Yeast Transcription Factor GCN4 Research Articles

A systematic genome-wide account of binding sites for the model transcription factor Gcn4

Sequence-specific DNA-binding transcription factors are central to gene regulation. They are often associated with consensus binding sites that predict far more genomic sites than are bound in vivo. One explanation is that most sites are blocked by nucleosomes, such that only sites in nucleosome-depleted regulatory regions are bound. We compared the binding of the yeast transcription factor Gcn4 in vivo using published ChIP-seq data (546 sites) and in vitro, using a modified SELEX method (“G-SELEX”), which utilizes short genomic DNA fragments to quantify binding at all sites. We confirm that Gcn4 binds strongly to an AP-1-like sequence (TGACTCA) and weakly to half-sites. However, Gcn4 binds only some of the 1078 exact matches to this sequence, even in vitro. We show that there are only 166 copies of the high-affinity RTGACTCAY site (exact match) in the yeast genome, all occupied in vivo, largely independently of whether they are located in nucleosome-depleted or nucleosomal regions. Generally, RTGACTCAR/YTGACTCAY sites are bound much more weakly and YTGACTCAR sites are unbound, with biological implications for determining induction levels. We conclude that, to a first approximation, Gcn4 binding can be predicted using the high-affinity site, without reference to chromatin structure. We propose that transcription factor binding sites should be defined more precisely using quantitative data, allowing more accurate genome-wide prediction of binding sites and greater insight into gene regulation.

Two Variants of Recombinant Human Bone Morphogenetic Protein-2 (rhBMP-2) with Additional Protein Domains: Synthesis in an Escherichia coli Heterologous Expression System.

Two variants of recombinant human bone morphogenetic protein-2 (rhBMP-2) with additional N-terminal protein domains were obtained by expression in E. coli. The N-terminal domains weres-tag (15-a.a. oligopeptide from bovine pancreatic ribonuclease A) and lz (leucine zipper dimerization domain from yeast transcription factor GCN4).The s-tag-BMP-2 and lz-BMP-2 were purified by a procedure that excluded a long refolding stage. The resulting dimeric proteins displayed higher solubility compared to rhBMP-2 without additional protein domains. Biological activity of both proteins was demonstrated in vitro by induction of alkaline phosphatase in C2C12 cells, and the activity ofs-tag-BMP-2 in vivo was shown in various experimental animal models.

Conformational Dynamics of the Partially Disordered Yeast Transcription Factor GCN4

Molecular dynamics (MD) simulations have been employed to study the conformational dynamics of the partially disordered DNA binding basic leucine zipper domain of the yeast transcription factor GCN4. We demonstrate that back-calculated NMR chemical shifts and spin-relaxation data provide complementary probes of the structure and dynamics of disordered protein states and enable comparisons of the accuracy of multiple MD trajectories. In particular, back-calculated chemical shifts provide a sensitive probe of the populations of residual secondary structure elements and helix capping interactions, while spin-relaxation calculations are sensitive to a combination of dynamic and structural factors. Back calculated chemical shift and spin-relaxation data can be used to evaluate the populations of specific interactions in disordered states and identify regions of the phase space that are inconsistent with experimental measurements. The structural interactions that favor and disfavor helical conformations in the disordered basic region of the GCN4 bZip domain were analyzed in order to assess the implications of the structure and dynamics of the apo form for the DNA binding mechanism. The structural couplings observed in these experimentally validated simulations are consistent with a mechanism where the binding of a preformed helical interface would induce folding in the remainder of the protein, supporting a hybrid conformational selection / induced folding binding mechanism.

