Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1992 Dec;12(12):5801–5815. doi: 10.1128/mcb.12.12.5801

Mutations activating the yeast eIF-2 alpha kinase GCN2: isolation of alleles altering the domain related to histidyl-tRNA synthetases.

M Ramirez 1, R C Wek 1, C R Vazquez de Aldana 1, B M Jackson 1, B Freeman 1, A G Hinnebusch 1
PMCID: PMC360520  PMID: 1448107

Abstract

The protein kinase GCN2 stimulates expression of the yeast transcriptional activator GCN4 at the translational level by phosphorylating the alpha subunit of translation initiation factor 2 (eIF-2 alpha) in amino acid-starved cells. Phosphorylation of eIF-2 alpha reduces its activity, allowing ribosomes to bypass short open reading frames present in the GCN4 mRNA leader and initiate translation at the GCN4 start codon. We describe here 17 dominant GCN2 mutations that lead to derepression of GCN4 expression in the absence of amino acid starvation. Seven of these GCN2c alleles map in the protein kinase moiety, and two in this group alter the presumed ATP-binding domain, suggesting that ATP binding is a regulated aspect of GCN2 function. Six GCN2c alleles map in a region related to histidyl-tRNA synthetases, and two in this group alter a sequence motif conserved among class II aminoacyl-tRNA synthetases that directly interacts with the acceptor stem of tRNA. These results support the idea that GCN2 kinase function is activated under starvation conditions by binding uncharged tRNA to the domain related to histidyl-tRNA synthetase. The remaining GCN2c alleles map at the extreme C terminus, a domain required for ribosome association of the protein. Representative mutations in each domain were shown to depend on the phosphorylation site in eIF-2 alpha for their effects on GCN4 expression and to increase the level of eIF-2 alpha phosphorylation in the absence of amino acid starvation. Synthetic GCN2c double mutations show greater derepression of GCN4 expression than the parental single mutations, and they have a slow-growth phenotype that we attribute to inhibition of general translation initiation. The phenotypes of the GCN2c alleles are dependent on GCN1 and GCN3, indicating that these two positive regulators of GCN4 expression mediate the inhibitory effects on translation initiation associated with activation of the yeast eIF-2 alpha kinase GCN2.

