Interaction of effecting ligands with lac repressor and repressor-operator complex.

The equilibrium association constants for the binding of a wide variety of effecting ligands of the lac repressor were measured by equilibrium dialysis. Also, detailed investigations of the apparent rate of dissociation of repressor-operator comples as a function of ligand concentration were carried out for several inducers and anti-inducers. The affinity of repressor-ligand comples for operator DNA was evaluated from the specific rate constants at saturating concentrations of effecting ligand. By fitting the experimental data depicting the functional dependence of the rate of dissociation upon ligand concentrations to calculated curves, assuming simple models of the induction mechanism, the equilibrium association constant for the binding of effecting ligand to repressor-operator comples was determined. Inducers reduce the affinity of lac repressor for operator DNA by a factor of approximately 1000 under standard conditions; the extent of destabilization depends on Mg2+ ion concentration. Anti-inducers increase the affinity of repressor for operator at most a factor of five. Only one neutral ligand, which binds to repressor without altering the stability of repressor-operator comples, was found. No homotropic or heterotropic interactions in the binding of effecting ligands either to repressor or to repressor-operator complex are evident.     

Interaction of tight binding repressors with lac operators. An analysis by DNA-footprinting

To increase our understanding of protein-DNA interaction in general, and in particular that of lac repressor with lac operator, we have investigated the interaction of tight binding (Itb) repressors with wild type (WT) operator and Oc operators. Nine Oc and a WT operator were cloned and sequenced. Three different Oc and an O+ were then chosen for the footprint analysis of six Itb repressors and WT repressor. Distinct protection patterns for the various repressor-operator pairs were observed at low repressor concentrations whereas, at high repressor concentrations, a stretch of 24 bases of the lower strand of the four different operators was protected in most cases. This protection pattern at high repressor concentration was almost completely redundant for all repressor-operator pairs, in spite of the fact that the affinities of the various pairs differed by more than three orders of magnitude. Two exceptions to this general observation were the two tight binding repressors R67 and R78a. These had been mapped in a region that codes for amino acid residues involved in subunit interaction. The two repressors showed reduced protection of O+ and of some Oc operators at the 3' (right) end of the lower strand. Dimethylsulfoxide, which is known to increase the affinity of O+ for repressor, did not increase the number of bases protected by WT repressor on the lower strand of O+. The footprinting results presented here clearly demonstrate that lac repressor can maximally protect about 24 bases of the lower strand of the operator and that the number and kind of interactions occurring in this region determine the strength of the repressor-operator interaction.     

Plasticity in protein-DNA recognition: lac repressor interacts with its natural operator 01 through alternative conformations of its DNA-binding domain

The lac repressor-operator system is a model system for understanding protein-DNA interactions and allosteric mechanisms in gene regulation. Despite the wealth of biochemical data provided by extensive mutations of both repressor and operator, the specific recognition mechanism of the natural lac operators by lac repressor has remained elusive. Here we present the first high-resolution structure of a dimer of the DNA-binding domain of lac repressor bound to its natural operator 01. The global positioning of the dimer on the operator is dramatically asymmetric, which results in a different pattern of specific contacts between the two sites. Specific recognition is accomplished by a combination of elongation and twist by 48 degrees of the right lac subunit relative to the left one, significant rearrangement of many side chains as well as sequence-dependent deformability of the DNA. The set of recognition mechanisms involved in the lac repressor-operator system is unique among other protein-DNA complexes and presents a nice example of the adaptability that both proteins and DNA exhibit in the context of their mutual interaction.     

Hinge-helix formation and DNA bending in various lac repressor-operator complexes

The hinge-region of the lac repressor plays an important role in the models for induction and DNA looping in the lac operon. When lac repressor is bound to a tight-binding symmetric operator, this region forms an alpha-helix that induces bending of the operator. The presence of the hinge-helices is questioned by previous data that suggest that the repressor does not bend the wild-type operator. We show that in the wild-type complex the hinge-helices are formed and the DNA is bent, similar to the symmetric complex. Furthermore, our data show differences in the binding of the DNA binding domains to the half-sites of the wild-type operator and reveal the role of the central base-pair of the wild-type operator in the repressor-operator interaction. The differences in binding to the operator half-sites are incorporated into a model that explains the relative affinities of the repressor for various lac operator sequences that contain left and right half-sites with different spacer lengths.     

