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標題: | 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 Study of interaction between frameshift-stimulating mRNA pseudoknots and ribosomes by single-molecule FRET |
作者: | Yi-Ting Lee 李翊廷 |
指導教授: | 溫進德(Jin-Der Wen) |
關鍵字: | 偽結,核醣體,單分子,螢光能量共振轉移技術,轉譯作用, Pseudoknot,Ribosome,Frameshifting,Single-molecule Förster Resonance Energy Transfer (smFRET), |
出版年 : | 2020 |
學位: | 碩士 |
摘要: | -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。 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. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/8226 |
DOI: | 10.6342/NTU202003194 |
全文授權: | 同意授權(全球公開) |
電子全文公開日期: | 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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U0001-1308202007000800.pdf 此日期後於網路公開 2025-08-17 | 6.57 MB | Adobe PDF |
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