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: 09235234
• 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): 1997
• Project Status: Completed (Fiscal Year 1997)
• Budget Amount: ¥2,100,000 (Direct Cost: ¥2,100,000); Fiscal Year 1997: ¥2,100,000 (Direct Cost: ¥2,100,000)
• Keywords: 転写制御 / 転写装置 / 蛋白質間相互作用 / 蛋白質DNA相互作用 / RNAポリメラーゼ / 人工プロテアーゼ / 人工ヌクレアーゼ

Research Abstract

大腸菌RNAポリメラーゼは、α_2ββ'σのサブユニット構造をもち、亜鉛を含む金属酵素である。転写調節蛋白因子やDNAの転写調節シグナルとの相互作用による、その機能特異性、特に転写対象遺伝子選択特性変換機構の解明を目的として、各種の転写因子やDNA調節シグナルとの接触実体の解明を目指した。まず分子間接触に異常をもつRNAポリメラーゼ変異体を単離し、変異部位の分析から、RNAポリメラーゼ分子上にそれらの接点を推定した。これら遺伝解析から得られた結果をより直接的に検証する目的で、本年度はふたつのサブユニットα及びσ上の推定接点領域のアミノ酸をシステインに置換し、本研究で開発した核酸及び蛋白質切断活性をもつ化学試薬Fe-BABEを結合した。サブユニット結合Fe-BABEによる接触相手蛋白質またはDNA分子の切断部位を配列分析から決定することに成功した。これらの成果は、転写研究に新たな突破口を開いたのみならず、生体における分子間相互作用解析のために、新たな研究方法を開発した意義としても評価された。

Research Products

• [Publications] Buyukuslu,N.: "Physical mapping of a collection of Mael-generating amber mutations and the functional effect of internal deletions constructed through their manipulation." Genes and Function. 1(2). 119-130 (1997)
• [Publications] Chugani,S.A.: "Activation of the catBCA promoter: Probing the interaction of CatR and RNA polymerase through in vitro transcription." J.Bacteriol.179(7). 2221-2227 (1997)
• [Publications] Choy,H.E.: "Repression and activation of prooter-bound RNA polymerase activity by Gal repressor." J.Mol.Biol.272(3). 293-300 (1997)
• [Publications] Greiner,D.P.: "Synthesis of the protein cutting reagent iron (S)-1(p-bromoacetamidebenzym)ethylenediaminetetraacetate and conjucation to cysteine side chains." Bioconjugate Chemistry. 8(1). 44-48 (997)
• [Publications] Ishihama,A.: "Adaptation of gene expression in stationary phase bacteria." Curr.Opn.Genet.Devel.7(5). 582-588 (1997)
• [Publications] Jeon,Y.H.: "Flexible linker in the RNA polymerase α subunit facilitates the independent motion of the C-terminal activator contact domain." J.Mol.Biol.267. 953-962 (1997)
• [Publications] Murakami,K.: "Positioning of two alpha subunits carboxy-terminal domains of RNA polymerase at promoters by two transcription factors." Proc.Natl.Acad.Sci.USA. 94(21). 11274-11278 (1997)
• [Publications] Negre,D.: "DNA flexibility of the UP element is a major determinant for transcriptional activation at the Escherichia coli acetate promoter." Nucleic Acids Res.25(4). 713-718 (1997)
• [Publications] Ozoline,O.N.: "Specific fluorescent labeling of two functional domains in RNA polymerase alpha subunit." PROTEINS:Structure,Function,and Genetics. 30(1). 1-10 (1997)
• [Publications] Rajkumari,K.: "Evidence for transcription attenuation rendering cryptic σ^S-dependent promoter for the osmotically regulated proU operon of Salmonella typhimurium" J.Bacteriol.179(22). 7169-7173 (1997)
• [Publications] Yang,J.: "Amino acid residues in the α subunit C-terminal domain of Escherichia coli RNA polymerase involved in interactions with an UP-like sequence and the TyrR protein in the control of transcription from the mtr promoter." J.Bacteriol.179(19). 6187-6191 (1997)
• [Publications] Chatterji,D.: "The mediator for stringent control,ppGpp, binds to the β-subunit of Escherichia coli RNA polymerase." Genes to Cells. (in press).
• [Publications] Jishage,M.: "Variation in RNA polymerase sigma subunit composition within different stocks of Escherichia coli strain W3110." J.Bacteriol.179(3). 959-963 (1997)
• [Publications] Kundu,T.K.: "Promoter selectivity of Escherichia coli RNA polymerase σ^Fholoenzyme involved in transcription of flagellar and chemotaxis genes." J.Bacteriol.179(13). 4264-4269 (1997)
• [Publications] Kusano,S.: "Stimulatory effect of trehalose on the formation and activity of Escherichia coli RNA polymerase Eσ^<38> holoenzyme." J.Bacteriol.179(11). 3649-3654 (1997)
• [Publications] Kusano,S.: "Functional interaction of Escherichia coli RNA polymerase with inorganic polyphosphate." Genes to Cells. 2(7). 433-441 (1997)
• [Publications] McHall,S.M.: "DNase I footprinting,DNA bending and in vitro transcription analysis of ClcR and CatR on the clcABD promoter: Evidence of a conserved transcriptional activation mechanism." Mol.Microbiol.24(5). 965-976 (1997)
• [Publications] Murakami,K.: "The two alpha subunits of Escherichia coli RNA polymerase are asymmetrically arranged and contact diferent halves of the DNA UP element." Proc.Natl.Acad.Sci.USA. 94(5). 1709-1714 (1997)
• [Publications] Ishihama,A.: "Subunits of yeast RNA polymerases in structure and function." Curr.Opn.Microbiol.1(2). 190-196 (1998)
• [Publications] Jishage,M.: "A stationary phase protein in Escherichia coli with binding activity to the major σ subunit of RNA polymerase." Proc.Natl.Acad.Sci.USA. (in press).
• [Publications] Miyake,R.: "The dimeric association of E.coli RNA polymerase alpha subunits, studied by cleavage of Fe-BABE conjugated single-cysteine alpha subunits." Biochemistry. 37(5). 1344-1349 (1998)
• [Publications] Owens,J.T.: "Mapping the Sigma-70 Subunit Mapping the Contact Sites on the Core Enzyme Subunits of Escherichia coli RNA Polymerase by Site-Specific Cleavage with Sigma-Conjugated Chemical Protease." Prpc.Natl.Acad.Sci.USA. (in press).
• [Publications] Ozoline,O.N.: "Topology of the Escherichia coli RNA polymerase surface involved in interaction with promoter upsteam enhancer elements." Eur.J.Biochem.(in press).
• [Publications] Ozoline,O.N.: "Specific fluorescent labeling of two functional domains in RNA polymerase alpha subunit." PROTEINS:Structure,Function,and Genetics. 30. 183-192 (1998)
• [Publications] Ishihama,A.: "Nucleic Acids & Molecular Biology,Vol.11,Mechanism of Transcription" Springer-Verlag, 327 (1997)
• [Publications] Ishihama,A.: "Biomolecular Interactions in DNA and Proteins" Nova Sci.Pubi., (1997)