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M T Record

Publications and source records attributed to M T Record.

At least 91 records · Page 5Linked to original sources
PubMed1985

Ions as regulators of protein-nucleic acid interactions in vitro and in vivo.

The key feature of the kinetics and equilibria of both specific and non-specific noncovalent interactions of proteins with nucleic acids is their sensitivity to the details of the ionic environment. Investigation of the effects of ion concentrations provides detailed and otherwise unobtainable information about the thermodynamics and mechanisms of these interactions. We discuss the molecular and thermodynamic basis of the contribution to these ion effects from electrolyte-nucleic acid interactions, and demonstrate that a simple ion exchange formalism, involving the stoichiometric participation of individual ions, is the appropriate basis for interpreting these profound effects at a thermodynamic level. Since the in vivo ionic environment is both complex and variable, we propose that variations in intracellular concentrations of individual ions play both global and specific roles in the control of the protein-nucleic acid interactions responsible for nucleoprotein structure and gene expression.

DNA, Bacterial↗
PubMed1984

23Na NMR relaxation study of the effects of conformation and base composition on the interactions of counterions with double-helical DNA.

NMR relaxation rates (T1(-1) and T2(-1)) have been determined for 23Na in aqueous salt solutions containing various types of helical double-stranded deoxyribonucleic acids. These measurements were performed on three synthetic polynucleotides having different overall conformations, poly-(dA-dT).poly(dA-dT) (alternating B-DNA), poly(dG-dC).poly(dG-dC) at low salt (B-DNA), and Br-poly(dG-dC).Br-poly(dG-dC) (left-handed Z-DNA), and on four types of natural DNA differing in base composition, Clostridium perfringens (26% GC), calf thymus (40% GC), Escherichia coli (50% GC), and Micrococcus lysodeikticus (72% GC). For all types of DNA investigated, except poly(dA-dT).poly(dA-dT), the 23Na NMR spectra measured at 21 degrees C and an applied field of 4.7 T are non-Lorentzian. These non-Lorentzian spectra were analyzed on the basis of the two-state model and the standard theory of nonexponential quadrupolar relaxation processes in order to obtain estimates of the correlation times (tau c) characteristic of the sodium nuclei associated with the various nucleic acids. All of the correlation times estimated in this way are in the range of nanoseconds. The magnitudes of these correlation times show a significant dependence on the overall conformation of the nucleic acid (B vs. Z) but not on its base composition. To investigate the concentration dependence of tau c, sodium or magnesium salts were added to solutions of Br-poly(dG-dC).Br-poly(dG-dC) (Z-DNA).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Kinetics and mechanism of the interaction of Escherichia coli RNA polymerase with the lambda PR promoter.

The kinetics of formation and dissociation of specific (open) complexes between active Escherichia coli RNA polymerase holoenzyme (RNAP) and the lambda PR promoter have been studied by selective nitrocellulose filter binding assays at two temperatures (25 degrees C, 37 degrees C) and over a range of ionic conditions. Competition with a polyanion (heparin) or stabilization of open promoter complexes at PR by incubation with specific combinations of nucleoside triphosphates was employed to obtain selectivity in the filter assay. This study provides a useful example of how information about mechanism may be obtained from the quantitative analysis of the effects of salt concentration and temperature on the rate constants of a protein-DNA interaction. The association reaction between RNAP and lambda PR was investigated under ionic conditions where the process is essentially irreversible, and under pseudo first-order conditions of excess polymerase. The pseudo first-order rate constant is directly proportional to the concentration of active polymerase over the entire range investigated (2 to 10 nM) at both 25 degrees C and 37 degrees C, within experimental uncertainty. Second-order association rate constants (ka), calculated from these data at standard ionic conditions (0.12 M-KCl, 0.01 M-MgCl2, 0.04 M-Tris (pH 8)), were strongly temperature-dependent: ka = (2.6 +/- 0.4) X 10(6) M-1 S-1 at 37 degrees C and ka = (7.2 +/- 1.4) X 10(5) M-1 s-1 at 25 degrees C, corresponding to an activation energy of the association reaction of approximately 20 +/- 5 kcal. In addition, ka decreases strongly with increasing KCl concentration, corresponding to the net release of the thermodynamic equivalent of at least nine monovalent ions prior to or during the rate-limiting step of the association reaction. This strong dependence of ka on the ionic environment suggests that inorganic cations should be considered as possible regulators of in vivo transcription initiation. Dissociation rate constants (kd) were also measured under irreversible reaction conditions. At the standard ionic conditions, kd = (2.2 +/- 0.3) X 10(-5) s-1 at 37 degrees C and kd = (4.0 +/- 0.4) X 10(-5) s-1 at 25 degrees C. The increase in kd with decreasing temperature corresponds to a negative activation energy of dissociation (-9 +/- 4 kcal). In addition, kd increases with increasing KCl concentration, corresponding to the net uptake of the thermodynamic equivalent of at least six monovalent ions in or prior to the rate-limiting step of the dissociation reaction.(ABSTRACT TRUNCATED AT 400 WORDS)

