Sun Lei, Harris Michael E
Center for RNA Molecular Biology, Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA.
RNA. 2007 Sep;13(9):1505-15. doi: 10.1261/rna.571007. Epub 2007 Jul 25.
The RNA subunit (P RNA) of the bacterial RNase P ribonucleoprotein is a ribozyme that catalyzes the Mg-dependent hydrolysis of pre-tRNA, but it requires an essential protein cofactor (P protein) in vivo that enhances substrate binding affinities and catalytic rates in a substrate dependent manner. Previous studies of Bacillus subtilis RNase P, containing a Type B RNA subunit, showed that its cognate protein subunit increases the affinity of metal ions important for catalysis, but the functional role of these ions is unknown. Here, we demonstrate that the Mg2+ dependence of the catalytic step for Escherichia coli RNase P, which contains a more common Type A RNA subunit, is also modulated by its cognate protein subunit (C5), indicating that this property is fundamental to P protein. To monitor specifically the binding of active site metal ions, we analyzed quantitatively the rescue by Cd2+ of an inhibitory Rp phosphorothioate modification at the pre-tRNA cleavage site. The results show that binding of C5 protein increases the apparent affinity of the rescuing Cd2+, providing evidence that C5 protein enhances metal ion affinity in the active site, and thus is likely to contribute significantly to rate enhancement at physiological metal ion concentrations.
细菌核糖核酸酶P核糖核蛋白的RNA亚基（P RNA）是一种核酶，可催化前体tRNA的镁依赖性水解，但在体内它需要一种必需的蛋白质辅因子（P蛋白），该辅因子以底物依赖性方式增强底物结合亲和力和催化速率。先前对含有B型RNA亚基的枯草芽孢杆菌核糖核酸酶P的研究表明，其同源蛋白质亚基增加了对催化重要的金属离子的亲和力，但这些离子的功能作用尚不清楚。在这里，我们证明了含有更常见的A型RNA亚基的大肠杆菌核糖核酸酶P催化步骤对Mg2+的依赖性也受到其同源蛋白质亚基（C5）的调节，这表明该特性是P蛋白的基本特性。为了特异性监测活性位点金属离子的结合，我们定量分析了Cd2+对前体tRNA切割位点抑制性Rp硫代磷酸酯修饰的挽救作用。结果表明，C5蛋白的结合增加了挽救性Cd2+的表观亲和力，这证明C5蛋白增强了活性位点中的金属离子亲和力，因此可能在生理金属离子浓度下对速率提高有显著贡献。
