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At least 217 records · Page 12Linked to original sources
PubMed1989

Characterization of a new prokaryotic transcriptional activator and its DNA recognition site.

The expression of the Bacillus subtilis phage phi 29 DNA is controlled by the viral gene 4 product, which is required for the initiation of transcription at the unique late promoter A3. Protein p4 binds specifically to a phi 29 DNA fragment containing the A3 promoter. DNase I footprinting analysis has shown that the DNA binding region for protein p4 is located between nucleotides -50 and -100 relative to the transcription start site. Methylation interference assays suggest that two eight base-pair long inverted repeats located within this binding region are the protein p4 recognition sequence. These results, together with the fact that the protein p4-dependent in vitro transcription requires the B. subtilis sigma 43-RNA polymerase, indicate that protein p4 is a transcriptional activator. The protein p4 DNA recognition region is statically bent as suggested by gel retardation and chemical cleavage assays. A model of protein p4 binding to its DNA target site is proposed.

Bacillus subtilis↗
PubMed2006

Principles of protein-DNA recognition revealed in the structural analysis of Ndt80-MSE DNA complexes.

The Saccharomyces cerevisiae transcription factor Ndt80 selectively binds a DNA consensus sequence (the middle sporulation element [MSE]) to activate gene expression after the successful completion of meiotic recombination. Here we report the X-ray crystal structures of Ndt80 bound to ten distinct MSE variants. Comparison of these structures with the structure of Ndt80 bound to a consensus MSE reveals structural principles that determine the DNA binding specificity of this transcription factor. The 5' GC-rich end of the MSE contains distinct 5'-YpG-3' steps that are recognized by arginine side chains through a combination of hydrogen bonding and cation-pi interactions. The 3' AT-rich region is recognized via minor groove contacts that sterically exclude the N2 atom of GC base pairs. The conformation of the AT-rich region is fixed by interactions with the protein that favor recognition of poly(A)-poly(T) versus mixed AT sequences through an avoidance of major groove steric clashes at 5'-ApT-3' steps.

Base Composition↗
PubMed1994

Specific DNA recognition and intersite spacing are critical for action of the bicoid morphogen.

We examined DNA site recognition by Bicoid and its importance for pattern formation in developing Drosophila embryos. Using altered DNA specificity Bicoid mutants and appropriate reporter genes, we show that Bicoid distinguishes among related DNA-binding sites in vivo by a specific contact between amino acid 9 of its recognition alpha-helix (lysine 50 of the homeodomain) and bp 7 of the site. This result is consistent with our earlier results using Saccharomyces cerevisiae but differs from that predicted by crystallographic analysis of another homeodomain-DNA interaction. Our results also demonstrate that Bicoid binds directly to those genes whose transcription it regulates and that the amino acid 9 contact is necessary for Bicoid to direct anterior pattern formation. In both Drosophila embryos and yeast cells, Bicoid requires multiple binding sites to activate transcription of target genes. We find that the distance between binding sites is critical for Bicoid activation but that, unexpectedly, this critical distance differs between Drosophila and S. cerevisiae. This result suggests that Bicoid activation in Drosophila might require an ancillary protein(s) not present in S. cerevisiae.

Animals↗
PubMed2001

The structural basis of damaged DNA recognition and endonucleolytic cleavage for very short patch repair endonuclease.

Endonucleases in DNA repair must be able to recognize damaged DNA as well as cleave the phosphodiester backbone. These functional prerequisites are manifested in very short patch repair (Vsr) endonuclease through a common endonuclease topology that has been tailored for recognition of TG mismatches. Structural and biochemical comparison with type II restriction enzymes illustrates how Vsr resembles these endonucleases in overall topology but also how Vsr diverges in terms of the detailed catalytic mechanism. A histidine and two metal-water clusters catalyze the phosphodiester cleavage. The mode of DNA damage recognition is also unique to Vsr. All other structurally characterized DNA damage-binding enzymes employ a nucleotide flipping mechanism for substrate recognition and for catalysis. Vsr, on the other hand, recognizes the TG mismatch as a wobble base pair and penetrates the DNA with three aromatic residues on one side of the mismatch. Thus, Vsr endonuclease provides important counterpoints in our understanding of endonucleolytic mechanisms and of damaged DNA recognition.

