Characteristics and variations of B-type DNA conformations in solution: a quantitative analysis of Raman band intensities of eight DNAs

Raman spectra were obtained from four bacterial DNAs varying in GC content and four periodic DNA polymers in 0.1 M NaCl at 25 degrees C. A curve fitting procedure was employed to quantify and compare Raman band characteristics (peak location, height, and width) from 400 to 1600 cm-1. This procedure enabled us to determine the minimum number of Raman bands in regions with overlapping peaks. Quantitative comparison of the Raman bands of the eight DNAs provided several new results. All of the DNAs examined required bands near 809 (+/- 7) and 835 (+/- 5) cm-1 to accurately reproduce the experimental spectra. Since bands at these frequencies are associated with A-family and B-family conformations, respectively, this result indicates that all DNAs in solution have a mixture of conformations on the time scale of the Raman scattering process. Band characteristics in the 800-850-cm-1 region exhibited some dependence on CG content and base pair sequence. As previously noted by Thomas and Peticolas [Thomas, G. A., & Peticolas, W. L. (1983) J. Am. Chem. Soc. 105, 993], the poly[d(A)].poly[d(T)] spectra were qualitatively distinct in this region. The A-family band is clearly observed at 816 cm-1. The intensity of this band and that of the B-family band at 841 cm-1 were similar, however, to intensities in the natural DNA spectra. Three bands at 811, 823, and 841 cm-1 were required to reproduce the 800-850-cm-1 region of the poly[d(A-T)].poly[d(A-T)] spectra. This may indicate the presence of three backbone conformations in this DNA polymer. Analysis of intensity vs. GC content for 42 Raman bands confirmed previous assignments of base and backbone vibrations and provided additional information on a number of bands.     

Raman spectroscopic studies of the DNA cro binding site conformation, free and bound to cro protein.

Raman spectra of the DNA binding site for cro repressor protein were obtained in the presence and absence of bound cro protein. The 17 base pair fragment is a consensus sequence of the six cro binding sites in phage lambda, except that the second base to the right of the center of pseudosymmetry is altered. Analysis of the spectrum of the free DNA indicates that the molecule exists in a B-like conformation with deviations from the usual B form occurring mainly in the bands assigned to A-T vibrations. The spectrum of the bound DNA was obtained by subtracting the spectrum of free cro from the spectrum of the complex which was estimated to be 90% bound. The DNA undergoes significant structural changes upon binding to the protein; most notable of these changes is a destacking of the G-C bases reflected by increases in the 1240, 1262, and 1320 cm-1 bands. A decrease in the 1361 cm-1 band that occurs has also been assigned to a destacking in guanine bases. The appearance of a 705 cm-1 band and the decrease and downshift of the 670 cm-1 band are consistent with the appearance of A-like character in the A-T region of the binding site when the protein binds; however, the spectra indicate that the entire binding site remains in a distorted B-like conformation. We use the 705 cm-1 band to estimate A-like character because the 800-850 cm-1 region is obscured by interference from strong protein bands. Other shifts in both intensity and position cannot be assigned to characteristic changes in conformation and therefore must be attributed to the protein influencing the structure in a novel way.     

Raman spectroscopy study of the B-Z transition in (dG-dC)n.(dG-dC)n and a DNA restriction fragment

The B to Z conformational transition of (dG-dC)n.(dG-dC)n and a 157 bp DNA restriction fragment were followed using Raman spectroscopy. The 157 bp DNA has a 95 bp segment from the E. coli lactose operon sandwiched between 26 and 32 bp of (dC-dG) sequences. Raman spectra of the DNAs were obtained at varying sodium chloride concentrations through the region of the transition. A data analysis procedure was developed to subtract the background curves and quantify Raman vibrational bands. Profiles of relative intensity vs. sodium chloride concentration are shown for bands at 626, 682, 831-833 and 1093 cm-1. Both (dG-dC)n.(dG-dC)n and the 157 bp DNA show changes in the guanine vibration at 682 cm-1 and backbone band at 831-3 cm-1 preceding a highly cooperative change in the 1093 cm-1 PO2- vibration. This result indicates that there are at least two conformational steps in the B to Z conformational pathway. We review the effect of the (dC-dG) portion of the 157 bp DNA on the 95 bp segment. Comparison of Raman spectra of the 157 bp DNA, the 95 bp fragment and (dG-dC)n.(dG-dC)n indicate that in 4.5 M NaCl the (dC-dG) segments are in a Z-conformation. Base stacking in the 95 bp portion of the 157 bp DNA appears to maintain a B-type conformation. However, a substantial portion of this region no longer has a B-type backbone vibration.     

