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首页> 外文期刊>Biochemistry >Two Divalent Metal Ions and Conformational Changes Play Roles in the Hammerhead Ribozyme Cleavage Reaction
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Two Divalent Metal Ions and Conformational Changes Play Roles in the Hammerhead Ribozyme Cleavage Reaction

机译：锤头核酶裂解反应中的两个二价金属离子和构象变化

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The hammerhead ribozyme is a self-cleaving RNA broadly dispersed across all kingdoms of life. Although it was the first of the small, nudeolytic ribozymes discovered, the mechanism by which it catalyzes its reaction remains elusive. The nudeobase of G12 is well positioned to be a general base, but it is unclear if or how this guanine base becomes activated for proton transfer. Metal ions have been implicated in the chemical mechanism, but no interactions between divalent metal ions and the cleavage site have been observed crystallographically. To better understand how this ribozyme functions, we have solved crystal structures of wild-type and G12A mutant ribozymes. We observe a pH-dependent conformational change centered around G12, consistent with this nucleotide becoming deprotonated. Crystallographic and kinetic analysis of the G12A mutant reveals a Zn2+ specificity switch suggesting a direct interaction between a divalent metal ion and the purine at position 12. The metal ion specificity switch and the pH rate profile of the G12A mutant suggest that the minor imino tautomer of A12 serves as the general base in the mutant ribozyme. We propose a model in which the hammerhead ribozyme rearranges prior to the cleavage reaction, positioning two divalent metal ions in the process. The first metal ion, positioned near G12, becomes directly coordinated to the 06 keto oxygen, to lower the pK(a) of the general base and organize the active site. The second metal ion, positioned near G10.1, bridges the N7 of G10.1 and the scissile phosphate and may participate directly in the cleavage reaction.
机译：锤头状核酶是一种自我切割的RNA，广泛分布于生活的各个王国。尽管它是发现的第一个小的核酶核酶，但是它催化其反应的机理仍然难以捉摸。 G12的裸碱基可以很好地定位为通用碱基，但尚不清楚该鸟嘌呤碱基是否或如何被激活以进行质子转移。金属离子与化学机理有关，但是在晶体学上未观察到二价金属离子与切割位点之间的相互作用。为了更好地了解这种核酶的功能，我们解决了野生型和G12A突变核酶的晶体结构。我们观察到围绕G12的pH依赖的构象变化，与该核苷酸去质子化一致。 G12A突变体的晶体学和动力学分析表明，Zn2 +特异性开关表明二价金属离子与12位嘌呤之间存在直接相互作用。G12A突变体的金属离子特异性开关和pH速率曲线表明，亚氨基的互变异构体是A12充当突变核酶的通用碱基。我们提出了一个模型，在该模型中，锤头状核酶在裂解反应之前会重新排列，在此过程中放置​​两个二价金属离子。位于G12附近的第一个金属离子直接与06酮氧配位，以降低一般碱的pK（a）并组织活性位点。位于G10.1附近的第二种金属离子桥接了G10.1的N7和易裂磷酸根，并可能直接参与裂解反应。

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