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. 1994 May 15;13(10):2464–2471. doi: 10.1002/j.1460-2075.1994.tb06531.x

Many of the conserved nucleotides of tRNA(Phe) are not essential for ternary complex formation and peptide elongation.

I A Nazarenko 1, K M Harrington 1, O C Uhlenbeck 1
PMCID: PMC395112  PMID: 8194535

Abstract

An RNase protection assay was used to show that the dissociation rate constants and equilibrium constants of unmodified yeast and Escherichia coli phenylalanyl-tRNA(Phes) to elongation factor Tu from E.coli were very similar to each other and to their fully modified counterparts. The affinity of aminoacylated tRNA to elongation factor Tu was substantially lower when GTP analogues were used in place of GTP, emphasizing the importance of the beta-gamma phosphate linkage in the function of G-proteins. Fourteen different mutations in conserved and semi-conserved nucleotides of yeast phenylalanyl-tRNA(Phe) were tested for binding to elongation factor Tu.GTP and assayed for activity in the ribosomal A- and P-sites. Most of the mutations did not severely impair the function of these tRNAs in any of the assays. This suggests that the translational machinery does not form sequence-specific interactions with the conserved nucleotides of tRNA.

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Selected References

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  1. Abdurashidova G. G., Tsvetkova E. A., Budowsky E. I. Direct tRNA-protein interactions in ribosomal complexes. Nucleic Acids Res. 1991 Apr 25;19(8):1909–1915. doi: 10.1093/nar/19.8.1909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Abrahamson J. K., Laue T. M., Miller D. L., Johnson A. E. Direct determination of the association constant between elongation factor Tu X GTP and aminoacyl-tRNA using fluorescence. Biochemistry. 1985 Jan 29;24(3):692–700. doi: 10.1021/bi00324a023. [DOI] [PubMed] [Google Scholar]
  3. Adkins H. J., Miller D. L., Johnson A. E. Changes in aminoacyl transfer ribonucleic acid conformation upon association with elongation factor Tu-guanosine 5'-triphosphate. fluorescence studies of ternary complex conformation and topology. Biochemistry. 1983 Mar 1;22(5):1208–1217. doi: 10.1021/bi00274a034. [DOI] [PubMed] [Google Scholar]
  4. Arai K., Kawakita M., Kaziro Y. Studies on the polypeptide elongation factors from E. coli. V. Properties of various complexes containing EF-Tu and EF-Ts. J Biochem. 1974 Aug;76(2):293–306. doi: 10.1093/oxfordjournals.jbchem.a130571. [DOI] [PubMed] [Google Scholar]
  5. Baer M. F., Reilly R. M., McCorkle G. M., Hai T. Y., Altman S., RajBhandary U. L. The recognition by RNase P of precursor tRNAs. J Biol Chem. 1988 Feb 15;263(5):2344–2351. [PubMed] [Google Scholar]
  6. Baksht E., de Groot N., Sprinzl M., Cramer F. The behaviour of phenylalanine transfer ribonucleic acid with 3'-terminal formycin in protein biosynthesis using a rabbit reticulocyte cell-free system. FEBS Lett. 1975 Jul 15;55(1):105–108. doi: 10.1016/0014-5793(75)80970-7. [DOI] [PubMed] [Google Scholar]
  7. Baron C., Böck A. The length of the aminoacyl-acceptor stem of the selenocysteine-specific tRNA(Sec) of Escherichia coli is the determinant for binding to elongation factors SELB or Tu. J Biol Chem. 1991 Oct 25;266(30):20375–20379. [PubMed] [Google Scholar]
  8. Behlen L. S., Sampson J. R., DiRenzo A. B., Uhlenbeck O. C. Lead-catalyzed cleavage of yeast tRNAPhe mutants. Biochemistry. 1990 Mar 13;29(10):2515–2523. doi: 10.1021/bi00462a013. [DOI] [PubMed] [Google Scholar]
