RNAポリメラーゼの分子解剖:転写因子作用部位のマッピング

1995 Fiscal Year Annual Research Report

• Project/Area Number: 06454672
• Research Institution: National Institute of Genetics
• Principal Investigator: 石浜 明 国立遺伝学研究所, 分子遺伝研究系, 教授 (80019869)
• Co-Investigator(Kenkyū-buntansha): 藤田 信之 国立遺伝学研究所, 分子遺伝研究系, 助手 (90173434)
• Keywords: 転写調節 / RNAポリメラーゼ / 転写因子 / 分子集合 / 蛋白構造 / 分子間相互作用 / 分子解剖 / 遺伝解析

Research Abstract

遺伝子の転写制御の基本機構は、転写酵素RNAポリメラーゼが、さまざまの転写因子と相互作用した結果、転写をする対象遺伝子の選択を変換することによっていることが、明かになり初めている。転写因子とRNAポリメラーゼの分子間コミュニケーションの実体を解明する目的で、本研究では、RNAポリメラーゼ上の転写因子接点の同定を目指した。RNAポリメラーゼ各サブユニットに変異を導入することで、転写因子との接触不能となり、遺伝子制御が出来なくなることを指標にしたスクリーニングを行った。その結果、アルファサブユニットのC端領域に一群の転写因子(クラス1転写因子と命名)との接点を同定し、その領域各アミノ酸の役割を決定した。また、この領域を含む蛋白ドメインの三次元構造を決定し、転写因子と接触するアミノ酸の配置を決めた。その上に、この領域が、DNAのエンハンサーをも認識するサイトであることを実証した。これは、蛋白因子とDNA制御シグナルのいずれをも認識する、新しい蛋白機能域の発見となった。一方、RNAポイメラーゼの他のサブユニット、シグマ、ベタ-およびベタ-プライム上にも、他の転写因子との接点のクラスターが存在する予備的結果を得た。この間の研究で、大腸菌の約100種類の転写因子全ての接点を同定する方向の研究の基礎を築き、軌道に乗せた。

Research Products

• [Publications] Asano,Y.: "Molecular anatomy of influenza virus RNA polymerase:Photoaffinity labeling of PBI subunit with 8-azido GTP.21GC01:J.Biochem." 117. 677-682 (1995)
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• [Publications] Lawley,B.: "The TyrR protein of Escherichia coli is a class I transcription activator." J.Bacteriol.177. 238-241 (1995)
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• [Publications] Jair,K.-W.: "ambidextrous transcription activation by SoxS:Requirement for the C-terminal domain of the RNA polymerase alpha subunit at a subset of supperoxide-inducible genes." Mol.Microbiol.19. 307-314 (1996)
• [Publications] Jeon,Y.H.: "Solution structure of the C-terminal domain of RNA polymerase α subunit responsible for the contact with trans-acting transcription factors and cis-acting UP element of promoter." Science. 270. 1495-1997 (1995)
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• [Publications] Kawagishi-Kobayashi,M.: "Mutational analysis of the RNase-like domain in subunit 2 of fission yeast RNA polymerase II." Mol.Gen.Genet.205. 1-5 (1996)
• [Publications] Kimura,M.: "Functional map of the alpha subunit of Escherichia coli RNA polymerase:Insertion analysis of the amino-terminal assembly domain." J.Mol.Biol.248. 756-767 (1995)
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• [Publications] Nagai,S.: "The cyclic AMP receptor protein(CRP)gene function is required for expression of α (cell-bound)-type hemolysis in an avian pathogenic Escherichia coli strain." Infect.Immun.
• [Publications] Nakagawa,Y.: "RNA polymerase PB2 subunit is not required for replication of the influenza virus genome,but is involved in capped RNA synthesis." J.Virol.69. 728-733 (1995)
• [Publications] Negishi,T.: "Structural map of the alpha subunit of Escherichia coli RNA polymerase:Structural domains identified by proteolytic cleavage." J.Mol.Biol.248. 723-728 (1995)
• [Publications] Shinkawa,H.: "Purification and characterization of RNA polymerase holoenzyme(Eσ^B)from vegetative-phase mycelia of Streptomyce griseus." J.Biochem.118. 488-495 (1995)
• [Publications] Tanaka,K.: "Promoter determinants for Escherichia coli RNA polymerase holoenzyme containing σ^<38>(the rpoS gene product)." Nucleic Acids Res.23. 827-834 (1995)
• [Publications] Tao,K.: "Mapping of the OxyR protein contact site in the C-terminal region of RNA polymerase α subunit." J.Bacteriol.177. 6740-6744 (1995)
• [Publications] Toyoda,T.: "Molecular dissection of influenza virus RNA polymerase:PBI subunit alone is able to catalyze RNA synthesis.21GC25:Virus.Genes"
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• [Publications] Jair,K.-W.: "Purification and regulatory properties of MarA protein:a tarnscriptional activator of Escherichia coli multiple antibiotic and superoxide resistance promoters." J.Bacteriol.177. 7100-7104 (1995)
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