nit-4, a pathway-specific regulatory gene of Neurospora crassa, encodes a protein with a putative binuclear zinc DNA-binding domain.

nit-4, a pathway-specific regulatory gene in the nitrogen circuit of Neurospora crassa, is required for the expression of nit-3 and nit-6, the structural genes which encode nitrate and nitrite reductase, respectively. The complete nucleotide sequence of the nit-4 gene has been determined. The predicted NIT4 protein contains 1,090 amino acids and appears to possess a single Zn(II)2Cys6 binuclear-type zinc finger, which may mediate DNA binding. Site-directed mutagenesis studies demonstrated that cysteine and other conserved amino acid residues in this possible DNA-binding domain are necessary for nit-4 function. A stretch of 27 glutamines, encoded by a CAGCAA repeating sequence, occurs in the C terminus of the NIT4 protein, and a second glutamine-rich domain occurs further upstream. A NIT4 protein deleted for the polyglutamine region was still functional in vivo. However, nit-4 function was abolished when both the polyglutamine region and the glutamine-rich domain were deleted, suggesting that the glutamine-rich domain might function in transcriptional activation. The homologous regulatory gene from Aspergillus nidulans, nirA, encodes a protein whose amino-terminal half has approximately 60% amino acid identity with NIT4 but whose carboxy terminus is completely different. A hybrid nit-4-nirA gene was constructed and found to function in N. crassa.

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Mutational analysis of the DNA-binding domain of the CYS3 regulatory protein of Neurospora crassa.

Molecular and Cellular Biology ◽

10.1128/mcb.11.9.4356 ◽

1991 ◽

Vol 11 (9) ◽

pp. 4356-4362 ◽

Cited By ~ 9

Author(s):

M N Kanaan ◽

G A Marzluf

Keyword(s):

Dna Binding ◽

Neurospora Crassa ◽

Mutational Analysis ◽

Regulatory Gene ◽

Regulatory Protein ◽

Binding Activity ◽

Binding Domain ◽

Site Directed Mutagenesis ◽

Dna Binding Domain ◽

Dna Binding Activity

cys-3, the major sulfur regulatory gene of Neurospora crassa, activates the expression of a set of unlinked structural genes which encode sulfur catabolic-related enzymes during conditions of sulfur limitation. The cys-3 gene encodes a regulatory protein of 236 amino acid residues with a leucine zipper and an upstream basic region (the b-zip region) which together may constitute a DNA-binding domain. The b-zip region was expressed in Escherichia coli to examine its DNA-binding activity. The b-zip domain protein binds to the promoter region of the cys-3 gene itself and of cys-14, the sulfate permease II structural gene. A series of CYS3 mutant proteins obtained by site-directed mutagenesis were expressed and tested for function, dimer formation, and DNA-binding activity. The results demonstrate that the b-zip region of cys-3 is critical for both its function in vivo and specific DNA-binding in vitro.

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Mutational analysis of the DNA-binding domain of the CYS3 regulatory protein of Neurospora crassa

Molecular and Cellular Biology ◽

10.1128/mcb.11.9.4356-4362.1991 ◽

1991 ◽

Vol 11 (9) ◽

pp. 4356-4362

Author(s):

M N Kanaan ◽

G A Marzluf

Keyword(s):

Dna Binding ◽

Neurospora Crassa ◽

Mutational Analysis ◽

Regulatory Gene ◽

Regulatory Protein ◽

Binding Activity ◽

Binding Domain ◽

Site Directed Mutagenesis ◽

Dna Binding Domain ◽

Dna Binding Activity

cys-3, the major sulfur regulatory gene of Neurospora crassa, activates the expression of a set of unlinked structural genes which encode sulfur catabolic-related enzymes during conditions of sulfur limitation. The cys-3 gene encodes a regulatory protein of 236 amino acid residues with a leucine zipper and an upstream basic region (the b-zip region) which together may constitute a DNA-binding domain. The b-zip region was expressed in Escherichia coli to examine its DNA-binding activity. The b-zip domain protein binds to the promoter region of the cys-3 gene itself and of cys-14, the sulfate permease II structural gene. A series of CYS3 mutant proteins obtained by site-directed mutagenesis were expressed and tested for function, dimer formation, and DNA-binding activity. The results demonstrate that the b-zip region of cys-3 is critical for both its function in vivo and specific DNA-binding in vitro.

