Atom-Specific Mutagenesis Reveals Structural and Catalytic Roles for an Active-Site Adenosine and Hydrated Mg2+ in Pistol Ribozymes
Atom-Specific Mutagenesis Reveals Structural and Catalytic Roles for an Active-Site Adenosine and Hydrated Mg2+ in Pistol Ribozymes
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原子特异性诱变揭示了手枪核酶中活性位点腺苷和水合 Mg(2) 的结构和催化作用
DOI：
10.1002/anie.201708679
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发表时间：
2017-12-11
影响因子：
16.6
通讯作者：
Micura, Ronald
中科院分区：
文献类型：
--
作者：
Neuner, Sandro;Falschlunger, Christoph;Micura, Ronald
手枪RNA基序代表了一类新的自切割核酶，其生物学功能尚不清楚。我们最近对该RNA催化前状态的晶体结构显示，鸟苷G40和腺苷A32靠近G53-U54裂解位点。虽然G40的N1在模型G53攻击可裂磷酸盐的2-OH基团的3.4埃范围内，因此表明在一般酸碱催化中起直接作用，但A32的功能不太清楚。我们从原子特异性诱变中获得的证据表明，A32的N1和N3碱基位置都不参与催化。相比之下，A32的核糖2-OH似乎对G40通过氢键网络的正确定位至关重要，该网络涉及G42作为A32和G40之间的桥接单元。我们还发现，G33的活性位点Mg2+阳离子与N7的内球协调的破坏使核酶的速度大大减慢。提出了A32在解理过程中起结构作用，水合Mg2+在解理过程中起催化作用的机理。
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 angstrom 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 Mg2+ cation to N7 of G33 makes the ribozyme drastically slower. A mechanistic proposal is suggested, with A32 playing a structural role and hydrated Mg2+ playing a catalytic role in cleavage.