Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1990 Mar;10(3):1134–1144. doi: 10.1128/mcb.10.3.1134

Bidirectional RNA helicase activity of eucaryotic translation initiation factors 4A and 4F.

F Rozen 1, I Edery 1, K Meerovitch 1, T E Dever 1, W C Merrick 1, N Sonenberg 1
PMCID: PMC360981  PMID: 2304461

Abstract

The mechanism of ribosome binding to eucaryotic mRNAs is not well understood, but it requires the participation of eucaryotic initiation factors eIF-4A, eIF-4B, and eIF-4F and the hydrolysis of ATP. Evidence has accumulated in support of a model in which these initiation factors function to unwind the 5'-proximal secondary structure in mRNA to facilitate ribosome binding. To obtain direct evidence for initiation factor-mediated RNA unwinding, we developed a simple assay to determine RNA helicase activity, and we show that eIF-4A or eIF-4F, in combination with eIF-4B, exhibits helicase activity. A striking and unprecedented feature of this activity is that it functions in a bidirectional manner. Thus, unwinding can occur either in the 5'-to-3' or 3'-to-5' direction. Unwinding in the 5'-to-3' direction by eIF-4F (the cap-binding protein complex), in conjunction with eIF-4B, was stimulated by the presence of the RNA 5' cap structure, whereas unwinding in the 3'-to-5' direction was completely cap independent. These results are discussed with respect to cap-dependent versus cap-independent mechanisms of ribosome binding to eucaryotic mRNAs.

Full text

PDF
1134

Images in this article

1137Image
on p.1137
1137Image
on p.1137
1138Image
on p.1138
1138Image
on p.1138
1139Image
on p.1139
1140Image
on p.1140
1141Image
on p.1141

