Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:Q02556 (DNA-binding domain)
 6,431 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The bovine papillomavirus E2 protein regulates viral transcription by binding as a dimer to the DNA sequence ACCGN4CGGT. The dimerization and DNA-binding properties are localized within its carboxy-terminal 85 amino acids (325-410). Utilizing random mutagenesis coupled with phenotypic selection in yeast, functionally important amino acids in the DNA-binding domain were identified. Four trans-activation defective point mutants within a short segment (amino acids 337-344) were DNA binding defective but dimeric. The mutation of a conserved tryptophan to serine also eliminated DNA binding, but loss of dimerization was implicated because addition of dimeric monoclonal antibody complemented this defect. A simple assay for E2 dimerization was developed using UV irradiation to produce an interchain cross-link within a dimer. No heterodimeric complexes were formed when pools of E2 of varying lengths were mixed, and only proteins with tryptophan at position 360 could be UV cross-linked. Peptide mapping of irradiated E2 protein localized the cross-link to an 18-amino-acid region bracketing this tryptophan. Substitutions for this tryptophan demonstrated the requirement for a hydrophobic residue at this position, but surprisingly, even alanine was functional. Replacement of this tryptophan with three polar amino acids or glycine eliminated DNA-binding activity, but addition of dimeric monoclonal antibody restored this function. The amino acids that were identified as being involved in DNA contact and dimerization imply that these functions are mediated by novel binding motifs.
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PMID:Amino acids necessary for DNA contact and dimerization imply novel motifs in the papillomavirus E2 trans-activator. 130 14

The cAMP receptor protein (CRP) of Escherichia coli is a dimer of a two-domain subunit. It requires binding of cAMP for a conformational change in order to function as a site-specific DNA-binding protein that regulates gene activity. The hinge region connecting the cAMP-binding domain to the DNA-binding domain is involved in the cAMP-induced allosteric change. We studied the structural changes in CRP that are required for gene regulation by making a large number of single and double amino acid substitutions at four different positions in or near the hinge. To achieve cAMP-independent transcription by CRP, amino acid residues 138 (located within the hinge region) and 141 (located in the D alpha-helix adjacent to the hinge) must be polar. This need for polar residues at positions 138 and 141 suggests an interaction that causes the C and D alpha-helices to come together. As a consequence, the F alpha-helix is released from the D alpha-helix and can interact with DNA. At position 144 in the D alpha-helix and within interacting distances of the F alpha-helix, replacement of alanine by an amino acid with a larger side chain, regardless of its nature, allows cAMP independence. This result indicates that pushing against the F alpha-helix may be a way of making the helix available for DNA binding. We believe that the cAMP-induced allosteric change involves similar hinge reorientation to adjust the C and D alpha-helices, allowing outward movement of the F alpha-helix.
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PMID:Allosteric changes in the cAMP receptor protein of Escherichia coli: hinge reorientation. 140 86

The complete nucleotide sequences of the lexA genes from Salmonella typhimurium, Erwinia carotovora, Pseudomonas aeruginosa and Pseudomonas putida were determined; the DNA sequences of the lexA genes from these bacteria were 86%, 76%, 61% and 59% similar, respectively, to the Escherichia coli K12 gene. The predicted amino acid sequences of the S. typhimurium, E. carotovora and P. putida LexA proteins are 202 residues long whereas that of P. aeruginosa is 204. Two putative LexA repressor binding sites were localized upstream of each of the heterologous genes, the distance between them being 5 bp in S. typhimurium and E. carotovora, as in the lexA gene of E. coli, and 3 bp in P. putida and P. aeruginosa. The first lexA site present in the lexA operator of all five bacteria is very well conserved. However, the second lexA box is considerably more variable. The Ala-84--Gly-85 bond, at which the LexA repressor of E. coli is cleaved during the induction of the SOS response, is also found in the LexA proteins of S. typhimurium and E. carotovora. Likewise, the amino acids Ser-119 and Lys-156 are present in all of these three LexA repressors. These residues also exist in the LexA proteins of P. putida and P. aeruginosa, but they are displaced by 4 and 6 residues, respectively. Furthermore, the structure and sequence of the DNA-binding domain of the LexA repressor of E. coli are highly conserved in the S. typhimurium, E. carotovora, P. aeruginosa and P. putida LexA proteins.
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PMID:Nucleotide sequence analysis and comparison of the lexA genes from Salmonella typhimurium, Erwinia carotovora, Pseudomonas aeruginosa and Pseudomonas putida. 149 43

