Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/23477
標題: DU177三鹼基對突變的 -1PRF效率受pH調控之研究
Study on the pH–dependency of the -1 PRF activity of DU177 base-triple mutants
作者: 孔獻弘
Kung, Hsien-Hung
關鍵字: DU177;DU177;三鹼基對;突變;-1;框架位移;pH;base;triple;mutant;-1PRF;frameshifting;pH
出版社: 生物化學研究所
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摘要: 
-1計劃性核糖體框架位移(-1 programmed ribosomal frameshifting, -1PRF)是為核糖體在轉譯作用的延長階段的位移現象,而滑動區域(slippery site)和其下游的假結結構(pseudoknot)是引起位移的兩個關鍵元件。本實驗室已經證明人類端粒酶 RNA中的假結結構 hTPK-DU177能夠造成約 50%的 -1PRF,也證明此假結結構特定三鹼基的突變能夠改變 -1PRF效率,顯示三鹼基對間的交互作用對假結結構的穩定性扮演重要的角色,其中三鹼基對突變 C-G.C+因形成 Hoogsteen配對需要額外的氫離子參與,因此環境的 pH值具有決定性的作用。熱動力學穩定測試證明在不同 pH下,會影響假結結構的穩定性;在 murine leukemia virus假結的研究也發現,低 pH值的環境會產生質子化的三鹼基對,使假結更加的穩定,進而提昇 reading-through的效率。
本研究針對 DU177的 stem 2 – loop 1上的 174T、175T、176T三鹼基對,及其組合 174-175T、175-176T、174-176T,在不同的 pH值下執行 in vitro translation assay,發現雖然 DU177的 -1PRF不具有 pH-dependency、而其突變型具有 pH-dependency的特性;即會隨 pH不同而使 –1PRF效率改變。此外,pH-dependency會因為三鹼基的組數而具有加成的作用。最後,利用 3D預測模型預測,pH改變並不會破壞 DU177結構,暗示 pH值對三鹼基穩定度的影響是改變 -1PRF效率的主要因子。

-1 programmed ribosomal frameshifting ( -1PRF ) is the frameshifting of ribosome in the elongation stage of translation. The two major elements responsible for frameshifting are slippery site and downstream RNA pseudoknot. It has been demonstrated that hTPK-DU177, derived from human telomerase RNA, induced 50% -1PRF efficiency, and DU177’s base-triple mutation alter
-1PRF efficiency. It suggests that the interaction between base-triple is important for pseudoknot stability and thus the frameshifting efficiency. The base-triple (C-G.C+) needs to associate H+ for formation of Hoogsteen base pair, and therefore pH may play a key role in frameshifting efficiency for mutants with such type of base-triple. Indeed, thermodynamic stability experiment demonstrates that the pseudoknot stability can be affected by different pH. Investigation in murine leukemia virus pseudoknot also shows that protonation of base triple in low pH causes pseudoknot structure rearrangement and enhances reading through efficiency.
The -1PRF efficiency of several base triple mutants of DU177, such as 174T, 175T, 176T, 174-175T, 175-176T, and 174-176T, with base-triple in stem 2 – loop1, are measured by in-vitro translation assay on different pH. It shows that -1PRF efficiency of DU177 is pH-dependent, whereas -1PRF efficiency of base-triple mutants involving C-G.C+ are characteristic of pH-dependent. In addition, number of C-G.CC+ triple also affect the pH-dependency. Finally, 3D structure model prediction suggests pH-dependent triple formation will not affect DU177 structure, implicating the stability change is the major cause of variation in -1PRF efficiency.
URI: http://hdl.handle.net/11455/23477
其他識別: U0005-2308201215510700
Appears in Collections:生物化學研究所

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