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研究生:黃呈彥
論文名稱:竹嵌紋病毒3'端非轉譯區與其核醣核酸複製酵素之結合分析
論文名稱(外文):The binding analyses of the 3' untranslated region of bamboo mosaic potexvirus RNA with the over-expressed RNA dependent RNA polymerase domain
指導教授:蔡慶修蔡慶修引用關係
學位類別:博士
校院名稱:國立中興大學
系所名稱:農業生物科技學研究所
學門:農業科學學門
學類:農業技術學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:72
中文關鍵詞:竹嵌紋病毒3'端非轉譯區結合核醣核酸複製酵素
外文關鍵詞:Bamboo mosaic virus3' untranslated regionbindingRNA dependent RNA polymerase
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竹嵌紋病毒( Bamboo Mosaic Potexvirus, BaMV )屬於potexvirus group,為單一正股RNA病毒。一般相信在負股RNA生合成之起始上,BaMV基因體RNA的3´端非轉譯區( 3´ UTR )扮演重要角色。此區域的三級結構已經由實驗證明,並且被認為是BaMV複製酵素複合物所認識的區域。由於BaMV第一個轉譯架構的C端為核醣核酸聚合酵素的核心區域,因此利用BaMV RNA的3´ UTR與純化自Escherichia coli大量表現之核醣核酸聚合酵素截斷蛋白(Δ893 )進行electrophoretic mobility shift以及competition的實驗,結果證明BaMV 3´ UTR與Δ893的結合具有專一性,而反應時鹽類離子的濃度會強烈影響RNA與蛋白質結合的穩定性。經由competition的實驗發現取自牛肝臟的tRNAs、poly (C) RNA以及雙股的poly (I) (C) homopolymer都無法將與Δ893結合的BaMV 3´ UTR競爭掉,而胡瓜嵌紋病毒( cucumber mosaic virus, CMV ) 3´ UTR則稍具競爭能力。比較poly (A)、poly (C)以及poly (G)三種RNA的競爭能力為poly (A) >> poly (G) > poly (C)。此外利用不同區域之BaMV 3´ UTR進行competition的實驗,以及含有D及E domains的BaMV 3´ UTR進行footprinting實驗,結果發現Δ893應具有兩個結合位置,分別在含有六個保留性核苷酸的D stem-loop以及poly (A) tail。利用glutathione S-transferase (GST)融合893蛋白(G893)進行electrophoretic mobility shift以及UV-cross-linking實驗發現G893蛋白對BaMV 3´ UTR的結合具專一性,因此進一步截短G893蛋白研究其與D stem-loop的結合關係,發現只存在A、B及C motifs的蛋白區段即與G893具有相同的結合能力,而一般被認為與模板、引子結合相關的motif B扮演重要角色。

Bamboo mosaic potexvirus (BaMV) is a single-stranded plus-sense RNA virus. The 3´ untranslated region (UTR) of BaMV genomic RNA is believed to be important in the initiation of minus-sense RNA synthesis. The tertiary structure of this region has been identified and is recognized by BaMV replicase complex for an in vitro replication assay. Electrophoretic mobility shift and competition assays demonstrated that the Escherichia coli over-expressed truncated portion (Δ893) of BaMV open reading frame 1 containing the core domain of the RNA-dependent RNA polymerase could specifically bind to the 3´ UTR of BaMV RNA. The stability of the RNA-protein complex was mainly maintained by the factors of salt concentration but not pH or temperature. Results derived from competition assay revealed that the interaction between the BaMV RdRp and the 3´ UTR could not be competed with the bovine liver tRNAs, poly (C) homopolymer, and the double-stranded poly (I) (C) nucleic acids, and could be competed with cucumber mosaic virus 3´ UTR with less efficiency. The competitive efficiency of the three homopolymer was poly (A) >> poly (G) > poly (C). Competition analysis with RNA molecules comprising different domains of the 3´ UTR of BaMV RNA revealed that two RNA binding sites of Δ893 was located at D stem-loop and the poly (A) tail. G893 was an E. coli-expressed glutathione S-transferase (GST) fusion RdRp protein. The binding specificity was determined by electrophoretic mobility shift and UV-cross-linking assay with the 3´ UTR of BaMV RNA. For localization of the binding regions responding to interact with D domain of BaMV 3´ UTR, truncated mutants of G893 were designed. Results indicated that the shifting activity of motif A, B, and C was the same as the full-length G893, and motif B could be the major determinant in responding the binding as been proposed to be involved in template and/or primer positioning.

目 錄
論文中文摘要 -------------------------------------------------1
論文英文摘要 -------------------------------------------------2
第一章 前人研究 ----------------------------------------------3
I.竹嵌紋病毒之研究背景 ---------------------------------------3
II.正股RNA病毒3´端非轉譯區之結構與功能 ----------------------4
III.病毒複制酵素之研究進展 --------------------------------8
第二章
Sequences at the 3' Untranslated Region of Bamboo Mosaic Potexvirus RNA Interact with the Viral RNA-Dependent RNA Polymerase
ABSCRACT ---------------------------------------------------12
INTRODUCTION -------------------------------------------13
METERIAILS AND METHODS ----------------------------------15
Expression and purification of recombinant RdRp ------15
Preparation of RNA transcripts -------------------------15
EMSA of the interactions between 893 RdRp and the labeled nucleic acids ---16
Quantitation of bands on autoradiographs ----------------17
DEPC footprinting analysis -------------------------17
RESULTS ----------------------------------------------------18
Specific binding of recombinant 893 RdRp to the 3' UTR of
BaMV RNA -------------------------------------------18
The poly(A) tail in the 3' UTR of BaMV RNA is involved in
893 RdRp binding------------------------------------------20
Stem-loop D is another region of the binding sites for 893
RdRp ----------------------------------------------------21
The potexviral conserved hexamer motif interacts with RdRp
---------------------------------------------------------21
DISCUSSION--------------------------------------------------23
TABLES AND FIGURES------------------------------------------26
第三章
Localization of the Region in Polymerase Domain of Bamboo Mosaic Potexvirus ORF1 Responding to the RNA Binding Activity with the 3' Untranslated Region
ABSCRACT ---------------------------------------------------33
INTRODUCTION -----------------------------------------------34
METERIAILS AND METHODS---------------------------------------36
Plasmid Construction-----------------------------------------36
Expression and purification of recombinant RdRp--------------36
Protein gel electrophoresis and Western blot analysis -------37
Preparation of RNA transcripts-------------------------------37
EMSA of the interactions between 893 RdRp and the labeled
nucleic acids------------------------------------------------38
Quantitation of bands on autoradiographs ----------------38
UV-cross-linking assay ---------------------------------39
RESULTS ----------------------------------------------------40
Construction and expression of plasmids----------------------40
Binding of G893 to the viral 3´ UTR ------------------------41
Mapping of the binding domains of G893 to the D domain of BaMV
3´ UTR -----------------------------------------------------41
DISCUSSION--------------------------------------------------43
TABLES AND FIGURES ------------------------------------------45
結論與未來展望 -------------------------------------------54
參考文獻 ---------------------------------------------------55

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