Batey R T, Williamson J R
Department of Chemistry, Massachusetts Institute of Technology, Cambridge 02139, USA.
RNA. 1998 Aug;4(8):984-97. doi: 10.1017/s1355838298980426.
The Bacillus stearothermophilus ribosomal protein S15 binds to a phylogenetically conserved three-way junction formed by the intersection of helices 20, 21, and 22 of eubacterial 16S ribosomal RNA, inducing a large conformational change in the RNA. Like many RNA structures, this three-way junction can also be folded by the addition of polyvalent cations such as magnesium, as demonstrated by comparing the mobilities of the wild-type and mutant junctions in the absence and presence of polyvalent cations in nondenaturing polyacrylamide gels. Using a modification interference assay, critical nucleotides for folding have been identified as the phylogenetically conserved nucleotides in the three-way junction. NMR spectroscopy of the junction reveals that the conformations induced by the addition of magnesium or S15 are extremely similar. Thus, the folding of the junction is determined entirely by RNA elements within the phylogenetically conserved junction core, and the role of Mg2+ and S15 is to stabilize this intrinsically unstable structure. The organization of the junction by Mg2+ significantly enhances the bimolecular association rate (k(on)) of S15 binding, suggesting that S15 binds specifically to the folded form of the three-way junction via a tertiary structure capture mechanism.
嗜热栖热放线菌核糖体蛋白S15与真细菌16S核糖体RNA的螺旋20、21和22相交形成的系统发育保守的三向接头结合，诱导RNA发生大的构象变化。与许多RNA结构一样，通过比较野生型和突变型接头在非变性聚丙烯酰胺凝胶中有无多价阳离子时的迁移率可以证明，这种三向接头也可以通过添加镁等多价阳离子进行折叠。使用修饰干扰试验，已确定折叠的关键核苷酸为三向接头中系统发育保守的核苷酸。接头的核磁共振光谱显示，添加镁或S15诱导的构象极其相似。因此，接头的折叠完全由系统发育保守接头核心内的RNA元件决定，Mg2+和S15的作用是稳定这种内在不稳定的结构。Mg2+对接头的组织显著提高了S15结合的双分子缔合速率（k(on)），表明S15通过三级结构捕获机制特异性结合三向接头的折叠形式。
