Chen Hao, Ercanbrack Carson, Wang Tony, Gan Qinglei, Fan Chenguang
Cell and Molecular Biology Program, University of Arkansas, Fayetteville, AR, United States.
Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR, United States.
Front Bioeng Biotechnol. 2020 Jun 24;8:623. doi: 10.3389/fbioe.2020.00623. eCollection 2020.
Aminoacyl-tRNA synthetases (AARSs) play key roles in maintaining high fidelity of protein synthesis. They charge cognate tRNAs with corresponding amino acids and hydrolyze mischarged tRNAs by editing mechanisms. Impairment of AARS editing activities can reduce the accuracy of tRNA aminoacylation to produce mischarged tRNAs, which cause mistranslation and cell damages. To evaluate the mistranslation rate of threonine codons in living cells, in this study, we designed a quantitative reporter derived from the green fluorescent protein (GFP). The original GFP has multiple threonine codons which could affect the accuracy of measurement, so we generated a GFP variant containing only one threonine residue to specifically quantify mistranslation at the threonine codon. To validate, we applied this single-threonine GFP reporter to evaluate mistranslation at the threonine codon with mutations or modifications of threonine-tRNA synthetase and compared it with other methods of mistranslation evaluation, which showed that this reporter is reliable and facile to use.
氨酰-tRNA合成酶（AARSs）在维持蛋白质合成的高保真度方面发挥着关键作用。它们将相应的氨基酸加载到同源tRNA上，并通过编辑机制水解错配的tRNA。AARS编辑活性的受损会降低tRNA氨酰化的准确性，从而产生错配的tRNA，导致错误翻译和细胞损伤。为了评估活细胞中苏氨酸密码子的错误翻译率，在本研究中，我们设计了一种源自绿色荧光蛋白（GFP）的定量报告基因。原始的GFP有多个苏氨酸密码子，这可能会影响测量的准确性，因此我们生成了一个仅含有一个苏氨酸残基的GFP变体，以特异性地定量苏氨酸密码子处的错误翻译。为了进行验证，我们应用这种单苏氨酸GFP报告基因来评估苏氨酸-tRNA合成酶发生突变或修饰时苏氨酸密码子处的错误翻译，并将其与其他错误翻译评估方法进行比较，结果表明该报告基因可靠且使用方便。
