Hatchet ribozyme structure and implications for cleavage mechanism
Hatchet ribozyme structure and implications for cleavage mechanism
复制标题
Hatchet核酶结构及其对裂解机制的影响
DOI：
10.1073/pnas.1902413116
复制
发表时间：
2019
影响因子：
11.1
通讯作者：
Ren Aiming
中科院分区：
文献类型：
--
作者：
Zheng Luqian;Falschlunger Christoph;Huang Kaiyi;Mairhofer Elisabeth;Yuan Shuguang;Wang Juncheng;Patel Dinshaw J;Micura Ronald;Ren Aiming
小的自切割核酶催化其自身磷酸二酯骨架的位点特异性切割，这与病毒基因组复制、前mRNA加工和可变剪接有关。我们报告的2.1-hatchet核酶产品，它采用了一个紧凑的假对称二聚体支架，与每个单体稳定的远程相互作用，涉及高度保守的核苷酸带来了紧密接近的易裂的磷酸盐的晶体结构。引人注目的是，催化口袋包含一个能够容纳模拟的易分裂磷酸盐及其侧翼5 '核苷的空腔。所得到的模型化的预催化构象在易分裂的磷酸盐处并入了分开的排列，从而提供了对在线切割机制的基于结构的见解。我们确定了一个鸟嘌呤衬里的催化口袋定位，以促进裂解化学。基于结构的见解到斧头核酶催化的功能相关性得到了切割测定的强烈支持，该测定监测所选核碱基和原子特异性突变对核酶活性的影响。
Small self-cleaving ribozymes catalyze site-specific cleavage of their own phosphodiester backbone with implications for viral genome replication, pre-mRNA processing, and alternative splicing. We report on the 2.1-Å crystal structure of the hatchet ribozyme product, which adopts a compact pseudosymmetric dimeric scaffold, with each monomer stabilized by long-range interactions involving highly conserved nucleotides brought into close proximity of the scissile phosphate. Strikingly, the catalytic pocket contains a cavity capable of accommodating both the modeled scissile phosphate and its flanking 5′ nucleoside. The resulting modeled precatalytic conformation incorporates a splayed-apart alignment at the scissile phosphate, thereby providing structure-based insights into the in-line cleavage mechanism. We identify a guanine lining the catalytic pocket positioned to contribute to cleavage chemistry. The functional relevance of structure-based insights into hatchet ribozyme catalysis is strongly supported by cleavage assays monitoring the impact of selected nucleobase and atom-specific mutations on ribozyme activity.