生体金属による遺伝子発現制御

• 研究課題/領域番号: 08249103
• 研究種目: 特定領域研究(A)
• 配分区分: 補助金
• 研究機関: 広島大学
• 研究代表者: 木村 栄一 (木村 榮一) 広島大学, 医学部, 教授 (30034010)
• 研究分担者: 石浜 明 国立遺伝学研究所, 分子遺伝研究系, 教授 (80019869) ; 杉浦 幸雄 京都大学, 化学研究所, 教授 (40025698) ; 塩谷 光彦 岡崎国立共同研究機構, 分子科学研究所, 教授 (60187333) ; 青木 伸 広島大学, 医学部, 助手 (00222472)
• 研究期間 (年度): 1999
• 研究課題ステータス: 完了 (1999年度)
• 配分額: 121,100千円 (直接経費: 121,100千円); 1999年度: 18,700千円 (直接経費: 18,700千円); 1998年度: 32,300千円 (直接経費: 32,300千円); 1997年度: 44,300千円 (直接経費: 44,300千円); 1996年度: 25,800千円 (直接経費: 25,800千円)
• キーワード: 大環状ポリアミン / DNaseI / 亜鉛錯体 / Znフィンガー / 塩基配列認識 / プロモーターDNA / TATAボックス / RNAポリメラーゼ / 人工β-cヌクレオチド / 亜鉛フィンガー / 生体金属 / 遺伝子発現制御 / Zn-フィンガー / 亜鉛 / 核酸塩基 / 転写因子 / 抗HIV活性 / 遺伝子プロモーター領域 / DNA融解温度

研究概要

1.核酸-合成金属錯体相互作用の研究:
12員環テトラアミン(cyclen)の亜鉛錯体が、核酸塩基中イミド基をもつチミン(又はウラシル)だけを選択的に認識しComplexをつくることを発見したが、それを大きく展開し、DNA(例:大腸菌プラスミドDNA(150bp))中のTと選択的に結合することをDNaseI及びmicrococcal nuclease footprint法により証明した。更に、Zn^<2+>-cyclen錯体は、DNA TATAboxに選択的に結合し、転写因子TFIIDの活性化機構を抑え、今までに例のない全く新しいタイプの遺伝子発現制御分子となることが明らかとなった。また、それらの化学的、生化学的機構の結果、抗菌活性を示すことも発見した。(木村栄一)
2.蛋白-核酸相互作用に関与する生体金属の研究:
転写因子Spl中のZnフィンガー(転写因子)Cys_2-His_2型亜鉛配位構造motifを3倍に拡大した新たな9つのZn-フィンガーからなるペプチドを合成し、DNAの30塩基対配列を選択的に認識する分子を創造した。また、二つのSplDNA結合ドメインを適当な長さのグリシンポリペプチドでlinkした分子を合成し、それらがDNAをおれ曲げることを発見、新たな遺伝子発現制御小工分子となりうることを示した。(杉浦幸雄)
(Fe^<2+>-EDTA)を大腸菌RNAポリメラーゼのCysやLysに共有結合させる方法を考案し、接触相手の転写因子蛋白やDNAとの接点部固定した。RNAポリメラーゼのシグマサブユニットのC端ドメインがDNAエンハンサー部と接触すること、サブユニット間の接点、シグマサブユニットのプロモーターDNAとの接点、多数の転写因子のポリメラーゼ上の接点等も明らかとなった。FeのO_2との反応を利用した分子間相互作用を解析する本法は、転写研究に新たなbreakthroughをもたらしたのみならず、他成分分子集合体や、多分子相互作用が主要な研究対象となって来た現代生命科学で、大変有用なtoolとなろう。(石浜明)

