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研究生:黃亦樂
研究生(外文):Yih Leh Huang
論文名稱:竹嵌紋病毒複製酵素之RNA戴帽酵素區域之純化與其催化反應步驟之研究
論文名稱(外文):Purification and Functional Analysis of Capping Enzyme Domain of Bamboo Mosaic Virus Replicase
指導教授:孟孟孝
指導教授(外文):Menghsiao Meng
學位類別:博士
校院名稱:國立中興大學
系所名稱:生物科技學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2003
畢業學年度:92
語文別:中文
論文頁數:104
中文關鍵詞:植物病毒竹嵌紋病毒戴帽酵素複製酵素
外文關鍵詞:capping enzymecapbamvrna replicasemethyltransferaseguanylyltransferaserna virus
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中文摘要
竹嵌紋病毒(Bamboo mosaic virus, BaMV)主要感染竹亞科植物,屬於馬鈴薯X病毒群之成員(potexvirus group),歸類於似a病毒上科(alphavirus-like superfamily)。BaMV之基因體為單股之正意RNA,約6.4-kb,包含5’端cap,5’端非轉譯區(untranslated region, UTR),5個轉譯架構 (open reading frame, ORF),3’ UTR與poly(A) tail。BaMV之ORF1可轉譯出一155-kDa之replicase,此蛋白質由N端至C端可區分為capping enzyme domain、helicase-like domain與RNA-dependent RNA polymerase domain (RdRp) domain。BaMV RNA之5’端帽(cap) 結構的形成,是由helicase-like domain所提供之RNA triphosphatase活性與capping enzyme domain之AdoMet-dependent guanylyltransferase活性依照順序反應完成。為了能夠深入解析BaMV capping enzyme之催化機制,在本研究中進行了capping enzyme domain的純化、突變蛋白質的活性比較,與探討capping enzyme domain對RNA受質專一性的初步實驗。
為了精確分析capping enzyme domain 的活性,所以本論文第一部份的目的為純化由酵母細胞表達之BaMV capping enzyme domain。由於酵母細胞表現的BaMV capping enzyme domain為膜蛋白質,經由不同界面活性劑的測試,發現離子性的sarkosyl (0.6 %) 將此膜蛋白質水溶性化的效果最好。將C端融合了His-tag之capping enzyme domain以sarkosyl水溶性化後,可以由金屬親和性層析純化得到。經由融合螢光重組蛋白質的觀察,發現capping enzyme位於酵母細胞中之某種胞器。隨即建構capping enzyme domain之N端或C端截斷蛋白,並且融合於EGFP; 藉由螢光分佈觀察capping enzyme domain在酵母細胞中分佈的位置,且配合高速離心之水溶性測試,以鑑定capping enzyme domain與膜依附之區域 (membrane-associated region)。
第二部分在探討capping enzyme domain中重要性胺基酸對本身methyltransferase活性與/或guanylyltransferase活性的影響,故設計了單點胺基酸突變為alanine之16個突變株,並比較突變後的酵素活性。當單點突變分別在保留性胺基酸 (D122,R125及Y213) 與非保留性之H66,則造成methyltransferase與guanylyltransferase活性的喪失; 當突變發生在保留性胺基酸H68,此突變株具有完整的GTP methyltransferase活性,但guanylyltransferase卻完全喪失; 而其他突變蛋白質的兩種活性呈現相似的減弱之趨勢。藉由UV-cross linking分析每個突變株對AdoMet的結合能力,再和它們的活性趨勢比較,發現活性趨勢與AdoMet的結合能力具有良好的相關性。H68A是一個特別的突變株,具GTP methyltransferase活性,但喪失guanylyltransferase活性; 進一步分析H68A與AdoMet和GTP作用之後形成的產物,證實為m7GTP。然而,此m7GTP只有在AdoHcy存在之下,才可被轉移至野生株capping enzyme上,而形成共價鍵結之m7GMP-enzyme intermediate,反映出AdoMet/AdoHcy的結合對m7GMP-enzyme intermediate的形成極為重要。
第三部份在探討m7GMP:RNA transguanylation之反應。根據BaMV capping enzyme的研究顯示,m7GMP-enzyme intermediate形成後,推測capping enzyme domain將所共價結合之m7GMP轉移至RNA之5’ diphosphate終端,而形成capped RNA; 而在本實驗中發現m7GMP基團可以直接被轉移至GDP,而形成 m7GpppG之cap結構。在未來的工作,將比較m7GMP-enzyme轉移其m7GMP至GDP與至5’ diphosphated-RNA的效率,並進行競爭性試驗,以證實RNA上被BaMV capping enzyme所辨識的部位。
Abstract
Bamboo mosaic virus (BaMV), a potexvirus in alphavirus-like superfamily, infects primarily members of the Bambusoideae. The approximate 6,400-nt positive-stranded RNA genome of BaMV comprises 5’ cap structure, 5’UTR, 5 ORFs, 3’UTR and poly(A) tail. The 155-kDa polypeptide, encoded by ORF1 of BaMV, consists of a capping enzyme domain, an RNA helicase-like domain, and an RNA-dependent RNA polymerase (RdRp) domain, in order of from N to C termini. Formation of the 5’ cap structure of BaMV is accomplished by sequential activities of RNA 5’ triphosphatase and AdoMet-depensent guanylyltransferase, which reside in the helicase-like domain and the capping enzyme domain, respectively. The aim of this study is to provide deeper biochemical analyses for gaining insights into the capping mechanism of BaMV.
