Study on tRNA recognition mechanism by aminoacyl-tRNA synthetase from archaea

• Project/Area Number: 13680705
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Functional biochemistry
• Research Institution: Yamagata University
• Principal Investigator: HASEGAWA Tsunemi Yamagata University, Faculty of Science, Professor, 理学部, 教授 (60095023)
• Co-Investigator(Kenkyū-buntansha): 久野 敦 山形大学, 理学部, 助手 (50302287)
• Project Period (FY): 2001 – 2003
• Project Status: Completed (Fiscal Year 2003)
• Budget Amount: ¥3,500,000 (Direct Cost: ¥3,500,000); Fiscal Year 2003: ¥1,000,000 (Direct Cost: ¥1,000,000); Fiscal Year 2002: ¥1,000,000 (Direct Cost: ¥1,000,000); Fiscal Year 2001: ¥1,500,000 (Direct Cost: ¥1,500,000)
• Keywords: Archaea identity / Archaea / Aeropyrum pernix K1 / Aminoacyl-tRNA synthetase / Evolution / Threonine tRNA / Proline tRNA / Tryptophan tRNA / Aeropyrum pernix / トリプトファニル-tRNA合成酵素 / トリプトファンRNA / 分子進化 / チロシル-tRNA合成酵素 / チロシンtRNA / in vitro転写反応 / プロリル-tRNA合成酵素 / T7RNAポリメラーゼ

Research Abstract

Although the tRNA identity elements have been widely investigated in eubacterial system including Escherichia coli, little is known about tRNA recognition sites for aminoacyl-tRNA synthetase (ARS) from archaea, which is classified as a third kingdom. To study the molecular mechanism of recognition of threonine, proline and tryptophan tRNA for each ARS from extreme thermophilic archaeon, Aeropyrum pernix K1, each ARS gene was cloned and overexpressed. Aminoacylation experiments using in vitro wild and mutant tRNA transcripts and overexpressed ARSs were examined. In the threonine system, the results support our previous finding that A.pernix ThrRs does not recognize the discriminator base of threonine tRNA. This result indicated the diversity of importance of the discriminator base among archaea because the discriminator base of threonine tRNA from halophilic archaeon, Haloferax volcanii, is a strong determinant by cognate ThrRS. It was also found that A.pernix ThrRs did recognize the ac ceptor stem region of threonine tRNA as well as other species ThrRS, in spite of N-terminal truncated unique structure. Furthermore, it was shown that in vitro transcript of proline tRNA whose second and third pairs of acceptor stem was mutated to C-G in addition to transplantation of G36U was threonylated by A.pernix ThrRS. This result of switching from proline tRNA to threonine tRNA by transplanting only five bases indicates that the major identity elements of A.pernix threonine tRNA are acceptor stem region and anticodon. In the proline system, it was found that the discriminator base A73, G1-C72 base pair, and anticodon G35 and G36 were strong determinants for A.pernix ProRs. Recognition of G1-C72 base pair, and anticodon G35 and G36 were strong determinants for A.pernix ProRS. Recognition of G1-C72 base pair was quite different from E.coli ProRS system. In the tryptophan system, results showed that anticodon C34, C35 and A36, discriminator base G73, G1-C62 and G2-C71base pairs of acceptor stem end were base specifically recognized by A.pernix TrpRS. These results indicate that the tRNA recognition mechanisms by ARS is different among three kingdomes.

