Base pairing in wheat germ ribosomal 5S RNA as measured by ultraviolet absorption, circular dichroism, and Fourier-transform infrared spectrometry

Ultraviolet absorption (UV) and circular dichroism (CD) spectra of wheat germ 5S RNA, when compared to tRNAPhe, indicate a largely base-paired and base-stacked helical structure, containing up to 36 base pairs. Fourier-transform infrared (FT-IR) spectra of tRNAPhe and wheat germ ribosomal 5S RNA have been acquired at 30 and 90 degrees C. From the difference of the FT-IR spectra between 90 and 30 degrees C, the number of base pairs in both RNAs was determined by modification of a previously published procedure [Burkey, K. O., Marshall, A. G., & Alben, J. O. (1983) Biochemistry 22, 4223-4229]. The base-pair composition and total base-pair number from FT-IR data are now consistent for the first time with optical (UV, CD, Raman) and NMR results for ribosomal 5S RNA. Without added Mg2+, tRNAPhe gave 18 +/- 2 base pairs [7 A-U and 11 G-C], in good agreement with the number of secondary base pairs from X-ray crystallography [8 A-U, 12 G-C, and 1 G-U]. Within the 10% precision of the FT-IR method, wheat germ 5S RNA exhibits essentially the same number of base pairs [14 A-U, 17 G-C, and 5 G-U; for a total of 36] in the absence of Mg2+ as in the presence of Mg2+ [14 A-U, 18 G-C, and 3 G-U; for a total of 35], in agreement with the UV hyperchromism estimate of G-C/(A-U + G-C) = 0.58.(ABSTRACT TRUNCATED AT 250 WORDS)     

Base pairing in Bacillus subtilis ribosomal 5S RNA as measured by ultraviolet absorption and Fourier-transform infrared spectrometry

Ultraviolet (260 and 280 nm) and Fourier-transform infrared (FT-IR) spectra of Bacillus subtilis ribosomal 5S RNA have been acquired between 20 and 90 degrees C. In the presence of added Mg2+, the average UV melting midpoint, Tm, is 60 (A260) or 62 degrees C (A280), resolving into two components (Tm = 54 and 68 degrees C). In the presence of 10 mM Mg2+, the normalized A260 increases by about 5%, and the average Tm increases to 70 degrees C (A260 or A280), resolving into components at 63 and 73 degrees C at 260 nm but not resolved at 280 nm. From the difference of the 5S RNA FT-IR spectra between 90 and 30 degrees C, the number of base pairs in B. subtilis 5S RNA was determined by the procedure outlined in the accompanying paper [Li, S.-J., Burkey, K. O., Luoma, G. A., Alben, J. O., & Marshall, A. G. (1984) Biochemistry (preceding paper in this issue)]. Addition of 10 mM Mg2+ increases the number of A-U pairs by 1 (from 11 to 12) and the number of G-C pairs by 2 (from 15 to 17). FT-IR melting curve midpoints show that addition of Mg2+ increases the melting point for both A-U and G-C pairs in B. subtilis 5S RNA. The A-U pairs melt before G-C pairs (56 vs. 64 degrees C) in the absence of Mg2+, but both types of pairs melt at the same temperature (67 vs. 70 degrees C) in the presence of Mg2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Photoreactivating enzyme from Escherichia coli: isolated enzyme lacks absorption in its actinic wavelength region and its ribonucleic acid cofactor is partially double stranded when associated with apoprotein

Isolated photoreactivating enzyme (PRE) from Escherichia coli exhibits some optical density at wavelengths greater than 300 nm. After correcting for the effects of light scattering, however, we find no true absorption in the spectral region that is required for enzymatic activity (320-450 nm). At shorter wavelengths, there is an absorption maximum near 260 nm that is due primarily to an RNA cofactor. Heating to 60 degrees C and subsequently cooling to 4 degrees C release the RNA cofactor from association with apoprotein and result in hyperchromicity. Circular dichroism indicates that the RNA associated with native enzyme is partially double stranded. At low ionic strength (mu = 0.01), heating to 15 degrees C or protease treatment at 4 degrees C results in irreversible loss of part of the double strandedness. We show that the difference spectrum at 4 degrees C between the absorption spectra of native enzyme and heat-treated enzyme can be fit by a superposition of reference spectra for denaturation of A-U and G-C base pairs derived from model polynucleotides. The coefficients of the linear combination of reference spectra were used to calculate the fraction of A-U and G-C base pairs. We find that both A-U and G-C base pairs are present in equal concentrations and that about 20% are in a double-stranded conformation in the native enzyme.     

