Reporter gene analysis confirms that sui1-L96P decreases translation of the CPA1 and AHK1 main CDS by increasing uORF translation and increases utilization of NCC start codons for the GRS1 amino-terminal extension. (A) Plasmid-borne luciferase reporters with FLUC CDS fused to the 15th codon of the CPA1 CDS with either the WT uORF (top) or a mutated uORF with the AUG start codon altered to UAG (bottom) (depicted schematically on the left), were introduced into WT and sui1-L96P strains. Transformants were cultured in SC−Leu−Ura medium at 30°C, diluted to A600 ∼ 0.1, and cultured for ∼3 additional doublings. Firefly luciferase activities were assayed in WCEs and reported in relative light units (RLUs) per mg of total protein, as means (±SEM) determined from four independent transformants. (B) RPFs and mRNA reads on the AHK1 gene in WT and L96P cells, presented as in Figure 1A, showing the ΔRRO values for each of three nested uORFs all in the same reading frame (listed below the uORF schematics) and average ΔRRO for all 3 uORFs (shown on the right). (C,D) Plasmid-borne luciferase reporters with FLUC CDS fused to the 15th codon of the AHK1 CDS with either the WT 5′UTR (top) or mutated 5′UTR with an internal deletion of all three uORFs (bottom) (depicted on the left), were assayed for luciferase expression in WT and L96P transformants as in A. The AHK1-FLUC expression ratio for the WT versus truncated 5′UTR is plotted for WT and L96P cells. (E) GRS1-FLUC reporters with FLUC CDS (beginning at the second codon, i.e., lacking the AUG start codon) fused to the CDS for the amino-terminal extension (NTE) 6 nt downstream from the last in-frame NCC start codon (AUU), containing either the WT NTE CDS (first row) or mutated NTE CDSs (rows 2–4) with the indicated substitutions of the four in-frame NCC start codons (shown as gray vs. red ovals). Expression of firefly luciferase in WT or L96P transformants was determined as in A. Expression of the GRS1-FLUC_UUC(1–4m) construct, lacking all four NCC start codons, is approximately sixfold above the background signal of ∼4.5 units.