  9. Berchtold H., Reshetnikova L., Reiser C. O., Schirmer N. K., Sprinzl M., Hilgenfeld R. Crystal structure of active elongation factor Tu reveals major domain rearrangements. Nature. 1993 Sep 9;365(6442):126–132. doi: 10.1038/365126a0. [DOI] [PubMed] [Google Scholar]
  10. Bhuta P., Chládek S. Stimulation of Escherichia coli elongation factor Tu-dependent GTP hydrolysis by aminoacyl oligonucleotides in the presence of aurodox. FEBS Lett. 1980 Dec 15;122(1):113–116. doi: 10.1016/0014-5793(80)80414-5. [DOI] [PubMed] [Google Scholar]
  11. Boutorin A. S., Clark B. F., Ebel J. P., Kruse T. A., Petersen H. U., Remy P., Vassilenko S. A study of the interaction of Escherichia coli elongation factor-Tu with aminoacyl-tRNAs by partial digestion with cobra venom ribonuclease. J Mol Biol. 1981 Nov 5;152(3):593–608. doi: 10.1016/0022-2836(81)90271-0. [DOI] [PubMed] [Google Scholar]
  12. Delaria K., Guillen M., Louie A., Jurnak F. Stabilization of the Escherichia coli elongation factor Tu-GTP-aminoacyl-tRNA complex. Arch Biochem Biophys. 1991 Apr;286(1):207–211. doi: 10.1016/0003-9861(91)90029-i. [DOI] [PubMed] [Google Scholar]
  13. Derwenskus K. H., Sprinzl M. Interaction of cinnamyl-tRNAPhe with Escherichia coli elongation factor Tu. FEBS Lett. 1983 Jan 10;151(1):143–147. doi: 10.1016/0014-5793(83)80360-3. [DOI] [PubMed] [Google Scholar]
  14. Dix D. B., Wittenberg W. L., Uhlenbeck O. C., Thompson R. C. Effect of replacing uridine 33 in yeast tRNAPhe on the reaction with ribosomes. J Biol Chem. 1986 Aug 5;261(22):10112–10118. [PubMed] [Google Scholar]
  15. Drabkin H. J., RajBhandary U. L. Site-specific mutagenesis on a human initiator methionine tRNA gene within a sequence conserved in all eukaryotic initiator tRNAs and studies of its effects on in vitro transcription. J Biol Chem. 1985 May 10;260(9):5580–5587. [PubMed] [Google Scholar]
  16. Eccleston J. F., Dix D. B., Thompson R. C. The rate of cleavage of GTP on the binding of Phe-tRNA.elongation factor Tu.GTP to poly(U)-programmed ribosomes of Escherichia coli. J Biol Chem. 1985 Dec 25;260(30):16237–16241. [PubMed] [Google Scholar]
  17. Eccleston J. F., Messerschmidt R. G., Yates D. W. A simple rapid mixing device. Anal Biochem. 1980 Jul 15;106(1):73–77. doi: 10.1016/0003-2697(80)90120-7. [DOI] [PubMed] [Google Scholar]
  18. Edqvist J., Stråby K. B., Grosjean H. Pleiotrophic effects of point mutations in yeast tRNA(Asp) on the base modification pattern. Nucleic Acids Res. 1993 Feb 11;21(3):413–417. doi: 10.1093/nar/21.3.413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Erdmann V. A., Sprinzl M., Pongs O. The involvement of 5S RNA in the binding of tRNA to ribosomes. Biochem Biophys Res Commun. 1973 Oct 1;54(3):942–948. doi: 10.1016/0006-291x(73)90785-7. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Greer C. L., Söll D., Willis I. Substrate recognition and identification of splice sites by the tRNA-splicing endonuclease and ligase from Saccharomyces cerevisiae. Mol Cell Biol. 1987 Jan;7(1):76–84. doi: 10.1128/mcb.7.1.76. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Harrington K. M., Nazarenko I. A., Dix D. B., Thompson R. C., Uhlenbeck O. C. In vitro analysis of translational rate and accuracy with an unmodified tRNA. Biochemistry. 1993 Aug 3;32(30):7617–7622. doi: 10.1021/bi00081a003. [DOI] [PubMed] [Google Scholar]
  23. Hazlett T. L., Johnson A. E., Jameson D. M. Time-resolved fluorescence studies on the ternary complex formed between bacterial elongation factor Tu, guanosine 5'-triphosphate, and phenylalanyl-tRNAPhe. Biochemistry. 1989 May 2;28(9):4109–4117. doi: 10.1021/bi00435a073. [DOI] [PubMed] [Google Scholar]
