請用此 Handle URI 來引用此文件：
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/59480完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 溫進德 | |
| dc.contributor.author | Zhen-An Wu | en |
| dc.contributor.author | 吳鎮安 | zh_TW |
| dc.date.accessioned | 2021-06-16T09:25:06Z | - |
| dc.date.available | 2017-08-25 | |
| dc.date.copyright | 2017-08-25 | |
| dc.date.issued | 2017 | |
| dc.date.submitted | 2017-06-15 | |
| dc.identifier.citation | Agalarov, S. C., G. Sridhar Prasad, P. M. Funke, C. D. Stout and J. R. Williamson (2000). 'Structure of the S15,S6,S18-rRNA complex: assembly of the 30S ribosome central domain.' Science 288(5463): 107-113.
Ashkin, A., J. M. Dziedzic, et al. (1986). 'Observation of a single-beam gradient force optical trap for dielectric particles.' Opt Lett 11(5): 288 Ban, N., P. Nissen, J. Hansen, P. B. Moore and T. A. Steitz (2000). 'The complete atomic structure of the large ribosomal subunit at 2.4 A resolution.' Science 289(5481): 905-920. Block, S. M., L. S. Goldstein and B. J. Schnapp (1990). 'Bead movement by single Kinesin molecules studied with optical tweezers.' Nature 348(6299): 348-352 Bornhorst, J. A. and J. J. Falke (2000). ' Purification of proteins using polyhistidine affinity tags.' Methods Enzymol 326: 245-254. Bustamante, C., Y. R. Chemla, N. R. Forde and D. Izhaky (2004). 'Mechanical processes in biochemistry.' Annu Rev Biochem 73: 705-748. Chen, G., J. D. Wen and I. Tinoco, Jr. (2007). 'Single-molecule mechanical unfoldinf and folding of a pseudoknot in human telomerase RNA.' RNA 13(12): 2175-2188. Dumont, S., W. Cheng, V. Serebrov, R. K. Beran, I. Tinoco, Jr., A. M. Pyle and C. Bustamante (2006). 'RNA translocation and unwinding mechanism of HCV NS3 helicase and its coordination by ATP.' Nature 439(7072): 105-108. Greenleaf, W. J., K. L. Frieda, D. A. Foster, M. T. Woodside and S. M. Block (2008). 'Direct observation of hierarchical folding in single riboswitch aptamers.' Science 319(5863): 630-633 Lang, M. J. and S. M. Block (2003). 'Resource Letter: LBOT-1: Laser-based optical tweezers.' Am J Phys 71(3): 201-215. Laursen, B. S., H. P. Sorensen, K. K. Mortensen and H. U. Sperling-Petersen (2005). 'Initiation of protein synthesis in bacteria.' Microbiol Mol Biol Rev 69(1): 101-123. Li, P. T., C. Bustamante and I. Tinoco, Jr. (2007). 'Real-time control of the energy landscape by force directs the folding of RNA molecules.' Proc Natl Acad Sci U S A 104(17): 7039-7044. Li, P. T. and I. Tinoco, Jr. (2009). 'Mechanical unfolding of two DIS RNA kissing complex from HIV-1.' J Mol Biol 386(5): 1343-1356 Maitra, U., E. A. Stringer, et al. (1982). 'Initiation factors in protein biosynthesis.' Annu Rev Biochem 51: 869-900 Mammen, M., K. Helmerson, R. Kishore, S. K. Choi, W. D. Phillips and G. M. Whitesides (1996). 'Optically controlled collisions of biological object to evaluate potent polyvalent inhibitors of virus-cell adhesion.' Chem Biol 3(9): 757-763. Marzi, S., A. G. Myasnikov, A. Serganov, C. Ehresmann, P. Romby, M. Yusupov and B. P. Klaholz (2007). 