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I类内含子编码的RNA成熟酶/DNA归巢内切核酸酶的功能不同的核酸结合位点。

Functionally distinct nucleic acid binding sites for a group I intron encoded RNA maturase/DNA homing endonuclease.

作者信息

Chatterjee Piyali, Brady Kristina L, Solem Amanda, Ho Yugong, Caprara Mark G

机构信息

Center for RNA Molecular Biology, Case Western Reserve University School of Medicine, Cleveland, OH 44106-4960, USA.

出版信息

J Mol Biol. 2003 May 30;329(2):239-51. doi: 10.1016/s0022-2836(03)00426-1.

Abstract

A large number of group I introns encode a family of homologous proteins that either promote intron splicing (maturases) or are site-specific DNA endonucleases that function in intron mobility (a process called "homing"). Genetic studies have shown that some of these proteins have both activities, yet how a single protein carries out both functions remains obscure. The similarity between respective DNA-binding sites and the RNA structure near the 5' and 3' splice sites has fueled speculation that such proteins may use analogous interactions to perform both functions. The Aspergillus nidulans mitochondrial COB group I intron encodes a bi-functional protein, I-AniI, that has both RNA maturase and site-specific DNA endonuclease activities in vitro. Here, we show that I-AniI shows distinctive features of the endonuclease family to which it belongs, including highly specific, tight binding and sequential DNA strand cleavage. Competition experiments demonstrate that I-AniI binds the COB intron RNA even in saturating concentrations of its DNA target site substrate, suggesting that the protein has a separate binding site for RNA. In addition, we provide evidence that two different DNA-binding site mutants of I-AniI have little effect on the protein's RNA maturation activity. Since RNA splicing is likely a secondary adaptation of the protein, these observations support a model in which homing endonucleases may have developed maturase function by utilizing a hitherto "non-functional" protein surface.

摘要

大量的I类内含子编码一族同源蛋白,这些蛋白要么促进内含子剪接(成熟酶),要么是在内含子移动(一种称为“归巢”的过程)中起作用的位点特异性DNA内切酶。遗传学研究表明,其中一些蛋白具有这两种活性,然而单个蛋白如何执行这两种功能仍不清楚。5'和3'剪接位点附近各自的DNA结合位点与RNA结构之间的相似性引发了这样的推测:此类蛋白可能利用类似的相互作用来执行这两种功能。构巢曲霉线粒体COB I类内含子编码一种双功能蛋白I-AniI,该蛋白在体外具有RNA成熟酶和位点特异性DNA内切酶活性。在此,我们表明I-AniI表现出其所属内切酶家族的独特特征,包括高度特异性、紧密结合和连续的DNA链切割。竞争实验表明,即使在其DNA靶位点底物饱和浓度下,I-AniI仍能结合COB内含子RNA,这表明该蛋白对RNA有一个独立的结合位点。此外,我们提供证据表明,I-AniI的两种不同DNA结合位点突变体对该蛋白的RNA成熟活性影响很小。由于RNA剪接可能是该蛋白的一种次生适应性变化,这些观察结果支持了一种模型,即归巢内切酶可能通过利用迄今“无功能”的蛋白表面发展出了成熟酶功能。

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