TransferRNA分子におけるクローバーリーフ構造の安定化戦略の解明

• Project/Area Number: 14704011
• Research Category: Grant-in-Aid for Young Scientists (A)
• Allocation Type: Single-year Grants
• Research Field: 応用微生物学・応用生物化学
• Research Institution: Toyohashi University of Technology
• Principal Investigator: 田中 照通 豊橋技術科学大学, 工学部, 助教授 (30273337)
• Project Period (FY): 2002 – 2004
• Project Status: Completed (Fiscal Year 2004)
• Budget Amount: ¥29,770,000 (Direct Cost: ¥22,900,000、Indirect Cost: ¥6,870,000); Fiscal Year 2004: ¥4,550,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥1,050,000); Fiscal Year 2003: ¥4,550,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥1,050,000); Fiscal Year 2002: ¥20,670,000 (Direct Cost: ¥15,900,000、Indirect Cost: ¥4,770,000)
• Keywords: Ribonuclease P / tRNA / cloverleaf / intron / extra variable loop / coevolution / ribonuclease P / Ribozyme / long extra loop

Research Abstract

tRNAはタンパク質生合成系におけるアミノ酸の運び手であり遺伝暗号の担い手でもある基本的な生体分子である。この分子は細胞内に約50種存在するが、その構造は対応するアミノ酸や由来する生物種を超えて共通のクローバーリーフ構造をとっている。この構造の維持は効率的なタンパク質合成においては必須の条件であるため、それぞれの生物種においてそれぞれのtRNA分子は何らかの構造安定化戦略をとっているものと考えられる。tRNA分子の安定化に種々の塩基修飾が寄与していることは既に知られているが、本申請研究では、塩基修飾を受ける以前のtRNA前駆体における安定性を1つの評価の指標とした。tRNA前駆体分子の安定性は、きちんとした成熟化tRNAの生成の条件であり、重要な評価の基準となる。tRNA分子の安定性の評価の方法としては、「大多数」を評価するための統計的な状態を反映する方法と、「微量な」変性を検出するための方法とを採用した。
「微量な」構造変性を検出する方法として真正細菌リボヌクレアーゼPを採用した。この酵素はtRNA成熟化酵素の1つであり、「きちんと」フォールディングしたtRNA前駆体を基質とするが、同時に、高濃度Mgイオンの存在下ではCCA付きのヘアピン型RNAをも基質として認識できる。反応条件をコントロールすることで、変性したもしくはフォールディングに失敗したtRNAを検出することができる。この酵素による検出系では切断産物の蓄積を通して、微量な存在比のものでも検出ができるところにある。この方法によって既に幾つかのtRNAの安定性評価を行い、由来する生物種によってtRNAの構造安定性に差異があることを報告してきた。また、これらの実験結果は同じ細胞内におけるtRNA分子とリボヌクレアーゼP分子とにおける共進化モデルを与えた。
また、上記の方法論によってtRNAイントロンはtRNA前駆体における構造安定化装置ではなく別の意味を持つことや、数種のtRNA分子に見られる長いエクルトラループという部分構造がtRNA分子の安定化に大きく寄与していることなどが明らかとなった。
本研究においては併せて道具としてのリボヌクレアーゼPの酵素学的な性質を目指したが、研究の成果として真正細菌酵素の基質認識が反応条件や基質の形状、及び反応溶液中におけるリボゾームコンポーネントの存在などによっても変化することを明らかにすることができた。

