Reconstitution of archaeal ribonuclease P from RNA and four protein components
Reconstitution of archaeal ribonuclease P from RNA and four protein components
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DOI：
10.1016/s0006-291x(03)01034-9
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发表时间：
2003-07-04
影响因子：
3.1
通讯作者：
Kimura, M
中科院分区：
文献类型：
--
作者：
Kouzuma, Y;Mizoguchi, M;Kimura, M
核糖核酸酶P(RNaseP)是一种负责产生成熟tRNA分子5‘端的内切酶。对嗜热考古菌Horikoshii OT3基因组数据库的同源性搜索表明，这4个基因PH1481、PH1601、PH1771和PH1877分别与编码人类RNaseP蛋白亚基hpop5、rpp21、rpp29和rpp30的基因具有显著的同源性。这些基因在大肠杆菌细胞中表达，所产生的蛋白Ph1481p、Ph1601p、Ph1771p和Ph1877p在一系列柱层析中得到明显的均一。这四种蛋白质的特征是它们与平菇的同源RNase P RNA结合的能力。这四种蛋白都具有与RNaseP RNA结合的活性。用体外转录的horikoshii RNase P RNA对4种可能的RNase P蛋白进行了体外重组，发现Ph1481p、Ph1601p和Ph1771p三种蛋白以及RNase P RNA是RNase P活性的最低组分。然而，第四个蛋白质Ph1877P的加入强烈地刺激了酶的活性，表明所有四种蛋白质和RNase P RNA都是RNase P最适活性所必需的。这些数据将为阐明古生物和真核生物RNaseP(C)2003 Elsevier Science(USA)的反应机制铺平道路。版权所有。
Ribonuclease P (RNase P) is an endonuclease responsible for generating the 5' end of matured tRNA molecules. A homology search of the hyperthermophilic archaeon Pyrococcus horikoshii OT3 genome database revealed that the four genes, PH1481, PH1601, PH1771, and PH1877, have a significant homology to those encoding RNase P protein subunits, hpop5, Rpp21, Rpp29, and Rpp30, of human, respectively. These genes were expressed in Escherichia coli cells, and the resulting proteins Ph1481p, Ph1601p, Ph1771p, and Ph1877p were purified to apparent homogeneity in a set of column chromatographies. The four proteins were characterized in terms of their capability to bind the cognate RNase P RNA from P. horikoshii. All four proteins exhibited the binding activity to the RNase P RNA. In vitro reconstitution of four putative RNase P proteins with the in vitro transcripted P. horikoshii RNase P RNA revealed that three proteins Ph1481p, Ph1601p, and Ph1771p, and RNase P RNA are minimal components for the RNase P activity. However, addition of the fourth protein Ph1877p strongly stimulated enzymatic activity, indicating that all four proteins and RNase P RNA are essential for optimal RNase P activity. The present data will pave the way for the elucidation of the reaction mechanism for archaeal as well as eukaryotic RNase P. (C) 2003 Elsevier Science (USA). All rights reserved.