Transcription Factor GCN4 Research Articles

Expression of an arabidopsis aspartate Kinase/Homoserine dehydrogenase gene is metabolically regulated by photosynthesis-related signals but not by nitrogenous compounds

Although the control of carbon fixation and nitrogen assimilation has been studied in detail, relatively little is known about the regulation of carbon and nitrogen flow into amino acids. In this paper we report our study of the metabolic regulation of expression of an Arabidopsis aspartate kinase/homoserine dehydrogenase (AK/HSD) gene, which encodes two linked key enzymes in the biosynthetic pathway of aspartate family amino acids. Northern blot analyses, as well as expression of chimeric AK/HSD-beta-glucuronidase constructs, have shown that the expression of this gene is regulated by the photosynthesis-related metabolites sucrose and phosphate but not by nitrogenous compounds. In addition, analysis of AK/HSD promoter deletions suggested that a CTTGACTCTA sequence, resembling the binding site for the yeast GCN4 transcription factor, is likely to play a functional role in the expression of this gene. Nevertheless, longer promoter fragments, lacking the GCN4-like element, were still able to confer sugar inducibility, implying that the metabolic regulation of this gene is apparently obtained by multiple and redundant promoter sequences. The present and previous studies suggest that the conversion of aspartate into either the storage amino acid asparagine or aspartate family amino acids is subject to a coordinated, reciprocal metabolic control, and this biochemical branch point is a part of a larger, coordinated regulatory mechanism of nitrogen and carbon storage and utilization.

Computer analysis of the entire budding yeast genome for putative targets of the GCN4 transcription factor.

The completion of the yeast genome project enables an analysis of various phenomena for a whole eukaryotic genome. We aimed at characterizing a full spectrum of target genes for a transcription activator, and specifically characterized putative targets for GCN4 in the budding yeast. The results suggest that about 1% of the genes are regulated by GCN4 and that these genes code for proteins involved in amino-acid and nucleotide metabolism. Our analysis proposes that, when enough data about the binding nature of a transcription factor exists, it is possible to identify its putative targets and also to try and assign a physiological role for this transcription factor.

Electrostatic effects in DNA bending by GCN4 mutants.

DNA architecture has been shown to be important for cellular processes such as activation of transcription, recombination, and replication. Many proteins reconfigure the shape of duplex DNA upon binding. Previous experiments have shown that some members of the eukaryotic bZIP family of DNA binding proteins appear to bend DNA, while others do not. We are exploring the role of electrostatic effects in DNA bending by bZIP proteins. The yeast bZIP transcription factor GCN4 does not induce DNA bending in vitro. Previously we substituted basic residues for three neutral amino acids in GCN4 to produce a GCN4 derivative that bends DNA by approximately 15 degrees. This result is consistent with a model of induced DNA bending wherein excess positive charge in proximity to one face of the double helix neutralizes local phosphate diester anions resulting in a laterally-asymmetric charge distribution along the DNA. Such an unbalanced charge distribution can result in collapse of the DNA toward the neutralized surface. We now present a more comprehensive analysis of electrostatic effects in DNA bending by GCN4 derivatives. It is shown that the direction and extent of DNA bending by these derivatives are a linear function of the charges of the amino acids adjacent to the basic domain of the protein. This relation holds over the charge range +6 (16 degrees bend toward the minor groove) to -6 (25 degrees bend toward the major groove).

Effect of Bcr sequences on the cellular function of the Bcr-Abl oncoprotein.

In Philadelphia chromosome (Ph1)-positive human leukemia, the c-Abl tyrosine kinase is activated by fusion to sequences encoded by the breakpoint cluster region (bcr) gene. Two major types of Bcr-Abl fusion proteins have been found in human leukemia. Fusion of the N-terminal 426 amino acids of Bcr generates p190(Bcr-Abl) which is mostly found in acute lymphocytic leukemia (ALL), whereas fusion of the N-terminal 902 or 927 amino acids of Bcr generates p210(Bcr-Abl) mostly found with chronic myelogenous leukemia (CML). Previous studies have demonstrated that both the Bcr and the Abl functional domains contribute to the oncogenic activity of Bcr-Abl proteins. Present in both p190 and p210 is the N-terminal coiled-coil of Bcr (aa 1-63), which is shown here to be functionally replaceable with the leucine zipper of the yeast transcription factor GCN4. The ZIP-Bcr-Abl protein transforms Rat-1/myc cells, is autophosphorylated on tyrosine and localized predominantly to actin filaments. Thus, formation of homo-oligomers through either Bcr or GCN4 coiled-coil can activate the tyrosine kinase and F-actin binding functions of Abl. We also found that a Bcr-Abl fusion containing only Bcr amino acids (1-191) can efficiently transform Rat-1/myc cells. Fusion of additional Bcr sequences (aa 192-923) did not affect the transformation of Rat-1/myc cells but progressively reduced the disruptive effect on the actin cytoskeleton. In particular, the Dbl homology domain present in p210(Bcr-Abl) but not in p190(Bcr-Abl) contributes to the stabilization of actin fibers. The modulatory effect of Bcr sequences on actin structure may underlie the apparent pathogenic variations between the different Bcr-Abl fusion proteins.