Research Products

• [Journal Article] The P3 domain of E.coil ribonuclease P RNA is able to be truncated and replaceable. (2004)
  • Author(s): Terumichi Tanaka, et al.
  • Journal Title: FEBS Letters 577 Pages: 101-104
• [Journal Article] Mutational analyses on the length of J3/4 domain of Escherichia coli ribonuclease P ribozyme. (2004)
  • Author(s): Shinnosuke Haga, et al.
  • Journal Title: Bioscience, Biotechnology, and Biochemistry 68 Pages: 2630-2632
• [Journal Article] Extracellular RNAs produced by a marine photosynthetic bacterium Rhodovulum sulfidophilum. (2004)
  • Author(s): Tomoaki Ando, et al.
  • Journal Title: Nucleic Acids Symposium Series No.48 Pages: 165-166
• [Journal Article] Mutational analyses of neighboring domains of active center of RNase P ribozyme. (2004)
  • Author(s): Terumichi Tanaka, et al.
  • Journal Title: Nucleic Acids Symposium Series No.48 Pages: 201-202
• [Journal Article] Natural intron sequence of human tyrosine pre-tRNA is not a temporal stabilizer for cloverleaf structure. (2004)
  • Author(s): Tomoaki Ando, et al.
  • Journal Title: Bioscience, Biotechnology, and Biochemistry 68 Pages: 1782-1785
• [Journal Article] Examining the bases of J3/4 domain of E. coli ribonuclease P (2004)
  • Author(s): Terumichi Tanaka, et al.
  • Journal Title: Bioscience, Biotechnology, and Biochemistry 68 Pages: 1388-1392
• [Journal Article] Substrate shape specificity of E. coli RNase P ribozyme is dependent on the concentration of magnesium ion (2003)
  • Author(s): Tomoaki Ando et al.
  • Journal Title: Journal of Biochemistry 133 Pages: 445-451
• [Journal Article] E.coli tRNAs are resistant to the hyperprocessing reaction of homologous E.coli ribonuclease P ribozyme. (2003)
  • Author(s): Terumichi Tanaka et al.
  • Journal Title: Bioscience, Biotechnology, and Biochemistry 67 Pages: 1172-1176
• [Journal Article] Revisiting the substrate recognition of bacterial ribonuclease P --- in the view of the recognition of the base N73 in the substrate. (2003)
  • Author(s): Terumichi Tanaka et al.
  • Journal Title: Nucleic Acids Research Suppl.No.3 Pages: 275-276
• [Journal Article] Bacterial ribonuclease P reaction is affected by substrate shape and magnesium ion concentration. (2003)
  • Author(s): Tomoaki Ando et al.
  • Journal Title: Nucleic Acids Research Suppl.No.3 Pages: 293-294
• [Journal Article] Extracellular DNA and RNA produced by a marine photosynthetic bacterium Rhodovulum sulfidophilum. (2003)
  • Author(s): Seiichiro Nishimura et al.
  • Journal Title: Nucleic Acids Research Suppl.No.3 Pages: 279-280
• [Journal Article] Recognition of tRNA variants by bacterial ribonuclease P. (2003)
  • Author(s): Yasuhiro Nagai et al.
  • Journal Title: Nucleic Acids Research Suppl.No.3 Pages: 281-282
• [Journal Article] Comparative analyses on hairpin substrate recognition by E.coli and B.subtilis RNase P ribozymes. (2003)
  • Author(s): Tomoaki Ando et al.
  • Journal Title: Bioscience, Biotechnology, and Biochemistry 67 Pages: 1823-1827
• [Journal Article] The protein component of bacterial RNase P flickers the metal ion response of the substrate shape preference of the ribozyme. (2003)
  • Author(s): Tomoaki Ando et al.
  • Journal Title: Bioscience, Biotechnology, and Biochemistry 67 Pages: 2294-2296
• [Journal Article] Another cut for lysine tRNA : Application of the hyperprocessing reaction reveals another stabilization strategy in metazaon lysine tRNAs. (2002)
  • Author(s): Terumichi Tanaka et al.
  • Journal Title: Journal of Biochemistry 131 Pages: 839-847
• [Journal Article] Kinetic analysis on hyperprocessing reaction of human tyrosine tRNA by E.coli ribonuclease P ribozyme. (2002)
  • Author(s): Tomoaki Ando et al.
  • Journal Title: Bioscience, Biotechnology, and Biochemistry 66 Pages: 1967-1971
• [Journal Article] Regulation of bacterial RNase P ribozyme by divalent cation and guide DNA. (2002)
  • Author(s): Tomoaki Ando et al.
  • Journal Title: Nucleic Acids Research Suppl.No.2 Pages: 271-272
• [Journal Article] Porphyrins and porphines inhibit the ribonuclease P reaction in vitro. (2002)
  • Author(s): Yoshiaki Hori et al.
  • Journal Title: Nucleic Acids Research Suppl.No.2 Pages: 111-112
• [Journal Article] Guide DNA technique in bacterial ribonuclease P reaction for effective processing of tRNA precursor. (2002)
  • Author(s): Terumichi Tanaka et al.
  • Journal Title: Biotechnology and Applied Biochemistry 36 Pages: 85-88
