A truncated aminoacyl-tRNA synthetase modifies RNA.
PDF (613K)
Journal: 2004/July - Proceedings of the National Academy of Sciences of the United States of America
ISSN: 0027-8424
Abstract:
Aminoacyl-tRNA synthetases are modular enzymes composed of a central active site domain to which additional functional domains were appended in the course of evolution. Analysis of bacterial genome sequences revealed the presence of many shorter aminoacyl-tRNA synthetase paralogs. Here we report the characterization of a well conserved glutamyl-tRNA synthetase (GluRS) paralog (YadB in Escherichia coli) that is present in the genomes of >40 species of proteobacteria, cyanobacteria, and actinobacteria. The E. coli yadB gene encodes a truncated GluRS that lacks the C-terminal third of the protein and, consequently, the anticodon binding domain. Generation of a yadB disruption showed the gene to be dispensable for E. coli growth in rich and minimal media. Unlike GluRS, the YadB protein was able to activate glutamate in presence of ATP in a tRNA-independent fashion and to transfer glutamate onto tRNA(Asp). Neither tRNA(Glu) nor tRNA(Gln) were substrates. In contrast to canonical aminoacyl-tRNA, glutamate was not esterified to the 3'-terminal adenosine of tRNA(Asp). Instead, it was attached to the 2-amino-5-(4,5-dihydroxy-2-cyclopenten-1-yl) moiety of queuosine, the modified nucleoside occupying the first anticodon position of tRNA(Asp). Glutamyl-queuosine, like canonical Glu-tRNA, was hydrolyzed by mild alkaline treatment. Analysis of tRNA isolated under acidic conditions showed that this novel modification is present in normal E. coli tRNA; presumably it previously escaped detection as the standard conditions of tRNA isolation include an alkaline deacylation step that also causes hydrolysis of glutamyl-queuosine. Thus, this aminoacyl-tRNA synthetase fragment contributes to standard nucleotide modification of tRNA.
Open in
PUBMED | DOI | PMC | Google Scholar | Wikipedia
Relations:
Content
Citations
(35)
References
(40)
Drugs
(2)
Chemicals
(5)
Genes
(1)
Affiliates
(1)
Similar articles
Articles by the same authors
Discussion board
Proc Natl Acad Sci U S A 101(20): 7536-7541
PMID: 15096612

A truncated aminoacyl–tRNA synthetase modifies RNA

Departments of Molecular Biophysics and Biochemistry, and Chemistry, Yale University, New Haven, CT 06520-8114; and Departments of Medicinal Chemistry and Biochemistry, University of Utah, Salt Lake City, UT 84112-5820
To whom correspondence should be addressed. E-mail: ude.elay.mehc.anrt@llos.
Contributed by Dieter Söll, March 21, 2004
Contributed by Dieter Söll, March 21, 2004
Copyright © 2004, The National Academy of Sciences

Abstract

Aminoacyl–tRNA synthetases are modular enzymes composed of a central active site domain to which additional functional domains were appended in the course of evolution. Analysis of bacterial genome sequences revealed the presence of many shorter aminoacyl–tRNA synthetase paralogs. Here we report the characterization of a well conserved glutamyl–tRNA synthetase (GluRS) paralog (YadB in Escherichia coli) that is present in the genomes of >40 species of proteobacteria, cyanobacteria, and actinobacteria. The E. coli yadB gene encodes a truncated GluRS that lacks the C-terminal third of the protein and, consequently, the anticodon binding domain. Generation of a yadB disruption showed the gene to be dispensable for E. coli growth in rich and minimal media. Unlike GluRS, the YadB protein was able to activate glutamate in presence of ATP in a tRNA-independent fashion and to transfer glutamate onto tRNA. Neither tRNA nor tRNA were substrates. In contrast to canonical aminoacyl–tRNA, glutamate was not esterified to the 3′-terminal adenosine of tRNA. Instead, it was attached to the 2-amino-5-(4,5-dihydroxy-2-cyclopenten-1-yl) moiety of queuosine, the modified nucleoside occupying the first anticodon position of tRNA. Glutamyl–queuosine, like canonical Glu–tRNA, was hydrolyzed by mild alkaline treatment. Analysis of tRNA isolated under acidic conditions showed that this novel modification is present in normal E. coli tRNA; presumably it previously escaped detection as the standard conditions of tRNA isolation include an alkaline deacylation step that also causes hydrolysis of glutamyl–queuosine. Thus, this aminoacyl–tRNA synthetase fragment contributes to standard nucleotide modification of tRNA.

