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National Chung Hsing University Institutional Repository - NCHUIR > 生命科學院 > 生物化學研究所 > 依資料類型分類 > 碩博士論文 >  -1計畫性核醣體框架轉移減弱子於不同細胞內功能之研究

Please use this identifier to cite or link to this item: http://nchuir.lib.nchu.edu.tw/handle/309270000/152224

標題: -1計畫性核醣體框架轉移減弱子於不同細胞內功能之研究
The functional study of -1 programmed ribosomal frameshifting attenuator in different cells
作者: 陳政裕
Chen, Cheng-Yu
Contributors: 張功耀 教授
Kung-Yao Chang
生物化學研究所
關鍵字: 計畫性核醣體框架轉移減弱子
-1 programmed ribosomal frameshifting attenuator
日期: 2012
Issue Date: 2013-11-07 13:16:31 (UTC+8)
Publisher: 生物化學研究所
摘要: 生物體在產生蛋白質的過程中,遵守著遺傳學中心教條,由DNA轉錄成mRNA,接著由mRNA經轉譯作用產生對應蛋白。而轉譯過程可能會受到-1計畫性核醣體框架轉移(-1 programmed ribosomal frameshifting)機制的調控來產生其他所需蛋白,目前已知是由mRNA序列中的滑動序列(XXXYYYZ)以及刺激RNA所引起。我們已知DU177假結結構會刺激-1計畫性核醣體框架轉移的發生,以及6BPGC髮夾序列出現在滑動序列上游時,扮演-1計畫性核醣體框架轉移減弱子的角色,會降低-1計畫性核醣體框架轉移發生機率。我進一步想知道當DU177假結結構及6BPGC序列出現在不同細胞內,是否一樣具有調控-1計畫性核醣體框架轉移的功能。
之前研究-1計畫性核醣體框架轉移的方法通常是使用放射性標定以及Dual luciferase系統來進行,但是缺乏安全性且昂貴,因此想發展一套取代的GST-RLuc系統。首先要確認GST-RLuc系統應用的可行性,所以利用已知會刺激-1計畫性核醣體框架轉移發生的SD-like sequence來驗證(Weiss, R. B. et al 1988),不論從GST酵素活性及RLuc冷光強度分析,或是western blot結果都顯示當SD-like sequence表現在GST-RLuc系統,確實能得到跟已發表結果相同趨勢的結論,所以印證應用在-1PRF研究的可行性。最後我們將DU177假結結構及減弱子6BPGC序列建構在GST-RLuc系統中,藉由表現在不同的細胞內,一方面觀察減弱子6BPGC序列在不同細胞內所扮演的角色,另一方面跟之前in vitro translation結果作比對,進一步應用GST-RLuc系統於-1PRF相關研究中。
The translation processes in distinct organisms follow the genetic central dogma that messenger RNAs (mRNA) are transcribed from DNA, and proteins are translated from messenger RNA. However, translational process can be regulated by -1 programmed ribosomal frameshifting (-1 PRF) to produce particular fusion proteins, and is caused by the slippery sequence (XXXYYYZ) and stimulator RNA structure within mRNA sequences. We already demonstrated that the DU177 pesudoknot structure could stimulate -1PRF and an RNA hairpin 6BPGC can serve as a -1 PRF attenuator to reduce the -1 PRF efficiency when it appears on the upstream of the slippery site. In this work, I want to know if the function of the DU177 pesudoknot and 6BPGC hairpin are conserved in different cells.
Previous methods in the study of -1 PRF efficiency are based on radioactive substance and dual luciferase assay. However they are either dangerous or inconvenient. Therefore, we want to establish a GST-Renilla luciferase (GST-RLuc) system which is safe and convenient, as an alternative method for the study of -1 PRF. We used the Shine-Dalgarno-like sequence (SD-like sequence) which had been known to regulate the -1 PRF to examine the usage of GST-RLuc system as an reliable reporter system. In my study, both the enzyme activity based assay of GST and the luminescence intensity of RLuc and the western blot reslults suggest that the GST-RLuc system is useful. Finally, I construct the DU177 pesudoknot structure and 6BPGC attenuator in GST-RLuc system, and espress them in different cells to analyze their roles in different cells.
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