Mutations in domain V of the 23S ribosomal RNA of Bacillus subtilis that inactivate its protein folding property in vitro
Mutations in domain V of the 23S ribosomal RNA of Bacillus subtilis that inactivate its protein folding property in vitro
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DOI：
10.1093/nar/30.5.1278
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发表时间：
2002-03-01
影响因子：
14.9
通讯作者：
DasGupta, C
中科院分区：
文献类型：
--
作者：
Chowdhury, S;Pal, S;DasGupta, C
蛋白质折叠反应的活性位点位于细菌核糖体中的 23S rRNA 及其在其他生物体中的同源物的 V 域。该结构域长期以来被称为肽基转移酶中心。枯草芽孢杆菌的结构域 V 分为两部分，即更保守的大肽基转移酶环 (RNA1) 和其余部分 (RNA2)。这两个片段一起充当蛋白质折叠调节剂以及完整的 V 域 RNA。从这些位点参与蛋白质合成各个步骤的报道中获取线索，在枯草芽孢杆菌的 RNA1 和 RNA2 中引入了许多定点突变。例如，大肠杆菌RNA1区中的62505、02506、02584和02585等位点在高Mg2+浓度下受到脱酰基tRNA的保护，而当P位点仍然被占据时，A2602受到氨酰基tRNA的保护。 RNA1 区域中的突变 A2058G 和 A2059G 导致大肠杆菌中的核糖体 Ery(r) 和烟草叶绿体中的 Lnc(r)。大肠杆菌中 P 环 62252 和 62253 中的位点受到保护，免受 P 位点结合 tRNA 的 CCA 末端的修饰。在枯草芽孢杆菌结构域V的RNA1和RNA2中的相应核苷酸中引入突变。使用未折叠碳酸酐酶的未折叠蛋白结合测定来测试突变体的重折叠。在蛋白质折叠测定中，突变体显示出部分或完全丧失这种活性。在 RNA 重折叠蛋白复合物的过滤结合测定中，突变体表现出一定程度的蛋白结合，与其蛋白折叠活性非常吻合。
The active site of a protein folding reaction is in domain V of the 23S rRNA in the bacterial ribosome and its homologs in other organisms. This domain has long been known as the peptidyl transferase center. Domain V of Bacillus subtilis is split into two segments, the more conserved large peptidyl transferase loop (RNA1) and the rest (RNA2). These two segments together act as a protein folding modulator as well as the complete domain V RNA. A number of site-directed mutations were introduced in RNA1 and RNA2 of B.subtilis, taking clues from reports of these sites being involved in various steps of protein synthesis. For example, sites like 62505, 02506, 02584 and 02585 in Escherichia coli RNA1 region are protected by deacylated tRNA at high Mg2+ concentration and A2602 is protected by amino acyl tRNA when the P site remains occupied already. Mutations A2058G and A2059G in the RNA1 region render the ribosome Ery(r) in E.coli and Lnc(r) in tobacco chloroplast. Sites in P loop 62252 and 62253 in E coli are protected against modification by the CCA end of the P site bound tRNA. Mutations were introduced in corresponding nucleotides in B.subtilis RNA1 and RNA2 of domain V. The mutants were tested for refolding using unfolded protein binding assays with unfolded carbonic anhydrase. In the protein folding assay, the mutants showed partial to complete loss of this activity. In the filter binding assay for the RNA-refolding protein complex, the mutants showed an extent of protein binding that agreed well with their protein folding activity.