  24. Helk B., Sprinzl M. Interaction of unfolded tRNA with the 3'-terminal region of E. coli 16S ribosomal RNA. Nucleic Acids Res. 1985 Sep 11;13(17):6283–6298. doi: 10.1093/nar/13.17.6283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Jekowsky E., Schimmel P. R., Miller D. L. Isolation, characterization and structural implications of a nuclease-digested complex of aminoacyl transfer RNA and Escherichia coli elongation factor Tu. J Mol Biol. 1977 Aug 15;114(3):451–458. doi: 10.1016/0022-2836(77)90262-5. [DOI] [PubMed] [Google Scholar]
  26. Jonák J., Smrt J., Holý A., Rychlík I. Interaction of Escherichia coli EF-Tu.GTP and EF-Tu.GDP with analogues of the 3' terminus of aminoacyl-tRNA. Eur J Biochem. 1980 Apr;105(2):315–320. doi: 10.1111/j.1432-1033.1980.tb04503.x. [DOI] [PubMed] [Google Scholar]
  27. Joshi R. L., Faulhammer H., Chapeville F., Sprinzl M., Haenni A. L. Aminoacyl RNA domain of turnip yellow mosaic virus Val-RNA interacting with elongation factor Tu. Nucleic Acids Res. 1984 Oct 11;12(19):7467–7478. doi: 10.1093/nar/12.19.7467. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lee C. P., Dyson M. R., Mandal N., Varshney U., Bahramian B., RajBhandary U. L. Striking effects of coupling mutations in the acceptor stem on recognition of tRNAs by Escherichia coli Met-tRNA synthetase and Met-tRNA transformylase. Proc Natl Acad Sci U S A. 1992 Oct 1;89(19):9262–9266. doi: 10.1073/pnas.89.19.9262. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lill R., Robertson J. M., Wintermeyer W. Affinities of tRNA binding sites of ribosomes from Escherichia coli. Biochemistry. 1986 Jun 3;25(11):3245–3255. doi: 10.1021/bi00359a025. [DOI] [PubMed] [Google Scholar]
  30. Lill R., Wintermeyer W. Quantitative indicator assay of tRNA binding to the ribosomal P and A sites. Methods Enzymol. 1988;164:597–611. doi: 10.1016/s0076-6879(88)64072-9. [DOI] [PubMed] [Google Scholar]
  31. Lin F. L., Kahan L., Ofengand J. Crosslinking of phenylalanyl-tRNA to the ribosomal A site via a photoaffinity probe attached to the 4-thiouridine residue is exclusively to ribosomal protein S19. J Mol Biol. 1984 Jan 5;172(1):77–86. doi: 10.1016/0022-2836(84)90415-7. [DOI] [PubMed] [Google Scholar]
  32. Louie A., Jurnak F. Kinetic studies of Escherichia coli elongation factor Tu-guanosine 5'-triphosphate-aminoacyl-tRNA complexes. Biochemistry. 1985 Nov 5;24(23):6433–6439. doi: 10.1021/bi00344a019. [DOI] [PubMed] [Google Scholar]
  33. Louie A., Ribeiro N. S., Reid B. R., Jurnak F. Relative affinities of all Escherichia coli aminoacyl-tRNAs for elongation factor Tu-GTP. J Biol Chem. 1984 Apr 25;259(8):5010–5016. [PubMed] [Google Scholar]
  34. Mattoccia E., Baldi I. M., Gandini-Attardi D., Ciafrè S., Tocchini-Valentini G. P. Site selection by the tRNA splicing endonuclease of Xenopus laevis. Cell. 1988 Nov 18;55(4):731–738. doi: 10.1016/0092-8674(88)90231-0. [DOI] [PubMed] [Google Scholar]
  35. Mitchell P., Stade K., Osswald M., Brimacombe R. Site-directed cross-linking studies on the E. coli tRNA-ribosome complex: determination of sites labelled with an aromatic azide attached to the variable loop or aminoacyl group of tRNA. Nucleic Acids Res. 1993 Feb 25;21(4):887–896. doi: 10.1093/nar/21.4.887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Moazed D., Noller H. F. Binding of tRNA to the ribosomal A and P sites protects two distinct sets of nucleotides in 16 S rRNA. J Mol Biol. 1990 Jan 5;211(1):135–145. doi: 10.1016/0022-2836(90)90016-F. [DOI] [PubMed] [Google Scholar]