Abstract

Faithful translation of the mRNA genetic information into proteins relies on the correct attachment of an amino acid to its corresponding tRNA(s) by enzymes collectively known as aminoacyl–tRNA synthetases (aaRSs). These essential enzymes are structurally conserved (1, 2) and easily detected in the analysis of genome sequences (e.g., ref. 3). Prokaryotes usually have one gene for each aaRS, although the presence of two functional paralogous genes for a synthetase (e.g., lysS and lysU for Escherichia coli lysyl–tRNA synthetase; ref. 4) is well known. Analysis of the ever larger number of bacterial genome sequences revealed the occurrence of many aaRS paralogs containing regional deletions (compared to the full-length genes also present in the genome). Given the modular nature of the aaRS proteins (5), the presence of synthetase gene pieces in a genome was not surprising, because they are likely remnants of a complex evolutionary history (2, 3, 6, 7). Although these truncated aaRSs were considered to be pseudogenes (8), more recent attention to particular aaRSs paralogs revealed their involvement in important biological processes. For instance, HisZ, a histidyl–tRNA synthetase-like protein lacking the anti-codon domain, was shown to be essential for histidine biosynthesis in a number of bacteria (9). Likewise, an archaeal asparaginyl–tRNA synthetase paralog, lacking the anticodon-binding domain, is able to carry out tRNA-independent asparagine synthesis (10). The function of aaRS paralogs is not restricted to amino acid biosynthesis. Truncated alanyl–, prolyl–, and threonyl–tRNA synthetase-like proteins were shown to possess esterase function and be capable of specific hydrolysis of misacylated tRNA in trans (11, 12), a mechanism that should increase the fidelity of the translation process.

Here we report the characterization of another widely distributed aaRS paralog, a glutamyl–tRNA synthetase (GluRS)-related protein encoded by yadB in E. coli.

Acknowledgments

We thank Hiroaki Nakano for help with the yadB deletion and Steven Pomerantz for assistance with the liquid chromatography MS analyses. This work was supported by grants from the National Institute of General Medical Sciences (to J.A.M. and D.S.) and the Department of Energy (to D.S.).

Acknowledgments

Notes

Abbreviations: aaRS, aminoacyl–tRNA synthetase; GluRS, glutamyl–tRNA synthetase; AspRS, aspartyl–tRNA synthetase.

See Commentary on page 7493.

Notes
Abbreviations: aaRS, aminoacyl–tRNA synthetase; GluRS, glutamyl–tRNA synthetase; AspRS, aspartyl–tRNA synthetase.
See Commentary on page 7493.

