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Molecular mechanism of DNA translocation during head assembly of T3

Research Project

Project/Area Number 04454609
Research Category

Grant-in-Aid for General Scientific Research (B)

Allocation TypeSingle-year Grants
Research Field 分子生物学
Research InstitutionKYOTO UNIVERSITY

Principal Investigator

FUJISAWA Hisao  Kyoto Univ, Dept.Sci., Professor, 理学部, 教授 (00025347)

Project Period (FY) 1992 – 1993
Project Status Completed (Fiscal Year 1993)
Budget Amount *help
¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1993: ¥600,000 (Direct Cost: ¥600,000)
KeywordsT3, T7 phages / Head Assembly / DNA Packaging / DNA Recognition / Transcription / DNA Translocation / Energetic Mechanism / T3,T7ファージ
Research Abstract

During head assembly of double-stranded DNA phages, phage DNA is selected and translocated into a preformed protein shell, called the prohead, by hydrolysis of ATP.The molecular mechanisms of the selective packaging are specific for each phages and the energetic mechanism of the DNA translocation is common among phages.
T3 DNA is synthesized as a concatemer in which unit-length molecules are jointed in head-to-tail fashion through terminally redundant sequences. The concatemeric DNA is processed and packaged into the prohead with the aid of noncapsid proteins, gp18 and gp19. We showed that recombinant plasmids carrying DNA sequences necessary for DNA packaging(Pac sequences)can be packaged into transducing particles. The pac sequence has a bipartite structure consisting of the cleavage sites for processing of concatemeric DNA and the sequence containing a promoter sequence for T3 RNA polymerase. Promoter activity is necessary for DNA packaging and the transcriptional specificity determi … More nes the DNA packaging specificity of T3 and T7.
We have developed a defined system, composed of purified gp18, gp19 and proheads, and a crude system, composed of lysates of T3 infected cells, in which DNA is packaged by a way of a concatemer as an intermediate, for in vitro packaging of T3 DNA.The defined system displays an ATPase activity, composed of DNA packaging-dependent and -independent ATPases. Approximately one molecule of ATP was hydrolyzed during the translocation of 1.8 bp of T3 DNA.gp19 has ATP binding activity and three ATP binfding and two Mg^<2+> binding consensus motifs in its amino acid sequence. We have studied the roles of gp19 in the DNA packaging reaction by molecular genetic analysis by site-directed mutagenesis or deletion mutations. The results indicate that gp19 plays crucial roles in DNA translocation and in procssing of concatemric DNA.gp19 undergoes conformational changes in multiple steps of DNA packaging process through interaction with ATP.We have proposed a model for DNA translocation in which the gp19-connector located at the portal vertex would translocate DNA into the prohead by a cyclic change in the conformation of gp19 mediated by ATP. Less

Report

(3 results)
  • 1993 Annual Research Report   Final Research Report Summary
  • 1992 Annual Research Report
  • Research Products

    (23 results)

All Other

All Publications (23 results)

  • [Publications] 橋本主税: "T3,T7ファージの複製と遺伝子発現" 蛋白質核酸酵素. 37. 228-237 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Hashimoto,C.: "DNA sequences necessary for packaging bacteriophage T3" Virology. 187. 788-795 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Valpuesta,J.M.: "Three-dimensional structure of T3 connector purified from overexpressing bacteria" J.Mol.Biol.224. 103-112 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Hashimoto,C.: "Transcription dependence of DNA packaging of bacteriophage T3 and T7" Virology. 191. 246-250 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] 加藤 尚子: "T3ファージを用いた巨大DNAのクローニングと構造解析" 蛋白質核酸酵素. 38. 618-625 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Morita,M.: "DNA packaging ATPase of bacteriophage T3" Virology. 193. 748-752 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Morita,M.: "Analysis of functional domains of the packaging protein of bacteriophage T3 by site-directed mutagenesis" J.Mol.Biol.235. 248-259 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Fujisawa,H.: "Structure,function and specificity of the DNA packaging signals in double-stranded DNA viruses" Seminars in Virology. 5 (in press). (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Hashimoto, C.: "DNA sequences necessary for packaging of bacteriophage T3 DNA" Virology. 187. 788-796 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Hashimoto, C.: "Transcription dependence of DNA packageing of bacteriophages T3 and T7." Virology. 191. 245-250 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Valpuesta, J.M.: "Three-dimensional structure of T3 connector purified from over-expressing bacteria." J.Mol.Biol. 224. 103-112 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Morita, M.: "Analysis of functional domains of the packaging proteins of bacteriophage T3 by site-directed mutagenesis." J.Mol.Biol.235. 248-259 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Fujisawa, H.: "Structure, function and specificity of the DNA packaging signals in double-stranded DNA viruses." Seminars in Virology. 5, (in press). (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] 加藤 尚子: "T3ファージを用いた巨大DNAのクローニングと構造解析" 蛋白質核酸酵素. 38. 618-625 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] Morita,M.: "DNA packaging ATPase of bacteriophage T3" Virology. 193. 748-752 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] Morita,M.: "Analysis of functional domains of the packaging proteins of bacteriophage T3 by site-directed mutagenesis" J.Mol.Biol.235. 248-259 (1994)

    • Related Report
      1993 Annual Research Report
  • [Publications] Fujisawa,H.: "Structure,function and specificity of the DNA packaging signals in double-straded DNA viruses" Seminars in Virology. 5(in press). (1994)

    • Related Report
      1993 Annual Research Report
  • [Publications] Hashimoto,C.: "DNA sequences necessary for packaging of bacteriophage T3" VIROLOGY. 187. 788-796 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] Hashimoto,C.: "Transcription dependence of DNA packaging of bacteriophages T3 and T7" VIROLOGY. 191. 245-250 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] Valpuesta,J.M.: "Three-dimensional structure of T3 connector purified from over-expressing bacteria" J.Mol.Biol.224. 103-112 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] 橋本 主税: "T3,T7ファージの複製と遺伝子発現" 蛋白質・核酸・酵素. 37. 2560-2569 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] Morita,M.: "DNA packaging ATPase of bacteriophage T3" VIROLOGY. 193. (1993)

    • Related Report
      1992 Annual Research Report
  • [Publications] 加藤 尚子: "T3ファージを用いた巨大DNAのクローニング解析" 蛋白質・核酸・酵素. 38. 618-625 (1993)

    • Related Report
      1992 Annual Research Report

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Published: 1993-04-01   Modified: 2016-04-21  

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