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A specific and unusual nuclear localization signal in the DNA binding domain of the Rev-erb orphan receptors

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0300197 ◽

2003 ◽

Vol 30 (2) ◽

pp. 197-211 ◽

Cited By ~ 17

Author(s):

S Chopin-Delannoy ◽

S Thenot ◽

F Delaunay ◽

E Buisine ◽

A Begue ◽

...

Keyword(s):

Amino Acid ◽

Dna Binding ◽

Fusion Protein ◽

Nuclear Receptors ◽

Nuclear Localization ◽

Binding Domain ◽

Dna Binding Domain ◽

Protein Fusion ◽

Orphan Receptors ◽

Amino Terminal

The orphan receptors Rev-erbalpha and Rev-erbbeta are members of the nuclear receptors superfamily and act as transcriptional repressors. Rev-erbalpha is expressed with a robust circadian rhythm and is involved in liver metabolism through repression of the ApoA1 gene, but no role has been yet defined for Rev-erbbeta. To gain better understanding of their function and mode of action, we characterized the proteins encoded by these two genes. Both Rev-erbalpha and Rev-erbbeta proteins were nuclear when transiently transfected in COS-1 cells. The major nuclear location signal (NLS) of Rev-erbalpha is in the amino-terminal region of the protein. Fusion of green fluorescent protein (GFP) to the amino terminus of Rev-erbalpha deletion mutants showed that the NLS is located within a 53 amino acid segment of the DNA binding domain (DBD). The homologous region of Rev-erbbeta fused to GFP also targeted the fusion protein to the nucleus, suggesting that the location of this NLS is conserved among all the Rev-erb group members. Interestingly, members of the phylogenetically closest nuclear orphan receptor group (ROR), which exhibit 58% amino acid identity with Rev-erb in the DBD, do not have their NLS located within the DBD. GFP/DBD. RORalpha or GFP/DBD.RORbeta remained cytoplasmic, in contrast to GFP/DBD. Rev-erb fusion proteins. Alignment of human Rev-erb and ROR DBD amino acid sequences predicted that the two basic residues, K167 and R168, located just upstream from the second zinc finger, could play a critical part in the nuclear localization of Rev-erb proteins. Substitution of these two residues with those found in ROR, in the GFP/DBD. Rev-erb context, resulted in cytoplasmic proteins. In contrast, the reverse mutation of the GFP/DBD. RORalpha towards the Rev-erbalpha residues targeted the fusion protein to the nucleus. Our data demonstrate that Rev-erb proteins contain a functional NLS in the DBD. Its location is unusual within the nuclear receptor superfamily and suggests that Rev-erb orphan receptors control their intracellular localization via a mechanism different from that of other nuclear receptors.

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Mutational Analysis of Conserved Residues in the Putative DNA-Binding Domain of the Response Regulator Spo0A ofBacillus subtilis

Journal of Bacteriology ◽

10.1128/jb.182.24.6975-6982.2000 ◽

2000 ◽

Vol 182 (24) ◽

pp. 6975-6982 ◽

Cited By ~ 4

Author(s):

Janet K. Hatt ◽

Philip Youngman

Keyword(s):

Dna Binding ◽

Transcriptional Activation ◽

Mutational Analysis ◽

Response Regulator ◽

Gene Activation ◽

Binding Domain ◽

Site Directed Mutagenesis ◽

Specific Gene ◽

Dna Binding Domain ◽

Conserved Residues

ABSTRACT The Spo0A protein of Bacillus subtilis is a DNA-binding protein that is required for the expression of genes involved in the initiation of sporulation. Spo0A binds directly to and both activates and represses transcription from the promoters of several genes required during the onset of endospore formation. The C-terminal 113 residues are known to contain the DNA-binding activity of Spo0A. Previous studies identified a region of the C-terminal half of Spo0A that is highly conserved among species of endospore-formingBacillus and Clostridium and which encodes a putative helix-turn-helix DNA-binding domain. To test the functional significance of this region and determine if this motif is involved in DNA binding, we changed three conserved residues, S210, E213, and R214, to Gly and/or Ala by site-directed mutagenesis. We then isolated and analyzed the five substitution-containing Spo0A proteins for DNA binding and sporulation-specific gene activation. The S210A Spo0A mutant exhibited no change from wild-type binding, although it was defective in spoIIA and spoIIE promoter activation. In contrast, both the E213G and E213A Spo0A variants showed decreased binding and completely abolished transcriptional activation of spoIIA and spoIIE, while the R214G and R214A variants completely abolished both DNA binding and transcriptional activation. These data suggest that these conserved residues are important for transcriptional activation and that the E213 residue is involved in DNA binding.

