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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 effects of the binding of the corepressor L-tryptophan and an operator oligonucleotide to Escherichia coli trp repressor have been studied, using selective 15N labelling to permit observation of the backbone amide resonances of 50 of the 107 residues of the protein monomer. Repressor molecules selectively labelled in turn with [15N]alanine, [15N]glutamate, [15N]isoleucine, [15N]leucine and [15N]methionine were prepared by isolating them from prototrophic E. coli cells grown in media containing a mixture of unlabelled and the appropriate 15N-enriched amino acids. Analysis of the heteronuclear correlation spectra of the labelled repressors shows the value of selective labelling in resolving the crosspeaks of, for example, the 19 leucine and 12 glutamate residues. All 50 residues studied show measurable changes in amide 1H and/or 15N chemical shift on the binding of tryptophan and/or the operator oligonucleotide, showing clearly that ligand binding has effects which are transmitted throughout almost the whole protein. Large chemical shift changes on ligand binding are seen in residues in the tryptophan binding site and in the 'helix-turn-helix' DNA-binding domain, but also in residues in helices C and F remote from the ligand binding sites. On operator binding there is selective broadening of the signals of residues in the N-terminal region of the protein and in the DNA-binding domain, perhaps reflecting a conformational equilibrium.
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PMID:The interactions of Escherichia coli trp repressor with tryptophan and with an operator oligonucleotide. NMR studies using selectively 15N-labelled protein. 795 74

The protein p10 of bacteriophage phi 29 assembled into connectors exhibit an intrinsic fluorescence with an emission peak centered at 335 nm, which suggests a hydrophobic environment of the three tryptohan residues that the protein contains. Upon incubation with linear DNA (but not with circular DNA), a decrease in the connector intrinsic fluorescence is measured which does not show any sequence specificity. The decrease in fluorescence is not observed when DNA is incubated with proteolyzed connectors, which lack the DNA-binding domain, suggesting that the fluorescence quenching is related to the binding of DNA to the phi 29 connectors. Acrylamide quenching studies reveal a higher accessibility of tryptophan residues to the quencher when the connector is bound to DNA. Protein denaturation by guanidine hydrochloride occurs at lower denaturant concentrations in the presence of linear DNA (but not circular DNA) than in its absence, suggesting a conformational change of phi 29 connector upon binding to linear DNA. This hypothesis is supported by the fact that the proteolyzed connectors, which do not bind DNA, are denatured at the same denaturant concentration, regardless of the presence of DNA. phi 29 connectors also bind RNA, but this interaction does not exert any effect on acrylamide quenching or guanidine hydrochloride denaturation. This result, together with that showing that proteolyzed connectors are able to interact with RNA, reinforces the idea that phi 29 connectors have two independent domains for interaction with DNA and RNA.
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PMID:An intrinsic-tryptophan-fluorescence study of phage phi 29 connector/nucleic acid interactions. 795 90

The N-terminal DNA-binding domain of c-myb oncoprotein binds to DNA in a sequence-specific manner. The domain, consisting of three imperfect tandem repeats, has tryptophan residues at very regular intervals and this is believed to be of some significance in the DNA-binding activity of the protein. We have found that the hydrophobic-site-specific probe 6-p-toluidino-2-naphthalenesulfonate (TNS) binds to the bacterially expressed DNA-binding domain of Drosophila c-myb protein (R123). TNS has a single binding site on this protein with an apparent dissociation constant in the range of (5-8) x 10(-7) M. When the TNS-protein complex was treated with an oligomeric DNA duplex having a cognate myb-binding site, the TNS was displaced from the complex. Nonspecific DNA duplex oligomers were ineffective, indicating that TNS displacement was a sequence-specific process. We examined further some features of the TNS-binding site on the protein, taking advantage of the fluorescence properties of the protein and the bound TNS. Our data indicate that the TNS binding occurs in a peripheral site on the protein in a manner that allows the bound TNS to be solvent accessible. Furthermore, there are indications that tyrosine(s) and tryptophans of the protein mediate resonance energy transfer to the bound TNS. From fluorescence-quenching data of the protein and protein-TNS complex, we could assess that both solvent-accessible and internal tryptophans are in the vicinity of the bound TNS. (ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Sequence-specific DNA displaces 6-p-toluidino-2-naphthalenesulfonate bound to a hydrophobic site on the DNA-binding domain of Drosophila c-myb. 800 78

