Paul R, Lazarev D, Altman S
Department of Molecular, Cellular and Developmental Biology, Yale University, PO Box 208103, New Haven, CT 06520-8103, USA.
Nucleic Acids Res. 2001 Feb 15;29(4):880-5. doi: 10.1093/nar/29.4.880.
The protein subunit of RNase P from a thermophilic bacterium, Thermotoga maritima, was overexpressed in and purified from Escherichia coli. The cloned protein was reconstituted with the RNA subunit transcribed in vitro. The temperature optimum of the holoenzyme is near 50 degrees C, with no enzymatic activity at 65 degrees C or above. This finding is in sharp contrast to the optimal growth temperature of T.maritima, which is near 80 degrees C. However, in heterologous reconstitution experiments in vitro with RNase P subunits from other species, we found that the protein subunit from T.maritima was responsible for the comparative thermal stability of such complexes.
来自嗜热细菌海栖热袍菌(Thermotoga maritima)的核糖核酸酶P(RNase P)的蛋白质亚基在大肠杆菌中实现了过表达并从中纯化出来。克隆得到的蛋白质与体外转录的RNA亚基进行了重组。全酶的最适温度接近50摄氏度,在65摄氏度及以上没有酶活性。这一发现与海栖热袍菌接近80摄氏度的最适生长温度形成了鲜明对比。然而,在体外使用来自其他物种的RNase P亚基进行的异源重组实验中,我们发现来自海栖热袍菌的蛋白质亚基赋予了此类复合物相对较高的热稳定性。