'Structured mRNAs regulate translation initiation by binding to the platform of the ribosome.' Cell 130(6): 1019-1031. Mikhailenko, S. V., Y. Oguchi and S. Ishiwata (2010). 'Insights into the mechanisms of myosin and kinesin molecular motors from the single-molecule unbinding force measurements.' J R Soc Interface 7 Suppl 3: S295-306 Neuman, K. C. and S. M. Block (2004). 'Optical traping.' Rev Sci Instrum 75(9): 2787-2809 Onoa, B., S. Dumont, J. Liphardt, S. B. Smith, I. Tinoco, Jr. and C. Bustamante (2003). 'Identifying kinetic barriers to mechanical unfolding of the T. thermophila ribozyme.' Science 299(5614): 1892-1895 Park, J. H., H. E. Johansson, et al. (2012). 'Post-translational modification by beta-lysylation is required for activity of Escherichia coli elongation factor P(EF-P).' J Biol Chem 287(4): 2579-2590. Philippe, C., F. Eyermann, L. Benard, C. Portier, B. Ehresmann and C. Ehresmann (1993). 'Ribosomal protein S15 from Escherichia coli modulates its own translation by trapping the ribosome on the mRNA initiation loading site.' Proc Natl Acad Sci U S A 90(10): 4394-4398. Philippe, C., C. Portier, M. Mougel, M. Grunberg-Manago, J. P. Ebel, B. Ehresmann and C. Ehresmann (1990). 'Target site of Escherichia coli ribosomal protein S15 on its messenger RNA. Conformation and interaction with the protein.' J Mol Biol 211(2): 415-426. Portier, C., C. Philippe, L. Dondon, M. Grunberg-Manago, J. P. Ebel, B. Ehresmann and C. Ehresmann (1990). 'Translational control of ribosomal protein S15.' Biochim Biophys Acta 1050(1-3): 328-336. Rice, S. E., T. J. Purcell and J. A. Spudich (2003). 'Building and using optical traps to study properties of molecular motors.' Methods Enzymol 361: 112-133. Rittie, L. and B. Perbal (2008). 'Enzymes used in molecular biology: a useful guide.' J Cell Commun Signal 2(1-2): 25-45 Sambrook, J. and D. W. Russell (2006). 'Labeling 3' Termini of Double-stranded DNA with Bacteriophage T4 DNA Polymerase.' CSH Protoc 2006(1). Schlax, P. J. and D. J. Worhunsky (2003). 'Translational repression mechanisms in prokaryotes.' Mol Microbiol 48(5): 1157-1169. Schmitt, J., H. Hess and H. G. Stunnenberg (1993). 'Affinity purification of histidine-tagged proteins.' Mol Biol Rep 18(3): 223-230. Shine, J., L. Dalgarno and J. A. Hunt (1974). 'Fingerprinting of eukaryotic ribosomal RNA labelled with tritiated nucleosides.' Anal Biochem 59(2): 360-365. Smith, D. E., S. J. Tans, S. B. Smith, S. Grimes, D. L. Anderson and C. Bustamante (2001). 'The bacteriophage straight phi29 portal motor can package DNA against a large internal force.' Nature 413(6857): 748-752. Soto, A. M., V. Misra and D. E. Draper (2007). 'Tertiary structure of an RNA pseudoknot is stabilized by 'diffuse' Mg2+ ions.' Biochemistry 46(11): 2973-2983. Stryer, L. and R. P. Haugland (1967). 'Energy transfer: a spectroscopic ruler.' Proc Natl Acad Sci U S A 58(2): 719-726. Svoboda, K. and S. M. Block (1994). 