Detection of a key tertiary interaction in the highly conserved GTPase center of large subunit ribosomal RNA.
Detection of a key tertiary interaction in the highly conserved GTPase center of large subunit ribosomal RNA.
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检测大亚基核糖体 RNA 高度保守的 GTP 酶中心的关键三级相互作用。
DOI：
10.1073/pnas.88.14.6308
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发表时间：
1991
影响因子：
11.1
通讯作者：
Draper,DE
中科院分区：
文献类型：
--
作者：
Ryan,PC;Draper,DE
核糖体RNA序列的补偿性碱基变化，保持沃森-克里克碱基配对的测序导致了这些RNA保守的二级结构的详细模型。原则上，三级相互作用也可以通过搜索遗传共变碱基来检测。在大亚基核糖体RNA的一个高度保守的区域内，称为“GTclock中心”，碱基G-1056-U-1082.A-1086在所有真细菌中发现（大肠杆菌编号），而A-1056.C-1082.G-1086在真核生物中的同源位置发现;古细菌属于这两类，但有一些例外。任何一个序列都可以潜在地形成连接3个碱基的一组相似的氢键。为了确定这3个碱基对RNA三级结构的贡献，在覆盖GTdR中心的RNA片段中制备序列变体。通过测量识别RNA三级结构的两种不同配体的结合亲和力来测定RNA片段的正确折叠：高度保守的核糖体蛋白L11，其通常与GTdR中心RNA相关，以及肽抗生素硫链丝菌素，其抑制真细菌和一些古细菌核糖体的GTdR活性。结果强烈支持在位置1082和1086之间存在碱基对：在任一位置的单突变使L11和硫链丝菌素结合减弱约10倍或更多，而补偿性双突变几乎与野生型E一样结合配体。coli序列。位置1056处的变体对L11或硫链丝菌素结合几乎没有影响;因此，这些实验不支持3碱基相互作用。位置1082和1086之间的碱基对强烈地限制了几何形状，三个螺旋片段在GTdR中心的中间连接。
Searches of ribosomal RNA sequences for compensatory base changes preserving Watson-Crick base pairing have led to detailed models of the conserved secondary structures of these RNAs. In principle, tertiary interactions can also be detected by searches for phylogenetically covariant bases. Within a highly conserved region of the large subunit ribosomal RNA termed the "GTPase center," the bases G-1056-U-1082.A-1086 are found in all eubacteria (Escherichia coli numbering), while A-1056.C-1082.G-1086 are found at the homologous positions in eukaryotes; archaebacteria fall into either category with some exceptions. Either sequence can potentially form a similar set of hydrogen bonds connecting the 3 bases. To determine the contribution of these 3 bases to RNA tertiary structure, sequence variants were made in RNA fragments covering the GTPase center. Correct folding of the RNA fragments was assayed by measuring the binding affinities of two different ligands that recognize the RNA tertiary structure: the highly conserved ribosomal protein L11, which is normally associated with the GTPase center RNA, and the peptide antibiotic thiostrepton, which inhibits the GTPase activity of eubacterial and some archaebacterial ribosomes. The results strongly support the existence of a base pair between positions 1082 and 1086: single mutations at either position weaken both L11 and thiostrepton binding by approximately 10-fold or more, while compensatory double mutations bind the ligands nearly as well as the wild-type E. coli sequence. Variants at position 1056 have little effect on either L11 or thiostrepton binding; a 3-base interaction is therefore not supported by these experiments. A base pair between positions 1082 and 1086 strongly constrains the geometry with which three helical segments join in the middle of the GTPase center.
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影响因子：
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