scholarly journals Mutations that alter ligand-induced switches and dimerization activities in the retinoid X receptor.

1994 ◽  
Vol 14 (6) ◽  
pp. 4311-4323 ◽  
Author(s):  
X K Zhang ◽  
G Salbert ◽  
M O Lee ◽  
M Pfahl
Keyword(s):  
Amino Acids ◽  
Retinoic Acid ◽  
Amino Acid ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Activation Function ◽  
Retinoid X Receptor ◽  
Single Amino Acid ◽  
Independent Manner ◽  
Present Evidence

The retinoid X receptor (RXR) heterodimerizes with a variety of nuclear receptors. In addition, RXR forms homodimers in the presence of its ligand, 9-cis-retinoic acid. From deletion and point mutation analysis we present evidence that a short region (amino acids 413 to 443) in the carboxy terminus of RXR alpha is critical for both homo- and heterodimeric interactions as well as for diverse functional activities. In addition, we present evidence that homo- and heterodimer functions can be separated. The deletion of 19 amino acids from the C-terminal end of RXR dramatically reduced the transcriptional activation function of RXR. The removal of 10 additional amino acids resulted in a receptor (delta RXR3) that had completely lost its ligand-dependent homodimer function but retained its heterodimer activities. Heterodimer function was abolished by the deletion of an additional 20 amino acids. Single amino acid substitutions in the region generated receptors with altered RXR homodimer DNA binding, while simultaneous mutation of three Leu residues (Leu-418, -419 and -422) completely abolished both RXR homodimer and heterodimer DNA binding activities. Mutation of Leu-430 to Phe (L430-F) resulted in a receptor that bound to DNA strongly as homodimers in a ligand-independent manner, while another single amino acid exchange (L422-Q) led to a mutant that behaved in a manner exactly opposite to that of wild-type RXR in that the homodimerization of the mutant occurred in the absence of ligand and was inhibited by 9-cis-retinoic acid. In transfection assays, both L422-Q and L430-F failed to act as homodimers but retained their heterodimer function. Our studies demonstrate the unique properties of the RXR ligand binding domain and point to specific residues that mediate homo- and heterodimer activities and ligand-induced conformational switches.

Mutations that alter ligand-induced switches and dimerization activities in the retinoid X receptor

1994 ◽  
Vol 14 (6) ◽  
pp. 4311-4323
Author(s):  
X K Zhang ◽  
G Salbert ◽  
M O Lee ◽  
M Pfahl
Keyword(s):  
Amino Acids ◽  
Retinoic Acid ◽  
Amino Acid ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Activation Function ◽  
Retinoid X Receptor ◽  
Single Amino Acid ◽  
Independent Manner ◽  
Present Evidence

The retinoid X receptor (RXR) heterodimerizes with a variety of nuclear receptors. In addition, RXR forms homodimers in the presence of its ligand, 9-cis-retinoic acid. From deletion and point mutation analysis we present evidence that a short region (amino acids 413 to 443) in the carboxy terminus of RXR alpha is critical for both homo- and heterodimeric interactions as well as for diverse functional activities. In addition, we present evidence that homo- and heterodimer functions can be separated. The deletion of 19 amino acids from the C-terminal end of RXR dramatically reduced the transcriptional activation function of RXR. The removal of 10 additional amino acids resulted in a receptor (delta RXR3) that had completely lost its ligand-dependent homodimer function but retained its heterodimer activities. Heterodimer function was abolished by the deletion of an additional 20 amino acids. Single amino acid substitutions in the region generated receptors with altered RXR homodimer DNA binding, while simultaneous mutation of three Leu residues (Leu-418, -419 and -422) completely abolished both RXR homodimer and heterodimer DNA binding activities. Mutation of Leu-430 to Phe (L430-F) resulted in a receptor that bound to DNA strongly as homodimers in a ligand-independent manner, while another single amino acid exchange (L422-Q) led to a mutant that behaved in a manner exactly opposite to that of wild-type RXR in that the homodimerization of the mutant occurred in the absence of ligand and was inhibited by 9-cis-retinoic acid. In transfection assays, both L422-Q and L430-F failed to act as homodimers but retained their heterodimer function. Our studies demonstrate the unique properties of the RXR ligand binding domain and point to specific residues that mediate homo- and heterodimer activities and ligand-induced conformational switches.

