Consecutive Major Grooves Research Articles

Structural insight into DNA recognition by bacterial transcriptional regulators of the SorC/DeoR family.

The SorC/DeoR family is a large family of bacterial transcription regulators that are involved in the control of carbohydrate metabolism and quorum sensing. To understand the structural basis of DNA recognition, structural studies of two functionally characterized SorC/DeoR family members from Bacillus subtilis were performed: the deoxyribonucleoside regulator bsDeoR and the central glycolytic genes regulator bsCggR. Each selected protein represents one of the subgroups that are recognized within the family. Crystal structures were determined of the N-terminal DNA-binding domains of bsDeoR and bsCggR in complex with DNA duplexes representing the minimal operator sequence at resolutions of 2.3 and 2.1 Å, respectively. While bsDeoRDBD contains a homeodomain-like HTH-type domain, bsCggRDBD contains a winged helix-turn-helix-type motif. Both proteins form C2-symmetric dimers that recognize two consecutive major grooves, and the protein-DNA interactions have been analyzed in detail. The crystal structures were used to model the interactions of the proteins with the full DNA operators, and a common mode of DNA recognition is proposed that is most likely to be shared by other members of the SorC/DeoR family.

Interaction of the pioneer transcription factor GATA3 with nucleosomes

During cellular reprogramming, the pioneer transcription factor GATA3 binds chromatin, and in a context-dependent manner directs local chromatin remodeling and enhancer formation. Here, we use high-resolution nucleosome mapping in human cells to explore the impact of the position of GATA motifs on the surface of nucleosomes on productive enhancer formation, finding productivity correlates with binding sites located near the nucleosomal dyad axis. Biochemical experiments with model nucleosomes demonstrate sufficiently stable transcription factor-nucleosome interaction to empower cryo-electron microscopy structure determination of the complex at 3.15 Å resolution. The GATA3 zinc fingers efficiently bind their target 5′-GAT-3′ sequences in the nucleosome when they are located in solvent accessible, consecutive major grooves without significant changes in nucleosome structure. Analysis of genomic loci bound by GATA3 during reprogramming suggests a correlation of recognition motif sequence and spacing that may distinguish productivity of new enhancer formation.

Fatty Acid Regulation of Virulence in Vibrio cholerae: A Structural Analysis of Unbound, Ligand‐Bound, and DNA‐Bound ToxT

Vibrio cholerae is an infectious bacterium that causes activation of transmembrane ion channels responsible for mediating the tonicity of cells in the small intestine. Activation of these ion channels results in osmotic movement into the small intestine, thus dehydrating cells and the infected individual. The transcription factor, ToxT, upregulates production of cholera toxin (CT) and the toxin coregulated pilus (TCP), key virulence factors in the progression of cholera. ToxT, a member of the AraC family of transcriptional regulators, has been crystallized in the presence of an unsaturated fatty acid (UFA) revealing a repressed form of the transcription factor in a conformation that prevents DNA binding. Two □‐helices in the DNA binding domain (DBD), □6 and □9, must be parallel to bind consecutive major grooves in the DNA. In the UFA‐bound structure, these helices are non‐parallel, supporting the model that ligand binding to ToxT prevents DNA binding. ToxT has been shown biochemically to be homodimeric when bound to DNA. However, crystallization of the DNA bound form of ToxT has been challenging due to instability of the complex. Recently, a natural variant of ToxT with increased solubility has been crystallized in the presence and absence of UFA. Crystallization of the apo form of ToxT required a lysine to alanine substitution at position 231 in the ligand‐binding pocket of the protein to prevent UFA binding. Although the structure of apo‐ToxT is highly similar to the UFA‐bound structure, the apo version was shown to be more flexible than ligand‐bound ToxT, supporting a model in which □6 and □9 in each subunit of dimeric ToxT are parallel and capable of binding to DNA. Using the structures described in this study, The Pingry School SMART (Students Modeling A Research Topic) Team used a 3D‐printer from the Milwaukee School of Engineering (MSOE) to model the apo structure and the dimeric DNA‐bound model of ToxT. These models support an in‐depth analysis of ToxT structural conformations in support of the proposed mechanism of regulation. A detailed structural understanding of ToxT may support future development of vaccines and therapeutics for cholera and other pathogens whose toxicity is regulated by AraC family members.

