金属応答の遺伝子制御機構の解明

2004 Fiscal Year Annual Research Report

• Project/Area Number: 12147209
• Research Institution: Nippon Institute for Biological Science
• Principal Investigator: 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)
• Co-Investigator(Kenkyū-buntansha): 本田 文江 財団法人日本生物科学研究所, 研究部, 研究員 (80343747)
• Keywords: 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

Research Abstract

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。

Research Products

• [Journal Article] The fission yeast protein, Ker1p, is an ortholog of RNA polymerase I subunit A14 in Saccharomyces cerevisiae, and is required for stable association of Rrm3 and RPA21 in Pol I. (2005)
  • Author(s): Imazawa, Y., Hisatake, K., Mitsuzawa, H., Matsumoto, M., Tsukui, T., Nakagawa, K., Nakadai, T., Shimada, M., Ishihama, A., Nogi, Y.
  • Journal Title: J.Biol.Chem. 280(12) Pages: 11467-11474
• [Journal Article] Glyceraldehyde-3-phosphate dehydrogenase associated with RNA polymerase II ad interacts with its Rpb6 subunit. (2005)
  • Author(s): Mitsuzawa, H., Kimura, M., Kanda, E., Ishihama, A
  • Journal Title: FEBS Letters 579 Pages: 48-52
• [Journal Article] uvrA and pric genes involved in DNA repair and replication are negatively regulated by ArcA in Escherichia coli K12. (2005)
  • Author(s): Ogasawara, H., Teramoto, J., Hirao, K., Yamamoto, K., Ishihama, A., Utsumi, R.
  • Journal Title: J.Bactereiol. (In press)
• [Journal Article] Systematic search for the Cra-binding promoters using genomic SELEX. (2005)
  • Author(s): Shimada, T., Fujita, N., Maeda, M., Ishihama, A.
  • Journal Title: Genes Cells (In press)
• [Journal Article] Transcriptional response of Escherichia coli to external copper. (2005)
  • Author(s): Yamamoto, K., Ishihama, A.
  • Journal Title: Mol.Microbiol. 55(1) Pages: 215-227
• [Journal Article] Cross-talks in signal transduction network among all two-component systems in Escherichia coli. (2005)
  • Author(s): Yamamoto, K., Hirano, K., Ohshima, T., Aiba, H., Utsumi, R., Ishihama, A.
  • Journal Title: J.Biol.Chem. 280(2) Pages: 1448-1456
• [Journal Article] Signal transduction cascade between EvgA/EvgS and PhoP/PhoQ two-component systems of Escherichia coli. (2004)
  • Author(s): Eguchi, Y., Okada, T., Minagawa, S., Oshima, T., Mori, H., Yamamoto, K., Ishihama, A., Utsumi, R
  • Journal Title: J.Bacteriol. 186(10) Pages: 3006-3014
• [Journal Article] High level activity of (2',3'9-oligoadenylate synthetase in dog serum. (2004)
  • Author(s): Iwata, A., Yamamoto, A., Fujino, M., Sato, I., Hosokawa-Kanai, T., Tuchiya, K., Ishihama, A., Sokawa, Y
  • Journal Title: J.Vet.Med.Sci. 66(6) Pages: 721-724
• [Journal Article] Fundamental structural units of the Escherichia coli nucleoid revealed by atomic force microscopy. (2004)
  • Author(s): Kim, J., Yoshimura, S.H., Hizume, K., Ohniwa, R.L., Ishihama, A., Takeyasu, K.
  • Journal Title: Nucleic Acids Res. 32(6) Pages: 1982-1992
• [Journal Article] A regulatory trade-off as a source of strain variation in the species Escherichia coli. (2004)
  • Author(s): King, T., Ishihama, A., Kori, A., Ferrenci, T
  • Journal Title: J.Bacteriol. 186(17) Pages: 5614-5620
• [Journal Article] Structure-Function Relationships of Influenza Virus RNA Polymerase: Primer-binding Site on PB1 Subunit. (2004)
  • Author(s): Kolpashchilkov, D., Honda, A., Ishihama, A
  • Journal Title: Biochemistry 43(19) Pages: 5882-5887
• [Journal Article] RNA polymerase II transcription apparatus in Schizosaccharomyces pombe. (2004)
  • Author(s): Mitsuzawa, H., Ishihama, A.
  • Journal Title: Curr.Genet. 44(6) Pages: 287-294
• [Journal Article] Negative regulation of the DNA repair gene (ung) epression by CpxR/cxA two-components system in Escherichia coli and induction of mutations by increased expression of CpxR. (2004)
  • Author(s): Ogasawara, H., Teramoto, J., Yamamoto, K., Ishihama, A., Utsumi, R
  • Journal Title: J.Bacteriol. 186(24) Pages: 8317-8325
• [Journal Article] Quantitative analysis of Escherichia coli promoter activity: Stationary-phase gene-associated promoters. (2004)
  • Author(s): Shimada, T., Makinoshima, H., Ogawa, N., Maeda, M., Ishihama, A.
  • Journal Title: J.Bacteriol. 186(21) Pages: 7112-7122
• [Journal Article] Genome architecture studied by nano-scale imaging: Analyses among bacterial phyla and their implications to eukaryotic genome folding. (2004)
  • Author(s): Takeyasu, K., Kim, J., Ohniwa, R.L., Kobori, T., Inose, Y., Morikawa, K., Ohta, T., Ishihama, A., Yoshimura, S.H
