Advertisement

Functional and Structural Studies on Artemia Polypeptide Chain Initiation Factor 2. Cloning and Sequencing of eIF-2α cDNA

  • J. N. Dholakia
  • N. S. Reddy
  • A. J. Wahba
Part of the NATO ASI Series book series (NSSA, volume 174)

Abstract

The eukaryotic polypeptide chain initiation factor 2 (eIF-2) plays a significant role in the regulation of protein synthesis [1,2]. The first step in polypeptide chain initiation is the formation of a ternary complex containing eIF-2, GTP and the initiator tRNA. This complex is transferred to a 40S ribosomal subunit [1–4] which later combines with a 60S ribosomal subunit and other initiation factors to form an 80S initiation complex, thereby setting the stage for polypeptide chain elongation. Upon formation of the 80S initiation complex, GTP is hydrolyzed and eIF-2 is released as the eIF-2·GDP binary complex [5,6]. In mammalian systems, this binary complex is stable in the presence of Mg2+ and is functionally inactive [7,8]. Regeneration of eIF-2·GTP·Met-tRNAf species require the guanine nucleotide exchange factor (GEF), which facilitates the exchange of elF-2-bound GDP for GTP and facilitates the recycling of eIF-2 [9–12]. It is at this point in the eIF-2 cycle (Fig. 1) that regulation of polypeptide chain initiation occurs. Most of our understanding about this step in protein synthesis in eukaryotes is derived from studies with rabbit reticulocyte lysates. We have previously observed that dormant and developing embryos of Artemia contain equivalent amounts of eIF-2 activity [13]. Similar to reticulocyte eIF-2, the Artemia factor eIF-2 has 100 to 300-fold higher affinity for GDP than GTP [13] and its α-subunit gets phosphorylated by the rabbit reticulocyte heme-controlled repressor (HCR) [14].

