Mechanism for expanding the decoding capacity of transfer RNAs by modification of uridines

Article metrics

Abstract

One of the most prevalent base modifications involved in decoding is uridine 5-oxyacetic acid at the wobble position of tRNA. It has been known for several decades that this modification enables a single tRNA to decode all four codons in a degenerate codon box. We have determined structures of an anticodon stem-loop of tRNAVal containing the modified uridine with all four valine codons in the decoding site of the 30S ribosomal subunit. An intramolecular hydrogen bond involving the modification helps to prestructure the anticodon loop. We found unusual base pairs with the three noncomplementary codon bases, including a G·U base pair in standard Watson-Crick geometry, which presumably involves an enol form for the uridine. These structures suggest how a modification in the uridine at the wobble position can expand the decoding capability of a tRNA.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: The degeneracy of the genetic code.
Figure 2: cmo5U base-pairs with all four bases at the wobble position of the mRNA.
Figure 3: Comparison of cmo5U·G with standard U·G wobble.
Figure 4: The two pyrimidine-pyrimidine base pairs.

Accession codes

Primary accessions

Protein Data Bank

References

  1. 1

    Crick, F.H.C. The origin of the genetic code. J. Mol. Biol. 38, 367–379 (1968).

  2. 2

    Sprinzl, M., Horn, C., Brown, M., Ioudovitch, A. & Steinberg, S. Compilation of tRNA sequences and sequences of tRNA genes. Nucleic Acids Res. 26, 148–153 (1998).

  3. 3

    Mitra, S.K., Lustig, F., Akesson, B. & Lagerkvist, U. Codon-anticodon recognition in the valine codon family. J. Biol. Chem. 252, 471–478 (1977).

  4. 4

    Mitra, K. & Frank, J. Ribosome dynamics: insights from atomic structure modeling into cryo-electron microscopy naps. Annu. Rev. Biophys. Biomol. Struct. 35, 299–317 (2006).

  5. 5

    Nasvall, S.J., Chen, P. & Bjork, G.R. The modified wobble nucleoside uridine-5-oxyacetic acid in tRNAPro(cmo5UGG) promotes reading of all four proline codons in vivo. RNA 10, 1662–1673 (2004).

  6. 6

    Agris, P.F., Vendeix, F.A. & Graham, W.D. tRNA's wobble decoding of the genome: 40 years of modification. J. Mol. Biol. 366, 1–13 (2007).

  7. 7

    Ogle, J.M. & Ramakrishnan, V. Structural insights into translational fidelity. Annu. Rev. Biochem. 74, 129–177 (2005).

  8. 8

    Rodnina, M.V. & Wintermeyer, W. Fidelity of aminoacyl-tRNA selection on the ribosome: kinetic and structural mechanisms. Annu. Rev. Biochem. 70, 415–435 (2001).

  9. 9

    Ogle, J.M. et al. Recognition of cognate transfer RNA by the 30S ribosomal subunit. Science 292, 897–902 (2001).

  10. 10

    Ogle, J.M., Murphy, F.V., Tarry, M.J. & Ramakrishnan, V. Selection of tRNA by the ribosome requires a transition from an open to a closed form. Cell 111, 721–732 (2002).

  11. 11

    Murphy, F.V., IV, Ramakrishnan, V., Malkiewicz, A. & Agris, P.F. The role of modifications in codon discrimination by tRNA(Lys)UUU. Nat. Struct. Mol. Biol. 11, 1186–1191 (2004).

  12. 12

    Selmer, M. et al. Structure of the 70S ribosome complexed with mRNA and tRNA. Science 313, 1935–1942 (2006).

  13. 13

    Hillen, W., Egert, E., Lindner, H.J., Gassen, H.G. & Vorbrüggen, H. 5-Methoxyuridine: the influence of 5-substituents on the keto-enol tautomerism of the 4-carbonyl group. J. Carbohydr. Nucleosides Nucleotides 5, 23–32 (1978).

  14. 14

    Dabkowska, I., Gutowski, M. & Rak, J. Interaction with glycine increases stability of a mutagenic tautomer of uracil. A density functional theory study. J. Am. Chem. Soc. 127, 2238–2248 (2005).

  15. 15

    Dirheimer, G., Keith, G., Dumas, P. & Westhof, E. The base pair directory. in tRNA: Structure, Biosynthesis, and Function (eds. Söll, D. & RajBhandary, U.L.) 111–112 (American Society for Microbiology, Washington, DC, 1995).

  16. 16

    Tinoco, I.J. The base pair directory. in The RNA World (eds. Gesteland, R.F. & Atkins, J.F.) 603–607 (Cold Spring Harbor Laboratory Press, New York, 1993).

  17. 17

    Cruse, W.B. et al. Structure of a mispaired RNA double helix at 1.6-A resolution and implications for the prediction of RNA secondary structure. Proc. Natl. Acad. Sci. USA 91, 4160–4164 (1994).

