Abstract：
Termination of translation is essential but hinders applications of genetic code engineering, e.g., unnatural amino acids incorporation and codon randomization mediated saturation mutagenesis. Here, for the first time, it is demonstrated that E. coli Pth and ArfB together play an efficient translation termination without codon preference in the absence of class-I release factors. By degradation of the targeted protein, both essential and alternative termination types of machinery are completely removed to disable codon-dependent termination in cell extract. Moreover, a total of 153 engineered tRNAs are screened for efficient all stop-codons decoding to construct a codon-dependent termination defect in vitro protein synthesis with all 64 sense-codons, iPSSC. Finally, this full sense genetic code achieves significant improvement in the incorporation of distinct unnatural amino acids at up to 12 positions and synthesis of protein encoding consecutive NNN codons. By decoding all information in nucleotides to amino acids, iPSSC may hold great potential in building artificial protein synthesis beyond the cell.
摘要：
翻译的终止是必不可少的，但阻碍了遗传密码工程的应用，例如，非天然氨基酸掺入和密码子随机化介导的饱和诱变。这里,第一次,已经证明,在不存在I类释放因子的情况下,大肠杆菌Pth和ArfB在没有密码子偏好的情况下一起发挥有效的翻译终止作用。通过降解目标蛋白，基本和替代终止类型的机器都被完全去除，以禁用细胞提取物中的密码子依赖性终止。此外，总共筛选了153个工程tRNA，用于有效解码所有终止密码子，以构建具有所有64个有义密码子的密码子依赖性终止缺陷的体外蛋白质合成，iPSSC.最后,这种全义遗传密码在多达12个位置的不同非天然氨基酸的掺入和编码连续NNN密码子的蛋白质的合成方面实现了显着改善。通过将核苷酸中的所有信息解码为氨基酸，iPSSC在构建细胞以外的人工蛋白质合成方面可能具有巨大潜力。