Raman spectroscopy of cytoplasmic muscle fiber proteins. Orientational order.

The polarized Raman spectra of glycerinated and intact single muscle fibers of the giant barnacle were obtained. These spectra show that the conformation-sensitive amide I, amide III, and C-C stretching vibrations give Raman bands that are stronger when the electric field of both the incident and scattered radiation is parallel to the fiber axis (Izz). The detailed analysis of the amide I band by curve fitting shows that approximately 50% of the alpha-helical segments of the contractile proteins are oriented along the fiber axis, which is in good agreement with the conformation and composition of muscle fiber proteins. Difference Raman spectroscopy was also used to highlight the Raman bands attributed to the oriented segments of the alpha-helical proteins. The difference spectrum, which is very similar to the spectrum of tropomyosin, displays amide I and amide III bands at 1,645 and 1,310 cm-1, respectively, the bandwidth of the amide I line being characteristic of a highly alpha-helical biopolymer with a small dispersion of dihedral angles. A small dichroic effect was also observed for the band due to the CH2 bending mode at 1,450 cm-1 and on the 1,340 cm-1 band. In the C-C stretching mode region, two bands were detected at 902 and 938 cm-1 and are both assigned to the alpha-helical conformation.     

The catabolite activator protein stabilizes its binding site in the E. coli lactose promoter.

The effect of catabolite activator protein, CAP, on the thermal stability of DNA was examined. Site specific binding was studied with a 62 bp DNA restriction fragment containing the primary CAP site of the E. coli lactose (lac) promoter. A 144 bp DNA containing the lac promoter region and a 234 bp DNA from the pBR322 plasmid provided other DNA sites. Thermal denaturation of protein-DNA complexes was carried out in a low ionic strength solvent with 40% dimethyl sulfoxide, DMSO. In this solvent free DNA denatured below the denaturation temperature of CAP. The temperature stability of CAP for site specific binding was monitored using an acrylamide gel electrophoresis assay. Results show that both specific and non-specific CAP binding stabilize duplex DNA. Site specific binding to the 62 bp DNA produced a 13.3 degrees C increase in the transition under conditions where non-specific binding stabilized this DNA by 2-3 degrees C.     

Conformation of d(GGGATCCC)2 in crystals and in solution studied by X-ray diffraction, Raman spectroscopy and molecular modelling.

In the crystal, d(GGGATCCC)2 forms an A-DNA double helix as known from a single crystal X-ray diffraction study. Accordingly, in the Raman spectra of crystals the A-family marker bands at 664, 705, 807 and 1101 cm-1 and the spectral characteristics in the region 1200 to 1500 cm-1 clearly demonstrate the A-form as the dominant conformation. Bands at 691, 850, and 1080 cm-1, however, indicate that a minor fraction of the octamer molecules in the crystal is in an unusual, still not unequivocally identified conformation possibly belonging to the B-family. In solution, the octamer is in B-like conformation as shown by the presence of B-DNA Raman marker bands at 685, 837, 1094 and 1421 cm-1. Molecular modelling techniques lead to three structures with slightly different B-form geometries as the lowest energies models when a sigmoidal dielectric function with the bulk dielectric constant epsilon = 78 and the value q = -0.5e for the effective phosphate charges was used in the calculations. An A-form structure bearing a strong resemblance to the experimentally determined crystal structure becomes the lowest energy model structure when the electrostatic parameters are changed to epsilon = 30 and q = -0.25e, respectively.     

