SHAPE analysis of long-range interactions reveals extensive and thermodynamically preferred misfolding in a fragile group I intron RNA.
SHAPE analysis of long-range interactions reveals extensive and thermodynamically preferred misfolding in a fragile group I intron RNA.
复制标题
DOI：
10.1021/bi800207b
复制
发表时间：
2008-08-19
期刊：
影响因子：
2.9
通讯作者：
Weeks KM
中科院分区：
文献类型：
--
作者：
Duncan CD;Weeks KM
大多数功能性 RNA 需要蛋白质来促进其活性结构的形成。就酵母 bI3 I 组内含子而言，剪接需要两种蛋白质的结合，即内含子编码的 bI3 成熟酶和核编码的 Mrs1。在这里，我们使用引物延伸 (SHAPE) 化学分析结合点突变体分析来分析选择性 2'-羟基酰化，以绘制该 RNA 中的长程相互作用图谱。该分析揭示了游离 RNA 状态的两个关键特征。首先，催化内含子通过稳定的锚定螺旋与侧翼外显子分离。这种锚定螺旋为内含子创建了一个自主结构域，并起到防止侧翼外显子错误折叠的作用。其次，游离RNA的热力学最稳定的结构与催化活性构象不一致，因为系统发育上保守的元件形成稳定的非天然结构。这些结果强调了脆弱的 bI3 RNA，其与蛋白质辅因子的结合可促进广泛的二级结构重排，这是形成催化活性三级结构的必要先决条件。
Most functional RNAs require proteins to facilitate formation of their active structures. In the case of the yeast bI3 group I intron, splicing requires binding by two proteins, the intron-encoded bI3 maturase and the nuclear encoded Mrs1. Here, we use selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry coupled with analysis of point mutants to map long-range interactions in this RNA. This analysis reveals two critical features of the free RNA state. First, the catalytic intron is separated from the flanking exons via a stable anchoring helix. This anchoring helix creates an autonomous structural domain for the intron and functions to prevent misfolding with the flanking exons. Second, the thermodynamically most stable structure for the free RNA is not consistent with the catalytically active conformation as phylogenetically conserved elements form stable, non-native structures. These results highlight a fragile bI3 RNA for which binding by protein cofactors functions to promote extensive secondary structure rearrangements that are an obligatory prerequisite for forming the catalytically active tertiary structure.