O6-Methylguanosine leads to position-dependent effects on ribosome speed and fidelity.
O6-Methylguanosine leads to position-dependent effects on ribosome speed and fidelity.
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DOI：
10.1261/rna.052464.115
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发表时间：
2015-09
期刊：
影响因子：
--
通讯作者：
Zaher HS
中科院分区：
文献类型：
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作者：
Hudson BH;Zaher HS
核酸不断受到内源和环境因素的攻击，这些因素改变了它们的物理和化学性质。鸟苷(M6G)的O6甲基化特别值得注意的是，在DNA复制过程中，它与T配对，具有高度的突变性。然而，虽然m6G同时在DNA和RNA中积累，但人们对其对RNA的影响知之甚少。在这里，我们使用一个重组的细菌翻译系统来研究m6G对解码过程的影响。密码子第一位和第三位的m6G降低了tRNA选择的准确性。核糖体通过形成m6G-尿苷密码子-反密码子对，容易结合近源氨基酰-tRNAs(AA-tRNAs)。令人惊讶的是，在密码子的第二个位置引入m6G并没有促进错误编码，相反，在没有改变近源AA-tRNA的速率的情况下，将同源AA-tRNA的肽键形成速度减慢了1000倍。这些体外观察在真核细胞提取物和HEK293细胞中得到了概括。有趣的是，第二位的类似修饰N6-甲基腺苷(M6A)对tRNA选择的影响很小，这表明m6G对tRNA选择的影响是由于碱基对的几何结构改变所致。鉴于m6G：U碱基对被预测与Watson-Crick碱基对几乎无法区分，我们的数据表明核糖体的解码中心对第二位的变化极其敏感。我们的数据除了强调这些加合物对细胞健康的有害影响外，还对解码和核糖体识别密码子-反密码子对的过程提供了新的见解。
Nucleic acids are under constant assault from endogenous and environmental agents that alter their physical and chemical properties. O6-methylation of guanosine (m6G) is particularly notable for its high mutagenicity, pairing with T, during DNA replication. Yet, while m6G accumulates in both DNA and RNA, little is known about its effects on RNA. Here, we investigate the effects of m6G on the decoding process, using a reconstituted bacterial translation system. m6G at the first and third position of the codon decreases the accuracy of tRNA selection. The ribosome readily incorporates near-cognate aminoacyl-tRNAs (aa-tRNAs) by forming m6G-uridine codon–anticodon pairs. Surprisingly, the introduction of m6G to the second position of the codon does not promote miscoding, but instead slows the observed rates of peptide-bond formation by >1000-fold for cognate aa-tRNAs without altering the rates for near-cognate aa-tRNAs. These in vitro observations were recapitulated in eukaryotic extracts and HEK293 cells. Interestingly, the analogous modification N6-methyladenosine (m6A) at the second position has only a minimal effect on tRNA selection, suggesting that the effects on tRNA selection seen with m6G are due to altered geometry of the base pair. Given that the m6G:U base pair is predicted to be nearly indistinguishable from a Watson-Crick base pair, our data suggest that the decoding center of the ribosome is extremely sensitive to changes at the second position. Our data, apart from highlighting the deleterious effects that these adducts pose to cellular fitness, shed new insight into decoding and the process by which the ribosome recognizes codon–anticodon pairs.