MECHANISM OF INITIATION OF PROTEIN BIOSYNTHESIS

Summary

Principal Investigator: JOHN HERSHEY
Affiliation: University of California
Country: USA
Abstract: The broad goal of the project is to elucidate the molecular mechanisms of initiation and control of protein synthesis in eukaryotic cells. Special focus is on the structure/function of mammalian and yeast initiation factors involved in Met-tRNAi or mRNA binding to ribosomes, on the phosphorylation of initiation factors as a general method to regulate translation rates, and on the mobilization or masking of mRNPs. Specifically, human eIF2 assembly and GTP binding will be elucidated by site-directed mutagenesis of the three subunit's cDNAs already cloned in the laboratory. Chemical crosslinking and cDNA cloning will be directed to elucidating the structure of the large multi-subunit protein complex, eIF3. The yeast Prt1 protein will be purified by stimulating a Prtl-defective lysate,and the putative initiation factor will be characterized biochemically and genetically. A detailed study of mammalian eIF4F, eIF4A, eIF4B and eIF3 binding to mRNAs will employ methods to locate the positions of bound proteins on the mRNA. mRNA derivatives with fluorescent reporter groups at specific positions along the RNA will be used to measure the kinetics of 40S ribosome or initiation factor movement during the mRNA scanning process. Phosphorylation of initiation factors is implicated in malignant transformation and cell growth control. The multiple sites of phosphorylation of eIF4B and eIF3 will be identified and altered to Ala by site-directed mutagenesis of the cDNAs to prevent phosphorylation, and the effects of the mutant forms will be assessed by transfection studies. Furthermore, the specific kinases responsible for eIF4B and eIF3 phosphorylation will be identified, since enhanced phosphorylation of these proteins correlates with mitogenic activation of mammalian cells. Lastly, the mechanism whereby mRNPs are functionally masked or repressed will be addressed. The 50 kDa mRNP protein that inhibits the translation of beta-globin mRNA in rabbit reticulocytes will be characterized. Its cDNA will be cloned on the basis of partial amino acid sequence information, and the detailed mechanism of its inhibitory activity will be studied in transfected cells and in highly purified translation assay systems. Thus by a combination of in vitro studies with purified components and in vivo approaches utilizing recombinant DNA techniques, detailed mechanisms of initiation factor action and translational control will be elucidated.
Funding Period: 1978-08-01 - 1997-07-31
more information: NIH RePORT

Top Publications

  1. ncbi Changes in ribosomal binding activity of eIF3 correlate with increased translation rates during activation of T lymphocytes
    Suzanne Miyamoto
    Department of Biochemistry and Molecular Medicine, School of Medicine, University of California Davis, Davis, California 95616, USA
    J Biol Chem 280:28251-64. 2005
  2. ncbi Movement in ribosome translocation
    Christopher S Fraser
    Department of Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley, CA 947020, USA
    J Biol 4:8. 2005
  3. ncbi Decreased expression of eukaryotic initiation factor 3f deregulates translation and apoptosis in tumor cells
    J Shi
    Department of Pathology, University of Arizona, Tucson, AZ 85724, USA
    Oncogene 25:4923-36. 2006
  4. ncbi The mTOR/PI3K and MAPK pathways converge on eIF4B to control its phosphorylation and activity
    David Shahbazian
    Department of Biochemistry, McGill Cancer Centre, McGill University, Montreal, Quebec, Canada
    EMBO J 25:2781-91. 2006
  5. ncbi Rabies virus matrix protein interplay with eIF3, new insights into rabies virus pathogenesis
    Anastassia V Komarova
    Unité de la Régulation de la Traduction Eucaryote et Virale, CNRS URA 1966, Unité de Génétique Papillomavirus et Cancer Humain, Plate forme de Biophysique des Macromolecules et de leurs interactions, Institute Pasteur, 75015 Paris, France
    Nucleic Acids Res 35:1522-32. 2007
  6. ncbi An oncogenic role for the phosphorylated h-subunit of human translation initiation factor eIF3
    Lili Zhang
    Department of Biochemistry and Molecular Medicine, School of Medicine, University of California Davis, CA 95616, USA
    J Biol Chem 283:24047-60. 2008
  7. ncbi Phosphorylation of the eukaryotic initiation factor 3f by cyclin-dependent kinase 11 during apoptosis
    Jiaqi Shi
    Department of Surgery, Arizona Cancer Center, University of Arizona, Tucson, AZ 85724, USA
    FEBS Lett 583:971-7. 2009

