Operator DNA Sequence Research Articles

Molecular Dynamics Simulation in Solvent of the Bacteriophage 434 cI Repressor Protein DNA Binding Domain Amino Acids (R1–69) in Complex with its Cognate Operator (OR1) DNA Sequence

We investigated protein/DNA interactions, using molecular dynamics simulations computed between a 10 Angstom water layer model of the 434 cl Repressor protein DNA binding domain (DBD) amino acids (R1–69) and DNA of operator (OR1) and its flanks consisting of 28 nucleotide base pairs. Hydrogen bonding interactions were monitored. In addition, van der Waals and electrostatic interaction energies were calculated. Amino acids of the 434 cl repressor DNA recognition helix 3 formed both direct and water mediated hydrogen bonds at cognate codon-anticodon nucleotide base and backbone sites within the ORl DNA major groove halfsites and flanking regions. In addition, hydrophilic amino acids within the loop between helix 3 and helix 4 have strong electrostatic attraction to codon-anticodon nucleotides located within the central nucleotides of the minor groove between the ORI major groove halfsites These interactions together induced significant structural changes in the operator DNA manifested by overtwisting of the central nucleotide base pairs and narrowing of the minor groove between the DNA major groove halfsites. Finally, these findings offer a code for site specific DNA recognition by the 434 cl repressor protein.

Quantitative analysis of electrophoresis data: novel curve fitting methodology and its application to the determination of a protein-DNA binding constant.

A computer program, GelExplorer, which uses a new methodology for obtaining quantitative information about electrophoresis has been developed. It provides a straightforward, easy-to-use graphical interface, and includes a number of features which offer significant advantages over existing methods for quantitative gel analysis. The method uses curve fitting with a nonlinear least-squares optimization to deconvolute overlapping bands. Unlike most curve fitting approaches, the data is treated in two dimensions, fitting all the data across the entire width of the lane. This allows for accurate determination of the intensities of individual, overlapping bands, and in particular allows imperfectly shaped bands to be accurately modeled. Experiments described in this paper demonstrate empirically that the Lorentzian lineshape reproduces the contours of an individual gel band and provides a better model than the Gaussian function for curve fitting of electrophoresis bands. Results from several fitting applications are presented and a discussion of the sources and magnitudes of uncertainties in the results is included. Finally, the method is applied to the quantitative analysis of a hydroxyl radical footprint titration experiment to obtain the free energy of binding of the lambda repressor protein to the OR1 operator DNA sequence.

Analysis of CRP-CytR interactions at the Escherichia coli udp promoter.

Multiprotein complexes regulate the transcription of certain bacterial genes in a sensitive, physiologically responsive manner. In particular, the transcription of genes needed for utilization of nucleosides in Escherichia coli is regulated by a repressor protein, CytR, in concert with the cyclic AMP (cAMP) activated form of cAMP receptor protein (CRP). We studied this regulation by selecting and characterizing spontaneous constitutive mutations in the promoter of the udp (uridine phosphorylase) gene, one of the genes most strongly regulated by CytR. We found deletions, duplications, and point mutations that affect key regulatory sites in the udp promoter, insertion sequence element insertions that activated cryptic internal promoters or provided new promoters, and large duplications that may have increased expression by udp gene amplification. Unusual duplications and deletions that resulted in constitutive udp expression that depended on the presence of CytR were also found. Our results support the model in which repression normally involves the binding of CytR to cAMP-CRP to form a complex which binds to specific sites in the udp promoter, without direct interaction between CytR protein and a specific operator DNA sequence, and in which induction by specific inducer cytidine involves dissociation of CytR from cAMP-CRP and the RNA polymerase interaction with cAMP-CRP bound to a site upstream of then transcription start point. The stimulation of udp expression by CytR in certain mutants may reflect its stabilization of cAMP-CRP binding to target DNA and illustrates that only modest evolutionary changes could allow particular multiprotein complexes to serve as either repressors or transcriptional activators.