Specific binding of lac repressor to linear versus circular polyoperator molecules

Gel shift assays were used to examine the binding of the lactose (lac) repressor to polyoperator DNA molecules. Specific binding was differentiated from nonspecific DNA association by (i) equilibrating repressor-operator complexes below the nonspecific association constant and (ii) demonstrating the effects of the inducer isopropyl beta-D-thiogalactoside (IPTG) on the formation of repressor-operator complexes. With the linear polyoperator molecules, all eight operator sites could be simultaneously bound by distinct repressors. However, with circular molecules, the eight operator sites were saturable by repressor only in the nicked circular state and not in the covalently closed circular form. Under the experimental conditions used, there was no evidence of bifunctional repressor binding or loop formation. The results suggest that the conformational perturbation of DNA that occurs upon specific repressor binding was retained in topologically closed molecules and could modify other operator sites so as to make them unavailable for specific binding.     

Targeting the Escherichia coli lac repressor to the mammalian cell nucleus

We have previously shown that about 90% of total Escherichia coli lac repressor synthesized in mammalian cells is located in the cytoplasm [Hu and Davidson, Cell 48 (1987) 555-566]. To target a functional lac repressor to the nucleus, we mutated 10 nucleotides at the 3' end of the coding sequence, thus adding the nuclear localization signal of the simian virus 40 large-T antigen to the C terminus of the repressor. The mutant lacI gene and the wild-type (wt) gene, both in standard animal cell expression vectors, driven by the promoter of the Rous sarcoma virus long terminal repeat, were stably transfected into three rodent cell lines. In confirmation of our previous results, only about 10% of the wt repressor, but all of the mutant protein, was localized in the nucleus. DNase I footprint analyses showed that the mutant repressor retained the same operator DNA-binding specificity as wt repressor. Furthermore, both repressor-operator complexes could be dissociated by addition of isopropyl-beta-D-thiogalactopyranoside in vitro. However, the ratio of number of repressor molecules per nucleus that, by in vitro assay, could bind to the operator sequence to the number of monomer repressor polypeptides per nucleus, as determined by Western blotting, was about 1:4 for the wt repressor and about 1:30 for the mutant repressor. This suggests that: (a) the mutant repressor assembles into tetramers inefficiently; and/or (b) it has reduced binding affinity to the operator sequence; and/or (c) it has higher binding affinity to nonspecific DNA.     

Analysis of trp repressor-operator interaction by filter binding.

A filter binding assay was developed that allows measurement of specific binding of trp repressor to operator DNA. The most important feature of this procedure is the concentration and type of salt present in the binding buffer. Using this assay the dissociation constant of the repressor-operator complex was determined to be 2.6 X 10(-9) M, and 1.34 repressor dimers were found to be bound to each operator-containing DNA molecule. These values agree with those obtained by more complex methods. The dissociation constant of the repressor for the corepressor L-tryptophan in the presence of operator DNA was shown to be 2.5 X 10(-5) M. A synthetic 48 bp operator fragment was used to determine the repressor-operator dissociation constant in the presence of tryptophan or tryptophan analogs which have higher or lower affinities for aporepressor. The rate of dissociation of repressor from operator DNA also was determined. Our findings indicate that dissociation is influenced by the concentration of tryptophan or tryptophan analogs and suggest that release of the corepressor may be the first step in dissociation of the repressor-operator complex.     