Scientific Experts

  • John W B Hershey
  • Jiaqi Shi
  • Lili Zhang
  • Anastassia V Komarova
  • David Shahbazian
  • J Shi
  • Christopher S Fraser
  • Suzanne Miyamoto
  • Mark A Nelson
  • Zeljka Smit-McBride
  • Xiaoyu Pan
  • Jeanette Rheinhardt
  • Eleonore Real
  • Noel Tordo
  • Katherine M Kean
  • Michele Brocard
  • Yves Jacob
  • Patrick England
  • Andrew M Borman
  • J A Warneke
  • Michael S Cohen
  • Virginie Mieulet
  • Philippe P Roux
  • Jack Taunton
  • John Blenis
  • Nahum Sonenberg
  • Brian Raught
  • Mario Pende
  • B M Honchak
  • S P L Leong
  • M A Nelson
  • A Kahle
  • J W B Hershey
  • Purvi Patel

Detail Information

Publications7

  1. ncbi Changes in ribosomal binding activity of eIF3 correlate with increased translation rates during activation of T lymphocytes
    Suzanne Miyamoto
    Department of Biochemistry and Molecular Medicine, School of Medicine, University of California Davis, Davis, California 95616, USA
    J Biol Chem 280:28251-64. 2005
    ..We conclude that the complex formation of eIF3 and its association with the ribosomes might contribute to increased translation rates during T lymphocyte activation...
  2. ncbi Movement in ribosome translocation
    Christopher S Fraser
    Department of Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley, CA 947020, USA
    J Biol 4:8. 2005
    ..A new study reports that EF-G binds to ribosomes as an EF-G.GDP complex and that GTP is exchanged for GDP on the ribosome. Together with cryo-electron microscopy, this unexpected finding helps clarify the role of GTP in translocation...
  3. ncbi Decreased expression of eukaryotic initiation factor 3f deregulates translation and apoptosis in tumor cells
    J Shi
    Department of Pathology, University of Arizona, Tucson, AZ 85724, USA
    Oncogene 25:4923-36. 2006
    ..We propose that eIF3f may play a role in ribosome degradation during apoptosis. These data provide critical insights into the cellular function of eIF3f and in linking translation initiation and apoptosis...
  4. ncbi The mTOR/PI3K and MAPK pathways converge on eIF4B to control its phosphorylation and activity
    David Shahbazian
    Department of Biochemistry, McGill Cancer Centre, McGill University, Montreal, Quebec, Canada
    EMBO J 25:2781-91. 2006
    ..Phosphorylation of eIF4B on Ser422 by RSK and S6K is physiologically significant, as it increases the interaction of eIF4B with the eukaryotic translation initiation factor 3...
  5. ncbi Rabies virus matrix protein interplay with eIF3, new insights into rabies virus pathogenesis
    Anastassia V Komarova
    Unité de la Régulation de la Traduction Eucaryote et Virale, CNRS URA 1966, Unité de Génétique Papillomavirus et Cancer Humain, Plate forme de Biophysique des Macromolecules et de leurs interactions, Institute Pasteur, 75015 Paris, France
    Nucleic Acids Res 35:1522-32. 2007
    ....
  6. ncbi An oncogenic role for the phosphorylated h-subunit of human translation initiation factor eIF3
    Lili Zhang
    Department of Biochemistry and Molecular Medicine, School of Medicine, University of California Davis, CA 95616, USA
    J Biol Chem 283:24047-60. 2008
    ..The results provide compelling evidence that high eIF3h levels directly stimulate protein synthesis, resulting in the establishment and maintenance of the malignant state in cells...
  7. ncbi Phosphorylation of the eukaryotic initiation factor 3f by cyclin-dependent kinase 11 during apoptosis
    Jiaqi Shi
    Department of Surgery, Arizona Cancer Center, University of Arizona, Tucson, AZ 85724, USA
    FEBS Lett 583:971-7. 2009
    ..Phosphorylation of eIF3f enhances the association of eIF3f with the core eIF3 subunits during apoptosis. Our data suggested that eIF3f may inhibit translation by increasing the binding to the eIF3 complex during apoptosis...