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1992 Fiscal Year Final Research Report Summary

Expression of mRNA cap-binding protein and structure analysis of its cap recognition mechanism

Research Project

Project/Area Number 02453146
Research Category

Grant-in-Aid for General Scientific Research (B)

Allocation TypeSingle-year Grants
Research Field Physical pharmacy
Research InstitutionOsaka University of Pharmaceutical Sciences

Principal Investigator

ISHIDA Toshimasa  Osaka University of Pharmaceutical Sciences Prof., 薬学部, 教授 (00111021)

Co-Investigator(Kenkyū-buntansha) TOMOO Koji  Osaka University of Pharmaceutical Sciences Assistant, 薬学部, 助手
KAFUKU Yayoi  Osaka University of Pharmaceutical Sciences Assistant, 薬学部, 助手
IN Yasuko  Osaka University of Pharmaceutical Sciences Assistant, 薬学部, 助手
Project Period (FY) 1990 – 1992
Keywordscap-binding protein / IF-4E / mRNA cap structure / gene expression / recognition / structure analysis
Research Abstract

Cap-binding protein (named CBP or IF-4E), the content of which is very small amount in living cell, plays an important role in initiating the protein synthesys on ribosome. In order to elucidate the biochemical and structural nature of this protein and to make clear the selective recognition of mRNA cap structure, we carried out this research project. The main results obtained so far are as follows:
(1) We succeeded in the chemical synthsis of gene of human cap-binding protein (hCBP)(total base pairs of 660).
(2) We established the inclusion of this gene into pBR322 vector and the large amount of expression of hCBP as the inclusion body with human growth hormone.
(3) We established the separation of hCBP from the confused protein and the HPLC purification method.
(4) The functional amino acid residues which probably functions in the selective recognition for the mRNA cap structure characterized by 7-methylguanine base (m7G) were surveyed by a series of model interaction studies.
(5) Based on the above insight, the functional amino acids of hCBP were transformed to the non-functional amino acids by the site-directed mutagenesis. The methods for the gene mutations and the isolation and purification of mutant hCBP proteins were established.
(6) A series of mRNA cap analogues were chemically synthesized.
(7) The interaction modes of these cap analogues with the native and mutant hCBP proteins were investigated by UV and fluorescence spectroscopies. As a result, it was made clear that the His-33, His-37, Trp-102 and Glu-105 residues play important role in interaction with mRNA cap structure. Further, the same results were obtained from the binding experiments of the affinity of m7G-containing column with native and mutant hCBPs.
(8) The X-ray crystal and NMR solution analyses are now in progress.

  • Research Products

    (20 results)

All Other

All Publications (20 results)

