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. 1995 May 9;92(10):4274–4278. doi: 10.1073/pnas.92.10.4274

Molecular cloning and characterization of cDNA encoding the alpha subunit of the rat protein synthesis initiation factor eIF-2B.

K M Flowers 1, S R Kimball 1, R C Feldhoff 1, A G Hinnebusch 1, L S Jefferson 1
PMCID: PMC41926  PMID: 7753796

Abstract

Eukaryotic initiation factor 2B (eIF-2B) is an essential component of the pathway of peptide-chain initiation in mammalian cells, yet little is known about its molecular structure and regulation. To investigate the structure, regulation, and interactions of the individual subunits of eIF-2B, we have begun to clone, characterize, and express the corresponding cDNAs. We report here the cloning and characterization of a 1510-bp cDNA encoding the alpha subunit of eIF-2B from a rat brain cDNA library. The cDNA contains an open reading frame of 918 bp encoding a polypeptide of 305 aa with a predicted molecular mass of 33.7 kDa. This cDNA recognizes a single RNA species approximately 1.6 kb in length on Northern blots of RNA from rat liver. The predicted amino acid sequence contains regions identical to the sequences of peptides derived from bovine liver eIF-2B alpha subunit. Expression of this cDNA in vitro yields a peptide which comigrates with natural eIF-2B alpha in SDS/polyacrylamide gels. The predicted amino acid sequence exhibits 42% identity to that deduced for the Saccharomyces cerevisiae GCN3 protein, the smallest subunit of yeast eIF-2B. In addition, expression of the rat cDNA in yeast functionally complements a gcn3 deletion for the inability to induce histidine biosynthetic genes under the control of GCN4. These results strongly support the hypothesis that mammalian eIF-2 alpha and GCN3 are homologues. Southern blots indicate that the eIF-2B alpha cDNA also recognizes genomic DNA fragments from several other species, suggesting significant homology between the rat eIF-2B alpha gene and that from other species.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alani E., Cao L., Kleckner N. A method for gene disruption that allows repeated use of URA3 selection in the construction of multiply disrupted yeast strains. Genetics. 1987 Aug;116(4):541–545. doi: 10.1534/genetics.112.541.test. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Birnstiel M. L., Busslinger M., Strub K. Transcription termination and 3' processing: the end is in site! Cell. 1985 Jun;41(2):349–359. doi: 10.1016/s0092-8674(85)80007-6. [DOI] [PubMed] [Google Scholar]
  3. Bokoch G. M., Der C. J. Emerging concepts in the Ras superfamily of GTP-binding proteins. FASEB J. 1993 Jun;7(9):750–759. doi: 10.1096/fasebj.7.9.8330683. [DOI] [PubMed] [Google Scholar]
  4. Bushman J. L., Foiani M., Cigan A. M., Paddon C. J., Hinnebusch A. G. Guanine nucleotide exchange factor for eukaryotic translation initiation factor 2 in Saccharomyces cerevisiae: interactions between the essential subunits GCD2, GCD6, and GCD7 and the regulatory subunit GCN3. Mol Cell Biol. 1993 Aug;13(8):4618–4631. doi: 10.1128/mcb.13.8.4618. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cigan A. M., Bushman J. L., Boal T. R., Hinnebusch A. G. A protein complex of translational regulators of GCN4 mRNA is the guanine nucleotide-exchange factor for translation initiation factor 2 in yeast. Proc Natl Acad Sci U S A. 1993 Jun 1;90(11):5350–5354. doi: 10.1073/pnas.90.11.5350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cleveland D. W., Fischer S. G., Kirschner M. W., Laemmli U. K. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J Biol Chem. 1977 Feb 10;252(3):1102–1106. [PubMed] [Google Scholar]
