Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P51532 (transcriptional activator)
 6,546 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Affinity cleaving proteins have been synthesized based on the DNA-binding domain of the yeast transcriptional activator GCN4 with the DNA cleaving moiety Fe.EDTA attached at the NH2 terminus [Oakley, M. G., & Dervan, P. B. (1990) Science 248, 847]. Cleavage patterns generated by Fe-EDTA-GCN4(226-281) bound to the DNA sites 5'-CTGACTAAT-3' and 5'-ATGACTCTT-3' reveal that the NH2 termini of the GCN4 DNA-binding domain are located in the major groove of DNA, 9-10 base pairs apart, consistent with a Y-shaped dimeric structure. 1-Methylimidazole-2-carboxamide netropsin (2-ImN) is a designed synthetic peptide which binds in the minor groove of DNA at 5'-TGACT-3' sites as an antiparallel, side-by-side dimer [Mrksich, M., Wade, W. S., Dwyer, T. J., Geierstanger, B. H., Wemmer, D.E., & Dervan, P. B. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 7586]. Through the use of Fe.EDTA-GCN4(226-281) as a sequence-specific footprinting agent, it is shown that the dimeric protein GCN4-(226-281) and the dimeric peptide 2-ImN can simultaneously occupy their common binding site in the major and minor grooves of DNA, respectively. The association constants for 2-ImN in the presence and in the absence of Fe.EDTA-GCN4(226-281) are found to be similar, suggesting that the binding of the two dimers is not cooperative.
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PMID:Evidence that a minor groove-binding peptide and a major groove-binding protein can simultaneously occupy a common site on DNA. 144 35

NtrC is the transcriptional activator for nitrogen-regulated promoters and, as a response regulator, belongs to the protein family of two-component systems. The activity of all response regulators is modulated by phosphorylation of the conserved N-terminal receiver domain. Phosphorylation of the dimeric NtrC has two consequences: (i) a strong increase in the cooperative binding of NtrC to two adjacent binding sites and (ii) activation of NtrC as an ATPase. Here we show that phosphorylation of NtrC is not sufficient for activation of NtrC. At low protein concentrations (50 nM), phosphorylated NtrC was only active as an ATPase upon cooperative binding to DNA. At high protein concentrations (above 50 nM), NtrC was active in the absence of DNA, and activation occurred in parallel with the formation of high-molecular-weight aggregates. We infer that activation of NtrC involves an interaction between two NtrC-P dimers and proceeds in two steps. The first step is the phosphorylation of NtrC. The second step is the interaction between two NtrC-P dimers. This interaction induces the conformational change in NtrC-P to the active conformation.
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PMID:Mechanism of activation of a response regulator: interaction of NtrC-P dimers induces ATPase activity. 766 84

A 33 membered polypeptide corresponding to the leucine zipper region of the yeast transcriptional activator GCN4 was synthesized by solid phase chemical synthesis and characterized. Asparagine in the hydrophobic core of the molecule is replaced by valine in the synthetic variant. The correctness of amino acid sequence of the preparation is corroborated by direct sequencing. High-speed equilibrium ultracentrifugation, ultraviolet circular dichroism spectroscopy and scanning microcalorimetry have been employed to demonstrate that in solution the peptide forms a highly stable triple-stranded alpha-helical coiled coil. The stability of the mutant form is 40 degrees C higher than the dimeric form of natural peptide under similar conditions. It was proposed that location of some polar groups in the 'a' and 'd' positions of natural two-stranded coiled coils may be regarded as protection against alternative triple- and multistranded conformations.
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PMID:Synthesis and properties of the peptide corresponding to the mutant form of the leucine zipper of the transcriptional activator GCN4 from yeast. 783 Dec 80

