Moore M J, Sharp P A
Center for Cancer Research, Massachusetts Institute of Technology, Cambridge 02139.
Nature. 1993 Sep 23;365(6444):364-8. doi: 10.1038/365364a0.
Excision of introns from nuclear precursors to messenger RNAs (pre-mRNAs) by the spliceosome requires two distinct phosphodiester transfer (transesterification) reactions: exchange of a 3'-5' for a 2'-5' bond in the first step (lariat formation) and exchange of one 3'-5' phosphodiester for another in the second step (exon ligation). We report here determination of the stereochemical course of each step using splicing substrates that contained a chiral phosphorothioate. This has provided strong evidence that both steps occur as single 'in-line' SN2 nucleophilic displacement reactions, analogous to the mechanism of group I self-splicing introns. Additionally, because both steps are strongly inhibited by the RP phosphorothioate diastereomer, but not by SP, the spliceosome probably shifts between two active sites in catalysis of the two steps. Chemical and stereochemical similarities suggest that the catalytic site for the second step of spliceosomal processing is related to that of group I self-splicing introns.
剪接体从核内信使核糖核酸前体（前体mRNA）中切除内含子需要两个不同的磷酸二酯转移（转酯反应）反应：第一步是3'-5'键被2'-5'键取代（套索状结构形成），第二步是一个3'-5'磷酸二酯被另一个取代（外显子连接）。我们在此报告使用含有手性硫代磷酸酯的剪接底物确定每个步骤的立体化学过程。这提供了有力证据，表明两个步骤均作为单一的“线性”SN2亲核取代反应发生，类似于I类自我剪接内含子的机制。此外，由于两个步骤均受到RP硫代磷酸酯非对映体的强烈抑制，但不受SP的抑制，因此剪接体在催化这两个步骤时可能在两个活性位点之间转换。化学和立体化学相似性表明，剪接体加工第二步的催化位点与I类自我剪接内含子的催化位点相关。
