1991 Fiscal Year Annual Research Report

蛋白質/DNA複合体の構造

Research Project

Project/Area Number	03259102
Research Institution	Osaka University
Principal Investigator	箱嶋敏雄大阪大学, 薬学部, 助手 (00164773)
Co-Investigator(Kenkyū-buntansha)	広瀬進国立遺伝学研究所, 助教授 (90022730) 月原冨武徳島大学, 工学部, 教授 (00032277) 田中勲北海道大学, 理学部, 助教授 (70093052) 白木原康雄兵庫教育大学, 自然系物理, 助教授 (20150287) 郷通子名古屋大学, 理学部, 教授 (70037290)
Keywords	蛋白質構造 / DNA構造 / 分子認識 / 三次元構造 / 高次構造 / 転写制御 / 塩基配列特異性 / DNA結合蛋白質
Research Abstract	本重点領域研究の他の班員を含む分子生物学者との緊密な連携の下に、延べ12種類のDNA結合蛋白質の大量調製し結晶化を試みることができた。また、30種類以上のDNAオリゴマ-を大量合成しDNAと複合体結晶の可能性も検討した。これらの蛋白質には、大腸菌の単鎖DNA結合蛋白質SSB、ヌクレオイド系の蛋白質であるHーNS、転写因子PhoB、CamR及びラットDNAポリメラ-ゼβがあり、DNAとの複合体としては分裂酵母のAPー1様転写因子であるpap1、出芽酵母のヘリックス・ル-プ・ヘリックスモチ-フをもつといわれる転写因子であるPHO4、DNAポリメラ-ゼβ、マウスのがん原遺伝子産物Myb及びヌクレオイド蛋白質であるHUの結晶化が成功している。得られた結晶中には、本格的な結晶構造解析に着手可能な良質な結晶(SSB、ラットDNAポリメラ-ゼβ)も見いだされ始めており、再現性の改良とともに、重原子置換誘導結晶の調製を開始した。エンドヌクレア-ゼVについては三次元分子構造を決定し、活性部位の同定および逆転写酵素との機能構造上の類似性について明らかにできた。また、既に構造解析に成功しているエンドヌクレア-ゼVについては基質アナログとの複合体の結晶化が進行している。DNA超構造形成因子としては超らせん因子のクロ-ニングに成功し、TFIID/プロモ-タ複合体形成における生化学的解析の基礎をつくれた。また、RecAの会合に必須な部位も同定され、機能との関係を解析する土台ができた。また、DNA結合モチ-フとしてのヘリックス・タ-ン・ヘリックス構造が特殊な構造ではないことを蛋白質構造のデ-タベ-スの検索およびモジュ-ルの概念を導入した解析より証明できた。

Research Products

(22 results)

All Other

All Publications (22 results)

