A divalent cation-dependent variant of the glmS ribozyme with stringent Ca(2+) selectivity co-opts a preexisting nonspecific metal ion-binding site.
A divalent cation-dependent variant of the glmS ribozyme with stringent Ca(2+) selectivity co-opts a preexisting nonspecific metal ion-binding site.
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DOI：
10.1261/rna.059824.116
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发表时间：
2017-03
期刊：
影响因子：
--
通讯作者：
Ferré-D'Amaré AR
中科院分区：
文献类型：
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作者：
Lau MW;Trachman RJ 3rd;Ferré-D'Amaré AR
核酶使用二价阳离子用于结构稳定，作为催化辅因子，或两者兼而有之。由于Ca ~（2+）在细胞内信号传导中的重要作用，具有严格Ca ~（2+）选择性的工程化核酶在生物技术中具有重要意义。野生型glmS核酶（glmSWT）需要6-磷酸葡萄糖胺（GlcN 6 P）作为催化辅因子。先前，鉴定了具有三个腺苷突变的glmS核酶变体（glmSAAA），其不含GlcN 6P，而是使用二价阳离子作为位点特异性RNA切割的辅因子，选择性很小。我们现在报道了一种由glmSAAA进化而来的Ca 2+特异性核酶（glmSCa），其在Ca 2+中的活性比Mg 2+高10，000倍，即使在100 mM Mg 2+中也无活性，并且对GlcN 6P无反应。这种严格的选择性，让人想起来自葡萄球菌的蛋白质核酸酶，允许使用Ca 2+螯合剂如EGTA快速和选择性的核酶失活。由于glmSCa在生理相关的Ca 2+浓度下发挥作用，因此它可以形成将Ca 2+水平与RNA切割偶联的细胞内传感器的基础。glmSCa的生化分析表明，它已增选选择性的Ca 2+结合的非特异性阳离子结合位点负责glmSWT和glmSAAA的结构稳定。微调的阳离子位点的选择性允许重新利用这一预先存在的分子特征。
Ribozymes use divalent cations for structural stabilization, as catalytic cofactors, or both. Because of the prominent role of Ca2+ in intracellular signaling, engineered ribozymes with stringent Ca2+ selectivity would be important in biotechnology. The wild-type glmS ribozyme (glmSWT) requires glucosamine-6-phosphate (GlcN6P) as a catalytic cofactor. Previously, a glmS ribozyme variant with three adenosine mutations (glmSAAA) was identified, which dispenses with GlcN6P and instead uses, with little selectivity, divalent cations as cofactors for site-specific RNA cleavage. We now report a Ca2+-specific ribozyme (glmSCa) evolved from glmSAAA that is >10,000 times more active in Ca2+ than Mg2+, is inactive in even 100 mM Mg2+, and is not responsive to GlcN6P. This stringent selectivity, reminiscent of the protein nuclease from Staphylococcus, allows rapid and selective ribozyme inactivation using a Ca2+ chelator such as EGTA. Because glmSCa functions in physiologically relevant Ca2+ concentrations, it can form the basis for intracellular sensors that couple Ca2+ levels to RNA cleavage. Biochemical analysis of glmSCa reveals that it has co-opted for selective Ca2+ binding a nonspecific cation-binding site responsible for structural stabilization in glmSWT and glmSAAA. Fine-tuning of the selectivity of the cation site allows repurposing of this preexisting molecular feature.