The nucleotide sequence of nirA, mediating nitrate induction in Aspergillus nidulans, has been determined. Alignment of the cDNA and the genomic DNA sequence indicates that the gene contains four introns and encodes a protein of 892 amino acids. The deduced NIRA protein displays all characteristics of a transcriptional activator. A putative double-stranded DNA-binding domain in the amino-terminal part comprises six cysteine residues, characteristic for the GAL4 family of zinc finger proteins. An amino-terminal highly acidic region and two proline-rich regions are also present. The nucleotide sequences of two mutations were determined after they were mapped by transformation with overlapping DNA fragments, amplified by the polymerase chain reaction. nirA87, a mutation conferring noninducibility by nitrate and nitrite, has a -1 frameshift at triplet 340, which eliminates 549 C-terminal amino acids from the polypeptide. Under the assumption that the truncated polypeptide is stable, it comprises the zinc finger domain and the acidic region, which seem not sufficient for transcriptional activation. nirAd-106, an allele conferring nitrogen metabolite derepression of nitrate and nitrite reductase activity, includes two transitions, changing a glutamic acid to a lysine and a valine to an alanine, situated between a basic and a proline-rich region of the protein. Northern (RNA) analysis of the wild type and of constitutive (nirAc) and derepressed (nirAd) mutants show that the nirA transcript does not vary between these strains, being in all cases constitutively expressed. On the other hand, transcript levels of structural genes (niaD and niiA) do vary, being highly inducible in the wild type but constitutively expressed in the nirAc mutant. The nirAd mutant appears phenotypically derepressed, because the niaD and niiA transcript levels are overinduced in the presence of nitrate but are still partially repressed in the presence of ammonium.
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PMID:nirA, the pathway-specific regulatory gene of nitrate assimilation in Aspergillus nidulans, encodes a putative GAL4-type zinc finger protein and contains four introns in highly conserved regions. 192 75

Efficient transcription of many Saccharomyces cerevisiae genes requires the GAL11 Protein. GAL11 belongs to a class of transcription activator that lacks a DNA-binding domain. Such proteins are thought to activate specific genes by complexing with DNA-bound proteins. To begin to understand the domain structure-function relationships of GAL11 we cloned and sequenced a homologue from the yeast Kluyveromyces lactis, Kl-GAL11. The two predicted GAL11 proteins show high overall amino acid conservation and an unusual amino acid composition including 18% glutamine, 10% asparagine (S. cerevisiae) or 7% (K. lactis), and 8% proline (K. lactis) or 5% (S. cerevisiae) residues. Both proteins have runs of pure glutamines. Sc-GAL11 has glutamine-alanine runs but in Kl-GAL11 the alanines in such runs are replaced by proline and other residues. The primary sequence similarity is reflected in functional similarity since a gal11 mutation in K. lactis creates phenotypes similar to those seen previously in gal11-defective S. cerevisiae. In addition, Kl-GAL11 complements a gal11-defect in S. cerevisiae by partially restoring induction of GAL1 expression, growth on nonfermentable carbon sources, and phosphorylation of GAL4.
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PMID:Sequence conservation in the Saccharomyces and Kluveromyces GAL11 transcription activators suggests functional domains. 192 18

We have previously identified three regions (called elements) in the DNA-binding domain of simian virus 40 large tumor (T) antigen which are critical for binding of the protein to the recognition pentanucleotides GAGGC at the viral replication origin. These are elements A (residues 147 to 159), B1 (185 to 187), and B2 (203 to 207). In this study, we generated mutants of simian virus 40 in order to make single-point substitution mutations at nearly every site in these three elements. Each mutation was tested for its effect on virus replication, and T antigen was produced from all replication-negative mutants. The mutant proteins were assayed for binding to several different DNA substrates and for helicase activity. We found that within each element, mutations at some sites had major effects on DNA binding while mutations at other sites had moderate, mild, or minimal effects, suggesting that some residues are more important than others in mediating DNA binding. Furthermore, we provide evidence that certain residues in elements A and B2 (Ala-149, Phe-159, and His-203) participate in nonspecific double-stranded and helicase substrate (single-stranded) DNA binding while others (Ser-147, Ser-152, Asn-153, Thr-155, Arg-204, Val-205, and Ala-207) are involved in sequence-specific binding at the origin. The residues in element B1 (primarily Ser-185 and His-187) take part only in nonspecific DNA binding. The amino acids important for nonspecific DNA binding are also required for helicase activity, and we hypothesize that they make contact with the sugar-phosphate backbone of DNA. On the other hand, those involved in sequence-specific binding are not needed for helicase activity. Finally, our analysis showed that three residues (Asn-153 and Thr-155 in element A and Arg-204 in element B2) may be the most important for sequence-specific binding. They are likely to make direct or indirect contacts with the pentanucleotide sequences at the origin.
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PMID:Identification of simian virus 40 T-antigen residues important for specific and nonspecific binding to DNA and for helicase activity. 216 72