Obtaining a pure preparation of the BaMV capping enzyme is the main task in the first part of this study. A variety of detergents were tried to solubilize the recombinant BaMV capping enzyme from yeast membrane fractions. Ionic detergents such as 0.6% sarkosyl buffer were found to be able to extract efficiently the enzyme from membrane fractions. Following the extraction, the capping enzyme domain, with a Hisx6-tag fused at the C terminus, was purified by metal affinity chromatography. The targeting of the recombinant BaMV capping enzyme to some unidentified organelles inside yeast cells was also visualized by fusing a EGFP to the enzyme. To identify the membrane-associated region on BaMV capping enzyme, the enzyme domain and series of its truncation versions from either the N or the C terminus were fused to EGFP, and their intracellular locations were detected by confocal microscopy. Membrane-cetrifugation assay was used to determine the solubility of the proteins. The region from residue 56 to 203 and that from 281 to 409 are thus proposed to associate with yeast membrane.
In the second part, to understand the critical residuals responsible for methyltransferase and/or guanylyltransfersae activities of BaMV capping enzyme, the site-directed mutagenesis was performed and the mutants’s activities were compared with each other. Mutations at conserved residuals (D122, R125, and Y213) and H66 resulted in abolishment of both methyltransferase and guanylyltransferase activities. The other mutants having reduced activities exhibited similar decreasing trends of mutational effects on methyltransferase and guanylyltransferase activities. The affinity to AdeMet of various BaMV capping enzymes, expect H68A, was found in good correlations with the magnitudes of methyltransferase and guanylyltransferase activities. H68A, a unique mutant, has intact methyltransferase but dead guanylyltransferase activities, as being evidenced by the production of m7GTP from GTP and AdoMet. m7GTP produced by H68A could support the formation of m7GMP-enzyme covalent intermediate in an AdoHcy-dependent manner reflecting that the affinity of AdoMet/AdoHcy to the capping enzyme is critical for forming the m7GMP-enzyme covalent intermediate.
The third part is to characterize the RNA: m7GMP transguanylation reaction. Previous studies showed that the BaMV capping enzyme could catalyze the transfer of m7GMP from the m7GMP-enzyme covalent intermediate to the 5’diphosphate end of RNA. In this study, we observed that the m7GMP could also be transferred to GDP molecules and formed the typical cap analog m7G(5’)ppp(5’)G. In future works, the transferring efficiency of the m7GMP from the m7GMP-enzyme covalent intermediate to GDP and to 5’ diphosphated-RNA will be compared, and the competition experiment will be taken place to demonstrate where would be the portion on RNA that is recognized by the BaMV capping enzyme.
