Capturing splicing complexes to study structure and mechanism
Capturing splicing complexes to study structure and mechanism
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DOI：
10.1016/s1046-2023(02)00240-2
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发表时间：
2002-11-01
期刊：
影响因子：
4.8
通讯作者：
Moore, MJ
中科院分区：
文献类型：
--
作者：
Jurica, MS;Moore, MJ
在最基本的水平上，前 mRNA 剪接可以描述为两个协调的核酸酶反应，在两端切割内含子并导致侧翼外显子的连接。事实上，这些反应是由类似于 3-MDa 的蛋白质和 RNA 庞然大物（剪接体）催化的，这一事实对目前用于表征较小尺寸酶的大多数生化和结构方法提出了挑战。除了这种分子复杂性之外，剪接复合物的高度动态性质为机械研究或三维结构测定提供了额外的障碍。因此，用于研究剪接体的方法经常探测机器的个体特性，但尚未出现完整的、高分辨率的剪接催化图片。为了促进天然剪接复合物的生化和结构研究，我们最近描述了剪接体催化形式（称为 C 复合物）的纯化。这种天然复合物适用于通过单粒子方法测定电子显微结构。在本文中，我们详细描述了纯化过程，并讨论了捕获和分析其他剪接复合物的其他方法。 (C) 2002 年爱思唯尔科学（美国）。版权所有。
At its most basic level, pre-mRNA splicing can be described as two coordinated nuclease reactions that cleave an intron at either end and result in ligation of the flanking exons. The fact that these reactions are catalyzed by a similar to3-MDa behemoth of protein and RNA (the spliceosome) challenges most biochemical and structural approaches currently used to characterize lesser-sized enzymes. In addition to this molecular complexity, the highly dynamic nature of splicing complexes provides additional hurdles for mechanistic studies or three-dimensional structure determination. Thus, the methods used to study the spliceosome often probe individual properties of the machine, but no complete, high-resolution picture of splicing catalysis has yet emerged. To facilitate biochemical and structural studies of native splicing complexes, we recently described purification of the catalytic form of the spliceosome (known as C complex). This native complex is suitable for electron microscopic structure determination by single-particle methods. In this paper, we describe the purification in detail and discuss additional methods for trapping and analyzing other splicing complexes. (C) 2002 Elsevier Science (USA). All rights reserved.