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1.  Differential gene expression signatures for cell wall integrity found in chitin synthase II (chs2Δ) and myosin II (myo1Δ) deficient cytokinesis mutants of Saccharomyces cerevisiae 
BMC Research Notes  2009;2:87.
Background
Myosin II-dependent contraction of the cytokinetic ring and primary septum formation by chitin synthase II are interdependent processes during cytokinesis in Saccharomyces cerevisiae. Hence, null mutants of myosin II (myo1Δ) and chitin synthase II (chs2Δ) share multiple morphological and molecular phenotypes. To understand the nature of their interdependent functions, we will seek to identify genes undergoing transcriptional regulation in chs2Δ strains and to establish a transcription signature profile for comparison with myo1Δ strains.
Results
A total of 467 genes were commonly regulated between myo1Δ and chs2Δ mutant strains (p ≤ 0.01). Common regulated biological process categories identified by Gene Set Enrichment Analysis (GSEA) in both gene expression profiles were: protein biosynthesis, RNA processing, and stress response. Expression of 17/20 genes in the main transcriptional fingerprint for cell wall stress was confirmed in the chs2Δ strain versus 5/20 for the myo1Δ strain. One of these genes, SLT2/MPK1, was up-regulated in both strains and both strains accumulated the hyperphosphorylated form of Slt2p thereby confirming that the PKC1 cell wall integrity pathway (CWIP) was activated by both mutations. The SLT2/MPK1 gene, essential for myo1Δ strains, was not required in the chs2Δ strain.
Conclusion
Comparison of the chs2Δ and myo1Δ gene expression profiles revealed similarities in the biological process categories that respond to the chs2Δ and myo1Δ gene mutations. This supports the view that these mutations affect a common function in cytokinesis. Despite their similarities, these mutants exhibited significant differences in expression of the main transcriptional fingerprint for cell wall stress and their requirement of the CWIP for survival.
doi:10.1186/1756-0500-2-87
PMCID: PMC2684100  PMID: 19426543
2.  Global mRNA expression analysis in myosin II deficient strains of Saccharomyces cerevisiae reveals an impairment of cell integrity functions 
BMC Genomics  2008;9:34.
Background
The Saccharomyces cerevisiae MYO1 gene encodes the myosin II heavy chain (Myo1p), a protein required for normal cytokinesis in budding yeast. Myo1p deficiency in yeast (myo1Δ) causes a cell separation defect characterized by the formation of attached cells, yet it also causes abnormal budding patterns, formation of enlarged and elongated cells, increased osmotic sensitivity, delocalized chitin deposition, increased chitin synthesis, and hypersensitivity to the chitin synthase III inhibitor Nikkomycin Z. To determine how differential expression of genes is related to these diverse cell wall phenotypes, we analyzed the global mRNA expression profile of myo1Δ strains.
Results
Global mRNA expression profiles of myo1Δ strains and their corresponding wild type controls were obtained by hybridization to yeast oligonucleotide microarrays. Results for selected genes were confirmed by real time RT-PCR. A total of 547 differentially expressed genes (p ≤ 0.01) were identified with 263 up regulated and 284 down regulated genes in the myo1Δ strains. Gene set enrichment analysis revealed the significant over-representation of genes in the protein biosynthesis and stress response categories. The SLT2/MPK1 gene was up regulated in the microarray, and a myo1Δslt2Δ double mutant was non-viable. Overexpression of ribosomal protein genes RPL30 and RPS31 suppressed the hypersensitivity to Nikkomycin Z and increased the levels of phosphorylated Slt2p in myo1Δ strains. Increased levels of phosphorylated Slt2p were also observed in wild type strains under these conditions.
Conclusion
Following this analysis of global mRNA expression in yeast myo1Δ strains, we conclude that 547 genes were differentially regulated in myo1Δ strains and that the stress response and protein biosynthesis gene categories were coordinately regulated in this mutant. The SLT2/MPK1 gene was confirmed to be essential for myo1Δ strain viability, supporting that the up regulated stress response genes are regulated by the PKC1 cell integrity pathway. Suppression of Nikkomycin Z hypersensitivity together with Slt2p phosphorylation was caused by the overexpression of ribosomal protein genes RPL30 and RPS31. These ribosomal protein mRNAs were down regulated in the myo1Δ arrays, suggesting that down regulation of ribosomal biogenesis may affect cell integrity in myo1Δ strains.
doi:10.1186/1471-2164-9-34
PMCID: PMC2253530  PMID: 18215314
3.  Functional and genetic interactions of TOR in the budding yeast Saccharomyces cerevisiae with myosin type II-deficiency (myo1Δ) 
BMC Cell Biology  2012;13:13.
Background
Yeast has numerous mechanisms to survive stress. Deletion of myosin type II (myo1Δ) in Saccharomyces cerevisiae results in a cell that has defective cytokinesis. To survive this genetically induced stress, this budding yeast up regulates the PKC1 cell wall integrity pathway (CWIP). More recently, our work indicated that TOR, another stress signaling pathway, was down regulated in myo1Δ strains. Since negative signaling by TOR is known to regulate PKC1, our objectives in this study were to understand the cross-talk between the TOR and PKC1 signaling pathways and to determine if they share upstream regulators for mounting the stress response in myo1Δ strains.
Results
Here we proved that TORC1 signaling was down regulated in the myo1Δ strain. While a tor1Δ mutant strain had increased viability relative to myo1Δ, a combined myo1Δtor1Δ mutant strain showed significantly reduced cell viability. Synthetic rescue of the tor2-21ts lethal phenotype was observed in the myo1Δ strain in contrast to the chs2Δ strain, a chitin synthase II null mutant that also activates the PKC1 CWIP and exhibits cytokinesis defects very similar to myo1Δ, where the rescue effect was not observed. We observed two pools of Slt2p, the final Mitogen Activated Protein Kinase (MAPK) of the PKC1 CWIP; one pool that is up regulated by heat shock and one that is up regulated by the myo1Δ stress. The cell wall stress sensor WSC1 that activates PKC1 CWIP under other stress conditions was shown to act as a negative regulator of TORC1 in the myo1Δ mutant. Finally, the repression of TORC1 was inversely correlated with the activation of PKC1 in the myo1Δ strain.