Conserved unpaired adenine residues are important for ordered structures of 5S ribosomal RNA. An infrared study of the secondary and tertiary structure of Thermus thermophilus 5S rRNA

An improved set of infrared calibration spectra for the determination of G X C and A X U base pairs leads to 32 +/- 3 G X C (+ G X U) and 4 +/- 1 A X U base pairs for Thermus thermophilus 5S RNA in the presence and absence of Mg2+. These results give further support for the consensus secondary structure of 5S RNA recently proposed by several groups. T. thermophilus 5S RNA shows, in the presence of Mg2+, a distinct two-step thermal melting of its ordered structure. Based on new data about the stacking dependence of infrared intensities of unpaired ribonucleotides the spectral changes of the low-temperature transition should be explained by melting of stacked arrangements of unpaired bases and/or non-standard base pairs. Striking is the reduction in A stacking, which is not related to the melting of A X U base pairs, indicating the importance of the mostly conserved unpaired adenines for the Mg2+ stabilized higher-order structures especially within internal loops of 5S RNA.     

A.U and G.C base pairs in synthetic RNAS have characteristic vacuum UV CD bands.

The vacuum UV CD spectra of GpC, CpG, GpG, poly[r(A)], poly[r(C)], poly[r(U)], poly[r(A-U)], poly[r(G).r(C)], poly[r(A).r(U)], and poly[r(A-U).r(A-U)] were measured down to at least 174 nm. These spectra, together with the published spectra of poly[r(G-C).r(G-C)], CMP, and GMP, were sufficient to estimate the CD changes upon base pairing for four double-stranded RNAs. The vacuum UV CD bands of poly[r(A)], poly[r(C)], and the dinucleotides GpC and CpG were temperature dependent, suggesting that they were due to intrastrand base stacking. The dinucleotide sequence isomers GpC and CpG had very different vacuum UV CD bands, indicating that the sequence can play a role in the vacuum UV CD of single-stranded RNA. The vacuum UV CD bands of the double-stranded (G.C)-containing RNAs, poly[r(G).r(C)] and poly[r(G-C).r(G-C)], were larger than the measured or estimated vacuum UV CD bands of their constituent single-stranded RNAs and were similar in having an exceptionally large positive band at about 185 nm and negative bands near 176 and 209 nm. These similarities were enhanced in difference-CD spectra, obtained by subtracting the CD spectra of the single strands from the CD spectra of the corresponding double strands. The (A.U)-containing double-stranded RNAs poly[r(A).r(U)] and poly[r(A-U).r(A-U)] were similar only in that their vacuum UV CD spectra had a large positive band at 177 nm. The spectrum of poly[r(A).r(U)] had a shoulder at 188 nm and a negative band at 206 nm, whereas the spectrum of poly[r(A-U).r(A-U)] had a positive band at 201 nm. On the other hand, difference spectra of both of the (A.U)-containing polymers had positive bands at about 177 and 201 nm. Thus, the difference-CD spectra revealed CD bands characteristic of A.U and G.C base pairing. (ABSTRACT TRUNCATED AT 250 WORDS)     

Circular dichroism calculations for double-stranded polynucleotides of repeating sequence

Optical property calculations are presented for poly(A·U), poly[(A-U)·(A-U)], poly(G·C), and poly[(G-C)·(G-C)] in RNA, B-DNA, and C-DNA conformations. An all-order classical coupled oscillator polarizability theory was used, and an effective dielectric constant of 2 was assumed. The calculated CD spectra were found to be sensitive to both geometry and sequence. Agreement with the measured CD spectra of poly(A·U), poly(G·C), and poly(dG·dC) is very good. Calculations for other sequences and geometries are less satisfactory and are particularly poor for poly[(G-C)·(G-C)] in RNA geometry and poly(A·T) in B-DNA geometry. Attempts to improve agreement with measured spectra by varying monomer properties have been only partially successful for these calculations, but they illustrate the types of changes that may prove to be necessary. Calculations using other published X-ray coordinates for certain deoxypolynucleotides of simple sequence, some of which are quite different from B-DNA coordinates, did not result in better agreement with measured spectra. Finally, the dependence of the calculated CD on chain length is examined. Results show that non-nearest neighbor interactions can be important when runs of 3 or more identical base pairs appear in a given sequence.     

