Skip to main content
PMC 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
. 1991 Jul 15;88(14):6132–6136. doi: 10.1073/pnas.88.14.6132

Interaction of Escherichia coli tRNA(Ser) with its cognate aminoacyl-tRNA synthetase as determined by footprinting with phosphorothioate-containing tRNA transcripts.

D Schatz 1, R Leberman 1, F Eckstein 1
PMCID: PMC52036  PMID: 2068094

Abstract

A footprinting technique using phosphorothioate-containing RNA transcripts has been developed and applied to identify contacts between Escherichia coli tRNA(Ser) and its cognate aminoacyl-tRNA synthetase. The cloned gene for the tRNA was transcribed in four reactions in which a different NTP was complemented by 5% of the corresponding nucleoside 5'-O-(1-thiotriphosphate). The phosphorothioate groups of such transcripts are cleaved by reaction with iodine to permit sequencing of the transcripts. Footprinting was achieved by performing the same reaction with the phosphorothioate-tRNA-enzyme complex. At 1 mM iodine, selective protection of the tRNA transcripts in the cognate system was observed, with strong protection at positions 52 and 68 and weak protection at positions 46, 53, 67, 69, and 70. It is suggested that these regions of the tRNA interact with the helical arm of the synthetase.

Full text

PDF
6132

Images in this article

6134Image
on p.6134
6134Image
on p.6134
6135Image
on p.6135

Selected References

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

  1. 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]
  2. Dock-Bregeon A. C., Garcia A., Giegé R., Moras D. The contacts of yeast tRNA(Ser) with seryl-tRNA synthetase studied by footprinting experiments. Eur J Biochem. 1990 Mar 10;188(2):283–290. doi: 10.1111/j.1432-1033.1990.tb15401.x. [DOI] [PubMed] [Google Scholar]
  3. Dock-Bregeon A. C., Moras D. Conformational changes and dynamics of tRNAs: evidence from hydrolysis patterns. Cold Spring Harb Symp Quant Biol. 1987;52:113–121. doi: 10.1101/sqb.1987.052.01.016. [DOI] [PubMed] [Google Scholar]
  4. Dock-Bregeon A. C., Westhof E., Giegé R., Moras D. Solution structure of a tRNA with a large variable region: yeast tRNASer. J Mol Biol. 1989 Apr 20;206(4):707–722. doi: 10.1016/0022-2836(89)90578-0. [DOI] [PubMed] [Google Scholar]
  5. Garcia A., Giegé R., Behr J. P. New photoactivatable structural and affinity probes of RNAs: specific features and applications for mapping of spermine binding sites in yeast tRNA(Asp) and interaction of this tRNA with yeast aspartyl-tRNA synthetase. Nucleic Acids Res. 1990 Jan 11;18(1):89–95. doi: 10.1093/nar/18.1.89. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Garret M., Labouesse B., Litvak S., Romby P., Ebel J. P., Giegé R. Tertiary structure of animal tRNATrp in solution and interaction of tRNATrp with tryptophanyl-tRNA synthetase. Eur J Biochem. 1984 Jan 2;138(1):67–75. doi: 10.1111/j.1432-1033.1984.tb07882.x. [DOI] [PubMed] [Google Scholar]
  7. Gish G., Eckstein F. DNA and RNA sequence determination based on phosphorothioate chemistry. Science. 1988 Jun 10;240(4858):1520–1522. doi: 10.1126/science.2453926. [DOI] [PubMed] [Google Scholar]
