Authentic interdomain communication in an RNA helicase reconstituted by expressed protein ligation of two helicase domains
Authentic interdomain communication in an RNA helicase reconstituted by expressed protein ligation of two helicase domains
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DOI：
10.1111/j.1742-4658.2006.05593.x
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发表时间：
2007-01-01
期刊：
影响因子：
5.4
通讯作者：
Klostermeier, Dagmar
中科院分区：
文献类型：
--
作者：
Karow, Anne R.;Theissen, Bettina;Klostermeier, Dagmar
RNA 解旋酶以 ATP 水解为代价介导 RNA 或 RNA-蛋白质复合物的结构重排。 DEAD 盒解旋酶家族的成员由两个灵活连接的解旋酶结构域组成。它们共有 9 个保守序列基序，参与核苷酸结合和水解、RNA 结合和解旋酶活性。大多数这些基序排列在两个解旋酶结构域之间的裂缝中，RNA 解旋需要它们之间的广泛通讯。枯草芽孢杆菌RNA解旋酶YxiN的两个解旋酶结构域分别作为内含肽融合体产生，并通过表达的蛋白连接产生功能性RNA解旋酶。连接的解旋酶以与野生型蛋白质非常相似的亲和力结合腺嘌呤核苷酸。重要的是，其本质上较低的 ATP 酶活性是由 RNA 刺激的，并且 Michaelis-Menten 参数与野生型相似。最后，连接的 YxiN 将最小的 RNA 底物解旋至与野生型解旋酶相当的程度，从而证实了真正的域间通信。
RNA helicases mediate structural rearrangements of RNA or RNA-protein complexes at the expense of ATP hydrolysis. Members of the DEAD box helicase family consist of two flexibly connected helicase domains. They share nine conserved sequence motifs that are involved in nucleotide binding and hydrolysis, RNA binding, and helicase activity. Most of these motifs line the cleft between the two helicase domains, and extensive communication between them is required for RNA unwinding. The two helicase domains of the Bacillus subtilis RNA helicase YxiN were produced separately as intein fusions, and a functional RNA helicase was generated by expressed protein ligation. The ligated helicase binds adenine nucleotides with very similar affinities to the wild-type protein. Importantly, its intrinsically low ATPase activity is stimulated by RNA, and the Michaelis-Menten parameters are similar to those of the wild-type. Finally, ligated YxiN unwinds a minimal RNA substrate to an extent comparable to that of the wild-type helicase, confirming authentic interdomain communication.