Atypical DNA recognition mechanism used by the EspR virulence regulator of Mycobacterium tuberculosis

The human pathogen Mycobacterium tuberculosis requires the ESX-1 secretion system for full virulence. EspR plays a key role in ESX-1 regulation via direct binding and transcriptional activation of the espACD operon. Here, we describe the crystal structures of EspR, a C-terminally truncated form, EspRΔ10, as well as an EspR-DNA complex. EspR forms a dimer with each monomer containing an N-terminal helix-turn-helix DNA binding motif and an atypical C-terminal dimerization domain. Structural studies combined with footprinting experiments, atomic force microscopy and molecular dynamic simulations allow us to propose a model in which a dimer of EspR dimers is the minimal functional unit with two subunits binding two consecutive major grooves. The other two DNA binding domains are thus free to form higher-order oligomers and to bridge distant DNA sites in a cooperative way. These features are reminiscent of nucleoid-associated proteins and suggest a more general regulatory role for EspR than was previously suspected.

Transcriptional repressor CopR: the structured acidic C terminus is important for protein stability

The transcriptional repressor CopR is one of the two copy-number control components of plasmid pIP501. CopR binds as a dimer at two consecutive major grooves on the same face of the DNA. Previously, equilibrium dissociation constants of CopR dimers and the CopR-DNA complex and the intracellular CopR concentration were calculated. Amino acid residues involved in DNA binding and dimerization were determined. Here, we provide a detailed analysis of the acidic C terminus of CopR. A series of C-terminally truncated CopR mutants were analysed with regard to activity and half-life in vivo and DNA binding, dimerization, structure and stability in vitro. The last 29 amino acid residues of CopR were not essential for DNA binding and dimerization but for protein stability. However, whereas CopΔ20 was, in spite of drastically shortened half-life, still 100 % active in vivo, CopΔ24 and CopΔ27 retained only 20 % activity. In vivo stability could be restored only partially by adding a C-terminal tail previously shown to stabilize the λ repressor N terminus. However, substitution of seven Glu residues by Lys within the last 20 residues drastically reduced half-life. Our results clearly demonstrate that the acidic C terminus is important for the stability of CopR. Using CD-measurements we show that the C terminus of CopR is structured.

Plasmid pIP501 encoded transcriptional repressor CopR binds to its target DNA as a dimer

The CopR protein is one of the two regulators of pIP501 copy number. It acts as transcriptional repressor at the essential repR promoter pII. Previously, we found that CopR contacts two consecutive major grooves (site I and site II) on the same face of the DNA. In spite of identical sequence motifs in these sites, neighboring bases were contacted differently. Furthermore, we showed that CopR can dimerize in solution. We demonstrate by two independent methods that CopR binds the DNA as a dimer. We present data that suggest that the sigmoidal CopR-DNA binding curve published previously is the result of two coupled equilibria: dimerization of CopR monomers and CopR dimer-DNA binding. A K D-value of 1.44(±0.49) × 10 −6 M for CopR dimers was determined by analytical ultracentrifugation. Based on this value and the binding curve, the equilibrium dissociation constant K 2 for the CopR-DNA complex was calculated to be 4(±1.3) × 10 −10 M. Quantitative Western blot analysis was used to determine the intracellular concentration of CopR in Bacillus subtilis. This value, 20 × 10 −6 to 30 × 10 −6 M, is 10 to 20-fold higher than the equilibrium constant for dimer dissociation, suggesting that CopR binds in vivo as a preformed dimer.