DNA bending by GCN4 mutants bearing cationic residues.

Transcription activation is thought to require DNA bending to promote the interaction of upstream activators and the basal transcription machinery. Previous experiments have shown that some members of the bZIP family of DNA binding proteins bend DNA, while others do not. We are exploring the possibility that electrostatic effects play a role in these differences. The yeast bZIP transcription factor GCN4 does not induce DNA bending in vitro. Substitution of basic residues for three neutral amino acids of GCN4 confers the ability to bend DNA. This result is consistent with a model of induced DNA bending wherein excess positive charge in proximity to one face of the double helix neutralizes local phosphate diester anions resulting in a laterally asymmetric charge distribution along the DNA. Previous data suggest that such an unbalanced charge distribution results in collapse of the DNA toward the neutralized surface. Interpretations of the present data are discussed. Our result supports the hypothesis that electrostatic interactions can play a key role in DNA bending by bZIP proteins.

A self-replicating peptide.

The production of amino acids and their condensation to polypeptides under plausibly prebiotic conditions have long been known. But despite the central importance of molecular self-replication in the origin of life, the feasibility of peptide self-replication has not been established experimentally. Here we report an example of a self-replicating peptide. We show that a 32-residue alpha-helical peptide based on the leucine-zipper domain of the yeast transcription factor GCN4 can act autocatalytically in templating its own synthesis by accelerating the thioester-promoted amide-bond condensation of 15- and 17-residue fragments in neutral, dilute aqueous solutions. The self-replication process displays parabolic growth pattern with the initial rates of product formation correlating with the square-foot of initial template concentration.

Synthesis of a peptide-intercalator hybrid based on the bZIP motif from GCN4

An artificial peptide, designed to combine the DNA-recognition portion of the bZIP motif from the yeast transcription factor GCN4 with an intercalating portion, 9-aminoacridine, has been synthesised.

Coupled folding and site-specific binding of the GCN4-bZIP transcription factor to the AP-1 and ATF/CREB DNA sites studied by microcalorimetry.

The site-specific interaction of the basic leucine zipper protein C62GCN4, which corresponds to the C-terminal sequence 220-281 of the yeast transcription factor GCN4, with the AP-1 and ATF/ CREB DNA recognition sites was analyzed by isothermal titration microcalorimetry. Free C62GCN4 is a dimer composed of a C-terminal leucine zipper and a basic, mainly unstructured DNA binding domain. Upon association with the target DNA, C62GCN4 folds to a fully alpha-helical dimer [Ellenberger et al. (1992) Cell 71, 1223-1237; König and Richmond (1993) J. Mol. Biol. 233, 139-154]. The protein-bound AP-1 site is straight, and the protein-bound ATF/CREB site is bent by 20 degrees toward the leucine zipper domain. The coupling between protein folding and DNA association resulting in two conformationally different complexes with C62GCN4 poses interesting thermodynamic problems. The association was strongly exothermic for both DNA target sites. The free energies of binding were indistinguishable in buffers of low salt concentration, and no change of the protonation state of C62GCN4 and/or the DNA target site occurred on formation of the complexes. Both complexes exhibited large and negative heat capacity changes. The empirical correlation between buried nonpolar and polar surfaces and the reduction in heat capacity concomitant to complexation did hold for the reaction with the AP-1 site at low salt concentration. However, in the case of the ATF/CREB site, the change in heat capacity was larger than could be accounted for by the burial of solvent-accessible surface. Potential sources of the extra decrement in the heat capacity could be restrictions in the vibrational modes of polar groups and of bound water molecules at the protein-DNA interface, thought to result from the bending of the ATF/CREB site. In the presence of high concentrations of glutamate and NaCl, the complex with the ATF/CREB site was significantly weaker than the complex with the AP-1 site.

Characterization of leucine zipper complexes by electrospray ionization mass spectrometry.

