Structural basis for template-independent RNA polymerization
Structural basis for template-independent RNA polymerization
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DOI：
10.1038/nature02712
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发表时间：
2004-08-05
期刊：
影响因子：
64.8
通讯作者：
Nureki, O
中科院分区：
文献类型：
--
作者：
Tomita, K;Fukai, S;Nureki, O
转运RNA的3 '末端CCA核苷酸序列（位置74 - 76）对于蛋白质合成期间氨基酸附着（1）和与核糖体相互作用（2 - 4）是必需的。使用CTP和ATP作为底物，通过模板非依赖性RNA聚合酶"CCA添加酶"从头合成和/或修复CCA序列（5）。尽管进行了结构和生物化学研究（5 - 8），但添加CCA的酶在没有核酸模板的情况下合成确定序列的机制仍然难以捉摸。在这里，我们提出了Aquifex aeolicus CCA添加酶的晶体结构，结合到缺乏末端腺苷和输入ATP类似物的引物tRNA，在2.8埃分辨率。这种酶包裹tRNA分子的受体T螺旋。在催化口袋中，C75与ATP相邻，并且它们的碱基部分堆叠。用于识别tRNA的C74-C75的互补口袋形成倒数第二个核苷酸的“蛋白质模板”，模拟模板依赖性聚合酶使用的核苷酸模板。这些结果得到了突变体系统分析的支持。我们的结构代表了选择进入的核苷酸的“插入前”阶段，并为模板无关的RNA聚合的机制提供了结构基础。
The 3'-terminal CCA nucleotide sequence ( positions 74 - 76) of transfer RNA is essential for amino acid attachment(1) and interaction with the ribosome(2-4) 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 substrates(5). Despite structural and biochemical studies(5-8), 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 Angstrom 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.