Catalysis↗
PubMed2003

DNA recognition by the androgen receptor: evidence for an alternative DNA-dependent dimerization, and an active role of sequences flanking the response element on transactivation.

The androgen receptor has a subset of target DNA sequences, which are not recognized by any other steroid receptors. The androgen selectivity of these sequences was proposed to be the consequence of the ability of the androgen receptor to dimerize on direct repeats of 5'-TGTTCT-3'-like sequences. This is in contrast with the classical non-selective elements consisting of inverted repeats of the 5'-TGTTCT-3' elements separated by three nucleotides and which are recognized by other steroid receptors in addition to the androgen receptor. We demonstrate that while the DNA-binding domain of the oestrogen receptor is unable to dimerize on direct repeats, dimeric binding can be rescued by replacing the second Zn finger and part of the hinge region by the corresponding fragment of the androgen receptor, but not the glucocorticoid receptor. In this study, we investigate the androgen receptor binding to all natural androgen-selective response elements described so far. We show that a 12-amino acid C-terminal extension of the DNA-binding domain is required for high-affinity binding of the androgen receptor to all these elements. For one androgen-specific low-affinity binding site, the flanking sequences do not contribute to the in vitro affinity of the androgen receptor DNA-binding domain. Surprisingly, however, they control the transcriptional activity of the androgen receptor in transient transfection experiments. In conclusion, we give evidence that the alternative DNA-dependent dimerization of the androgen receptor on direct repeats is a general mechanism for androgen specificity in which the second Zn finger and hinge region are involved. In addition, the sequences flanking an androgen-response element can control the activity of the androgen receptor.

Animals↗
PubMed2002

Zinc ions inhibit the protein-DNA complex formation between cyanobacterial transcription factor SmtB and its recognition DNA sequences.

SmtB is a trans-acting dimeric repressor of the metal-regulated smtA gene, and the release of SmtB from the smtA operator/promoter region is essential for the tolerance to Zn(2+) stress by SmtA expression. Gel retardation assaying demonstrated that different sizes of SmtB-DNA complexes were formed depending on the DNA sequences, and the amounts of these complexes decreased in the presence of Zn(2+). Here, we present the first direct evidence that Zn(2+ )(>4 micro M) inhibits the SmtB-DNA complex formation in vitro, which ensures the physiological functions of SmtB as a Zn(2+) sensor and a transcription factor.

Bacterial Proteins↗
PubMed1993

Functional analysis of the amino acids in the DNA recognition alpha-helix of the human thyroid hormone receptor.

The roles in DNA binding and transcriptional activation of individual amino acids in the putative recognition alpha-helix of the first zinc finger of the beta-isoform of the human thyroid hormone receptor (hT3R beta) have been probed by site-directed mutagenesis. Alanine substitutions of highly conserved residues involved in the folding of this zinc finger abolished the binding of hT3R beta to various thyroid response elements. A similar effect was observed for alanine substitutions of those conserved residues in hT3R beta that were expected to make specific contacts to DNA bases common to all hormone response elements. The three P-box amino acids have previously been shown to be essential for discrimination of the base pairs that differ between the DNA binding sites for related steroid/thyroid hormone receptors. In hT3R beta, the P-box residues are E, G, and G; the results of this study show that alanine substitution of the glutamic acid dramatically reduces DNA binding activity by hT3R beta, while the substitution of either glycine has little or no effect on DNA binding. The effects of alanine substitutions on hT3R beta transcriptional activation properties were consistent with the effect of these substitutions on DNA binding properties, with the exception of the second P-box amino acid. T3R beta substituted with alanine at this position is substantially more defective in transcriptional activation than it is in specific DNA binding. These results indicate that there are two separate mechanisms of response element discrimination by P-box amino acids of steroid/thyroid hormone receptors, one which operates at the level of DNA recognition and a second which operates at the level of transcriptional activation.

