Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/53416
標題: 核醣體轉譯位移與讀取軌道定位之生化與單分子分析-核醣體轉譯位移與讀取軌道定位之生化分析-總計畫暨子計畫一(I)
Biochemical and Single-Molecule Analysis of Ribosomal Translocation and Reading-Frame Maintenance (I)
作者: 張功耀
關鍵字: 其他(理);基礎研究;protein translation;蛋白質轉譯作用;核醣體讀取軌道移轉與解旋功能;-1 轉譯框架移轉;單分子分析技術;光鉗;單分子螢光探針系統;ribosome;translocation and mRNA unwinding;-1 ribosomal frameshifting;optical tweezer;single-molecule FRET
摘要: 
本跨領域計畫長程目標為利用嶄新之單分子(single-molecule)分析技術並配合生化與分子生物學的工具,探究核醣體各個組成份子於蛋白質轉譯作用(protein translation)中所扮演之功能與機制。在這個三年期計畫中,則將著重探討不同實驗系統平臺之建立(從大腸桿菌到酵母菌),以及針對一些特定核醣核酸二級結構所引起之-1 轉譯框架移轉(-1 ribosomal frameshifting)及其對核醣體讀取軌道移轉(translocation)與 mRNA 解旋活性所造成之影響,進行深入之單分子實驗分析與探討。子計畫一著重在運用生化方法建立適合以光鉗與螢光探針技術進行單分子研究的RNA 偽節結構的典範系統,並將著重於會造成–1 框架移轉的偽節結構以及其生化功能之分析。此外我們將建立一套可以適用於各種細菌之胞內核醣體純化流程以提昇細菌核醣體單分子研究的效率與延伸性。並以此基礎,建立酵母菌核醣體單分子研究的系統平臺。子計畫二將著重在利用先進的光鉗技術探討–1 框架移轉效率與不同類型偽節結構承受機械抗力之能力的關係,以及利用光鉗來追蹤其對核醣體讀取軌道移轉功能之影響。同時亦將運用單分子螢光探針系統觀測–1 框架移轉對核醣體mRNA 解旋活性所造成之影響。藉由此兩種互補的單分子分析系統,並配合相關生化活性之研究,來提昇對–1 框架移轉機制與核醣體運作方式的瞭解。

The long-term objective of this inter-disciplines proposal is to combine the single-moleculemethods with biochemical approaches to study the role of different ribosomal components in theprocess of protein translation. This three-years project will focus on the setup of two experimentalplatforms (E. Coli as well as yeast ribosome) and single-molecule analysis of the effect of -1programmed ribosomal frameshifting (-1 PRF) on the translocation and mRNA unwinding activity ofthe ribosomeThe subproject 1 will apply biochemical approach to construct model system containing the -1PRF module suitable for optical tweezer-based and FRET-based single molecule analysis, and thestudy of related biochemical property. In addition, a procedure to purify the ribosome from prokaryoticsystem will be developed to speed up its application in single-molecule study, and be used as theframework to purify yeast ribosome for single-molecule analysis。The subproject 2 will use opticaltweezer to investigate the potential correlation between the mechanical stability of different -1 PRFRNA stimulators and their frameshifting efficiencies. Furthermore, the effect of -1 PRF module on thetranslocation activity of the ribosome will also be tracked by an established optical tweezer approach.At the same time, single-molecule FRET approach will also be applied to observe the dynamicproperty of the mRNA unwinding activity of the ribosome in the presence of a -1 PRF module.Together, these two complementary approaches will help revealing the mechanism of -1 PRF as wellas the dynamic properties of the translocation and unwinding activity of the ribosome.
URI: http://hdl.handle.net/11455/53416
其他識別: NSC99-2627-M005-002
Appears in Collections:生物化學研究所

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