Two Amino Acid Residues from the DNA-binding Domain of MalT Play a Crucial Role in Transcriptional Activation

1996 ◽  
Vol 262 (1) ◽  
pp. 1-11 ◽  
Author(s):  
Olivier Danot ◽  
Dominique Vidal-Ingigliardi ◽  
Olivier Raibaud
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Crucial Role ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Amino Acid Residues

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

1991 ◽  
Vol 11 (11) ◽  
pp. 5735-5745 ◽  
Author(s):  
G F Yuan ◽  
Y H Fu ◽  
G A Marzluf
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Neurospora Crassa ◽  
Transcriptional Activation ◽  
Regulatory Gene ◽  
Binding Domain ◽  
Site Directed Mutagenesis ◽  
Dna Binding Domain ◽  
Amino Terminal ◽  
Rich 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.

scholarly journals Structural analysis of the DNA-binding domain of alternatively spliced steroid receptors

2002 ◽  
Vol 173 (3) ◽  
pp. 429-436 ◽  
Author(s):  
L Wickert ◽  
J Selbig
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Splice Variants ◽  
Steroid Receptor ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Amino Acid Residues ◽  
Receptor Function ◽  
Alternatively Spliced

We have generated 24 DNA-binding domain structure models of alternatively spliced or mutated steroid receptor variants by homology-based modeling. Members of the steroid receptor family dispose of a DNA-binding domain which is built from two zinc fingers with a preserved sequence homology of about 90%. Data from crystallographic analysis of the glucocorticoid receptor DNA-binding domain are therefore appropriate to serve as a template structure. We inserted or deleted amino acid residues in order to study the structural details of the glucocorticoid, mineralocorticoid, and androgen receptor splice variants. The receptor variants are created by QUANTA- and MODELLER-based modeling. Subsequently, the resulting energy-minimized models were compared with each other and with the wild-type receptor respectively. A prediction for the receptor function based mainly on the preservation or destruction of secondary structures has been carried out. The simulations showed that amino acid insertions of one, four or nine additional residues of existing steroid receptor splice variants were tolerated without destruction of the secondary structure. In contrast, a deletion of four amino acids at the splice site junction leads to modifications in the secondary structure of the DNA-recognition helix which apparently disturb the receptor-DNA interaction. Furthermore, an insertion of 23 amino acid residues between the zinc finger of the androgen receptor leads to a large loop with an additional alpha-helical structure which seems to disconnect a specific contact from its hormone response element. Thereafter, the prediction of receptor function based on the molecular models was compared with the available experimental results from the in vitro function tests. We obtained a close correspondence between the molecular modeling-based predictions and the conclusions of receptor function drawn from in vitro studies.

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

1991 ◽  
Vol 7 (2) ◽  
pp. 89-96 ◽  
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.

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

1991 ◽  
Vol 11 (11) ◽  
pp. 5735-5745
Author(s):  
G F Yuan ◽  
Y H Fu ◽  
G A Marzluf
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Neurospora Crassa ◽  
Transcriptional Activation ◽  
Regulatory Gene ◽  
Binding Domain ◽  
Site Directed Mutagenesis ◽  
Dna Binding Domain ◽  
Amino Terminal ◽  
Rich 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.

scholarly journals The C6 zinc finger and adjacent amino acids determine DNA-binding specificity and affinity in the yeast activator proteins LAC9 and PPR1.

1990 ◽  
Vol 10 (10) ◽  
pp. 5128-5137 ◽  
Author(s):  
M M Witte ◽  
R C Dickson
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Dna Binding ◽  
Zinc Finger ◽  
Binding Specificity ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Transcription Activator ◽  
Activator Proteins ◽  
Dna Binding Specificity

LAC9 is a DNA-binding protein that regulates transcription of the lactose-galactose regulon in Kluyveromyces lactis. The DNA-binding domain is composed of a zinc finger and nearby amino acids (M. M. Witte and R. C. Dickson, Mol. Cell. Biol. 8:3726-3733, 1988). The single zinc finger appears to be structurally related to the zinc finger of many other fungal transcription activator proteins that contain positively charged residues and six conserved cysteines with the general form Cys-Xaa2-Cys-Xaa6-Cys-Xaa6-9-Cys-Xaa2-Cys-Xaa 6-Cys, where Xaan indicates a stretch of the indicated number of any amino acids (R. M. Evans and S. M. Hollenberg, Cell 52:1-3, 1988). The function(s) of the zinc finger and other amino acids in DNA-binding remains unclear. To determine which portion of the LAC9 DNA-binding domain mediates sequence recognition, we replaced the C6 zinc finger, amino acids adjacent to the carboxyl side of the zinc finger, or both with the analogous region from the Saccharomyces cerevisiae PPR1 or LEU3 protein. A chimeric LAC9 protein, LAC9(PPR1 34-61), carrying only the PPR1 zinc finger, retained the DNA-binding specificity of LAC9. However, LAC9(PPR1 34-75), carrying the PPR1 zinc finger and 14 amino acids on the carboxyl side of the zinc finger, gained the DNA-binding specificity of PPR1, indicating that these 14 amino acids are necessary for specific DNA binding. Our data show that C6 fingers can substitute for each other and allow DNA binding, but binding affinity is reduced. Thus, in a qualitative sense C6 fingers perform a similar function(s). However, the high-affinity binding required by natural C6 finger proteins demands a unique C6 finger with a specific amino acid sequence. This requirement may reflect conformational constraints, including interactions between the C6 finger and the carboxyl-adjacent amino acids; alternatively or in addition, it may indicate that unique, nonconserved amino acid residues in zinc fingers make sequence-specifying or stabilizing contacts with DNA.