The Native GCN4 Leucine-Zipper Domain Does Not Uniquely Specify a Dimeric Oligomerization State

The dimerization domain of the yeast transcription factor GCN4, one of the first coiled-coil proteins to be structurally characterized at high resolution, has served as the basis for numerous fundamental studies on α-helical folding. Mutations in the GCN4 leucine zipper are known to change its preferred oligomerization state from dimeric to trimeric or tetrameric; however, the wild-type sequence has been assumed to encode a two-chain assembly exclusively. Here we demonstrate that the GCN4 coiled-coil domain can populate either a dimer or trimer fold, depending on environment. We report high-resolution crystal structures of the wild-type sequence in dimeric and trimeric assemblies. Biophysical measurements suggest populations of both oligomerization states under certain experimental conditions in solution. We use parallel tempering molecular dynamics simulations on the microsecond time scale to compare the stability of the dimer and trimer folded states in isolation. In total, our results suggest that the folding behavior of the well-studied GCN4 leucine-zipper domain is more complex than was previously appreciated. Our results have implications in ongoing efforts to establish predictive algorithms for coiled-coil folds and the selection of coiled-coil model systems for design and mutational studies where oligomerization state specificity is an important consideration.

Complex networks govern coiled-coil oligomerization--predicting and profiling by means of a machine learning approach.

Understanding the relationship between protein sequence and structure is one of the great challenges in biology. In the case of the ubiquitous coiled-coil motif, structure and occurrence have been described in extensive detail, but there is a lack of insight into the rules that govern oligomerization, i.e. how many α-helices form a given coiled coil. To shed new light on the formation of two- and three-stranded coiled coils, we developed a machine learning approach to identify rules in the form of weighted amino acid patterns. These rules form the basis of our classification tool, PrOCoil, which also visualizes the contribution of each individual amino acid to the overall oligomeric tendency of a given coiled-coil sequence. We discovered that sequence positions previously thought irrelevant to direct coiled-coil interaction have an undeniable impact on stoichiometry. Our rules also demystify the oligomerization behavior of the yeast transcription factor GCN4, which can now be described as a hybrid—part dimer and part trimer—with both theoretical and experimental justification.

Signal perception and transmission in histidine autokinases: Insights from the Agrobacterium tumefaciens VirA/VirG system

VirA, the histidine autokinase found in Agrobacterium tumefaciens, is activated in response to several signaling cues, among which the plant‐derived phenolic molecule being the most critical. The cytoplasmic linker domain is responsible for phenol sensing, and a coiled‐coil was predicted to bridge the linker domain to the kinase. Using yeast transcription factor GCN4, the possible signal‐induced rotational motion in the coiled‐coil was examined. Through different insertions between GCN4 and the coiled‐coil, the signal‐independent ratcheted activities were observed. Random mutagenesis and functional library screening also indicated several constitutively on mutants in the coiled‐coil, which enabled the dimerization interface to be established. Together with the structure model of the linker domain, generated by homology modeling, and the possible phenol binding site, probed by computational docking, the model for phenol perception and the associated motion in the signaling module of VirA is proposed.This work is supported by NSF‐IOS‐0818613.

Homodimerization of Marek's Disease Virus-Encoded Meq Protein Is Not Sufficient for Transformation of Lymphocytes in Chickens

Marek's disease virus (MDV), the etiologic agent of Marek's disease, is a potent oncogenic herpesvirus. MDV is highly contagious and elicits a rapid onset of malignant T-cell lymphomas in chickens within several weeks after infection. MDV genome codes an oncoprotein, Meq, which shares resemblance with the Jun/Fos family of bZIP transcription factors. Similar to Jun, the leucine zipper region of Meq allows the formation of homo- and heterodimers. Meq homo- and heterodimers have different DNA binding affinities and transcriptional activity; therefore, they may differentially regulate transcription of viral and cellular genes. In this study we investigated the role of Meq homodimers in the pathogenicity of MDV by generating a chimeric meq gene, which contains the leucine zipper region of the yeast transcription factor GCN4 (meqGCN). A recombinant virus (rMd5-MeqGCN) containing the chimeric meqGCN gene in place of parental meq was generated with overlapping cosmid clones of Md5, a very virulent MDV strain. The rMd5-MeqGCN virus replicated in vitro and in vivo but was unable to transform T cells in infected chickens. These data provide the first in vivo evidence that Meq homodimers are not sufficient for MDV-induced transformation.

