Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1994 Aug 11;22(15):2963–2969. doi: 10.1093/nar/22.15.2963

Seryl-tRNA synthetase from Escherichia coli: implication of its N-terminal domain in aminoacylation activity and specificity.

F Borel 1, C Vincent 1, R Leberman 1, M Härtlein 1
PMCID: PMC310262  PMID: 8065908

Abstract

Escherichia coli seryl-tRNA synthetase (SerRS) a dimeric class II aminoacyl-tRNA synthetase with two structural domains charges specifically the five iso-acceptor tRNA(ser) as well as the tRNA(sec) (selC product) of E. coli. The N-terminal domain is a 60 A long arm-like coiled coil structure built of 2 long antiparallel a-h helices, whereas the C-terminal domain is a alpha-beta structure. A deletion of the N-terminal arm of the enzyme does not affect the amino acid activation step of the reaction, but reduces dramatically amino-acylation activity. The Kcat/Km value for the mutant enzyme is reduced by more than 4 orders of magnitude, with a nearly 30 fold increased Km value for tRNA(ser). An only slightly truncated mutant form (16 amino acids of the tip of the arm replaced by a glycine) has an intermediate aminoacylation activity. Both mutant synthetases have lost their specificity for tRNA(ser) and charge also non-cognate type 1 tRNA(s). Our results support the hypothesis that class II synthetases have evolved from an ancestral catalytic core enzyme by adding non-catalytic N-terminal or C-terminal tRNA binding (specificity) domains which act as determinants for cognate and anti-determinants for non-cognate tRNAs.

