Ohki Y, Ikawa Y, Shiraishi H, Inoue T
Graduate School of Biostudies, Kyoto University, 606-8502, Kyoto, Japan.
FEBS Lett. 2001 Mar 30;493(2-3):95-100. doi: 10.1016/s0014-5793(01)02279-7.
The Tetrahymena group I ribozyme requires a hierarchical folding process to form its correct three-dimensional structure. Ribozyme activity depends on the catalytic core consisting of two domains, P4-P6 and P3-P7, connected by a triple-helical scaffold. The folding proceeds in the following order: (i) fast folding of the P4-P6 domain, (ii) slow folding of the P3-P7 domain, and (iii) structure rearrangement to form the active ribozyme structure. The third step is believed to directly determine the conformation of the active catalytic domain, but as yet the precise mechanisms remain to be elucidated. To investigate the folding kinetics of this step, we analyzed mutant ribozymes having base substitution(s) in the triple-helical scaffold and found that disruption of the scaffold at A105G results in modest slowing of the P3-P7 folding (1.9-fold) and acceleration of step (iii) by 5.9-fold. These results suggest that disruption or destabilization of the scaffold is a normal component in the formation process of the active structure of the wild type ribozyme.
嗜热四膜虫I组核酶需要一个分级折叠过程来形成其正确的三维结构。核酶活性取决于由两个结构域P4 - P6和P3 - P7组成的催化核心，这两个结构域由一个三螺旋支架连接。折叠按以下顺序进行：（i）P4 - P6结构域的快速折叠，（ii）P3 - P7结构域的缓慢折叠，以及（iii）结构重排以形成活性核酶结构。第三步被认为直接决定了活性催化结构域的构象，但到目前为止，确切机制仍有待阐明。为了研究这一步骤的折叠动力学，我们分析了在三螺旋支架中具有碱基替换的突变核酶，发现A105G处支架的破坏导致P3 - P7折叠适度减慢（1.9倍），并使步骤（iii）加速5.9倍。这些结果表明，支架的破坏或不稳定是野生型核酶活性结构形成过程中的正常组成部分。
