Identification of sites of cysteine misincorporation during in vivo synthesis of bacteriophage T7 0.3 protein.
The 0.3 protein encoded by coliphage T7 does not normally contain cysteine residues. Incorporation of [35S]cysteine can therefore be used to assay mistranslation. We have purified 0.3 protein, synthesized in the presence of [35S]cysteine, from T7 infected cells of E. coli and determined the locations of misincorporated cysteine residues. Analysis of the molecular weights (Mr) of [35S]cysteine-labeled tryptic peptides of 0.3 protein demonstrated that cysteine (encoded by UGU or UGC) is not extensively misincorporated, as might be predicted by substitution for arginine residues (encoded by CGU or CGC). Edman degradation of the amino-terminal 50 residues of [35S]cysteine-labeled 0.3 protein determined that cysteine was most frequently misincorporated at position 15, which is correctly occupied by a tyrosine residue (encoded by UAC). There are four other tyrosine codons (1 UAU; 3 UAC) in the region of the 0.3 protein studied, but these were not mistranslated. The context in which a codon is located must therefore be more important in causing mistranslation than the sequence of the codon itself. Misincorporation of [35S]cysteine was also found at positions 9 (ACC, asparagine), 16 (GAA, glutamic acid), 41 (GCC, alanine) and 42 (GAU, aspartic acid). One mistranslation event appears to increase the likelihood that the following codon will also be mistranslated. This clustering of misincorporated [35S]cysteine residues was accentuated in 0.3 protein synthesized in the presence of streptomycin.
D000595:Amino Acid Sequence; D001120:Arginine; D002851:Chromatography, High Pressure Liquid; D003062:Codon; D003545:Cysteine / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D008715:Methionine / Q000378:metabolism; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism; D014176:Protein Biosynthesis / Q000187:drug effects; D013307:Streptomycin / Q000494:pharmacology; D013462:Sulfur Radioisotopes; D013604:T-Phages / Q000235:genetics; D014357:Trypsin; D014764:Viral Proteins / Q000096:biosynthesis* / Q000235:genetics
C041424:0.3 protein, Enterobacteria phage T7; D003062:Codon; D010446:Peptide Fragments; D013462:Sulfur Radioisotopes; D014764:Viral Proteins; D001120:Arginine; D008715:Methionine; D014357:Trypsin; D003545:Cysteine; D013307:Streptomycin