'Force and velocity measured for single kinesin molecules.' Cell 77(5): 773-784. Svoboda, K., C. F. Schmidt, B. J. Schnapp and S. M. Block (1993). 'Direct observation of kinesin stepping by optical trapping interferometry.' Nature 365(6448): 721-727. Takata, R., T. Mukai, M. Aoyagi and K. Hori (1984). ' Nucleotide sequence of the gene for Escherichia coli ribosomal protein S15 (rpsO).' Mol Gen Genet 197(2): 225-229. Veigel, C. and C. F. Schmidt (2011). 'Moving into the cell: single-molecule studies of molecular motors in complex environments.' Nat Rev Mol Cell Biol 12(3): 163-176. Warner, J. R., P. M. Knopf, et al. (1963). 'A multiple ribosomal structure in protein synthesis.' Proc Natl Acad Sci U S A 49: 122-129. Wen, J. D., L. Lancaster, C. Hodges, A. C. Zeri, S. H. Yoshimura, H. F. Noller, C. Bustamante and I. Tinoco (2008). 'Following translation by single ribosomes one codon at a time.' Nature 452(7187): 598-603. Wimberly, B. T., D. E. Brodersen, W. M. Clemons, Jr., R. J. Morgan-Warren, A. P. Carter, C. Vonrhein, T. Hartsch and V. Ramakrishnan (2000). 'Structure of the 30S ribosomal subunit.' Nature 407(6802): 327-339. Wu, Y. J. (2013). Using Optical Tweezers to Study the Mechanism of Structural Rearrangement in 5'UTR of rpsO mRNA. Master Thesis, National Taiwan University. Wu, Y. J., C. H. Wu, A. Y. Yeh and J. D. Wen (2014). 'Folding a stable RNA pseudoknot through rearrangement of two hairpin structures.' Nucleic Acids Res 42(7): 4505-4515. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/59480 | - |
| dc.description.abstract | 在細胞中，信使核醣核酸（mRNA）的轉譯起始作用需要受到高度調控。在此，我們聚焦於大腸桿菌（Escherichia coli）rpsO 基因所轉譯的核醣體蛋白 S15（ecS15）與其自身信使核醣核酸5’端未轉譯區之交互作用，當細胞中S15過量時此作用將導致其合成受到抑制。rpsO 基因的信使核醣核酸5’端未轉譯區可以摺疊成雙髮夾（double-hairpin）或假結（pseudoknot）兩種結構，但唯有後者可與核醣體或S15產生交互作用，而此信使核醣核酸的SD（Shine-Dalgarno）序列只會暴露於假結結構，當S15結合時，會阻擋核醣體解開此結構而抑制轉譯作用。在過去，假結結構已被廣泛研究，然而雙髮夾的作用仍了解得不夠完全。
在本篇研究，我們嘗試使用光鉗技術探究S15對rpsO 基因信使核醣核酸5’端未轉譯區之結構動力學的影響，光鉗技術使我們能夠在單分子層次即時觀察核醣核酸的構形改變。而我們發現S15的存在不只使假結結構出現的比例增加，更使解開結構所需的力大幅上升。我們推測S15能夠藉此穩定假結結構，使得核醣體無法將其解開而進行轉譯起始作用。在此實驗中，我們還嘗試重新設計光鉗實驗使用的DNA handles，使其具有大量洋地黃毒（digoxigenin）提高與珠體結合的穩定性，以因應S15結合時解開假結結構所需的較高額外施力，而我們也成功合成之，提供未來進行相關實驗時一項有用的工具。 | zh_TW |
| dc.description.abstract | Translation initiation of mRNA is a highly regulated process in the cell. Here, we have focused on the initiation between Escherichia coli ribosomal protein S15 (ec15, encoded by the rpsO gene) and the 5’ untranslated region (5’ UTR) of its own mRNA. The reaction results in repressing its biosynthesis when ecS15 is in excess in the cell. The 5’ UTR of rpsO mRNA can fold into two alternative structures, double-hairpin and pseudoknot, but only the latter can interact with the ribosome and S15. The Shine-Dalgarno(SD) sequence of rpsO mRNA is only exposed on the pseudoknot structure. When S15 binds to the pseudoknot, the ribosome is blocked from unfolding the structure, consequently repressing its own translation. In previous research, the pseudoknot form has been extensively studied, but the role of the double-hairpin is still not well understood.
