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M T Record

Publications and source records attributed to M T Record.

At least 55 records · Page 3Linked to original sources
PubMed1993

In vivo thermodynamic analysis of repression with and without looping in lac constructs. Estimates of free and local lac repressor concentrations and of physical properties of a region of supercoiled plasmid DNA in vivo.

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.

DNA, Bacterial↗
PubMed1993

Two open complexes and a requirement for Mg2+ to open the lambda PR transcription start site.

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.

Barium↗
PubMed1992

Thermodynamic stoichiometries of participation of water, cations and anions in specific and non-specific binding of lac repressor to DNA. Possible thermodynamic origins of the "glutamate effect" on protein-DNA interactions.

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.

Anions↗
PubMed1992

Roles of Mg2+ in the mechanism of formation and dissociation of open complexes between Escherichia coli RNA polymerase and the lambda PR promoter: kinetic evidence for a second open complex requiring Mg2+.

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)

Acetates↗
PubMed1992

Polypeptides containing highly conserved regions of transcription initiation factor sigma 70 exhibit specificity of binding to promoter DNA.

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.

Bacterial Proteins↗
PubMed1992

Use of liquid hydrocarbon and amide transfer data to estimate contributions to thermodynamic functions of protein folding from the removal of nonpolar and polar surface from water.

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)

Amides↗
PubMed1992

Origins of the osmoprotective properties of betaine and proline in Escherichia coli K-12.

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.

Betaine↗
PubMed1991

Characterization of the cytoplasm of Escherichia coli K-12 as a function of external osmolarity. Implications for protein-DNA interactions in vivo.

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)

Biophysical Phenomena↗
PubMed1991

Relative affinities of divalent polyamines and of their N-methylated analogues for helical DNA determined by 23Na NMR.

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.

Bis-Trimethylammonium Compounds↗
PubMed1991

Contribution to the thermodynamics of protein folding from the reduction in water-accessible nonpolar surface area.

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.

Protein Conformation↗
PubMed1991

Importance of oligoelectrolyte end effects for the thermodynamics of conformational transitions of nucleic acid oligomers: a grand canonical Monte Carlo analysis.

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.

Base Sequence↗
PubMed1991

Analysis of equilibrium and kinetic measurements to determine thermodynamic origins of stability and specificity and mechanism of formation of site-specific complexes between proteins and helical DNA.

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)

Anions↗
PubMed1990

E. coli promoter spacer regions contain nonrandom sequences which correlate to spacer length.

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.

Base Sequence↗
PubMed1989

Intermediates in the formation of the open complex by RNA polymerase holoenzyme containing the sigma factor sigma 32 at the groE promoter.

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).

Bacterial Proteins↗
PubMed1989

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

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

Binding Sites↗
PubMed1989

In an Escherichia coli coupled transcription-translation system, expression of the osmoregulated gene proU is stimulated at elevated potassium concentrations and by an extract from cells grown at high osmolality.

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.

Acetates↗