Thermally induced structural transition in the d(TTTTATAATAAA) d(TTTATTATAAAA) heteroduplex is characterized by UV-spectroscopy and differential scanning calorimetry. At low salt (less than 0.1 M) the occurrence of a cooperative transition in the lower temperature range, followed by a broad transition connected with small increase in absorbance is observed. At high salt (greater than or equal to 0.2 M) a single, monophasic transition appears. Linear dependence of the latter on log of salt concentration (dTm:dlogM = 14.2 degrees C) and of 1/Tm on log of oligomer concentration [derived therefrom delta H (v.H.) = 77.1 kcal/mole (duplex)] allows relating it to the melting of the heteroduplex helix. The non-cooperative transition, independent of oligomer concentration and similar to that of the single chain, was attributed to melting of short hairpin helices upon heteroduplex dissociation. Calorimetric enthalpy: 75.6 kcal/mole (duplex) proved significantly lower than predicted from known calorimetric data for poly[d(AT)] and poly d(A) X poly d(T). 相似文献
We have investigated the equilibrium binding of racemic 7r,8t,9t,10c-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene to the double-stranded, synthetic polynucleotides poly[d(A-T)], poly[d(G-C)], and poly[d(G-m5C)] at low binding ratios. Difference absorption spectroscopy shows a 10-nm red shift for binding to poly[d(A-T)] and an 11-nm red shift for binding to either poly[d(G-C)] or poly[d(G-m5C)]. The value of delta epsilon for binding is approximately the same for all three hydrocarbon-polynucleotide complexes. Binding of this neutral polycyclic aromatic hydrocarbon derivative to these polynucleotides is dependent upon ionic strength and temperature. Analysis of complex formation employing polyelectrolyte theory shows a greater release of counterions associated with binding to poly[d(A-T)] than with the other two polynucleotides (0.5 and ca. 0.36, respectively). Thus, sequence-selective binding of this hydrocarbon in DNA would be expected to change depending on salt concentration. The temperature dependence of binding was studied at 100 mM Na+ where the equilibrium binding constants for poly[d(A-T)] and poly[d(G-m5C)] are roughly equivalent and 6-fold greater than the binding affinity for poly[d(G-C)]. The binding to poly[d(A-T)] and poly[d(G-C)] is characterized by a delta H omicron = -7.0 kcal/mol, and the large difference in affinity constants arises from differences in negative entropic contributions. Formation of hydrocarbon-poly[d(G-m5C)] complexes is accompanied by a delta H = -9.1 kcal/mol. However, the affinity for poly[d-(G-m5C)] is the same as that for poly[d(A-T)] due to the much more negative entropy associated with binding to poly[d(G-m5C)]. 相似文献
The interaction of Escherichia coli RNA polymerase with poly[d(A-T)] and poly[d-(I-C)] was studied by difference absorption spectroscopy at temperatures, from 5 to 45 degrees C in the absence and presence of Mg2+. The effect of KCl concentration, at a fixed temperature, was studied from 12.5 to 400 mM. Difference absorption experiments permitted calculation of the extent of DNA opening induced by RNA polymerase and estimation of the equilibrium constant associated with the isomerization from a closed to an open RNA polymerase-DNA complex. delta H0 and delta S0 for the closed-to-open transition with poly[d(A-T)] or poly[d(I-C)] complexed with RNA polymerase are significantly lower than the values associated with the helix-to-coil transition for the free polynucleotides. For the RNA polymerase complexes with poly[d(A-T)] and poly[d(I-C)] in 50 mM KCl, delta H0 approximately 15-16 kcal/mol (63-67 kJ/mol) and delta S0 approximately 50-57 cal/K per mol (209-239 J/K per mol). The presence of Mg2+ does not change these parameters appreciably for the RNA polymerase-poly[d(A-T)] complex, but for the RNA polymerase-poly[d(I-C)] complex in the presence of Mg2+, the delta H0 and delta S0 values are larger and temperature-dependent, with delta H0 approximately 22 kcal/mol (92 kJ/mol) and delta S0 approximately 72 cal/K per mol (approx. 