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 journalArticlepeer-review

80 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

ASJC Scopus subject areas

  • General

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