Kinetics and mechanism in the reaction of gene regulatory proteins with DNA

We have measured the kinetic properties of the Escherichia coli cAMP receptor protein (CAP) and lac repressor interacting with lac promoter restriction fragments. Under our reaction conditions (10 mM-Tris X HCl (pH 8.0 at 21 degrees C), 1 mM-EDTA, 10 microM-cAMP, 50 micrograms bovine serum albumin/ml, 5% glycerol), the association of CAP is at least a two-step process, with an initial, unstable complex formed with rate constant kappa a = 5(+/- 2.5) X 10(7) M-1 s-1. Subsequent formation of a stable complex occurs with an apparent bimolecular rate constant kappa a = 6.7 X 10(6) M-1 s-1. At low total DNA concentration, the dissociation rate constant for the specific CAP-DNA complex is 1.2 X 10(-4) s-1. The ratio of formation and dissociation rate constants yields an estimate of the equilibrium constant, Keq = 5 X 10(10) M-1, in good agreement with static results. We observed that the dissociation rate constant of both CAP-DNA and repressor-DNA complexes is increased by adding non-specific "catalytic" DNA to the reaction mixture. CAP dissociation by the concentration-dependent pathway is second-order in added non-specific DNA, consistent with either the simultaneous or the sequential participation of two DNA molecules in the reaction mechanism. The results imply a role for distal DNA in assembly-disassembly of specific CAP-DNA complexes, and are consistent with a model in which the subunits in the CAP dimer separate in the assembly-disassembly process. The dissociation of lac repressor-operator complexes was found to be DNA concentration-dependent as well, although in contrast to CAP, the reaction is first-order in catalytic DNA. Added excess operator-rich DNA gave more rapid dissociation than equivalent concentrations of non-specific DNA, indicating that the sequence content of the competing DNA influences the rate of repressor dissociation. The simplest interpretation of these observations is that lac repressor can be transferred directly from one DNA molecule to another. A comparison of the translocation rates calculated for direct transfer with those predicted by the one-dimensional sliding model indicates that direct transfer may play a role in the binding site search of lac repressor.     

The solution structure of Lac repressor headpiece 62 complexed to a symmetrical lac operator

BACKGROUND: Lactose repressor protein (Lac) controls the expression of the lactose metabolic genes in Escherichia coli by binding to an operator sequence in the promoter of the lac operon. Binding of inducer molecules to the Lac core domain induces changes in tertiary structure that are propagated to the DNA-binding domain through the connecting hinge region, thereby reducing the affinity for the operator. Protein-protein and protein-DNA interactions involving the hinge region play a crucial role in the allosteric changes occurring upon induction, but have not, as yet, been analyzed in atomic detail. RESULTS: We have used nuclear magnetic resonance (NMR) spectroscopy and restrained molecular dynamics (rMD) to determine the structure of the Lac repressor DNA-binding domain (headpeice 62; HP62) in complex with a symmetrized lac operator. Analysis of the structures reveals specific interactions between Lac repressor and DNA that were not found in previously investigated Lac repressor-DNA complexes. Important differences with the previously reported structures of the HP56-DNA complex were found in the loop following the helix-turn-helix (HTH) motif. The protein-protein and protein-DNA interactions involving the hinge region and the deformations in the DNA structure could be delineated in atomic detail. The structures were also used for comparison with the available crystallographic data on the Lac and Pur repressor-DNA complexes. CONCLUSIONS: The structures of the HP62-DNA complex provide the basis for a better understanding of the specific recognition in the Lac repressor-operator complex. In addition, the structural features of the hinge region provide detailed insight into the protein-protein and protein-DNA interactions responsible for the high affinity of the repressor for operator DNA.     

Methylphosphonates as probes of protein-nucleic acid interactions.

Deoxydinucleoside methylphosphonates were prepared by chemical synthesis and were introduced stereospecifically into the lac operator at two sites. These sites within d(ApApTpTpGpTpGpApGpCpGpGpApTpApApCpApApTpT), segment I, and d(ApApTpTpGpTpTpApTpCpCpGpCpTpCpApCpApApTpT), segment II, are indicated by p. Each segment containing a chiral methylphosphonate was annealed to the complementary unmodified segment. The interactions of these four modified lac operators with lac repressor were analyzed by the nitrocellulose filter binding assay. Introduction of either chiral phosphonate in segment II had little effect on the stability of the repressor-operator complex. When methylphosphonates were introduced into segment I, the affinity of lac repressor for the modified operators was shown to be dependent on the stereochemical configuration of the methylphosphonate.     

How Lac repressor finds lac operator in vitro.

