Lai Lien B, Tanimoto Akiko, Lai Stella M, Chen Wen-Yi, Marathe Ila A, Westhof Eric, Wysocki Vicki H, Gopalan Venkat
Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA.
Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA.
Nucleic Acids Res. 2017 Jul 7;45(12):7432-7440. doi: 10.1093/nar/gkx388.
RNase P is primarily responsible for the 5΄ maturation of transfer RNAs (tRNAs) in all domains of life. Archaeal RNase P is a ribonucleoprotein made up of one catalytic RNA and five protein cofactors including L7Ae, which is known to bind the kink-turn (K-turn), an RNA structural element that causes axial bending. However, the number and location of K-turns in archaeal RNase P RNAs (RPRs) are unclear. As part of an integrated approach, we used native mass spectrometry to assess the number of L7Ae copies that bound the RPR and site-specific hydroxyl radical-mediated footprinting to localize the K-turns. Mutagenesis of each of the putative K-turns singly or in combination decreased the number of bound L7Ae copies, and either eliminated or changed the L7Ae footprint on the mutant RPRs. In addition, our results support an unprecedented 'double K-turn' module in type A and type M archaeal RPR variants.
核糖核酸酶P主要负责生命所有领域中转运RNA（tRNA）的5΄成熟过程。古细菌核糖核酸酶P是一种核糖核蛋白，由一个催化RNA和五个蛋白质辅因子组成，其中包括L7Ae，已知它能结合扭结环（K-turn），这是一种导致轴向弯曲的RNA结构元件。然而，古细菌核糖核酸酶P RNA（RPR）中K-turn的数量和位置尚不清楚。作为综合方法的一部分，我们使用了天然质谱法来评估与RPR结合的L7Ae拷贝数，并使用位点特异性羟基自由基介导的足迹法来定位K-turn。对每个假定的K-turn进行单独或组合诱变，会减少结合的L7Ae拷贝数，并消除或改变突变RPR上的L7Ae足迹。此外，我们的结果支持了A型和M型古细菌RPR变体中前所未有的“双K-turn”模块。