300 J/K per mol) at 25 degrees C, and delta Cp0 approximately 2 kcal/K per mol (approx. 8.3 kJ/K per mol). The circular dichroism (CD) changes observed for helix opening induced by RNA polymerase are qualitatively consistent with the thermally induced changes observed for the free polynucleotides, supporting the difference absorption method. The salt-dependent studies indicate that two monovalent cations are released upon helix opening. For poly[d(A-T)], the temperature-dependence of enzyme activity correlates well with the helix opening, implying this step to be the rate-determining step. In the case of poly[d(I-C)], the same is not true, and so the rate-determining step must be a process subsequent to helix opening. 相似文献
gp32 I is a protein with a molecular weight of 27 000. It is obtained by limited hydrolysis of T4 gene 32 coded protein, which is one of the DNA melting proteins. gp32 I itself appears to be also a melting protein. It denatures poly[d(A-T)].poly[d(A-T)] and T4 DNA at temperatures far (50-60 degrees C) below their regular melting temperatures. Under similar conditions gp32 I will denature poly[d(A-T).poly[d(A-T)] at temperatures approximately 12 degrees C lower than those measured for the intact gp32 denaturation. For T4 DNA gp32 shows no melting behavior while gp32 I shows considerable denaturation (i.e., hyperchromicity) even at 1 degree C. In this paper the denaturation of poly[d(A-T)].poly[d(A-T)] and T4 DNA by gp32 I is studied by means of circular dichroism. It appears that gp32 I forms a complex with poly[d(A-T)]. The conformation of the polynucleotide in the complex is equal to that of one strand of the double-stranded polymer in 6 M LiCl. In the gp32 I DNA complex formed upon denaturation of T4 DNA, the single-stranded DNA molecule has the same conformation as one strand of the double-strand T4 DNA molecule in the C-DNA conformation. 相似文献
Gene 32 protein (g32P), the single-stranded DNA binding protein from bacteriophage T4, contains 1 mol of Zn(II) bound in a tetrahedral complex to -S- ligands, proposed on spectral evidence to include Cys-77, Cys-87, and Cys-90 [Giedroc, D. P., Keating, K. M., Williams, K. R., Konigsberg, W. H., & Coleman, J. E. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 8452]. The Zn(II) can be completely removed by treatment with the mercurial reagent p-(hydroxymercuri)benzenesulfonate and ethylenediaminetetraacetic acid. The resultant apo-g32P is rapidly digested by trypsin in contrast to the zinc protein which undergoes specific limited proteolysis to yield a resistant DNA-binding core. Rebinding of Zn(II) to the apoprotein restores the same limited susceptibility to proteolysis displayed by the native Zn(II) protein. In the presence of 150 mM NaCl, Zn(II) g32P reduces the melting temperature Tm of poly[d(A-T)] by 47 degrees C, while apo-g32P is unable to melt poly[d(A-T)] at this salt concentration, as the protein thermally unfolds before melting can take place. At 25 mM NaCl, however, apo-g32P lowers the Tm of poly[d(A-T)] by 36 degrees C, but the melting curve is broad compared to the steep cooperative melting induced by Zn(II) g32P. Association constants Ka calculated from the poly[d(A-T)] melting curves for Zn(II) and apo-g32P differ by 3 orders of magnitude, 4.8 X 10(10) M-1 and 4.3 X 10(7) M-1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
