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Cotranscriptional methylation of histone H3 lysines 4 and 36 by Set1 and Set2, respectively, stimulates interaction between nucleosomes and histone deacetylase complexes to block cryptic transcription in budding yeast. We previously showed that loss of all H3K4 and H3K36 methylation in a set1Δset2Δ mutant reduces interaction between native nucleosomes and the NuA4 lysine acetyltransferase (KAT) complex. We now provide evidence that NuA4 preferentially binds H3 tails mono- and dimethylated on H3K4 and di- and trimethylated on H3K36, an H3 methylation pattern distinct from that recognized by the RPD3C(S) and Hos2/Set3 histone deacetylase complexes (HDACs). Loss of H3K4 or H3K36 methylation in set1Δ or set2Δ mutants reduces NuA4 interaction with bulk nucleosomes in vitro and in vivo, and reduces NuA4 occupancy of transcribed coding sequences at particular genes. We also provide evidence that NuA4 acetylation of lysine residues in the histone H4 tail stimulates SAGA interaction with nucleosomes and its recruitment to coding sequences and attendant acetylation of histone H3 in vivo. Thus, H3 methylation exerts opposing effects of enhancing nucleosome acetylation by both NuA4 and SAGA as well as stimulating nucleosome deacetylation by multiple HDACs to maintain the proper level of histone acetylation in transcribed coding sequences., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

eIF4B has been implicated in attachment of the 43 S preinitiation complex (PIC) to mRNAs and scanning to the start codon. We recently determined that the internal seven repeats (of ∼26 amino acids each) of Saccharomyces cerevisiae eIF4B (yeIF4B) compose the region most critically required to enhance mRNA recruitment by 43 S PICs in vitro and stimulate general translation initiation in yeast. Moreover, although the N-terminal domain (NTD) of yeIF4B contributes to these activities, the RNA recognition motif is dispensable. We have now determined that only two of the seven internal repeats are sufficient for wild-type (WT) yeIF4B function in vivo when all other domains are intact. However, three or more repeats are needed in the absence of the NTD or when the functions of eIF4F components are compromised. We corroborated these observations in the reconstituted system by demonstrating that yeIF4B variants with only one or two repeats display substantial activity in promoting mRNA recruitment by the PIC, whereas additional repeats are required at lower levels of eIF4A or when the NTD is missing. These findings indicate functional overlap among the 7-repeats and NTD domains of yeIF4B and eIF4A in mRNA recruitment. Interestingly, only three highly conserved positions in the 26-amino acid repeat are essential for function in vitro and in vivo. Finally, we identified conserved motifs in the NTD and demonstrate functional overlap of two such motifs. These results provide a comprehensive description of the critical sequence elements in yeIF4B that support eIF4F function in mRNA recruitment by the PIC.

Recognition of the translation initiation codon is thought to require dissociation of eIF1 from the 40 S ribosomal subunit, enabling irreversible GTP hydrolysis (Pi release) by the eIF2·GTP·Met-tRNAi ternary complex (TC), rearrangement of the 40 S subunit to a closed conformation incompatible with scanning, and stable binding of Met-tRNAi to the P site. The crystal structure of a Tetrahymena 40 S·eIF1 complex revealed several basic amino acids in eIF1 contacting 18 S rRNA, and we tested the prediction that their counterparts in yeast eIF1 are required to prevent premature eIF1 dissociation from scanning ribosomes at non-AUG triplets. Supporting this idea, substituting Lys-60 in helix α1, or either Lys-37 or Arg-33 in β-hairpin loop-1, impairs binding of yeast eIF1 to 40 S·eIF1A complexes in vitro, and it confers increased initiation at UUG codons (Sui(-) phenotype) or lethality, in a manner suppressed by overexpressing the mutant proteins or by an eIF1A mutation (17-21) known to impede eIF1 dissociation in vitro. The eIF1 Sui(-) mutations also derepress translation of GCN4 mRNA, indicating impaired ternary complex loading, and this Gcd(-) phenotype is likewise suppressed by eIF1 overexpression or the 17-21 mutation. These findings indicate that direct contacts of eIF1 with 18 S rRNA seen in the Tetrahymena 40 S·eIF1 complex are crucial in yeast to stabilize the open conformation of the 40 S subunit and are required for rapid TC loading and ribosomal scanning and to impede rearrangement to the closed complex at non-AUG codons. Finally, we implicate the unstructured N-terminal tail of eIF1 in blocking rearrangement to the closed conformation in the scanning preinitiation complex.

Accurate recognition of the start codon in an mRNA by the eukaryotic translation preinitiation complex (PIC) is essential for proper gene expression. The process is mediated by eukaryotic translation initiation factors (eIFs) in conjunction with the 40 S ribosomal subunit and (initiator) tRNA(i). Here, we provide evidence that the C-terminal tail (CTT) of eIF1A, which we previously implicated in start codon recognition, moves closer to the N-terminal domain of eIF5 when the PIC encounters an AUG codon. Importantly, this movement is coupled to dissociation of eIF1 from the PIC, a critical event in start codon recognition, and is dependent on the scanning enhancer elements in the eIF1A CTT. The data further indicate that eIF1 dissociation must be accompanied by the movement of the eIF1A CTT toward eIF5 in order to trigger release of phosphate from eIF2, which converts the latter to its GDP-bound state. Our results also suggest that release of eIF1 from the PIC and movement of the CTT of eIF1A are triggered by the same event, most likely accommodation of tRNA(i) in the P site of the 40 S subunit driven by base pairing between the start codon in the mRNA and the anticodon in tRNA(i). Finally, we show that the C-terminal domain of eIF5 is responsible for the factor's activity in antagonizing eIF1 binding to the PIC. Together, our data provide a more complete picture of the chain of molecular events that is triggered when the scanning PIC encounters an AUG start codon in the mRNA.

Translation initiation factor eIF4F (eukaryotic initiation factor 4F), composed of eIF4E, eIF4G, and eIF4A, binds to the m(7)G cap structure of mRNA and stimulates recruitment of the 43S preinitiation complex and subsequent scanning to the initiation codon. The HEAT domain of eIF4G stabilizes the active conformation of eIF4A required for its RNA helicase activity. Mammalian eIF4B also stimulates eIF4A activity, but this function appears to be lacking in yeast, making it unclear how yeast eIF4B (yeIF4B/Tif3) stimulates translation. We identified Ts(-) mutations in the HEAT domains of yeast eIF4G1 and eIF4G2 that are suppressed by overexpressing either yeIF4B or eIF4A, whereas others are suppressed only by eIF4A overexpression. Importantly, suppression of HEAT domain substitutions by yeIF4B overexpression was correlated with the restoration of native eIF4A·eIF4G complexes in vivo, and the rescue of specific mutant eIF4A·eIF4G complexes by yeIF4B was reconstituted in vitro. Association of eIF4A with WT eIF4G in vivo also was enhanced by yeIF4B overexpression and was impaired in cells lacking yeIF4B. Furthermore, we detected native complexes containing eIF4G and yeIF4B but lacking eIF4A. These and other findings lead us to propose that yeIF4B acts in vivo to promote eIF4F assembly by enhancing a conformation of the HEAT domain of yeast eIF4G conducive for stable binding to eIF4A.

In eukaryotes, phosphorylation of translation initiation factor 2α (eIF2α) by the kinase Gcn2 (general control nonderepressible 2) is a key response to amino acid starvation. Sensing starvation requires that Gcn2 directly contacts its effector protein Gcn1, and both must contact the ribosome. We have proposed that Gcn2 is activated by uncharged tRNA bound to the ribosomal decoding (A) site, in a manner facilitated by ribosome-bound Gcn1. Protein synthesis requires cyclical association of eukaryotic elongation factors (eEFs) with the ribosome. Gcn1 and Gcn2 are large proteins, raising the question of whether translation and monitoring amino acid availability can occur on the same ribosome. Part of the ribosome-binding domain in Gcn1 has homology to one of the ribosome-binding domains in eEF3, suggesting that these proteins utilize overlapping binding sites on the ribosome and consequently cannot function simultaneously on the same ribosome. Supporting this idea, we found that eEF3 overexpression in Saccharomyces cerevisiae diminished growth on amino acid starvation medium (Gcn(-) phenotype) and decreased eIF2α phosphorylation, and that the growth defect associated with constitutively active Gcn2 was diminished by eEF3 overexpression. Overexpression of the eEF3 HEAT domain, or C terminus, was sufficient to confer a Gcn(-) phenotype, and both fragments have ribosome affinity. eEF3 overexpression did not significantly affect Gcn1-ribosome association, but it exacerbated the Gcn(-) phenotype of Gcn1-M7A that has reduced ribosome affinity. Together, this suggests that eEF3 blocks Gcn1 regulatory function on the ribosome. We propose that the Gcn1-Gcn2 complex only functions on ribosomes with A-site-bound uncharged tRNA, because eEF3 does not occupy these stalled complexes.

During eukaryotic translation initiation, the 43 S ribosomal pre-initiation complex is recruited to the 5'-end of an mRNA through its interaction with the 7-methylguanosine cap, and it subsequently scans along the mRNA to locate the start codon. Both mRNA recruitment and scanning require the removal of secondary structure within the mRNA. Eukaryotic translation initiation factor 4A is an essential component of the translational machinery thought to participate in the clearing of secondary structural elements in the 5'-untranslated regions of mRNAs. eIF4A is part of the 5'-7-methylguanosine cap-binding complex, eIF4F, along with eIF4E, the cap-binding protein, and the scaffolding protein eIF4G. Here, we show that Saccharomyces cerevisiae eIF4F has a strong preference for unwinding an RNA duplex with a single-stranded 5'-overhang versus the same duplex with a 3'-overhang or without an overhang. In contrast, eIF4A on its own has little RNA substrate specificity. Using a series of deletion constructs of eIF4G, we demonstrate that its three previously elucidated RNA binding domains work together to provide eIF4F with its 5'-end specificity, both by promoting unwinding of substrates with 5'-overhangs and inhibiting unwinding of substrates with 3'-overhangs. Our data suggest that the RNA binding domains of eIF4G provide the S. cerevisiae eIF4F complex with a second mechanism, in addition to the eIF4E-cap interaction, for directing the binding of pre-initiation complexes to the 5'-ends of mRNAs and for biasing scanning in the 5' to 3' direction.

