The Experts below are selected from a list of 288 Experts worldwide ranked by ideXlab platform

Hans Rudolf Bosshard - One of the best experts on this subject based on the ideXlab platform.

  • Monomeric and dimeric bZIP Transcription Factor GCN4 bind at the same rate to their target DNA site.
    Biochemistry, 2004
    Co-Authors: Susanne Cranz, Ilian Jelesarov, Christine Berger, Antonio Baici, Hans Rudolf Bosshard
    Abstract:

    Basic leucine zipper (bZIP) Transcription Factors are dimeric proteins that recognize dyadic and mostly palindromic DNA sites. Dimerization of bZIP Transcription Factor GCN4 is linked to the folding of its C-terminal leucine zipper domain. However, monomeric GCN4, lacking a folded leucine zipper, also recognizes the DNA site with dimerization taking place on the DNA. Here we report the kinetics of DNA recognition by unfolded monomeric and folded dimeric derivatives of GCN4 using a 19 bp double-stranded DNA containing a palindromic CRE site. The rate of DNA binding of both monomeric and dimeric GCN4 has a bimolecular rate constant of 3−5 × 108 M-1 s-1, which is near the diffusion limit. Because the rate of dimerization of GCN4 is slower (1.7 × 107 M-1 s-1) than the rate of DNA association, the formation of the dimeric GCN4−DNA complex through consecutive binding of two monomers (monomer pathway) is faster when starting from free monomers. Thus, the results presented here support facilitated and rapid targe...

  • Energetics of coiled coil folding: the nature of the transition states.
    Biochemistry, 2001
    Co-Authors: Hans Rudolf Bosshard, Eberhard Durr, Thomas Hitz, Ilian Jelesarov
    Abstract:

    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 [Durr, 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.

  • Thermodynamics and kinetics of the reaction of a single-chain antibody fragment (scFv) with the leucine zipper domain of Transcription Factor GCN4.
    Biochemistry, 1998
    Co-Authors: Susanne Weber-bornhauser, Ilian Jelesarov, Christine Berger, Jolanda Eggenberger, André Bernard, Hans Rudolf Bosshard
    Abstract:

    Single-chain Fv (scFv) fragments of antibodies have become important analytical and therapeutic tools in biology and medicine. The reaction of scFv fragments has not been well-characterized with respect to the energetics and kinetics of antigen binding. This paper describes the thermodynamic and kinetic behavior of the high-affinity scFv fragment SW1 directed against the dimeric leucine zipper domain of the yeast Transcription Factor GCN4. The scFv fragment was selected by the phage display technique from the immune repertoire of a mouse that had been immunized with the leucine zipper domain of GCN4. The scFv fragment was produced in high yield in Escherichia coli inclusion bodies and refolded from the denatured state. Differential scanning calorimetry showed that SW1 was stable up to about 50 degreesC, but the subsequent thermal denaturation was irreversible (Tm approximately 68 degreesC). The scFv fragment specifically recognized the dimeric leucine zipper conformation. Two scFv fragments bound to the GCN4 dimer to form the complex (scFv)2-GCN4. Because of its repetitive structure, the rod-shaped GCN4 leucine zipper may present two similar epitopes for the scFv fragment. Surprisingly, the binding reaction was highly cooperative, that is, the species (scFv)2-GCN4 dominated over scFv-GCN4 even in the presence of a large excess of the antigen GCN4. It is speculated that cooperativity resulted from direct interaction between the two GCN4-bound scFv fragments. At 25 degreesC, the average binding enthalpy for a scFv fragment was favorable (-61 kJ mol-1), the entropy change was unfavorable, and the change in heat capacity was -1.27 +/- 0.14 kJ mol-1 K-1. As a result of enthalpy-entropy compensation, the free binding energy was virtually independent of temperature in the physiological temperature range. Antigen binding in solution could be described by a single-exponential reaction with an apparent rate constant of 1 x 10(6) M-1 s-1. Binding followed in a biosensor with the dimeric GCN4 coupled to the surface of the metal oxide sensor chip was 20 times slower.

