Site-specific DNA-affinity chromatography of the lac repressor.

To test the feasibility of site-specific DNA-affinity chromatography, E. coli lac repressor was bound to an operator-containing DNA column, and in parallel to a non-operator DNA column. Salt gradient elution shows: 1) elution from non-operator DNA was near 250mM KCl or NaCl; interpretation of this result suggests the usefulness of the procedure for studying salt-dependence of DNA-protein affinities; 2) elution from operator-containing DNA was delayed (average elution = 1000mM salt), demonstrating a feasibility of site-specific DNA-affinity chromatography, if one provides a sufficiently favorable ratio of specific to non-specific DNA binding sites; 3) repressor eluted from operator-containing DNA over a very broad salt range, which may represent chromatography-generated repressor heterogeneity.     

Wrapping of flanking non-operator DNA in lac repressor-operator complexes: implications for DNA looping

In our studies of lac repressor tetramer (T)-lac operator (O) interactions, we observed that the presence of extended regions of non-operator DNA flanking a single lac operator sequence embedded in plasmid DNA produced large and unusual cooperative and anticooperative effects on binding constants (Kobs) and their salt concentration dependences for the formation of 1:1 (TO) and especially 1:2 (TO2) complexes. To explore the origin of this striking behavior we report and analyze binding data on 1:1 (TO) and 1:2 (TO2) complexes between repressor and a single O(sym) operator embedded in 40 bp, 101 bp, and 2514 bp DNA, over very wide ranges of [salt]. We find large interrelated effects of flanking DNA length and [salt] on binding constants (K(TO)obs, K(TO2)obs) and on their [salt]-derivatives, and quantify these effects in terms of the free energy contributions of two wrapping modes, designated local and global. Both local and global wrapping of flanking DNA occur to an increasing extent as [salt] decreases. Global wrapping of plasmid-length DNA is extraordinarily dependent on [salt]. We propose that global wrapping is driven at low salt concentration by the polyelectrolyte effect, and involves a very large number (>/similar 20) of coulombic interactions between DNA phosphates and positively charged groups on lac repressor. Coulombic interactions in the global wrap must involve both the core and the second DNA-binding domain of lac repressor, and result in a complex which is looped by DNA wrapping. The non-coulombic contribution to the free energy of global wrapping is highly unfavorable ( approximately +30-50 kcal mol(-1)), which presumably results from a significant extent of DNA distortion and/or entropic constraints. We propose a structural model for global wrapping, and consider its implications for looping of intervening non-operator DNA in forming a complex between a tetrameric repressor (LacI) and one multi-operator DNA molecule in vivo and in vitro. The existence of DNA wrapping in LacI-DNA interactions motivates the proposal that most if not all DNA binding proteins may have evolved the capability to wrap and thereby organize flanking regions of DNA.     

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   

Sequence-specific interactions of the tight-binding I12-X86 lac repressor with non-operator DNA.

The tight-binding I12-X86 lac repressor binds to non-operator DNA in a sequence-specific fashion. Using the DNA of the E. coli I gene we have investigated these sequence-specific interactions and compared them to the operator binding of wild-type repressor. The specific, non-operator DNA interactions are sensitive to the inducer IPTG. One strong binding site in the I gene DNA was found to be one of two expected on the basis of their homology with the lac operator. The binding of I12-X86 repressor to this site was visualized using the footprinting technique, and found to be consistent with an operator-like binding configuration. The protection pattern extends into an adjacent sequence suggesting that two repressor tetramers are bound in tandem.     

TET repressor.tet operator complex formation induces conformational changes in the tet operator DNA.

The structural changes of the tet operator DNA upon binding of the TET repressor protein are examined by circular dichroism. For this purpose a 70 bp DNA fragment was prepared which contains both tet operators. About 67% of the base pairs of this DNA are involved in specific interaction with the TET repressor. A rather large change in the CD of the DNA is induced by binding of the TET repressor. The shape of the CD difference spectrum is similar to the respective difference found for the lac operator DNA upon complex formation with the lac repressor. However, the effect induced by the TET repressor on tet operator DNA seems to comprise both the specific and non-specific effect of the lac repressor on the structure of DNA [Culard, F. and Maurizot, J.C. (1981) Nucl. Acids Res. 9, 5157-5184]. Specificity of binding is confirmed by the lack of any effect of the TET repressor on the CD of a 95 bp lac operator containing DNA fragment, by the reduced mobility of TET repressor.tet operator complexes on polyacrylamide gels under CD conditions, and by a titration experiment of tet operator DNA with TET repressor employing the CD change. The latter experiment reveals a stoichiometry of four TET repressors per tet operon control region.     

