Structural basis for template-independent RNA polymerization

Kozo Tomita, Shuya Fukai, Ryuichiro Ishitani, Takuya Ueda, Nono Takeuchi, Dmitry G. Vassylyev and Osamu Nureki ()
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Kozo Tomita: Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology
Shuya Fukai: Tokyo Institute of Technology
Ryuichiro Ishitani: the University of Tokyo
Takuya Ueda: University of Tokyo
Nono Takeuchi: University of Tokyo
Dmitry G. Vassylyev: RIKEN Harima Institute at SPring-8
Osamu Nureki: Tokyo Institute of Technology

Nature, 2004, vol. 430, issue 7000, 700-704

Abstract: 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.

Date: 2004
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DOI: 10.1038/nature02712

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