Conclusions
Regulated expression of TOR1 was important in the activation of the PKC1 CWIP in a myo1Δ strain and hence its survival. We found evidence that the PKC1 and TORC1 pathways share a common upstream regulator associated with the cell wall stress sensor WSC1. Surprisingly, essential TORC2 functions were not required in the myo1Δ strain. By understanding how yeast mounts a concerted stress response, one can further design pharmacological cocktails to undermine their ability to adapt and to survive.
doi:10.1186/1471-2121-13-13
PMCID: PMC3470973  PMID: 22646158
PKC1; SLT2/MPK1; WSC1; Tor2-21; Fungal cell wall
4.  The 4E-BP Caf20p Mediates Both eIF4E-Dependent and Independent Repression of Translation 
PLoS Genetics  2015;11(5):e1005233.
Translation initiation factor eIF4E mediates mRNA selection for protein synthesis via the mRNA 5’cap. A family of binding proteins, termed the 4E-BPs, interact with eIF4E to hinder ribosome recruitment. Mechanisms underlying mRNA specificity for 4E-BP control remain poorly understood. Saccharomyces cerevisiae 4E-BPs, Caf20p and Eap1p, each regulate an overlapping set of mRNAs. We undertook global approaches to identify protein and RNA partners of both 4E-BPs by immunoprecipitation of tagged proteins combined with mass spectrometry or next-generation sequencing. Unexpectedly, mass spectrometry indicated that the 4E-BPs associate with many ribosomal proteins. 80S ribosome and polysome association was independently confirmed and was not dependent upon interaction with eIF4E, as mutated forms of both Caf20p and Eap1p with disrupted eIF4E-binding motifs retain ribosome interaction. Whole-cell proteomics revealed Caf20p mutations cause both up and down-regulation of proteins and that many changes were independent of the 4E-binding motif. Investigations into Caf20p mRNA targets by immunoprecipitation followed by RNA sequencing revealed a strong association between Caf20p and mRNAs involved in transcription and cell cycle processes, consistent with observed cell cycle phenotypes of mutant strains. A core set of over 500 Caf20p-interacting mRNAs comprised of both eIF4E-dependent (75%) and eIF4E-independent targets (25%), which differ in sequence attributes. eIF4E-independent mRNAs share a 3’ UTR motif. Caf20p binds all tested motif-containing 3’ UTRs. Caf20p and the 3’UTR combine to influence ERS1 mRNA polysome association consistent with Caf20p contributing to translational control. Finally ERS1 3’UTR confers Caf20-dependent repression of expression to a heterologous reporter gene. Taken together, these data reveal conserved features of eIF4E-dependent Caf20p mRNA targets and uncover a novel eIF4E-independent mode of Caf20p binding to mRNAs that extends the regulatory role of Caf20p in the mRNA-specific repression of protein synthesis beyond its interaction with eIF4E.
Author Summary
In eukaryotic cells protein synthesis initiation factor eIF4E controls mRNA selection by interacting with the mRNA 5’ cap. A family of binding proteins, termed the 4E-BPs, interact with eIF4E to hinder ribosome recruitment and repress translation of their target mRNAs. The yeast Saccharomyces cerevisiae has two 4E-BPs Caf20p and Eap1p that regulate distinct but overlapping sets of mRNAs. Here, we describe genome wide experiments to identify protein and RNA partners of each 4E-BP, with a greater focus on Caf20p. We present evidence that the 4E-BPs unexpectedly bind to ribosomes, an interaction that is not dependent on eIF4E binding. We also define a core set of over 500 Caf20p target mRNAs that fall into two classes with distinct features. One mRNA class, representing 25% of the targets, binds Caf20p independently of its eIF4E interaction and instead via a novel 3’ UTR interaction. Our data indicate that these proteins can repress mRNA-specific protein synthesis independently of their known role as eIF4E-binding proteins.
doi:10.1371/journal.pgen.1005233
PMCID: PMC4431810  PMID: 25973932
5.  Heterologous Expression of Membrane and Soluble Proteins Derepresses GCN4 mRNA Translation in the Yeast Saccharomyces cerevisiae 
Eukaryotic Cell  2006;5(2):248-261.
This paper describes the first physiological response at the translational level towards heterologous protein production in Saccharomyces cerevisiae. In yeast, the phosphorylation of eukaryotic initiation factor 2α (eIF-2α) by Gcn2p protein kinase mediates derepression of GCN4 mRNA translation. Gcn4p is a transcription factor initially found to be required for transcriptional induction of genes responsible for amino acid or purine biosynthesis. Using various GCN4-lacZ fusions, knockout yeast strains, and anti-eIF-2α-P/anti-eIF-2α antibodies, we observed that heterologous expression of the membrane-bound α1β1 Na,K-ATPase from pig kidney, the rat pituitary adenylate cyclase seven-transmembrane-domain receptor, or a 401-residue soluble part of the Na,K-ATPase α1 subunit derepressed GCN4 mRNA translation up to 70-fold. GCN4 translation was very sensitive to the presence of heterologous protein, as a density of 1‰ of heterologous membrane protein derepressed translation maximally. Translational derepression of GCN4 was not triggered by misfolding in the endoplasmic reticulum, as expression of the wild type or temperature-sensitive folding mutants of the Na,K-ATPase increased GCN4 translation to the same extent. In situ activity of the heterologously expressed protein was not required for derepression of GCN4 mRNA translation, as illustrated by the expression of an enzymatically inactive Na,K-ATPase. Two- to threefold overexpression of the highly abundant and plasma membrane-located endogenous H-ATPase also induced GCN4 translation. Derepression of GCN4 translation required phosphorylation of eIF-2α, the tRNA binding domain of Gcn2p, and the ribosome-associated proteins Gcn1p and Gcn20p. The increase in Gcn4p density in response to heterologous expression did not induce transcription from the HIS4 promoter, a traditional Gcn4p target.
doi:10.1128/EC.5.2.248-261.2006
PMCID: PMC1405899  PMID: 16467466
6.  Modulation of tRNA(iMet), eIF-2, and eIF-2B expression shows that GCN4 translation is inversely coupled to the level of eIF-2.GTP.Met-tRNA(iMet) ternary complexes. 
Molecular and Cellular Biology  1995;15(11):6351-6363.
