Recoding the Genetic Code with Selenocysteine

Abstract

Selenocysteine (Sec) is naturally incorporated into proteins by recoding the stop codon UGA. Sec is not hardwired to UGA, as we found the Sec insertion machinery to be able to site-specifically incorporate Sec directed by 58 of the 64 codons. For 15 sense codons, complete conversion of the codon meaning from canonical amino acid to Sec was observed along with a 10-fold increase in selenoprotein yield compared to Sec insertion at the three stop codons. This high-fidelity sense-codon recoding mechanism was demonstrated for Escherichia coli formate dehydrogenase and recombinant human thioredoxin reductase and confirmed by independent biochemical and biophysical methods. Although Sec insertion at UGA is known to compete against protein termination, it is surprising that the Sec machinery has the ability to outcompete abundant aminoacyl-tRNAs in decoding sense codons. The findings have implications for the process of translation and the information storage capacity of the biological cell.

Selenocysteine Insertion at a Predefined UAG Codon in a Release Factor 1 (RF1)-depleted Escherichia coli Host Strain Bypasses Species Barriers in Recombinant Selenoprotein Translation

“…6B). Additional codon-anticodon variations have been reported, but have not yet been analyzed at depth regarding yields and efficiencies in final products of recombinant selenoproteins (29,49). Importantly, here we found that if UAG-directed TrxR1 production is performed in the RF1-depleted C321.⌬A host strain with a maintained variant SECIS element, almost full Sec content is achieved due to the fact that termination no longer occurs (Fig.…”

Polyspecific pyrrolysyl-tRNA synthetases from directed evolution

“…Genetic code expansion technologies are moving toward genetically encoding multiple diverse ncAAs. Cross-reactivity between engineered tRNA synthetases and diverse ncAA substrates will become a limiting factor in creating high-fidelity orthogonal translation systems with multiple varied ncAAs (16,54,55). The extent of protein engineering is limited by the fitness of the organism.…”

“…The active site of the o-AARS is usually redesigned via directed evolution (6), including positive and negative selective rounds, to produce an enzyme that is assumed to be specific for an ncAA and not active with the 20 canonical amino acids. Genetic code expansion technology is rapidly evolving (13), and the ability to incorporate multiple ncAAs into a protein using quadruplet-codon decoding (14) or sense-codon recoding (15)(16)(17)(18)(19) is now becoming feasible. Protein synthesis with multiple ncAAs will require o-AARSs that are able to discriminate their ncAA substrate not only from canonical amino acids in the cell but from other ncAAs that are added to the cell.…”

Towards Reassignment of the Methionine Codon AUG to Two Different Noncanonical Amino Acids in Bacterial Translation

“…[23] Recently, O'Donoghue et al showed that the machinery of selenocysteine can be modulated to recognize 58 of the 64 naturally occurring codons, in many cases completely outcompeting the endogenous tRNA, although other codons resulted in ambiguous translation. [24] Söll and co-workers showed that the serine sense codon AGU can be reassigned to 3-iodo-Lphenylalanine (3-I-Phe), [25] while Kwon and co-workers recently showed that it is possible to achieve ambiguous reading of the leucine UUG codon with 2-naphthylalanine. [26] In general, amino acids encoded by several codons (e.g., serine, leucine and arginine) offer more chances to be recoded since the wobble pairing at the 3 rd base could be outcompeted by a more energetically favorable WatsonCrick base pairing with the orthogonal tRNA.…”