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For order-disorder transitions of double- and triple-stranded nucleic acid helices, the midpoint temperatures Tm depend strongly on a +/-, the mean ionic activity of uniunivalent salt. Experimental determinations of dTm/d ln a +/- and of the enthalpy change (delta H(o)) accompanying the transition in excess salt permit evaluation of delta gamma, the stoichiometrically weighted combination of preferential interaction coefficients, each of which reflects thermodynamic effects of interactions of salt ions with a reactant or product of the conformational transition (formula; see text) Here delta H(o) is defined per mole of nucleotide by analogy to delta gamma. Application of Eq. 1 to experimental values of delta H(o) and Tm yields values of delta gamma for the denaturation of B-DNA over the range of NaCl concentrations 0.01-0.20 M (Privalov et al. (1969), Biopolymers 8,559) and for each of four order-disorder transitions of poly rA.(poly rU)n, n = 1, 2 over the range of NaCl concentrations 0.01-1.0 M (Krakauer and Sturtevant (1968), Biopolymers 6, 491). For denaturation of duplexes and triplexes, delta gamma is negative and not significantly dependent on a +/-, but delta gamma is positive and dependent on a +/- for the disproportionation transition of poly rA.poly rU duplexes. Quantitative interpretations of these trends and magnitudes of delta gamma in terms of coulombic and excluded volume effects are obtained by fitting separately each of the two sets of thermodynamic data using Eq. 1 with delta gamma PB evaluated from the cylindrically symmetric Poisson-Boltzmann (PB) equation for a standard model of salt-polyelectrolyte solutions. The only structural parameters required by this model are: b, the mean axial distance between the projections of adjacent polyion charges onto the cylindrical axis; and a, the mean distance of closest approach between a salt ion center and the cylindrical axis. Fixing bMS and aMS for the multi-stranded (ordered) conformations, we determined the corresponding best fitted values of bSS and aSS for single-stranded RNA and DNA. The resulting best fitted values of aSS are systematically less than aDS by 2-4 A. Uncertainty in the best-fitted values of bSS is significantly lower than in the aSS, because bMS is known with relatively high precision and because the larger uncertainty in aMS has a relatively small effect on the best-fitted values of bSS:bSS = 3.2 +/- 0.6 A for single-stranded poly rA and poly rU; and bSS = 3.4 +/- 0.2 A for single-stranded DNA. These values are approximately one-halt of those expected for a fully extended single-stranded conformation. With the best fitted values of ass and bss, our calculations of delta gamma PB are in close quantitative agreement with experimental observations on each of five nucleic acid order-disorder transitions.

Thermodynamic studies have demonstrated the central importance of a large negative heat capacity change (delta C degree assoc) in site-specific protein-DNA recognition. Dissection of the large negative delta C degree assoc and the entropy change of protein-ligand and protein-DNA complexation provide a thermodynamic signature identifying processes in which local folding is coupled to binding. Estimates of the number of residues that fold on binding obtained from this analysis agree with structural data. Structural comparisons indicate that these local folding transitions create key parts of the protein-DNA interface. The energetic implications of this "induced fit" model for DNA site recognition are considered.

The extraordinary strength of the Escherichia coli rRNA promoter rrnB P1 derives primarily from sequences upstream of the core (-10, -35) region. We find that sequences between -40 and -60 increase the activity of this promoter at least 30-fold in vitro and in vivo. This region, which we refer to as the upstream (UP) element, is located between the -35 consensus hexamer and the previously characterized binding sites for the rRNA transcription factor Fis. The effect of the UP element is independent of Fis in vivo, and independent of any other proteins besides RNA polymerase (RNAP) in vitro. The UP element increases the overall second-order rate constant for association of RNAP with the promoter (ka) and probably the apparent overall first-order isomerization constant (ki). Together with the previously reported protection of the UP element region by RNAP in footprinting experiments, these results indicate that rrnB P1 has an "extended" promoter structure, consisting of the UP element and the core promoter region. We find that the UP element is a separable promoter module that can function to increase the activity of the lac core promoter in an rrnB P1-lac hybrid promoter construct. A functional UP element is not absolutely essential for stimulation of rrnB P1 by the Fis protein.

A strong-binding primary (O1) lac operator located 100 to 200 base-pairs (bp) upstream from a lac promoter control region reduces expression from a lac promoter controlled by a weaker-binding (Oc) lac operator between 3 and 20-fold on a multicopy plasmid in E. coli. We attribute this effect to loop formation in which a thermodynamically stable complex is formed between bidentate lac repressor tetramers and the O1 and Oc operators. A thermodynamic model for repression is developed to interpret these data in terms of the composite effects of free lac repressor concentration and of local repressor concentration (from looping) at the Oc site. The local repressor concentration is found to vary periodically with the distance in base-pairs between the O1 and the Oc operators, ranging from 2 to 20-fold larger than the free concentration (i.e. the bulk thermodynamic activity) of repressor in this F'Iq overproducing strain (estimated to be approximately less than 0.5 microM). The amplitude of the periodic variation in expression and in local concentration appears to decrease with increasing interoperator distance in the range examined. Quantitative analysis of the looping data provides estimates of the physical properties of the intervening DNA region in vivo. For distances in the range 127 to 197 bp, the periodicity of modulation is uniformly 11.28(+/- 0.04) bp, which we interpret as the helical repeat of this region of supercoiled plasmid DNA in vivo. Possible origins of this altered helical repeat include the global linking deficit of the supercoiled DNA and any local linking deficit induced by divergent transcription from promoters bracketing the interoperator region. DNA cyclization analysis yields an apparent in vivo persistence length of this interoperator region of 64(+/- 26) A (which is approximately 15% of the in vivo result) and an in vivo torsional rigidity constant of 1.1(+/- 0.1) x 10(-19) erg cm, which is at the lower end of the range of values found in vitro.

