Download MendeleyStructural basis for template-independent RNA polymerization.
Tomita, K., Fukai, S., Ishitani, R., Ueda, T., Takeuchi, N., Vassylyev, D.G., Nureki, O.(2004) Nature 430: 700-704
- PubMed: 15295603
- DOI: https://doi.org/10.1038/nature02712
- Primary Citation of Related Structures:
1VFG - PubMed 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.
Organizational Affiliation:
Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology, 1-1-1, Higashi, Tsukuba-shi, Ibaragi 305-8666, Japan.