We have studied the circular dichroism and ultraviolet difference spectra of T7 bacteriophage DNA and various synthetic polynucleotides upon addition of Escherichia coli RNA polymerase. When RNA polymerase binds nonspecifically to T7 DNA, the CD spectrum shows a decrease in the maximum at 272 but no detectable changes in other regions of the spectrum. This CD change can be compared with those associated with known conformational changes in DNA. Nonspecific binding to RNA polymerase leads to an increase in the winding angle, theta, in T7 DNA. The CD and UV difference spectra for poly[d(A-T)] at 4 degrees C show similar effects. At 25 degrees C, binding of RNA polymerase to poly[d(A-T)] leads to hyperchromicity at 263 nm and to significant changes in CD. These effects are consistent with an opening of the double helix, i.e. melting of a short region of the DNA. The hyperchromicity observed at 263 nm for poly[d(A-T)] is used to determine the number of base pairs disrupted in the binding of RNA polymerase holoenzyme. The melting effect involves about 10 base pairs/RNA polymerase molecule. Changes in the CD of poly(dT) and poly(dA) on binding to RNA polymerase suggest an unstacking of the bases with a change in the backbone conformation. This is further confirmed by the UV difference spectra. We also show direct evidence for differences in the template binding site between holo- and core enzyme, presumably induced by the sigma subunit. By titration of the enzyme with poly(dT) the physical site size of RNA polymerase on single-stranded DNA is approximately equal to 30 bases for both holo- and core enzyme. Titration of poly[d(A-T)] with polymerase places the figure at approximately equal to 28 base pairs for double-stranded DNA. 相似文献
The equilibrium between the right- and left-handed conformations of poly[d(G-C)] in aqueous NaCl shifts towards the right-handed (B) form with increasing pressure. The optical density at 290 and 260 nm was determined at 50 and 180 MPa for solutions in which approximately equal amounts of the two conformations were present at 0.1 MPa (atmospheric pressure). Interpretation of the observed changes in terms of a two-state unimolecular reaction mechanism results in an average molar reaction volume (delta V0) equal to 26 cm3 mol-1 at 22 degrees C; that is, the partial molar volume of B form poly[d(G-C)] is smaller than that of the left-handed (Z) form. Based upon the thermodynamics of ion-pair formation in polar solvents, it is proposed that the positive delta V0 reflects a favorable entropy change for the reaction toward the Z conformation. The larger entropy change of the Z form may derive from the release of water molecules from the hydration spheres of the cation and the poly[d(G-C)] due to the formation of ionic interactions with the Z conformer. The delta V0 of the transition is similar in sign and magnitude to the calculated molar volume change of the interaction of Na+ with H2PO4- in water. 相似文献
CD spectra were obtained for eight synthetic double-stranded DNA polymers down to at least 175 nm in the vacuum uv. Three sets of sequence isomers were studied: (a) poly[d(A-C).d(G-T)] and poly[d(A-G).d(C-T)], (b) poly[d(A-C-C).d(G-G-T)] and poly[d(A-C-G).d(C-G-T)], and (c) poly[d(A).d(T)], poly[d(A-T).d(A-T)], poly[d(A-A-T).d(A-T-T)], and poly[d(A-A-T-T).d(A-A-T-T)]. There were significant differences in the CD spectra at short wavelengths among each set of sequence isomers. The (G.C)-containing sequences had the largest vacuum uv bands, which were positive and in the wavelength range of 180-191 nm. There were no large negative bands at longer wavelengths, consistent with the polymers all being in right-handed conformations. Among the set of sequences containing only A.T base pairs, poly[d(A).d(T)] had the largest vacuum uv CD band, which was at 190 nm. This CD band was not present in the spectra of the other (A.T)-rich polymers and was absent from two first-neighbor estimations of the poly[d(A).d(T)] spectrum obtained from the other three sequences. We concluded that the sequence dependence of the vacuum uv spectra of the (A.T)-rich polymers was due in part to the fact that poly[d(A).d(T)] exists in a noncanonical B conformation. 相似文献