References

  • 1. Arnez, J. G. & Moras, D. (1997) Trends Biochem. Sci.22, 211-216. [[PubMed]
  • 2. O'Donoghue, P. & Luthey-Schulten, Z. (2003) Microbiol. Mol. Biol. Rev.67, 550-573.
  • 3. Woese, C. R., Olsen, G. J., Ibba, M. & Söll, D. (2000) Microbiol. Mol. Biol. Rev.64, 202-236.
  • 4. Leveque, F., Plateau P, Dessen P. & Blanquet, S. (1990) Nucleic Acids Res.18, 305-312.
  • 5. Schimmel, P. & Ripmaster, T. (1995) Trends Biochem. Sci.20, 333-334. [[PubMed]
  • 6. Rould, M. A., Perona, J. J., Söll, D. & Steitz, T. A. (1989) Science246, 1135-1142. [[PubMed]
  • 7. Ribas de Pouplana, L. & Schimmel, P. (2000) Cell. Mol. Life Sci.57, 865-870. [[PubMed]
  • 8. Lamour, V., Quevillon, S., Diriong, S., N′Guyen, V. C., Lipinski, M. & Mirande, M. (1994) Proc. Natl. Acad. Sci. USA91, 8670-8674.
  • 9. Sissler, M., Delorme, C., Bond, J., Ehrlich, S. D., Renault, P. & Francklyn, C. (1999) Proc. Natl. Acad. Sci. USA96, 8985-8990.
  • 10. Roy, H., Becker, H. D., Reinbolt, J. & Kern, D. (2003) Proc. Natl. Acad. Sci. USA100, 9837-9842.
  • 11. Ahel, I., Korencic, D., Ibba, M. & Söll, D. (2003) Proc. Natl. Acad. Sci. USA100, 15422-15427.
  • 12. Wong, F. C., Beuning, P. J., Silvers, C. & Musier-Forsyth, K. (2003) J. Biol. Chem.278, 52857-52864. [[PubMed]
  • 13. Noguchi, S., Nishimura, Y., Hirota, Y. & Nishimura, S. (1982) J. Biol. Chem.257, 6544-6550. [[PubMed]
  • 14. Datsenko, K. A. & Wanner, B. L. (2000) Proc. Natl. Acad. Sci. USA97, 6640-6645.
  • 15. Raczniak, G. Becker, H. D., Min, B. & Söll, D. (2001) J. Biol. Chem.276, 45862-45867. [[PubMed]
  • 16. Ribeiro, S., Nock, S. & Sprinzl, M. (1995) Anal. Biochem.228, 330-335. [[PubMed]
  • 17. Sampson, J. R. & Uhlenbeck, O. C. (1988) Proc. Natl. Acad. Sci. USA85, 1033-1037.
  • 18. Choi, H., Gabriel, K., Schneider, J., Otten, S. & McClain, W. H. (2003) RNA9, 386-393.
  • 19. Pomerantz, S. C. & McCloskey, J. A. (1990) Methods Enzymol.193, 796-824. [[PubMed]
  • 20. Sekine, S., Nureki, O., Dubois, D. Y., Bernier, S., Chênevert, R., Lapointe, J., Vassylyev, D. G. & Yokoyama, S. (2003) EMBO J.22, 676-688.
  • 21. Gerdes, S. Y., Scholle, M. D., Campbell, J. W., Balazsi, G., Daugherty, M. D., Somera, A. L., Kyrpides, N. C., Anderson, I., Gelfand, M. S., Bhattacharya, A., et al. (2003) J. Bacteriol.185, 5673-5684.
  • 22. Campanacci, V., Dubois, D. Y., Becker, H. D., Kern, D., Spinelli, S., Valencia, C., Pagot, F., Salomoni, A., Grisel, S., Vincentelli, R., et al. (2004) J. Mol. Biol.337, 273-283. [[PubMed]
  • 23. Ravel, J. M., Wang, S.-F., Heinemeyer, C. & Shive, W. (1965) J. Biol. Chem.240, 432-438. [[PubMed]
  • 24. Kern, D. & Lapointe, J. (1979) Biochemistry18, 5809-5818. [[PubMed]
  • 25. Skouloubris, S., Ribas de Pouplana, L., De Reuse, H. & Hendrickson, T. L. (2003) Proc. Natl. Acad. Sci. USA100, 11297-11302.
  • 26. Salazar, J. C., Ahel, I., Orellana, O., Tumbula-Hansen, D., Krieger, R., Daniels, L. & Söll, D. (2003) Proc. Natl. Acad. Sci. USA100, 13863-13868.
  • 27. Cayama, E., Yepez, A., Rotondo, F., Bandeira, E., Ferreras, A. C. & Triana-Alonso, F. J. (2000) Nucleic Acids Res.28, E64.
  • 28. Dubois, D. Y., Blaise, M., Becker, H. D., Campanacci, V., Keith, G., Giegé, R., Cambillau, C., Lapointe, J. & Kern, D. (2004) Proc. Natl. Acad. Sci. USA101, 7530-7535.
  • 29. Whitfeld, P. R. (1954) Biochem. J.58, 390-396.
  • 30. Sekiya, T., Mori, M., Takahashi, N. & Nishimura, S. (1980) Nucleic Acids Res.8, 3809-3827.
  • 31. Kasai, H., Ohashi, Z., Harada, F., Nishimura, S., Oppenheimer, N. J., Crain, P. F., Liehr, J. G., Von Minden, D. L. & McCloskey, J. A. (1975) Biochemistry14, 4198-4208. [[PubMed]
  • 32. Kasai, H., Nakanishi, K., Macfarlane, R. D., Torgerson, D. F., Ohashi, Z., McCloskey, J. A., Gross, H. J. & Nishimura, S. (1976) J. Am. Chem. Soc.98, 5044-5046. [[PubMed]
  • 33. Nakanishi, S., Ueda, T., Hori, H., Yamazaki, N., Okada, N. & Watanabe, K. (1994) J. Biol. Chem.269, 32221-32225. [[PubMed]
  • 34. Curnow, A. W. & Garcia, G. A. (1995) J. Biol. Chem.270, 17264-17267. [[PubMed]
  • 35. Soma, A., Ikeuchi, Y., Kanemasa, S., Kobayashi, K., Ogasawara, N., Ote, T., Kato, J., Watanabe, K., Sekine, Y. & Suzuki, T. (2003) Mol. Cell12, 689-698. [[PubMed]
  • 36. Körner, A. & Söll, D. (1974) FEBS Lett.39, 301-306. [[PubMed]
  • 37. Elkins, B. N. & Keller, E. B. (1974) Biochemistry13, 4622-4628. [[PubMed]
  • 38. Iwata-Reuyl, D(2003) Bioorg. Chem.31, 24-43. [[PubMed][Google Scholar]
  • 39. Durand, J. M., Dagberg, B., Uhlin, B. E. & Björk, G. R. (2000) Mol. Microbiol.35, 924-935. [[PubMed]
  • 40. Costa, A., Païs de Barros, J. P., Keith, G., Baranowski, W. & Desgrès, J. (2004) J. Chromatogr. B Analyt. Technol. Biomed. Life Sci.801, 237-247. [[PubMed]
Collaboration tool especially designed for Life Science professionals.Drag-and-drop any entity to your messages.