Acquisition of NFKB1-selective DNA binding by substitution of four amino acid residues from NFKB1 into RelA

1993 ◽  
Vol 13 (7) ◽  
pp. 3850-3859
Author(s):  
T A Coleman ◽  
C Kunsch ◽  
M Maher ◽  
S M Ruben ◽  
C A Rosen
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Dna Binding ◽  
Fusion Protein ◽  
Binding Specificity ◽  
Single Amino Acid ◽  
Nf Kappa B ◽  
Dna Binding Specificity ◽  
Dna Motif ◽  
Single Amino Acid Change

The subunits of NF-kappa B, NFKB1 (formerly p50) and RelA (formerly p65), belong to a growing family of transcription factors that share extensive similarity to the c-rel proto-oncogene product. The homology extends over a highly conserved stretch of approximately 300 amino acids termed the Rel homology domain (RHD). This region has been shown to be involved in both multimerization (homo- and heterodimerization) and DNA binding. It is now generally accepted that homodimers of either subunit are capable of binding DNA that contains a kappa B site originally identified in the immunoglobulin enhancer. Recent studies have demonstrated that the individual subunits of the NF-kappa B transcription factor complex can be distinguished by their ability to bind distinct DNA sequence motifs. By using NFKB1 and RelA subunit fusion proteins, different regions within the RHD were found to confer DNA-binding and multimerization functions. A fusion protein that contains 34 N-terminal amino acids of NFKB1 and 264 amino acids of RelA displayed preferential binding to an NFKB1-selective DNA motif while dimerizing with the characteristics of RelA. Within the NFKB1 portion of this fusion protein, a single amino acid change of His to Arg altered the DNA-binding specificity to favor interaction with the RelA-selective DNA motif. Furthermore, substitution of four amino acids from NFKB1 into RelA was able to alter the DNA-binding specificity of the RelA protein to favor interaction with the NFKB1-selective site. Taken together, these findings demonstrate the presence of a distinct subdomain within the RHD involved in conferring the DNA-binding specificity of the Rel family of proteins.

scholarly journals Identification of amino acids essential for DNA binding and dimerization in p67SRF: implications for a novel DNA-binding motif

1993 ◽  
Vol 13 (1) ◽  
pp. 123-132
Author(s):  
A D Sharrocks ◽  
H Gille ◽  
P E Shaw
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Serum Response Factor ◽  
Alpha Helix ◽  
Site Directed Mutagenesis ◽  
Binding Motif ◽  
Amino Acid Peptide ◽  
Serum Response

The serum response factor (p67SRF) binds to a palindromic sequence in the c-fos serum response element (SRE). A second protein, p62TCF binds in conjunction with p67SRF to form a ternary complex, and it is through this complex that growth factor-induced transcriptional activation of c-fos is thought to take place. A 90-amino-acid peptide, coreSRF, is capable for dimerizing, binding DNA, and recruiting p62TCF. By using extensive site-directed mutagenesis we have investigated the role of individual coreSRF amino acids in DNA binding. Mutant phenotypes were defined by gel retardation and cross-linking analyses. Our results have identified residues essential for either DNA binding or dimerization. Three essential basic amino acids whose conservative mutation severely reduced DNA binding were identified. Evidence which is consistent with these residues being on the face of a DNA binding alpha-helix is presented. A phenylalanine residue and a hexameric hydrophobic box are identified as essential for dimerization. The amino acid phasing is consistent with the dimerization interface being presented as a continuous region on a beta-strand. A putative second alpha-helix acts as a linker between these two regions. This study indicates that p67SRF is a member of a protein family which, in common with many DNA binding proteins, utilize an alpha-helix for DNA binding. However, this alpha-helix is contained within a novel domain structure.

scholarly journals tau4/tau c/AF-2 of the thyroid hormone receptor relieves silencing of the retinoic acid receptor silencer core independent of both tau4 activation function and full dissociation of corepressors.

