Translation Reinitiation Relies on the Interaction between eIF3a/TIF32 and Progressively Folded cis-Acting mRNA Elements Preceding Short uORFs
Figure 5
The extreme NTD of a/TIF32 contains two distal regions that promote efficient REI in the 5′ enhancer-dependent manner.
(A) Schematic representation of the first 200 amino acid residues of a/TIF32 shown as numbered circles (Boxes 1–20), each of them composed of 10 consecutive residues that were substituted with a stretch of 10 alanines. The sequence of Boxes 6, 8, and 17 is given below the schematic. (B) The a/tif32-Boxes 6, 8, and 17 impart a strong Gcn- phenotype. YBS52 (GCN2 a/tif32Δ) was transformed with individual YCplac111-based plasmids carrying the indicated a/TIF32 alleles and the resident YCpTIF32-His-U plasmid was evicted on 5-FOA. The resulting strains, together with isogenic strains H2880 (GCN2 a/TIF32; row 1) and H2881 (gcn2Δ a/TIF32; row 2), were then spotted in five serial 10-fold dilutions on SD (left panel) or SD containing 30 mM 3-AT (right panel) and incubated at 30°C for 3 and 6 days, respectively. (C) The failure of the a/tif32-NTD-Box mutations to derepress GCN4 is caused by a defect in resumption of scanning of post-termination 40S ribosomes on uORF1. Selected strains described in section B were introduced with the GCN4-lacZ constructs p180 (i.), pG67 (ii.), pM199 (iii.), and p209 (iv.), respectively, and analyzed as in Figure 2B. To induce the GCN4-lacZ expression (section i.), the transformants grown at the minimal media for 2 hrs after dilution were treated with 10 mM 3-AT for 6 hrs (a/TIF32) or overnight (Box mutants). An asterisk indicates data taken from [10] for comparison purposes. (D) Indicated strains described in section B were introduced with the GCN4-lacZ deletion constructs described in Figure 1D and Figure 4A–4C and analyzed as in Figure 2B. The RPEs affected by individual mutations are indicated above the bar diagram. wt, construct 1111; bg*, construct 4411.