We are studying how the yeast Saccharomyces cerevisiae regulates the expression of its genome according to nutrient availability. In response to starvation for an amino acid, purine, or glucose, a global regulatory mechanism known as general amino acid control mediates the transcriptional induction of about one-fifth of the genome, including most genes encoding amino acid or purine biosynthetic enzymes. Synthesis of the transcriptional activator GCN4 is stimulated under starvation conditions by a translational control mechanism involving short open reading frames (uORFs) in the GCN4 mRNA leader and the general translation initiation factors eIF2 and eIF2B. The guanine nucleotide exchange factor eIF2, which recycles inactive eIF2-GDP to active eIF2-GTP after each round of initiation, forms a ternary complex (TC) with initiator tRNAiMet and GTP and binds to the 40S ribosomal subunit. The induction of GCN4 translation depends on phosphorylation of the a-subunit of eIF2 on serine-51 by the protein kinase GCN2, converting eIF2 from a substrate to inhibitor of eIF2B. Phosphorylation of eIF2 by GCN2 reduces the rate of general protein synthesis but simultaneously induces translation of GCN4 mRNA. In this way, the cell can decrease the demand for amino acids while increasing its capacity for the acids' de novo production.

Importance of Direct eIF2-eIF3 Contact in the Multifactor Complex for Translation Initiation in Vivo
Valasek, Nielsen, Hinnebusch
Assembly of the 80S translation initiation complex is a multiple-step process involving a large number of soluble eukaryotic initiation factors (eIFs). According to current models, the TC binds to the 40S ribosome with the help of eIFs 1, 1A, and 3. The 43S preinitiation complex thus formed interacts with mRNA in a manner stimulated by eIF4F and poly(A)-binding pro-tein, and the resulting 48S complex scans the mRNA until the Met-tRNAiMet base-pairs with the AUG start codon. On AUG recognition, the eIF5 stimulates GTP hydrolysis by eIF2, the eIFs are ejected, and the 60S subunit joins with the 40S-Met-tRNAiMet-mRNA complex in a reaction stimulated by eIF5B.

We showed previously that yeast eIF3 contains only five subunits (TIF32, PRT1, NIP1, TIF35, and TIF34). We also demonstrated that eIF5, the GTPase-activating protein (GAP) for the TC, and eIF1, a factor regulating AUG start codon selection, both copurify with eIF3 (Phan et al., 2001; Asano et al., 2001). Through protein interaction assays with recombinant subunits, we produced a subunit interaction map for eIF3 in which PRT1 is the scaffold of the complex. The two small-est subunits (TIF34/TIF35) bind to the extreme C-terminal domain (CTD) of PRT1 and to one another, and the largest subunit (TIF32) binds to the N-terminal domain (NTD) in PRT1. The remaining subunit NIP1 binds to TIF32 but not to PRT1. Both eIF5 and eIF1 interact with the NIP1 NTD (Fig. 23) (Valasek et al., 2001; Asano et al., 2001).

FIGURE 23

Schematic representation of the Multifactor Complex comoprised of eukaryotic initiation factors, 1, 3, 5, and the eIF2/GTP/Met-tRNAiMet ternary complex

We demonstrated that the eIF5 CTD contains a conserved motif that can bind simultaneously to the N-terminus of the b-subunit of eIF2 and the NIP1-NTD. These interactions enable eIF5 to bridge an interaction between eIF2 and eIF3 in vivo, and a multifactor complex (MFC) containing eIFs 1, 2, 3, and 5 and Met-tRNAiMet was purified from cell extracts (Fig. 23). The integrity of the MFC depends on the bridging function of eIF5, which is disrupted by a mutation in the eIF5 CTD (tif5-7A). As tif5-7A also impairs translation initiation in vivo and cell growth, we proposed that the MFC is an important intermediate in translation initiation and that eIF5 plays an important role in initiation complex assembly beyond its conventional function as a GAP (Asano et al., 2001).

