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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/52525完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 溫進德(Jin-Der Wen) | |
| dc.contributor.author | Chieh Han | en |
| dc.contributor.author | 韓捷 | zh_TW |
| dc.date.accessioned | 2021-06-15T16:17:28Z | - |
| dc.date.available | 2018-08-25 | |
| dc.date.copyright | 2015-08-25 | |
| dc.date.issued | 2015 | |
| dc.date.submitted | 2015-08-17 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/52525 | - |
| dc.description.abstract | 在大腸桿菌裡，dnaX基因的初級轉錄本(transcript)可以藉由-1核醣體框架位移(-1 ribosomal frameshifting)的方式，轉譯成兩種去氧核醣核酸聚合酶III(DNA polymerase III)所需之次單元：gamma (γ) 和tau (τ)。要能夠造成-1核醣體框架位移，mRNA上就必需要有三個重要的因素，分別為內部SD序列(internal Shine- Dalgarno sequence，後稱iSD序列)、滑動序列(slippery sequence)以及一個二級結構。當一個核醣體的轉譯作用進行到滑動序列的時候，核醣體的小次單元(30S)中的16S核醣體RNA會和iSD序列產生交互作用；於此同時，位於下游的二級結構也會給予核醣體一個阻力。當這兩種因素產生的物理效應一起作用，就可能會使該核醣體暫時停滯而發生框架位移(frameshifting)，這種停滯稱為核醣體暫停(ribosome pausing)。因此為了探討這兩種因素與核醣體暫停彼此之間的關係，我們同時使用了大腸桿菌細胞內(in vivo)以及細胞外(in vitro)實驗來進行。
本實驗著重在大腸桿菌細胞外實驗，並以細胞內實驗來作為輔助實驗。我們使用dnaX序列來當作實驗的模型並修改了滑動序列，讓核醣體沒辦法進行-1框架位移；這麼一來，我們就可以直接觀察iSD序列和二級結構對核醣體暫停的影響。再者，為了避免聚核醣體(polyribosome)的形成，我們縮短了起始密碼子(start codon)和二級結構之間的距離，當核醣體因二級結構而暫停時，下一個核醣體不會結合於起始密碼子，因此整體的轉譯速率會下降。透過觀察蛋白的表現量，我們發現iSD序列和二級結構都會造成核醣體暫停；而我們更進一步發現，二級結構是造成核醣體暫停的主因。除此之外，我們也發現30S會結合在iSD序列，但是並不會表現蛋白，卡住的30S也會阻礙下一個核醣體進行正常的起始作用，使得蛋白的表現量降低。而卡住的30S有可能會自動與mRNA分開，或是被另一個即將進行起始作用的核醣體給置換掉。另外，若我們讓兩個核醣體可以同時結合在起始密碼子和二級結構之間，則會讓核醣體暫停的效果大大地降低。 | zh_TW |
| dc.description.abstract | In E. coli, the transcript of dnaX gene is translated into two subunits of DNA polymerase III, gamma (γ) and tau (τ), through the regulation of programmed -1 ribosomal frameshifting. There are three important elements on RNA that lead to -1 frameshifting: an internal Shine-Dalgarno (SD) sequence, a slippery sequence and a secondary structure. When a ribosome translates to the slippery sequence, 16S rRNA may interact with the internal SD. At the same time, the secondary structure may form a road block to the ribosome. These two factors may cause the ribosomes to pause and undergo frameshifting. In order to study the correlation between ribosome pausing and these two factors, we used both in vivo and in vitro methods to conduct the experiments.
In our study, we used the dnaX -1 frameshifting motif as a model system. We mutated the slippery sequence to abolish frameshifting to directly observe the influence of internal SD and hairpin on ribosome pausing. To prevent polyribosome formation, we shortened the distance between the start codon (AUG) and the secondary structure to ensure that no ribosomes can initiate at the start codon when a ribosome is paused by the structure. Our results suggest that both the internal SD and secondary structure led to ribosome pausing and the secondary structure was the dominant element. Moreover, if two or more ribosomes can accommodate between the AUG and secondary structure, the frequency of ribosome pausing was decreased considerably. We also discovered the ribosome could bind to the internal SD and inhibit initiation of ribosomes on the start codon. The internal SD-bound ribosomes may drop off spontaneously or be displaced by the upstream, newly initiated ribosomes. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T16:17:28Z (GMT). No. of bitstreams: 1 ntu-104-R02b43025-1.pdf: 3953686 bytes, checksum: ad0ea78a6443a336e18caeaca835bcf1 (MD5) Previous issue date: 2015 | 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 vii 表目錄 viii 第一章 前言 1 1.1 核醣體 1 1.2 轉譯作用 2 1.2.1 起始作用 2 1.2.2 延長作用 3 1.2.3 終止作用 3 1.3 框架位移 4 1.4 核醣體暫停 5 1.5 DNA聚合酶III 5 1.6 dnaX -1框架位移 6 1.7 聚核醣體 7 1.8 研究動機 7 第二章 材料與方法 9 2.1 材料 9 2.1.1 細菌株 9 2.1.2 質體 9 2.1.3 試劑 10 2.1.4 藥品 10 2.1.5 酵素 11 2.1.6 抗體 11 2.1.7 核苷酸序列 12 2.1.8 緩衝溶液 14 2.2 方法 14 2.2.1 質體構築 14 2.2.2 蛋白表現 15 2.2.3 西方點墨法及定量 15 2.2.4 細胞外轉錄 16 2.2.5 細胞外轉譯 16 2.2.6 螢光酶檢測法(Luciferase assay) 17 2.2.7 單因子變異數分析和事後檢定 17 第三章 結果 18 3.1 iSD序列和二級結構皆會造成核醣體暫停 18 3.2 iSD序列和二級結構造成不同的核醣體暫停效果 19 3.3 30S會結合在iSD序列 19 3.4 二級結構並不影響30S直接結合至iSD序列 21 3.5 聚核醣體會降低核醣體暫停的效果 22 3.6 mRNA和蛋白質在細胞外轉譯過程中會被降解 23 第四章 討論 25 4.1 30S是否會結合在iSD序列 25 4.2 mRNA的DB與16S rRNA互補可能增加蛋白表現量 26 4.3 與iSD序列結合的30S能否被移除 26 4.4 修改滑動序列會使核醣體提前結束轉譯作用 27 4.5 細胞外轉譯容易受到外在環境影響 28 4.6 核醣體暫停是否和框架位移效率成正相關 29 參考文獻 30 | |
| dc.language.iso | zh-TW | |
| dc.subject | dnaX | zh_TW |
| dc.subject | 框架位移 | zh_TW |
| dc.subject | 核醣體暫停 | zh_TW |
| dc.subject | iSD序列序列 | zh_TW |
| dc.subject | 聚核醣體 | zh_TW |
| dc.subject | dnaX | en |
| dc.subject | frameshifting | en |
| dc.subject | ribosome pausing | en |
| dc.subject | polyribosome | en |
| dc.subject | internal SD | en |
| dc.title | 利用誘導框架位移的RNA序列來探討核醣體暫停 | zh_TW |
| dc.title | The Study of Ribosome Pausing by
Frameshift-stimulating RNA Sequence | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 103-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 黃筱鈞(Hsiao-Chun Huang),張功耀(Kung-Yao Chang) | |
| dc.subject.keyword | dnaX,框架位移,核醣體暫停,iSD序列序列,聚核醣體, | zh_TW |
| dc.subject.keyword | dnaX,frameshifting,ribosome pausing,internal SD,polyribosome, | en |
| dc.relation.page | 67 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2015-08-17 | |
| 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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