Bacteriophage lambda↗
PubMed1983

Direct evidence for the preferential binding of Escherichia coli RNA polymerase holoenzyme to the ends of deoxyribonucleic acid restriction fragments.

Escherichia coli RNA polymerase holoenzyme has been observed to form a variety of nonpromoter complexes with DNA restriction fragments in experiments performed with the nitrocellulose filter assay [Melançon, P., Burgess, R. R., & Record, M. T., Jr. (1982) Biochemistry 21, 4318-4331]. Here we report the use of this assay to investigate aspects of the weak (heparin-sensitive) interactions of RNA polymerase core and holoenzyme with a 1600 base pair (bp) fragment of T7 DNA which contains no promoters or TB (tight binding; heparin-resistant) sites. Under the ionic conditions investigated [50 mM NaCl/10 mM MgCl2/10 mM sodium N-(2-hydroxyethyl)piperazine-N'-ethanesulfonic acid (pH 7.7)], both core and holoenzyme bind to the linear DNA fragment and cause comparable levels of filter retention. When the DNA fragment is self-ligated into a circular molecule (nonsupercoiled), the extent of binding of holoenzyme (but not that of core) is dramatically reduced. This directly proves our previous hypotheses that holoenzyme recognizes and preferentially binds to the ends of DNA fragments and that this mode of binding is responsible for most of the heparin-sensitive filter retention of nonpromoter fragments. The residual mode of binding of holoenzyme detected with the circular DNAs was considered in determining the amount of protein bound at ends only. To calculate end-binding constants (KE), the amount of protein bound nonspecifically (which does not appear to cause efficient filter retention) was also taken into consideration. At 0 degrees C, we obtain a value for KE of (2.1 +/- 0.5) X 10(8) M-1, in good agreement with that determined earlier.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA↗
PubMed1982

Aggregation equilibria of Escherichia coli RNA polymerase: evidence for anion-linked conformational transitions in the protomers of core and holoenzyme.

The aggregation equilibria of Escherichia coli RNA polymerase core and holoenzyme have been studied by velocity sedimentation as a function of [NaCl] both in the presence and in the absence of MgCl2. Effects of other anions (F- and I-), pH, and temperature have also been examined. Diffusion coefficients obtained by quasi-elastic light scattering (QLS) at high and low salt concentrations were used in conjunction with sedimentation coefficients under these conditions to obtain molecular weights of the protomer and aggregates of the core enzyme. At low salt concentration, core aggregates to a tetramer in the absence of MgCl2 and to an octamer in the presence of MgCl2. Some ambiguity exists in the interpretation of the sedimentation and QLS data for holoenzyme. The sedimentation results are consistent with the formation of dimers at low salt, both in the presence and in the absence of MgCl2. In all cases, equilibrium constants were calculated assuming a simple monomer--j-mer stoichiometry. These equilibrium constants are extremely sensitive functions of the concentration and type of monovalent anion. In Cl-, aggregation of both core and holoenzyme begins abruptly when the salt concentration is reduced below approximately 0.2 M (at a protein concentration of approximately 0.30 mg/mL); for core, substitution of I- for Cl- suppresses aggregation while F- enhances aggregation at a fixed anion concentration. No specific effect of monovalent cations (Na+, NH4+) is observed; Mg2+ has no effect on holoenzyme dimerization and has little effect on the salt range of core aggregation, though the stoichiometries of the core aggregates in the presence and absence of Mg2+ differ. Anion effects on these equilibria were modeled by assuming that a class of anion-binding sites on the protomer is not present in the aggregate, so that anion release accompanies aggregation. Analytical expressions for several models of the effect of anions on the aggregation equilibria were derived by using the method of binding polynomials. The salt dependence of the aggregation equilibria in the absence of Mg2+ appears inconsistent with a model in which the anion-binding sites on the protomer are independent (noncooperative), but it is well described by a model in which anion binding to the protomers occurs in a completely cooperative manner. The molecular basis of this apparent cooperative effect of anions on the aggregation equilibria is proposed to be an allosteric effect of anions on conformational equilibria of the protomers of core polymerase and the holoenzyme. Implications of such a salt-dependent conformational transition for the DNA-binding interactions of the enzyme are considered.

