金属応答の遺伝子制御機構の解明

• 研究課題/領域番号: 12147209
• 研究種目: 特定領域研究
• 配分区分: 補助金
• 審査区分: 生物系
• 研究機関: (財)日本生物科学研究所 (2002-2004); 国立遺伝学研究所 (2000-2001)
• 研究代表者: 石浜 明 財団法人日本生物科学研究所, 研究部, 主任研究員 (80019869)
• 研究分担者: 本田 文江 財団法人日本生物科学研究所, 研究部, 研究員 (80343747) ; 藤田 信之 国立遺伝学研究所, 分子遺伝, 助手 (90173434)
• 研究期間 (年度): 2003 – 2004
• 研究課題ステータス: 完了 (2004年度)
• 配分額: 44,300千円 (直接経費: 44,300千円); 2004年度: 11,500千円 (直接経費: 11,500千円); 2003年度: 12,400千円 (直接経費: 12,400千円); 2002年度: 10,200千円 (直接経費: 10,200千円); 2001年度: 10,200千円 (直接経費: 10,200千円)
• キーワード: RNAポリメラーゼ / 転写因子 / 金属応答 / プロモーター / 転写制御 / マグネシウム / 亜鉛 / 銅 / 金属 / 転写 / 蛋白-蛋白相互作用 / FeBABE / 転写調節 / 大腸菌 / 生体金属 / 機能制御 / 金属結合蛋白 / サブユニット / 金属蛋白質 / 金属酵素 / 金属結合 / 転写装置

研究概要

生体内で金属は蛋白質に結合して存在し、遊離の金属は極めて少なく、一定濃度に維持されている。外界に直接触れている大腸菌は、環境中の各種金属濃度を感知し、それに応答して、金属の取り込みと排出に関わる一群の生体装置の形成と活性制御の遺伝子プログラムを発動し、細胞内の金属のホメオステーテスの維持を行っている。その分子機構の解明を目的として、環境中の各種金属への応答に係る遺伝子群の転写包括制御の全体像の解明を目指して本研究を実施した。
1)大腸菌には外界の9種類の金属の濃度を感知し応答する転写因子が少なくとも12種類存在する。マグネシウム、亜鉛、銅に応答する遺伝子群をDNAチィップを利用したマイクロアレイ解析で同定した。
2)これら金属応答遺伝子群を支配する転写因子を解析し、マグネシウム応答のPhoP、亜鉛応答のZntR、rとCpxR、銅応答のCueR, CusR及びCpxRを同定した。加えて、機能未知であたYedWも金属応答に関与している可能性を示唆した。
3)金属応答遺伝子群からプロモーターを単離し、新たに開発した蛍光蛋白二成分ベクターに挿入し、各転写因子欠損変異系統大腸菌でのプロモーターの転写シグナルとしての活性と制御を解析し、同定した転写因子による支配を実証した。
4)金属応答転写因子支配下遺伝子の単離プロモーターと、全転写因子の純化標品を用いて、試験管内転写反応で転写因子作用様式を系統的に解析する。試験管内転写反応系を利用して、転写因子の金属に応答し活性制御機構を解析した。
5)金属応答転写因子各成分に対する抗体を作製し、イムノブロット法を用いて、外界金属濃度変化に相した金属応答転写因子群の大腸菌内濃度の変化を系統的に観測した。
これら結果を総合して、大腸菌の外界金属変化への応答の遺伝子制御の全体像を提案した。

研究成果

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• [雑誌論文] Structure-Function Relationships of Influenza Virus RNA Polymerase: Primer-binding Site on PB1 Subunit. (2004)
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• [雑誌論文] Involvement of region 4 of the sigma 70 subunit of RNA polymerase in transcription activation of the lux operon during quorum sensing. (2003)
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• [雑誌論文] Recruitment of σ^<54>-RNA polymerase to the Pu promoter of Pseudomonas putida through integration host factor-mediated positioning switch of subunit carboxy-terminal domain on an UP-like element. (2003)
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• [雑誌論文] Identification and molecular characterization of Mg(II)-regulon of Escherichia coli. (2003)
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• [雑誌論文] Rpb7 subunit of RNA polymerase II interacts with an RNA-binding protein involved in processing of transcripts. (2003)
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• [雑誌論文] The fission yeast RPA51 is a functional homolog of the budding yeast A49 subunit of RNA polymerase I and required for maximizing transcription of ribosomal RNA. (2003)
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• [雑誌論文] Two different modes of transcription repression of the Escherichia coli acetate operon by lclR. (2003)
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• [図書] International Congress Series: Options for Control of Influenza Virus, V. (Y.Kawaoka. ed.) (2004)
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  • 総ページ数: 175
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• [図書] The Molecular Biology of Schizosaccharomyces pombe. (2003)
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• [図書] Meth.Enzymol. (2003)
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  • 総ページ数: 330
  • 出版者: Academic Press, New York
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• [文献書誌] Yang, J., Hwang, S., Irawaty, W., Ishihama, A., Pittard, J.: "Modes of action of the TyrR protein, which result in repression and activation of the tyrP promoter of Escherichia coli."Mol.Microbiol.. 52(1). 243-256 (2004)
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• [文献書誌] Makinoshima H.: "Fractionation of Escherichia coli cell populations at different stages during growth transition to stationary phase"Mol. Microbiol.. 43(2). 269-279 (2002)
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