Full text

PDF
2963

Images in this article

2967Image
on p.2967
2968Image
on p.2968

Selected References

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

  1. Abrahams J. P., Kraal B., Bosch L. Zone-interference gel electrophoresis: a new method for studying weak protein-nucleic acid complexes under native equilibrium conditions. Nucleic Acids Res. 1988 Nov 11;16(21):10099–10108. doi: 10.1093/nar/16.21.10099. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Avis J. M., Day A. G., Garcia G. A., Fersht A. R. Reaction of modified and unmodified tRNA(Tyr) substrates with tyrosyl-tRNA synthetase (Bacillus stearothermophilus). Biochemistry. 1993 May 25;32(20):5312–5320. doi: 10.1021/bi00071a005. [DOI] [PubMed] [Google Scholar]
  3. Bilderback D. H., Hoffman S. A., Thiel D. J. Nanometer spatial resolution achieved in hard x-ray imaging and Laue diffraction experiments. Science. 1994 Jan 14;263(5144):201–203. doi: 10.1126/science.8284671. [DOI] [PubMed] [Google Scholar]
  4. Biou V., Yaremchuk A., Tukalo M., Cusack S. The 2.9 A crystal structure of T. thermophilus seryl-tRNA synthetase complexed with tRNA(Ser). Science. 1994 Mar 11;263(5152):1404–1410. doi: 10.1126/science.8128220. [DOI] [PubMed] [Google Scholar]
  5. Borel F., Härtlein M., Leberman R. In vivo overexpression and purification of Escherichia coli tRNA(ser). FEBS Lett. 1993 Jun 14;324(2):162–166. doi: 10.1016/0014-5793(93)81385-d. [DOI] [PubMed] [Google Scholar]
  6. Calendar R., Berg P. Purification and physical characterization of tyrosyl ribonucleic acid synthetases from Escherichia coli and Bacillus subtilis. Biochemistry. 1966 May;5(5):1681–1690. doi: 10.1021/bi00869a033. [DOI] [PubMed] [Google Scholar]
  7. Campbell J. L., Richardson C. C., Studier F. W. Genetic recombination and complementation between bacteriophage T7 and cloned fragments of T7 DNA. Proc Natl Acad Sci U S A. 1978 May;75(5):2276–2280. doi: 10.1073/pnas.75.5.2276. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cusack S., Berthet-Colominas C., Härtlein M., Nassar N., Leberman R. A second class of synthetase structure revealed by X-ray analysis of Escherichia coli seryl-tRNA synthetase at 2.5 A. Nature. 1990 Sep 20;347(6290):249–255. doi: 10.1038/347249a0. [DOI] [PubMed] [Google Scholar]
  9. Cusack S., Härtlein M., Leberman R. Sequence, structural and evolutionary relationships between class 2 aminoacyl-tRNA synthetases. Nucleic Acids Res. 1991 Jul 11;19(13):3489–3498. doi: 10.1093/nar/19.13.3489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dibbelt L., Pachmann U., Zachau H. G. Serine activation is the rate limiting step of tRNASer aminoacylation by yeast seryl tRNA synthetase. Nucleic Acids Res. 1980 Sep 11;8(17):4021–4039. doi: 10.1093/nar/8.17.4021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Eriani G., Cavarelli J., Martin F., Dirheimer G., Moras D., Gangloff J. Role of dimerization in yeast aspartyl-tRNA synthetase and importance of the class II invariant proline. Proc Natl Acad Sci U S A. 1993 Nov 15;90(22):10816–10820. doi: 10.1073/pnas.90.22.10816. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Eriani G., Delarue M., Poch O., Gangloff J., Moras D. Partition of tRNA synthetases into two classes based on mutually exclusive sets of sequence motifs. Nature. 1990 Sep 13;347(6289):203–206. doi: 10.1038/347203a0. [DOI] [PubMed] [Google Scholar]
  13. Himeno H., Hasegawa T., Ueda T., Watanabe K., Miura K., Shimizu M. Role of the extra G-C pair at the end of the acceptor stem of tRNA(His) in aminoacylation. Nucleic Acids Res. 1989 Oct 11;17(19):7855–7863. doi: 10.1093/nar/17.19.7855. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hou Y. M., Schimmel P. A simple structural feature is a major determinant of the identity of a transfer RNA. Nature. 1988 May 12;333(6169):140–145. doi: 10.1038/333140a0. [DOI] [PubMed] [Google Scholar]
  15. Härtlein M., Madern D., Leberman R. Cloning and characterization of the gene for Escherichia coli seryl-tRNA synthetase. Nucleic Acids Res. 1987 Feb 11;15(3):1005–1017. doi: 10.1093/nar/15.3.1005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kumazawa Y., Himeno H., Miura K., Watanabe K. Unilateral aminoacylation specificity between bovine mitochondria and eubacteria. J Biochem. 1991 Mar;109(3):421–427. doi: 10.1093/oxfordjournals.jbchem.a123397. [DOI] [PubMed] [Google Scholar]
