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首页> 外文期刊>Biochemistry >Trans insertion-splicing: Ribozyme-catalyzed insertion of targeted sequences into RNAs
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Trans insertion-splicing: Ribozyme-catalyzed insertion of targeted sequences into RNAs

机译：反式插入剪接：核酶催化的目标序列插入RNA

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摘要

A group I intron-derived ribozyme from Pneumocystis carinii has been previously shown to bind an exogenous RNA substrate, splice out an internal segment, and then ligate the two ends back together (the trans excision-splicing reaction). We demonstrate that this same ribozyme can perform a trans insertion-splicing (TIS) reaction, where the ribozyme binds two exogenous RNA substrates and inserts one directly into the other. Reactions were optimized for both yield and rate, with optimum reactions carried out in 10 mM MgCl2 for 2 h. Reaction products are stable, with no visible loss at extended times. The ribozyme recognizes the two substrates primarily through base pairing and requires an omega G on the ribozyme and an omega G on the sequence being inserted. We give evidence that the reaction mechanism is not the reverse of the trans excision-splicing reaction, but is composed of three steps, with intermediates attached to the ribozyme. Surprisingly, the internal guide sequence of the ribozyme is utilized to sequentially bind both substrates, forming independent P1 helices. This is an indication that ribozymes with essentially the native intron sequence can catalyze reactions significantly more dynamic and complex than self-splicing. The implications of group I intron-derived ribozymes being able to catalyze this unique reaction, and via this mechanism, are discussed.
机译：先前已显示卡氏肺孢子虫的I组内含子来源的核酶与外源RNA底物结合，剪接一个内部片段，然后将两端连接在一起（反式切除剪接反应）。我们证明了相同的核酶可以进行反式插入-剪接（TIS）反应，其中核酶结合两个外源RNA底物并将一个直接插入另一个。优化了反应的产率和速率，并在10 mM MgCl2中进行了2h的最佳反应。反应产物稳定，长时间无可见损失。核酶主要通过碱基配对识别两种底物，并需要核酶上的ωG和插入序列上的ωG。我们提供的证据表明，该反应机理并非与反式切除剪接反应相反，而是由三个步骤组成，中间体与核酶相连。出人意料的是，核酶的内部引导序列被用来顺序结合两个底物，形成独立的P1螺旋。这表明具有天然内含子序列的核酶可以比自剪接更显着地动态和复杂地催化反应。讨论了能够催化这种独特反应的I类内含子衍生核酶的意义，并通过这种机制。

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