The arginine repressor of Escherichia coli K-12 makes direct contacts to minor and major groove determinants of the operators

In order to gain further insight into the molecular mechanism of arginine-dependent operator recognition by the hexameric Escherichia coli arginine repressor we have probed protein-DNA interactions in vitro and in vivo. We have extensively applied the chemical modification-protection and premodification-interference approach to two operators, the natural operator overlapping the P2 promoter of the carAB operon and a fully symmetrical consensus sequence. Backbone contacts were revealed by hydroxyl radical footprinting and phosphate ethylation interference. Base-specific contacts to purines and pyrimidines were revealed by methylation protection and premodification interference, KMnO4 and NH2OH.HCl-specific modification of thymine and cytosine residues, base-removal (depurination and depyrimidation), and base substitution (uracil and inosine). Additional information on the groove specificity of repressor binding was obtained by small ligand binding interference (distamycin and methyl green). In vivo, we measured the effects on the repressibility of 24 single base-pair substitutions obtained by saturation mutagenesis of half an Arg box in the carAB operator. The results of these experiments point to the conclusion that a hexameric arginine repressor molecule covers four turns of the helix, makes base-specific contacts to at least one guanine (G4 or G4′) and two thymine (T3, T13′, or T3′, T13) residues in each one of four consecutive major grooves on one face of the helix and with four A-T/T-A base-pairs, comprising the adenine residues A9, 9′, 12, 12′ and the thymine residues T10, 10′, 11, 11′, in the two outermost minor grooves of the operator, on the very same face of the DNA molecule. The hydrophobic 5-methyl groups of four thymine residues (T3, 3′, 13, 13′) in each Arg box contribute to major groove-specific recognition via hydrophobic and/or van der Waals interactions. The importance of minor groove contacts was further supported by the drastic effect of distamycin binding interference. In vivo, the most pronounced drops in repressibility were occasioned by mutations at positions 10 (A → G or C), 11 (T → A or G) and 12 (A → G, T or C).

The Na+-specific interaction between the LysR-type regulator, NhaR, and the nhaA gene encoding the Na+/H+ antiporter of Escherichia coli.

We used partially purified NhaR and a highly purified His-tagged NhaR derivative to identify the cis-regulatory sequences of nhaA recognized by NhaR and to study the specific effect of Na+ on this interaction. Gel retardation assay with DNase I footprinting analysis showed that NhaR binds a region of nhaA which spans 92 bp and contains three copies of the conserved LysR-binding motif. Na+, up to 100 mM, had no effect on the binding of NhaR to nhaA. The dimethylsulfate methylation protection assay in vivo and in vitro, showed that bases G-92, G-60, G-29 and A-24 form direct contacts with NhaR; in the absence of added Na+ in vivo, these bases were protected but became exposed to methylation in a DeltanhaR strain; accordingly, these bases were protected in vitro by the purified His-tagged NhaR. 100 mM Na+, but not K+, removed the protection of G-60 conferred by His-tagged NhaR in vitro. Exposure of intact cells to 100 mM Na+, but not K+, exposed G-60. The maximal effect of Na+ in vitro was observed at 20 mM and was pH dependent, vanishing below pH 7.5. In contrast to G-60, G-92 was exposed to methylation by the ion only in vivo, suggesting a requirement for another factor existing only in vivo for this interaction. We suggest that NhaR is both sensor and transducer of the Na+ signal and that it regulates nhaA expression by undergoing a conformational change upon Na+ binding which modifies the NhaR-nhaA contact points.

Plasmid pIP501 encoded transcriptional repressor CopR binds asymmetrically at two consecutive major grooves of the DNA

Replication of the streptococcal plasmid pIP501 is regulated by the CopR protein and an antisense-RNA (RNAIII). CopR acts as transcriptional repressor at the essential repR promoter pII by binding to inverted repeat IR1 upstream of pII. To further characterize the interaction of CopR with its target, footprinting studies were performed. Methylation interference identified three guanine bases (G240, G242 and G251) in the top strand and two (G252 and G254) in the bottom strand contacted by CopR in the major groove of the DNA. Missing base interference revealed the contribution of the bases in the neighbourhood of these guanine bases to the specific DNA-protein contacts. Phosphate residues essential for CopR binding were determined by ethylation interference. The recognition sequence was localized at the centre of inverted repeat IR1. CopR contacts two consecutive major grooves (site I and II) on the same face of the DNA. Although the two sites share a common sequence motif, neighbouring bases are contacted differently. DNA fragments carrying single mutations in site I or II were analysed by band shift assays. Gel filtration and native gel electrophoresis demonstrated that CopR exists only as a dimer. A sigmoidal binding curve of CopR to its DNA target was observed and allowed the determination of the apparent dissociation constant KD. The significance of the relatively high apparent KD for the role of CopR in pIP501 copy number regulation is discussed.

