1887

Abstract

strains harbouring elongator tRNAs that insert amino acids in response to a termination codon during elongation have been generated for various applications. Additionally, it was shown that expression of an initiator tRNA containing a CUA anticodon from a multicopy plasmid in resulted in initiation from an amber codon. Even though the initiation-based system remedies toxicity-related drawbacks, its usefulness has remained limited for want of a strain with a chromosomally encoded initiator tRNA ‘suppressor’. K strains possess four initiator tRNA genes: the , and genes, located at a single locus, encode tRNA , and a distantly located gene encodes a variant, tRNA . In this study, a stable strain of K-12 that affords efficient initiation from an amber initiation codon was isolated. Genetic analysis revealed that the gene in this strain acquired mutations to encode tRNA with a CUA anticodon (a U35A36 mutation). The acquisition of the mutations depended on the presence of a plasmid-borne copy of the mutant and host background. The mutations were observed when the plasmid-borne gene encoded tRNA (U35A36) with additional changes in the acceptor stem (G72; G72G73) but not in the anticodon stem (U29C30A31/U35A36/ψ39G40A41). The usefulness of this strain, and a possible role for multiple tRNA genes in in safeguarding their intactness, are discussed.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.27915-0
2005-06-01
2020-08-08
Loading full text...

Full text loading...

/deliver/fulltext/micro/151/6/mic1511741.html?itemId=/content/journal/micro/10.1099/mic.0.27915-0&mimeType=html&fmt=ahah