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.     

Interaction of mutant lambda repressors with operator and non-operator DNA

We have described a set of mutations that alter side-chains on the operator binding surface of lambda repressor. In this paper, we study the interactions of 12 purified mutant repressors with operator and non-operator DNA. The mutant proteins have operator affinities that are reduced from tenfold to greater than 10,000-fold compared to wild-type. Nine of the mutants have affinities for non-operator DNA that are similar to wild-type, two mutants show decreased non-specific binding, and one mutant has increased affinity for non-operator DNA. We discuss these findings in terms of the structural and energetic contributions of side-chain--DNA interactions, and show that certain contacts between the repressor and the operator backbone contribute both energy and specificity to the interaction.     

Interaction between the lac operator and the lac repressor headpiece: fluorescence and circular dichroism studies

The interaction between the lac repressor headpiece and a small operator DNA fragment has been examined by fluorescence and circular dichroism (c.d.) measurements. Binding of the headpiece to the DNA fragment induces a strong quenching of the fluorescence of its tyrosine residues. Quantitative analysis of the fluorescence data demonstrates that, in a first step, two headpieces bind very strongly to the DNA fragment then weaker binding occurs. C.d. demonstrates that the binding induces conformational changes of the DNA. The c.d. change produced upon binding of the first two headpieces differs from that induced upon binding of two further headpieces . Binding of the second pair of headpieces is similar to non-specific binding to non-operator DNA. The conformation of the operator DNA in the presence of two headpieces differs drastically from that in presence of lac repressor. Addition of the core to the lac operator does not induce any conformational change of the nucleic acids. These results are discussed with respect to the relative roles of core and headpieces in the lac repressor-lac operator interaction.     

Preferential protection of the minor groove of non-operator DNA by lac repressor against methylation by dimethyl sulphate.

The binding of lactose repressor to non-operator DNA was studied by the modification of several DNA's, including glycosylated DNA, with dimethyl sulphate, which affects the minor and major grooves of DNA and single stranded DNA regions. The non-specific binding of the repressor to DNA protected the minor groove but apparently not the major groove of the DNA double helix against methylation and did not increase the content of single stranded DNA regions. This suggests that the repressor on binding to non-operator DNA makes contacts mainly in the minor groove of DNA and does not uncoil the DNA double helix. This is different from the interaction of the repressor with lactose operator DNA which occurs, as shown by Gilbert et al. (1), along both the major and the minor groove.     

Role of the hydrophobic effect in stability of site-specific protein-DNA complexes

The site-specific binding interaction of lac repressor with a symmetric operator sequence and of EcoRI endonuclease with its specific recognition site both exhibit a characteristic dependence of equilibrium binding constant (Kobs) on temperature, in which Kobs attains a relative maximum in the physiologically relevant temperature range. This behavior, which appears to be quite general for site-specific protein-DNA interactions, is indicative of a large negative standard heat capacity change (delta C0P,obs) in the association process. By analogy with model compound transfer studies and protein folding data, we propose that this delta C0P,obs results primarily from the removal of non-polar surface from water in the association process. From delta C0P,obs we obtain semiquantitative information regarding the change in water-exposed non-polar surface area (delta Anp) and the corresponding hydrophobic driving force for association (delta G0hyd): delta G0hyd approximately equal to 8(+/- 1) x 10(1) delta C0P,obs approximately equal to -22(+/- 5) delta Anp. We propose that removal of non-polar surface from water (the hydrophobic effect) and release of cations (the polyelectrolyte effect) drive the thermodynamically unfavorable process (e.g. conformational distortions) necessary to achieve mutually complementary recognition surfaces (at a steric and functional-group level) in the specific complex.     

Specific and non-specific interactions of integration host factor with DNA: thermodynamic evidence for disruption of multiple IHF surface salt-bridges coupled to DNA binding.

