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New Fluorescent Techniques Applied to σ Functions.

Research Project

Project/Area Number 09044238
Research Category

Grant-in-Aid for Scientific Research (A).

Allocation TypeSingle-year Grants
Section一般
Research Field Biophysics
Research InstitutionNational Institute of Genetic

Principal Investigator

SHIMAMOTO Nobuo  National Institute of Genetics, Structural Biology Center, Professor, 構造遺伝学研究センター, 教授 (20127658)

Co-Investigator(Kenkyū-buntansha) SOGAWA Kumiko  National Institute of Genetics, Structural Biology Center, Assistant, 構造遺伝学研究センター, 助手 (20291073)
NAGAI Hiroki  National Institute of Genetics, Structural Biology Center, Assistant, 構造遺伝学研究センター, 助手 (80222173)
HAYWARD Rich  英国エジンバラ大学細胞分子生物学研究所, 教授
CHATTERJI Di  インド細胞分子生物学研究センター, 教授
Project Period (FY) 1997 – 1999
Project Status Completed (Fiscal Year 1999)
Budget Amount *help
¥12,800,000 (Direct Cost: ¥12,800,000)
Fiscal Year 1999: ¥4,100,000 (Direct Cost: ¥4,100,000)
Fiscal Year 1998: ¥4,100,000 (Direct Cost: ¥4,100,000)
Fiscal Year 1997: ¥4,600,000 (Direct Cost: ¥4,600,000)
KeywordsRNA polymerase / major sigma factor / protein footprinting / omega subunit / Esherichia coli / Bacillus subtilis / transcription initiation / RNA polymerase / major sigma factor / omega subunit / Esherichia coli / transcription initiation / major sigma / protein footpronting / sigma subyunit / omega subyunit / GroE / trascription initiation
Research Abstract

All bacteria have major sigma factors, which enable RNA polymerase to recognize most of their promoters, and σィイD170ィエD1 is the major sigma factor of E. coli. The largest finding in this research is the aggregation of σィイD170ィエD1 in response to environment such as high physiological temperature and the end of log phase. The purified σィイD170ィエD1 also makes aggregates at similar temperature, while a mutant protein with deletion of aa 130-374 exists as a mixture of monomer and aggregates at all temperatures. The monomeric mutant protein is as active as the wild-type σィイD170ィエD1 in purified reconstituted transcription system, while oligomers have at most 15% of the activity. Thus the domain of aa 130-374 is not essential for transcription reaction but has essential role to prevent aggregation of σィイD170ィエD1. We have first succeeded to constructed a disruptant of rpoD (the gene of σィイD170ィエD1) and the mutant σィイD170ィエD1 and SigA, the major sigma of B. subtilis lacking aa 130-374, complement the disruption in limited conditions, proving that aa 130-374 is not required for minimal transcription in vivo.
By cleaving σィイD170ィエD1 with hydroxide radical, the exposed surfaces of σィイD170ィエD1 were identified in monomeric and aggregated forms. The regions protected upon aggregation sandwich the regions exposed upon aggregation, suggesting the existence of more than two interacting sites for aggregation.
We have determined the role of omega subunit of E. coli RNA polymerase. The core enzyme prepared from omega-depleted cells binds GroEL shaperonin, and the removal of GroEL destroys the ability of core enzyme to bind σィイD170ィエD1. This proves the role of omega is to maturate core enzyme, and explain why the depletion of omega does not bring any phenotype. This proves the role of omega is to maturate core enzyme, and explain why the depletion of omega does not bring any phenotype.

Report

(4 results)
  • 1999 Annual Research Report   Final Research Report Summary
  • 1998 Annual Research Report
  • 1997 Annual Research Report
  • Research Products

    (55 results)

All Other

All Publications (55 results)