1997 ◽  
Vol 17 (8) ◽  
pp. 4259-4271 ◽  
Author(s):  
A Baniahmad ◽  
D Thormeyer ◽  
R Renkawitz
Keyword(s):  
Amino Acids ◽  
Retinoic Acid ◽  
Thyroid Hormone ◽  
Transcriptional Activation ◽  
Hormone Receptors ◽  
Thyroid Hormone Receptor ◽  
Retinoic Acid Receptor ◽  
Activation Function ◽  
Acid Receptor ◽  
C Terminus

Members of the thyroid hormone (TR)-retinoic acid receptor (RAR) subfamily of nuclear hormone receptors silence gene expression in the absence of hormone. Addition of cognate ligands leads to dissociation of corepressors, association of coactivators, and transcriptional activation. Here, we used the hRAR alpha silencer core, which encompasses the ligand binding domain, including receptor regions D and E of RAR alpha without the activation function called tau4/tau c/AF-2 and without the F region, to analyze the mechanisms by which transcriptional silencing is relieved. Although the RAR silencer core is able to bind ligand, it acts as a constitutive transcriptional silencer. We have fused various small activation domains to the C terminus of the silencer core and analyzed hormone-dependent changes in receptor function. We show that nine amino acids derived from the hTRbeta are sufficient to transform the RAR silencer core into a hormone-dependent activator. Lengthening the linker between the silencer core and these nine amino acids is not critical for mediating ligand-induced relief of silencing and activation. In addition, we show that a transactivation function at the C terminus is not required for relief of silencing by the hormone, but it is required for transcriptional activation. Furthermore, we created functional silencer fusions which lose their repressive function upon addition of hormone, although the corepressors SMRT and N-CoR remain attached to the receptor.

scholarly journals A 10-amino-acid sequence in the N-terminal A/B domain of thyroid hormone receptor alpha is essential for transcriptional activation and interaction with the general transcription factor TFIIB.

1995 ◽  
Vol 15 (8) ◽  
pp. 4507-4517 ◽  
Author(s):  
E Hadzic ◽  
V Desai-Yajnik ◽  
E Helmer ◽  
S Guo ◽  
S Wu ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Thyroid Hormone ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Thyroid Hormone Receptor ◽  
Alpha Helix ◽  
Binding Domain ◽  
Terminal Region ◽  
Binding Studies

The effects of the thyroid hormone (3,5,3'-triiodo-L-thyronine [T3]) on gene transcription are mediated by nuclear T3 receptors (T3Rs). alpha- and beta-isoform T3Rs (T3R alpha and -beta) are expressed from different genes and are members of a superfamily of ligand-dependent transcription factors that also includes the receptors for steroid hormones, vitamin D, and retinoids. Although T3 activates transcription by mediating a conformational change in the C-terminal approximately 220-amino-acid ligand-binding domain (LBD), the fundamental mechanisms of T3R-mediated transcriptional activation remain to be determined. We found that deletion of the 50-amino-acid N-terminal A/B domain of chicken T3R alpha (cT3R alpha) decreases T3-dependent stimulation of genes regulated by native thyroid hormone response elements about 10- to 20-fold. The requirement of the A/B region for transcriptional activation was mapped to amino acids 21 to 30, which contain a cluster of five basic amino acids. The A/B region of cT3R alpha is not required for T3 binding or for DNA binding of the receptor as a heterodimer with retinoid X receptor. In vitro binding studies indicate that the N-terminal region of cT3R alpha interacts efficiently with TFIIB and that this interaction requires amino acids 21 to 30 of the A/B region. In contrast, the LBD interacts poorly with TFIIB. The region of TFIIB primarily involved in the binding of cT3R alpha includes an amphipathic alpha helix contained within residues 178 to 201. Analysis using a fusion protein containing the DNA-binding domain of GAL4 and the entire A/B region of cT3R alpha suggests that this region does not contain an intrinsic activation domain. These and other studies indicate that cT3R alpha mediates at least some of its effects through TFIIB in vivo and that the N-terminal region of DNA-bound cT3R alpha acts to recruit and/or stabilize the binding of TFIIB to the transcription complex. T3 stimulation could then result from ligand-mediated changes in the LBD which may lead to the interaction of other factors with cT3R alpha, TFIIB, and/or other components involved in the initiation of transcription.

scholarly journals GAL4 mutations that separate the transcriptional activation and GAL80-interactive functions of the yeast GAL4 protein.

