Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1993 Nov 1;90(21):10046–10050. doi: 10.1073/pnas.90.21.10046

Diversified sequences of peptide epitope for same-RNA recognition.

S Kim 1, L Ribas de Pouplana 1, P Schimmel 1
PMCID: PMC47710  PMID: 7694278

Abstract

We replaced an essential RNA-binding, 30-amino acid helix-loop in an Escherichia coli tRNA synthetase with an inactive and simplified "generic" sequence having 23 of the 30 amino acids as alanine and serine. Wild-type residues were restored in random combinations to generate a library with a sequence complexity of about 1.9 x 10(7). Active molecules were obtained by genetic selection at a frequency of approximately 1% and contained variants with as many as 11 alanine/serine replacements and a total of 17 alanine/serine residues. These variants have activities which are thermodynamically competitive with that of the native protein and therefore are functionally and, most likely, conformationally equivalent.

Full text

PDF
10046

Images in this article

10047Fig. 2
on p.10047
10049Fig. 4
on p.10049

Selected References

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

  1. Barker D. G., Ebel J. P., Jakes R., Bruton C. J. Methionyl-tRNA synthetase from Escherichia coli. Primary structure of the active crystallised tryptic fragment. Eur J Biochem. 1982 Oct;127(3):449–457. [PubMed] [Google Scholar]
  2. Bowie J. U., Lüthy R., Eisenberg D. A method to identify protein sequences that fold into a known three-dimensional structure. Science. 1991 Jul 12;253(5016):164–170. doi: 10.1126/science.1853201. [DOI] [PubMed] [Google Scholar]
  3. Brunie S., Zelwer C., Risler J. L. Crystallographic study at 2.5 A resolution of the interaction of methionyl-tRNA synthetase from Escherichia coli with ATP. J Mol Biol. 1990 Nov 20;216(2):411–424. doi: 10.1016/S0022-2836(05)80331-6. [DOI] [PubMed] [Google Scholar]
  4. Burbaum J. J., Schimmel P. Assembly of a class I tRNA synthetase from products of an artificially split gene. Biochemistry. 1991 Jan 15;30(2):319–324. doi: 10.1021/bi00216a002. [DOI] [PubMed] [Google Scholar]
  5. Chaudhury A. M., Smith G. R. A new class of Escherichia coli recBC mutants: implications for the role of RecBC enzyme in homologous recombination. Proc Natl Acad Sci U S A. 1984 Dec;81(24):7850–7854. doi: 10.1073/pnas.81.24.7850. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Clarke L., Carbon J. A colony bank containing synthetic Col El hybrid plasmids representative of the entire E. coli genome. Cell. 1976 Sep;9(1):91–99. doi: 10.1016/0092-8674(76)90055-6. [DOI] [PubMed] [Google Scholar]
  7. Dardel F., Fayat G., Blanquet S. Molecular cloning and primary structure of the Escherichia coli methionyl-tRNA synthetase gene. J Bacteriol. 1984 Dec;160(3):1115–1122. doi: 10.1128/jb.160.3.1115-1122.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Francklyn C., Shi J. P., Schimmel P. Overlapping nucleotide determinants for specific aminoacylation of RNA microhelices. Science. 1992 Feb 28;255(5048):1121–1125. doi: 10.1126/science.1546312. [DOI] [PubMed] [Google Scholar]
  9. Ghosh G., Kim H. Y., Demaret J. P., Brunie S., Schulman L. H. Arginine-395 is required for efficient in vivo and in vitro aminoacylation of tRNAs by Escherichia coli methionyl-tRNA synthetase. Biochemistry. 1991 Dec 24;30(51):11767–11774. doi: 10.1021/bi00115a005. [DOI] [PubMed] [Google Scholar]
  10. Ghosh G., Pelka H., Schulman L. H. Identification of the tRNA anticodon recognition site of Escherichia coli methionyl-tRNA synthetase. Biochemistry. 1990 Mar 6;29(9):2220–2225. doi: 10.1021/bi00461a003. [DOI] [PubMed] [Google Scholar]
  11. Jasin M., Schimmel P. Deletion of an essential gene in Escherichia coli by site-specific recombination with linear DNA fragments. J Bacteriol. 1984 Aug;159(2):783–786. doi: 10.1128/jb.159.2.783-786.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kim S., Schimmel P. Function independence of microhelix aminoacylation from anticodon binding in a class I tRNA synthetase. J Biol Chem. 1992 Aug 5;267(22):15563–15567. [PubMed] [Google Scholar]
  13. Kollman P., van Gunsteren W. F. Molecular mechanics and dynamics in protein design. Methods Enzymol. 1987;154:430–449. doi: 10.1016/0076-6879(87)54089-7. [DOI] [PubMed] [Google Scholar]
  14. Mechulam Y., Schmitt E., Panvert M., Schmitter J. M., Lapadat-Tapolsky M., Meinnel T., Dessen P., Blanquet S., Fayat G. Methionyl-tRNA synthetase from Bacillus stearothermophilus: structural and functional identities with the Escherichia coli enzyme. Nucleic Acids Res. 1991 Jul 11;19(13):3673–3681. doi: 10.1093/nar/19.13.3673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Nureki O., Muramatsu T., Suzuki K., Kohda D., Matsuzawa H., Ohta T., Miyazawa T., Yokoyama S. Methionyl-tRNA synthetase gene from an extreme thermophile, Thermus thermophilus HB8. Molecular cloning, primary-structure analysis, expression in Escherichia coli, and site-directed mutagenesis. J Biol Chem. 1991 Feb 15;266(5):3268–3277. [PubMed] [Google Scholar]
  17. Pabo C. Molecular technology. Designing proteins and peptides. Nature. 1983 Jan 20;301(5897):200–200. doi: 10.1038/301200a0. [DOI] [PubMed] [Google Scholar]
  18. Richmond T. J., Richards F. M. Packing of alpha-helices: geometrical constraints and contact areas. J Mol Biol. 1978 Mar 15;119(4):537–555. doi: 10.1016/0022-2836(78)90201-2. [DOI] [PubMed] [Google Scholar]
  19. Sander C., Schneider R. Database of homology-derived protein structures and the structural meaning of sequence alignment. Proteins. 1991;9(1):56–68. doi: 10.1002/prot.340090107. [DOI] [PubMed] [Google Scholar]
  20. Toth M. J., Schimmel P. Internal structural features of E. coli glycyl-tRNA synthetase examined by subunit polypeptide chain fusions. J Biol Chem. 1986 May 25;261(15):6643–6646. [PubMed] [Google Scholar]
  21. Tzagoloff A., Vambutas A., Akai A. Characterization of MSM1, the structural gene for yeast mitochondrial methionyl-tRNA synthetase. Eur J Biochem. 1989 Feb 1;179(2):365–371. doi: 10.1111/j.1432-1033.1989.tb14562.x. [DOI] [PubMed] [Google Scholar]
  22. Walter P., Gangloff J., Bonnet J., Boulanger Y., Ebel J. P., Fasiolo F. Primary structure of the Saccharomyces cerevisiae gene for methionyl-tRNA synthetase. Proc Natl Acad Sci U S A. 1983 May;80(9):2437–2441. doi: 10.1073/pnas.80.9.2437. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES