Gössringer Markus, Kretschmer-Kazemi Far Rosel, Hartmann Roland K
Institut für Pharmazeutische Chemie, Philipps-Universität Marburg, Marbacher Weg 6, D-35037 Marburg, Germany.
J Bacteriol. 2006 Oct;188(19):6816-23. doi: 10.1128/JB.00756-06.
Bacterial RNase P is composed of an RNA subunit and a single protein subunit (encoded by the rnpB and rnpA genes, respectively). We constructed Bacillus subtilis mutant strains that conditionally express the RNase P protein under control of the xylose promoter (P(xyl)). In one strain (d7), rnpA expression was efficiently repressed in the absence of the inducer xylose, leading to cell growth arrest. Growth could be restored by a second functional rnpA allele. This is the first RNase P protein knockdown strain, providing the first direct proof that the rnpA gene is essential in B. subtilis and, by inference, in other bacteria. We further show (i) that, in the wild-type context, rnpA expression is attenuated by transcriptional polarity and (ii) that translation of rnpA mRNA in B. subtilis can be initiated at two alternative start codons. His-tagged RNase P protein variants are functional in vivo and permit purification of in vivo-assembled holoenzymes by affinity chromatography. Simultaneous expression of plasmid-encoded RNase P RNA and His-tagged protein increased RNase P holoenzyme yields. Massive overproduction of RNase P protein in strain d7 is compatible with cell viability.
细菌核糖核酸酶P由一个RNA亚基和一个单一蛋白质亚基组成（分别由rnpB和rnpA基因编码）。我们构建了枯草芽孢杆菌突变菌株，其在木糖启动子（P(xyl)）的控制下条件性表达核糖核酸酶P蛋白。在一个菌株（d7）中，在没有诱导剂木糖的情况下，rnpA表达被有效抑制，导致细胞生长停滞。通过第二个功能性rnpA等位基因可以恢复生长。这是第一个核糖核酸酶P蛋白敲低菌株，首次直接证明rnpA基因在枯草芽孢杆菌中是必需的，由此推断在其他细菌中也是必需的。我们进一步表明：（i）在野生型背景下，rnpA表达因转录极性而减弱；（ii）枯草芽孢杆菌中rnpA mRNA的翻译可以在两个不同的起始密码子处起始。带有组氨酸标签的核糖核酸酶P蛋白变体在体内具有功能，并允许通过亲和色谱法纯化体内组装的全酶。质粒编码的核糖核酸酶P RNA和带有组氨酸标签的蛋白的同时表达提高了核糖核酸酶P全酶的产量。菌株d7中核糖核酸酶P蛋白的大量过量生产与细胞活力是相容的。
