EVIDENCE FOR 2 ACTIVE-SITES IN THE SPLICEOSOME PROVIDED BY STEREOCHEMISTRY OF PREMESSENGER RNA SPLICING
EVIDENCE FOR 2 ACTIVE-SITES IN THE SPLICEOSOME PROVIDED BY STEREOCHEMISTRY OF PREMESSENGER RNA SPLICING
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DOI：
10.1038/365364a0
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发表时间：
1993-09-23
期刊：
影响因子：
64.8
通讯作者：
SHARP, PA
中科院分区：
文献类型：
--
作者：
MOORE, MJ;SHARP, PA
剪接体从核前体到信使RNA(前mRNAs)的内含子的切除需要两个不同的磷酸二酯转移(酯交换)反应：第一步(套索形成)中3‘-5’交换为2‘-5’键，第二步(外显子连接)1-3交换一个3‘-5’磷酸二酯。我们在这里报告了使用含有手性硫代磷酸盐的剪接底物来确定每一步的立体化学过程。这提供了强有力的证据，证明这两个步骤都是以单一的“串联”SN2亲核置换反应发生的，类似于I族自剪接内含子4，5。此外，由于这两个步骤都被R(P)硫代非对映异构体强烈抑制，而不是被S(P)所抑制，剪接体可能在两个步骤的催化作用中在两个活性部位之间移动。化学和立体化学的相似性表明，剪接体加工的第二步的催化部位与I组自剪接内含子的催化部位有关。
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)1-3. 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 introns4,5. Additionally, because both steps are strongly inhibited by the R(P) phosphorothioate diastereomer, but not by S(P), 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.