The eukaryotic elongation factor 1A (eEF1A) delivers aminoacyl-tRNAs to the ribosomal A-site during protein synthesis. To ensure a continuous supply of amino acids, cells harbor the kinase Gcn2 and its effector protein Gcn1. The ultimate signal for amino acid shortage is uncharged tRNAs. We have proposed a model for sensing starvation, in which Gcn1 and Gcn2 are tethered to the ribosome, and Gcn1 is directly involved in delivering uncharged tRNAs from the A-site to Gcn2 for its subsequent activation. Gcn1 and Gcn2 are large proteins, and these proteins as well as eEF1A access the A-site, leading us to investigate whether there is a functional or physical link between these proteins. Using Saccharomyces cerevisiae cells expressing His(6)-eEF1A and affinity purification, we found that eEF1A co-eluted with Gcn2. Furthermore, Gcn2 co-immunoprecipitated with eEF1A, suggesting that they reside in the same complex. The purified GST-tagged Gcn2 C-terminal domain (CTD) was sufficient for precipitating eEF1A from whole cell extracts generated from gcn2Δ cells, independently of ribosomes. Purified GST-Gcn2-CTD and purified His(6)-eEF1A interacted with each other, and this was largely independent of the Lys residues in Gcn2-CTD known to be required for tRNA binding and ribosome association. Interestingly, Gcn2-eEF1A interaction was diminished in amino acid-starved cells and by uncharged tRNAs in vitro, suggesting that eEF1A functions as a Gcn2 inhibitor. Consistent with this possibility, purified eEF1A reduced the ability of Gcn2 to phosphorylate its substrate, eIF2α, but did not diminish Gcn2 autophosphorylation. These findings implicate eEF1A in the intricate regulation of Gcn2 and amino acid homeostasis.

Yeast Yih1 protein and its mammalian ortholog IMPACT, abundant in neurons, are inhibitors of Gcn2, a kinase involved in amino acid homeostasis, stress response, and memory formation. Like Gcn2, Yih1/IMPACT harbors an N-terminal RWD domain that mediates binding to the Gcn2 activator Gcn1. Yih1 competes with Gcn2 for Gcn1 binding, thus inhibiting Gcn2. Yih1 also binds G-actin. Here, we show that Yih1-actin interaction is independent of Gcn1 and that Yih1-Gcn1 binding does not require actin. The Yih1 RWD (residues 1-132) was sufficient for Gcn2 inhibition and Gcn1 binding, but not for actin binding, showing that actin binding is dispensable for inhibiting Gcn2. Actin binding required Yih1 residues 68-258, encompassing part of the RWD and the C-terminal "ancient domain"; however, residues Asp-102 and Glu-106 in helix3 of the RWD were essential for Gcn1 binding and Gcn2 inhibition but dispensable for actin binding. Thus, the Gcn1- and actin-binding sites overlap in the RWD but have distinct binding determinants. Unexpectedly, Yih1 segment 68-258 was defective for inhibiting Gcn2 even though it binds Gcn1 at higher levels than does full-length Yih1. This and other results suggest that Yih1 binds with different requirements to distinct populations of Gcn1 molecules, and its ability to disrupt Gcn1-Gcn2 complexes is dependent on a complete RWD and hindered by actin binding. Modeling of the ancient domain on the bacterial protein YigZ showed peculiarities to the eukaryotic and prokaryotic lineages, suggesting binding sites for conserved cellular components. Our results support a role for Yih1 in a cross-talk between the cytoskeleton and translation.

Mediator is a multisubunit coactivator required for initiation by RNA polymerase II. The Mediator tail subdomain, containing Med15/Gal11, is a target of the activator Gcn4 in vivo, critical for recruitment of native Mediator or the Mediator tail subdomain present in sin4Delta cells. Although several Gal11 segments were previously shown to bind Gcn4 in vitro, the importance of these interactions for recruitment of Mediator and transcriptional activation by Gcn4 in cells was unknown. We show that interaction of Gcn4 with the Mediator tail in vitro and recruitment of this subcomplex and intact Mediator to the ARG1 promoter in vivo involve additive contributions from three different segments in the N terminus of Gal11. These include the KIX domain, which is a critical target of other activators, and a region that shares a conserved motif (B-box) with mammalian coactivator SRC-1, and we establish that B-box is a critical determinant of Mediator recruitment by Gcn4. We further demonstrate that Gcn4 binds to the Gal11 KIX domain directly and, by NMR chemical shift analysis combined with mutational studies, we identify the likely binding site for Gcn4 on the KIX surface. Gcn4 is distinctive in relying on comparable contributions from multiple segments of Gal11 for efficient recruitment of Mediator in vivo.

The ABCE1 gene is a member of the ATP-binding cassette (ABC) multigene family and is composed of two nucleotide binding domains and an N-terminal Fe-S binding domain. The ABCE1 gene encodes a protein originally identified for its inhibition of ribonuclease L, a nuclease induced by interferon in mammalian cells. The protein is also required for the assembly of the HIV and SIV gag polypeptides. However, ABCE1 is one of the most highly conserved proteins and is found in one or two copies in all characterized eukaryotes and archaea. Yeast ABCE1/RLI1 is essential to cell division and interacts with translation initiation factors in the assembly of the pre-initiation complex. We show here that the human ABCE1 protein is essential for in vitro and in vivo translation of mRNA and that it binds to eIF2alpha and eIF5. Inhibition of the Xenopus ABCE1 arrests growth at the gastrula stage of development, consistent with a block in translation. The human ABCE1 gene contains 16 introns, and the extremely high degree of amino acid identity allows the evolution of its introns to be examined throughout eukaryotes. The demonstration that ABCE1 plays a role in vertebrate translation initiation extends the known functions of this highly conserved protein. Translation is a highly regulated process important to development and pathologies such as cancer, making ABCE1 a potential target for therapeutics. The evolutionary analysis supports a model in which an ancestral eukaryote had large number of introns and that many of these introns were lost in non-vertebrate lineages.

