K. A. Hartman

Raman studies of nucleic acids VIII estimation of RNA secondary structure from raman scattering by phosphate-group vibrations

Abstract Analysis of the Raman spectra of a large number of ribonucleotide monomers and polymers indicates that the frequencies and intensities characteristic of the P-O stretching vibrations provide a basis for the quantitative determination of RNA secondary structure. The intensity ratio of the Raman lines at 815 and 1100 cm−1 is shown to have a value of 1.64 ± 0.04 in completely ordered structures and a value of zero in disordered structures. Ordered structures in this context include double-helical configurations containing A · U or G · C pairs and ordered single-stranded configurations (such as occur in poly(rA) and poly(rC)) but exclude other type of base pairing as well as random-chain configurations. The percentages of RNA nucleotides in such ordered configurations, as computed from Raman intensity measurements, are 16-S RNA ( 95 ± 5 ), 23-S RNA ( 85 ± 5 ), tRNAfMet ( 84 ± 3 ), tRNAVal ( 84 ± 3 ), tRNAPhe ( 85 ± 3 ) and R17 RNA ( 87 ± 3 % ). Since the present method makes use of spectral transitions originating in the RNA backbone and not in the bases, no limitations are imposed by lack of knowledge of the RNA base sequence. In combination with data obtained from quantitative infrared spectroscopy the Raman results are also shown to yield information on the percentages of RNA nucleotides in ordered single-stranded configurations.

The infrared spectrum and structure of the type I complex of silver and DNA

Infrared spectroscopy was used to study films of the type I complex of Ag+ and DNA as a function of hydration with the following conclusions. Ag+ binds to guanine residues but not to cytosine or thymine residues. Cytosine becomes protonated as Ag+ binds to guanine. (These conclusions confirm previous models.) The type I complex remains in the B family of structures with slight modifications of the sugar-phosphate geometry. This modified B structure remains stable at lower values of hydration for which pure DNA is in the A form. Binding of Ag+ to PO2-, O-P-O or the deoxyribose oxygen is excluded.

Raman studies of nucleic acids X. Conformational structures of Escherichia coli transfer RNAs in aqueous solution

Abstract Laser-excited Raman spectra of aqueous solutions of purified samples of Escherichia coli tRNA Glu , tRNA Arg , tRNA Val , tRNA fMet and tRNA 2 Phe are compared. The characteristic Raman frequencies and intensities, from scattering by symmetrical stretching vibrations of the phosphodiester groups, reveal that the total secondary structure of each tRNA is qualitatively and quantitatively the same. The percentage of nucleotide residues in each tRNA which exist in a highly ordered configuration is 84 ± 4 %, consistent with a cloverleaf structure provided two-thirds of the unpaired bases in loops are also in ordered configurations. The remaining nucleotides (16 ± 4 %) must occur in randomly oriented or highly-strained configurations of the tRNA chain. The Raman spectra also reveal specific differences in the amount of stacking of A and G residues in different tRNAs. These results provide strong evidence for the presence of specific tertiary structure (folding) in aqueous tRNA. A study of the melting transition of tRNA Glu over the range 32–90 °C reveals that appreciable order persists in the tRNA backbone at 90 °C and is attributable to residual stacking of the purines. Gross changes in the conformation of tRNA Val are also detected when excess Mg 2+ is present. The improved spectral sensitivity in this work demonstrates that the Raman spectrum of tRNA may be used to detect the presence of the minor nucleoside, dihydrouridine, and that the frequency region 1150–1450 cm −1 is highly sensitive to the base composition, base sequence and base-stacked secondary structure of tRNA.

Raman studies of nucleic acids VI. Conformational structures of tRNAfMet, tRNAVal and tRNAPhe2☆

Abstract Laser-excited Raman spectra of tRNA fMet , tRNA Val and tRNA Phe 2 from Escherichia coli indicate that each tRNA has a similar conformation in aqueous solution. The spectra of tRNA fMet and tRNA Val show further that their specific base-paired and base-stacked secondary structures are consistent with the proposed clover-leaf models. The onset of melting in tRNA fMet and tRNA Val occurs at about 40°C. At 70°C all base pairs are broken. Above 70°C and up to 90°C, significant order is retained in the tRNA backbones and appreciable stacking of G residues persists. In the presence of a 3-fold molar excess of Mg 2+ , the onset of melting occurs at about 60°C. Comparison of Raman spectra of tRNA and rRNA reveals that the latter molecules have a more ordered ribose-phosphate backbone and contain more A but less G residues in stacked configurations at 32°C.

Structure of RNA in Ribosomes

The 50S and 30S ribosomes and 23S and 16S RNA were hydrolyzed with ribonuclease A. The rate constants and number of fragments produced were determined for each reaction. The conformation of 23S RNA changes when the RNA is extracted from the ribosome. Specific regions of the RNA in 50S and 30S ribosomes are protected from hydrolysis by the ribosomal proteins.

Secondary Structure of Ribosomal RNA

Infrared spectra were obtained for 16S and for 23S ribosomal RNAs in D2O solutions. The percentage of each base in the paired and unpaired regions of the RNA was determined from the spectra. The secondary structures of 16S and 23S ribosomal RNAs (from Escherichia coli) are significantly different from each other and are also different from those of yeast ribosomal RNA, formylmethionyl-transfer RNA, and the anticodon fragment of this transfer RNA.

