Skip to main content
PMC home page
RNA logoLink to RNA
. 2002 Mar;8(3):382–397. doi: 10.1017/s1355838202029527

Development and characterization of a reconstituted yeast translation initiation system.

Mikkel A Algire 1, David Maag 1, Peter Savio 1, Michael G Acker 1, Salvador Z Tarun Jr 1, Alan B Sachs 1, Katsura Asano 1, Klaus H Nielsen 1, Deanne S Olsen 1, Lon Phan 1, Alan G Hinnebusch 1, Jon R Lorsch 1
PMCID: PMC1370259  PMID: 12008673

Abstract

To provide a bridge between in vivo and in vitro studies of eukaryotic translation initiation, we have developed a reconstituted translation initiation system using components from the yeast Saccharomyces cerevisiae. We have purified a minimal set of initiation factors (elFs) that, together with yeast 80S ribosomes, GTP, and initiator methionyl-tRNA, are sufficient to assemble active initiation complexes on a minimal mRNA template. The kinetics of various steps in the pathway of initiation complex assembly and the formation of the first peptide bond in vitro have been explored. The formation of active initiation complexes in this system is dependent on ribosomes, mRNA, Met-tRNAi, GTP hydrolysis, elF1, elF1A, elF2, elF5, and elF5B. Our data indicate that elF1 and elF1A both facilitate the binding of the elF2 x GTP x Met-tRNAi complex to the 40S ribosomal subunit to form the 43S complex. elF5 stimulates a step after 43S complex formation, consistent with its proposed role in activating GTP hydrolysis by elF2 upon initiation codon recognition. The presence of elF5B is required for the joining of the 40S and 60S subunits to form the 80S initiation complex. The step at which each of these factors acts in this reconstituted system is in agreement with previous data from in vivo studies and work using reconstituted mammalian systems, indicating that the system recapitulates fundamental events in translation initiation in eukaryotic cells. This system should allow us to couple powerful yeast genetic and molecular biological experiments with in vitro kinetic and biophysical experiments, yielding a better understanding of the molecular mechanics of this central, complex process.

Full Text

The Full Text of this article is available as a PDF (2.1 MB).

