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. 1985 Jan;82(2):330–333. doi: 10.1073/pnas.82.2.330

Isolation and characterization of factors from wheat germ that exhibit eukaryotic initiation factor 4B activity and overcome 7-methylguanosine 5'-triphosphate inhibition of polypeptide synthesis.

S Lax, W Fritz, K Browning, J Ravel
PMCID: PMC397031  PMID: 3855554

Abstract

Three highly purified preparations (preparations I, II-1, and II-2) have been obtained from wheat germ and shown to support in vitro polypeptide synthesis directed by capped or uncapped mRNAs in a eukaryotic initiation factor 4B (eIF-4B)-deficient system. The three preparations differ, however, in polypeptide composition and in the ability to overcome the inhibitory effect of 7-methylguanosine 5'-triphosphate (m7GTP) on in vitro polypeptide synthesis. Preparation I contains two polypeptides (Mr = 80,000 and 28,000), which are present in a 1:1 molar ratio and are associated in a high molecular weight complex. Preparation II-1 contains two major polypeptides (Mr = 220,000 and 26,000) and preparation II-2 also contains two major polypeptides (Mr = 110,000 and 26,000). Preparations II-1 and II-2 are high molecular weight complexes; neither contains detectable amounts of a Mr 80,000 or a Mr 50,000 component. Preparations II-1 and II-2 both overcome m7GTP inhibition, whereas preparation I does not. These findings raise several questions with regard to the identity of eIF-4B and its relationship to cap recognition factors.

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Selected References

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  1. Benne R., Luedi M., Hershey J. W. Purification and characterization of initiation factors IF-E4 and IF-E6 from rabbit reticulocytes. J Biol Chem. 1977 Aug 25;252(16):5798–5803. [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  3. Brown-Luedi M. L., Meyer L. J., Milburn S. C., Yau P. M., Corbett S., Hershey J. W. Protein synthesis initiation factors from human HeLa cells and rabbit reticulocytes are similar: comparison of protein structure, activities, and immunochemical properties. Biochemistry. 1982 Aug 31;21(18):4202–4206. doi: 10.1021/bi00261a002. [DOI] [PubMed] [Google Scholar]
  4. Checkley J. W., Cooley L., Ravel J. M. Characterization of initiation factor eIF-3 from wheat germ. J Biol Chem. 1981 Feb 25;256(4):1582–1586. [PubMed] [Google Scholar]
  5. Clark J. M., Jr, Klein W. H. In vitro translation of STNV-RNA. Methods Enzymol. 1974;30:754–761. doi: 10.1016/0076-6879(74)30074-2. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. 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]
  9. Hellmann G. M., Chu L. Y., Rhoads R. E. A polypeptide which reverses cap analogue inhibition of cell-free protein synthesis. Purification and binding to capped oligonucleotides. J Biol Chem. 1982 Apr 25;257(8):4056–4062. [PubMed] [Google Scholar]
  10. 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]
  11. Merrick W. C. Purification of protein synthesis initiation factors from rabbit reticulocytes. Methods Enzymol. 1979;60:101–108. doi: 10.1016/s0076-6879(79)60010-1. [DOI] [PubMed] [Google Scholar]
  12. Schreier M. H., Erni B., Staehelin T. Initiation of mammalian protein synthesis. I. Purification and characterization of seven initiation factors. J Mol Biol. 1977 Nov;116(4):727–753. doi: 10.1016/0022-2836(77)90268-6. [DOI] [PubMed] [Google Scholar]
  13. Seal S. N., Schmidt A., Marcus A. Fractionation and partial characterization of the protein synthesis system of wheat germ. I. Resolution of two elongation factors and five initiation factors. J Biol Chem. 1983 Jan 25;258(2):859–865. [PubMed] [Google Scholar]
  14. 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]
  15. Sonenberg N., Rupprecht K. M., Hecht S. M., Shatkin A. J. Eukaryotic mRNA cap binding protein: purification by affinity chromatography on sepharose-coupled m7GDP. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4345–4349. doi: 10.1073/pnas.76.9.4345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Sonenberg N., Trachsel H., Hecht S., Shatkin A. J. Differential stimulation of capped mRNA translation in vitro by cap binding protein. Nature. 1980 May 29;285(5763):331–333. doi: 10.1038/285331a0. [DOI] [PubMed] [Google Scholar]
  17. Spremulli L. L., Walthall B. J., Lax S. R., Ravel J. M. Partial purification of the factors required for the initiation of protein synthesis in wheat germ. J Biol Chem. 1979 Jan 10;254(1):143–148. [PubMed] [Google Scholar]
  18. 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]
  19. Trachsel H., Erni B., Schreier M. H., Braun L., Staehelin T. Purification of seven protein synthesis initiation factors from Krebs II ascites cells. Biochim Biophys Acta. 1979 Feb 27;561(2):484–490. doi: 10.1016/0005-2787(79)90156-4. [DOI] [PubMed] [Google Scholar]
  20. Walthall B. J., Spremulli L. L., Lax S. R., Ravel J. M. Isolation and purification of protein synthesis initiation factors from wheat germ. Methods Enzymol. 1979;60:193–204. doi: 10.1016/s0076-6879(79)60016-2. [DOI] [PubMed] [Google Scholar]
  21. Webb N. R., Chari R. V., DePillis G., Kozarich J. W., Rhoads R. E. Purification of the messenger RNA cap-binding protein using a new affinity medium. Biochemistry. 1984 Jan 17;23(2):177–181. doi: 10.1021/bi00297a001. [DOI] [PubMed] [Google Scholar]

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