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Synthesis of an enzymatically active FLP recombinase in vitro: search for a DNA-binding domain

Molecular and Cellular Biology ◽

10.1128/mcb.9.5.1987-1995.1989 ◽

1989 ◽

Vol 9 (5) ◽

pp. 1987-1995

Author(s):

A A Amin ◽

P D Sadowski

Keyword(s):

Amino Acid ◽

Dna Binding ◽

In Vitro Transcription ◽

Binding Activity ◽

Binding Domain ◽

Translation System ◽

Dna Binding Domain ◽

Amino Terminal ◽

Dna Binding Activity

We have used an in vitro transcription and translation system to synthesize an enzymatically active FLP protein. The FLP mRNA synthesized in vitro by SP6 polymerase is translated efficiently in a rabbit reticulocyte lysate to produce enzymatically active FLP. Using this system, we assessed the effect of deletions and tetrapeptide insertions on the ability of the respective variant proteins synthesized in vitro to bind to the FLP recognition target site and to carry out excisive recombination. Deletions of as few as six amino acids from either the carboxy- or amino-terminal region of FLP resulted in loss of binding activity. Likewise, insertions at amino acid positions 79, 203, and 286 abolished DNA-binding activity. On the other hand, a protein with an insertion at amino acid 364 retained significant DNA-binding activity but had no detectable recombination activity. Also, an insertion at amino acid 115 had no measurable effect on DNA binding, but recombination was reduced by 95%. In addition, an insertion at amino acid 411 had no effect on DNA binding and recombination. On the basis of these results, we conclude that this approach fails to define a discrete DNA-binding domain. The possible reasons for this result are discussed.

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Synthesis of an enzymatically active FLP recombinase in vitro: search for a DNA-binding domain.

Molecular and Cellular Biology ◽

10.1128/mcb.9.5.1987 ◽

1989 ◽

Vol 9 (5) ◽

pp. 1987-1995 ◽

Cited By ~ 10

Author(s):

A A Amin ◽

P D Sadowski

Keyword(s):

Amino Acid ◽

Dna Binding ◽

In Vitro Transcription ◽

Binding Activity ◽

Binding Domain ◽

Translation System ◽

Dna Binding Domain ◽

Amino Terminal ◽

Dna Binding Activity

We have used an in vitro transcription and translation system to synthesize an enzymatically active FLP protein. The FLP mRNA synthesized in vitro by SP6 polymerase is translated efficiently in a rabbit reticulocyte lysate to produce enzymatically active FLP. Using this system, we assessed the effect of deletions and tetrapeptide insertions on the ability of the respective variant proteins synthesized in vitro to bind to the FLP recognition target site and to carry out excisive recombination. Deletions of as few as six amino acids from either the carboxy- or amino-terminal region of FLP resulted in loss of binding activity. Likewise, insertions at amino acid positions 79, 203, and 286 abolished DNA-binding activity. On the other hand, a protein with an insertion at amino acid 364 retained significant DNA-binding activity but had no detectable recombination activity. Also, an insertion at amino acid 115 had no measurable effect on DNA binding, but recombination was reduced by 95%. In addition, an insertion at amino acid 411 had no effect on DNA binding and recombination. On the basis of these results, we conclude that this approach fails to define a discrete DNA-binding domain. The possible reasons for this result are discussed.

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Genetic variation of the glucocorticoid receptor from a steroid-resistant primate

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0070089 ◽

1991 ◽

Vol 7 (2) ◽

pp. 89-96 ◽

Cited By ~ 31

Author(s):

D. D. Brandon ◽

A. J. Markwick ◽

M. Flores ◽

K. Dixon ◽

B. D. Albertson ◽

...

