Department of Chemistry, Virginia Commonwealth University, Richmond, Virginia 23220, United States.
Massey Cancer Center, Virginia Commonwealth University, Richmond, Virginia 23220, United States.
ACS Synth Biol. 2022 Jun 17;11(6):2193-2201. doi: 10.1021/acssynbio.2c00150. Epub 2022 May 12.
The reprogramming of the genetic code through the introduction of noncanonical amino acids (ncAAs) has enabled exciting advances in synthetic biology and peptide drug discovery. Ribosomes that function with high efficiency and fidelity are necessary for all of these efforts, but for challenging ncAAs, the competing processes of near-cognate readthrough and peptidyl-tRNA dropoff can be issues. Here we uncover the surprising extent of these competing pathways in the PURE translation system using mRNAs encoding peptides with affinity tags at the N- and C-termini. We also show that hyperaccurate or error restrictive ribosomes with mutations in ribosomal protein S12 lead to significant improvements in yield and fidelity in the context of both canonical AAs and a challenging α,α-disubstituted ncAA. Hyperaccurate ribosomes also improve yields for quadruplet codon readthrough for a tRNA containing an expanded anticodon stem-loop, although they are not able to eliminate triplet codon reading by this tRNA. The impressive improvements in fidelity and the simplicity of introducing this mutation alongside other efforts to engineer the translation apparatus make hyperaccurate ribosomes an important advance for synthetic biology.
通过引入非规范氨基酸（ncAAs）来重新编程遗传密码，为合成生物学和肽类药物发现带来了令人兴奋的进展。核糖体的高效和保真度对于所有这些努力都是必要的，但对于具有挑战性的 ncAAs，近同义和肽酰-tRNA 脱落的竞争过程可能是问题所在。在这里，我们使用在 N 端和 C 端带有亲和标签的编码肽的 mRNA 在 PURE 翻译系统中揭示了这些竞争途径的惊人程度。我们还表明，带有核糖体蛋白 S12 突变的超精确或错误限制核糖体导致在规范氨基酸和具有挑战性的α，α-二取代 ncAA 的情况下产量和保真度的显著提高。超精确的核糖体还提高了含有扩展反密码子茎环的 tRNA 的四联体密码通读的产量，尽管它们不能通过该 tRNA 消除三联体密码的读取。在保真度方面的显著提高以及与其他工程翻译装置的努力一起引入这种突变的简单性使得超精确的核糖体成为合成生物学的重要进展。