研究成果

• [文献書誌] E.Kikuta et al.: "Novel Recognition of Thymine Base in Double-Stranded DNA by Zinc(II)-Macrocyclic Tetraamine Complexes Appended with Aromatic Groups"J.Am.Chem.Soc.. 121(23). 5426-5436 (1999)
• [文献書誌] H.Kurosaki et al.: "New Robust Bleomycin Analogues : Synthesis, Spectroscopy, and Cristal Structures of the Copper(II) Complexes"Inorg.Chem.. 38. 2824-2832 (1999)
• [文献書誌] E.Kikuta et al.: "Natural and synthetic double-stranded DNA binding studies of macrocyclictetraamine Zinc(II) complexes appended with polyaromatic groups"J.Biol.Inorg.Chem.. 4. 431-440 (1999)
• [文献書誌] E.Kimura et al.: "Selective and Efficient Recognition of Thymidylylthymidine (TpT) by Bis(Zn^<2+>-cyclen) and Thymidylythymidylythymidine(TpTpT) by Tris(Zn^<2+>-cyclen) at Neutral pH in Aqueous Solution"Chem.Eur.J.. 5(11). 3113-3123 (1999)
• [文献書誌] H.Kurosaki et al.: "Synthesis, spectroscopic, redox properties, and DNA cleavage activity of low-spin iron(III) complexes of bleomycin analogues"Inorg.Chimica.Acta.. 294. 56-61 (1999)
• [文献書誌] S.Aoki et al.: "Three-Dimensional Supermolecules Assembled from a Tris(Zn^<2+>-cyclen)Complex and Di- and Trianionic Cyanuric Acid in Aqueous Solution (Cyclen=1, 4, 7, 10-Tetraazacyclododecane)"J.Am.Chem.Soc.. 122(4). 576-584 (2000)
• [文献書誌] Y.Sugiura et al.: "Comparison of the Sequence-Selective DNA Binding by Peptide Dimers with Covalent and Noncovalent Dimerization Domains"Biochemistry. 38. 1626-1632 (1999)
• [文献書誌] Y.Sugiura et al.: "Fluorescence Property of Oxazole Yellow-linked Oligonucleotide : Triple Helix Formation and Photocleavage of Double-standed DNA in the Presence of Spermine"Bioog.Med.Chem.. 7. 1207-1211 (1999)
• [文献書誌] Y.Sugiura et al.: "Fluorescence Property of Oxazole Yellow-linked Oligonucleotide : Triple Helix Formation and Photocleavage of Double-standed DNA in the Presence of Spermine"Bioog.Med.Chem.. 7. 1207-1211 (1999)
• [文献書誌] A.Ishihama et al.: "Organisation of open complexes at Escherichia coli promoters : mapping the promoter DNA sites close to region 2.5 of the σ^<70> subunit of RNA polymerase"J.Biol.Chem.. 274. 2263-2270 (1999)
• [文献書誌] 木村栄一: "The First Selective and Efficient Transport of Imide-Containing Nucleosides and Nucleotides by Lipophilic Cyclen-Zinc (II) Complexes." J.Am.Chem.Soc.120. 10018-10026 (1998)
• [文献書誌] 木村栄一: "Efficient Inhibition of Photo [2+2] cycloaddition of Thymidilyl (3'-5') Thymidine and Promotion of Photosplitting of the cis-syn-Cyclobutane Thymine Dimer by Dimeric Zinc (II) -Cyclen Complexes Containing m-and p-Xylyl Spacers." J.Am.Chem.Soc.120. 10094-10102 (1998)
• [文献書誌] 木村栄一: "Dynamic Anion Recognition by Macrocyclic Polyamines in Neutral pH Aqueous Solution : Development from Static Anion Complexes to an Enolate Complex." J.Chem.Soc.Chem.Comm.1495-1500 (1998)
• [文献書誌] 杉浦幸雄: "Artificial Nine Zinc-Finger Peptide with 30 Base pair Binding Sites" Biochenistry. 37. 13827-13834 (1998)
• [文献書誌] 塩谷光彦: "Macrocyclic Zinc (II) Complexes for Selective Recognition of Nucleobases in Single- and Double-Stranded Polynucleotides." J.Bioimorg.Chem.3. 259-267 (1998)
• [文献書誌] 石浜明: "Organisation of open complexes at Escherichia coli promoters : mapping the promoter DNA sites close to region 2.5 of the σ^<70> subunit of RNA polymerase." J.Biol.Chem.274. 2263-2270 (1999)
• [文献書誌] Eiichi Kimura: "Structure and O_2 uptake preperties of a novel nickel (II) wmplex of pyridyl-pendant dioxocyclam [1-(2-pyridyl) methyl-5,7-dioxo-1,4,8,11-tetraazacyclo tetradecane" J.Biol.Inorg.Chem.2・1. 74-82 (1997)
• [文献書誌] Eiichi Kimura: "A tris(Zn^<II>-1,4,7,10-tetraazacyclodade cannel complex as a new receptor for phosphate dianions in aqueous solution" J.Am.Chem.Soc.119・13. 3068-3076 (1997)
• [文献書誌] Xian He Bu: "Crystal structure and properties of a new copper^<(II)> wmplex of dioxocyclam appended with 8-methyl quinolire (Dioxocyclam = 1,4,8,11-tetraazacyclotetradecane-5,7-dione)" J.Incul.Phenom.Mol.Recog.Chem. 27.3. 245-258 (1997)
