RNAポリメラーゼの金属をとりまく局所の構造と機能

• Project/Area Number: 08249243
• Research Category: Grant-in-Aid for Scientific Research on Priority Areas
• Allocation Type: Single-year Grants
• 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)
• Project Period (FY): 1996
• Project Status: Completed (Fiscal Year 1996)
• Budget Amount: ¥1,800,000 (Direct Cost: ¥1,800,000); Fiscal Year 1996: ¥1,800,000 (Direct Cost: ¥1,800,000)
• Keywords: RNAポリメラーゼ / 転写因子 / 転写シグナル / 生体金属 / 鉄 / DNA-蛋白質相互作用 / 蛋白質-蛋白質相互作用 / 転写制御

Research Abstract

RNAポリメラーゼは、転写調節蛋白因子や、DNAの調節シグナルと相互作用をすることによって、転写対象の遺伝子の選択認識特性を変える。遺伝解析によって我々は、大腸菌RNAポリメラーゼ上に、各種の転写因子やDNA調節シグナルとの接点のマッピングを行った。蛋白-蛋白、蛋白-DNA接触の接点が示唆されたが、変異にともなう間接的影響を排除できない。そこで、分子間接触の直接的証明を得る目的で、Fe-BABE(p-Bromoacetamidobenzyl-EDTA)をシステイン残基特異的に導入し、還元剤添加によって鉄原子から発生したラジカルによる、至近距離の蛋白質や核酸の主鎖切断点を同定することによって、接触点を決定する試みを実施した。クラス1転写因子、DNAエンハンサーの接点が推定されていたアルファサブユニットC端ドメインの1個所にだけFe-BABEを結合させ、修飾サブユニットからRNAポリメラーゼを再構成して、DNAとの相互作用を調べたところ、先にエンハンサーと推定していた場所だけが、切断を受け、アルファサブニットのDNA上の接点が同定できた。しかも、2分子のアルファサブユニットの一方にだけFe-BABEを導入する、画期的方策を確立し、それぞれのアルファサブユニットのDNA上の接点を別々に同定することが出来た。本研究のよって、転写制御の分子機構の新たな突破口を切り開くことが出来た。

Research Products

• [Publications] Artsimovitch,I.: "Transcription activation by the bacteriophage Mu Mor protein requires the C-terminal regions of both α and σ^<70> subunits of Escherichiacoli RNA polymerase." J.Biol.Chem.271. 32343-32348 (1996)
• [Publications] Belyaeva,T.: "Location of the C-terminal domain of the RNA polymerase alpha subunit in diggerent open complexes at the Escherichiacoli galactose operon regulatory region." Nucleic Acids Res.24・12. 2243-2251 (1996)
• [Publications] Eichenberger,P.: "The influence of the location of the activator binding site on the geometry of a transcriptional activation the geometry of a transcriptional activation complex in Eschericiacoli." Biochemistry. 35・48. 15302-15312 (1996)
• [Publications] Giladi,H.: "Identification of an UP element within the IHF binding site at the P_L1-P_L2 tandem promoter of bacteriophase λ." J.Mol.Biol.260. 484-491 (1996)
• [Publications] Imamura,R.: "Identification of the cdpA gene encoding cyclic 3′,5′-adenosine phosophate phosphodiesterase in Escherichiacoli." J.Biol.Chem.271・41. 25423-25429 (1996)
• [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 superoxide inducible genes." Mol.Microbiol.19・2. 307-314 (1996)
• [Publications] Rajkumari,K.: "Effects of H-HS and potassium glutamate on σ^S- and σ^<70>-directed transcription in vitro from osmotically regulated P1 and P2 promoters of proU in Escherichiacoli." J.Bacteriol.178・14. 4176-4181 (1996)
• [Publications] Tang,Y.: "Upstream interactions at the lambda P_<RM> promoter are sequence-nonspecific and activate the promoter to a lesser extent than an introduced UP element of an rRNA promoter." J.Bacteriol.178・23. 6945-6951 (1996)
• [Publications] Greiner,D.P.: "Synthesis of the protein cutting reagent iron (S)-1-(p-bromoacetamidebenzym)ethylenediam inetetraaceate and conjucation to cysteine side chains." Bioconjugate Chemistry. 8・1. 44-48 (1997)
• [Publications] Jishage,M.: "Variation in RNA polymerase sigma subunit composition within different stocks Escherichiacoli strain W3110." J.Bacteriol.179・3. 959-963 (1997)
• [Publications] Kusano,S.: "Promoter selectivity control of Escherichiacoli RNA polyerase Eσ^<38> holoenzyme by trehalose." J.Bacteriol.(in press).
• [Publications] Murakami,K.: "The two alpha subunits of Escherichiacoli RNA polymerase are asymmetrically arranged and contact different halves of the DNA UP element." Proc.Nat.Acad.Sci.(in press).
• [Publications] Jair,K.-W.: "Transcription activation of promoters of the superoxide and multiple antibiotic resistance regulation by Rob,a binding protein of the Escherichiacoli origin of chromosomal replication." J.Bacteriol.178・9. 2507-2513 (1996)
• [Publications] Jishage,M.: "Regulation of RNA polymerase sigma subunit synthesis in Escherichiacoli : Intracellular levels of four species of sigma subunit under various growth conditons." J.Bacteriol.178・18. 5447-5451 (1996)
• [Publications] Kimura,M.: "Subunit assembly in vivo of Escherichiacoli RNA polymerase : Role of the amino-terminal assembly domain of alpha subunit." Genes to Cells. 1・6. 517-528 (1996)
• [Publications] Kusano,S.: "Promoter selectivity of Escherichiacoli RNA polymerase Eσ^<70> and Eσ^<38> holoenzymes : Effect of DNA supercoiling." J.Biol.Chem.271・4. 1998-2004 (1996)
• [Publications] Luo,J.: "Molecular anatomy of the β′subunit of the E.coli RNA polymerase : Identification of region involved in polymerase assemblymerase : Identification of regions involved in polymerase assembly." Genes to Cells. 1・9. 819-828 (1996)
• [Publications] Murakami,K.: "Transcription factor recognition surface on the RNA polymerase α subunit is involved in contact with the DNA enhancer element." EMBOJ.15・16. 4358-4367 (1996)
• [Publications] Negre,D.: "DNA flexibility of the UP element is a major determinant for transcriptional activation at the Escherichiacoli aoetate promoter." Nucleic Acids Res.25・4. 713-718 (1997)