Amino Acid Sequence↗
PubMed1995

Specific DNA recognition by EcoRV restriction endonuclease induced by calcium ions.

In the presence of Mg2+, the EcoRV restriction endonuclease cleaves DNA specifically at its recognition sequence, but in the absence of divalent metal ions, it binds DNA without any specificity: gel-shift experiments had revealed multiple EcoRV-DNA complexes, due to the binding of one, two, three, or more molecules of protein per molecule of DNA, with the same equilibrium constant for each association. In this study, the binding of EcoRV to DNA was measured by gel shift in the presence of Ca2+, an ion that perturbs the Mg(2+)-dependent activity of EcoRV but that fails to support DNA cleavage. With Ca2+, and at a lower concentration of EcoRV protein than that required for binding in the absence of divalent metal ions, a single complex was observed with DNA containing the EcoRV recognition site. This complex was not formed with DNA that had been methylated at the EcoRV site nor with an isogenic DNA lacking the EcoRV recognition site. The single complex thus is due to the specific binding of EcoRV to its recognition site on the DNA. From gel shifts with a permuted set of DNA fragments, the degree of DNA bending by EcoRV at its recognition site was estimated to be 53 degrees +/- 4 degrees. This angle is similar to that seen in the crystal structure of the cognate DNA-protein complex. Calcium ions thus appear to mimic the role of Mg2+ in generating a specific protein-metal-DNA complex, but in contrast to Mg2+, Ca2+ gives a stable ternary complex in which the DNA-bound nuclease cannot cleave the DNA.

Calcium↗
PubMed1989

Scissors-grip model for DNA recognition by a family of leucine zipper proteins.

C/EBP is a sequence-specific DNA binding protein that regulates gene expression in certain mammalian cells. The region of the C/EBP polypeptide required for specific recognition of DNA is related in amino acid sequence to other regulatory proteins, including the Fos and Jun transforming proteins. It has been proposed that these proteins bind DNA via a bipartite structural motif, consisting of a dimerization interface termed the "leucine zipper" and a DNA contact surface termed the "basic region." An evaluation of the properties of conserved amino acids within the basic region of 11 deduced protein sequences, coupled with the observation that they are located at an invariant distance from the leucine zipper, has led to the formulation of a "scissors-grip" model for DNA binding. The architectural features of this model are well suited for interaction with directly abutted, dyadsymmetric DNA sequences. Data supportive of the model were obtained with chemical probes of protein: DNA complexes.

Amino Acid Sequence↗
PubMed1996

Distinct phosphate backbone contacts revealed by some mutant peptides of zinc finger protein Spl: effect of protein-induced bending on DNA recognition.

By using some mutant peptides of transcription factor Spl, phosphate backbone contacts with the DNA binding protein containing three zinc fingers have been investigated by alkylation interference, circular permutation, DNase I footprinting, and methylation protection methods. The ethylation interference analyses of Spl(R565S) and Spl(K595S) mutants demonstrate that arginine at 565 position and lysine at 595 position interact with the phosphate between G(3) and G(4) and with the phosphate between G(9) and G(10) in GC-box DNA, respectively. On the basis of the experimental results for Spl(K535G), Sp1(537-623), and Sp1(530-623), lysine and glutamine at 535 and 536 positions have been clarified to be in contacts with phosphate between G(7) and G(8) and with phosphate outside GC-box, respectively. In particular, glutamine at the N-terminal side of zinc finger 1 is a key amino acid residue to induce DNA bending and also participates in total base specificity of Sp1. The present study strongly indicates that (1) each zinc finger is not independent for the DNA interaction with Sp1 and (2) DNA base recognition of the zinc finger protein is influenced by local conformational change of DNA induced by the protein binding.

Amino Acid Sequence↗
PubMed1995

Atomic model of a pyrimidine dimer excision repair enzyme complexed with a DNA substrate: structural basis for damaged DNA recognition.

