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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 溫進德(Jin-Der Wen) | |
| dc.contributor.author | An-I Lee | en |
| dc.contributor.author | 李安益 | zh_TW |
| dc.date.accessioned | 2021-06-17T04:43:57Z | - |
| dc.date.available | 2018-08-13 | |
| dc.date.copyright | 2018-08-13 | |
| dc.date.issued | 2018 | |
| dc.date.submitted | 2018-08-03 | |
| dc.identifier.citation | Agranovsky, A. A., Dolja, V. V., & Atabekov, J. G. (1982). Structure of the 3’ extremity of barley stripe mosaic virus RNA: evidence for internal poly(A) and a 3'-terminal tRNA-like structure. Virology, 119, 51–58.
Ban, N., Nissen, P., Hansen, J., Moore, P. B., & Steitz, T. A. (2000). The complete atomic structure of the large ribosomal subunit at 2.4 A resolution. Science, 289, 905–920. Berk, V., Zhang, W., Pai, R. D., Cate, J. H. D., & Cate, J. H. D. (2006). Structural basis for mRNA and tRNA positioning on the ribosome. Proceedings of the National Academy of Sciences of the United States of America, 103, 15830–15834. Carter, A. P., Clemons, W. M., Brodersen, D. E., Morgan-Warren, R. J., Wimberly, B. T., & Ramakrishnan, V. (2000). Functional insights from the structure of the 30S ribosomal subunit and its interactions with antibiotics. Nature, 407, 340–348. Carter, A. P., Clemons, W. M., Jr, Brodersen, D. E., Morgan-Warren, R. J., Hartsch, T., Wimberly, B. T., & Ramakrishnan, V. (2001). Crystal structure of an initiation factor bound to the 30S ribosomal subunit. Science, 291, 498–501. Chang, K,-C. (2018). Single-Molecule and Theoretical Approaches Reveal Translation Recoding Guided by mRNA Dynamics. In Institute of Molecular and Cellular Biology (National Taiwan University), pp.1-357. Chang, Y,-C. (2016). Engineering Fluorescently Labled Ribosome for Observing mRNA-Ribosome Interaction at Single-Molecule Level. In Institute of Molecular and Cellular Biology (National Taiwan University), pp.1-93. Chen, G., Chang, K.-Y., Chou, M.-Y., Bustamante, C., & Tinoco, I. (2009). Triplex structures in an RNA pseudoknot enhance mechanical stability and increase efficiency of--1 ribosomal frameshifting. Proceedings of the National Academy of Sciences, 106, 12706–12711. Chou, M. Y., & Chang, K. Y. (2009). An intermolecular RNA triplex provides insight into structural determinants for the pseudoknot stimulator of− 1 ribosomal frameshifting. Nucleic Acids Research 38, 1676-1685. Doty, P., Boedtker, H., Fresco, J. R., Haselkorn, R., & Litt, M. (1959). SECONDARY STRUCTURE IN RIBONUCLEIC ACIDS. Proceedings of the National Academy of Sciences of the United States of America, 45, 482–499. Forster, T. (1948). Zwischenmolekulare Energiewanderung und Fluoreszenz. Annalen der Physik 437, 55-75. Giedroc, D. P., & Cornish, P. V. (2009). Frameshifting RNA pseudoknots: structure and mechanism. Virus Research, 139, 193–208. Gualerzi, C. O., & Pon, C. L. (1990). Initiation of mRNA translation in prokaryotes. Biochemistry, 29, 5881–5889. Gualerzi, C., Risuleo, G., & Pon, C. L. (1977). Initial rate kinetic analysis of the mechanism of initiation complex formation and the role of initiation factor IF-3. Biochemistry, 16, 1684–1689. Ha, T. (2001). Single-molecule fluorescence resonance energy transfer. Methods , 25(1), 78–86. Hirokawa, G., Demeshkina, N., Iwakura, N., Kaji, H., & Kaji, A. (2006). The ribosome-recycling step: consensus or controversy? Trends in Biochemical Sciences, 31, 143–149. Hsieh, P.