  7. Dever T. E., Chen J. J., Barber G. N., Cigan A. M., Feng L., Donahue T. F., London I. M., Katze M. G., Hinnebusch A. G. Mammalian eukaryotic initiation factor 2 alpha kinases functionally substitute for GCN2 protein kinase in the GCN4 translational control mechanism of yeast. Proc Natl Acad Sci U S A. 1993 May 15;90(10):4616–4620. doi: 10.1073/pnas.90.10.4616. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dever T. E., Feng L., Wek R. C., Cigan A. M., Donahue T. F., Hinnebusch A. G. Phosphorylation of initiation factor 2 alpha by protein kinase GCN2 mediates gene-specific translational control of GCN4 in yeast. Cell. 1992 Feb 7;68(3):585–596. doi: 10.1016/0092-8674(92)90193-g. [DOI] [PubMed] [Google Scholar]
  9. Dholakia J. N., Francis B. R., Haley B. E., Wahba A. J. Photoaffinity labeling of the rabbit reticulocyte guanine nucleotide exchange factor and eukaryotic initiation factor 2 with 8-azidopurine nucleotides. Identification of GTP- and ATP-binding domains. J Biol Chem. 1989 Dec 5;264(34):20638–20642. [PubMed] [Google Scholar]
  10. Dholakia J. N., Wahba A. J. Phosphorylation of the guanine nucleotide exchange factor from rabbit reticulocytes regulates its activity in polypeptide chain initiation. Proc Natl Acad Sci U S A. 1988 Jan;85(1):51–54. doi: 10.1073/pnas.85.1.51. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Frohman M. A., Dush M. K., Martin G. R. Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8998–9002. doi: 10.1073/pnas.85.23.8998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Goss D. J., Parkhurst L. J., Mehta H. B., Woodley C. L., Wahba A. J. Studies on the role of eukaryotic nucleotide exchange factor in polypeptide chain initiation. J Biol Chem. 1984 Jun 25;259(12):7374–7377. [PubMed] [Google Scholar]
  13. Hannig E. M., Hinnebusch A. G. Molecular analysis of GCN3, a translational activator of GCN4: evidence for posttranslational control of GCN3 regulatory function. Mol Cell Biol. 1988 Nov;8(11):4808–4820. doi: 10.1128/mcb.8.11.4808. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hannig E. M., Williams N. P., Wek R. C., Hinnebusch A. G. The translational activator GCN3 functions downstream from GCN1 and GCN2 in the regulatory pathway that couples GCN4 expression to amino acid availability in Saccharomyces cerevisiae. Genetics. 1990 Nov;126(3):549–562. doi: 10.1093/genetics/126.3.549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Harashima S., Hinnebusch A. G. Multiple GCD genes required for repression of GCN4, a transcriptional activator of amino acid biosynthetic genes in Saccharomyces cerevisiae. Mol Cell Biol. 1986 Nov;6(11):3990–3998. doi: 10.1128/mcb.6.11.3990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hershey J. W. Translational control in mammalian cells. Annu Rev Biochem. 1991;60:717–755. doi: 10.1146/annurev.bi.60.070191.003441. [DOI] [PubMed] [Google Scholar]
  17. Hinnebusch A. G. Gene-specific translational control of the yeast GCN4 gene by phosphorylation of eukaryotic initiation factor 2. Mol Microbiol. 1993 Oct;10(2):215–223. doi: 10.1111/j.1365-2958.1993.tb01947.x. [DOI] [PubMed] [Google Scholar]
  18. Kimball S. R., Karinch A. M., Feldhoff R. C., Mellor H., Jefferson L. S. Purification and characterization of eukaryotic translational initiation factor eIF-2B from liver. Biochim Biophys Acta. 1994 Dec 15;1201(3):473–481. doi: 10.1016/0304-4165(94)90079-5. [DOI] [PubMed] [Google Scholar]