The c-myb protooncogene codes for a sequence-specific DNA-binding protein that appears to act as a transcriptional regulator and is highly conserved through evolution. The DNA-binding domain of Myb has been shown to contain three imperfectly conserved repeats of 52 amino acids that constitute the amino-terminal end. Within each repeat, there are three tryptophans that are separated by 18 or 19 amino acids and are flanked by basic amino acids. To determine the role of tryptophans and the flanking basic amino acids in the DNA-binding activity of Myb proteins, we have selectively mutagenized individual tryptophans as well as some of the amino acid residues that flank these tryptophans. Replacement of these tryptophans with glycine, proline, or arginine abolished the DNA-binding activity whereas replacement with other aromatic amino acids or leucine or alanine did not appreciably affect this activity. On the other hand the replacement of two amino acids, asparagine and lysine, that flank the last tryptophan with acidic amino acids completely abolished their DNA-binding activity. These results are consistent with a model we present in which the tryptophans form a hydrophobic scaffold that plays a crucial role in maintaining the helix-turn-helix structure of the DNA binding domain. Basic and polar amino acids adjacent to these tryptophans seem to participate directly in DNA binding.
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PMID:Role of tryptophan repeats and flanking amino acids in Myb-DNA interactions. 223 54

In the DNA-binding domain of the c-myb protooncogene product (c-Myb) which consists of three repeats of 51-52 amino acids, there are 3 perfectly conserved tryptophans in each repeat. Site-directed mutagenesis of these tryptophans showed that any single or multiple mutations of tryptophan to hydrophilic residues or alanine abolished or greatly reduced the sequence-specific DNA-binding activity, but mutations to hydrophobic amino acids retained considerable activity. Raman spectroscopic study showed that these tryptophans were buried in the protein core. These 3 tryptophans are proposed to form a cluster in the hydrophobic core in each repeat. This hypothetical structure is referred to as the "tryptophan cluster," and it may represent a characteristic property of a group of DNA-binding proteins including the myb- and ets-related proteins.
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PMID:The tryptophan cluster: a hypothetical structure of the DNA-binding domain of the myb protooncogene product. 224 75

A threonine residue at the beginning of each DNA-binding domain of HMG-I (residue numbers 21, 53, and 78) is conserved among mammalian species and proposed to help stabilize the A.T-hook DNA-binding motif. Phosphorylation of threonines number 53 and 78 of human HMG-I(Y) both in vivo and in vitro leads to a 20 fold reduction in the proteins DNA binding affinity. Recombinant human HMG-I proteins were engineered to contain alanine instead of the conserved threonine in each DNA-binding domain. The DNA dissociation constant of each protein was assayed at various salt concentrations by competition with the fluorescent dye Hoechst 33258 for an AT-rich DNA substrate. Replacement of these threonines did not affect the equilibrium binding of these proteins to DNA as compared with wild-type HMG-I and HMG-Y. Molecular modelling of analogous peptides supported this finding. We conclude that these threonines are not directly important for A.T-hook DNA-binding and are conserved phosphorylation sites for down regulation of DNA binding by the A.T-hook motif in the HMG-I(Y) proteins.
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PMID:Replacement of conserved threonines by alanine residues in high mobility group protein HMG-I(Y): effect on DNA binding affinity. 753 3

Full-length wild type and deletion mutant human androgen receptors (AR) were transiently expressed in monkey kidney COS cells to identify the phosphorylated amino acid residues. Phosphoamino acid analysis indicated serine (Ser) and threonine (Thr) residues as the major sites of phosphorylation. Both NH2- and carboxyl-terminal fragments containing the DNA-binding domain were highly phosphorylated, suggesting the presence of phosphorylation sites throughout the protein. Site-directed mutagenesis of wild type and deletion mutant AR at proline-directed consensus phosphorylation sites replaced Ser or Thr residues with Ala; wild type and mutant ARs were expressed in the presence of [32P]orthophosphate and isolated by immunoprecipitation using AR-specific antipeptide antibodies. Three proline-directed phosphorylation sites were identified: Ser 81 and 94 in the NH2-terminal region and Ser 650 in the hinge region. Expression of a series of NH2-terminal AR fragments provided evidence for additional sites in the NH2-terminal region. The effect of loss of each phosphorylation site on receptor function was determined by introducing the Ser to Ala mutations into full-length AR. Substituting Ser 81 and 94 with Ala had little effect on transcriptional activity when assayed by transient cotransfection. Substituting Ser 650 with Ala in the hinge region reduced transcriptional activity up to 30%. The results suggest at least three proline-directed phosphorylation sites in AR, one of which, serine 650, contributes to optimal gene activation by AR.
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PMID:Identification of three proline-directed phosphorylation sites in the human androgen receptor. 756 7


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