Enhanced Codon–Anticodon Interaction at In-Frame UAG Stop Codon Improves the Efficiency of Non-Natural Amino Acid Mutagenesis
journal contributionposted on 2022-05-09, 14:05 authored by Purnima Mala, Ishu Saraogi
The introduction of non-natural amino acids into proteins through the stop codon readthrough methodology has been used to design proteins for diverse applications. However, this method suffers from low yields of the modified protein, as the suppressor tRNA that recognizes the stop codon is unable to compete effectively with release factor 1 (RF1), which terminates translation. We reasoned that a suppressor tRNA with improved interaction with the UAG stop codon on the mRNA will be able to compete more effectively with RF1. To test this idea, we inserted two 2,6-diaminopurine (D) units in the tRNA anticodon stem loop, including one at the third position of the tRNA anticodon. The modified suppressor tRNA could potentially form additional H-bonds between the N2-exocyclic amine of D and the C2 carbonyl group of uracil, thereby enhancing mRNA–tRNA interaction and/or altering tRNA conformation. The stronger interaction at the codon–anticodon interface resulted in improved UAG decoding efficiency and a higher yield of the modified protein containing a non-natural amino acid at multiple sites. Our findings are consistent with the importance of hydrogen bonding and tRNA conformation at the tRNA–mRNA duplex interface during in-frame UAG suppression, which improves protein translation at multiple UAG stop sites. This work provides valuable inputs toward improved non-natural amino acid mutagenesis for creating designer proteins.
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release factor 1natural amino acidsnatural amino acidinserted two 2frame uag suppressionc2 carbonyl groupmodified protein containingimproves protein translationuag stop codoncreating designer proteinsaltering trna conformationmodified proteinstop codontrna conformationterminates translationtrna anticodonsuppressor trnathird positionstronger interactionmultiple sitesmethod sufferslow yieldsincluding oneimproved interactionhydrogen bondinghigher yielddiverse applicationsdesign proteins