Genetics ◽  
1990 ◽  
Vol 125 (1) ◽  
pp. 21-27 ◽  
Author(s):  
J M Salmeron ◽  
K K Leuther ◽  
S A Johnston
Keyword(s):  
Amino Acid ◽  
Transcriptional Activation ◽  
Regulatory Protein ◽  
Activation Function ◽  
Single Amino Acid ◽  
Carboxyl Terminus ◽  
Wild Type ◽  
Wild Type Phenotype ◽  
Transcriptional Activator Protein ◽  
Carboxy Terminal

Abstract The carboxy-terminal 28 amino acids of the Saccharomyces cerevisiae transcriptional activator protein GAL4 execute two functions--transcriptional activation and interaction with the negative regulatory protein, GAL80. Here we demonstrate that these two functions are separable by single amino acid changes within this region. We determined the sequences of four GAL4C-mutations, and characterized the abilities of the encoded GAL4C proteins to activate transcription of the galactose/melibiose regulon in the presence of GAL80 and superrepressible GAL80S alleles. One of the GAL4C mutations can be compensated by a specific GAL80S mutation, resulting in a wild-type phenotype. These results support the idea that while the GAL4 activation function tolerates at least minor alterations in the GAL4 carboxyl terminus, the GAL80-interactive function is highly sequence-specific and sensitive even to single amino acid alterations. They also argue that the GAL80S mutations affect the affinity of GAL80 for GAL4, and not the ability of GAL80 to bind inducer.

scholarly journals Balance between mutually exclusive traits shifted by variants of a yeast transcription factor

2017 ◽  
Author(s):  
Michael W. Dorrity ◽  
Josh T. Cuperus ◽  
Jolie A. Carlisle ◽  
Stanley Fields ◽  
Christine Queitsch
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcription Factor ◽  
Amino Acid ◽  
Dna Binding ◽  
Single Amino Acid ◽  
Environmental Cues ◽  
Dna Binding Domain ◽  
Independent Manner ◽  
Half Site

AbstractIn Saccharomyces cerevisiae, the decision to mate or invade relies on environmental cues that converge on a shared transcription factor, Ste12. Specificity toward invasion occurs via Ste12 binding cooperatively with the co-factor Tec1. Here, we characterize the in vitro binding preferences of Ste12 to identify a defined spacing and orientation of dimeric sites, one that is common in pheromone-regulated genes. We find that single amino acid changes in the DNA-binding domain of Ste12 can shift the preference of yeast toward either mating or invasion. These mutations define two distinct regions of this domain, suggesting alternative modes of DNA binding for each trait. Some exceptional Ste12 mutants promote hyperinvasion in a Tec1-independent manner; these fail to bind cooperative sites with Tec1 and bind to unusual dimeric Ste12 sites that contain one highly degenerate half site. We propose a model for how activation of invasion genes could have evolved with Ste12 alone.

scholarly journals Identification of amino acids essential for DNA binding and dimerization in p67SRF: implications for a novel DNA-binding motif.

1993 ◽  
Vol 13 (1) ◽  
pp. 123-132 ◽  
Author(s):  
A D Sharrocks ◽  
H Gille ◽  
P E Shaw
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Serum Response Factor ◽  
Alpha Helix ◽  
Site Directed Mutagenesis ◽  
Binding Motif ◽  
Amino Acid Peptide ◽  
Serum Response

