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. 1994 Jul 1;13(13):3166–3169. doi: 10.1002/j.1460-2075.1994.tb06615.x

The exchange of the discriminator base A73 for G is alone sufficient to convert human tRNA(Leu) into a serine-acceptor in vitro.

K Breitschopf 1, H J Gross 1
PMCID: PMC395208  PMID: 8039509

Abstract

Transfer RNA (tRNA) identify is maintained by the highly specific interaction of a few defined nucleotides or groups of nucleotides, called identity elements, with the cognate aminoacyl-tRNA synthetase, and by nonproductive interactions with the other 19 aminoacyl-tRNA synthetases. Most tRNAs have a set of identity elements in at least two locations, commonly in the anticodon loop or in the acceptor stem, and at the discriminator base position 73. We have used T7 RNA polymerase transcribed tRNAs to demonstrate that the sole replacement of the discriminator base A73 of human tRNA(Leu) with the tRNA(Ser)-specific G generates a complete identity switch to serine acceptance. The reverse experiment, the exchange of G73 in human tRNA(Ser) for the tRNA(Leu-specific A, causes a total loss of serine specificity without creating any leucine acceptance. These results suggest that the discriminator base A73 of human tRNA(Leu) alone protects this tRNA against serylation by seryl-tRNA synthetase. This is the first report of a complete identity switch caused by an exchange of the discriminator base alone.

