Release Factors: In Vitro Assay and Purification

The release of formyl[³H]methionine from fMet-tRNA AUG ribosome intermediates requires both terminator codon and protein release factor, and the expression of each codon requires one of two protein release factors (R1 or R2). This chapter emphasizes on the method of quantitating R activity and the purification of R1 and R2. These protein molecules apparently recognize trinucleotide codons for they participate in formylmethionine release and bind to ribosomes with codon specificity. Formylmethionine-tRNA is the species of tRNA participating in initiation of Escherichia coli protein synthesis, and aminoacyl-tRNA or fMet-tRNA bound to the ribosomal A site are unreactive ribosomal intermediates for in vitro release. The release of formyl[³H]methionine from fMet-tRNA AUG ribosome intermediates, dependent upon terminator codon corresponds to in vitro peptide chain termination. Formylmethionine release that occurs in the absence of codon is because of chemical and enzymatic deacylation unrelated to R activity. Both R1 and R2 can be purified by preparative disc gel electrophoresis with good recovery.