A DExH/D-box protein coordinates the two steps of splicing in a group I intron.
A DExH/D-box protein coordinates the two steps of splicing in a group I intron.
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DOI：
10.1016/j.jmb.2008.08.070
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发表时间：
2008-11-14
影响因子：
5.6
通讯作者：
Caprara, Mark G.
中科院分区：
文献类型：
--
作者：
Bifano, Abby L.;Caprara, Mark G.
DExH/D家族的蛋白是在体外可以解开双链RNA的ATPase。这个家族的个体成员在体内协调核糖核蛋白酶组装和催化的许多步骤，但这些辅助因子的作用是如何指定和精确计时的在很大程度上尚不清楚。作为生物化学解决这一问题的第一步，我们描述了一种新的蛋白质依赖的I族内含子剪接系统的开发，该系统需要这样的ATPase来协调剪接中的连续步骤。虽然在酵母中的遗传分析表明，线粒体AI5β组I内含子的剪接至少需要五个核编码蛋白，但我们证明了在体外有效的AI5β剪接只需其中两个辅助因子，而且它们在体外实现了不同的功能。Mrs1p蛋白稳定RNA结构，促进剪接的第一步。相反，DExH/D蛋白Mss 116p在第一步后起作用，并利用ATP水解，特别提高外显子连接的效率。对Mss 116p突变的分析表明，ATP水解的效率是促进外显子连接的主要决定因素，突变会削弱其RNA刺激的ATP水解活性或降低其解开双链的能力。这些观察表明，Mss 116p通过催化β/蛋白质剪接中间体结构的变化来促进第二步，从而在AI5RNA剪接中发挥作用。更广泛地说，这些观察结果与一个模型是一致的，在该模型中，DExH/D-box蛋白的作用可以通过其底物的特定构象来指定，这是由于其他辅助因子的“上游”活性。
Proteins of the DExH/D family are ATPases that can unwind duplex RNA in vitro. Individual members of this family coordinate many steps in ribonucleoprotein enzyme assembly and catalysis in vivo, but it is largely unknown how the action of these co-factors is specified and precisely timed. As a first step to address this question biochemically, we describe the development of a new protein-dependent group I intron splicing system that requires such an ATPase for coordinating successive steps in splicing. While genetic analysis in yeast have shown that at least five nuclear-encoded proteins are required for splicing of the mitochondrial aI5β group I intron, we show that efficient in vitro splicing of aI5β occurs with only two of these co-factors, and furthermore, they fulfill distinct functions in vitro. The Mrs1p protein stabilizes RNA structure and promotes the first step in splicing. In contrast, a DExH/D protein, Mss116p, acts after the first step and, utilizing ATP hydrolysis, specifically enhances the efficiency of exon ligation. An analysis of Mss116p variants with mutations that impair its RNA-stimulated ATP hydrolysis activity or reduce its ability to unwind duplexes show that the efficiency of ATP hydrolysis is a major determinant in promoting exon ligation. These observations suggest that Mss116p acts in aI5β splicing by catalyzing changes in the structure of the RNA/protein splicing intermediate that promote the second step. More broadly, these observations are consistent with a model in which the “functional-timing” of DExH/D-box protein action can be specified by a specific conformation of its substrate due to the “upstream” activity of other co-factors.
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影响因子：
14.9
作者：
Kaspar BJ;Bifano AL;Caprara MG
通讯作者：
Caprara MG
影响因子：
16
作者：
Konarska, MM;Vilardell, J;Query, CC
通讯作者：
Query, CC
影响因子：
4.5
作者：
Downing, ME;Brady, KL;Caprara, MG
通讯作者：
Caprara, MG
影响因子：
2.9
作者：
Emerick, VL;Pan, J;Woodson, SA
通讯作者：
Woodson, SA
DOI：
10.1073/pnas.0407896101
发表时间：
2005-01-04
影响因子：
11.1
作者：
Huang, HR;Rowe, CE;Perlman, PS
通讯作者：
Perlman, PS