Combined Conformational Search and Finite-Difference Poisson?Boltzmann Approach for Flexible Docking: Application to an Operator Mutation in the λ Repressor-Operator Complex

The N-terminal domain of the phage λ repressor binds as a dimer to its palindromic DNA operator sequence. In addition to a helix-turn-helix DNA recognition motif, the first six amino acids of the phage λ repressor form a flexible peptide segment which wraps around DNA. Site-directed mutagenesis studies have shown that amino acid replacements or partial removal of the arm structure, or changes in the DNA sequence contacting the N-terminal arm, can lower the repressor-operator binding affinity by several orders of magnitude. The finite-difference Poisson-Boltzmann approach in combination with a conformational search procedure was used to study energetic contributions of the λ arm to repressor-operator recognition based on the high resolution X-ray structure. It allows for the local relaxation of the structure upon changing the DNA sequence in the λ arm binding region. A simplified potential energy function including torsional, truncated Lennard-Jones and approximate electrostatic terms is used in the initial step to screen out energetically unfavorable structures. The electrostatic energy of selected conformations is subsequently calculated more accurately using the finite-difference Poisson-Boltzmann approach. The method was applied to study the effect of a C→T mutation at position 6 of the consensus half-site of the operator. This base-pairs contacts Lys4 which is part of the arm segment. Keeping only the Lys4 side-chain mobile and with the wild-type DNA operator sequence, several conformations close to the X-ray structure were identified as those with lowest energy. In the case of the DNA mutation, lowest energy conformations differed significantly from those selected for the wild-type sequence. These initial calculations indicate that the approach might be a useful tool to estimate conformational and energetic effects upon mutagenesis of protein-DNA complexes.

Wild-type operator binding and altered cooperativity for inducer binding of lac repressor dimer mutant R3.

Substitution of the C-terminal leucine heptad repeat region of the normally tetrameric lactose repressor by the leucine heptad repeat dimerization domain of GCN4 protein resulted in cell extracts containing protein, designated R3, which behaved as a dimer based on gel retardation analysis of DNA binding (Alberti, S., Oehler, S., von Wilcken-Bergmann, B., and Müller-Hill, B. (1993) EMBO J. 12, 3227-3236). We have purified this R3 protein and characterized its properties in comparison with the wild-type repressor. R3 protein elutes from a molecular sieve with a Stokes radius characteristic of a dimer and a deduced molecular mass of 66 kDa. Unlike other dimeric repressors, produced by deletion or mutation in the leucine heptad repeat region, which display reduced apparent operator affinity, R3 binds to operator DNA sequences with wild-type equilibrium and kinetic properties. Although inducer affinity at neutral pH is similar for R3 and wild-type protein, at elevated pH the R3 protein undergoes a slightly smaller decrease in affinity and exhibits minimal cooperativity in sugar binding compared with the wild-type protein. Interestingly, in the presence of operator DNA, a state in which inducer binding to wild-type repressor is also of reduced affinity and slightly cooperative, R3 binding affinity is decreased to a greater extent, and the protein displays higher cooperativity than wild-type repressor. Consistent with inducer binding data in the presence of operator, the release of operator from R3 protein requires a higher sugar concentration than wild-type protein. These results are interpreted in the context of alterations involving the subunit interface which affect the allosteric behavior of the repressor protein.

Trp repressor mutations alter DNA complex stoichiometry.

We have examined the interaction of a series of mutant trp repressors with various operator DNA sequences using gel retardation. Binding to 40 base pairs (bp) TrpEDCBA operator yielded patterns distinct from the wild-type protein for superrepressors EK13, EK18, and EK49, with a protein-DNA complex of higher stoichiometry (three dimers/operator) than observed for wild-type repressor (two dimers/operator). This higher stoichiometry complex may contribute to the enhanced binding affinity and higher protein-operator stability observed for the superrepressors. In contrast, DN46 displayed the same complexes characteristic of the wild-type protein, although the complex of a single dimer with operator was more prominent in the DN46 binding pattern than wild-type despite higher apparent affinity of this protein for TrpEDCBA operator than wild-type protein. The binding of AV77 was indistinguishable from the wild-type protein. Similar patterns to that found for TrpEDCBA were also observed for the 40-bp aroH operator and symmetrized derivatives of TrpEDCBA for these superrepressors. Binding of EK13, EK18, and EK49 superrepressors to half-site DNAs, composed of 20 bp of TrpEDCBA sequence coupled with 20 bp of lac operator sequence, yielded 2:1 complex as the primary product with no detectable 3:1 complex; thus, two half-sites appear to be required for generation of the 3:1 complex. Mutation in the tryptophan-binding site can also generate higher order complexes with TrpEDCBA DNA as demonstrated by the binding of VA58; the presence of 3:1 complex with this protein was also dependent on the presence of two half-sites. In addition to effects of sequence changes in the protein, the ligand employed can influence the binding pattern, as demonstrated for EK49 and VA58 using 5-methyl-tryptophan; the 3:1 complex is produced more prominently and at lower protein concentration for both mutants. It is apparent from these data that binding of the trp repressor to DNA is influenced by the operator sequence, the nature of the corepressor, as well as interactions (perhaps involving the N-terminal regions) that occur within and between the dimeric structure of this protein.

