The evolution of tRNA multigene families remains poorly understood, exhibiting unusual phenomena such as functional conversions of tRNA genes through anticodon shift substitutions. We improved FlyBase tRNA gene annotations from twelve Drosophila species, incorporating previously identified ortholog sets to compare substitution rates across tRNA bodies at single-site and base-pair resolution. All rapidly evolving sites fell within the same metal ion-binding pocket that lies at the interface of the two major stacked helical domains. We applied our tRNA Structure–Function Mapper (tSFM) method independently to each Drosophila species and one outgroup species Musca domestica and found that, although predicted tRNA structure–function maps are generally highly conserved in flies, one tRNA Class-Informative Feature (CIF) within the rapidly evolving ion-binding pocket—Cytosine 17 (C17), ancestrally informative for lysylation identity—independently gained asparaginylation identity and substituted in parallel across tRNA^Asn paralogs at least once, possibly multiple times, during evolution of the genus. In D. melanogaster, most tRNA^Lys and tRNA^Asn genes are co-arrayed in one large heterologous gene cluster, suggesting that heterologous gene conversion as well as structural similarities of tRNA-binding interfaces in the closely related asparaginyl-tRNA synthetase (AsnRS) and lysyl-tRNA synthetase (LysRS) proteins may have played a role in these changes. A previously identified Asn-to-Lys anticodon shift substitution in D. ananassae may have arisen to compensate for the convergent and parallel gains of C17 in tRNA^Asn paralogs in that lineage. Our results underscore the functional and evolutionary relevance of our tRNA structure–function map predictions and illuminate multiple genomic and structural factors contributing to rapid, parallel and compensatory evolution of tRNA multigene families.

tRNA 多基因家族的进化仍然知之甚少，表现出不寻常的现象，例如通过反密码子移位替换实现 tRNA 基因的功能转换。我们改进了来自 12 个果蝇物种的 FlyBase tRNA 基因注释，纳入了之前确定的直系同源集，以在单位点和碱基对分辨率下比较 tRNA 体之间的取代率。所有快速进化的位点都位于位于两个主要堆叠螺旋结构域界面处的同一金属离子结合袋内。我们将我们的 tRNA 结构-功能图谱 (tSFM) 方法独立地应用于每个果蝇物种和一种外群物种家蝇，并发现，尽管预测的 tRNA 结构-功能图谱在果蝇中通常高度保守，但一种 tRNA 类信息特征 (CIF)在快速进化的离子结合口袋——胞嘧啶 17 (C17) 中，细胞嘧啶 17 (C17) 为赖氨酸化身份提供了祖先信息——在该属的进化过程中，独立获得了天冬酰胺化身份，并在 tRNA ^Asn旁系同源物中平行取代至少一次，可能多次。在黑腹果蝇中，大多数 tRNA ^Lys和 tRNA ^Asn基因共同排列在一个大的异源基因簇中，这表明密切相关的天冬酰胺酰-tRNA 合成酶 (AsnRS) 和 tRNA 结合界面的异源基因转换以及 tRNA 结合界面的结构相似性。赖氨酰-tRNA 合成酶 (LysRS) 蛋白可能在这些变化中发挥了作用。先前在D. ananassae中发现的 Asn 到 Lys 反密码子移位替换可能是为了补偿该谱系中 tRNA ^Asn旁系同源物中 C17 的收敛和平行增益。我们的结果强调了 tRNA 结构功能图谱预测的功能和进化相关性，并阐明了有助于 tRNA 多基因家族快速、平行和补偿进化的多个基因组和结构因素。