Wegscheid Barbara, Hartmann Roland K
Institut für Pharmazeutische Chemie, Philipps-Universität Marburg, Marbacher Weg 6, D-35037 Marburg, Germany.
Nucleic Acids Res. 2007;35(6):2060-73. doi: 10.1093/nar/gkm005. Epub 2007 Mar 13.
For catalysis by bacterial type B RNase P, the importance of a specific interaction with p(recursor)tRNA 3'-CCA termini is yet unclear. We show that mutation of one of the two G residues assumed to interact with 3'-CCA in type B RNase P RNAs inhibits cell growth, but cell viability is at least partially restored at increased RNase P levels due to RNase P protein overexpression. The in vivo defects of the mutant enzymes correlated with an enzyme defect at low Mg(2+) in vitro. For Bacillus subtilis RNase P, an isosteric C259-G(74) bp fully and a C258-G(75) bp slightly rescued catalytic proficiency, demonstrating Watson-Crick base pairing to tRNA 3'-CCA but also emphasizing the importance of the base identity of the 5'-proximal G residue (G258). We infer the defect of the mutant enzymes to primarily lie in the recruitment of catalytically relevant Mg(2+), with a possible contribution from altered RNA folding. Although with reduced efficiency, B. subtilis RNase P is able to cleave CCA-less ptRNAs in vitro and in vivo. We conclude that the observed in vivo defects upon disruption of the CCA interaction are either due to a global deceleration in ptRNA maturation or severe inhibition of 5'-maturation for a ptRNA subset.
对于细菌B型核糖核酸酶P的催化作用而言，与前体tRNA 3'-CCA末端发生特异性相互作用的重要性尚不清楚。我们发现，在B型核糖核酸酶P RNA中，假定与3'-CCA相互作用的两个G残基之一发生突变会抑制细胞生长，但由于核糖核酸酶P蛋白的过表达，在核糖核酸酶P水平升高时细胞活力至少会部分恢复。突变酶在体内的缺陷与体外低镁离子浓度下的酶缺陷相关。对于枯草芽孢杆菌核糖核酸酶P，一个等排的C259-G(74)碱基对完全恢复了催化活性，一个C258-G(75)碱基对则略微恢复了催化活性，这表明与tRNA 3'-CCA形成了沃森-克里克碱基对，但同时也强调了5'-近端G残基（G258）碱基同一性的重要性。我们推断突变酶的缺陷主要在于催化相关镁离子的募集，RNA折叠改变可能也有一定作用。尽管效率有所降低，但枯草芽孢杆菌核糖核酸酶P在体外和体内均能够切割不含CCA的前体tRNA。我们得出结论，破坏CCA相互作用后在体内观察到的缺陷，要么是由于前体tRNA成熟的整体减速，要么是由于前体tRNA子集的5'-成熟受到严重抑制。
