In vitro interaction of the Escherichia coli cyclic AMP receptor protein with the lactose repressor

Sedimentation equilibrium studies show that the Escherichia coli cyclic AMP receptor protein (CAP) and lactose repressor associate to form a 2:1 complex in vitro. This is, to our knowledge, the first demonstration of a direct interaction of these proteins in the absence of DNA. No 1:1 complex was detected over a wide range of CAP concentrations, suggesting that binding is highly cooperative. Complex formation is stimulated by cAMP, with a net uptake of 1 equivalent of cAMP per molecule of CAP bound. Substitution of the dimeric lacI-18 mutant repressor for tetrameric wild-type repressor completely eliminates detectable binding. We therefore propose that CAP binds the cleft between dimeric units in the repressor tetramer. CAP-lac repressor interactions may play important roles in regulatory events that take place at overlapping CAP and repressor binding sites in the lactose promoter.     

The catabolite activator protein stabilizes its binding site in the E. coli lactose promoter.

The effect of catabolite activator protein, CAP, on the thermal stability of DNA was examined. Site specific binding was studied with a 62 bp DNA restriction fragment containing the primary CAP site of the E. coli lactose (lac) promoter. A 144 bp DNA containing the lac promoter region and a 234 bp DNA from the pBR322 plasmid provided other DNA sites. Thermal denaturation of protein-DNA complexes was carried out in a low ionic strength solvent with 40% dimethyl sulfoxide, DMSO. In this solvent free DNA denatured below the denaturation temperature of CAP. The temperature stability of CAP for site specific binding was monitored using an acrylamide gel electrophoresis assay. Results show that both specific and non-specific CAP binding stabilize duplex DNA. Site specific binding to the 62 bp DNA produced a 13.3 degrees C increase in the transition under conditions where non-specific binding stabilized this DNA by 2-3 degrees C.     

The Role of Protein-Ligand Contacts in Allosteric Regulation of the Escherichia coli Catabolite Activator Protein

Allostery is a fundamental process by which ligand binding to a protein alters its activity at a distant site. Both experimental and theoretical evidence demonstrate that allostery can be communicated through altered slow relaxation protein dynamics without conformational change. The catabolite activator protein (CAP) of Escherichia coli is an exemplar for the analysis of such entropically driven allostery. Negative allostery in CAP occurs between identical cAMP binding sites. Changes to the cAMP-binding pocket can therefore impact the allosteric properties of CAP. Here we demonstrate, through a combination of coarse-grained modeling, isothermal calorimetry, and structural analysis, that decreasing the affinity of CAP for cAMP enhances negative cooperativity through an entropic penalty for ligand binding. The use of variant cAMP ligands indicates the data are not explained by structural heterogeneity between protein mutants. We observe computationally that altered interaction strength between CAP and cAMP variously modifies the change in allosteric cooperativity due to second site CAP mutations. As the degree of correlated motion between the cAMP-contacting site and a second site on CAP increases, there is a tendency for computed double mutations at these sites to drive CAP toward noncooperativity. Naturally occurring pairs of covarying residues in CAP do not display this tendency, suggesting a selection pressure to fine tune allostery on changes to the CAP ligand-binding pocket without a drive to a noncooperative state. In general, we hypothesize an evolutionary selection pressure to retain slow relaxation dynamics-induced allostery in proteins in which evolution of the ligand-binding site is occurring.     

DNA bending and expression of the divergent nagE-B operons.

Repression of the divergent nagE - B operons requires NagC binding to two operators which overlap the nagE and nagB promoters, resulting in formation of a DNA loop. Binding of the cAMP/CAP activator to its site, adjacent to the nagE operator, stabilizes the DNA loop in vitro. The DNA of the nagE-B intergenic region is intrinsically bent, with the bend centred on the CAP site. We show that displacement of the CAP site by 6 bp results in complete derepression of the two operons. This derepression is observed even in the absence of cAMP/CAP binding and despite the fact that the two NagC operators are still in phase, demonstrating that the inherently bent structure of the DNA loop is important for repression. Since no interaction between NagC and CAP has been detected, we propose that the role of CAP in the repression loop is architectural, stabilizing the intrinsic bend. The cAMP/CAP complex is necessary for activation of the nagE-B promoters. In this case protein-protein contacts between CAP and RNA polymerase are necessary for full activation, but at least a part of the activation is likely due to an effect of CAP binding altering DNA structure.     

