Institute of Biochemistry and Molecular Biology, University of Hamburg, Hamburg, Germany.
Center for Bioinformatics, University of Hamburg, Hamburg, Germany.
Nat Commun. 2021 Jun 22;12(1):3850. doi: 10.1038/s41467-021-24076-x.
Three stop codons (UAA, UAG and UGA) terminate protein synthesis and are almost exclusively recognized by release factors. Here, we design de novo transfer RNAs (tRNAs) that efficiently decode UGA stop codons in Escherichia coli. The tRNA designs harness various functionally conserved aspects of sense-codon decoding tRNAs. Optimization within the TΨC-stem to stabilize binding to the elongation factor, displays the most potent effect in enhancing suppression activity. We determine the structure of the ribosome in a complex with the designed tRNA bound to a UGA stop codon in the A site at 2.9 Å resolution. In the context of the suppressor tRNA, the conformation of the UGA codon resembles that of a sense-codon rather than when canonical translation termination release factors are bound, suggesting conformational flexibility of the stop codons dependent on the nature of the A-site ligand. The systematic analysis, combined with structural insights, provides a rationale for targeted repurposing of tRNAs to correct devastating nonsense mutations that introduce a premature stop codon.
三个终止密码子（UAA、UAG 和 UGA）终止蛋白质合成，几乎完全被释放因子识别。在这里，我们设计了从头合成的转移 RNA（tRNA），可在大肠杆菌中有效地解码 UGA 终止密码子。tRNA 的设计利用了有义密码子解码 tRNA 的各种功能保守方面。在 TΨC-茎中进行优化以稳定与延伸因子的结合，在增强抑制活性方面显示出最有效的效果。我们以 2.9 Å 的分辨率确定了核糖体与在 A 位结合 UGA 终止密码子的设计 tRNA 复合物的结构。在抑制 tRNA 的情况下，UGA 密码子的构象类似于有义密码子，而不是当结合规范的翻译终止释放因子时，这表明终止密码子的构象灵活性取决于 A 位配体的性质。系统分析与结构见解相结合，为靶向重新利用 tRNA 以纠正引入过早终止密码子的破坏性无义突变提供了依据。
