1997 年度実績報告書

RNA触媒機能の分子基盤

研究課題

研究課題/領域番号	09278103
研究種目	重点領域研究
研究機関	京都大学
研究代表者	井上丹京都大学, 大学院・理学研究科, 教授 (40114855)
研究分担者	新田至東京大学, 工学系研究科, 助手 (30272404) 白石英秋京都大学, 大学院・理学研究科, 講師 (90202118) 谷時雄九州大学, 理学部, 助教授 (80197516) 多比良和誠筑波大学, 応用生物化学系, 教授 (10261778) 小松康雄北海道大学, 薬学部, 助手 (30271670)
キーワード	RNA / 酵素 / リボザイム / 分子設計 / 反応機構 / スプライシング / catalyticRNA / リボソーム
研究概要	・触媒活性をもつRNAの反応の分子機構の解析を,グループIイントロンRNAを材料として行い,イントロンRNAの機能構造単位の一つであるP2.1がtrans-activatorとして高効率でかつ新規なメカニズムで作用することを明らかにした.in vitro/in vivo selection法により新たにリボザイムを構築する計画を実施し、既知のRNAの高次構造を基盤としてリボザイムを作成するシステムの構築を試み,このシステムの作成に成功した.(井上) ・天然型のヘアピン型リボザイムより,3つのドメイン構造をもつ新規ヘアピン型リボザイムの構築に成功した.天然型よりも高い切断活性を示すヘアピン型リボザイムを構築した.(小松) ・ハンマーヘッド型リボザイムの反応機構として、一般的には"single-metal-ion"メカニズムが受け入れられているが、実際には"double-metal-ion"メカニズムであることを証明した。(多比良) ・分裂酵母mRNAスプライシング変異株の解析から、U4/U6snRNPの構成因子と考えられるPrp1が、G0/G1期変換に関与する因子と同一であることを明らかにした。(谷) ・大腸菌RNase Pは、活性中心をもつM1 RNAとC5タンパク質から成る複合体酵素である。C5タンパク質の機能を架橋実験によって解析し、これがM1 RNAとtRNAの位置関係を変化させる働きを持っていることを明らかにした。(白石) ・試験管内転写により調製された大腸菌由来の23SリボソームRNAを用いることにり、リボソームタンパク質を一切必要とすることなく、ペプチド結合生成反応を進行させることに成功し、リボソームRNA上に活性中心が存在することを証明した。(新田) ・新規のグアニン塩基結合RNAアプタマ-の単離に成功した.(坂本)

研究成果

(19件)

すべてその他

すべて文献書誌 (19件)

[文献書誌] Ikawa,Y.: "Long-range interaction between the P2.1 and P9.1 peripheral domains of the Tetrahymena ribozyme." Nucleic Acids Research. 25. 1761-1765 (1997)
[文献書誌] Ikawa,Y.: "Idertification of the nucleotides in the A-rich bulge of the Tetrahymena ribozyme responsible for an efficient self-splicing." J.Biochem. 122. 878-882 (1997)
[文献書誌] Ikawa,Y.: "Trans-activation of the Tetrahymena ribozyme by its P2-2.1 domains" J.Biochem. 123. 528-533 (1998)
[文献書誌] Y.Naito: "P5abc of the Tetrahymena ribozyme consists of three functionally independent elements" RNA. (in press).
[文献書誌] Komatsu,Y.: "A new type of hairpin ribozyme consisting of three domains." Biochemistry. 36. 9935-9940 (1997)
[文献書誌] Komatsu,Y.: "Construction of hairpin ribozymes with a three-way junction." Bioorg.Med.Chem.5. 11063-11069 (1997)
[文献書誌] Urushiyama,S.: "The prp1+gene required for pre-mRNA splicing in Schizosaccharomyces pombe encodes a protein that contains TPR motifs and is similar to Prp6p of budding yeast." Genetics. 147. 101-115 (1997)
[文献書誌] J-H.Park: "Structure and expression of the gsa-1 gene encoding a G protein a (s) subunit in C. elegans" Gene. 194. 183-190 (1997)
[文献書誌] A.K.Azed: "Isolation and molecular characterization of mRNA transport mutants in Schizosaccharomyces pombe" Mol.Biol.Cell. 8. 825-841 (1997)
[文献書誌] Nitta,I.: "Possible involvement of Escherichia coli 23S ribosomal RNA in peptide bond formation." RNA. 4・3. 257-267 (1998)
[文献書誌] Nitta,I.: "A novel cell-free system for peptide synthesis driven by pyridine." Bio.Chem.(in press).
[文献書誌] D.Kiga,: "An RNA aptamer to the xanthine/guanine base with a distinctive mode of purine recognition" Nucleic Acids Research. (in press).
[文献書誌] Koizumi,M.: "Design and anti-HIV-1 activity of hammerhead and hairpin ribozymes containing a stable loop." Nucleosides & Nucleotides. (in press).
[文献書誌] Warashina,M.: "Entropically driven enhancement of cleavage activity of aDNA-armed hammerhead ribozyme : mechanism of action of hammerhead ribozymes." J.Org.Chem.62. 9138-9147 (1997)
[文献書誌] Fujita,S.: "Discrimination of a single base change in a ribozyme using the gene for dihydrofolate reductase as a selective marker in Escherichia coli." Proc.Natl.Acad.Sci.USA. 94. 391-396 (1997)
[文献書誌] Zhou,D.-M.: "Explanation by thedouble-metal-ion mechanism of catalysis for the differential metal ion effects on the cleavage rates of 5'-oxy and 5'-thio substrates by a hammerhead ribozyme." Proc.Natl.Acad.Sci.USA. 94. 14343-14348 (1997)
[文献書誌] Kuwabara,T.: "Comparison of the specificities and catalytic activities of hammerhead ribozymes and DNA enzymes with respect to the cleavage of BCR-ABL chimeric L6 (b2a2) mRNA." Nucleic Acids Res.25. 3074-3082 (1997)
[文献書誌] Imamura,O.: "Cloning and characterization of a human DEAH-box RNA helicase,a functional homolog of fission yeast Cdc28/Prp8." Nucleic Acids Res.(in press).
[文献書誌] 新田至, 上田卓也, 渡辺公綱: "リボソームとRNAワールド" 化学と生物 36(4), 92 (1998)