Lattice model simulation of interchain protein interactions and the folding dynamics and dimerization of the GCN4 Leucine zipper

The highest level in the hierarchy of protein structure and folding is the formation of protein complexes through protein-protein interactions. We have made modifications to a well established computer lattice model to expand its applicability to two-protein dimerization and aggregation. Based on Brownian dynamics, we implement translation and rotation moves of two peptide chains relative to each other, in addition to the intrachain motions already present in the model. We use this two-chain model to study the folding dynamics of the yeast transcription factor GCN4 leucine zipper. The calculated heat capacity curves agree well with experimental measurements. Free energy landscapes and median first passage times for the folding process are calculated and elucidate experimentally measured characteristics such as the multistate nature of the dimerization process.

Computational Study of a Single Surface-Immobilized Two-Stranded Coiled-Coil Polypeptide

The equilibrium structure and dynamics of a two-stranded coiled-coil polypeptide are investigated via Langevin dynamics simulations. An off-lattice model of the polypeptide chain is employed, which gives rise to a well-defined helical dimer native state and two-state folding kinetics. The behavior of the freely diffusing and surface-immobilized polypeptide is studied under different surface and denaturation conditions. The effect of surface immobilization on the distributions of structural and dynamical properties is considered in detail. The relationship between the simulation results and recent single-molecule fluorescence resonance energy transfer experiments performed on the two-stranded coiled-coil from the yeast transcription factor GCN4 (Jia et al. Chem. Phys. 1999, 247, 69; Talaga et al. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 13021) is discussed.

Association of RNA Helicase A with Human Immunodeficiency Virus Type 1 Particles

RNA helicase A (RHA) belongs to the DEAH family of proteins that are capable of unwinding double-stranded RNA structure. In addition to its involvement in the metabolism of cellular RNA, RHA has been shown to stimulate RNA transcription from the long terminal repeat promoter of human immunodeficiency virus type 1 (HIV-1) as well as to enhance Rev/Rev response element-mediated gene expression. In this study, we provide evidence that RHA associates with HIV-1 Gag in an RNA-dependent manner. This interaction results in specific incorporation of RHA into HIV-1 particles. Knockdown of endogenous RHA in virus producer cells leads to generation of HIV-1 particles that are less infectious in part as a result of restricted reverse transcription. Therefore, RHA represents the first example of cellular RNA helicases that participate in HIV-1 particle production and promote viral reverse transcription.

Opposing effects of the M368A point mutation and deletion of the SP1 region on membrane binding of human immunodeficiency virus type 1 Gag

The Gag protein of human immunodeficiency virus type 1 (HIV-1) contains a 14-amino-acid region termed SP1 that is located between the capsid (CA) and nucleocapsid (NC) domains. It has been previously observed that either a M368A substitution within SP1 or the ΔSP1 deletion impaired virus production. In this study, we further showed that the M368A point mutation, but not the ΔSP1 deletion, severely diminished the levels of membrane-associated Gag proteins. This membrane binding defect associated with M368A was corrected either by changing NC to the leucine zipper (LZ) motif derived from the yeast transcription factor GCN4 or by a L364A second-site mutation in the context of the first four residues of SP1. Yet, neither the L364A mutation nor the LZ substitution restored wild type levels of particle production to the M368A Gag. These results suggest that SP1 affects both Gag–membrane binding and the subsequent events of virus assembly such as capsid morphogenesis or virus budding.

DNA-mediated assembly of weakly interacting DNA-binding protein subunits: in vitro recruitment of phage 434 repressor and yeast GCN4 DNA-binding domains.