The GCN2 protein kinase coordinates protein synthesis with levels of amino acid stores by phosphorylating eukaryotic translation initiation factor 2. The autoinhibited form of GCN2 is activated in cells starved of amino acids by binding of uncharged tRNA to a histidyl-tRNA synthetase-like domain. Replacement of Arg-794 with Gly in the PK domain (R794G) activates GCN2 independently of tRNA binding. Crystal structures of the GCN2 protein kinase domain have been determined for wild-type and R794G mutant forms in the apo state and bound to ATP/AMPPNP. These structures reveal that GCN2 autoinhibition results from stabilization of a closed conformation that restricts ATP binding. The R794G mutant shows increased flexibility in the hinge region connecting the N- and C-lobes, resulting from loss of multiple interactions involving Arg794. This conformational change is associated with intradomain movement that enhances ATP binding and hydrolysis. We propose that intramolecular interactions following tRNA binding remodel the hinge region in a manner similar to the mechanism of enzyme activation elicited by the R794G mutation.

Translational control directed by the eukaryotic translation initiation factor 2 alpha-subunit (eIF2alpha) kinase GCN2 is important for coordinating gene expression programs in response to nutritional deprivation. The GCN2 stress response, conserved from yeast to mammals, is critical for resistance to nutritional deficiencies and for the control of feeding behaviors in rodents. The mouse protein IMPACT has sequence similarities to the yeast YIH1 protein, an inhibitor of GCN2. YIH1 competes with GCN2 for binding to a positive regulator, GCN1. Here, we present evidence that IMPACT is the functional counterpart of YIH1. Overexpression of IMPACT in yeast lowered both basal and amino acid starvation-induced levels of phosphorylated eIF2alpha, as described for YIH1 (31). Overexpression of IMPACT in mouse embryonic fibroblasts inhibited phosphorylation of eIF2alpha by GCN2 under leucine starvation conditions, abolishing expression of its downstream target genes, ATF4 (CREB-2) and CHOP (GADD153). IMPACT bound to the minimal yeast GCN1 segment required for interaction with yeast GCN2 and YIH1 and to native mouse GCN1. At the protein level, IMPACT was detected mainly in the brain. IMPACT was found to be abundant in the majority of hypothalamic neurons. Scattered neurons expressing this protein at higher levels were detected in other regions such as the hippocampus and piriform cortex. The abundance of IMPACT correlated inversely with phosphorylated eIF2alpha levels in different brain areas. These results suggest that IMPACT ensures constant high levels of translation and low levels of ATF4 and CHOP in specific neuronal cells under amino acid starvation conditions.

The protein kinase GCN2 mediates translational control of gene expression in amino acid-starved cells by phosphorylating eukaryotic translation initiation factor 2alpha. In Saccharomyces cerevisiae, activation of GCN2 by uncharged tRNAs in starved cells requires its direct interaction with both the GCN1.GCN20 regulatory complex and ribosomes. GCN1 also interacts with ribosomes in cell extracts, but it was unknown whether this activity is crucial for its ability to stimulate GCN2 function in starved cells. We describe point mutations in two conserved, noncontiguous segments of GCN1 that lead to reduced polyribosome association by GCN1.GCN20 in living cells without reducing GCN1 expression or its interaction with GCN20. Mutating both segments simultaneously produced a greater reduction in polyribosome binding by GCN1.GCN20 and a stronger decrease in eukaryotic translation initiation factor 2alpha phosphorylation than did mutating in one segment alone. These findings provide strong evidence that ribosome binding by GCN1 is required for its role as a positive regulator of GCN2. A particular mutation in the GCN1 domain, related in sequence to translation elongation factor 3 (eEF3), decreased GCN2 activation much more than it reduced ribosome binding by GCN1. Hence, the eEF3-like domain appears to have an effector function in GCN2 activation. This conclusion supports the model that an eEF3-related activity of GCN1 influences occupancy of the ribosomal decoding site by uncharged tRNA in starved cells.

RLI1 is an essential yeast protein closely related in sequence to two soluble members of the ATP-binding cassette family of proteins that interact with ribosomes and function in translation elongation (YEF3) or translational control (GCN20). We show that affinity-tagged RLI1 co-purifies with eukaryotic translation initiation factor 3 (eIF3), eIF5, and eIF2, but not with other translation initiation factors or with translation elongation or termination factors. RLI1 is associated with 40 S ribosomal subunits in vivo, but it can interact with eIF3 and -5 independently of ribosomes. Depletion of RLI1 in vivo leads to cessation of growth, a lower polysome content, and decreased average polysome size. There was also a marked reduction in 40 S-bound eIF2 and eIF1, consistent with an important role for RLI1 in assembly of 43 S preinitiation complexes in vivo. Mutations of conserved residues in RLI1 expected to function in ATP hydrolysis were lethal. A mutation in the second ATP-binding cassette domain of RLI1 had a dominant negative phenotype, decreasing the rate of translation initiation in vivo, and the mutant protein inhibited translation of a luciferase mRNA reporter in wild-type cell extracts. These findings are consistent with a direct role for the ATP-binding cassettes of RLI1 in translation initiation. RLI1-depleted cells exhibit a deficit in free 60 S ribosomal subunits, and RLI1-green fluorescent protein was found in both the nucleus and cytoplasm of living cells. Thus, RLI1 may have dual functions in translation initiation and ribosome biogenesis.

