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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(Jin-Der Wen) | |
dc.contributor.author | Yi-Ting Lee | en |
dc.contributor.author | 李翊廷 | zh_TW |
dc.date.accessioned | 2021-05-20T00:50:23Z | - |
dc.date.available | 2025-08-17 | |
dc.date.available | 2021-05-20T00:50:23Z | - |
dc.date.copyright | 2020-09-15 | |
dc.date.issued | 2020 | |
dc.date.submitted | 2020-08-13 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/8226 | - |
dc.description.abstract | -1 框架位移是指核醣體在轉譯過程中重複讀取一個核苷酸,導致核苷酸密碼子重新排列組合而做出不同的蛋白質,在RNA 病毒常用來使自身的基因體有效利用的機制,發生的條件需要訊息核醣核酸 (mRNA) 的上游有滑動序列以及距離滑動序列下游5-6個核苷酸有核醣核酸的二級結構。在此實驗中我們選用∆U177偽結作為實驗材料,∆U177偽結是人類端粒酶核酸的衍生物,∆U177偽結有兩個莖、兩個環的結構以及五組三重鹼基對,在前人研究中顯示刺激-1 框架位移的效率約有52%,非常適合作為研究-1 框架位移的材料。 我們利用單分子螢光能量共振轉移技術探討在發生-1 框架位移時核醣體和下游偽結的交互作用,我們發現在核醣體解開下游偽結的過程中,偽結結構在距離核醣體P位點11-12個核苷酸時會形成扭曲的過渡態,且在核醣體中的mRNA會被拉長。若是將在第一個環和第二個莖上的三組三重鹼基對移除,偽結就不會形成扭曲的過渡態,且-1 框架位移的效率只剩4%。綜合以上結果三重鹼基對於穩定偽結結構,以及形成過度態扮演非常重要的角色,這兩個特性存在可以刺激-1 框架位移的發生。 | zh_TW |
dc.description.abstract | Minus-one programmed ribosomal frameshifting (-1 PRF) occurs when the ribosome rereads a nucleotide and consequently changes the reading frame in order to properly express their genomes in many RNA viruses. To stimulate -1 PRF, the mRNA usually contains a slippery sequence with a pattern of X-XXY-YYZ and a downstream pseudoknot (PK). The ∆U177 sequence, derived from the human telomerase RNA, is folded into a pseudoknot structure with two overlapping stem-loop structures, including three major groove base triples. Previous studies have shown that the ∆U177 pseudoknot is an efficient -1 PRF stimulator. However, details about how the ribosome unwinds the downstream pseudoknot are unknown. Here, we used single-molecule Förster Resonance Energy Transfer (smFRET) to elucidate the interaction between mRNA pseudoknots and ribosomes. We found that the PK was twisted into a compact intermediate structure when the first stem of the PK was 11 to 12 nucleotides away from the P site of the ribosome and the mRNA inside the ribosome was stretched during the unwinding process. Furthermore, after disrupting the three base triples of the PK, the -1 PRF efficiency was decreased from 52% to 4% in in vitro translation experiment, the first stem of the mutant ∆U177 pseudoknot was less resistant to unwinding, and the compact intermediate structure was not observed. These results indicate that base triples would serve as a key element for the formation of intermediate structures and the maintenance of the structure stability which are important for stimulating -1 PRF. In conclusion, the flexibility of the downstream mRNA structure could greatly enhance the -1 PRF efficiency. | en |
dc.description.provenance | Made available in DSpace on 2021-05-20T00:50:23Z (GMT). No. of bitstreams: 1 U0001-1308202007000800.pdf: 6724596 bytes, checksum: 7772ffa58d565d9e63872f8b8555bf9a (MD5) Previous issue date: 2020 | en |
dc.description.tableofcontents | 口試委員會審定書 i 誌謝 ii 中文摘要 iii ABSTRACT iv CONTENTS v LIST OF FIGURES vii ABBREVIATION ix Chapter 1 Introduction 1 1.1 Translation 1 1.1.1 Initiation 1 1.1.2 Elongation 1 1.1.3 Termination 2 1.2 Minus-one programmed ribosomal frameshifting 3 1.3 ∆U177 pseudoknots 4 1.4 Single molecule technology 5 1.4.1 Förster Resonance Energy Transfer (FRET) 5 1.5 Motive and purpose 6 Chapter 2 The Formats of the Thesis 7 2.1 Materials 7 2.1.1 Buffers 7 2.1.2 Chemicals 10 2.1.3 Constructs 12 2.1.4 E. coli stocks 14 2.1.5 Enzymes 14 2.1.6 Kits 14 2.1.7 Sequence 15 2.2 Methods 18 2.2.1 Ribosome purification and labeling 18 2.2.2 RNA construction and labeling 21 2.2.3 Aminoacyl-tRNA preparation 26 2.2.4 Purification of elongation factors (EF-G and EF-Tu) 28 2.2.5 smFRET experiment 30 Chapter 3 Results 33 3.1 PK H_MFKEY 33 3.1.1 PK H_MFKEY_L1C3 33 3.1.2 PK H_MFKEY_L2C3 35 3.2 PK H_MFKEY U3C 36 3.2.1 PK H_MFKEY U3C_L1C3 36 3.2.2 PK H_MFKEY U3C_L2C3 37 3.3 PK H_MVKKFY 38 3.3.1 PK H_MVKKFY_L1C3 38 3.3.2 PK H_MVKKFY_L2C3 39 3.4 PK H_MRLKSY_ L1C3 40 3.5 PK MFKEY_L1C3L2C5 and MFKEY-1_L1C3L2C5 41 3.6 Other related experiments - blocking stem 1 may help PK folding 42 Chapter 4 Discussion 43 REFERENCES 45 | |
dc.language.iso | en | |
dc.title | 利用單分子螢光共振轉移技術探討引起框架位移之RNA偽結與核醣體間交互作用 | zh_TW |
dc.title | Study of interaction between frameshift-stimulating mRNA pseudoknots and ribosomes by single-molecule FRET | en |
dc.type | Thesis | |
dc.date.schoolyear | 108-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 楊立威(Lee-Wei Yang),張功耀(Kung-Yao Chang) | |
dc.subject.keyword | 偽結,核醣體,單分子,螢光能量共振轉移技術,轉譯作用, | zh_TW |
dc.subject.keyword | Pseudoknot,Ribosome,Frameshifting,Single-molecule Förster Resonance Energy Transfer (smFRET), | en |
dc.relation.page | 72 | |
dc.identifier.doi | 10.6342/NTU202003194 | |
dc.rights.note | 同意授權(全球公開) | |
dc.date.accepted | 2020-08-13 | |
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 |
dc.date.embargo-lift | 2025-08-17 | - |
顯示於系所單位: | 分子與細胞生物學研究所 |
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