Site-specific DNA binding of architectural protein integration host factor (IHF) is involved in formation of functional multiprotein-DNA assemblies in Escherichia coli, while non-specific binding of IHF and other histone-like proteins serves to structure the nucleoid. Here, we report an isothermal titration calorimetry study of the thermodynamics of binding IHF to a 34 bp fragment composed entirely of the specific H' site from lambda-phage DNA. At low to moderate [K(+)] (60-100 mM), strong competition is observed between specific and non-specific binding as a result of a low specificity ratio (approximately 10(2)) and a very small non-specific site size. In this [K(+)] range, both specific and non-specific binding are enthalpy-driven, with large negative enthalpy, entropy and heat capacity changes and binding constants that are insensitive to [K(+)]. Above 100 mM K(+), only specific binding is observed, and both the binding constant and the magnitudes of enthalpy, entropy and heat capacity changes all decrease strongly with increasing [K(+)]. When interpreted in the context of the structure of the specific complex, the thermodynamics provide compelling evidence for a previously unrecognized design principle by which proteins that form extensive binding interfaces with nucleic acids control binding constants, binding site sizes and effects of temperature and ion concentrations on stability and specificity. We propose that up to 22 of the 23 IHF cationic side-chains that are located within 6 A of DNA phosphate oxygen atoms in the complex, are masked in the absence of DNA by pairing with anionic carboxylate groups in intramolecular salt-bridges (dehydrated ion-pairs). These salt-bridges increase in stability with increasing temperature and decreasing [K(+)]. To explain the unusual thermodynamics of IHF-DNA interactions, we propose that both specific and non-specific binding at low [K(+)] require disruption of salt-bridges (as many as 18 for specific binding) whereupon many of the unmasked charged groups hydrate and the cationic groups interact with DNA. From structural or thermodynamic parallels with IHF, we propose that large-scale coupling of disruption of protein salt-bridges to DNA binding is significant for other large-interface DNA wrapping proteins including the nucleosome, lac repressor core tetramer, RNA polymerase core protein, HU and SSB.     

Isolation of amino-terminal fragment of lactose repressor necessary for DNA binding.

lac repressor can be dissected by trypsin into a homogenous tetrameric core (accounting for residues 60 to 347), carrying inducer binding activity, and the monomeric amino-terminal peptides ("headpieces") accounting for residues 1 to 59 and 1 to 51, respectively. This restriction of the action of trypsin on lac repressor is obtained in 1 M Tris-HCl (pH 7.5)-30% in glycerol at 25 degrees C since only the peptide bonds at lysine-59 and to a lesser extent after at arginine-51 are cleaved under these conditions. The headpieces can be purified by gel filtration. They have ordered secondary structure as revealed by circular dichroism studies. The monomeric headpieces show the relatively weak binding to nonoperator DNA but not the highly specific and strong binding to operator DNA typical for tetrameric lac repressor.     

Affinities of tight-binding lactose repressors for wild-type and pseudo-operators

Five tight-binding (Itb) mutants of the Escherichia coli lactose (lac) repressor have been characterized with regard to their non-specific affinity for DNA and their specific affinity for the wild-type operator and several sequence-altered (pseudo-) operators. Repressor-operator association rates were determined in the presence or absence of competitor DNA, dissociation rates of repressor from various DNA fragments were measured, and equilibrium competition for repressor binding was examined for several pseudo-operator DNAs. The mutant repressors exhibited increased non-specific affinity for DNA, and variable increases in affinity for sequence-altered operators. The known positions of amino acid substitutions for three of these Itb repressors support suggestions that residues 51 to 64 are important for operator recognition in addition to residues 1 to 50.     

Cooperative and Anticooperative Effects in Binding of the First and Second Plasmid OsymOperators to a LacI Tetramer: Evidence for Contributions of Non-operator DNA Binding by Wrapping and Looping