Anions↗
PubMed1980

Binding of Escherichia coli ribonucleic acid polymerase holoenzyme to a bacteriophage T7 promoter-containing fragment: selectivity exists over a wide range of solution conditions.

The selectivity of binding of Escherichia coli RNA polymerase holoenzyme to a promoter-containing fragment of T7 DNA has been investigated over a range of solution conditions by using a double-label nitrocellulose filter binding assay. A 32P-labeled HaeIII restriction fragment of T7 D111 DNA containing the A1 and D promoters for the E. coli enzyme and a 3H-labeled nonpromoter HaeIII fragment of comparable size were incubated with sigma-saturated holoenzyme and filtered through a nitrocellulose membrane filter. We find that the extent of binding of polymerase to the promoter-containing fragment decreases dramatically with increasing salt concentrations and with increasing pH and increases moderately with increasing temperature in the range 0-37 degrees C. By contrast, the nonspecific interaction of polymerase with the nonpromoter fragment is known to be relatively insensitive to pH and temperature, though a strong function of salt concentration [deHaseth, O. L., Lohman, T. M., Burgess, R. R., & Record, M. T., Jr. (1978) Biochemistry 17, 1612-1622]. Selectivity of binding of RNA polymerase in our assay is demonstrated by a greater fractional retention of the promoter-containing fragment than of the nonpromoter fragment on the filter. We observe selective binding over the temperature range from 0 to 37 degrees C near neutral pH and over a wide range of Na+ concentrations, in the presence or absence of Mg2+. Because of the different dependences of promoter and nonpromoter binding on pH and temperature, the extent of selectivity increases with increasing temperature and decreases with increasing pH. Quantitative treatment of these binding data [Strauss, H. S., Burgess, R. R., & Record, M. t., Jr. (1980) Biochemistry (second paper of four in this issue)] confirms these conclusions and shows that selectivity is a function of ion concentration as well.

DNA Restriction Enzymes↗
PubMed1980

Binding of Escherichia coli ribonucleic acid polymerase holoenzyme to a bacteriophage T7 promoter-containing fragment: evaluation of promoter binding constants as a function of solution conditions.

In this paper we obtain thermodynamic and molecular information about the specific complexes formed between Escherichia coli RNA polymerase holoenzyme and a restriction fragment of T7 D111 DNA carrying the A1 and D promoters. Specific binding was observed at both 0 and 37 degrees C over a side range of pH values and ion concentrations [Strauss, H. S., Burgess, R. R., & Record, M. T., Jr. (1980) Biochemistry (first paper of four in this issue)]. The specific complexes formed at these two temperatures may correspond to the closed and open promoter complexes discussed by Chamberlin [Chamberlin, M. J. (1976) RNA Polymerase (Losick, R., & Chamberlin, M., Eds.) pp 159-161, Cold Spring Harbor Laboratory, cold Spring Harbor, NY]. Promoter binding constants KobsdRP are obtained from competition filter binding data by using a statistical analysis and previously determined values of the nonspecific holoenzyme-DNA binding constant KobsdRD. From the magnitudes of KobsdRP at 0 and 37 degrees C, and the dependences of these binding constants on pH and ion concentrations, we conclude that, under physiological ionic conditions, both the 0 and the 37 degrees C complexes are stabilized to a large extent by the formation of ionic interactions and the accompanying release of counterions and that one or two protonation events (pK approximately 7.4) are required for complex formation in both cases. However, the 0 and 37 degrees C complexes differ in their sensitivity to ion concentrations as well as in the magnitude of KobsdRP, and we conclude that the two complexes are distinct. (More counterion release accompanies formation of the 37 degrees C complex). Comparisons of the two complexes with one another and with nonspecific holoenzyme-DNA complexes are drawn from the binding data. We have also examined the equilibrium selectivity ratio (KobsdRP/DobsdRD) and find it to be a sensitive function of temperature and ionic conditions. Selectivity of holoenzyme for promoter sites on the promoter-containing fragment is higher at 37 degrees C than at 0 degrees C under the conditions investigated. Selectivity at either temperature is increased by reducing the pH (in the range 6.1-8.6). At 37 degrees C, selectivity is increased by reducing the salt concentration. Under approximately physiological conditions (0.2 M NaCl and 0.003 M MgCl2, pH 7.4, 37 degrees C), the equilibrium selectivity ratio is found to be of order of magnitude 10(4).