  • Diffusion-controlled DNA recognition by an unfolded, monomeric bZIP Transcription Factor
    FEBS letters, 1998
    Co-Authors: Christine Berger, Luciano Piubelli, Ursula Haditsch, Hans Rudolf Bosshard
    Abstract:

    Basic leucine zipper (bZIP) Transcription Factors are dimers that recognize mainly palindromic DNA sites. It has been assumed that bZIP Factors have to form a dimer in order to bind to their target DNA. We find that DNA binding of both monomeric and dimeric bZIP Transcription Factor GCN4 is diffusion-limited and that, therefore, the rate of dimerization of the bZIP domain does not affect the rate of DNA recognition and GCN4 need not dimerize in order to bind to its specific DNA site. The results have implications for the mechanism by which bZIP Transcription Factors find their target sites for Transcriptional regulation.

  • Coupled folding and site-specific binding of the GCN4-bZIP Transcription Factor to the AP-1 and ATF/CREB DNA sites studied by microcalorimetry.
    Biochemistry, 1996
    Co-Authors: Christine Berger, Ilian Jelesarov, Hans Rudolf Bosshard
    Abstract:

    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; Konig 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.

Christine Berger - One of the best experts on this subject based on the ideXlab platform.

  • Monomeric and dimeric bZIP Transcription Factor GCN4 bind at the same rate to their target DNA site.
    Biochemistry, 2004
    Co-Authors: Susanne Cranz, Ilian Jelesarov, Christine Berger, Antonio Baici, Hans Rudolf Bosshard
    Abstract:

    Basic leucine zipper (bZIP) Transcription Factors are dimeric proteins that recognize dyadic and mostly palindromic DNA sites. Dimerization of bZIP Transcription Factor GCN4 is linked to the folding of its C-terminal leucine zipper domain. However, monomeric GCN4, lacking a folded leucine zipper, also recognizes the DNA site with dimerization taking place on the DNA. Here we report the kinetics of DNA recognition by unfolded monomeric and folded dimeric derivatives of GCN4 using a 19 bp double-stranded DNA containing a palindromic CRE site. The rate of DNA binding of both monomeric and dimeric GCN4 has a bimolecular rate constant of 3−5 × 108 M-1 s-1, which is near the diffusion limit. Because the rate of dimerization of GCN4 is slower (1.7 × 107 M-1 s-1) than the rate of DNA association, the formation of the dimeric GCN4−DNA complex through consecutive binding of two monomers (monomer pathway) is faster when starting from free monomers. Thus, the results presented here support facilitated and rapid targe...

  • Thermodynamics and kinetics of the reaction of a single-chain antibody fragment (scFv) with the leucine zipper domain of Transcription Factor GCN4.
    Biochemistry, 1998
    Co-Authors: Susanne Weber-bornhauser, Ilian Jelesarov, Christine Berger, Jolanda Eggenberger, André Bernard, Hans Rudolf Bosshard
    Abstract:

    Single-chain Fv (scFv) fragments of antibodies have become important analytical and therapeutic tools in biology and medicine. The reaction of scFv fragments has not been well-characterized with respect to the energetics and kinetics of antigen binding. This paper describes the thermodynamic and kinetic behavior of the high-affinity scFv fragment SW1 directed against the dimeric leucine zipper domain of the yeast Transcription Factor GCN4. The scFv fragment was selected by the phage display technique from the immune repertoire of a mouse that had been immunized with the leucine zipper domain of GCN4. The scFv fragment was produced in high yield in Escherichia coli inclusion bodies and refolded from the denatured state. Differential scanning calorimetry showed that SW1 was stable up to about 50 degreesC, but the subsequent thermal denaturation was irreversible (Tm approximately 68 degreesC). The scFv fragment specifically recognized the dimeric leucine zipper conformation. Two scFv fragments bound to the GCN4 dimer to form the complex (scFv)2-GCN4. Because of its repetitive structure, the rod-shaped GCN4 leucine zipper may present two similar epitopes for the scFv fragment. Surprisingly, the binding reaction was highly cooperative, that is, the species (scFv)2-GCN4 dominated over scFv-GCN4 even in the presence of a large excess of the antigen GCN4. It is speculated that cooperativity resulted from direct interaction between the two GCN4-bound scFv fragments. At 25 degreesC, the average binding enthalpy for a scFv fragment was favorable (-61 kJ mol-1), the entropy change was unfavorable, and the change in heat capacity was -1.27 +/- 0.14 kJ mol-1 K-1. As a result of enthalpy-entropy compensation, the free binding energy was virtually independent of temperature in the physiological temperature range. Antigen binding in solution could be described by a single-exponential reaction with an apparent rate constant of 1 x 10(6) M-1 s-1. Binding followed in a biosensor with the dimeric GCN4 coupled to the surface of the metal oxide sensor chip was 20 times slower.