  • [Publications] T.Ishida: "Prominent stacking interaction with aromatic amino acid by N-quarternization of nucleic acid base:X-Ray crystallographic characteristics and biological implication" Archi.Biochem.Biophys.278. 217-227 (1990)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] T.Ishida: "Selective binding of guanine base by a tryptophan-con-taining dipeptide" J.Chem.Soc.Chem.Commun.217-218 (1991)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] T.Ishida: "Importance of simultaneous cooperation of hydrogen bond pairing and stacking interaction for recognition of guanine base by a peptide:X-Ray crystal..." J.Chem.Soc.Perkin trans.1. 1847-1853 (1991)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] H.Ueda: "Epression of a synthetic gene for human cap binding protein (human IF-4E) in Escherichia coli and fluorescence studies on interaction with mRNA cap strcuture.." J.Biochem.(Tokyo). 109. 882-889 (1991)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] H.Ueda: "Combination of Trp anf Glu resicues for recognition of mRNA cap structure:Analysis of m7G base recognition site of human cap binding protein(IF-4E)by site-..." FEBS Lett.280. 207-210 (1991)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] H.Iyo: "Sequence-dependent interaction of acidic amino acid with gaunine base in tryptophan-containing dipeptides:Spectroscopic studies" Chem.Pharm.Bull.39. 2483-2486 (1991)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Y.Kafuku: "Spectroscopic study on interaction of nucleic acid base with tryptophan-containing tripeptides:acetylTrp-X-Trp-NHCH_3(X=Gly,Asn,Asp,Gln and Glu)" Chem.Pharm.Bull.39. 2487-2490 (1991)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] H.Ueda: "Cooperative stacking and hydrogen bond pairing interactions of fragment peptide in cap binding protein with mRNA cap structure" Biochim.Biophys.Acta. 1075. 181-186 (1991)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Y.Kafuku: "Recognition of nucleic acid base by tryptophan-containing peptides: Spectroscopic study on interaction of N-AcetylTrp-(Gly)m-Asp-(Gly)n-TrpNHCH_3(m=0-2,n=0-2).." Chem.Pharm.Bull.41. 231-234 (1993)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] T.Ishida: "Advances in Pharmaceutical Sciences" The Research Foundation for Pharmaceutical Sciences, 27 (1992)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] T.Ishida, H.Ueda, K.Segawa, M.Doi, M.Inoue: "Prominent stacking interaction with aromatic amino acid by N-quarternization of nucleic acid base: X-Ray crystallographic characteristics and biological implication" Arch.Biochem.Biophys. 278. 217-227 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] T.Ishida, H.Iyo, H.Ueda, M.Doi, M.Inoue: "Selective binding of guanine base by a tryptophan-containing dipeptide" J.Chem.Soc.Chem.Commun. 217-218 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] T.Ishida, H.Iyo, H.Ueda, M.Doi, M.Inoue, S.Nishimura, K.Kitamura: "Importance of simultaneous cooperation of hydrogen bond pairing and stacking interaction for recognition of guanine base by a peptide: X-Ray crystal analysis of 7-methylguanine-5'-phosphate-tryptophanylglutamic acid complex" J.Chem.Soc.Perkin Trans. 1. 1847-1853 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] H.Ueda, H.Maruyama, M.Doi, M.Inoue, T.Ishida, H.Morioka, S.Nishikawa, S.Uesugi: "Expression of a syntheric gene for human cap binding protein (human IF-4E) in Escherichia coli and fluorescence studies on interaction with mRNA cap structure analogues" J.Biochem. 109. 882-889 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] H.Ueda, H.Iyo, M.Doi, M.Inoue, T.Ishida, H.Morioka, T.Tanaka, S.Nishikawa, S.Uesugi: "Combination of Trp and Glu residues for recognition of mRNA cap structure: Analysis of m7G base recognition site of human cap binding protein (IF-4E) by site-directed mutagenesis" FEBS Lett. 280. 207-210 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] H.Iyo, H.Ueda, Y.Usami, Y.Kafuku, M.Doi, M.Inoue, T.Ishida: "Sequence-dependent interaction of acidic amino acid with guanine base in tryptophan-containing dipeptides: Spectroscopic studies" Chem.Pharm.Bull. 39. 2483-2486 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Y.Kafuku, Y.Matsui, J.Ohtani, Y.Usami, H.Ueda, M.Doi, M.Inoue, T.Ishida: "Spectroscopic study on interaction of nucleic acid base with tryptophan-containing tripeptides: Acetyl-Trp-X-Trp-" Chem.Pharm.Bull. 39. 2487-2490 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] H.Ueda, H.Iyo, M.Doi, M.Inoue, T.Ishida: "Cooperative stacking and hydrogen bond pairing interactions of fragment peptide in cap binding protein with mRNA cap structure" Biochim.Biophys.Acta. 1075. 181-186 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] T.Ishida: "Molecular design of oligopeptides possessing the binding ability selective for a target nucleic acid base" Advances in Pharmaceutical Sciences. 8. 55-81 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Y.Kafuku, J.Ohnishi, M.Doi, M.Inoue, T.Ishida: "Recognition of nucleic acid base by tryptophan-containing peptides: Spectroscopic study on interaction of N-Acetyl-Trp-(Gly)m-Asp-(Gly)n-TrpNHCH_3(m=0 - 2, n=0 - 2) with guanine base" Chem.Pharm.Bull. 41. 231-234 (1993)

    • Description
      「研究成果報告書概要(欧文)」より

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Published: 1994-03-24  

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