T4 endonuclease V is a DNA repair enzyme from bacteriophage T4 that catalyzes the first reaction step of the pyrimidine dimer-specific base excision repair pathway. The crystal structure of this enzyme complexed with a duplex DNA substrate, containing a thymine dimer, has been determined at 2.75 A resolution. The atomic structure of the complex reveals the unique conformation of the DNA duplex, which exhibits a sharp kink with a 60 degree inclination at the central thymine dimer. The adenine base complementary to the 5' side of the thymine dimer is completely flipped out of the DNA duplex and trapped in a cavity on the protein surface. These structural features allow an understanding of the catalytic mechanism and implicate a general mechanism of how other repair enzymes recognize damaged DNA duplexes.

Adenine↗
PubMed2002

Probabilistic code for DNA recognition by proteins of the EGR family.

A recognition code for protein-DNA interactions would allow for the prediction of binding sites based on protein sequence, and the identification of binding proteins for specific DNA targets. Crystallographic studies of protein-DNA complexes showed that a simple, deterministic recognition code does not exist. Here, we present a probabilistic recognition code (P-code) that assigns energies to all possible base-pair-amino acid interactions for the early growth response factor (EGR) family of zinc-finger transcription factors. The specific energy values are determined by a maximum likelihood method using examples from in vitro randomisation experiments (namely, SELEX and phage display) reported in the literature. The accuracy of the model is tested in several ways, including the ability to predict in vivo binding sites of EGR proteins and other non-EGR zinc-finger proteins, and the correlation between predicted and measured binding affinities of various EGR proteins to several different DNA sites. We also show that this model improves significantly upon the prediction capabilities of previous qualitative and quantitative models. The probabilistic code we develop uses information about the interacting positions between the protein and DNA, but we show that such information is not necessary, although it reduces the number of parameters to be determined. We also employ the assumption that the total binding energy is the sum of the energies of the individual contacts, but we describe how that assumption can be relaxed at the cost of additional parameters.

Algorithms↗
PubMed2002

Is there a code for protein-DNA recognition? Probab(ilistical)ly. . .

Transcriptional regulation of all genes is initiated by the specific binding of regulatory proteins called transcription factors to specific sites on DNA called promoter regions. Transcription factors employ a variety of mechanisms to recognise their DNA target sites. In the last few decades, attempts have been made to describe these mechanisms by general sets of rules and associated models. We give an overview of these models, starting with a historical review of the somewhat controversial issue of a "recognition code" governing protein-DNA interaction. We then present a probabilistic framework in which advantages and disadvantages of various models can be discussed. Finally, we conclude that simplifying assumptions about additivity of interactions are sufficiently justified in many situations (and can be suitably extended in other situations) to allow a unifying concept of a "probabilistic code" for protein-DNA recognition to be defined.

Animals↗
PubMed1999

Myb-DNA recognition: role of tryptophan residues and structural changes of the minimal DNA binding domain of c-Myb.

The Myb oncoprotein specifically binds DNA by a domain composed of three imperfect repeats, R1, R2, and R3, each containing 3 tryptophans. The tryptophan fluorescence of the minimal binding domain, R2R3, of c-Myb was used to monitor structural flexibility changes occurring upon DNA binding to R2R3. The quenching of the Trp fluorescence by DNA titration shows that four out of the six tryptophans are involved in the formation of the specific R2R3-DNA complex and the environment of the tryptophan residues becomes more hydrophobic in the complex. The fluorescence intensity quenching of the tryptophans by binding of R2R3 to DNA is consistent with the decrease of the decay time: 1.46 ns for free R2R3 to 0.71 ns for the complexed protein. In the free R2R3, the six tryptophans are equally accessible to the iodide and acrylamide quenchers with a high collisional rate constant (4 x 10(9) and 3 x 10(9) M-1 s-1, respectively), indicating that R2R3 in solution is very flexible. In the R2R3-DNA complex, no Trp fluorescence quenching is observed with iodide whereas all tryptophan residues remain accessible to acrylamide with a collisional rate constant slightly slower than that in the free state. These results indicate that (i) a protein structural change occurs and (ii) the R2R3 molecule keeps a high mobility in the complex. The complex formation presents a two-step kinetics: a fast step corresponding to the R2R3-DNA association (7 x 10(5) M-1 s-1) and a slower one (0.004 s-1), which should correspond to a structural reorganization of the protein including a reordering of the water molecules at the protein-DNA interface.

