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. 1993 Jul;175(14):4507–4514. doi: 10.1128/jb.175.14.4507-4514.1993

Importance of formylability and anticodon stem sequence to give a tRNA(Met) an initiator identity in Escherichia coli.

J M Guillon 1, Y Mechulam 1, S Blanquet 1, G Fayat 1
PMCID: PMC204892  PMID: 8331078

Abstract

In bacteria, the free amino group of the methionylated initiator tRNA is specifically modified by the addition of a formyl group. The functional relevance of such a formylation for the initiation of translation is not yet precisely understood. Advantage was taken here of the availability of the fmt gene, encoding the Escherichia coli Met-tRNA(fMet) formyltransferase, to measure the influence of variations in the level of formyltransferase activity on the involvement of various mutant tRNA(fMet) and tRNA(mMet) species in either initiation or elongation in vivo. The data obtained established that formylation plays a dual role, firstly, by dictating tRNA(fMet) to engage in the initiation of translation, and secondly, by preventing the misappropriation of this tRNA by the elongation apparatus. The importance of formylation in the initiator identity of tRNA(fMet) was further shown by the demonstration that elongator tRNA(fMet) may be used in initiation and no longer in elongation, provided that it is mutated into a formylatable species and is given the three G.C base pairs characteristic of the anticodon stem of initiator tRNAs.