• [文献書誌] Eiichi Kimura: "Fcile and selective electrostatic stabilization of urocil N (1) -anion by a proximale protonated amine : An implication for why urocil N (1) is chosen by glycosylazien site" J.Am.Chem.Soc.119・45. 10909-10919 (1997)
• [文献書誌] Takashi Morii: "Factors govering the sequence-selective DNA binding of geometrically constrained peptide dimers" J.Am.Chem.Soc. 119・16. 3649-3655 (1997)
• [文献書誌] Yukio Sugiura: "Selective cleavage of tRNAphe with secondary and tertiary structures by enedyne antitumor antibiotics" Bioorg.Med.Chem. 5・6. 1229-1234 (1997)
• [文献書誌] Eiichi Kimura: "Metal Sites in Proteins and Models Phosphatases,Lewis Acids and Vanadium" Springer-Verlag, 28 (1997)
• [文献書誌] H. Fujioka et. al.: "A new Bis (Zinc (II) -Cyclen) Complex as a Novel Chelator for Barbiturates and Phosphates" Heterocycles. 42 (2). 775-787 (1996)
• [文献書誌] E. Kimura et. al: "Zinc Complexes as Targeting Agents for Nucleic Acids" "Metal Ions in Biological Systems" ed. by A. Sigel and H. Sigel. Marcel Dekker. Inc.33. 29-52 (1996)
• [文献書誌] Y. Inouye et. al: "Differential Contribution of Metal Complexation and Dimerization to the Chemotherapeutic Potential of Bicyclen-Zn2^<II> Complex against Human Immunodeficiency Virus" Biol. Pharm. Bull.19 (3). 456-458 (1996)
• [文献書誌] T. Koike et. al: "Novel Properties of Cooperative Dinuclear Zinc (II) Ions : The Selective Recognition of Phosphomonoesters and Their P-O Ester Bond Cleavage by a New Dinuclear Zinc (II) Cryptate" J. Am. Chem. Soc. 118. 3091-3099 (1996)
• [文献書誌] T. Koike et. al: "Bis (Zn^<II>-Cyclen) Complex as a Novel Receptor of Barbiturates in Aqueous Solution." Chem. Europ. J. 2. 617-623 (1996)
• [文献書誌] X. H. Bu et.al: "Stabilization of Nickel (III) ion by New Macrocyclic Diozotetraamines Bearing Functional Pendant Donors" Polyhedron. 15. 161-163 (1996)
• [文献書誌] Y. Kodama et. al: "A New Cyclam with a Triphenylphosphine-pendant and its Metal Complexes" Inorg. Chim. Acta.246. 151-158 (1996)
• [文献書誌] T. Koike et. al: "Metalloenzyme Models that Answer Mysteries Surrounding the Intrinsic Properties of Zn" Comprehensive Supramoleculaar Chemistry' 10, Pergamon. 429-444 (1996)
• [文献書誌] E. Kimura: "Creation of new Supramoleculaar Science with Macrocyclic Polyamines" (1996 Pharmaceutical Society of Japan Award Review) Yakugaku Zasshi. 116. 587-605 (1996)
• [文献書誌] H. Hashimoto et. al: "Hydrolysis of Lipophilic Esters Catalyzed by a Zinc (II) Complex of a Long Alkyl-Pendant Macrocyclic Teraamine in Micellar Solution" J. Am. Chem. Soc.118. 10963-10970 (1996)
• [文献書誌] X. H. Bu et. al: "Synthesis, Crystal Structure and Properties of the Copper (II) Complex of a New Dioxocyclam Appended with Two 8-Methylquinolines as Additional Donor Pendants" Inorg. Chim. Acta.249. 125-130 (1996)
• [文献書誌] T. Koike et. al: "Intrinsic Properties of Zinc (II) Ion Pertinent to Zinc Enzymes" Advance in Inorganic Chemistry', Ed. by A. G. Sykes, Academic Press,. 44. 229-261 (1996)
• [文献書誌] T. Watanabe: "A Novel Biomimetic Zinc (II)-Fluorophore, Dansylamin oethyl-Pendant Macrocyclic Tetraamine 1, 4, 7, 10-Tetraazacyclododecane (Cyclen)" J. Am. Chem. Soc.118. 12696-12730 (1996)
• [文献書誌] S. Aoki et. al: "A This (Zinc (II) -1. 4. 7. 10-Cecraazacyclododecane) Complex as a New Receptor for Phosphate Dianions in Aqueous Solution" J. Am. Chem. Soc.119(in press). (1997)
• [文献書誌] M. Shionoya et. al: "Zinc Complexes as Targeting Agents for Nucleic Acids" "Metal Ions in Biological Systems" ed. by A. Sigel and H. Sigel, Marcel Dekker, Inc,. 33. 29-52 (1996)
• [文献書誌] M. Shionoya et. al: "Macrocyclic Metal Complexes for Selective Recognition of Nucleic Acid Bases and Manipulation of Gene Expression." Pure & Appl. chem.(in press).
• [文献書誌] Y. Sugiura et. al: "Distinct Phosphate Backbone Contacts revealed by Some Mutant Peptides of Zinc Finger Protein Sp1 : Effect of Protein-Induced Bending on DNA Recognition" Biochemistry. 35. 8761-8768 (1996)