Insertion of a homing endonuclease creates a genes-in-pieces ribonudeotide reductase that retains function
Insertion of a homing endonuclease creates a genes-in-pieces ribonudeotide reductase that retains function
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DOI：
10.1073/pnas.0609915104
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发表时间：
2007-04-10
影响因子：
11.1
通讯作者：
Edgell, David R.
中科院分区：
文献类型：
--
作者：
Friedrich, Nancy C.;Torrents, Eduard;Edgell, David R.
在细菌和噬菌体基因组中，编码区有时被自我剪接内含子或内含肽中断，其可以编码促进迁移的归巢核酸内切酶。归巢核酸内切酶基因也被发现在噬菌体基因组中是独立的（不是内含子或内含肽编码的），其中它们被插入基因间区域。一个例子是HNH家族内切核酸酶，mobE，插入在T-偶联T4、11132、111133、RB 15和LZ 7的需氧核糖核苷酸还原酶（RNR）的大亚基（nrdA）和小亚基（nrdB）基因之间。在这里，我们描述了一个插入到nrdA基因的气单胞菌噬菌体Aeh 1的mobE。插入创建了一个独特的基因片段排列，其中nrdA被分成两个独立的基因，nrdA-a和nrdA-b，每个基因编码对应于不间断NrdA蛋白的活性位点残基的半胱氨酸残基。值得注意的是，mobE插入不破坏NrdA功能，尽管插入不是自剪接内含子或内含肽。我们从Aeh 1感染的细胞中共纯化了NrdA-a、NrdA-b和NrdB蛋白作为复合物，并且还表明重构的复合物具有RNR活性。因此，噬菌体Aeh 1中的I类RNR活性是由相互作用形成复合活性位点的单独蛋白质组装而成的，这表明mobE插入在表型上是中性的，因为其作为间插序列的存在不会破坏周围基因的功能。
In bacterial and phage genomes, coding regions are sometimes interrupted by self-splicing introns or inteins, which can encode mobility-promoting homing endonucleases. Homing endonuclease genes are also found free-standing (not intron- or intein-encoded) in phage genomes where they are inserted in intergenic regions. One example is the HNH family enclonuclease, mobE, inserted between the large (nrdA) and small (nrdB) subunit genes of aerobic ribonucleotide reductase (RNR) of T-even phages T4, 11132, 111133, RB15, and LZ7. Here, we describe an insertion of mobE into the nrdA gene of Aeromonas hydrophila phage Aeh1. The insertion creates a unique genes-in-pieces arrangement, where nrdA is split into two independent genes, nrdA-a and nrdA-b, each encoding cysteine residues that correspond to the active-site residues of uninterrupted NrdA proteins. Remarkably, the mobE insertion does not inactivate NrdA function, although the insertion is not a self-splicing intron or intein. We copurified the NrdA-a, NrdA-b, and NrdB proteins as complex from Aeh1-infected cells and also showed that a reconstituted complex has RNR activity. Class I RNR activity in phage Aeh1 is thus assembled from separate proteins that interact to form a composite active site, demonstrating that the mobE insertion is phenotypically neutral in that its presence as an intervening sequence does not disrupt the function of the surrounding gene.