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TRANSCRIPTIONAL AND TRANSLATIONAL CONTROL
MECHANISMS IN NUTRIENT REGULATION OF GENE EXPRESSION
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Alan G. Hinnebusch, PhD, Head, Section on Nutrient Control of Gene Expression Vera Cherkasova, PhD, Senior Research Fellow |
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| We are studying regulatory mechanisms in the yeast Saccharomyces cerevisiae that stimulate transcription of amino acid, vitamin, and purine biosynthetic genes in response to nutrient limitation. Translation of transcriptional activator GCN4 is stimulated in starved cells by a mechanism involving short open reading frames (uORFs) in the mRNA leader and phosphorylation of initiation factor eIF2. Bound to GTP, eIF2 delivers initiator tRNAiMet to the 40S ribosome. Phosphorylation of eIF2 by the kinase GCN2 inhibits formation of the eIF2-GTP-tRNAiMet ternary complex (TC), reducing general protein synthesis but stimulating translation of GCN4. We are analyzing the physical and functional interactions of eIF2 with other initiation factors (eIFs-1, -1A, -3, and -5) and the 40S ribosome that promote TC recruitment, ribosomal scanning, and recognition of AUG codons during general and GCN4-specic translation. We are also studying the regulation of GCN2 kinase activity by uncharged tRNA (the starvation signal), a protein that associates with GCN2 on translating ribosomes (GCN1), and the TOR signaling pathway. Finally, we are analyzing the coactivators required for transcriptional activation by GCN4 to define the molecular program for the recruitment of chromatin-remodeling enzymes and adaptor proteins that deliver TATA-binding protein and RNA polymerase to target genes in vivo.
Analysis of eIF2-eIF3 contacts and ribosome-binding domains in the multifactor complex that are required for general and GCN4-specic translation Valasek, Nielsen, Szamecz, Jivotovskaya, Hinnebusch According to current models, the TC binds to the 40S ribosome with the help of eIFs 1, 1A, and 3. Interaction of the 43S preinitiation complex thus formed with mRNA is stimulated by eIF4F and poly(A)-binding protein, with the resulting 48S complex scanning 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 are probing the relative importance of eIFs-1, -1A, and -3 in the recruitment of TC and mRNA to the 40S ribosome, scanning, and AUG selection by generating mutations in these factors and examining the consequences on the rate of translation initiation, 43S/48S complex assembly, and GCN4 translational control in living cells. We previously showed that eIF3 contains five subunits and exists in a multifactor complex (MFC) with eIFs-1, -5, and the eIF2-GTP-Met-tRNAiMet ternary complex, produced a detailed subunit-interaction map for the MFC components, and showed that MFC integrity depends on simultaneous interaction of the eIF5 C-terminal domain (CTD) with eIF1, eIF3c/NIP1, and eIF2-beta. Furthermore, we showed that disrupting these interactions by the tif5-7A mutation in the eIF5-CTD impairs translation initiation and cell growth. To obtain further support for our model, we incorporated an affinity tag into TIF32, PRT1, and NIP1, the three largest eIF3 subunits, and deleted predicted binding domains in each tagged protein. We confirmed all binding interactions predicted by our model and uncovered a new interaction between the TIF32 CTD and eIF2-beta that is required for eIF2-eIF3 association. Overexpressing a CTD-less form of TIF32 exacerbates the translation initiation defect of the tif5-7A mutation and reduces eIF2 binding to 40S subunits, which implies that the two eIF2-eIF3 contacts in the MFC additively enhance the efficiency of TC recruitment and the rate of translation initiation (Valasek et al., 2002).
Reducing the rate of TC binding to 40S ribosomes is expected to derepress GCN4 translation (Gcd- phenotype), which we observed for a mutant lacking the CTD of eIF1A (Olsen et al., 2003). By contrast, we did not observe a Gcd- phenotype in tif5-7A cells overexpressing CTD-less TIF32, despite the reduction in levels of 40S-associated TC, indicating that eIF2-eIF3 contacts in the MFC also contribute to functions downstream of TC recruitment, such as scanning and AUG recognition, and that defects in these processes suppress the effects of reduced impaired TC recruitment on GCN4 translation. We recently obtained additional support for our conclusion by showing that mutants with conditionally lethal mutations in eIF3 subunits accumulate 48S preinitiation complexes containing TC, mRNA, and all relevant eIFs under nonpermissive conditions that impair translation initiation. Thus, it appears that eIF3 is critical for one or more post-assembly functions of the 48S complex. We are testing this hypothesis further by examining the effects of eIF3 mutations on GCN4 translational control and the stringency of AUG selection during the scanning process.