The general amino acid control (GAAC) enables yeast cells to overcome amino acid deprivation by activation of the alpha subunit of translation initiation factor 2 (eIF2alpha) kinase GCN2 and consequent induction of GCN4, a transcriptional activator of amino acid biosynthetic genes. Binding of GCN2 to GCN1 is required for stimulation of GCN2 kinase activity by uncharged tRNA in starved cells. Here we show that YIH1, when overexpressed, dampens the GAAC response (Gcn- phenotype) by suppressing eIF2alpha phosphorylation by GCN2. The overexpressed YIH1 binds GCN1 and reduces GCN1-GCN2 complex formation, and, consistent with this, the Gcn- phenotype produced by YIH1 overexpression is suppressed by GCN2 overexpression. YIH1 interacts with the same GCN1 fragment that binds GCN2, and this YIH1-GCN1 interaction requires Arg-2259 in GCN1 in vitro and in full-length GCN1 in vivo, as found for GCN2-GCN1 interaction. However, deletion of YIH1 does not increase eIF2alpha phosphorylation or derepress the GAAC, suggesting that YIH1 at native levels is not a general inhibitor of GCN2 activity. We discovered that YIH1 normally resides in a complex with monomeric actin, rather than GCN1, and that a genetic reduction in actin levels decreases the GAAC response. This Gcn- phenotype was partially suppressed by deletion of YIH1, consistent with YIH1-mediated inhibition of GCN2 in actin-deficient cells. We suggest that YIH1 resides in a YIH1-actin complex and may be released for inhibition of GCN2 and stimulation of protein synthesis under specialized conditions or in a restricted cellular compartment in which YIH1 is displaced from monomeric actin.

The translation elongation machinery in fungi differs from other eukaryotes in its dependence upon eukaryotic elongation factor 3 (eEF3). eEF3 is essential in vivo and required for each cycle of the translation elongation process in vitro. Models predict eEF3 affects the delivery of cognate aminoacyl-tRNA, a function performed by eEF1A, by removing deacylated tRNA from the ribosomal Exit site. To dissect eEF3 function and its link to the A-site activities of eEF1A, we have identified a temperature-sensitive allele of the YEF3 gene. The F650S substitution, located between the two ATP binding cassettes, reduces both ribosome-dependent and intrinsic ATPase activities. In vivo this mutation increases sensitivity to aminoglycosidic drugs, causes a 50% reduction of total protein synthesis at permissive temperatures, slows run-off of polyribosomes, and reduces binding to eEF1A. Reciprocally, excess eEF3 confers synthetic slow growth, increased drug sensitivity, and reduced translation in an allele specific fashion with an E122K mutation in the GTP binding domain of eEF1A. In addition, this mutant form of eEF1A shows reduced binding of eEF3. Thus, optimal in vivo interactions between eEF3 and eEF1A are critical for protein synthesis.

Protein kinase GCN2 regulates translation initiation by phosphorylating eukaryotic initiation factor 2alpha (eIF2alpha), impeding general protein synthesis but specifically inducing translation of GCN4, a transcriptional activator of amino acid biosynthetic genes in Saccharomyces cerevisiae. GCN2 activity is stimulated in amino acid-deprived cells through binding of uncharged tRNA to a domain related to histidyl tRNA synthetase. We show that GCN2 is phosphorylated by another kinase on serine 577, located N-terminal to the kinase domain. Mutation of Ser-577 to alanine produced partial activation of GCN2 in nonstarved cells, increasing the level of phosphorylated eIF2alpha, derepressing GCN4 expression, and elevating the cellular levels of tryptophan and histidine. The Ala-577 mutation also increased the tRNA binding affinity of purified GCN2, which can account for the elevated kinase activity of GCN2-S577A in nonstarved cells where uncharged tRNA levels are low. Whereas Ser-577 remains phosphorylated in amino acid-starved cells, its dephosphorylation could mediate GCN2 activation in other stress or starvation conditions by lowering the threshold of uncharged tRNA required to activate the protein.

eIF3j/Hcr1p, a protein associated with eIF3, was shown to bind to, and stabilize, the multifactor complex containing eIFs 1, 2, 3, and 5 and Met-tRNA(i)(Met), whose formation is required for an optimal rate of translation initiation. Here we present evidence that eIF3j/Hcr1p is an RNA binding protein that enhances a late step in 40 S ribosome maturation involving cleavage of the 20 S precursor of 18 S rRNA in the cytoplasm. Immunofluorescence staining shows that eIF3j/Hcr1p is localized predominantly in the cytoplasm. The hcr1Delta mutant exhibits a decreased amount of 40 S subunits, hypersensitivity to paromomycin, and increased levels of 20 S pre-rRNA. Combining the hcr1Delta mutation with drs2Delta or rps0aDelta, deletions of two other genes involved in the same step of 40 S subunit biogenesis, produced a synthetic growth defect. p35, the human ortholog of eIF3j/Hcr1p, partially complemented the slow growth phenotype conferred by hcr1Delta when overexpressed in yeast. heIF3j/p35 was found physically associated with yeast eIF3 and 43 S initiation complexes in vitro and in vivo. Because it did not complement the 40 S biogenesis defect of hcr1Delta, it appears that heIF3j can substitute for eIF3j/Hcr1p only in translation initiation. We conclude that eIF3j/Hcr1p is required for rapid processing of 20 S to 18 S rRNA besides its role in translation initiation, providing an intriguing link between ribosome biogenesis and translation.

