1. Leaky scanning past the CPA1 and arg‐2 uORFs.
Leaky scanning past the CPA1 and arg‐2 uORFs. Equal amounts (120 ng) of synthetic RNA transcripts were used to program translation mixtures derived from S.cerevisiae or N.crassa. (A) Saccharomyces cerevisiae CPA1–LUC RNA containing the wild‐type CPA1 uORF and intergenic region in the 5′‐leader was incubated at 25°C for 20 min in S.cerevisiae translation extracts. Reaction mixtures were supplemented with cycloheximide added either prior to 25°C incubation (T0) or after 15 min of incubation at 25°C (T15), and contained either 10 μM Arg (−) or 500 μM Arg (+) as indicated. (B) Predicted improvement of the arg‐2 uORF initiation context increases initiation at that site and decreases downstream initiation. N.crassa arg‐2–LUC RNA containing the wild‐type arg‐2 uORF in a poor wild‐type (wt) initiation context or in an improved initiation context (↑) were incubated in S.cerevisiae or N.crassa translation extracts at 25°C for 20 min; reaction mixtures were as for panel A for 20 min. Reaction mixtures containing 10 μM arginine were supplemented with cycloheximide added either prior to incubation (T0) or after 5 min of incubation (T5). Toeprinting and analysis with controls were performed as described in Figure 3. The positions of cDNA extension products corresponding to the mRNA 5′‐end, uORF initiation and termination codons, and the LUC initiation codon, are indicated diagrammatically in each panel.
Anthony Gaba et al. EMBO J. 2001;20:
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