DNA, Viral↗
PubMed1980

Use of difference boundary sedimentation velocity to investigate nonspecific protein-nucleic acid interactions.

The difference boundary sedimentation velocity technique of Schachman and co-workers is demonstrated to be applicalbe to the measurement of binding constants (Kobsd) in the range 10(2)-10(5) M(-1) for the nonspecific interactions of proteins with DNA. The difference technique can reproducibly detect a 2% change in the sedimentation coefficient of the DNA upon binding ligands, corresponding to average extents of association as low as 10 molecules of protein (in the cases of Escherichia coli lac repressor and E. coli RNA polymerase) per molecule of bacteriophage T7 DNA. At these low binding densities, it is plausible to assume that the primary effect of ligand binding is on the buoyant mass of the complex and not on the frictional coefficient of the flexible DNA coil. Binding constants calculated by using this assumption agree well with literature values for the nonspecific interactions of RNase and lac repressor proteins with double-stranded DNA. Advantages of the method are that it is relatively rapid, requires the optical detection of the DNA only, and can be performed on small amounts of sample. The method appears useful for surveying (to an accuracy of +/-50% in Kobsd or +/-10% in log Kobsd) the effects of solution variables on Kobsd of protein-DNA interactions. Applications of the method to the nonspecific interactions of RNA polymerase core and holoenzymes with T7 DNA are discussed.

Animals↗
PubMed1980

The relationship between the poisson-boltzmann model and the condensation hypothesis: an analysis based on the low salt form of the Donnan coefficient.

Two common models for the interaction of counterions with cylindrical polyions are considered in the context of the Donnan membrane equilibrium. General analytic expressions are obtained from the Poisson-Boltzmann equation for the Donnan coefficient in terms of the potential at the surface of the polyion or the local concentration of unbound ions at the surface. Analysis based on these expressions shows that if, and only if, the polyion charge density exceeds a certain critical value a large local concentration of ions will persist near the polyion surface at low ionic strengths. We therefore conclude that this principal hypothesis of the condensation model is consistent with the characteristics of the Poisson-Boltzmann potential at the surface of the polyion.

Journal Article↗
PubMed1980

Mg NMR in DNA solutions: Dominance of site binding effects.

(25)Mg NMR spectroscopy is applied to a study of magnesium ion interactions with DNA, which is considered as a model for a linear polyelectrolyte. It is demonstrated that the magnesium ion spectrum is complicated by a non-Lorent-zian line shape and is dominated by the effects of chemical exchange with macromolecule binding sites. A distinction is made between specific-site interactions in which the magnesium ion loses a water molecule from the first coordination sphere on binding and those interactions, referred to as territorial binding, in which the ion maintains its first coordination sphere complement of solvent. The first type of site-binding interactions are shown to dominate the magnesium ion NMR spectrum, based on a consideration of the magnitudes of the observed (25)Mg relaxation rates compared with (23)Na relaxation rates, the clear contributions of chemical exchange-limited relaxation, and an ion displacement experiment employing sodium.

Journal Article↗
PubMed1980

Relative binding affinities of monovalent cations for double-stranded DNA.

The competition between sodium and various other monovalent cations that bind to helical DNA in aqueous solution has been studied by (23)Na NMR. Variations in the sodium linewidth with the concentration of the other ion have been analyzed with an equation that describes the competitive binding in terms of two parameters: r, the total extent of counterion binding, and D, a measure of the binding affinity of a cation relative to sodium. The concentration dependence of these parameters was found to be minimal. In the absence of a competing cation the constancy of r has been demonstrated over a range of DNA phosphate concentrations (0.0025-0.015 M) and NaCl concentrations (0.003-1.3 M). For the cations investigated the range in D values is small (0.5-0.9), and the relative binding affinities follow the order: NH(4) (+) > Cs(+) > K(+) > Li(+) > Na(+).

Journal Article↗
PubMed1978

Nonspecific interactions of Escherichia coli RNA polymerase with native and denatured DNA: differences in the binding behavior of core and holoenzyme.