Circular dichroism (CD) spectra of poly(dA), poly(dT), poly(dA)·poly(dT), and poly[d(A-T)]·poly[d(T-A)] have been measured as a function of temperature. From these data difference spectra have been calculated by subtracting the spectrum measured at low temperature from the spectra measured at higher temperatures. The CD difference spectra obtained upon melting of the two double-stranded polymers are very similar. From a comparison of these difference spectra with calculated ones it is shown that optical transitions near 272 nm (on A) and 288 nm (most probably on T) are present. The premelting changes of the CD spectrum of poly[d(A-t)]·poly[d(T-A)] are due to a change in conformation in which the secondary structure goes from a C- to B-type spectrum by increasing the A-type nature of the polymer. Such a change is not observed for poly(dA)·poly(dT). Instead, a transition between two different B-type geometries occurs. 相似文献
The gene 5 protein (g5p) from Ff filamentous virus is a model single-stranded DNA (ssDNA) binding protein that has an oligonucleotide/oligosaccharide binding (OB)-fold structure and binding properties in common with other ssDNA-binding proteins. In the present work, we use circular dichroism (CD) spectroscopy to analyze the effects of amino acid substitutions on the binding of g5p to double-stranded DNA (dsDNA) compared to its binding to ssDNA. CD titrations of poly[d(A). d(T)] with mutants of each of the five tyrosines of the g5p showed that the 229-nm CD band of Tyr34, a tyrosine at the interface of adjacent protein dimers, is reversed in sign upon binding to the dsDNA, poly[d(A). d(T)]. This effect is like that previously found for g5p binding to ssDNAs, suggesting there are similarities in the protein-protein interactions when g5p binds to dsDNA and ssDNA. However, there are differences, and the possible perturbation of a second tyrosine, Tyr41, in the complex with dsDNA. Three mutant proteins (Y26F, Y34F, and Y41H) reduced the melting temperature of poly[d(A). d(T)] by 67 degrees C, but the wild-type g5p only reduced it by 2 degrees C. This enhanced ability of the mutants to denature dsDNA suggests that their binding affinities to dsDNA are reduced more than are their binding affinities to ssDNA. Finally, we present evidence that when poly[d(A). d(T)] is melted in the presence of the wild-type, Y26F, or Y34F proteins, the poly[d(A)] and poly[d(T)] strands are separately sequestered such that renaturation of the duplex is facilitated in 2 mM Na(+). 相似文献
We report the temperature and salt dependence of the volume change (DeltaVb) associated with the binding of ethidium bromide and netropsin with poly(dA).poly(dT) and poly[d(A-T)].poly[d(A-T)]. The DeltaV(b) of binding of ethidium with poly(dA).poly(dT) was much more negative at temperatures approximately 70 degrees C than at 25 degrees C, whereas the difference is much smaller in the case of binding with poly[d(A-T)].poly[d(A-T)]. We also determined the volume change of DNA-drug interaction by comparing the volume change of melting of DNA duplex and DNA-drug complex. The DNA-drug complexes display helix-coil transition temperatures (Tm several degrees above those of the unbound polymers, e.g., the Tm of the netropsin complex with poly(dA)poly(dT) is 106 degrees C. The results for the binding of ethidium with poly[d(A-T)].poly[d(A-T)] were accurately described by scaled particle theory. However, this analysis did not yield results consistent with our data for ethidium binding with poly(dA).poly(dT). We hypothesize that heat-induced changes in conformation and hydration of this polymer are responsible for this behavior. The volumetric properties of poly(dA).poly(dT) become similar to those of poly[d(A-T)].poly[d(A-T)] at higher temperatures. 相似文献
We have measured the thermal melting profile for poly[d(AT)].poly[d(TA)] as a function of concentration of three trivalent cations: spermidine, me8spermidine, and hexammine cobalt(III). Using McGhee's (1976) theory of DNA melting in the presence of ligands, we have estimated association constants Kh, Kc and binding site sizes nh, nc for binding to double-helical (h) and single-stranded (c) polynucleotide. The results are as follows: (table; see text) The binding parameters for spermidine and hexammine cobalt(III) to double helical molecules agree fairly well with direct equilibrium dialysis measurements, and are in reasonable accord with predictions of counterion condensation theory. However, despite their identical charges, the three ligands bind to single-stranded DNA with quite different affinities. Estimates of the charge spacing of single-stranded DNA suggest that poly[d(AT)] is less elongated in the presence of spermidine and hexammine cobalt(III) than it is when complexed with me8spermidine. 相似文献