Animals↗
PubMed2001

DNA recognition by the herpes simplex virus transactivator VP16: a novel DNA-binding structure.

Upon infection, the herpes simplex virus (HSV) transcriptional activator VP16 directs the formation of a multiprotein-DNA complex-the VP16-induced complex-with two cellular proteins, the host cell factor HCF-1 and the POU domain transcription factor Oct-1, on TAATGARAT-containing sequences found in the promoters of HSV immediate-early genes. HSV VP16 contains carboxy-terminal sequences important for transcriptional activation and a central conserved core that is important for VP16-induced complex assembly. On its own, VP16 displays little, if any, sequence-specific DNA-binding activity. We show here that, within the VP16-induced complex, however, the VP16 core has an important role in DNA binding. Mutation of basic residues on the surface of the VP16 core reveals a novel DNA-binding surface with essential residues which are conserved among VP16 orthologs. These results illuminate how, through association with DNA, VP16 is able to interpret cis-regulatory signals in the DNA to direct the assembly of a multiprotein-DNA transcriptional regulatory complex.

Amino Acid Substitution↗
PubMed2006

Crystal structures of the DNA-binding domain of Escherichia coli proline utilization A flavoprotein and analysis of the role of Lys9 in DNA recognition.

PutA (proline utilization A) from Escherichia coli is a 1320-amino-acid residue protein that is both a bifunctional proline catabolic enzyme and an autogenous transcriptional repressor. Here, we report the first crystal structure of a PutA DNA-binding domain along with functional analysis of a mutant PutA defective in DNA binding. Crystals were grown using a polypeptide corresponding to residues 1-52 of E. coli PutA (PutA52). The 2.1 Angstrom resolution structure of PutA52 mutant Lys9Met was determined using Se-Met MAD phasing, and the structure of native PutA52 was solved at 1.9 Angstrom resolution using molecular replacement. Residues 3-46 form a ribbon-helix-helix (RHH) substructure, thus establishing PutA as the largest protein to contain an RHH domain. The PutA RHH domain forms the intertwined dimer with tightly packed hydrophobic core that is characteristic of the RHH family. The structures were used to examine the three-dimensional context of residues conserved in PutA RHH domains. Homology modeling suggests that Lys9 and Thr5 contact DNA bases through the major groove, while Arg15, Thr28, and His30 may interact with the phosphate backbone. Lys9 is shown to be essential for specific recognition of put control DNA using gel shift analysis of the Lys9Met mutant of full-length PutA. Lys9 is disordered in the PutA52 structure, which implies an induced-fit binding mechanism in which the side chain of Lys9 becomes ordered through interaction with DNA. These results provide new insights into the structural basis of DNA recognition by PutA and reveal three-dimensional structural details of the PutA dimer interface.

Bacterial Proteins↗
PubMed1998

Sequence-specific DNA recognition by the myb-like domain of the human telomere binding protein TRF1: a model for the protein-DNA complex.

Telomeres consist of tandem arrays of short G-rich sequence motifs packaged by specific DNA binding proteins. In humans the double-stranded telomeric TTAGGG repeats are specifically bound by TRF1 and TRF2. Although telomere binding proteins from evolutionarily distant species are not sequence homologues, they share a Myb-like DNA binding motif. Here we have used gel retardation, primer extension and DNase I footprinting analyses to define the binding site of the isolated Myb-like domain of TRF1 and present a three-dimensional model for its interaction with human telomeric DNA. Our results suggest that the Myb-like domain of TRF1 recognizes a binding site centred on the sequence GGGTTA and that its DNA binding mode is similar to that of the homeodomain-like motifs of the yeast telomere binding protein RAP1. The implications of these findings for recognition of telomeric DNA in general are discussed.

Amino Acid Sequence↗