Selected References

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

  1. Abramson R. D., Dever T. E., Lawson T. G., Ray B. K., Thach R. E., Merrick W. C. The ATP-dependent interaction of eukaryotic initiation factors with mRNA. J Biol Chem. 1987 Mar 15;262(8):3826–3832. [PubMed] [Google Scholar]
  2. Abramson R. D., Dever T. E., Merrick W. C. Biochemical evidence supporting a mechanism for cap-independent and internal initiation of eukaryotic mRNA. J Biol Chem. 1988 May 5;263(13):6016–6019. [PubMed] [Google Scholar]
  3. Banerjee A. K. 5'-terminal cap structure in eucaryotic messenger ribonucleic acids. Microbiol Rev. 1980 Jun;44(2):175–205. doi: 10.1128/mr.44.2.175-205.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brennan C. A., Dombroski A. J., Platt T. Transcription termination factor rho is an RNA-DNA helicase. Cell. 1987 Mar 27;48(6):945–952. doi: 10.1016/0092-8674(87)90703-3. [DOI] [PubMed] [Google Scholar]
  5. Daniels-McQueen S., Detjen B. M., Grifo J. A., Merrick W. C., Thach R. E. Unusual requirements for optimum translation of polio viral RNA in vitro. J Biol Chem. 1983 Jun 10;258(11):7195–7199. [PubMed] [Google Scholar]
  6. Dever T. E., Glynias M. J., Merrick W. C. GTP-binding domain: three consensus sequence elements with distinct spacing. Proc Natl Acad Sci U S A. 1987 Apr;84(7):1814–1818. doi: 10.1073/pnas.84.7.1814. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Duncan R., Milburn S. C., Hershey J. W. Regulated phosphorylation and low abundance of HeLa cell initiation factor eIF-4F suggest a role in translational control. Heat shock effects on eIF-4F. J Biol Chem. 1987 Jan 5;262(1):380–388. [PubMed] [Google Scholar]
  8. Edery I., Hümbelin M., Darveau A., Lee K. A., Milburn S., Hershey J. W., Trachsel H., Sonenberg N. Involvement of eukaryotic initiation factor 4A in the cap recognition process. J Biol Chem. 1983 Sep 25;258(18):11398–11403. [PubMed] [Google Scholar]
  9. Edery I., Lee K. A., Sonenberg N. Functional characterization of eukaryotic mRNA cap binding protein complex: effects on translation of capped and naturally uncapped RNAs. Biochemistry. 1984 May 22;23(11):2456–2462. doi: 10.1021/bi00306a021. [DOI] [PubMed] [Google Scholar]
  10. Etchison D., Hansen J., Ehrenfeld E., Edery I., Sonenberg N., Milburn S., Hershey J. W. Demonstration in vitro that eucaryotic initiation factor 3 is active but that a cap-binding protein complex is inactive in poliovirus-infected HeLa cells. J Virol. 1984 Sep;51(3):832–837. doi: 10.1128/jvi.51.3.832-837.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Ford M. J., Anton I. A., Lane D. P. Nuclear protein with sequence homology to translation initiation factor eIF-4A. Nature. 1988 Apr 21;332(6166):736–738. doi: 10.1038/332736a0. [DOI] [PubMed] [Google Scholar]
  12. Frisby D., Eaton M., Fellner P. Absence of 5' terminal capping in encephalomyocarditis virus RNA. Nucleic Acids Res. 1976 Oct;3(10):2771–2787. doi: 10.1093/nar/3.10.2771. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gehrke L., Auron P. E., Quigley G. J., Rich A., Sonenberg N. 5'-Conformation of capped alfalfa mosaic virus ribonucleic acid 4 may reflect its independence of the cap structure or of cap-binding protein for efficient translation. Biochemistry. 1983 Oct 25;22(22):5157–5164. doi: 10.1021/bi00291a015. [DOI] [PubMed] [Google Scholar]
  14. Geider K., Hoffmann-Berling H. Proteins controlling the helical structure of DNA. Annu Rev Biochem. 1981;50:233–260. doi: 10.1146/annurev.bi.50.070181.001313. [DOI] [PubMed] [Google Scholar]
  15. Goetz G. S., Dean F. B., Hurwitz J., Matson S. W. The unwinding of duplex regions in DNA by the simian virus 40 large tumor antigen-associated DNA helicase activity. J Biol Chem. 1988 Jan 5;263(1):383–392. [PubMed] [Google Scholar]