The interaction of RNA polymerase and lac repressor with the lac control region.

We have examined the interactions of lac repressor and RNA polymerase with the DNA of the lac control region, using a method for direct visualization of the regions of DNA protected by proteins from DNAase attack. The repressor protects the operator essentially as reported by Gilbert and Maxam (1) with some small modifications. However, the evidence reported here concerning the binding of RNA polymerase to the DNA of the promoter mutant UV5 indicates that : 1) the RNA polymerase molecule binds asymmetrically to the promoter DNA, 2) RNA polymerase protects DNA sequences to within a few bases of the CAP binding site, suggesting direct interaction between polymerase and the CAP protein at this site, 3) RNA polymerase still binds to the promoter when repressor is bound to the operator, but fails to form the same extensive complex.     

Effect of the G.T mismatch on backbone and sugar conformations of Z-DNA and B-DNA: analysis by Raman spectroscopy of crystal and solution structures of d(CGCGTG) and d(CGCGCG)

The Z-DNA crystal structures of d(CGCGTG) and d(CGCGCG) are compared by laser Raman spectroscopy. Raman bands originating from vibrations of the phosphodiester groups and sensitive to the DNA backbone conformation are similar for the two structures, indicating no significant perturbation to the Z-DNA backbone as a result of the incorporation of G.T mismatches. Both Z structures also exhibit Raman markers at 625 and 670 cm-1, assigned respectively to C3'-endo/syn-dG (internal) and C2'-endo/syn-dG conformers (3' terminus). Additional Raman intensity near 620 and 670 cm-1 in the spectrum of the d(CGCGTG) crystal is assigned to C4'-exo/syn-dG conformers at the mismatch sites (penultimate from the 5' terminus). A Raman band at 1680 cm-1, detected only in the d(CGCGTG) crystal, is assigned to the hydrogen-bonded dT residues and is proposed as a definitive marker of the Z-DNA wobble G.T pair. For aqueous solutions, the Raman spectra of d(CGCGTG) and d(CGCGCG) are those of B-DNA, but with significant differences between them. For example, the usual B-form marker band at 832 cm-1 in the spectrum of d(CGCGTG) is about 40% less intense than the corresponding band in the spectrum of d(CGCGCG), and the former structure exhibits a companion band at 864 cm-1 not observed for d(CGCGCG). The simplest interpretation of these results is that the conventional B-form OPO geometry occurs for only 6 of the 10 OPO groups of d(CGCGTG). The remaining four OPO groups, believed to be those at or near the mismatch site, are in an "unusual B" conformation which generates the 864 cm-1 band.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA bending induced by the catabolite activator protein allows ring formation of a 144 bp DNA

The effect of the catabolite activator protein, CAP, on the ligation of a 144 bp DNA was examined. This DNA has EcoRI ends and contains the lac operon CAP site and promoter-operator region. At low DNA concentrations (nM) and 37 degrees C the presence of CAP and cAMP enables T4 ligase to convert the linear duplex to a covalently closed ring. Nuclease digestion and sedimentation equilibrium studies show that the ring is a monomer circle. Ring formation does not occur in the absence of either CAP or cAMP. The kinetics of ring closure, and the bimolecular joining of two fragments were measured. The presence of CAP decreased the rate of bimolecular joining of the EcoRI ends of linear DNAs. Thus the measured rates of ring closure are likely to be a lower limit for this process. Closure reactions carried out with ethidium bromide indicate that CAP induced bending rather than twisting is responsible for ring formation. The all or none nature of the closure reaction suggests that persistence length DNAs may be useful in a simple assay for protein induced DNA bending.     

