Molecular reconstruction of a fungal genetic code alteration.

Fungi of the CTG clade translate the Leu CUG codon as Ser. This genetic code alteration is the only eukaryotic sense-to-sense codon reassignment known to date, is mediated by an ambiguous serine tRNA (tRNA(CAG)(Ser)), exposes unanticipated flexibility of the genetic code and raises major questions about its selection and fixation in this fungal lineage. In particular, the origin of the tRNA(CAG)(Ser) and the evolutionary mechanism of CUG reassignment from Leu to Ser remain poorly understood. In this study, we have traced the origin of the tDNA(CAG)(Ser) gene and studied critical mutations in the tRNA(CAG)(Ser) anticodon-loop that modulated CUG reassignment. Our data show that the tRNA(CAG)(Ser) emerged from insertion of an adenosine in the middle position of the 5-CGA-3anticodon of a tRNA(CGA)(Ser) ancestor, producing the 5-CAG-3 anticodon of the tRNA(CAG)(Ser), without altering its aminoacylation properties. This mutation initiated CUG reassignment while two additional mutations in the anticodon-loop resolved a structural conflict produced by incorporation of the Leu 5-CAG-3anticodon in the anticodon-arm of a tRNA(Ser). Expression of the mutant tRNA(CAG)(Ser) in yeast showed that it cannot be expressed at physiological levels and we postulate that such downregulation was essential to maintain Ser misincorporation at sub-lethal levels during the initial stages of CUG reassignment. We demonstrate here that such low level CUG ambiguity is advantageous in specific ecological niches and we propose that misreading tRNAs are targeted for degradation by an unidentified tRNA quality control pathway.