Keywords

Casein Kinase Guanine Nucleotide Exchange Factor Rabbit Reticulocyte Lysate Protein Synthesis Initiation Artemia Embryo 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    A. J. Wahba and C. L. Woodley, Molecular aspects of development in the brine shrimp Artemia, in: “Progress in Nucleic Acids Research and Molecular Biology,” Vol. 31, W. Cohn and K. Moldave, eds., Academic Press, New York (1984).Google Scholar
  2. 2.
    V. M. Pain, Initiation of protein synthesis in mammalian cells, Biochem. J. 235:625 (1986).PubMedGoogle Scholar
  3. 3.
    K. Moldave, Eukaryotic protein synthesis, Ann. Rev. Biochem. 54:1109 (1985).PubMedCrossRefGoogle Scholar
  4. 4.
    S. Ochoa, Regulation of protein synthesis initiation in eukaryotes, Arch. Biochem. Biophys. 223:325 (1983).PubMedCrossRefGoogle Scholar
  5. 5.
    H. Trachsel and T. Staehelin, Binding and release of eukaryotic initiation factor eIF-2 and GTP during protein synthesis initiation, Proc. Natl. Acad. Sci. U.S.A. 75:204 (1978).PubMedCrossRefGoogle Scholar
  6. 6.
    M. S. Clemens, V. M. Pain, S. Wong and E. C. Henshaw, Phosphorylation inhibits guanine nucleotide exchange on eukaryotic initiation factor 2, Nature (London) 297:93 (1982).CrossRefGoogle Scholar
  7. 7.
    R. Panniers and E. C. Henshaw, A GDP/GTP exchange factor essential for eukaryotic initiation factor 2 cycling in Ehrlich ascites tumor cells and its regulation by eukaryotic initiation factor 2 phosphorylation, Biol. Chem. 258:7928 (1983).Google Scholar
  8. 8.
    G. M. Walton and G. N. Gill, Nucleotide regulation of a eukaryotic protein synthesis initiation complex, Biochim. Biophys. Acta 390: 231 (1975).PubMedCrossRefGoogle Scholar
  9. 9.
    J. Siekierka, L. Mauser and S. Ochoa, Mechanism of polypeptide chain initiation in eukaryotes and its control by phosphorylation of the α subunit of initiation factor 2, Proc. Natl. Acad. Sci. U.S.A. 79:2537 (1982)PubMedCrossRefGoogle Scholar
  10. 10.
    M. Salimans, H. Goumans, H. Amesz, R. Benne and H. O. Voorma, Regulation of protein synthesis in eukaryotes. Mode of action of eRF, an eIF-2-recycling factor from rabbit reticulocytes involved in GDP/GTP exchange, Eur. J. Biochem. 145:91 (1984).PubMedCrossRefGoogle Scholar
  11. 11.
    J. N. Dholakia and A. J. Wahba, The association of NADPH with the guanine nucleotide exchange factor from rabbit reticulocytes: A role of Pyridine dinucleotides in eukaryotic Polypeptide chain initiation, Proc. Natl. Acad. Sci. U.S.A. 83:6746 (1986).PubMedCrossRefGoogle Scholar
  12. 12.
    A. Konieczny and B. Safer, Purification of the eukaryotic initiation factor 2-eukaryotic initiation factor 2β complex and characterization of its guanine nucleotide exchange activity during protein synthesis initiation, J. Biol. Chem. 258:3402 (1983).PubMedGoogle Scholar
  13. 13.
    T. H. MacRae, M. Roychowdhury, K. J. Houston, C. L. Woodley and A. J. Wahba, Protein synthesis in brine shrimp embryos. Dormant and developing embryos of Artemia salina contain equivalent amounts of chain initiation factor 2, Eur. J. Biochem. 100:67 (1979).PubMedCrossRefGoogle Scholar
  14. 14.
    H. B. Mehta, J. N. Dholakia, W. W. Roty, B. S. Parekh, R. C. Montelaro, C. L. Woodley and A. J. Wahba, Structural studies on the eukaryotic chain initiation factor 2 from rabbit reticulocytes and brine shrimp Artemia embryos. Phosphorylation by the heme-controlled repressor and casein kinase II, J. Biol. Chem. 261:6705 (1986).PubMedGoogle Scholar
  15. 15.
    H. B. Mehta, C. L. Woodley and A. J. Wahba, Protein synthesis in brine shrimp embryos and rabbit reticulocytes, j. Biol. Chem. 256: 3438 (1983).Google Scholar
  16. 16.
    D. J. Goss, L. J. Parkhurst, H. B. Mehta, C. L. Woodley and A. J. Wahba, Studies on the role of eukaryotic nucleotide exchange factor in Polypeptide chain initiation, J. Biol. Chem. 259:7374 (1984).PubMedGoogle Scholar
  17. 17.
    C. L. Woodley, M. Roychowdhury, T. H. MacRae, K. W. Olsen and A. J. Wahba, Protein synthesis in brine shrimp embryos. Regulation of the formation of the ternary complex (Met-tRNA · eIF-2·GTP) by two purified protein factors and phosphorylation of Artemia eIF-2, Eur. J. Biochem. 117:543 (1981).PubMedCrossRefGoogle Scholar
  18. 18.
    J. N. Dholakia and A. J. Wahba, The isolation and characterization from rabbit reticulocytes of two forms of eukaryotic initiation factor 2 having different β-Polypeptides, J. Biol. Chem. 262:10164 (1987).PubMedGoogle Scholar
  19. 19.
    J. N. Dholakia and A. J. Wahba, Phosphorylation of the guanine nucleotide exchange factor from rabbit reticulocytes regulates its activity in Polypeptide chain initiation, Proc. Natl. Acad. Sci. U.S.A. 85:51 (1988).PubMedCrossRefGoogle Scholar
  20. 20.
    G. Kramer, J. M. Cimadevilla and B. Hardesty, Proc. Natl. Acad., Sci. U.S.A. 73:3078 (1976).CrossRefGoogle Scholar
  21. 21.
    C. L. Woodley, J. N. Dholakia and A. J. Wahba, Polypeptide chain initiation in Artemia embryos: functional and structural studies on chain initiation factor in Artemia, in: “Artemia Research and its Applications,” Vol. 2, W. Declair, L. Moens, H. Siegers, E. Jaspers and P. Sorgeloos, eds., Universa Press, Wetteren (1987).Google Scholar
  22. 22.
    H. A. Daum, III, P. W. Bragg, D. B. Sittman, J. N. Dholakia, C. L. Woodley and A. J. Wahba, The expression of a gene for eukaryotic elongation factor Tu in Artemia during development, J. Biol. Chem. 260:16347 (1985).PubMedGoogle Scholar
  23. 23.
    W. W. Roth, P. W. Bragg, M. V. Corrias, N. S. Reddy, J. N. Dholakia and A. J. Wahba, Expression of a gene for mouse eucaryotic elongation factor Tu during murine erythroleukemic cell differentiation, Mol. Cell Biol. 7:3929 (1987).PubMedGoogle Scholar
  24. 24.
    F. Sanger, S. Nicklen and A. R. Coulson, DNA sequencing with chainterminating inhibitors, Proc. Natl. Acad. Sci. U.S.A. 74:5463 (1977).PubMedCrossRefGoogle Scholar
  25. 25.
    Y. W. Hwang and D. L. Miller, A mutation that alters the nucleotide specificity of elongation factor Tu, a GTP regulatory protein, J. Biol. Chem. 262:13081 (1987).PubMedGoogle Scholar
  26. 26.
    J. S. Clegg, Interrelationghips between water and cellular metabolism in Artemia cysts. V. 14CO2 incorporation, Cell. Physiol. 89: 369 (1976).CrossRefGoogle Scholar
  27. 27.
    W. Filipowicz, J. M. Sierra and S. Ochoa, Polypeptide chain initiation in eukaryotes: initiation factor MP in Artemia salina embryos, Proc. Natl. Acad. Sci. U.S.A. 72:3947 (1975).PubMedCrossRefGoogle Scholar
  28. 28.
    W. Filipowicz, J. M. Sierra and S. Ochoa, Polypeptide chain initiation in eukaryotes: initiation factor requirements for translation of natural messengers, Proc. Natl. Acad. Sci. U.S.A. 73:44 (1976).PubMedCrossRefGoogle Scholar
  29. 29.
    H. Ernst, R. F. Duncan and J. W. B. Hershey, Cloning and sequencing of complementary DNAs encoding the α-subunit of translational initiation factor eIF-2. Characterization of the protein and its messenger RNA, J. Biol. Chem. 262:1206 (1987).PubMedGoogle Scholar
  30. 30.
    T. E. Dever, M. J. Glynias and W. C. Merrick, GTP-binding domain: Three consensus sequence elements with distinct spacing, Proc. Natl. Acad. Sci. U.S.A. 84:1814 (1987).PubMedCrossRefGoogle Scholar

Copyright information

© Plenum Press, New York 1989

Authors and Affiliations

  • J. N. Dholakia
    • 1
  • N. S. Reddy
    • 1
  • A. J. Wahba
    • 1
  1. 1.Department of BiochemistryThe University of Mississippi Medical CenterJacksonUSA

Personalised recommendations