Potassium permanganate (KMnO4) footprinting in the absence and presence of magnesium (Mg2+) at the lambda PR promoter identified two different open complexes with Escherichia coli E sigma 70 RNA polymerase (designated RPo1 and RPo2). The single-stranded region in RPo1 (formed in the absence of Mg2+) was at most 12 bases long, whereas that in RPo2 (formed in the presence of Mg2+) spanned at least 14 bases. Only in RPo2 did the single-stranded region extend to the start point of transcription (+1, +2). These results provide a structural basis for the requirement for uptake of Mg2+ in the formation of RPo2 from RPo1, as deduced from kinetic studies at this promoter.

The objective of this study is to quantify the contributions of cations, anions and water to stability and specificity of the interaction of lac repressor (lac R) protein with the strong-binding symmetric lac operator (Osym) DNA site. To this end, binding constants Kobs and their power dependences on univalent salt (MX) concentration (SKobs = d log Kobs/d log[MX]) have been determined for the interactions of lac R with Osym operator and with non-operator DNA using filter binding and DNA cellulose chromatography, respectively. For both specific and non-specific binding of lac R, Kobs at fixed salt concentration [KX] increases when chloride (Cl-) is replaced by the physiological anion glutamate (Glu-). At 0.25 M-KX, the increase in Kobs for Osym is observed to be approximately 40-fold, whereas for non-operator DNA the increase in Kobs is estimated by extrapolation to be approximately 300-fold. For non-operator DNA, SKobsRD is independent of salt concentration within experimental uncertainty, and is similar in KCl (SKobs,RDKCl = -9.8(+/- 1.0) between 0.13 M and 0.18 M-KCl) and KGlu (SKobs,RDKGlu = -9.3(+/- 0.7) between 0.23 M and 0.36 M-KGlu). For Osym DNA, SKobsRO varies significantly with the nature of the anion, and, at least in KGlu appears to decrease in magnitude with increasing [KGlu]. Average magnitudes of SKobsRO are less than SKobsRD, and, for specific binding decrease in the order [SKobsRO,KCl[>[SKobsRO,KAc[>[SKobsRO,KGlu[ . Neither KobsRO nor SKobsRO is affected by the choice of univalent cation M+ (Na+, K+, NH4+, or mixtures thereof, all as the chloride salt), and SKobsRO is independent of [MCl] in the range examined (0.125 to 0.3 M). This behavior of SKobsRO is consistent with that expected for a binding process with a large contribution from the polyelectrolyte effect. However, the lack of an effect of the nature of the cation on the magnitude of KobsRO at a fixed [MX] is somewhat unexpected, in view of the order of preference of cations for the immediate vicinity of DNA (NH4+ > K+ > Na+) observed by 23Na nuclear magnetic resonance. For both specific and non-specific binding, the large stoichiometry of cation release from the DNA polyelectrolyte is the dominant contribution to SKobs. To interpret these data, we propose that Glu- is an inert anion, whereas Ac- and Cl- compete with DNA phosphate groups in binding to lac repressor. A thermodynamic estimate of the minimum stoichiometry of water release from lac repressor and Osym operator (210(+/- 30) H2O) is determined from analysis of the apparently significant reduction in [SKobsRO,KGlu[ with increasing [KGlu] in the range 0.25 to 0.9 M. According to this analysis, SKobs values of specific and non-specific binding in KGlu differ primarily because of the release of water in specific binding. In KAc and KCl, we deduce that anion competition affects Kobs and SKobs to an extent which differs for different anions and for the different binding modes.

