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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 溫進德 | |
| dc.contributor.author | Cheng-Fu Chang | en |
| dc.contributor.author | 張成甫 | zh_TW |
| dc.date.accessioned | 2021-06-16T23:09:49Z | - |
| dc.date.available | 2012-08-10 | |
| dc.date.copyright | 2012-08-10 | |
| dc.date.issued | 2012 | |
| dc.date.submitted | 2012-08-03 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/64951 | - |
| dc.description.abstract | 病毒基因藉由-1核醣體框架位移(-1 ribosomal frameshifting)以表現各種不同功能的蛋白，包括結構蛋白及催化酵素。而在此機制中，訊息RNA(messenger RNAs)所形成的結構以及滑動序列(slippery sequences)皆為不可或缺的因子。而在病毒中，此RNA結構經常為一偽結(pseudoknot)。過去的許多實驗已證明了-1框架位移效率與偽結的結構穩定性有直接的關聯，當穩定性越高，則-1框架位移的比例就隨之上升。已知修改自人類端粒酶(human telomerase)的偽結結構：hTRΔU177，其具有引發框架位移的能力，且效率與其中的三重鹼基對(base-triple)形成為正相關，當三重鹼基對形成，可以穩定偽結，同時使-1框架位移效率提升；除此之外，若以一個髮夾結構及一個單股RNA(ssRNA oligo)產生鍵結組成類似的雙分子偽結結構(bimolecular pseudoknot)，也一樣具有促進框架位移的能力。
而在此實驗中，利用單分子技術，以雷射光鉗(optical tweezers)測量將類偽結結構打開所需要施加的外力，進以分析三重鹼基對的形成及在ssRNA上未參與鍵結序列的改變對類偽結之結構穩定性的影響。結果發現當三重鹼基對形成，且未鍵結序列存在時，ssRNA可將解開髮夾結構之所需外力，由約17pN提升到約21.5pN；對ssRNA的改變，無論是阻礙三重鹼基對的生成，或是未鍵結序列的改變甚至是移除，則降低了ssRNA影響的程度，解開結構之外力只需要約19pN。推測這是由於本來的未鍵結序列在類偽結結構中產生了新的三重鹼基對，額外的鍵結致使結構穩定度上升。實驗的最後對hTRΔU177偽結結構做了分析，也發現當loop 2的序列改變(相對應於ssRNA上的未鍵結序列)，雖不影響三重鹼基對生成，卻造成了解開結構需要的外力下降。即使機制不同於類偽結結構，卻可以肯定除了三重鹼基對的存在之外，loop 2也是影響結構穩定性的重要因子之一。 | zh_TW |
| dc.description.abstract | Virus-encoded proteins, including structural proteins and enzymes, are usually produced by -1 programmed ribosomal frameshifting. In this mechanism, the structures of the messenger RNA and the slippery sequences are required. Moreover, secondary structures of the viral mRNA usually exist in pseudoknots. Previous experiments have shown that -1 frameshifting efficiency is related to the stability of the pseudoknots.
In this project, I used hTR ΔU177, a modified pseudoknot structure derived from the human telomerase RNA. The pseudoknot can be mimicked by annealing a hairpin and an ssRNA oligo derived form hTR ΔU177 . The bimolecular pseudoknot has a reduced ability to induce -1 frameshifting as the original hTR ΔU177. By using optical tweezers, I measured the unfolding force of a series of hTR ΔU177-related structures and analyzed the relationship between the structural stability and formation of the triplex. The unfolding force of the hairpin was increased from about 17pN to 21.5pN in the presence of the ssRNA oligo. Furthermore, I found that the unfolding force was affected by the 5’ unpaired sequence of the ssRNA oligo as well. No matter whether the 5’ unpaired sequence were truncated or mutated, the unfolding force was decreased from 21.5pN to 19pN. I assume that there are extra hydrogen bonds formed between the unpaired nucleotides of the ssRNA and the hairpin G-C stem on the hairpin. Moreover, when the loop 2 sequence in the pseudoknot structure was mutated, the unfolding force dropped from 47pN to 41pN. All the results suggest that both the triplex and the loop 2 sequence (5’ unpaired nucleotides of the ssRNA oligo in the bimolecular pseudoknots) contribute significantly to the stabilities of the hTR ΔU177-related structures. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T23:09:49Z (GMT). No. of bitstreams: 1 ntu-101-R99b43026-1.pdf: 3715262 bytes, checksum: 7772ae45af2ad8a39fe40c8ae7cd9d13 (MD5) Previous issue date: 2012 | en |
| dc.description.tableofcontents | CONTENTS
