Base-pairing status and location of the internal AUG codon are not imperative for IRES activity. (A) Altering the base-pairing status by making mutations in domain 3 of the minimal URE2 IRES element does not affect levels of cap-independent translation initiation. Identity and location of the mutations are indicated on the x-axis. (B) The location of the internal AUG codon within the minimal URE2 IRES element is not a strict requirement for internal initiation of translation. The AUG codon was translocated by switching its position either one codon upstream (AUG→GAU) or one codon downstream (AUG→AGU). The sequences surrounding the AUG codon (for the wild-type, AUG→GAU, and AUG→AGU constructs) are shown in the inset, with the internal AUG codon underlined. The values in A and B are measured using the p281-4 reporter plasmid (Altmann et al. 1993) in both the wild-type (gray bars) and ΔeIF2A (2A-KO; black bars) yeast cells, and represented as in Figure 2.
