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M G Fried

Publications and source records attributed to M G Fried.

35 records · Page 2Linked to original sources
PubMed1993

Ion-exchange reactions of proteins during DNA binding.

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 displacement of cations from the DNA is the predominant form of the involvement of ions in the binding reaction. With this assumption, the graph of log K(obs) versus log [MX] is predicted to have a constant slope proportional to the number of ions displaced from the DNA upon protein binding [Record, M. T., Lohman, T. M. & deHaseth, P. L. (1976) J. Mol. Biol. 107, 145-158]. Experimental data often deviate from linearity, however, at lower salt concentrations. Such deviations can be due to differential cation binding, anion binding or changes in macromolecular hydration, or differential screening effects of the electrolyte on protein and/or DNA charges. Here the theoretical effects on K(obs) of a simple form of ion-protein interaction are examined. A model for binding interactions is used that includes a mass balance of ions bound to both protein and DNA as the protein is transferred from the salt concentration of bulk solvent to the typically higher cation and lower anion concentrations characteristic of the volume adjacent to the DNA. We show that models in which the cation and anion stoichiometries of a protein change as it associates with DNA are consistent with the curvature of plots of log K(obs) versus log [MX]. Such mechanisms could reduce the sensitivity of gene-regulatory interactions to changes in environmental salt concentration.

Anions↗
PubMed1993

Effects of anions on the binding of the cAMP receptor protein to the lactose promoter.

The DNA binding affinities of several gene-regulatory proteins, restriction endonucleases and the Escherichia coli RNA polymerase have previously been found to be dependent on the nature of the dominant buffer anion. To discover whether the E. coli cAMP receptor protein (CAP) exhibits a similar dependency, we measured its affinity for its primary lactose promoter binding site (lac site 1) in buffers in which the principal anion was chloride, phosphate, sulfate, acetate, or glutamate. We found that the affinity of CAP for lac site 1 is affected only slightly by changes in the dominant buffer anion. The binding of cAMP is similarly insensitive to buffer anion type, indicating that specific protein-anion interactions, if they occur, must be similar for the free and cAMP-bound forms of the protein. The effect of anion substitution on the ability of acrylamide to quench the intrinsic fluorescence of tryptophanyl residues of CAP is also small, suggesting that changes in buffer anion composition have minimal effect on the conformation of tryptophan-proximal regions of CAP. This conclusion is extended by the finding that anion substitution has a relatively small effect on the urea-concentration dependence of CAP denaturation. Taken together, these results support the notion that neither CAP nor CAP.cAMP nor the CAP.cAMP complex with lac promoter DNA interact selectively with anions present in the surrounding buffer. A possible role for this anion-insensitivity in the in vivo function of CAP is suggested.

Anions↗
PubMed1993

Measurement of binding kinetics using the gel electrophoresis mobility shift assay.

The gel electrophoresis mobility shift assay is a technique for the qualitative and quantitative analysis of protein-DNA complexes. The ability to resolve reactants, reaction intermediates and products makes this method particularly well-suited for the measurement of the assembly and dissociation rates of protein-nucleic acid complexes. Here we identify conditions that must be met and variations of the technique that are useful for the measurement of reaction rates.

Cyclic AMP Receptor Protein↗
PubMed1991

Prostatic acid phosphatase, beta-glucuronidase and prostate specific antigen assays in fine needle aspirates from benign and malignant prostates.

Enzymatic assays for tartrate-sensitive acid phosphatase and beta-glucuronidase, and radio-immunoassay for prostate-specific antigen, were modified for application to fine-needle aspirate samples from benign and malignant human prostates. When compared to samples from benign prostates, the ratio of acid phosphatase to beta-glucuronidase activities was significantly decreased in needle aspirates from malignant prostates. Prostate-specific antigen values in the aspirates did not correlate with malignancy.

Acid Phosphatase↗
PubMed1991

The binding of cyclic AMP receptor protein to two lactose promoter sites is not cooperative in vitro.

The lactose promoter-operator region of Escherichia coli contains two binding sites for cyclic AMP receptor protein (CAP), two for the lactose repressor, and two for RNA polymerase. The high density of binding sites makes cooperative interactions between these proteins likely. In this study, we used the gel electrophoresis mobility shift assay and binding partition analysis techniques to determine whether the secondary CAP site influences the binding of CAP to the principal CAP site in the lactose promoter when both are present on a linear DNA molecule. Such an effect could occur through the formation of a bridged DNA-CAP-DNA structure, through the interaction of CAP molecules bound to each of the sites, or through allosteric effects caused by CAP-mediated DNA bending. We found, however, that the interaction of CAP with these sites was not cooperative, indicating that CAP sites 1 and 2 bind CAP in an independent manner.

Base Sequence↗
PubMed1990

Co-operative interactions between the catabolite gene activator protein and the lac repressor at the lactose promoter.

The catabolite gene activator protein (CAP) and the lac repressor regulate the transcriptional activity of the lactose operon. An early step in the regulatory functions of these proteins is their binding to specific DNA sequences within the lac promoter-operator region. Using the gel electrophoresis mobility-shift technique, we have found that the ternary complex with CAP and repressor bound to their respective highest affinity sites is 4 to 11-fold more stable than is predicted from the affinities of the independently bound proteins. This favorable binding interaction is unexpected, because CAP and lac repressor exert opposing effects on lac operon transcription. Deoxyribonuclease I footprinting analyses show that interacting proteins remain bound to the sites occupied when the proteins bind singly. These sites have a center-to-center separation of 72 base-pairs (corresponding to 6.9 turns of a B-form DNA helix), and thus occupy the same "face" of the DNA cylinder. Such an orientation is compatible with models of the ternary complex in which DNA curvature facilitates the interaction of CAP and lac repressor.

