Structural basis for template-independent RNA polymerization.

Kozo Tomita; Shuya Fukai; Ryuichiro Ishitani; Takuya Ueda; Nono Takeuchi; Dmitry G Vassylyev; Osamu Nureki

doi:10.1038/nature02712

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PubMed · 15295603

Structural basis for template-independent RNA polymerization.

Kozo Tomita·Shuya Fukai·Ryuichiro Ishitani·Takuya Ueda·Nono Takeuchi·Dmitry G Vassylyev·Osamu Nureki

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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 A 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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Adenosine Monophosphate Adenosine Triphosphate Bacteria Binding Sites Biopolymers Crystallization Crystallography, X-Ray Cytidine Triphosphate DNA-Directed RNA Polymerases Geobacillus stearothermophilus Models, Molecular Protein Conformation RNA RNA Nucleotidyltransferases Structure-Activity Relationship Templates, Genetic Journal Article Research Support, Non-U.S. Gov't

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Kozo Tomita, Shuya Fukai, Ryuichiro Ishitani, Takuya Ueda, Nono Takeuchi, Dmitry G Vassylyev, Osamu Nureki. 2004-08-05. Structural basis for template-independent RNA polymerization.. https://doi.org/10.1038/nature02712

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