一个硫化叶菌病毒启动子的分离与鉴定
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摘要
几乎所有古菌病毒基因组中无RNA聚合酶(RNA polymerase,RNAP)等组成基本转录装置的同源蛋白编码序列,而且启动子活性对病毒感染过程中病毒基因的转录上可能具有重要的影响.为进一步揭示古菌病毒基因启动子的序列结构特点和活性之间的关系,首先基于硫化叶菌质粒pSeSD,将β-半乳糖苷酶编码基因lacS克隆到阿拉伯糖启动子araS下游多克隆位点,构建重组表达载体pSeSD-lacS.将pSeSD-lacS转化冰岛硫化叶菌(Sulfolobus islandicus)E233S菌株后的功能分析结果表明,lacS基因成功表达.在此基础上,利用硫化叶菌病毒STSV2衣壳蛋白编码基因ORF37上游500bp的潜在启动子片段P37替换pSeSD-lacS中的araS启动子,构建出新的重组表达质粒pSeSD-P37-lacS,进一步将pSeSD-P37-lacS转化E233S菌株进行启动子活性分析.β-半乳糖苷酶酶活结果显示,诱导后araS启动子酶活为14 345.7±422.3 mU,P37酶活为13 723.1±370.9 mU,表明P37片段具有启动子功能,而且活性与araS启动子相当.序列分析也显示,P37具有与硫化叶菌基因启动子类似的基础序列元件initiator、TATA-box及BRE等.本研究表明pSeSD-lacS可作为一个硫化叶菌病毒基因启动子筛选载体,而且高活性的基因启动子可能在STSV2病毒生命过程具有重要的作用.(图4表1参27)
Many studies have shown that there are no homologous protein-encoding sequences of eukaryotic basal transcriptional machinery such as RNA polymerase in the genomes of almost all archaeal viruses, and promoter activity may be important for the transcription of viral genes during infection. To further determine the relationship between the sequence structure characteristics and the activity of archaeal viral gene promoters, the present study first subcloned the β-galactosidase encoding gene lacS into a multiple clone site downstream of the arabinose promoter araS on vector pSeSD to generate the recombinant expression plasmid pSeSD-lacS, which was further transformed into a Sulfolobus islandicus strain E233 S for functional analysis. The results showed that the lacS gene was successfully expressed in the E233 S strain. Based on this, a new recombinant expression plasmid pSeSD-P37-lacS was constructed by replacing the araS promoter on the pSeSD-lacS with a potential promoter fragment P37, a 500-base pair sequence upstream of the capsid-encoding gene ORF37 of virus STSV2, and the promoter activity was further analyzed by transforming pSeSD-P37-lacS into the E233 S strain. β-galactosidase activity showed that the araS promoter activity was 14 345.7 ± 422.3 mU after induction and the P37 activity was 13 723.1 ± 370.9 mU,which indicated that P37 was a functional promoter fragment and exhibited almost equivalent activity with that of the araS promoter. Further sequence analysis showed that P37 contained basal promoter sequence elements of Sulfolobus genes, such as initiator, TATA-box, and BRE. These results indicate that pSeSD-lacS can be used as a screening vector for the identification of promoters from Sulfolobus viruses and the high activity of viral gene promoters may play an important role in the life process of the virus STSV2.
Many studies have shown that there are no homologous protein-encoding sequences of eukaryotic basal transcriptional machinery such as RNA polymerase in the genomes of almost all archaeal viruses, and promoter activity may be important for the transcription of viral genes during infection. To further determine the relationship between the sequence structure characteristics and the activity of archaeal viral gene promoters, the present study first subcloned the β-galactosidase encoding gene lacS into a multiple clone site downstream of the arabinose promoter araS on vector pSeSD to generate the recombinant expression plasmid pSeSD-lacS, which was further transformed into a Sulfolobus islandicus strain E233 S for functional analysis. The results showed that the lacS gene was successfully expressed in the E233 S strain. Based on this, a new recombinant expression plasmid pSeSD-P37-lacS was constructed by replacing the araS promoter on the pSeSD-lacS with a potential promoter fragment P37, a 500-base pair sequence upstream of the capsid-encoding gene ORF37 of virus STSV2, and the promoter activity was further analyzed by transforming pSeSD-P37-lacS into the E233 S strain. β-galactosidase activity showed that the araS promoter activity was 14 345.7 ± 422.3 mU after induction and the P37 activity was 13 723.1 ± 370.9 mU,which indicated that P37 was a functional promoter fragment and exhibited almost equivalent activity with that of the araS promoter. Further sequence analysis showed that P37 contained basal promoter sequence elements of Sulfolobus genes, such as initiator, TATA-box, and BRE. These results indicate that pSeSD-lacS can be used as a screening vector for the identification of promoters from Sulfolobus viruses and the high activity of viral gene promoters may play an important role in the life process of the virus STSV2.