真核基因转录激活的多位点协同调控

真核基因的转录激活具有协同性的特征,表现为多个转录调控位点的共同作用效果大于每个位点单独作用之和,受多个位点调控的基因转录呈S型曲线.多个激活蛋白之间的相互作用、激活蛋白与各DNA位点的协同结合以及激活蛋白与转录机器的协同作用,三种途径都对协同性转录激活行为产生影响.协同性转录激活的本质是多个结合在调控位点上的激活蛋白之间直接或间接的相互作用.多位点的协同转录调控机制有助于理解生物的多种调控过程和建立基因调控网络.     

Chemical probe and missing nucleoside analysis of Flp recombinase bound to the recombination target sequence.

The Flp protein catalyzes a site-specific recombination reaction between two 47 bp DNA sites without the assistance of any other protein or cofactor. The Flp recognition target (FRT) site consists of three nearly identical sequences, two of which are separated by an 8 bp spacer sequence. In order to gain insight into this remarkable protein-DNA interaction we used a variety of chemical probe methods and the missing nucleoside experiment to examine Flp binding. Hydroxyl radical footprints of Flp bound to a recombinationally-competent site fall on opposite faces of canonical B-DNA. The 8 bp spacer region between the two Flp binding sites becomes reactive towards 5-phenyl-1,10-phenanthroline.copper upon Flp binding, indicating that once bound by Flp, this segment of DNA is not in the B-form. Missing nucleoside analysis reveals that within each binding site the presence of two nucleosides on the top strand and four on the bottom, are required for formation of a fully-occupied FRT site. In contrast, loss of any nucleoside in the three binding sites in the FRT interferes with formation of lower-occupancy complexes. DNA molecules with gaps in the 8 bp spacer region are over-represented in complexes with either two or three binding sites occupied by Flp, evidence that DNA flexibility facilitates the cooperative interaction of Flp protomers bound to a recombinationally-active site.     

Consensus DNA site for the Escherichia coli catabolite gene activator protein (CAP): CAP exhibits a 450-fold higher affinity for the consensus DNA site than for the E. coli lac DNA site.

We have synthesized two 40 base pair DNA fragments; one fragment contains the consensus DNA site for CAP (fragment 'ICAP'); the other fragment contains the E. coli lac promoter DNA site for CAP (fragment 'LCAP'). We have investigated the binding of CAP to the two DNA fragments using the nitrocellulose filter binding assay. Under standard conditions [( NaCl] = 200 mM, pH = 7.3), CAP exhibits a 450-fold higher affinity for ICAP than for LCAP. The salt dependence of the binding equilibrium indicates that CAP makes eight ion pairs with ICAP, but only six ion pairs with LCAP. Approximately half of the difference in binding free energy for interaction of CAP with ICAP vs. LCAP is attributable to this difference in ion-pair formation. The pH dependence of the binding equilibrium indicates that the eight CAP-ICAP ion pairs and the six CAP-LCAP ion pairs do not involve His residues of CAP.     

Solution conformations of DNAs containing binding sites of the catabolite gene activator protein and lac repressor protein: characterization by Raman spectroscopy

