CGG: an unassigned or nonsense codon in Mycoplasma capricolum

CGG is an arginine codon in the universal genetic code. We previously reported that in Mycoplasma capricolum, a relative of Gram-positive eubacteria, codon CGG did not appear in coding frames, including termination sites, and tRNA(ArgCCG) pairing with codon CGG, was not detected. These facts suggest that CGG is a nonsense (unassigned and untranslatable) codon--i.e., not assigned to arginine or to any other amino acid. We have investigated whether CGG is really an unassigned codon by using a cell-free translation system prepared from M. capricolum. Translation of synthetic mRNA containing in-frame CGG codons does not result in "read-through" to codons beyond the CGG codons--i.e., translation ceases just before CGG. Sucrose-gradient centrifugation profiles of the reaction mixture have shown that the bulk of peptide that has been synthesized is attached to 70S ribosomes and is released upon further incubation with puromycin. The result suggests that the peptide is in the P site of ribosome in the form of peptidyl-tRNA, leaving the A site empty. When in-frame CGG codons are replaced by UAA codons in mRNA, no read-through occurs beyond UAA, just as in the case of CGG. However, the synthesized peptide is released from 70S ribosomes, presumably by release factor 1. These data suggest strongly that CGG is an unassigned codon and differs from UAA in that CGG is not used for termination.