  • Diffusion-controlled DNA recognition by an unfolded, monomeric bZIP Transcription Factor
    FEBS letters, 1998
    Co-Authors: Christine Berger, Luciano Piubelli, Ursula Haditsch, Hans Rudolf Bosshard
    Abstract:

    Basic leucine zipper (bZIP) Transcription Factors are dimers that recognize mainly palindromic DNA sites. It has been assumed that bZIP Factors have to form a dimer in order to bind to their target DNA. We find that DNA binding of both monomeric and dimeric bZIP Transcription Factor GCN4 is diffusion-limited and that, therefore, the rate of dimerization of the bZIP domain does not affect the rate of DNA recognition and GCN4 need not dimerize in order to bind to its specific DNA site. The results have implications for the mechanism by which bZIP Transcription Factors find their target sites for Transcriptional regulation.

  • Coupled folding and site-specific binding of the GCN4-bZIP Transcription Factor to the AP-1 and ATF/CREB DNA sites studied by microcalorimetry.
    Biochemistry, 1996
    Co-Authors: Christine Berger, Ilian Jelesarov, Hans Rudolf Bosshard
    Abstract:

    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; Konig 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.

  • coupled folding and site specific binding of the GCN4 bzip Transcription Factor to the ap 1 and atf creb dna sites studied by microcalorimetry
    Biochemistry, 1996
    Co-Authors: Christine Berger, Ilian Jelesarov, Hans Rudolf Bosshard
    Abstract:

    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; Konig 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.

Ilian Jelesarov - One of the best experts on this subject based on the ideXlab platform.

  • Monomeric and dimeric bZIP Transcription Factor GCN4 bind at the same rate to their target DNA site.
    Biochemistry, 2004
    Co-Authors: Susanne Cranz, Ilian Jelesarov, Christine Berger, Antonio Baici, Hans Rudolf Bosshard
    Abstract:

    Basic leucine zipper (bZIP) Transcription Factors are dimeric proteins that recognize dyadic and mostly palindromic DNA sites. Dimerization of bZIP Transcription Factor GCN4 is linked to the folding of its C-terminal leucine zipper domain. However, monomeric GCN4, lacking a folded leucine zipper, also recognizes the DNA site with dimerization taking place on the DNA. Here we report the kinetics of DNA recognition by unfolded monomeric and folded dimeric derivatives of GCN4 using a 19 bp double-stranded DNA containing a palindromic CRE site. The rate of DNA binding of both monomeric and dimeric GCN4 has a bimolecular rate constant of 3−5 × 108 M-1 s-1, which is near the diffusion limit. Because the rate of dimerization of GCN4 is slower (1.7 × 107 M-1 s-1) than the rate of DNA association, the formation of the dimeric GCN4−DNA complex through consecutive binding of two monomers (monomer pathway) is faster when starting from free monomers. Thus, the results presented here support facilitated and rapid targe...

  • Energetics of coiled coil folding: the nature of the transition states.
    Biochemistry, 2001
    Co-Authors: Hans Rudolf Bosshard, Eberhard Durr, Thomas Hitz, Ilian Jelesarov
    Abstract:

    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 [Durr, 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.

  • Thermodynamics and kinetics of the reaction of a single-chain antibody fragment (scFv) with the leucine zipper domain of Transcription Factor GCN4.
    Biochemistry, 1998
    Co-Authors: Susanne Weber-bornhauser, Ilian Jelesarov, Christine Berger, Jolanda Eggenberger, André Bernard, Hans Rudolf Bosshard
    Abstract:

    Single-chain Fv (scFv) fragments of antibodies have become important analytical and therapeutic tools in biology and medicine. The reaction of scFv fragments has not been well-characterized with respect to the energetics and kinetics of antigen binding. This paper describes the thermodynamic and kinetic behavior of the high-affinity scFv fragment SW1 directed against the dimeric leucine zipper domain of the yeast Transcription Factor GCN4. The scFv fragment was selected by the phage display technique from the immune repertoire of a mouse that had been immunized with the leucine zipper domain of GCN4. The scFv fragment was produced in high yield in Escherichia coli inclusion bodies and refolded from the denatured state. Differential scanning calorimetry showed that SW1 was stable up to about 50 degreesC, but the subsequent thermal denaturation was irreversible (Tm approximately 68 degreesC). The scFv fragment specifically recognized the dimeric leucine zipper conformation. Two scFv fragments bound to the GCN4 dimer to form the complex (scFv)2-GCN4. Because of its repetitive structure, the rod-shaped GCN4 leucine zipper may present two similar epitopes for the scFv fragment. Surprisingly, the binding reaction was highly cooperative, that is, the species (scFv)2-GCN4 dominated over scFv-GCN4 even in the presence of a large excess of the antigen GCN4. It is speculated that cooperativity resulted from direct interaction between the two GCN4-bound scFv fragments. At 25 degreesC, the average binding enthalpy for a scFv fragment was favorable (-61 kJ mol-1), the entropy change was unfavorable, and the change in heat capacity was -1.27 +/- 0.14 kJ mol-1 K-1. As a result of enthalpy-entropy compensation, the free binding energy was virtually independent of temperature in the physiological temperature range. Antigen binding in solution could be described by a single-exponential reaction with an apparent rate constant of 1 x 10(6) M-1 s-1. Binding followed in a biosensor with the dimeric GCN4 coupled to the surface of the metal oxide sensor chip was 20 times slower.

  • Coupled folding and site-specific binding of the GCN4-bZIP Transcription Factor to the AP-1 and ATF/CREB DNA sites studied by microcalorimetry.
    Biochemistry, 1996
    Co-Authors: Christine Berger, Ilian Jelesarov, Hans Rudolf Bosshard
    Abstract:

    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; Konig 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.

  • coupled folding and site specific binding of the GCN4 bzip Transcription Factor to the ap 1 and atf creb dna sites studied by microcalorimetry
    Biochemistry, 1996
    Co-Authors: Christine Berger, Ilian Jelesarov, Hans Rudolf Bosshard
    Abstract:

    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; Konig 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.

Daniel Kornitzer - One of the best experts on this subject based on the ideXlab platform.

  • Regulation of the Transcription Factor GCN4 by Pho85 cyclin PCL5.
    Molecular and cellular biology, 2002
    Co-Authors: Revital Shemer, Ariella Meimoun, Tsvi Holtzman, Daniel Kornitzer
    Abstract:

    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 SCFCDC4 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.

  • degradation of the Transcription Factor GCN4 requires the kinase pho85 and the scfcdc4 ubiquitin ligase complex
    Molecular Biology of the Cell, 2000
    Co-Authors: Ariella Meimoun, Tsvi Holtzman, Ziva Weissman, Helen J. Mcbride, David J. Stillman, Gerald R. Fink, Daniel Kornitzer
    Abstract:

    GCN4, a yeast Transcriptional activator that promotes the expression of amino acid and purine biosynthesis genes, is rapidly degraded in rich medium. Here we report that SCFCDC4, a recently characterized protein complex that acts in conjunction with the ubiquitin-conjugating enzyme Cdc34 to degrade cell cycle regulators, is also necessary for the degradation of the Transcription Factor GCN4. Degradation of GCN4 occurs throughout the cell cycle, whereas degradation of the known cell cycle substrates of Cdc34/SCFCDC4 is cell cycle regulated. GCN4 ubiquitination and degradation are regulated by starvation for amino acids, whereas the degradation of the cell cycle substrates of Cdc34/SCFCDC4 is unaffected by starvation. We further show that unlike the cell cycle substrates of Cdc34/SCFCDC4, which require phosphorylation by the kinase Cdc28, GCN4 degradation requires the kinase Pho85. We identify the critical target site of Pho85 on GCN4; a mutation of this site stabilizes the protein. A specific Pho85-Pcl complex that is able to phosphorylate GCN4 on that site is inactive under conditions under which GCN4 is stable. Thus, Cdc34/SCFCDC4 activity is constitutive, and regulation of the stability of its various substrates occurs at the level of their phosphorylation.

