Torsional constant of 27-mer DNA oligomers of different sequences.

We have studied the torsional elastic constant (alpha) of short DNA (27mer) oligomers of various sequence by fluorescence polarization anysotropy (FPA) measurements. The lowest alpha values were found in samples with sequence rich in AA dinucleotides or containing the alternating d(A-T) x d(A-T) motif. The torsional rigidity of our DNA samples was compared to that calculated according to the current values of twist angle fluctuations derived for ten dinucleotide steps by recent analyses of DNA crystal structure database. The values of torsional rigidity derived from crystals are higher than our experimental ones, obtained by FPA analysis, suggesting that packing force in crystals may notably hinder the dinucleotide twist angle fluctuations that occur in solution. This behaviour is more evident for samples containing AA, TA and AT steps. In all the samples there is about a twofold change of the alpha value in the 10-40 degrees C range. An activation enthalpy (Delta H (#)) of about 17.4 kJ mol(-1), on average, was obtained for the temperature dependence of eight of the ten samples studied. A correlation with the stacking energy is discussed.

Oligonucleotide labeling: synthesis of a new spin-labeled 2'-deoxyguanosine analogue.

In order to achieve an EPR sensitive probe for DNA, 3-carboxy-Proxyl free radical was linked to O-6 of dG through a five-atoms-tether. The modified base was incorporated into a 30-mer ODN, then annealed to its complementary DNA strand. Hydrodynamic parameters show only a slight destabilization with respect to the equivalent unlabeled hybrid. EPR could monitor the hybrid formation showing a progressive enlargement of the upfield signal in passing from the labeled ss- to the ds-30-mer.

DNA, RNA and hybrid RNA-DNA oligomers of identical sequence: structural and dynamic differences.

A 27-mer sequence was synthesised as DNA duplex (DD), RNA duplex (RR), and RNA-DNA (RD) hybrid in order to characterise their structural and dynamic features. The hydrodynamic radius (Rh) and the rise (b) values of the three samples were consistent with the conformations predicted by CD analysis. The value of the torsional constant (alpha) of the samples containing RNA was approximately twice that of the DD sample and followed the order: DD < RD < RR. The same order was observed in the thermodynamic stability and in the reduction of the electrophoretic mobility. gamma-Ray footprinting analysis was carried out to resolve the individual strand conformation in the hybrid. The RNA strand preserved its conformation, while the DNA strand showed local deformations mainly at TA and TG steps.

gamma-Ray footprinting and fluorescence polarization anisotropy of a 30-mer synthetic DNA fragment with one 2'-deoxy-7-hydro-8- oxoguanosine lesion.

The influence of the oxidative lesion 2'-deoxy-7-hydro-8-oxoguanosine (8-oxodG) on some conformational properties of DNA has been studied. Four 30-mer duplexes of the form [5'-GATCCTCTAGAGTC[G(*) or G]ACCTGCAGGCATGCA-3']:[3'-CTAGGAGATCTCAG[C or A]TGGACGTCCGTACGT-5'], in which G(*) is the 8-oxodG lesion, were synthesized in order to compare the effect of the GA mismatch and of the damaged G(*)C and G(*)A forms with the normal GC. Spectroscopic measurements performed by means of UV denaturation and circular dichroism experiments do not show gross changes of stability and overall structure in the damaged and mismatched samples. The control DNA and the samples containing GA mismatch show very similar gamma-rays cutting patterns, indicating that the introduction of the GA mismatch does not perturb the phosphate backbone geometry. In the samples containing the 8-oxodG there are some variations of the cleavage pattern near G(*) which are extended for almost one helical turn. Some differences are observed between G(*)C and G(*)A duplexes. In particular, in the G(*)C sample the reduced accessibility to OH radicals at the G15 site, observed in the control, spreads on the intrastrand adjacent bases and in the G(*)A sample a shift of the minimum is observed. The hydrodynamic radius R(h) derived by fluorescence polarization anisotropy decay exhibits a constant value of 11.4 +/- 0.2 A between 5 and 40 degrees C, in all the samples. The torsional constant alpha of each oligomer decreases when the temperature is raised and the alpha values of the damaged samples are higher than those of the normal ones.

Structural and dynamical properties of two DNA oligomers with the same base composition and different sequence.