Mammalian, plant, and Schizosaccharomyces pombe eukaryotic initiation factor-3 (eIF3) contains a protein homologous to the product of int-6 (eIF3e), a frequent integration site of mouse mammary tumor viruses. By contrast, Saccharomyces cerevisiae does not encode a protein closely related to eIF3e/Int-6. Here, we characterize a novel S. cerevisiae protein (Pci8p, Yil071cp) that contains a PCI (proteasome-COP9 signalosome-eIF3) domain conserved in eIF3e/Int-6. We show that both Pci8p and human eIF3e/Int-6 expressed in budding yeast interact with the yeast eIF3 complex in vivo and in vitro by binding to a discrete segment of its eIF3b subunit Prt1p and that human eIF3e/Int-6 interacts with the human eIF3b segment homologous to the Pci8p-binding site of yeast Prt1p. These results refine our understanding of subunit interactions in the eIF3 complex and suggest structural similarity between human eIF3e/Int-6 and yeast Pci8p. However, deletion of PCI8 had no discernible effect on cell growth or translation initiation as judged by polysome analysis, suggesting that Pci8p is not required for the essential function of eIF3 in translation initiation. Motivated by the involvement of Int-6 in transcriptional control, we investigated the effects of deleting PCI8 on the total mRNA expression profile by oligonucleotide microarray analysis and found reduced mRNA levels for a subset of heat shock proteins in the pci8Delta mutant. We discuss possible dual functions of Pci8p and Int-6 in transcriptional and translational control.

Protein kinase PKR is activated by double-stranded RNA (dsRNA) and phosphorylates translation initiation factor 2alpha to inhibit protein synthesis in virus-infected mammalian cells. PKR contains two dsRNA binding motifs (DRBMs I and II) required for activation by dsRNA. There is strong evidence that PKR activation requires dimerization, but the role of dsRNA in dimer formation is controversial. By making alanine substitutions predicted to remove increasing numbers of side chain contacts between the DRBMs and dsRNA, we found that dimerization of full-length PKR in yeast was impaired by the minimal combinations of mutations required to impair dsRNA binding in vitro. Mutation of Ala-67 to Glu in DRBM-I, reported to abolish dimerization without affecting dsRNA binding, destroyed both activities in our assays. By contrast, deletion of a second dimerization region that overlaps the kinase domain had no effect on PKR dimerization in yeast. Human PKR contains at least 15 autophosphorylation sites, but only Thr-446 and Thr-451 in the activation loop were found here to be critical for kinase activity in yeast. Using an antibody specific for phosphorylated Thr-451, we showed that Thr-451 phosphorylation is stimulated by dsRNA binding. Our results provide strong evidence that dsRNA binding is required for dimerization of full-length PKR molecules in vivo, leading to autophosphorylation in the activation loop and stimulation of the eIF2alpha kinase function of PKR.

The murine int-6 locus, identified as a frequent integration site of mouse mammary tumor viruses, encodes the 48-kDa eIF3e subunit of translation initiation factor eIF3. Previous studies indicated that the catalytically active core of budding yeast eIF3 consists of five subunits, all conserved in eukaryotes, but does not contain a protein closely related to eIF3e/Int-6. Whereas the budding yeast genome does not encode a protein closely related to murine Int-6, fission yeast does encode an Int-6 ortholog, designated here Int6. We found that fission yeast Int6/eIF3e is a cytoplasmic protein associated with 40 S ribosomes. FLAG epitope-tagged Tif35, a putative core eIF3g subunit, copurified with Int6 and all five orthologs of core eIF3 subunits. An int6 deletion (int6Delta) mutant was viable but grew slowly in minimal medium. This slow growth phenotype was accompanied by a reduction in the amount of polyribosomes engaged in translation and was complemented by expression of human Int-6 protein. These findings support the idea that human and Schizosaccharomyces pombe Int-6 homologs are involved in translation. Interestingly, haploid int6Delta cells showed unequal nuclear partitioning, possibly because of a defect in tubulin function, and diploid int6Delta cells formed abnormal spores. We propose that Int6 is not an essential subunit of eIF3 but might be involved in regulating the activity of eIF3 for translation of specific mRNAs in S. pombe.

Eukaryotic translation initiation factor 2B (eIF2B) is the heteropentameric guanine nucleotide exchange factor for translation initiation factor 2 (eIF2). Recent studies in the yeast Saccharomyces cerevisiae have served to characterize genetically the exchange factor. However, enzyme kinetic studies of the yeast enzyme have been hindered by the lack of sufficient quantities of protein suitable for biochemical analysis. We have purified yeast eIF2B and characterized its catalytic properties in vitro. Values for K(m) and V(max) were determined to be 12.2 nm and 250.7 fmol/min, respectively, at 0 degrees C. The calculated turnover number (K(cat)) of 43.2 pmol of GDP released per min/pmol of eIF2B at 30 degrees C is approximately 1 order of magnitude lower than values previously reported for the mammalian factor. Reciprocal plots at varying fixed concentrations of the second substrate were linear and intersected to the left of the y axis. This is consistent with a sequential catalytic mechanism and argues against a ping-pong mechanism similar to that proposed for EF-Tu/EF-Ts. In support of this model, our yeast eIF2B preparations bind guanine nucleotides, with an apparent dissociation constant for GTP in the low micromolar range.