The minor-groove ligand netropsin provides a sensitive probe of the hydration difference between poly(dA).poly(dT) and poly[d(AT)].poly[d(AT)]. We have measured the volume change delta V accompanying binding of netropsin to these polymers, using an improved magnetic suspension densimeter. For poly(dA).poly(dT) we find delta V = +97 mL/mol of bound netropsin at pH 7.0 and 10 mM sodium phosphate buffer. For poly[d(AT)].poly[d(AT)] we find delta V = -16 mL/mol of bound netropsin. This striking differential effect suggests that the poly(dA).poly(dT) duplex compresses more water (or is more extensively hydrated). From our enthalpy and entropy results we estimate the approximately 10 water molecules, immobilized in the minor groove of this system, are displaced by each netropsin bound. The volume increase, however, is substantially larger than can be explained by a simple melting of these immobilized water molecules in the minor groove. A decompression of at least 40 water molecules must attend the complexation to the poly(dA).poly(dT) duplex. This suggests that the conformation change attending the binding of the drug to this polymer duplex causes a further dehydration, whereas no such change in dehydration and configuration for the heteropolymer system is indicated. 相似文献
Pressure-jump chemical relaxation has been used to investigate the kinetics of ethidium bromide binding to the synthetic double-stranded polymers poly[d(G-C)] and poly[d(A-T)] in 0.1 M NaCl, 10 mM tris(hydroxymethyl)aminomethane hydrochloride, and 1 mM ethylenediaminetetraacetic acid, pH 7.2, at 24 degrees C. The progress of the reaction was followed by monitoring the fluorescence of the intercalated ethidium at wavelengths greater than 610 nm upon excitation at 545 nm. The concentration of DNA was varied from 1 to 45 microM and the ethidium bromide concentration from 0.5 to 25 microM. The data for both polymers were consistent with a single-step bimolecular association of ethidium bromide with a DNA binding site. The necessity of a proper definition of the ethidium bromide binding site is discussed: it is shown that an account of the statistically excluded binding phenomenon must be included in any adequate representation of the kinetic data. For poly[d(A-T)], the bimolecular association rate constant is k1 = 17 X 10(6) M-1 s-1, and the dissociation rate constant is k-1 = 10 s-1; in the case of poly[d(G-C)], k1 = 13 X 10(6) M-1 s-1, and k-1 = 30 s-1. From the analysis of the kinetic amplitudes, the molar volume change, delta V0, of the intercalation was calculated. In the case of poly[d(A-T)], delta V0 = -15 mL/mol, and for poly[d(G-C)], delta V0 = -9 mL/mol; that is, for both polymers, intercalation is favored as the pressure is increased.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
The pressure dependence of the helix–coil transition of poly(dA)∙poly(dT) and poly[d(A-T)]·poly[d(A-T)] in aqueous solutions of NaCl and CsCl at concentrations between 10 and 200 mM is reported and used to calculate the accompanying volume change. We also investigated the binding parameters and volume change of ethidium bromide binding with poly(dA)∙poly(dT) and poly[d(A-T)]·poly[d(A-T)] in aqueous solutions of these two salts. The volume change of helix–coil transition of poly(dA)∙poly(dT) in Cs+-containing solutions differs by less than 1 cm3 mol− 1 from the value measured when Na+ is the counter-ion. We propose that this insensitivity towards salt type arises if the counter-ions are essentially fully hydrated around DNA and the DNA conformation is not significantly altered by salt types. Circular dichroism spectroscopy showed that the previously observed large volumetric disparity for the helix–coil transition of poly[d(A-T)]·poly[d(A-T)] in solutions containing Na+ and Cs+ is likely result of a Cs+-induced conformation change that is specific for poly[d(A-T)]·poly[d(A-T)]. This cation-specific conformation difference is mostly absent for poly(dA)∙poly(dT) and EB bound poly[d(A-T)]·poly[d(A-T)]. 相似文献
