Complete crystallographic analysis of the dynamics of CCA sequence addition
Complete crystallographic analysis of the dynamics of CCA sequence addition
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DOI：
10.1038/nature05204
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发表时间：
2006-10-26
期刊：
影响因子：
64.8
通讯作者：
Nureki, Osamu
中科院分区：
文献类型：
--
作者：
Tomita, Kozo;Ishitani, Ryuichiro;Nureki, Osamu
加入CCA的聚合酶使转移RNA的必需的39-CCA末端成熟，而不需要任何核酸模板。然而，目前尚不清楚在每个反应步骤中如何选择正确的核苷酸三磷酸，以及蛋白质和RNA动力学如何驱动聚合。在这里，我们提供了六种复杂的CCA添加酶结构和四种不同的RNA底物的完整顺序快照，其中有或没有CTP(三磷酸胞苷)或ATP(三磷酸腺苷)。缺乏CCA的RNA茎延伸一个碱基对，迫使鉴别子核苷进入活性部位口袋，然后在掺入第一个胞嘧啶一磷酸(CMP)后回溯。第二个CTP的调节夹住了催化裂隙，诱导了β-转角的重新定位，这翻转了c74，使cMP被接受。相反，在加入第二个CMP之后，聚合酶和RNA引物被锁定在闭合状态，这指导了随后的A加成。在CTP和ATP结合阶段之间，Arg 224的侧链构象发生了显著的变化，这是由酶的全局运动和引物末端的位置控制的，并可能实现CTP/ATP的识别，这取决于聚合阶段。在整个CCA添加反应中，酶的尾部结构域牢固地锚定tRNA的TYC环，这确保了准确的聚合和终止。
CCA-adding polymerase matures the essential 39-CCA terminus of transfer RNA without any nucleic-acid template. However, it remains unclear how the correct nucleotide triphosphate is selected in each reaction step and how the polymerization is driven by the protein and RNA dynamics. Here we present complete sequential snapshots of six complex structures of CCA-adding enzyme and four distinct RNA substrates with and without CTP ( cytosine triphosphate) or ATP ( adenosine triphosphate). The CCA-lacking RNA stem extends by one base pair to force the discriminator nucleoside into the active-site pocket, and then tracks back after incorporation of the first cytosine monophosphate (CMP). Accommodation of the second CTP clamps the catalytic cleft, inducing a reorientation of the beta-turn, which flips C74 to allow CMP to be accepted. In contrast, after the second CMP is added, the polymerase and RNA primer are locked in the closed state, which directs the subsequent A addition. Between the CTP- and ATP-binding stages, the side-chain conformation of Arg 224 changes markedly; this is controlled by the global motion of the enzyme and position of the primer terminus, and is likely to achieve the CTP/ATP discrimination, depending on the polymerization stage. Throughout the CCA-adding reaction, the enzyme tail domain firmly anchors the TYC-loop of the tRNA, which ensures accurate polymerization and termination.