Reengineering CCA-adding enzymes to function as (U,G)- or dCdCdA-adding enzymes or poly(C,A) and poly(U,G) polymerases
Reengineering CCA-adding enzymes to function as (U,G)- or dCdCdA-adding enzymes or poly(C,A) and poly(U,G) polymerases
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DOI：
10.1073/pnas.0606961104
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发表时间：
2007-01-02
影响因子：
11.1
通讯作者：
Weiner, Alan M.
中科院分区：
文献类型：
--
作者：
Cho, HyunDae D.;Verlinde, Christophe L. M. J.;Weiner, Alan M.
CCA添加酶构建并修复tRNA的T末端CCA序列。这些不寻常的RNA聚合酶使用核糖核蛋白模板（I类）或纯蛋白模板（II类）与传入CTP和ATP的沃森-克里克边缘形成模拟碱基对。在II类嗜热脂肪芽孢杆菌CCA添加酶结构的指导下，我们引入了旨在逆转核碱基和蛋白质模板之间氢键极性的突变。我们能够将CCA添加酶转化为（U，G）添加酶，其掺入UTP和GTP而不是CTP和ATP;我们将相关的Aquifex aeolicus CC和A添加酶转化为UU和G添加酶，将大肠杆菌poly（A）聚合酶转化为poly（G）聚合酶;我们转化了B。通过螺旋J中的突变将嗜热脂肪酸菌CCA添加酶添加到聚（C，A）聚合酶中，基于脱辅基酶结构，所述突变似乎在空间上限制添加到CCA。我们还改造了B。通过突变与引入的核糖2'羟基相互作用的精氨酸，将嗜热脂肪菌CCA添加酶转化为dCdCdA添加酶。最重要的是，我们发现螺旋J中的突变可以影响核苷酸结合位点的特异性，这表明I类和II类酶的特异性可能由涉及蛋白质、引入的核苷酸和tRNA的3'端的氢键的复杂网络决定。RNA和蛋白质以核糖核蛋白模板的形式合作可能有助于解释核苷酸转移酶家族的进化多样性。
CCA-adding enzymes build and repair the T-terminal CCA sequence of tRNA. These unusual RNA polymerases use either a ribonucleoprotein template (class I) or pure protein template (class II) to form mock base pairs with the Watson-Crick edges of incoming CTP and ATP. Guided by the class II Bacillus stearothermophilus CCA-adding enzyme structure, we introduced mutations designed to reverse the polarity of hydrogen bonds between the nucleobases and protein template. We were able to transform the CCA-adding enzyme into a (U,G)-adding enzyme that incorporates UTP and GTP instead of CTP and ATP; we transformed the related Aquifex aeolicus CC- and A-adding enzymes into UU- and G-adding enzymes and Escherichia coli poly(A) polymerase into a poly(G) polymerase; and we transformed the B. stearothermophilus CCA-adding enzyme into a poly(C,A) polymerase by mutations in helix J that appear, based on the apoenzyme structure, to sterically limit addition to CCA. We also transformed the B. stearothermophilus CCA-adding enzyme into a dCdCdA-adding enzyme by mutating an arginine that interacts with the incoming ribose 2' hydroxyl. Most importantly, we found that mutations in helix J can affect the specificity of the nucleotide binding site some 20 A away, suggesting that the specificity of both class I and II enzymes may be dictated by an intricate network of hydrogen bonds involving the protein, incoming nucleotide, and 3' end of the tRNA. Collaboration between RNA and protein in the form of a ribonucleoprotein template may help to explain the evolutionary diversity of the nucleoticlyltransferase family.