The specificity of DNA-mediated protein assembly was studied in two in vitro systems, based on (i) the DNA-binding domain of bacteriophage 434 repressor cI (amino acid residues 1-69), or (ii) the DNA-binding domain of the yeast transcription factor GCN4, (amino acids 1-34) and their respective oligonucleotide cognates. In vivo, both of these peptides are part of larger protein molecules that also contain dimerization domains, and the resulting dimers recognize cognate palindromic DNA sequences that contain two half-sites of 4 bp each. The dimerization domains were not included in the peptides tested, so in solution-in the presence or absence of non-cognate DNA oligonucleotides-these molecules did not show appreciable dimerization, as determined by pyrene excimer fluorescence spectroscopy and oxidative cross-linking monitored by mass spectrometry. Oligonucleotides with only one 4 bp cognate half-site were able to initiate measurable dimerization, and two half-sites were able to select specific dimers even from a heterogeneous pool of molecules of closely related specificity (such as DNA-binding domains of the 434 repressor and their engineered mutants that mimic the binding helix of the related P22 phage repressor). The fluorescent technique allowed us to separately monitor the unspecific, ionic interaction of the peptides with DNA which produced a roughly similar signal in the case of both cognate and non-cognate oligonucleotides. But in the former case, a concomitant excimer fluorescence signal showed the formation of correctly positioned dimers. The results suggest that DNA acts as a highly specific template for the recruitment of weakly interacting protein molecules that can thus build up highly specific complexes.

Secretion of proteins with dimerization capacity by the haemolysin type I transport system of Escherichia coli.

The tolerance of the haemolysin transport system (Hly) for exporting dimeric protein substrates to the supernatants of Escherichia coli cultures was examined. A strong dimerization domain (i.e. an amphipathic alpha-helix capable of forming a leucine zipper in the yeast transcription factor GCN4) was inserted into an epitope-tagged version of the 23 kDa C-terminal secretion signal of haemolysin (EHlyA). The zipper-containing polypeptide (ZEHlyA) was effectively secreted by E. coli cells carrying the HlyBD transporter and accumulated in the culture media as a stable dimer as determined by gel filtration chromatography. In vivo protein cross-linking experiments and coexpression with a secretion-deficient derivative of ZEHlyA indicated that leucine zipper-dependent dimerization occurs following secretion. To test whether dimerization allows the correct folding of the secreted polypeptide, immunoglobulin V(HH)-domains obtained from camel antibodies were fused to EHlyA and ZEHlyA. Functional dimerization of the ZEHlyA hybrid was anticipated to increase the apparent binding affinity (i.e. avidity) of the V(HH) moiety, thus becoming an excellent reporter of correct protein folding and dimerization. Both V(HH)-EHlyA and V(HH)-ZEHlyA hybrids were quantitatively secreted and found in the extracellular medium as active monomers and dimers respectively. When compared with their monomeric counterparts, the dimeric V(HH)-ZEHlyA molecules showed superior binding properties to their cognate antigen, with a 10-fold increase in their avidity. These data reveal a non-anticipated permissiveness of the Hly type I transport machinery for the secretion of substrates with dimerization capacity.

Bacteriophage phi29 scaffolding protein gp7 before and after prohead assembly.

Three-dimensional structures of the double-stranded DNA bacteriophage phi29 scaffolding protein (gp7) before and after prohead assembly have been determined at resolutions of 2.2 and 2.8 A, respectively. Both structures are dimers that resemble arrows, with a four-helix bundle composing the arrowhead and a coiled coil forming the tail. The structural resemblance of gp7 to the yeast transcription factor GCN4 suggests a DNA-binding function that was confirmed by native gel electrophoresis. DNA binding to gp7 may have a role in mediating the structural transition from prohead to mature virus and scaffold release. A cryo-EM analysis indicates that gp7 is arranged inside the capsid as a series of concentric shells. The position of the higher density features in these shells correlates with the positions of hexamers in the equatorial region of the capsid, suggesting that gp7 may regulate formation of the prolate head through interactions with these hexamers.

Regulation of the transcription factor Gcn4 by Pho85 cyclin PCL5.