The yeast BAS1 protein is a transcriptional activator with an amino-terminal domain homologous to the DNA-binding domain of the oncoprotein Myb containing three imperfect tryptophan-rich repeats. In contrast to Myb-related transcription factors from higher eukaryotes, where the second and third repeat constitutes a minimal independent DNA-binding domain, all three repeats of BAS1 were found to be necessary for sequence-specific DNA binding. Moreover, an active DNA-binding subdomain was obtained only if the first repeat was enlarged in the amino-terminal direction to include 3 tryptophans and a 23-amino acid insertion and if 55 amino acids carboxyl-terminal to the third repeat were included. The BAS1 DNA-binding site was analyzed in detail and found to cover 8-9 base pairs with no similarity to the Myb recognition element. The binding site included a conserved hexameric TGACTC motif, the methylation of which abolished BAS1 binding, as well as a 3-base pair extension that seemed to have a modulatory effect on BAS1 affinity and where binding was less affected by methylation.
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PMID:DNA-binding domain and recognition sequence of the yeast BAS1 protein, a divergent member of the Myb family of transcription factors. 802 Dec 77

The oncoprotein Myb is a sequence-specific DNA-binding protein with a pivotal function in the development and proliferation of hematopoietic precursor cells. A minimal DNA-binding domain composed of two tryptophan-rich repeats R2 and R3 is responsible for sequence recognition. Based on model building and mutational analysis, Myb was proposed to recognise its target through a double helix-turn-helix (HTH)-related motif using two recognition helices, one in R2 and one in R3. We found, by mutational analysis, that the DNA-binding site for c-Myb is functionally bipartite. While the first half site is dominant and absolutely required for binding, the second half site is only modulatory and mainly affects the half life of the complex. This bipartite nature of the binding site parallels the proposed bipartite structure of R2R3 with two HTH-related domains. Analysis of the DNA-binding site of R2R3 by missing-base interference-footprint analysis showed that the protein interacted with a 9-bp region. The same was found with a larger protein containing all three repeats. The effect of adding R1 was mainly to stabilise the complex. The borders of the complex, as revealed by exonuclease III footprinting, did not change due to the presence of R1. However, both borders became more refractory to the nuclease when R1 was present, but with a difference that suggested a specific orientation of the repeat domains relative to the DNA-binding site. We propose that the first half site is recognised by R3, while the second modulatory half site interacts with the R2 repeat.
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PMID:Two functionally distinct half sites in the DNA-recognition sequence of the Myb oncoprotein. 820 Mar 35

Trp repressor (25 kDa) is a regulatory protein that controls transcription initiation in the tryptophan biosynthetic operon and at least four other operons in Escherichia coli. An alanine to valine mutation (AV77) in the DNA binding domain is known to increase repressor activity at the trp operator in vivo, but not in vitro. We report here the amide proton exchange rates for the DNA-binding domains of both the wild-type and AV77 proteins. We find that the alanine to valine change stabilizes the flexible DNA-binding domain of the repressor. We present in vivo data showing that, although the AV77 repressor is more inhibitory at the trp operator than the wild-type repressor, it does not have increased activity at the aroH or trpR operator; repression at the aroH operator is, in fact, reduced. Our results suggest that the flexibility exhibited by the wild-type repressor allows a broader range of repressor/DNA interactions, whereas the increased rigidity resulting from the AV77 change limits the repressor's effectiveness at some operators.
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PMID:Flexibility of DNA binding domain of trp repressor required for recognition of different operator sequences. 876 53