To understand how phosphorylation of eukaryotic translation initiation factor (eIF)-2 alpha in Saccharomyces cerevisiae stimulates GCN4 mRNA translation while at the same time inhibiting general translation initiation, we examined the effects of altering the gene dosage of initiator tRNA(Met), eIF-2, and the guanine nucleotide exchange factor for eIF-2, eIF-2B. Overexpression of all three subunits of eIF-2 or all five subunits of eIF-2B suppressed the effects of eIF-2 alpha hyperphosphorylation on both GCN4-specific and general translation initiation. Consistent with eIF-2 functioning in translation as part of a ternary complex composed of eIF-2, GTP, and Met-tRNA(iMet), reduced gene dosage of initiator tRNA(Met) mimicked phosphorylation of eIF-2 alpha and stimulated GCN4 translation. In addition, overexpression of a combination of eIF-2 and tRNA(iMet) suppressed the growth-inhibitory effects of eIF-2 hyperphosphorylation more effectively than an increase in the level of either component of the ternary complex alone. These results provide in vivo evidence that phosphorylation of eIF-2 alpha reduces the activities of both eIF-2 and eIF-2B and that the eIF-2.GTP. Met-tRNA(iMet) ternary complex is the principal component limiting translation in cells when eIF-2 alpha is phosphorylated on serine 51. Analysis of eIF-2 alpha phosphorylation in the eIF-2-overexpressing strain also provides in vivo evidence that phosphorylated eIF-2 acts as a competitive inhibitor of eIF-2B rather than forming an excessively stable inactive complex. Finally, our results demonstrate that the concentration of eIF-2-GTP. Met-tRNA(iMet) ternary complexes is the cardinal parameter determining the site of reinitiation on GCN4 mRNA and support the idea that reinitiation at GCN4 is inversely related to the concentration of ternary complexes in the cell.
PMCID: PMC230887  PMID: 7565788
7.  Eukaryotic Translation Initiation Factor 5 Is Critical for Integrity of the Scanning Preinitiation Complex and Accurate Control of GCN4 Translation 
Molecular and Cellular Biology  2005;25(13):5480-5491.
The integrity of eukaryotic translation initiation factor (eIF) interactions in ribosomal preinitiation complexes is critical for the proper regulation of GCN4 mRNA translation in response to amino acid availability. Increased phosphorylation of eIF2 under amino acid starvation conditions leads to a corresponding increase in GCN4 mRNA translation. The carboxyl-terminal domain (CTD) of eIF5 (eIF5-CTD) has been identified as a potential nucleation site for preinitiation complex assembly. To further characterize eIF5 and delineate its role in GCN4 translational control, we isolated mutations leading to temperature sensitivity (Ts− phenotype) targeted at TIF5, the structural gene encoding eIF5 in yeast (Saccharomyces cerevisiae). Nine single point mutations were isolated, in addition to an allele in which the last 15 amino acids were deleted. The nine point mutations clustered in the eIF5-CTD, which contains two conserved aromatic/acidic boxes. Six of the point mutations derepressed GCN4 translation independent of eIF2 phosphorylation (Gcd− phenotype) at a permissive temperature, directly implicating eIF5-CTD in the eIF2/GTP/Met-tRNAiMet ternary complex binding process required for GCN4 translational control. In addition, stronger restriction of eIF5-CTD function at an elevated temperature led to failure to derepress GCN4 translation (Gcn− phenotype) in all of the mutants, most likely due to leaky scanning of the first upstream open reading frame of GCN4 mRNA. This latter result directly implicates eIF5-CTD in the process of accurate scanning for, or recognition of, AUG codons. Taken together, our results indicate that eIF5-CTD plays a critical role in both the assembly of the 43S complex and the postassembly process in the 48S complex, likely during the scanning process.
doi:10.1128/MCB.25.13.5480-5491.2005
PMCID: PMC1156968  PMID: 15964804
8.  Mutations in the GCD7 subunit of yeast guanine nucleotide exchange factor eIF-2B overcome the inhibitory effects of phosphorylated eIF-2 on translation initiation. 
Molecular and Cellular Biology  1994;14(5):3208-3222.
Phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2 alpha) impairs translation initiation by inhibiting the guanine nucleotide exchange factor for eIF-2, known as eIF-2B. In Saccharomyces cerevisiae, phosphorylation of eIF-2 alpha by the protein kinase GCN2 specifically stimulates translation of GCN4 mRNA in addition to reducing general protein synthesis. We isolated mutations in several unlinked genes that suppress the growth-inhibitory effect of eIF-2 alpha phosphorylation catalyzed by mutationally activated forms of GCN2. These suppressor mutations, affecting eIF-2 alpha and the essential subunits of eIF-2B encoded by GCD7 and GCD2, do not reduce the level of eIF-2 alpha phosphorylation in cells expressing the activated GCN2c kinase. Four GCD7 suppressors were shown to reduce the derepression of GCN4 translation in cells containing wild-type GCN2 under starvation conditions or in GCN2c strains. A fifth GCD7 allele, constructed in vitro by combining two of the GCD7 suppressors mutations, completely impaired the derepression of GCN4 translation, a phenotype characteristic of deletions in GCN1, GCN2, or GCN3. This double GCD7 mutation also completely suppressed the lethal effect of expressing the mammalian eIF-2 alpha kinase dsRNA-PK in yeast cells, showing that the translational machinery had been rendered completely insensitive to phosphorylated eIF-2. None of the GCD7 mutations had any detrimental effect on cell growth under nonstarvation conditions, suggesting that recycling of eIF-2 occurs efficiently in the suppressor strains. We propose that GCD7 and GCD2 play important roles in the regulatory interaction between eIF-2 and eIF-2B and that the suppressor mutations we isolated in these genes decrease the susceptibility of eIF-2B to the inhibitory effects of phosphorylated eIF-2 without impairing the essential catalytic function of eIF-2B in translation initiation.
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PMCID: PMC358688  PMID: 8164676
9.  Glucose Limitation Induces GCN4 Translation by Activation of Gcn2 Protein Kinase 
Molecular and Cellular Biology  2000;20(8):2706-2717.
Phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF-2α) is a well-characterized mechanism regulating protein synthesis in response to environmental stresses. In the yeast Saccharomyces cerevisiae, starvation for amino acids induces phosphorylation of eIF-2α by Gcn2 protein kinase, leading to elevated translation of GCN4, a transcriptional activator of more than 50 genes. Uncharged tRNA that accumulates during amino acid limitation is proposed to activate Gcn2p by associating with Gcn2p sequences homologous to histidyl-tRNA synthetase (HisRS) enzymes. Given that eIF-2α phosphorylation in mammals is induced in response to both carbohydrate and amino acid limitations, we addressed whether activation of Gcn2p in yeast is also controlled by different nutrient deprivations. We found that starvation for glucose induces Gcn2p phosphorylation of eIF-2α and stimulates GCN4 translation. Induction of eIF-2α phosphorylation by Gcn2p during glucose limitation requires the function of the HisRS-related domain but is largely independent of the ribosome binding sequences of Gcn2p. Furthermore, Gcn20p, a factor required for Gcn2 protein kinase stimulation of GCN4 expression in response to amino acid starvation, is not essential for GCN4 translational control in response to limitation for carbohydrates. These results indicate there are differences between the mechanisms regulating Gcn2p activity in response to amino acid and carbohydrate deficiency. Gcn2p induction of GCN4 translation during carbohydrate limitation enhances storage of amino acids in the vacuoles and facilitates entry into exponential growth during a shift from low-glucose to high-glucose medium. Gcn2p function also contributes to maintenance of glycogen levels during prolonged glucose starvation, suggesting a linkage between amino acid control and glycogen metabolism.
PMCID: PMC85486  PMID: 10733573
10.  Guanine nucleotide exchange factor for eukaryotic translation initiation factor 2 in Saccharomyces cerevisiae: interactions between the essential subunits GCD2, GCD6, and GCD7 and the regulatory subunit GCN3. 
Molecular and Cellular Biology  1993;13(8):4618-4631.
Phosphorylation of eukaryotic translation initiation factor 2 (eIF-2) in amino acid-starved cells of the yeast Saccharomyces cerevisiae reduces general protein synthesis but specifically stimulates translation of GCN4 mRNA. This regulatory mechanism is dependent on the nonessential GCN3 protein and multiple essential proteins encoded by GCD genes. Previous genetic and biochemical experiments led to the conclusion that GCD1, GCD2, and GCN3 are components of the GCD complex, recently shown to be the yeast equivalent of the mammalian guanine nucleotide exchange factor for eIF-2, known as eIF-2B. In this report, we identify new constituents of the GCD-eIF-2B complex and probe interactions between its different subunits. Biochemical evidence is presented that GCN3 is an integral component of the GCD-eIF-2B complex that, while dispensable, can be mutationally altered to have a substantial inhibitory effect on general translation initiation. The amino acid sequence changes for three gcd2 mutations have been determined, and we describe several examples of mutual suppression involving the gcd2 mutations and particular alleles of GCN3. These allele-specific interactions have led us to propose that GCN3 and GCD2 directly interact in the GCD-eIF-2B complex. Genetic evidence that GCD6 and GCD7 encode additional subunits of the GCD-eIF-2B complex was provided by the fact that reduced-function mutations in these genes are lethal in strains deleted for GCN3, the same interaction described previously for mutations in GCD1 and GCD2. Biochemical experiments showing that GCD6 and GCD7 copurify and coimmunoprecipitate with GCD1, GCD2, GCN3, and subunits of eIF-2 have confirmed that GCD6 and GCD7 are subunits of the GCD-eIF-2B complex. The fact that all five subunits of yeast eIF-2B were first identified as translational regulators of GCN4 strongly suggests that regulation of guanine nucleotide exchange on eIF-2 is a key control point for translation in yeast cells just as in mammalian cells.
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PMCID: PMC360088  PMID: 8336705
11.  Eukaryotic Translation Initiation Factor 3 (eIF3) and eIF2 Can Promote mRNA Binding to 40S Subunits Independently of eIF4G in Yeast†  
Molecular and Cellular Biology  2006;26(4):1355-1372.
Recruitment of the eukaryotic translation initiation factor 2 (eIF2)-GTP-Met-tRNAiMet ternary complex to the 40S ribosome is stimulated by multiple initiation factors in vitro, including eIF3, eIF1, eIF5, and eIF1A. Recruitment of mRNA is thought to require the functions of eIF4F and eIF3, with the latter serving as an adaptor between the ribosome and the 4G subunit of eIF4F. To define the factor requirements for these reactions in vivo, we examined the effects of depleting eIF2, eIF3, eIF5, or eIF4G in Saccharomyces cerevisiae cells on binding of the ternary complex, other initiation factors, and RPL41A mRNA to native 43S and 48S preinitiation complexes. Depleting eIF2, eIF3, or eIF5 reduced 40S binding of all constituents of the multifactor complex (MFC), comprised of these three factors and eIF1, supporting a mechanism of coupled 40S binding by MFC components. 40S-bound mRNA strongly accumulated in eIF5-depleted cells, even though MFC binding to 40S subunits was reduced by eIF5 depletion. Hence, stimulation of the GTPase activity of the ternary complex, a prerequisite for 60S subunit joining in vitro, is likely the rate-limiting function of eIF5 in vivo. Depleting eIF2 or eIF3 impaired mRNA binding to free 40S subunits, but depleting eIF4G led unexpectedly to accumulation of mRNA on 40S subunits. Thus, it appears that eIF3 and eIF2 are more critically required than eIF4G for stable binding of at least some mRNAs to native preinitiation complexes and that eIF4G has a rate-limiting function at a step downstream of 48S complex assembly in vivo.
doi:10.1128/MCB.26.4.1355-1372.2006
PMCID: PMC1367198  PMID: 16449648
12.  Complex formation by positive and negative translational regulators of GCN4. 
Molecular and Cellular Biology  1991;11(6):3217-3228.
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 KCl. 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-2 alpha, 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.
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PMCID: PMC360174  PMID: 2038327
13.  Truncated protein phosphatase GLC7 restores translational activation of GCN4 expression in yeast mutants defective for the eIF-2 alpha kinase GCN2. 
Molecular and Cellular Biology  1992;12(12):5700-5710.