Selected References

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

  1. Benne R., Hershey J. W. The mechanism of action of protein synthesis initiation factors from rabbit reticulocytes. J Biol Chem. 1978 May 10;253(9):3078–3087. [PubMed] [Google Scholar]
  2. Chaudhuri J., Chowdhury D., Maitra U. Distinct functions of eukaryotic translation initiation factors eIF1A and eIF3 in the formation of the 40 S ribosomal preinitiation complex. J Biol Chem. 1999 Jun 18;274(25):17975–17980. doi: 10.1074/jbc.274.25.17975. [DOI] [PubMed] [Google Scholar]
  3. Chaudhuri J., Si K., Maitra U. Function of eukaryotic translation initiation factor 1A (eIF1A) (formerly called eIF-4C) in initiation of protein synthesis. J Biol Chem. 1997 Mar 21;272(12):7883–7891. doi: 10.1074/jbc.272.12.7883. [DOI] [PubMed] [Google Scholar]
  4. Choi S. K., Lee J. H., Zoll W. L., Merrick W. C., Dever T. E. Promotion of met-tRNAiMet binding to ribosomes by yIF2, a bacterial IF2 homolog in yeast. Science. 1998 Jun 12;280(5370):1757–1760. doi: 10.1126/science.280.5370.1757. [DOI] [PubMed] [Google Scholar]
  5. Erickson F. L., Hannig E. M. Ligand interactions with eukaryotic translation initiation factor 2: role of the gamma-subunit. EMBO J. 1996 Nov 15;15(22):6311–6320. [PMC free article] [PubMed] [Google Scholar]
  6. Huang H. K., Yoon H., Hannig E. M., Donahue T. F. GTP hydrolysis controls stringent selection of the AUG start codon during translation initiation in Saccharomyces cerevisiae. Genes Dev. 1997 Sep 15;11(18):2396–2413. doi: 10.1101/gad.11.18.2396. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Johnson A. E., Adkins H. J. Glycerol, sucrose, and other diol-containing reagents are not inert components in in vitro incubations containing aminoacyl-tRNA. Anal Biochem. 1984 Mar;137(2):351–359. doi: 10.1016/0003-2697(84)90097-6. [DOI] [PubMed] [Google Scholar]
  8. Lorsch J. R., Herschlag D. Kinetic dissection of fundamental processes of eukaryotic translation initiation in vitro. EMBO J. 1999 Dec 1;18(23):6705–6717. doi: 10.1093/emboj/18.23.6705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Matasova N. B., Myltseva S. V., Zenkova M. A., Graifer D. M., Vladimirov S. N., Karpova G. G. Isolation of ribosomal subunits containing intact rRNA from human placenta: estimation of functional activity of 80S ribosomes. Anal Biochem. 1991 Nov 1;198(2):219–223. doi: 10.1016/0003-2697(91)90416-q. [DOI] [PubMed] [Google Scholar]
  10. Morley S. J., Hershey J. W. A fractionated reticulocyte lysate retains high efficiency for protein synthesis. Biochimie. 1990 Apr;72(4):259–264. doi: 10.1016/0300-9084(90)90081-q. [DOI] [PubMed] [Google Scholar]
  11. Nika J., Yang W., Pavitt G. D., Hinnebusch A. G., Hannig E. M. Purification and kinetic analysis of eIF2B from Saccharomyces cerevisiae. J Biol Chem. 2000 Aug 25;275(34):26011–26017. doi: 10.1074/jbc.M003718200. [DOI] [PubMed] [Google Scholar]
  12. Palmiter R. D. Quantitation of parameters that determine the rate of ovalbumin synthesis. Cell. 1975 Mar;4(3):189–189. doi: 10.1016/0092-8674(75)90167-1. [DOI] [PubMed] [Google Scholar]
  13. Pavitt G. D., Ramaiah K. V., Kimball S. R., Hinnebusch A. G. eIF2 independently binds two distinct eIF2B subcomplexes that catalyze and regulate guanine-nucleotide exchange. Genes Dev. 1998 Feb 15;12(4):514–526. doi: 10.1101/gad.12.4.514. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Pestova T. V., Borukhov S. I., Hellen C. U. Eukaryotic ribosomes require initiation factors 1 and 1A to locate initiation codons. Nature. 1998 Aug 27;394(6696):854–859. doi: 10.1038/29703. [DOI] [PubMed] [Google Scholar]
  15. Pestova T. V., Lomakin I. B., Lee J. H., Choi S. K., Dever T. E., Hellen C. U. The joining of ribosomal subunits in eukaryotes requires eIF5B. Nature. 2000 Jan 20;403(6767):332–335. doi: 10.1038/35002118. [DOI] [PubMed] [Google Scholar]
  16. Phan L., Schoenfeld L. W., Valásek L., Nielsen K. H., Hinnebusch A. G. A subcomplex of three eIF3 subunits binds eIF1 and eIF5 and stimulates ribosome binding of mRNA and tRNA(i)Met. EMBO J. 2001 Jun 1;20(11):2954–2965. doi: 10.1093/emboj/20.11.2954. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Phan L., Zhang X., Asano K., Anderson J., Vornlocher H. P., Greenberg J. R., Qin J., Hinnebusch A. G. Identification of a translation initiation factor 3 (eIF3) core complex, conserved in yeast and mammals, that interacts with eIF5. Mol Cell Biol. 1998 Aug;18(8):4935–4946. doi: 10.1128/mcb.18.8.4935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Rosa M. D., Sigler P. B. Isolation and characterization of two methionine: tRNA ligases from wheat germ. Eur J Biochem. 1977 Aug 15;78(1):141–151. doi: 10.1111/j.1432-1033.1977.tb11723.x. [DOI] [PubMed] [Google Scholar]
  19. Thomas A., Goumans H., Voorma H. O., Benne R. The mechanism of action of eukaryotic initiation factor 4C in protein synthesis. Eur J Biochem. 1980;107(1):39–45. doi: 10.1111/j.1432-1033.1980.tb04621.x. [DOI] [PubMed] [Google Scholar]
  20. Thomas A., Spaan W., van Steeg H., Voorma H. O., Benne R. Mode of action of protein synthesis initiation factor eIF-1 from rabbit reticulocytes. FEBS Lett. 1980 Jul 11;116(1):67–71. doi: 10.1016/0014-5793(80)80530-8. [DOI] [PubMed] [Google Scholar]
  21. Trachsel H., Erni B., Schreier M. H., Staehelin T. Initiation of mammalian protein synthesis. II. The assembly of the initiation complex with purified initiation factors. J Mol Biol. 1977 Nov;116(4):755–767. doi: 10.1016/0022-2836(77)90269-8. [DOI] [PubMed] [Google Scholar]
  22. Valásek L., Phan L., Schoenfeld L. W., Valásková V., Hinnebusch A. G. Related eIF3 subunits TIF32 and HCR1 interact with an RNA recognition motif in PRT1 required for eIF3 integrity and ribosome binding. EMBO J. 2001 Feb 15;20(4):891–904. doi: 10.1093/emboj/20.4.891. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. van Hoof A., Lennertz P., Parker R. Three conserved members of the RNase D family have unique and overlapping functions in the processing of 5S, 5.8S, U4, U5, RNase MRP and RNase P RNAs in yeast. EMBO J. 2000 Mar 15;19(6):1357–1365. doi: 10.1093/emboj/19.6.1357. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from RNA are provided here courtesy of The RNA Society

RESOURCES