-S. (2016). Study of Interactions Between the mRNA Pseudoknot and the Ribosome with Single-Molecule FRET. In Institute of Molecular and Cellular Biology (National Taiwan University), pp.1-74. Jacks, T., & Varmus, H. E. (1985). Expression of the Rous sarcoma virus pol gene by ribosomal frameshifting. Science, 230, 1237–1242. Kapanidis, A. N., & Strick, T. (2009). Biology, one molecule at a time. Trends in Biochemical Sciences, 34, 234–243. La Teana, A., Gualerzi, C. O., & Brimacombe, R. (1995). From stand-by to decoding site. Adjustment of the mRNA on the 30S ribosomal subunit under the influence of the initiation factors. RNA , 1, 772–782. Luthe, D. S. (1983). A simple technique for the preparation and storage of sucrose gradients. Analytical Biochemistry, 135, 230-232. Laursen, B. S., Sørensen, H. P., Mortensen, K. K., & Sperling-Petersen, H. U. (2005). Initiation of protein synthesis in bacteria. Microbiology and Molecular Biology Reviews: MMBR, 69, 101–123. Liljas, A., & al-Karadaghi, S. (1997). Structural aspects of protein synthesis. Nature Structural Biology, 4, 767–771. Michalet, X., Weiss, S., & Jäger, M. (2006). Single-molecule fluorescence studies of protein folding and conformational dynamics. Chemical Reviews, 106, 1785–1813. Michiels, P., Versleijen, A., & Verlaan, P. W. (2001). Solution structure of the pseudoknot of SRV-1 RNA, involved in ribosomal frameshifting1. Journal of Molecular. 310, 1109-1123. Nixon, P. L., Rangan, A., Kim, Y.-G., Rich, A., Hoffman, D. W., Hennig, M., & Giedroc, D. P. (2002). Solution Structure of a Luteoviral P1–P2 Frameshifting mRNA Pseudoknot. Journal of Molecular Biology, 322, 621–633. Palade, G. E. (1955). A small particulate component of the cytoplasm. The Journal of Biophysical and Biochemical Cytology, 1, 59–68. Perrotta, A. T., & Been, M. D. (1991). A pseudoknot-like structure required for efficient self-cleavage of hepatitis delta virus RNA. Nature, 350, 434–436. Petrelli, D., LaTeana, A., Garofalo, C., Spurio, R., Pon, C. L., & Gualerzi, C. O. (2001). Translation initiation factor IF3: two domains, five functions, one mechanism? The EMBO Journal, 20, 4560–4569. Polacek, N., & Mankin, A. S. (2005). The ribosomal peptidyl transferase center: structure, function, evolution, inhibition. Critical Reviews in Biochemistry and Molecular Biology, 40, 285–311. Ramakrishnan, V. (2002). Ribosome structure and the mechanism of translation. Cell, 108, 557–572. Schluenzen, F., Tocilj, A., Zarivach, R., Harms, J., Gluehmann, M., Janell, D., … Yonath, A. (2000). Structure of functionally activated small ribosomal subunit at 3.3 angstroms resolution. Cell, 102, 615–623. Schürer, H., Lang, K., Schuster, J., & Mörl, M. (2002). A universal method to produce in vitro transcripts with homogeneous 3’ ends. Nucleic Acids Research, 30, e56. Scolnick, E., Tompkins, R., Caskey, T., & Nirenberg, M. (1968). Release factors differing in specificity for terminator codons. Proceedings of the National Academy of Sciences of the United States of America, 61, 768–774. Shen, L. X., & Tinoco, I., Jr. (1995). The structure of an RNA pseudoknot that causes efficient frameshifting in mouse mammary tumor virus. Journal of Molecular Biology, 247, 963–978. Steitz, T. A. (2008). A structural understanding of the dynamic ribosome machine. Nature Reviews. Molecular Cell Biology, 9, 242–253. Stryer, L., & Haugland, R. P. (1967). Energy transfer: a spectroscopic ruler. Proceedings of the National Academy of Sciences of the United States of America, 58, 