c-Jun and c-Fos belong to the bZIP class of transcriptional activator proteins, many of which have been implicated in the neoplastic transformation of cells. We are interested in engineering dominant-negative leucine zipper (LZ) peptides as a means of sequestering these proteins in vivo in order to suppress their transcriptional regulatory activity. Toward this end, we have developed a novel immunoassay for measuring the dimerization affinities of dimeric Jun and Fos complexes. This peptide-based ELISA relies on the fact that Jun and Fos preferentially form heterodimers via their leucine zipper domains. Recombinant Jun leucine zipper peptides (either native JunLZ or a V36 --> E point mutant) were labeled with biotin and specifically bound through a leucine zipper interaction to a FosLZ-glutathione S-transferase fusion protein adsorbed onto the wells of an ELISA tray. Jun:Fos complexes were subsequently detected using a recently developed streptavidin-based amplification system known as enzyme complex amplification [Wilson, M. R., & Easterbrook-Smith, S.B. (1993) Anal. Biochem. 209, 183-187]. This ELISA system can detect subnanomolar concentrations of Jun and Fos, thus allowing determination of the dissociation constants for complex formation. The dissociation constant for formation of the native JunLZ:FosLZ heterodimer at 37 degrees C was determined to be 0.99 +/- 0.30 nM, while that for JunLZ(V36E):FosLZ heterodimer was 0.90 +/- 0.13 microM. These results demonstrate that the novel peptide-based ELISA described herein is simple and sensitive and can be used to rapidly screen for potential dominant-negative leucine zipper peptides.
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PMID:Development of a sensitive peptide-based immunoassay: application to detection of the Jun and Fos oncoproteins. 870 10

It has previously been suggested that the DNA binding domain (residues 1 to 147) of the yeast transcriptional activator GAL4 exists in solution in dimeric form, with the region responsible for dimerisation somewhere between residues 74 and 147. In this study limited proteolysis and carboxy-terminal deletions of the DNA binding domain (residues 1 to 147) of the yeast transcriptional activator GAL4 followed by subsequent characterization by equilibrium sedimentation in the analytical ultracentrifuge have been used to define more precisely the regions required for DNA binding and protein self-association. Sedimentation equilibrium analyses confirmed that the 'hydrophobic region' of the protein (residues 54-97, which contains a larger proportion of alpha-helix), is essential for dimerisation, with an apparent dissociation constant K(D,app), of approximately 50 microM for the 1-94 residue peptide and approximately 20 microM for the 1-147 residue peptide. Our studies do not rule out the possible formation of small amounts of additional higher order complexes.
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PMID:A physico-chemical investigation of the self-association of the DNA binding domain of the yeast transcriptional activator GAL4. 876 12

The yeast transcriptional activator Gcn4 requires the Ada2/Gcn5/Ada3 co-activator complex to exert part of its activation potential. Here we show that the sequence of the DNA target modulates the function of Gcn4 by modifying this requirement. Promoter configurations were generated that rendered Gcn4-induced transcription either completely dependent or completely independent of the Ada2/Gcn5/Ada3 complex. The topological constraints imposed by these configurations suggest that Gcn4 makes multiple contacts with the basic transcription machinery that are subject to modification by the incident DNA target. We propose that these modifications further determine the direction on the chromosome in which an otherwise symmetric, dimeric transcription factor will activate.
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PMID:The DNA target sequence influences the dependence of the yeast transcriptional activator Gcn4 on co-factors. 907 89

A 42 residue artificial peptide that binds to the HIV-1 enhancers has been described previously. The specificity of interaction of the peptide with its target DNA sequence has been demonstrated by a variety of techniques. Naturally occurring regulatory proteins frequently bind to DNA as dimers, thereby increasing the strength and specificity of the interaction, the dimer interface often being provided by a leucine zipper type coiled coil. As a suitable binding site for this kind of system is located to the 5' end of the HIV enhancer region, it was decided to design and synthesize a fusion peptide that not only contained the DNA binding sequence of the original 42 residue peptide but also incorporated a leucine zipper based on that of the GCN4 transcriptional activator, that should, therefore, be capable of dimerizing. The resultant peptide, LZ66, has now been shown to be fully active in band shift and in vitro transcription assays and to exhibit about double the inhibitory activity of the parent 42 residue peptide. Preliminary CD measurements revealed that the peptide has a high alpha-helical content and that it adopts a stable conformation down to the low micromolar peptide concentration range. Sedimentation equilibrium studies confirmed that the principles involved in the design of the peptide are valid and that the peptide is indeed dimeric in solution.
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PMID:An artificial HIV enhancer-binding peptide is dimerized by the addition of a leucine zipper. 918 62