The development of "soft" ionization methods has enabled the mass spectrometric analysis of higher-order structural features of proteins. We have applied electrospray ionization mass spectrometry (ESI-MS) to the analysis of the number and composition of polypeptide chains in homomeric and heteromeric leucine zippers. In comparison with other methods that have been used to analyze leucine zippers, such as analytical ultracentrifugation, gel chromatography, or electrophoretic band shift assays, ESI-MS is very fast and highly sensitive and provides a straightforward way to distinguish between homomeric and heteromeric coiled-coil structures. ESI-MS analyses were carried out on the parallel dimeric leucine zipper domain GCN4-p1 of the yeast transcription factor GCN4 and on three synthetic peptides with the sequences Ac-EYEALEKKLAAX1EAKX2QALEKKLEALEHG-amide: peptide LZ (X1, X2 = Leu), peptide LZ(12A) (X1 = Ala, X2 = Leu), and peptide LZ(16N) (X1 = Leu, X2 = Asn). Equilibrium ultracentrifugation analysis showed that LZ forms a trimeric coiled coil and this could be confirmed unequivocally by ESI-MS as could the dimeric nature of GCN4-p1. The formation of heteromeric two- and three-stranded leucine zippers composed of chains from LZ and LZ(12A), or from GCN4-p1 and LZ, was demonstrated by ESI-MS and confirmed by fluorescence quenching experiments on fluorescein-labeled peptides. The results illustrate the adaptability and flexibility of the leucine zipper motif, properties that could be useful to the design of specific protein assemblies by way of coiled-coil domains.

Regulated degradation of the transcription factor Gcn4.

We report that Gcn4, a yeast transcriptional activator of the bZIP family involved in the regulation of the biosynthesis of amino acids and purines, is rapidly turned over. This degradation is inhibited under conditions of starvation for amino acids. Degradation is also inhibited by single amino acid alterations in a region adjacent to the Gcn4 activation domain. Furthermore, we show that degradation of Gcn4 proceeds through the ubiquitin pathway, a major proteolytic system for cytoplasmic proteins, and is dependent on two specific ubiquitin conjugating enzymes, Cdc34 (Ubc3) and Rad6 (Ubc2). As a first step towards reconstituting the Gcn4 degradation pathway in vitro, we show that purified Cdc34 and Rad6 proteins are able to direct the specific ubiquitination of Gcn4.

The plant transcription factor TGA1 stimulates expression of the CaMV 35S promoter in Saccharomyces cerevisiae

We have previously shown that two CRE elements situated on a 31 bp region of the cauliflower mosaic virus (CaMV) 35S promoter activate gene expression in the yeast Saccharomyces cerevisiae and are regulated by cAMP. Studies with the yeast transcription factors GCN4, SKO1 and YAP1, which bind CRE-like sequences, showed no influence on expression of the 35S promoter indicating that a yet unknown factor is involved in activation. Band shift experiments with the 31 bp promoter region revealed binding of similar factors in yeast and plant protein extracts. In a previous study this promoter region was shown to confer tissue-specific expression in plants and to interact with the transcription factor TGA1. To test whether expression of TGA1 in yeast also stimulates transcription of the 35S promoter, we co-transformed yeast cells with a cDNA clone of this transcription factor and a 35S promoter/reporter gene construct. Promoter activity studies revealed that TGA1 confers enhanced expression of a reporter gene under the control of the 35S promoter in yeast cells. Yeast cells that were transformed with a 35S promoter construct that containing a mutated TGA1-binding site showed that both TGA1 and the intact binding site are necessary for this activation. These results suggest that stimulation of the 35S promoter by TGA1 is mediated by competition with an endogenous down-regulating yeast factor that is modulated by the nutritional state of the cells.