  37. Moazed D., Noller H. F. Sites of interaction of the CCA end of peptidyl-tRNA with 23S rRNA. Proc Natl Acad Sci U S A. 1991 May 1;88(9):3725–3728. doi: 10.1073/pnas.88.9.3725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Moazed D., Robertson J. M., Noller H. F. Interaction of elongation factors EF-G and EF-Tu with a conserved loop in 23S RNA. Nature. 1988 Jul 28;334(6180):362–364. doi: 10.1038/334362a0. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. Ofengand J., Chen C. M. Inactivation of T u factor-guanosine triphosphate recognition and ribosome-binding ability by terminal oxidation-reduction of yeast phenylalanine transfer ribonucleic acid. J Biol Chem. 1972 Apr 10;247(7):2049–2058. [PubMed] [Google Scholar]
  41. Ofengand J., Henes C. The function of pseudouridylic acid in transfer ribonucleic acid. II. Inhibition of amino acyl transfer ribonucleic acid-ribosome complex formation by ribothymidylyl-pseudouridylyl-cytidylyl-guanosine 3'-phosphate. J Biol Chem. 1969 Nov 25;244(22):6241–6253. [PubMed] [Google Scholar]
  42. Parlato G., Guesnet J., Crechet J. B., Parmeggiani A. The GTPase activity of elongation factor Tu and the 3'-terminal end of aminoacyl-tRNA. FEBS Lett. 1981 Mar 23;125(2):257–260. doi: 10.1016/0014-5793(81)80733-8. [DOI] [PubMed] [Google Scholar]
  43. Peterson E. T., Blank J., Sprinzl M., Uhlenbeck O. C. Selection for active E. coli tRNA(Phe) variants from a randomized library using two proteins. EMBO J. 1993 Jul;12(7):2959–2967. doi: 10.1002/j.1460-2075.1993.tb05958.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Peterson E. T., Uhlenbeck O. C. Determination of recognition nucleotides for Escherichia coli phenylalanyl-tRNA synthetase. Biochemistry. 1992 Oct 27;31(42):10380–10389. doi: 10.1021/bi00157a028. [DOI] [PubMed] [Google Scholar]
  45. Pingoud A., Block W., Wittinghofer A., Wolf H., Fischer E. The elongation factor Tu binds aminoacyl-tRNA in the presence of GDP. J Biol Chem. 1982 Oct 10;257(19):11261–11267. [PubMed] [Google Scholar]
  46. Rasmussen N. J., Wikman F. P., Clark B. F. Crosslinking of tRNA containing a long extra arm to elongation factor Tu by trans-diamminedichloroplatinum(II). Nucleic Acids Res. 1990 Aug 25;18(16):4883–4890. doi: 10.1093/nar/18.16.4883. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Reyes V. M., Abelson J. Substrate recognition and splice site determination in yeast tRNA splicing. Cell. 1988 Nov 18;55(4):719–730. doi: 10.1016/0092-8674(88)90230-9. [DOI] [PubMed] [Google Scholar]
  48. Rheinberger H. J., Schilling S., Nierhaus K. H. The ribosomal elongation cycle: tRNA binding, translocation and tRNA release. Eur J Biochem. 1983 Aug 15;134(3):421–428. doi: 10.1111/j.1432-1033.1983.tb07584.x. [DOI] [PubMed] [Google Scholar]
  49. Romby P., Carbon P., Westhof E., Ehresmann C., Ebel J. P., Ehresmann B., Giegé R. Importance of conserved residues for the conformation of the T-loop in tRNAs. J Biomol Struct Dyn. 1987 Dec;5(3):669–687. doi: 10.1080/07391102.1987.10506419. [DOI] [PubMed] [Google Scholar]
  50. Rose S. J., 3rd, Lowary P. T., Uhlenbeck O. C. Binding of yeast tRNAPhe anticodon arm to Escherichia coli 30 S ribosomes. J Mol Biol. 1983 Jun 15;167(1):103–117. doi: 10.1016/s0022-2836(83)80036-9. [DOI] [PubMed] [Google Scholar]
  51. Sampson J. R., DiRenzo A. B., Behlen L. S., Uhlenbeck O. C. Nucleotides in yeast tRNAPhe required for the specific recognition by its cognate synthetase. Science. 1989 Mar 10;243(4896):1363–1366. doi: 10.1126/science.2646717. [DOI] [PubMed] [Google Scholar]