目錄
頁次
中文摘要………………………………………………………….…….1
英文摘要………………………………………………………….…….3
第一章 緒言……………………...……………………………………5
1.竹嵌紋毒…………………………………………………………………......5
2.BaMV ORF1之複製酵素(replicase)……….………………………...……..6
3.BaMV之capping活性的發現…………………………………………..….7
4.已知的RNA capping enzymes………………….………………….…….….8
真核細胞、DNA virus與dsRNA virus…………………………..…...….8
Alphavirus-like病毒……………………………………………….….…10
5.研究BaMV capping enzyme的重要性……….………………….………...12
圖……………………………………………………………...……..14
Fig.1. Genome organization of Bamboo mosaic virus……….…………….….14
Fig.2. Schematic representation of genome organization of alpha-like viruses………………………………………………….….……….…....15
第二章 竹嵌紋病毒capping enzyme domain之純化與結合於膜之區域鑑定
中文摘要……………………………………………….....…………16
英文摘要…………………………………………....…………...…..17
前言…………………………………………………....…….………18
材料與方法…………………………………………………………20
1. 質體的構築………………………………………….….……………20
2. 質體的轉形………………………………………………….……….20
3. 於酵母菌中誘導病毒蛋白的表現……………………….……….…21
4. 破菌………………………………………………………….……….21
5. 超高速離心之膜漂浮法(membrane floatation)……………...….…..22
6. 膜蛋白質之依附性測試…………………………..….……….……..22
7. 膜蛋白質水溶性化之測試及純化……………………………..……23
8. 金屬親和性管柱層析……………………………………….……….23
9. Capping enzyme domain於酵母菌中的位置………………………..23
10. AdoMet-dependent guanylyltransferase之活性分析……………….24
結果…………………………………………….………………...…25
Capping enzyme domain於酵母菌中之表現及活性……………..……25
以超高速離心之膜漂浮法鑑定BaMV之 capping enzyme domain是否為膜蛋白質…………………………………………..………………….....25
觀察capping enzyme domain 在酵母細胞中的分佈位置………...….26
鑑定BaMV capping enzyme domain與膜依附的形式……...………...26
以不同界面活性劑使capping enzyme domain水溶性化………..……26
純化已水溶性化之capping enzyme domain及其AdoMet-dependent guanylyltransferase活性測試…………………………………………...27
BaMV capping enzyme domain與膜依附之區域探討……………..….27
討論……………………………………………..………………..…29
圖…………………………………………………………..…..……33
FIG.1. BaMV capping enzyme domain於酵母菌表現之情形………….33
FIG.2. BaMV capping enzyme domain之膜漂浮測試…………….….…34
FIG.3. BaMV之 replicase以及capping enzyme domain於酵母細胞之位置……………………………………..…………………..….……35
FIG.4. Capping enzyme domain與膜的依附測試…………………….....36
FIG.5.Capping enzyme domain以不同之界面活性劑水溶性化測試……………………………………………………………..…...37
FIG.6. 水溶性化之capping enzyme dimain經金屬親和性管柱純化及活性分析…………………………………………………………….38
FIG.7. 膜蛋白質的模型……………………………………………….…39
FIG. 8.鑑定BaMV capping enzyme domain與膜依附之區域.……....40-41
第三章 竹嵌紋病毒複製酵素之capping enzyme domain中重要胺基酸對於methyltransferase活性與形成m7GMP-enzyme複合體能力之影響
中文摘要……………………………………………….……………42
英文摘要………………………………………………………...…..44
背景介紹…………………………………………………….………46
材料與方法……………………………………………………….…48
1. 質體的構築………………………………………………….…..….48
2. 於酵母菌中誘導病毒蛋白的表現…………………………...….....48
3. 蛋白質的純化……………………………………………….……...48
4. Capping enzyme之酵素活性之偵測…………………….…….…..50
(1) m7GMP-enzyme intermediate形成之偵測………………..….50
(2) Methyltransferase活性之偵測………………………………..51
(3) Capping enzyme對AdoMet或GTP結合能力之偵測……...51
(4) 以m7GTP為基質所進行之m7GMP-enzyme intermediate形成之活性測試……………………………………….………...…52
5. 以TLC薄層色層分析法分析AdoMet-dependent guanylyltransferase 活性反應中之產物……………….……………………………………....52
6. m7GTP之純化與鑑定………………………………………………....52
結果…………………………………………………………..………54
1. Capping enzyme之單點突變……………………………….…..……54
2. Capping enzyme的純化…………………………………….………..55
3. 點突變對形成共價鍵結m7GMP-enzyme intermediate的影響(以GTP與AdoMet為反應基質)………………………..……………….…...…55
4. 點突變對GTP或AdoMet結合能力的影響………………….……...56
