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. 1996 Aug;16(8):4248–4256. doi: 10.1128/mcb.16.8.4248

The A1 x U72 base pair conserved in eukaryotic initiator tRNAs is important specifically for binding to the eukaryotic translation initiation factor eIF2.

D Farruggio 1, J Chaudhuri 1, U Maitra 1, U L RajBhandary 1
PMCID: PMC231423  PMID: 8754825

Abstract

The formation of a specific ternary complex between eukaryotic initiation factor 2 (eIF2), the initiator methionyl-tRNA (Met-tRNA), and GTP is a critical step in translation initiation in the cytoplasmic protein-synthesizing system of eukaryotes. We show that the A1 x U72 base pair conserved at the end of the acceptor stem in eukaryotic and archaebacterial initiator methionine tRNAs plays an important role in this interaction. We changed the A1 x U72 base pair of the human initiator tRNA to G1 x C72 and expressed the wild-type and mutant tRNA genes in the yeast Saccharomyces cerevisiae by using constructs previously developed in our laboratory for expression of the human initiator tRNA gene in yeasts. We show that both the wild-type and mutant human initiator tRNAs are aminoacylated well in vivo. We have isolated the wild-type and mutant human initiator tRNAs in substantially pure form, free of the yeast initiator tRNA, and have analyzed their properties in vitro. The G1 x C72 mutation affects specifically the binding affinity of eIF2 for the initiator tRNA. It has no effect on the subsequent formation of 40S or 80S ribosome initiator Met-tRNA-AUG initiation complexes in vitro or on the puromycin reactivity of the Met-tRNA in the 80S initiation complex.

