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標題: 利用酵母菌系統探討竹嵌紋病毒基因體3''端非轉譯區與其核醣核酸聚合酵素之間的交互作用
The Study of the Interaction between RNA Dependent RNA Polymerase and the 3''Untranslated Region of Bamboo Mosaic Virus RNA in vivo
作者: 林鈺倍
Lin, Yu-Pei
關鍵字: BaMV RdRp;竹嵌紋病毒核醣核酸聚合酵素;yeast three-hybrid system;酵母菌三雜交系統
出版社: 生物科技學研究所
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竹嵌紋病毒(BaMV)屬於potexvirus group,為單一正股RNA病毒。病毒基因體的轉譯框架可轉譯出一個分子量約155 kDa的複製酵素,此酵素的C端為核酸聚合酵素的核心區域。其核酸基因體3''端非轉譯區(3''UTR)為病毒複製時合成負股RNA的啟動子,在結構上已被證實存在有一特殊的三級結構。經由前人的試管內研究結果已知,核酸聚合酵素截斷蛋白(∆893 RdRp)與BaMV 3''UTR的結合具有專一性。本研究用yeast three-hybrid系統來了解在in vivo狀態下,BaMV 3''UTR與 ∆893 RdRp (以下內文皆以RdRp表示) 之間的結合關係。當核酸與蛋白之間有交互作用時,將會活化報導基因,利用酵母菌的表現型和產生的酵素量來做分析。根據C型肝炎病毒的研究,HCV NS5B是膜相關蛋白且其C端為疏水性區域,因此我們將BaMV RdRp C端疏水區刪去 (稱為RdRp∆C17)。利用此系統,表現RdRp∆C17可以與BaMV 3''UTR有專一性交互作用。為了增加交互作用的機會,我們設計二重複的BaMV 3''UTR (2×3''UTR),它能提高報導基因的表現。另外,因為3''UTR的轉錄啟動子為RNA聚合脢Ш,顧及BaMV 3''UTR序列中含有連續性U,可能會中止轉錄。因此對其序列做點突變,破壞連續性uridine,經過測試後,與RdRp∆C17結合能力並未提高。前人依據RdRp的保守性motifs,設計七種截短蛋白,研究其與3''UTR的結合關係,發現motif B可能扮演重要角色。擷取其constructs,進一步利用酵母菌系統作測試,結果偵測不到結合能力,可能是蛋白質三級結構的改變,導致RdRp無法與3''UTR結合。接下來要對RdRp∆C17保守的motifs做點突變,減少改變蛋白三級結構的可能性,能進一步利用酵母菌系統做分析。

Bamboo mosaic virus (BaMV) is a single-strand positive-sense RNA virus. The 3' untranslated region (UTR) of BaMV genomic RNA contains the promoter for the initiation of minus-strand RNA synthesis. The tertiary structure of this region has been identified. Results derived from previous experiments showed that the E. coli expressed ORF1 truncated polypeptide (∆893), containing the core domain of the RdRp (RNA-dependent RNA polymerase), could specifically interact with the 3'UTR of BaMV. Here, a yeast three-hybrid system which can be analyzed using simple phenotypic or enzymatic assays to detect the interaction between BaMV 3'UTR and ∆893 RdRp (following named RdRp in this thesis) in vivo. Interaction of these elements results in the activation of a reporter gene in the yeast Saccharomyces cerevisiae. Based on the structure of HCV NS5B (Hepatitis C Virus Non-Structural Protein B), which is membrane-associated, and C-terminal hydrophobic, we deleted C-terminal 17 amino acids of RdRp. Using this system, we have shown that the BaMV RdRp∆C17 binds specifically to BaMV 3'UTR. Mutation in the pseudoknot E domain of 3'UTR (3'UTRMut) reduced the activation of the reporter gene and the tandem repeat 3'UTR (2×3'UTR) increased the activation of the reporter gene. According to conserved motifs of RdRp, seven mutants were previously constructed on ∆893 RdRp to test the interaction with RNA in vitro. We subcloned the constructs, for three-hybrid assay, probably because the tertiary structure of protein was disrupted, we couldn't detect the binding of truncated protein to 3'UTR. Next, we will design site-directed mutations on conserved motifs of BaMV RdRp∆C17 for further investigation.
其他識別: U0005-2007200622300900
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