Physical evidence for distinct mechanisms of translational control by upstream open reading frames

The Saccharomyces cerevisiae GCN4 mRNA 5'-leader contains four upstream ope n reading frames (uORFS) and the CPA1 leader contains a single uORF. To det ermine how these uORFs control translation, we examined mRNAs containing th ese leaders in cell-free translation extracts to determine where ribosomes were loaded first and where they were loaded during steady-state translatio n. Ribosomes predominantly loaded first at GCN4 uORF1. Following its transl ation, but not the translation of uORF4, they efficiently reinitiated prote in synthesis at Gcn4p. Adding purified eIF2 increased reinitiation at uORFs 3 or 4 and reduced reinitiation at Gcn4p. This indicates that eIF2 affects the site of reinitiation following translation of GCN4 uORF1 in vitro. In contrast, for mRNA containing the CPA1 uORF, ribosomes reached the downstre am start codon by scanning past the uORF. Addition of arginine caused ribos omes that had synthesized the uORF polypeptide to stall at its termination codon, reducing loading at the downstream start codon, apparently by blocki ng scanning ribosomes, and not by affecting reinitiation. The GCN4 and CPA1 uORFs thus control translation in fundamentally different ways.