Full text

PDF
5801

Images in this article

5806Image
on p.5806
5807Image
on p.5807
5807Image
on p.5807

Selected References

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

  1. Abastado J. P., Miller P. F., Jackson B. M., Hinnebusch A. G. Suppression of ribosomal reinitiation at upstream open reading frames in amino acid-starved cells forms the basis for GCN4 translational control. Mol Cell Biol. 1991 Jan;11(1):486–496. doi: 10.1128/mcb.11.1.486. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Anselme J., Härtlein M. Tyr-426 of the Escherichia coli asparaginyl-tRNA synthetase, an amino acid in a C-terminal conserved motif, is involved in ATP binding. FEBS Lett. 1991 Mar 11;280(1):163–166. doi: 10.1016/0014-5793(91)80228-u. [DOI] [PubMed] [Google Scholar]
  3. Bischoff J. R., Samuel C. E. Mechanism of interferon action. The interferon-induced phosphoprotein P1 possesses a double-stranded RNA-dependent ATP-binding site. J Biol Chem. 1985 Jul 15;260(14):8237–8239. [PubMed] [Google Scholar]
  4. Boeke J. D., Trueheart J., Natsoulis G., Fink G. R. 5-Fluoroorotic acid as a selective agent in yeast molecular genetics. Methods Enzymol. 1987;154:164–175. doi: 10.1016/0076-6879(87)54076-9. [DOI] [PubMed] [Google Scholar]
  5. Castilho-Valavicius B., Yoon H., Donahue T. F. Genetic characterization of the Saccharomyces cerevisiae translational initiation suppressors sui1, sui2 and SUI3 and their effects on HIS4 expression. Genetics. 1990 Mar;124(3):483–495. doi: 10.1093/genetics/124.3.483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chen J. J., Throop M. S., Gehrke L., Kuo I., Pal J. K., Brodsky M., London I. M. Cloning of the cDNA of the heme-regulated eukaryotic initiation factor 2 alpha (eIF-2 alpha) kinase of rabbit reticulocytes: homology to yeast GCN2 protein kinase and human double-stranded-RNA-dependent eIF-2 alpha kinase. Proc Natl Acad Sci U S A. 1991 Sep 1;88(17):7729–7733. doi: 10.1073/pnas.88.17.7729. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cigan A. M., Foiani M., Hannig E. M., Hinnebusch A. G. Complex formation by positive and negative translational regulators of GCN4. Mol Cell Biol. 1991 Jun;11(6):3217–3228. doi: 10.1128/mcb.11.6.3217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cigan A. M., Pabich E. K., Feng L., Donahue T. F. Yeast translation initiation suppressor sui2 encodes the alpha subunit of eukaryotic initiation factor 2 and shares sequence identity with the human alpha subunit. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2784–2788. doi: 10.1073/pnas.86.8.2784. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Colthurst D. R., Campbell D. G., Proud C. G. Structure and regulation of eukaryotic initiation factor eIF-2. Sequence of the site in the alpha subunit phosphorylated by the haem-controlled repressor and by the double-stranded RNA-activated inhibitor. Eur J Biochem. 1987 Jul 15;166(2):357–363. doi: 10.1111/j.1432-1033.1987.tb13523.x. [DOI] [PubMed] [Google Scholar]
  10. Cusack S., Berthet-Colominas C., Härtlein M., Nassar N., Leberman R. A second class of synthetase structure revealed by X-ray analysis of Escherichia coli seryl-tRNA synthetase at 2.5 A. Nature. 1990 Sep 20;347(6290):249–255. doi: 10.1038/347249a0. [DOI] [PubMed] [Google Scholar]
  11. Cusack S., Härtlein M., Leberman R. Sequence, structural and evolutionary relationships between class 2 aminoacyl-tRNA synthetases. Nucleic Acids Res. 1991 Jul 11;19(13):3489–3498. doi: 10.1093/nar/19.13.3489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Eriani G., Delarue M., Poch O., Gangloff J., Moras D. Partition of tRNA synthetases into two classes based on mutually exclusive sets of sequence motifs. Nature. 1990 Sep 13;347(6289):203–206. doi: 10.1038/347203a0. [DOI] [PubMed] [Google Scholar]
  15. Feng G. S., Chong K., Kumar A., Williams B. R. Identification of double-stranded RNA-binding domains in the interferon-induced double-stranded RNA-activated p68 kinase. Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5447–5451. doi: 10.1073/pnas.89.12.5447. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Foiani M., Cigan A. M., Paddon C. J., Harashima S., Hinnebusch A. G. GCD2, a translational repressor of the GCN4 gene, has a general function in the initiation of protein synthesis in Saccharomyces cerevisiae. Mol Cell Biol. 1991 Jun;11(6):3203–3216. doi: 10.1128/mcb.11.6.3203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Freedman R., Gibson B., Donovan D., Biemann K., Eisenbeis S., Parker J., Schimmel P. Primary structure of histidine-tRNA synthetase and characterization of hisS transcripts. J Biol Chem. 1985 Aug 25;260(18):10063–10068. [PubMed] [Google Scholar]