Reconstitution of transcriptional activation domains by reiteration of short peptide segments reveals the modular organization of a glutamine-rich activation domain

1994 ◽  
Vol 14 (9) ◽  
pp. 6056-6067
Author(s):  
M Tanaka ◽  
W Herr
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Activation Domain ◽  
Activation Domains ◽  
Transcriptional Activation Domain ◽  
Pou Domain

The POU domain activator Oct-2 contains an N-terminal glutamine-rich transcriptional activation domain. An 18-amino-acid segment (Q18III) from this region reconstituted a fully functional activation domain when tandemly reiterated and fused to either the Oct-2 or GAL4 DNA-binding domain. A minimal transcriptional activation domain likely requires three tandem Q18III segments, because one or two tandem Q18III segments displayed little activity, whereas three to five tandem segments were active and displayed increasing activity with increasing copy number. As with natural Oct-2 activation domains, in our assay a reiterated activation domain required a second homologous or heterologous activation domain to stimulate transcription effectively when fused to the Oct-2 POU domain. These results suggest that there are different levels of synergy within and among activation domains. Analysis of reiterated activation domains containing mutated Q18III segments revealed that leucines and glutamines, but not serines or threonines, are critical for activity in vivo. Curiously, several reiterated activation domains that were inactive in vivo were active in vitro, suggesting that there are significant functional differences in our in vivo and in vitro assays. Reiteration of a second 18-amino-acid segment from the Oct-2 glutamine-rich activation domain (Q18II) was also active, but its activity was DNA-binding domain specific, because it was active when fused to the GAL4 than to the Oct-2 DNA-binding domain. The ability of separate short peptide segments derived from a single transcriptional activation domain to activate transcription after tandem reiteration emphasizes the flexible and modular nature of a transcriptional activation domain.

Conserved DNA binding and self-association domains of the Drosophila zeste protein

1992 ◽  
Vol 12 (2) ◽  
pp. 598-608
Author(s):  
J D Chen ◽  
C S Chan ◽  
V Pirrotta
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Amino Acid Sequence ◽  
Coiled Coil ◽  
Helical Structure ◽  
Binding Activity ◽  
Binding Domain ◽  
Drosophila Virilis ◽  
Predicted Amino Acid Sequence ◽  
Dna Binding Domain

The zeste gene product is involved in two types of genetic effects dependent on chromosome pairing: transvection and the zeste-white interaction. Comparison of the predicted amino acid sequence with that of the Drosophila virilis gene shows that several blocks of amino acid sequence have been very highly conserved. One of these regions corresponds to the DNA binding domain. Site-directed mutations in this region indicate that a sequence resembling that of the homeodomain DNA recognition helix is essential for DNA binding activity. The integrity of an amphipathic helical region is also essential for binding activity and is likely to be responsible for dimerization of the DNA binding domain. Another very strongly conserved domain of zeste is the C-terminal region, predicted to form a long helical structure with two sets of heptad repeats that constitute two long hydrophobic ridges at opposite ends and on opposite faces of the helix. We show that this domain is responsible for the extensive aggregation properties of zeste that are required for its role in transvection phenomena. A model is proposed according to which the hydrophobic ridges induce the formation of open-ended coiled-coil structures holding together many hundreds of zeste molecules and possibly anchoring these complexes to other nuclear structures.

scholarly journals Role of GCR2 in transcriptional activation of yeast glycolytic genes.

1992 ◽  
Vol 12 (9) ◽  
pp. 3834-3842 ◽  
Author(s):  
H Uemura ◽  
Y Jigami
Keyword(s):  
Dna Binding ◽  
Fusion Protein ◽  
Transcriptional Activation ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Gene Products ◽  
Lacz Expression ◽  
Genetic Method ◽  
Potential Interactions

The Saccharomyces cerevisiae GCR2 gene affects expression of most of the glycolytic genes. We report the nucleotide sequence of GCR2, which can potentially encode a 58,061-Da protein. There is a small cluster of asparagines near the center and a C-terminal region that would be highly charged but overall neutral. Fairly homologous regions were found between Gcr2 and Gcr1 proteins. To test potential interactions, the genetic method of S. Fields and O. Song (Nature [London] 340:245-246, 1989), which uses protein fusions of candidate gene products with, respectively, the N-terminal DNA-binding domain of Gal4 and the C-terminal activation domain II, assessing restoration of Gal4 function, was used. In a delta gal4 delta gal80 strain, double transformation by plasmids containing, respectively, a Gal4 (transcription-activating region)/Gcr1 fusion and a Gal4 (DNA-binding domain)/Gcr2 fusion activated lacZ expression from an integrated GAL1/lacZ fusion, indicating reconstitution of functional Gal4 through the interaction of Gcr1 and Gcr2 proteins. The Gal4 (transcription-activating region)/Gcr1 fusion protein alone complemented the defects of both gcr1 and gcr2 strains. Furthermore, a Rap1/Gcr2 fusion protein partially complemented the defects of gcr1 strains. These results suggest that Gcr2 has transcriptional activation activity and that the GCR1 and GCR2 gene products function together.

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