GCN2 is a protein kinase in Saccharomyces cerevisiae that is required for increased expression of the transcriptional activator GCN4 in amino acid-starved cells. GCN2 stimulates GCN4 synthesis at the translational level by phosphorylating the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2). We identified a truncated form of the GLC7 gene, encoding the catalytic subunit of a type 1 protein phosphatase, by its ability to restore derepression of GCN4 expression in a strain containing the partially defective gcn2-507 allele. Genetic analysis suggests that the truncated GLC7 allele has a dominant negative phenotype, reducing the level of native type 1 protein phosphatase activity in the cell. The truncated form of GLC7 does not suppress the regulatory defect associated with a gcn2 deletion or a mutation in the phosphorylation site of eIF-2 alpha (Ser-51). In addition, the presence of multiple copies of wild-type GLC7 impairs the derepression of GCN4 that occurs in response to amino acid starvation or dominant-activating mutations in GCN2. These findings suggest that the phosphatase activity of GLC7 acts in opposition to the kinase activity of GCN2 in modulating the level of eIF-2 alpha phosphorylation and the translational efficiency of GCN4 mRNA. This conclusion is supported by biochemical studies showing that the truncated GLC7 allele increases the level of eIF-2 alpha phosphorylation in the gcn2-507 mutant to a level approaching that seen in wild-type cells under starvation conditions. The truncated GLC7 allele also leads to reduced glycogen accumulation, indicating that this protein phosphatase is involved in regulating diverse metabolic pathways in yeast cells.
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PMCID: PMC360510  PMID: 1333044
14.  Mutations activating the yeast eIF-2 alpha kinase GCN2: isolation of alleles altering the domain related to histidyl-tRNA synthetases. 
Molecular and Cellular Biology  1992;12(12):5801-5815.
The protein kinase GCN2 stimulates expression of the yeast transcriptional activator GCN4 at the translational level by phosphorylating the alpha subunit of translation initiation factor 2 (eIF-2 alpha) in amino acid-starved cells. Phosphorylation of eIF-2 alpha reduces its activity, allowing ribosomes to bypass short open reading frames present in the GCN4 mRNA leader and initiate translation at the GCN4 start codon. We describe here 17 dominant GCN2 mutations that lead to derepression of GCN4 expression in the absence of amino acid starvation. Seven of these GCN2c alleles map in the protein kinase moiety, and two in this group alter the presumed ATP-binding domain, suggesting that ATP binding is a regulated aspect of GCN2 function. Six GCN2c alleles map in a region related to histidyl-tRNA synthetases, and two in this group alter a sequence motif conserved among class II aminoacyl-tRNA synthetases that directly interacts with the acceptor stem of tRNA. These results support the idea that GCN2 kinase function is activated under starvation conditions by binding uncharged tRNA to the domain related to histidyl-tRNA synthetase. The remaining GCN2c alleles map at the extreme C terminus, a domain required for ribosome association of the protein. Representative mutations in each domain were shown to depend on the phosphorylation site in eIF-2 alpha for their effects on GCN4 expression and to increase the level of eIF-2 alpha phosphorylation in the absence of amino acid starvation. Synthetic GCN2c double mutations show greater derepression of GCN4 expression than the parental single mutations, and they have a slow-growth phenotype that we attribute to inhibition of general translation initiation. The phenotypes of the GCN2c alleles are dependent on GCN1 and GCN3, indicating that these two positive regulators of GCN4 expression mediate the inhibitory effects on translation initiation associated with activation of the yeast eIF-2 alpha kinase GCN2.
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PMCID: PMC360520  PMID: 1448107
15.  Selective control of amino acid metabolism by the GCN2 eIF2 kinase pathway in Saccharomyces cerevisiae 
BMC Biochemistry  2010;11:29.
Background
When eukaryotic cells are deprived of amino acids, uncharged tRNAs accumulate and activate the conserved GCN2 protein kinase. Activated Gcn2p up-regulates the general amino acid control pathway through phosphorylation of the translational initiation factor eIF2. In Saccharomyces cerevisiae, Gcn2p is the only kinase that phosphorylates eIF2 to regulate translation through this mechanism. We addressed changes in yeast growth and tRNA aminoacylation, or charging, during amino acid depletion in the presence and absence of GCN2. tRNA charging was measured using a microarray technique which simultaneously measures all cytosolic tRNAs. A fully prototrophic strain, and its isogenic gcn2Δ counterpart, were used to study depletion for each of the 20 amino acids, with a focus on Trp, Arg, His and Leu, which are metabolically distinct and together provide a good overview on amino acid metabolism.
Results
While the wild-type strain had no observable phenotype upon depletion for any amino acid, the gcn2Δ strain showed slow growth in media devoid of only Trp or Arg. Consistent with the growth phenotypes, profiles of genome-wide tRNA charging revealed significant decrease in cognate tRNA charging only in the gcn2Δ strain upon depletion for Trp or Arg. In contrast, there was no change in tRNA charging during His and Leu depletion in either the wild-type or gcn2Δ strains, consistent with the null effect on growth during loss of these amino acids. We determined that the growth phenotype of Trp depletion is derived from feedback inhibition of aromatic amino acid biosynthesis. By removing Phe and Tyr from the media in addition to Trp, regular growth was restored and tRNATrp charging no longer decreased. The growth phenotype of Arg depletion is derived from unbalanced nitrogen metabolism. By supplementing ornithine upon Arg depletion, both growth and tRNAArg charging were partially restored.
Conclusion
Under mild stress conditions the basal activity of Gcn2p is sufficient to allow for proper adaptation to amino acid depletion. This study highlights the importance of the GCN2 eIF2 kinase pathway for maintaining metabolic homeostasis, contributing to appropriate tRNA charging and growth adaptation in response to culture conditions deficient for the central amino acids, tryptophan and arginine.
doi:10.1186/1471-2091-11-29
PMCID: PMC2921344  PMID: 20684782
16.  The Yeast Eukaryotic Initiation Factor 4G (eIF4G) HEAT Domain Interacts with eIF1 and eIF5 and Is Involved in Stringent AUG Selection 
Molecular and Cellular Biology  2003;23(15):5431-5445.
