Two modes of amber codon read-through in vitro

Read-through translation of bacteriophage R17 amB2 coat cistron carrying an amber mutation at the seventh codon was studied in vitro using the crude cell extract (S30) derived from an Escherichia coli nonsuppressor strain. Despite the presence of termination factors as well as ribosome-releasing factor (RRF) which prevent the read-through translation [M. Ryoji, J. W. Karpen, and A. Kaji (1981) J. Biol. Chem. 256, 5798-5801], synthesis of coat-like protein still persists at a low level in this system. Characterization of this protein by peptide fingerprinting and amino acid sequencing was performed to reexamine the generally accepted notion that it is produced by amino acid misinsertion to the amber mutation codon. The results indicated, however, that the major population of this coat-like protein is produced as a result of reinitiation of translation from the eighth codon. Read-through by amino acid misinsertion in this system becomes predominant only when the Mg2+ concentration is higher than 16 mM.