A kinetic framework for tRNA ligase and enforcement of a 2′-phosphate requirement for ligation highlights the design logic of an RNA repair machine
A kinetic framework for tRNA ligase and enforcement of a 2′-phosphate requirement for ligation highlights the design logic of an RNA repair machine
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DOI：
10.1261/rna.038406.113
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发表时间：
2013-05-01
期刊：
影响因子：
4.5
通讯作者：
Shuman, Stewart
中科院分区：
文献类型：
--
作者：
Remus, Barbara S.;Shuman, Stewart
tRNA 连接酶是信息和应激反应途径的重要组成部分，需要修复 2',3'-环磷酸和 5'-OH 末端的 RNA 断裂。植物和真菌 tRNA 连接酶包含三个催化结构域。磷酸二酯酶和激酶模块修复断裂的末端，生成连接酶密封所需的 3'-OH、2'-PO4 和 5'-PO4。我们利用具有不同末端的 RNA 底物来定义植物连接酶 AtRNL 修复途径中各个步骤或步骤子集的速率。结果强调了限速交易、活性位点突变如何影响修复，以及 RNA 改变如何绕过突变。通过证明 AtRNL 在将 AMP 转移到 pRNA(OH) 形成 AppRNA(OH) 方面存在缺陷，但在密封预腺苷酸化的 AppRNA(OH) 方面，我们获得了对 2'-PO4 特异性的见解。这种区分 2'-PO4 与 2'-OH 末端的策略提供了一个质量控制检查点，以确保仅密封有目的的 RNA 断裂并避免 5'-PO4 末端的非特异性“加帽”。
tRNA ligases are essential components of informational and stress-response pathways entailing repair of RNA breaks with 2',3'-cyclic phosphate and 5'-OH ends. Plant and fungal tRNA ligases comprise three catalytic domains. Phosphodiesterase and kinase modules heal the broken ends to generate the 3'-OH, 2'-PO4, and 5'-PO4 required for sealing by the ligase. We exploit RNA substrates with different termini to define rates of individual steps or subsets of steps along the repair pathway of plant ligase AtRNL. The results highlight rate-limiting transactions, how repair is affected by active-site mutations, and how mutations are bypassed by RNA alterations. We gain insights to 2'-PO4 specificity by showing that AtRNL is deficient in transferring AMP to pRNA(OH) to form AppRNA(OH) but proficient at sealing pre-adenylylated AppRNA(OH). This strategy for discriminating 2'-PO4 versus 2'-OH ends provides a quality-control checkpoint to ensure that only purposeful RNA breaks are sealed and to avoid nonspecific "capping" of 5'-PO4 ends.