Having previously characterized MFC subcomplexes produced from affinity-tagged eIF3 subunits lacking discrete binding domains for other MFC components, we investigated whether these subcomplexes can bind to 40S ribosomes in vivo. The N- and C-terminal domains of NIP1/eIF3c, the N- and C-terminal domains of TIF32/eIF3a, and eIF5 all have critical functions in 40S binding, with eIF5 and the TIF32-CTD performing redundant functions. In accordance with these results, purified eIF3 and a trimeric complex consisting of NIP1, C-terminally truncated TIF32, and eIF5 can bind to purified 40S ribosomes in vitro. Given that removing the TIF32-NTD and NIP-CTD does not disrupt the MFC, these domains likely make direct contacts with the 40S ribosome. Furthermore, the TIF32-CTD interacts specifically in vitro with helices 16 to 18 of domain I in 18S rRNA, and the TIF32-NTD and NIP1 can interact with recombinant 40S subunit protein RPS0A. These results, together with the known positions of helices 16 to 18 and RPS0A in the three-dimensional structure of the 40S subunit, suggest that eIF3 binds to the solvent side of the 40S subunit in a way that provides access for the two eIF3 segments (NIP1-NTD and TIF32-CTD) with connections to eIFs 1, 5, and the eIF2/GTP/Met-tRNAiMet ternary complex (Valasek et al., 2003) (see Figure 13.1).
Algire MA, Maag D, Savio P, Acker MG, Tarun SZ Jr, Sachs AB, Asano K, Nielsen KH, Olsen DS, Phan L, Hinnebusch AG, Lorsh JR. Development and characterization of a reconstituted yeast translation initiation system. RNA 2002;8:382-397. Anand M, Chakraburtty K, Marton MJ, Hinnebusch AG, Kinzy TG. Functional interactions between yeast translation eukaryotic elongation factor (eEF)1A and eEF3. J Biol Chem 2003;278:6985-6991. Olsen DS, Savner EM, Mathew A, Zhang F, Krishnamoorthy T, Phan L, Hinnebusch AG. Domains of eIF1A that mediate binding to eIF2, eIF3 and eIF5B and promote ternary complex recruitment in vivo. EMBO J 2003;22:193-204. Valasek L, Mathew AA, Shin BS, Nielsen K H, Szamecz B, Hinnebusch AG. The yeast eIF3 subunits TIF32/a, NIP1/c, and eIF5 make critical connections with the 40S ribosome in vivo. Genes Dev 2003;17:786-799. Valasek L, Nielsen KH, Hinnebusch AG. Direct eIF2-eIF3 contact in the multifactor complex is important for translation initiation in vivo. EMBO J 2002;21:5886-5898. The yeast TOR proteins impede activation of GCN2 in nutrient-replete cells by promoting phosphorylation of Ser577 Cherkasova, Garcia-Barrio,* Dong, Zhang, Hinnebusch; in collaboration with Qin Through mass-spectrometric analysis of purified, catalytically inactive GCN2, we identified Ser577 as a site of phosphorylation by another kinase in vivo. It appears that Ser577 phosphorylation inhibits GCN2 because the substitution with alanine (S577A mutation) results in constitutive eIF2alpha phosphorylation and derepressed GCN4 translation. The S577A mutation increases the affinity of GCN2 for uncharged tRNA in vitro, suggesting that Ser577 phosphorylation impedes GCN2 activation in rich medium by preventing the binding of uncharged tRNAs present at basal concentrations in nonstarved cells (Garcia-Barrio et al., 2002). Because Ser577 remains phosphorylated in amino acid-starved cells, we asked whether it is dephosphorylated in other conditions under which GCN2 is activated. We found that activation of GCN2 by the antibiotic rapamycin is associated with dephosphorylation of Ser577. The S577A mutation dampened the effect of rapamycin on eIF2alpha phosphorylation and GCN4 translation, consistent with the idea that activation of GCN2 by rapamycin requires dephosphorylation of Ser577. The stimulatory effect of rapamycin requires tRNA binding by GCN2, suggesting that rapamycin increases the affinity of GCN2 for uncharged tRNA through Ser577 dephosphorylation, allowing kinase activation by the basal levels of uncharged tRNA present in nutrient-replete cells (Cherkasova and Hinnebusch, 2003). Our genetic analysis indicates that rapamycin stimulates eIF2alpha phosphorylation by GCN2 through the inhibition of TOR1 and TOR2, two PI3-kinase-related proteins that stimulate protein synthesis and repress pathways for utilization of poor nitrogen sources in nutrient-rich medium. We demonstrated that, in yeast, TOR proteins inhibit GCN2 function rather than stimulating the eIF2alpha phosphatase. In addition, we showed that rapamycin-induced dephosphorylation of GCN2-Ser577 involves TAP42, a regulator of type 2A-related protein phosphatases (PPases) implicated previously as a regulator of certain targets of the TOR pathway. Thus, it seems likely that TOR promotes GCN2-Ser577 phosphorylation in nutrient-replete cells by inhibiting the dephosphorylation of this residue by a type 2A-related PPase (Cherkasova and Hinnebusch, 2003) (see Figure 13.2).
