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Structural basis for template-independent RNA polymerization

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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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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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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