Characterization of the functional role of nucleotides within the URE2 IRES element and the requirements for eIF2A-mediated repression

The poly(A)-rich nature of the domain 1 apical loop is not critical for optimal activity of the URE2 minimal IRES element. Using the monocistronic p281-4 reporter plasmid harboring a stable stem–loop upstream of a fusion between the URE2 and LacZ open reading frames (Altmann et al. 1993), deletions and mutations within the domain 1 apical loop were tested in wild-type (gray bars) and ΔeIF2A (2A-KO; black bars) yeast strains. Reporter activity is presented in Miller Units [(OD₄₂₀ × 1000)/(OD₆₀₀ × reaction time)] on the y-axis, while the identity of deletions and mutations present in the minimal URE2 IRES element are indicated on the x-axis. Minimal URE2 IRES deletions of 6, 12, and 18 nt in the domain 1 loop were reported previously (Reineke et al. 2008).