In this study, we used optical tweezers to characterize the structure dynamics of the 5’ UTR of the rpsO mRNA in the presence of S15. Optical tweezers allow us to observe RNA conformational change in real time at the single molecule level. In our results, we find that S15 not only increases the population of the pseudoknot form, but also stabilizes it such that a higher force is needed to unfold the structure. We suggest the S15 binds to the pseudoknot form to enhance the protein-RNA interaction, consequently blocking the ribosome from melting the structure to initiate translation. In addition, we also tried to design new handles used in optical tweezers experiment. The handles have many copies of digoxigenin to increase the stability betweem handles and beads. It allows us to use higher force to unfold the pseudoknot structure when S15 binds. We have successfully made the handles, which provides a useful tool in future works. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T09:25:06Z (GMT). No. of bitstreams: 1 ntu-106-R02b43028-1.pdf: 2768132 bytes, checksum: c9f6916417dea3a88788b0b8c5ebcf63 (MD5) Previous issue date: 2017 | en |
| dc.description.tableofcontents | 口試委員審定書...ii
致謝...iii 摘要...iv ABSTRACT...v 目錄...vii 圖目錄...ix 第一章 導論...1 1.1 大腸桿菌核醣體與其核醣體蛋白S15...1 1.2 核醣體蛋白S15的轉譯自調節...2 1.3 光鉗單分子技術...2 1.4 研究動機...3 第二章 材料與方法...5 2.1 材料...5 2.1.1 勝任細胞品系...5 2.1.2 質體...5 2.1.3 引子...5 2.1.4 酵素...6 2.1.5 藥品...7 2.1.6 試劑...9 2.1.7 緩衝溶液...9 2.2 方法...11 2.2.1 載體構築與純化...11 2.2.2 胞外轉錄作用（In vitro transcription）...11 2.2.3 聚合酶連鎖反應（Polymerase Chain Reaction，PCR）...11 2.2.4 Handles修飾...12 2.2.5 DNA handles與RNA之黏合（annealing）反應...12 2.2.6 光鉗實驗...13 2.2.7 大腸桿菌核醣體蛋白S15的純化...14 2.2.8 設計具有多DIG的5’端handles...16 第三章 結果...18 3.1 純化野生型大腸桿菌核醣體蛋白S15...18 3.1.1 載體構築、純化與轉殖...18 3.1.2 細胞培養並誘導蛋白質表達...19 3.1.3 FPLC純化、濃縮與透析...19 3.1.4 腸激酶反應與第二次FPLC純化...20 3.1.5 純化完成蛋白質的定量與功能性分析...21 3.2 目標RNA與DNA handles的合成與純化...22 3.3 藉由光鉗實驗觀察S15蛋白質與RNA之交互作用...22 3.4 合成具有多DIG的5’端handles...24 3.4.1 合成5’端handles與多DIG延長區域並進行接合反應...24 3.4.2 調整多DIG延長區域的合成並進行接合反應...25 3.4.3 尋找接合反應未成功的原因並解決之...26 第四章 討論...29 4.1 純化S15蛋白質時鎳管柱的非專一性結合...29 4.2 S15蛋白質之胺基酸組成對純化過程的影響...29 4.3 鎂離子濃度影響S15蛋白質對RNA之結合...30 4.4 S15蛋白質的結合明顯改變了RNA的結構特性...31 4.5 有關合成具有多DIG 5’端handles的討論...32 4.6 未來展望...33 參考文獻...34 | |
| dc.language.iso | zh-TW | |
| dc.subject | rpsO信使核醣核酸 | zh_TW |
| dc.subject | 結構重組 | zh_TW |
| dc.subject | 轉譯調控 | zh_TW |
| dc.subject | 單分子 | zh_TW |
| dc.subject | 光鉗 | zh_TW |
| dc.subject | rpsO mRNA | en |
| dc.subject | structural rearrangement | en |
| dc.subject | optical tweezers | en |
| dc.subject | single-molecule | en |
| dc.subject | translational regulation | en |
| dc.title | 以光鉗技術研究S15蛋白質與rpsO 基因 5’端未轉譯區核醣核酸之交互作用 | zh_TW |
| dc.title | Optical Tweezers Study of Interaction between S15 Protein and 5’UTR of rpsO mRNA | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 105-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 李以仁,李弘文 | |
| dc.subject.keyword | 轉譯調控,rpsO信使核醣核酸,結構重組,單分子,光鉗, | zh_TW |
| dc.subject.keyword | translational regulation,rpsO mRNA,structural rearrangement,single-molecule,optical tweezers, | en |
| dc.relation.page | 83 | |
| dc.identifier.doi | 10.6342/NTU201602534 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2017-06-16 | |
| 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 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-106-1.pdf 未授權公開取用 | 2.7 MB | Adobe PDF |
系統中的文件，除了特別指名其著作權條款之外，均受到著作權保護，並且保留所有的權利。