Mooers Blaine H M
Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, 975 NE 10th St., Stanton L. Young Biomedical Research Center Rm. 466, Oklahoma City, OK, 73104-5419, USA,
Methods Mol Biol. 2015;1240:191-216. doi: 10.1007/978-1-4939-1896-6_14.
Head-to-head fusions of two identical double-stranded fragments of RNA can be designed to self-assemble from a single RNA species and form a double-stranded helix with a twofold rotation axis relating the two strands. These symmetrical RNA molecules are more likely to crystallize without end-on-end statistical packing disorder because the two halves of the molecule are identical. This approach can be used to study many fragments of double-stranded RNA or many isolated helical domains from large single-stranded RNAs that may not yet be amenable to high-resolution studies by crystallography or NMR. We used fusion RNAs to study one (the U-helix) of three functional domains formed when guide RNA binds to its cognate pre-edited mRNA from the trypanosome RNA editing system. The U-helix forms when the 3' oligo(U) tail of the guide RNA (gRNA) binds to the purine-rich, pre-edited mRNA upstream from the current RNA editing site. Fusion RNAs 16-and 32-base pairs in length formed crystals that gave diffraction to 1.37 and 1.05 Å respectively. We provide the composition of a fusion RNA crystallization screen and describe the X-ray data collection, structure determination, and refinement of the crystal structures of fusion RNAs.
可以设计两个相同的双链RNA片段进行头对头融合，使其从单一RNA物种自组装，并形成具有双旋转轴的双链螺旋结构，该双旋转轴将两条链联系起来。这些对称的RNA分子更有可能结晶，且不会出现端对端的统计堆积紊乱，因为分子的两半是相同的。这种方法可用于研究许多双链RNA片段或来自大型单链RNA的许多孤立螺旋结构域，这些结构域可能尚未适合通过晶体学或核磁共振进行高分辨率研究。我们使用融合RNA来研究当引导RNA与其来自锥虫RNA编辑系统的同源预编辑mRNA结合时形成的三个功能结构域之一（U螺旋）。当引导RNA（gRNA）的3'寡聚（U）尾与当前RNA编辑位点上游富含嘌呤的预编辑mRNA结合时，U螺旋形成。长度为16和32个碱基对的融合RNA形成了晶体，其衍射分别达到1.37 Å和1.05 Å。我们提供了融合RNA结晶筛选的组成，并描述了融合RNA晶体结构的X射线数据收集、结构测定和精修。
