Several methanogenic archaea lack cysteinyl-transfer RNA (tRNA) synthetase (CysRS), the essential enzyme that provides Cys-tRNA(Cys) for translation in most organisms. Partial purification of the corresponding activity from Methanocaldococcus jannaschii indicated that tRNA(Cys) becomes acylated with O-phosphoserine (Sep) but not with cysteine. Further analyses identified a class II-type O-phosphoseryl-tRNA synthetase (SepRS) and Sep-tRNA:Cys-tRNA synthase (SepCysS). SepRS specifically forms Sep-tRNA(Cys), which is then converted to Cys-tRNA(Cys) by SepCysS. Comparative genomic analyses suggest that this pathway, encoded in all organisms lacking CysRS, can also act as the sole route for cysteine biosynthesis. This was proven for Methanococcus maripaludis, where deletion of the SepRS-encoding gene resulted in cysteine auxotrophy. As the conversions of Sep-tRNA to Cys-tRNA or to selenocysteinyl-tRNA are chemically analogous, the catalytic activity of SepCysS provides a means by which both cysteine and selenocysteine may have originally been added to the genetic code.

At the time of its discovery four decades ago, the genetic code was viewed as the result of a "frozen accident." Our current knowledge of the translation process and of the detailed structure of its components highlights the roles of RNA structure (in mRNA and tRNA), RNA modification (in tRNA), and aminoacyl-tRNA synthetase diversity in the evolution of the genetic code. The diverse assortment of codon reassignments present in subcellular organelles and organisms of distinct lineages has 'thawed' the concept of a universal immutable code; it may not be accidental that out of more than 140 amino acids found in natural proteins, only two (selenocysteine and pyrrolysine) are known to have been added to the standard 20-member amino acid alphabet. The existence of phosphoseryl-tRNA (in the form of tRNACys and tRNASec) may presage the discovery of other cotranslationally inserted modified amino acids.