Department of Biochemistry and Molecular Genetics, Center for RNA Biology, Ohio State University, Columbus, OH 43210, USA.
Proc Natl Acad Sci U S A. 2010 Aug 17;107(33):14573-8. doi: 10.1073/pnas.1005556107. Epub 2010 Jul 30.
To the mounting evidence of nonribosomal functions for ribosomal proteins, we now add L7Ae as a subunit of archaeal RNase P, a ribonucleoprotein (RNP) that catalyzes 5'-maturation of precursor tRNAs (pre-tRNAs). We first demonstrate that L7Ae coelutes with partially purified Methanococcus maripaludis (Mma) RNase P activity. After establishing in vitro reconstitution of the single RNA with four previously known protein subunits (POP5, RPP21, RPP29, and RPP30), we show that addition of L7Ae to this RNase P complex increases the optimal reaction temperature and k(cat)/K(m) (by approximately 360-fold) for pre-tRNA cleavage to those observed with partially purified native Mma RNase P. We identify in the Mma RNase P RNA a putative kink-turn (K-turn), the structural motif recognized by L7Ae. The large stimulatory effect of Mma L7Ae on RNase P activity decreases to <or= 4% of wild type upon mutating either the conserved nucleotides in this K-turn or amino acids in L7Ae shown to be essential for K-turn binding. The critical, multifunctional role of archaeal L7Ae in RNPs acting in tRNA processing (RNase P), RNA modification (H/ACA, C/D snoRNPs), and translation (ribosomes), especially by employing the same RNA-recognition surface, suggests coevolution of various translation-related functions, presumably to facilitate their coordinate regulation.
我们现在将核糖体蛋白的非核糖体功能的证据增加到越来越多，将 L7Ae 添加为古菌 RNase P 的亚基，RNase P 是一种催化前体 tRNA（pre-tRNA）5'成熟的核糖核蛋白（RNP）。我们首先证明 L7Ae 与部分纯化的 Methanococcus maripaludis（Mma）RNase P 活性共洗脱。在用先前已知的四个蛋白亚基（POP5、RPP21、RPP29 和 RPP30）体外重建单个 RNA 后，我们表明将 L7Ae 添加到该 RNase P 复合物中会提高最佳反应温度和 k(cat)/K(m)（约 360 倍），用于 pre-tRNA 切割与部分纯化的天然 Mma RNase P 观察到的一致。我们在 Mma RNase P RNA 中鉴定出一个假定的拐点（K 环），这是 L7Ae 识别的结构基序。Mma L7Ae 对 RNase P 活性的巨大刺激作用在突变该 K 环中的保守核苷酸或对 K 环结合至关重要的 L7Ae 氨基酸时降低至野生型的<或=4%。古菌 L7Ae 在参与 tRNA 加工（RNase P）、RNA 修饰（H/ACA、C/D snoRNPs）和翻译（核糖体）的 RNP 中的关键、多功能作用，特别是通过利用相同的 RNA 识别表面，表明各种翻译相关功能的共同进化，可能是为了促进它们的协调调节。