References

  1. Atlung T., Christensen B. B., Hansen F. G. 1999; Role of the rom protein in copy number control of plasmid pBR322 at different growth rates in Escherichia coli K-12. Plasmid41:110–119[CrossRef]
    [Google Scholar]
  2. Bain J. D., Glabe C. G., Dix J. A., Chamberlin A. R., Diala E. S. 1989; Biosynthetic site specific incorporation of a non-natural amino acid into a polypeptide. Biochemistry29:5881–5889
    [Google Scholar]
  3. Berlyn M. K. 1998; Linkage map of Escherichia coli K-12, edition 10: the traditional map. Microbiol Mol Biol Rev62:814–984
    [Google Scholar]
  4. Brenner S., Beckwith J. R. 1965; Ochre mutants, a new class of suppressible nonsense mutants. J Mol Biol13:629–637[CrossRef]
    [Google Scholar]
  5. Chattapadhyay R., Pelka H., Schulman L. H. 1990; Initiation of in vivo protein synthesis with non-methionine amino acids. Biochemistry29:4263–4268[CrossRef]
    [Google Scholar]
  6. Cornish V. W., Benson D. R., Altenbach C. A., Hideg K., Hubbell W. L., Schultz P. G. 1994; Site-specific incorporation of biophysical probes into proteins. Proc Natl Acad Sci U S A91:2910–2914[CrossRef]
    [Google Scholar]
  7. Craigen W. J., Caskey C. T. 1986; Expression of peptide chain release factor 2 requires high-efficiency frameshift. Nature322:273–275[CrossRef]
    [Google Scholar]
  8. Datsenko K. A., Wanner B. L. 2000; One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A97:6640–6645[CrossRef]
    [Google Scholar]
  9. Ellman J., Mendel D., Cahill S. A., Noren C. J., Schultz P. G. 1991; Biosynthetic method for introducing unnatural amino acids site specifically into proteins. Methods Enzymol202:301–336
    [Google Scholar]
  10. Gold L. 1988; Posttranscriptional regulatory mechanisms in Escherichia coli. Annu Rev Biochem57:199–233[CrossRef]
    [Google Scholar]
  11. Guillon J. M., Heiss S., Soutourina J., Mechulam Y., Laalami S., Grunberg-Monago M., Blanquet S. 1996; Interplay of methionine tRNAs with translation elongation factor Tu and translation initiation factor 2 in Escherichia coli. J Biol Chem271:22321–22325[CrossRef]
    [Google Scholar]
  12. Hashimoto J. G., Stevenson B. S., Schmidt T. M. 2003; Rates and consequences of recombination between rRNA operons. J Bacteriol185:966–972[CrossRef]
    [Google Scholar]
  13. Kenri T., Kohno K., Goshima N., Imamoto F., Kano Y. 1991; Construction and characterization of an Escherichia coli mutant with a deletion of the metZ gene encoding tRNAf1Met. Gene103:31–36[CrossRef]
    [Google Scholar]
  14. Kenri T., Imamoto F., Kano Y. 1992; Construction and characterization of an Escherichia coli mutant deficient in the metY gene encoding tRNA(f2Met): either tRNA(f1Met) or tRNA(f2Met) is required for cell growth. Gene114:109–114[CrossRef]
    [Google Scholar]
  15. Kleina L. G., Masson J. M., Normanly J., Abelson J., Miller J. H. 1990; Construction of Escherichia coli amber suppressor tRNA genes. II. Synthesis of additional tRNA genes and improvement of suppressor efficiency. J Mol Biol213:705–717[CrossRef]
    [Google Scholar]
  16. Kozak M. 1983; Comparison of initiation of protein synthesis in procaryotes, eucaryotes, and organelles. Microbiol Rev47:1–45
    [Google Scholar]
  17. Lee C. P., RajBhandary U. L. 1991; Structural and sequence elements important for recognition of Escherichia coli formylmethionine tRNA by methionyl-tRNA transformylase are clustered in the acceptor stem. J Biol Chem266:18012–18017
    [Google Scholar]
  18. Mahadevan S., Reynolds A. E., Wright A. 1987; Positive and negative regulation of the bgl operon in Escherichia coli. J Bacteriol169:2570–2578
    [Google Scholar]
  19. Mamaev S., Olejnik J., Olejnik E. K., Rothschild K. J. 2004; Cell-free N-terminal protein labeling using initiator suppressor tRNA. Anal Biochem326:25–32[CrossRef]
    [Google Scholar]
  20. Mandal N., RajBhandary U. L. 1992; Escherichia coli B lacks one of the two initiator tRNA species present inE. coli K-12. J Bacteriol174:7827–7830
    [Google Scholar]
  21. Mandal N., Mangroo D., Dalluge J. J., McCloskey J. A., RajBhandary U. L. 1996; Role of the three consecutive G : C base pairs conserved in the anticodon stem of initiator tRNAs in initiation of protein synthesis in Escherichia coli. RNA 5:473–482
    [Google Scholar]
  22. Mangroo D., RajBhandary U. L. 1995; Mutants of Escherichia coli initiator tRNA defective in initiation. Effects of overproduction of methionyl-tRNA transformylase and the initiation factors IF2 and IF3. J Biol Chem270:12203–12209[CrossRef]
    [Google Scholar]
  23. Mansell J. B., Guevremont D., Poole E. S., Tate W. P. 2001; A dynamic competition between release factor 2 and the tRNASec decoding UGA at the recoding site of Escherichia coli formate dehydrogenase H. EMBO J20:7284–7293[CrossRef]
    [Google Scholar]
  24. Mayer C., Stortchevoi A., Kohrer C., Varshney U., RajBhandary U. L. 2001; Initiator tRNA and its role in initiation of protein synthesis. Cold Spring Harbor Symp Quant Biol66:195–206[CrossRef]
    [Google Scholar]