Sequence-dependant polymorphism of DNA duplex in solutions

Raman spectra of six synthetic polydeoxyribonucleotide duplexes with different base sequences have been examined in aqueous solutions with different salt or nucleotide concentrations. Detailed conformational differences have been indicated between B and Z forms of poly[d(G-C)] X poly[d(G-C)], between B forms of poly[d(G-C)] X poly[d(G-C)] and poly[d(G-m5C)] X poly[d(G-m5C)], between A and B forms of poly(dG) X poly(dC), between B and "CsF" forms of poly[d(A-T)] X poly[d(A-T)], between B forms of poly[d(A-U)] X poly[d(A-U)] and poly[d(A-T)] X poly[d(A-T)], and between low- and high-salt (CsF) forms of poly(dA) X poly(dT). The Raman spectrum of calf-thymus DNA in aqueous solution was also observed and was compared with the Raman spectra of its fibers in A, B, and C forms.     

A study of the influence of magnesium ions on the conformation of ribosomal ribonucleic acid and on the stability of the larger subribosomal particle of rabbit reticulocytes.

Mg2+ was shown to affect the conformation of rRNA over the range of 0.03-1.2M-KCl. The species studies were Escherichia coli S-rRNA and L-rRNA (the RNA moieties of the smaller and larger subribosomal particles respectively) and rabbits S-rRNA and L-rRNA. 2. The addition of Mg2+ to rRNA in reconstitution buffer (0.35M-KCl0.01M-Tris/HCl, pH7.2) at 20 degrees C let to an increase in bihelical secondary structure through the formation of additional (mainly A-U) base-pairs (e.g. an additional approx. 58 A-U base-pairs per molecule of E. coli S-rRNA as judged by u.v. difference spectrophotometry...     

Several properties of complexes of polynucleotides with aromatic amino acid amides of oligonucleotides

Residues of Phe, Tyr and Trp in the complexes of their oligonucleotide amidates and polynucleotides of A-U of G-C nucleotide composition are most likely localized in the minor groove of the Watson--Crick part of the triple helix where they interact with bases but do not intercalate into the helix. Formation of the complexes is accompanied with a change in the relative localization of amino acids and bases. The major geometrical parameters of the triple helices of the complexes are not changed by the residues of aromatic amino acids (according to CD data). A slight violation of stacking interactions between bases is observed along with an increase of the cooperativity of melting of the complexes of A-U composition (according to UV absorption data). The effect of the residues of aromatic amino acids on the stability of triple helices is determined by the nucleotide composition of the latter, i.e. complexes of A-U composition are destabilized with the Phe, Tyr and Trp residues, whereas the Trp residue does not affect the stability of the complexes of G-C composition. The hydrophobic character of aromatic amino acids and their different affinity for bases of different structure seem to account for this difference in stability. The dependence of the thermal stability of RNH-dp(An).2poly(U)-complexes on the structure of the amide radical (residues of glycin, aromatic amino acids, alkyl- and arylalkyl amines) testifies the ability of the radical to "regulate" the interaction between the oligomer and the complementary polynucleotide. This capacity for "regulation" is not observed in the system of G-C composition.     

The hydrodynamic shape, conformation, and molecular model of Escherichia coli ribosomal 5 S RNA.