In amino acid-starved yeast cells, inhibition of the guanine nucleotide exchange factor eIF2B by phosphorylated translation initiation factor 2 results in increased translation of GCN4 mRNA. We isolated a suppressor of a mutant eIF2B. The suppressor prevents efficient GCN4 mRNA translation due to inactivation of the small ribosomal subunit protein Rps31 and results in low amounts of mutant 40 S ribosomal subunits. Deletion of one of two genes encoding ribosomal protein Rps17 also reduces the amounts of 40 S subunits but does not suppress eIF2B mutations or prevent efficient GCN4 translation. Our findings show that Rps31-deficient ribosomes are altered in a way that decreases the eIF2B requirement and that the small ribosomal subunit mediates the effects of low eIF2B activity on cell viability and translational regulation in response to eIF2 phosphorylation.

TFIID is a multiprotein complex composed of TBP and several TAFIIs. Small amino-terminal segments (TAF N-terminal domain (TAND)) of Drosophila TAFII230 (dTAFII230) and yeast TAFII145 (yTAFII145) bind strongly to TBP and inhibit TBP-DNA interactions. yTAFII145 TAND (yTAND) was divided into two subdomains, yTANDI10-37 and yTANDII46-71, that function cooperatively. Here, we identify dTANDII within the amino terminus of dTAFII230 at 118-143 amino acids in addition to dTANDI18-77, reported previously. dTANDII exhibits pronounced sequence similarity to yTANDII, and the two were shown to be functionally equivalent in binding to TBP and inhibiting TBP-DNA interactions in vitro. Alanine scanning mutation analysis demonstrated that Phe-57 (yTANDII) and Tyr-129 (dTANDII) are critically required for the interaction with TBP. Yeast strains containing mutant yTAFII145 lacking yTANDI or yTANDII showed a temperature-sensitive growth phenotype. The conserved core of dTANDII could substitute for the yTANDII core, and Phe-57 or Tyr-129 described above was critically required for the function of this segment in promoting normal cell growth at 37 degreesC. In these respects, the impact of yTANDII mutations on cell growth paralleled their effects on TBP binding in vitro, strongly suggesting that the yTAFII145-TBP interaction and its negative effects on TFIID binding to core promoters are physiologically important.

Nip1p is an essential Saccharomyces cerevisiae protein that was identified in a screen for temperature conditional (ts) mutants exhibiting defects in nuclear transport. New results indicate that Nip1p has a primary role in translation initiation. Polysome profiles indicate that cells depleted of Nip1p and nip1-1 cells are defective in translation initiation, a conclusion that is supported by a reduced rate of protein synthesis in Nip1p-depleted cells. Nip1p cosediments with free 40 S ribosomal subunits and polysomal preinitiation complexes, but not with free or elongating 80 S ribosomes or 60 S subunits. Nip1p can be isolated in an about 670-kDa complex containing polyhistidine-tagged Prt1p, a subunit of translation initiation factor 3, by binding to Ni2+-NTA-agarose beads in a manner completely dependent on the tagged form of Prt1p. The nip1-1 ts growth defect was suppressed by the deletion of the ribosomal protein, RPL46. Also, nip1-1 mutant cells are hypersensitive to paromomycin. These results suggest that Nip1p is a subunit of eukaryotic initiation factor 3 required for efficient translation initiation.

Based on characteristic amino acid sequences of kinases that phosphorylate the alpha subunit of eukaryotic translation initiation factor 2 (eIF2alpha kinases), degenerate oligonucleotide primers were constructed and used to polymerase chain reaction-amplify from genomic DNA of Neurospora crassa a sequence encoding part of a putative protein kinase. With this sequence an open reading frame was identified encoding a predicted polypeptide with juxtaposed eIF2alpha kinase and histidyl-tRNA synthetase-related domains. The 1646 amino acid sequence of this gene, called cpc-3, showed 35% positional identity over almost the entire sequence with GCN2 of yeast, which stimulates translation of the transcriptional activator of amino acid biosynthetic genes encoded by GCN4. Strains disrupted for cpc-3 were unable to induce increased transcription and derepression of amino acid biosynthetic enzymes in amino acid-deprived cells. The cpc-3 mutation did not affect the ability to up-regulate mRNA levels of cpc-1, encoding the GCN4 homologue and transcriptional activator of amino acid biosynthetic genes in N. crassa, but the mutation abolished the dramatic increase of CPC1 protein level in response to amino acid deprivation. These findings suggest that cpc-3 is the functional homologue of GCN2, being required for increased translation of cpc-1 mRNA in amino acid-starved cells.

The PRT1, TIF34, GCD10, and SUI1 proteins of Saccharomyces cerevisiae were found previously to copurify with eukaryotic translation initiation factor 3 (eIF3) activity. Although TIF32, NIP1, and TIF35 are homologous to subunits of human eIF3, they were not known to be components of the yeast factor. We detected interactions between PRT1, TIF34, and TIF35 by the yeast two-hybrid assay and in vitro binding assays. Discrete segments (70-150 amino acids) of PRT1 and TIF35 were found to be responsible for their binding to TIF34. Temperature-sensitive mutations mapping in WD-repeat domains of TIF34 were isolated that decreased binding between TIF34 and TIF35 in vitro. The lethal effect of these mutations was suppressed by increasing TIF35 gene dosage, suggesting that the TIF34-TIF35 interaction is important for TIF34 function in translation. Pairwise in vitro interactions were also detected between PRT1 and TIF32, TIF32 and NIP1, and NIP1 and SUI1. Furthermore, PRT1, NIP1, TIF34, TIF35, and a polypeptide with the size of TIF32 were specifically coimmunoprecipitated from the ribosomal salt wash fraction. We propose that all five yeast proteins homologous to human eIF3 subunits are components of a stable heteromeric complex in vivo and may comprise the conserved core of yeast eIF3.