  52. 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]
  53. Schilling-Bartetzko S., Franceschi F., Sternbach H., Nierhaus K. H. Apparent association constants of tRNAs for the ribosomal A, P, and E sites. J Biol Chem. 1992 Mar 5;267(7):4693–4702. [PubMed] [Google Scholar]
  54. Schulman L. H., Pelka H. Anticodon switching changes the identity of methionine and valine transfer RNAs. Science. 1988 Nov 4;242(4879):765–768. doi: 10.1126/science.3055296. [DOI] [PubMed] [Google Scholar]
  55. Seong B. L., RajBhandary U. L. Mutants of Escherichia coli formylmethionine tRNA: a single base change enables initiator tRNA to act as an elongator in vitro. Proc Natl Acad Sci U S A. 1987 Dec;84(24):8859–8863. doi: 10.1073/pnas.84.24.8859. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Sprinzl M., Kucharzewski M., Hobbs J. B., Cramer F. Specificity of elongation factor Tu from Escherichia coli with respect to attachment to the amino acid to the 2' or 3'-hydroxyl group of the terminal adenosine of tRNA. Eur J Biochem. 1977 Aug 15;78(1):55–61. doi: 10.1111/j.1432-1033.1977.tb11713.x. [DOI] [PubMed] [Google Scholar]
  57. Sprinzl M., Wagner T., Lorenz S., Erdmann V. A. Regions of tRNA important for binding to the ribosomal A and P sites. Biochemistry. 1976 Jul 13;15(14):3031–3039. doi: 10.1021/bi00659a015. [DOI] [PubMed] [Google Scholar]
  58. Thompson R. C., Dix D. B., Eccleston J. F. Single turnover kinetic studies of guanosine triphosphate hydrolysis and peptide formation in the elongation factor Tu-dependent binding of aminoacyl-tRNA to Escherichia coli ribosomes. J Biol Chem. 1980 Dec 10;255(23):11088–11090. [PubMed] [Google Scholar]
  59. Thompson R. C., Dix D. B., Karim A. M. The reaction of ribosomes with elongation factor Tu.GTP complexes. Aminoacyl-tRNA-independent reactions in the elongation cycle determine the accuracy of protein synthesis. J Biol Chem. 1986 Apr 15;261(11):4868–4874. [PubMed] [Google Scholar]
  60. Thurlow D. L., Shilowski D., Marsh T. L. Nucleotides in precursor tRNAs that are required intact for catalysis by RNase P RNAs. Nucleic Acids Res. 1991 Feb 25;19(4):885–891. doi: 10.1093/nar/19.4.885. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Weygand-Durasevic I., Kruse T. A., Clark B. F. The influence of elongation-factor-Tu . GTP and anticodon-anticodon interactions on the anticodon loop conformation of yeast tRNATyr. Eur J Biochem. 1981 May;116(1):59–65. doi: 10.1111/j.1432-1033.1981.tb05300.x. [DOI] [PubMed] [Google Scholar]
  62. Wikman F. P., Romby P., Metz M. H., Reinbolt J., Clark B. F., Ebel J. P., Ehresmann C., Ehresmann B. Crosslinking of elongation factor Tu to tRNA(Phe) by trans-diamminedichloroplatinum (II). Characterization of two crosslinking sites in the tRNA. Nucleic Acids Res. 1987 Jul 24;15(14):5787–5801. doi: 10.1093/nar/15.14.5787. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Wikman F. P., Siboska G. E., Petersen H. U., Clark B. F. The site of interaction of aminoacyl-tRNA with elongation factor Tu. EMBO J. 1982;1(9):1095–1100. doi: 10.1002/j.1460-2075.1982.tb01302.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Yount R. G., Babcock D., Ballantyne W., Ojala D. Adenylyl imidodiphosphate, an adenosine triphosphate analog containing a P--N--P linkage. Biochemistry. 1971 Jun 22;10(13):2484–2489. doi: 10.1021/bi00789a009. [DOI] [PubMed] [Google Scholar]

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