The yeast transcription factor Gcn4 is regulated by amino acid starvation at the levels of both protein synthesis and stability. Gcn4 degradation depends on the ubiquitination complex SCF(CDC4) and requires phosphorylation by the cyclin-dependent kinase Pho85. Here, we show that Pcl5 is the Pho85 cyclin specifically required for Gcn4 degradation. PCL5 is itself induced by Gcn4 at the level of transcription. However, even when PCL5 is constitutively overexpressed, Pho85-associated Gcn4 phosphorylation activity is reduced in starved cells and Gcn4 degradation is decreased. Under these conditions, the Pcl5 protein disappears because of rapid constitutive turnover. We suggest that, by virtue of its constitutive metabolic instability, Pcl5 may be a sensor of cellular protein biosynthetic capacity. The fact that PCL5 is transcriptionally induced in the presence of Gcn4 suggests that it is part of a homeostatic mechanism that reduces Gcn4 levels upon recovery from starvation.

A de Novo Designed Template for Generating Conformation-specific Antibodies That Recognize α-Helices in Proteins

The generation of antibodies directed toward the surface-exposed regions of a protein using synthetic peptides as immunogens representing surface loops and turns has been widely successful. However, peptides representing alpha-helical regions are typically unstructured in solution and unable to produce antibodies that recognize alpha-helices in native proteins. We describe a de novo designed parallel two-stranded alpha-helical coiled-coil template for immunization to prepare antibodies that recognize alpha-helical protein sequences in the native protein. This template was designed for maximum stability through an Ile/Leu hydrophobic core and an interchain disulfide bridge. Surface-exposed helical residues are inserted into the template and used for immunization to generate polyclonal antibodies. To demonstrate the feasibility of this approach, 15 residues of the yeast transcription factor GCN4 were inserted into this template, and the resultant antibodies were screened for conformational specificity. Peptide antigens that contain the same surface-exposed residues but differ in structure were used as competitors in a competition assay. Direct competition between the capture peptide immobilized on a biosensor chip, the peptide antigens, and the antibodies generated by the template demonstrated that the antibodies were specific for helical structure in the native coiled-coil (synthetic GCN4 residues 250-280). These antibodies were unable to recognize the same inserted sequence in an unstructured analog. The helix-specific antibodies were also able to identify native GCN4 (31.3 kDa) from yeast whole cell extracts.

A coiled coil with a fluorous core.

The design, synthesis, and structural characterization of a highly fluorinated peptide system based on the coiled coil region of the yeast transcription factor GCN4 is described. All four leucine residues (a position) and three valine residues (d position) were replaced by the unnatural amino acids 5,5,5-trifluoroleucine and 4,4,4-trifluorovaline, respectively. The peptide is highly alpha-helical at low micromolar concentrations as judged by circular dichroism spectra, sediments as a dimeric species in the 5-30 microM concentration range, and exhibits a dimer melting temperature that is 15 degrees C higher than a control peptide with a hydrocarbon core. Furthermore, the apparent free energy of unfolding as calculated from guanidinium hydrochloride denaturation experiments is larger by approximately 1.0 kcal/mol for the fluorinated peptide than its hydrocarbon counterpart. We conclude that additional stability is derived from sequestering the more hydrophobic trifluoromethyl groups from aqueous solvent. These studies introduce a new paradigm in the design of molecular self-assembling systems, one based on orthogonal solubility properties of liquid phases.

Energetics of coiled coil folding: the nature of the transition states.