  • Degradation of the Transcription Factor GCN4 Requires the Kinase Pho85 and the SCFCDC4 Ubiquitin–Ligase Complex
    Molecular biology of the cell, 2000
    Co-Authors: Ariella Meimoun, Tsvi Holtzman, Ziva Weissman, Helen J. Mcbride, David J. Stillman, Gerald R. Fink, Daniel Kornitzer
    Abstract:

    GCN4, a yeast Transcriptional activator that promotes the expression of amino acid and purine biosynthesis genes, is rapidly degraded in rich medium. Here we report that SCFCDC4, a recently characterized protein complex that acts in conjunction with the ubiquitin-conjugating enzyme Cdc34 to degrade cell cycle regulators, is also necessary for the degradation of the Transcription Factor GCN4. Degradation of GCN4 occurs throughout the cell cycle, whereas degradation of the known cell cycle substrates of Cdc34/SCFCDC4 is cell cycle regulated. GCN4 ubiquitination and degradation are regulated by starvation for amino acids, whereas the degradation of the cell cycle substrates of Cdc34/SCFCDC4 is unaffected by starvation. We further show that unlike the cell cycle substrates of Cdc34/SCFCDC4, which require phosphorylation by the kinase Cdc28, GCN4 degradation requires the kinase Pho85. We identify the critical target site of Pho85 on GCN4; a mutation of this site stabilizes the protein. A specific Pho85-Pcl complex that is able to phosphorylate GCN4 on that site is inactive under conditions under which GCN4 is stable. Thus, Cdc34/SCFCDC4 activity is constitutive, and regulation of the stability of its various substrates occurs at the level of their phosphorylation.

  • Regulated degradation of the Transcription Factor GCN4.
    The EMBO journal, 1994
    Co-Authors: Daniel Kornitzer, B. Raboy, R.g. Kulka, Gerald R. Fink
    Abstract:

    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.

Alan G Hinnebusch - One of the best experts on this subject based on the ideXlab platform.

  • chromatin remodeler ino80c acts independently of h2a z to evict promoter nucleosomes and stimulate Transcription of highly expressed genes in yeast
    Nucleic Acids Research, 2020
    Co-Authors: Emily Biernat, Răzvan V Chereji, Yashpal Rawal, David J Clark, Chhabi K Govind, Alan G Hinnebusch
    Abstract:

    The chromatin remodelers SWI/SNF and RSC function in evicting promoter nucleosomes at highly expressed yeast genes, particularly those activated by Transcription Factor GCN4. Ino80 remodeling complex (Ino80C) can establish nucleosome-depleted regions (NDRs) in reconstituted chromatin, and was implicated in removing histone variant H2A.Z from the -1 and +1 nucleosomes flanking NDRs; however, Ino80C's function in Transcriptional activation in vivo is not well understood. Analyzing the cohort of GCN4-induced genes in ino80Δ mutants has uncovered a role for Ino80C on par with SWI/SNF in evicting promoter nucleosomes and Transcriptional activation. Compared to SWI/SNF, Ino80C generally functions over a wider region, spanning the -1 and +1 nucleosomes, NDR and proximal genic nucleosomes, at genes highly dependent on its function. Defects in nucleosome eviction in ino80Δ cells are frequently accompanied by reduced promoter occupancies of TBP, and diminished Transcription; and Ino80 is enriched at genes requiring its remodeler activity. Importantly, nuclear depletion of Ino80 impairs promoter nucleosome eviction even in a mutant lacking H2A.Z. Thus, Ino80C acts widely in the yeast genome together with RSC and SWI/SNF in evicting promoter nucleosomes and enhancing Transcription, all in a manner at least partly independent of H2A.Z editing.

  • swi snf and rsc cooperate to reposition and evict promoter nucleosomes at highly expressed genes in yeast
    Genes & Development, 2018
    Co-Authors: Yashpal Rawal, Răzvan V Chereji, Sudha Ananthakrishnan, David J Clark, Chhabi K Govind, Alan G Hinnebusch
    Abstract:

    : The nucleosome remodeling complex RSC functions throughout the yeast genome to set the positions of -1 and +1 nucleosomes and thereby determines the widths of nucleosome-depleted regions (NDRs). The related complex SWI/SNF participates in nucleosome remodeling/eviction and promoter activation at certain yeast genes, including those activated by Transcription Factor GCN4, but did not appear to function broadly in establishing NDRs. By analyzing the large cohort of GCN4-induced genes in mutants lacking the catalytic subunits of SWI/SNF or RSC, we uncovered cooperation between these remodelers in evicting nucleosomes from different locations in the promoter and repositioning the +1 nucleosome downstream to produce wider NDRs-highly depleted of nucleosomes-during Transcriptional activation. SWI/SNF also functions on a par with RSC at the most highly transcribed constitutively expressed genes, suggesting general cooperation by these remodelers for maximal Transcription. SWI/SNF and RSC occupancies are greatest at the most highly expressed genes, consistent with their cooperative functions in nucleosome remodeling and Transcriptional activation. Thus, SWI/SNF acts comparably with RSC in forming wide nucleosome-free NDRs to achieve high-level Transcription but only at the most highly expressed genes exhibiting the greatest SWI/SNF occupancies.

  • SWI/SNF and RSC cooperate to reposition and evict promoter nucleosomes at highly expressed genes in yeast
    Genes & development, 2018
    Co-Authors: Yashpal Rawal, Răzvan V Chereji, Sudha Ananthakrishnan, David J Clark, Chhabi K Govind, Hongfang Qiu, Alan G Hinnebusch
    Abstract:

    The nucleosome remodeling complex RSC functions throughout the yeast genome to set the positions of -1 and +1 nucleosomes and thereby determines the widths of nucleosome-depleted regions (NDRs). The related complex SWI/SNF participates in nucleosome remodeling/eviction and promoter activation at certain yeast genes, including those activated by Transcription Factor GCN4, but did not appear to function broadly in establishing NDRs. By analyzing the large cohort of GCN4-induced genes in mutants lacking the catalytic subunits of SWI/SNF or RSC, we uncovered cooperation between these remodelers in evicting nucleosomes from different locations in the promoter and repositioning the +1 nucleosome downstream to produce wider NDRs-highly depleted of nucleosomes-during Transcriptional activation. SWI/SNF also functions on a par with RSC at the most highly transcribed constitutively expressed genes, suggesting general cooperation by these remodelers for maximal Transcription. SWI/SNF and RSC occupancies are greatest at the most highly expressed genes, consistent with their cooperative functions in nucleosome remodeling and Transcriptional activation. Thus, SWI/SNF acts comparably with RSC in forming wide nucleosome-free NDRs to achieve high-level Transcription but only at the most highly expressed genes exhibiting the greatest SWI/SNF occupancies.

  • 7 Translational Control of GCN4: Gene-specific Regulation by Phosphorylation of elF2
    Cold Spring Harbor Monograph Archive, 1996
    Co-Authors: Alan G Hinnebusch
    Abstract:

    OVERVIEW OF GCN4 TRANSLATIONAL CONTROL When subjected to various kinds of starvation, stress, or certain viral infections, mammalian cells respond by reducing the overall rate of protein synthesis by phosphorylating the α-subunit of translation initiation Factor-2 (eIF2) (see Clemens; Mathews; Katze; Duncan; Schneider; all this volume). This down-regulation of protein synthesis is presumably a means of conserving resources and limiting cell division under adverse growth conditions or of preventing virus multiplication. Studies on the yeast Saccharomyces cerevisiae have shown that eIF2α becomes phosphorylated when cells are deprived of an amino acid or purine and that this event leads to an inhibition of translation by the same mechanism that operates in mammalian cells. Interestingly, it also regulates the translation of a specific mRNA encoding the Transcription Factor GCN4, causing increased synthesis of GCN4 protein under conditions in which the translation of other yeast messenger RNAs is being reduced. GCN4 activates Transcription of at least 40 different genes encoding amino acid biosynthetic enzymes (Hinnebusch 1988); thus, the induction of GCN4 alleviates the limitation for nutrients that triggers phosphorylation of eIF2 in yeast. The extent of eIF2 phosphorylation required to induce GCN4 translation is too low to cause a significant reduction in the rate of general protein synthesis. Consequently, GCN4 expression is a very sensitive indicator of the activity of eIF2 and associated translation initiation Factors. The unique induction of GCN4 translation under starvation conditions is mediated by four short upstream open reading frames (uORFs) in the leader of GCN4 mRNA, located between...