Structure-Guided Engineering of the Regioselectivity of RNA Ligase Ribozymes
Structure-Guided Engineering of the Regioselectivity of RNA Ligase Ribozymes
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DOI：
10.1021/ja8067325
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发表时间：
2009-03-18
影响因子：
15
通讯作者：
Ferre-D'Amare, Adrian R.
中科院分区：
文献类型：
--
作者：
Pitt, Jason N.;Ferre-D'Amare, Adrian R.
核酶催化的RNA合成是RNA世界假说的核心。目前还没有发现天然的RNA聚合酶核酶。然而，通过体外选择，已经分离出催化所需化学的核酶，通过RNA的末端羟基攻击三磷酸活化的寡核苷酸的α-磷酸生成新的磷酸二酯。这些实验经常产生产生2‘-5’磷酸二酯的核酶，而不是传统的3‘-5’键。我们测定了具有代表性的2‘-5’RNA连接酶核酶的模板片段、II类连接酶及其连接产物形成的双链的晶体结构。结构显示在连接连接处有G-A对的产物-模板双链。这个剪切的对在一侧被一个由两个未配对的核苷酸组成的微小的凹槽加宽的楔形所组成。报道了一种独立分离的3‘-5’连接酶核酶L1连接酶的结构，显示了与II类连接酶共享G-A对的产物-模板双链。然而，这对G-A是由G-U摆动的侧翼，而不是未配对的楔形。我们通过构建嵌合体证明了底物-模板双链体的这些结构差异是导致这两种核酶区域选择性不同的主要原因，而与它们的催化部分无关。具有II类底物-模板双链的L1连接酶显示2‘-5’键的合成增加了30倍，而具有L1底物-模板双链的II类连接酶仅产生3‘-5’键。这些结果表明，底物-模板双链固有的局部几何结构如何控制核酶催化的RNA连接反应的区域选择性。
Ribozyme-catalyzed RNA synthesis is central to the RNA world hypothesis. No natural RNA polymerase ribozymes have been discovered. However, ribozymes that catalyze the requisite chemistry, generating a new phosphodiester through attack of a terminal hydroxyl of an RNA on the a-phosphate of a triphosphate-activated oligonucleotide, have been isolated by in vitro selection. These experiments often yield ribozymes that generate 2'-5' phosphodiesters rather than conventional 3'-5' linkages. We have determined crystal structures of the duplex formed by the template segment of a representative 2'-5' RNA ligase ribozyme, the class II ligase, and its ligation product. The structures reveal a product-template duplex with a G-A pair at the ligation junction. This sheared pair is flanked on one side by a minor groove-broadening wedge comprised of two unpaired nucleotides. The reported structure of an independently isolated 3'-5' ligase ribozyme, the L1 ligase, shows a product-template duplex that shares the G-A pair with the class II ligase. However, this G-A pair is flanked by G-U wobbles, rather than an unpaired wedge. We demonstrate that these structural differences of the substrate-template duplexes are largely responsible for the divergent regioselectivity of the two ribozymes, independent of their catalytic moieties, by constructing chimeras. The L1 ligase with a class II substrate-template duplex shows a 30-fold increase in 2'-5' bond synthesis, while the class II ligase with an L1 substrate-template duplex produces 3'-5' bonds exclusively. These results demonstrate how local geometry inherent to the substrate-template duplexes controls the regioselectivity of ribozyme-catalyzed RNA ligation reactions.