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Computer modeling of eubacterial SRP RNA by NMR-based study

基于 NMR 的研究对真细菌 SRP RNA 进行计算机建模

基本信息

批准号：
12660065
负责人：
NAKAMURA Kouji
金额：
$ 2.3万
依托单位：
University of Tsukuba
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12660065/
关键词：
SRP RNA NMR-based structural analysis protein translocation small cytoplasmic RNA Nuclear magnetic resonance translation riboregulation elongation factor G (EF-G)NMR 細胞内低分子RNA 大腸菌タンパク質相互作用構造解析 RNA 4.5S RNA

项目摘要

Consisting of 114 nucleotides, 4.5S RNA is structurally homologous to mammalian 7S RNA, and it plays an essential role in targeting proteins containing signal peptide to the membrane by forming an SRP-like particle by binding with Ffh. It also binds independently to protein elongation factor (EF-G) and functions in the translation process. This RNA contains a phylogenetically conserved RNA domain, the predicted secondary structure of which consists of a hairpin motif with two bulges. We examined the binding activity of mutants with systematic deletions to define the minimal functional domain of 4.5S RNA that can interact with EF-G. This domain consisted of 35-nucleotides extending from 36 to 70 nucleotides of mature 4.5S RNA and contained two conserved bulges in which mutations of A47, A60, G61, C62, A63, and A67 diminished binding to EF-G, whereas those at A39, C40, C41, A48, and G49 did not affect binding. These data suggested that the 10 nucleotides in 4.5S RNA, which are conserved between 4.5S RNA and 23S rRNA, have a key role for EF-G biding. Based on the NMR-derived structure of mutant A47U causes striking structure changes and the loss of the symmetrical bulge. These results indicate the mechanism by which EF-G interacts with 4.5S RNA and the importance of the bulge structure for EF-G binding.

4.5S RNA由114个核苷酸组成，在结构上与哺乳动物的7S RNA同源，通过与FFH结合形成SRP样颗粒，在将含有信号肽的蛋白质靶向细胞膜上发挥重要作用。它还独立地与蛋白质延伸因子(EF-G)结合，并在翻译过程中发挥作用。该RNA包含一个在系统发育上保守的RNA结构域，其预测的二级结构由一个带有两个凸起的发夹基序组成。我们检测了系统缺失的突变体的结合活性，以确定4.5S RNA与EF-G相互作用的最小功能域。该结构域由成熟4.5S RNA的36-70个核苷酸组成，包含两个保守的突起，其中A47、A60、G61、C62、A63和A67的突变降低了与EF-G的结合，而A39、C40、C41、A48和G49的突变不影响结合。这些数据表明，4.5S RNA中的10个核苷酸在EF-G结合中起关键作用，它们保守于4.5S RNA和23S rRNA之间。根据核磁共振衍生的结构，突变体A47U会导致显着的结构变化和对称性凸起的丧失。这些结果表明了EF-G与4.5S RNA相互作用的机制以及凸起结构对EF-G结合的重要性。