We compared the structural and dynamical properties of two DNA fragments, 27 bp long, having the same base composition but a different sequence. This work aims to understand how the base sequence on a purine rich strand in a double helix, which is important for many biological functions, is related to structural features and to measurable physical quantities. Structural characterization of the two samples was performed both by conventional spectroscopic methods (circular dichroism and UV denaturation experiments) and by means of a gamma-ray footprinting technique which gives information on fine conformational differences. Dynamical features of the samples were studied by fluorescence polarization anisotropy (FPA) measurements which allow the evaluation of some hydrodynamic parameters, such as the hydrodynamic radius and the elastic torsion constant of DNA. Using a gamma-ray footprinting technique, we observed that the interruption of the long homopurine-homopyrimidine run in the control sample, due to the 'scrambling' operation, alters the DNA three-dimensional structure, also at nucleotide level. Besides, an increase in thermal stability and in the torsional rigidity of the 'scrambled' sample was observed. A possible association between base-stacking interaction and torsional rigidity was inferred from the comparison of the two samples.

Small angle neutron scattering analysis of thermal stability of 23S rRNA and the intact 50S subunits of Sulfolobus solfataricus.

The ribosomes of the extremely thermophilic archaebacterium, Sulfolobus solfataricus, are very resistant to thermal denaturation (optimal growth temperature 87 degrees C), remaining essentially intact up to above 90 degrees C. However, the separate ribosomal components (rRNA and r-proteins) are less thermally stable than the ribosome as a whole, indicating that the mode of interaction of all of the components within the ribonucleoprotein particle play an essential role in determining thermal stability. To get some insight into the structural features of the thermophilic ribosome, we performed small angle neutron scattering (SANS) measurements at various temperatures on Sulfolobus solfataricus intact large ribosomal subunits (50S) and deproteinated large ribosomal subunit RNA (23S). Even if the scattering profiles suggest the presence of supramolecular aggregates in all of the samples and at all of the investigated temperatures, the measured form factors indicated for both samples that, at temperatures above 70 degrees C, the suspended particles underwent a structural rearrangement. This finding is likely to reflect single particles' properties, since S. solfataricus ribosomes are known to be biologically activated only above 60 degrees C, and there are indications that such activation requires a conformational rearrangement of the particle. A remarkable superimposition of the percentage variation of the volume from neutron scattering and of the absorbency increment with respect to temperature supports this view.

Triple helix DNA oligomer melting measured by fluorescence polarization anisotropy.

A synthetic DNA triple helix sequence was formed by annealing a pyrimidinic 21 mer single strand sequence onto the complementary purinic sequence centred on a 27 mer duplex DNA. Melting of the third strand was monitored by UV spectrophotometry in the temperature range 10-90 degrees C. The T(m) of the triplex, 37 degrees C, was well separated from the onset of duplex melting. When the same triple helix was formed on the duplex bearing one nick in the center of the pyrimidinic sequence the T(m) of the triplex was shifted to approximately 32 degrees C and overlapped the melting of the duplex. We have used fluorescence polarization anisotropy (FPA) measurements of ethidium bromide (EB) intercalated in duplex and triplex samples to determine the hydrodynamic parameters in the temperature range 10-40 degrees C. The fluorescence lifetime of EB in the samples of double and triple stranded DNA is the same (21.3 +/- 0.5 ns) at 20 degrees C, indicating that the geometries of the intercalation sites are similar. The values for the hydration radii of the duplex, normal triplex, and nicked triplex samples were 10.7 +/- 0.2, 12.2 +/- 0.2, and 12.0 +/- 0.2 A. FPA measurements on normal triplex DNA as a function of temperature gave a melting profile very similar to that derived by UV absorption spectroscopy. For the triplex carrying a nick, the melting curve obtained using FPA showed a clear shift compared with that obtained for the normal triplex sample. The torsional rigidity of the triplex forms was found to be higher than that of the duplex form.

Effects of magnesium and temperature on the conformation and reassociation of Escherichia coli and Sulfolobus solfataricus ribosomes.

