利用合成 RNA 探测细菌 Pnkp/Hen1 RNA 修复系统的底物特异性。
Probing the substrate specificity of the bacterial Pnkp/Hen1 RNA repair system using synthetic RNAs.
机构信息
Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
出版信息
RNA. 2012 Feb;18(2):335-44. doi: 10.1261/rna.030502.111. Epub 2011 Dec 21.
Ribotoxins cleave essential RNAs involved in protein synthesis as a strategy for cell killing. RNA repair systems exist in nature to counteract the lethal actions of ribotoxins, as first demonstrated by the RNA repair system from bacteriophage T4 25 yr ago. Recently, we found that two bacterial proteins, named Pnkp and Hen1, form a stable complex and are able to repair ribotoxin-cleaved tRNAs in vitro. However, unlike the well-studied T4 RNA repair system, the natural RNA substrates of the bacterial Pnkp/Hen1 RNA repair system are unknown. Here we present comprehensive RNA repair assays with the recombinant Pnkp/Hen1 proteins from Anabaena variabilis using a total of 33 different RNAs as substrates that might mimic various damaged forms of RNAs present in living cells. We found that unlike the RNA repair system from bacteriophage T4, the bacterial Pnkp/Hen1 RNA repair system exhibits broad substrate specificity. Based on the experimental data presented here, a model of preferred RNA substrates of the Pnkp/Hen1 repair system is proposed.
核糖核酸酶通过切割参与蛋白质合成的关键 RNA 来实现细胞杀伤，这是一种常见的策略。自然界中存在 RNA 修复系统来对抗核糖核酸酶的致命作用，这一发现最早是在 25 年前由噬菌体 T4 的 RNA 修复系统证明的。最近，我们发现两种名为 Pnkp 和 Hen1 的细菌蛋白可以形成稳定的复合物，并能够在体外修复被核糖核酸酶切割的 tRNA。然而，与研究充分的 T4 RNA 修复系统不同，细菌 Pnkp/Hen1 RNA 修复系统的天然 RNA 底物尚不清楚。在这里，我们使用总共 33 种不同的 RNA 作为底物，对来自可变鱼腥藻的重组 Pnkp/Hen1 蛋白进行了全面的 RNA 修复实验，这些 RNA 可能模拟了存在于活细胞中的各种受损形式的 RNA。我们发现，与噬菌体 T4 的 RNA 修复系统不同，细菌 Pnkp/Hen1 RNA 修复系统具有广泛的底物特异性。基于这里呈现的实验数据，我们提出了 Pnkp/Hen1 修复系统偏好性 RNA 底物的模型。
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