FIS (factor for inversion stimulation) is a small dimeric DNA-bending protein which both stimulates DNA inversion and activates transcription at stable RNA promoters in Escherichia coli. Both these processes involve the initial formation of a complex nucleoprotein assembly followed by local DNA untwisting at a specific site. We have demonstrated previously that at the tyrT promoter three FIS dimers are required to form a nucleoprotein complex with RNA polymerase. We now show that this complex is structurally dynamic and that FIS, uniquely for a prokaryotic transcriptional activator, facilitates sequential steps in the initiation process, enabling efficient polymerase recruitment, untwisting of DNA at the transcription startpoint and finally the escape of polymerase from the promoter. Activation of all these steps requires that the three FIS dimers bind in helical register. We suggest that FIS acts by stabilizing a DNA microloop whose topology is coupled to the local topological transitions generated during the initiation of transcription.
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PMID:FIS activates sequential steps during transcription initiation at a stable RNA promoter. 921 6

We measure the effects of low concentrations of helix-stabilizing cosolvents, including 2,2,2-trifluoroethanol (TFE), on the thermodynamics and kinetics of folding of the dimeric alpha-helical coiled coil derived from the leucine zipper region of bZIP transcriptional activator GCN4. The change in kinetic behavior upon addition of 5% (v/v) TFE indicates that it stabilizes the transition state to the same degree as the fully helical native state. However, folding rates are largely insensitive to alanine to glycine mutagenesis, indicating that the majority of helical structure is formed after the transition state. Equilibrium hydrogen isotope partitioning measurements indicate that intramolecular hydrogen bonds are not strengthened by TFE and that amide hydrogen bonds in the transition state are nearly the same strength as those in the unfolded state. Thus, the mechanism by which TFE exerts its helix-stabilizing effects can be divorced from helix formation and does not depend on the strengthening of intrahelical hydrogen bonds. Rather, TFE increases the structure of the binary alcohol/water solvent, thereby increasing the energetic cost associated with solvation of the polypeptide backbone. At low concentrations, TFE destabilizes the unfolded species and thereby indirectly enhances the kinetics and thermodynamics of folding of the coiled coil. A high degree of polypeptide backbone desolvation, and not the formation of regular helical structure and native strength hydrogen bonds, is the critical feature of the transition state for folding of this small dimeric protein.
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PMID:Trifluoroethanol promotes helix formation by destabilizing backbone exposure: desolvation rather than native hydrogen bonding defines the kinetic pathway of dimeric coiled coil folding. 977 90

We measured whether solvent viscosity, and hence chain diffusion, plays a role in the rate-limiting step of the folding reactions of GCN4-p2', a simple alpha-helical coiled coil derived from the leucine zipper region of bZIP transcriptional activator GCN4. To deconvolute the dual effects of viscosogenic solvents on both viscosity, eta, and stability, earlier attempts assumed that the cosolvent and denaturant interact to the same degree in the transition state. Applying this analysis to GCN4-p2' yielded a nearly 1/eta dependence between folding rates and viscosity for both the dimeric and the cross-linked, monomeric versions of the coiled coil, but it revealed no such coherent relationship for cytochrome c. We also developed a method to determine the relative viscosity dependence of the dimeric and monomeric forms of the coiled coil independent of the assumption concerning the transition state's relative interaction with cosolvents and denaturants. Application of this method indicated that the effect of viscosity on both the folding and the unfolding rates was the same for the dimeric and monomeric versions, further supporting the view that the folding of the dimeric version is folding-limited rather than encounter-limited. The finding that GCN4-p2' folding appears to exhibit a 1/eta viscosity dependence implies that the rate-limiting step in folding is opposed predominantly by solvent-derived rather than internal frictional forces. These results are interpreted in relation to various models for protein folding.
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PMID:Viscosity dependence of the folding kinetics of a dimeric and monomeric coiled coil. 1002 55


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