Analysis of rate-determining conformational changes during self-splicing of the Tetrahymena intron
Analysis of rate-determining conformational changes during self-splicing of the Tetrahymena intron
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DOI：
10.1021/bi960865i
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发表时间：
1996-10-15
期刊：
影响因子：
2.9
通讯作者：
Woodson, SA
中科院分区：
文献类型：
--
作者：
Emerick, VL;Pan, J;Woodson, SA
RNA 催化反应在体外通常受到 RNA 结构重排速率的限制。对四膜虫前核糖体 RNA 的分子内和分子间剪接的分析揭示了两个很好解析的动力学阶段，在 30 摄氏度下速率常数约为 2.5 和 0.02 min(-1)。数据与第二阶段由前 rRNA 缓慢重折叠产生的模型一致。点突变导致 RNA 在不同构象之间重新分布，这可以通过天然凝胶电泳检测到。在 GTP 存在的情况下，活性前 rRNA 迅速发展为产物复合物。连接的外显子的释放比 30°C 下的剪接稍慢（0.3 -0.5 分钟(-1)）。相比之下，第一步剪接后的中间复合物解离速度要慢得多（5 x 10(-3) min(-1)），这说明了野生型前 rRNA 剪接过程中通常看到的内含子 3' 外显子中间体含量较低的原因。这些结果为研究四膜虫内含子从核糖体 RNA 中切除所伴随的构象变化提供了一个初步框架。
RNA catalyzed reactions are often limited in vitro by the rate of structural rearrangements in the RNA. Analysis of intra- and intermolecular splicing of the Tetrahymena preribosomal RNA revealed two well resolved kinetic phases with rate constants of approximately 2.5 and 0.02 min(-1) at 30 degrees C. The data are consistent with a model in which the second phase results from slow refolding of the pre-rRNA. Point mutations result in redistribution of the RNA among different conformations that can be detected by native gel electrophoresis. The active pre-rRNA rapidly progresses to a product complex in the presence of GTP. Release of the ligated exons is slightly slower than splicing at 30 degrees C (0.3 -0.5 min(-1)). In contrast, the intermediate complex after the first step of splicing dissociates much more slowly (5 x 10(-3) min(-1)), accounting for the low amount of intron-3' exon intermediate typically seen during splicing of wild type pre-rRNA. These results provide an initial framework for studying conformational changes that accompany excision of the Tetrahymena intron from ribosomal RNA.