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標題: | 以單分子螢光共振能量轉移技術觀測rpsO 5’非轉譯區與S15之間的交互作用 Observing interactions between the ribosomal protein S15 and rpsO 5’UTR by single molecule FRET |
作者: | Pei-Hsuan Wang 王培軒 |
指導教授: | 溫進德(Jin-Der Wen) |
關鍵字: | 核醣蛋白S15,rpsO,autoregulation,單分子,螢光共振能量轉移, Ribosomal protein S15,rpsO,autoregulation,single molecule,FRET, |
出版年 : | 2019 |
學位: | 碩士 |
摘要: | 在大腸桿菌中,rpsO所轉譯出的S15核醣蛋白是參與核醣體組裝的重要蛋白質之一。S15在過量表現的情況下會結合rpsO轉錄本5’非轉譯區 (RPSOutr) 的偽結結構 (pseudoknot structure) 使核醣體無法進入轉譯前起始階段,以自體調節 (autoregulate) 其生合成。目前對於S15蛋白與RPSOutr結合的交互作用以及對其結構所造成的影響至今仍不甚清楚。
本篇研究利用單分子螢光共振能量轉移技術 (smFRET) 觀測S15與RPSOutr之間的結合強度、動態分析,以及利用螢光分子標定在S15上,試圖獲取S15與RPSOutr結合的直接證據。研究結果發現S15會專一的與野生型的RPSOutr或有能力形成偽結的RPSOutr結合,並使偽結結構從原本的中轉移效率變成高轉移效率,意味著S15不只是單純地結合在RPSOutr上,而是結合後可能會造成RPSOutr的偽結結構更扭曲或使構型更緊密,此現象與S15結合在16S rRNA上時造成H20與H22之間的角度變小的狀況可能相似,但目前為止均沒有相關研究證實S15會改變RPSOutr的結構,因此我們假設S15亦可以對RPSOutr造成類似的作用,未來也希望可以釐清S15對RPSOutr是否具有此相似的影響。 In Escherichia coli, ribosomal protein S15, encoded by rpsO, is one of the critical components in ribosome assembling. When S15 is over-expressed in the cell, it would inhibit its expression by binding the pseudoknot structure of rpsO transcript at the 5’ untranslated region (RPSOutr), and form an obstacle that stalls the ribosome at pre-initiation state, a mechanism called “autoregulation”. However, the mechanism of interactions between S15 and RPSOutr as well as the influence to the structure is still unclear. This study attempts to observe the binding strength, dynamics between S15 and RPSOutr with smFRET, and to provide the direct evidence of S15 binding RPSOutr by labeling fluorescence dye on S15. In this thesis, we have investigated the interactions between S15 and RPSOutr by Förster resonance energy transfer at single molecule level (smFRET). We identfied that S15 specifically binds the wild-type RPSOutr, or the RPSOutr mutant that is capable of forming pseudoknot structure, causing the pseudoknot structure to shift from middle FRET to high FRET. This implies that S15 not only binds RPSOutr, but also results a more distorted pseudoknot structure. We hypothesize that this phenomenon resembles the decrease of angle between H20 and H22 on 16S rRNA after S15 binding, therefore, we will clarify if S15 possess similar effect to RPSOutr in the near future. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/73823 |
DOI: | 10.6342/NTU201903714 |
全文授權: | 有償授權 |
顯示於系所單位: | 分子與細胞生物學研究所 |
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