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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 溫進德 | |
dc.contributor.author | Tsung-Tse Wu | en |
dc.contributor.author | 吳宗澤 | zh_TW |
dc.date.accessioned | 2021-06-17T04:46:04Z | - |
dc.date.available | 2018-08-08 | |
dc.date.copyright | 2018-08-08 | |
dc.date.issued | 2018 | |
dc.date.submitted | 2018-08-01 | |
dc.identifier.citation | Aitken, C. E., A. Petrov and J. D. Puglisi. (2010). Single ribosome dynamics and the mechanism of translation. Annual Review of Biophysics 39: 491-513.
Ashkin, A. (1970). Acceleration and Trapping of Particles by Radiation Pressure. Phys. Rev. Lett. 24 (4): 156–159. Ashkin A., Dziedzic JM., Bjorkholm JE., Chu S. (1986). Observation of a single-beam gradient force optical trap for dielectric particles. Opt. Lett. 11 (5): 288–290. Cenik C., Cenik ES., Byeon GW., Grubert F., Candille SI., Spacek D., Alsallakh B., Tilgner H., Araya CL., Tang H., Ricci E., Snyder MP. (2015). Integrative analysis of RNA, translation, and protein levels reveals distinct regulatory variation across humans. Genome Research. 25 (11): 1610–21 Chen JL, Greider CW. (2005). Functional analysis of the pseudoknot structure in human telomerase RNA. Proc Natl Acad Sci USA 102(23): 8080–5 Chen, G., Wen, J.-D. and Tinoco, I., Jr. (2007). Single-molecule mechanical unfolding and folding of a pseudoknot in human telomerase RNA. RNA 13, 2175-2188 Dustin B. Ritchie., Daniel A. N. Foster., and Michael T. Woodside. (2012). Programmed −1 frameshifting efficiency correlates with RNA pseudoknot conformational plasticity, not resistance to mechanical unfolding. Proceedings of the National Academy of Sciences 109 (40), 16167-1617 Hunseung Kang and Ignacio Tinoco Jr. (1997). A mutant RNA pseudoknot that promotes ribosomal frameshifting in mouse mammary tumor virus. Nucleic Acids Research, Vol. 25, No. 10 1943–1949. Hunseung Kang., Jennifer V. Hines., and Ignacio Tinoco Jr. (1996). Conformation of a Non-frameshifting RNA Pseudoknot from Mouse Mammary Tumor Virus Hunseung Kang. J. Mol. Biol. 259, 135–147 Juette, MF., Terry, DS., Wasserman, MR., Zhou, Z., Altman, RB; Zheng, Q., Blanchard, SC. (2014). The bright future of single-molecule fluorescence imaging Curr Opin Chem Biol. 20: 103–11. Ling X. Shen and Ignacio Tinoco, Jr. (1995). The Structure of an RNA Pseudoknot that Causes Efficient Frameshifting in Mouse Mammary Tumor Virus. J. Mol. Biol. 247, 963–978 Meulenberg JJ., Hulst MM., de Meijer EJ., Moonen PL., den Besten A., de Kluyver EP., Wensvoort G., Moormann RJ. (1993). Lelystad virus, the causative agent of porcine epidemic abortion and respiratory syndrome (PEARS), is related to LDV and EAV. Virolog. 192(1):62-72. Moffitt JR., Chemla YR., Smith SB., Bustamante C. (2008). Recent advances in optical tweezers. Annu Rev Biochem. 