Doherty E A, Doudna J A
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520, USA.
Biochemistry. 1997 Mar 18;36(11):3159-69. doi: 10.1021/bi962428+.
The active site of group I self-splicing introns occurs at the interface of two proposed structural domains. In the Tetrahymena intron, half of the catalytic core resides within the independently-folding P4-P6 domain while the other half belongs to a putative domain that includes helices P3, P7, P8, and P9 (P3-P9). To determine whether the P3-P9 region of the intron can also fold independently, we used Fe(II)-EDTA and dimethyl sulfate to probe the solvent accessibility of separate fragments of the Tetrahymena intron. These RNAs self-assemble into an active complex in trans, enabling analysis of their structural features both alone and within the complex. Our results show that while the P3-P9 region of the intron retains its secondary structure, most of the tertiary interactions within this region do not form stably in the absence of the P4-P6 domain. This indicates that the P4-P6 domain induces folding in the P3-P9 region, organizing the catalytic cleft between them. Thus the P4-P6 domain provides a scaffold for the folding of the Tetrahymena intron core.
I 类自剪接内含子的活性位点位于两个假定结构域的界面处。在嗜热四膜虫内含子中，催化核心的一半位于独立折叠的P4 - P6结构域内，而另一半属于一个假定结构域，该结构域包括螺旋P3、P7、P8和P9（P3 - P9）。为了确定内含子的P3 - P9区域是否也能独立折叠，我们使用Fe（II）-EDTA和硫酸二甲酯来探测嗜热四膜虫内含子不同片段的溶剂可及性。这些RNA在反式中自组装成活性复合物，从而能够单独以及在复合物中分析它们的结构特征。我们的结果表明，虽然内含子的P3 - P9区域保留了其二级结构，但在没有P4 - P6结构域的情况下，该区域内的大多数三级相互作用不能稳定形成。这表明P4 - P6结构域诱导P3 - P9区域折叠，在它们之间组织催化裂隙。因此，P4 - P6结构域为嗜热四膜虫内含子核心的折叠提供了一个支架。
