lac operator DNA modification in the presence of proteolytic fragments of the repressor protein

Singly end-labeled DNA fragments containing the lactose operator were methylated in the presence of the lactose repressor and homogeneous preparations of its proteolytic fragments. Binding of core protein produced by mild trypsin digestion yielded a methylation perturbation pattern that differed significantly from that elicited by binding to intact repressor, although similarities in the patterns for these related proteins were noted in the central, asymmetric region of the operator. An NH2-terminal peptide (residues 1 to 56) from lac repressor bound operator fragments in a nitrocellulose filter assay, but failed to perturb DNA methylation significantly relative to the pattern in the absence of peptide. Binding of hybrid tetramers of core and intact repressor monomers produced related but unique methylation patterns for the purines on the operator fragment. The general pattern of perturbation observed suggests preferred binding of a single NH2 terminus to the promoter-distal region of the operator and asymmetric interaction of the core region with the operator sequence. Differences in purine methylation patterns produced by the presence of effector complexes of repressor and core protein suggest the possible nature of changes in protein topology that result in the affinity changes accompanying induction.     

Energetic differences between the specific binding of a 40 bp DNA duplex and the lac promoter to lac repressor protein

The energetics of LRP binding to a 104 bp lac promoter determined from ITC measurements were compared to the energetics of binding to a shorter 40 bp DNA duplex with the 21 bp promoter binding site sequence. The promoter binding affinity of 2.47 +/- 0.0 1x 10(7) M(-1) was higher than the DNA binding affinity of 1.81 +/- 0.67 x 10(7) M(-1) while the binding enthalpy of -804 +/- 41 kJ mol(-1) was lower than that of the DNA binding enthalpy of -145 +/- 16 kJ mol(-1) at 298.15 K. Both the promoter and DNA binding reactions were exothermic in phosphate buffer but endothermic in Tris buffer that showed the transfer of four protons to LRP in the former reaction but only two in the latter. A more complicated dependence of these parameters on temperature was observed for promoter binding. These energetic differences are attributable to additional LRP-promoter interactions from wrapping of the promoter around the LRP.     

Efficient purification of DNA fragments using a protein binding membrane

A novel and efficient method has been developed for isolation of correctly digested DNA fragments without the use of classic size-dependent electrophoretic separation methods. To achieve this, DNA fragments are end-labelled by haptens. After specific endonuclease digestion of the hapten-labelled DNA, the DNA is incubated with a protein that specifically binds to the hapten. The incubation mixture is then passed through a cartridge containing a protein-binding membrane that does not bind DNA. Undigested and partly digested DNA are retained on the membrane, while correctly digested DNA is selectively recovered for use in further downstream applications.     

Affinity capture of specific DNA fragments with short synthetic sequences

The ability of short peptide nucleic acid (PNA) oligomers and oligonucleotides containing modified residues of 5-methylcitidine, 2-aminoadenosine, and 5-propynyl-2′-deoxyuridine (strong binding oligonucleotides, SBO) to affinity capture the target double-stranded DNA fragment from mixture by means of the end invasion was compared. Both types of probes were highly effective at the conditions used. The SBO-based probes may represent a handy and easily prepared alternative to PNA for selection of target DNA fragments in mixtures.     

In vivo interaction of Escherichia coli lac repressor N-terminal fragments with the lac operator

Escherichia coli lac repressor is a tetrameric protein composed of 360 amino acid subunits. Considerable attention has focused on its N-terminal region which is isolated by cleavage with proteases yielding N-terminal fragments of 51 to 59 amino acid residues. Because these short peptide fragments bind operator DNA, they have been extensively examined in nuclear magnetic resonance structural studies. Longer N-terminal peptide fragments that bind DNA cannot be obtained enzymatically. To extend structural studies and simultaneously verify proper folding in vivo, the DNA sequence encoding longer N-terminal fragments were cloned into a vector system with the coliphage T7 RNA polymerase/promoter. In addition to the wild-type lacI gene sequence, single amino acid substitutions were generated at positions 3 (Pro3----Tyr) and 61 (Ser61----Leu) as well as the double substitution in a 64 amino acid N-terminal fragment. These mutations were chosen because they increase the DNA binding affinity of the intact lac repressor by a factor of 10(2) to 10(4). The expression of these lac repressor fragments in the cell was verified by radioimmunoassays. Both wild-type and mutant lac repressor N termini bound operator DNA as judged by reduced beta-galactosidase synthesis and methylation protection in vivo. These observations also resolve a contradiction in the literature as to the location of the operator-specific, inducer-dependent DNA binding domain.     