Missing-base and ethylation interference footprinting of P1 plasmid replication initiator.

RepA, the replication initiator protein of plasmid P1, binds to specific 19 bp sequences on the plasmid DNA. Earlier footprinting studies with dimethylsulfate identified the guanines that contact RepA through the major groove of DNA. In this study, base elimination was used to identify the contribution of all four bases to the binding reaction. Depurination and depyrimidation of any base in the neighborhood of the contacting guanines was found to decrease RepA binding. These results are consistent with the notion that RepA contacts bases of two consecutive major grooves on the same face of DNA. We also observed that depurination but not methylation of three guanines (G3, G8 and G9) affected binding. We identified the DNA phosphate groups (3 in the top strand, one of which mapped between G8 and G9, and 4 in the bottom strand, one of which was adjacent to C3) that strongly interfered with RepA binding upon ethylation. These results indicate that certain bases (e.g. G3, G8 and G9) may not contact RepA directly but contribute to base and backbone contacts by maintaining proper structure of the binding site.

(+)-CC-1065 as a structural probe of Mu transposase-induced bending of DNA: overcoming limitations of hydroxyl-radical footprinting

Phage Mu transposase (A-protein) is primarily responsible for transposition of the Mu genome. The protein binds to six att sites, three at each end of Mu DNA. At most att sites interaction of a protein monomer with DNA is seen to occur over three minor and two consecutive major grooves and to result in bending up to about 90 degrees. To probe the directionality and locus of these A-protein-induced bends, we have used the antitumor antibiotic (+)-CC-1065 as a structural probe. As a consequence of binding within the minor groove, (+)-CC-1065 is able to alkylate N3 of adenine in a sequence selective manner. This selectivity is partially determined by conformational flexibility of the DNA sequence, and the covalent adduct has a bent DNA structure in which narrowing of the minor groove has occurred. Using this drug in experiments in which either gel retardation or DNA strand breakage are used to monitor the stability of the A-protein--DNA complex or the (+)-CC-1065 alkylation sites on DNA (att site L3), we have demonstrated that of the three minor grooves implicated in the interaction with A-protein, the peripheral two are 'open' or accessible to drug bonding following protein binding. These drug-bonding sites very likely represent binding at at least two A-protein-induced bending sites. Significantly, the locus of bending at these sites is spaced approximately two helical turns apart, and the bending is proposed to occur by narrowing of the minor groove of DNA. The intervening minor groove between these two peripheral sites is protected from (+)-CC-1065 alkylation. The results are discussed in reference to a proposed model for overall DNA bending in the A-protein att L3 site complex. This study illustrates the utility of (+)-CC-1065 as a probe for protein-induced bending of DNA, as well as for interactions of minor groove DNA bending proteins with DNA which may be masked in hydroxyl radical footprinting experiments.

Arginine regulon of Escherichia coli K-12 : A study of repressor-operator interactions and of in vitro binding affinities versus in vivo repression

The 12 genes which in E. coli K-12 constitute the arginine regulon are organized in nine transcriptional units all of which contain in their 5′ non-coding region two 18 bp partially conserved imperfect palindromes (ARG boxes) which are the target sites for binding of the represser, a hexameric protein. In vitro binding experiments with purified repressor (a gift from W. K. Maas) were performed on the operator sites of four genes, argA, argD, argF, argG, and of two operons, carAB and the bipolar argECBH cluster. A compilation of results obtained by DNase I and hydroxyl radical footprinting clearly indicates that in each case the repressor binds symmetrically to four helical turns covering adjacent pairs of boxes separated by 3 bp, but to one face of the DNA only. Methylation protection experiments bring to light major base contacts with four highly conserved G residues symmetrically distributed in four consecutive major grooves. Symmetrical contacts in the minor groove with A residues have also been identified. Stoichiometry experiments suggest that a single hexameric repressor molecule binds to a pair of adjacent ARG boxes. Although the wildtype operator consists of a pair of adjacent ARG boxes separated by 3 bp (except argR where there are only 2 bp), repressor can bind to a single box but with a greatly reduced affinity. Therefore, adjacent boxes behave co-operatively with respect to the Arg repressor binding, in the sense that the presence of one box largely stimulates the binding of the properly located second box. The optimal distance separating two boxes is 3 bp, but one bp more or less does not abolish this stimulation effect. However, it is completely abolished by the introduction of two or more additional bp unless a full helical turn is introduced. Large variations in the in vivo repression response between individual arginine genes or a wild-type gene and cognate O c type mutants are not reflected by similar differences in the in vitro binding results where only small differences are observed. The significance of this lack of correlation is discussed.