Comparative studies of the effects of Mg2+ vs Na+ and of acetate (OAc-) vs Cl- on the kinetics of formation and dissociation of E. coli RNA polymerase (E sigma 70)-lambda PR promoter open complexes have been used to probe the mechanism of this interaction. Composite second-order association rate constants ka and first-order dissociation rate constants kd, and their power dependences on salt concentration SKa (SKa identical to d log ka/d log [salt]) and Skd (Skd identical to d log kd/d log [salt]), were determined in MgCl2 and NaOAc to compare with the results of Roe and Record (1985) in NaCl. Replacement of NaCl by MgCl2 reduces the magnitude of Ska 2-fold (Ska = -11.9 +/- 1.1 in NaCl; Ska = -5.2 +/- 0.3 in MgCl2) and (by extrapolation) drastically reduces the magnitude of ka at any specified salt concentration (e.g., approximately 10(6)-fold at 0.2 M). Replacement of NaCl by NaOAc does not significantly affect Ska (Ska = -12.0 +/- 0.7 in NaOAc) and (by extrapolation) increased ka by approximately 80-fold at any fixed [Na+]. In the absence of Mg2+, replacement of NaCl by NaOAc is found to increase the half-life of the open complex by approximately 560-fold at fixed [Na+] without affecting Skd [Skd = 7.6 +/- 0.1 in NaOAc; in NaCl, Skd = 7.7 +/- 0.2 (Roe & Record, 1985)]. Replacement of NaCl by MgCl2 drastically reduces both Skd and the half-life of the open complex at any salt concentration below approximately 0.2 M. Strikingly, Skd = 0.4 +/- 0.1 in MgCl2, indicating that the net uptake of Mg2+ ions in the kinetically significant steps in dissociation of the open complex is much smaller than that expected by analogy with the uptake of approximately 8 Na+ ions in the corresponding steps in NaCl. In NaCl/MgCl2 mixtures, at a constant [NaCl] in the range 0.1-0.2 M, initial addition of MgCl2 (0.5 mM less than or equal to [MgCl2] less than or equal to 1 mM) increases the half-life of the open complex; further addition of MgCl2 causes the half-life to decrease, though the effect of [MgCl2] on kd is always less than that predicted by a simple competitive model. The observed effects of MgCl2 on Skd and kd differ profoundly from those expected from the behavior of kd and Skd in NaCl and NaOAc and indicate that the role of Mg2+ in dissociation is not merely that of a nonspecific divalent competitor with RNAP for interactions with DNA phosphates and of a DNA helix-stabilizer, both of which should cause kd to increase monotonically with increasing [Mg2+].(ABSTRACT TRUNCATED AT 400 WORDS)

The sigma 70 subunit of E. coli RNA polymerase is required for sequence-specific recognition of promoter DNA. Genetic studies and sequence analysis have indicated that sigma 70 contains two specific DNA-binding domains that recognize the two conserved portions of the prokaryotic promoter. However, intact sigma 70 does not bind to DNA. Using C-terminal and internal polypeptides of sigma 70, carrying one or both putative DNA-binding domains, we demonstrate that sigma 70 does contain two DNA-binding domains, but that N-terminal sequences inhibit the ability of intact sigma 70 to bind to DNA. Thus, we propose that sigma 70 is a sequence-specific DNA-binding protein that normally functions through an allosteric interaction with the core subunits of RNA polymerase.

This extension of the liquid hydrocarbon model seeks to quantify the thermodynamic contributions to protein stability from the removal of nonpolar and polar surface from water. Thermodynamic data for the transfer of hydrocarbons and organic amides from water to the pure liquid phase are analyzed to obtain contributions to the thermodynamics of folding from the reduction in water-accessible surface area. Although the removal of nonpolar surface makes the dominant contribution to the standard heat capacity change of folding (delta C0fold), here we show that inclusion of the contribution from removal of polar surface allows a quantitative prediction of delta C0fold within the uncertainty of the calorimetrically determined value. Moreover, analysis of the contribution of polar surface area to the enthalpy of transfer of liquid amides provides a means of estimating the contributions from changes in nonpolar and polar surface area as well as other factors to the enthalpy of folding (delta H0fold). In addition to estimates of delta H0fold, this extension of the liquid hydrocarbon model provides a thermodynamic explanation for the observation [Privalov, P. L., & Khechinashvili, N. N. (1974) J. Mol. Biol. 86, 665-684] that the specific enthalpy of folding (cal g-1) of a number of globular proteins converges to a common value at approximately 383 K. Because amounts of nonpolar and polar surface area buried by these proteins upon folding are found to be linear functions of molar mass, estimates of both delta C0fold and delta H0fold may be obtained given only the molar mass of the protein of interest.(ABSTRACT TRUNCATED AT 250 WORDS)

The amounts of cytoplasmic water and of all osmotically significant cytoplasmic solutes were determined for Escherichia coli K-12 grown in 3-(N-morpholino)propane sulfonate (MOPS)-buffered glucose-minimal medium containing 0.5 M NaCl in the presence and absence of the osmoprotectants betaine and proline. The goal of this work is to correlate the effects of osmoprotectants on the composition of the cytoplasm with their ability to increase the growth rate of osmotically stressed cells. At a concentration of 1 mM in the growth medium, betaine increases the growth rate more than does proline; choline, which is converted to betaine by E. coli, appears to have an intermediate effect on growth rate. The accumulation of either betaine or proline reduces the cytoplasmic amounts of K+, glutamate, trehalose, and MOPS (the major cytoplasmic osmolytes accumulated in the absence of osmoprotectants), so that at this external osmolarity the total amount of cytoplasmic solutes is essentially the same in the presence or absence of either osmoprotectant. More betaine than proline is accumulated, so the extent of replacement of cytoplasmic solutes is greater for betaine than for proline. Accumulation of these osmoprotectants is accompanied by a large (20 to 50%) increase in the volume of cytoplasmic water per unit of cell dry weight (Vcyto). This effect, which appears to result from an increase in the volume of free water, Vf (as opposed to water of hydration, or bound water), is greater for betaine than for proline. Taken together, these results indicate that the molar effects of betaine and proline on water activity and on the osmotic pressure of the cytoplasm must be significantly larger than those of the solutes they replace. Cayley et al. (S. Cayley, B. A. Lewis, H. J. Guttman, and M. T. Record, Jr., J. Mol. Biol. 222:281-300, 1991) observed that, in cells grown in the absence of osmoprotectants, both growth rate and Vcyto decreased, whereas the amount of cytoplasmic K+ (nK+) increased, with increasing external osmolarity. We predicted that the observed changes in nK+ and Vcyto would have large and approximately compensating effects on key protein-nucleic acid interactions of gene expression, and we proposed that Vf was the fundamental determinant of growth rate in osmotically stressed cells. The properties of cells cultured in the presence of betaine and proline appear completely consistent with our previous work and proposals. Accumulation of betaine and, to a lesser extent, proline shifts the set of linked physiological parameters (nK+, Vcyto, growth rate) to those characteristic of growth at lower osmolarity in the absence of osmoprotectants. Models for the thermodynamic basis and physiological consequences of the effect of osmoprotectants on Vcyto and Vf are discussed.

