Structural domains involved in the RNA folding activity of RNA helicase II/Gu protein.
Structural domains involved in the RNA folding activity of RNA helicase II/Gu protein.
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DOI：
10.1046/j.1432-1327.2000.01727.x
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发表时间：
2000-11
期刊：
影响因子：
--
通讯作者：
B. Valdez
中科院分区：
文献类型：
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作者：
B. Valdez
RNA解旋酶II/Gu（RH II/Gu）是一种核仁蛋白，其在5'至3'方向上解旋dsRNA，并将二级结构引入ssRNA中。解旋酶结构域位于分子的N-末端四分之三，折叠酶结构域位于C-末端四分之一。RH II/Gu的RNA折叠活性不仅仅是其与RNA结合的人工产物。这项研究将RNA折叠酶结构域缩小到蛋白质C末端的氨基酸749-801。通过缺失和定点诱变对该区域的解剖显示，四个FRGQR重复以及C末端独立地结合RNA。这些并置的亚结构域对于RH II/Gu的RNA折叠酶活性都很重要。重复序列2或重复序列4的突变，或RH II/Gu的C-末端的Lys 792、Arg 793和Lys 797同时突变为丙氨酸抑制RNA折叠酶活性。RH II/Gu的最后17个氨基酸可以被核仁蛋白p120的RNA结合基序取代，而对其折叠酶活性没有有害影响。提出了一个模型来解释RH II/Gu如何结合和折叠RNA底物。
RNA helicase II/Gu (RH II/Gu) is a nucleolar protein that unwinds dsRNA in a 5' to 3' direction, and introduces a secondary structure into a ssRNA. The helicase domain is at the N-terminal three-quarters of the molecule and the foldase domain is at the C-terminal quarter. The RNA folding activity of RH II/Gu is not a mere artifact of its binding to RNA. This study narrows down the RNA foldase domain to amino acids 749-801 at the C-terminus of the protein. Dissection of this region by deletion and site-directed mutagenesis shows that the four FRGQR repeats, as well as the C-terminal end bind RNA independently. These juxtaposed subdomains are both important for the RNA foldase activity of RH II/Gu. Mutation of either repeat 2 or repeat 4, or simultaneous mutation of Lys792, Arg793 and Lys797 at the C-terminal end of RH II/Gu to alanines inhibits RNA foldase activity. The last 17 amino acids of RH II/Gu can be replaced by an RNA binding motif from nucleolar protein p120 without a deleterious effect on its foldase activity. A model is proposed to explain how RH II/Gu binds and folds an RNA substrate.