Structural Forms and Transitions of Poly(dG-dC)with Cd(II), Ag(I) and NaN03

Infrared spectroscopy was used to study the structures and transitions in hydrated gels of double-helical poly(dG-dC) complexed with the metal carcinogens Cd(II) and Ag(I). For one Cd(II) per ten nucleotides (r = 0.1), the B structure was stable at high and moderate hydrations with D2O and the B and Z structures coexisted at low hydrations. For poly(dG-dC) with Cd(II) at r = 0.2 to 0.35, the Z structure was stable at high hydrations (94% r.h. for r = 0.2). At a given value of hydration, H2O gave a higher content of Z structure than D2O. Cd(II) most likely binds to guanine residues at N7 in both the B and Z forms of poly(dG-dC) but binding to guanine N3 can not be excluded. It is unlikely that Cd(II) binds to cytosine residues at the r values studied and the cytosine residues did not protonate at N3 as Cd(II) bound to guanine residues. Poly(dG-dC) with Ag(I) at r = 0.2 to 0.36, existed in a B-family structure which is different from the B-family structure of the type I complex of Ag(I) and calf-thymus DNA. Poly(dG-dC) with Ag(I) did not assume the Z structure at lower hydrations even though NO3- was present in the sample. Ag(I) differs from other soft-metal acids which promote the Z structure. Ag(I) most likely binds to the guanine N7 or N3 and not to cytosine residues. Cytosine residues did not protonate at N3 as Ag(I) was bound to guanine.(ABSTRACT TRUNCATED AT 250 WORDS)

Existence of the C Structure in Poly(dA-dC) · Poly(dG-dT)

We show that the lithium salt of calf-thymus DNA can assume the C structure in nonoriented, hydrated gels. The transitions between the B and C structures showed little hysteresis and none of the metastable structural states which occur in oriented gels. Therefore crystal-lattice forces are not needed to stabilize the C structure. The occurrence of the alternative structures of the Li, Na and K salts of poly(dA-dC).poly(dG-dT) was measured as a function of hydration for nonoriented gels. Poly(dA-dC).poly(dG-dT).Li exists in the B structure at high hydrations and in the C structure at moderate hydrations with no A or Z structure at any hydration tested. The Na salt of poly(dA-dC).poly(dG-dT) exists in the B structure at high hydration, as mixtures of B and C at moderate hydrations and in the A structure at lower hydrations. The potassium salt behaves similarly except that mixtures of the C and A structures exist at lower hydrations. ZnCl2 and NaNO3, which promote the Z structure in duplex poly(dG-dC), promote the C structure in poly(dA-dC).poly(dG-dT). Information contained in the sequence of base pairs and not specific ionic interactions appear to determine the stability of the alternative structures of polynucleotides as hydration is changed.

Structural forms and transitions for the complex of mercury(II) with poly(dG-dC).

Infrared spectroscopy was used to study hydrated double-helical poly(dG-dC) complexed with varying amounts of mercury(II). For one Hg(II) per ten nucleotide residues (r = 0.1), the B structure was stabilized and the B* structure was absent at high hydration. The Z structure did not form as hydration was reduced. For r = 0.2, the B and Z structures coexisted at high hydration and the transition to total Z structure was broad as hydration was reduced. Hg(II) was bound exclusively to the guanine residues probably at N3 or N7 for r less than or equal to 0.25. The cytosine residue did not protonate (at N3) as Hg(II) was bound to guanine. The addition of NaCl together with Hg(II) reduced the binding of Hg(II), stabilized the B structure at the highest hydration and caused a sharp transition between the B and Z structures as hydration was lowered. Hydration with D2O stabilized the Z structure for poly(dG-dC) complexed with HgCl2.

Structures of Poly(dG-dC) and Poly(dA-dT) Stabilized by Anions

Infrared spectra were used to show that the sodium salts of acetate, sulfate and phosphate (pH 7.2) selectively stabilize some of the alternative structures of poly(dG-dC).Na and poly(dA-dT).Na as a function of hydration in nonoriented gels. NaCl was used as a reference. Each anion was present at 0.36 mole per mole of nucleotide residue. The weak absorption bands from these anions did not interfere with conclusive interpretation of the IR spectra of the polynucleotides. Poly(dG-dC).Na assumed the usual B* structure with each of the anions at high hydrations (r.h. of the ambient air > or = 94%). Lowering the hydration gave the following results. With acetate, the B* structure remained with only a small fraction of a modified Z or some other unusual structure present. With sulfate or phosphate, a sharp transition to the Z structure occurred (essentially complete by 86% r.h.). With reference to chloride ions, acetate favors the B* while sulfate and phosphate (pH 7.2) favors the Z structure. Poly(dA-dT).Na assumed the usual B structure with each of the anions at high hydrations. Lowering the hydration gave the following results. With acetate, the A structure was observed at the same hydrations as with chloride. With sulfate, a sharper transition to the A structure occurred (complete by 80% r.h.). With phosphate, a still sharper transition to the A structure occurred (complete by 86% r.h.). With reference to chloride, acetate shows little difference but sulfate and phosphate (pH 7.2) promote the A over the B structure. These results are compared with past results for NaNO3.