Raman spectra from three subfragments of the Escherichia coli lactose promoter region were obtained in 0.1 M NaCl. The three DNAs are 21, 40, and 62 bp in length. The 21 and 62 bp DNAs contain the binding site for the catabolite gene activator protein (CAP). The 40 bp DNA contains the binding site for the lac repressor. A quantitative analysis of Raman band characteristics indicates an overall B-type conformation for these gene regulatory sites. Bands which correspond to A-family (807 cm-1) and B-family (834 cm-1) deoxyribose phosphate vibrations have the same intensities as bands found in heterogeneous DNAs. The spectra of the 21 bp CAP site have, however, a small band at 867 cm-1 and several other small differences similar to some characteristics observed in C-DNA spectra. Several dG nucleosides in the CAP site appear to be altered from the conventional C2'-endo/anti conformation. At 45 degrees C, well below the melting region of these DNAs, small changes occur in the spectra of the 40 bp lac repressor site which are not observed in the other DNAs. A weak band occurs at 705 cm-1, and intensity changes are observed at 497, 682, and 792 cm-1. The changes suggest that the conformations of several dG nucleosides are altered and that a small region may exist with characteristics of an A-family backbone. This conformational change at 45 degrees C coincides with previous NMR observations indicating an enhanced imino proton exchange rate at a GTG sequence within the lac operator site.     

Separate domains in E1 and E2 proteins serve architectural and productive roles for cooperative DNA binding

The E1 and E2 proteins from bovine papillomavirus bind cooperatively to binding sites in the viral origin of DNA replication. The DNA-binding domains (DBDs) of the two proteins interact with each other, and the E2 transactivation domain interacts with the helicase domain of E1. Mutations that disrupt the interaction between the two DBDs also disrupt the interaction between the E2 activation domain and the E1 helicase domain, demonstrating interdependence of the two interactions. Cooperative binding of the two DBDs generates a sharp bend in the DNA that is required for interaction between the E2 activation domain and E1. This indicates that interaction between the two DBDs plays an architectural role, 'triggering' a productive interaction between the E2 transactivation domain and E1 through introduction of a sharp bend in the DNA. This two-step mechanism may be a required feature for cooperative DNA binding to proximal binding sites.     

Binding of SeqA protein to hemi-methylated GATC sequences enhances their interaction and aggregation properties

The SeqA protein regulates chromosome initiation and is involved in segregation in Escherichia coli. One SeqA protein binds to two hemi-methylated GATC sequences to form a stable SeqA-DNA complex. We found that binding induced DNA bending, which was pronounced when the two sequences were on the same face of the DNA. Two SeqA molecules bound cooperatively to each pair of hemi-methylated sites when the spacing between the sites was < or = 30 bp. This cooperative binding was able to stabilize the binding of a wild type to a single hemi-methylated site, or mutant form of SeqA protein to hemi-methylated sites, although such binding did not occur without cooperative interaction. Two cooperatively bound SeqA molecules interacted with another SeqA bound up to 185 bp away from the two bound SeqA proteins, and this was followed by aggregation of free SeqA proteins onto the bound proteins. These results suggest that the stepwise interaction of SeqA proteins with hemi-methylated GATC sites enhances their interaction and leads to the formation of SeqA aggregates. Cooperative interaction followed by aggregation may be the driving force for formation of the SeqA foci that appear to be located behind replication forks.     

CAP binding to B and Z forms of DNA.

We have examined the interaction between the cyclic AMP receptor protein (CAP) and a small DNA fragment containing its specific recognition sequence by circular dichroism spectroscopy. The binding of CAP to this fragment induces a B to "C-like" change in the CD spectrum, which is different from that observed for non-specific binding. A one-to-one (CAP dimer to DNA) binding stoichiometry was deduced from spectroscopic titration data, as was a non-specific binding site size of 17 bp/dimer. In addition, we have compared the non-specific binding affinity of CAP for the B and Z forms of synthetic DNA copolymers. A slight preference for the B form was found. These results do not support the recent specific suggestion that CAP binds to a left-handed form of DNA (1), but indicate more generally that an optically detectable conformational change takes place in DNA on binding CAP.     