719–726. Takyar, S., Hickerson, R. P., & Noller, H. F. (2005). mRNA helicase activity of the ribosome. Cell, 120, 49–58. Theimer, C. A., Blois, C. A., & Feigon, J. (2005). Structure of the human telomerase RNA pseudoknot reveals conserved tertiary interactions essential for function. Molecular Cell, 17, 671–682. Thill, G., Vasseur, M., & Tanner, N. K. (1993). Structural and sequence elements required for the self-cleaving activity of the hepatitis delta virus ribozyme. Biochemistry, 32, 4254-4262. Valle, M., Zavialov, A., Sengupta, J., Rawat, U., Ehrenberg, M., & Frank, J. (2003). Locking and unlocking of ribosomal motions. Cell, 114, 123–134. Walter, N. G., Huang, C.-Y., Manzo, A. J., & Sobhy, M. A. (2008). Do-it-yourself guide: how to use the modern single-molecule toolkit. Nature Methods, 5, 475–489. Weinger, J. S., Parnell, K. M., Dorner, S., Green, R., & Strobel, S. A. (2004). Substrate-assisted catalysis of peptide bond formation by the ribosome. Nature Structural & Molecular Biology, 11, 1101–1106. Wimberly, B. T., Brodersen, D. E., Clemons, W. M., Jr, Morgan-Warren, R. J., Carter, A. P., Vonrhein, C., … Ramakrishnan, V. (2000). Structure of the 30S ribosomal subunit. Nature, 407, 327–339. Yusupova, G. Z., Yusupov, M. M., Cate, J. H., & Noller, H. F. (2001). The path of messenger RNA through the ribosome. Cell, 106, 233–241. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/70922 | - |
| dc.description.abstract | 偽結為一RNA二級結構，其參與許多重要的生物反應過程如RNA的複製、轉錄和轉譯等。此外，研究也發現如將偽結結構上游放置一段滑動序列，能有效刺激核醣體轉譯時發生-1框架位移，許多病毒如HIV、IBV皆是透過此方式轉譯出特定蛋白。本實驗利用由人類端粒酶RNA修改之DU177偽結做為模板，其結構上包含了兩個部分配對的莖-環，以及維持整個結構高度穩定的三重鹼基配對。在先前研究已發現當轉譯過程中，部分偽結序列進入核醣體時，會觀察到偽結結構被扭曲成一緊密的中間構形。同時，部分核醣體會因無法克服此穩定的二級結構而暫停轉譯。在本篇研究中，我們透過標定特定螢光於偽結結構上，以單分子螢光共振能量轉移技術去觀察轉譯過程中不同條件下偽結結構變化。結果發現，當去除結構中三重鹼基配對時，核醣體可輕易解開偽結，且轉譯過程中不再出現中間構形，此結果也呼應之前研究發現將三重鹼基對去除時，核醣體被誘導之-1框架位移效率為零。而在另一組實驗，我們保持偽結的三重鹼基配對但添加一段滑動序列於結構上游，在給予足夠tRNA情況下，我們預期核醣體有更多機會去解開偽結結構。結果顯示，在轉譯過程中被扭轉的中間構形同樣會出現，多數核醣體如預期皆能順利解開結構，沒有出現暫停轉譯的現象。綜合實驗結果，我們發現三重鹼基配對對於偽結來說是重要的，其能讓核醣體在轉譯過程逐步解旋時扭曲成緊密的中間構形。此外，當核醣體位於滑動序列時，會受到下游偽結影響而發生-1框架位移，幫助核醣體將偽結結構打開使轉譯不至完全停止。 | zh_TW |
| dc.description.abstract | Pseudoknots are a type of RNA structures that participate in many biologically important processes. They can program the ribosome to frameshift when positioned downstream of a slippery sequence. Here we use the DU177 pseudoknot, which is derived from the human telomerase RNA, as a model system. The structure of DU177 contains two partially overlapping stem-loop, and several base triples that strongly stabilize the overall structure. Our lab previously showed that during translation and ribosomal unwinding of DU177, the pseudoknot is twisted into a compact intermediate structure. The ribosome also tends to pause due to the presence of this stable “roadblock”. In my study, I performed single-molecule Förster resonance energy transfer (smFRET) to further study how base triples of DU177 induce frameshifting. I found that for the pseudoknot mutant without base triples, the ribosome unwinds the structure easily, and that the compact intermediate does not form during ribosomal unwinding. Alternatively, when a slippery sequence is inserted upstream of DU177, the intermediate structure can only form transiently as the ribosome slips and attempts to unwind DU177 multiple times on a sub-second time scale. Notably, despite the mechanical resistance of base triples, most of the ribosome molecules successfully unwind the structure after multiple attempts on the tract of the slippery sequence.