Research Products

• [Publications] Yokozawa, J., Hasegawa, T.et al.: "Recognition of tRNA by aminoacyl-tRNA synthetase from hyperthermophilic archaeon, Aeropyrum pernix K1."Nucleic Acids Research Supplement. 1. 117-118 (2001)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Fukuda, K, Hasegawa, T. et al.: "Construction of the dual-functional RNA ligand against HCV protease and helicase."Nucleic Acids Research Supplement. 1. 147-145 (2001)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Nagaoka, Y., Hasegawa, T.et al.: "Molecular recognition of threonine tRNA by threonyl-tRNA synthetase from an extreme thermophilic archaeon, Aeropyrum pernix K1."Nucleic Acids Research Supplement. 2. 81-82 (2002)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Taki, M., Hasegawa, T.et al.: "A novel fluorescent nonnatural amino acid that can be incorporated into a specific position of streptavidin."Nucleic Acids Research Supplement. 2. 203-204 (2002)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Iwaki, J., Hasegawa, T.et al.: "Differences in tyrosine tRNA identity between Escherichia coil and archaeon, Aeropyrum pernix K1."Nucleic Acids Research Supplement. 2. 225-226 (2002)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Kikuchi, K, Hasegawa, T. et al.: "In vitro selection of RNA aptamers that bind to domain II of HCV IRES."Nucleic Acids Research Supplement. 2. 267-268 (2002)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Nagaoka, Y., Hasegawa, T.et al.: "Evolution and tRNA recognition of threonyl-tRNA synthetase from an extreme thermophilic archaeon, Aeropyrum pernix K1."Viva Origino. 31・1. 62-70 (2003)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Kikuchi, K., Hasegawa, T.et al.: "RNA aptamers targeted domain II of hepatitis C IRES could bind its apical loop region."Journal of Biochemistry. 133・3. 263-270 (2003)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Kikuchi, K., Hasegawa, T.et al.: "Structure-inhibition analysis of RNA aptamers that bind to HCV IRES."Nucleic Acids Research Supplement. 3. 291-292 (2003)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Kuno, A., Hasegawa, T.et al.: "Leucyl/Phenylalanyl(L/F)-tRNA-protein transferase-mediated N-terminal specific labeling to a protein in vitro."Nucleic Acids Research Supplement. 3. 259-260 (2003)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Yokozawa, J., Hasegawa, T.et al.: "Molecular recognition of proline tRNA by prolyl-tRNA synthetase from hyperthermophilic archaeon, Aeropyrum pernix K1."Nucleic Acids Research Supplement. 3. 247-248 (2003)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Hemmi, H., Hasegawa, T.et al.: "^1H, ^<13>C and ^<15>N chemical shift assignment of xylan-binding domain from Streptomyces olivaceoviridis E-86 β-xylanase."Journal of Biomolecular NMR. 27・1. 91-92 (2003)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Ikeda, Y., Hasegawa, T.et al.: "Synthesis of novel histidine analogue and its efficient incorporation into a protein in vivo"Protein Engineering. 16・9. 699-706 (2003)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Ito, S., Hasegawa, T.et al.: "Rational affinity purification of native Streptomyces family 10 xylanase."Journal of Biotechnology. (in press). (2004)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Kuno, A, Hasegawa, T. et al.: "Kinetics and binding mechenism of a family 13 xylan-binding domain from Streptomyces olivaceoviridis E-86."Mie Bioforum 2003 Proceeding. (in press). (2004)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Ito, S., Hasegawa, T.et al.: "Binding specificity of family13 carbohydrate binding module connected to Streptomyces family 10 xylanase."Mie Bioforum 2003 Proceeding. (in press). (2004)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Suzuki R, Hasegawa, T. et al.: "Mutagenic study of Streptomyces family 10 xylanase."Mie Bioforum 2003 Proceeding. (in press). (2004)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Yokozawa, J., Nagaoka, Y., Umehara, T., Iwaki, J., Kawarabayasi, Y., Koyama, Y., Sako, Y., Wakagi, T., Kuno, A., Hasegawa, T.: "Recognition of tRNA by aminoacyl-tRNA synthetase from hyperthermophilic archaeon, Aeropyrum pernix K1."Nucleic Acids Research Supplement. 