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人工細胞モデル構築のためのRNA転写後修飾複合体装置の試験管内再構成

Research Project

Project/Area Number 18048032
Research Category

Grant-in-Aid for Scientific Research on Priority Areas

Allocation TypeSingle-year Grants
Review Section Science and Engineering
Research InstitutionEhime University

Principal Investigator

堀 弘幸  Ehime University, 大学院・理工学研究科, 教授 (20256960)

Co-Investigator(Kenkyū-buntansha) 渡邊 洋一  東京大学, 大学院・医学研究科, 准教授 (90323568)
富田 耕造  独立行政法人産業技術総合研究所, 生物機能工学研究部門, 研究グループ長 (00345274)
Project Period (FY) 2006 – 2007
Project Status Completed (Fiscal Year 2007)
Budget Amount *help
¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 2007: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 2006: ¥1,500,000 (Direct Cost: ¥1,500,000)
Keywords核酸 / 酵素 / 生体分子 / ゲノム / 進化
Research Abstract

生体内で、RNAが生理作用を発揮するためには、RNA修飾、スプライシング、プロセシングといった様々な編集・加工の工程(転写後修飾)を経る必要があります。転写後修飾は、様々なタンパク質が経時的に前駆体RNAにアッセンブリし、作用することにより起こります。本申請は、この転写後修飾装置を試験管内で再構成し、その基本システムを解明することを目標としました。
(1)TrmH-AdoMet-tRNA三者複合体を調製するため、TrmHの認識部位の部がデオキシ体となったキメラRNA-DNA核酸を合成した。これらのうち、いくつかは触媒反応の進行をくい止める効果があり、反応中間体に近い複合体形成に成功した。この成果を受け、平成19年度はX線結晶構造解析に着手し、結晶化条件をいくつか見いだした。
(2)この複合体を用いて、ハイドロキシラジカルプロービングをおこない、TrmHのC末端(tRNAI選択部位)と接触する部位がtRNAの三次元コア内部にあることを見いだし報告した。
(3)Aquifex aeolicus由来TrmBのC末端構造が二量体インターフェースの一部であることを確認し、高温環境下でのタンパク質構造の安定性およびメチル基転移部位の選択性に寄与することを報告した。
(4)無細胞翻訳系により、ヘテロサブユニットRNA修飾酵素(Trm8-Trm82)の試験管内合成に成功し、その詳細について報告した。

Report

(2 results)
  • 2007 Annual Research Report
  • 2006 Annual Research Report
  • Research Products

    (23 results)

All 2008 2007 2006 Other

All Journal Article (14 results) (of which Peer Reviewed: 6 results) Presentation (8 results) Remarks (1 results)

  • [Journal Article] The C-terminal region of thermophilic tRNA (m^7G46) methyltransferase (TrmB) stabilizes the dimer structure and enhances fidelity of methylation.2008

    • Author(s)
      C. Tomikawa, A. Ochi, and H.Hori
    • Journal Title

      Proteins: Structure, Function and Bioinformtics 71

      Pages: 1400-1408

    • Related Report
      2007 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Production of Hetero Subunits RNA Modification Enzyme by Wheat Germ Cell-Free Translation System2008

    • Author(s)
      K. Matsumoto, C. Tomikawa, T.Toyooka, A. Ochi, Y. Takano, N.Takayanagi, M. Abe, Y. Endo, and H. Hori
    • Journal Title

      J.of Biotechnol. 133

      Pages: 453-460

    • Related Report
      2007 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Hetero subunit interaction and RNA recognition of yeast tRNA (m7G46) methyltransferase synthesized in a wheat germ cell-free translation system2007

    • Author(s)
      Y. Muneyoshi, K. Matsumoto, C.Tomikawa, T. Toyooka, A. Ochi, T. Masaoka, Y. Endo, and H. Hori
    • Journal Title

      Nucleic Scids Res. Symp. Ser. 51

      Pages: 359-360

    • Related Report
      2007 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Hydroxyl Radical Probing of tRNA (Gm18) methyltransferase [TrmH]-AdoMet-artificial tRNA ternary complex2007

    • Author(s)
      A. Ochi, and H. Hori
    • Journal Title

      Nucleic Scids Res. Svtnp. Ser 51

      Pages: 373-374

    • Related Report
      2007 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Differences in substrate selectivity of the new SPOUT superfamily of methyltransferases2007

    • Author(s)
      T. Toyooka, and H. Hori
    • Journal Title

      Nucleic Scids Res. Symp. Ser 51

      Pages: 445-446

    • Related Report
      2007 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Complex Formations between Artificial RNA-DNA Chimera Nucleic Acids and RNA Modification Enzymes2007

    • Author(s)
      A. Ochi, and H. Hori
    • Journal Title

      Micro-NanoMechatronics and Human Science2007 CEP07768-CDR

      Pages: 113-118

    • Related Report
      2007 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Detection of structural changes in a cofactor binding protein by using a wheat germ cell-free protein synthesis system coupled with unnatural amino acid probing2007

    • Author(s)
      M.Abe
    • Journal Title

      Proteins : Structure, Function and Bioinformatics (In press)

    • Related Report
      2006 Annual Research Report
  • [Journal Article] RNA recognition mechanism of eukaryote tRNA (m^7G46) methyltransferase (Trm8-Trm82 complex)2007

    • Author(s)
      K.Matsumoto
    • Journal Title

      FEBS Lett (In press)

