Institute of Organic Chemistry and Center for Molecular Biosciences (CMBI), Leopold-Franzens University, Innrain 80-82, 6020, Innsbruck, Austria.
Life Science Institute, Zhejiang University, Hangzhou, 310058, China.
Angew Chem Int Ed Engl. 2017 Dec 11;56(50):15954-15958. doi: 10.1002/anie.201708679. Epub 2017 Nov 15.
The pistol RNA motif represents a new class of self-cleaving ribozymes of yet unknown biological function. Our recent crystal structure of a pre-catalytic state of this RNA shows guanosine G40 and adenosine A32 close to the G53-U54 cleavage site. While the N1 of G40 is within 3.4 Å of the modeled G53 2'-OH group that attacks the scissile phosphate, thus suggesting a direct role in general acid-base catalysis, the function of A32 is less clear. We present evidence from atom-specific mutagenesis that neither the N1 nor N3 base positions of A32 are involved in catalysis. By contrast, the ribose 2'-OH of A32 seems crucial for the proper positioning of G40 through a H-bond network that involves G42 as a bridging unit between A32 and G40. We also found that disruption of the inner-sphere coordination of the active-site Mg cation to N7 of G33 makes the ribozyme drastically slower. A mechanistic proposal is suggested, with A32 playing a structural role and hydrated Mg playing a catalytic role in cleavage.
手枪 RNA 基序代表了一类新的具有未知生物学功能的自我切割核酶。我们最近的该 RNA 前催化态晶体结构表明，鸟苷 G40 和腺苷 A32 靠近 G53-U54 切割位点。虽然 G40 的 N1 距离 G53 2'-OH 基团 3.4Å，该基团攻击磷酸酯键，因此提示其在广义酸碱催化中具有直接作用，但 A32 的功能不太清楚。我们提供的证据表明，A32 的 N1 或 N3 碱基位置都不参与催化。相比之下，A32 的核糖 2'-OH 似乎通过一个涉及 G42 作为 A32 和 G40 之间桥连单元的氢键网络对于正确定位 G40 至关重要。我们还发现，破坏活性位点 Mg 阳离子与 G33 的 N7 的内球配位使核酶的速度急剧下降。提出了一种机制假说，其中 A32 发挥结构作用，水合 Mg 在切割中发挥催化作用。
