RNAポリメラーゼの分子解剖転写因子接点の同定と構造解析

1997 Fiscal Year Annual Research Report

• Project/Area Number: 08458224
• 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

遺伝子発現制御の本質は、多種多様の転写調節蛋白因子や転写調節DNAシグナルとの相互作用によって、RNAポリメラーゼの特異性が変化し、その結果、転写対象遺伝子が変動にすることである。RNAポリメラーゼの特異性変化の機構を解明する目的で、大腸菌RNAポリメラーゼ分子上に、約100種存在すると推定される大腸菌各種転写因子との接点の同定を目指した。まず、接触不能となったRNAポリメラーゼ変異体を単離し、変異の位置から、接点を予測した。予測は、分子間クロスリンク法、接点切断法など、化学反応を利用して実証を試みた。特に、我々が最近開発した、人工プロテアーゼ活性、人工ヌクレアーゼ活性をもつFe-BABEをRNAポリメラーゼ各サブユニットに共有結合で固定し、その領域と接触する相手蛋白質またはDNAを接点で切断し、切断部位を配列から同定することで、接点の直接証明する方策が成果をもたらした。この成果は、転写制御研究に突破口をもたらしたのみならず、生命現象各分野での、分子間コミュニケーション研究に広く適用できるために、高い評価を得た。

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-bromoacetamide-benzym) ethylenediaminetetraacetate and conjucation to cysteine side chains." Bioconjugate Chemistry. 8(1). 44-48 (1997)
• [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 redering 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^F holoenzyme 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:" Proc.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.: "BiomolecularInteractions in DNA and Proteins" Nova Sci.Publ., (1997)