  16. Goss D. J., Woodley C. L., Wahba A. J. A fluorescence study of the binding of eucaryotic initiation factors to messenger RNA and messenger RNA analogues. Biochemistry. 1987 Mar 24;26(6):1551–1556. doi: 10.1021/bi00380a009. [DOI] [PubMed] [Google Scholar]
  17. Grabowski P. J., Padgett R. A., Sharp P. A. Messenger RNA splicing in vitro: an excised intervening sequence and a potential intermediate. Cell. 1984 Jun;37(2):415–427. doi: 10.1016/0092-8674(84)90372-6. [DOI] [PubMed] [Google Scholar]
  18. Grifo J. A., Abramson R. D., Satler C. A., Merrick W. C. RNA-stimulated ATPase activity of eukaryotic initiation factors. J Biol Chem. 1984 Jul 10;259(13):8648–8654. [PubMed] [Google Scholar]
  19. Grifo J. A., Tahara S. M., Morgan M. A., Shatkin A. J., Merrick W. C. New initiation factor activity required for globin mRNA translation. J Biol Chem. 1983 May 10;258(9):5804–5810. [PubMed] [Google Scholar]
  20. Herman R. C. Internal initiation of translation on the vesicular stomatitis virus phosphoprotein mRNA yields a second protein. J Virol. 1986 Jun;58(3):797–804. doi: 10.1128/jvi.58.3.797-804.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hewlett M. J., Rose J. K., Baltimore D. 5'-terminal structure of poliovirus polyribosomal RNA is pUp. Proc Natl Acad Sci U S A. 1976 Feb;73(2):327–330. doi: 10.1073/pnas.73.2.327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hirling H., Scheffner M., Restle T., Stahl H. RNA helicase activity associated with the human p68 protein. Nature. 1989 Jun 15;339(6225):562–564. doi: 10.1038/339562a0. [DOI] [PubMed] [Google Scholar]
  23. Jagus R., Anderson W. F., Safer B. The regulation of initiation of mammalian protein synthesis. Prog Nucleic Acid Res Mol Biol. 1981;25:127–185. doi: 10.1016/s0079-6603(08)60484-5. [DOI] [PubMed] [Google Scholar]
  24. Jang S. K., Kräusslich H. G., Nicklin M. J., Duke G. M., Palmenberg A. C., Wimmer E. A segment of the 5' nontranslated region of encephalomyocarditis virus RNA directs internal entry of ribosomes during in vitro translation. J Virol. 1988 Aug;62(8):2636–2643. doi: 10.1128/jvi.62.8.2636-2643.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Konarska M. M., Padgett R. A., Sharp P. A. Recognition of cap structure in splicing in vitro of mRNA precursors. Cell. 1984 Oct;38(3):731–736. doi: 10.1016/0092-8674(84)90268-x. [DOI] [PubMed] [Google Scholar]
  26. Kozak M. Leader length and secondary structure modulate mRNA function under conditions of stress. Mol Cell Biol. 1988 Jul;8(7):2737–2744. doi: 10.1128/mcb.8.7.2737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lawson T. G., Cladaras M. H., Ray B. K., Lee K. A., Abramson R. D., Merrick W. C., Thach R. E. Discriminatory interaction of purified eukaryotic initiation factors 4F plus 4A with the 5' ends of reovirus messenger RNAs. J Biol Chem. 1988 May 25;263(15):7266–7276. [PubMed] [Google Scholar]
  28. Lawson T. G., Lee K. A., Maimone M. M., Abramson R. D., Dever T. E., Merrick W. C., Thach R. E. Dissociation of double-stranded polynucleotide helical structures by eukaryotic initiation factors, as revealed by a novel assay. Biochemistry. 1989 May 30;28(11):4729–4734. doi: 10.1021/bi00437a033. [DOI] [PubMed] [Google Scholar]
  29. Lee M. S., Marians K. J. The Escherichia coli primosome can translocate actively in either direction along a DNA strand. J Biol Chem. 1989 Aug 25;264(24):14531–14542. [PubMed] [Google Scholar]
  30. Linder P., Lasko P. F., Ashburner M., Leroy P., Nielsen P. J., Nishi K., Schnier J., Slonimski P. P. Birth of the D-E-A-D box. Nature. 1989 Jan 12;337(6203):121–122. doi: 10.1038/337121a0. [DOI] [PubMed] [Google Scholar]
  31. Linder P., Slonimski P. P. An essential yeast protein, encoded by duplicated genes TIF1 and TIF2 and homologous to the mammalian translation initiation factor eIF-4A, can suppress a mitochondrial missense mutation. Proc Natl Acad Sci U S A. 1989 Apr;86(7):2286–2290. doi: 10.1073/pnas.86.7.2286. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Marcus A. Tobacco mosaic virus ribonucleic acid-dependent amino acid incorporation in a wheat embryo system in vitro. Analysis of the rate-limiting reaction. J Biol Chem. 1970 Mar 10;245(5):955–961. [PubMed] [Google Scholar]