Base Sequence↗
PubMed1990

A new DNA binding mode for CAP.

In the absence of cyclic AMP, the Escherichia coli cyclic AMP receptor protein (CAP) binds without detectable sequence specificity to restriction fragments containing lac and crp promoter sequences. Under standard conditions (10 mM Tris, 1 mM EDTA, pH 8.0), our estimates of the equilibrium constant and cooperativity parameter for complex formation are 114,000 +/- 1400 M-1 and 1.3 +/- 0.8, respectively. Thus, this interaction lacks the substantial cooperativity previously reported for CAP binding to genomic DNAs. Using the electrophoresis mobility shift assay, we find that complexes of increasing CAP content differ by a highly uniform mobility decrement. This result is most consistent with a binding mode in which little or no DNA bending occurs. The ability of CAP to distinguish between restriction fragments and genomic DNA, shown by the difference in binding cooperativity, suggests the existence of previously unsuspected DNA sequences or structures that modulate its binding cooperativity.

Binding Sites↗
PubMed1990

Cooperative interactions in transcriptional regulation.

Cooperative interactions between regulatory proteins and RNA polymerase are a common feature of transcriptional systems. We have developed a method, based on the electrophoresis mobility shift assay, for the measurement of cooperative effects in the binding of proteins to DNA restriction fragments. Using this approach we have identified a hitherto unknown interaction between the E. coli lactose repressor and CAP proteins. We suggest that this interaction plays a role in the control of the lactose operon that is not predicted by current regulatory models.

Bacterial Proteins↗
PubMed1984

Equilibrium studies of the cyclic AMP receptor protein-DNA interaction.

The binding of the Escherichia coli cyclic AMP receptor protein (CAP) to restriction fragments containing the lac promoter-operator region has been investigated as a function of cAMP concentration, using a sensitive gel electrophoresis assay. Under standard conditions (13 mM ionic strength), the equilibrium constant for CAP binding to its primary site on a 203 base-pair lac promoter fragment is 6.3 X 10(8) M-1 at 0.2 microM-cAMP, and increases to 8.4 X 10(10) M-1 at 5.0 microM-cAMP. The latter is about 10(5) times larger than the equilibrium constant for binding to an isolated, non-specific site. The L8 mutation, which renders the lac promoter unresponsive to CAP in vivo, lowers this binding affinity by five- to tenfold. Analysis of the cAMP dependency of binding over the concentration range of 0.2 microM to 10 microM reveals that uptake of a single equivalent of cAMP is required for site-specific binding. Similarly, the transfer of CAP from a non-specific DNA site to a specific site requires the net uptake of a single molecule of cAMP. In contrast, co-operative non-specific binding to DNA was found to be independent of cAMP concentration with an equilibrium binding constant of 6 X 10(6) M-1. We conclude that the cAMP affinity of the two CAP subunits in the specific promoter complex is not equal, and that the complex structure therefore deviates significantly from twofold symmetry. A model for the regulation of the lac promoter by the intracellular cAMP concentration is proposed on the basis of the equilibrium binding results.

Binding Sites↗
PubMed1984

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.

Bacterial Proteins↗
PubMed1983

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.

Base Composition↗
PubMed1983

CAP and RNA polymerase interactions with the lac promoter: binding stoichiometry and long range effects.

The binding stoichiometries of the complexes formed when the E. coli cyclic AMP receptor protein (CAP) binds to 203 bp lac promoter-operator restriction fragments have been determined. Under quantitative binding conditions, a single dimer of CAP occupies each of two sites in the promoter. Different electrophoretic mobilities are observed for 1:1 complexes formed with L8-UV5 mutant, L305 mutant, and wild type promoter fragments, indicating sequence-specific structural differences between the complexes. The differences in gel mobility between L8-UV5 and wild type complexes disappear when the promoter fragments are cleaved with Hpa II restriction endonuclease. Models in which CAP alters DNA conformation or in which CAP forms a transient intramolecular bridge between two domains of a DNA molecule could account for these observations. The selective binding of RNA polymerase to CAP-promoter complexes is demonstrated: the binding of a single CAP dimer to the promoter is sufficient to stimulate subsequent polymerase binding. Functional CAP molecules are not released from the promoter on polymerase binding.

Carrier Proteins↗
PubMedDate not supplied

Measurement of protein-DNA interaction parameters by electrophoresis mobility shift assay.

Native gel electrophoresis (mobility shift) assays may be used to obtain quantitative information about the site distribution, equilibria and kinetics of protein-DNA interactions. These applications depend on the ability of the electrophoretic system to resolve the reaction components, and on their stabilities during the separation process. Factors which affect the lifetimes and mobilities of protein-DNA complexes during electrophoresis include reaction and electrophoresis buffer composition, pH, and ionic strength; the presence of low molecular weight effectors and enzymatic substrates; the nature and concentration of the gel matrix; the temperature; the molecular weights of protein and DNA; the stoichiometric ratios of their complexes; and the possibility of conformational and configurational isomerization of reaction components. We discuss how these factors influence the acquisition of quantitative data from electrophoretic patterns and band intensities, and present formulas for the estimation of equilibrium constants and rate constants for prototypical DNA-protein interactions.

Binding, Competitive↗