The Runt domain is the DNA-binding domain defining a small family of transcription factors that are involved in important developmental processes. Developmental pathways controlled by Runt domain proteins include sex determination, neurogenesis, segmentation, and eye development in Drosophila and hematopoiesis in mammals. In addition to binding DNA, the Runt domain also mediates heterodimerization with another subunit called the core-binding factor beta (CBFbeta) subunit. In this study we overexpress the Runt domain from the mouse CBFalpha2 (AML1) protein in Escherichia coli, and purify it from the insoluble fraction. We determine the equilibrium constants for Runt domain binding to two different DNA sequences by surface plasmon resonance technology. Circular dichroism spectroscopy demonstrates that the Runt domain is a folded beta-domain with essentially no alpha-helical content. The single tryptophan residue in the CBFalpha2 Runt domain at amino acid 79 is shown by tryptophan fluorescence spectroscopy to reside in a polar environment. Finally, we demonstrate that ATP can be UV cross-linked to the Runt domain and that ATP binding is sensitive to an amino acid substitution in the putative Kinase-1a motif (P-loop).
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PMID:Biochemical and biophysical properties of the core-binding factor alpha2 (AML1) DNA-binding domain. 882 75

The DNA-binding domain of the oncoprotein c-Myb consists of three imperfect tryptophan-rich repeats, R1, R2 and R3. Each repeat forms an independent mini-domain with a helix-turn-helix related motif and they are connected by linkers containing highly conserved residues. The location of the linker between two DNA-binding units suggests a function analogous to a dimerisation motif with a critical role in positioning the recognition helices of each mini-domain. Mutational analysis of the minimal DNA-binding domain of chicken c-Myb (R2 and R3), revealed that besides the recognition helices of each repeat, the linker connecting them was of critical importance in maintaining specific DNA-binding. A comparison of several linker sequences from different Myb proteins revealed a highly conserved motif of four amino acids in the first half of the linker: LNPE (L138 to E141 in chicken c-Myb R2R3). Substitution of residues within this sequence led to reduced stability of protein-DNA complexes and even loss of DNA-binding. The two most affected mutants showed increased accessibility to proteases, and fluorescence emission spectra and quenching experiments revealed greater average exposure of tryptophans which suggests changes in conformation of the proteins. From the structure of R2R3 we propose that the LNPE motif provides two functions: anchorage to the first repeat (through L) and determination of the direction of the bridge to the next repeat (through P).
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PMID:The importance of the linker connecting the repeats of the c-Myb oncoprotein may be due to a positioning function. 891 2

Hepatitis C virus (HCV) core protein forms the internal viral coat that encapsidates the genomic RNA and is enveloped in a host cell-derived lipid membrane. As the single capsid protein, core should be capable of multimerization but attempts to produce virus-like particles following expression of HCV structural proteins have not been successful. In this study, we have analysed the interaction capacity of full-length and truncated HCV core using the yeast two-hybrid system. Full-length core containing or lacking the translocation signal for the E1 glycoprotein did not interact with full-length or truncated core proteins. Truncation to the N-terminal 122 aa revealed an interaction domain which was mapped to the tryptophan-rich sequence from aa 82-102 and was termed the main homotypic interaction domain. The C-terminal hydrophobic fragment of core (aa 122-172) was incapable of interacting with itself but interacted with the main homotypic interaction domain in trans (the weak heterotypic interaction domain). Core proteins truncated at their N and C termini (aa 46-102) were trans-activating when fused to the DNA-binding domain of GAL4. Based on our results, we suggest that the C-terminal segment may interact in cis with the main homotypic interaction domain and thereby prevent multimerization. Core-core interaction was also observed for in vitro-translated proteins bound to truncated immobilized core 102. However, interaction was less specific in this system suggesting that protein interaction and possibly conformational alteration of core may be dependent on the experimental system.
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PMID:Analysis of hepatitis C virus core protein interaction domains. 919 26

The crystal structure of a DNA-binding domain of PHO4 complexed with DNA at 2.8 A resolution revealed that the domain folds into a basic-helix-loop-helix (bHLH) motif with a long but compact loop that contains a short alpha-helical segment. This helical structure positions a tryptophan residue into an aromatic cluster so as to make the loop compact. PHO4 binds to DNA as a homodimer with direct reading of both the core E-box sequence CACGTG and its 3'-flanking bases. The 3'-flanking bases GG are recognized by Arg2 and His5. The residues involved in the E-box recognition are His5, Glu9 and Arg13, as already reported for bHLH/Zip proteins MAX and USF, and are different from those recognized by bHLH proteins MyoD and E47, although PHO4 is a bHLH protein.
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PMID:Crystal structure of PHO4 bHLH domain-DNA complex: flanking base recognition. 930 13


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