Photo-CIDNP study of the interaction between lac repressor headpiece and lac operator DNA

Lac repressor headpiece (HP) and intact lac repressor have been studied using the photo-CIDNP method. At neutral pH histidine 29, tyrosines 7, 12 and 17 and methionine 1 are polarised. His-29 polarizations are weaker and broader in HP59 than in HP51 indicating that the C-terminal octapeptide in HP59 adopts a conformation that allows an interaction with His-29. The photo-CIDNP spectra of intact lac repressor and HP51 are very similar, showing that the same residues are accessible to the photo-excited flavin. An equimolar mixture of HP51 and a 14 base pair lac operator fragment strongly suppresses the photo-CIDNP effect of tyrosines 7 and 17 and abolishes the His-29 polarizations. The results are compared with earlier photo-CIDNP measurements on a complex of headpiece with poly[d(AT)] and with a model derived from a 2D NMR study on a lac headpiece-operator complex.     

Thermal denaturation of the core protein of lac repressor

The thermal denaturation of the core protein of lac repressor was studied alone and in the presence of the inducer isopropyl beta-D-thiogalactoside (IPTG) and the antiinducer o-nitrophenyl beta-D-fucoside (ONPF) by means of high-sensitivity differential scanning calorimetry. The denaturation that takes place at about 65 degrees C is apparently irreversible; i.e., a rescan of a previously scanned sample of protein solution shows no denaturational endotherm. Despite this irreversibility, the denaturation appeared to follow quantitatively the dictates of equilibrium thermodynamics as embodied in the van't Hoff equation. The results obtained indicate clearly that the tetrameric protein dissociates to monomers during denaturation and that the ligands are not dissociated until denaturation takes place. The enthalpy of denaturation of the protein is 4.57 +/- 0.25 cal g-1 and is independent of temperature. The enthalpies of dissociation of IPTG and ONPF at the denaturation temperature are very large, 37 and 42 kcal (mol of ligand)-1, respectively.     

Determination of protein structures from nuclear magnetic resonance data using a restrained molecular dynamics approach: the lac repressor DNA binding domain

A procedure is described to determine from NMR data the three-dimensional structure of biomolecules. This procedure combines model building with a restrained Molecular Dynamics algorithm, in which distance information from NOEs is incorporated in the form of pseudo potentials. The method has been applied to the N-terminal DNA-binding domain or "headpiece" (amino acids 1-51) of the lac repressor from E. coli, for which no crystal structure is available. The spatial structure of the headpiece is discussed in terms of known physical and biochemical data and of its DNA binding properties.     

lac repressor forms loops with linear DNA carrying two suitably spaced lac operators.

Tetrameric lac repressor may bind to two lac operators on one DNA fragment and induce the intervening DNA to form a loop. Electron microscopy, non-denaturing polyacrylamide gel electrophoresis, and DNase I protection experiments were used to demonstrate such DNA loops, where the distance between the centres of symmetry of the two lac operators varies between 63 and 535 bp. Formation of a DNA loop is favoured by correct phasing of the two lac operators and a low concentration of both components of the reaction. When a large excess of lac repressor over DNA is used, a 'tandem' structure is observed, in which both lac operators are occupied independently by two repressor tetramers. When the concentrations of both lac repressor and lac operator are high, a 'sandwich' structure is observed, in which two DNA molecules are connected by two lac repressor tetramers in trans.     

