Structural basis of specific tRNA aminoacylation by a small in vitro selected ribozyme

Abstract

In modern organisms, protein enzymes are solely responsible for the aminoacylation of transfer RNA. However, the evolution of protein synthesis in the RNA world required RNAs capable of catalysing this reaction. Ribozymes that aminoacylate RNA by using activated amino acids have been discovered through selection in vitro1,2,3,4,5. Flexizyme is a 45-nucleotide ribozyme capable of charging tRNA in trans with various activated l-phenylalanine derivatives. In addition to a more than 105 rate enhancement and more than 104-fold discrimination against some non-cognate amino acids, this ribozyme achieves good regioselectivity: of all the hydroxyl groups of a tRNA, it exclusively aminoacylates the terminal 3′-OH5,6,7. Here we report the 2.8-Å resolution structure of flexizyme fused to a substrate RNA. Together with randomization of ribozyme core residues and reselection, this structure shows that very few nucleotides are needed for the aminoacylation of specific tRNAs. Although it primarily recognizes tRNA through base-pairing with the CCA terminus of the tRNA molecule, flexizyme makes numerous local interactions to position the acceptor end of tRNA precisely. A comparison of two crystallographically independent flexizyme conformations, only one of which appears capable of binding activated phenylalanine, suggests that this ribozyme may achieve enhanced specificity by coupling active-site folding to tRNA docking. Such a mechanism would be reminiscent of the mutually induced fit of tRNA and protein employed by some aminoacyl-tRNA synthetases8,9 to increase specificity.

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Figure 1: Overall structure of the flexizyme–tRNA minihelix fusion.
Figure 2: Comparison of protein and RNA aminoacyl-tRNA synthetases.
Figure 3: Structure and sequence requirements of the active site.
Figure 4: Conjectural coupling of tRNA docking to active-site folding.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factor amplitudes for aminoacyl-tRNA synthetase ribozyme–minihelix fusions refined against crystal II and crystal III data have been deposited with the Protein Data Bank with accession codes 3CUL and 3CUN, respectively.

References

  1. 1

    Illangasekare, M., Sanchez, G., Nickles, T. & Yarus, M. Aminoacyl-RNA synthesis catalyzed by an RNA. Science 267, 643–647 (1995)

    ADS  Article  CAS  PubMed  Google Scholar 

  2. 2

    Illangasekare, M. & Yarus, M. A tiny RNA that catalyzes both aminoacyl-RNA and peptidyl-RNA synthesis. RNA 5, 1482–1489 (1999)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. 3

    Illangasekare, M. & Yarus, M. Specific, rapid synthesis of Phe-RNA by RNA. Proc. Natl Acad. Sci. USA 96, 5470–5475 (1999)

    ADS  Article  CAS  PubMed  Google Scholar 

  4. 4

    Lee, N., Bessho, Y., Wei, K., Szostak, J. W. & Suga, H. Ribozyme-catalyzed tRNA aminoacylation. Nature Struct. Biol. 7, 28–33 (2000)

    Article  CAS  PubMed  Google Scholar 

  5. 5

    Saito, H., Kourouklis, D. & Suga, H. An in vitro evolved precursor tRNA with aminoacylation activity. EMBO J. 20, 1797–1806 (2001)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. 6

    Saito, H. & Suga, H. A ribozyme exclusively aminoacylates the 3′-hydroxyl group of the tRNA terminal adenosine. J. Am. Chem. Soc. 123, 7178–7179 (2001)

    Article  CAS  PubMed  Google Scholar 

  7. 7

    Murakami, H., Saito, H. & Suga, H. A versatile tRNA aminoacylation catalyst based on RNA. Chem. Biol. 10, 655–662 (2003)

    Article  CAS  PubMed  Google Scholar 

  8. 8

    Cusack, S., Yaremchuk, A. & Tukalo, M. The crystal structure of the ternary complex of T. thermophilus seryl-tRNA synthetase with tRNASer and a seryl-adenylate analogue reveals a conformational switch in the active site. EMBO J. 15, 2834–2842 (1996)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. 9

