Translation initiation at an ACG triplet in mammalian cells.
The initiator AUC of the mouse dihydrofolate reductase gene (dhfr) was converted to ACG by site-directed mutagenesis and assayed for expression in cultured monkey cells using an SV40 recombinant called SVGT5dhfr26m2. Synthesis of apparently full-length dihydrofolate reductase (DHFR) protein was significantly reduced compared to wild-type, but not entirely abolished, suggesting that the ACG triplet was being utilized for translation initiation. In addition, a truncated form of DHFR was produced, apparently by initiation at the next in-frame AUG downstream. This result was confirmed in vitro. Transcripts of the dhfr sequence were produced by SP6 RNA polymerase in the presence of m7GpppG and translated in vitro using reticulocyte lysates and wheat germ extracts. The results paralleled those observed in vivo. Synthesis of full-length DHFR was reduced, but not eliminated, and a new species was produced by initiation at an internal site. Amino acid sequence analysis of the products of in vitro translation demonstrated that translation does indeed initiate at the ACG triplet and that it initiates with methionine. Additional mutations were introduced which altered the sequence context of the ACG triplet. Mutation of the translation initiation consensus sequence by substitution of the A residue at position -3, or of the G at +4 resulted in a significant decrease in initiation at the ACG and an increase in the level of the internal initiation product. Thus, translation initiation at a non-AUG triplet depends on a favorable sequence context.
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D051379:Mice; D009154:Mutation; D010442:Peptide Chain Initiation, Translational; D014176:Protein Biosynthesis; D011995:Recombination, Genetic; D013539:Simian virus 40 / Q000235:genetics; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*; D014158:Transcription, Genetic
D013762:Tetrahydrofolate Dehydrogenase; D012321:DNA-Directed RNA Polymerases