Polypeptide chain termination in vitro: isolation of a release factor.

The growing polypeptide chain remains bound to the ribosome-messenger RNA complex through the sRNA carrying the last amino acid incorporated into the polypeptide chain.' On completion of the polypeptide chain a mechanism must exist for releasing it from the protein-synthesizing machinery. To date, most of the effort has been concentrated on establishing the coded information required for this event.2-5 Very little is known about the biochemical mechanism of releasing the finished protein. The chain-terminating codon has to be read and the last peptidyl-sRNA bond must be cleaved. What components are required for this process remains to be determined. It appears that a number of codons (UAA, UAG, UGA) can code for polypeptide chain termination.6-9 If by genetic accident such a codon arises in the middle of a cistron, premature polypeptide chain termination occurs at the site of the mutation.'0 The RNA genome from the bacteriophage R17, carrying such a mutation, has been used to develop a specific assay for polypeptide chain termination. In this mutant, the first glutamine codon (CAG) in the R17 coat protein cistron has mutated to the chain-terminating codon UAG."1 In a cell-free system, RNA from this mutant directs the synthesis of a small NH2-terminal coat protein fragment, N-formyl-met-ala-ser-aspn-phe-thr, which is released into the supernatant.'2 We can stop the synthesis of this peptide before it reaches the UAG codon by starvation for a chosen amino acid. This allows us to control the very last step of protein synthesis, the release of the completed polypeptide chain. Exploiting this procedure a protein component required for polypeptide chain termination has been isolated.