Romaniuk P J, de Stevenson I L, Ehresmann C, Romby P, Ehresmann B
Department of Biochemistry and Microbiology, University of Victoria, British Columbia, Canada.
Nucleic Acids Res. 1988 Mar 25;16(5):2295-312. doi: 10.1093/nar/16.5.2295.
The secondary and tertiary structures of Xenopus oocyte and somatic 5S rRNAs were investigated using chemical and enzymatic probes. The accessibility of both RNAs towards single-strand specific nucleases (T1, T2, A and S1) and a helix-specific ribonuclease from cobra venom (RNase V1) was determined. The reactivity of nucleobase N7, N3 and N1 positions towards chemical probes was investigated under native (5 mM MgCl2, 100 mM KCl, 20 degrees C) and semi-denaturing (1 mM EDTA, 20 degrees C) conditions. Ethylnitrosourea was used to identify phosphates not reactive towards alkylation under native conditions. The results obtained confirm the presence of the five helical stems predicted by the consensus secondary structure model of 5S rRNA. The chemical reactivity data indicate that loops C and D are involved in a number of tertiary interactions, and loop E folds into an unusual secondary structure. A comparison of the data obtained for the two types of Xenopus 5S rRNA indicates that the conformations of the oocyte and somatic 5S rRNAs are very similar. However, the data obtained with nucleases under native conditions, and chemical probes under semi-denaturing conditions, reveal that helices III and IV in the somatic 5S rRNA are less stable than the same structures in oocyte 5S rRNA. Using chimeric 5S rRNAs, it was possible to demonstrate that the relative resistance of oocyte 5S rRNA to partial denaturation in 4 M urea is conferred by the five oocyte-specific nucleotide substitutions in loop B/helix III. In contrast, the superior stability of oocyte 5S rRNA in the presence of EDTA is related to a single C substitution at position 79.
利用化学和酶促探针研究了非洲爪蟾卵母细胞和体细胞5S rRNA的二级和三级结构。测定了这两种RNA对单链特异性核酸酶（T1、T2、A和S1）以及眼镜蛇毒中的螺旋特异性核糖核酸酶（RNase V1）的可及性。研究了在天然条件（5 mM MgCl2、100 mM KCl、20℃）和半变性条件（1 mM EDTA、20℃）下，核碱基N7、N3和N1位置对化学探针的反应性。使用乙基亚硝基脲来鉴定在天然条件下对烷基化无反应的磷酸酯。所得结果证实了5S rRNA共有二级结构模型所预测的五个螺旋茎的存在。化学反应性数据表明，环C和环D参与了许多三级相互作用，环E折叠成一种不寻常的二级结构。对两种非洲爪蟾5S rRNA所得数据的比较表明，卵母细胞和体细胞5S rRNA的构象非常相似。然而，在天然条件下用核酸酶以及在半变性条件下用化学探针获得的数据显示，体细胞5S rRNA中的螺旋III和螺旋IV比卵母细胞5S rRNA中的相同结构更不稳定。利用嵌合5S rRNA，有可能证明卵母细胞5S rRNA对4 M尿素中部分变性的相对抗性是由环B/螺旋III中的五个卵母细胞特异性核苷酸取代赋予的。相反，卵母细胞5S rRNA在EDTA存在下的更高稳定性与79位的单个C取代有关。