In conclusion, we show that base triples serve as a key element for the formation of the compact pseudoknot intermediate that resists ribosomal unwinding and finally leads to -1 frameshifting in the presence of a slippery sequence. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T04:43:57Z (GMT). No. of bitstreams: 1 ntu-107-R05b43010-1.pdf: 7168260 bytes, checksum: 723ba65a8c0ef7cdd1138f280878370a (MD5) Previous issue date: 2018 | 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啟始階段 2 1.2.2 延長階段 3 1.2.3 終止階段 3 1.3 偽結 (PSEUDOKNOT) 4 1.4 滑動序列 (SLIPPERY SEQUENCE) 5 1.5 單分子技術 5 1.5.1 簡介我 5 1.5.2 螢光共振能量轉移 (Fluorescence resonance energy transfer) 6 1.6 D型肝炎病毒核酶 (HEPATITIS DELTA VIRUS RIBOZYME) 6 1.7 研究動機 7 第二章 材料與方法 8 2.1材料 8 2.1.1 序列設計 8 2.1.2 酵素 11 2.1.3 藥品 11 2.1.4 試劑 13 2.1.5 溶液 13 2.1.6 樣品配置 14 2.2方法 16 2.2.1 核醣體製備 16 2.2.2 RNA序列製備 19 2.2.3 聚合酶連鎖反應 (Polymerase Chain Reaction, PCR) 19 2.2.4 RNA螢光標定及純化 20 2.2.5 DNA夾板輔助RNA連接反應 21 2.2.6 RNA凝膠電泳純化 21 2.2.7 單分子螢光共振能量轉移實驗 (smFRET) 22 第三章 結果 25 3.1 核醣體製備與純化 25 3.2 RNA樣品製備 25 3.2.1 製備具Shine-Dalgarno序列及起始密碼子之RNA片段 25 3.2.2 RNA螢光標定及HPLC純化 26 3.2.3 DNA夾板輔助RNA連接反應 26 3.3 RNA樣本之SMFRET實驗 26 3.4 核醣體轉譯過程中偽結結構之FRET效率變化 27 3.4.1 PK MFKEY U3C 27 3.4.2 PK MVKKFY 28 3.4.3 PK MVKKFY(dG) 29 3.5 嘗試篩選出有進行轉譯作用的核醣體 30 3.6 減少FRET背景訊號及核醣體活性測試 30 第四章 討論 32 4.1 轉譯過程之偽結結構變化 32 4.1.1 PK MFKEY U3C 32 4.1.2 PK MVKKFY 32 4.1.3 PK MVKKFY(dG) 33 4.2 無法有效觀測螢光分子的動態變化 34 4.3 使用生物素化修飾之DNA引子無法有效篩選掉不具活性的核醣體 34 第五章 結論與未來展望 35 參考文獻 36 | |
| dc.language.iso | zh-TW | |
| dc.subject | 偽結 | zh_TW |
| dc.subject | 核醣體 | zh_TW |
| dc.subject | 轉譯作用 | zh_TW |
| dc.subject | 單分子 | zh_TW |
| dc.subject | 螢光共振能量轉移 | zh_TW |
| dc.subject | single-molecule | en |
| dc.subject | ribosome | en |
| dc.subject | frameshifting | en |
| dc.subject | fluorescence resonance energy transfer(FRET) | en |
| dc.subject | pseudoknot | en |
| dc.title | 利用單分子螢光共振能量轉移技術探討不同修飾之RNA偽結與核醣體間交互作用 | zh_TW |
| dc.title | Study of Interactions Between Different Modifications of mRNA Pseudoknots and the Ribosome by Single-Molecule FRET | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 106-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 楊立威(Lee-Wei Yang),張功耀(Kung-Yao Chang),李盼(Pan Li) | |
| dc.subject.keyword | 偽結,核醣體,轉譯作用,單分子,螢光共振能量轉移, | zh_TW |
| dc.subject.keyword | pseudoknot,ribosome,frameshifting,single-molecule,fluorescence resonance energy transfer(FRET), | en |
| dc.relation.page | 75 | |
| dc.identifier.doi | 10.6342/NTU201802348 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2018-08-03 | |
| 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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