To obtain in vivo support for our model of the MFC, we incorporated an affinity tag into TIF32, PRT1, and NIP1, the three largest eIF3 subunits, and deleted predicted binding domains in each tagged protein. By purifying and characterizing the mutant subcomplexes, we confirmed all binding interactions predicted by our model and uncovered a new interaction between NIP1 and PRT1. In addition to the confirmed contact between eIF2 and the NIP1 NTD bridged by eIF5, we found that the TIF32 CTD binds to eIF2 directly (Fig. 23) and is required for eIF2-eIF3 association in vivo. Overexpressing a form of TIF32 lacking CTD exacerbated the translation initiation defect of the tif5-7A mutation, weakening the NIP1/eIF5/eIF2 connection. Thus, the two independent eIF2-eIF3 contacts identified here have additive effects on the efficiency of translation in vivo.

Overexpressing the NIP1-NTD sequestered eIF1/eIF5/eIF2 in a defective subcomplex that derepressed GCN4 mRNA translation (Gcd¯ phenotype). GCN4 translation is normally repressed by its four upstream open reading frames (uORFs). Ribosomes that translate uORF1 and resume scanning reinitiate at uORF4 and fail to reach GCN4 unless the concentration of TC is reduced by eIF2a phosphorylation or the ability of TC to rebind to ribosomes scanning between uORFs 1 and 4 is impaired by mutation. Under these circumstances, the ribosomes bypass uORF4 and reinitiate at GCN4 instead. The Gcd- phenotype of overexpressing the NIP-NTD was enhanced by overexpressing the TIF32-CTD, which also sequesters eIF2 in a distinct subcomplex, and was suppressed by overproducing the constituents of the TC. These results provide the first evidence that physical association with eIF3 promotes binding of TC to 40S ribosomes in vivo (Valasek et al., 2002).

Domains in eIF1A That Interact with eIF2, eIF3, and eIF5B and Promote Ribosome Bind-ing of the Ternary Complex
Olsen, Savner, Mathew, Zhang, Hinnebusch
Because of its homology to bacterial translation initiation factor IF1, the eukaryotic factor eIF1A is predicted to bind in the decoding (A) site of the 40S ribosome, and mammalian eIF1A has been implicated in recruitment of TC and ribosomal scanning. We showed previously that yeast eIF1A interacts in vivo and in vitro with eIF5B, the ortholog of bacterial IF2 that promotes 40S-60S subunit joining in vitro. The fact that overexpression of eIF1A exacerbated the growth defect of a strain lacking eIF5B led us to propose that eIF1A's interaction with eIF5B is instrumental in release of eIF1A from the A-site following subunit joining. Consistent with this hypothesis, we have mapped the binding domain for eIF5B to the last 24 residues of the unstructured C-terminus of eIF1A and shown that removal of these residues diminishes transla-tion initiation in vivo. A larger C-terminal truncation that removes a predicted helix 310 in eIF1A derepresses GCN4 translation (Gcd- phenotype), a derepression that can be suppressed by overex-pressing the TC. These latter results provide the first evidence that eIF1A promotes TC binding to 40S ribosomes in vivo. The unstructured N-terminus of eIF1A interacts directly with eIF2 and eIF3 and is required for optimum translation at a step following TC recruitment, especially at low growth temperatures. We propose a modular organization for eIF1A in which a core ribosome-binding domain is flanked by flexible segments that mediate its interactions with other factors involved in TC recruitment and release of eIF1A at subunit joining.

Activation of the Intrinsically Defective Ki-nase Domain in GCN2 by Mutations That Bypass tRNA Binding
Qiu, Hu, Dong, Hinnebusch
GCN2 down-regulates general translation and specifically induces GCN4 translation by phosphorylating eIF2a and thereby impeding TC formation. GCN2 contains regulatory domains located both N-terminal and C-terminal to the protein kinase (PK) domain, including a region related to histidyl-tRNA synthetase (HisRS), a C-terminal ribosome-binding and dimerization domain (Cterm), and an N-terminal domain that mediates binding of the positive effectors GCN1 and GCN20. The HisRS-related domain can bind to tRNA in vitro and mediates the activation of GCN2 by uncharged tRNA in amino acid starved cells. The mechanism of kinase activation is not well understood but appears to depend on physical contact between the PK domain and the N-terminal portion of the HisRS region (HisRS-N) bound to uncharged tRNA. In addition, interactions between the PK and Cterm play a role in preventing tRNA binding and kinase activation at low, basal concentrations of uncharged tRNA present in nonstarved cells (Qiu et al., 2001).