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Selected References

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  1. Beggs J. D. Transformation of yeast by a replicating hybrid plasmid. Nature. 1978 Sep 14;275(5676):104–109. doi: 10.1038/275104a0. [DOI] [PubMed] [Google Scholar]
  2. Botstein D., Falco S. C., Stewart S. E., Brennan M., Scherer S., Stinchcomb D. T., Struhl K., Davis R. W. Sterile host yeasts (SHY): a eukaryotic system of biological containment for recombinant DNA experiments. Gene. 1979 Dec;8(1):17–24. doi: 10.1016/0378-1119(79)90004-0. [DOI] [PubMed] [Google Scholar]
  3. Chakrabarti A., Maitra U. Function of eukaryotic initiation factor 5 in the formation of an 80 S ribosomal polypeptide chain initiation complex. J Biol Chem. 1991 Jul 25;266(21):14039–14045. [PubMed] [Google Scholar]
  4. Chapman K. B., Byström A. S., Boeke J. D. Initiator methionine tRNA is essential for Ty1 transposition in yeast. Proc Natl Acad Sci U S A. 1992 Apr 15;89(8):3236–3240. doi: 10.1073/pnas.89.8.3236. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chaudhuri A., Stringer E. A., Valenzuela D., Maitra U. Characterization of eukaryotic initiation factor 2 containing two polypeptide chains of Mr = 48,000 and 38,000. J Biol Chem. 1981 Apr 25;256(8):3988–3994. [PubMed] [Google Scholar]
  6. Chaudhuri J., Das K., Maitra U. Purification and characterization of bacterially expressed mammalian translation initiation factor 5 (eIF-5): demonstration that eIF-5 forms a specific complex with eIF-2. Biochemistry. 1994 Apr 26;33(16):4794–4799. doi: 10.1021/bi00182a007. [DOI] [PubMed] [Google Scholar]
  7. Chevesich J., Chaudhuri J., Maitra U. Characterization of mammalian translation initiation factor 5 (eIF-5). Demonstration that eIF-5 is a phosphoprotein and is present in cells as a single molecular form of apparent M(r) 58,000. J Biol Chem. 1993 Sep 25;268(27):20659–20667. [PubMed] [Google Scholar]
  8. Cigan A. M., Feng L., Donahue T. F. tRNAi(met) functions in directing the scanning ribosome to the start site of translation. Science. 1988 Oct 7;242(4875):93–97. doi: 10.1126/science.3051379. [DOI] [PubMed] [Google Scholar]
  9. Despons L., Walter P., Senger B., Ebel J. P., Fasiolo F. Identification of potential amino acid residues supporting anticodon recognition in yeast methionyl-tRNA synthetase. FEBS Lett. 1991 Sep 9;289(2):217–220. doi: 10.1016/0014-5793(91)81073-h. [DOI] [PubMed] [Google Scholar]
  10. Dever T. E., Yang W., Aström S., Byström A. S., Hinnebusch A. G. Modulation of tRNA(iMet), eIF-2, and eIF-2B expression shows that GCN4 translation is inversely coupled to the level of eIF-2.GTP.Met-tRNA(iMet) ternary complexes. Mol Cell Biol. 1995 Nov;15(11):6351–6363. doi: 10.1128/mcb.15.11.6351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dholakia J. N., Wahba A. J. The isolation and characterization from rabbit reticulocytes of two forms of eukaryotic initiation factor 2 having different beta-polypeptides. J Biol Chem. 1987 Jul 25;262(21):10164–10170. [PubMed] [Google Scholar]
  12. Donahue T. F., Cigan A. M., Pabich E. K., Valavicius B. C. Mutations at a Zn(II) finger motif in the yeast eIF-2 beta gene alter ribosomal start-site selection during the scanning process. Cell. 1988 Aug 26;54(5):621–632. doi: 10.1016/s0092-8674(88)80006-0. [DOI] [PubMed] [Google Scholar]
  13. Drabkin H. J., Helk B., RajBhandary U. L. The role of nucleotides conserved in eukaryotic initiator methionine tRNAs in initiation of protein synthesis. J Biol Chem. 1993 Nov 25;268(33):25221–25228. [PubMed] [Google Scholar]
  14. Drabkin H. J., RajBhandary U. L. Attempted expression of a human initiator tRNA gene in Saccharomyces cerevisiae. J Biol Chem. 1985 May 10;260(9):5596–5602. [PubMed] [Google Scholar]
  15. Erhart E., Hollenberg C. P. The presence of a defective LEU2 gene on 2 mu DNA recombinant plasmids of Saccharomyces cerevisiae is responsible for curing and high copy number. J Bacteriol. 1983 Nov;156(2):625–635. doi: 10.1128/jb.156.2.625-635.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Francis M. A., Rajbhandary U. L. Expression and function of a human initiator tRNA gene in the yeast Saccharomyces cerevisiae. Mol Cell Biol. 1990 Sep;10(9):4486–4494. doi: 10.1128/mcb.10.9.4486. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gillum A. M., Roe B. A., Anandaraj M. P., RajBhandary U. L. Nucleotide sequence of human placenta cytoplasmic initiator tRNA. Cell. 1975 Nov;6(3):407–413. doi: 10.1016/0092-8674(75)90190-7. [DOI] [PubMed] [Google Scholar]
  18. Gillum A. M., Urquhart N., Smith M., RajBhandary U. L. Nucleotide sequence of salmon testes and salmon liver cytoplasmic initiator tRNA. Cell. 1975 Nov;6(3):395–405. doi: 10.1016/0092-8674(75)90189-0. [DOI] [PubMed] [Google Scholar]
  19. Gupta R. Halobacterium volcanii tRNAs. Identification of 41 tRNAs covering all amino acids, and the sequences of 33 class I tRNAs. J Biol Chem. 1984 Aug 10;259(15):9461–9471. [PubMed] [Google Scholar]
  20. Hershey J. W. Protein phosphorylation controls translation rates. J Biol Chem. 1989 Dec 15;264(35):20823–20826. [PubMed] [Google Scholar]
  21. Housman D., Jacobs-Lorena M., Rajbhandary U. L., Lodish H. F. Initiation of haemoglobin synthesis by methionyl-tRNA. Nature. 1970 Aug 29;227(5261):913–918. doi: 10.1038/227913a0. [DOI] [PubMed] [Google Scholar]