5. 點突變對methyltransferase活性的影響………………….………....56
6. 鑑定H68A之反應產物為m7GTP………………………………...…57
7. 點突變對形成共價鍵結之m7GMP-enzyme中間產物的影響……..58
8. AdoMet-dependent 與AdoHcy-dependent 形成m7GMP-enzyme intermediate的活性之比較………………….………………………….59
9. Capping enzyme domain 結合GTP的分析….…………..…………..59
討論………………………………………………………………..…61
表1: Mutational effects of capping enzyme domain on GTP methylation, guanylation of the protein, and AdoMet-binding ability…………………………65
圖…………………………………………………………………………………67
FIG.1. Primary and predicted secondary structures of the BaMV capping enzyme…………………………………………………………………..67
FIG.2. Protein purification, and formation of the covalent m7GMP-enzyme intermediate…………………………………………………………..…68
FIG.3. Time course of the formation of the covalent m7GMP-enzyme intermediate of the wild-type enzyme….……………………………….69
FIG.4. Effects of mutations on the formation of the covalent m7GMP-enzyme intermediate and the AdoMet-binding ability…….…..70
FIG.5. Effects of mutations on the GTP methyltransferase activity…………………………………………………………….….…71
FIG.6. Substrate specificity of the methyltransferase activity……………………………………..………………………..…..72
FIG.7. Product analysis of the reactions containing various BaMV capping enzymes, [a-32P]GTP, and AdoMet by TLC……………..……73
FIG.8. Production of m7GTP by H68A………………………………...74
FIG.9. Separation of the reaction products of H68A by HPLC…………………………………………………………………...75
FIG.10. Reaction conditions for the formation of the covalent m7GMP-enzyme intermediate when m7GTP was used as the substrate…………………………………………….……………….…76
FIG.11. Effects of mutations of capping enzyme on the formation of the covalent m7GMP-enzyme intermediate………………………….….…77
FIG.12. Reaction efficiency of formation of the covalent m7GMP-enzyme intermediate using GTP and AdoMet, or m7GTP and AdoHcy…………………………………………………………….…..78
FIG.13. Binding activity of capping enzyme to GTP in the presence of AdoMet or AdoHcy……………………………………………………..79
Figure 14. Schematic representation of the formation of capped RNA in BaMV…………………………………………………………………...80
第四章 m7GMP自capping enzyme轉移至RNA 5’端之活性分析及受質專一性探討
中文摘要…………… ………..…………………………………..…81
英文摘要…………………………………….………..……………..82
前言……………………………………………………...…………..83
材料與方法……………………………………….……………….…84
備製m7GMP-enzyme intermediate……………………………………84
RNA受質之備製…………………………………………………….…84
m7GMP轉移至受質測試………………………………………………85
m7GMP轉移至受質5’ diphosphated-RNA 之測試………………..…85
結果…………………………………….………………………………86
m7GMP轉移至受質測試………………………………………………...86
Cap 結構的產生…………………………………………………………86
m7GMP轉移至5’ diphosphated-RNA ( 5’ GDP-RNA)受質測試……....86
討論…………………….………………………………..……………..88
圖……………………………………………….………………………90
FIG. 1. [a-32P]m7GMP-enzyme intermediate的純化…………………..90
FIG.2. [a-32P]m7GMP-enzyme之[a-32P]m7GMP的轉移……………..91
FIG.3. Cap 結構m7GpppG的產生…………………………………….92
FIG. 4. [a-32P]m7GMP-enzyme與GDP的反應時間測試………….….93
FIG. 5. m7GMP轉移至5’ diphosphate-RNA………….………………..94
參考文獻……………………………………………………………………………95
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1. 竹嵌紋病毒複製酵素之功能特性分析
2. 竹嵌紋病毒複製酵素之N端區域的功能分析
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4. 一、竹嵌紋病毒戴帽酵素中參與SAM結合與催化甲基轉移能力之氨基酸探討。二、建立竹嵌紋病毒於酵母菌中之完整複製系統。
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