The structural response of the ribosomes of the extremely thermophilic archaeon Sulfolobus solfataricus was analysed and compared to that of the mesophilic (E. coli) ribosomes by assaying ethidium bromide (EB) binding to the native 70S particles as a function of magnesium concentration. We found that the thermophilic ribosomes bound more EB than their mesophilic counterparts; on the other hand, inhibition of EB binding by Mg2+ ions was more effective in the E. coli 70S particle. In Sulfolobus, the separated 30S and 50S subunits and the 70S particle bound the drug in a similar fashion, whereas the E. coli 70S had a reduced number of binding sites with respect to the subunits. Light scattering measurements as a function of Mg2+ concentration were carried out at various temperatures to study the interaction between the ribosomal subunits from the thermophilic and the mesophilic bacteria. As expected, the association of ribosomal subunits in E. coli was magnesium dependent and could be observed also at low temperature. By contrast, the interaction between Sulfolobus ribosomal subunits was obligatorily dependent upon both magnesium ions and a temperature of at least 80 degrees C, close to the physiological optimum for cell growth (87 degrees C).

Effect of thymine dimer introduction in a 21 base pair oligonucleotide.

It is well known that the pyrimidine dimers are the main damage produced by UV radiation on the DNA structure. However, while studies on the photoproduct structure have been carried out extensively, uncertainties still exist on the implication that a single damaging event has on the overall conformation. In particular, the extension of the damage influence on the polynucleotide chain is a matter of debate. This problem is especially important to understanding some steps of the repair mechanisms. In this study we performed a chemical-physical characterization of 21 base pair oligonucleotides containing a single thymine dimer in one strand. Thermodynamic parameters were determined by means of thermal denaturation experiments, and static fluorescence measurements were performed to unequivocally define the primary structure-conformation relationship in this specific case. We used hydroxyl radicals, produced by means of gamma-irradiation of the sample solution, to detect fine structure changes. Our data show that the introduction of a single thymine dimer might cause only a slight distortion of the helix geometry, as judged by the evaluation of the enthalpic and the entropic terms and by the small changes observed in the binding of ethidium bromide to DNA. The modifications in the sugar phosphate backbone subsequent to the damaging event are especially evident, near the thymine dimer, toward the 5'-end direction in the strand containing the dimer.

Effects of magnesium ions on ribosomes: a fluorescence study.

Fluorescence intensity measurements of ethidium bromide (EB) bound to ribosomal RNA (rRNA) in suspensions of 30S and 50S subunits, of 70S ribosomal particles and of protein-free extracted rRNA are presented. Changes in the intercalation of EB reflect changes in conformation and degree of exposure of rRNA. The effect of removal of magnesium ions on the binding of EB is compared in protein-free rRNA and in ribosomal particles by a Scatchard plot analysis. In free ribosomal RNA the number of bound EBs do not depend on magnesium content, only the association constant is affected. In intact 70S particles and both in the separated 50S and 30S subunits the presence of magnesium greatly reduces binding of EB and no saturation of the fluorescence intensity with rRNA concentration is observed, preventing a Scatchard plot analysis. Removal of magnesium restores a strong EB intercalation. Then magnesium ions induce a conformational change in the 70S particles as well as in the separated subunits. The different behavior of the free-rRNA and of the ribosomal particles indicates that ribosomal proteins are relevant to the structural changes induced by magnesium ions. The comparison of the number of excluded sites and of the association constant in the 30S, 50S subunits and in the 70S particles indicates that even without Mg2+ ions the two subunits still interact, at variance with the commonly shared opinion that subunits dissociation takes place at low magnesium concentration.

Influence of defects on the electrophoretic, thermodynamic and dielectric properties of a 21 base pair DNA in solution.

The thermodynamic and dielectric properties of a 21 base pair DNA have been evaluated and compared with those of samples with some defects. In particular, fragments in which the absence of a phosphate group (nick) or of two nucleotides (gap) causes chain interruptions were studied. Measurements of ultraviolet absorption as a function of temperature at different oligomer concentrations and at various ionic strengths were performed. Dielectric spectroscopy at radiofrequencies (1 MHz-1 GHz) was applied on aqueous solutions of the duplexes at 5 degrees C, where the solutes are thermally stable. Dielectric dispersions with 30-40 MHz characteristic frequencies were defined. The results of melting experiments indicate a thermal destabilization of the oligomers containing the defects. Electrophoretic data and the dielectric results show that the conformations of the nicked and control samples are very similar, while the oligomer with a gap is more compact with a different charge distribution at the ends.

Radiofrequency dielectric spectroscopy of ribosome suspensions.

