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. 1999 Feb;5(2):188–194. doi: 10.1017/s1355838299981529

Single atom modification (O-->S) of tRNA confers ribosome binding.

S S Ashraf 1, E Sochacka 1, R Cain 1, R Guenther 1, A Malkiewicz 1, P F Agris 1
PMCID: PMC1369751  PMID: 10024171

Abstract

Escherichia coli tRNALysSUU, as well as human tRNALys3SUU, has 2-thiouridine derivatives at wobble position 34 (s2U*34). Unlike the native tRNALysSUU, the full-length, unmodified transcript of human tRNALys3UUU and the unmodified tRNALys3UUU anticodon stem/loop (ASLLys3UUU) did not bind AAA- or AAG-programmed ribosomes. In contrast, the completely unmodified yeast tRNAPhe anticodon stem/loop (ASLPheGAA) had an affinity (Kd = 136+/-49 nM) similar to that of native yeast tRNAPheGmAA (Kd = 103+/-19 nM). We have found that the single, site-specific substitution of s2U34 for U34 to produce the modified ASLLysSUU was sufficient to restore ribosomal binding. The modified ASLLysSUU bound the ribosome with an affinity (Kd = 176+/-62 nM) comparable to that of native tRNALysSUU (Kd = 70+/-7 nM). Furthermore, in binding to the ribosome, the modified ASLLys3SUU produced the same 16S P-site tRNA footprint as did native E. coli tRNALysSUU, yeast tRNAPheGmAA, and the unmodified ASLPheGAA. The unmodified ASLLys3UUU had no footprint at all. Investigations of thermal stability and structure monitored by UV spectroscopy and NMR showed that the dynamic conformation of the loop of modified ASLLys3SUU was different from that of the unmodified ASLLysUUU, whereas the stems were isomorphous. Based on these and other data, we conclude that s2U34 in tRNALysSUU and in other s2U34-containing tRNAs is critical for generating an anticodon conformation that leads to effective codon interaction in all organisms. This is the first example of a single atom substitution (U34-->s2U34) that confers the property of ribosomal binding on an otherwise inactive tRNA.

