Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1990 Dec 11;18(23):7109–7118. doi: 10.1093/nar/18.23.7109

Aspartyl-tRNA synthetase from Escherichia coli: cloning and characterisation of the gene, homologies of its translated amino acid sequence with asparaginyl- and lysyl-tRNA synthetases.

G Eriani 1, G Dirheimer 1, J Gangloff 1
PMCID: PMC332776  PMID: 2129559

Abstract

By screening of an Escherichia coli plasmidic library using antibodies against aspartyl-tRNA synthetase (AspRS) several clones were obtained containing aspS, the gene coding for AspRS. We report here the nucleotide sequence of aspS and the corresponding primary structure of the aspartyl-tRNA synthetase, a protein of 590 amino acid residues with a Mr 65,913, a value in close agreement with that observed for the purified protein. Primer extension analysis of the aspS mRNA using reverse transcriptase located its 5'-end at 94 nucleotides upstream of the translation initiation AUG; nuclease S1 analysis located the 3'-end at 126 nucleotides downstream of the stop codon UGA. Comparison of the DNA-derived protein sequence with known aminoacyl-tRNA sequences revealed important homologies with asparaginyl- and lysyl-tRNA synthetases from E.coli; more than 25% of their amino acid residues are identical, the homologies being distributed preferencially in the first part and the carboxy-terminal end of the molecule. Mutagenesis directed towards a consensus tetrapeptide (Gly-Leu-Asp-Arg) and the carboxy-terminal end showed that both domains could be implicated in catalysis as well as in ATP binding.