Dielectric measurements on different ribosome suspensions were carried out in the frequency range from 10 kHz to 1 GHz. In intact ribosomes two dispersions were detected: one around 100 kHz and the other one in the MHz region. In separated ribosomal subunits and in ribosomes resuspended in a buffer with no magnesium ions (relaxed ribosomes) only the MHz dispersion was observed. Electrical conductivities of the samples at 1 kHz were also measured. The temperature dependence of the two dispersions was investigated and a tentative attribution was proposed.

Temperature dependence of DNA dielectric dispersion at radiofrequency.

We have studied the dielectric behavior of DNA aqueous solutions at various ionic strengths and in the presence of the specific DNA ligand ethidium bromide, in the frequency range 1 MHz-1 GHz, at different temperatures ranging from 5 to 40 degrees C. The activation enthalpies of the dielectric relaxations studied were obtained by Arrhenius plots of In(tau T)-1 vs. T-1. The results are consistent with a counterion fluctuation model as previously developed by Mandel and colleagues.

Complex dielectric constant of arginine-DNA and protamine-DNA aqueous systems at 10 GHz.

The complex dielectric constant of arginine and protamine from herring sperm (clupeine) and their complexes with herring sperm DNA was measured at 10 GHz in the temperature range -20 to +45 degrees C by a microwave cavity perturbation method. The experimental results were analysed in terms of a three-component equation (solute molecules, interfacial water and bulk water) to calculate the fractional volume of modified water and hence the specific hydration of the samples. A fourfold reduction of the specific hydration is observed for the clupeine molecule as compared to the free monomers. This is consistent with a folded conformation of the protein in solution. The specific hydration of the complex between clupeine and DNA is reduced by 50% with respect to the weighted average for the uncomplexed components. This result indicates an intimate contact between clupeine and DNA with exclusion of water molecules and is consistent with the highly condensed form of nucleoprotamines which is known in vivo.

Hydration properties of DNA-lysine gels by microwave dielectric measurements as a function of temperature.

Dielectric measurements by a cavity perturbation method at 10 GHz in the temperature range from -20 degrees C to +45 degrees C are reported for aqueous gels of herring sperm DNA in the presence of 1 or 3 lysine molecules per nucleotide. Measurements for lysine-water and DNA-water systems are also reported. The experimental results can be accounted for by the presence of interfacial water, with dielectric properties different from those of bulk water, and are analyzed in terms of a three component equation (solute molecules, interfacial water and bulk water) to calculate hydration parameters of the systems. The lysine molecule is found to coordinate a particular number of water molecules, in agreement with the literature. The specific hydration of DNA is reduced by the presence of lysine, indicating a direct interaction between the polyion and the aminoacid: a decrease to about 50% was observed at a ratio of one molecule of lysine per nucleotide. A suggestion is made that the interaction is mainly electrostatic in nature.

Effect of ions on counterion fluctuation in low-molecular weight DNA dielectric dispersions.

The dielectric permittivity of aqueous solutions of low-molecular weight DNA (Mr = 3.2 X 10(5) ) in the presence of MgCl2 and AgNO3 has been measured in the frequency range from 5 kHz to 30 MHz, at a temperature of 25 degrees C. The DNA concentration was 3.5 X 10(-4) M in terms of phosphate and the salt concentration was varied from 1 X 10(-5) to 2 X 10(-4) M. The dielectric results have been analyzed in terms of two contiguous dielectric dispersions, and characteristic parameters have been discussed on the basis of polyelectrolyte theories which deal with counterion fluctuation. Some molecular parameters of the DNA molecule in electrolyte solutions are estimated.

Stimulation of yeast RNA polymerase II transcription by critical values of supercoiling.

RNA chains of discrete length were obtained in vitro by yeast RNA polymerase II directed transcription of a supercoiled plasmid. On the basis of the amount and the molecular weight of the RNA chains synthesized in the absence of reinitiation events, the number of actively transcribing RNA polymerase molecules has been calculated. A stimulation of transcriptional activity was found to be related to the torsional strength of negative supercoiling of the template. The DNA unwinding angle measured in the complexes formed with the enzyme in the presence of three ribonucleoside triphosphates equals 485 +/- 30 degrees, marking a melting effect of 14 base pairs per bound enzyme molecule.