Kouzuma Yoshiaki, Mizoguchi Masashi, Takagi Hisanori, Fukuhara Hideo, Tsukamoto Masayo, Numata Tomoyuki, Kimura Makoto
Laboratory of Biochemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, Hakozaki 6-10-1, Higashi-ku, 812-8581, Fukuoka, Japan.
Biochem Biophys Res Commun. 2003 Jul 4;306(3):666-73. doi: 10.1016/s0006-291x(03)01034-9.
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.
核糖核酸酶P（RNase P）是一种内切核酸酶，负责生成成熟tRNA分子的5′端。对嗜热古菌堀越火球菌OT3基因组数据库进行同源性搜索发现，PH1481、PH1601、PH1771和PH1877这四个基因分别与人源核糖核酸酶P蛋白亚基hpop5、Rpp21、Rpp29和Rpp30的编码基因具有显著同源性。这些基因在大肠杆菌细胞中表达，通过一系列柱层析将产生的蛋白Ph1481p、Ph1601p、Ph1771p和Ph1877p纯化至表观均一。对这四种蛋白结合来自堀越火球菌的同源核糖核酸酶P RNA的能力进行了表征。所有四种蛋白均表现出与核糖核酸酶P RNA的结合活性。用体外转录的堀越火球菌核糖核酸酶P RNA对四种假定的核糖核酸酶P蛋白进行体外重组，结果表明，三种蛋白Ph1481p、Ph1601p和Ph1771p以及核糖核酸酶P RNA是核糖核酸酶P活性的最小组成成分。然而，添加第四种蛋白Ph1877p可强烈刺激酶活性，表明所有四种蛋白和核糖核酸酶P RNA对最佳核糖核酸酶P活性都是必需的。目前的数据将为阐明古菌以及真核生物核糖核酸酶P的反应机制铺平道路。
