We have isolated and characterized two suppressor genes, SUI4 and SUI5, that can initiate translation in the absence of an AUG start codon at the HIS4 locus in Saccharomyces cerevisiae. Both suppressor genes are dominant in diploid cells and lethal in haploid cells. The SUI4 suppressor gene is identical to the GCD11 gene, which encodes the ␥ subunit of the eIF-2 complex and contains a mutation in the G 2 motif, one of the four signature motifs that characterizes this subunit to be a G-protein. The SUI5 suppressor gene is identical to the TIF5 gene that encodes eIF-5, a translation initiation factor known to stimulate the hydrolysis of GTP bound to eIF-2 as part of the 43S preinitiation complex. Purified mutant eIF-5 is more active in stimulating GTP hydrolysis in vitro than wild-type eIF-5, suggesting that an alteration of the hydrolysis rate of GTP bound to the 43S preinitiation complex during ribosomal scanning allows translation initiation at a non-AUG codon. Purified mutant eIF-2␥ complex is defective in ternary complex formation and this defect correlates with a higher rate of dissociation from charged initiator-tRNA in the absence of GTP hydrolysis. Biochemical characterization of SUI3 suppressor alleles that encode mutant forms of the ␤ subunit of eIF-2 revealed that these mutant eIF-2 complexes have a higher intrinsic rate of GTP hydrolysis, which is eIF-5 independent. All of these biochemical defects result in initiation at a UUG codon at the his4 gene in yeast. These studies in light of other analyses indicate that GTP hydrolysis that leads to dissociation of eIF-2 ⅐ GDP from the initiator-tRNA in the 43S preinitiation complex serves as a checkpoint for a 3-bp codon/anticodon interaction between the AUG start codon and the initiator-tRNA during the ribosomal scanning process.

GCN4 is a transcriptional activator of amino acid biosynthetic genes in Saccharomyces cerevisiae whose expression is regulated by amino acid availability at the translational level. GCD1 and GCD2 are negative regulators required for the repression of GCN4 translation under nonstarvation conditions that is mediated by upstream open reading frames (uORFs) in the leader of GCN4 mRNA. GCD factors are thought to be antagonized by the positive regulators GCN1, GCN2, and GCN3 in amino acid-starved cells to allow for increased GCN4 protein synthesis. Previous genetic studies suggested that GCD1, GCD2, and GCN3 have closely related functions in the regulation of GCN4 expression that involve translation initiation factor 2 (eIF-2). In agreement with these predictions, we show that GCD1, GCD2, and GCN3 are integral components of a high-molecular-weight complex of approximately 600,000 Da. The three proteins copurified through several biochemical fractionation steps and could be coimmunoprecipitated by using antibodies against GCD1 or GCD2. Interestingly, a portion of the eIF-2 present in cell extracts also cofractionated and coimmunoprecipitated with these regulatory proteins but was dissociated from the GCD1/GCD2/GCN3 complex by 0.5 M KCI. Incubation of a temperature-sensitive gcdl-101 mutant at the restrictive temperature led to a rapid reduction in the average size and quantity of polysomes, plus an accumulation of inactive 80S ribosomal couples; in addition, excess amounts of eIF-2a, GCD1, GCD2, and GCN3 were found comigrating with free 40S ribosomal subunits. These results suggest that GCD1 is required for an essential function involving eIF-2 at a late step in the translation initiation cycle. We propose that lowering the function of this high-molecular-weight complex, or of eIF-2 itself, in amino acid-starved cells leads to reduced ribosomal recognition of the uORFs and increased translation initiation at the GCN4 start codon. Our results provide new insights into how general initiation factors can be regulated to affect gene-specific translational control.The GCN4 protein of the yeast Saccharomyces cerevisiae is a transcriptional activator of amino acid biosynthetic genes that are subject to general amino acid control. Transcription of these genes is stimulated by GCN4 in response to starvation for any amino acid. The expression of GCN4 itself is regulated by amino acid availability, but at the translational level. Four short open reading frames (uORFs) in the long leader sequence of GCN4 mRNA function as cis-acting regulatory elements that couple the rate of GCN4 translation to amino acid levels. Under nonstarvation conditions, the uORFs restrict scanning ribosomes from reaching the GCN4 start codon; in amino acid-starved cells, this translational barrier is overcome, leading to increased GCN4 protein synthesis (reviewed in reference 26).Multiple trans-acting factors have also been implicated in translational control of GCN4 expression. GCD genes were defined genetically as negative regulatory factors that are r...