  • [Publications] R. Sen, H. Nagai, N.Simamoto: "Polymerase-arrest at the λPR promoter during transcription initiation"J. Biol. Chem.. 275(in press). (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] T. Yamamoto 他4名: "Molecular surgery of DNA based on electrostatic micromanipulation"IEEE Trans. Ind. Appl.. (in press). (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] K. Mukherjee 他3名: "GroEL is involved in activation of Escherichia coil RNA polymerase devoid of polimerase devoid of omega subunit in vivo"Eur. J. Biochem.. 266. 15293-15296 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] N.Shimamoto: "On dimensional diffusion of protein along DNA : its biological and chemical significance revealed by single-molecule measurements"J. Biol. Chem.. 274. 15293-15296 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] 嶋本伸雄、杵渕隆: "DNA上のタンパク質の滑り運動:DNA結合蛋白質の遠攻近交戦略"科学(岩波). 69. 42-50 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] R. Sen 他3名: "Reduction in abortive transcription from the λPR promoter by mutations in region 3 of the σ70 subunit of E.coil RNA polimerace"J. Biol. Chem.. 273. 9872-9877 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] N.Shimamoto,他3名: "Efficient solubilization of proteins overproduced as inclusion bodies by use of an extreme concentration of glycerol"Tech. Tips Online.. t01576 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] 杵渕隆、嶋本伸雄: "DNA上におけるタンパク質のスライディング"科学と生物. 36. 278-280 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] 永井宏樹: "蛋白質のフットプリント法"生物物理. 217. 116-118 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] T. Kinebuchi,他4名: "Functional domain of Ensherichia coil Single-stranded DNA binding protein as assessed by analysis of the deletion mutants"Biochemistry. 36. 6732-6738 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] T. Kubori, N. Shimamoto: "Physical interference between EsherichiacoliRNA polymerase molecules transcribing in trademenhances abortive synthesis and misincorporation"Nuc. Acids Res.. 25. 2640-2647 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] H. Nagai, N. Shimamoto: "Most conserved regions of the E.coil primary sigma factor are involved in interaction with RNA polymerase core enzyme"Gene. Cells. 2. 725-734 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] 久堀智子、嶋本伸雄: "転写開始とその調節の新モデル"生物物理. 214. 249-253 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] 加畑博幸、嶋本伸雄: "DNA・蛋白質相互作用の可視化とスライディング運動"蛋白質・核酸・酵素. 増刊42. 1181-1186 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] 嶋本伸雄: "Bioscience新用語ライブラリー転写因子"大腸菌RNAポリメラーゼ. 227 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] 嶋本伸雄: "GreAGreB"GreAGreB. 227 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] 嶋本伸雄、郷通子編: "遺伝子の構造生物学(ニューバイオフィジックス)"共立出版(1998). 196 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] 嶋本伸雄: "Bioscience新用語ライブラリー転写因子"GreAGreB. 227 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] T.Yamamoto, O.Kurosawa, H.Kabata, N.Shimamoto, M.Washizu: "Molecular surgery of DNA based on electrostatic micromanipuration."IEEE Trans. On Indst. Appl.. (in press). (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] R.Sen, H.Nagai , N.Shimamoto: "Polymerase-arrest at the IPR promoter during transcription initiation."J. Biol. Chem.. 275(in press). (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] K.Mukherjee, H.Nagai, N.Shimamoto, D.Chatterji: "GroEL is involved in activation of Escherichia coli RNA polymerase devoid of omega subunit in vivo."Eur. J. Biochem.. 266. 228-235 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] N.Shimanoto: "One dimensional diffusion of proteins along DNA: its biological and chemical significance revealed by single-molecule measurements"J. Biol. Chem.. 274. 15293-15296 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] N.Shimamoto, T.Kinebuchi: "Protein sliding along DNA: A clever strategy of DNA binding proteins. (in Japanese)"Kagaku. 69. 42-50 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] R.Sen, H.Nagai, V.J. Hernandez, N.Shimamoto: "Reduction in abortive transcription from the λPR promoter by mutations in region 3 of the s70 subunit of E. coli RNA polymerase."J. Biol. Chem.. 273. 9872-9877 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] N.Shimamoto, T.Kaxciukovich, H.Nagai, R.S.Hayward: "Efficient solubilization of proteins overproduced as inclusion bodies by use of an extreme concentration of glycerol."Tech. Tips Online. t01576. (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] T.Kinebuchi, N.Shimamoto: "Protein sliding along DNA. (in Japanese)"Chem. Biol.. 36. 278-280 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] H.Nagai: "Protein footprinting (in Japanese)"Biophysics. 217. 116-118 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] T.Kinebuchi, H.Shindo, H.Nagai, N.Shimamoto, M.Shimizu: "Functional domain of Esherichia coli Single-stranded DNA binding protein as assessed by analysis of the deletion mutants."Biochemistry. 36. 6732-6738 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] T.Kubori, N.Shimamoto: "Physical interference between Esherichia RNA polymerase molecules transcribing in tandem enhances abortive synthesis and misincorporation."Nuc. Acids Res.. 25. 2640-2647 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] H.Nagai, N.Shimamoto: "Most conserved regions of the E. coli primary sigma factor are involved in interaction with RNA polymerase enzyme."Gene. Cells. 2. 725-734 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] T.Kubori, N.Shimamoto: "A new model for transcription initiation and its regulation. (in Japanese)"Biophysics. 218. 249-253 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] H.Kabata, N.Shimamoto: "Visualization of DNA-protein interaction and sliding of Proteins. (in Japanese)"Prot. Nuc. Acids. Enz. Extr.. 42. 1181-1186 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] N.Shimamoto: "E. coli RNA polymerase in Bioscience New Library of Bioscience Words: Transcription factors. eds, T. Tamura, M. Yamamoto, K. Yasuda (in Japanese)"Youdo-sha. 227 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] N.Shimamoto: "GreA GreB in Bioscience New Library of Bioscience Words: Transcription factors. eds, T. Tamura, M. Yamamoto, K. Yasuda"Youdou-sha. 227 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] N.Shimamoto, M.Go: "Edition, Structural Biology of the gene. In the series of New Biophysics (in Japanese)"Kyoritsu Publication. 196 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] R. Sen, H. Nagai, N. Shimamoto: "Polymerase-arrest at the λPR promoter during transcription initiation"J. Biol. Chem.. 275(in press). (2000)