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Selected References

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  1. Agris P. F., Guenther R., Ingram P. C., Basti M. M., Stuart J. W., Sochacka E., Malkiewicz A. Unconventional structure of tRNA(Lys)SUU anticodon explains tRNA's role in bacterial and mammalian ribosomal frameshifting and primer selection by HIV-1. RNA. 1997 Apr;3(4):420–428. [PMC free article] [PubMed] [Google Scholar]
  2. Agris P. F., Malkiewicz A., Kraszewski A., Everett K., Nawrot B., Sochacka E., Jankowska J., Guenther R. Site-selected introduction of modified purine and pyrimidine ribonucleosides into RNA by automated phosphoramidite chemistry. Biochimie. 1995;77(1-2):125–134. doi: 10.1016/0300-9084(96)88115-6. [DOI] [PubMed] [Google Scholar]
  3. Agris P. F., Söll D., Seno T. Biological function of 2-thiouridine in Escherichia coli glutamic acid transfer ribonucleic acid. Biochemistry. 1973 Oct 23;12(22):4331–4337. doi: 10.1021/bi00746a005. [DOI] [PubMed] [Google Scholar]
  4. Cusack S., Yaremchuk A., Tukalo M. The crystal structures of T. thermophilus lysyl-tRNA synthetase complexed with E. coli tRNA(Lys) and a T. thermophilus tRNA(Lys) transcript: anticodon recognition and conformational changes upon binding of a lysyl-adenylate analogue. EMBO J. 1996 Nov 15;15(22):6321–6334. [PMC free article] [PubMed] [Google Scholar]
  5. Dao V., Guenther R., Malkiewicz A., Nawrot B., Sochacka E., Kraszewski A., Jankowska J., Everett K., Agris P. F. Ribosome binding of DNA analogs of tRNA requires base modifications and supports the "extended anticodon". Proc Natl Acad Sci U S A. 1994 Mar 15;91(6):2125–2129. doi: 10.1073/pnas.91.6.2125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ericson G., Minchew P., Wollenzien P. Structural changes in base-paired region 28 in 16 S rRNA close to the decoding region of the 30 S ribosomal subunit are correlated to changes in tRNA binding. J Mol Biol. 1995 Jul 21;250(4):407–419. doi: 10.1006/jmbi.1995.0386. [DOI] [PubMed] [Google Scholar]
  7. Faulstich K., Wörner K., Brill H., Engels J. W. A sequencing method for RNA oligonucleotides based on mass spectrometry. Anal Chem. 1997 Nov 1;69(21):4349–4353. doi: 10.1021/ac961186g. [DOI] [PubMed] [Google Scholar]
  8. Grossenbacher A. M., Stadelmann B., Heyer W. D., Thuriaux P., Kohli J., Smith C., Agris P. F., Kuo K. C., Gehrke C. Antisuppressor mutations and sulfur-carrying nucleosides in transfer RNAs of Schizosaccharomyces pombe. J Biol Chem. 1986 Dec 15;261(35):16351–16355. [PubMed] [Google Scholar]
  9. 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]
  10. Heurgué-Hamard V., Mora L., Guarneros G., Buckingham R. H. The growth defect in Escherichia coli deficient in peptidyl-tRNA hydrolase is due to starvation for Lys-tRNA(Lys). EMBO J. 1996 Jun 3;15(11):2826–2833. [PMC free article] [PubMed] [Google Scholar]
  11. Heyer W. D., Thuriaux P., Kohli J., Ebert P., Kersten H., Gehrke C., Kuo K. C., Agris P. F. An antisuppressor mutation of Schizosaccharomyces pombe affects the post-transcriptional modification of the "wobble" base in the anticodon of tRNAs. J Biol Chem. 1984 Mar 10;259(5):2856–2862. [PubMed] [Google Scholar]
  12. Houssier C., Degée P., Nicoghosian K., Grosjean H. Effect of uridine dethiolation in the anticodon triplet of tRNA(Glu) on its association with tRNA(Phe). J Biomol Struct Dyn. 1988 Jun;5(6):1259–1266. doi: 10.1080/07391102.1988.10506468. [DOI] [PubMed] [Google Scholar]
  13. Igloi G. L. Interaction of tRNAs and of phosphorothioate-substituted nucleic acids with an organomercurial. Probing the chemical environment of thiolated residues by affinity electrophoresis. Biochemistry. 1988 May 17;27(10):3842–3849. doi: 10.1021/bi00410a048. [DOI] [PubMed] [Google Scholar]
  14. Jacks T., Madhani H. D., Masiarz F. R., Varmus H. E. Signals for ribosomal frameshifting in the Rous sarcoma virus gag-pol region. Cell. 1988 Nov 4;55(3):447–458. doi: 10.1016/0092-8674(88)90031-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kumar R. K., Davis D. R. Synthesis and studies on the effect of 2-thiouridine and 4-thiouridine on sugar conformation and RNA duplex stability. Nucleic Acids Res. 1997 Mar 15;25(6):1272–1280. doi: 10.1093/nar/25.6.1272. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Muramatsu T., Nishikawa K., Nemoto F., Kuchino Y., Nishimura S., Miyazawa T., Yokoyama S. Codon and amino-acid specificities of a transfer RNA are both converted by a single post-transcriptional modification. Nature. 1988 Nov 10;336(6195):179–181. doi: 10.1038/336179a0. [DOI] [PubMed] [Google Scholar]
  19. Rance M., Sørensen O. W., Bodenhausen G., Wagner G., Ernst R. R., Wüthrich K. Improved spectral resolution in cosy 1H NMR spectra of proteins via double quantum filtering. Biochem Biophys Res Commun. 1983 Dec 16;117(2):479–485. doi: 10.1016/0006-291x(83)91225-1. [DOI] [PubMed] [Google Scholar]
  20. Rould M. A., Perona J. J., Steitz T. A. Structural basis of anticodon loop recognition by glutaminyl-tRNA synthetase. Nature. 1991 Jul 18;352(6332):213–218. doi: 10.1038/352213a0. [DOI] [PubMed] [Google Scholar]
  21. Sen G. C., Ghosh H. P. Role of modified nucleosides in tRNA: effect of modification of the 2-thiouridine derivative located at the 5'-end of the anticodon of yeast transfer RNA Lys2. Nucleic Acids Res. 1976 Mar;3(3):523–535. doi: 10.1093/nar/3.3.523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Sprinzl M., Horn C., Brown M., Ioudovitch A., Steinberg S. Compilation of tRNA sequences and sequences of tRNA genes. Nucleic Acids Res. 1998 Jan 1;26(1):148–153. doi: 10.1093/nar/26.1.148. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sylvers L. A., Rogers K. C., Shimizu M., Ohtsuka E., Söll D. A 2-thiouridine derivative in tRNAGlu is a positive determinant for aminoacylation by Escherichia coli glutamyl-tRNA synthetase. Biochemistry. 1993 Apr 20;32(15):3836–3841. doi: 10.1021/bi00066a002. [DOI] [PubMed] [Google Scholar]
  24. Vormbrock R., Morawietz R., Gassen H. G. Codon-anticodon interaction studies with trinucleoside diphosphates containing 2-thiouridine, 4-thiouridine, 2,4-diethiouridine, or 2-thiocytidine. Biochim Biophys Acta. 1974 Mar 27;340(3):348–358. [PubMed] [Google Scholar]
  25. von Ahsen U., Green R., Schroeder R., Noller H. F. Identification of 2'-hydroxyl groups required for interaction of a tRNA anticodon stem-loop region with the ribosome. RNA. 1997 Jan;3(1):49–56. [PMC free article] [PubMed] [Google Scholar]

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