Molecular mechanism and physiological roles of trans-translation mediated by tmRNA

• Project/Area Number: 14380322
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Molecular biology
• Research Institution: Hirosaki University
• Principal Investigator: MUTO Akira Hirosaki University, Faculty of Agriculture and Life Science, Professor, 農学生命科学部, 教授 (80034635)
• Co-Investigator(Kenkyū-buntansha): HIMENO Hyouta Hirosaki University, Faculty of Agriculture and Life Science, Associate Professor, 農学生命科学部, 助教授 (80208785)
• Project Period (FY): 2002 – 2004
• Project Status: Completed (Fiscal Year 2004)
• Budget Amount: ¥14,200,000 (Direct Cost: ¥14,200,000); Fiscal Year 2004: ¥3,200,000 (Direct Cost: ¥3,200,000); Fiscal Year 2003: ¥3,200,000 (Direct Cost: ¥3,200,000); Fiscal Year 2002: ¥7,800,000 (Direct Cost: ¥7,800,000)
• Keywords: tmRNA / tran-tranalation / Escherichia coli / Bacillus subtilis / antibiotics / ribosome / tag-peptide / spore / RNA / 翻訳 / 遺伝子 / SmpB

Research Abstract

a)Molecular mechanism of trans-translation mediated by tmRNA
To investigate the functional structure of tmRNA in trans-translation reaction, we have developed an in vitro trans-translation system using E.coli extracts. Many mutant tmRNAs were constructed by in vitro mutagenesis, and tested their activities as tRNA, mRNA and rribosome binding. From the results, we could identify the structures and sequences in tmRNA required for each function. The effect of several antibiotics on the trans-translation was investigated : we found that paromomycin directly binds to tmRNA causing the frame-shift of translation of tab peptides.
b)Physiological roles of trans-translation
We have constructed an in vivo system in which trans-translation products can be detected in Bacillus subtilis and E.coli. Using this system, several reaction products were isolated and identified by the N-terminal amino acid sequence analyzes. The sites of trans-translation in some of the products were determined by MAS-spectrometry. It was shown that the trans-translation is involved in various cellular activities, such as catabolite repression, amino acid metabolism, stress responses, sporulation and so on.
Endspore formation of Bacillus subtilis strain is significantly impaired by deleting the gene for tmRNA. The genetic and biochemical experimental results revealed that the synthesis of sigma-Kduring the late stage of sporulation process is significantly impaired in the mutant cells.

