Doherty E A, Herschlag D, Doudna J A
Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University, New Haven, Connecticut 06520, USA.
Biochemistry. 1999 Mar 9;38(10):2982-90. doi: 10.1021/bi982113p.
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 (EDeltaP5abc). Equilibrium gel shift experiments show that the affinity of the P5abc and EDeltaP5abc RNAs is exceptionally strong, with a Kd of approximately 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.
I 组内含子RNA包含一个由外围结构域侧翼包围的高度保守螺旋核心，这些外围结构域可稳定核心结构。在嗜热四膜虫I组核酶中，核心的P4、P5和P6螺旋紧密堆积在一个称为P5abc的三螺旋亚结构域上。化学足迹法和嗜热四膜虫内含子P4 - P6结构域的晶体结构表明，该结构域这两部分之间的三级相互作用形成了一个广泛的溶剂不可及界面。我们通过将P5abc片段反式提供给一个缺乏P5abc的嗜热四膜虫核酶构建体（EDeltaP5abc），研究了这个三级界面的形成和稳定性。平衡凝胶迁移实验表明，P5abc和EDeltaP5abc RNA的亲和力异常强，在10 mM MgCl2（37摄氏度）下Kd约为100 pM。化学和酶足迹法表明，RNA在复合物组装之前基本已折叠。溶剂可及性图谱显示，在没有P5abc的情况下，内含子RNA保持类似天然的折叠，但其活性位点螺旋没有紧密堆积。在P5abc结合后，催化核心通过三级界面形成的间接作用变得更加紧密堆积。这个双组分系统有助于对稳定折叠内含子的各个三级接触进行定量研究。