[Publications] T.Hakoshima: "Three-dimensional structure of a mutant ribonuclease T_1 (Y45W) complexed with non-cognizable ribonucleotide,2'AMP,and its comparison with specific complex with 2'GMP." J.Mol.Biol.223. 1013-1028 (1992)
[Publications] T.Hakoshima: "Hydrophobic effects on protein/nucleic acid interaction: Enhancement of substrate binding by mutating tyrosine 45 to tryptophan in ribonuclease T_1." Protein Eng.4. 793-799 (1991)
[Publications] T.Hakoshima: "Non-cognizable ribonucleotide,2'AMP,binds to a mutant ribonuclease T_1 (Y45W) at a new base-binding site but not at the guanine-recognition site." FEBS Lett.290. 216-220 (1991)
[Publications] T.Itoh: "Conformation of 2'GMP bound to a mutant ribonuclease T_1 (Y45W) determined by X-ray and NMR studies." J.Biochem.(Tokyo). 110. 677-680 (1991)
[Publications] M.Satoh: "Molecular cloning,nucleotide sequence and expression of elongation factor Tu from Thermus thermophilus HB8." FEBS Lett.288. 98-100 (1991)
[Publications] H.Ohishi: "Interaction between the left-handed Z-DNA and polyamine: The crystal structure of the d(CG)_3 and N-(2-aminoethyl)-1,4-diaminobutane complex." FEBS Lett.284. 238-244 (1991)
[Publications] 箱嶋敏雄: "蛋白質の結晶構造と溶液構造" 日本の科学と技術. 32. 54-69 (1991)
[Publications] T.Horii: "Inhibitory effects on N- and C-terminal truncated E.coli recA gene products on functions of wild-type recA gene." J.Mol.Biol.223. 105-114 (1992)
[Publications] T.Horii: "Mapping of functional domains by truncated RecA proteins" J.Mol.Biol.,in press.
[Publications] S.Tateishi: "C-terminal truncated E.coli RecA protein,RecA_<5327>,has enhanced DNA binding affinities to single- and double- stranded DNAs: Reguratory role of C-terminal region in DNA binding." J.Mol.Biol.223. 115-129 (1992)
[Publications] T.Shirai: "RNase-like domain in DNA-directed RNA polymerase II." Proc.Natl.Acad.Sci.USA.88. 9056-9060 (1991)
[Publications] N.Watanabe (eds.T.Fukui,H.Kagamiyama,K.Soda & H.Wada,Pergamon Press): "Crystal structure of ω-amino acid:pyruvate aminotransferase." in Enzymes Dependent on Pyridoxal Phosphate and Other Carbonyl Compounds as Cofactors. 121-124 (1991)
[Publications] N.Sakabe: "X-Ray diffraction data collection system for modern protein crystallography with a Weissenberg camera and imaging plate using synchrotron radiation." Nucle.Instr.& Meth.A303. 448-463 (1991)
[Publications] H.Mizuno: "Crystallization and preliminary X-ray study of a double-shelled spherical virus,rice dwarf virus." J.Mol.Biol.219. 665-669 (1991)
[Publications] K.Ura: "Possible role of DNA topoisomerase II on transcription of the homeobox gene Hox-2.1 in F9 embryonal carcinoma cells." Nucle.Acids Res.19. 6087-6092 (1991)
[Publications] M.Mizutani: "DNA superhelicity affects the formation of transcription preinitiation complex on eukaryotic genes differently." Nucle.Acids Res.19. 2907-2911 (1991)
[Publications] H.Ueda: "Defining the sequence recognized with BmFTZ-F1,a sequence specific DNA binding factor in the silkworm,Bombyx mori,as revealed by direct sequencing of bound oligonucleotides and gel mobility shift competition analysis." Nucle.Acids Res.19. 3689-3693 (1991)
[Publications] M.Mizutani: "Negative supercoling of DNA facilitates an interaction between transcription factor IID and the fibroin gene promotor." Proc.Natl.Acad.Sci.USA. 88. 718-722 (1991)
[Publications] K.Terada: "Demonstration of extrinsic DNA from immune complexes in plasma of a patient with systemic lupus erythematosus." Biochem.Biophys.REs.Commun.174. 323-330 (1991)
[Publications] K.Okada: "Negative supercoling of DNA by eukaryotic DNA topoisomerase II and dextran sulfate." J.Biochem (Tokyo).109. 365-369 (1991)
[Publications] 箱嶋敏雄: "新生化学実験講座第2巻"核酸II構造と性質" (日本生化学会編)、第二章 X線回折" 東京化学同人, 36 (1991)
[Publications] 小川智子: "新生化学実験講座核酸の化学I (生化学会編) 密度勾配法" 東京化学同人, (1991)

1991 Fiscal Year Annual Research Report

蛋白質/DNA複合体の構造

Principal Investigator

箱嶋 敏雄 大阪大学, 薬学部, 助手 (00164773)

Research Products

[Publications] T.Hakoshima: "Three-dimensional structure of a mutant ribonuclease T_1 (Y45W) complexed with non-cognizable ribonucleotide,2'AMP,and its comparison with specific complex with 2'GMP." J.Mol.Biol.223. 1013-1028 (1992)

[Publications] T.Hakoshima: "Hydrophobic effects on protein/nucleic acid interaction: Enhancement of substrate binding by mutating tyrosine 45 to tryptophan in ribonuclease T_1." Protein Eng.4. 793-799 (1991)

[Publications] T.Hakoshima: "Non-cognizable ribonucleotide,2'AMP,binds to a mutant ribonuclease T_1 (Y45W) at a new base-binding site but not at the guanine-recognition site." FEBS Lett.290. 216-220 (1991)

[Publications] T.Itoh: "Conformation of 2'GMP bound to a mutant ribonuclease T_1 (Y45W) determined by X-ray and NMR studies." J.Biochem.(Tokyo). 110. 677-680 (1991)

