Mistranslation, incorporating an amino acid not specified by the "standard" genetic code, has applications in research and synthetic biology. Since mistranslation is toxic, its level must be modulated. Using a serine tRNA with a proline anticodon, we identify... Transfer RNAs (tRNAs) read the genetic code, translating nucleic acid sequence into protein. For tRNASer the anticodon does not specify its aminoacylation. For this reason, mutations in the tRNASer anticodon can result in amino acid substitutions, a process called mistranslation. Previously, we found that tRNASer with a proline anticodon was lethal to cells. However, by incorporating secondary mutations into the tRNA, mistranslation was dampened to a nonlethal level. The goal of this work was to identify second-site substitutions in tRNASer that modulate mistranslation to different levels. Targeted changes to putative identity elements led to total loss of tRNA function or significantly impaired cell growth. However, through genetic selection, we identified 22 substitutions that allow nontoxic mistranslation. These secondary mutations are primarily in single-stranded regions or substitute G:U base pairs for Watson-Crick pairs. Many of the variants are more toxic at low temperature and upon impairing the rapid tRNA decay pathway. We suggest that the majority of the secondary mutations affect the stability of the tRNA in cells. The temperature sensitivity of the tRNAs allows conditional mistranslation. Proteomic analysis demonstrated that tRNASer variants mistranslate to different extents with diminished growth correlating with increased mistranslation. When combined with a secondary mutation, other anticodon substitutions allow serine mistranslation at additional nonserine codons. These mistranslating tRNAs have applications in synthetic biology, by creating "statistical proteins," which may display a wider range of activities or substrate specificities than the homogenous form.

中文翻译：

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调节酿酒酵母中 tRNA 的误译潜力。


错误翻译，即掺入“标准”遗传密码未指定的氨基酸，在研究和合成生物学中具有应用。由于误译是有毒的，因此必须调节其水平。使用丝氨酸 tRNA 和脯氨酸反密码子，我们识别出……转移 RNA (tRNA) 读取遗传密码，将核酸序列翻译成蛋白质。对于 tRNASer，反密码子不指定其氨酰化。因此，tRNASer 反密码子的突变可能导致氨基酸替换，这一过程称为误译。此前，我们发现带有脯氨酸反密码子的 tRNASer 对细胞具有致命性。然而，通过将二次突变整合到 tRNA 中，错误翻译被抑制到非致命水平。这项工作的目标是确定 tRNASer 中的第二个位点替换，将错误翻译调节到不同的水平。对假定的身份元件进行有针对性的改变会导致 tRNA 功能完全丧失或细胞生长显着受损。然而，通过基因选择，我们发现了 22 个允许无毒误译的替换。这些二次突变主要发生在单链区域或用 G:U 碱基对替代 Watson-Crick 对。许多变体在低温下和损害快速 tRNA 衰减途径时毒性更大。我们认为大多数二次突变会影响细胞中 tRNA 的稳定性。 tRNA 的温度敏感性允许有条件的误译。蛋白质组学分析表明，tRNASer 变体不同程度地误译，生长减弱与误译增加相关。当与二次突变结合时，其他反密码子取代会导致其他非丝氨酸密码子处的丝氨酸错误翻译。 这些错误翻译的 tRNA 通过产生“统计蛋白质”而在合成生物学中得到应用，这些蛋白质可能比同质形式表现出更广泛的活性或底物特异性。