The structure of ribosomal 5 S RNA has been examined using several physical biochemical techniques. Hydrodynamic measurements yield a s020,omega and [eta] of 5.5 x 10(-13) x and 6.9 ml/g, respectively. Other parameters calculated from these values indicate the shape of 5 S RNA is consistent with that of a prolate ellipsoid 160 A in length and 32 A wide. Sedimentation equilibrium results show that 5 S RNA exists as a monomer in the reconstitution buffer with an apparent molecular weight of 44,000. Ultraviolet absorption difference spectra show that approximately 75% of the bases in 5 S RNA are involved in base pairing, and of these base pairs 70% are G-C and 30% are A-U. These results on the overall shape and secondary structure of 5 S RNA have been incorporated with the results of other investigators as to the possible location of single-stranded and double-stranded helical regions, and a molecular model for 5 S RNA is proposed. The molecular model consists of three double helices in the shape of a prolate ellipsoid, with two of the double helical regions at one end of the molecule. The structure is consistent with the available data on the structure and function of 5 S RNA and bears similarity to the molecular model proposed by Osterberg et al. ((1976) Eur. J. Biochem. 68, 481-487) based on small angle x-ray scattering results and the secondary structure proposed by Madison ((1968) Annu. Rev. Biochem. 37, 131-148).     

On the stability of nucleic acid structures in solution: enthalpy-entropy compensations, internal rotations and reversibility.

The thermodynamics of self-association (stacking) of free bases and nucleotides, intramolecular stacking in dinucleotides, nearest-neighbour base pair stacking interactions in duplex DNA and RNA, and the formation of hairpin loops illustrate enthalpy/entropy compensations. Large stacking exothermicities are associated with large negative entropy changes that ensure that delta G is small, permitting readily reversible associations in solution. We rationalise enthalpy/entropy compensations with reference to residual motions and torsional vibrations which make a larger entropic contribution to binding when - delta H approximately kT (thermal energy at room temperature), than when - delta H >> kT. We present a factorisation of experimental free energies for helix formation in terms of approximate contributions from the restriction of rotations, hydrophobic interactions, electrostatic interactions due to base stacking, and contributions from hydrogen bonding, and estimate the adverse free energy cost per rotor (mainly entropy) of ordering the phosphate backbone as between 1.9 and 5.4 kJ mol-1 [averaged over 12 rotors per base pair for A-U on A-U stacking (lower limit), and G-C on C-G stacking (upper limit)]. The largest cost is associated with the most exothermic stacking interactions, while the range of values is consistent with earlier conclusions from data on the fusion of hydrocarbon chains (lower value), and with entropy changes in covalent isomerisations of small molecules involving severe restrictions (upper value).     

Spectroscopic analysis of the interaction of Escherichia coli DNA-dependent RNA polymerase with T7 DNA and synthetic polynucleotides.

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.     

Growth properties associated with A-U replacement of specific G-C base pairs in 16S rRNA from Escherichia coli.

Mutations that disrupt each of seven specific G-C base pairs in 16S rRNA from Escherichia coli confer loss of expression of a plasmid-encoded 16S rRNA selectable marker (spectinomycin resistance). However, A-U replacement of G-C base pairs at nucleotides 359/52 or 1292/1245 in 16S rRNA permits normal expression of the marker. By contrast, A-U replacements at 146/176, 153/168, 350/339, or 1293/1244 are associated with loss of expression of the marker. These genetic studies are designed to determine the importance of specific base pairs by assessment of the structural and functional impairments of 16S rRNA molecules resulting from expression of base pair substitutions at these positions.     

Evidence for Z-form RNA by vacuum UV circular dichroism.

Circular dichroism (CD) spectra in the vacuum UV region for different conformations of poly d(G-C) X poly d(G-C) and poly r(G-C) X poly r(G-C) are very characteristic. The CD of the RNA in the A-form (6 M NaClO4 and 22 degrees C) is very similar to that of the DNA in 80% alcohol where it is believed to be in the A-form. With the exception of the longest wavelength transition, the CD of the RNA in 6 M NaClO4 at 46 degrees C is similar to the CD of the DNA under conditions where it is believed to be in the Z-form (2 M NaClO4). This substantiates that poly r(G-C) X poly r(G-C) assumes a left-handed Z-conformation in 6 M NaClO4 above 35 degrees C. CD spectra for the left-handed Z-forms of both the RNA and DNA are characterized by an intense negative peak at 190-195 nm, a crossover at about 184 nm, and an intense positive peak below 180 nm. The right-handed A- and B-forms of RNA and DNA all have an intense positive peak in their CD spectra near 186 nm. The large difference in CD in the range 185-195 nm for right- and left-handed conformations of nucleic acids can be used to identify the sense of helix winding.     

