Structural basis for template-independent RNA polymerization

Kozo Tomita, Shuya Fukai, Ryuichiro Ishitani, Takuya Ueda, Hono Takeuchi, Dmitry G. Vassylyev, Osamu Nureki

Research output: Contribution to journalArticle

79 Citations (Scopus)

Abstract

The 3′-terminal CCA nucleotide sequence (positions 74-76) of transfer RNA is essential for amino acid attachment and interaction with the ribosome 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 substrates. Despite structural and biochemical studies, 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.

Original languageEnglish
Pages (from-to)700-704
Number of pages5
JournalNature
Volume430
Issue number7000
DOIs
Publication statusPublished - 2004 Aug 5
Externally publishedYes

Fingerprint

Transfer RNA
Polymerization
RNA
Nucleotides
Adenosine Triphosphate
Cytidine Triphosphate
Essential Amino Acids
DNA-Directed RNA Polymerases
Ribosomes
Adenosine
Nucleic Acids
Proteins
Enzymes
tRNA nucleotidyltransferase

ASJC Scopus subject areas

  • Medicine(all)
  • General

Cite this

Tomita, K., Fukai, S., Ishitani, R., Ueda, T., Takeuchi, H., Vassylyev, D. G., & Nureki, O. (2004). Structural basis for template-independent RNA polymerization. Nature, 430(7000), 700-704. https://doi.org/10.1038/nature02712

Structural basis for template-independent RNA polymerization. / Tomita, Kozo; Fukai, Shuya; Ishitani, Ryuichiro; Ueda, Takuya; Takeuchi, Hono; Vassylyev, Dmitry G.; Nureki, Osamu.

In: Nature, Vol. 430, No. 7000, 05.08.2004, p. 700-704.

Research output: Contribution to journalArticle

Tomita, K, Fukai, S, Ishitani, R, Ueda, T, Takeuchi, H, Vassylyev, DG & Nureki, O 2004, 'Structural basis for template-independent RNA polymerization', Nature, vol. 430, no. 7000, pp. 700-704. https://doi.org/10.1038/nature02712
Tomita K, Fukai S, Ishitani R, Ueda T, Takeuchi H, Vassylyev DG et al. Structural basis for template-independent RNA polymerization. Nature. 2004 Aug 5;430(7000):700-704. https://doi.org/10.1038/nature02712
Tomita, Kozo ; Fukai, Shuya ; Ishitani, Ryuichiro ; Ueda, Takuya ; Takeuchi, Hono ; Vassylyev, Dmitry G. ; Nureki, Osamu. / Structural basis for template-independent RNA polymerization. In: Nature. 2004 ; Vol. 430, No. 7000. pp. 700-704.
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