How Does Tobacco Etch Viral mRNA Get Translated? A Fluorescence Study of Competition, Stability and Kinetics

Part of the Reviews in Fluorescence book series (RFLU, volume 2010)


Fluorescence techniques have been used to describe protein-protein and protein-nucleic acid interactions that lead to a competitive advantage for translation of tobacco etch viral mRNA. Using both quenching of intrinsic protein fluorescence and labeling of RNA, equilibrium and thermodynamic parameters were determined to gain insight into preferential binding of protein synthesis initiation factors (eIFs) to tobacco etch virus (TEV) mRNA and the mechanism of binding. Equilibrium data showed that the eIF4F complex binding to TEV mRNA was enthalpically favored and that the complex binds to TEV with greater stability than the cap complex. Kinetic studies using changes in fluorescence anisotropy further characterized the eIF4F-RNA interaction as a bi-molecular, single-step reaction. However, ionic strength dependence of the reaction revealed a possible conformational change after initial binding. These studies provide insight into how viral RNA can successfully compete with host cell mRNA through increasing stability of complexes and kinetic competition.


Internal Ribosome Entry Site Association Rate Tobacco Etch Virus Ionic Strength Dependence eIF4F Complex 
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.



This work was supported by NSF grant MCB 0814051. The author wishes to thank the many members of her research group who contributed to these studies, in particular Dr. Mateen Khan, Hasan Yumak, Shemaila Sultana, Sibnath Ray, Artem Domashevskiy, Sumeyra Yumak and Dr. John Trujillo.


