Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599-3290, USA.
Biochemistry. 2010 Jul 6;49(26):5418-25. doi: 10.1021/bi100267g.
Proteins play diverse and critical roles in cellular ribonucleoproteins (RNPs) including promoting formation of and stabilizing active RNA conformations. Yet, the conformational changes required to convert large RNAs into active RNPs have proven difficult to characterize fully. Here we use high-resolution approaches to monitor both local nucleotide flexibility and solvent accessibility for nearly all nucleotides in the bI3 group I intron RNP in four assembly states: the free RNA, maturase-bound RNA, Mrs1-bound RNA, and the complete six-component holocomplex. The free RNA is misfolded relative to the secondary structure required for splicing. The maturase and Mrs1 proteins each stabilized long-range tertiary interactions, but neither protein alone induced folding into the functional secondary structure. In contrast, simultaneous binding by both proteins results in large secondary structure rearrangements in the RNA and yielded the catalytically active group I intron structure. Secondary and tertiary folding of the RNA component of the bI3 RNP are thus not independent: RNA folding is strongly nonhierarchical. These results emphasize that protein-mediated stabilization of RNA tertiary interactions functions to pull the secondary structure into an energetically disfavored, but functional, conformation and emphasize a new role for facilitator proteins in RNP assembly.
蛋白质在细胞核糖核蛋白（RNP）中发挥着多样化和关键的作用，包括促进 RNA 构象的形成和稳定。然而，将大型 RNA 转化为活性 RNP 所需的构象变化很难被完全描述。在这里，我们使用高分辨率的方法来监测四个组装状态下 bI3 组 I 内含子 RNP 中几乎所有核苷酸的局部核苷酸灵活性和溶剂可及性：游离 RNA、成熟酶结合的 RNA、Mrs1 结合的 RNA 和完整的六组分全复合物。游离 RNA 相对于剪接所需的二级结构是错误折叠的。成熟酶和 Mrs1 蛋白各自稳定长程三级相互作用，但单独的任何一种蛋白都不能诱导 RNA 折叠成功能性二级结构。相比之下，两种蛋白的同时结合导致 RNA 中发生大的二级结构重排，并产生具有催化活性的组 I 内含子结构。因此，bI3 RNP 的 RNA 部分的二级和三级折叠不是独立的：RNA 折叠强烈的是非层次化的。这些结果强调了蛋白质介导的 RNA 三级相互作用的稳定作用，将二级结构拉入能量不利但功能上有利的构象，并强调了促进蛋白在 RNP 组装中的新作用。