The water-accessible volumes, the amounts of all significant osmolytes, and the protein concentration in the cytoplasm of aerobically grown Escherichia coli K-12 have been determined as a function of the osmolarity of the minimal growth medium. The volume of cytoplasmic water (Vcyto) decreases linearly with increasing osmolarity from 2.23(+/- 0.12) microliters/mg dry weight in cells grown at 0.10 OSM to 1.18(+/- 0.06) microliters/mg dry weight at 1.02 OSM. Above 0.28 OSM, growth rate decreases linearly with increasing osmolarity. The growth rate extrapolates to zero at an osmolarity of approximately 1.8, corresponding to an estimated Vcyto of 0.5(+/- 0.2) microliters/mg dry weight. Measurements of Vcyto in titrations of non-growing cells with the plasmolyzing agent NaCl were used to obtain volumes of "bound" water (presumably water of macromolecular hydration) and cytoplasmic osmotic coefficients for cells grown in medium of low (0.10 OSM) and moderate (0.28 OSM) osmolarity. The volume of bound water Vb is similar in the two osmotic conditions (Vb = 0.40(+/- 0.04) microliters/mg dry wt), and corresponds to approximately 0.5 g H2O/g cytoplasmic macromolecule. Since Vcyto decreases with increasing osmolarity, whereas Vb appears to be independent of osmolarity, water of hydration becomes a larger fraction of Vcyto as the osmolarity of the growth medium increases. Growth appears to cease at the osmolarity where Vcyto is approximately equal to Vb. K+ and glutamate (Glu-) are the only significant cytoplasmic osmolytes in cells grown in medium of low osmolarity. The amount of K+ greatly exceeds that of Glu-. Analysis of cytoplasmic electroneutrality indicates that the cytoplasm behaves like a concentrated solution of the K+ salt of cytoplasmic polyanions, in which the amount of additional electrolyte (K+ Glu-) increases with increasing osmolarity. As the osmolarity of the growth medium becomes very low, the cytoplasm approaches an electrolyte-free K+-polyanion solution. In vivo osmotic coefficients were determined from the variation of Vcyto with external osmolarity in plasmolysis titrations of non-growing cells. The values obtained (phi = 0.54(+/- 0.06) for cells grown at 0.10 OSM and phi = 0.71(+/- 0.11) at 0.28 OSM) indicate a high degree of non-ideality of intracellular ions arising from coulombic interactions between K+ and cytoplasmic polyanions. Analysis of these osmotic coefficients using polyelectrolyte theory indicates that the thermodynamic activity of cytoplasmic K+ increases from approximately 0.14 M in cells grown at an external osmolarity of 0.10 OSM to approximately 0.76 M at 1.02 OSM.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of salt concentration on the stabilities of oligonucleotide helices are analyzed directly in terms of delta gamma N----yN identical to gamma denyN - gamma natN, the difference in the salt-nucleotide phosphate preferential interaction coefficients for the denatured state, having yN phosphate charges, and for the native state, having N phosphate charges (y = 1 for hairpin denaturation and y = 0.5 for dimer denaturation). Previous experimental studies of the denaturation of hairpin oligonucleotides (having 18 less than N less than 44) indicate significant differences between delta gamma N----N and delta gamma infinity, the value determined for the denaturation of the corresponding polynucleotide. These differences are thermodynamic manifestations of the oligoelectrolyte end effect. In contrast, the available data on the denaturation of oligonucleotide dimer helices (N less than or equal to 22) imply that differences between delta gamma infinity and delta gamma N----0.5N, and hence oligoelectrolyte end effects, are small or negligible. To determine the origin of these apparently conflicting implications concerning the importance of oligoelectrolyte end effects, we have calculated the N dependence of gamma N from grand canonical Monte Carlo simulations for an idealized model of the structure and charge distribution of each oligomer conformation. Our calculations are in quantitative agreement with the experimental finding for d(TA) hairpin oligomers that -delta gamma N----N decreases linearly as N-1 increases, and with the extant experimental determinations of delta gamma N----0.5N. These results provide an illustration of how the large electrostatic end effects exhibited by the hairpin denaturation data are masked when delta gamma infinity is compared with values of delta gamma N----0.5N for short dimer helices (N less than or equal to 22). For 0.5N greater than 24, -delta gamma N----0.5N is predicted to be a linear function of N-1 whose slope has the opposite sign from, and is more salt-concentration dependent than, the corresponding slope of -delta gamma N----N as a function of N-1. Our calculations also yield predictions about the N dependences of the individual values of gamma N that can be tested by determining Donnan coefficients from membrane dialysis equilibrium experiments. For long enough hairpin and dimer oligonucleotides (yN greater than or equal to 24), in either native or denatured forms, we predict that the (positive) difference gamma infinity - gamma N increases linearly with increasing N-1. For smaller values of N the difference gamma infinity - gamma N continues to increase with increasing N-1.