  1. 1.
    Fitzgerald K, Semler B (2009) Bridging IRES elements in mRNAs to the eukaryotic ­translation apparatus. Biochim Biophys Acta 1789:518–528PubMedGoogle Scholar
  2. 2.
    Pestova T, Kolupaeva V, Lomakin I, Pilipenko E, Shatsky I, Agol V, Hellen C (2001) Molecular mechanisms of translation initiation in eukaryotes. Proc Natl Acad Sci USA 98:7029–7036PubMedCrossRefGoogle Scholar
  3. 3.
    Sonenberg N, Dever TE (2003) Eukaryotic translation initiation factors and regulators. Curr Opin Struct Biol 13:56–63PubMedCrossRefGoogle Scholar
  4. 4.
    Le H, Tanguay RL, Balasta ML, Wei C-C, Browning KS, Metz AM, Goss DJ, Gallie DR (1997) The translation initiation factors eIF-iso4G and eIF4B interact with the poly(A)-binding protein and increase its RNA binding affinity. J Biol Chem 272:16247–16255PubMedCrossRefGoogle Scholar
  5. 5.
    Wei C-C, Balasta ML, Ren J, Goss DJ (1998) Wheat germ poly(A) binding protein enhances the binding affinity of eukaryotic initiation factor 4F and (iso)4F for cap analogs. Biochemistry 37:1910–1916PubMedCrossRefGoogle Scholar
  6. 6.
    Hellen CUT, Sarnow P (2001) Internal ribosome entry sites in eukaryotic mRNA molecules. Genes Dev 15:1593–1612PubMedCrossRefGoogle Scholar
  7. 7.
    Kean KM (2003) The role of mRNA 5′-noncoding and 3′-end sequences on 40S ribosomal subunit recruitment, and how RNA viruses successfully compete with cellular mRNAs to ensure their own protein synthesis. Biol Cell 95:129–139PubMedCrossRefGoogle Scholar
  8. 8.
    Jang S, Krausslich H-G, Nicklin M, Duke G, Palmenberg A, Wimmer E (1988) A segment of the 5′ nontranslated region of encephalomyocarditis virus RNA directs internal entry of ­ribosomes during in vitro translation. J Virol 62:2636–2643PubMedGoogle Scholar
  9. 9.
    Pelletier J, Sonenberg N (1988) Internal initiation of translation of eukaryotic mRNA directed by a sequence derived from poliovirus RNA. Nature 334:320–325PubMedCrossRefGoogle Scholar
  10. 10.
    Pestova T, Hellen C, Shatsky I (1996) Canonical eukaryotic initiation factors determine ­initiation of translation by internal ribosomal entry. Mol Cell Biol 16:6859–6869PubMedGoogle Scholar
  11. 11.
    Carrington J, Freed D (1990) Cap-independent enhancement of translation by a plant ­potyvirus 5′ nontranslated region. J Virol 64:1590–1597PubMedGoogle Scholar
  12. 12.
    Gallie D, Tanguay R, Leathers V (1995) The tobacco etch viral 5′ leader and poly(A) tail are synergistic regulators of translation. Gene 165:233–238PubMedCrossRefGoogle Scholar
  13. 13.
    Palmenberg A (1987) Comparative organization and genome structure in picornaviruses. UCLS Symp Mol Cell Biol 54:25–34Google Scholar
  14. 14.
    Gallie D (2001) Cap-Independent translation conferred by the 5′ leader of tobacco etch virus is eukaryotic initiation factor 4G dependent. J Virol 75:12141–12152PubMedCrossRefGoogle Scholar
  15. 15.
    Ray S, Yumak H, Domashevskiy A, Khan M, Gallie DR, Goss DJ (2006) Tobacco etch virus mRNA preferentially binds wheat germ eukaryotic initiation factor (eIF)4G rather than ­eIFiso4G. J Biol Chem 281:35826–35834PubMedCrossRefGoogle Scholar
  16. 16.
    Zeenko V, Gallie DR (2005) Cap-independent translation of tobacco etch virus is conferred by an RNA pseudoknot in the 5′-leader. J Biol Chem 280:26813–26824PubMedCrossRefGoogle Scholar
  17. 17.
    Thivierge K, Nicaise V, Dufresne P, Cotton S, Laliberte J-F, Le Gall O, Fortin M (2005) Plant virus RNAs. Coordinated recruitment of conserved host functions by (+) ssRNA viruses ­during early infection events. Plant Physiol 138:1822–1827PubMedCrossRefGoogle Scholar
  18. 18.
    Mohr I (2006) Phosphorylation and dephosphorylation events that regulate viral mRNA ­translation. Virus Res 119:89–99PubMedCrossRefGoogle Scholar
  19. 19.
    Dougherty W, Carrington J (1988) Expression and function of potyviral gene products. Ann Rev Phytopathol 26:123–143CrossRefGoogle Scholar
  20. 20.
    Pettersson R, Flanegan J, Rose J, Baltimore D (1977) 5′-terminal nucleotide sequences of polio virus polyribosomal RNA and virion RNA are identical. Nature 268:270–272PubMedCrossRefGoogle Scholar
  21. 21.
    Nomoto A, Kitamura N, Golini F, Wimmer E (1977) The 5′-terminal structures of poliovirion RNA and poliovirus mRNA differ only in the genom-linked protein VPg. Proc Natl Acad Sci USA 73:5345–5349CrossRefGoogle Scholar
  22. 22.
    Hewlett M, Rose J, Baltimore D (1976) 5′-terminal structure of poliovirus polyribosomal RNA is pUp. Proc Natl Acad Sci USA 73:327–330PubMedCrossRefGoogle Scholar
  23. 23.
    Ehrenfeld E (1996) Initiation of translation by picornavirus RNAs. Translational control. Cold Spring Harbor, New York, pp 549–574Google Scholar