The guanine nucleotide exchange activity of eIF2B plays a key regulatory role in the translation initiation phase of protein synthesis. The activity is markedly inhibited when the substrate, i. e. eIF2, is phosphorylated on Ser51 of its alpha-subunit. Genetic studies in yeast implicate the alpha-, beta-, and delta-subunits of eIF2B in mediating the inhibition by substrate phosphorylation. However, the mechanism involved in the inhibition has not been defined biochemically. In the present study, we have coexpressed the five subunits of rat eIF2B in Sf9 cells using the baculovirus system and have purified the recombinant holoprotein to >90% homogeneity. We have also expressed and purified a four-subunit eIF2B complex lacking the alpha-subunit. Both the five- and four-subunit forms of eIF2B exhibit similar rates of guanine nucleotide exchange activity using unphosphorylated eIF2 as substrate. The five-subunit form is inhibited by preincubation with phosphorylated eIF2 (eIF2(alphaP)) and exhibits little exchange activity when eIF2(alphaP) is used as substrate. In contrast, eIF2B lacking the alpha-subunit is insensitive to inhibition by eIF2(alphaP) and is able to exchange guanine nucleotide using eIF2(alphaP) as substrate at a faster rate compared with five-subunit eIF2B. Finally, a double point mutation in the delta-subunit of eIF2B has been identified that results in insensitivity to inhibition by eIF2(alphaP) and exhibits little exchange activity when eIF2(alphaP) is used as substrate. The results provide the first direct biochemical evidence that the alpha- and delta-subunits of eIF2B are involved in mediating the effect of substrate phosphorylation.

The mammalian translation initiation factor 3 (eIF3), is a multiprotein complex of approximately 600 kDa that binds to the 40 S ribosome and promotes the binding of methionyl-tRNAi and mRNA. cDNAs encoding 5 of the 10 subunits, namely eIF3-p170, -p116, -p110, -p48, and -p36, have been isolated previously. Here we report the cloning and characterization of human cDNAs encoding the major RNA binding subunit, eIF3-p66, and two additional subunits, eIF3-p47 and eIF3-p40. Each of these proteins is present in immunoprecipitates formed with affinity-purified anti-eIF3-p170 antibodies. Human eIF3-p66 shares 64% sequence identity with a hypothetical Caenorhabditis elegans protein, presumably the p66 homolog. Deletion analyses of recombinant derivatives of eIF3-p66 show that the RNA-binding domain lies within an N-terminal 71-amino acid region rich in lysine and arginine. The N-terminal regions of human eIF3-p40 and eIF3-p47 are related to each other and to 17 other eukaryotic proteins, including murine Mov-34, a subunit of the 26 S proteasome. Phylogenetic analyses of the 19 related protein sequences, called the Mov-34 family, distinguish five major subgroups, where eIF3-p40, eIF3-p47, and Mov-34 are each found in a different subgroup. The subunit composition of eIF3 appears to be highly conserved in Drosophila melanogaster, C. elegans, and Arabidopsis thaliana, whereas only 5 homologs of the 10 subunits of mammalian eIF3 are encoded in S. cerevisiae.

Adenine repression of the purine nucleotide biosynthetic genes in Saccharomyces cerevisiae involves down-regulation of the activator protein BAS1 or BAS2 by an unknown mechanism. To determine the minimal cis-acting requirements for adenine regulation, hybrid promoter constructs were made between ADE5,7 promoter fragments and a CYC1-lacZ reporter. A 139-nucleotide fragment containing two BAS1 binding sites was sufficient to confer adenine regulation on the CYC1-lacZ reporter. Analysis of deletion and substitution mutations led to the conclusion that the proximal BAS1 binding site is both necessary and sufficient for regulation, whereas the distal site augments the function of the proximal site. By performing saturation mutagenesis, we found two essential regions that flank the proximal site. An ABF1 consensus sequence is within one of these regions, and mutations that impaired in vitro ABF1 binding impaired promoter activity in vivo. A second region is AT-rich and appears to bind BAS2. No substitution mutations led to high level constitutive promoter activity as would be expected from removal of an upstream repression sequence. Our results indicate that ABF1, BAS1, and BAS2 are required for ADE5,7 promoter function and that adenine repression most likely involves activator modification or a negative regulator that does not itself bind DNA.

The HIS1 gene of Saccharomyces cerevisiae encodes ATP phosphoribosyltransferase, the first enzyme in the pathway of histidine biosynthesis. We have cloned this gene by complementation of a his1 auxotroph. The HIS1 coding region was localized within the cloned segment by assay of subcloned fragments for their ability to complement a his1 auxotroph. We determined the DNA sequence of the HIS1 region defined by this complementation test. S1 nuclease and exonuclease VII mapping of the 5' and 3' termini of HIS1 mRNA reveal considerable heterogeneity at both ends of the transcript, especially the 5' end which displays 13 different termini that span a 110-base pair region. Northern analysis shows that derepression of HIS1 enzyme activity under conditions of amino acid deprivation can be accounted for by an increase in the steady state level of HIS1 mRNA. There are no large differences between the relative levels of HIS1 mRNA molecules with different 5' termini in repressed and derepressed cells. In the DNA sequence upstream from the 5' termini of HIS1 mRNA we have found four closely related copies of a 9-base pair sequence. This sequence is also repeated in the 5' noncoding regions of HIS4, HIS3, and TRP5. Closely related sequences are not found flanking a number of other yeast genes, suggesting that the repeated sequence plays a role in the regulation of amino acid biosynthetic genes subject to the general amino acid control.