口試委員會審定書………………………………………………………………………i 誌謝…………………………………………………………………………………………………ii 摘要…………………………………………………………………………………………………iii ABSTRACT……………………………………………………………………………………v TABLE LIST………………………………………………………………………………ix FIGURE LIST……………………………………………………………………………ix CHAPTER 1 INTRODUCTION 1.1 Human Telomerase RNA……………………………………………………………………………1 1.1.1 Telomerase…………………………………………………………………………………………………1 1.1.2 Pseudoknot and -1 Programmed Ribosomal Frameshifting…………………………………………………………………………………………2 1.1.3 Human Telomerase RNA (ΔU177) and Bimolecular Pseudoknot…………………………………………………………………………………………………3 1.2 Single-Molecule Techniques……………………………………………………………4 1.2.1 Overview………………………………………………………………………………………………………4 1.2.2 Application of Single-Molecule Techniques………………5 1.2.3 Optical Tweezers…………………………………………………………………………………5 1.3 Specific Aims………………………………………………………………………………………………6 CHAPTER 2 EXPERIMENTAL PROCEDURES 2.1 Materials…………………………………………………………………………………………………………8 2.1.1 Bacterial Strains & Plasmids…………………………………………………8 2.1.2 Kits…………………………………………………………………………………………………………………8 2.1.3 Chemicals……………………………………………………………………………………………………8 2.1.4 Enzymes…………………………………………………………………………………………………………9 2.1.5 Primers and RNA Oligos…………………………………………………………………10 2.1.6 Buffers…………………………………………………………………………………………………………11 2.2 Methods………………………………………………………………………………………………………………12 2.2.1 Plasmid Constructions……………………………………………………………………12 2.2.2 In Vitro Transcription…………………………………………………………………13 2.2.3 DNA Handles Production & Modification…………………………14 2.2.4 Anneal RNA and DNA Handles………………………………………………………16 2.2.5 hTR RNA 5’ Hairpin Structure Construction………………16 2.2.6 Mini-Tweezers Pulling Experiments……………………………………17 2.2.7 RNA Hairpin and Single-Strand RNA Oligo Hybrid Construction……………………………………………………………………………………………19 CHAPTER 3 RESULTS 3.1 hTR 5’ hairpin……………………………………………………………………………………………20 3.2 Adding hTR 3’ ssRNA Oligo to the hTR 5’ Hairpin Construct…………………………………………………………………………………………………………21 3.3 Mutation on the hTR 3’ ssRNA Oligo………………………………………23 3.3.1 Truncated hTR 3’ ssRNA Oligo (ThTR 3’ ssRNA Oligo)…………………………………………………………………………………………………………………23 3.3.2 Double Truncated hTR 3’ ssRNA Oligo (TThTR 3’ ssRNA Oligo)…………………………………………………………………………………………………24 3.3.3 Mutation on the 5’ Nucleotides of hTR 3’ ssRNA (hTR 3’ ssU)…………………………………………………………………………………………………25 3.4 Mutation on the hTR 5’ Hairpin…………………………………………………25 3.5 hTR Pseudoknot……………………………………………………………………………………………27 3.6 hTR Pseudoknot-CUU…………………………………………………………………………………28 CHAPTER 4 DISCUSSION 4.1 The Role of the Base Triple…………………………………………………………29 4.2 The Role of the 5’ Unbinding Nucleotides of the 3’ ssRNA Oligo……………………………………………………………………………………………………30 4.3 Both Triplex and Loop 2 Sequence Affect Stability of hTR Pseudoknot……………………………………………………………………………………………31 4.4 Refolding Force of Different Constructs…………………………31 CHAPTER 5 PERSPECTIVES 5.1 Nucleotides Interactions in the Bimolecular Pseudoknot………………………………………………………………………………………………………33 5.2 Unfolding Force of hTR Hairpin/ ThTR 3’ ssRNA Oligo Complex………………………………………………………………………………………………………………33 5.3 The Unfolding Force Not Equals to the Stability……34 5.4 Construct Stability and -1 PRF Efficiency……………………35 REFERENCES……………………………………………………………………………………………………………………36 | |
| dc.language.iso | en | |
| dc.subject | 核糖核酸 | zh_TW |
| dc.subject | 髮夾結構 | zh_TW |
| dc.subject | 偽結 | zh_TW |
| dc.subject | 三重鹼基對 | zh_TW |
| dc.subject | 單分子 | zh_TW |
| dc.subject | 雷射光鉗 | zh_TW |
| dc.subject | optical tweezers | en |
| dc.subject | RNA | en |
| dc.subject | hairpin | en |
| dc.subject | pseudoknot | en |
| dc.subject | base-triple | en |
| dc.subject | single-molecule | en |
| dc.title | 雷射光鉗應用於端粒酶核醣核酸二級結構之動力學分析 | zh_TW |
| dc.title | Optical Tweezers Analysis on Structural Dynamics of the Truncated Human Telomerase RNA Structure | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 100-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 張功耀,范秀芳 | |
| dc.subject.keyword | 核糖核酸,髮夾結構,偽結,三重鹼基對,單分子,雷射光鉗, | zh_TW |
| dc.subject.keyword | RNA,hairpin,pseudoknot,base-triple,single-molecule,optical tweezers, | en |
| dc.relation.page | 59 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2012-08-06 | |
| 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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