Ajuh P M, Maden E B
Department of Biochemistry, University of Liverpool, UK.
Biochim Biophys Acta. 1994 Sep 13;1219(1):89-97. doi: 10.1016/0167-4781(94)90250-x.
The large ribosomal subunit (LSU) RNA or 28S rRNA of vertebrates is characterized by two highly conserved and methylated regions towards the 3' end of the molecule that extend from domains IV to V of the molecule. In this report we describe the probing of the secondary structure of these two highly methylated regions in Xenopus laevis LSU RNA by chemical modification using the single-strand nucleotide specific probes; dimethyl sulphate (DMS) and 1-cyclo-hexyl-3-(2-morpholinoethyl-carbodiimide metho-p-toluene sulphonate (CMCT) followed by primer extension. The same regions in in vitro synthesized unmethylated X. laevis 28S rRNA were also probed for comparison. Our results in general tend to support the theoretically determined secondary structure model for the probed domains. From the results obtained, methylated cellular LSU RNA appears to be relatively more reactive than the in vitro transcript to the chemical probes. Accessibility to the probes was found to be similar at most sites for cellular and in vitro transcript LSU RNAs. This implies that structural destabilization due to 2'-O-methylations in cellular LSU RNA is not significant.
脊椎动物的大核糖体亚基（LSU）RNA或28S rRNA的特征在于，在分子的3'端有两个高度保守且甲基化的区域，这些区域从分子的结构域IV延伸到V。在本报告中，我们描述了通过使用单链核苷酸特异性探针进行化学修饰，对非洲爪蟾LSU RNA中这两个高度甲基化区域的二级结构进行探测；硫酸二甲酯（DMS）和1-环己基-3-（2-吗啉代乙基）碳二亚胺对甲苯磺酸盐（CMCT），然后进行引物延伸。还对体外合成的未甲基化的非洲爪蟾28S rRNA中的相同区域进行了探测以作比较。我们的结果总体上倾向于支持所探测结构域的理论确定的二级结构模型。从获得的结果来看，甲基化的细胞LSU RNA对化学探针的反应似乎比体外转录本相对更活跃。发现细胞和体外转录本LSU RNA在大多数位点对探针的可及性相似。这意味着细胞LSU RNA中由于2'-O-甲基化导致的结构不稳定并不显著。