Origin of the Asymmetrical Contact between lac Repressor and lac Operator DNA

The Escherichia coli lac operator DNA contains two sequence repeats related by a pseudo-dyad axis. Deviations from symmetry, in the central 21 bp sequence, occur at two pairs of symmetrically related sites (+15/+7, +13/+9) and at the central base-pair +11. Mutational analysis and DNA protection studies have suggested asymmetric interactions of lac repressor along this sequence. Previous biophysical studies on the lac repressor-operator system have typically employed symmetrized operator sequences to simplify analysis. As a result, it has remained difficult to assess the importance of the naturally occurring sequence deviations from symmetry. Here, 19F-NMR is used to determine if the wild-type E. coli lac operator DNA sequence itself specifies a pair of distinct half-site interactions with lac repressor DNA binding domains. To observe protein interactions simultaneously at operator half-sites using 19 F-NMR, three pairs of naturally occurring, symmetry related thymine residues (at +6/+16, +8/+14 and +1/+21) were substituted pair wise by 5-fluoro-deoxyuridines (5-FdU). Two polypeptides corresponding to the N-terminal DNA binding domain of lac repressor "headpiece", residues 1 to 56 and 1 to 64, were employed to remove the steric constraints of subunit interaction in the wild-type tetramer. Spectral changes associated with headpiece binding to left side DNA sequences differ from those caused by binding to equivalent sequences on the right half-site. These results are similar to non-symmetric intact tetramer repressor interactions specified by the DNA sequence. Three mutant lac operator sequences with increased symmetry, bearing FdU substitutions were used to identify the relative importance of the three naturally asymmetric positions. Symmetrizing one pair of these sites alone or in addition to removing the central base-pair failed to produce identical NMR signal changes characteristic of symmetric headpiece—DNA complexes. However, symmetrizing both asymmetric pairs gave chemical shift changes expected from symmetric protein—DNA complexes. We propose that key interactions with the left side +9 (G · C) are altered at the symmetrically related right side +13 (A · T). The data show that the DNA sequence at +13 influences interactions three base-pairs away.

Energy coupling between DNA binding and subunit association is responsible for the specificity of DNA-Arc interaction.

The effects of several DNA molecules on the free energy of subunit association of Arc repressor were measured. The association studies under equilibrium conditions were performed by the dissociating perturbation of hydrostatic pressure. The magnitude of stabilization of the subunit interaction was determined by the specificity of the protein-DNA interaction. Operator DNA stabilized the free energy of association by about 2.2 kcal/mol of monomeric unit, whereas poly(dG-dC) stabilized the subunit interaction by only 0.26 kcal. Measurements of the stabilizing free energy at different DNA concentrations revealed a stoichiometry of two dimers per 21 bp for the operator DNA sequence and for the nonspecific DNA poly(dA-dT). However, the maximum stabilization was much larger for operator sequence (delta p = 1,750 bar) as compared for poly(dA-dT) (delta p = 750 bar). The importance of the free-energy linkage for the recognition process was corroborated by its absence in a mutant Arc protein (PL8) that binds to operator and nonspecific DNA sequences with equal, low affinity. We conclude that the coupling accounts for the high specificity of the Arc-operator DNA interaction. We hypothesize a mutual coupling between the protein subunits and the two DNA strands, in which the much higher persistency of the associated form when Arc is bound to operator would stabilize the interactions between the two DNA strands.