  • Journal Title: Cytogenet.Genome Res. 107(1-2) Pages: 38-48
• [Journal Article] Identification and characterization of antibacterial agents that inhibit the essential histidine kinase, YycG, from Baccilus subtilis and Staphylococcus aureus. (2004)
  • Author(s): Watanabe, T., Hashimoto, Y., Yamamoto, K., Hirao, K., Ishihama, A., Fukamizo, T., Hino, M., Utsumi, R.
  • Journal Title: J.Antibiot. 56(12) Pages: 1045-1052
• [Journal Article] Modes of action of the TyrR protein, which result in repression and activation of the tyrP promoter of Escherichia coli. (2004)
  • Author(s): Yang, J., Hwang, S., Camakaris, H., Irawaty, W., Ishihama, A., Pittard, J.
  • Journal Title: Mol.Microbiol. 52(1) Pages: 243-256
• [Journal Article] A unifying model for the role of polyamines in bacterial cell growth: the polyamine modulon. (2004)
  • Author(s): Yoshida, M., Kashiwagi, K., Shigemasa, A., Shigemasa, A., Taniguchi, S., Yamamoto, K., Makinoshima, H., Ishihama, A., Igarashi, K
  • Journal Title: J.Biol.Chem. 279(44) Pages: 46008-46013
• [Journal Article] Role of a nonnatural b-C-nucleotide unit in DNA as template for DNA and RNA syntheses and as substrates for nucleolytic digestion. (2003)
  • Author(s): Aketani, S., Tanaka, K., Yamamoto, K., Ishihama, A., Cao, H., Tengenji, A., Shionoya, M.
  • Journal Title: Eur.J.Pharm.Sci. 20 Pages: 43-51
• [Journal Article] Mapping sigma 54-RNA polymerase interactions at the -24 consensus promoter element. (2003)
  • Author(s): Burrows, P.C., Severinov, K., Ishihama, A., Buck, M., Wigneshweraraj, S.R.
  • Journal Title: J.Biol.Chem. 278(32) Pages: 29728-29743
• [Journal Article] Transcription regulation of the drug efflux genes by EvgAs, a two-component system in Escherichia coli. (2003)
  • Author(s): Eguchi, Y., Oshima, T., Mori, H., Aono, R., Yamamoto, K., Ishihama, A., Utsumi, R.
  • Journal Title: Microbiology 149(10) Pages: 2819-2828
• [Journal Article] In vitro properties of RpoS (α^S) mutants of Escherichia coli with postulated N-terminal subregion 1.1 of C-terminal region 4 deleted. (2003)
  • Author(s): Gowrishankar, J., Yamamoto, K., Subbarayan, P.R., Ishihama, A
  • Journal Title: J.Bacteriol. 185(8) Pages: 2673-2679
• [Journal Article] Involvement of region 4 of the sigma 70 subunit of RNA polymerase in transcription activation of the lux operon during quorum sensing. (2003)
  • Author(s): Johnson, D.C., Ishihama, A., Stevens, A.M
  • Journal Title: FEMS Microbiol.Lett. 228(2) Pages: 193-201
• [Journal Article] 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)
  • Author(s): Macchi, R., Montesissa, L., Murakami, K., Ishihama, A., de Lorenzo, V., Bertoni, G.
  • Journal Title: J.Biol.Chem. 278(30) Pages: 27695-27702
• [Journal Article] Growth phase-coupled alterations in cell structure and function of Escherichia coli. (2003)
  • Author(s): Makinoshima, H., Aizawa, S., Hayashi, H., Miki, T., Nishimura, A., Ishihama, A
  • Journal Title: J.Bacteriol. 185(4) Pages: 1338-1345
• [Journal Article] Identification and molecular characterization of Mg(II)-regulon of Escherichia coli. (2003)
  • Author(s): Minagawa, S., Ogasawara, H., Kato, A., Yamamoto, K., Eguchi, T., Oshima, T., Mori, H., Ishihama, A., Utsumi, R.
  • Journal Title: J.Bacteriol. 185(13) Pages: 3696-3702
• [Journal Article] Rpb7 subunit of RNA polymerase II interacts with an RNA-binding protein involved in processing of transcripts. (2003)
  • Author(s): Mitsuzawa, H., Kanda, E., Ishihama, A.
  • Journal Title: Nucleic Acids Res. 31(16) Pages: 4696-4701
• [Journal Article] 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)
  • Author(s): Nakagawa, K., Hisatake, K., Imazawa, Y., Ishiguro, A., Matsumoto, M., Pape, L., Ishihama, A., Nogi, Y.
  • Journal Title: Genes Genet.Syst. 78 Pages: 199-209
• [Journal Article] Two different modes of transcription repression of the Escherichia coli acetate operon by lclR. (2003)
  • Author(s): Yamamoto, K., Ishihama, A.
  • Journal Title: Mol.Microbiol. 47(1) Pages: 183-194
• [Book] International Congress Series: Options for Control of Influenza Virus, V. (Y.Kawaoka. ed.) (2004)
  • Author(s): Honda, A., Ishihama, A.
  • Total Pages: 175
  • Publisher: Elsevier B.V., Amsterdam
• [Book] The Molecular Biology of Schizosaccharomyces pombe. (2003)
  • Author(s): Kimura, M., Mitsuzawa, H., Ishihama, A.
  • Total Pages: 445
• [Book] Meth.Enzymol. (2003)
  • Author(s): Meares, C.F., Datwyler, S.A., Schmidt, B.D., Owens, J., Ishihama, A.
  • Total Pages: 330
  • Publisher: Academic Press, New York