Raman spectra of the model B-DNA oligomer d(CGCGAATTCGCG)2 and of the DNA in living salmon sperm show that both have very similar B-type conformations

Raman spectra were obtained from aqueous solutions of the deoxyoligonucleotide d(CGCGAATTCGCG)2 (I), which has been suggested as a model for B-type DNA conformation. These spectra were compared with the Raman spectra of the aqueous solutions of several DNAs of natural origin taken under identical solution conditions. Since the model sequence has a high percent GC (66%), the Raman spectrum was compared with the Raman spectrum of the DNA from Micrococcus lysodeikticus (72% GC), and the spectra of the two different DNAs were found to be rather similar in both 50 mM salt and 6 M salt solutions. Computer-aided band-shape analysis of the backbone vibrational region of the Raman spectra shows the existence of several bands corresponding to different furanose ring puckers. This appears to indicate a heterogeneity of furanose ring pucker in both the model dodecamer and the native DNA. Significant differences were found in the intensity of the conformational marker band at 810 cm-1, which indicates corresponding differences in furanose ring pucker heterogeneities in these two high GC content DNAs. The Raman spectrum of the dodecamer (I) was used to analyze the Raman spectrum of the DNA inside the head of living intact salmon sperm. Sperm spectra were taken with both our conventional Raman spectrograph and a newly developed intracavity laser Raman microscope system. Although the DNA in the sperm head is required by packing considerations to be in a highly compact and condensed state, the Raman spectra of the intact sperm are almost identical with that of the model dodecamer (I) if the difference in base composition is taken into account.(ABSTRACT TRUNCATED AT 250 WORDS)     

Raman spectra of single crystals of r(GCG)d(CGC) and d(CCCCGGGG) as models for A DNA, their structure transitions in aqueous solution, and comparison with double-helical poly(dG).poly(dC)

The self-complementary oligonucleotides [r(CGC)d(CGC)]2 and [d(CCCCGGGG)]2 in single-crystal and solution forms have been investigated by Raman spectroscopy. Comparison of the Raman spectra with results of single-crystal X-ray diffraction and with data from polynucleotides permits the identification of a number of Raman frequencies diagnostic of the A-helix structure for GC sequences. The guanine ring frequency characteristic of C3'-endo pucker and anti base orientation is assigned at 668 +/- 2 cm-1 for both dG and rG residues of the DNA/RNA hybrid [r(GCG)d(CGC)]2. The A-helix backbone of crystalline [r(GCG)d(CGC)]2 is altered slightly in the aqueous structure, consistent with the conversion of at least two residues to the C2'-endo/anti conformation. For crystalline [d(CCCCGGGG)]2, the Raman and X-ray data indicate nucleosides of alternating 2'-endo-3'-endo pucker sandwiched between terminal and penultimate pairs of C3'-endo pucker. The A-A-B-A-B-A-A-A backbone of the crystalline octamer is converted completely to a B-DNA fragment in aqueous solution with Raman markers characteristic of C2'-endo/anti-G (682 +/- 2) and the B backbone (826 +/- 2 cm-1). In the case of poly(dG).poly(dC), considerable structural variability is detected. A 4% solution of the duplex is largely A DNA, but a 2% solution is predominantly B DNA. On the other hand, an oriented fiber drawn at 75% relative humidity reveals Raman markers characteristic of both A DNA and a modified B DNA, not unlike the [d-(CCCCGGGG)]2 crystal. A comparison of Raman and CD spectra of the aqueous [d(CCCCGGGG)]2 and poly(dG).poly(dC) structures suggests the need for caution in the interpretation of CD data from G clusters in DNA.     

A Raman scattering study of the helix-destabilizing gene-5 protein with adenine-containing nucleotides.

Raman spectra of gp5 and complexes of gp5 with poly(rA) and poly(dA) have been determined and analysed. From a fit of the amide I-band with model spectra it follows that the secondary structure of gp5 contains 52% beta-sheet, 28% undefined conformation and 19% alpha-helix. The band at 1032 cm-1 due to phenylalanine has an anomalous intensity both in the spectra of the complexes and the free protein. This possibly indicates a stacked structure present in the protein. Binding of gp5 to poly(rA) and poly(dA) influences the intensity of bands near 1338 and 1480 cm-1 which are considered to be marker-bands for the phosphate-sugar-base conformer. A change in conformation of the nucleotides is also reflected by vibrations originating in the phosphate- and sugar-residues of the backbone. In the spectrum of complexed poly(rA) the intensity of the conformation sensitive band at 813 cm-1, which is due to the phosphodiester group, is zero. It seems that gp5 forces poly(rA) and poly(dA) to a similar conformation. A marker band for stacking interaction in poly(rA) indicates that stacking interactions in the complex have increased.     