    • Related Report
      2006 Annual Research Report
  • [Journal Article] Production of Hetero Subunits RNA Modification Enzyme by Wheat Germ Cell-Free Translation System2007

    • Author(s)
      K.Matsumoto
    • Journal Title

      J. of Biotechnol. (In press)

    • Related Report
      2006 Annual Research Report
  • [Journal Article] The substrate specificity of tRNA (m^1G37) methyltransferase (TrmD), from Aquifex aeolicus2006

    • Author(s)
      H.Takeda
    • Journal Title

      Genes To Cells 11

      Pages: 1353-1365

    • Related Report
      2006 Annual Research Report
  • [Journal Article] Functional categorization of the conserved basic amino acid residues in TrmH [tRNA (Gm18) methyltansferase] enzymes2006

    • Author(s)
      K.Watanabe
    • Journal Title

      J. Biol. Chem. 281

      Pages: 34630-34639

    • Related Report
      2006 Annual Research Report
  • [Journal Article] The core domain of Aquifex aeolicus tRNA (m^7G46) methyltransferast has the methyl-transfer activity to tRNA2006

    • Author(s)
      C.Tomikawa
    • Journal Title

      Nucleic Scids Res. Symp. Ser. 50

      Pages: 245-246

    • Related Report
      2006 Annual Research Report
  • [Journal Article] Amount changes of tRNA modification enzymes in Thermus thermophilus HB8 cells according to culture temperatures.2006

    • Author(s)
      C.Iwashita
    • Journal Title

      Nucleic Scids Res. Symp. Ser. 50

      Pages: 247-248

    • Related Report
      2006 Annual Research Report
  • [Journal Article] Hetero subunits assembly study of RNA modification enzyme by wheat germ cell-free translation system2006

    • Author(s)
      K.Matsumoto
    • Journal Title

      IEEE : Micro-NanoMechatronics and Human Science 2006

      Pages: 355-360

    • Related Report
      2006 Annual Research Report
  • [Presentation] Transfer RNA methylation in ThemtHS thermophilus2007

    • Author(s)
      H. Hori, C. Iwashita, A. Shinkai, C. Tomikawa, Y. Terui C. Nakamoto, K. Watanabe, N. Shigi, T. Suzuki, Y. Endo, T. Oshima, and K. Watanabe
    • Organizer
      tRNA workshop 2007
    • Place of Presentation
      Uppsala, Sweden
    • Year and Date
      2007-11-02
    • Related Report
      2007 Annual Research Report
  • [Presentation] Chemical probing of tRNA-protein interface in TrmH-AdoMet-artificialtRNA ternary complex2007

    • Author(s)
      A. Ochi, and H. Hori
    • Organizer
      tRNA workshop 2007
    • Place of Presentation
      Uppsala, Sweden
    • Year and Date
      2007-11-02
    • Related Report
      2007 Annual Research Report
  • [Presentation] Functions of C-terminal region of thermophilic tRNA (m^7G46) methyltransferase (TrmB2007

    • Author(s)
      C. Tomikawa, and H. Hori
    • Organizer
      tRNA workshop 2007
    • Place of Presentation
      Uppsala, Sweden
    • Year and Date
      2007-11-02
    • Related Report
      2007 Annual Research Report
  • [Presentation] Substrate Recognition of Trml [tRNA(m^2_2G26) methyltransferase] from Aquifex aeolicus.2007

    • Author(s)
      T. Awai, Ihsanawati, T. Toyooka, Y. Bessho, S. Yokoyama, and H. Hori
    • Organizer
      tRNA workshop 2007
    • Place of Presentation
      Uppsala, Sweden
    • Year and Date
      2007-11-02
    • Related Report
      2007 Annual Research Report
  • [Presentation] 翻訳伸長因子EF-TuとtRNA修飾酵素の相互憚用解析系の構築2007

    • Author(s)
      中村 仁, 堀 弘幸
    • Organizer
      高度好熱菌丸ごと一匹プロジェクト第6回連携研究会
    • Place of Presentation
      兵庫
    • Year and Date
      2007-08-03
    • Related Report
      2007 Annual Research Report
  • [Presentation] RNAメチル化酵素とキメラ核酸の複合体形成メカニズム2007

    • Author(s)
      越智 杏奈, 堀 弘幸
    • Organizer
      分子・細胞・組織操作を目指したシステム細胞工学 女性研究者による医学・理学・工学融合分野
    • Place of Presentation
      東京
    • Year and Date
      2007-08-03
    • Related Report
      2007 Annual Research Report
  • [Presentation] 生命ドメインの変遷に伴うtRNA (m^7G46)メチル化酵素の分子進化2007

    • Author(s)
      冨川千恵, 松本啓介, 豊岡 峻, 越智杏奈, 高柳直行, 高野 義孝, 遠藤弥重太, 堀 弘幸
    • Organizer
      第9回日本RNA学会年会
    • Place of Presentation
      名古屋
    • Year and Date
      2007-07-30
    • Related Report
      2007 Annual Research Report
  • [Presentation] SPOUTスーパーファミリーメチル化酵素の構造と機能の相関2007

    • Author(s)
      豊岡 峻, 堀 弘幸
    • Organizer
      第9回日本RNA学会年会
    • Place of Presentation
      名古屋
    • Year and Date
      2007-07-29
    • Related Report
      2007 Annual Research Report
  • [Remarks]

    • URL

      http://www.ehime-u.ac.jp/~achem/bchem/

    • Related Report
      2007 Annual Research Report

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Published: 2006-04-01   Modified: 2018-03-28  

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