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1998 Fiscal Year Final Research Report Summary

Structural Study on the RNA recognition mechanism by the Drosophila Sex-lethal protein

Research Project

Project/Area Number 08680648
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Structural biochemistry
Research InstitutionThe University of Tokyo

Principal Investigator

MUTO Yutaka  The University of Tokyo, Graduate School of Science, Lecturer, 大学院・理学系研究科, 講師 (30192769)

Project Period (FY) 1996 – 1998
KeywordsRNA binding protein / Sxl protein / structural biology / NMR / X-ray chrystallography / U2AF65 / ELAV-family / HuC protein
Research Abstract

The Sex-lethal (Sxl) protein of Drosophila melanogaster regulates alternative splicing of the transformer (tra) mRNA precursor by binding to the tra polypyrimidine tract in the sex determination cascade. The Sxl protein has two tandemly-linked RNA binding domains. As the amino-terminal RBD (RBD1) of the Sxl protein exhibits low sequence homology to the typical RBDs, paticularly at the putative functional residues, it was difficult to unambiguously locate the RNP1 and RNP2 motifs. Therefore, at first, we defined the amino and carboxy-terminal borders of the first RNA-binding domain (RBD1) of the Sxl protein by limited tryptic digestion. By replacement of Phe 166 by Tyr, we constructed a highly soluble mutant, which exhibits the same RNA-binding properties as those of the wild-type. Using this mutant protein, we performed NMR measurements, andelucidated the secondary and tertiary structures of the Sxl RBD1 in solution.
Second, we have determined the crystal structure of the complex between the two tandemly-arranged RNA binding domains of the Sxl protein and a 12-nucleotide, single-stranded RNa derived from the tra polypyrimidine tract. The two RNA-binding domains have their beta-sheet platforms facing each other, to form a V-shaped cleft. The RNA is characteristically extended and bound in this cleft, where the UGUUUUUUU sequence is specifically recognised by the protein. This result provides the first insight into the mechanism by which a protein binds specifically to a cognate RNA that has no intramolecular base pairs.

  • Research Products

    (14 results)

All Other

All Publications (14 results)

  • [Publications] K.Sakamoto: "NMR Studies of the effects of the 5'-Phosphate group on Conformational Properties of 5-Methylaminomethyluridine found in the firstposition of the anticodon of E.coli" Biochemistry. 35. 6533-6538 (1996)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] S.Watanabe: "An RNA fragment consisting of the P7 and P9.0 stems and the 3'-terminal guanosine of the Tetrahymena group I intron" Nucleic Acids Research. 24. 1337-1344 (1996)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] M.Inoue: "A Characteristic Arrangement of Aromatic Amino Acid Residues in the Solution Structure of the Amino-terminal RNA-binding Domain of Drosophila Sex-lethal" J.Mol.Biol.272. 82-94 (1997)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] I.Kim: "NMR analysis of the hydrogen bonding interactions of the RNA-binding domains of the D.Sex-lethal protein with target RNA fragmentswith site specific[3-15N]uridines" Nucleic Acids Research. 25. 1565-1569 (1997)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] S.Chi: "Chemical Shift perturbation studies of the interactions of the second RNA-binding domain of the D.Sex-lethalprotein with the tra pre-mRNAPPT and 3'splice-site" Eur.J.Biochem.259. 1-13 (1999)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] N.Handa: "Structural basis for tra mRNA precursor recognition by the Sex-lethal protein" Nature. (in press).

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] K.Sakmoto, G.Kawai, S.Watanabe, T.Niimi, N.Hayashi, Y.Muto, K.Watanabe, T.Satoh, M.Sekine, and S.Yokoyama: "NMR Studies of the Effects of the 5'-Phosphate group on Conformational Properties of 5-Methylaminomethyluridine found in the First Position of the Anticodon of Escherichia coli tRNA4Arg" Biochemistry. 35. 6533-6538 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] T.Taguchi, M.Iwasaki, Y.Muto, K.Kitajima, S.Inoue, K-H.Khoo, H.R.Morris, A.Dell & S.Inoue: "Occurence and structural analysis of highly sulfated multiantennary N-linked glycan chains derived from a fertilization-associated carbohydrate-rich glycoprotein in unfertilized eggs of Triibolodon hakonesis" Eur.J.Biochem.238. 357-367 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] S.Watanabe, G.Kawai, Y.Muto, K.Watanabe, T.Inoue & S.Yokoyama: "An RNA fragment consisting of the P7 and P9.0 stems and the 3'-terminal guanosine of the Tetrahymena group I intron" Nucleic Acids Research. 24. 1337-1344 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Y.Ito, K.Yamasaki, J.Iwahara, T.Terada, A.Kamiya, M.Shirouzu, Y.Muto, G.Kawai, S.Yokoyama, E.D.Laue, M.Waelchli, T.Shibata, S.Nishimura, & T.Miyazawa: "Regional Polysterism in the GTP-Bound Form of the Human c-Ha-Ras Protein" Biochemistry. 36. 9109-9119 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] M.Inoue, Y.Muto, H.Sakamoto, T.Kigawa, K.Takio, Y.Shimura, & S.Yokoyama: "A Characteristic Arrangement of Aromatic Amino Acid Residues in the Solution Structure of the Amino-terminal RNA-binding Domain of Drosophila Sex-lethal" J.Mol.Biol.272. 82-94 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] I.Kim, Y.Muto, M.Inoue, S.Watanabe, A.Kitamura, S.Yokoyama, K.Hosono, H.Takaku, A.Ono, M.Kainosho, H.Sakamoto, & Y.Shimura: "NMR analysis of the hydrogen bonding interactions of the RNA-binding domains of the Drosophila Sex-lethal protein with target RNA fragments with site-specific [3-15N] uridine substitutions" Nucleic Acids Research. 25. 1565-1569 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] S.Chi, Y.Muto, M.Inoue, I.Kim, H.Sakamoto, Y.Shimura, S.Yokoyama, B.Choi & H.Kim: "Chemical shift perturbation studies of the interactions of the second RNA-binding domain of the Drosophila Sex-lethal protein with the transformer pre-mRNA polyuridine tract and 3' splice-site sequences" Eur.J.Biochem.259. 1-13 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] N.Handa, O.Nureki, K.Kurimoto, I.Kim, H.Sakamoto, Y.Shimura, Y.Muto & S.Yokoyama: "Structural basis for tra mRNA precursor recognition by the Sex-lethal protein." Nature. (accepted).

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

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Published: 1999-12-08  

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