Coiled coils are simple models for studying the association of two polypeptide chains to form a folded protein. Previous work has shown that the folding of a coiled coil can be described by a two-state transition between two unfolded monomeric peptide chains and a folded coiled coil dimer. Here we report the thermodynamic activation parameters for the folding and unfolding of two unrelated coiled coils: C62GCN4 and A(2). C62GCN4 corresponds to the 62 C-terminal residues of yeast transcription factor GCN4. The peptide forms a dimeric coiled coil through its 33 C-terminal residues. A(2) is a designed 30-residue dimeric coiled coil whose folding is induced by low pH [Dürr, E., Jelesarov, I., and Bosshard, H. R. (1999) Biochemistry 38, 870-880]. Folding and unfolding were assessed under identical native buffer conditions so that the microscopic reversibility applied and the transition state was the same for folding and unfolding. The time course of folding was followed from the self-quenching of a C-terminal fluorescent label (Texas Red). The overall folding of both peptides is enthalpy-driven and opposed by a loss of entropy. The main energetic changes occur after the system has passed the transition state. In the folding of C62GCN4, only 10-20% of the heat capacity change is attained between the monomeric state and the dimeric transition state. For coiled coil A(2), the fractional heat capacity change preceding the transition state is 30-40%. The results indicate that the activated states of folding of coiled coils are not well structured and differ considerably from the folded coiled coil conformation. These findings are in agreement with a rate-limiting transition state in which the coiled coil helices and the hydrophobic coiled coil interface are poorly developed.

Arginine-rich Peptides: AN ABUNDANT SOURCE OF MEMBRANE-PERMEABLE PEPTIDES HAVING POTENTIAL AS CARRIERS FOR INTRACELLULAR PROTEIN DELIVERY

A basic peptide derived from human immunodeficiency virus (HIV)-1 Tat protein (positions 48-60) has been reported to have the ability to translocate through the cell membranes and accumulate in the nucleus, the characteristics of which are utilized for the delivery of exogenous proteins into cells. Based on the fluorescence microscopic observations of mouse macrophage RAW264.7 cells, we found that various arginine-rich peptides have a translocation activity very similar to Tat-(48-60). These included such peptides as the d-amino acid- and arginine-substituted Tat-(48-60), the RNA-binding peptides derived from virus proteins, such as HIV-1 Rev, and flock house virus coat proteins, and the DNA binding segments of leucine zipper proteins, such as cancer-related proteins c-Fos and c-Jun, and the yeast transcription factor GCN4. These segments have no specific primary and secondary structures in common except that they have several arginine residues in the sequences. Moreover, these peptides were able to be internalized even at 4 degrees C. These results strongly suggested the possible existence of a common internalization mechanism ubiquitous to arginine-rich peptides, which is not explained by a typical endocytosis. Using (Arg)(n) (n = 4-16) peptides, we also demonstrated that there would be an optimal number of arginine residues (n approximately 8) for the efficient translocation.

Role for homodimerization in growth deregulation by E2a fusion proteins.

The oncogenic transcription factor E2a-Pbx1 is expressed in some cases of acute lymphoblastic leukemia as a result of chromosomal translocation 1;19. The early observation that E2a-Pbx1 incorporates transcriptional activation domains from E2a and a DNA-binding homeodomain from Pbx1 inspired a model in which E2a-Pbx1 promotes leukemogenic transformation of lymphoid progenitor cells through transcriptional induction of target genes defined by the Pbx1 portion of the molecule. However, the subsequent demonstration that the only known DNA-binding module on the molecule, the Pbx1 homeodomain, is dispensable for the induction of lymphoblastic lymphoma in transgenic mice called into question the contribution made by the Pbx1 portion. In this study, we have used a domain swap approach coupled with a fibroblast-based focus formation assay to evaluate further the requirement for PBX1-encoded peptide elements in growth deregulation by E2a-Pbx1. No impairment of focus formation was observed when the entire Pbx1 portion was replaced with DNA-binding/dimerization domains derived from yeast transcription factor GAL4 or GCN4. Furthermore, replacement of Pbx1 with tandem FKBP domains that mediate homodimerization in the presence of a synthetic ligand led to striking growth deregulation exclusively in the presence of the dimerizing agent. N-terminal elements encoded by E2A, including the AD1 transcriptional activation domain, were required for dimerization-induced focus formation. We conclude that transcriptional target genes defined by heterologous C-terminal DNA-binding modules are not required in growth deregulation by E2a fusion proteins. We speculate that interactions between N-terminal E2a elements and undefined proteins that could function as components of a transcriptional coactivator complex may be more important.