Eukaryotic initiation factor 4G (eIF4G) promotes mRNA recruitment to the ribosome by binding to the mRNA cap- and poly(A) tail-binding proteins eIF4E and Pap1p. eIF4G also binds eIF4A at a distinct HEAT domain composed of five stacks of antiparallel α-helices. The role of eIF4G in the later steps of initiation, such as scanning and AUG recognition, has not been defined. Here we show that the entire HEAT domain and flanking residues of Saccharomyces cerevisiae eIF4G2 are required for the optimal interaction with the AUG recognition factors eIF5 and eIF1. eIF1 binds simultaneously to eIF4G and eIF3c in vitro, as shown previously for the C-terminal domain of eIF5. In vivo, cooverexpression of eIF1 or eIF5 reverses the genetic suppression of an eIF4G HEAT domain Ts− mutation by eIF4A overexpression. In addition, excess eIF1 inhibits growth of a second eIF4G mutant defective in eIF4E binding, which was also reversed by cooverexpression of eIF4A. Interestingly, excess eIF1 carrying the sui1-1 mutation, known to relax the accuracy of start site selection, did not inhibit the growth of the eIF4G mutant, and sui1-1 reduced the interaction between eIF4G and eIF1 in vitro. Moreover, a HEAT domain mutation altering eIF4G moderately enhances translation from a non-AUG codon. These results strongly suggest that the binding of the eIF4G HEAT domain to eIF1 and eIF5 is important for maintaining the integrity of the scanning ribosomal preinitiation complex.
doi:10.1128/MCB.23.15.5431-5445.2003
PMCID: PMC165723  PMID: 12861028
17.  eIF2β is critical for eIF5-mediated GDP-dissociation inhibitor activity and translational control 
Nucleic Acids Research  2016;44(20):9698-9709.
In protein synthesis translation factor eIF2 binds initiator tRNA to ribosomes and facilitates start codon selection. eIF2 GDP/GTP status is regulated by eIF5 (GAP and GDI functions) and eIF2B (GEF and GDF activities), while eIF2α phosphorylation in response to diverse signals is a major point of translational control. Here we characterize a growth suppressor mutation in eIF2β that prevents eIF5 GDI and alters cellular responses to reduced eIF2B activity, including control of GCN4 translation. By monitoring the binding of fluorescent nucleotides and initiator tRNA to purified eIF2 we show that the eIF2β mutation does not affect intrinsic eIF2 affinities for these ligands, neither does it interfere with eIF2 binding to 43S pre-initiation complex components. Instead we show that the eIF2β mutation prevents eIF5 GDI stabilizing nucleotide binding to eIF2, thereby altering the off-rate of GDP from eIF2•GDP/eIF5 complexes. This enables cells to grow with reduced eIF2B GEF activity but impairs activation of GCN4 targets in response to amino acid starvation. These findings provide support for the importance of eIF5 GDI activity in vivo and demonstrate that eIF2β acts in concert with eIF5 to prevent premature release of GDP from eIF2γ and thereby ensure tight control of protein synthesis initiation.
doi:10.1093/nar/gkw657
PMCID: PMC5175340  PMID: 27458202
18.  Stress-Induced C/EBP Homology Protein (CHOP) Represses MyoD Transcription to Delay Myoblast Differentiation 
PLoS ONE  2011;6(12):e29498.
When mouse myoblasts or satellite cells differentiate in culture, the expression of myogenic regulatory factor, MyoD, is downregulated in a subset of cells that do not differentiate. The mechanism involved in the repression of MyoD expression remains largely unknown. Here we report that a stress-response pathway repressing MyoD transcription is transiently activated in mouse-derived C2C12 myoblasts growing under differentiation-promoting conditions. We show that phosphorylation of the α subunit of the translation initiation factor 2 (eIF2α) is followed by expression of C/EBP homology protein (CHOP) in some myoblasts. ShRNA-driven knockdown of CHOP expression caused earlier and more robust differentiation, whereas its constitutive expression delayed differentiation relative to wild type myoblasts. Cells expressing CHOP did not express the myogenic regulatory factors MyoD and myogenin. These results indicated that CHOP directly repressed the transcription of the MyoD gene. In support of this view, CHOP associated with upstream regulatory region of the MyoD gene and its activity reduced histone acetylation at the enhancer region of MyoD. CHOP interacted with histone deacetylase 1 (HDAC1) in cells. This protein complex may reduce histone acetylation when bound to MyoD regulatory regions. Overall, our results suggest that the activation of a stress pathway in myoblasts transiently downregulate the myogenic program.
doi:10.1371/journal.pone.0029498
PMCID: PMC3248460  PMID: 22242125
19.  Evidence that GCN1 and GCN20, translational regulators of GCN4, function on elongating ribosomes in activation of eIF2alpha kinase GCN2. 
Molecular and Cellular Biology  1997;17(8):4474-4489.
In the yeast Saccharomyces cerevisiae, phosphorylation of translation initiation factor eIF2 by protein kinase GCN2 leads to increased translation of the transcriptional activator GCN4 in amino acid-starved cells. The GCN1 and GCN20 proteins are components of a protein complex required for the stimulation of GCN2 kinase activity under starvation conditions. GCN20 is a member of the ATP-binding cassette (ABC) family, most of the members of which function as membrane-bound transporters, raising the possibility that the GCN1/GCN20 complex regulates GCN2 indirectly as an amino acid transporter. At odds with this idea, indirect immunofluorescence revealed cytoplasmic localization of GCN1 and no obvious association with plasma or vacuolar membranes. In addition, a fraction of GCN1 and GCN20 cosedimented with polysomes and 80S ribosomes, and the ribosome association of GCN20 was largely dependent on GCN1. The C-terminal 84% of GCN20 containing the ABCs was found to be dispensable for complex formation with GCN1 and for the stimulation of GCN2 kinase function. Because ABCs provide the energy-coupling mechanism for ABC transporters, these results also contradict the idea that GCN20 regulates GCN2 as an amino acid transporter. The N-terminal 15 to 25% of GCN20, which is critically required for its regulatory function, was found to interact with an internal segment of GCN1 similar in sequence to translation elongation factor 3 (EF3). Based on these findings, we propose that GCN1 performs an EF3-related function in facilitating the activation of GCN2 by uncharged tRNA on translating ribosomes. The physical interaction between GCN20 and the EF3-like domain in GCN1 could allow for modulation of GCN1 activity, and the ABC domains in GCN20 may be involved in this regulatory function. A human homolog of GCN1 has been identified, and the portion of this protein most highly conserved with yeast GCN1 has sequence similarity to EF3. Thus, similar mechanisms for the detection of uncharged tRNA on translating ribosomes may operate in yeast and human cells.
PMCID: PMC232301  PMID: 9234705
20.  Regulation of Eukaryotic Initiation Factor 4AII by MyoD during Murine Myogenic Cell Differentiation 
PLoS ONE  2014;9(1):e87237.
