Specific binding of 8-oxoguanine-containing RNA to polynucleotide phosphorylase protein
Specific binding of 8-oxoguanine-containing RNA to polynucleotide phosphorylase protein
复制标题
DOI：
10.1021/bi010595q
复制
发表时间：
2001-08-21
期刊：
影响因子：
2.9
通讯作者：
Sekiguchi, M
中科院分区：
文献类型：
--
作者：
Hayakawa, H;Kuwano, M;Sekiguchi, M
8-氧代鸟嘌呤是鸟嘌呤的一种氧化形式，有可能与胞嘧啶和腺嘌呤配对，因此，该碱基在信使 RNA 中的持续存在会导致翻译错误。为了防止这种结果，生物体可能有一种机制来识别携带 8-氧代鸟嘌呤的 RNA 分子，并阻止它们进入细胞翻译机制。我们现在报告说，大肠杆菌细胞拥有与携带 8-氧代鸟嘌呤的 RNA 特异性结合的蛋白质。与无细胞提取物一起孵育后，含 8-氧代鸟嘌呤的 RNA 是稳定的，而正常 RNA 会被细胞核酸酶降解。 RNase 保护测定和凝胶位移测定表明，一些蛋白质与含有 8-氧代鸟嘌呤的 RNA 特异性结合，从而防止核酸酶攻击。在检测到的复合物中，一种具有 77 kDa 蛋白质的复合物表现出 RNA 和蛋白质成分之间的紧密结合。该蛋白质被鉴定为多核苷酸磷酸化酶，由 pnp 基因编码。 pnp(-) 突变体对百草枯高度耐药，百草枯是一种诱导细胞氧化应激的药物。 Pnp 蛋白与含有 8-氧代鸟嘌呤的 RNA 结合会抑制细胞生长，可能是由于此类 RNA 从翻译机器中撤出。因此，Pnp 蛋白可以区分氧化的 RNA 分子和正常的 RNA 分子，从而实现高保真度的翻译。
8-Oxoguanine, an oxidized form of guanine, has the potential to pair with both cytosine and adenine, and thus, the persistence of this base in messenger RNA would cause translational errors. To prevent such an outcome, organisms probably have a mechanism for recognizing RNA molecules carrying 8-oxoguanine and prevent them from entering into the cellular translational machinery. We now report that the Escherichia coli cell possesses proteins that bind specifically to RNA carrying 8-oxoguanine. On incubation with a cell-free extract, 8-oxoguanine-containing RNA is stable while normal RNA is degraded by cellular nucleases. The RNase protection assay and gel shift assay revealed that some proteins bind specifically to 8-oxoguanine-containing RNA, hence preventing nuclease attacks. Among the complexes that were detected, one with a 77 kDa protein exhibits tight binding between RNA and protein components. This protein was identified as polynucleotide phosphorylase, encoded by the pnp gene. pnp(-) mutants are hyperresistant to paraquat, a drug that induces oxidative stress in the cell. Binding of Pnp protein to 8-oxoguanine-containing RNA would inhibit cell growth, probably due to withdrawal of such RNA from the translational machinery. The Pnp protein may, therefore, discriminate between an oxidized RNA molecule and a normal one, thus contributing a high fidelity of translation.