞外轉譯作用實驗設計及結果 71 圖23. 核醣體蛋白S15與rpsO 5’UTR結合位置示意圖 72 圖24. 核醣體蛋白S15存在時rpsO 5’UTR的時間軌跡及FRET效率圖 73 表目錄 表1. 實驗中使用的所有RNA結構之序列 74 表2. 每個結構在各實驗條件下的FRET效率 75 | |
| dc.language.iso | zh-TW | |
| dc.title | 利用單分子螢光共振能量轉移技術探討核醣體在rpsO基因轉錄本上對轉譯起始的影響 | zh_TW |
| dc.title | Observing Translation Initiation of the Ribosome on rpsO Transcript by smFRET | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 103-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 張功耀(Kung-Yao Chang),黃筱鈞(Hsiao-Chun Huang) | |
| dc.subject.keyword | 核醣體,轉譯起始作用,單分子,螢光共振能量轉移技術,rpsO基因, | zh_TW |
| dc.subject.keyword | ribosome,translation initiation,single-molecule,FRET,rpsO gene, | en |
| dc.relation.page | 75 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2015-07-27 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 分子與細胞生物學研究所 | zh_TW |
| 顯示於系所單位： | 分子與細胞生物學研究所 | |
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