Draper D E, Xing Y, Laing L G
Department of Chemistry, Johns Hopkins University, Baltimore, MD 21218, USA.
J Mol Biol. 1995 Jun 2;249(2):231-8. doi: 10.1006/jmbi.1995.0291.
RNAs with interesting secondary and tertiary structures tend to melt in several broad and overlapping transitions over a wide temperature range, and it has been consequently difficult to resolve the thermodynamics of individual unfolding steps. In the case that a ligand selectively binds a single folded state of the RNA, it is possible to obtain reliable thermodynamic parameters for both RNA unfolding and RNA-ligand binding simply from the hyperchromicity of RNA denaturation. The analysis procedure involves fitting a three-dimensional surface to absorbance data collected as a function of both temperature and ligand concentration. Analysis of the unfolding of a fragment of the large subunit ribosomal RNA (Escherichia coli sequence 1051 to 1109) is presented; both an antibiotic (thiostrepton) and ammonium ion specifically stabilize a tertiary structure within this RNA. A consistent set of thermodynamic parameters (delta H and tm) for the first two sequentially linked unfolding transitions is obtained from the experiments, and the binding constants obtained for the two ligands are consistent with other independent measurements. The approach is applicable to a variety of RNAs that specifically bind proteins, antibiotics, ions or other ligands.
具有有趣二级和三级结构的RNA往往会在很宽的温度范围内经历几个宽泛且重叠的转变过程中发生解链，因此，很难解析各个解链步骤的热力学情况。如果一种配体选择性地结合RNA的单一折叠状态，那么仅通过RNA变性的增色效应，就有可能获得RNA解链和RNA-配体结合的可靠热力学参数。分析过程包括将一个三维曲面拟合到作为温度和配体浓度函数收集的吸光度数据上。本文展示了对大亚基核糖体RNA（大肠杆菌序列1051至1109）片段解链的分析；一种抗生素（硫链丝菌素）和铵离子都能特异性地稳定该RNA内的一种三级结构。从实验中获得了前两个连续相连的解链转变的一组一致的热力学参数（ΔH和Tm），并且为两种配体获得的结合常数与其他独立测量结果一致。该方法适用于各种能特异性结合蛋白质、抗生素、离子或其他配体的RNA。