    Moulinier, L. et al. The structure of an AspRS-tRNAAsp complex reveals a tRNA-dependent control mechanism. EMBO J. 20, 5290–5301 (2001)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. 10

    Schimmel, P., Giegé, R., Moras, D. & Yokoyama, S. An operational code for amino acids and possible relationship to genetic code. Proc. Natl Acad. Sci. USA 90, 8763–8768 (1993)

    ADS  Article  CAS  PubMed  Google Scholar 

  11. 11

    Ramaswamy, K., Saito, H., Murakami, H., Shiba, K. & Suga, H. Designer ribozymes: programming the tRNA specificity into flexizyme. J. Am. Chem. Soc. 126, 11454–11455 (2004)

    Article  CAS  PubMed  Google Scholar 

  12. 12

    Saito, H., Watanabe, K. & Suga, H. Concurrent molecular recognition of the amino acid and tRNA by a ribozyme. RNA 7, 1867–1878 (2001)

    PubMed  PubMed Central  CAS  Google Scholar 

  13. 13

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

    ADS  Article  CAS  PubMed  Google Scholar 

  14. 14

    Kirsebom, L. A. & Svard, S. G. Base pairing between Escherichia coli RNase P RNA and its substrate. EMBO J. 13, 4870–4876 (1994)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. 15

    Eriani, G., Delarue, M., Poch, O., Gangloff, J. & Moras, D. Partition of tRNA synthetases into two classes based on mutually exclusive sets of sequence motifs. Nature 347, 203–206 (1990)

    ADS  Article  CAS  PubMed  Google Scholar 

  16. 16

    Rould, M. A., Perona, J. J., Söll, D. & Steitz, T. A. Structure of E. coli glutaminyl-tRNA synthetase complexed with tRNAGln at 2.8 Å resolution: implications for tRNA discrimination. Science 246, 1135–1142 (1989)

    ADS  Article  CAS  PubMed  Google Scholar 

  17. 17

    Ruff, M. et al. Class II aminoacyl transfer RNA synthetases: crystal structure of yeast aspartyl-tRNA synthetase complexed with tRNAAsp . Science 252, 1682–1689 (1991)

    ADS  Article  CAS  PubMed  Google Scholar 

  18. 18

    Nissen, P., Ippolito, J. A., Ban, N., Moore, P. B. & Steitz, T. A. RNA tertiary interactions in the large ribosomal subunit: the A-minor motif. Proc. Natl Acad. Sci. USA 98, 4899–4903 (2001)

    ADS  Article  CAS  PubMed  Google Scholar 

  19. 19

    Saenger, W. Principles of Nucleic Acid Structure (Springer, New York, 1984)

    Google Scholar 

  20. 20

    Burley, S. K. & Petsko, G. A. Weakly polar interactions in proteins. Adv. Protein Chem. 39, 125–192 (1988)

    Article  CAS  PubMed  Google Scholar 

  21. 21

    Murakami, H., Ohta, A., Ashiai, H. & Suga, H. A highly flexible tRNA acylation method for non-natural polypeptide synthesis. Nature Methods 3, 357–359 (2006)

    Article  CAS  PubMed  Google Scholar 

  22. 22

    Perona, J. J. & Hou, Y. M. Indirect readout of tRNA for aminoacylation. Biochemistry 46, 10419–10432 (2007)

    Article  CAS  PubMed  Google Scholar 

  23. 23

    Weiner, A. M. & Maizels, N. tRNA-like structures tag the 3′ ends of genomic RNA molecules for replication: implications for the origin of protein synthesis. Proc. Natl Acad. Sci. USA 84, 7383–7387 (1987)

    ADS  Article  CAS  PubMed  Google Scholar 

  24. 24

    Orgel, L. E. The origin of polynucleotide-directed protein synthesis. J. Mol. Evol. 29, 465–474 (1989)

    ADS  Article  CAS  PubMed  Google Scholar 

  25. 25

    Wong, J. T. Origin of genetically encoded protein synthesis: a model based on selection for RNA peptidation. Orig. Life Evol. Biosph. 21, 165–176 (1991)