    • Related Report
      1999 Annual Research Report
  • [Publications] T. Yamamoto 他4名: "Molecular surgery of DNA based on electrostatic micromanipulation"IEEE Trans. Ind. Appl.. (in press). (2000)

    • Related Report
      1999 Annual Research Report
  • [Publications] K. Mukherjee 他3名: "GroEL is involved in activation of Escherichia coli RNA polymerase devoid of polymerase devoid of omega subunit in vivo"Eur. J. Biochem.. 266. 15293-15296 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] 嶋本伸雄,杵渕隆: "DNA上のタンパク質の滑り運動:DNA結合蛋白質の遠攻近交戦略"科学(岩波). 69. 42-50 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] 嶋本伸雄: "Bioscience 新用語ライブラリー 転写因子"大腸菌RNAポリメラーゼ. 227 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] 嶋本伸雄: "Bioscience 新用語ライブラリー 転写因子"GreAGreB. 227 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] N.Shimamoto: "One dimentional diffusion of proteins along DNA: its biological and chemical significance revealed by single-molecule measurements" J. Biol. Chem.275(in press). (1999)

    • Related Report
      1998 Annual Research Report
  • [Publications] N.Shimamoto, T.Kasciukovich, H.Nagai, R.S.Hayward: "Efficient solubilization of proteins overproduced as inclusion bodies by use of an extreme cncentration of glycerol." Tech. Tips Online. (1999)

    • Related Report
      1998 Annual Research Report
  • [Publications] R.Sen, H.Nagai, V.J.Hemandez, N.Shimamoto: "Reduction in abortive transcription from the λPR promoter by mutations in region 3 of the σ70 subunit of E. coli RNA polymerase" J. Biol. Chem.273. 9872-9877 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] H.Nagai, N.Shimamoto: "Most conserved regions of the E. coli primary sigma factor are involved in interaction with RNA polymerase core enzyme." Gene. Cells.2. 725-734 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] 嶋本伸雄,杵渕 隆: "DNA上のタンパク質の滑り運動 : DNA結合蛋白質の遠攻近交戦略" 科学. 69. 42-50 (1999)

    • Related Report
      1998 Annual Research Report
  • [Publications] 杵渕 隆,嶋本伸雄: "DNA上におけるタンパク質のスライディング" 化学と生物. 36. 278-280 (1999)

    • Related Report
      1998 Annual Research Report
  • [Publications] 嶋本伸雄,郷 通子: "遺伝子の構造生物学" 共立出版, 196 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] R.Sen, H.Nagai, V.J.Hernandez, N.Shimamoto: "Reduction in abortive transcription from the λPR promoter by mutations in region 3 of the σ70 subunit of E.coli RNA polymerase." J.Biol.Chem.273(in press). (1998)

    • Related Report
      1997 Annual Research Report
  • [Publications] H.Nagai, N.Shimamoto: "Most conserved regions of the E. coli primary sigma factor are involved in interaction with RNA polymerase core enzyme." Gene.Cells.1998 (2(in press))

    • Related Report
      1997 Annual Research Report
  • [Publications] T.Kubori, N.Shimamoto: "Physical interference between Esherichia coli RNA polymerase molecules transcribing in tandem enhances abortive synthesis and misincorporation." Nuc.Acids Res.25. 2640-2647 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] V.Gopa1, D.Chatterji.: "Mutations in the 1.1 sub‐domain of E.coli σ70 and disruption of its overa11 structure." Eur.J.Biochem.244. 613-618 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] K.Mukherjee, D.Chatteji.: "Studies on the 'ω '-subunit of E.coli RNA polymerase : Its role in recovery of denatured enzyme activity." Eur.J.Biochem.247. 884-889 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] S.Pahari, D.Chatterji.: "Interaction of bacteriophage T4 AsiA protein with E.coli σ70 and its variant." FEBS Letts. 411. 60-62 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] 嶋本 伸雄、郷 通子: "遺伝学の構造生物学" 共立出版, 196 (1998)

    • Related Report
      1997 Annual Research Report

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Published: 1997-04-01   Modified: 2016-04-21  

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