Towards Sense Codon Reassignment in Escherichia Coli

Abstract

Expansion of the genetic code through engineering of the translation machinery has vastly increased the chemical repertoire of the proteome. Incorporation of non-canonical amino acids (ncAAs) entails use of engineered aminoacyl-tRNA synthetase (AARS)•transfer RNA (tRNA) pairs that do not interact with the canonical aminoacylation machinery. Although engineered AARSs are selected for their ability to utilize ncAAs and discriminate against the twenty canonical amino acids, many utilize other ncAAs – a phenomenon known as polyspecificity. Specific incorporation of multiple ncAAs in a single polypeptide requires knowledge of the polyspecificities of engineered and wild-type AARSs, which this thesis explores. Recoding of the UAG and UGA stop codons has facilitated the incorporation of more than 150 ncAAs. Given the abundance of degenerate sense codons, potentially many more ncAAs can be incorporated. This thesis investigates the malleability of sense codons, and presents the findings from two engineered systems that change the amino acid designations of sense codons. In bacteria, an mRNA minihelix (selenocysteine insertion sequence, SECIS) recruits a dedicated elongation factor SelB that incorporates selenocysteine (Sec) at a UGA stop codon. Replacement of the UGA codon revealed that at least 58 of the 64 codons could be recoded to Sec, thus demonstrating that the Sec machinery is able to outcompete cognate aminoacyl-tRNAs. Next, an engineered pyrrolysyl-tRNA synthetase developed to incorporate 3-iodo-L-phenylalanine (3-I-Phe) was used to reassign sense codons in wild-type E. coli. To quantify the amino acids incorporated at a specific serine AGU codon in a super-folder GFP reporter protein, a selected reaction monitoring (SRM) experiment was performed – the incorporation yields (65±17% 3-I-Phe, 33±17% serine, 1±1% phenylalanine, and 1±1% threonine) revealed the intricacies of tRNA competition during aminoacylation and decoding. Reassignments of other serine (AGC, UCG) and leucine (CUG, UUG, UUA) codons were less efficient, thus providing a guideline for the choice of sense codons to reassign in future studies. Since the engineered AARS•tRNA pair so efficiently reassigns a serine AGU codon in wild-type E. coli, it should provide complete reassignment in genomically recoded organisms that feature an “open” codon.