Full text

PDF
7109

Images in this article

7112Image
on p.7112
7114Image
on p.7114
7115Image
on p.7115

Selected References

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

  1. Akesson B., Lundvik L. Simultaneous purification and some properties of aspartate: tRNA ligase and seven other amino-acid:tRNA ligases from Escherichia coli. Eur J Biochem. 1978 Feb 1;83(1):29–36. doi: 10.1111/j.1432-1033.1978.tb12064.x. [DOI] [PubMed] [Google Scholar]
  2. Anselme J., Härtlein M. Asparaginyl-tRNA synthetase from Escherichia coli has significant sequence homologies with yeast aspartyl-tRNA synthetase. Gene. 1989 Dec 14;84(2):481–485. doi: 10.1016/0378-1119(89)90524-6. [DOI] [PubMed] [Google Scholar]
  3. Berger S. L., Wallace D. M., Puskas R. S., Eschenfeldt W. H. Reverse transcriptase and its associated ribonuclease H: interplay of two enzyme activities controls the yield of single-stranded complementary deoxyribonucleic acid. Biochemistry. 1983 May 10;22(10):2365–2372. doi: 10.1021/bi00279a010. [DOI] [PubMed] [Google Scholar]
  4. Berk A. J., Sharp P. A. Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of S1 endonuclease-digested hybrids. Cell. 1977 Nov;12(3):721–732. doi: 10.1016/0092-8674(77)90272-0. [DOI] [PubMed] [Google Scholar]
  5. Bhat T. N., Blow D. M., Brick P., Nyborg J. Tyrosyl-tRNA synthetase forms a mononucleotide-binding fold. J Mol Biol. 1982 Jul 15;158(4):699–709. doi: 10.1016/0022-2836(82)90255-8. [DOI] [PubMed] [Google Scholar]
  6. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Brune M., Schumann R., Wittinghofer F. Cloning and sequencing of the adenylate kinase gene (adk) of Escherichia coli. Nucleic Acids Res. 1985 Oct 11;13(19):7139–7151. doi: 10.1093/nar/13.19.7139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Carter P. J., Winter G., Wilkinson A. J., Fersht A. R. The use of double mutants to detect structural changes in the active site of the tyrosyl-tRNA synthetase (Bacillus stearothermophilus). Cell. 1984 Oct;38(3):835–840. doi: 10.1016/0092-8674(84)90278-2. [DOI] [PubMed] [Google Scholar]
  9. Cirakoglu B., Waller J. P. Do yeast aminoacyl-tRNA synthetases exist as soluble enzymes within the cytoplasm? Eur J Biochem. 1985 Jun 3;149(2):353–361. doi: 10.1111/j.1432-1033.1985.tb08933.x. [DOI] [PubMed] [Google Scholar]
  10. Dale R. M., McClure B. A., Houchins J. P. A rapid single-stranded cloning strategy for producing a sequential series of overlapping clones for use in DNA sequencing: application to sequencing the corn mitochondrial 18 S rDNA. Plasmid. 1985 Jan;13(1):31–40. doi: 10.1016/0147-619x(85)90053-8. [DOI] [PubMed] [Google Scholar]
  11. Dang C. V., Dang C. V. Multienzyme complex of aminoacyl-tRNA synthetases: an essence of being eukaryotic. Biochem J. 1986 Oct 15;239(2):249–255. doi: 10.1042/bj2390249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ehresmann B., Imbault P., Weil J. H. Spectrophotometric determination of protein concentration in cell extracts containing tRNA's and rRNA's. Anal Biochem. 1973 Aug;54(2):454–463. doi: 10.1016/0003-2697(73)90374-6. [DOI] [PubMed] [Google Scholar]
  14. Fersht A. R. Dissection of the structure and activity of the tyrosyl-tRNA synthetase by site-directed mutagenesis. Biochemistry. 1987 Dec 15;26(25):8031–8037. doi: 10.1021/bi00399a001. [DOI] [PubMed] [Google Scholar]
  15. Gampel A., Tzagoloff A. Homology of aspartyl- and lysyl-tRNA synthetases. Proc Natl Acad Sci U S A. 1989 Aug;86(16):6023–6027. doi: 10.1073/pnas.86.16.6023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Heck J. D., Hatfield G. W. Valyl-tRNA synthetase gene of Escherichia coli K12. Primary structure and homology within a family of aminoacyl-TRNA synthetases. J Biol Chem. 1988 Jan 15;263(2):868–877. [PubMed] [Google Scholar]
  17. Hountondji C., Blanquet S., Lederer F. Methionyl-tRNA synthetase from Escherichia coli: primary structure at the binding site for the 3'-end of tRNAfMet. Biochemistry. 1985 Feb 26;24(5):1175–1180. doi: 10.1021/bi00326a018. [DOI] [PubMed] [Google Scholar]
  18. Jacobo-Molina A., Peterson R., Yang D. C. cDNA sequence, predicted primary structure, and evolving amphiphilic helix of human aspartyl-tRNA synthetase. J Biol Chem. 1989 Oct 5;264(28):16608–16612. [PubMed] [Google Scholar]
  19. Jasin M., Regan L., Schimmel P. Modular arrangement of functional domains along the sequence of an aminoacyl tRNA synthetase. Nature. 1983 Dec 1;306(5942):441–447. doi: 10.1038/306441a0. [DOI] [PubMed] [Google Scholar]
