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リボソームタンパク質―RNAの相互作用機構の解析

Research Project

Project/Area Number 11169228
Research Category

Grant-in-Aid for Scientific Research on Priority Areas

Allocation TypeSingle-year Grants
Review Section Biological Sciences
Research InstitutionKyushu University

Principal Investigator

木村 誠  九州大学, 農学研究院, 教授 (10204992)

Project Period (FY) 2001 – 2002
Project Status Completed (Fiscal Year 2002)
Budget Amount *help
¥22,500,000 (Direct Cost: ¥22,500,000)
Fiscal Year 2002: ¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 2001: ¥2,400,000 (Direct Cost: ¥2,400,000)
Fiscal Year 2000: ¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1999: ¥15,700,000 (Direct Cost: ¥15,700,000)
Keywordsリボソーム / リボソームタンパク質 / RNA結合タンパク質 / タンパク質-RNA相互作用 / 表面プラズモン共鳴法 / リボソーム蛋白質 / RNA / 蛋白質-RNA 相互作用 / 高次構造 / X線解析 / リボソームRNA
Research Abstract

昨年度までに、リボソームタンパク質BstL5と5S rRNAとの相互作用機構を明らかにするため、5S rRNAとの結合に関与するアミノ酸を特定するとともに、その役割について解析した。その結果、βシート上のアミノ酸はL5とRNAとの複合体の安定化に貢献していること、一方、ループ上のアミノ酸はRNAの認識に関与していることが明らかになった。
本年度では、BstL5のRNP2領域(Glu-^<32>-Lys-Ile-Val-Ile-Asn-Met^<38>)に無作為に変異を導入し、ファージ提示法によりRNA結合能を維持した変異タンパク質BstL5を選択することにより、RNP2のアミノ酸残基の重要性を検討した。変異体L5遺伝子は(VNN)7を含むプライマー(72mer)を用いたPCRにより作成し、ファージミドpLUCK2000を用いたファージ提示法でRNA結合能を保持した変異体を選択した。その結果、野生型と同程度の結合活性を持っ11個の変異体が選択され、その内7種は33位にLysもしくはArg、37位にAsnを持つこと、また34〜36位に疎水性アミノ酸が位置していることを見出した。以上の結果より、33位の塩基性アミノ酸と37位のAsn、その間の3残基の疎水性アミノ酸が、RNP2のRNA結合活性に重要であることが示唆された。

Report

(4 results)
  • 2002 Annual Research Report
  • 2001 Annual Research Report
  • 2000 Annual Research Report
  • 1999 Annual Research Report
  • Research Products

    (14 results)

All Other

All Publications (14 results)

  • [Publications] Hino et al.: "Expression of Nicotiana glutinosa ribonucleases in Escherichia coli"Biosci. Biotechnol. Biochem.. 66. 910-912 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] Kawano et al.: "Guanine binding site of the Nicotiana glutinosa ribonuclease NW revealed by X-ray crystallography"Biochemistry. 41. 15195-15202 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] T.Nakashima 等: "Ribosomal protein L5 has a highly twisted concave surface and flexible arms responsible for rRNA binding"RNA. 7. 692-701 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] H.Hosaka 等: "The structure of the archaebacterial ribosomal protein S7 and its possible interaction with 1 6S rRNA"J. Biochem.. 130. 695-701 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] K.Iwasaki: "On the interaction of ribosomal protein L5 with 5S rRNA"Biosci. Biotechnol. Biochem.,. 66. 103-109 (2002)

    • Related Report
      2001 Annual Research Report
  • [Publications] T.Hayashi 等: "Requirement for C-terminal extension to the RNA binding domain for efficient RNA binding by ribosomal protein L2"Biosci. Biotechnol. Biochem.,. 66. 682-684 (2002)

    • Related Report
      2001 Annual Research Report
  • [Publications] Numata 等: "Expression and mutational analysis of amino acid residues involved in catalytic activity in a ribonuclease"Biosci.Biotechnol.Biochem.. 64・3. 603-605 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Kouzuma 等: "Molecular cloning and functional expression of cDNA encoding the cycteine proteinase inhibitor"J.Biochem.. 128. 161-166 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Suzuki 等: "Cystal structure of ribonuclease MCl from bitter gourd seeds complexed with 1'-UMP"Biochem.Biophys.Res.Commun.. 275. 572-576 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Numata 等: "Amino acid residues in ribonuclease MCl from bitter gourd seeds which are essential"Biochemistry. 40・2. 524-530 (2001)

    • Related Report
      2000 Annual Research Report
  • [Publications] Nakagawa 等: "The-thtee-dimensional structure of the RNA-binding domain of ribosomal protein LZ;a protein at the pepticlyl transferase center of theribosome"The EMBO Journal. 18・6. 1459-1467 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Miyamoto 等: "Role of the N-terminal region of ribosomal protein S7 in its interaction with 16S rRNA which binds to the concavity formed by the β-ribbon arm and the α-hel"Eur.J.Biochem.. 266. 591-598 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Saitoh 等: "Arginine-55 in the β-arm is essential for the activity of DNA binding protein HU from Bacillus stearothermophilus"Biosci.Biotechnol.Biochem.. 63-12. 2232-2235 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Nakagawa 等: "Crystal structure of a ribonuclease From the seeds of bitter gourd at 1.75A"Biochim.Biophysi.Acta. 1433. 253-260 (1999)

    • Related Report
      1999 Annual Research Report

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Published: 1999-04-01   Modified: 2018-03-28  

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