Interactions of divalent polyamines with double-helical DNA in aqueous solution are investigated by monitoring the decrease in 23Na NMR relaxation rates as NaDNA is titrated with H3N(+)-(CH2)m-+NH3, where m = 3, 4, 5, or 6. Analogous measurements are made for the same homologous series of methylated polyamines (methonium ions). The dependence of the 23Na relaxation rates on the amount of added divalent cation (M2+) is analyzed quantitatively in terms of a two-state model. The sodium ions are assumed to be in rapid exchange between a "bound" state, where they are close enough to DNA so that it affects their relaxation rate, and a "free" state in bulk solution, where their relaxation rate is the same as in solutions containing no DNA. The distribution of Na+ and M2+ between these states is described quantitatively in terms of an ion-exchange parameter: DM = (pMB)(1-pNaB)n/(pNaB)n(1-pMB), where pNaB and pMB are the fractions of Na+ and M2+ that are close enough to DNA to be considered bound (by the NMR criterion), and n is the number of sodium ions displaced from DNA by the binding of one M2+ ion. For each of the polyamines and methonium ions investigated here, equations derived from this two-state model yield acceptable fittings of the titration curves if roNa, the number of sodium ions bound per DNA phosphate when no competing cations are present, is assigned a value between 0.6 and 1.00. Within this range, changing the value assigned to roNa does change the best-fitted values of DM determined for these polyamines (DH) and for the methonium ions (DMe) but does not alter the following conclusions about the trends in these parameters. (1) For polyamines and methonium ions of the same m, DH exceeds DMe by factors that are significantly larger for m = 3 and 4 than for m = 5 and 6. (2) DH for m = 3 and 4 is larger than DH for m = 5 and 6. (3) DMe for m = 3 and 4 is smaller than DMe for m = 5 and 6.

Protein folding and the transfer of hydrocarbons from a dilute aqueous solution to the pure liquid phase are thermodynamically similar in that both processes remove nonpolar surface from water and both are accompanied by anomalously large negative heat capacity changes. On the basis of a limited set of published surface areas, we previously proposed that heat capacity changes (delta C degrees p) for the transfer of hydrocarbons from water to the pure liquid phase and for the folding of globular proteins exhibit the same proportionality to the reduction in water-accessible nonpolar surface area (delta Anp) [Spolar, R.S., Ha, J.H., & Record, M.T., Jr. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 8382-8385]. The consequence of this proposal is that the experimental delta C degrees p for protein folding can be used to obtain estimates of delta Anp and of the contribution to the stability of the folded state from removal of a nonpolar surface from water. In this paper, a rigorous molecular surface area algorithm [Richmond, T.J. (1984) J. Mol. Biol. 178, 63-89] is applied to obtain self-consistent values of the water-accessible nonpolar surface areas of the native and completely denatured states of the entire set of globular proteins for which both crystal structures and delta C degrees p of folding have been determined and for the set of liquid and liquefiable hydrocarbons for which delta C degrees p of transfer are known. Both processes (hydrocarbon transfer and protein folding) exhibit the same direct proportionality between delta C degrees p and delta Anp. We conclude that the large negative heat capacity changes observed in protein folding and other self-assembly processes involving proteins provide a quantitative measure of the reduction in the water-accessible nonpolar surface area and of the contribution of the hydrophobic effect to the stability of the native state and to protein assembly.

The concentration and nature of the electrolyte are key factors determining (1) the equilibrium extent of binding of oligocations or proteins to DNA, (2) the distribution of bound protein between specific and nonspecific sites, and (3) the kinetics of association and dissociation of both specific and nonspecific complexes. Salt concentration may therefore be used to great advantage to probe the thermodynamic basis of stability and specificity of protein-DNA complexes, and the mechanisms of association and dissociation. Cation concentration serves as a thermodynamic probe of the contributions to stability and specificity from neutralization of DNA phosphate charges and/or reduction in phosphate charge density. Cation concentration also serves as a mechanistic probe of the kinetically significant steps in association and dissociation that involve cation uptake. In general, effects of electrolyte concentration on equilibrium constants (quantified by SKobs) and rate constants (quantified by Skobs) are primarily cation effects that result from the cation-exchange character of the interactions of proteins and oligocations with polyanionic DNA. The competitive effects of Mg2+ or polyamines on the equilibria and kinetics of protein-DNA interactions are interpretable in the context of the cation-exchange model. The nature of the anion often has a major effect on the magnitude of the equilibrium constant (Kobs) and rate constant (kobs) of protein-DNA interactions, but a minor effect on SKobs and Skobs, which are dominated by the cation stoichiometry. The order of effects of different anions generally follows the Hofmeister series and presumably reflects the relative extent of preferential accumulation or exclusion of these anions from the relevant surface regions of DNA-binding proteins. The question of which anion is most inert (i.e., neither accumulated nor excluded from the relevant regions of these proteins) remains unanswered. The characteristic effects of temperature on equilibrium constants and rate constants for protein-DNA interactions also serve as diagnostic probes of the thermodynamic origins of stability and specificity and of the mechanism of the interaction, since large changes in thermodynamic and activation heat capacities accompany processes with large changes in the amount of water-accessible nonpolar surface area.(ABSTRACT TRUNCATED AT 400 WORDS)

