Template-independent terminal ribonucleotide transferases (TENTs) catalyze the addition of nucleotide monophosphates to the 3′-end of RNA molecules regulating their fate. TENTs include poly(U) polymerases (PUPs) with a subgroup of 3′ CUCU-tagging enzymes, such as CutA in Aspergillus nidulans. CutA preferentially incorporates cytosines, processively polymerizes only adenosines and does not incorporate or extend guanosines. The basis of this peculiar specificity remains to be established. Here, we describe crystal structures of the catalytic core of CutA in complex with an incoming non-hydrolyzable CTP analog and an RNA with three adenosines, along with biochemical characterization of the enzyme. The binding of GTP or a primer with terminal guanosine is predicted to induce clashes between 2-NH₂ of the guanine and protein, which would explain why CutA is unable to use these ligands as substrates. Processive adenosine polymerization likely results from the preferential binding of a primer ending with at least two adenosines. Intriguingly, we found that the affinities of CutA for the CTP and UTP are very similar and the structures did not reveal any apparent elements for specific NTP binding. Thus, the properties of CutA likely result from an interplay between several factors, which may include a conformational dynamic process of NTP recognition.

中文翻译：

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CutA，RNA核苷酸转移酶的结构和机制具有对胞嘧啶的异常偏爱。

独立于模板的末端核糖核苷酸转移酶（TENT）催化将单磷酸核苷酸添加到调节其命运的RNA分子的3'端。TENTs包括带有3'CUCU标签酶亚类的聚（U）聚合酶（PUP），例如构巢曲霉中的CutA。CutA优先掺入胞嘧啶，仅聚合腺苷，不掺入或扩展鸟苷。这种特殊特异性的基础仍有待建立。在这里，我们描述了与传入的不可水解的CTP类似物和带有三个腺苷的RNA配合的CutA催化核心的晶体结构，以及该酶的生化特性。预测GTP或引物与末端鸟苷的结合会诱导2-NH ₂之间的碰撞鸟嘌呤和蛋白质的特性，这可以解释为什么CutA无法使用这些配体作为底物。进行性腺苷聚合可能是由于以至少两个腺苷为末端的引物的优先结合所致。有趣的是，我们发现CutA对CTP和UTP的亲和力非常相似，并且该结构并未揭示任何与特定NTP结合的明显成分。因此，CutA的属性可能是由于几个因素之间的相互作用而产生的，其中可能包括NTP识别的构象动态过程。