  20. Konigsberg W., Godson G. N. Evidence for use of rare codons in the dnaG gene and other regulatory genes of Escherichia coli. Proc Natl Acad Sci U S A. 1983 Feb;80(3):687–691. doi: 10.1073/pnas.80.3.687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  22. Leatherbarrow R. J., Fersht A. R., Winter G. Transition-state stabilization in the mechanism of tyrosyl-tRNA synthetase revealed by protein engineering. Proc Natl Acad Sci U S A. 1985 Dec;82(23):7840–7844. doi: 10.1073/pnas.82.23.7840. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Leberman R., Berthet-Colominas C., Cusack S., Härtlein M. Crystals of seryl-tRNA synthetase from Escherichia coli. Preliminary crystallographic data. J Mol Biol. 1987 Jan 20;193(2):423–425. doi: 10.1016/0022-2836(87)90232-4. [DOI] [PubMed] [Google Scholar]
  24. Leon O., Schulman L. H. tRNA recognition site of Escherichia coli methionyl-tRNA synthetase. Biochemistry. 1987 Aug 25;26(17):5416–5422. doi: 10.1021/bi00391a030. [DOI] [PubMed] [Google Scholar]
  25. Lorber B., Kern D., Dietrich A., Gangloff J., Ebel J. P., Giegé R. Large scale purification and structural properties of yeast aspartyl-tRNA synthetase. Biochem Biophys Res Commun. 1983 Nov 30;117(1):259–267. doi: 10.1016/0006-291x(83)91569-3. [DOI] [PubMed] [Google Scholar]
  26. Lévêque F., Plateau P., Dessen P., Blanquet S. Homology of lysS and lysU, the two Escherichia coli genes encoding distinct lysyl-tRNA synthetase species. Nucleic Acids Res. 1990 Jan 25;18(2):305–312. doi: 10.1093/nar/18.2.305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Maxam A. M., Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci U S A. 1977 Feb;74(2):560–564. doi: 10.1073/pnas.74.2.560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mirande M., Waller J. P. Molecular cloning and primary structure of cDNA encoding the catalytic domain of rat liver aspartyl-tRNA synthetase. J Biol Chem. 1989 Jan 15;264(2):842–847. [PubMed] [Google Scholar]
  29. Mulvey R. S., Gualtieri R. J., Beychok S. Composition, fluorescence, and circular dichroism of rat lysozyme. Biochemistry. 1974 Feb 12;13(4):782–787. doi: 10.1021/bi00701a021. [DOI] [PubMed] [Google Scholar]
  30. Prevost G., Eriani G., Kern D., Dirheimer G., Gangloff J. Study of the arrangement of the functional domains along the yeast cytoplasmic aspartyl-tRNA synthetase. Eur J Biochem. 1989 Mar 15;180(2):351–358. doi: 10.1111/j.1432-1033.1989.tb14655.x. [DOI] [PubMed] [Google Scholar]
  31. Risler J. L., Zelwer C., Brunie S. Methionyl-tRNA synthetase shows the nucleotide binding fold observed in dehydrogenases. Nature. 1981 Jul 23;292(5821):384–386. doi: 10.1038/292384a0. [DOI] [PubMed] [Google Scholar]
  32. Rosenberg M., Court D. Regulatory sequences involved in the promotion and termination of RNA transcription. Annu Rev Genet. 1979;13:319–353. doi: 10.1146/annurev.ge.13.120179.001535. [DOI] [PubMed] [Google Scholar]
  33. Rould M. A., Perona J. J., Söll D., Steitz T. A. Structure of E. coli glutaminyl-tRNA synthetase complexed with tRNA(Gln) and ATP at 2.8 A resolution. Science. 1989 Dec 1;246(4934):1135–1142. doi: 10.1126/science.2479982. [DOI] [PubMed] [Google Scholar]
  34. Rubin J., Blow D. M. Amino acid activation in crystalline tyrosyl-tRNA synthetase from Bacillus stearothermophilus. J Mol Biol. 1981 Jan 25;145(3):489–500. doi: 10.1016/0022-2836(81)90541-6. [DOI] [PubMed] [Google Scholar]
  35. Ruff M., Cavarelli J., Mikol V., Lorber B., Mitschler A., Giege R., Thierry J. C., Moras D. A high resolution diffracting crystal form of the complex between yeast tRNAAsp and aspartyl-tRNA synthetase. J Mol Biol. 1988 May 5;201(1):235–236. doi: 10.1016/0022-2836(88)90450-0. [DOI] [PubMed] [Google Scholar]
  36. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Schimmel P. R., Söll D. Aminoacyl-tRNA synthetases: general features and recognition of transfer RNAs. Annu Rev Biochem. 1979;48:601–648. doi: 10.1146/annurev.bi.48.070179.003125. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. Sellami M., Fasiolo F., Dirheimer G., Ebel J. P., Gangloff J. Nucleotide sequence of the gene coding for yeast cytoplasmic aspartyl-tRNA synthetase (APS); mapping of the 5' and 3' termini of AspRS mRNA. Nucleic Acids Res. 1986 Feb 25;14(4):1657–1666. doi: 10.1093/nar/14.4.1657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Shine J., Dalgarno L. Determinant of cistron specificity in bacterial ribosomes. Nature. 1975 Mar 6;254(5495):34–38. doi: 10.1038/254034a0. [DOI] [PubMed] [Google Scholar]
  41. Tabor S., Richardson C. C. DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc Natl Acad Sci U S A. 1987 Jul;84(14):4767–4771. doi: 10.1073/pnas.84.14.4767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Webster T., Tsai H., Kula M., Mackie G. A., Schimmel P. Specific sequence homology and three-dimensional structure of an aminoacyl transfer RNA synthetase. Science. 1984 Dec 14;226(4680):1315–1317. doi: 10.1126/science.6390679. [DOI] [PubMed] [Google Scholar]
  43. Young R. A., Davis R. W. Yeast RNA polymerase II genes: isolation with antibody probes. Science. 1983 Nov 18;222(4625):778–782. doi: 10.1126/science.6356359. [DOI] [PubMed] [Google Scholar]

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

RESOURCES