The guanine nucleotide-exchange factor, eIF-2B

Eukaryotic initiation factor eIF-2B catalyses the exchange of guanine nucleotides on another translation initiation factor, eIF-2, which itself mediates the binding of the initiator Met-tRNA to the 40S ribosomal subunit during translation initiation. eIF-2B promotes the release of GDP from inactive [eIF-2·GDP] complexes, thus allowing formation of the active [eIF-2·GTP] species which subsequently binds the Met-tRNA. This guanine nucleotide-exchange, step, and thus eIF-2B activity, are known to be an important control point for translation initiation. The activity of eIF-2B can be modulated in several ways. The best characterised of these involves the phosphorylation of the α-subunit of eIF-2 by specific protein kinases regulated by particular ligands. Phosphorylation of eIF-2α leads to inhibition of eIF-2B. This mechanism is involved in the control of translation under a variety of conditions, including amino acid deprivation in yeast ( Saccharomyces cereviseae) where it causes translational upregulation of the transcription factor GCN4, and in virus-infected animal cells, where it involves a protein kinase activated by double-stranded RNA. There is now also growing evidence for direct regulation of eIF-2B. This appears likely to involve the phosphorylation of its largest subunit. Under certain circumstances eIF-2B may also be regulated by allosteric mechanisms. eIF-2B is a heteropentamer (subunits termed α, β, γ, δ and ϵ) and is thus more complex than most other guanine nucleotide-exchange factors. The genes encoding all five subunits have been cloned in yeast (exploiting the GCN4 regulatory system): all but the α appear to be essential for eIF-2B activity. However, this subunit may confer sensitivity to eIF-2α phosphorylation. cDNAs encoding the α, β, δ and ϵ subunits have been cloned from mammalian sources. There is substantial homology between the yeast and mammalian sequences. Attention is now directed towards understanding the roles of individual subunits in the function and regulation of eIF-2B.

Thermodynamic characterization of the structural stability of the coiled-coil region of the bZIP transcription factor GCN4.

The thermal stability of a 56 amino acid fragment of GCN4 has been studied by high-sensitivity differential scanning calorimetry and circular dichroism spectroscopy. This fragment contains the leucine zipper and part of the basic region. The thermal unfolding of GCN4-56 is a reversible process and can be well represented by a reaction of the form N2<-->2U, indicating that the unfolding of the leucine zipper is a two-state process in which the helices are only stable when they are in the coiled-coil conformation. As expected, the transition temperature is concentration dependent. At pH 7.06 and a protein concentration of 5 x 10(-4) M the transition temperature is close to 70 degrees C while at 5 x 10(-6) M it is close to 50 degrees C. The enthalpy change for unfolding is 31.5 kcal mol-1 at 70 degrees C. Since the isolated helices are unstable, interactions at the interface between the two helices play a key role in the stabilization of the native dimer. These interactions primarily involve the burial of apolar surface from the solvent (hydrophobic effect) and electrostatic interactions. Structural thermodynamic calculations have permitted a dissection of the magnitude of the various contributions to the total Gibbs free energy of stabilization.

Identification of three residues in the basic regions of the bZIP proteins GCN4, C/EBP and TAF-1 that are involved in specific DNA binding.

The bZIP regions of the eukaryotic transcription factors GCN4 and C/EBP have similar protein sequences but they recognize different DNA sequences. In order to understand their specificity, a vector was constructed which permits overexpression in Escherichia coli of those domains of GCN4 that are necessary and sufficient for specific DNA binding i.e. the basic region and the leucine zipper. Specific DNA binding was monitored with gel shift experiments. The residues of the basic region of GCN4 were systematically replaced by those of C/EBP to transform GCN4 into C/EBP with respect to DNA binding. Residues -17, -16 and -14 were found to be responsible for switching GCN4 to C/EBP binding specificity (we define as residue +1 the first leucine of the first leucine heptad repeat of GCN4). We broadened the specificity of GCN4 to TAF-1 by replacing residues -15 and -17 and we changed the specificity of C/EBP to TAF-1 by swapping residue -17 of a particular hybrid. Thus residues positioned from -14 to -17 of the basic region play a key role in recognizing specific DNA sequences.

The glycophorin A transmembrane domain dimer: Sequence-specific propensity for a right-handed supercoil of helixes

Recent studies suggest specific roles for transmembrane helix association in a range of functions, but understanding of the conformation and energetics of these interactions has been elusive. We have studied the specific dimerization of the transmembrane helix of glycophorin A by calculating the minimized interaction energies of a large number of conformations using simulated annealing techniques and tested the models against mutational analysis data. We find that the dimer is best modeled as a right-handed supercoil with an extensive region of close packing along the dimer interface. Furthermore, we observe a sequence-specific propensity for a right-handed supercoil to form when starting the simulated annealing modeling from a dimer of helices with parallel axes, in contrast with the dimerization region of the transcription factor GCN4 which shows a high propensity for the more prevalent left-handed supercoiling.

What is the pitch of the alpha-helical coiled coil?

The alpha-helical, coiled-coil protein motif is increasingly recognized in a variety of functional classes of proteins. The pitch of a coiled coil, or rate of winding of the alpha-helices around each other, is a key determinant of both intra- and intermolecular interactions. Experimental measurements of the pitch of parallel two-stranded coiled coils of muscle proteins, and examination of the recently determined structure of another two-stranded coiled coil, the GCN4 transcription factor protein, suggest that the pitch has an average value of about 140 A. This value is consistent with the observed number of residues per turn in alpha-helices of globular proteins, the determinant of the interhelical packing within the coiled-coil motif. An understanding of the structural determinants of this value for the pitch and possible variations will be important in defining the interactions of coiled-coil proteins with other macromolecules.