  19. Kozak M. The scanning model for translation: an update. J Cell Biol. 1989 Feb;108(2):229–241. doi: 10.1083/jcb.108.2.229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Mateu M. G., Sierra J. M. Protein synthesis in Drosophila melanogaster embryos. Two mechanisms for guanine nucleotide exchange on eukaryotic initiation factor 2. Eur J Biochem. 1987 Jun 15;165(3):507–513. doi: 10.1111/j.1432-1033.1987.tb11468.x. [DOI] [PubMed] [Google Scholar]
  21. Mellor H., Flowers K. M., Kimball S. R., Jefferson L. S. Cloning and characterization of cDNA encoding rat hemin-sensitive initiation factor-2 alpha (eIF-2 alpha) kinase. Evidence for multitissue expression. J Biol Chem. 1994 Apr 8;269(14):10201–10204. [PubMed] [Google Scholar]
  22. Oldfield S., Proud C. G. Purification, phosphorylation and control of the guanine-nucleotide-exchange factor from rabbit reticulocyte lysates. Eur J Biochem. 1992 Aug 15;208(1):73–81. doi: 10.1111/j.1432-1033.1992.tb17160.x. [DOI] [PubMed] [Google Scholar]
  23. Price N. T., Francia G., Hall L., Proud C. G. Guanine nucleotide exchange factor for eukaryotic initiation factor-2. Cloning of cDNA for the delta-subunit of rabbit translation initiation factor-2B. Biochim Biophys Acta. 1994 Mar 1;1217(2):207–210. doi: 10.1016/0167-4781(94)90037-x. [DOI] [PubMed] [Google Scholar]
  24. Proud C. G. Protein phosphorylation in translational control. Curr Top Cell Regul. 1992;32:243–369. doi: 10.1016/b978-0-12-152832-4.50008-2. [DOI] [PubMed] [Google Scholar]
  25. Rhoads R. E. Regulation of eukaryotic protein synthesis by initiation factors. J Biol Chem. 1993 Feb 15;268(5):3017–3020. [PubMed] [Google Scholar]
  26. Riis B., Rattan S. I., Clark B. F., Merrick W. C. Eukaryotic protein elongation factors. Trends Biochem Sci. 1990 Nov;15(11):420–424. doi: 10.1016/0968-0004(90)90279-k. [DOI] [PubMed] [Google Scholar]
  27. Rowlands A. G., Panniers R., Henshaw E. C. The catalytic mechanism of guanine nucleotide exchange factor action and competitive inhibition by phosphorylated eukaryotic initiation factor 2. J Biol Chem. 1988 Apr 25;263(12):5526–5533. [PubMed] [Google Scholar]
  28. Sulston J., Du Z., Thomas K., Wilson R., Hillier L., Staden R., Halloran N., Green P., Thierry-Mieg J., Qiu L. The C. elegans genome sequencing project: a beginning. Nature. 1992 Mar 5;356(6364):37–41. doi: 10.1038/356037a0. [DOI] [PubMed] [Google Scholar]
  29. Trachsel H., Staehelin T. Binding and release of eukaryotic initiation factor eIF-2 and GTP during protein synthesis initiation. Proc Natl Acad Sci U S A. 1978 Jan;75(1):204–208. doi: 10.1073/pnas.75.1.204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Wek R. C., Jackson B. M., Hinnebusch A. G. Juxtaposition of domains homologous to protein kinases and histidyl-tRNA synthetases in GCN2 protein suggests a mechanism for coupling GCN4 expression to amino acid availability. Proc Natl Acad Sci U S A. 1989 Jun;86(12):4579–4583. doi: 10.1073/pnas.86.12.4579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wek R. C., Ramirez M., Jackson B. M., Hinnebusch A. G. Identification of positive-acting domains in GCN2 protein kinase required for translational activation of GCN4 expression. Mol Cell Biol. 1990 Jun;10(6):2820–2831. doi: 10.1128/mcb.10.6.2820. [DOI] [PMC free article] [PubMed] [Google Scholar]

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