The effects of tetraalkylammonium salts on helix-coil transition parameters in natural and synthetic ribo- and deoxyribo-polynucleotides

Thermal transition profiles were recorded for a variety of natural and synthetic DNA and double-stranded RNA preparations in the presence of tetramethylammonium (TMA+) and tetraethylammonium (TEA+) cations. Double-stranded RNAs of natural origin, with GC contents of 50% exhibited the same profiles and Tm values as native DNA containing normal bases. Hence the tetraalkylammonium cations liquidate not only the effects of base composition, and the difference in stability between A-T and A-U base pairs (further confirmed by measurements with uracil-containing DNA from phage PBS-2), but also that of the 2'OH. In the presence of TMA+ cations, there is very marked enhancement of the stability of U-U base pairs in poly(rU) and poly(Um). In 2.4 M TEA, the 1:1 complex of poly(G) with poly (C) formed readily and melted reversibly with a Tm as low as 87 degrees C. At concentrations of TMA and TEA for which dTm/dXGC = 0, the Tm values for various phage DNA preparations containing atypical bases (phages T2, T4, phi e, phi W-14, PBS-2) differ appreciably from those with 'normal bases'. Analysis of these findings indicates that the selective interaction of TMA and TEA cations with A-T base pairs occurs in the minor groove of the DNA helix. The overall results show that the action of these quaternary ammonium cations is not due exclusively to preferential binding to A-T base pairs, but must involve other factors, including modifications of solvent structure. They also underline the utility of TMA and TEA solvent systems for placing in evidence transition profiles not accessible in other solvent systems.     

Intensity changes of base and backbone Raman modes in the B to Z transition of poly(dG-dm5C)

Raman spectroscopy was employed to investigate the temperature-induced B to Z transition of poly(dG-dm5C). The transition midpoint was about 37 degrees C for a solvent containing 20 mM Mg2+. A 10-fold change in Mg2+ concentration altered the transition midpoint by at least 60 degrees C. Raman spectra of the B and Z forms of poly(dG-dm5C) exhibited characteristics similar to those observed with poly(dG-dC). The 682 cm-1 guanine mode and 835 cm-1 backbone mode were present in the B conformation. In the Z form the intensities of these two bands decrease substantially and new peaks were observed at 621 cm-1, 805 and 819 cm-1. Several bands unique to poly(dG-dm5C) were also observed. Transition profiles of band intensity vs. temperature were determined for fourteen Raman bands. The curves of all of the base vibrations and one backbone mode had the same slope and midpoint. This indicates that conformational changes in the guanine and methycytosine bases occur concurrently.     

Secondary structure of ovalbumin messenger RNA.

The secondary structure of highly purified ovalbumin mRNA was studied by automated thermal denaturation techniques and the data were subjected to computer processing. Comparative studies with 20 natural and synthetic model nucleic acids suggested that the secondary structure of ovalbumin mRNA possesses the following features: the extent of base pairing of ovalbumin mRNA is similar to that found in tRNAs or ribosomal RNAs; the secondary structure of ovalbumin mRNA is more thermolabile than any of the model compounds tested, including the copolymer poly(A-U); ovalbumin mRNA does not have extensive G-C rich stems as found in tRNAs or ribosomal RNAs; the base composition of the double-stranded regions reveals 54% G-C residues which was significantly higher than that noted in the whole molecule (approximately 41.5% G-C). The presence of 46% A-U pairs in short stems of about five base pairs would have a very large destabilizing effect on the secondary structure of ovalbumin mRNA. However, at 0.175 M monovalent cations and 36 degrees C most of the secondary structure of ovalbumin mRNA is preserved. These data suggest that the double-stranded regions in ovalbumin mRNA are of sufficient length to provide the necessary stability for maintaining the open loop regions in an appropriate conformation which may be required for the biological function of ovalbumin mRNA. Furthermore, the lability of the double-stranded regions in ovalbumin mRNA may also be important for the biological function of this mRNA.