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. 1998 Aug 17;17(16):4798–4808. doi: 10.1093/emboj/17.16.4798

Cooperative modulation by eIF4G of eIF4E-binding to the mRNA 5' cap in yeast involves a site partially shared by p20.

M Ptushkina 1, T von der Haar 1, S Vasilescu 1, R Frank 1, R Birkenhäger 1, J E McCarthy 1
PMCID: PMC1170809  PMID: 9707439

Abstract

Interaction between the mRNA 5'-cap-binding protein eIF4E and the multiadaptor protein eIF4G has been demonstrated in all eukaryotic translation assemblies examined so far. This study uses immunological, genetic and biochemical methods to map the surface amino acids on eIF4E that contribute to eIF4G binding. Cap-analogue chromatography and surface plasmon resonance (SPR) analyses demonstrate that one class of mutations in these surface regions disrupts eIF4E-eIF4G association, and thereby polysome formation and growth. The residues at these positions in wild-type eIF4E mediate positive cooperativity between the binding of eIF4G to eIF4E and the latter's cap-affinity. Moreover, two of the mutations confer temperature sensitivity in eIF4G binding to eIF4E which correlates with the formation of large numbers of inactive ribosome 80S couples in vivo and the loss of cellular protein synthesis activity. The yeast 4E-binding protein p20 is estimated by SPR to have a ten times lower binding affinity than eIF4G for eIF4E. Investigation of a second class of eIF4E mutations reveals that p20 shares only part of eIF4G's binding site on the cap-binding protein. The results presented provide a basis for understanding how cycling of eIF4E and eIF4G occurs in yeast translation and explains how p20 can act as a fine, but not as a coarse, regulator of protein synthesis.