The -10 and -35 regions of E. coli promoter sequences are separated by a spacer region which has a consensus length of 17 base-pairs. This region is thought to contribute to promoter function by correctly positioning the two conserved regions. We have performed a statistical evaluation of 224 spacer sequences and found that spacers which deviate from the 17 base-pair consensus length have nonrandom sequences in their upstream ends. Spacer regions which are shorter than 17 base-pairs in length have a significantly higher than expected frequency of purine-purine and pyrimidine-pyrimidine homo-dinucleotides at the six upstream positions. Spacer regions which are longer than 17 base-pairs in length have a significantly higher than expected frequency of purine-pyrimidine and pyrimidine-purine hetero-dinucleotides at these positions. This suggests that the nature of the purine-pyrimidine sequence at the upstream end of spacer regions affect promoter function in a manner which is related to the spacer length. We examine the spacer sequences as a function of spacer length and discuss some possible explanations for the observed relationship between sequence and length.

We have investigated the effects of mixed Na+: Mg2+ ionic solutions on the stability of the nonspecific lac repressor-DNA complex. The effects of Mg2+ are simply interpreted in terms of its role as a competitor (with repressor) for DNA sites. From these studies, the binding constant of the Mg-DNA complex can be determined as a function of the concentration of Na+. We have used this information to interpret the data of Riggs and collaborators (Riggs, A.D., et al. (1970), J. Mol. Biol. 48, 67-83; 53, 401-417) on the ion dependence of the repressor-operator interaction. We find that there are approximately 70% as many ionic interactions in the repressor-operator complex as in the nonspecific complex. Our best estimate is that 8 +/- 1 ion pairs are formed. We calculate that the release of counterions in the formation of the specific complex contributes approximately 40% of the favorable free energy change in the association reaction under in vivo ionic conditions. Implications of these findings for the control of the lac operon and for the molecular relationship between the specific and nonspecific complexes are considered.

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)

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)

We have investigated the nonspecific interaction of lac repressor protein with DNA by a quantitative application of DNA-cellulose chromatography (deHaseth, P.L., et al. (1977), Biochemistry 16 (third of five papers in a series in this issue)). The observed association constant for the interaction, KRD obsd, is a sensitive function of ion concentrations and pH. Application of binding theory to interpret these effects gives the results that 11 +/- 2 monovalent ions are released in the interaction and two groups on repressor must be protonated for repressor to bind to DNA. We argue that much of the ion release results from the displacement of cations from the DNA, and estimate on this basis that 12 +/- 2 phosphates are involved in ionic interactions with the protein. Ion release drives the protonation reaction and the overall repressor-DNA interaction. The major role of low molecular weight ions in the repressor-DNA interaction suggests that ion concentration changes must be considered in discussing mechanisms of control of gene expression.

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.

Although protein-nucleic acid interactions exhibit dramatic dependences on both ion concentration and type in vitro, large variations in intracellular ion concentrations can occur in Escherichia coli and other organisms without apparent effects on gene expression in vivo. E. coli accumulates K+ and glutamate as cytoplasmic osmolytes. The cytoplasmic K+ concentration in E. coli varies from less than 0.2 to greater than 0.9 m as a function of external osmolarity; corresponding cytoplasmic glutamate concentrations range from less than 0.03 to greater than 0.25 m. Only low levels of chloride occur in the cytoplasm of E. coli at all osmotic conditions. Since most in vitro studies have been performed in chloride salts, whereas glutamate is the more relevant physiological anion, we have measured the effects of the substitution of potassium glutamate (KGlu) for KCl on the kinetics and equilibria of a variety of site-specific protein-DNA interactions in vitro. Both the interaction of E. coli RNA polymerase with two phage lambda promoters and the interactions of various restriction enzymes with their DNA cleavage sites are enhanced by this substitution. Using the abortive initiation assay, we find a greater than 30-fold increase in the second-order rate constant for open complex formation at the lambda PR promoter and a 10-fold increase at the lambda PR' promoter, when KGlu is substituted for KCl. Replacement of KCl by KGlu does not affect the strong salt dependences of these interactions; increasing either KCl or KGlu concentrations decreases both reaction rates and extents. Substitution of glutamate for chloride does, however, shift the range of salt concentrations over which these interactions are observable to higher K+ concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

We have applied the formalism developed previously for the kinetics of domain-localized reactions [S. Mazur and M. T. Record, Jr. (1986) Biopolymers 25, 985-1008] to describe complex mechanisms of association of a protein with a specific site on a large DNA molecule also containing many nonspecific binding sites. These nonspecific sites participate in the mechanism of formation of the specific complex through competitive binding and the facilitating mechanisms of sliding and transfer. The effects of localizing the sites in a domain are represented by a simple algebraic expression, and the sequence of interactions within the domain are described by equations closely related to a conventional, homogeneous solution mechanism. We apply this formalism to examine the interplay between sliding and direct transfer in domain-localized interactions in general and in the lac repressor-lac operator interaction in particular. Experimental investigation of the effect of the molecular location of the specific site (e.g., end vs middle of the polymer chain) on the kinetics of association may allow the contributions of sliding and direct transfer to be resolved.

