Translational Regulation of Yeast GCN4

Abstract

When subjected to starvation, stress, or viral infections, mammalian cells down-regulate general protein synthesis by phosphorylating the α subunit of eukaryotic translation initiation factor 2 (eIF2) 1The abbreviations used are: eIF2, eukaryotic translation initiation factor 2; uORF, upstream open reading frame; HRI, heme-regulated inhibitor protein kinase; PKR, double-stranded RNA-dependent protein kinase; HisRS, histidyl-tRNA synthetase; EF, elongation factor.1The abbreviations used are: eIF2, eukaryotic translation initiation factor 2; uORF, upstream open reading frame; HRI, heme-regulated inhibitor protein kinase; PKR, double-stranded RNA-dependent protein kinase; HisRS, histidyl-tRNA synthetase; EF, elongation factor. (1Hershey J.W.B. Annu. Rev. Biochem. 1991; 60: 717-755Crossref PubMed Scopus (840) Google Scholar). eIF2 functions in translation initiation by delivering charged initiator tRNAMet (Met-tRNAiMet) 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) (2Merrick W.C. Hershey J.W.B. Hershey J.W.B. Matthews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1996: 31-69Google Scholar). 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 α subunit of eIF2 (eIF2α) on Ser-51 prevents the recycling of eIF2 by eIF2B; in addition, the phosphorylated complex eIF2(αP)·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 (2Merrick W.C. Hershey J.W.B. Hershey J.W.B. Matthews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1996: 31-69Google Scholar). In Saccharomyces cerevisiae, eIF2α 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 (3Hinnebusch A.G. Broach J.R. Jones E.W. Pringle J.R. The Molecular and Cellular Biology of the Yeast Saccharomyces: Gene Expression. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1992: 319-414Google Scholar).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 GCN4expression under both starvation and non-starvation conditions without altering the mRNA (4Mueller P.P. Hinnebusch A.G. Cell. 1986; 45: 201-207Abstract Full Text PDF PubMed Scopus (355) Google Scholar). Thus, the uORFs inhibit GCN4translation 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 onGCN4 translation. When present alone, uORF4 reducesGCN4 translation to only 1% of the level seen in the absence of all four uORFs, under both starvation and non-starvation conditions (4Mueller P.P. Hinnebusch A.G. Cell. 1986; 45: 201-207Abstract Full Text PDF PubMed Scopus (355) Google Scholar). In this situation, it appears that all ribosomes translate uORF4 and then dissociate from the mRNA. In contrast, uORF1 alone reduces GCN4 translation by only 50%, presumably because half of the ribosomes that translate uORF1 resume scanning and reinitiate at GCN4. When uORF1 is present upstream of uORF4 and amino acids are abundant, all the ribosomes that resume scanning following uORF1 translation will reinitiate at uORF4 and dissociate from the mRNA, preventing GCN4translation. Under starvation conditions, however, half of the ribosomes that resume scanning after uORF1 translation will bypass the uORF4 start site (and also those at uORFs 2–3 when present) and reinitiate at GCN4 instead (Fig. 1) (5Abastado J.P. Miller P.F. Jackson B.M. Hinnebusch A.G. Mol. Cell. Biol. 1991; 11: 486-496Crossref PubMed Scopus (165) Google Scholar). Thus, prior translation of uORF1 allows ribosomes to overcome the strong translational barrier at uORF4 by a reinitiation mechanism.We proposed that under conditions where GCN4 is repressed, the ribosomes that resume scanning following uORF1 translation are forced to reinitiate at uORF4 because they rebind the eIF2·GTP·Met-tRNAiMet ternary complex before reaching the uORF4 start codon. Under starvation conditions, phosphorylation of eIF