Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/24072
標題: DU177 RNA假結刺激程式化-1轉譯框架移轉的重要結構特徵探討
The important structural features of the DU177 RNA pseudoknot in stimulating programmed -1 ribosomal frameshifting
作者: 周銘源
Chou, Ming-Yuan
關鍵字: frameshifting;轉譯框架移轉;-1PRF;RNA pseudoknot;ribosome translocation;程式化-1轉譯框架移轉;核醣核酸假結;核醣體轉譯位移
出版社: 生物化學研究所
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摘要: 
程式化-1轉譯框架位移作用,同時涉及到改變轉譯中核醣體位移的距離與方向,目前所知是由mRNA上的滑動序列與刺激因子序列所造成。當轉譯複合體在轉譯進行到滑動序列上時易於發生-1轉譯框架位移,並且由於滑動序列的特性使轉譯複合體中的tRNAs在位移之後仍能穩定形成基因碼與反基因碼的結合。刺激因子序列與滑動序列相隔一段適當的距離,被認為是主要使轉譯複合體停留在滑動序列,並且造成程式化-1轉譯框架移轉作用的原因,是控制位移作用效率的關鍵。
我們發現DU177假結(一個由人類端粒酶RNA所衍生的假結)能刺激程式化-1轉譯框架移轉作用。藉由其已發表的詳細結構資料,我們對其序列進行突變,並量測-1轉譯框架移轉作用效率,以試圖了解DU177假結的結構特性,在其刺激程式化-1轉譯框架移轉作用中的重要性。我們的結果顯示在DU177假結構造中,假結柄部與圈部間的作用力,以及所同時涉及的二柄部螺旋的疊合,對其刺激程式化-1轉譯框架移轉特性的影響,較發生程式化-1轉譯框架移轉時位於RNA進入通道入口的假結圈部序列為大。我們將該DU177假結構造分為mRNA上的髮夾構造與單股RNA,進一步發現了假結構成的髮夾構造部分,在施加完整假結柄部與圈部間的作用力後,即足以刺激程式化-1轉譯框架移轉作用發生;除了同時驗證了我們在突變假結序列的實驗中所觀察到的現象外,我們也等同架構了可以用單股RNA調控的RNA髮夾構造程式化-1轉譯框架移轉刺激因子。
我們所發現的可調控程式化-1轉譯框架移轉刺激因子序列,是在與其反應性分子結合後,從不具有-1轉譯框架移轉效果,變成能刺激顯著的程式化-1轉譯框架移轉作用;其本身適於進一步研究的程式化-1轉譯框架移轉作用的機制。並且由於程式化-1轉譯框架移轉作用與轉譯作用機制的關係,很可能會對轉譯位移機制的細節之了解有幫助。除此之外,可調控轉譯程式化-1轉譯框架移轉刺激因子序列也可能直接用於生物體,進一步研發成為可調控程式化轉譯框架位移作用的生物科技系統,應用於生物檢測與醫療等方面。

Programmed -1 ribosomal frameshifting (-1 PRF), which is known to be caused by the slippery sequence and the stimulator sequence, modulates the moving direction and distance of a translating ribosome. Upon reaching the slippery sequence, the translation complex tends to shift into the -1 reading frame. However, the tRNAs in the translation complex can still form stable codon-anticodon interactions after shifting into the -1 frame because of the composition of the slippery sequence. The stimulator sequence, properly distant from the slippery sequence, is regarded to be the main source to pause the translation complex on the slippery sequence and cause -1 PRF, and thus the lever that controls -1 PRF efficiency.
We found DU177, an RNA pseudoknot derived from the human telomerase RNA, can stimulate -1 PRF. With its detailed structural data, we tried to understand the important structural features of the DU177 pseudoknot responsible for its -1 PRF efficiency through mutagenesis. Our results indicated both the interactions between stem and loop and the stacking junction of the two stem helixes have greater effect on the -1 PRF stimulating property of the DU177 pseudoknot than the loop nucleotides near the RNA entry channel of the ribosome when -1 PRF occurred. We divided the DU177 pseudoknot into a hairpin structure on mRNA and an oligo RNA, and we found -1 PRF occurred after the stem loop interactions applying on the hairpin structure. In addition to confirming the importance of stem loop interactions and junction stacking in -1 PRF, we also found an single-strand RNA inducible -1 PRF stimulator.
The inducible -1 PRF stimulator was converted to stimulate -1 PRF after the binding of its responsive molecule and is suitable for application in the study of -1 PRF mechanism. Because of the correlation between translation elongation mechanism and -1 PRF, the study might help in understanding the details of translation translocation. Besides, the inducible -1 PRF stimulator might directly be applied on living organisms and has the potential to become an inducible translation control system in bioassay and medicine.
URI: http://hdl.handle.net/11455/24072
其他識別: U0005-2707201016092700
Appears in Collections:生物化學研究所

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