Non-Standard Genetic Codes Define New Concepts for Protein Engineering

Abstract

The essential feature of the genetic code is the strict one-to-one correspondence between codons and amino acids. The canonical code consists of three stop codons and 61 sense codons that encode 20% of the amino acid repertoire observed in nature. It was originally designated as immutable and universal due to its conservation in most organisms, but sequencing of genes from the human mitochondrial genomes revealed deviations in codon assignments. Since then, alternative codes have been reported in both nuclear and mitochondrial genomes and genetic code engineering has become an important research field. Here, we review the most recent concepts arising from the study of natural non-standard genetic codes with special emphasis on codon re-assignment strategies that are relevant to engineering genetic code in the laboratory. Recent tools for synthetic biology and current attempts to engineer new codes for incorporation of non-standard amino acids are also reviewed in this article.

Keywords: amino acids; biotechnology; codon reassignment; evolution; genetic code.

Figure 1

(A) An expanded wobble rule; (B) Possible pairings between the wobble nucleoside of tRNA and the codon third nucleoside of mRNA found in animal mitochondria. U *: cmnm⁵(s²)U, mnm⁵U, τm⁵U, τm⁵s²U (adapted from [64]).

Figure 2

tRNA secondary structures. (A,B) A purine at position 33 (G33) in the C. albicans tRNA ^Ser_CAG anticodon loop replaces a conserved pyrimidine found in all other tRNAs and is a key structural element in the reassignment of the CUG codon from leucine to serine. Two other nucleotides in the anticodon loop, A35 and G37, are important for leucylation, and the discriminator base, G73, functions as a negative identity determinant for leucyl-tRNA synthetase (A73 is required for leucylation); (C) tRNAs^Sec from all domains of life are unusual in both length (>90 nt) and structure. Most tRNAs have 7 bp in the acceptor stem and 5 in the TΨC arm, while eukaryal and archaeal tRNAs^Sec exhibit a 9 bp in the acceptor stem and 4 in the TΨC arm. Eukaryotic and archaeal tRNA^Sec species have 6 or 7 bp D-stems, respectively. Molecular modeling suggested that a 7 bp D-stem in archaeal tRNA^Sec would compensate for the short 4 bp T-stem thus allowing for the normal interaction between the D- and T-loops; (D) tRNAs^Pyl has a smaller D-loop (4–5 bp). Only one base is found between the acceptor and D-stems, rather than two bases, and the almost universally conserved G-purine sequence in the D-loop and TΨC sequence in the T loop are lacking. The anticodon stem forms with six, rather than five, base pairs, leaving only a very short (three base only) variable loop (adapted from [3]).

Figure 3

(A) Aminoacylation with canonical amino acids. tRNA aminoacylation is catalyzed by the corresponding aminoacyl-tRNA synthetase responsible for charging the tRNA with the cognate amino acid; (B) Stop codon suppression methods use heterologous orthogonal AARS:tRNA pairs to incorporate an orthogonal amino acid in response to a stop or quadruplet codon. This orthogonal amino acid is not a substrate for the endogenous tRNA and AARS (adapted from [104]).