Translational regulation of yeast GCN4. A window on factors that control initiator-tRNA binding to the ribosome

When subjected to starvation, stress, or viral infections, mammalian cells down-regulate general protein synthesis by phosphorylating the alpha subunit of eukaryotic translation initiation factor 2 (eIF2). eIF2 functions in translation initiation by delivering charged initiator tRNA super(Met) (Met-tRNA sub(i) super(Met)) in a ternary complex with GTP to the 40 S ribosomal subunit, forming a 43 S preinitiation complex. In the translation of most mRNAs, the 43 S complex binds near the capped 5' end, migrates downstream, and upon reaching the first AUG codon, joins with the 60 S subunit to form an 80 S initiation complex (the scanning mechanism). Following AUG recognition, the GTP bound to eIF2 is hydrolyzed and eIF2 is released as an inactive eIF2-GDP binary complex. Exchange of the GDP bound to eIF2 with GTP is catalyzed by eIF2B. Phosphorylation of the alpha subunit of eIF2 (eIF2 alpha ) on Ser-51 prevents the recycling of eIF2 by eIF2B; in addition, the phosphorylated complex eIF2( alpha P)-GDP has a higher affinity than non-phosphorylated eIF2-GDP for eIF2B, such that GDP-GTP exchange on non-phosphorylated eIF2 is also impaired and ternary complex formation is blocked. In Saccharomyces cerevisiae, eIF2 alpha is phosphorylated when cells are deprived of an amino acid or purine, and interestingly, this leads to increased translation of a specific mRNA encoding GCN4, a transcriptional activator of at least 40 genes encoding amino acid biosynthetic enzymes. The unique induction of GCN4 translation in response to eIF2 phosphorylation is mediated by four short open reading frames (uORFs) in the leader of GCN4 mRNA located 150-360 nucleotides upstream of the authentic initiation codon. Eliminating the start codons of all four uORFs results in high level GCN4 expression under both starvation and non-starvation conditions without altering the mRNA. Thus, the uORFs inhibit GCN4 translation in non-starved cells by restricting the progression of scanning ribosomes through the leader to the GCN4 start codon. The first and fourth uORFs (from the 5' end), which are sufficient for nearly wild-type regulation, have different effects on GCN4 translation. When present alone, uORF4 reduces GCN4 translation to only 1% of the level seen in the absence of all four uORFs, under both starvation and non-starvation conditions. In this situation, it appears that all ribosomes translate uORF4 and then dissociate from the mRNA. In contrast, uORF1 alone reduces GCN4 translation by on