Systematic data on the dependence of the melting curve parameters of DNA from different organisms on the concentration of salt (C2H5)5NBr have been obtained. The melting curves were studied by spectrophotometric as well as by microcalorimetric methods. The DNA melting range width is shown to pass through the minimum value delta0T = 0.6 +/- 0.1 degrees at the point of inversion of relative stability of AT and GC pairs that corresponds to the concentration of (C2H5)4NBr equal to 2.9 +/- 0.1 M. This concentration, as well as the value of delta0T, are the same for different DNA's of common chemical structure. The T2 and T4 DNA containing hydroxymethylated and glucosylated cytosine residues show an anomalous behaviour. The enthalpy of melting falls very slowly as the salt concentration increases. The possible causes of the observed value of delta0T are discussed. A conclusion is drawn that the main factor which governs the DNA melting process in the region of inversion of the relative stability of AT and GC pairs is the heterogeneity of stacking interaction between different base pairs. 相似文献
The two non-complementary synthetic DNAs, poly[d(G-G-A)] and poly[d(T-C)] can form a duplex structure at moderate ionic strengths in which every other T is extrahelical: Only this structure is consistent with the stoichiometry of formation and with the production of C? photodimers on ultraviolet light irradiation. Further characterization with respect to melting temperature and interaction with nuclease or damage-recognizing proteins is described. 相似文献
The thermal transition of poly[d(A-r5U)] polydeoxynucleotides (where r was a hydrogen atom, or a methyl, ethyl, n-propyl, n-butyl or n-pentyl group) was studied by measuring the derivative melting profiles of the polymers in the range of 0.01--0.36 M K+, at pH 6.8. According to the Tm values, polydeoxynucleotide analogues show lower thermal stability than poly[d(A-T)] at any counterion concentration applied. At a given salt concentration, Tm of the alkyl analogues decreased as the number of carbon atoms (n) in the r substituent of poly[d(A-r5U)] increased. 1/Tm plotted against against 1/n yielded a linear relationship. Cooperativity of the melting of all poly[d(A--U)] analogues decreased with the increase of salt concentration in the solution. This change depended again on 5-substitution of the uracil moiety of poly[d(A-U)]. Smallest decrease was observed in the case of poly[d(A--U)] whereas largest decrease was shown by poly[d(A-pe5U)] (pe=pentyl group). 相似文献
The alpha-form of poly[d(A)].poly[d(T)], observed in fibers at high (greater than 80%) relative humidity, is a 10-fold double-helical structure of pitch 3.2 nm. This new X-ray analysis shows that the two strands of the double helix are of the same kind conformationally and both B-like in containing C-2'-endo-puckered deoxyribose rings. Nevertheless, the two strands are different enough for the overall morphology of the duplex to resemble that of the heteromerous model for the drier (beta) form of poly[d(A)].poly[d(T)] in which one strand has C-2'-endo rings and the other C-3'-endo. Since the orientations of the bases in poly[d(A)].poly[d(T)] are persistently different from those of classical B-DNA it is likely that there will be local bending (about 10 degrees) at the junctions between general sequence tracts and the oligo[d(A)].oligo[d(T)] tracts that occur in some native DNAs. The conclusions about the structure of alpha-poly[d(A)].poly[d(T)] are reinforced by independent analyses of similar X-ray diffraction patterns from poly[d(A)].poly[d(U)] and poly[d(A-I)].poly[d(C-T)]. 相似文献