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Selected References

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

  1. Calos M. P., Lebkowski J. S., Botchan M. R. High mutation frequency in DNA transfected into mammalian cells. Proc Natl Acad Sci U S A. 1983 May;80(10):3015–3019. doi: 10.1073/pnas.80.10.3015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chattapadhyay R., Pelka H., Schulman L. H. Initiation of in vivo protein synthesis with non-methionine amino acids. Biochemistry. 1990 May 8;29(18):4263–4268. doi: 10.1021/bi00470a001. [DOI] [PubMed] [Google Scholar]
  4. Csonka L. N., Clark A. J. Construction of an Hfr strain useful for transferring recA mutations between Escherichia coli strains. J Bacteriol. 1980 Jul;143(1):529–530. doi: 10.1128/jb.143.1.529-530.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Eisenstadt J., Lengyel P. Formylmethionyl-tRNA dependence of amino acid incorporation in extracts of trimethoprim-treated Escherichia coli. Science. 1966 Oct 28;154(3748):524–527. [PubMed] [Google Scholar]
  6. Guillon J. M., Mechulam Y., Schmitter J. M., Blanquet S., Fayat G. Disruption of the gene for Met-tRNA(fMet) formyltransferase severely impairs growth of Escherichia coli. J Bacteriol. 1992 Jul;174(13):4294–4301. doi: 10.1128/jb.174.13.4294-4301.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Guillon J. M., Meinnel T., Mechulam Y., Lazennec C., Blanquet S., Fayat G. Nucleotides of tRNA governing the specificity of Escherichia coli methionyl-tRNA(fMet) formyltransferase. J Mol Biol. 1992 Mar 20;224(2):359–367. doi: 10.1016/0022-2836(92)91000-f. [DOI] [PubMed] [Google Scholar]
  8. Hamilton C. M., Aldea M., Washburn B. K., Babitzke P., Kushner S. R. New method for generating deletions and gene replacements in Escherichia coli. J Bacteriol. 1989 Sep;171(9):4617–4622. doi: 10.1128/jb.171.9.4617-4622.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hartz D., Binkley J., Hollingsworth T., Gold L. Domains of initiator tRNA and initiation codon crucial for initiator tRNA selection by Escherichia coli IF3. Genes Dev. 1990 Oct;4(10):1790–1800. doi: 10.1101/gad.4.10.1790. [DOI] [PubMed] [Google Scholar]
  10. Hartz D., McPheeters D. S., Gold L. Selection of the initiator tRNA by Escherichia coli initiation factors. Genes Dev. 1989 Dec;3(12A):1899–1912. doi: 10.1101/gad.3.12a.1899. [DOI] [PubMed] [Google Scholar]
  11. Janiak F., Dell V. A., Abrahamson J. K., Watson B. S., Miller D. L., Johnson A. E. Fluorescence characterization of the interaction of various transfer RNA species with elongation factor Tu.GTP: evidence for a new functional role for elongation factor Tu in protein biosynthesis. Biochemistry. 1990 May 8;29(18):4268–4277. doi: 10.1021/bi00470a002. [DOI] [PubMed] [Google Scholar]
  12. Kung H. F., Eskin B., Redfield B., Weissbach H. DNA-directed in vitro synthesis of beta-galactosidase: requirement for formylation of methionyl-tRNAf. Arch Biochem Biophys. 1979 Jul;195(2):396–400. doi: 10.1016/0003-9861(79)90366-7. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Lee C. P., Seong B. L., RajBhandary U. L. Structural and sequence elements important for recognition of Escherichia coli formylmethionine tRNA by methionyl-tRNA transformylase are clustered in the acceptor stem. J Biol Chem. 1991 Sep 25;266(27):18012–18017. [PubMed] [Google Scholar]
  15. Meinnel T., Mechulam Y., Fayat G., Blanquet S. Involvement of the size and sequence of the anticodon loop in tRNA recognition by mammalian and E. coli methionyl-tRNA synthetases. Nucleic Acids Res. 1992 Sep 25;20(18):4741–4746. doi: 10.1093/nar/20.18.4741. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Meinnel T., Mechulam Y., Fayat G. Fast purification of a functional elongator tRNAmet expressed from a synthetic gene in vivo. Nucleic Acids Res. 1988 Aug 25;16(16):8095–8096. doi: 10.1093/nar/16.16.8095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Meinnel T., Mechulam Y., Lazennec C., Blanquet S., Fayat G. Critical role of the acceptor stem of tRNAs(Met) in their aminoacylation by Escherichia coli methionyl-tRNA synthetase. J Mol Biol. 1993 Jan 5;229(1):26–36. doi: 10.1006/jmbi.1993.1005. [DOI] [PubMed] [Google Scholar]
  18. Meinnel T., Mechulam Y., Le Corre D., Panvert M., Blanquet S., Fayat G. Selection of suppressor methionyl-tRNA synthetases: mapping the tRNA anticodon binding site. Proc Natl Acad Sci U S A. 1991 Jan 1;88(1):291–295. doi: 10.1073/pnas.88.1.291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Meinnel T., Schmitt E., Mechulam Y., Blanquet S. Structural and biochemical characterization of the Escherichia coli argE gene product. J Bacteriol. 1992 Apr;174(7):2323–2331. doi: 10.1128/jb.174.7.2323-2331.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Mellot P., Mechulam Y., Le Corre D., Blanquet S., Fayat G. Identification of an amino acid region supporting specific methionyl-tRNA synthetase: tRNA recognition. J Mol Biol. 1989 Aug 5;208(3):429–443. doi: 10.1016/0022-2836(89)90507-x. [DOI] [PubMed] [Google Scholar]
  21. Miller J. H., Albertini A. M. Effects of surrounding sequence on the suppression of nonsense codons. J Mol Biol. 1983 Feb 15;164(1):59–71. doi: 10.1016/0022-2836(83)90087-6. [DOI] [PubMed] [Google Scholar]
  22. Normanly J., Kleina L. G., Masson J. M., Abelson J., Miller J. H. Construction of Escherichia coli amber suppressor tRNA genes. III. Determination of tRNA specificity. J Mol Biol. 1990 Jun 20;213(4):719–726. doi: 10.1016/S0022-2836(05)80258-X. [DOI] [PubMed] [Google Scholar]
  23. Petersen H. U., Røll T., Grunberg-Manago M., Clark B. F. Specific interaction of initiation factor IF2 of E. coli with formylmethionyl-tRNA f Met. Biochem Biophys Res Commun. 1979 Dec 14;91(3):1068–1074. doi: 10.1016/0006-291x(79)91989-2. [DOI] [PubMed] [Google Scholar]
  24. Schulman L. H., Pelka H., Sundari R. M. Structural requirements for recognition of Escherichia coli initiator and non-initiator transfer ribonucleic acids by bacterial T factor. J Biol Chem. 1974 Nov 25;249(22):7102–7110. [PubMed] [Google Scholar]
  25. Seong B. L., Lee C. P., RajBhandary U. L. Suppression of amber codons in vivo as evidence that mutants derived from Escherichia coli initiator tRNA can act at the step of elongation in protein synthesis. J Biol Chem. 1989 Apr 15;264(11):6504–6508. [PubMed] [Google Scholar]
  26. Seong B. L., RajBhandary U. L. Escherichia coli formylmethionine tRNA: mutations in GGGCCC sequence conserved in anticodon stem of initiator tRNAs affect initiation of protein synthesis and conformation of anticodon loop. Proc Natl Acad Sci U S A. 1987 Jan;84(2):334–338. doi: 10.1073/pnas.84.2.334. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Seong B. L., RajBhandary U. L. Mutants of Escherichia coli formylmethionine tRNA: a single base change enables initiator tRNA to act as an elongator in vitro. Proc Natl Acad Sci U S A. 1987 Dec;84(24):8859–8863. doi: 10.1073/pnas.84.24.8859. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Simons R. W., Houman F., Kleckner N. Improved single and multicopy lac-based cloning vectors for protein and operon fusions. Gene. 1987;53(1):85–96. doi: 10.1016/0378-1119(87)90095-3. [DOI] [PubMed] [Google Scholar]
  29. Springer M., Graffe M., Dondon J., Grunberg-Manago M. tRNA-like structures and gene regulation at the translational level: a case of molecular mimicry in Escherichia coli. EMBO J. 1989 Aug;8(8):2417–2424. doi: 10.1002/j.1460-2075.1989.tb08372.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sprinzl M., Hartmann T., Weber J., Blank J., Zeidler R. Compilation of tRNA sequences and sequences of tRNA genes. Nucleic Acids Res. 1989;17 (Suppl):r1–172. doi: 10.1093/nar/17.suppl.r1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sundari R. M., Stringer E. A., Schulman L. H., Maitra U. Interaction of bacterial initiation factor 2 with initiator tRNA. J Biol Chem. 1976 Jun 10;251(11):3338–3345. [PubMed] [Google Scholar]
  32. Varshney U., Lee C. P., RajBhandary U. L. From elongator tRNA to initiator tRNA. Proc Natl Acad Sci U S A. 1993 Mar 15;90(6):2305–2309. doi: 10.1073/pnas.90.6.2305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Varshney U., Lee C. P., Seong B. L., RajBhandary U. L. Mutants of initiator tRNA that function both as initiators and elongators. J Biol Chem. 1991 Sep 25;266(27):18018–18024. [PubMed] [Google Scholar]
  34. 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]
  35. Varshney U., RajBhandary U. L. Role of methionine and formylation of initiator tRNA in initiation of protein synthesis in Escherichia coli. J Bacteriol. 1992 Dec;174(23):7819–7826. doi: 10.1128/jb.174.23.7819-7826.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Wrede P., Woo N. H., Rich A. Initiator tRNAs have a unique anticodon loop conformation. Proc Natl Acad Sci U S A. 1979 Jul;76(7):3289–3293. doi: 10.1073/pnas.76.7.3289. [DOI] [PMC free article] [PubMed] [Google Scholar]

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