The downstream enhancer and inhibitory elements can be isolated to smaller regions within the URE2 coding region. (A) Small deletions within the URE2 coding region were made from the 3′ end to determine the 3′ boundary of the enhancer element downstream from the minimal URE2 IRES element. Nucleotide numbers correspond to the URE2 nucleotides present in the reporter constructs being examined. Nucleotide 1 is the adenosine nucleotide in the AUG initiation codon in the prion forming domain of URE2. (B) Consecutive 100 nt internal deletions within the URE2 coding region were used to examine the distance dependence of the enhancer element between nucleotides 1008 and 1061. These deletions were then tested using the previously described p281-4 construct (Altmann et al. 1993). The deletions were made in the “variable region” illustrated in the inset. For each deletion, the minimal IRES (dotted region, nucleotides 205–309; inset) and the enhancer element (lined region, nucleotides 897–1061; inset) remained constant. The nucleotide numbers on the x-axis indicate which nucleotides within the variable region are present in the respective construct. (C) Small deletions were made from the 3′ end of the inhibitory element directly downstream from the minimal URE2 IRES element. In A–C, activity of each deletion was measured using the p281-4 reporter plasmid (Altmann et al. 1993) in both wild-type (gray bars) and ΔeIF2A (2A-KO; black bars) yeast cells, and is represented as in Figure 2. Furthermore, the nucleotides within the URE2 coding region present in the constructs represented in A–C are depicted on the x-axis.