  22. Kaempfer R., Hollender R., Abrams W. R., Israeli R. Specific binding of messenger RNA and methionyl-tRNAfMet by the same initiation factor for eukaryotic protein synthesis. Proc Natl Acad Sci U S A. 1978 Jan;75(1):209–213. doi: 10.1073/pnas.75.1.209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Keeling P. J., Doolittle W. F. Archaea: narrowing the gap between prokaryotes and eukaryotes. Proc Natl Acad Sci U S A. 1995 Jun 20;92(13):5761–5764. doi: 10.1073/pnas.92.13.5761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Keith G., Heitzler J., el Adlouni C., Glasser A. L., Fix C., Desgrès J., Dirheimer G. The primary structure of cytoplasmic initiator tRNA(Met) from Schizosaccharomyces pombe. Nucleic Acids Res. 1993 Jun 25;21(12):2949–2949. doi: 10.1093/nar/21.12.2949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Konieczny A., Safer B. Purification of the eukaryotic initiation factor 2-eukaryotic initiation factor 2B complex and characterization of its guanine nucleotide exchange activity during protein synthesis initiation. J Biol Chem. 1983 Mar 10;258(5):3402–3408. [PubMed] [Google Scholar]
  26. Kozak M. Comparison of initiation of protein synthesis in procaryotes, eucaryotes, and organelles. Microbiol Rev. 1983 Mar;47(1):1–45. doi: 10.1128/mr.47.1.1-45.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kozak M. The scanning model for translation: an update. J Cell Biol. 1989 Feb;108(2):229–241. doi: 10.1083/jcb.108.2.229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lane D., Prentki P., Chandler M. Use of gel retardation to analyze protein-nucleic acid interactions. Microbiol Rev. 1992 Dec;56(4):509–528. doi: 10.1128/mr.56.4.509-528.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lee C. P., Dyson M. R., Mandal N., Varshney U., Bahramian B., RajBhandary U. L. Striking effects of coupling mutations in the acceptor stem on recognition of tRNAs by Escherichia coli Met-tRNA synthetase and Met-tRNA transformylase. Proc Natl Acad Sci U S A. 1992 Oct 1;89(19):9262–9266. doi: 10.1073/pnas.89.19.9262. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Manchester K. L. Evaluation and significance of kinetic parameters governing function of protein synthesis initiation factors eIF-2 and eIF-2B. FEBS Lett. 1985 Mar 11;182(1):15–19. doi: 10.1016/0014-5793(85)81144-3. [DOI] [PubMed] [Google Scholar]
  31. Merrick W. C. Assays for eukaryotic protein synthesis. Methods Enzymol. 1979;60:108–123. doi: 10.1016/s0076-6879(79)60011-3. [DOI] [PubMed] [Google Scholar]
  32. Merrick W. C. Mechanism and regulation of eukaryotic protein synthesis. Microbiol Rev. 1992 Jun;56(2):291–315. doi: 10.1128/mr.56.2.291-315.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. RajBhandary U. L., Ghosh H. P. Studies on polynucleotides. XCI. Yeast methionine transfer ribonucleic acid: purification, properties, and terminal nucleotide sequences. J Biol Chem. 1969 Mar 10;244(5):1104–1113. [PubMed] [Google Scholar]
  34. RajBhandary U. L. Initiator transfer RNAs. J Bacteriol. 1994 Feb;176(3):547–552. doi: 10.1128/jb.176.3.547-552.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Roy A. L., Chakrabarti D., Datta B., Hileman R. E., Gupta N. K. Natural mRNA is required for directing Met-tRNA(f) binding to 40S ribosomal subunits in animal cells: involvement of Co-eIF-2A in natural mRNA-directed initiation complex formation. Biochemistry. 1988 Oct 18;27(21):8203–8209. doi: 10.1021/bi00421a033. [DOI] [PubMed] [Google Scholar]
  36. Schulman L. H., Pelka H. In vitro conversion of a methionine to a glutamine-acceptor tRNA. Biochemistry. 1985 Dec 3;24(25):7309–7314. doi: 10.1021/bi00346a043. [DOI] [PubMed] [Google Scholar]
  37. Schulman L. H. Recognition of tRNAs by aminoacyl-tRNA synthetases. Prog Nucleic Acid Res Mol Biol. 1991;41:23–87. [PubMed] [Google Scholar]
  38. Simsek M., RajBhandary U. L., Boisnard M., Petrissant G. Nucleotide sequence of rabbit liver and sheep mammary gland cytoplasmic initiatory transfer RNAs. Nature. 1974 Feb 22;247(5442):518–520. doi: 10.1038/247518a0. [DOI] [PubMed] [Google Scholar]
  39. Simsek M., RajBhandary U. L. The primary structure of yeast initiator transfer ribonucleic acid. Biochem Biophys Res Commun. 1972 Oct 17;49(2):508–515. doi: 10.1016/0006-291x(72)90440-8. [DOI] [PubMed] [Google Scholar]
  40. Smith A. E., Marcker K. A. Cytoplasmic methionine transfer RNAs from eukaryotes. Nature. 1970 May 16;226(5246):607–610. doi: 10.1038/226607a0. [DOI] [PubMed] [Google Scholar]
  41. Sonenberg N. Remarks on the mechanism of ribosome binding to eukaryotic mRNAs. Gene Expr. 1993;3(3):317–323. [PMC free article] [PubMed] [Google Scholar]
  42. 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]
  43. Steinberg S., Misch A., Sprinzl M. Compilation of tRNA sequences and sequences of tRNA genes. Nucleic Acids Res. 1993 Jul 1;21(13):3011–3015. doi: 10.1093/nar/21.13.3011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Varshney U., Lee C. P., RajBhandary U. L. Direct analysis of aminoacylation levels of tRNAs in vivo. Application to studying recognition of Escherichia coli initiator tRNA mutants by glutaminyl-tRNA synthetase. J Biol Chem. 1991 Dec 25;266(36):24712–24718. [PubMed] [Google Scholar]
  45. Varshney U., RajBhandary U. L. Initiation of protein synthesis from a termination codon. Proc Natl Acad Sci U S A. 1990 Feb;87(4):1586–1590. doi: 10.1073/pnas.87.4.1586. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Wagner T., Gross M., Sigler P. B. Isoleucyl initiator tRNA does not initiate eucaryotic protein synthesis. J Biol Chem. 1984 Apr 25;259(8):4706–4709. [PubMed] [Google Scholar]
  47. von Pawel-Rammingen U., Aström S., Byström A. S. Mutational analysis of conserved positions potentially important for initiator tRNA function in Saccharomyces cerevisiae. Mol Cell Biol. 1992 Apr;12(4):1432–1442. doi: 10.1128/mcb.12.4.1432. [DOI] [PMC free article] [PubMed] [Google Scholar]

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