Comparison of the structures of operator DNA free and in complex with λ repressor

The structures of operator DNA unbound and in complex with lambda repressor protein are compared. The conformation of the left 10 base pairs of a lambda right regulatory operator DNA sequence has been previously determined in solution using nuclear magnetic resonance techniques and the structure of a homologous left regulatory operator DNA bound to lambda repressor N-terminal domain had been previously solved using X-ray crystallography. The DNA adopts an overall linear B-form DNA both in the absence and presence of lambda repressor. Superimpositioning of the DNA structures reveals small differences between them that are due to the binding of protein and not to the different techniques used for their determination.

General colorimetric method for DNA diagnostics allowing direct solid-phase genomic sequencing of the positive samples.

A system for rapid colorimetric detection of specific genome DNA fragments amplified by the polymerase chain reaction (PCR) is described that has been designed to allow direct solid-phase sequencing of positive samples. The amplified material is immobilized on magnetic beads by using the biotin streptavidin system. An Escherichia coli lac operator DNA sequence is incorporated in the amplified material during the second step of a nested primer procedure. This 21-base-pair sequence is used for a general colorimetric detection with a fusion protein consisting of the E. coli Lac repressor and beta-galactosidase. Positive samples can be treated subsequently with alkali to obtain a single-stranded DNA template suitable for direct genomic sequencing. This method to detect immobilized amplified nucleic acids (DIANA) is well adapted for automated or semiautomated clinical assays. Here, we show that it can be used to detect and sequence Chlamydia trachomatis genomic DNA in clinical samples.

Sequence-dependent variations in the 31P NMR spectra and backbone torsional angles of wild-type and mutant Lac operator fragments.

Assignment of the 31P resonances of a series of six sequenced-related tetradecamer DNA duplexes, d(TGTGAGCGCTCACA)2, d(TATGAGCGCTCATA)2, d(TCTGAGCGCTCAGA)2, d(TGTGTGCGCACACA)2, d(TGTGACGCGTCACA)2 and d(CACAGTATACTGTG)2, related to the lac operator DNA sequence was determined either by site-specific 17O labeling of the phosphoryl groups or by two-dimensional 1H-31P pure absorption phase constant time (PAC) heteronuclear correlation spectroscopy. J(H3'-P) coupling constants for each of the phosphates of the tetradecamers were obtained from 1H-31P J-resolved selective proton flip 2D spectra. By use of a modified Karplus relationship the C4'-C3'-O3'-P torsional angles (epsilon) were obtained. Comparison of the 31P chemical shifts and J(H3'-P) coupling constants of these sequences has allowed greater insight into those various factors responsible for 31P chemical shift variations in oligonucleotides and provided an important probe of the sequence-dependent structural variation of the deoxyribose phosphate backbone of DNA in solution. These sequence-specific variations in the conformation of the DNA sugar phosphate backbone of various lac operator DNA sequences can possibly explain the sequence-specific recognition of DNA by DNA binding proteins, as mediated through direct contacts between the phosphates and the protein.

DNA bending by negative regulatory proteins: Gal and Lac repressors.

The ability of two negative regulatory proteins, Gal and Lac repressors, to induce DNA bending was tested by the respective cloning of gal and lac operator DNA sequences into a sensitive vector, pBend2. pBend2 can generate a large number of DNA fragments in each of which a cloned operator is present in circularly permuted positions along the length of the DNA. Gel electrophoresis of these DNA fragments individually complexed to a repressor shows that the Gal repressor bends both of the gal operators, OE and OI. Similarly, the Lac repressor induces a bend to a lac operator DNA. In each case, the center of the average bent segment is located at or close to the dyad symmetry axis of the operator sequence. In view of these findings, we discuss how these negative regulatory proteins may function by a more dynamic mechanism than was perceived previously.

NMR Investigation of Palindromic LAC Operator DNA Sequences of Varying Size: Influences of Complex Formation with the LAC Repressor Headpiece on the DNA1H Resonances

Abstract 1H-NMR studies on s3rmmetrical lac operators were performed to determine the minimum size of a lac operator for tight complex formation with the lac repressor headpiece. Four operators of varying size from 18 base pairs to 24 base pairs were chemically synthesized. The data obtained suggest that for a synthetic lac operator to form a tight complex with the lac headpiece it should be at least 22 base pairs long.

