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

23/28S rRNA中的GTP酶相关结构域是所有生物中最保守的功能区之一。我们在家蚕的GTP酶相关结构域中发现了一个独特的核苷酸序列，其中1094和1098位的碱基(编号来自大肠杆菌23S rRNA)分别是C和G，而不是其他普遍保守的碱基U和A。在其他四种蛾身上也观察到了这些变化，但除了蛾之外，其他生物没有观察到这些变化。在没有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两条涂抹带。然而，这些条带移动到一个清晰的条带，即与RNA结合的家蚕核糖体蛋白P0、P1、P2和EL12，这表明家蚕28S rRNA的GTPase相关结构域具有不稳定的结构特征，该结构特征通过核糖体蛋白的结合而稳定。为了了解协变bas…的功能意义为了使家蚕rRNA发生更多的变化，我们尝试用携带大肠杆菌rDNA(RrnB)的质粒将C-1094和G-1098导入到大肠杆菌核糖体中。到目前为止，我们在分离的核糖体中检测到突变的rRNA，表明突变的核糖体是在体内组装的。我们还利用天然凝胶电泳法研究了核糖体蛋白P0、P1和P2之间的相互作用。P_1和P_2混合形成P_1-P_2杂二聚体。该异二聚体与P0紧密结合，形成一个五聚体P0(P1-P2)_2。我们通过将这些蛋白质整合到大肠杆菌核糖体中，证实了核糖体蛋白的功能，即用家蚕P0(P1-P2)_2复合体和EL12取代了与GTP酶相关的RNA结构域上的大肠杆菌蛋白L10-L7/L12复合体和L11。蛋白质的替代导致了核糖体与家蚕翻译因子的活性。这种蛋白质依赖的核糖体活性的诱导在没有P1或P2的情况下都不会发生。这些结果表明，P1-P2异源二聚体的形成对于蛋白质在rRNA结构域上的组装和功能rRNA结构的稳定至关重要。较少

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