Anticodon sequence determines the impact of mistranslating tRNA^Ala variants

Transfer RNAs (tRNAs) maintain translation fidelity through accurate charging by their cognate aminoacyl-tRNA synthetase and codon:anticodon base pairing with the mRNA at the ribosome. Mistranslation occurs when an amino acid not specified by the genetic message is incorporated into proteins and has applications in biotechnology, therapeutics and is relevant to disease. Since the alanyl-tRNA synthetase uniquely recognizes a G3:U70 base pair in tRNA^Ala and the anticodon plays no role in charging, tRNA^Ala variants with anticodon mutations have the potential to mis-incorporate alanine. Here, we characterize the impact of the 60 non-alanine tRNA^Ala anticodon variants on the growth of <i>Saccharomyces cerevisiae</i>. Overall, 36 tRNA^Ala anticodon variants decreased growth in single- or multi-copy. Mass spectrometry analysis of the cellular proteome revealed that 52 of 57 anticodon variants, not decoding alanine or stop codons, induced mistranslation when on single-copy plasmids. Variants with G/C-rich anticodons resulted in larger growth deficits than A/U-rich variants. In most instances, synonymous anticodon variants impact growth differently, with anticodons containing U at base 34 being the least impactful. For anticodons generating the same amino acid substitution, reduced growth generally correlated with the abundance of detected mistranslation events. Differences in decoding specificity, even between synonymous anticodons, resulted in each tRNA^Ala variant mistranslating unique sets of peptides and proteins. We suggest that these differences in decoding specificity are also important in determining the impact of tRNA^Ala anticodon variants.转运RNA（Transfer RNAs, tRNAs）通过与其对应的氨基酰-tRNA合成酶（aminoacyl-tRNA synthetase）进行精准氨酰化，并在核糖体（ribosome）上与信使RNA（mRNA）完成密码子-反密码子碱基配对，从而维持翻译保真度。错译（mistranslation）指未按遗传信息编码的氨基酸被掺入蛋白质的现象，该现象在生物技术、治疗领域具有应用价值，且与疾病发生密切相关。由于丙氨酰-tRNA合成酶（alanyl-tRNA synthetase）仅识别丙氨酸转运RNA（tRNA^Ala）上的G3:U70碱基对，且反密码子不参与氨酰化过程，因此携带反密码子突变的丙氨酸转运RNA变体有掺入丙氨酸造成错译的潜力。本研究针对60种非丙氨酸型丙氨酸转运RNA反密码子变体，解析其对酿酒酵母（Saccharomyces cerevisiae）生长的影响。总体而言，36种丙氨酸转运RNA反密码子变体在单拷贝或多拷贝质粒载体中会抑制酿酒酵母的生长。对细胞蛋白质组的质谱分析法（mass spectrometry）分析显示，在单拷贝质粒中，57种非解码丙氨酸或终止密码子的反密码子变体中有52种可诱导错译。携带G/C富集型反密码子的变体，其生长缺陷程度显著高于A/U富集型变体。多数情况下，同义反密码子变体对生长的影响存在差异，其中反密码子第34位携带U的变体影响最弱。对于能引发相同氨基酸替换的反密码子变体，生长抑制程度通常与检测到的错译事件丰度呈正相关。即便在同义反密码子之间，解码特异性的差异也会使每种丙氨酸转运RNA变体产生独特的肽段和蛋白质错译谱。我们认为，解码特异性的这些差异同样是决定丙氨酸转运RNA反密码子变体影响程度的关键因素。