Characterization of I-Ppo, an intron-encoded endonuclease that mediates homing of a group I intron in the ribosomal DNA of Physarum polycephalum.
Characterization of I-Ppo, an intron-encoded endonuclease that mediates homing of a group I intron in the ribosomal DNA of Physarum polycephalum.
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I-Ppo 的表征，I-Ppo 是一种内含子编码的核酸内切酶，可介导 I 组内含子在多头绒泡菌核糖体 DNA 中的归巢。
DOI：
10.1128/mcb.10.7.3386-3396.1990
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发表时间：
1990
影响因子：
5.3
通讯作者：
Vogt,VM
中科院分区：
文献类型：
--
作者：
Muscarella,DE;Ellison,EL;Ruoff,BM;Vogt,VM
一类新的且最近才被认识的酶由一些 I 族硝基编码的位点特异性核酸内切酶组成。我们已经表征了 I-Ppo 的几个方面，I-Ppo 是介导多头绒泡菌核糖体 DNA 中内含子 3 移动性的核酸内切酶。该内含子在移动组 I 内含子中是独特的，因为它位于核 DNA 中。我们发现 I-Ppois 由内含子 3 的 5' 半部分中的开放阅读框编码，位于 I 组内含子自剪接所需序列的上游。两个 AUG 起始密码子中的任何一个都可以启动该阅读框，一个位于内含子的开头，另一个位于上游外显子，从而产生 138 和 160 个氨基酸残基的预测多肽。较长的多肽是网织红细胞提取物中体外翻译的主要形式。通过对部分缺失 DNA 体外合成的蛋白质进行核酸酶测定，我们得出结论，这两种多肽都具有核酸内切酶活性。我们还使用噬菌体 T7 RNA 聚合酶表达系统表达了 I-Ppoin 大肠杆菌。较长的多肽也是该系统中产生的主要形式。它在细菌体内显示出酶活性，如携带靶位点的质粒的切割所证明的那样。与其他几种内含子编码的核酸内切酶一样，I-Ppo 在其核糖体 DNA 靶序列中进行四碱基交错切割，非常靠近内含子 3 整合到包含内含子 3 和缺乏内含子 3 的绒泡菌菌株杂交中的位点。
A novel and only recently recognized class of enzymes is composed of the site-specific endonucleases encoded by some group I nitrons. We have characterized several aspects of I-Ppo, the endonuclease that mediates the mobility of intron 3 in the ribosomal DNA ofPhysarum polycephalum.This intron is unique among mobile group I introns in that it is located in nuclear DNA. We found that I-Ppois encoded by an open reading frame in the 5' half of intron 3, upstream of the sequences required for self-splicing of group I introns. Either of two AUG initiation codons could start this reading frame, one near the beginning of the intron and the other in the upstream exon, leading to predicted polypeptides of 138 and 160 amino acid residues. The longer polypeptide was the major form translated in vitro in a reticulocyte extract. From nuclease assays of proteins synthesized in vitro with partially deleted DNAs, we conclude that both polypeptides possess endonuclease activity. We also have expressed I-PpoinEscherichia coli, using a bacteriophage T7 RNA polymerase expression system. The longer polypeptide also was the predominant form made in this system. It showed enzymatic activity in bacteria in vivo, as demonstrated by the cleavage of a plasmid carrying the target site. Like several other intron-encoded endonucleases, I-Ppomakes a four-base staggered cut in its ribosomal DNA target sequence, very near the site where intron 3 becomes integrated in crosses of intron 3-containing and intron 3-lackingPhysarumstrains.