Gene expression during muscle cell differentiation is tightly regulated at multiple levels, including translation initiation. The PI3K/mTOR signalling pathway exerts control over protein synthesis by regulating assembly of eukaryotic initiation factor (eIF) 4F, a heterotrimeric complex that stimulates recruitment of ribosomes to mRNA templates. One of the subunits of eIF4F, eIF4A, supplies essential helicase function during this phase of translation. The presence of two cellular eIF4A isoforms, eIF4AI and eIF4AII, has long thought to impart equivalent functions to eIF4F. However, recent experiments have alluded to distinct activities between them. Herein, we characterize distinct regulatory mechanisms between the eIF4A isoforms during muscle cell differentiation. We find that eIF4AI levels decrease during differentiation whereas eIF4AII levels increase during myofiber formation in a MyoD-dependent manner. This study characterizes a previously undefined mechanism for eIF4AII regulation in differentiation and highlights functional differences between eIF4AI and eIF4AII. Finally, RNAi-mediated alterations in eIF4AI and eIF4AII levels indicate that the myogenic process can tolerate short term reductions in eIF4AI or eIF4AII levels, but not both.
doi:10.1371/journal.pone.0087237
PMCID: PMC3900710  PMID: 24466343
21.  An upstream ORF with non-AUG start codon is translated in vivo but dispensable for translational control of GCN4 mRNA 
Nucleic Acids Research  2011;39(8):3128-3140.
Genome-wide analysis of ribosome locations in mRNAs of Saccharomyces cerevisiae has revealed the translation of upstream open reading frames that initiate with near-cognate start codons in many transcripts. Two such non-translation initiation codon (AUG)-initiated upstream open reading frames (uORFs) (nAuORFs 1 and 2) occur in GCN4 mRNA upstream of the four AUG-initiated uORFs (uORFs 1–4) that regulate GCN4 translation. We verified that nAuORF2 is translated in vivo by demonstrating β-galactosidase production from lacZ coding sequences fused to nAuORF2, in a manner abolished by replacing its non-AUG initiation codon (AUA) start codon with the non-cognate triplet AAA, whereas translation of nAuORF1 was not detected. Importantly, replacing the near-cognate start codons of both nAuORFs with non-cognate triplets had little or no effect on the repression of GCN4 translation in non-starved cells, nor on its derepression in response to histidine limitation, nutritional shift-down or treatment with rapamycin, hydrogen peroxide or methyl methanesulfonate. Additionally, we found no evidence that initiation from the AUA codon of nAuORF2 is substantially elevated, or dependent on Gcn2, the sole eIF2α kinase of yeast, in histidine-deprived cells. Thus, although nAuORF2 is translated in vivo, it appears that this event is not stimulated by eIF2α phosphorylation nor significantly influences GCN4 translational induction under various starvation or stress conditions.
doi:10.1093/nar/gkq1251
PMCID: PMC3082883  PMID: 21227927
22.  Translation of 5′ leaders is pervasive in genes resistant to eIF2 repression 
eLife  null;4:e03971.
Eukaryotic cells rapidly reduce protein synthesis in response to various stress conditions. This can be achieved by the phosphorylation-mediated inactivation of a key translation initiation factor, eukaryotic initiation factor 2 (eIF2). However, the persistent translation of certain mRNAs is required for deployment of an adequate stress response. We carried out ribosome profiling of cultured human cells under conditions of severe stress induced with sodium arsenite. Although this led to a 5.4-fold general translational repression, the protein coding open reading frames (ORFs) of certain individual mRNAs exhibited resistance to the inhibition. Nearly all resistant transcripts possess at least one efficiently translated upstream open reading frame (uORF) that represses translation of the main coding ORF under normal conditions. Site-specific mutagenesis of two identified stress resistant mRNAs (PPP1R15B and IFRD1) demonstrated that a single uORF is sufficient for eIF2-mediated translation control in both cases. Phylogenetic analysis suggests that at least two regulatory uORFs (namely, in SLC35A4 and MIEF1) encode functional protein products.
DOI: http://dx.doi.org/10.7554/eLife.03971.001
eLife digest
Proteins carry out essential tasks for living cells and genes contain the instructions to make proteins within their DNA. These instructions are copied to make a molecule of mRNA, and a molecular machine known as a ribosome then reads and translates the mRNA to build the protein.
The first step in the translation process is called ‘initiation’ and requires a protein called eIF2 to work together with the ribosome. This step involves identifying an instruction called the start codon that marks the beginning of the mRNA's coding sequence. The section of an mRNA molecule before the start codon is not normally translated by the ribosome and is hence called the 5′ untranslated region.
Building proteins requires energy and resources, and so it is carefully regulated. If a cell is stressed, such as by being exposed to harmful chemicals, it makes fewer proteins in order to conserve its resources. This down-regulation of protein production is achieved in part by the cell chemically modifying its eIF2 proteins to make them less able to initiate translation. However, stressed cells still continue to make more of certain proteins that help them to combat stress. The mRNA molecules for some of these proteins contain at least one other start codon in the 5′ untranslated region. The sequence that would be translated from such a start codon is known as an upstream open reading frame (or uORF for short)—and this feature is thought to help certain proteins to still be expressed despite low levels of active eIF2. Andreev, O'Connor et al. have now analysed which mRNAs are translated in human cells that have been treated with a chemical that induces stress and makes the eIF2 protein less able to initiate translation. To do so, a technique called ribosome profiling was used to identify all of the mRNA molecules bound to ribosomes shortly after treatment with this chemical.
Overall translation of most mRNAs in stressed cells was reduced to a quarter of the normal level. However, Andreev, O'Connor et al. observed that the translation of a few mRNAs continued almost as normal, or even increased, after the chemical treatment. Notably, most of these mRNAs encoded regulatory proteins, which are not required in large amounts. With one exception, all of these resistant mRNAs contained uORFs. In unstressed cells, these uORFs were efficiently translated, while the same mRNA's coding sequences were translated less efficiently. Andreev, O'Connor et al. suggest that these two features could be used to identify mRNAs that are still translated into working proteins when cells are stressed. Further work is now needed to explore the mechanisms by which translation of these uORFs allows mRNAs to resist the stress.