We have investigated the nonspecific interactions of Escherichia coli RNA polymerase core and holoenzyme with double-stranded (ds) and single-stranded (ss) DNA. Binding constants for these interactions as functions of such solution variables as monovalent and/or divalent cation concentration, temperature, or pH were determined by the method of deHaseth et a. [deHaseth, P.L., Gross, C.A., Burgess, R.R. and Record, M.T. (1977), Biochemistry 16, 4777--4783] from analysis of the elution of the proteins from small columns containing immobilized DNA. This technique, although as yet empirical, has been demonstrated to yield accurate binding constants fot the nonspecific interation of lac repressor with ds DNA. We find that observed binding constants (Kobsd) are extraordinarily sensitive functions of the monovalent cation concentration for the interactions of both core and holoenzyme with ds DNA. In the absence of divalent cations, the derivatives --(d log Kobsd/d log [Na+]) are 11 +/- 2 for the holo--ds DNA interaction and 21 +/- 3 for the core--ds DNA interaction. Consequently, approximately 11 and 21 low-molecular-weight ions are released, iin the thermodynamic sense, in the formation of the holo--ds and core--ds complexes, respectively (Record, M.T., Jr., Lohman, T.M., and deHaseth, P.L. (1976), J. Mol. Biol. 107, 145--158; Record, M.T., Jr., Anderson, C.F., and Lohman, T.M. (1978), Q. Rev. Biophys., in press). Ion release is a thermodynamic driving force for these nonspecific interactions and causes the stability of the complexes to increase very substantially with a reduction in monovalent ion concnetration. Possible molecular models which account for the different salt sensitivities of the holo--ds and core--ds complexes are discussed. Effects of the competitive ligand Mg2+ on these interactions are also examined. Substantial ion release (approximately 18 monovalent ions) also accompanies the interaction of either holo or core polymerase with ss DNA. Over the range of ion concentrations investigated the holo--ss interaction is substantially stronger than the core--ss interaction; furthermore, we conclude that the interactions of polymerase with ss DNA are, in general, stronger than the nonspecific interations of the enzyme with ds DNA. It is likely that the nonspecific interactions of RNA polymerase with DNA have physiological relevance. Not only is it plausible to assume that the same regions of the protein are involved in both specific and nonspecific interactions, but in addition nonspecific interactions of RNA polymerase and DNA may play role in determining the availability of this protein, in both the thermodynamic and the kinetic sense, for promoter binding and RNA chain initiation [von Hippel. P.H., Revzin, A., Gross, C.A., and Wang, A.C. (1974), Proc. Natl. Acad. Sci U.S.A. 71, 4808--4812]. Consequently, the strong dependences of the nonspecific interactions of RNA polymerase on ionic conditions suggest the possibility of a modulating role of ion concentrations in the control of transcription.

DNA↗
PubMed1978

Analysis of ion concentration effects of the kinetics of protein-nucleic acid interactions. Application to lac repressor-operator interactions.

The effects of monovalent and divalent cations on the bimolecular rate constant of the reaction of a positively charged ligand with a nucleic acid polyanion are analyzed for two possible reaction mechanisms. One mechanism postulates that the association reaction occurs without intermediates, and that ion effects on the rate constant result entirely from the screening of the charged reactants by ionic atmospheres of low molecular weight ions (a screening-controlled mechanism). This mechanism is analyzed by analogy with the Bronsted-Bjerrum theory for the kinetics of interaction of low molecular weight ions. The second mechanism to be considered here postulates the existence of a ligand-DNA intermediate which is in rapid equilibrium with the reactants (pre-equilibrium mechanism). Ion concentration effects on the association rate constants for the pre-equilibrium mechanism result mainly from the release of counterions from the DNA upon formation of the intermediate. Both of the above mechanisms predict that the logarithm of the association rate constant, ka, will be a linear function of the logarithm of the monovalent cation concentration, [M+] (in the absence of competition by divalent cations or anions). Knowledge of the salt dependences of ka and of the observed equilibrium constance Kobs of the ligand-nucleic acid interaction should usually be sufficient to determine whether a screening controlled mechanism or a pre-equilibrium mechanism is suitable to describe the process. If the association reaction can be described by a pre-equilibrium mechanism, the number of ionic interactions involved in the ligand-nucleic acid intermediate can be estimated. This analysis, extended to include the effects of divalent cations on screening or on the pre-equilibrium step, is applied to literature data on the salt dependence of the kinetics of the interaction of lac repressor with lac operator DNA. When the operator is present on bacteriophage lambda DNA, the observed reaction kinetics are consistent with the formation of an intermediate repressor-DNA complex in a pre-equilibrium step. On the other hand, the kinetics of association of lac repressor with synthetic lac operator fragments may be an example of a screening-controlled reaction.

Binding Sites↗