Zarrinkar P P, Sullenger B A
Center for Genetic and Cellular Therapies, Department of Surgery, Duke University Medical Center, Durham, North Carolina 27710, USA.
Biochemistry. 1998 Dec 22;37(51):18056-63. doi: 10.1021/bi982193x.
One largely unexplored question about group I intron splicing is how the cleavage and ligation steps of the reaction are coordinated. We describe a simple in vitro trans-splicing model system in which both steps take place, including the exchange of ligands in the guanosine-binding site that must occur between the two steps. Using this model system, we show that the switch is accomplished by modulating the relative affinity of the binding site for the two ligands. While the terminal guanosine of the intron (omegaG) and exogenous guanosine compete for binding during the first step of splicing, no competition is apparent during the second step, when omegaG is bound tightly. These results help explain how the ribozyme orchestrates progression through the splicing reaction. In addition to providing a new tool to ask basic questions about RNA catalysis, the trans-splicing model system will also facilitate the development of therapeutically useful group I ribozymes that can repair mutant mRNAs.
关于I组内含子剪接，一个很大程度上未被探索的问题是该反应的切割和连接步骤是如何协调的。我们描述了一个简单的体外反式剪接模型系统，其中两个步骤都能发生，包括在两步之间鸟苷结合位点中配体的交换。利用这个模型系统，我们表明这种转换是通过调节结合位点对两种配体的相对亲和力来实现的。在内含子的末端鸟苷（ωG）和外源鸟苷在剪接的第一步中竞争结合时，而在第二步中，当ωG紧密结合时，没有明显的竞争。这些结果有助于解释核酶如何在剪接反应中协调进程。除了提供一个询问有关RNA催化基本问题的新工具外，反式剪接模型系统还将促进开发可修复突变mRNA的具有治疗用途的I组核酶。