The rate of formation of transcriptionally competent open complexes between Escherichia coli RNA polymerase (RNAP) and the lambda PR promoter is extraordinarily sensitive to the nature and concentration of the electrolyte ions in the solution. The pseudo-first-order time constant of open complex formation tau obsd, determined in excess RNAP at 25 degrees C as a function of NaCl concentration, is proportional to the concentration product [Na+]12 [RNAP]-1. Consequently, tau obsd is far more sensitive to changes in the salt concentration than to changes in the concentration of RNAP. The origin of this effect is the release of the thermodynamic equivalent of 12 monovalent ions in the process of closed complex formation at the lambda PR promoter. In more complex ionic mixtures, ion-specific stoichiometric effects on tau obsd are observed. These are not ionic strength effects but are instead both valence and species specific. Both the association and dissociation rate constants of RNAP at the lambda PR promoter are strongly salt dependent, varying (in NaCl) as [Na+]-12 and [Na+]8, respectively. Consequently, the equilibrium constant characterizing open complex formation at this promoter varies with [Na+]-20. Electrostatic interactions and counterion release are the major contributors to the binding free energy driving open complex formation in a dilute salt solution. Since the in vivo ionic environment of E. coli (and other cells) is highly variable, these large salt effects are almost certainly of physiological significance. Variations in the intracellular concentrations of inorganic and organic ions, including polyamines, must exert both global and also promoter-specific regulatory effects on the initiation of transcription, as well as on numerous other protein-nucleic acid interactions.

In this paper we characterize the effect of varying the solution conditions and filter-binding protocols on the extent and selectivity of DNA retention on nitrocellulose filters by DNA-binding proteins. These effects are illustrated by the binding interaction of Escherichia coli RNA polymerase with lambda and T7 phage DNA restriction fragments. We present procedures which will help enhance the selective retention of some DNA restriction fragments over others. These include increasing the pH and salt concentration, decreasing the enzyme-to-DNA ratio, and including an appropriate washing step. Selective binding is not dependent on the presence of Mg2+. Although we only show data for RNA polymerase-DNA interactions, many of the principles discussed are likely to find practical applications in studying selective DNA-protein binding in general.

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.

The interaction of E sigma 32 with the groE promoter at temperatures between 0 degrees C and 37 degrees C was studied using DNase I footprinting and dimethyl sulfate methylation. Three distinct complexes were observed. At 0 degrees C E sigma 32 fully protected sequences between -60 and -5 from DNase I digestion on the top (non-template) strand of the promoter. At 16 degrees C the majority of the E sigma 32 promoter complexes had a DNase I footprint almost identical with that seen at 37 degrees C, protecting the DNA from about -60 to +20; however, little DNA strand separation had occurred, and the changes in sensitivity of guanine residues to dimethyl sulfate methylation caused by E sigma 32 differed from those seen at 37 degrees C. DNA strand separation, and changes in the pattern of protections from and enhancements of methylation by dimethyl sulfate to those characteristic of the open complex, occurred at temperatures between 16 degrees C and 27 degrees C. It is plausible to assume that these temperature-dependent isomerizations are analogous to the time-dependent sequence of intermediates on the pathway to open complex formation at 37 degrees C. Therefore we propose that the formation of an open complex by E sigma 32 at the groE promoter involves three classes of steps: E sigma 32 initially binds to the promoter in a closed complex (RPC1) in which the enzyme interacts with a smaller region of the DNA than in the open complex. E sigma 32 then isomerizes to form a second closed complex (RPC2) in which the enzyme interacts with the same region of the DNA as in the open complex. Finally, a process of local DNA denaturation (strand opening) leads to formation of the open complex (RPO).

59Co NMR is demonstrated to provide a useful probe of the interactions of Co(NH3)6(3+) with helical B-DNA. The association of Co(NH3)6(3+) with B-DNA produces relatively modest effects on the relaxation rate and chemical shift of 59Co, which indicate that the octahedral coordination shell remains intact and that no significant number of long-lived "outer-sphere" complexes are formed at specific sites on the DNA surface. Under conditions where essentially all of the cobalt complex is associated with DNA, the chemical shift of 59Co appears to depend on its binding density. This effect could be due to magnetic heterogeneity in the environments of Co(NH3)6(3+) adjacent to DNA. The local exchange reaction between Co(NH3)6(3+) and Na+ in the vicinity of DNA has been investigated by measuring 59Co chemical shifts and 23Na line widths concurrently. The number of sodium ions displaced by the association of one Co(NH3)6(3+) with DNA cannot be uniquely determined, but the data indicate that this number remains constant over at least the initial stage of a titration of NaDNA with NaCl. 59Co chemical shifts have been analyzed to construct binding isotherms for the association of cobalt hexaammine with DNA over a range of salt (NaCl) concentrations. The magnitudes of the resulting binding constants and their salt dependence are similar to those previously reported for the association of structurally diverse trivalent ligands, such as spermidine and trilysine, with helical nucleic acids. Therefore, these association equilibria appear to be governed primarily by electrostatic interactions.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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)