To elucidate the mechanism of kinase activation, we isolated two point mutations in the protein kinase (PK) domain, R794G and F842L, that permit strong kinase activity in the absence of tRNA binding. These mutations bypass the requirement for ribosome binding, dimeriza-tion, and association with the GCN1/GCN20 regulatory complex, suggesting that all these functions facilitate tRNA binding to wild-type GCN2. While the isolated wild-type PK domain was completely inert, the mutant PK was highly active in vivo and in vitro. These results identify an inhibitory structure intrinsic to the PK domain that must be overcome on tRNA binding by interactions with a regulatory region, most likely the HisRS-N/tRNA module. As R794 and F842 are predicted to lie close to one another and to the active site, they may participate directly in misaligning active site residues. Consistent with this idea, many wild-type protein kinases contain Gly and Leu residues at the positions corresponding to the R794G and F842L activating mutations in GCN2. Autophosphorylation of two Thr residues in the activation loop of the kinase domain is required for GCN2 activation. Autophosphorylation of these residues was stimulated by R794G and F842L, and the autophosphorylation sites remained critical for GCN2 function in the presence of the activating mutations. Hence, our results imply a two-step mechanism for activation by uncharged tRNA: (1) interaction between the PK domain and HisRS-N/tRNA module elicits a conformational change that corrects the intrinsic defect of the PK domain, leading to (2) autophos-phorylation of the activation loop and activation of the eIF2± kinase function of the PK domain (Qiu et al., 2002).

Multiplicity of Coactivators Required by GCN4p at Individual Promoters in Vivo
Swanson, Qiu, Krueger, Sumibcay, Kim, Zhang, Hinnebusch
Eukaryotic transcriptional regulation requires sequence-specific DNA binding proteins to activate transcription from target genes that are organized in repressive chromatin. Activators recruit multisubunit coactivators harboring activities that alter chromatin structure and recruit the general transcription factors and RNA polymerase to the promoter. Yeast cells respond to amino acid starvation by inducing the activator of amino acid biosynthetic genes, GCN4. Our previous studies showed that the SAGA, SWI/SNF, and SRB/MED mediator complexes can interact with GCN4 in vitro and that mutations in subunits of these complexes decrease activation by GCN4 in vivo (Gcn- phenotype). SAGA contains the histone acetyltransferase (HAT) Gcn5p, which acetylates lysines in the tails of histone H3, plus additional subunits involved in recruiting TATA binding protein (TBP) to the promoter. SWI/SNF is an ATP-dependent chromatin-remodeling complex, and the SRB/MED mediator is associated with RNA polymerase in a holoenzyme complex, transducing the effects of activators and repressors on polymerase function.

We have conducted a comprehensive analysis of coactivator requirements by testing all viable mutants from the Saccharomyces Deletion Project for defects in activation by GCN4 in vivo. Our data confirm that GCN4 requires SAGA, SWI/SNF, and SRB/MED and identify the key nonessential subunits in these complexes required for activation in vivo. Among the numerous histone acetyltransferases examined, GCN5 was uniquely required for activation by GCN4. We also uncovered a strong dependence on the CCR4/NOT complex and significant requirements for RSC and the Paf1 complex, but we could detect no role for ISW1/ISW2 in activation by GCN4. RSC and ISW are ATP-dependent chromatin-remodeling complexes, the Paf1 complex is an alternative form of mediator, and CCR4/NOT has been implicated in controlling TBP binding and holoenzyme function. In vitro binding experiments indicate that the GCN4 activation domain interacts specifically with CCR4-NOT and RSC in addition to SAGA, SWI/SNF, and SRB/MED, but not with the Paf1 complex. Chromatin immunoprecipitation experiments show that GCN4 recruits all six of these coactivators to one of its target genes (ARG1) in living cells. Deletions of certain SAGA and SRB/MED subunits with strong activation defects in vivo disrupt binding of GCN4 to these complexes in vitro. In contrast, deletions of other subunits with Gcn- phenotypes do not reduce coactivator binding to GCN4 and hence may impair a biochemical function of the coactivator at the promoter.