Cruciform transitions in DNA.

The rates of transition between the cruciform and linear conformations of a perfectly inverted repeated lac operator DNA sequence have been measured using a trimethylpsoralen intrastrand cross-linking assay. The rate and extent of the linear to cruciform transition were dependent on temperature and on the superhelical density of the DNA. Apparent half-lives for this transition were between 4-9 min at 37 degrees C for supercoiled DNAs as isolated from cells. The half-life for the cruciform to linear transition in relaxed DNA was about 30 s at 37 degrees C. Mg2+ stabilized both conformations but stabilized the linear form to a greater degree than the cruciform. The rates of transition were temperature dependent suggesting enthalpies of activation of 26.3 kcal mol-1 for the cruciform to linear transition and 33.4 kcal mol-1 for the linear to cruciform transition. The rate of the linear to cruciform transition was slower at 50 than 37 degrees C. Heating above 70 degrees C resulted in the loss of the cruciform structure.

Perfect palindromic lac operator DNA sequence exists as a stable cruciform structure in supercoiled DNA in vitro but not in vivo.

A perfect palindromic 66-base pair (bp) DNA sequence derived from the lac operator and cloned into plasmid pMB9 [Betz, J. L. & Sadler, J. R. (1981) Gene 13, 1-12] can exist in a 66-bp linear form or as two 33-bp cruciform arms. The fraction of the sequence in the cruciform depends on the superhelical density of the plasmid DNA. Relaxed DNA contains no cruciforms. The palindrome in the cruciform structure is cut by EcoRI endonuclease at the base of the cruciform arms, releasing 33-bp fragments; when in the linear form only 66-bp fragments are produced. The cruciform structure is fixed by trimethylpsoralen crosslinks in the cruciform arms. This together with the EcoRI cutting provides an assay for the cruciform structures in the DNA of living cells. Using this assay we show that the cruciform structure rarely if ever exists in vivo, but after DNA isolation greater than 90% of the sequence is in cruciforms. Results suggest that the plasmid DNA as organized in vivo either lacks sufficient torsional tension to form this cruciform or the palindrome is restrained in the linear form by other bound molecules.

Repressor-operator interaction in the lac operon : III. Nuclear magnetic resonance observations with altered amino-terminal DNA binding domains

We have examined the N-terminal 56 amino acid fragment, the domain that can bind DNA independently, from 3-fluorotyrosine-substituted Escherichia coli lac repressor by 19F-nuclear magnetic resonance. The fragments or “headpieces” from four altered repressers missing each of the tyrosines in turn were examined in parallel. When the wild-type N-terminal fragment is titrated with a 36 base-pair lac operator DNA sequence, the 19F resonances undergo changes in their chemical shifts that are different from those changes when the N-terminal fragment is titrated with non-specific DNA fragments. By looking at these operator-induced changes as well as pH-dependent effects with all four altered N-terminal fragments, we show systematic correlations with the genetic data. The data lead us to conclude that upon operator DNA binding: (1) tyrosine 7 is displaced to a less polar environment and the higher than normal p K value of the phenolic OH group is decreased; (2) tyrosine 12 does not change much in either its mobility or environment; and (3) tyrosine 17 is involved, as suggested by the genetic data, when the headpiece forms a complex with operator DNA.

Lac repressor-lac operator interaction: NMR observations.

We show here the changes in the NMR spectra of the Escherichia coli lac repressor when bound to isolated lac operator DNA. The observations focus on the aromatic residues--four tyrosines and a single histidine--in the amino-terminal DNA binding domain of the lac repressor. There is a good correlation between chemical shift changes seen by 19F NMR when compared with 1 H NMR of otherwise identical repressor--DNA complexes. The results suggest that the tyrosines do not intercalate in the DNA. The NMR spectral changes with similarly sized DNA fragments, not containing the lac operator DNA sequence, are different. Thus, the amino-terminal domain of the lac repressor is independently capable of discriminating between lac operator and nonspecific DNA sequences. There can be two amino-terminal fragments per operator in the specific complex.