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Structural basis for template-independent RNA polymerization

Nature volume 430, pages 700–704 (2004)Cite this article

Abstract

The 3′-terminal CCA nucleotide sequence (positions 74–76) of transfer RNA is essential for amino acid attachment1 and interaction with the ribosome2,3,4 during protein synthesis. The CCA sequence is synthesized de novo and/or repaired by a template-independent RNA polymerase, ‘CCA-adding enzyme’, using CTP and ATP as substrates5. Despite structural and biochemical studies5,6,7,8, the mechanism by which the CCA-adding enzyme synthesizes the defined sequence without a nucleic acid template remains elusive. Here we present the crystal structure of Aquifex aeolicus CCA-adding enzyme, bound to a primer tRNA lacking the terminal adenosine and an incoming ATP analogue, at 2.8 Å resolution. The enzyme enfolds the acceptor T helix of the tRNA molecule. In the catalytic pocket, C75 is adjacent to ATP, and their base moieties are stacked. The complementary pocket for recognizing C74-C75 of tRNA forms a ‘protein template’ for the penultimate two nucleotides, mimicking the nucleotide template used by template-dependent polymerases. These results are supported by systematic analyses of mutants. Our structure represents the ‘pre-insertion’ stage of selecting the incoming nucleotide and provides the structural basis for the mechanism underlying template-independent RNA polymerization.

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Figure 1: Overall structure of the ternary complex of Aa.LC, tRNA-CC and AMPcPP.
Figure 2: Stereoview of the primer C74-C75 and the incoming ATP.
Figure 3: In vitro AMP incorporation.
Figure 4: Comparison of template-independent and template-dependent RNA polymerases.

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References

  1. Sprinzl, M. & Cramer, F. The -C-C-A end of tRNA and its role in protein biosynthesis. Prog. Nucleic Acid Res. Mol. Biol. 22, 1–69 (1979)

    Article  CAS  Google Scholar 

  2. Green, R. & Noller, H. F. Ribosomes and translation. Annu. Rev. Biochem. 66, 679–716 (1997)

    Article  CAS  Google Scholar 

  3. Kim, D. F. & Green, R. Base-pairing between 23S rRNA and tRNA in the ribosomal A site. Mol. Cell 4, 859–864 (1999)

    Article  CAS  Google Scholar 

  4. Nissen, P., Hansen, J., Ban, N., Moore, P. B. & Steitz, T. A. The structural basis of ribosome activity in peptide bond synthesis. Science 289, 920–930 (2000)

    Article  ADS  CAS  Google Scholar 

  5. Deutscher, M. P. in Enzymes of Nucleic Acid Synthesis and Modification. RNA Enzymes vol. 2 (ed. Jacob, S. T.) 159–183 (CRC, Boca Raton, Fl, 1983)

    Google Scholar 

  6. Yue, D., Weiner, A. M. & Maizels, N. The CCA-adding enzyme has a single active site. J. Biol. Chem. 273, 29693–29700 (1998)

    Article  CAS  Google Scholar 

  7. Shi, P. Y., Maizels, N. & Weiner, A. M. CCA addition by tRNA nucleotidyltransferase: polymerization without translocation? EMBO J. 17, 3197–3206 (1998)

    Article  CAS  Google Scholar 

  8. Hou, Y. M. Unusual synthesis by the Escherichia coli CCA-adding enzyme. RNA 6, 1031–1043 (2000)

    Article  CAS  Google Scholar 

  9. Holm, L. & Sander, C. DNA polymerase β belongs to an ancient nucleotidyltransferase superfamily. Trends Biochem. Sci. 20, 345–347 (1995)

    Article  CAS  Google Scholar 

  10. Martin, G. & Keller, W. Mutational analysis of mammalian poly(A) polymerase identifies a region for primer binding and catalytic domain, homologous to the family X polymerases, and to other nucleotidyltransferases. EMBO J. 15, 2593–2603 (1996)

    Article  CAS  Google Scholar 

  11. Yue, D., Maizels, N. & Weiner, A. M. CCA-adding enzymes and poly(A) polymerases are all members of the same nucleotidyltransferase superfamily: characterization of the CCA-adding enzyme from the archaeal hyperthermophile Sulfolobus shibatae. RNA 2, 895–908 (1996)

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Li, F. et al. Crystal structures of the Bacillus stearothermophilus CCA-adding enzyme and its complexes with ATP or CTP. Cell 111, 815–824 (2002)

    Article  CAS  Google Scholar 

  13. Okabe, M. et al. Divergent evolutions of trinucleotide polymerization revealed by an archaeal CCA-adding enzyme structure. EMBO J. 22, 5918–5927 (2003)

    Article  CAS  Google Scholar 

  14. Xiong, Y., Li, F., Wang, J., Weiner, A. M. & Steitz, T. A. Crystal structures of an archaeal class I CCA-adding enzyme and its nucleotide complexes. Mol. Cell 12, 1165–1172 (2003)

