Translation of the genetic message, IV. UAA as a chain termination codon.

The discovery that certain mutations result in the production of unfinished, amino-terminal peptide fragments of the corresponding wild-type proteins suggested that "nonsense" codons may normally signal termination of polypeptide chain synthesis.'-3 :\Ioreover, the finding that these "amber" mutations are suppressible, whereby the resulting polypeptide contains serine (codon, UCG) in place of glutamine (CAG) or tryptophan (UGG),4-6 led to the prediction2 3 that UAG is the chain terminator of "amber" mutants. The study of other nonsense ("ochre") mutations suggested further that UAA also functions as a chain termination codon.3 These suggestions were in accord with the fact that no amino acids are coded by UAG or UAA.7' 8 There are indications that UAA rather than UAG may be the codon normally concerned with chain termination.9 In vitro experiments with synthetic polynucleotides of random base sequence lent some support to the view that UAA is involved in chain termination. The polypeptides synthesized under the direction of Uand A-containing copolymers or natural messengers consist largely of free, i.e., released peptide chains, whereas the peptide chains formed with polynucleotides which do not contain U and A residues are attached to tRNA.10-12 In order to determine unambiguously whether UAA is a chain-termination codon, we have studied the in vitro translation of a series of oligonucleotides having, along with the chain-initiating formylmethionine codon (AUG) and lysine codons (AAA), leucine (UUA), phenylalanine (UUU), or "ochre" (UAA) codons in specified sequences as follows: (a) AUGAAAAAAAAA... AAA (AUGA,));(b) AUGUAAAAAAAA...AAA (AUGUA.); (c) AUGUUAAAAAAA... AAA (ATGU2A.); (d) AUGUUUAAAAAA... AAA (AUGU3A.); (e) AUGUUUUAAAAA... AAA (AUGU4An). The (a) polymers promoted the incorporation into acid-insoluble products of methionine and lysine; the (c) polymers, that of methionine, leucine, and lysine; and the (d) polymers, that of methionine, phenylalanine, and lysine. On the other hand, the (b) and (e) polymers promoted the incorporation of only small amounts of lysine, with little or no incorporation of methionine or phenylalanine. The inference that the (e) oligonucleotides directed the synthesis and release of acidsoluble formylmethionyl-phenylalanine was proved correct by isolation of the dipeptide. This work provides conclusive proof that UAA is a chain-termination codon. It also provides a suitable system for study of the mechanism of chain termination. Materials and Methods.-These were as in previous work13 14 unless otherwise noted. Amino acid incorporation: The low-nuclease system consisting of purified Escherichia coli Q13 ribosomes and Lactobacillus arabinosus supernatant,15 supplemented with initiation factors,13 18 was used throughout. In experiment 1 of Table 1 the samples contained the following components in a final volume of 0.125 ml: Tris-HCl buffer, pH 7.8, 60 mM; ammoniumn chloride, 50 mM;