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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 溫進德(Jin-Der Wen) | |
| dc.contributor.author | Po-Szu Hsieh | en |
| dc.contributor.author | 謝伯思 | zh_TW |
| dc.date.accessioned | 2021-06-15T12:39:40Z | - |
| dc.date.available | 2021-08-24 | |
| dc.date.copyright | 2016-08-24 | |
| dc.date.issued | 2016 | |
| dc.date.submitted | 2016-07-28 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/50412 | - |
| dc.description.abstract | 偽結是一種在 RNA 中重要而複雜的結構，在各種生物反應中例如 RNA 的複製、轉錄與轉譯等等，皆扮演著重要的角色，許多 RNA 病毒更透過偽結作為刺激物，進而造成計畫性核醣體框架位移 (Programmed Ribosomal Frameshifting)。先前研究利用人類端粒酶 RNA 中所衍生的 DU177 偽結作為研究模板，其結構為兩個具有鹼基配對區域的螺旋莖 (helical stem) 透過兩個單股環 (single-stranded loop) 連接，研究發現若將此段偽結結構放置於一段滑動序列 (slippery sequence) 下游，在進行轉譯作用的過程中，可有效刺激 -1 計畫性框架位移的發生。另外， mRNA 必須呈現單股狀態才能進行轉譯作用，先前研究指出核醣體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，但目前對於偽結結構如何被核醣體解開的分子機制尚未明瞭。
本篇研究透過單分子螢光共振能量轉移技術來探討偽結結構與核醣體間的交互作用。研究結果發現核醣體在進行轉譯的過程中，當部分偽結結構序列進入核醣體時，會因受到核醣體的影響使其結構產生扭曲，這現象可能和框架位移的發生有所關聯。但隨著轉譯作用的持續進行，核醣體將會克服結構屏障進而將偽結中第一個 stem 的鹼基對全數解開。根據此結果推測，由於此偽結含有數個提高其穩定度的三重鹼基對 (base triples)，即使部分偽結的序列進入核醣體時，可能依舊保持偽結結構狀態。 | zh_TW |
| dc.description.abstract | Pseudoknots are important and complex RNA structures. They play critical roles in many biological processes, such as RNA replication, transcription and translation. Several viruses use a pseudoknot structure to induce programmed ribosomal frameshifting during translation. Previous research used an H-type pseudoknot derived from the human telomerase RNA (DU177) as a model system, which has two helical stems connected by two single-stranded loops. DU177 can function as a -1 ribosomal frameshifting stimulator when it is positioned downstream of a slippery sequence. In addition, mRNA structures must be unfolded to a single-stranded form to be translated. Previous research has revealed that the ribosome has intrinsic helicase activity during translation. However, the detailed molecular mechanism of how a pseudoknot is unwound by the ribosome remains unclear.
In this study, we use single-molecule fluorescence resonance energy transfer (smFRET) to elucidate how a pseudoknot is unwound by the ribosome. Our results show that when a partial pseudoknot sequence enters the ribosome during translation, the pseudoknot structure is distorted by the ribosome. The structural distortion may play a role in inducing the ribosome to undergo frameshifting. However, the ribosome can overcome the steric barrier to unwind the first stem of the pseudoknot completely when translation proceeds further. According to our results, we propose that, due to the presence of several base triples that stabilize the whole RNA structure, the folding of the pseudoknot may be maintained even if a partial sequence enters the ribosome. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T12:39:40Z (GMT). No. of bitstreams: 1 ntu-105-R03b43004-1.pdf: 2451227 bytes, checksum: 730d6ed7105edde519ae51666f6a33ec (MD5) Previous issue date: 2016 | en |