1. 117-118 (2001)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Fukuda, K., Sekiya, S., Kikuchi, K., Funaji, K., Kuno, A., Hasegawa, T., Nishikawa, S.: "Construction of the dual-functional RNA ligand against HCV protease and helicase."Nucleic Acisds Research Supplement. 1. 147-148 (2001)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Nagaoka, Y., Yokozawa, J., Umehara, T., Iwaki, J., Okamoto, K., Kawarabayasi, Y., Koyama, Y., Sako, Y., Wakagi, T., Kuno, A., Hasegawa, T.: "Molecular recognition of threonine tRNA by threonyl-tRNA synthetase from an extreme thermophilic archaeon, Aeropyrum pernix K1."Nucleic Acids Research Supplement. 2. 81-82 (2002)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Taki, M., Hahsaka, T., Murakami, H., Kuno, A., Hasegawa, T., Taira, K., Sisido, M.: "A novel fluorescent nonnatural amino acid that can be incorporated into a specific position of streptavidin."Nucleic Acids Research Supplment. 2. 225-226 (2002)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Iwaki, J., Asahara, H., Nagaoka, Y., Yokozawa, J., Umehara, T., Kawarabayasi, Y., Koyama, Y., Sako, Y., Kuno, A., Hasegawa, T.: "Differences in tyrosine tRNA identity between Escherichia coli and archaeon, Aeropyrum pernix K1."Nucleic Acids Research Supplement. 2. 225-226 (2002)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Kikuchi, K., FUkuda, K., Umehara, T., Hwang, J., Kuno, A., Hasegawa, T., Nishikawa, S.: "In vitro selection of RNA aptamers that bind to domain II of HCV IRES."Nucleic Acids Research Supplement. 2. 267-268 (2002)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Nagaoka, Y., Ishikura, K., Kuno, A., Hasegawa, T.: "Evolution and tRNA recognition of threonyl-tRNA synthetase from an extreme thermophilic archaeon, Aeropyrum pernix K1."Viva Origino. 31(1). 62-70 (2003)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Kikuchi, K., Umehara, T., Fukuda, K., Hwang, J., Kuno, A., Hasegawa, T., Nishikawa, S.: "RNA aptamers targeted domain II of hepatitis CIRES could bind its apical loop region."Journal of Biochemistry. 133(3). 263-270 (2003)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Kikuchi, K., Umehara, T., Fukuda, K., Hwang, J., Kuno, A., Hasegawa, T., Nishikawa, S.: "Structure-inhibition analysis of RNA aptamers that bind to HCV IRES."Nucleic Acids Research Supplement. 3. 291-292 (2003)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Kuno, A., Taki, M., Kaneko, S., Taira, K., Hasegawa, T.: "Leucyl/phynylalanyl(L/F)-tRNA-protein transferase-mediated N-terminal specific labeling to a protein in vitro."Nucleic Acids Research Supplement. 3. 247-248 (2003)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Yokozawa, J., Okamoto, K., Kawarabayasi, Y., Kuno, A., Hasegawa, T.: "Molecular recognition of proline tRNA by prolyl-tRNA synthetase from hyperthermophilic archaeon, Aeropyrum pernix K1."Nucleic Acids Research Supplement. 3. 247-248 (2003)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Hemmi, H., Kuno A., Ito, S., Kaneko, S., Hasegawa, T.: "^1H, ^<13>C and ^<15>N chemical shift assignment of xylan-binding domain from Streptomyces olivaceoviridis E-86 β-xylanase."Journal of Biomolecular NMR. 27(1). 91-92 (2003)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Ikeda, Y., Kawahara, S., Taki, M., Kuno, A., Hasegawa, T., Taira, K.: "Synthesis of novel histidine analogue and its officient incorporation into a protein in vivo."Protein Engineering. 