    ADS  Article  CAS  PubMed  Google Scholar 

  26. 26

    Golden, B. L., Gooding, A. R., Podell, E. R. & Cech, T. R. A preorganized active site in the crystal structure of the Tetrahymena ribozyme. Science 282, 259–264 (1998)

    ADS  Article  CAS  PubMed  Google Scholar 

  27. 27

    Serganov, A. et al. Structural basis for Diels–Alder ribozyme catalyzed carbon–carbon bond formation. Nature Struct. Mol. Biol. 12, 218–224 (2005)

    Article  CAS  Google Scholar 

  28. 28

    Klein, D. J. & Ferré-D’Amaré, A. R. Structural basis of glmS ribozyme activation by glucosamine-6-phosphate. Science 313, 1752–1756 (2006)

    ADS  Article  CAS  PubMed  Google Scholar 

  29. 29

    Giegé, R., Sissler, M. & Florentz, C. Universal rules and idiosyncratic features in tRNA identity. Nucleic Acids Res. 26, 5017–5035 (1998)

    Article  PubMed  PubMed Central  Google Scholar 

  30. 30

    Saito, H. & Suga, H. Outersphere and innersphere coordinated metal ions in an aminoacyl-tRNA synthetase ribozyme. Nucleic Acids Res. 30, 5151–5159 (2002)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. 31

    Ferré-D’Amaré, A. R. & Doudna, J. A. Crystallization and structure determination of a hepatitis delta virus ribozyme: use of the RNA-binding protein U1A as a crystallization module. J. Mol. Biol. 295, 541–556 (2000)

    Article  CAS  PubMed  Google Scholar 

  32. 32

    Ferré-D’Amaré, A. R. & Doudna, J. A. Use of cis- and trans-ribozymes to remove 5′ and 3′ heterogeneities from milligrams of in vitro transcribed RNA. Nucleic Acids Res. 24, 977–978 (1996)

    Article  PubMed  PubMed Central  Google Scholar 

  33. 33

    Rupert, P. B. & Ferré-D’Amaré, A. R. Crystallization of the hairpin ribozyme: illustrative protocols. Methods Mol. Biol. 252, 303–311 (2004)

    PubMed  CAS  Google Scholar 

  34. 34

    Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997)

    Article  CAS  PubMed  Google Scholar 

  35. 35

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

    Article  PubMed  Google Scholar 

  36. 36

    McCoy, A. J., Grosse-Kunstleve, R. W., Storoni, L. C. & Read, R. J. Likelihood-enhanced fast translation functions. Acta Crystallogr. D 61, 458–464 (2005)

    Article  CAS  PubMed  Google Scholar 

  37. 37

    Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 (1991)

    Article  PubMed  Google Scholar 

  38. 38

    DeLano, W. L. The PyMOL Molecular Graphics System (DeLano Scientific, San Carlos, CA, 2002)

    Google Scholar 

  39. 39

    Carson, M. Ribbons. Methods Enzymol. 277, 493–505 (1997)

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank the staff at ALS beamline 5.0.2 and J. Bolduc for assistance with synchrotron and in-house X-ray data collection, respectively, and T. Edwards, T. Hamma, D. Klein, J. Pitt, J. Posakony, A. Roll-Mecak and B. Shen for discussions. A.R.F. is a Distinguished Young Scholar in Medical Research of the W. M. Keck Foundation. This work was supported by grants from research and development projects of the Industrial Science and Technology Program in the New Energy and Industrial Technology Development Organization (to H.S.) and the W. M. Keck Foundation (to A.R.F.).

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Correspondence to Adrian R. Ferré-D’Amaré.

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The file contains Supplementary Figures 1-8 with Legends, Supplementary Table 1 and additional references. (PDF 1067 kb)

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Xiao, H., Murakami, H., Suga, H. et al. Structural basis of specific tRNA aminoacylation by a small in vitro selected ribozyme. Nature 454, 358–361 (2008). https://doi.org/10.1038/nature07033

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