Raman spectroscopic investigations of sarcoplasmic reticulum membranes.

Raman spectra are presented for sarcoplasmic reticulum membranes. Interpretation of the 1000-1130 cm-1 region of the spectrum indicates that the sarcoplasmic reticulum membrane may be more fluid than erythrocyte membranes that have been examined by the I portion of the membrane spectrum with a strong 1658 cm-1 band characteristic of C=C stretching in hydrocarbon side chains exhibiting cis conformation. This band is unaltered in intensity and position in H2O and in 2H2O thus obscuring amide I protein conformation. Of particular interest is the appearance of strong, resonantly enhanced bands at 1160 and 1527 cm-1 attributable to membrane-associated carotenoids.     

NMR study of the interaction between the lac repressor and the lac operator

Binding of the lac repressor headpiece, the N-terminal region of the lac repressor, to the lac operator of Escherichia coli was studied by 1H-NMR spectroscopy. Two DNA fragments, of 51 base pairs and 62 base pairs, containing the lac operator region, were investigated. The signals of their hydrogen-bonded imino protons were well resolved in the 500-MHz NMR spectra. The spectra of the free lac operator DNA are similar to those obtained from ring-current-shift calculations for a B-DNA structure. Complex formation with the headpiece led to small but nevertheless characteristic changes in the spectra. The fact that very few imino resonances shifted upon addition of headpiece, as well as the variety in direction and size of these chemical shifts, indicate the formation of a specific complex between the lac repressor and the lac operator. The observed changes in the resonance positions exclude the intercalation of tyrosine residues of the headpiece between adjacent base pairs of the lac operator as well as the formation of a cruciform structure. They rather reflect a small conformational transition in the DNA itself, caused for example by an alteration in the tilt of a few base pairs or a shift of the keto-enol tautomeric equilibrium of the bases towards the enolic form.     

DNA melting investigated by differential scanning calorimetry and Raman spectroscopy.

Thermal denaturation of the B form of double-stranded DNA has been probed by differential scanning calorimetry (DSC) and Raman spectroscopy of 160 base pair (bp) fragments of calf thymus DNA. The DSC results indicate a median melting temperature Tm = 75.5 degrees C with calorimetric enthalpy change delta Hcal = 6.7 kcal/mol (bp), van't Hoff enthalpy change delta HVH = 50.4 kcal/mol (cooperative unit), and calorimetric entropy change delta Scal = 19.3 cal/deg.mol (bp), at the experimental conditions of 55 mg DNA/ml in 5 mM sodium cacodylate at pH 6.4. The average cooperative melting unit (nmelt) comprises 7.5 bp. The Raman signature of 160 bp DNA is highly sensitive to temperature. Analyses of several conformation-sensitive Raman bands indicate the following ranges for thermodynamic parameters of melting: 43 < delta HVH < 61 kcal/mol (cooperative unit), 75 < Tm < 80 degrees C and 6 < (nmelt) < 9 bp, consistent with the DSC results. The changes observed in specific Raman band frequencies and intensities as a function of temperature reveal that thermal denaturation is accompanied by disruption of Watson-Crick base pairs, unstacking of the bases and disordering of the B form backbone. These three types of structural change are highly correlated throughout the investigated temperature range of 20 to 93 degrees C. Raman bands diagnostic of purine and pyrimidine unstacking, conformational rearrangements in the deoxyribose-phosphate moieties, and changes in environment of phosphate groups have been identified. Among these, bands at 834 cm-1 (due to a localized vibration of the phosphodiester group), 1240 cm-1 (thymine ring) and 1668 cm-1 (carbonyl groups of dT, dG and dC), are shown by comparison with DSC results to be the most reliable quantitative indicators of DNA melting. Conversely, the intensities of Raman marker bands at 786 cm-1 (cytosine ring), 1014 cm-1 (deoxyribose ring) and 1092 cm-1 (phosphate group) are largely invariant to melting and are proposed as appropriate standards for intensity normalizations.     

