A C-terminal fragment of an intron-encoded maturase is sufficient for promoting group I intron splicing
A C-terminal fragment of an intron-encoded maturase is sufficient for promoting group I intron splicing
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DOI：
10.1261/rna.7225205
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发表时间：
2005-04-01
期刊：
影响因子：
4.5
通讯作者：
Caprara, MG
中科院分区：
文献类型：
--
作者：
Downing, ME;Brady, KL;Caprara, MG
I 组内含子通常编码催化位点特异性 DNA 水解的蛋白质。其中一些蛋白质已经获得了促进其同源内含子剪接的能力，但这两种活性是否存在于蛋白质的不同区域仍然不清楚。 I-AniI（一种双功能内含子编码蛋白）的晶体结构表明，该蛋白具有两个大小相等的伪对称结构域。每个结构域在两个切割位点的两侧接触其 DNA 底物。作为鉴定 RNA 结合表面的第一步，分别表达 I-AniI 的 N 端和 C 端结构域并测试其促进线粒体 (mt) COB 前 RNA 剪接的情况。 N 端蛋白没有表现出剪接激活或 RNA 结合，表明该结构域在活性中发挥的作用很小或者折叠不正确。值得注意的是，16-kDa C 末端的一半促进内含子剪接，其速度与全长蛋白质相似。 C 端片段和全长蛋白均与 COB 内含子紧密结合。 RNase 足迹显示 C 端和全长蛋白结合相同区域并在 COB 内含子中诱导相同的构象变化。总之，这些结果表明 I-AniI 的 C 端片段对于成熟酶活性是必要且充分的，并表明 I-AniI 通过利用可能代表新型 RNA 结合基序的相对较小的蛋白质表面获得剪接功能。 I-AniI 的这个片段代表了迄今为止所描述的最小的 I 组内含子剪接辅因子。
Group I introns often encode proteins that catalyze site-specific DNA hydrolysis. Some of these proteins have acquired the ability to promote splicing of their cognate intron, but whether these two activities reside in different regions of the protein remains obscure. A crystal structure of I-AniI, a dual function intron-encoded protein, has shown that the protein has two pseudo-symmetric domains of equal size. Each domain contacts its DNA substrate on either side of two cleavage sites. As a first step to identify the RNA binding surface, the N- and C-terminal domains of I-AniI were separately expressed and tested for promoting the splicing of the mitochondrial (mt) COB pre-RNA. The N-terminal protein showed no splicing activation or RNA binding, suggesting that this domain plays a minimal role in activity or is improperly folded. Remarkably, the 16-kDa C-terminal half facilitates intron splicing with a rate similar to that of the full-length protein. Both the C-terminal fragment and full-length proteins bind tightly to the COB intron. RNase footprinting shows that the C-terminal and full-length proteins bind to the same regions and induce the same conformational changes in the COB intron. Together, these results show that the C-terminal fragment of I-AniI is necessary and sufficient for maturase activity and suggests that I-AniI acquired splicing function by utilizing a relatively small protein surface that likely represents a novel RNA binding motif. This fragment of I-AniI represents the smallest group I intron splicing cofactor described to date.