    Article  CAS  Google Scholar 

  15. Tomita, K. & Weiner, A. M. Collaboration between CC- and A-adding enzymes to build and repair the 3′-terminal CCA of tRNA in Aquifex aeolicus. Science 294, 1334–1336 (2001)

    Article  ADS  CAS  Google Scholar 

  16. Tomita, K. & Weiner, A. M. Closely related CC- and A-adding enzymes collaborate to construct and repair the 3′-terminal CCA of tRNA in Synechocystis sp. and Deinococcus radiodurans. J. Biol. Chem. 277, 48192–48198 (2003)

    Article  Google Scholar 

  17. Augustin, M. A. et al. Crystal structure of the human CCA-adding enzyme: insights into template-independent polymerization. J. Mol. Biol. 328, 985–994 (2003)

    Article  CAS  Google Scholar 

  18. Brautigam, C. A. & Steitz, T. A. Structural and functional insights provided by crystal structures of DNA polymerases and their substrate complexes. Curr. Opin. Struct. Biol. 8, 54–63 (1998)

    Article  CAS  Google Scholar 

  19. Hegg, L. A. & Thurlow, D. L. Cytidines in tRNAs that are required intact by ATP/CTP:tRNA nucleotidyltransferases from Escherichia coli and Saccharomyces cerevisiae. Nucleic Acids Res. 18, 5975–9597 (1990)

    Article  CAS  Google Scholar 

  20. Xiong, Y. & Steitz, T. A. Mechanism of transfer RNA maturation by CCA-adding enzyme without using an oligonucleotide template. Nature doi:10.1038/nature02711 (this issue)

  21. Shi, P. Y., Weiner, A. M. & Maizels, N. A top-half tDNA minihelix is a good substrate for the eubacterial CCA-adding enzyme. RNA 4, 276–284 (1998)

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Temiakov, D. et al. Structural basis for substrate selection by T7 RNA polymerase. Cell 116, 381–391 (2004)

    Article  CAS  Google Scholar 

  23. Yin, Y. W. & Steitz, T. A. The structural mechanism of translocation and helicase activity in T7 RNA polymerase. Cell 116, 393–404 (2004)

    Article  CAS  Google Scholar 

  24. Pelletier, H., Sawaya, M. R., Kumar, A., Wilson, S. H. & Kraut, J. Structures of ternary complexes of rat DNA polymerase β, a DNA template-primer, and ddCTP. Science 264, 1891–1903 (1994)

    Article  ADS  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  26. Weeks, C. M. & Miller, R. The design and implementation of SnB version 2.0. J. Appl. Crystallogr. 32, 120–124 (1999)

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  28. Terwilliger, T. C. Maximum-likelihood density modification. Acta Crystallogr. D 56, 965–972 (2000)

    Article  CAS  Google Scholar 

  29. 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  Google Scholar 

  30. Rice, L. M. & Brunger, A. T. Torsion angle dynamics: reduced variable conformational sampling enhances crystallographic structure refinement. Proteins Struct. Funct. Genet. 19, 277–290 (1994)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank A. M. Weiner for the Aa.LC plasmids; and M. Kawamoto and H. Sakai for help with data collection at SPring-8. This work was supported by Kurata Memorial Hitachi Science and Technology Foundation, Takeda Science Foundation, Foundation of Advanced Technology Institute and a Grant-in-aid for Young Scientists (to K.T.); by Asahi Glass Foundation (to S.F.); by a grant from the Ministry of Education, Culture, Sports, Science and Technology (to N.T.); and by a PRESTO Program grant from Japan Science and Technology and a Naito Foundation grant (to O.N.).

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Author notes

  1. Kozo Tomita and Shuya Fukai: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology, 1-1-1, Higashi, Tsukuba-shi, Ibaragi, 305-8666, Japan

    Kozo Tomita

  2. Department of Integrated Bioscience, Graduate School of Frontier Science, University of Tokyo, 5-1-5, Kashiwanoha, Kashiwa-shi, 277-8562, Chiba, Japan

    Kozo Tomita, Takuya Ueda & Nono Takeuchi

  3. Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa, 226-8501, Japan

    Shuya Fukai & Osamu Nureki

  4. Department of Biotechnology, Graduate School of Agricultural and Life Sciences, the University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, 113-8657, Tokyo, Japan

    Ryuichiro Ishitani

  5. Cellular Signaling Laboratory, RIKEN Harima Institute at SPring-8, 1-1-1 Kouto Mikazuki-cho, Sayo-gun, Hyogo, 679-5148, Japan

    Dmitry G. Vassylyev

  6. PRESTO, JST, Honcho, Kawaguchi-shi, Saitama, 332-0012, Japan

    Osamu Nureki

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Correspondence to Osamu Nureki.

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Supplementary information

Supplementary Table 1

Data collection and refinement statistics (DOC 56 kb)

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Tomita, K., Fukai, S., Ishitani, R. et al. Structural basis for template-independent RNA polymerization. Nature 430, 700–704 (2004). https://doi.org/10.1038/nature02712

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