1997 年度 実績報告書

RNA触媒機能の分子基盤

研究代表者

井上 丹 京都大学, 大学院・理学研究科, 教授 (40114855)

研究成果

[文献書誌] Ikawa,Y.: "Long-range interaction between the P2.1 and P9.1 peripheral domains of the Tetrahymena ribozyme." Nucleic Acids Research. 25. 1761-1765 (1997)

[文献書誌] Ikawa,Y.: "Idertification of the nucleotides in the A-rich bulge of the Tetrahymena ribozyme responsible for an efficient self-splicing." J.Biochem. 122. 878-882 (1997)

[文献書誌] Ikawa,Y.: "Trans-activation of the Tetrahymena ribozyme by its P2-2.1 domains" J.Biochem. 123. 528-533 (1998)

[文献書誌] Y.Naito: "P5abc of the Tetrahymena ribozyme consists of three functionally independent elements" RNA. (in press).

[文献書誌] Komatsu,Y.: "A new type of hairpin ribozyme consisting of three domains." Biochemistry. 36. 9935-9940 (1997)

[文献書誌] Komatsu,Y.: "Construction of hairpin ribozymes with a three-way junction." Bioorg.Med.Chem.5. 11063-11069 (1997)

[文献書誌] Urushiyama,S.: "The prp1+gene required for pre-mRNA splicing in Schizosaccharomyces pombe encodes a protein that contains TPR motifs and is similar to Prp6p of budding yeast." Genetics. 147. 101-115 (1997)

[文献書誌] J-H.Park: "Structure and expression of the gsa-1 gene encoding a G protein a (s) subunit in C. elegans" Gene. 194. 183-190 (1997)

[文献書誌] A.K.Azed: "Isolation and molecular characterization of mRNA transport mutants in Schizosaccharomyces pombe" Mol.Biol.Cell. 8. 825-841 (1997)

[文献書誌] Nitta,I.: "Possible involvement of Escherichia coli 23S ribosomal RNA in peptide bond formation." RNA. 4・3. 257-267 (1998)

[文献書誌] Nitta,I.: "A novel cell-free system for peptide synthesis driven by pyridine." Bio.Chem.(in press).

[文献書誌] D.Kiga,: "An RNA aptamer to the xanthine/guanine base with a distinctive mode of purine recognition" Nucleic Acids Research. (in press).

[文献書誌] Koizumi,M.: "Design and anti-HIV-1 activity of hammerhead and hairpin ribozymes containing a stable loop." Nucleosides & Nucleotides. (in press).

[文献書誌] Warashina,M.: "Entropically driven enhancement of cleavage activity of aDNA-armed hammerhead ribozyme : mechanism of action of hammerhead ribozymes." J.Org.Chem.62. 9138-9147 (1997)

[文献書誌] Fujita,S.: "Discrimination of a single base change in a ribozyme using the gene for dihydrofolate reductase as a selective marker in Escherichia coli." Proc.Natl.Acad.Sci.USA. 94. 391-396 (1997)

[文献書誌] Zhou,D.-M.: "Explanation by thedouble-metal-ion mechanism of catalysis for the differential metal ion effects on the cleavage rates of 5'-oxy and 5'-thio substrates by a hammerhead ribozyme." Proc.Natl.Acad.Sci.USA. 94. 14343-14348 (1997)

[文献書誌] Kuwabara,T.: "Comparison of the specificities and catalytic activities of hammerhead ribozymes and DNA enzymes with respect to the cleavage of BCR-ABL chimeric L6 (b2a2) mRNA." Nucleic Acids Res.25. 3074-3082 (1997)

[文献書誌] Imamura,O.: "Cloning and characterization of a human DEAH-box RNA helicase,a functional homolog of fission yeast Cdc28/Prp8." Nucleic Acids Res.(in press).

[文献書誌] 新田至, 上田卓也, 渡辺公綱: "リボソームとRNAワールド" 化学と生物 36(4), 92 (1998)

1997 年度実績報告書

井上丹京都大学, 大学院・理学研究科, 教授 (40114855)