CAP binding sites reveal pyrimidine-purine pattern characteristic of DNA bending

To investigate the intrinsic bending of DNA at sites where proteins bind, we analyzed catabolite gene activator protein (CAP) binding sites and various operators from the viewpoint of DNA bending flexibility. Theoretical conformational analysis. DNase I digestion and x-ray crystallography data indicate that bending of B-DNA is highly anisotropic and sequence-dependent. Certain dimers prefer to bend into the major groove ("major-philic") and others prefer to bend into the minor groove ("minor-philic" dimers). From these data we considered TA, CG, CA:TG and GG:CC as major-philic dimers and AT,AA:TT and GT:AC as minor-philic ones. Analysis of 31 CAP binding sites has identified strong major-philic tendencies 5-7 base pairs (bp) away from the center. In addition, we found minor-philic poly-A tracts extending 4-5 bp away from the proposed major-philic bends. Finally, to analyze the central regions we followed the lead of Shumilov and classified the DNA sites by their spacer lengths [V.Y. Shumilov, Mol. Biol. (Mosk) 21, 168-187 (1987)]. In this way, we identified two subsets of CAP binding sites: one with 6 bp between the TGTGA:TCACA consensus boxes (N6-set) and one with 8 central bp (N8-set). We discovered that the dimer at the center of an N6-set site was usually major-philic, whereas at the center of an N8-set site more often minor-philic. Analysis of phages 434, P22 lambda and trp operators revealed similar results. In conclusion, our data show that CAP binding sites have major-philic and minor-philic dimers at specific positions; the location of these dimers may facilitate wrapping of DNA around CAP. A similar pattern is seen in nucleosomes.     

A model for the non-specific binding of catabolite gene activator protein to DNA

The binding of E. coli catabolite gene activator protein (CAP) to non-specific sequences of DNA has been modelled as an electrostatic interaction between four basic side chains of the CAP dimer and the charged phosphates of DNA. Calculation of the electrostatic contribution to the binding free energy at various separations of the two molecules shows that complex formation is favored when CAP and DNA are separated by as much as 12 A. Thus, the long range electrostatic interactions may provide the initial energy for complex formation and also the correct relative orientation of CAP and DNA. The non-specific complex does not involve the penetration of amino acid side chains into the major grooves of DNA and permits 'sliding' of the protein along DNA, which would enhance the rate of association of CAP with the specific site as has been proposed previously for lac repressor. We propose that, as it 'slides', CAP is moving in and out of the major grooves in order to sample the DNA sequence. Recognition of the specific DNA site is achieved by a complementarity in structure and hydrogen bonding between amino acids and the edges of base pairs exposed in the major grooves of DNA.     

Cation binding linked to a sequence-specific CAP-DNA interaction

The equilibrium association constant observed for many DNA-protein interactions in vitro (K(obs)) is strongly dependent on the salt concentration of the reaction buffer ([MX]). This dependence is often used to estimate the number of ionic contacts between protein and DNA by assuming that release of cations from the DNA is the dominant involvement of ions in the binding reaction. With this assumption, the graph of logK(obs) versus log[MX] is predicted to have a constant slope proportional to the number of ions released from the DNA upon protein binding. However, experimental data often deviate from log-linearity at low salt concentrations. Here we show that for the sequence-specific interaction of CAP with its primary site in the lactose promoter, ionic stoichiometries depend strongly on cation identity and weakly on anion identity. This outcome is consistent with a simple linkage model in which cation binding by the protein accompanies its association with DNA. The order of ion affinities deduced from analysis of DNA binding is the same as that inferred from urea-denaturation experiments performed in the absence of DNA, suggesting that ion binding to free CAP contributes significantly to the ionic stoichiometry of DNA binding. In living cells, the coupling of ion-uptake and DNA binding mechanisms could reduce the sensitivity of gene-regulatory interactions to changes in environmental salt concentration.     

The transmission of stability or instability from site specific protein-DNA complexes.

Theoretical calculations were made to determine the influence of side specific 'melting' and 'stabilizing' proteins on the thermal stability of nearby base pairs (bp). A DNA sequence 999bp. long containing the 123 bp. lactose operon control region in the center was examined. Melting curves of base pairs near the binding sites of the catabolite activator protein, CAP, the lactose repressor, and RNA polymerase were calculated in the absence and presence of each protein. The empirical loop entropy model of the helix-coil transition of DNA was employed. Calculations show that melting and stabilizing proteins alter the tm of base pairs 20 to 100 bp-away. The magnitude and range of the effect is strongly influenced by the base pair composition and sequence of the protein site and the immediately adjacent DNA regions.