DNA supercoiling changes the spacing requirement of two lac operators for DNA loop formation with lac repressor.

We have used a gel retardation assay to investigate the influence of DNA supercoiling on loop formation between lac repressor and two lac operators. A series of 15 DNA minicircles of identical size (452 bp) was constructed carrying two lac operators at distances ranging from 153 to 168 bp. Low positive or negative supercoiling (sigma = +/- 0.023) changed the spacing between the two lac operators required for the formation of the most stable loops. This reveals the presence of altered double helical repeats (ranging from 10.3 to 10.7 bp) in supercoiled DNA minicircles. At elevated negative supercoiling (sigma = -0.046) extremely stable loops were formed at all operator distances tested, with a slight spacing periodicity remaining. After relaxation of minicircle-repressor complexes with topoisomerase I one superhelical turn was found to be constrained in those minicircles which carry operators at distances corresponding to a non-integral number of helical turns. This indicates that DNA loop formation can define local DNA domains with altered topological properties of the DNA helix.     

lac repressor forms stable loops in vitro with supercoiled wild-type lac DNA containing all three natural lac operators

We have analyzed protein-DNA complexes formed between lac repressor and linear or differently supercoiled lac DNA (802 or 816 base-pairs in length), which carry all three natural lac operators (O1, O2 and O3) in their wild-type sequence context and spacing and compared them with constructs that contain specifically mutated "pseudo-operators" O2 or O3. We used gel retardation assays to identify the nature of the complexes according to their characteristic electrophoretic mobility and dissociation rate measurements to determine their stability. With linear DNA we found only indirect evidence for loop formation between O1 and O2. In covalently closed DNA minicircles the formation of a loop between O1 and O2 could be demonstrated by the observation that O1-O2 containing DNA with low negative supercoiling (sigma = -0.013 and less) is constricted by binding of lac repressor, resulting in an increased electrophoretic mobility. At elevated negative supercoiling (sigma = -0.025, -0.037, -0.05) O1-O2 containing DNA complexed with lac repressor migrates significantly slower than the corresponding O1-DNA, indicating loop formation. The dissociation of lac repressor-operator complexes is decreased with increasing negative supercoiling for all tested operator combinations of O1, O2 and O3. However, in the presence of at least two natural lac operators on the same DNA minicircle the enhancement of stability is particularly large. This indicates that a DNA loop is formed between these two lac operators, O1 and O2 as well as O1 and O3, since negative supercoiling is known specifically to promote the formation of looped structures. Additionally, we observe a dependence of dissociation rate on the spatial alignment of the operators as a result of changing helical periodicity in differently supercoiled DNA and consider this to be further evidence for loop formation between O1 and O2 as well as O1 and O3.     

Specificity of the interaction between lambda cro repressor protein and operator DNA fragments

Cro repressor protein is known to interact with specific sites in the operator DNA. The cro protein of lambda phage was isolated and the mode of its interaction with three different DNA fragment, lambda-OR3 17mer, phi 80-OR2 19mer and CAP binding site 22-mer, were examined by the use of proton NMR. Some of the imino proton resonances of lambda-OR3 shifted and were broadened remarkably on addition of lambda-cro protein, which indicated the induction of conformational change with complexation. In the spectrum of phi 80-OR2 which has a six base pair sequence common to lambda-OR3 the signals of the common base pairs revealed slight shifts on addition of lambda-cro protein. The imino proton signals of the CAP site DNA, however, did not show any change at all on mixing with lambda-cro. Combining the data of photo CIDNP of lambda-cro, we could postulate the mode of interaction between lambda-cro repressor and operator DNA.     

Affinity chromatography of a sequence-specific DNA binding protein using Teflon-linked oligonucleotides

An affinity matrix was constructed by synthesis of a DNA oligonucleotide on a Teflon fiber support followed by deblocking and hybridization of the complementary strand. It was used to purify a sequence-specific binding protein at least 100-fold to near homogeneity. This matrix is easily fabricated on an automated DNA synthesizer, contains high levels of attached DNA, and has superior mechanical properties. It should be generally useful for affinity chromatography of DNA binding proteins.     