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Selected References

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  1. Altmann M., Edery I., Trachsel H., Sonenberg N. Site-directed mutagenesis of the tryptophan residues in yeast eukaryotic initiation factor 4E. Effects on cap binding activity. J Biol Chem. 1988 Nov 25;263(33):17229–17232. [PubMed] [Google Scholar]
  2. Altmann M., Müller P. P., Pelletier J., Sonenberg N., Trachsel H. A mammalian translation initiation factor can substitute for its yeast homologue in vivo. J Biol Chem. 1989 Jul 25;264(21):12145–12147. [PubMed] [Google Scholar]
  3. Altmann M., Schmitz N., Berset C., Trachsel H. A novel inhibitor of cap-dependent translation initiation in yeast: p20 competes with eIF4G for binding to eIF4E. EMBO J. 1997 Mar 3;16(5):1114–1121. doi: 10.1093/emboj/16.5.1114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Belev T. N., Singh M., McCarthy J. E. A fully modular vector system for the optimization of gene expression in Escherichia coli. Plasmid. 1991 Sep;26(2):147–150. doi: 10.1016/0147-619x(91)90056-3. [DOI] [PubMed] [Google Scholar]
  5. Browning K. S. The plant translational apparatus. Plant Mol Biol. 1996 Oct;32(1-2):107–144. doi: 10.1007/BF00039380. [DOI] [PubMed] [Google Scholar]
  6. Carberry S. E., Rhoads R. E., Goss D. J. A spectroscopic study of the binding of m7GTP and m7GpppG to human protein synthesis initiation factor 4E. Biochemistry. 1989 Oct 3;28(20):8078–8083. doi: 10.1021/bi00446a017. [DOI] [PubMed] [Google Scholar]
  7. Craig A. W., Haghighat A., Yu A. T., Sonenberg N. Interaction of polyadenylate-binding protein with the eIF4G homologue PAIP enhances translation. Nature. 1998 Apr 2;392(6675):520–523. doi: 10.1038/33198. [DOI] [PubMed] [Google Scholar]
  8. Goyer C., Altmann M., Lee H. S., Blanc A., Deshmukh M., Woolford J. L., Jr, Trachsel H., Sonenberg N. TIF4631 and TIF4632: two yeast genes encoding the high-molecular-weight subunits of the cap-binding protein complex (eukaryotic initiation factor 4F) contain an RNA recognition motif-like sequence and carry out an essential function. Mol Cell Biol. 1993 Aug;13(8):4860–4874. doi: 10.1128/mcb.13.8.4860. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gradi A., Imataka H., Svitkin Y. V., Rom E., Raught B., Morino S., Sonenberg N. A novel functional human eukaryotic translation initiation factor 4G. Mol Cell Biol. 1998 Jan;18(1):334–342. doi: 10.1128/mcb.18.1.334. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Grossman A. D., Erickson J. W., Gross C. A. The htpR gene product of E. coli is a sigma factor for heat-shock promoters. Cell. 1984 Sep;38(2):383–390. doi: 10.1016/0092-8674(84)90493-8. [DOI] [PubMed] [Google Scholar]
  11. Haghighat A., Sonenberg N. eIF4G dramatically enhances the binding of eIF4E to the mRNA 5'-cap structure. J Biol Chem. 1997 Aug 29;272(35):21677–21680. doi: 10.1074/jbc.272.35.21677. [DOI] [PubMed] [Google Scholar]
  12. Haile D. J., Hentze M. W., Rouault T. A., Harford J. B., Klausner R. D. Regulation of interaction of the iron-responsive element binding protein with iron-responsive RNA elements. Mol Cell Biol. 1989 Nov;9(11):5055–5061. doi: 10.1128/mcb.9.11.5055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  14. Lamphear B. J., Kirchweger R., Skern T., Rhoads R. E. Mapping of functional domains in eukaryotic protein synthesis initiation factor 4G (eIF4G) with picornaviral proteases. Implications for cap-dependent and cap-independent translational initiation. J Biol Chem. 1995 Sep 15;270(37):21975–21983. doi: 10.1074/jbc.270.37.21975. [DOI] [PubMed] [Google Scholar]
  15. Lang V., Zanchin N. I., Lünsdorf H., Tuite M., McCarthy J. E. Initiation factor eIF-4E of Saccharomyces cerevisiae. Distribution within the cell, binding to mRNA, and consequences of its overproduction. J Biol Chem. 1994 Feb 25;269(8):6117–6123. [PubMed] [Google Scholar]
  16. Lanker S., Müller P. P., Altmann M., Goyer C., Sonenberg N., Trachsel H. Interactions of the eIF-4F subunits in the yeast Saccharomyces cerevisiae. J Biol Chem. 1992 Oct 15;267(29):21167–21171. [PubMed] [Google Scholar]
  17. Le H., Tanguay R. L., Balasta M. L., Wei C. C., Browning K. S., Metz A. M., Goss D. J., Gallie D. R. Translation initiation factors eIF-iso4G and eIF-4B interact with the poly(A)-binding protein and increase its RNA binding activity. J Biol Chem. 1997 Jun 27;272(26):16247–16255. doi: 10.1074/jbc.272.26.16247. [DOI] [PubMed] [Google Scholar]
  18. Mader S., Lee H., Pause A., Sonenberg N. The translation initiation factor eIF-4E binds to a common motif shared by the translation factor eIF-4 gamma and the translational repressors 4E-binding proteins. Mol Cell Biol. 1995 Sep;15(9):4990–4997. doi: 10.1128/mcb.15.9.4990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Marcotrigiano J., Gingras A. C., Sonenberg N., Burley S. K. Cocrystal structure of the messenger RNA 5' cap-binding protein (eIF4E) bound to 7-methyl-GDP. Cell. 1997 Jun 13;89(6):951–961. doi: 10.1016/s0092-8674(00)80280-9. [DOI] [PubMed] [Google Scholar]