• [Book] 「生体の科学」53巻2号・特集「RNA」「RNA酵素に生命の謎解きができるか?」 (2002)
  • Author(s): 田中照通, 菊池洋
  • Publisher: (財)金原一郎記念医学医療振興財団
• [Publications] Tomoaki Ando et al.: "Substrate shape specificity of E. coli RNase P ribozyme is dependent on the concentration of magnesium ion."Journal of Biochemistry. 133. 445-451 (2003)
• [Publications] Terumichi Tanaka et al.: "E. coli tRNAs are resistant to the hyperprocessing reaction of homologous E. coli ribonuclease P ribozyme."Bioscience, Biotechnology, and Biochemistry. 67. 1172-1176 (2003)
• [Publications] Terumichi Tanaka et al.: "Revisiting the substrate recognition of bacterial ribonuclease P --- in the view of the recognition of the base N73 in the substrate."Nucleic Acids Research. Suppl.No.3. 275-276 (2003)
• [Publications] Tomoaki Ando et al.: "Bacterial ribonuclease P reaction is affected by substrate shape and magnesium ion concentration."Nucleic Acids Research. Suppl.No.3. 293-294 (2003)
• [Publications] Seiichiro Nishimura et al.: "Extracellular DNA and RNA produced by a marine photosynthetic bacterium Rhodovulum sulfidophilum."Nucleic Acids Research. Suppl.No.3. 279-280 (2003)
• [Publications] Yasuhiro Nagai et al.: "Recognition of tRNA variants by bacterial ribonuclease P."Nucleic Acids Research. Suppl.No.3. 281-282 (2003)
• [Publications] Tomoaki Ando et al.: "Comparative analyses on hairpin substrate recognition by E. coli and B. subtilis RNase P ribozymes."Bioscience, Biotechnology, and Biochemistry. 67. 1823-1827 (2003)
• [Publications] Tomoaki Ando et al.: "The protein component of bacterial RNase P flickers the metal ion response of the substrate shape preference of the ribozyme."Bioscience, Biotechnology, and Biochemistry. 67. 2294-2296 (2003)
• [Publications] Terumichi Tanaka et al.: "Another cut for lysine tRNA : Application of the hyperprocessing reaction reveals another stabilization strategy in metazaon lysine tRNAs."Journal of Biochemistry. 131. 839-847 (2002)
• [Publications] Tomoaki Ando et al.: "Kinetic analysis on hyperprocessing reaction of human tyrosine tRNA by E. coli ribonuclease P ribozyme."Bioscience, biotechnology, and Biochemistry. 66. 1967-1971 (2002)
• [Publications] Tomoaki Ando et al.: "Regulation of bacterial RNase P ribozyme by divalent cation and guide DNA."Nucleic Acids Research. Suppl.No.2. 271-272 (2002)
• [Publications] Yoshiaki Hori et al.: "Porphyrins and porphines inhibit the ribonuclease P reaction in vitro."Nucleic Acids Research. Suppl.No.2. 111-112 (2002)
• [Publications] Terumichi Tanaka et al.: "Guide DNA technique in bacterial ribonuclease P reaction for effective processing of tRNA precursor."Biotechnology and Applied Biochemistry. 36. 85-88 (2002)
• [Publications] 田中照通, 菊池洋: "「生体の科学」53巻2号・特集「RNA」「RNA酵素に生命の謎解きができるか?」"(財)金原一郎記念医学医療振興財団. 136-141 (2002)
• [Publications] Terumichi Tanaka et al.: "Guide DNA technique in bacterial ribonuclease P reaction for effective processing of tRNA precursor"Biotechnology and Applied Biochemistry. 36. 85-88 (2002)
• [Publications] Terumichi Tanak et al.: "Another cut for lysine tRNA : Application of the hyperprocessing reaction reveals another stabilization strategy in metazaon lysine tRNAs"Journal of Biochemistry. 131. 839-847 (2002)
• [Publications] Tomoaki Ando et al.: "Kinetic analysis on hyperprocessing reaction of human tyrosine tRNA by E.coli ribonuclease P ribozyme"Bioscience, Biotechnology and Biochemistry. 66. 1967-1971 (2002)
• [Publications] Tomoaki Ando et al.: "Regulation of bacterial RNase P ribozyme by divalent cation and guide DNA"Nucleic Acids Research. Suppl.No.2. 271-272 (2002)
• [Publications] Yoshiaki Hori et al.: "Porphyrins and porphines inhibit the ribonuclease P reaction in vitro"Nucleic Acids Research. Suppl.No.2. 111-112 (2002)
• [Publications] Tomoaki Ando et al.: "Substrate shape specificity of E.coli RNase P ribozyme is dependent on the concentration of magnesium ion"Journal of Biochemistry. 133(No.4)(in press). (2003)
• [Publications] Terumichi Tanaka et al.: "E.coli tRNAs are resistant to the hyperprocessing reaction by homologous E.coli ribonuclease P ribozyme"Bioscience, Biotechnology and Biochemistry. 67(No.5)(in press). (2003)
• [Publications] Terumichi Tanaka et al.: "Revisiting the substrate recognition of bacterial ribonuclease P ---in the view of the recognition of the base N73 in the substrate"Nucleic Acids Research. Suppl.No.3(in press). (2003)
• [Publications] 田中照通, 菊池洋: "「生命の科学」53巻2号・特集「RNA」「RNA酵素に生命の謎解きができるか?」"(財)金原一郎記念医学医療振興財団. 6 (2002)