  18. Galabru J., Hovanessian A. Autophosphorylation of the protein kinase dependent on double-stranded RNA. J Biol Chem. 1987 Nov 15;262(32):15538–15544. [PubMed] [Google Scholar]
  19. Gatti D. L., Tzagoloff A. Structure and evolution of a group of related aminoacyl-tRNA synthetases. J Mol Biol. 1991 Apr 5;218(3):557–568. doi: 10.1016/0022-2836(91)90701-7. [DOI] [PubMed] [Google Scholar]
  20. Gimble F. S., Sauer R. T. Mutations in bacteriophage lambda repressor that prevent RecA-mediated cleavage. J Bacteriol. 1985 Apr;162(1):147–154. doi: 10.1128/jb.162.1.147-154.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hanks S. K., Quinn A. M. Protein kinase catalytic domain sequence database: identification of conserved features of primary structure and classification of family members. Methods Enzymol. 1991;200:38–62. doi: 10.1016/0076-6879(91)00126-h. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  24. Hartwell L. H., McLaughlin C. S. A mutant of yeast apparently defective in the initiation of protein synthesis. Proc Natl Acad Sci U S A. 1969 Feb;62(2):468–474. doi: 10.1073/pnas.62.2.468. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Hinnebusch A. G. A hierarchy of trans-acting factors modulates translation of an activator of amino acid biosynthetic genes in Saccharomyces cerevisiae. Mol Cell Biol. 1985 Sep;5(9):2349–2360. doi: 10.1128/mcb.5.9.2349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Hinnebusch A. G. Evidence for translational regulation of the activator of general amino acid control in yeast. Proc Natl Acad Sci U S A. 1984 Oct;81(20):6442–6446. doi: 10.1073/pnas.81.20.6442. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Hinnebusch A. G., Fink G. R. Repeated DNA sequences upstream from HIS1 also occur at several other co-regulated genes in Saccharomyces cerevisiae. J Biol Chem. 1983 Apr 25;258(8):5238–5247. [PubMed] [Google Scholar]
  29. Hinnebusch A. G. Mechanisms of gene regulation in the general control of amino acid biosynthesis in Saccharomyces cerevisiae. Microbiol Rev. 1988 Jun;52(2):248–273. doi: 10.1128/mr.52.2.248-273.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Kast P., Hennecke H. Amino acid substrate specificity of Escherichia coli phenylalanyl-tRNA synthetase altered by distinct mutations. J Mol Biol. 1991 Nov 5;222(1):99–124. doi: 10.1016/0022-2836(91)90740-w. [DOI] [PubMed] [Google Scholar]
  32. Knighton D. R., Zheng J. H., Ten Eyck L. F., Xuong N. H., Taylor S. S., Sowadski J. M. Structure of a peptide inhibitor bound to the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. Science. 1991 Jul 26;253(5018):414–420. doi: 10.1126/science.1862343. [DOI] [PubMed] [Google Scholar]
  33. Lorber B., Mejdoub H., Reinbolt J., Boulanger Y., Giegé R. Properties of N-terminal truncated yeast aspartyl-tRNA synthetase and structural characteristics of the cleaved domain. Eur J Biochem. 1988 May 16;174(1):155–161. doi: 10.1111/j.1432-1033.1988.tb14076.x. [DOI] [PubMed] [Google Scholar]
  34. Lucchini G., Hinnebusch A. G., Chen C., Fink G. R. Positive regulatory interactions of the HIS4 gene of Saccharomyces cerevisiae. Mol Cell Biol. 1984 Jul;4(7):1326–1333. doi: 10.1128/mcb.4.7.1326. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Messenguy F., Delforge J. Role of transfer ribonucleic acids in the regulation of several biosyntheses in Saccharomyces cerevisiae. Eur J Biochem. 1976 Aug 16;67(2):335–339. doi: 10.1111/j.1432-1033.1976.tb10696.x. [DOI] [PubMed] [Google Scholar]
  36. Meurs E., Chong K., Galabru J., Thomas N. S., Kerr I. M., Williams B. R., Hovanessian A. G. Molecular cloning and characterization of the human double-stranded RNA-activated protein kinase induced by interferon. Cell. 1990 Jul 27;62(2):379–390. doi: 10.1016/0092-8674(90)90374-n. [DOI] [PubMed] [Google Scholar]
  37. Mirande M., Waller J. P. The yeast lysyl-tRNA synthetase gene. Evidence for general amino acid control of its expression and domain structure of the encoded protein. J Biol Chem. 1988 Dec 5;263(34):18443–18451. [PubMed] [Google Scholar]
  38. Moldave K. Eukaryotic protein synthesis. Annu Rev Biochem. 1985;54:1109–1149. doi: 10.1146/annurev.bi.54.070185.005333. [DOI] [PubMed] [Google Scholar]
  39. Mueller P. P., Hinnebusch A. G. Multiple upstream AUG codons mediate translational control of GCN4. Cell. 1986 Apr 25;45(2):201–207. doi: 10.1016/0092-8674(86)90384-3. [DOI] [PubMed] [Google Scholar]
  40. Natsoulis G., Hilger F., Fink G. R. The HTS1 gene encodes both the cytoplasmic and mitochondrial histidine tRNA synthetases of S. cerevisiae. Cell. 1986 Jul 18;46(2):235–243. doi: 10.1016/0092-8674(86)90740-3. [DOI] [PubMed] [Google Scholar]