  33. Milligan J. F., Groebe D. R., Witherell G. W., Uhlenbeck O. C. Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. Nucleic Acids Res. 1987 Nov 11;15(21):8783–8798. doi: 10.1093/nar/15.21.8783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Moldave K. Eukaryotic protein synthesis. Annu Rev Biochem. 1985;54:1109–1149. doi: 10.1146/annurev.bi.54.070185.005333. [DOI] [PubMed] [Google Scholar]
  35. Nielsen P. J., McMaster G. K., Trachsel H. Cloning of eukaryotic protein synthesis initiation factor genes: isolation and characterization of cDNA clones encoding factor eIF-4A. Nucleic Acids Res. 1985 Oct 11;13(19):6867–6880. doi: 10.1093/nar/13.19.6867. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Nomoto A., Lee Y. F., Wimmer E. The 5' end of poliovirus mRNA is not capped with m7G(5')ppp(5')Np. Proc Natl Acad Sci U S A. 1976 Feb;73(2):375–380. doi: 10.1073/pnas.73.2.375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Pain V. M. Initiation of protein synthesis in mammalian cells. Biochem J. 1986 May 1;235(3):625–637. doi: 10.1042/bj2350625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Peabody D. S., Subramani S., Berg P. Effect of upstream reading frames on translation efficiency in simian virus 40 recombinants. Mol Cell Biol. 1986 Jul;6(7):2704–2711. doi: 10.1128/mcb.6.7.2704. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Pelletier J., Sonenberg N. Insertion mutagenesis to increase secondary structure within the 5' noncoding region of a eukaryotic mRNA reduces translational efficiency. Cell. 1985 Mar;40(3):515–526. doi: 10.1016/0092-8674(85)90200-4. [DOI] [PubMed] [Google Scholar]
  40. Pelletier J., Sonenberg N. Internal initiation of translation of eukaryotic mRNA directed by a sequence derived from poliovirus RNA. Nature. 1988 Jul 28;334(6180):320–325. doi: 10.1038/334320a0. [DOI] [PubMed] [Google Scholar]
  41. Ray B. K., Lawson T. G., Kramer J. C., Cladaras M. H., Grifo J. A., Abramson R. D., Merrick W. C., Thach R. E. ATP-dependent unwinding of messenger RNA structure by eukaryotic initiation factors. J Biol Chem. 1985 Jun 25;260(12):7651–7658. [PubMed] [Google Scholar]
  42. Rhoads R. E. Cap recognition and the entry of mRNA into the protein synthesis initiation cycle. Trends Biochem Sci. 1988 Feb;13(2):52–56. doi: 10.1016/0968-0004(88)90028-x. [DOI] [PubMed] [Google Scholar]
  43. Rozen F., Pelletier J., Trachsel H., Sonenberg N. A lysine substitution in the ATP-binding site of eucaryotic initiation factor 4A abrogates nucleotide-binding activity. Mol Cell Biol. 1989 Sep;9(9):4061–4063. doi: 10.1128/mcb.9.9.4061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Sarkar G., Edery I., Sonenberg N. Photoaffinity labeling of the cap-binding protein complex with ATP/dATP. Differential labeling of free eukaryotic initiation factor 4A and the eukaryotic initiation factor 4A component of the cap-binding protein complex with [alpha-32P]ATP/dATP. J Biol Chem. 1985 Nov 5;260(25):13831–13837. [PubMed] [Google Scholar]
  45. Scheffner M., Knippers R., Stahl H. RNA unwinding activity of SV40 large T antigen. Cell. 1989 Jun 16;57(6):955–963. doi: 10.1016/0092-8674(89)90334-6. [DOI] [PubMed] [Google Scholar]
  46. Shatkin A. J. Capping of eucaryotic mRNAs. Cell. 1976 Dec;9(4 Pt 2):645–653. doi: 10.1016/0092-8674(76)90128-8. [DOI] [PubMed] [Google Scholar]
  47. Shatkin A. J. mRNA cap binding proteins: essential factors for initiating translation. Cell. 1985 Feb;40(2):223–224. doi: 10.1016/0092-8674(85)90132-1. [DOI] [PubMed] [Google Scholar]