引文
1 Woese CR,Fox GE.Phylogenetic structure of the prokaryotic domain:the primary kingdom[J].PNAS,1977,74:5088-5090
2 Woese CR,Kandler O,Wheelis ML.Towards a natural system of organisms:proposal for the domains archaea,Bacteria and Eucarya[J].PNAS,1990,87(12):4576-4579
3 Sheppard C,Werner F.Structure and mechanisms of viral transcription factors in archaea[J].Extremophiles,2017,21(5):829
4敖翔.硫化叶菌启动子Initiator元件功能研究[D].武汉:华中农业大学,2013[Ao X.Study on the function of Initiator element of Sulfolobus promoter[D].Wuhan:Huazhong Agricultural University,2013]
5 Nikolov DB,Chen H,Halay ED,Usheva AA,Hisatake K,Lee DK,Roeder RG,Burley SK.Crystal structure of a TFIIB-TBP-TATA-element ternary complex[J].Nature,1995,377(6545):119-128
6 Kosa PF,Ghosh G,DeDecker BS,Sigler PB.The 2.1-A crystal structure of an archaeal preinitiation complex:TATA-box-binding protein/transcription factor(II)B core/TATA-box[J].PNAS,1997,94(12):6042-6047
7 Reiter WD,Palm P,Zillig W.Analysis of transcription in the archaebacterium Sulfolobus indicates that archaebacterial promoters are homologous to eukaryotic pol II promoters[J].Nucl Acids Res,1988,16(1):1-19
8 Qureshi SA,Jackson SP.Sequence-specific DNA binding by the S.shibatae TFIIB homolog,TFB,and its effect on promoter strength[J].Mol Cell,1998,1(3):389-400
9张琦,胡彬彬,季秀玲,林连兵,魏云林.细菌噬菌体转录调节机制的研究进展[J].昆明理工大学学报(自然科学版),2013(2):84-87[Zhang Q,Hu BB,Ji XL,Lin LB,Wei YL.Advances in the regulation mechanism of bacteriophage transcriptional regulation[J].J Kunming Univ Sci Technol(Nat Sci Ed),2013,38(2):84-87]
10 Hsieh ML,James TD,Knipling L,Waddell MB,White S,Hinton DM.Architecture of the bacteriophage T4 activator MotA/promoter DNAinteraction during sigma appropriation[J].J Biol Chem,2013,288(38):27607-27618
11 Parks AR,Court C,Lubkowska L,Jin DJ,Kashlev M,Court DL.Bacteriophage lambda N protein inhibits transcription slippage by Escherichia coli RNA polymerase[J].Nucl Acids Res,2014,42(9):5823-5829
12 Twist KA,Campbell EA,Deighan P,Nechaev S,Jain V,Geiduschek EP,Hochschild A,Darst SA.Crystal structure of the bacteriophage T4 late-transcription coactivator gp33 with the?2-subunit flap domain of Escherichia coli RNA polymerase[J].PNAS,2011,108(50):19961-19966
13 Bohn-Wippert K,Tevonian EN,Megaridis MR,Dar RD.Similarity in viral and host promoters couples viral reactivation with host cell migration[J].Nat Commun,2017,8:15006
14 Krupovic M,White MF,Forterre P,Prangishvili D.Postcards from the edge:structural genomics of archaeal viruses[J].Adv Virus Res,2012,82:33-62
15 Ren Y,She Q,Huang L.Transcriptomic analysis of the SSV2 infection of Sulfolobus solfataricus,with and without the integrative plasmid pSSVi[J].Virology,2013,441(2):126-134
16 Munsonmcgee JH,Snyder JC,Young MJ.Archaeal viruses from hightemperature environments[J].Genes,2018,9(3):128
17党亚锋,陈波,张琦,黄晓星,魏云林,林连兵.腾冲热海高温酸性热泉类病毒颗粒的多样性[J].应用与环境生物学报,2012,18(2):256-261[Dang YF,Chen B,Zhang Q,Huang XX,Wei YL,Lin LB.Diversity of high temperature acidic hot spring virus particles in Tengchong Thermal Sea[J].Chin J Appl Environ Biol,2012,18(2):256-261]
18 Erdmann S,Chen B,Huang X,Deng L,Liu C,Shah SA,Le Moine Bauer S,Sobrino CL,Wang H,Wei Y,She Q,Garrett RA,Huang L,Lin L.A novel single-tailed fusiform Sulfolobus virus STSV2 infecting model Sulfolobus species[J].Extremophiles,2014,18(1):51-60
