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Studies on a unique rRNA sequence found in the highly conserved functional region of silkworm ribosomes

家蚕核糖体高度保守功能区中独特rRNA序列的研究

基本信息

批准号：
12660053
负责人：
UCHIUMI Toshio
金额：
$ 2.24万
依托单位：
Shinshu University
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 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12660053/
关键词：
ribosome 28S rRNA silkworm ribosomal protein GTPase center site-directed mutagenesis covariant base change protein biosynthesis 28SrRNA 部位特異的塩基異変翻訳装置リボソームRNA GTPaseドメイン鱗翅類昆虫 RNA-蛋白質相互作用 P蛋白質

项目摘要

The GTPase-associated domain in 23/28S rRNA is one of the most highly conserved functional regions throughout all organisms. We detected a unique nucleotide sequence within the GTPase-associated domain in silkworm species, in which the bases at positions 1094 and 1098 (numbering from Escherichia coli 23S rRNA) are C and G instead of the otherwise universally conserved bases U and A, respectively. These changes were also observed in four other moths, but not in organisms other than the moths. The RNA fragment covering the silkworm GTPase-associated domain showed two smearing bands in native gel electrophoresis in the absence of ribosomal proteins. However, these bands shifted to a single clear band, wheh silkworm ribosomal proteins P0, P1, P2, and eL12 bound to the RNA, suggesting that the GTPase-associated domain of silkworm 28S rRNA has a labile structural feature that is stabilized by binding of the ribosomal proteins. In order to know the functional significance of the covariant bas … More e change in the silkworm rRNA, we attempted the introduction of C-1094 and G-1098 into E. coli ribosomes by using a plasmid carrying E. coli rDNA (rrnB). So far, we detected the mutant rRNA in isolated ribosomes, suggesting that the mutant ribosomes were assembled in vivo. Further analyses of function of the mutant ribosomes were remained.We also investigated interactions among ribosomal proteins P0, P1, and P2 by native gel electrophoresis. Mixing of P1 and P2 formed the P1-P2 heterodimer. This heterodimer, but neither P1 nor P2 alone, tightly bound to P0 and formed a pentameric complex P0(P1-P2)_2, We confirmed the functions of the ribosomal proteins by incorporating the proteins into E. coli ribosomes, i.e., by replacing E. coli proteins L10-L7/L12 complex and L11 on the GTPase-associated RNA domain with silkworm P0(P1-P2)_2 complex and eL12. The protein replacement caused ribosomal activity with silkworm translation factors. This protein-dependent induction of the ribosomal activity did not occur in the absence either of P1 or P2. These results suggest that formation of P1-P2 heterodimer is crucial for assembly of the proteins on the rRNA domain and for the stabilization of the functional rRNA structure. Less

23/28 S rRNA中的GTP酶相关结构域是所有生物体中最高度保守的功能区域之一。我们检测到一个独特的核苷酸序列内的GTP酶相关的结构域中的家蚕物种，其中在位置1094和1098（编号从大肠杆菌23 S rRNA）的碱基是C和G，而不是其他普遍保守的碱基U和A，分别。在其他四种蛾中也观察到了这些变化，但在蛾以外的生物中没有观察到。在不存在核糖体蛋白的情况下，覆盖家蚕GTP酶相关结构域的RNA片段在天然凝胶电泳中显示出两条涂抹带。然而，这些条带移动到一个单一的清晰的带，当家蚕核糖体蛋白P0，P1，P2，和eL 12结合的RNA，表明GTP酶相关的结构域的家蚕28 S rRNA具有不稳定的结构特征，是稳定的核糖体蛋白的结合。为了了解协变基的函数意义， ...更多信息利用家蚕rRNA基因的突变，我们尝试将C-1094和G-1098基因导入E.大肠杆菌核糖体的表达。coli rDNA（rrnB）。到目前为止，我们在分离的核糖体中检测到了突变的rRNA，这表明突变的核糖体是在体内组装的。我们还利用非变性凝胶电泳研究了核糖体蛋白P0、P1和P2之间的相互作用。P1和P2的混合形成P1-P2异二聚体。这种异源二聚体与P0紧密结合，形成五聚体复合物P0（P1-P2）_2。大肠杆菌核糖体，即，取代E。大肠杆菌蛋白L10-L7/L12复合物和L11与家蚕P0（P1-P2）_2复合物和eL 12在GTP酶相关RNA结构域上的结合。蛋白质替换引起了家蚕翻译因子的核糖体活性。这种蛋白质依赖性的诱导核糖体活性没有发生在P1或P2的情况下。这些结果表明，P1-P2异源二聚体的形成是至关重要的蛋白质的装配上的rRNA结构域和功能的rRNA结构的稳定。少