  48. Smith K. R., Yancey J. E., Matson S. W. Identification and purification of a protein that stimulates the helicase activity of the Escherichia coli Rep protein. J Biol Chem. 1989 Apr 15;264(11):6119–6126. [PubMed] [Google Scholar]
  49. Sonenberg N. ATP/Mg++-dependent cross-linking of cap binding proteins to the 5' end of eukaryotic mRNA. Nucleic Acids Res. 1981 Apr 10;9(7):1643–1656. doi: 10.1093/nar/9.7.1643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Sonenberg N. Cap-binding proteins of eukaryotic messenger RNA: functions in initiation and control of translation. Prog Nucleic Acid Res Mol Biol. 1988;35:173–207. doi: 10.1016/s0079-6603(08)60614-5. [DOI] [PubMed] [Google Scholar]
  51. Sonenberg N., Guertin D., Cleveland D., Trachsel H. Probing the function of the eucaryotic 5' cap structure by using a monoclonal antibody directed against cap-binding proteins. Cell. 1981 Dec;27(3 Pt 2):563–572. doi: 10.1016/0092-8674(81)90398-6. [DOI] [PubMed] [Google Scholar]
  52. Sonenberg N., Morgan M. A., Merrick W. C., Shatkin A. J. A polypeptide in eukaryotic initiation factors that crosslinks specifically to the 5'-terminal cap in mRNA. Proc Natl Acad Sci U S A. 1978 Oct;75(10):4843–4847. doi: 10.1073/pnas.75.10.4843. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Sonenberg N., Shatkin A. J. Nonspecific effect of m7GMP on protein-RNA interactions. J Biol Chem. 1978 Oct 10;253(19):6630–6632. [PubMed] [Google Scholar]
  54. Stahl H., Dröge P., Knippers R. DNA helicase activity of SV40 large tumor antigen. EMBO J. 1986 Aug;5(8):1939–1944. doi: 10.1002/j.1460-2075.1986.tb04447.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Séraphin B., Simon M., Boulet A., Faye G. Mitochondrial splicing requires a protein from a novel helicase family. Nature. 1989 Jan 5;337(6202):84–87. doi: 10.1038/337084a0. [DOI] [PubMed] [Google Scholar]
  56. Tahara S. M., Morgan M. A., Shatkin A. J. Two forms of purified m7G-cap binding protein with different effects on capped mRNA translation in extracts of uninfected and poliovirus-infected HeLa cells. J Biol Chem. 1981 Aug 10;256(15):7691–7694. [PubMed] [Google Scholar]
  57. Thomas K. R., Capecchi M. R. Introduction of homologous DNA sequences into mammalian cells induces mutations in the cognate gene. Nature. 1986 Nov 6;324(6092):34–38. doi: 10.1038/324034a0. [DOI] [PubMed] [Google Scholar]
  58. Trachsel H., Sonenberg N., Shatkin A. J., Rose J. K., Leong K., Bergmann J. E., Gordon J., Baltimore D. Purification of a factor that restores translation of vesicular stomatitis virus mRNA in extracts from poliovirus-infected HeLa cells. Proc Natl Acad Sci U S A. 1980 Feb;77(2):770–774. doi: 10.1073/pnas.77.2.770. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Wahle E., Lasken R. S., Kornberg A. The dnaB-dnaC replication protein complex of Escherichia coli. II. Role of the complex in mobilizing dnaB functions. J Biol Chem. 1989 Feb 15;264(5):2469–2475. [PubMed] [Google Scholar]
  60. Walker J. E., Saraste M., Runswick M. J., Gay N. J. Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J. 1982;1(8):945–951. doi: 10.1002/j.1460-2075.1982.tb01276.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Wickner S., Hurwitz J. Interaction of Escherichia coli dnaB and dnaC(D) gene products in vitro. Proc Natl Acad Sci U S A. 1975 Mar;72(3):921–925. doi: 10.1073/pnas.72.3.921. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Wold M. S., Kelly T. Purification and characterization of replication protein A, a cellular protein required for in vitro replication of simian virus 40 DNA. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2523–2527. doi: 10.1073/pnas.85.8.2523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Wood E. R., Matson S. W. Purification and characterization of a new DNA-dependent ATPase with helicase activity from Escherichia coli. J Biol Chem. 1987 Nov 5;262(31):15269–15276. [PubMed] [Google Scholar]

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

RESOURCES