Transcriptional activation of yeast nucleotide biosynthetic gene ADE4 by GCN4.

The yeast transcriptional regulator protein GCN4 harbors the bZIP DNA binding motif, which is common to a family of DNA-binding proteins in eukaryotic organisms from yeast to man. GCN4 and the mammalian activator protein AP-1 (jun/fos) regulate transcription by binding the same consensus DNA sequence ATGA (C/G)TCAT. GCN4 positively regulates the production of precursors of protein synthesis in yeast cells in response to the environmental signal "amino acid starvation." We find three GCN4 responsive elements (GCREs) in the 5'-flanking region of the purine biosynthetic gene ADE4 and demonstrate that GCN4 efficiently activates transcription of ADE4. Two GCREs are essential to synergistically activate ADE4 transcription by binding GCN4. The distal GCRE1 is also required for basal transcription of ADE4. Therefore, transcription factor GCN4 affects, in addition to protein biosynthesis, also nucleotide biosynthesis and, comparable to its mammalian counterpart AP-1, has a more general function within the yeast cell than previously assumed.

Multiple protein factors bind to a rice glutelin promoter region

Our goal is to identify cis-acting elements in the regulatory region of the major seed storage protein gene in rice. A glutelin gene (pGL5-1) has been cloned by screening a rice genomic DNA library with synthetic oligonucleotides and with an amplified DNA fragment. A transient expression assay using immature rice seeds shows that its 5' flanking sequence can direct the synthesis of beta-glucuronidase (GUS) when fused upstream of the GUS coding region. Gel-retardation assays were performed to study protein-DNA interactions between putative regulatory sequences of pGL5-1 and nuclear proteins from immature rice seeds. We demonstrate that at least six protein-DNA complexes are formed between the 5' flanking sequence of pGL5-1 (-677 to -45) and nuclear protein factors. By subsequent DNase I-footprinting analyses we defined several protein-binding regions. Two of the protein-binding sequences contain the TGAGTCA motif, which is also present in the -300 element found in the 5' flanking sequences of several storage protein genes of other crop plants, and to which the transcription factors jun and GCN4 bind.

Solution structure of the DNA-binding domain of the yeast transcriptional activator protein GCN4

The solution structure of an active synthetic peptide containing both the leucine zipper and the adjacent basic domain of the yeast transcription factor GCN4 (residues 220-280) was determined by NMR. The two domains show structurally distinct behaviours. In the absence of DNA, the basic domain is, although very flexible, structured and fluctuating around a helical conformation. The leucine zipper region forms a long, uninterrupted helix. From a suitable set of NMR distances the three-dimensional structure of the leucine zipper monomeric sub-domain was calculated by distance geometry algorithms. The structure of the symmetrical parallel dimer was obtained by model building using the NMR information. A smaller peptide with the sequence of the isolated basic region (residues 1-35 of the 61 residue peptide) was also synthesized. Circular dichroism studies showed 30-40% helicity. A flexible helix spans the region between residues 8 and 21. The comparison of our results with suggested models is discussed in detail.

Cyclic AMP-responsive DNA-binding protein: structure based on a cloned placental cDNA.

Cyclic AMP (cAMP) is an intracellular second messenger that activates transcription of many cellular genes. A palindromic consensus DNA sequence, TGACGTCA, functions as a cAMP-responsive transcriptional enhancer (CRE). The CRE binds a cellular protein of 38 kD in placental JEG-3 cells. A placental lambda gt11 library was screened for expression of specific CRE-binding proteins with the CRE sequence as a radioactive probe. A cDNA encoding a protein of 326 amino acids with the binding properties of a specific CRE-binding protein (CREB) was isolated. The protein contains a COOH-terminal basic region adjacent to a sequence similar to the "leucine zipper" sequence believed to be involved in DNA binding and in protein-protein contacts in several other DNA-associated transcriptional proteins including the products of the c-myc, c-fos, and c-jun oncogenes and GCN4. The CREB protein also contains an NH2-terminal acidic region proposed to be a potential transcriptional activation domain. The putative DNA-binding domain of CREB is structurally similar to the corresponding domains in the phorbol ester-responsive c-jun protein and the yeast transcription factor GCN4.