  17. Leberman R., Antonsson B., Giovanelli R., Guariguata R., Schumann R., Wittinghofer A. A simplified procedure for the isolation of bacterial polypeptide elongation factor EF-Tu. Anal Biochem. 1980 May 1;104(1):29–36. doi: 10.1016/0003-2697(80)90272-9. [DOI] [PubMed] [Google Scholar]
  18. Low B., Gates F., Goldstein T., Söll D. Isolation and partial characterization of temperature-sensitive Escherichia coli mutants with altered leucyl- and seryl-transfer ribonucleic acid synthetases. J Bacteriol. 1971 Nov;108(2):742–750. doi: 10.1128/jb.108.2.742-750.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Madern D., Anselme J., Härtlein M. Asparaginyl-tRNA synthetase from the Escherichia coli temperature-sensitive strain HO202. A proline replacement in motif 2 is responsible for a large increase in Km for asparagine and ATP. FEBS Lett. 1992 Mar 24;299(1):85–89. doi: 10.1016/0014-5793(92)80106-q. [DOI] [PubMed] [Google Scholar]
  20. Moras D. Structural and functional relationships between aminoacyl-tRNA synthetases. Trends Biochem Sci. 1992 Apr;17(4):159–164. doi: 10.1016/0968-0004(92)90326-5. [DOI] [PubMed] [Google Scholar]
  21. Perona J. J., Swanson R. N., Rould M. A., Steitz T. A., Söll D. Structural basis for misaminoacylation by mutant E. coli glutaminyl-tRNA synthetase enzymes. Science. 1989 Dec 1;246(4934):1152–1154. doi: 10.1126/science.2686030. [DOI] [PubMed] [Google Scholar]
  22. Price S., Cusack S., Borel F., Berthet-Colominas C., Leberman R. Crystallization of the seryl-tRNA synthetase:tRNAS(ser) complex of Escherichia coli. FEBS Lett. 1993 Jun 14;324(2):167–170. doi: 10.1016/0014-5793(93)81386-e. [DOI] [PubMed] [Google Scholar]
  23. Rosenberg A. H., Lade B. N., Chui D. S., Lin S. W., Dunn J. J., Studier F. W. Vectors for selective expression of cloned DNAs by T7 RNA polymerase. Gene. 1987;56(1):125–135. doi: 10.1016/0378-1119(87)90165-x. [DOI] [PubMed] [Google Scholar]
  24. Ruff M., Krishnaswamy S., Boeglin M., Poterszman A., Mitschler A., Podjarny A., Rees B., Thierry J. C., Moras D. Class II aminoacyl transfer RNA synthetases: crystal structure of yeast aspartyl-tRNA synthetase complexed with tRNA(Asp). Science. 1991 Jun 21;252(5013):1682–1689. doi: 10.1126/science.2047877. [DOI] [PubMed] [Google Scholar]
  25. Sampson J. R., Saks M. E. Contributions of discrete tRNA(Ser) domains to aminoacylation by E.coli seryl-tRNA synthetase: a kinetic analysis using model RNA substrates. Nucleic Acids Res. 1993 Sep 25;21(19):4467–4475. doi: 10.1093/nar/21.19.4467. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Schimmel P. Aminoacyl tRNA synthetases: general scheme of structure-function relationships in the polypeptides and recognition of transfer RNAs. Annu Rev Biochem. 1987;56:125–158. doi: 10.1146/annurev.bi.56.070187.001013. [DOI] [PubMed] [Google Scholar]
  27. Schulman L. H., Pelka H. An anticodon change switches the identity of E. coli tRNA(mMet) from methionine to threonine. Nucleic Acids Res. 1990 Jan 25;18(2):285–289. doi: 10.1093/nar/18.2.285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Shimizu M., Asahara H., Tamura K., Hasegawa T., Himeno H. The role of anticodon bases and the discriminator nucleotide in the recognition of some E. coli tRNAs by their aminoacyl-tRNA synthetases. J Mol Evol. 1992 Nov;35(5):436–443. doi: 10.1007/BF00171822. [DOI] [PubMed] [Google Scholar]
  29. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  30. Sundharadas G., Katze J. R., Söll D., Konigsberg W., Lengyel P. On the recognition of serine transfer RNA's specific for unrelated codons by the same seryl-transfer RNA synthetase. Proc Natl Acad Sci U S A. 1968 Oct;61(2):693–700. doi: 10.1073/pnas.61.2.693. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Ueda T., Yotsumoto Y., Ikeda K., Watanabe K. The T-loop region of animal mitochondrial tRNA(Ser)(AGY) is a main recognition site for homologous seryl-tRNA synthetase. Nucleic Acids Res. 1992 May 11;20(9):2217–2222. doi: 10.1093/nar/20.9.2217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. de Bruijn M. H., Schreier P. H., Eperon I. C., Barrell B. G., Chen E. Y., Armstrong P. W., Wong J. F., Roe B. A. A mammalian mitochondrial serine transfer RNA lacking the "dihydrouridine" loop and stem. Nucleic Acids Res. 1980 Nov 25;8(22):5213–5222. doi: 10.1093/nar/8.22.5213. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES