Polynucleotides. XI. Thermodynamics of (A) N -2(U) infinity from the dependence of T m N on oligomer length

The variation in the helix-coil transition temperature, T_m^N, with oligomer length, N, for the system ((I)) has been examined. The results for N = 4-13, measured in 0.2M Na+, have been analyzed in terms of the expression of Blake (1972): ((II)) where c_m is the free oligomer concentration at T_m^N, and V_r^f is the thermodynamic free volume available to a helical base-triplet residue. The correlation coefficient for the fit to expression (II) of data obtained over a 50° temperature range is 0.997 when ΔH_r = ?12.6 kcal/mole of base-triplets (independent of oligomer length (N ? 4) or temperature), the value previously obtained from both calorimetry of (A)_∞·2(U)_∞ and (A)₄ concentration dependence of T_m. It is found that V_r^f = 8.0 × 10^?4 1/mole (± 30%) or 1.33 ų per helical base-triplet, and is constant with temperature. A maximum value for V_r^f of 21.0 × 10^?4 1/M (± 1.3%), equivalent to 3.54 ų per helical basetriplet is obtained by the same treatment of the helix-coil transition data for the three-stranded helix formed by adenosine (N = 1) and 2(U)_∞ obtained by Davies and Davidson (1971).     

Pressure dependence of the helix-coil transition temperature for polynucleic acid helices

Thermal denaturation of DNA's and the corresponding helix–coil transformation of artificial polyribonucleic and polydeoxyribonucleic acids have been studied extensively both theoretically^1–13 and experimentally. ^14–30 Much less work has been carried out on the properties of these polynucleic acids at high pressure, and in particular, on the presure dependence of the helix–coil transition temperature.^31–33 Light-scattering techniques have been used in this study to measure the pressure dependence of the helix–coil transition temperature of the two- and three-stranded helices of polyriboadenylic and polyribouridilic acids and of calf thymus DNA. From the slopes of the transition temperature vs. pressure curves and heats of transition obtained from the literature,^20,34 the following volume changes from these helix–coil transitions have been obtained: (a) ?0.96 cc/mole of nucleotide base pairs for the poly (A + U) transition, (b) +0.35 cc/mole of nucleotide base trios for the poly (A + 2U) transition, and (c) +2.7 cc/mole of nucleotide base pairs for the DNA transition. The relative magnitudes and signs of these volume changes which show that poly (A + U) is destabilized by increased pressure, whereas poly (A + 2U) and calf thymus DNA are stabilized by increased pressure, indicates that further development of the helix–coil transition theory for polynucleotides is needed.     

Enthalpy changes in the helix-coil transition of poly(gamma-benzyl L-glutamate)

The helix-coil transition temperature T_c of poly(γ-benzyl L -glutamate) in binary solvent mixtures of dichloroacetic acid and 1,4-dichlorobutane, 1-chlorooctane, or 1-chlorododecane have been measured. A treatment is presented with which the transition enthalpy can be calculated from the observed dependence of T_c on solvent composition. Results are compared with previously obtained calorimetric data. The underlying assumptions of the calculation are discussed.     

Enthalpy of helix-coil transition of DNA: dependence on Na+ concentration and GC-content

The enthalpy of helix-coil transition of DNA (delta H) is determined from the experiments on DNA melting with ligands by means of absolutely general formula, which contains only values directly known from the experiment (M.D. Frank-Kamenetskii, and A.T. Karapetian, Mol. Biol. USSR 6, 621 (1972)) with the combination of the "area" method (P.O. Vardevanian, et al., Biophysica 28, 130 (1983)). The experimentally obtained data show that delta H depends on both concentration of Na+ in solution and GC-content of DNA and is of high accuracy.     

Kinetic studies on the helix-coil transition of fluorescent labeled poly(-L-lysine) by the temperature-jump technique

Fluorescent dansyl labels were covalently attached to poly (L-lysine) (poly(Lys)) with a degree of polymerization of 300 to 600. The degree of labeling was 0.01 to 0.085 (mol label to mol amino acid residues). From the decay of the anisotropy of fluorescence it was concluded that the labels were highly mobile both in the coiled and helical state. A decrease of fluorescence intensity accompanied the helix-coil transition. Identical pH induced transition curves were measured by circular dichroism and fluorescence. The midpoint of the transition was at pH 10.2. The kinetics of the transition were studied by temperature-jump relaxation using fluorescence detection. A single relaxation phase was observed. The relaxation time tau exhibited a distorted bell shaped dependence on the degree of helicity f with a maximum value tau(max) = 15 micros at f = 0.3 and 20 degrees C. It was independent of polymer concentration and of the degree of labeling. A rate constant of helix propagation kF = 10(7) s(-1) was calculated from tau(max) and published values of the nucleation parameter sigma. The activation energy was 16 kJ mol . The observed rate constant is comparable to that of poly(L-glutamic acid) but two orders of magnitude smaller than that found for polyamino acids with nonionizable side chains.     

NMR and optical activity observations on the helix-coil transition in poly(gamma-benzyl L-glutamate) solutions

The helix–coil transition of poly(γ-benzyl L -glutamate) was studied by comparing proton magnetic relaxation behavior with optical activity studies. The transition temperature as determined by magnetic relaxation was lower than that obtained by optical activity. The concentration dependence of the transition was also studied. The relationship of these experiments with previous NMR studies and also with calorimetric investigation of the transition is developed.     

Helix formation in poly (N epsilon,N epsilon,N epsilon-trimethyl-L-lysine) and poly (L-lysine): dependence on concentration and molecular weight.

Helix formation in (Lys)n.HClO4 and poly(N epsilon,N epsilon,N epsilon-trimethyl-L-lysine).HClO4 +AD(LysMe3)n.HClO4+BD is dependent on peptide concentration and on molecular weight. For (LysMe3)n.HClO4 of degree of polymerization (DP) 2510 the midpoint of the coil-to-helix transition is 2 mM and for DP of 190 it is 5 mM. For (Lys)n.HClO4 the peptide concentration for half-helix is 30-60 times as high, and is only weakly dependent, if at all, on molecular weight. Helix formation is an intermolecular process. The use of methylated (Lys)n as the perchlorate permits study of the intermolecular coil-helix transition at low concentration, instead of the high concentration (ca. 1-2 M) required for (Lys)n.HBr. At constant peptide concentration helix content increases with added NaClO4. The higher the peptide concentration, the less NaClO4 is needed to induce helix.     

Solvent dependence of the helix-coil transition of poly-gamma-benzyl-L-glutamate. A PMR study

The coil to helix conformational transitions undergone by poly-γ-benzyl-_L-glutamate in solutions of haloacetic acids and various cosolvents were studied by means of proton magnetic resonance. The results indicate a very small solvent dependence of the α-CH Helix–coil chemical shift difference. The helical stabilities of PBLG in different solvent mixtures were interpreted in terms of modifications of the “solvent structure.”     

Thermodynamics of the B to Z transition in poly(dGdC)

The thermodynamics of the B to Z transition in poly(dGdC) was examined by differential scanning calorimetry, temperature-dependent absorbance spectroscopy, and CD spectroscopy. In a buffer containing 1 mM Na cacodylate, 1 mM MgCl₂, pH 6.3, the B to Z transition is centered at 76.4°C, and is characterized by ΔH_cal = 2.02 kcal (mol base pair)^?1 and a cooperative unit of 150 base pairs (bp). The t_m of this transition is independent of both polynucleotide and Mg²⁺ concentrations. A second transition, with ΔH_cal = 2.90 cal (mol bp)^?1, follows the B to Z conversion, the t_m of which is dependent upon both the polynucleotide and the Mg²⁺ concentrations. Turbidity changes are concomitant with the second transition, indicative of DNA aggregation. CD spectra recorded at a temperature above the second transition are similar to those reported for ψ(–)-DNA. Both the B to Z transition and the aggregation reaction are fully and rapidly reversible in calorimetric experiments. The helix to coil transition under these solution conditions is centered at 126°C, and is characterized by ΔH_cal = 12.4 kcal (mol bp)^?1 and a cooperative unit of 290 bp. In 5 mM MgCl₂, a single transition is seen centered at 75.5°C, characterized by ΔH_cal = 2.82 kcal (mol bp)^?1 and a cooperative unit of 430 bp. This transition is not readily reversible in calorimetric experiments. Changes in turbidity are coincident with the transition, and CD spectra at a temperature just above the transition are characteristic of ψ(–)-DNA. A transition at 124.9°C is seen under these solution conditions, with ΔH_cal = 10.0 kcal (mol bp)^?1 and which requires a complex three-step reaction mechanism to approximate the experimental excess heat capacity curve. Our results provide a direct measure of the thermodynamics of the B to Z transition, and indicate that Z-DNA is an intermediate in the formation of the ψ-(–) aggregate under these solution conditions.     

Effects of pressure on the helix-coil transitions of the poly A-poly U system

Studies were made of the influence of hydrostatic pressure on the helix–coil transitions of poly (A + U) and poly (A + 2U). The results were analyzed by a thermodynamic treatment which emphasized the cooperative aspect of the transitions. The helix-to-coil volume changes were found to be small and negative indicating pressure stabilization of the coil form. The significance of the results with respect to other denaturation measurements was discussed.     

U2AF modulates poly(A) length control by the poly(A)-limiting element

The poly(A)-limiting element (PLE) restricts the length of the poly(A) tail to <20 nt when present in the terminal exon of a pre-mRNA. We previously identified a 65 kDa protein that could be cross-linked to a functional PLE, but not to an inactive mutant element. This binding was competed by poly(U) and poly(C), but not poly(A) or poly(G). Selectivity for the pyrimidine-rich portion of the PLE was demonstrated by RNase footprinting of the binding activity in total nuclear extract. A 65 kDa protein that selectively cross-linked to the functional PLE was purified by conventional chromatography and identified as the large subunit of U2 snRNP auxiliary factor (U2AF). Overexpression of U2AF65 in cells transfected with a PLE-containing reporter construct resulted in the appearance of a population of mRNAs with heterogeneous poly(A) tails. However, this effect was lost following deletion of the C-terminal RNA recognition motifs (RRMs). A C-->G mutation following the AG dinucleotide in the PLE resulted in mRNA with poly(A) ranging from 25-50 nt. This reverted to a discrete, <20 nt poly(A) tail in cells expressing U2AF65. Our results suggest that U2AF modulates the function of the PLE, perhaps by facilitating the binding of another protein to the element.     

Potentiometric titrations and the helix-coil transition of poly(L-glutamic acid) and poly-L-lysine in aqueous salt solutions

The objective have been to establish if those ions which are known to change the stability of the structure of proteins, have any influence on the properties of ionizable polypeptides. Potentiometric titrations and complementary optical rotation data are presented for aqueous solutions of poly-L -lysine (PLL) in the presence of KSCN, KCl, and KF, and for poly(L -glutamic acid) (PLGA) in the presence of KSCN, KCl, and LiCl. The following measured quantities which are affected by salt concentration were obtained: intrinsic pK (pK₀), slope of pK_app versus degree of ionization (α) curves, the degree of ionization at which the helix to coil transition occurs, and the free energy of this transition for the uncharged molecule (δG°_hel). The effects of nonspecific salts (KCl and LiCl for PLL and KSCN and KCl for PLGA) are small, and about, as expected from general electrostatic considerations. In line with the observations made with isoelectric and cat ionic collagen, specific, effects were noted with KSCN–PLL and with LiCl–PLGA. In the presence of KSCN, the poly-L -lysine helix becomes stabilized at much lower degree of ionization than in the presence of KCl, and the slope of the pK_app versus α plots is greatly reduced. However, ΔG°_hel (for the uncharged molecule) is not affected, and pK₀ is only slightly higher. We interpret these data in terms of binding of SCN^? primarily to the side-chain amino groups (both to R? NH₃⁺ and to R? NH₂) solutions. (L -glutamic acid) in LiCl solution has its transition at the same α value as in KCl solution. However, both the slopes of the pK_app versus α plots and the absolute values of ΔG°_hel are lower than in KCl solution. We interpret these results in terms of binding of Li⁺ to side chains as well as to the peptide bond.     

Study of specific interactions of amino acid esters with the synthetic polynucleotides poly(A) x 2 poly(U), poly(A) x poly(U) and poly(A) by thermal denaturation

The interactions of amino acid esters with poly(A)x2poly(U) and poly(A)xpoly(U) have been investigated by means of thermal denaturation of these polynucleotides. The esters under consideration raised the melting point, revealing the preferable binding to helical polynucleotide structures. The melting point shifts demonstrate the following sequence of the stabilities of these complexes: Arg greater than Lys much greater than His greater than Met greater than Ser greater than Gly. The same stability order is observed when studying the polynucleotide renaturation in the presence of esters. This order coincides with that previously obtained for the nucleotide base--amino acid ester complexes excepting basic amino acid esters. The ester interactions with poly(A) and poly(U) also reveal the specificity of monomer--monomer interactions. Some dynamic contributions into the studied specificity are also discussed.     

Kinetic properties and the electric field effect of the helix-coil transition of poly(gamma-benzyl L-glutamate) determined from dielectric relaxation measurements

Dielectric relaxation of poly(γ-benzyl L -glutamate) in solution has been studied in the 5 kcps-10 Mcps range for various values of the helix content. The results give first experimental evidence for three effects of major significance. (1) The system exhibits dielectric relaxation due to a chemical rate process (namely helix formation). This confirms recent theoretical predictions. (2) The mean relaxation time τ* of the helix–coil transition could be evaluated as a function of the degree of transition. The results are in excellent agreement with a previously developed theory. At the midpoint of transition it is found τ*_max = 5 × 10^?7 sec. The elementary process of helical growth turns out to be practically diffusion-controlled (with a rate constant of hydrogen bond formation of 1.3 × 10¹⁰ sec^?1). (3) There is a considerable electric field effect of the helix–coil transition. This indicates that conformation changes in biological systems could be potentially caused by direct action of an electric field.     

Structure of Poly (U).poly (A).poly (U)

The molecular structure of poly (U).poly (A).poly (U) has been determined and refined using the continuous x-ray intensity data on layer lines in the diffraction pattern obtained from an oriented fiber of the RNA. The final R-value for the preferred structure is 0.24, far lower than that for the plausible alternatives. The polymer forms an 11-fold right-handed triple-helix of pitch 33.5A and each base triplet is stabilized by Crick-Watson-Hoogsteen hydrogen bonds. The ribose rings in the three strands have C3'-endo, C2'-endo and C2'-endo conformations, respectively. The helix derives additional stability through systematic interchain hydrogen bonds involving ribose hydroxyls and uracil bases. The relatively grooveless cylindrical shape of the triple-helix is consistent with the lack of lateral organization.