We have studied the in vitro expression of the osmoregulated proU promoter in Escherichia coli coupled transcription-translation (S-30) extracts as a function of the osmolality of the culture medium used to grow the cells and of the salt concentration added to the extract. These variables represent novel extensions of the use of S-30 extracts to investigate gene regulatory phenomena in vitro. The concentrations of potassium acetate and of the physiologically relevant osmolyte potassium glutamate for maximal expression of the proU promoter are approximately 2-fold higher than the concentrations of these salts providing maximal expression of the lacUV5 promoter. The relative promoter activity of proU with respect to lacUV5 increases more than 30-fold with an increase in salt concentration from 50 to 300 mM. In comparative studies with S-30 extracts prepared from cells grown at high and low osmolalities, we find that at fixed salt concentrations expression of proU is increased 10-fold in the S-30 extract prepared from high osmolality-grown cells, whereas the expression of lacUV5 is increased less than 2-fold. Addition of anti-sigma 70 monoclonal antibodies or purified sigma 70 to the S-30 extract demonstrated that the major proU promoter(s) used in the S-30 extracts is sigma 70-dependent.

Large negative standard heat capacity changes (delta CP degree much less than 0) are the hallmark of processes that remove nonpolar surface from water, including the transfer of nonpolar solutes from water to a nonaqueous phase and the folding, aggregation/association, and ligand-binding reactions of proteins [Sturtevant, J. M. (1977) Proc. Natl. Acad. Sci. USA 74, 2236-2240]. More recently, Baldwin [Baldwin, R. L. (1986) Proc. Natl. Acad. Sci. USA 83, 8069-8072] proposed that the delta CP degree of protein folding could be used to quantify the contribution of the burial of nonpolar surface (the hydrophobic effect) to the stability of a globular protein. We demonstrate that identical correlations between the delta CP degree and the change in water-accessible nonpolar surface area (delta Anp) are obtained for both the transfer of nonpolar solutes from water to the pure liquid phase and the folding of small globular proteins: delta CP degree/delta Anp = -(0.28 +/- 0.05) (where delta Anp is expressed in A2 and delta CP degree is expressed in cal.mol-1.K-1; 1 cal = 4.184 J). The fact that these correlations are identical validates the proposals by both Sturtevant and Baldwin that the hydrophobic effect is in general the dominant contributor to delta CP degree and provides a straightforward means of estimating the contribution of the hydrophobic driving force (delta Ghyd degree) to the standard free energy change of a noncovalent process characterized by a large negative delta CP degree in the physiological temperature range: delta Ghyd degree congruent to (80 +/- 10)delta CP degree.

Plasmid constructs containing a wild-type (O+) lac operator upstream of an operator-constitutive (Oc) lac control element exhibit a length-dependent, oscillatory pattern of repression of expression of the regulated gene as interoperator spacing is varied from 115 to 177 base pairs (bp). Both the length dependence and the periodicity of repression are consistent with a thermodynamic model involving a stable looped complex in which bidentate lac repressor interacts simultaneously with both O+ and Oc operators. The oscillatory pattern of repression with distance occurs with a period approximating the helical repeat of DNA and presumably reflects the necessity for proper alignment of interacting operators along the helical face of the DNA. In the length regime examined, the presence of the upstream operator enhances repression between 6-fold and 50-fold depending upon phasing. This reflects a torsional rigidity of DNA in vivo that is consistent with in vitro measurements. The oscillatory pattern of repression is best fit with a period of either 9.0 or 11.7 bp/cycle but not 10.5 bp/cycle. This periodicity is interpreted as reflecting the average helical repeat of the 40-bp interoperator region of plasmid DNA in vivo, suggesting that the local helical repeat of DNA in vivo may differ significantly from 10.5 bp/turn. The apparent persistence length needed to fit the data (aapp) is only one-fifth the standard in vitro value. This low value of aapp may be due in part to DNA bending induced by catabolite activator protein (CAP) bound to its site between the interacting operators.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

Applications of the grand canonical Monte Carlo method demonstrate the importance of end effects on fundamental molecular and thermodynamic properties of oligoelectrolyte solutions. Simulations are carried out for a series of solutions containing double-helical DNA oligomers of varying numbers of phosphate charges N (8 less than or equal to N less than or equal to 100) and univalent electrolyte at fixed activity (a +/- = 1.76 mmol/dm3). These results are used to evaluate as follows: C+N(a), the local concentration of cations at various axial positions along the oligomer surface; C+N(a), the axial average of these concentrations; TN, the preferential interaction coefficient expressed per oligomer charge, which is directly related to the fractional thermodynamic extent of association of counterions. A sufficiently long oligomer (N greater than or equal to 48 under the conditions simulated) is characterized by an interior region over which C+N(a) is uniform and equal to C+ infinity (a), the polyion limit. This interior region is flanked by two symmetric terminal regions, in which C+N(a) varies linearly with axial position from the end of the oligomer to a distance approximately 18 monomer units (approximately 3.1 nm) from that end. For long oligomers, the characteristics of the terminal regions [length and axial profile of C+N(a)] do not vary with N and, by inference, also pertain to the polyion under the same conditions. Both C+N(a) and TN approach their polyelectrolyte limits as linear functions of 1/N. These linear dependences can be attributed to the increasing predominance of the contribution due to the polyion-like interior of the oligomer as N increases.

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).