Assembly of an exceptionally stable RNA tertiary interface in a group I ribozyme
Assembly of an exceptionally stable RNA tertiary interface in a group I ribozyme
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DOI：
10.1021/bi982113p
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发表时间：
1999-03-09
期刊：
影响因子：
2.9
通讯作者：
Doudna, JA
中科院分区：
文献类型：
--
作者：
Doherty, EA;Herschlag, D;Doudna, JA
I组内含子rna包含一个高度保守的螺旋核心，两侧是稳定核心结构的外周结构域。在四膜虫I群核酶中，核心的P4、P5和P6螺旋紧密排列在一个称为P5abc的三螺旋亚结构域上。四膜虫内含子P4-P6结构域的化学足迹和晶体结构表明，这两个结构域之间的tertiary相互作用产生了一个广泛的溶剂不可接近的界面。我们通过将P5abc片段转化为缺乏P5abc的四膜核酶结构体（E-Delta P5abc），研究了该三级界面的形成和稳定性。平衡凝胶转移实验表明，P5abc和E-Delta P5abc rna的亲和力非常强，在10 mM MgCl2（37℃）下的K-d近似于100 PM。化学和酶印迹表明，rna在复合物组装之前基本上折叠。溶剂可及性图谱显示，在缺乏P5abc的情况下，内含子RNA保持原生折叠，但其活性位点螺旋不紧密排列。结合P5abc后，催化核通过三级界面形成的间接作用变得更加紧密。这种双组分系统有助于定量检查稳定折叠内含子的单个三级接触。
Group I intron RNAs contain a core of highly conserved helices flanked by peripheral domains that stabilize the core structure. In the Tetrahymena group I ribozyme, the P4, P5, and P6 helices of the core pack tightly against a three-helix subdomain called P5abc. Chemical footprinting and the crystal structure of the Tetrahymena intron P4-P6 domain revealed that tertiary interactions between these two parts of the domain create an extensive solvent-inaccessible interface. We have examined the formation and stability of this tertiary interface by providing the P5abc segment in trans to a Tetrahymena ribozyme construct that lacks P5abc (E-Delta P5abc). Equilibrium gel shift experiments show that the affinity of the P5abc and E-Delta P5abc RNAs is exceptionally strong, with a K-d of similar to 100 PM at 10 mM MgCl2 (at 37 degrees C). Chemical and enzymatic footprinting shows that the RNAs are substantially folded prior to assembly of the complex. Solvent accessibility mapping reveals that, in the absence of P5abc, the intron RNA maintains a nativelike fold but its active-site helices are not tightly packed. Upon binding of P5abc, the catalytic core becomes more tightly packed through indirect effects of the tertiary interface formation. This two-component system facilitates quantitative examination of individual tertiary contacts that stabilize the folded intron.