[Publications] M.Satoh: "Molecular cloning,nucleotide sequence and expression of elongation factor Tu from Thermus thermophilus HB8." FEBS Lett.288. 98-100 (1991)

[Publications] H.Ohishi: "Interaction between the left-handed Z-DNA and polyamine: The crystal structure of the d(CG)_3 and N-(2-aminoethyl)-1,4-diaminobutane complex." FEBS Lett.284. 238-244 (1991)

[Publications] 箱嶋 敏雄: "蛋白質の結晶構造と溶液構造" 日本の科学と技術. 32. 54-69 (1991)

[Publications] T.Horii: "Inhibitory effects on N- and C-terminal truncated E.coli recA gene products on functions of wild-type recA gene." J.Mol.Biol.223. 105-114 (1992)

[Publications] T.Horii: "Mapping of functional domains by truncated RecA proteins" J.Mol.Biol.,in press.

[Publications] S.Tateishi: "C-terminal truncated E.coli RecA protein,RecA_<5327>,has enhanced DNA binding affinities to single- and double- stranded DNAs: Reguratory role of C-terminal region in DNA binding." J.Mol.Biol.223. 115-129 (1992)

[Publications] T.Shirai: "RNase-like domain in DNA-directed RNA polymerase II." Proc.Natl.Acad.Sci.USA.88. 9056-9060 (1991)

[Publications] N.Watanabe (eds.T.Fukui,H.Kagamiyama,K.Soda & H.Wada,Pergamon Press): "Crystal structure of ω-amino acid:pyruvate aminotransferase." in Enzymes Dependent on Pyridoxal Phosphate and Other Carbonyl Compounds as Cofactors. 121-124 (1991)

[Publications] N.Sakabe: "X-Ray diffraction data collection system for modern protein crystallography with a Weissenberg camera and imaging plate using synchrotron radiation." Nucle.Instr.& Meth.A303. 448-463 (1991)

[Publications] H.Mizuno: "Crystallization and preliminary X-ray study of a double-shelled spherical virus,rice dwarf virus." J.Mol.Biol.219. 665-669 (1991)

[Publications] K.Ura: "Possible role of DNA topoisomerase II on transcription of the homeobox gene Hox-2.1 in F9 embryonal carcinoma cells." Nucle.Acids Res.19. 6087-6092 (1991)

[Publications] M.Mizutani: "DNA superhelicity affects the formation of transcription preinitiation complex on eukaryotic genes differently." Nucle.Acids Res.19. 2907-2911 (1991)

[Publications] H.Ueda: "Defining the sequence recognized with BmFTZ-F1,a sequence specific DNA binding factor in the silkworm,Bombyx mori,as revealed by direct sequencing of bound oligonucleotides and gel mobility shift competition analysis." Nucle.Acids Res.19. 3689-3693 (1991)

[Publications] M.Mizutani: "Negative supercoling of DNA facilitates an interaction between transcription factor IID and the fibroin gene promotor." Proc.Natl.Acad.Sci.USA. 88. 718-722 (1991)

[Publications] K.Terada: "Demonstration of extrinsic DNA from immune complexes in plasma of a patient with systemic lupus erythematosus." Biochem.Biophys.REs.Commun.174. 323-330 (1991)

[Publications] K.Okada: "Negative supercoling of DNA by eukaryotic DNA topoisomerase II and dextran sulfate." J.Biochem (Tokyo).109. 365-369 (1991)

[Publications] 箱嶋 敏雄: "新生化学実験講座 第2巻"核酸II構造と性質" (日本生化学会編)、第二章 X線回折" 東京化学同人, 36 (1991)

[Publications] 小川 智子: "新生化学実験講座 核酸の化学I (生化学会編) 密度勾配法" 東京化学同人, (1991)

箱嶋敏雄大阪大学, 薬学部, 助手 (00164773)

[Publications] 箱嶋敏雄: "蛋白質の結晶構造と溶液構造" 日本の科学と技術. 32. 54-69 (1991)

[Publications] 箱嶋敏雄: "新生化学実験講座第2巻"核酸II構造と性質" (日本生化学会編)、第二章 X線回折" 東京化学同人, 36 (1991)

[Publications] 小川智子: "新生化学実験講座核酸の化学I (生化学会編) 密度勾配法" 東京化学同人, (1991)