The serum response factor (p67SRF) binds to a palindromic sequence in the c-fos serum response element (SRE). A second protein, p62TCF binds in conjunction with p67SRF to form a ternary complex, and it is through this complex that growth factor-induced transcriptional activation of c-fos is thought to take place. A 90-amino-acid peptide, coreSRF, is capable for dimerizing, binding DNA, and recruiting p62TCF. By using extensive site-directed mutagenesis we have investigated the role of individual coreSRF amino acids in DNA binding. Mutant phenotypes were defined by gel retardation and cross-linking analyses. Our results have identified residues essential for either DNA binding or dimerization. Three essential basic amino acids whose conservative mutation severely reduced DNA binding were identified. Evidence which is consistent with these residues being on the face of a DNA binding alpha-helix is presented. A phenylalanine residue and a hexameric hydrophobic box are identified as essential for dimerization. The amino acid phasing is consistent with the dimerization interface being presented as a continuous region on a beta-strand. A putative second alpha-helix acts as a linker between these two regions. This study indicates that p67SRF is a member of a protein family which, in common with many DNA binding proteins, utilize an alpha-helix for DNA binding. However, this alpha-helix is contained within a novel domain structure.

scholarly journals Acquisition of NFKB1-selective DNA binding by substitution of four amino acid residues from NFKB1 into RelA.

1993 ◽  
Vol 13 (7) ◽  
pp. 3850-3859 ◽  
Author(s):  
T A Coleman ◽  
C Kunsch ◽  
M Maher ◽  
S M Ruben ◽  
C A Rosen
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Dna Binding ◽  
Fusion Protein ◽  
Binding Specificity ◽  
Single Amino Acid ◽  
Nf Kappa B ◽  
Dna Binding Specificity ◽  
Dna Motif ◽  
Single Amino Acid Change

The subunits of NF-kappa B, NFKB1 (formerly p50) and RelA (formerly p65), belong to a growing family of transcription factors that share extensive similarity to the c-rel proto-oncogene product. The homology extends over a highly conserved stretch of approximately 300 amino acids termed the Rel homology domain (RHD). This region has been shown to be involved in both multimerization (homo- and heterodimerization) and DNA binding. It is now generally accepted that homodimers of either subunit are capable of binding DNA that contains a kappa B site originally identified in the immunoglobulin enhancer. Recent studies have demonstrated that the individual subunits of the NF-kappa B transcription factor complex can be distinguished by their ability to bind distinct DNA sequence motifs. By using NFKB1 and RelA subunit fusion proteins, different regions within the RHD were found to confer DNA-binding and multimerization functions. A fusion protein that contains 34 N-terminal amino acids of NFKB1 and 264 amino acids of RelA displayed preferential binding to an NFKB1-selective DNA motif while dimerizing with the characteristics of RelA. Within the NFKB1 portion of this fusion protein, a single amino acid change of His to Arg altered the DNA-binding specificity to favor interaction with the RelA-selective DNA motif. Furthermore, substitution of four amino acids from NFKB1 into RelA was able to alter the DNA-binding specificity of the RelA protein to favor interaction with the NFKB1-selective site. Taken together, these findings demonstrate the presence of a distinct subdomain within the RHD involved in conferring the DNA-binding specificity of the Rel family of proteins.

scholarly journals Mutational Analysis of the Transcriptional Regulator GcvA: Amino Acids Important for Activation, Repression, and DNA Binding

1998 ◽  
Vol 180 (18) ◽  
pp. 4865-4871 ◽  
Author(s):  
Alissa D. Jourdan ◽  
George V. Stauffer
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Dna Binding ◽  
Mutational Analysis ◽  
Transcriptional Regulator ◽  
Single Amino Acid ◽  
Amino Acid Substitutions ◽  
Constitutive Activation ◽  
Carboxy Terminal

ABSTRACT The GcvA protein is required for both glycine-mediated activation and purine-mediated repression of the gcvTHP operon. Random and site-directed PCR mutagenesis was used to create nucleotide changes in gcvA to identify residues of the protein involved in activation, repression, and DNA binding. Single amino acid substitutions at L30 and F31 cause a defect in activation of agcvT-lacZ fusion but have no effect on repression or DNA binding. Single amino acid substitutions at V32 and S38 cause the loss of binding of GcvA to DNA. A deletion of the carboxy-terminal 14 amino acids of GcvA results in the loss of purine-mediated repression and, consequently, a constitutive activation of a gcvT-lacZfusion. The results of this study partially define regions of GcvA involved in activation, repression, and DNA binding and demonstrate that these functions of GcvA are genetically separable.

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