| Summary: | 碩士 === 國立中興大學 === 生物科技學研究所 === 94 === 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.
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