GCN4 encodes a transcriptional activator of amino acid biosynthetic genes in Saccharomyces cerevisiae. The GCN3 product is a positive regulator required for increased synthesis of GCN4 protein in amino acid-starved cells. GCN3 appears to act indirectly by antagonizing GCD-encoded negative regulators of GCN4 expression under starvation conditions; however, GCN3 can also suppress the effects of gedi and gcdl2 mutations under nonstarvation conditions. These results imply that the GCN3 product can promote either repression or activation of GCN4 expression depending on amino acid availability. We present a complete physical description of the GCN3 gene and its transcript, plus measurements of GCN3 expression at the transcriptional and translational levels under different growth conditions. GCN3 encodes a 305-amino-acid polypeptide with no significant homology to any other known protein sequence. GCN3 mRNA contains no leader AUG codons, and no potential GCN4 binding sites were found in GCN3 5' noncoding DNA. In accord with the absence of these regulatory sequences found at other genes in the general control system, GCN3 mRNA and a GCN3-lacZ fusion enzyme are present at similar levels under both starvation and nonstarvation conditions. These data suggest that modulation of GCN3 regulatory function in response to amino acid availability occurs posttranslationaliy. A gcn3 deletion leads to unconditional lethality in a gcdl-1O1 mutant, supporting the idea that GCN3 is expressed under normal growth conditions and cooperates with the GCD1 product under these circumstances to carry out an essential cellular function. We describe a point mutation that adds three amino acids to the carboxyl terminus of GCN3, which inactivates its positive regulatory function required under starvation conditions without impairing its ability to promote functions carried out by GCD12 under nonstarvation conditions.Expression of amino-acid-biosynthetic genes in the yeast Saccharomyces cerevisiae is regulated by at least two mechanisms. The first involves pathway-specific repression by the amino acid end products of certain pathways. A second mechanism, known as general amino acid control, leads to increased transcription of at least 30 genes encoding enzymes in nine different pathways in response to starvation for any single amino acid. The products of nine GCN genes are required for derepression of structural genes subject to general control under starvation conditions. The products of 12 GCD genes are required for repression of these genes under normal growth conditions. Studies of epistasis relationships among regulatory mutations suggest that the products of GCNI, GCN2, and GCN3 act indirectly as positive effectors by negative regulation of GCD gene products (for a review, see reference 19). GCN4, identified by this genetic analysis as the most direct positive regulator in the general control system, was shown to function as a transcriptional activator by binding to regulatory sequences located upstream from structural genes subject to the ge...

Eukaryotic translation initiation factor (eIF) 5 is crucial for the assembly of the eukaryotic preinitiation complex. This activity is mediated by the ability of its C-terminal HEAT domain to interact with eIF1, eIF2, and eIF3 in the multifactor complex and with eIF4G in the 48S complex. However, the binding sites for these factors on eIF5-C-terminal domain (CTD) have not been known. Here we present a homology model for eIF5-CTD based on the HEAT domain of eIF2B. We show that the binding site for eIF2␤ is located in a surface area containing aromatic and acidic residues (aromatic͞ acidic boxes), that the binding sites for eIF1 and eIF3c are located in a conserved surface region of basic residues, and that eIF4G binds eIF5-CTD at an interface overlapping with the acidic area. Mutations in these distinct eIF5 surface areas impair GCN4 translational control by disrupting preinitiation complex interactions. These results indicate that the eIF5 HEAT domain is a critical nucleation core for preinitiation complex assembly and function.general amino acid control ͉ ribosome preinitiation complex ͉ translation initiation ͉ translational control I n eukaryotic translation initiation, the 40S ribosomal subunit binds Met-tRNA i Met , 5Ј-capped mRNA, and the 60S subunit in a coordinated manner, setting up the 80S initiation complex with the anticodon of Met-tRNA i Met base-paired at the ribosomal P site to the first start codon of the mRNA (for review, see ref. 1). At least 11 eukaryotic initiation factors (eIFs) mediate this process. MettRNA iMet binds the 40S subunit in a ternary complex (TC) with eIF2 and GTP to form the 43S preinitiation complex. Subsequent joining of the 43S particle to the mRNA͞eIF4F assembly produces the 48S preinitiation complex, which then scans for the first AUG codon. Correct AUG pairing with the Met-tRNA i Met anticodon triggers eIF5-dependent GTP hydrolysis for eIF2, leading to dissociation of the eIFs and formation of the 40S initiation complex. The GDPbound eIF2 that is released after GTP hydrolysis is recycled to eIF2-GTP by the pentameric guanylate exchange factor eIF2B.The C-terminal domain (CTD) of eIF5 is an important nucleation core of the preinitiation complex assembly and mediates formation of the multifactor complex (MFC) with eIF1, eIF2 TC, and eIF3 (2, 3). It contains unique aromatic͞acidic boxes (AA boxes) 1 and 2. These are also found in the CTDs of eIF2B (the catalytic subunit of eIF2B) and mammalian eIF4G (4). The AA boxes in eIF5 and eIF2B are required for binding to the lysine-rich segment [lysine box (K box)] present in the N-terminal domain of the common substrate, the ␤ subunit of eIF2 (4). The ability of eIF5-CTD to bind eIF3c is strongly enhanced by its interaction with the eIF2␤ K box, then leading to rapid and tight MFC assembly (5).The integrity of the translation initiation machinery is critical for proper cellular response to different stress stimuli (6). In yeast, amino acid starvation activates Gcn2p kinase to phosphorylate eIF2, rendering eIF2 a competitive inhibit...