77:205-28 Nakamoto, T. (2011). Mechanisms of the initiation of protein synthesis: In reading frame binding of ribosomes to mRNA. Molecular Biology Reports, 38(2), 847-855 Rivas E., Eddy S. (1999). A dynamic programming algorithm for RNA structure prediction including pseudoknots. J Mol Biol 285(5): 2053–2068. Staple DW., Butcher SE. (2005). Pseudoknots: RNA structures with diverse functions. PLoS Biol. 3 (6): e213 W. E. Moerner and L. Kador. (1989). Optical detection and spectroscopy of single molecules in a solid, Phys. Rev. Lett. 62, 2535 - 2538 Y. Roiter and S. Minko. (2005). AFM Single Molecule Experiments at the Solid-Liquid Interface: In Situ Conformation of Adsorbed Flexible Polyelectrolyte Chains. Journal of the American Chemical Society, vol. 127, iss. 45, pp. 15688–15689 | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/70964 | - |
dc.description.abstract | 框架位移作用係發生於細胞內部一種轉譯重編碼現象,病毒經常會利用-1框架位移來表現特定的蛋白以及酵素(例如:SARS-CoV, HIV以及IBV),當核醣體在進行轉譯作用時,mRNA上的特殊二級結構,例如偽結結構,可能會刺激框架位移的發生,而且框架位移的效率與偽結結構的穩定性呈現正相關。
MMTV是源自於老鼠乳腺腫瘤病毒的偽結構造,先前已經有研究團隊對它進行分析,也有利用此偽結結構進行框架位移的觀察,惟對於MMTV詳細的摺疊機制目前仍非十分清楚,且亦尚未有研究實際利用核醣體針對MMTV進行框架位移的探究,是以本實驗欲透過實際加入30S去探討對於不同框架位移效率的偽結結構,會產生何種差異性。 本研究利用單分子技術,以雷射光鉗對MMTV以及其突變體APK施加外力,並且測量解開結構所需要之外力與打開長度,進而推導其中的詳細機制,此外我們也實際加入30S以及tRNAfMet形成pre-initiation complex (pre-IC),想去觀察其對於結構穩定性會造成何種影響。我們從實驗中發現,30S對於偽結結構的確會有實際的影響,並且在不同框架位移效率的偽結影響力也不相同,另外我們亦發現若僅加入30S反而對於結構的影響力會比pre-IC更顯著。在Constant force實驗中我們發現30S可以穩定並且幫助結構的摺疊,而且MMTV偽結結構的摺疊會分成兩步進行,此外,偽結上的第一個髮夾與結構的摺疊也有著一定的關聯性。綜合上述結果,我們推斷MMTV偽結結構的摺疊為兩步摺疊,並且是先疊第二個髮夾接著才是第一個髮夾,且在有加入30S的情況下,可以更加快速的幫助結構摺疊以及穩定。 | zh_TW |
dc.description.abstract | Frameshifting is a recording translation mechanism. Viruses often use -1 frame-shifting to express specific proteins and enzymes (eg, SARS-CoV, HIV, and IBV). When the ribosome is undergoing translation, specially secondary structures on mRNA, such as pseudoknot structures, may stimulate the occurrence of frameshifting, and the efficiency of frameshifting is proportional with the stability of the pseudoknot structure.
MMTV is an RNA pseudoknot derived from the mouse mammary tumor virus. It has been analyzed previously for frameshifting. However, the detailed folding mechanism of MMTV is not well understood, and no related research has been done by using ribosomes to observe the behavior of the MMTV frameshifting so far. Therefore, the main purpose of this study is dedicated on investigating the features of different frameshift-stimulating RNA pseudoknots and their responses to the action of ribosomal 30S subunit.. In this study, optical tweezers had been applied to exert external force to the MMTV and the APK. From the unfolding force and opened length, we can deduce the detailed mechanism. In addition, 30S and tRNAfMet were added to form pre-initiation complexes to observe the impact on structural stability. From the experimental outcome, 30S has prominent influence on the pseudoknot structure, and the influence can be divided into different levels according to the pseudoknot with different frameshifting efficiencies. On the other hand, we also found that if merely added 30S, the influence on the structure was more dramatic than pre-initiation complex. In experiment of Jump-drop, 30S can play a role in structure stabilization and assisting folding. Moreover, the refolding of MMTV pseudoknot occurred in two steps. Concluded by previous mention, the mechanism of refolding can be assumed by two steps. The pathway of pseudoknot formation is hp2 folded firstly, followed hp1. At the same time , the presence of 30S can accelerate the refolding and stabilize structure. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T04:46:04Z (GMT). No. of bitstreams: 1 ntu-107-R05b43026-1.pdf: 3764683 bytes, checksum: 0140f0a6b4acbbee9b34ba8a90fb0e1a (MD5) Previous issue date: 2018 | en |