Two helix DNA binding motif of CAP found in lac repressor and gal repressor.

Comparison of both the DNA and protein sequences of catabolite gene activator protein (CAP) with the sequences of lac and gal repressors shows significant homologies between a sequence that forms a two alpha-helix motif in CAP and sequences near the amino terminus of both repressors. This two-helix motif is thought to be involved in specific DNA sequence recognition by CAP. The region in lac repressor to which CAP is homologous contains many i-d mutations that are defective in DNA binding. Less significant sequence homologies between CAP and phage repressors and activators are also shown. The amino acid residues that are critical to the formation of the two-helix motif are conserved, while those residues expected to interact with DNA are variable. These observations suggest the lac and gal repressors also have a two alpha-helix structural motif which is involved in DNA binding and that this two helix motif may be generally found in many bacterial and phage repressors. We conclude that one major mechanism by which proteins can recognize specific base sequences in double stranded DNA is via the amino acid side chains of alpha-helices fitting into the major groove of B-DNA.     

Role of hydration in the binding of lac repressor to DNA

The osmotic stress technique was used to measure changes in macromolecular hydration that accompany binding of wild-type Escherichia coli lactose (lac) repressor to its regulatory site (operator O1) in the lac promoter and its transfer from site O1 to nonspecific DNA. Binding at O1 is accompanied by the net release of 260 +/- 32 water molecules. If all are released from macromolecular surfaces, this result is consistent with a net reduction of solvent-accessible surface area of 2370 +/- 550 A. This area is only slightly smaller than the macromolecular interface calculated for a crystalline repressor dimer-O1 complex but is significantly smaller than that for the corresponding complex with the symmetrical optimized O(sym) operator. The transfer of repressor from site O1 to nonspecific DNA is accompanied by the net uptake of 93 +/- 10 water molecules. Together these results imply that formation of a nonspecific complex is accompanied by the net release of 165 +/- 43 water molecules. The enhanced stabilities of repressor-DNA complexes with increasing osmolality may contribute to the ability of Escherichia coli cells to tolerate dehydration and/or high external salt concentrations.     

Binding of E.coli lac repressor to non-operator DNA*

It is shown by melting profile analysis of lac repressor-DNA complexes that repressor binds tightly and preferentially (relative to single-stranded DNA) to double-stranded non-operator DNA. This binding stabilizes the DNA against melting and the repressor against thermal denaturation. Analysis of the extent of stabilization and the rate of dissociation of repressor from non-operator DNA as a function of sodium ion concentration shows, in confirmation of other studies,(3,4) that the binding constant (K(RD)) is very ionic strength dependent; K(RD) increases from approximately 10(6) M(-1) at approximately 0.1 M Na(+) to values in excess of 10(10) M(-1) at 0.002 M Na(+). Repressor bound to non-operator DNA is not further stabilized against thermal denaturation by inducer binding, indicating that the inducer and DNA binding sites probably represent separately stabilized local conformations. Transfer melting experiments are used to measure the rate of dissociation of repressor from operator DNA. These experiments show that most of the ionic strength dependence of the binding constant is in the dissociation process; the estimated dissociation rate constant decreases from greater than 10(-1) sec(-1) at [Na(+)] >/= 0.02 M to less than 10(-4) sec(-1) at [Na(+)] 相似文献   

Affinity chromatography of DNA fragments and P-modified oligonucleotides

The wide possibilities for use of affinity chromatography are demonstrated by two examples: (i) isolation of a single-stranded fragment of the tick-borne encephalitis virus DNA (302-mer) and an oligonucleotide (34 bases) from reaction mixtures and (ii) fractionation of mixtures of diastereoisomers of octathymidylates with modified internucleotide phosphates. All affinity sorbents are constructed by the covalent attachment of the oligonucleotides to solid supports and can be used repeatedly.