| dc.description.tableofcontents | 口試委員審定書 i
致謝 ii 摘要 iii ABSTRACT iv 目錄 v 圖目錄 viii 表目錄 ix 縮寫對照表 x 第一章 緒論 1 1.1 核醣體 1 1.1.1 簡介 1 1.1.2 解旋機制 2 1.2 轉譯作用 2 1.2.1 起始階段 2 1.2.2 延長階段 3 1.2.3 終止階段 4 1.3 RNA 偽結 (RNA Pseudoknot) 5 1.4 單分子技術 5 1.4.1 簡介 5 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 9 2.1 材料 9 2.1.1 勝任細胞品系 9 2.1.2 質體 9 2.1.3 載體構築序列及引子設計 9 2.1.4 酵素 11 2.1.5 藥品 11 2.1.6 試劑 13 2.1.7 溶液 13 2.1.7 樣本配製 15 2.2 方法 16 2.2.1 質體構築及純化 16 2.2.2 細胞外轉錄作用 (in vitro transcription) 16 2.2.3 RNA 螢光標定及純化 17 2.2.4 DNA 夾板 RNA 連接反應 (DNA - splinted RNA ligation) 18 2.2.5 DNA 引子及 RNA 黏合反應 19 2.2.6 單分子螢光共振能量轉移實驗 19 第三章 結果 23 3.1 RNA 樣本製備 23 3.1.1 製備具 SD 序列及起始密碼子之 RNA 片段 - pPK RNA 23 3.1.2 RNA 螢光標定及純化 23 3.1.3 DNA 夾板 RNA 連接反應 24 3.2 確認 RNA 樣本 FRET 效率 25 3.3 觀測 PK 結構受核醣體逐步解開之 FRET 效率變化 26 3.4 減少實驗中可能存在的背景訊號 28 第四章 討論 29 4.1 IVT 產物無法進行接合反應 29 4.2 偽結受核醣體作用產生的結構變化 29 4.3 核醣體受偽結結構的影響 31 4.4 實驗可能具背景訊號 31 4.5 引子無法有效與偽結結構中 stem 1 間鹼基配對競爭 32 4.6 未來展望 32 參考文獻 34 附錄 – Intermolecular RNA triplex 69 圖 1. 樣本製備流程示意圖 40 圖 2. pT7SP6-PK 載體構築示意圖及部分序列示意圖 41 圖 3. 確認細胞外轉錄產物片段大小及品質 42 圖 4. RNA 螢光標定與 HPLC 純化 43 圖 5. IVT 產物 pPK RNA 無法進行連接反應 44 圖 6. DNA 夾板 RNA 連接反應 45 圖 7. 單分子螢光共振能量轉移實驗設置 46 圖 8. 偽結(PK MFKEY full)及髮夾(PK stem 1(-) full)結構序列示意圖及FRET效率 47 圖 9. 預期偽結於轉譯過程中受核醣體解開鍵結之序列及結構狀態示意圖。 49 圖 10. PK MFKEY-IC 轉換效率分佈圖及時間軌跡圖 51 圖 11. PK MFKEY-IC_TM(F) 轉換效率分佈圖及時間軌跡圖 52 圖 12. PK MFKEY-IC_TM(FK) 轉換效率分佈圖及時間軌跡圖 54 圖 13. PK MFKEY-IC_TM(FKE) 轉換效率分佈圖及時間軌跡圖 56 圖 14. PK MFKEY-IC_TM(FKEY) 轉換效率分佈圖及時間軌跡圖 58 圖 15. PK 結構受核醣體逐步解開之 FRET 效率變化及各反應狀態下 FRET 效率間轉換頻率 60 圖 16. 反應條件中加入 50S 及 IF-2，PK 結構受核醣體逐步解開之 FRET 效率變化及各反應狀態下 FRET 效率間轉換頻率 62 圖 17. 以引子與偽結結構中 stem 1 間鹼基配對競爭 64 表 1. 各單分子實驗樣本 RNA 全長序列 66 表 2. 各反應狀態下 FRET 效率統整 67 表 3. PK MFKEY full RNA 各反應狀態下 FRET 效率轉換頻率統整 68 | |
| dc.language.iso | zh-TW | |
| dc.title | 利用單分子螢光共振能量轉移技術探討 RNA 偽結與核醣體間交互作用 | zh_TW |
| dc.title | Study of Interactions Between the mRNA Pseudoknot and the Ribosome with Single-Molecule FRET | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 104-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 張功耀(Kung-Yao Chang),楊立威(Lee-Wei Yang) | |
| dc.subject.keyword | 偽結,核醣體,轉譯作用,單分子,螢光共振能量轉移技術, | zh_TW |
| dc.subject.keyword | pseudoknot,ribosome,translation,single-molecule,fluorescence resonance energy transfer (FRET), | en |
| dc.relation.page | 74 | |
| dc.identifier.doi | 10.6342/NTU201601522 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2016-07-28 | |
| 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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