Department of Biochemistry, The University of Western Ontario, London, Ontario, Canada.
Department of Genome Sciences, University of Washington, Seattle, WA, USA.
RNA Biol. 2023 Jan;20(1):791-804. doi: 10.1080/15476286.2023.2257471. Epub 2023 Sep 30.
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 and the anticodon plays no role in charging, tRNA variants with anticodon mutations have the potential to mis-incorporate alanine. Here, we characterize the impact of the 60 non-alanine tRNA anticodon variants on the growth of . Overall, 36 tRNA 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 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 anticodon variants.
转移 RNA（tRNA）通过其同源氨酰-tRNA 合成酶和密码子：反密码子与核糖体上的 mRNA 碱基配对来维持翻译准确性。当非遗传信息指定的氨基酸被掺入蛋白质中时，就会发生错译，这种现象在生物技术、治疗学中有应用，与疾病相关。由于丙氨酰-tRNA 合成酶独特地识别 tRNA 中的 G3:U70 碱基对，而反密码子在充电中不起作用，因此具有反密码子突变的 tRNA 变体有可能错误掺入丙氨酸。在这里，我们研究了 60 种非丙氨酸 tRNA 反密码子变体对. 生长的影响。总体而言，36 种 tRNA 反密码子变体在单拷贝或多拷贝时降低了生长速度。对细胞蛋白质组的质谱分析显示，在单拷贝质粒中，57 个反密码子变体中有 52 个不解码丙氨酸或终止密码子，导致错译。富含 G/C 的反密码子变体比富含 A/U 的变体导致更大的生长缺陷。在大多数情况下，同义反密码子变体对生长的影响不同，反密码子第 34 位含有 U 的变体影响最小。对于产生相同氨基酸取代的反密码子，生长减少通常与检测到的错译事件的丰度相关。即使在同义反密码子之间，解码特异性的差异也会导致每个 tRNA 变体翻译独特的肽和蛋白质组。我们认为，这些解码特异性的差异对于确定 tRNA 反密码子变体的影响也很重要。