DOI: http://dx.doi.org/10.7554/eLife.03971.002
doi:10.7554/eLife.03971
PMCID: PMC4383229  PMID: 25621764
upstream open reading frame (uORF); eukaryotic initiation factor 2 (eIF2); integrated stress response (ISR); ribosome profiling; 5′ leader translation; bicistronic mRNA; human
23.  Sequential Eukaryotic Translation Initiation Factor 5 (eIF5) Binding to the Charged Disordered Segments of eIF4G and eIF2β Stabilizes the 48S Preinitiation Complex and Promotes Its Shift to the Initiation Mode 
Molecular and Cellular Biology  2012;32(19):3978-3989.
During translation initiation in Saccharomyces cerevisiae, an Arg- and Ser-rich segment (RS1 domain) of eukaryotic translation initiation factor 4G (eIF4G) and the Lys-rich segment (K-boxes) of eIF2β bind three common partners, eIF5, eIF1, and mRNA. Here, we report that both of these segments are involved in mRNA recruitment and AUG recognition by distinct mechanisms. First, the eIF4G-RS1 interaction with the eIF5 C-terminal domain (eIF5-CTD) directly links eIF4G to the preinitiation complex (PIC) and enhances mRNA binding. Second, eIF2β-K-boxes increase mRNA binding to the 40S subunit in vitro in a manner reversed by the eIF5-CTD. Third, mutations altering eIF4G-RS1, eIF2β-K-boxes, and eIF5-CTD restore the accuracy of start codon selection impaired by an eIF2β mutation in vivo, suggesting that the mutual interactions of the eIF segments within the PIC prime the ribosome for initiation in response to start codon selection. We propose that the rearrangement of interactions involving the eIF5-CTD promotes mRNA recruitment through mRNA binding by eIF4G and eIF2β and assists the start codon-induced release of eIF1, the major antagonist of establishing tRNAiMet:mRNA binding to the P site.
doi:10.1128/MCB.00376-12
PMCID: PMC3457530  PMID: 22851688
24.  GCD2, a translational repressor of the GCN4 gene, has a general function in the initiation of protein synthesis in Saccharomyces cerevisiae. 
Molecular and Cellular Biology  1991;11(6):3203-3216.
The GCD2 protein is a translational repressor of GCN4, the transcriptional activator of multiple amino acid biosynthetic genes in Saccharomyces cerevisiae. We present evidence that GCD2 has a general function in the initiation of protein synthesis in addition to its gene-specific role in translational control of GCN4 expression. Two temperature-sensitive lethal gcd2 mutations result in sensitivity to inhibitors of protein synthesis at the permissive temperature, and the gcd2-503 mutation leads to reduced incorporation of labeled leucine into total protein following a shift to the restrictive temperature of 36 degrees C. The gcd2-503 mutation also results in polysome runoff, accumulation of inactive 80S ribosomal couples, and accumulation of at least one of the subunits of the general translation initiation factor 2 (eIF-2 alpha) in 43S-48S particles following a shift to the restrictive temperature. The gcd2-502 mutation causes accumulation of 40S subunits in polysomes, known as halfmers, that are indicative of reduced 40S-60S subunit joining at the initiation codon. These phenotypes suggest that GCD2 functions in the translation initiation pathway at a step following the binding of eIF-2.GTP.Met-tRNA(iMet) to 40S ribosomal subunits. consistent with this hypothesis, we found that inhibiting 40S-60S subunit joining by deleting one copy (RPL16B) of the duplicated gene encoding the 60S ribosomal protein L16 qualitatively mimics the phenotype of gcd2 mutations in causing derepression of GCN4 expression under nonstarvation conditions. However, deletion of RPL16B also prevents efficient derepression of GCN4 under starvation conditions, indicating that lowering the concentration of 60S subunits and reducing GCD2 function affect translation initiation at GCN4 in different ways. This distinction is in accord with a recently proposed model for GCN4 translational control in which ribosomal reinitiation at short upstream open reading frames in the leader of GCN4 mRNA is suppressed under amino acid starvation conditions to allow for increased reinitiation at the GCN4 start codon.
Images
PMCID: PMC360173  PMID: 2038326
25.  Analysing GCN4 translational control in yeast by stochastic chemical kinetics modelling and simulation 
BMC Systems Biology  2011;5:131.
Background
The yeast Saccharomyces cerevisiae responds to amino acid starvation by inducing the transcription factor Gcn4. This is mainly mediated via a translational control mechanism dependent upon the translation initiation eIF2·GTP·Met-tRNAiMet ternary complex, and the four short upstream open reading frames (uORFs) in its 5' mRNA leader. These uORFs act to attenuate GCN4 mRNA translation under normal conditions. During amino acid starvation, levels of ternary complex are reduced. This overcomes the GCN4 translation attenuation effect via a scanning/reinitiation control mechanism dependent upon uORF spacing.
Results
Using published experimental data, we have developed and validated a probabilistic formulation of GCN4 translation using the Chemical Master Equation (Model 1). Model 1 explains GCN4 translation's nonlinear dependency upon uORF placements, and predicts that an as yet unidentified factor, which was proposed to regulate GCN4 translation under some conditions, only has pronounced effects upon GCN4 translation when intercistronic distances are unnaturally short. A simpler Model 2 that does not include this unidentified factor could well represent the regulation of a natural GCN4 mRNA. Using parameter values optimised for this algebraic Model 2, we performed stochastic simulations by Gillespie algorithm to investigate the distribution of ribosomes in different sections of GCN4 mRNA under distinct conditions. Our simulations demonstrated that ribosomal loading in the 5'-untranslated region is mainly determined by the ratio between the rates of 5'-initiation and ribosome scanning, but was not significantly affected by rate of ternary complex binding. Importantly, the translation rate for codons starved of cognate tRNAs is predicted to be the most significant contributor to the changes in ribosomal loading in the coding region under repressing and derepressing conditions.
Conclusions
Our integrated probabilistic Models 1 and 2 explained GCN4 translation and helped to elucidate the role of a yet unidentified factor. The ensuing stochastic simulations evaluated different factors that may impact on the translation of GCN4 mRNA, and integrated translation status with ribosomal density.
doi:10.1186/1752-0509-5-131
PMCID: PMC3201031  PMID: 21851603
mRNA translation; GCN4; Gillespie algorithm; stochastic model

Results 1-25 (1099532)