Skip to main content
RNA logoLink to RNA
. 2000 May;6(5):680–686. doi: 10.1017/s1355838200000029

Effects of anticodon 2'-O-methylations on tRNA codon recognition in an Escherichia coli cell-free translation.

A Satoh 1, K Takai 1, R Ouchi 1, S Yokoyama 1, H Takaku 1
PMCID: PMC1369948  PMID: 10836789

Abstract

The methylation of 2'-hydroxyl groups is one of the most common posttranscriptional modifications of naturally occurring stable RNA molecules. Some tRNA species have a 2'-O-methyl nucleoside at the first position of the anticodon, and it was suggested that this modification stabilizes the codon-anticodon duplex. However, no tRNA species have been found to have the modification at the second or third position of the anticodon. In the present study, we measured the effects of anticodon 2'-O-methylation on the codon-reading efficiencies of the anticodon variants of the unmodified forms of Escherichia coli tRNA1(Ser), using a cell-free protein synthesis assay. The modification of C in the first position of the anticodon into 2'-O-methylcytidine increased the efficiency of reading the G-ending codon. On the other hand, the modifications of the second and/or third positions were detrimental to the codon-reading activity. Thus, 2'-hydroxyl groups at the second and third positions of the anticodon may have some role in the translation reaction, and this may be the reason why 2'-O-methyl nucleosides are not found in these positions within natural tRNA species.

Full Text

The Full Text of this article is available as a PDF (199.5 KB).