  25. Mayer C., Kohrer C., Kenny E., Prusko C., RajBhandary U. L. 2003; Anticodon sequence mutants of Escherichia coli initiator tRNA: effects of overproduction of aminoacyl tRNA synthetases, methionyl-tRNA formyltransferase and initiation factor 2 on activity in initiation. Biochemistry42:4787–4799[CrossRef]
    [Google Scholar]
  26. Miller J. H. 1972; Generalized transduction: use of P1 in strain construction. In Experiments in Molecular Genetics pp201–205 Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  27. Murgola E. J., Prather N. E., Pagel F. T., Mims B. H., Hijazi K. A. 1984; Missense and nonsense suppressors derived from a glycine tRNA by nucleotide insertion and deletion in vivo. Mol Gen Genet193:76–81[CrossRef]
    [Google Scholar]
  28. Normanly J., Abelson J. 1989; tRNA identity. Annual Rev Biochem58:1029–1049[CrossRef]
    [Google Scholar]
  29. Normanly J., Kleina L. G., Masson J. M., Abelson J., Miller J. H. 1990; Construction of Escherichia coli amber suppressor tRNA genes. III. Determination of tRNA specificity. J Mol Biol213:719–726[CrossRef]
    [Google Scholar]
  30. O'Connor M., Gregory S. T., RajBhandary U. L., Dahlberg A. E. 2001; Altered discrimination of start codons and initiator tRNAs by mutant initiation factor 3. RNA 7:969–978[CrossRef]
    [Google Scholar]
  31. Pallanck L., Schulman L. H. 1991; Anticodon-dependent aminoacylation of a noncognate tRNA with isoleucine, valine, and phenylalanine in vivo. Proc Natl Acad Sci U S A88:3872–3876[CrossRef]
    [Google Scholar]
  32. RajBhandary U. L. 1994; Initiator transfer RNAs. J Bacteriol176:547–552
    [Google Scholar]
  33. RajBhandary U. L., Chow C. M. 1995; Initiator tRNAs and initiation of protein synthesis. In tRNA: Structure, Biosynthesis, and Function p.511 Edited by Soll D., RajBhandary U. L.. Washington, DC: American Society for Microbiology;
    [Google Scholar]
  34. Sambrook J., Fritsch E. F., Maniatis T. 1989; Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  35. Sarin P. S., Zamecnik P. C. 1964; On the stability of aminoacyl soluble ribonucleic acid to nucleophilic catalysis. Biochim Biophys Acta91:653–655
    [Google Scholar]
  36. Schulman L. H., Pelka H. 1985; In vitro conversion of a methionine to a glutamine-acceptor tRNA. Biochemistry24:7309–7314[CrossRef]
    [Google Scholar]
  37. Seong B. L., RajBhandary U. L. 1987; Escherichia coli formylmethionine tRNA: mutations in GGG : CCC sequence conserved in anticodon stem of initiator tRNAs affect initiation of protein synthesis and conformation of anticodon loop. Proc Natl Acad Sci U S A84:334–338[CrossRef]
    [Google Scholar]
  38. Seong B. L., Lee C. P., RajBhandary U. L. 1989; 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 Chem246:6504–6508
    [Google Scholar]
  39. Shaw W. V. 1983; Chloramphenicol acetyltransferase: enzymology and molecular biology. CRC Crit Rev Biochem14:1–46
    [Google Scholar]
  40. Singer M., Baker T. A., Schnitzler G.. 7 other authors 1989; A collection of strains containing genetically linked alternating antibiotic resistance elements for genetic mapping of Escherichia coli. Microbiol Rev53:1–24
    [Google Scholar]
  41. Snyder L., Champness W. 1997; Mutations in bacteria. In Molecular Genetics of Bacteria pp75–103 Washington, DC: American Society for Microbiology;
    [Google Scholar]
  42. Steege D. A., Soll D. G. 1979; Suppression. In Biological Regulation and Development vol1 pp433–485 Edited by Goldberger R. F.. New York: Plenum;
    [Google Scholar]
  43. Tate W. P., Mannering S. A. 1996; Three, four or more: the translational stop signal at length. Mol Microbiol21:213–219[CrossRef]
    [Google Scholar]
  44. Thanedar S., Kumar N. V., Varshney U. 2000; The fate of the initiator tRNAs is sensitive to the critical balance between interacting proteins. J Biol Chem275:20361–20367[CrossRef]
    [Google Scholar]
  45. Varshney U., RajBhandary U. L. 1990; Initiation of protein synthesis from a termination codon. Proc Natl Acad Sci U S A87:1586–1590[CrossRef]
    [Google Scholar]
  46. Varshney U., RajBhandary U. L. 1992; Role of methionine and formylation of initiator tRNA in initiation of protein synthesis in Escherichia coli. J Bacteriol174:7819–7826
    [Google Scholar]
  47. Varshney U., Lee C. P., RajBhandary U. L. 1991a; 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 Chem266:24712–24718
    [Google Scholar]
  48. Varshney U., Lee C. P., Seong B. L., RajBhandary U. L. 1991b; Mutants of initiator tRNA that function both as initiators and elongators. J Biol Chem266:18018–18024
    [Google Scholar]
  49. Wu X. Q., RajBhandary U. L. 1997; Effect of the amino acid attached to Escherichia coli initiator tRNA on its affinity for the initiation factor IF2 and on the IF2 dependence of its binding to the ribosome. J Biol Chem272:1891–1895[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.27915-0
Loading
/content/journal/micro/10.1099/mic.0.27915-0
Loading

Data & Media loading...

Most cited this month Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error