  8. Griffiths A. D., Potter B. V., Eperon I. C. Stereospecificity of nucleases towards phosphorothioate-substituted RNA: stereochemistry of transcription by T7 RNA polymerase. Nucleic Acids Res. 1987 May 26;15(10):4145–4162. doi: 10.1093/nar/15.10.4145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Himeno H., Hasegawa T., Ueda T., Watanabe K., Shimizu M. Conversion of aminoacylation specificity from tRNA(Tyr) to tRNA(Ser) in vitro. Nucleic Acids Res. 1990 Dec 11;18(23):6815–6819. doi: 10.1093/nar/18.23.6815. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Latham J. A., Cech T. R. Defining the inside and outside of a catalytic RNA molecule. Science. 1989 Jul 21;245(4915):276–282. doi: 10.1126/science.2501870. [DOI] [PubMed] [Google Scholar]
  12. Normanly J., Ogden R. C., Horvath S. J., Abelson J. Changing the identity of a transfer RNA. Nature. 1986 May 15;321(6067):213–219. doi: 10.1038/321213a0. [DOI] [PubMed] [Google Scholar]
  13. Park S. J., Schimmel P. Evidence for interaction of an aminoacyl transfer RNA synthetase with a region important for the identity of its cognate transfer RNA. J Biol Chem. 1988 Nov 15;263(32):16527–16530. [PubMed] [Google Scholar]
  14. Romaniuk P. J., de Stevenson I. L., Wong H. H. Defining the binding site of Xenopus transcription factor IIIA on 5S RNA using truncated and chimeric 5S RNA molecules. Nucleic Acids Res. 1987 Mar 25;15(6):2737–2755. doi: 10.1093/nar/15.6.2737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Romby P., Moras D., Bergdoll M., Dumas P., Vlassov V. V., Westhof E., Ebel J. P., Giegé R. Yeast tRNAAsp tertiary structure in solution and areas of interaction of the tRNA with aspartyl-tRNA synthetase. A comparative study of the yeast phenylalanine system by phosphate alkylation experiments with ethylnitrosourea. J Mol Biol. 1985 Aug 5;184(3):455–471. doi: 10.1016/0022-2836(85)90294-3. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Roy K. L., Söll D. Purification of five serine transfer ribonucleic acid species from Escherichia coli and their acylation by homologous and heterologous seryl transfer ribonucleic acid synthetases. J Biol Chem. 1970 Mar 25;245(6):1394–1400. [PubMed] [Google Scholar]
  18. Sampson J. R., Uhlenbeck O. C. Biochemical and physical characterization of an unmodified yeast phenylalanine transfer RNA transcribed in vitro. Proc Natl Acad Sci U S A. 1988 Feb;85(4):1033–1037. doi: 10.1073/pnas.85.4.1033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Sayers J. R., Schmidt W., Eckstein F. 5'-3' exonucleases in phosphorothioate-based oligonucleotide-directed mutagenesis. Nucleic Acids Res. 1988 Feb 11;16(3):791–802. doi: 10.1093/nar/16.3.791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Schulman L. H., Abelson J. Recent excitement in understanding transfer RNA identity. Science. 1988 Jun 17;240(4859):1591–1592. doi: 10.1126/science.2454505. [DOI] [PubMed] [Google Scholar]
  21. Sprinzl M., Sternbach H., von der Haar F., Cramer F. Enzymatic incorporation of ATP and CTP analogues into the 3' end of tRNA. Eur J Biochem. 1977 Dec;81(3):579–589. doi: 10.1111/j.1432-1033.1977.tb11985.x. [DOI] [PubMed] [Google Scholar]
  22. Theobald A., Springer M., Grunberg-Manago M., Ebel J. P., Giege R. Tertiary structure of Escherichia coli tRNA(3Thr) in solution and interaction of this tRNA with the cognate threonyl-tRNA synthetase. Eur J Biochem. 1988 Aug 15;175(3):511–524. doi: 10.1111/j.1432-1033.1988.tb14223.x. [DOI] [PubMed] [Google Scholar]
  23. Vlassov V. V., Kern D., Romby P., Giegé R., Ebel J. P. Interaction of tRNAPhe and tRNAVal with aminoacyl-tRNA synthetases. A chemical modification study. Eur J Biochem. 1983 May 16;132(3):537–544. doi: 10.1111/j.1432-1033.1983.tb07395.x. [DOI] [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