Transposase contacts with mu DNA ends.

We have investigated the interaction between phage Mu transposase (A protein) and the ends (att sites) of Mu by chemical and nuclease protection and interference studies. These studies define a 24-base pair contact region at five of the six att sites (L1, L3 at att L and R1, R2, R3 at att R). Hydroxyl radical footprints show that the transposase binds to one face of the DNA helix and covers two consecutive major grooves. Binding specificity is achieved primarily through the major groove. Strong contacts are found with 3 guanines which are conserved at five of the sites. Two of these guanines are missing in the weakest binding site (L2) where 13 base pairs are mainly contacted. A pair of DNAase I hypersensitive sites, one on each strand, appear at the back of only one of the two contacted major grooves at most sites except at L2, and can be correlated with the degree of A protein-induced bend (Kuo, C.-F., Zou, A., Jayaram, M., Getzoff, E. D., and Harshey, R. M. (1991) EMBO J. 10, 1585-1591) at these sites. No contacts are observed for 4-5 base pairs in the vicinity of L1 and R1, where the A protein nicks DNA during transposition.

Identification of Protein Contact Sites within the Glucocorticoid/Progestin Response Element

The glucocorticoid receptor (GR) and the progestin receptor (PR) bind specifically to a variety of DNA sequences, glucocorticoid/progestin response elements (GRE/PRE), located in the proximity of responsive gene promoters. Using the isolated recombinant GR DNA-binding domain (DBD), it has recently been shown that GR interacts with the GRE/PRE, a 15-basepair partially palindromic consensus sequence, as a dimer. In this study an investigation into the GR-GRE/PRE and PR-GRE/PRE interaction has been performed using missing base contact analysis with the tyrosine aminotransferase GREII (TATII) and recombinant GR DBD as well as a fusion protein consisting of the PR DBD fused to Staph. aureus protein-A. GR and PR had identical base contact points, localized within two consecutive major grooves, binding to the same face of the DNA. Ethylation interference was also performed on the GR DBD-TATII interaction. The contact points with the backbone phosphate groups flank the contacts within the major groove for each of the two half-sites. Knowledge of the contact points within the DNA sequence together with the three-dimensional structure of the protein enables modelling of the protein-DNA interaction.

Probing the Escherichia coli glnALG upstream activation mechanism in vivo.

In vivo "footprints" of the glnA regulatory region under activating conditions demonstrate that the three most upstream activator sequences bind the protein NRI in the cell. Together, protections at these sites span six of seven consecutive major grooves and lie on the same helix face. E sigma 54 protects two major grooves of DNA approximately 60 base pairs downstream at the glnAp2 promoter and primarily on the opposite helix face. Experiments using potassium permanganate to probe open complex formation in vivo demonstrate that NRI is absolutely required for E sigma 54 to open the promoter DNA. Together, the dimethyl sulfate and permanganate studies verify [Reitzer, L. J., Bueno, R., Cheng, W. D., Abrams, S. A., Rothstein, D. M., Hunt, T. P., Tyler, B. & Magasanik, B. (1987) J. Bacteriol. 169, 4279-4284] that E sigma 54 occupies the glnAp2 promoter in a closed complex in vivo even in the presence of excess nitrogen and the absence of NRI. Furthermore, the slow step in transcriptional activation is shown to be an NRI-dependent conformational change in the downstream promoter DNA, which results in DNA melting. These observations place interesting restrictions on models describing the mechanism by which NRI activates transcription from glnAp2 at a distance.