Figure 4. uORFs can control translation in response to metabolites.

(A) Structure of an mRNA with one main ORF (mORF, yellow) and three uORFs (blue), one of which overlaps the mORF. Start codons are highlighted with bent arrows.

(B) Reporter gene translation assay designed to quantify leaky scanning versus reinitiation on an mRNA with a single uORF. As compared to the wild-type allele (I), in allele (II) the start codon of the uORF has been mutated to prevent initiation. In allele (III), the stop codon of the uORF has been mutated so as to extend the uORF into the main ORF, which precludes reinitiation at the Reporter AUG. Additional mutations may be needed to remove any downstream in-frame stops in the 5’ leader and to ensure that the uORF is out of frame with the main ORF. The rates of leaky scanning past the uORF initiation codon (LS), of initiation at the uAUG (I), of initiation-reinitiation (I/R) and of failure to reinitiate (I/NR) are calculated from the empirical reporter gene expression activities A_I, A_II and A_III as shown in the table. Numbers shown are for illustration only.

(C) uORF-mediated metabolic control on the GGP (GDP-galactose phosphorylase) mRNA (after Laing et al., 2015). At a high ascorbate level, the non-canonical start codon (ACG) of the uORF is recognized and the encoded conserved peptide disallows reinitiation thus switching translation of the main ORF off. At low ascorbate the ACG codon is thought to be skipped by leaky scanning.

(D) uORF-mediated metabolic control on the AdoMetDC (adenosyl-methionine decarboxylase) mRNA (after Hanfrey et al., 2005 and Uchiyama-Kadokura et al., 2014). At a low polyamine level, ribosomes initiate on uORF1, thus bypass the start of uORF2, and reinitiate on the main ORF. At a high polyamine level, uORF1 is skipped and uORF2 is translated. uORF2 codes for a conserved peptide and is not conducive to reinitiation, thus inhibiting translation of AdoMetDC by negative feedback.