Selected References

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

  1. Ban N., Nissen P., Hansen J., Capel M., Moore P. B., Steitz T. A. Placement of protein and RNA structures into a 5 A-resolution map of the 50S ribosomal subunit. Nature. 1999 Aug 26;400(6747):841–847. doi: 10.1038/23641. [DOI] [PubMed] [Google Scholar]
  2. Clemons W. M., Jr, May J. L., Wimberly B. T., McCutcheon J. P., Capel M. S., Ramakrishnan V. Structure of a bacterial 30S ribosomal subunit at 5.5 A resolution. Nature. 1999 Aug 26;400(6747):833–840. doi: 10.1038/23631. [DOI] [PubMed] [Google Scholar]
  3. Dao V., Guenther R. H., Agris P. F. The role of 5-methylcytidine in the anticodon arm of yeast tRNA(Phe): site-specific Mg2+ binding and coupled conformational transition in DNA analogs. Biochemistry. 1992 Nov 17;31(45):11012–11019. doi: 10.1021/bi00160a010. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Gabashvili I. S., Agrawal R. K., Grassucci R., Squires C. L., Dahlberg A. E., Frank J. Major rearrangements in the 70S ribosomal 3D structure caused by a conformational switch in 16S ribosomal RNA. EMBO J. 1999 Nov 15;18(22):6501–6507. doi: 10.1093/emboj/18.22.6501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Glasser A. L., el Adlouni C., Keith G., Sochacka E., Malkiewicz A., Santos M., Tuite M. F., Desgrès J. Presence and coding properties of 2'-O-methyl-5-carbamoylmethyluridine (ncm5Um) in the wobble position of the anticodon of tRNA(Leu) (U*AA) from brewer's yeast. FEBS Lett. 1992 Dec 21;314(3):381–385. doi: 10.1016/0014-5793(92)81510-s. [DOI] [PubMed] [Google Scholar]
  7. Inoue H., Hayase Y., Imura A., Iwai S., Miura K., Ohtsuka E. Synthesis and hybridization studies on two complementary nona(2'-O-methyl)ribonucleotides. Nucleic Acids Res. 1987 Aug 11;15(15):6131–6148. doi: 10.1093/nar/15.15.6131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kawai G., Yamamoto Y., Kamimura T., Masegi T., Sekine M., Hata T., Iimori T., Watanabe T., Miyazawa T., Yokoyama S. Conformational rigidity of specific pyrimidine residues in tRNA arises from posttranscriptional modifications that enhance steric interaction between the base and the 2'-hydroxyl group. Biochemistry. 1992 Feb 4;31(4):1040–1046. doi: 10.1021/bi00119a012. [DOI] [PubMed] [Google Scholar]
  9. Kigawa T., Yabuki T., Yoshida Y., Tsutsui M., Ito Y., Shibata T., Yokoyama S. Cell-free production and stable-isotope labeling of milligram quantities of proteins. FEBS Lett. 1999 Jan 8;442(1):15–19. doi: 10.1016/s0014-5793(98)01620-2. [DOI] [PubMed] [Google Scholar]
  10. Lodmell J. S., Dahlberg A. E. A conformational switch in Escherichia coli 16S ribosomal RNA during decoding of messenger RNA. Science. 1997 Aug 29;277(5330):1262–1267. doi: 10.1126/science.277.5330.1262. [DOI] [PubMed] [Google Scholar]
  11. Mendel D., Cornish V. W., Schultz P. G. Site-directed mutagenesis with an expanded genetic code. Annu Rev Biophys Biomol Struct. 1995;24:435–462. doi: 10.1146/annurev.bb.24.060195.002251. [DOI] [PubMed] [Google Scholar]
  12. Moore P. B. The three-dimensional structure of the ribosome and its components. Annu Rev Biophys Biomol Struct. 1998;27:35–58. doi: 10.1146/annurev.biophys.27.1.35. [DOI] [PubMed] [Google Scholar]
  13. Murasugi A., Takemura S. Nucleotide sequence of leucine transfer RNA 1 from Candida (Torulopsis) utilis. J Biochem. 1978 Apr;83(4):1029–1038. doi: 10.1093/oxfordjournals.jbchem.a131991. [DOI] [PubMed] [Google Scholar]
  14. Noren C. J., Anthony-Cahill S. J., Griffith M. C., Schultz P. G. A general method for site-specific incorporation of unnatural amino acids into proteins. Science. 1989 Apr 14;244(4901):182–188. doi: 10.1126/science.2649980. [DOI] [PubMed] [Google Scholar]
  15. Ohtsuki T., Vinayak R., Watanabe Y., Kita K., Kawai G., Watanabe K. Automated chemical synthesis of biologically active tRNA having a sequence corresponding to Ascaris suum mitochondrial tRNA(Met) toward NMR measurements. J Biochem. 1996 Dec;120(6):1070–1073. doi: 10.1093/oxfordjournals.jbchem.a021522. [DOI] [PubMed] [Google Scholar]
  16. Païs de Barros J. P., Keith G., El Adlouni C., Glasser A. L., Mack G., Dirheimer G., Desgrès J. 2'-O-methyl-5-formylcytidine (f5Cm), a new modified nucleotide at the 'wobble' of two cytoplasmic tRNAs Leu (NAA) from bovine liver. Nucleic Acids Res. 1996 Apr 15;24(8):1489–1496. doi: 10.1093/nar/24.8.1489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Pixa G., Dirheimer G., Keith G. Sequence of tRNALeu CmAA from Bacillus stearothermophilus. Biochem Biophys Res Commun. 1983 Apr 29;112(2):578–585. doi: 10.1016/0006-291x(83)91503-6. [DOI] [PubMed] [Google Scholar]
  18. Rothschild K. J., Gite S. tRNA-mediated protein engineering. Curr Opin Biotechnol. 1999 Feb;10(1):64–70. doi: 10.1016/s0958-1669(99)80012-3. [DOI] [PubMed] [Google Scholar]
  19. Rozenski J., Crain P. F., McCloskey J. A. The RNA Modification Database: 1999 update. Nucleic Acids Res. 1999 Jan 1;27(1):196–197. doi: 10.1093/nar/27.1.196. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Shimotono K., Miura K. 5'-Terminal structure of messenger RNA transcribed by RNA polymerase of silkworm cytoplasmic polyhedrosis virus containing double-stranded RNA. J Mol Biol. 1974 Jun 15;86(1):21–30. doi: 10.1016/s0022-2836(74)80004-5. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Takai K., Takaku H., Yokoyama S. Codon-reading specificity of an unmodified form of Escherichia coli tRNA1Ser in cell-free protein synthesis. Nucleic Acids Res. 1996 Aug 1;24(15):2894–2899. doi: 10.1093/nar/24.15.2894. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Yabuki T., Kigawa T., Dohmae N., Takio K., Terada T., Ito Y., Laue E. D., Cooper J. A., Kainosho M., Yokoyama S. Dual amino acid-selective and site-directed stable-isotope labeling of the human c-Ha-Ras protein by cell-free synthesis. J Biomol NMR. 1998 Apr;11(3):295–306. doi: 10.1023/a:1008276001545. [DOI] [PubMed] [Google Scholar]
  24. Yoshizawa S., Fourmy D., Puglisi J. D. Recognition of the codon-anticodon helix by ribosomal RNA. Science. 1999 Sep 10;285(5434):1722–1725. doi: 10.1126/science.285.5434.1722. [DOI] [PubMed] [Google Scholar]

Articles from RNA are provided here courtesy of The RNA Society

RESOURCES