Evaluating Sense Codon Reassignment with a Simple Fluorescence Screen.

Area of Science:

• Molecular Biology
• Biochemistry
• Synthetic Biology

Background:

• The genetic code's fidelity is crucial for protein synthesis, but its degeneracy offers potential for modification.
• Reassigning sense codons allows for the expansion of the proteome with non-natural amino acids.
• Green fluorescent protein (GFP) fluorescence requires an active site tyrosine, making it a useful reporter for genetic code studies.

Purpose of the Study:

• To develop a screening method to assess the pliability of the genetic code's degeneracy.
• To evaluate the efficiency of sense codon reassignment in Escherichia coli.
• To identify improved orthogonal tRNA/aminoacyl tRNA synthetase pairs for sense codon reassignment.

Main Methods:

• A fluorescence-based screen was designed using green fluorescent protein (GFP) to detect tyrosine incorporation.
• Orthogonal tRNA/aminoacyl tRNA synthetase pairs from Methanocaldococcus jannaschii were engineered by altering tRNA anticodons.
• Sense codon reassignment was tested at four wobble positions in Escherichia coli.
• A high-throughput screen of a 10^9-member library was performed to find variants for Lys AAG codon reassignment.

Main Results:

• Sense codon reassignment efficiencies ranged from 1% (Phe UUU) to 6% (Lys AAG).
• Engineered tRNAs preferentially recognized wobble-paired codons, except for the AUG anticodon tRNA which recognized both His codons.
• A high-throughput screen identified improved variants for Lys AAG codon reassignment.

Conclusions:

• The developed screen effectively probes genetic code pliability and facilitates sense codon reassignment.
• This approach enables the expansion of the encoded amino acid repertoire for protein biosynthesis.
• The method aids in identifying and optimizing orthogonal pairs for efficient sense codon reassignment, advancing protein engineering.