The interaction oflacoperator DNA withlacrepressor (LacI) is a classic example of a genetic regulatory switch. To dissect the role of stoichiometry, subunit association, and effects of DNA length in positioning this switch, we have determined binding isotherms for the interaction of LacI with a high affinity (O^sym) operator on linearized plasmid (2500 bp) DNA over a wide range of macromolecular concentrations (10⁻¹⁴to 10⁻⁸M). Binding data were analyzed using a thermodynamic model involving four equilibria: dissociation of tetramers (T) into dimers (D), and binding of operator-containing plasmid DNA (O) to dimers and tetramers to form three distinct complexes, DO, TO, and TO₂. Over the range of con- centrations of repressor, operator, and salt (0.075 M K⁺to 0.40 M K⁺) investigated, we find no evidence for any significant thermodynamic effect of LacI dimers. Instead, all isotherms can be interpreted in terms of just two equilibria, involving only T and the TO and TO₂complexes. As a reference binding equilibrium, which we propose must approximate the DO binding interaction, we compare the plasmid O^symresults with our extensive studies of the binding of a 40 bp O^symDNA fragment to LacI. On this basis, we obtain a lower bound on the LacI dimer – tetramer equilibrium constant and values of the equilibrium constants for formation of TO and TO₂complexes.At a salt concentration of 0.40 M, the O^symplasmid binding data are consistent with a model with two independent and identical binding sites for operator per LacI tetramer, in which the binding to a site on the tetramer is only slightly more favorable than the reference binding interaction. Increasingly large deviations from the independent-site model are observed as the salt concentration is reduced; binding of a second operator to form TO₂becomes strongly disfavored relative to formation of TO at low salt concentrations (0.075 to 0.125 M). In addition, binding of both the first and second plasmid operator DNA molecules to the tetramer becomes increasingly more favorable than the reference binding interaction as [K⁺] is reduced from 0.40 M to 0.125 M. At 0.075 M K⁺, however, the strength of binding of the second plasmid operator DNA to the LacI tetramer is dramatically reduced; this interaction is much less favorable than binding the first plasmid operator DNA, and becomes much less favorable than the reference binding interaction. We propose that these differences arise from changes in the nature of the TO and TO₂complexes with decreasing salt concentration. At low salt concentration, we suggest the hypothesis that flanking non-operator sequences bind non-specifically (coulombically) by local wrapping, and that distant regions of non-operator DNA occupy the second operator-binding site by looping. We propose that wrapping stabilizes both 1:1 and 2:1 complexes at low salt concentration, and that looping stabilizes the 1:1 complex but competitively destabilizes the 2:1 TO₂complex at low salt concentration. These effects must play a role in adjusting the stability and structure of the LacI-lac operator repression complex as the cytoplasmic [K⁺] varies in response to changes in extracellular osmolarity.     

Better conditions for mammalian in vitro splicing provided by acetate and glutamate as potassium counterions

We demonstrate here that replacing potassium chloride (KCl) with potassium acetate (KAc) or potassium glutamate (KGlu) routinely enhances the yield of RNA intermediates and products obtained from in vitro splicing reactions performed in HeLa cell nuclear extract. This effect was reproducibly observed with multiple splicing substrates. The enhanced yields are at least partially due to stabilization of splicing precursors and products in the KAc and KGlu reactions. This stabilization relative to KCl reactions was greatest with KGlu and was observed over an extended potassium concentration range. The RNA stability differences could not be attributed to heavy metal contamination of the KCl, since ultrapure preparations of this salt yielded similar results. After testing various methods for altering the salts, we found that substitution of KAc or KGlu for KCl and MgAc₂ for MgCl₂ in splicing reactions is the simplest and most effective. Since the conditions defined here more closely mimic in vivo ionic concentrations, they may permit the study of more weakly spliced substrates, as well as facilitate more detailed analyses of spliceosome structure and function.     

Isolation and characterization of LexA mutant repressors with enhanced DNA binding affinity.

The LexA repressor from Escherichia coli is a sequence-specific DNA binding protein that shows no pronounced sequence homology with any of the known structural motifs involved in DNA binding. Since little is known about how this protein interacts with DNA, we have selected and characterized a great number of intragenic, second-site mutations which restored at least partially the activity of LexA mutant repressors deficient in DNA binding. In 47 cases, the suppressor effect of these mutations was due to an Ind- phenotype leading presumably to a stabilization of the mutant protein. With one exception, these second-site mutations are all found in a small cluster (amino acid residues 80 to 85) including the LexA cleavage site between amino acid residues 84 and 85 and include both already known Ind- mutations as well as new variants like GN80, GS80, VL82 and AV84. The remaining 26 independently isolated second-site suppressor mutations all mapped within the amino-terminal DNA binding domain of LexA, at positions 22 (situated in the turn between helix 1 and helix 2) and positions 57, 59, 62, 71 and 73. These latter amino acid residues are all found beyond helix 3, in a region where we have previously identified a cluster of LexA (Def) mutant repressors. In several cases the parental LexA (Def) mutation has been removed by subcloning or site-directed mutagenesis. With one exception, these LexA variants show tighter in vivo repression than the LexA wild-type repressor. The most strongly improved variant (LexA EK71, i.e. Glu71----Lys) that shows an about threefold increased repression rate in vivo, was purified and its binding to a short consensus operator DNA fragment studied using a modified nitrocellulose filter binding assay. As expected from the in vivo data, LexA EK71 interacts more tightly with both operator and (more dramatically) with non-operator DNA. A determination of the equilibrium association constants of LexA EK71 and LexA wild-type as a function of monovalent salt concentration suggests that LexA EK71 might form an additional ionic interaction with operator DNA as compared to the LexA wild-type repressor. A comparison of the binding of LexA to a non-operator DNA fragment further shows that LexA interacts with the consensus operator very selectively with a specificity factor of Ks/Kns of 1.4 x 10(6) under near-physiological salt conditions.     

