Probing the mechanisms of DEAD-Box proteins as general RNA chaperones: The C-terminal domain of CYT-19 mediates general recognition of RNA
Probing the mechanisms of DEAD-Box proteins as general RNA chaperones: The C-terminal domain of CYT-19 mediates general recognition of RNA
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DOI：
10.1021/bi0619472
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发表时间：
2007-03-20
期刊：
影响因子：
2.9
通讯作者：
Russell, Rick
中科院分区：
文献类型：
--
作者：
Grohman, Jacob K.;Del Campo, Mark;Russell, Rick
DEAD-box 蛋白 CYT-19 在体内折叠多个 I 组内含子以及在体外折叠不同的 I 组和 II 组 RNA。最近使用四膜虫 I 类核酶的研究表明，CYT-19 拥有第二个 RNA 结合位点，与解旋活性位点不同，该位点通过非特异性结合邻近的 RNA 结构来增强解旋活性。在这里，我们通过构建缺乏 49 个氨基酸并终止于结构域边界（如有限蛋白水解所定义的）的 C 端截短变体来探测负责这种结合的 CYT-19 区域。这种截短的蛋白质以接近野生型的效率解开四膜虫核酶的寡核苷酸底物和对应于核酶的内部引导序列的寡核苷酸之间形成的六碱基对双链体。然而，当双链体与核酶或单链或双链延伸共价连接时，截短的蛋白质的活化远低于野生型蛋白质。因此，RNA 解旋的活性位点在截短的 CYT-19 中仍然具有功能，但结合相邻 RNA 结构的位点已受到损害。平衡结合实验证实，截短的蛋白质与 RNA 的结合不如野生型蛋白质紧密。受损位点的 RNA 结合对于分子伴侣活性很重要，因为截短的蛋白质在促进体内需要 CYT-19 的 I 组内含子折叠方面活性较低。删除的区域包含富含精氨酸的序列，如在其他 RNA 结合蛋白中发现的那样，并且可能通过将 CYT-19 束缚到结构化 RNA 上来发挥作用，因此它可以有效地破坏暴露的非天然结构元件，使它们能够重新折叠。许多其他 DExD/H-box 蛋白也含有富含精氨酸的辅助结构域，其中一些结构域的功能可能类似于非特异性 RNA 结合元件，可增强一般 RNA 伴侣活性。
The DEAD-box protein CYT-19 functions in the folding of several group I introns in vivo and a diverse set of group I and group II RNAs in vitro. Recent work using the Tetrahymena group I ribozyme demonstrated that CYT-19 possesses a second RNA-binding site, distinct from the unwinding active site, which enhances unwinding activity by binding nonspecifically to the adjacent RNA structure. Here, we probe the region of CYT-19 responsible for that binding by constructing a C-terminal truncation variant that lacks 49 amino acids and terminates at a domain boundary, as defined by limited proteolysis. This truncated protein unwinds a six-base-pair duplex, formed between the oligonucleotide substrate of the Tetrahymena ribozyme and an oligonucleotide corresponding to the internal guide sequence of the ribozyme, with near-wild-type efficiency. However, the truncated protein is activated much less than the wild-type protein when the duplex is covalently linked to the ribozyme or single-stranded or double-stranded extensions. Thus, the active site for RNA unwinding remains functional in the truncated CYT-19, but the site that binds the adjacent RNA structure has been compromised. Equilibrium binding experiments confirmed that the truncated protein binds RNA less tightly than the wild-type protein. RNA binding by the compromised site is important for chaperone activity, because the truncated protein is less active in facilitating the folding of a group I intron that requires CYT-19 in vivo. The deleted region contains arginine-rich sequences, as found in other RNA-binding proteins, and may function by tethering CYT-19 to structured RNAs, so that it can efficiently disrupt exposed, non-native structural elements, allowing them to refold. Many other DExD/H-box proteins also contain arginine-rich ancillary domains, and some of these domains may function similarly as nonspecific RNA-binding elements that enhance general RNA chaperone activity.