Observation of the Fe4+ = O stretching Raman band for cytochrome oxidase compound B at ambient temperature

Resonance Raman and visible absorption spectra were simultaneously observed for cytochrome oxidase reaction intermediates at 5 degrees C by using the artificial cardiovascular system (Ogura, T., Yoshikawa, S., and Kitagawa, T. (1989) Biochemistry 28, 8022-8027) and a device for Raman/absorption simultaneous measurements (Ogura, T., and Kitagawa, T. (1988) Rev. Sci. Instrum. 59, 1316-1320). The Fe4+ = O stretching (nu FeO) Raman band was observed at 788 cm-1 for compound B for the first time. This band showed the 16O/18O isotopic frequency shift (delta nu FeO) by 40 cm-1, in agreement with that for horseradish peroxidase compound II (nu FeO = 787 cm-1 and delta nu FeO = 34 cm-1). In the time region when the FeII-O2 stretching band for compound A and the nu FeO band for compound B were coexistent, a Raman band assignable to the Fe3+-O-O-Cu2+ linkage was not recognized.     

Application of Raman spectroscopy to biological macromolecules.

The Raman spectra of biological macromolecules arise from molecular vibrations of either the backbone chains or the side chains. The frequencies of the Raman bands lie in a region between 200 cm-1 and 3000 cm-1. From certain frequencies of the vibrations of the backbone chains one can determine the conformation or secondary structure of a macromolecule. Thus for polypeptides and proteins the frequencies of the Amide I and Amide III vibrations allow one to determine the averge conformation of their backbone chain. In polynucleotides and nucleic acids, the frequency of the phosphate diester stretch of the phosphate furanose chain varies between 814 cm-1 for A conformation and 790 cm-1 for B conformation. Raman spectra of the bases in nucleic acids can be used to determine base stacking and hydrogen bonding interactions. Thus Raman spectroscopy is an important tool for determining the conformation structure of proteins and nucleic acids.     

The hydrogen-bonding structure in parallel-stranded duplex DNA is reverse Watson-Crick

Raman spectra of the parallel-stranded duplex formed from the deoxyoligonucleotides 5'-d-[(A)10TAATTTTAAATATTT]-3' (D1) and 5'-d[(T)10ATTAAAATTTATAAA]-3' (D2) in H2O and D2O have been acquired. The spectra of the parallel-stranded DNA are then compared to the spectra of the antiparallel double helix formed from the deoxyoligonucleotides D1 and 5'-d(AAATATTTAAAATTA-(T)10]-3' (D3). The Raman spectra of the antiparallel-stranded (aps) duplex are reminiscent of the spectra of poly[d(A)].poly[d(T)] and a B-form structure similar to that adopted by the homopolymer duplex is assigned to the antiparallel double helix. The spectra of the parallel-stranded (ps) and antiparallel-stranded duplexes differ significantly due to changes in helical organization, i.e., base pairing, base stacking, and backbone conformation. Large changes observed in the carbonyl stretching region (1600-1700 cm-1) implicate the involvement of the C(2) carbonyl of thymine in base pairing. The interaction of adenine with the C(2) carbonyl of thymine is consistent wtih formation of reverse Watson-Crick base pairing in parallel-stranded DNA. Phosphate-furanose vibrations similar to those observed for B-form DNA of heterogenous sequence and high A,T content are observed at 843 and 1092 cm-1 in the spectra of the parallel-stranded duplex. The 843-cm-1 band is due to the presence of a sizable population of furanose rings in the C2'-endo conformation. Significant changes observed in the regions from 1150 to 1250 cm-1 and from 1340 to 1400 cm-1 in the spectra of the parallel-stranded duplex are attributed to variations in backbone torsional and glycosidic angles and base stacking.