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Selected References

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  1. Achsel T., Gross H. J. Identity determinants of human tRNA(Ser): sequence elements necessary for serylation and maturation of a tRNA with a long extra arm. EMBO J. 1993 Aug;12(8):3333–3338. doi: 10.1002/j.1460-2075.1993.tb06003.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Asahara H., Himeno H., Tamura K., Hasegawa T., Watanabe K., Shimizu M. Recognition nucleotides of Escherichia coli tRNA(Leu) and its elements facilitating discrimination from tRNASer and tRNA(Tyr). J Mol Biol. 1993 May 20;231(2):219–229. doi: 10.1006/jmbi.1993.1277. [DOI] [PubMed] [Google Scholar]
  3. Capone J. P., Sharp P. A., RajBhandary U. L. Amber, ochre and opal suppressor tRNA genes derived from a human serine tRNA gene. EMBO J. 1985 Jan;4(1):213–221. doi: 10.1002/j.1460-2075.1985.tb02338.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Crothers D. M., Seno T., Söll G. Is there a discriminator site in transfer RNA? Proc Natl Acad Sci U S A. 1972 Oct;69(10):3063–3067. doi: 10.1073/pnas.69.10.3063. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dignam J. D., Lebovitz R. M., Roeder R. G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983 Mar 11;11(5):1475–1489. doi: 10.1093/nar/11.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Giegé R., Puglisi J. D., Florentz C. tRNA structure and aminoacylation efficiency. Prog Nucleic Acid Res Mol Biol. 1993;45:129–206. doi: 10.1016/s0079-6603(08)60869-7. [DOI] [PubMed] [Google Scholar]
  7. Green C. J., Sohel I., Vold B. S. The discovery of new intron-containing human tRNA genes using the polymerase chain reaction. J Biol Chem. 1990 Jul 25;265(21):12139–12142. [PubMed] [Google Scholar]
  8. 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]
  9. Leinfelder W., Zehelein E., Mandrand-Berthelot M. A., Böck A. Gene for a novel tRNA species that accepts L-serine and cotranslationally inserts selenocysteine. Nature. 1988 Feb 25;331(6158):723–725. doi: 10.1038/331723a0. [DOI] [PubMed] [Google Scholar]
  10. Li S., Pelka H., Schulman L. H. The anticodon and discriminator base are important for aminoacylation of Escherichia coli tRNA(Asn). J Biol Chem. 1993 Aug 25;268(24):18335–18339. [PubMed] [Google Scholar]
  11. McClain W. H., Foss K. Changing the acceptor identity of a transfer RNA by altering nucleotides in a "variable pocket". Science. 1988 Sep 30;241(4874):1804–1807. doi: 10.1126/science.2459773. [DOI] [PubMed] [Google Scholar]
  12. McClain W. H. Rules that govern tRNA identity in protein synthesis. J Mol Biol. 1993 Nov 20;234(2):257–280. doi: 10.1006/jmbi.1993.1582. [DOI] [PubMed] [Google Scholar]
  13. McClain W. H. Transfer RNA identity. FASEB J. 1993 Jan;7(1):72–78. doi: 10.1096/fasebj.7.1.8422977. [DOI] [PubMed] [Google Scholar]
  14. Milligan J. F., Uhlenbeck O. C. Synthesis of small RNAs using T7 RNA polymerase. Methods Enzymol. 1989;180:51–62. doi: 10.1016/0076-6879(89)80091-6. [DOI] [PubMed] [Google Scholar]
  15. Mizutani T., Narihara T., Hashimoto A. Purification and properties of bovine liver seryl-tRNA synthetase. Eur J Biochem. 1984 Aug 15;143(1):9–13. doi: 10.1111/j.1432-1033.1984.tb08331.x. [DOI] [PubMed] [Google Scholar]
  16. Normanly J., Abelson J. tRNA identity. Annu Rev Biochem. 1989;58:1029–1049. doi: 10.1146/annurev.bi.58.070189.005121. [DOI] [PubMed] [Google Scholar]
  17. Normanly J., Ollick T., Abelson J. Eight base changes are sufficient to convert a leucine-inserting tRNA into a serine-inserting tRNA. Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5680–5684. doi: 10.1073/pnas.89.12.5680. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Rogers M. J., Söll D. Discrimination between glutaminyl-tRNA synthetase and seryl-tRNA synthetase involves nucleotides in the acceptor helix of tRNA. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6627–6631. doi: 10.1073/pnas.85.18.6627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Saks M. E., Sampson J. R., Abelson J. N. The transfer RNA identity problem: a search for rules. Science. 1994 Jan 14;263(5144):191–197. doi: 10.1126/science.7506844. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Schimmel P. Parameters for the molecular recognition of transfer RNAs. Biochemistry. 1989 Apr 4;28(7):2747–2759. doi: 10.1021/bi00433a001. [DOI] [PubMed] [Google Scholar]
  22. Schulman L. H. Recognition of tRNAs by aminoacyl-tRNA synthetases. Prog Nucleic Acid Res Mol Biol. 1991;41:23–87. [PubMed] [Google Scholar]
  23. 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]
  24. Steinberg S., Misch A., Sprinzl M. Compilation of tRNA sequences and sequences of tRNA genes. Nucleic Acids Res. 1993 Jul 1;21(13):3011–3015. doi: 10.1093/nar/21.13.3011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sturchler C., Westhof E., Carbon P., Krol A. Unique secondary and tertiary structural features of the eucaryotic selenocysteine tRNA(Sec). Nucleic Acids Res. 1993 Mar 11;21(5):1073–1079. doi: 10.1093/nar/21.5.1073. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Wu X. Q., Gross H. J. The long extra arms of human tRNA((Ser)Sec) and tRNA(Ser) function as major identify elements for serylation in an orientation-dependent, but not sequence-specific manner. Nucleic Acids Res. 1993 Dec 11;21(24):5589–5594. doi: 10.1093/nar/21.24.5589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Yaniv M., Folk W. R., Berg P., Soll L. A single mutational modification of a tryptophan-specific transfer RNA permits aminoacylation by glutamine and translation of the codon UAG. J Mol Biol. 1974 Jun 25;86(2):245–260. doi: 10.1016/0022-2836(74)90016-3. [DOI] [PubMed] [Google Scholar]
  28. Zawadzki V., Gross H. J. Rapid and simple purification of T7 RNA polymerase. Nucleic Acids Res. 1991 Apr 25;19(8):1948–1948. doi: 10.1093/nar/19.8.1948. [DOI] [PMC free article] [PubMed] [Google Scholar]

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