Thompson J F, Hearst J E
Cell. 1983 Apr;32(4):1355-65. doi: 10.1016/0092-8674(83)90316-1.
E. coli 16S RNA in solution was photoreacted with hydroxymethyltrimethylpsoralen and long wave ultraviolet light. Positions of crosslinks were determined to high resolution by partially digesting the RNA with T1 RNase, separating the crosslinked fragments by two-dimensional gel electrophoresis, reversing the crosslink, and sequencing the separated fragments. This method yielded the locations of crosslinks to +/- 15 nucleotides. Even finer placement has been made on the basis of our knowledge of psoralen reactivity. Thirteen unique crosslinks were mapped. Seven crosslinks confirmed regions of secondary structure which had been predicted in published phylogenetic models, three crosslinks discriminated between phylogenetic models, and three proved the existence of new structures. The new structures were all long range interactions which appear to be in dynamic equilibrium with local secondary structure. Because this technique yields direct information about the secondary structure of large RNAs, it should prove invaluable in studying the structure of other RNAs of all sizes.
溶液中的大肠杆菌16S RNA与羟甲基三甲基补骨脂素及长波紫外线发生光反应。通过用T1核糖核酸酶对RNA进行部分消化、二维凝胶电泳分离交联片段、逆转交联以及对分离片段进行测序，将交联位置确定到高分辨率。该方法可将交联位置定位到正负15个核苷酸。基于我们对补骨脂素反应性的了解，已实现了更精确的定位。绘制了13个独特的交联图谱。7个交联证实了已发表的系统发育模型中预测的二级结构区域，3个交联区分了不同的系统发育模型，3个交联证明了新结构的存在。这些新结构均为长程相互作用，似乎与局部二级结构处于动态平衡。由于该技术可直接提供有关大型RNA二级结构的信息，因此在研究各种大小的其他RNA结构方面应具有极高价值。
