Orthogonal Pair Directed, Codon Specific, Sense Codon Reassignment: An Improved Tool for Evaluating the Plasticity of the E coli Genetic Code

Natural proteins are constructed out of 20 amino acids building blocks which contain a limited set of chemical functionalities. Expanding the set of genetically encoded amino acids introduces new chemical moieties that enable the specific modification, labeling, and cross-linking of proteins. Genetically encoding new amino acids with diverse chemical functionalities is limited by the fact that all 64 triplet codons have assigned functions in the genetic code. Two general methods of genetic code expansion are commonly employed, but each has limitations. Non-canonical amino acid residues can be incorporated in response to stop codons, but this strategy is limited to single site incorporation. Residue specific reassignment allows multisite incorporation, but requires complete substitution of one natural amino acid for a non-canonical residue. A third approach combining aspects of both non-sense and residue specific reassignment enables the expansion of the genetic code by reassigning the meaning of sense codons.

We have developed a fluorescence-based screen to quantify the extent to which the sense codons in E coli can be reassigned by introducing M. jannaschii tyrosine and M. barkeri pyrrolysine orthogonal aminoacyl tRNA synthetase/tRNA pairs directed to decode sense codons. The M. jannaschii tyrosine and M. barkeri pyrrolysine orthogonal pairs are commonly used to incorporate non-cannonical amino acids in response to nonsense codons. The fluorescence screen was employed to examine the complete set of codons read via wobble interactions as well as the set of rarely used codons in E coli. The method of using orthogonal pairs to precisely direct the incorporation of amino acids in response to sense codons allows a more focused examination of the physiological effects of substitutions than previous methods employing aminoacyl tRNA synthetases with defective editing sites or ribosomes with relaxed proofreading. We describe the efficiency and specificity of sense codon reassignment of E. coli codons and the ability to improve these qualities through the application of the fluorescence-based screen in a high throughput mode to select improved variants from large libraries. The main finding of these studies are that the E coli genetic code is readily reassignable and genome wide amino acid substitutions are well tolerated. We observe weak correlations between tRNA abundance and changes in amino acylation efficiency of the modified orthogonal system on sense codon reassignment. The relative energetics of codon -- anticodon interactions appear to be an important factor in reassignment efficiency. We identify four codons that have not been targeted for sense codon reassignment that appear to promising.