Directed Evolution of a SelB Variant that Does Not Require a Selenocysteine Insertion Sequence Element for Function.

Abstract

In bacteria, the incorporation of selenocysteine is achieved through the interaction of the selenocysteine specific elongation factor (SelB) with selenocysteine-charged tRNA^Sec and a selenocysteine insertion sequence (SECIS) element adjacent to an opal stop codon in an mRNA. The more generalized, SECIS-independent incorporation of selenocysteine is of interest because of the high nucleophilicity of selenium and the greater durability of diselenide bonds. It is likely that during the course of evolution, selenocysteine insertion originally arose without the presence of a SECIS element, relying only on SelB. Herein, we undertake experiments to evolve an ancestral version of SelB that is SECIS-independent and show that not only can this protein (SelB-v2) generally incorporate selenocysteine across from stop codons but also that the new, orthogonal translation factor can be repurposed to other amino acids, such as serine. Given the delicate energetic balancing act already performed by EF-Tu, this achievement raises the possibility that greatly expanded genetic codes that relied in part on SelB-based loading can now be contrived.