Mistranslation of the mRNA encoding bacteriophage T7 0.3 protein

We have devised an experimental system using the T7 phage 0.3 protein to accurately quantitate in vivo errors in protein synthesis. The 0.3 protein is well suited for mistranslation studies because it is easy to purify, its precise amino acid and RNA sequences are known, and it contains no cysteine. Utilizing [35S]cysteine as a precursor we found an average of 1 cysteine residue misincorporated for every 43.5 molecules of 0.3 protein synthesized. Since there are 116 amino acids in 0.3 protein, 1 cysteine residue was misincorporated /5000 codons translated. If all 20 amino acids were misincorporated at the same frequency, the overall frequency of misincorporation of amino acids into 0.3 protein would be 4 X 10(-3)/codon translated. Parallel experiments measuring [35S]methionine incorporation into 0.3 protein supported the accuracy of our findings for cysteine misincorporation. We found an average of 5.7 methionine residues incorporated/molecule of 0.3 protein synthesized; the actual number from sequence data is known to be 6. Antibiotics which stimulate mistranslation (gentamicin and streptomycin) caused a modest increase in the number of cysteine residues misincorporated into 0.3 protein. The use of Escherichia coli strains, identical except for mutations in ribosomal protein genes known to affect the fidelity of translation, supported the contention that the errors being quantitated were mainly due to mistranslation rather than mistranscription .