16(9). 699-706 (2003)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Ito, S., Kuno, A., Suzuki, R., Kaneko, S., Kawabata, Y., Kusakabe, I., Hasegawa, T.: "Rational affinity purification of native Streptomyces family 10 xylanase."Journal of Biotechnology. (in press). (2004)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Kuno, A., Ito, S., Fujimoto, Z., Hemmi, H., Kaneko, S., Mizuno, H., Kusakabe, I., Hasegawa, T.: "Kinetics and binding mechenism of a family 13 xylan-binding domain from Streptomyces olivaceoviridis E-86."Mie Bioforum 2003 Proceeding. (in press). (2004)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Ito, S., Kuno, A., Kanno, Y., Suzuki, R., Kaneko, S., Kawabata, Y., Kusakabe, I., Hasegawa, T.: "Binding specificity of family 13 carbohydrate binding module connected to Streptomyces family 10 xylanase."Mie Bioforum 2003 Proceeding. (In press). (2004)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Suzuki, R., Kuno, A., Ichikawa, T., Iwamatsu, S., Watanabe, M., Kaneko, S., FUjimoto, Z., Hasegawa, T.: "Mutagenic study of Streptomyces family 10 xylanase."Mie Bioforum 2003 Proceeding. (in press). (2004)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Nagaoka, Y., Ishikura, K., Kuno, A., Hasegawa, T.: "Evolution and tRNA recognition of threonyl-tRNA synthetase from an extreme thermophilic archaeon, Aeropyrum pernix K1"Viva Origino. 31・1. 62-70 (2003)
• [Publications] Kikuchi, K., Umehara, T., Fukuda, K., Hwang, J., Kuno, A., Hasegawa, T.: "RNA aptamers targeted domain II of hepatitis C IRES could bind its apical loop region"Journal of Biochemistry. 133・3. 263-270 (2003)
• [Publications] Kuno, A., Taki, M., Kaneko, S., Taira, K., Hasegawa, T.: "Leucyl/Phenylalanyl(L/F)-tRNA-protein transferase-mediated N-terminal specific labeling to a protein in vitro"Nucleic Acids Research Supplement. 3. 259-260 (2003)
• [Publications] Yokozawa, J., Okamoto, K., Kuno, A., Hasegawa, T.: "Molecular recognition of proline tRNA by prolyl-tRNA synthetase from hyperthermophilic archaeon, Aeropyrum pernix K1"Nucleic Acids Research Supplement. 3. 247-248 (2003)
• [Publications] Hemmi, H., Kuno, A., Ito, S., Kaneko, S., Hasegawa, T.: "1H, 13C 15N chemical shift assignment of xylan-binding domain from Streptomyces olivaceoviridis E-86 β-xylanase"Journal of Biomolecular NMR. 27・1. 91-92 (2003)
• [Publications] Ikeda, Y., Kawahara, S., Taki, M., Kuno, A., Hasegawa, T., Taira, K.: "Synthesis of novel histidine analogue and its efficient incorporation into a protein in vivo"Protein Engineering. 16・9. 699-706 (2003)
• [Publications] Nagaoka, Y., Hasegawa, T., et al.: "Molecular recognition of threonine tRNA by threomyl-tRNA synthetase from an extreme thermohilic archoen, Aeropyrum ptmix K1"Nucl. Acids Res. Suppl.. 2. 81-82 (2002)
• [Publications] Taki, M., Hasegawa, T., et al.: "A novel fluorescent nonnatural amino acid that can be incorporated into a specific position of streptavidin"Nucl. Acids Res. Suppl.. 2. 203-204 (2002)
• [Publications] Iwaki, J., Hasegawa, T., et al.: "Differences in tyrosine tRNA identity between Escherichia coli and archaeon, Aeropyrm permix K1"Nucl. Acids Res. Suppl.. 2. 225-226 (2002)
• [Publications] Kikuchi, K., Hasegawa, T., et al.: "In vitro selection of RNA aptamers that bind to domain II of HCV IRES"Nucl. Acids Res. Suppl.. 2. 267-268 (2002)
• [Publications] Nagaoka, Y., Hasegawa, T., et al.: "Evolution and tRNA recognition of threonyl-tRNA synthetase from an extreme thermophilie archeaeon, Aeropyram pernix K1"Viva Origino. 31・1. 62-70 (2003)
• [Publications] Kikuchi, K., Hasegawa, T., et al.: "RNA aptamers fargeted domain II of hepatitis C IRES could bint its apical loop region"J. Biochem.. 133(in press). (2003)