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Functional analysis of the Bacillus subtilis GTP-binding protein family

枯草芽孢杆菌 GTP 结合蛋白家族的功能分析

基本信息

批准号：
17201040
负责人：
OGASAWARA Naotake
金额：
$ 30.78万
依托单位：
Nara Institute of Science and Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17201040/
关键词：
Protein Genome Bacteria GTP-binding protein Bacillus subtilis Ribosome

项目摘要

GTP-binding proteins are highly conserved and play critical roles in various cellular processes. The Bacillus subtilis genome encodes 7 essential GTP binding proteins, Era, Obg, YphC, YsxC, YlqF, YqeH, and YloQ, belonging to the Obg/Era subfamily of small GTP binding proteins, which had been poorly characterized. We demonstrated that YlqF is co-fractionated with the 50S ribosome subunit, in the presence of noncleavable GTP analog. Moreover, the GTPase activity of YlqF was stimulated specifically by the 50S subunit in vitro. Dimethyl sulfate footprinting analysis disclosed that YlqF is positioned around the A-site and P-site on the 50S subunit. Next, we found that an N-terminal deletion mutant of YlqF (YlqF_N10) inhibits cell growth even in the presence of wild-type YlqF. The GTPase activity of this mutant was not stimulated by the 50S subunit and did not dissociate from the premature 50S subunit. Premature 50S subunit accumulated in YlqF_N10-overexpressing cells and in YlqF depleted cells, suggesting that YlqF is targeted to the premature 50S subunit lacking ribosomal proteins L16 and L27 to assemble functional 50S subunit through a GTPase activity-dependent conformational change of 23S rRNA. Furthermore, we investigated functional relationships among Obg, YsxC, YphC and YlqF, and proposed a model for their sequential action in the late step of 50S subunit biogenesis. In contrast, Era, YloQ and YqeH are involved in the 30S subunit biogenesis. We demonstrated a reduction in the 70S ribosome and accumulation of the free 50S subunit in YqeH-depleted cells and our results strongly suggested that YqeH and Era function at distinct checkpoints during 30S subunit assembly. In addition, we have found that that alternative localization of two types of ribosomal proteins in the B. subtilis ribosome depending on the intracellular concentration of zinc, zinc-binding motif containing type (RpmE or RpsN) and their homologues without the motif (YtiA or YhzA).

GTP结合蛋白是高度保守的，并且在各种细胞过程中发挥关键作用。枯草芽孢杆菌基因组编码7种必需的GTP结合蛋白，Era、Obg、YphC、YsxC、YlqF、YqeH和YloQ，属于Obg/Era小GTP结合蛋白亚家族，其特征很差。我们证明，YlqF与50 S核糖体亚基共分馏，在不可切割的GTP类似物的存在下。此外，在体外，50 S亚基特异性地刺激YlqF的GTdR活性。硫酸二甲酯足迹分析揭示YlqF位于50 S亚基上的A-位点和P-位点周围。接下来，我们发现YlqF的N-末端缺失突变体（YlqF_N10）即使在野生型YlqF存在下也抑制细胞生长。该突变体的GT3活性不受50 S亚基的刺激，也不与早熟50 S亚基分离。在YlqF_N10过表达的细胞和YlqF缺失的细胞中积累了早熟50 S亚基，表明YlqF靶向缺乏核糖体蛋白L16和L27的早熟50 S亚基，通过23 S rRNA的GTP酶活性依赖性构象变化组装功能性50 S亚基。此外，我们还研究了Obg、YsxC、YphC和YlqF之间的功能关系，并提出了它们在50 S亚基生物合成后期顺序作用的模型。相反，Era、YloQ和YqeH参与30 S亚基的生物发生。我们证明了减少70 S核糖体和积累的游离50 S亚基在YqeH耗尽的细胞和我们的结果强烈表明，YqeH和Era功能在不同的检查点在30 S亚基组装。此外，我们还发现两种类型核糖体蛋白在B中的交替定位。枯草杆菌核糖体依赖于细胞内锌的浓度，含锌结合基序的类型（RpmE或RpsN）及其不含基序的同源物（YtiA或YhzA）。