Complexes of Escherichia coli lac-repressor with non-operator DNA revealed by electron microscopy: two repressor molecules can share the same segment of DNA.

Complexes of Escherichia coli lac-repressor with non-operator DNA have been visualized in the electron microscope using high-resolution metal shadowing and negative staining. Under conditions of a high ratio of repressor to DNA, all the DNA molecules are covered by repressor molecules and the resulting complexes appear as flattened ribbons with a width of approximately 200 Å. The overall dimensions of these complexes and their substructure indicate that it is very likely that repressor molecules are tightly packed on both “sides” of the DNA helix. Thus two repressor molecules can share the same segment of non-operator DNA by binding to opposite sides of the DNA helix.     

A model for the binding of lac repressor to the lac operator

A model is suggested for the lac repressor binding to the lac operator in which the repressor polypeptide chain sequences from Gly 14 to Ala 32 and from Ala 53 to Leu 71 are involved in specific interaction with operator DNA. A correspondence between the protein and DNA sequences is found which explains specificity of the repressor binding to the lac operator. The model can be extended to describe specific binding of other regulatory proteins to DNA.     

"Second" and "third operator" of the lac operon: an investigation of their role in the regulatory mechanism.

The influence of the two operator-like regions lying within or near the lac regulatory region on the binding of lac repressor to lac operator has been investigated. λdlac phages deleted either for the “second operator” in the beginning of the Z gene or deleted for the “third operator” at the end of the I gene were constructed. In in vitro binding experiments it could be shown that the deletion of secondary repressor binding sites from the lac regulatory region does not significantly alter the stability of the repressor—operator complex. Measuring the rate constant of association of repressor with operator in the presence of a 150-fold excess of unspecific DNA, we observed a concentration-dependent effect of the unspecific DNA, although the ratio of operator to non-operator DNA was kept constant. A small effect of the secondary binding sites is seen on the rate of association of repressor with operator, indicating that the secondary binding sites might play a role in facilitating association of repressor with operator under _{in vivo} conditions.     

Interaction between the cyclic AMP receptor protein and DNA. Conformational studies

The binding of the cyclic adenosine 3',5' monophosphate receptor protein (CRP or CAP) of Escherichia coli to non-specific DNA and to a specific lac recognition sequence has been investigated by circular dichroism (c.d.) spectroscopy. The effect of cAMP and cGMP on the co-operative non-specific binding was also studied. For the non-specific binding in the absence of cAMP a c.d. change (decrease of the intensity of the positive band with a shift of its maximum to longer wavelength) indicates that the DNA undergoes a conformational change upon CRP binding. This change might reflect the formation of the solenoidal coil previously observed by electron microscopy. The amplitude of the c.d. change increases linearly with the degree of saturation of the DNA and does not depend on the size of the clusters of CRP bound. From the variation of the c.d. effect as a function of the ionic strength, the product K omega (K, the intrinsic binding constant and omega, the co-operativity parameter) could be determined. The number of ion pairs involved in complex formation between CRP and DNA was found to be six to seven. Experiments performed with several DNAs, including the alternating polymers poly[d(A-T)] and poly[d(G-C)], demonstrated that the conformational change does not depend on the DNA sequence. However, in the presence of cAMP the c.d. spectrum of the DNA shows only a small variation upon binding CRP. In contrast, in the presence of cGMP the conformational change of the DNA is similar to that observed when non-liganded CRP binds. For the specific lac operon binding, the c.d. change is different from those observed for non-specific binding in the presence or absence of cAMP. These results emphasize the high variability of the DNA structure upon binding the same protein.