Cuzic Simona, Hartmann Roland K
Institut für Pharmazeutische Chemie, Philipps-Universität Marburg, Marburg, Germany.
Biol Chem. 2007 Jul;388(7):717-26. doi: 10.1515/BC.2007.095.
We analyzed processing of precursor tRNAs carrying a single 2'-deoxy, 2'-OCH(3), or locked nucleic acid (LNA) modification at G+1 by Escherichia coli RNase P RNA in the absence and presence of its protein cofactor. The extra methyl or methylene group caused a substrate binding defect, which was rescued at higher divalent metal ion (M(2+)) concentrations (more efficiently with Mn(2+) than Mg(2+)), and had a minor effect on cleavage chemistry at saturating M(2+) concentrations. The 2'-OCH(3) and LNA modification at G+1 resulted in higher metal ion cooperativity for substrate binding to RNase P RNA without affecting cleavage site selection. This indicates disruption of an M(2+) binding site in enzyme-substrate complexes, which is compensated for by occupation of alternative M(2+) binding sites of lower affinity. The 2'-deoxy modification at G+1 caused at most a two-fold decrease in the cleavage rate; this mild defect relative to 2'-OCH(3) and LNA at G+1 indicates that the defect caused by the latter two is steric in nature. We propose that the 2'-hydroxyl at G+1 in the substrate is in the immediate vicinity of the M(2+) cluster at the phosphates of A67 to U69 in helix P4 of E. coli RNase P RNA.
我们分析了在有无蛋白质辅因子的情况下，大肠杆菌核糖核酸酶P RNA对在G +1位点携带单个2'-脱氧、2'-OCH(3)或锁核酸（LNA）修饰的前体tRNA的加工过程。额外的甲基或亚甲基导致底物结合缺陷，在较高的二价金属离子（M(2+)）浓度下该缺陷得以挽救（Mn(2+)比Mg(2+)更有效），并且在饱和M(2+)浓度下对切割化学有较小影响。G +1位点的2'-OCH(3)和LNA修饰导致底物与核糖核酸酶P RNA结合时具有更高的金属离子协同性，而不影响切割位点的选择。这表明酶-底物复合物中一个M(2+)结合位点被破坏，这可通过占据较低亲和力的替代M(2+)结合位点来补偿。G +1位点的2'-脱氧修饰最多使切割速率降低两倍；相对于G +1位点的2'-OCH(3)和LNA，这种轻微缺陷表明后两者造成的缺陷本质上是空间位阻性的。我们推测底物中G +1位点的2'-羟基紧邻大肠杆菌核糖核酸酶P RNA螺旋P4中A67至U69磷酸处的M(2+)簇。