Our results add a new dimension to the regulation of protein synthesis by TOR proteins and demonstrate cross-talk between two major pathways for nutrient control of gene expression. The TOR pathway stimulates translation in yeast by promoting ribosome biogenesis and the abundance of mRNA cap-binding factor eIF4F. We show that TOR also stimulates eIF2 function by preventing activation of GCN2. In addition to enhancing protein synthesis, inhibition of GCN2 by TOR maintains repression of GCN4 and the amino acid-biosynthetic genes under its control in nutrient-replete cells. Interestingly, the derepression of GCN4 that occurs when TOR is inhibited by rapamycin augments the transcriptional induction of genes required for utilization of poor nitrogen sources by the activator protein GLN3. of eIF2alpha kinase GCN2. Genes Dev 2003;17:859-872. Garcia-Barrio M, Dong J, Cherkasova VA, Zhang X, Zhang F, Ufano S, Lai R, Qin J, Hinnebusch AG. Serine 577 is phosphorylated and negatively affects the tRNA binding and eIF2alpha kinase activities of GCN2. J Biol Chem 2002;277:30675-30683. Qiu H, Hu C, Dong J, Hinnebusch AG. Mutations that bypass tRNA binding activate the intrinsically defective kinase domain in GCN2. Genes Dev 2002;16:1271-1280. A multiplicity of coactivators required for transcriptional activation by GCN4; requirements for SWI/SNF recruitment by GCN4 in vivo Swanson, Qiu, Yoon, Sumibcay, Kim, Zhang, Hu, Hinnebusch Transcriptional activation in eukaryotes typically involves sequence-specific DNA binding proteins that bind upstream of promoters and recruit multisubunit coactivator complexes with the capacity to stimulate assembly of a preinitiation complex (PIC) at the promoter. Some coactivators, including SWI/SNF and RSC, are ATP-dependent enzymes capable of remodeling the nucleosome structure of the promoter while others, such as the SAGA complex, contain histone acetyltransferase (HAT) activities that facilitate chromatin remodeling or mark promoter nucleosomes as binding sites for other coactivators. A third class of coactivators, including the Srb and Paf1 mediators, TFIID, and CCR4-NOT, are physically associated with TATA-binding protein (TBP), other general transcription factors (GTFs), or RNA polymerase II (Pol II) and are thought to function as adaptors between the activator and transcriptional machinery that promote PIC assembly. Our previous studies showed that SAGA, SWI/SNF, and Srb mediator complexes can interact specifically with GCN4 in vitro, dependent on bulky hydrophobic residues in the GCN4 activation domain that are required for transcriptional activation in vivo. In addition, we and others showed that mutations in various subunits of these three coactivators reduce activation by GCN4 in vivo (Gcn- phenotype). More recently, we 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 critical nonessential subunits in these complexes required for activation in vivo. Among the numerous histone acetyltransferases examined, GCN5 was the only one 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 in activation by GCN4 but found no role for the ISW1 and ISW2 chromatin remodeling complexes. We showed 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, whereas GCN4 recruits all six of these coactivators, plus the TBP adaptor protein MBF1, to the same target gene (ARG1) in living cells. Thus, GCN4 recruits an array of coactivators that promote its transcriptional activation function in vivo (Swanson et al., 2003). We wished to determine the roles of different subunits of SWI/SNF and the coactivators SAGA and Srb mediator in recruitment of SWI/SNF by GCN4 in vivo. To this end, we performed ChIP analysis on deletion mutants lacking different subunits of these coactivators and bearing epitope tags on six different SWI/SNF subunits. The results provide strong evidence that Gcn4p recruits the entire SWI/SNF complex to its target genes ARG1 and SNZ1 but that SWI/SNF is dispensable for GCN4 binding to these promoters. Previous work showed that the SWI/SNF subunits SNF2, SNF5, and SWI1 interact directly with GCN4 in vitro. However, we found that SNF2 is not required for recruitment of SWI/SNF by GCN4 and that SNF2 cannot be recruited independently of other SWI/SNF subunits in vivo. SNF5 also was not recruited as an isolated subunit but was required along with SNF6 and SWI3 for optimal recruitment of other SWI/SNF subunits. The results suggest that SNF2, SNF5, and SWI1 are recruited only as subunits of intact SWI/SNF, consistent with the idea that GCN4 makes multiple contacts with SWI/SNF in vivo. Optimal recruitment of SWI/SNF by GCN4 also requires specific subunits of the Srb mediator (GAL11, MED2, ROX3) and SAGA (ADA1 and ADA5) but is independent of the histone acetyltransferase in SAGA, GCN5. Based on these findings, we suggest that SWI/SNF recruitment is enhanced by cooperative interactions with subunits of Srb mediator and SAGA that are recruited by GCN4 to the same promoter but is insensitive to histone H3 acetylation by GCN5 (Yoon et al., in press). Experiments are now under way to probe the requirements for the recruitment of SAGA, Srb mediator, TBP, and Pol II by GCN4 and to define the kinetic order of recruitment of these factors and coactivators at various target genes in vivo. levels by diverse signals of starvation and stress. Eukaryotic Cell 2002;1:22-32. coactivators is required by Gcn4p at individual promoters in vivo. Mol Cell Biol 2003;23:2800-2820. Yoon S, Qiu H, Swanson MJ, Hinnebusch AG. Recruitment of SWI/SNF by Gcn4p does not require Snf2p or Gcn5p but depends strongly on SWI/SNF integrity, SRB mediator, and SAGA. Mol Cell Biol 2003; in press. COLLABORATORS For further information, contact ahinnebusch@nih.gov |
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