Toward a Quadruplet Codon Mitochondrial Genetic Code

Nature has evolved to exclusively use a genetic code consisting of triplet nucleotide codons. The translation system, however, is known to be compatible with 4-nucleotide frameshift or quadruplet codons. In this study, we begin to explore the possibility of a genome made up entirely of quadruplet codons using the minimal mitochondrial genome of <i>Saccharomyces cerevisiae</i> as a model system. We demonstrate that mitochondrial tryptophanyl- and tyrosyl-tRNAs with modified anticodons effectively suppress mutant <i>cox</i><i>3</i> genes containing a TAG stop or TAGA quadruplet codon, leading to the production of full-length COX3 and a respiratory-competent phenotype. This work provides a method for introducing heterologous tRNAs into the yeast mitochondria for genetic engineering applications and serves as a starting point for the development of a quadruplet codon genetic code.