dc.description.tableofcontents | 目錄
口試委員審定書 i 致謝 ii 摘要 iii Abstract iv 目錄 vi 圖表目錄 x 第一章 導論 1 1.1 轉譯作用 1 1.1.1 起始作用 1 1.1.2 延長作用 1 1.1.3 終止作用 1 1.1.4 轉譯障礙 1 1.2 計畫性框架位移 2 1.2.1 偽結結構 2 1.2.2 人類端粒酶RNA 2 1.3 單分子技術 3 1.3.1 介紹 3 1.3.2 單分子的應用 3 1.3.3 雷射光鉗 4 1.3.1.1 歷史應用 4 1.3.3.2 原理 4 1.4 研究動機 5 1.4.1 利用天然偽結結構作為研究標的 5 1.4.2 MMTV以及APK 5 第二章 材料與方法 6 2.1 材料 6 2.1.1 勝任細胞品系 6 2.1.2 質體 6 2.1.3 載體DNA序列及引子設計 6 2.1.4 試劑 8 2.1.5 藥品 11 2.1.6 酵素 11 2.1.7 溶液 12 2.2 方法 13 2.2.1 質體建立 13 2.2.2 細胞外轉錄作用 14 2.2.3 聚合酶連鎖反應製作DNA handle 14 2.2.4 DNA handle的修飾 15 2.2.5 DNA handle與RNA的黏合反應 15 2.2.6 單分子雷射光鉗 15 2.2.6.1 實驗樣本製備 15 2.2.6.2 力的遞增實驗(force ramp experiment) 16 2.2.6.3 定力實驗(constant force experiment) 16 2.2.6.3 實驗數據分析 16 第三章 結果 17 3.1 利用雷射光鉗探討MMTV以及APK摺疊機制 17 3.1.1 探討在wild type的MMTV以及APK力的分析以及分佈 17 3.1.2 利用5’handle的延長去模仿核醣體嘗試打開偽結的情況 17 3.1.2.1 5’handle延伸兩個核苷酸 17 3.1.2.2 5’handle延伸三個核苷酸 18 3.2 實際加入30S去觀察對於MMTV以及APK的影響 19 3.2.1 不同距離的序列設計 19 3.2.2 利用雷射光鉗觀察力的變化 19 3.2.2.1 MMTV-11以及MMTV-13 20 3.2.2.2 APK-11以及APK-13 20 3.2.3 利用Constant Force來觀察差異性 21 3.2.4 針對MMTV-et2進行Constant force實驗 22 3.3 分別對於MMTV hp1以及hp2進行實驗觀察 23 3.3.1 利用handle延長來產生hp1以及hp2 24 3.3.2 利用雷射光鉗觀察hp1以及hp2力的變化 24 第四章 討論 25 4.1 MMTV與APK摺疊情況 25 4.2 沒有加入tRNA的30S對於結構影響力更大 26 4.3 30S會穩定結構並幫助摺疊 28 4.4 MMTV摺疊分兩種型態 29 4.5 MMTV摺疊分兩種型態 30 4.6 未來展望 31 參考文獻 32 圖表目錄 圖1……………………………………………………………………………………..35 圖2……………………………………………………………………………………..37 圖3……………………………………………………………………………………..38 圖4……………………………………………………………………………………..41 圖5……………………………………………………………………………………..43 圖6……………………………………………………………………………………..46 圖7……………………………………………………………………………………..47 圖8……………………………………………………………………………………..49 圖9……………………………………………………………………………………..52 圖10……………………………………………………………………………………54 圖11……………………………………………………………………………………55 圖12……………………………………………………………………………………56 圖13……………………………………………………………………………………57 圖14……………………………………………………………………………………59 圖15……………………………………………………………………………………62 圖16……………………………………………………………………………………63 圖17……………………………………………………………………………………66 圖18……………………………………………………………………………………67 圖19……………………………………………………………………………………69 表1……………………………………………………………………………………..71 表2……………………………………………………………………………………..72 | |
dc.language.iso | zh-TW | |
dc.title | 探討核醣體30S對於能誘導框架位移之核酸偽結所產生的影響 | zh_TW |
dc.title | Exploring how the ribosomal 30S subunit affects two frameshift-stimulating pseudoknot RNA | en |
dc.type | Thesis | |
dc.date.schoolyear | 106-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 楊立威,李盼,張功耀 | |
dc.subject.keyword | 偽結結構,框架位移,核醣體,雷射光鉗,單分子技術, | zh_TW |
dc.subject.keyword | frameshifting,pseudoknot,ribosome,single-molecule,optical tweezers, | en |
dc.relation.page | 72 | |
dc.identifier.doi | 10.6342/NTU201802347 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2018-08-02 | |
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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