  20. Matsuo H., Li H., McGuire A. M., Fletcher C. M., Gingras A. C., Sonenberg N., Wagner G. Structure of translation factor eIF4E bound to m7GDP and interaction with 4E-binding protein. Nat Struct Biol. 1997 Sep;4(9):717–724. doi: 10.1038/nsb0997-717. [DOI] [PubMed] [Google Scholar]
  21. McCarthy J. E., Kollmus H. Cytoplasmic mRNA-protein interactions in eukaryotic gene expression. Trends Biochem Sci. 1995 May;20(5):191–197. doi: 10.1016/s0968-0004(00)89006-4. [DOI] [PubMed] [Google Scholar]
  22. Mikaelian I., Sergeant A. A general and fast method to generate multiple site directed mutations. Nucleic Acids Res. 1992 Jan 25;20(2):376–376. doi: 10.1093/nar/20.2.376. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Morino S., Hazama H., Ozaki M., Teraoka Y., Shibata S., Doi M., Ueda H., Ishida T., Uesugi S. Analysis of the mRNA cap-binding ability of human eukaryotic initiation factor-4E by use of recombinant wild-type and mutant forms. Eur J Biochem. 1996 Aug 1;239(3):597–601. doi: 10.1111/j.1432-1033.1996.0597u.x. [DOI] [PubMed] [Google Scholar]
  24. Morley S. J., Curtis P. S., Pain V. M. eIF4G: translation's mystery factor begins to yield its secrets. RNA. 1997 Oct;3(10):1085–1104. [PMC free article] [PubMed] [Google Scholar]
  25. Oliveira C. C., van den Heuvel J. J., McCarthy J. E. Inhibition of translational initiation in Saccharomyces cerevisiae by secondary structure: the roles of the stability and position of stem-loops in the mRNA leader. Mol Microbiol. 1993 Aug;9(3):521–532. doi: 10.1111/j.1365-2958.1993.tb01713.x. [DOI] [PubMed] [Google Scholar]
  26. Pause A., Belsham G. J., Gingras A. C., Donzé O., Lin T. A., Lawrence J. C., Jr, Sonenberg N. Insulin-dependent stimulation of protein synthesis by phosphorylation of a regulator of 5'-cap function. Nature. 1994 Oct 27;371(6500):762–767. doi: 10.1038/371762a0. [DOI] [PubMed] [Google Scholar]
  27. Ptashne M. Specific binding of the lambda phage repressor to lambda DNA. Nature. 1967 Apr 15;214(5085):232–234. doi: 10.1038/214232a0. [DOI] [PubMed] [Google Scholar]
  28. Ptushkina M., Fierro-Monti I., van den Heuvel J., Vasilescu S., Birkenhäger R., Mita K., McCarthy J. E. Schizosaccharomyces pombe has a novel eukaryotic initiation factor 4F complex containing a cap-binding protein with the human eIF4E C-terminal motif KSGST. J Biol Chem. 1996 Dec 20;271(51):32818–32824. doi: 10.1074/jbc.271.51.32818. [DOI] [PubMed] [Google Scholar]
  29. Rozen F., Edery I., Meerovitch K., Dever T. E., Merrick W. C., Sonenberg N. Bidirectional RNA helicase activity of eucaryotic translation initiation factors 4A and 4F. Mol Cell Biol. 1990 Mar;10(3):1134–1144. doi: 10.1128/mcb.10.3.1134. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sagliocco F. A., Vega Laso M. R., Zhu D., Tuite M. F., McCarthy J. E., Brown A. J. The influence of 5'-secondary structures upon ribosome binding to mRNA during translation in yeast. J Biol Chem. 1993 Dec 15;268(35):26522–26530. [PubMed] [Google Scholar]
  31. Sayle R. A., Milner-White E. J. RASMOL: biomolecular graphics for all. Trends Biochem Sci. 1995 Sep;20(9):374–374. doi: 10.1016/s0968-0004(00)89080-5. [DOI] [PubMed] [Google Scholar]
  32. Tarun S. Z., Jr, Sachs A. B. Association of the yeast poly(A) tail binding protein with translation initiation factor eIF-4G. EMBO J. 1996 Dec 16;15(24):7168–7177. [PMC free article] [PubMed] [Google Scholar]
  33. Tarun S. Z., Jr, Wells S. E., Deardorff J. A., Sachs A. B. Translation initiation factor eIF4G mediates in vitro poly(A) tail-dependent translation. Proc Natl Acad Sci U S A. 1997 Aug 19;94(17):9046–9051. doi: 10.1073/pnas.94.17.9046. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Ueda H., Maruyama H., Doi M., Inoue M., Ishida T., Morioka H., Tanaka T., Nishikawa S., Uesugi S. Expression of a synthetic gene for human cap binding protein (human IF-4E) in Escherichia coli and fluorescence studies on interaction with mRNA cap structure analogues. J Biochem. 1991 Jun;109(6):882–889. doi: 10.1093/oxfordjournals.jbchem.a123475. [DOI] [PubMed] [Google Scholar]
  35. Vasilescu S., Ptushkina M., Linz B., Müller P. P., McCarthy J. E. Mutants of eukaryotic initiation factor eIF-4E with altered mRNA cap binding specificity reprogram mRNA selection by ribosomes in Saccharomyces cerevisiae. J Biol Chem. 1996 Mar 22;271(12):7030–7037. doi: 10.1074/jbc.271.12.7030. [DOI] [PubMed] [Google Scholar]
  36. Zanchin N. I., McCarthy J. E. Characterization of the in vivo phosphorylation sites of the mRNA.cap-binding complex proteins eukaryotic initiation factor-4E and p20 in Saccharomyces cerevisiae. J Biol Chem. 1995 Nov 3;270(44):26505–26510. doi: 10.1074/jbc.270.44.26505. [DOI] [PubMed] [Google Scholar]
  37. Zhong T., Arndt K. T. The yeast SIS1 protein, a DnaJ homolog, is required for the initiation of translation. Cell. 1993 Jun 18;73(6):1175–1186. doi: 10.1016/0092-8674(93)90646-8. [DOI] [PubMed] [Google Scholar]
  38. de la Cruz J., Iost I., Kressler D., Linder P. The p20 and Ded1 proteins have antagonistic roles in eIF4E-dependent translation in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1997 May 13;94(10):5201–5206. doi: 10.1073/pnas.94.10.5201. [DOI] [PMC free article] [PubMed] [Google Scholar]

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