  24. 24.
    Gallie D, Walbot V (1992) Identification of the motifs within the tobacco mosaic virus 5′ leader responsible for enhancing translation. Nucleic Acids Res 20:4631–4638PubMedCrossRefGoogle Scholar
  25. 25.
    Gallie D (2002) The 5′-leader of tobacco mosaic virus promotes translation through enhanced recruitment of eIF4F. Nucleic Acids Res 30:3401–3411PubMedCrossRefGoogle Scholar
  26. 26.
    Gallie D, Sleat D, Watts J, Turner PC, Wilson TM (1987) The 5′-leader sequence of tobacco mosaic virus RNA enhances the expression of foreign gene transcripts in vitro and in vivo. Nucleic Acids Res 15:3257–3273PubMedCrossRefGoogle Scholar
  27. 27.
    Gallie D, Sleat D, Watts J, Turner P, Wilson T (1988) Mutational analysis of the tobacco mosaic virus 5′-leader for altered ability to enhance translation. Nucleic Acids Res 16:883–893PubMedCrossRefGoogle Scholar
  28. 28.
    Niepel M, Gallie D (1999) Identification and characterization of the functional elements within the tobacco etch virus 5′ leader required for cap-independent translation. J Virol 73: 9080–9088PubMedGoogle Scholar
  29. 29.
    Goyer C, Altmann M, Lee H, Blanc A, Deshmukh M, Woolford J Jr, Trachsel H, Sonenberg N (1993) 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 13:4860–4874PubMedGoogle Scholar
  30. 30.
    Gradi A, Svitkin Y, Imataka H, Sonenberg N (1998) Proteolysis of human eukaryotic translation initiation factor eIF4GII, but not eIF4GI, coincides with the shutoff of host protein ­synthesis after poliovirus infection. Proc Natl Acad Sci USA 95:11089–11094PubMedCrossRefGoogle Scholar
  31. 31.
    Browning KS (1996) The plant translational apparatus. Plant Mol Biol 32:107–144PubMedCrossRefGoogle Scholar
  32. 32.
    Imataka H, Gradi A, Sonenberg N (1998) A newly identified N-terminal amino acid sequence of human eIF4G binds poly(a)-binding protein and functions in poly(A)-dependent translation. EMBO J 17:7480–7489PubMedCrossRefGoogle Scholar
  33. 33.
    Gallie DR, Browning K (2001) EIF4G functionally differs from eIFiso4G in promoting ­internal initiation, cap-independent translation, and translation of structured mRNAs. J Biol Chem 276:36915–36960CrossRefGoogle Scholar
  34. 34.
    Rambo R, Doudna J (2004) Biochemistry 44:4510–4516Google Scholar
  35. 35.
    Khan M, Miyoshi H, Gallie DR, Goss DJ (2008) Potyvirus genome-linked protein, VPg, directly affects wheat germ in vitro translation: interactions with translation initiation factors eIF4F and eIFiso4F. J Biol Chem 283:1340–1349PubMedCrossRefGoogle Scholar
  36. 36.
    Khan MA, Yumak H, Gallie DR, Goss DJ (2008) Effects of poly(A)-binding protein on the interactions of translation initiation factor eIF4F and eIF4F-4B with internal ribosome entry site (IRES) of tobacco etch virus RNA. Biochim Biophys Acta 1779:622–627PubMedGoogle Scholar
  37. 37.
    Kohler J, Schepartz A (2001) Kinetic studies of Fos, Jun DNA complex formation: DNA binding prior to dimerization. Biochemistry 40:130–142PubMedCrossRefGoogle Scholar
  38. 38.
    Tayyab S, Khan N, Khan MA, Kumar Y (2003) Behavior of various mammalian albumins towards bilirubin binding and photochemical properties of different bilirubin–albumin ­complexes. Int J Biol Macromol 31:187–193PubMedCrossRefGoogle Scholar
  39. 39.
    Khan MA, Yumak H, Goss DJ (2009) Kinetic mechanism for the binding of eIF4F and tobacco etch virus internal ribosome entry site RNA: effects of eIF4B and Poly(A)-binding protein. J Biol Chem 284:35461–35470PubMedCrossRefGoogle Scholar
  40. 40.
    Sha M, Wang Y, Xiang T, van Heerden A, Browning K, Goss DJ (1995) Interaction of wheat germ protein synthesis initiation factor eIF-(iso)4F and its subunits p28 and p86 with m7GTP and mRNA analogs. J Biol Chem 270:29904–29909PubMedCrossRefGoogle Scholar
  41. 41.
    Khan M, Miyoshi H, Ray S, Natsuaki T, Suehiro N, Goss DJ (2006) Interaction of genome-linked protein (VPg) of turnip mosaic virus with wheat germ translation initiation factors eIFiso4E and eIFiso4F. J Biol Chem 281:2802–2810Google Scholar
  42. 42.
    Pilipenko E, Pestova T, Kolupaeva V, Khitrina E, Poperechnaya A, Agol V, Hellen C (2000) A cell cycle-dependent protein serves as a template-specific translation initiation factor. Genes Dev 14:2028–2045PubMedGoogle Scholar
  43. 43.
    Khan MA, Goss DJ (2005) Translation initiation factor (eIF) 4B affects the rates of binding of the mRNA m7G cap analogue to wheat germ eIFiso4F and eIFiso4F-PABP. Biochemistry 44:4510–4516PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Chemistry DepartmentHunter College, City University of New YorkNew YorkUSA

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