Research Products

• [Journal Article] Participation of 3'-to-5' Exoribonucleases in the turnover of Bacillus subtilis Messenger RNA (2005)
  • Author(s): Oussenko, I., Abe, T., Ujiie, H., Muto, A., Bechhofer, D.A.
  • Journal Title: J.Bacteriology 187 Pages: 2758-2767
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Participation of 3'-to-5' Exoribonucleases in the turnover of Bacillus subtilis Messenger RNA (2005)
  • Author(s): Oussenko, I., Abe, T., Ujiie, H., Muto, A., Bechhofer, D.A.
  • Journal Title: J.Bacteriol. 187 Pages: 2758-2767
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Participation of 3'-to-5' Exoribonucleases in the turnover of Bacillus subtilis Messenger RNA (2005)
  • Author(s): Oussenko, I., Abe, T., Ujiie, H., Muto, A., Bechhofer, D.A.
  • Journal Title: J.Bacteriology 187(in press)
• [Journal Article] A minimum structure of aminoglycosides that causes an initiation shift of trans-translation (2004)
  • Author(s): Konno, T., Takahashi, T., Kurita, D., Muto, A., Himeno, H
  • Journal Title: Nucleic Acids Res. 32 Pages: 4119-4126
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Isolation of eight novel Caenorhabditis elegans small RNAs (2004)
  • Author(s): Wachi, M., Ogawa, T.Yokoyama, K.Hokii, Y., Shimoyama, M., Muto, A., Ushida, C.
  • Journal Title: Gene 335 Pages: 47-56
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] A novel GTPase activated by small subunit of ribosome (2004)
  • Author(s): Himeno, H., Hanawa-Suetsugu, K., Kimura, T., Takagi, K., Sugiyama, W., Shirata, S., Mikami, T., Odagiri, F., Osanai, Y., Watanabe, D., Goto, S., Kalachnyuk, L., Ushida, C., Muto, A.
  • Journal Title: Nucleic Acids Res. 32 Pages: 5303-5309
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] 翻訳と共役した蛋白質品質管理システム:トランストランスレーション (2004)
  • Author(s): 姫野俵太, 武藤 あきら
  • Journal Title: 蛋白質核酸酵素 49 Pages: 1062-1066
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Isolation of eight novel Caenorhabditis elegans small RNAs (2004)
  • Author(s): Wachi, M., Ogawa, T., Yokoyama, K., Hokii, Y., Shimoyama, M., Muto, A., Ushida, C.
  • Journal Title: Gene 335 Pages: 47-56
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] A novel GTPase activated by the small subunit of ribosome (2004)
  • Author(s): Himeno, H., Hanawa-Suetsugu, K., Kimura, T., Takagi, K., Sugiyama, W., Shirata, S., Mikami, T., Odagiri, F., Osanai, Y., Watanabe, D., Goto, S., Kalachnyuk, L., Ushida, C., Muto, A.
  • Journal Title: Nucelic Acids Res. 32 Pages: 5303-5309
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Trans-tranlation : Co-translational protein quality control system (2004)
  • Author(s): Himeno, H., Muto, A.
  • Journal Title: Protein, Nucleic Acid and Enzyme 49(7)(suppl.) Pages: 1062-1066
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Isolation of eight novel Caenorhabditis elegans small RNAs (2004)
  • Author(s): Wachi, M., Ogawa, T., Yokoyama, K, Hokii, Y., Shimoyama, M., Muto, A., Ushida, C.
  • Journal Title: Gene 335 Pages: 47-56
• [Journal Article] A novel GTPase activated by the small subunit of ribosome (2004)
  • Author(s): Himeno, H., Hanawa-Suetsugu, K., Kimura, T., Takagi, K., Sugiyama, W., Shirata, S., Mikami, T., Odagiri, F., Osanai, Y., Watanabe, D., Goto, S., Kalachnyuk, L., Ushida, C., Muto, A.
  • Journal Title: Nucleic Acids Res. 32 Pages: 5303-5309
• [Publications] T.Takahashi, T.Konno, A.Muto, H.Himeno: "Various effects of paromomycin on tmRNA-mediated trans-translation"J.Biol.Chem.. 278. 27672-27680 (2003)
• [Publications] C.Ushida, A.Yoshida, Y.Miyakawa, Y.Ara, A.Muto: "Distribution of the MCS4 RNA genes in mycoplasmas belonging to the Mycoplasma mycoides cluster"Gene. 314. 149-155 (2003)
• [Publications] 武藤あきら, 姫野俵太: "tmRNAによるトランス翻訳機構と生理的機能"蛋白質核酸酵素. 48. 346-356 (2003)
• [Publications] T.Konno, T.Takahashi, A.Muto, H.Himeno: "Various effect of paramomycin on tmRNA-mediated trans-translation"Nucleic Acid Res. Supplement. 3. 235-236 (2003)
• [Publications] K.Takada, C.Hori-takemoto, M.Kawazoe, S.Lee, M.Shirouzu, M.Yakoyama, A.Muto, H.Himeno: "In vitro anaysis of the initiation steps of trans-translation"Nucleic Acid Res. Supplement. 3. 235-236 (2003)
• [Publications] 姫野 俵太: "タンパク質がわかる"竹縄忠臣編(羊土社). 26-35 (2003)
• [Publications] A.Fujihara, H.Tomatsu, S.Inagaki, C.Ushida, H.Himeno, A.Muto: "Detection of tmRNA-mediated trans-translation products in Bacillus subtilis"Genes to Cells. 7. 343-350 (2002)
• [Publications] K.Hanawa-Suetsugu, H.Himeno, A.Muto: "SmpB functions in various steps of trans-translation"Nucelic Acids Research. 30. 1620-1629 (2002)
• [Publications] K.Ito, T.Tadaki, S.Lee, K.Takada, A.Muto, H.Himeno: "Trans-translation mediated by Bacillus subtilis tmRNA"FEBS Letters. 516. 245-252 (2002)
• [Publications] 渡辺公綱, 姫野俵太: "生命科学II 遺伝子の働きとその応用"丸善株式会社. 255 (2002)