19 Deng L,Zhu H,Chen Z,Liang YX,She Q.Unmarked gene deletion and host-vector system for the hyperthermophilic crenarchaeon Sulfolobus islandicus[J].Extremophiles,2009,13(4):735-746
20 Ao X,Li Y,Wang F,Feng M,Lin Y,Zhao S,Liang Y,Peng N.The Sulfolobus initiator element is an important contributor to promoter strength[J].J Biotechnol,2013,195(22):5216-5222
21 Zhang C,Whitaker RJ.A broadly applicable gene knockout system for the thermoacidophilic archaeon Sulfolobus islandicus based on simvastatin selection[J].Microbiology,2012,158:1513-1522
22 Reese JC.Basal transcription factors[J].Curr Opin Gene Dev,2003,13(2):114-118
23 Soppa J.Normalized nucleotide frequencies allow the definition of archaeal promoter elements for different archaeal groups and reveal base-specific TFB contacts upstream of the TATA box[J].MolMicrobiol,1999,31(5):1589-1592
24 Brenneis M,Hering O,Lange C,Soppa J.Experimental characterization of Cis-acting elements important for translation and transcription in halophilic archaea[J].Genetics,2007,3(12):2450-2467
25 Peng N,Xia Q,Chen Z,Liang YX,She Q.An upstream activation element exerting differential transcriptional activation on an archaeal promoter[J].Mol Microbiol,2009,74(4):928
26彭楠.硫化叶菌阿拉伯糖启动子转录调控及其应用[D].武汉:华中农业大学,2009[Peng N.Transcriptional and application regulation of the arabinose promoter of sulfolobus[D].Wuhan:Huazhong Agricultural University,2009]
27 Jonuscheit M,Martusewitsch E,Stedman KM,Schleper C.A reporter gene system for the hyperthermophilic archaeon Sulfolobus solfataricus based on a selectable and integrative shuttle vector[J].Mol Microbiol,2003,48(5):1241-1252
2 Woese CR,Kandler O,Wheelis ML.Towards a natural system of organisms:proposal for the domains archaea,Bacteria and Eucarya[J].PNAS,1990,87(12):4576-4579
3 Sheppard C,Werner F.Structure and mechanisms of viral transcription factors in archaea[J].Extremophiles,2017,21(5):829
4敖翔.硫化叶菌启动子Initiator元件功能研究[D].武汉:华中农业大学,2013[Ao X.Study on the function of Initiator element of Sulfolobus promoter[D].Wuhan:Huazhong Agricultural University,2013]
5 Nikolov DB,Chen H,Halay ED,Usheva AA,Hisatake K,Lee DK,Roeder RG,Burley SK.Crystal structure of a TFIIB-TBP-TATA-element ternary complex[J].Nature,1995,377(6545):119-128
6 Kosa PF,Ghosh G,DeDecker BS,Sigler PB.The 2.1-A crystal structure of an archaeal preinitiation complex:TATA-box-binding protein/transcription factor(II)B core/TATA-box[J].PNAS,1997,94(12):6042-6047
7 Reiter WD,Palm P,Zillig W.Analysis of transcription in the archaebacterium Sulfolobus indicates that archaebacterial promoters are homologous to eukaryotic pol II promoters[J].Nucl Acids Res,1988,16(1):1-19
8 Qureshi SA,Jackson SP.Sequence-specific DNA binding by the S.shibatae TFIIB homolog,TFB,and its effect on promoter strength[J].Mol Cell,1998,1(3):389-400
9张琦,胡彬彬,季秀玲,林连兵,魏云林.细菌噬菌体转录调节机制的研究进展[J].昆明理工大学学报(自然科学版),2013(2):84-87[Zhang Q,Hu BB,Ji XL,Lin LB,Wei YL.Advances in the regulation mechanism of bacteriophage transcriptional regulation[J].J Kunming Univ Sci Technol(Nat Sci Ed),2013,38(2):84-87]
10 Hsieh ML,James TD,Knipling L,Waddell MB,White S,Hinton DM.Architecture of the bacteriophage T4 activator MotA/promoter DNAinteraction during sigma appropriation[J].J Biol Chem,2013,288(38):27607-27618
