在Tetrahymena ribozyme的非保存序列中的突变增加了活性和特异性
B Young1, D Herschlag, T R Cech
1Department of Molecular, Cellular and Developmental Biology, Howard Hughes Medical Institute, University of Colorado, Boulder 80309-0215.
Cell
|November 29, 1991
概括
在Tetrahymena ribozyme的J1/2区域的突变提高了RNA分裂的效率和特异性. 这些改进源于减少RNA结合亲和力,优化基质定位以实现高效的催化.
科学领域:
- 分子生物学分子生物学
- 生物化学 生物化学
- 在RNA催化过程中.
背景情况:
- 一个I组内的Tetrahymena ribozyme利用一个J1/2区域将RNA基质结合部位与催化核连接起来.
- 虽然J1/2序列在I组内基中没有保持,但修改显著影响了 ribozyme 的活性.
研究的目的:
- 研究J1/2区域中的小插入或删除如何影响 ribozyme-catalyzed RNA 分裂的周转数和序列特异性.
- 阐明由J1/2序列改变引起的增强的 ribozyme 活性的机械基础.
主要方法:
- 单个反应步骤的动态分析,以测量速率常数.
- 野生类型和突变的Tetrahymena ribozymes 的比较.
主要成果:
- 在J1/2区域的突变大大提高了营业额数和序列特异性.
- 矛盾的是,这些改善与对RNA基质的 afinity 降低有关.
- 发现J1/2序列将RNA基质定位为最佳的三级相互作用和裂解部位选择.
结论:
- 非保存的J1/2序列通过影响基质定位而不是直接催化增强,在优化 ribozyme 功能方面发挥着至关重要的作用.
- 虽然野生类型的核糖酶是生物适应的,但J1/2中的突变可以在多个周转场景中克服限制.
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