Filter-binding and gel mobility shift assays were used to analyse the kinetics of the interaction of Lac repressor with lac operator. A comparison of the two techniques reveals that filter-binding assays with tetrameric Lac repressor have often been misinterpreted. It has been assumed that all complexes of Lac repressor and lac operator DNA bind with equal affinity to nitrocellulose filters. This assumption is wrong. Sandwich or loop complexes where two lac operators bind to one tetrameric Lac repressor are not or are only badly retained on nitrocellulose filters under normal conditions. Taking this into account, dimeric and tetrameric Lac repressor do not show any DNA-length dependence of their association and dissociation rate constants when they bind to DNA fragments smaller than 2455 base-pairs carrying a single symmetric ideal lac operator. A ninefold increased association rate to ideal lac operator on lambda DNA is observed for tetrameric but not dimeric Lac repressor. It is presumably due to intersegment transfer involving lac operator-like sequences.     

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.     

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.     

Mutant lac repressors with new specificities hint at rules for protein--DNA recognition.

Proteins which recognize specific sequences of DNA play a fundamental role in the regulation of protein synthesis in all organisms. A particular helix of the bacterial protein lac repressor recognizes the bases in the major groove of the lac operator. We show that the first two residues of this recognition helix interact independently with two base pairs. This allows us in many cases to predict repression as an indicator of strength of the repressor-operator complex. Rules of recognition can be derived for 16 symmetric operators. They also apply to the gal repressor and possibly to other bacterial repressors.     

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   

Lac repressor purification without inactivation of DNA binding activity.

A procedure has been developed which eliminates the commonly observed inactivation of the DNA binding activity of the lac repressor during purification. The operator binding activity of the repressor obtained by this method is 100 +/- 10%. The repressor can be stored frozen indefinitely without losing its affinity for DNA.     

lac Operator nucleosomes. 2. lac Nucleosomes can change conformation to strengthen binding by lac repressor

We have shown previously that lac repressor binds specifically and quantitatively to lac operator restriction fragments which have been complexed with histones to form artificial nucleosomes (203 base pair restriction fragment) or core particles (144 base pair restriction fragment. We describe here a quantitative method for determining the equilibrium binding affinities of repressor for these lac reconstitutes. Quantitative analysis shows that the operator-histone reconstitutes may be grouped into two affinity classes: those with an affinity for repressor close to that of naked DNA and those with an affinity 2 or more orders of magnitude less than that of naked DNA. All particles in the lac nucleosome preparations bind repressor with high affinity, but the lac core particle preparations contain particles of both high and low affinities for repressor. Formaldehyde cross-linking causes all high-affinity species to suffer a 100-fold decrease in binding affinity. In contrast, there is no effect of cross-linking on species of low affinity. Therefore, the ability of a particle to be bound tightly by repressor depends on a property of the particle which is eliminated by cross-linking. Control experiments have shown that chemical damage to the operator does not accompany cross-linking. Therefore, the property sensitive to cross-linking must be the ability of the particle to change conformation. We infer that the particles of low native affinity, like cross-linked particles, are of low affinity because of an inability to facilitate repressor binding by means of this conformational change. Dimethyl suberimidate cross-linking experiments show that histone-histone cross-linking is sufficient to preclude high-affinity binding. Thus, the necessary conformational change involves a nucleosome histone core event. We find that the ability of a particle to undergo a repressor-induced facilitating conformational change appears to depend on the position of the operator along the DNA binding path of the nucleosome core. We present a general model which proposes that nucleosomes are divided into domains which function differentially to initiate conformational changes in response to physiological stimuli.     

Sequence-specific DNA-protein interaction: the lac represser

A model is proposed for lac repressor-lac operator binding which accounts for the tetrameric subunit structure of the lac repressor and for factors involved in the strength, specificity and regulation of repressor-operator interaction. The model employs a π-helix in the amino terminal 25 residues of the lac repressor whereby three tyrosine residues of each subunit intercalate between base pairs of the lac operator. For the outer palindromic sequences of the operator, base specificity is provided by amino acids adjacent to the carboxyl sides of the tyrosine residues of two of the subunits. The inner palindromic sequences which bind the other two subunits form stems of hairpin loops in the operator. Base specificity for these two subunits is provided by amino acids adjacent to the amino sides of the tyrosine residues. In addition to 12 intercalated tyrosine residues, the model provides for a total of at least eight electrostatic interactions and ten sequence-specific hydrogen bonds.