11 Parks AR,Court C,Lubkowska L,Jin DJ,Kashlev M,Court DL.Bacteriophage lambda N protein inhibits transcription slippage by Escherichia coli RNA polymerase[J].Nucl Acids Res,2014,42(9):5823-5829
12 Twist KA,Campbell EA,Deighan P,Nechaev S,Jain V,Geiduschek EP,Hochschild A,Darst SA.Crystal structure of the bacteriophage T4 late-transcription coactivator gp33 with the?2-subunit flap domain of Escherichia coli RNA polymerase[J].PNAS,2011,108(50):19961-19966
13 Bohn-Wippert K,Tevonian EN,Megaridis MR,Dar RD.Similarity in viral and host promoters couples viral reactivation with host cell migration[J].Nat Commun,2017,8:15006
14 Krupovic M,White MF,Forterre P,Prangishvili D.Postcards from the edge:structural genomics of archaeal viruses[J].Adv Virus Res,2012,82:33-62
15 Ren Y,She Q,Huang L.Transcriptomic analysis of the SSV2 infection of Sulfolobus solfataricus,with and without the integrative plasmid pSSVi[J].Virology,2013,441(2):126-134
16 Munsonmcgee JH,Snyder JC,Young MJ.Archaeal viruses from hightemperature environments[J].Genes,2018,9(3):128
17党亚锋,陈波,张琦,黄晓星,魏云林,林连兵.腾冲热海高温酸性热泉类病毒颗粒的多样性[J].应用与环境生物学报,2012,18(2):256-261[Dang YF,Chen B,Zhang Q,Huang XX,Wei YL,Lin LB.Diversity of high temperature acidic hot spring virus particles in Tengchong Thermal Sea[J].Chin J Appl Environ Biol,2012,18(2):256-261]
18 Erdmann S,Chen B,Huang X,Deng L,Liu C,Shah SA,Le Moine Bauer S,Sobrino CL,Wang H,Wei Y,She Q,Garrett RA,Huang L,Lin L.A novel single-tailed fusiform Sulfolobus virus STSV2 infecting model Sulfolobus species[J].Extremophiles,2014,18(1):51-60
19 Deng L,Zhu H,Chen Z,Liang YX,She Q.Unmarked gene deletion and host-vector system for the hyperthermophilic crenarchaeon Sulfolobus islandicus[J].Extremophiles,2009,13(4):735-746
20 Ao X,Li Y,Wang F,Feng M,Lin Y,Zhao S,Liang Y,Peng N.The Sulfolobus initiator element is an important contributor to promoter strength[J].J Biotechnol,2013,195(22):5216-5222
21 Zhang C,Whitaker RJ.A broadly applicable gene knockout system for the thermoacidophilic archaeon Sulfolobus islandicus based on simvastatin selection[J].Microbiology,2012,158:1513-1522
22 Reese JC.Basal transcription factors[J].Curr Opin Gene Dev,2003,13(2):114-118
23 Soppa J.Normalized nucleotide frequencies allow the definition of archaeal promoter elements for different archaeal groups and reveal base-specific TFB contacts upstream of the TATA box[J].MolMicrobiol,1999,31(5):1589-1592
24 Brenneis M,Hering O,Lange C,Soppa J.Experimental characterization of Cis-acting elements important for translation and transcription in halophilic archaea[J].Genetics,2007,3(12):2450-2467
25 Peng N,Xia Q,Chen Z,Liang YX,She Q.An upstream activation element exerting differential transcriptional activation on an archaeal promoter[J].Mol Microbiol,2009,74(4):928
26彭楠.硫化叶菌阿拉伯糖启动子转录调控及其应用[D].武汉:华中农业大学,2009[Peng N.Transcriptional and application regulation of the arabinose promoter of sulfolobus[D].Wuhan:Huazhong Agricultural University,2009]
27 Jonuscheit M,Martusewitsch E,Stedman KM,Schleper C.A reporter gene system for the hyperthermophilic archaeon Sulfolobus solfataricus based on a selectable and integrative shuttle vector[J].Mol Microbiol,2003,48(5):1241-1252
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