  18. 18

    Nagaswamy, U. et al. NCIR: a database of non-canonical interactions in known RNA structures. Nucleic Acids Res. 30, 395–397 (2002).

  19. 19

    Mitra, S.K. et al. Relative efficiency of anticodons in reading the valine codons during protein synthesis in vitro. J. Biol. Chem. 254, 6397–6401 (1979).

  20. 20

    Sorensen, M.A. et al. Over expression of a tRNA(Leu) isoacceptor changes charging pattern of leucine tRNAs and reveals new codon reading. J. Mol. Biol. 354, 16–24 (2005).

  21. 21

    Masquida, B. & Westhof, E. On the wobble G·U and related pairs. RNA 6, 9–15 (2000).

  22. 22

    Mizuno, H. & Sundaralingam, M. Stacking of Crick Wobble pair and Watson-Crick pair: stability rules of G-U pairs at ends of helical stems in tRNAs and the relation to codon-anticodon Wobble interaction. Nucleic Acids Res. 5, 4451–4461 (1978).

  23. 23

    Fersht, A.R. Structure and Mechanism in Protein Science (W.H. Freeman, New York, 1998).

  24. 24

    Doherty, E.A., Batey, R.T., Masquida, B. & Doudna, J.A. A universal mode of helix packing in RNA. Nat. Struct. Biol. 8, 339–343 (2001).

  25. 25

    Kothe, U. & Rodnina, M.V. Codon reading by tRNAAla with modified uridine in the wobble position. Mol. Cell 25, 167–174 (2007).

  26. 26

    Clemons, W.M., Jr et al. Crystal structure of the 30S ribosomal subunit from Thermus thermophilus: purification, crystallization and structure determination. J. Mol. Biol. 310, 827–843 (2001).

  27. 27

    Sproat, B.S. RNA synthesis using 2′-O-(tert-butyldimethylsilyl) protection. Methods Mol. Biol. 288, 17–32 (2005).

  28. 28

    Boudou, V. et al. Synthesis of the anticodon hairpin tRNAfMet containing N-{[9-(b-D-ribofuranosyl)-9H-purin-6-yl]carbamoyl}-L-threonine (=N6-{{[(1S,2R)-1-carboxy-2-hydroxypropyl]amino}-carbonyl}adenosine, t6A). Helv. Chim. Acta 83, 152–161 (2000).

  29. 29

    Gehrke, C.W. & Kuo, K.C. Ribonucleoside analysis by reversed-phase high-performance liquid chromatography. J. Chromatogr. 471, 3–36 (1989).

  30. 30

    Kabsch, W. Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. J. Appl. Cryst. 26, 795–800 (1993).

  31. 31

    Collaborative Computational Project Number 4. The CCP4 suite: Programs for protein crystallography. Acta Crystallogr. D. Biol. Crystallogr. 50, 760–763 (1994).

  32. 32

    Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

  33. 33

    Brünger, A.T. et al. Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54, 905–921 (1998).

  34. 34

    Kleywegt, G.J. & Jones, T.A. Databases in protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 54, 1119–1131 (1998).

  35. 35

    DeLano, W.L. The PyMOL Molecular Graphics System (Delano Scientific, San Carlos, California, USA, 2006).

  36. 36

    Masquida, B., Sauter, C. & Westhof, E. A sulfate pocket formed by three GoU pairs in the 0.97 Å resolution X-ray structure of a nonameric RNA. RNA 5, 1384–1395 (1999).

  37. 37

    Lu, X.J. & Olson, W.K. 3DNA: a software package for the analysis, rebuilding and visualization of three-dimensional nucleic acid structures. Nucleic Acids Res. 31, 5108–5121 (2003).

Download references

Acknowledgements

We thank B. Sproat for help and advice on the polymer chemistry, W.D. Graham for purification and analysis of ASLVal, A. Kelley for purification and crystallization of 30S subunits, C.M. Dunham and S. Petry for help with synchrotron data collection, R. Ravelli, J. McCarthy and G. Leonard for help with data collection on beamline ID14 at the European Synchrotron Radiation Facility, and L. Passmore and M. Schmeing for helpful comments. This work was funded by the UK Medical Research Council (V.R.) and grants from the US National Institutes of Health (P.F.A. and V.R.), the US National Science Foundation (P.F.A.), the Agouron Institute (V.R.), the Austrian Academy of Sciences (A.W.) and the Polish Ministry of Science and Education (A.M.).

Author information

Correspondence to Paul F Agris or V Ramakrishnan.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Derivatives of cmo5U. (PDF 59 kb)

Supplementary Fig. 2

Unbiased difference density for ribosomal A site. (PDF 113 kb)

Supplementary Fig. 3

Role of m6A37. (PDF 101 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Weixlbaumer, A., Murphy, F., Dziergowska, A. et al. Mechanism for expanding the decoding capacity of transfer RNAs by modification of uridines. Nat Struct Mol Biol 14, 498–502 (2007) doi:10.1038/nsmb1242

Download citation

Further reading