臺灣博碩士論文加值系統

病毒經常利用-1核醣體框架位移來表現特定的結構蛋白及酵素(例如：HIV, SARS-CoV 及IBV)，當核醣體在轉譯mRNA成蛋白質時，mRNA上的偽結結構會刺激框架位移，而且偽結結構穩定性與框架位移之效率呈現正相關。
DU177此偽結結構是由人類端粒酶RNA修改而來，我們利用DU177當model進行實驗操作，此結構包含兩個stem及兩個loop，且為重疊性的hairpin構成，而其中stem與loop之間有些核苷酸有Hoogsteen base pair的形成，並且產生5組三重鹼基對，進而增強了此三級結構的穩定性。
本研究利用單分子技術，以光鉗對DU177施加外力，並且測量解開此結構所需之外力與打開的長度，進而推導其原先的結構為何。此外，我們也建構不同DU177的突變序列和結構，進行實驗操作，來推測DU177的摺疊機制。我們在實驗中發現，大部分的DU177會形成hairpin或是一些較不穩定的intermediate結構，僅六分之一比例的DU177會形成完整穩定的偽結結構。在綜合一系列的實驗結果後，我們推論形成偽結結構有兩種主要途徑，第一種是先形成hairpin 1並且以此結構穩定存在著，且較不易進一步形成完整穩定的偽結結構；另一種為hairpin 2先形成，且hairpin 2一旦形成，即可迅速形成帶有三重鹼基的穩定偽結結構。

Viruses often use -1 ribosomal frameshifting to express specific enzymes and structural proteins (e.g. HIV, SARS-CoV and IBV). When the ribosome translates an mRNA, a pseudoknot structure on the mRNA can stimulate frameshifting, and the efficiency is positively correlated with its mechanical strength, instead of the thermodynamic stability.
Here we used the DU177 RNA pseudoknot, which is modified from the human telomerase RNA, as a model system to study the folding and unfolding mechanisms of a typical frameshift-stimulating RNA pseudoknot. DU177 consists of two overlapping hairpins, with five base triples in between that give extra stability to the whole structure.
We used optical tweezers to measure and analyze the forces and extensions of a series of DU177 mutants during unfolding and refolding. We found that in most cases DU177 folds into pseudoknot-like intermediate structures or hairpins alone. Only one sixth of the RNA molecules completely refold to the native structure from the fully unfolded state. With a series of related experiments, we propose a folding pathway of the pseudoknot, where DU177 is usually trapped in a stable hairpin-1-containing intermediate and rarely proceeds to form the native pseudoknot. On the other hand, if the less stable hairpin 2 is formed first, refolding toward the native state is favored, as no stable intermediates were detected from the experiments.

目錄
口試委員會審定書 i
謝誌 ii
摘要 iii
Abstract iv
目錄 v
圖目錄 viii
第一章 導論 1
1.1 計畫性框架位移 1
1.2 人類端粒酶RNA 1
1.2.1 簡介 1
1.2.2 結構分析 2
1.3 單分子技術 2
1.3.1 簡介 2
1.3.2 應用 3
1.3.3 雷射光鉗 3
1.4 研究動機 4
1.4.1 A173-U99在DU177偽結結構的helical junction的重要性 4
1.4.2 uu-DU177偽結結構的摺疊機制 5
第二章 材料與方法 7
2.1 材料 7
2.1.1 勝任細胞品系 7
2.1.2 質體 7
2.1.3 載體DNA序列及引子設計 7
2.1.4 試劑 9
2.1.5 藥品 9
2.1.6 酵素 11
2.1.7 溶液 13
2.2 方法 14
2.2.1 質體建構 14
2.2.2 細胞外轉譯作用 15
2.2.3 聚合酶連鎖反應製作DNA handle 15
2.2.4 DNA handle的修飾 16
2.2.5 DNA handle及RNA的黏合反應 16
2.2.6 單分子技術光鉗 16
第三章 結果 18
3.1 A173-U99 Hoogsteen base pair 在DU177偽結結構穩定性的重要性 18
3.2 利用雷射光鉗探討uu-DU177的摺疊機制 18
3.2.1 uu-DU177力的分析與分布情形 18
3.2.2 hairpin 1的先形成影響偽結結構的生成 20
3.2.3 hairpin 2的先形成影響偽結結構的生成 21
3.2.4 loop 2核苷酸的堆疊影響由hairpin 1所形成的偽結結構 22
3.2.5 干擾不同stem對DU177偽結結構生成的影響 24
3.2.6 干擾更多stem 2的核苷酸對DU177偽結結構生成的影響 27
3.2.7 干擾stem 2的核苷酸對uu-UUC偽結結構生成的影響 28
第四章 討論 30
4.1 uu-DU177摺疊情形 30
4.2 uu-175/176GC兩種偽結結構分布之關係與成因 31
4.3 uu-UUC2st2與uu-WT2st2之比較 32
4.4 uu-WT2st2與框架位移的相關性 32
4.5 從熱力學角度探討各種不同建構的結構 33
4.6 未來展望 33
參考文獻 34
<附錄一>hairpin的自由能計算平衡常數 71

 
圖目錄
圖1. -1框架位移 38
圖2 DU177結構示意圖 40
圖3 單分子光鉗實驗架設 41
圖4 A173-U99 hoogsteen base pair 的重要性 43
圖5 uu-DU177結構摺疊與分布情形 45
圖6 hairpin 結構分析圖 48
圖7 uu-DU177 摺疊機制的假設 49
圖8 uu-DU177在給予更多時間下的摺疊情形 50
圖9 模擬hairpin 1已形成的摺疊情形 52
圖10 uu-175/176GC 迫使hairpin 2先形成導致結構形成的影響 55
圖11 uu-GU/GC 結構摺疊情形 57
圖12 uu-UUC 增加loop2柔軟度影響結構摺疊 59
圖13 uu-WT2st1 干擾stem1影響結構摺疊機制 62
圖14 uu-WT2st2 干擾stem2影響結構摺疊 65
圖15 uu-WT4st2干擾更多stem2造成摺疊機制的改變 67
圖16 干擾stem 2的核苷酸對uu-UUC偽結結構生成的影響 69
圖17 uu-175/176GC 結構摺疊與打開的關係 70

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