Biochemical Characterization of Ribosome Assembly GTPase RbgA in Bacillus subtilis
Biochemical Characterization of Ribosome Assembly GTPase RbgA in Bacillus subtilis
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DOI：
10.1074/jbc.m111.331322
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发表时间：
2012-03-09
影响因子：
4.8
通讯作者：
Britton, Robert A.
中科院分区：
文献类型：
--
作者：
Achila, David;Gulati, Megha;Britton, Robert A.
核糖体生物发生GTgA蛋白RbgA参与枯草芽孢杆菌中核糖体大亚基的组装，并且RbgA的同系物涉及古细菌和真核生物中线粒体、叶绿体和细胞质核糖体的生物发生。RbgA如何促进核糖体组装的精确功能尚不清楚。RbgA的缺陷产生一个大的核糖体亚基，是不成熟的，并在45 S的蔗糖密度梯度迁移。在这里，我们报告一个详细的生化分析RbgA及其与核糖体的相互作用。我们发现，与大多数其他GTPases一样，RbgA表现出非常慢的k（cat）（14 h（-1））并且具有高K-m（90 μ M）。同源建模的RbgA开关I区使用的K-环GTdR MnmE作为模板表明，RbgA需要K+离子的GTdR活性，这是实验证实。与50个S亚基，但不是45个S中间体的相互作用，增加了GTdR活性的55倍。与50 S亚基和45 S中间体的稳定关联是核苷酸依赖性的，GDP不支持与任何一个亚基的强相互作用。GTP和鸟苷5 '-（β，γ-亚氨基）三磷酸（GMPPNP）足以促进与45 S中间体的结合，而只有GMPPNP能够支持与50 S亚基的结合，推测是由于GTP水解的刺激。这些结果支持一个模型，其中RbgA促进核糖体生物合成的后期步骤，GTP水解的作用之一是刺激RbgA从核糖体的解离。
The ribosome biogenesis GTPase A protein RbgA is involved in the assembly of the large ribosomal subunit in Bacillus subtilis, and homologs of RbgA are implicated in the biogenesis of mitochondrial, chloroplast, and cytoplasmic ribosomes in archaea and eukaryotes. The precise function of how RbgA contributes to ribosome assembly is not understood. Defects in RbgA give rise to a large ribosomal subunit that is immature and migrates at 45 S in sucrose density gradients. Here, we report a detailed biochemical analysis of RbgA and its interaction with the ribosome. We found that RbgA, like most other GTPases, exhibits a very slow k(cat) (14 h(-1)) and has a high K-m (90 mu M). Homology modeling of the RbgA switch I region using the K-loop GTPase MnmE as a template suggested that RbgA requires K+ ions for GTPase activity, which was confirmed experimentally. Interaction with 50 S subunits, but not 45 S intermediates, increased GTPase activity by similar to 55-fold. Stable association with 50 S subunits and 45 S intermediates was nucleotide-dependent, and GDP did not support strong interaction with either of the subunits. GTP and guanosine 5'-(beta,gamma-imido)triphosphate (GMPPNP) were sufficient to promote association with the 45 S intermediate, whereas only GMPPNP was able to support binding to the 50 S subunit, presumably due to the stimulation of GTP hydrolysis. These results support a model in which RbgA promotes a late step in ribosome biogenesis and that one role of GTP hydrolysis is to stimulate dissociation of RbgA from the ribosome.