Keyword(s):

Amino Acid ◽

Dna Binding ◽

Amino Acid Sequence ◽

Transcriptional Activation ◽

Primary Structure ◽

New World ◽

Binding Domain ◽

Dna Binding Domain ◽

Coding Region ◽

Nucleotide Substitutions

ABSTRACT The neotropical cotton-top marmoset (Saguinus oedipus) is a New World primate known to have markedly increased total and free plasma cortisol concentrations when compared with Old World primates including man. The relative end-organ 'resistance' to glucocorticoids found in various New World primates has been attributed to a glucocorticoid receptor (GR) with diminished affinity for glucocorticoids. It has been demonstrated that the marmoset GR has approximately tenfold lower binding affinity for dexamethasone when compared with the human GR. We have examined the primary structure of the marmoset GR by molecular cloning and sequencing of GR functional domains. A library of cDNA clones was constructed in the phage vector λgt10 using poly(A)+ RNA from a marmoset-derived lymphoid cell line, and screened using the human GR cDNA. DNA sequencing determined 76 individual nucleotide substitutions in the coding region of the marmoset GR. Comparison of the marmoset GR nucleotide sequence with the human GR cDNA coding region indicated an overall sequence homology of about 97%. Thirty of the nucleotide substitutions lead to alterations in the predicted amino acid sequence (28 amino acid substitutions) of the marmoset GR. The size of the marmoset GR predicted from the 778 amino acids is approximately 90 000 which is in agreement with previous size estimates of the human and marmoset GRs. Alterations of amino acid sequence in the marmoset GR were greatest towards the amino terminus, including the τ1 domain putatively involved in transcriptional activation. The DNA-binding domain contained an additional codon (arginine). Comparison of the DNA-binding domain of the marmoset GR with other members of the steroid receptor superfamily indicates that the additional arginine occurs in the same position as other amino acid insertions within the interfinger region of the human androgen receptor and the erb-A proto-oncogene. There are only four missense substitutions within the steroid-binding domain. Two of these substitutions occur within the transducing site which has been associated with binding of the GR to a 90 kDa heat shock protein. These data suggest that diminished GR affinity for glucocorticoids in the marmoset may be due to alterations in the primary structure of one or more functional domains of the GR gene. In addition, other important regulatory functions, such as transcriptional activation, DNA binding and receptor transduction, may also be affected.

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The POU domain of SCIP/Tst-1/Oct-6 is sufficient for activation of an acetylcholine receptor promoter.

Molecular and Cellular Biology ◽

10.1128/mcb.16.9.5004 ◽

1996 ◽

Vol 16 (9) ◽

pp. 5004-5014 ◽

Cited By ~ 20

Author(s):

D Fyodorov ◽

E Deneris

Keyword(s):

Dna Binding ◽

Acetylcholine Receptor ◽

Transcriptional Activation ◽

Binding Domain ◽

Dependent Manner ◽

Dna Binding Domain ◽

Wild Type ◽

Amino Terminal ◽

Pou Domain ◽

Synergistic Activation

In the PC12 neuroendocrine line, the neuronal nicotinic acetylcholine receptor alpha3 gene promoter is activated by SCIP/Tst-1/Oct-6, a POU domain transcription factor proposed to be important for regulating the development of specific neural cell populations. In this study, we have investigated the SCIP polypeptide domains involved in alpha3 promoter activation. The characteristics of activation by a chimeric effector in which the GAL4 DNA binding domain was substituted for the SCIP POU domain were dramatically different from those of wild-type SCIP. At low effector masses, the chimeric polypeptide weakly activated alpha3 in a GAL4 binding-site-dependent manner but then squelched transcription at higher masses. In contrast, wild-type SCIP activation was not modulated by the presence of multimerized SCIP binding sites, and squelching was not observed. Analysis of wild-type SCIP truncations revealed that deletion of the previously characterized SCIP amino-terminal activation domain did not destroy activity of the factor. Surprisingly, a truncation expressing nothing more than the POU domain was nearly as active as wild-type SCIP. Moreover, cotransfection of a GAL4-VP16 effector with an effector expressing just the SCIP POU domain resulted in synergistic activation of the promoter. Synergistic activation did not depend on an Sp1 motif that is the only functional alpha3 cis element outside the transcription start site region. Our results show that the DNA binding domain of a POU factor is capable of transcriptional activation probably through protein-protein interactions with components of the basal transcription complex.

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