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

Area of Science:

• Molecular Biology
• Biochemistry
• Synthetic Biology

Background:

• Selenocysteine incorporation in bacteria typically requires the selenocysteine-specific elongation factor (SelB), selenocysteine-charged tRNA^Sec, and a SECIS element.
• SECIS-independent selenocysteine incorporation is desirable due to selenium's high nucleophilicity and the stability of diselenide bonds.
• Evolutionary origins suggest selenocysteine insertion may have initially occurred without a SECIS element, relying solely on SelB.

Purpose of the Study:

• To evolve an ancestral, SECIS-independent version of the SelB protein.
• To investigate the generalized incorporation of selenocysteine at stop codons using the evolved SelB.
• To explore the potential for repurposing this orthogonal translation factor for other amino acids.

Main Methods:

• Experimental evolution of the SelB protein to achieve SECIS independence.
• Assaying the ability of the evolved SelB (SelB-v2) to incorporate selenocysteine at stop codons in mRNA.
• Testing the repurposing of SelB-v2 for the incorporation of alternative amino acids, such as serine.

Main Results:

• Successfully evolved a SECIS-independent SelB variant (SelB-v2).
• Demonstrated that SelB-v2 can generally incorporate selenocysteine opposite stop codons.
• Showed that SelB-v2 can be repurposed to incorporate other amino acids, like serine, creating an orthogonal translation system.

Conclusions:

• The engineered SelB-v2 protein enables generalized, SECIS-independent selenocysteine incorporation.
• This work validates the possibility of expanding the genetic code using SelB-based orthogonal translation.
• The findings open avenues for contriving greatly expanded genetic codes leveraging SelB-based amino acid loading.