  41. Niederberger P., Aebi M., Hütter R. Identification and characterization of four new GCD genes in Saccharomyces cerevisiae. Curr Genet. 1986;10(9):657–664. doi: 10.1007/BF00410913. [DOI] [PubMed] [Google Scholar]
  42. Parent S. A., Fenimore C. M., Bostian K. A. Vector systems for the expression, analysis and cloning of DNA sequences in S. cerevisiae. Yeast. 1985 Dec;1(2):83–138. doi: 10.1002/yea.320010202. [DOI] [PubMed] [Google Scholar]
  43. Pathak V. K., Schindler D., Hershey J. W. Generation of a mutant form of protein synthesis initiation factor eIF-2 lacking the site of phosphorylation by eIF-2 kinases. Mol Cell Biol. 1988 Feb;8(2):993–995. doi: 10.1128/mcb.8.2.993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Raben N., Borriello F., Amin J., Horwitz R., Fraser D., Plotz P. Human histidyl-tRNA synthetase: recognition of amino acid signature regions in class 2a aminoacyl-tRNA synthetases. Nucleic Acids Res. 1992 Mar 11;20(5):1075–1081. doi: 10.1093/nar/20.5.1075. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Ramirez M., Wek R. C., Hinnebusch A. G. Ribosome association of GCN2 protein kinase, a translational activator of the GCN4 gene of Saccharomyces cerevisiae. Mol Cell Biol. 1991 Jun;11(6):3027–3036. doi: 10.1128/mcb.11.6.3027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Roussou I., Thireos G., Hauge B. M. Transcriptional-translational regulatory circuit in Saccharomyces cerevisiae which involves the GCN4 transcriptional activator and the GCN2 protein kinase. Mol Cell Biol. 1988 May;8(5):2132–2139. doi: 10.1128/mcb.8.5.2132. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Ruff M., Krishnaswamy S., Boeglin M., Poterszman A., Mitschler A., Podjarny A., Rees B., Thierry J. C., Moras D. Class II aminoacyl transfer RNA synthetases: crystal structure of yeast aspartyl-tRNA synthetase complexed with tRNA(Asp). Science. 1991 Jun 21;252(5013):1682–1689. doi: 10.1126/science.2047877. [DOI] [PubMed] [Google Scholar]
  48. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Schürch A., Miozzari J., Hütter R. Regulation of tryptophan biosynthesis in Saccharomyces cerevisiae: mode of action of 5-methyl-tryptophan and 5-methyl-tryptophan-sensitive mutants. J Bacteriol. 1974 Mar;117(3):1131–1140. doi: 10.1128/jb.117.3.1131-1140.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Thireos G., Penn M. D., Greer H. 5' untranslated sequences are required for the translational control of a yeast regulatory gene. Proc Natl Acad Sci U S A. 1984 Aug;81(16):5096–5100. doi: 10.1073/pnas.81.16.5096. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Tsui F. W., Siminovitch L. Isolation, structure and expression of mammalian genes for histidyl-tRNA synthetase. Nucleic Acids Res. 1987 Apr 24;15(8):3349–3367. doi: 10.1093/nar/15.8.3349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Tzamarias D., Roussou I., Thireos G. Coupling of GCN4 mRNA translational activation with decreased rates of polypeptide chain initiation. Cell. 1989 Jun 16;57(6):947–954. doi: 10.1016/0092-8674(89)90333-4. [DOI] [PubMed] [Google Scholar]
  53. Tzamarias D., Thireos G. Evidence that the GCN2 protein kinase regulates reinitiation by yeast ribosomes. EMBO J. 1988 Nov;7(11):3547–3551. doi: 10.1002/j.1460-2075.1988.tb03231.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. 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]
  55. 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]
  56. Williams N. P., Hinnebusch A. G., Donahue T. F. Mutations in the structural genes for eukaryotic initiation factors 2 alpha and 2 beta of Saccharomyces cerevisiae disrupt translational control of GCN4 mRNA. Proc Natl Acad Sci U S A. 1989 Oct;86(19):7515–7519. doi: 10.1073/pnas.86.19.7515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Williams N. P., Mueller P. P., Hinnebusch A. G. The positive regulatory function of the 5'-proximal open reading frames in GCN4 mRNA can be mimicked by heterologous, short coding sequences. Mol Cell Biol. 1988 Sep;8(9):3827–3836. doi: 10.1128/mcb.8.9.3827. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Wolfner M., Yep D., Messenguy F., Fink G. R. Integration of amino acid biosynthesis into the cell cycle of Saccharomyces cerevisiae. J Mol Biol. 1975 Aug 5;96(2):273–290. doi: 10.1016/0022-2836(75)90348-4. [DOI] [PubMed] [Google Scholar]
  59. Yoon H. J., Donahue T. F. The suil suppressor locus in Saccharomyces cerevisiae encodes a translation factor that functions during tRNA(iMet) recognition of the start codon. Mol Cell Biol. 1992 Jan;12(1):248–260. doi: 10.1128/mcb.12.1.248. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES