Action of ribonuclease BS-1 on a DNA-RNA hybrid

Evidence has been obtained that ribonuclease BS-1, structurally and catalytically related to ribonuclease A, is capable of degrading a DNA-RNA hybrid. This finding has been discussed with regard to the recently reported biological effects of the enzyme.     

Crystallization and preliminary crystallographic investigation of a DNA-RNA hybrid

A complex between the complementary hexanucleotides ribo-C-A-A-A-A-G and deoxyribo-C-T-T-T-T-G has been crystallized. The space group was determined to be P2₁ with unit cell dimensions $\text{a = 26.9}\text{A}\text{?}\text{, b = 50.9}\text{A}\text{?}\text{, c = 62.0}\text{A}\text{?}\text{; α = γ = 90 °, β = 108 °}$. The crystals contain both the ribo- and the deoxyribo-hexanucleotide strands in a ratio of approximately 1:1. Each asymmetric unit contains the equivalent of four hybrid molecules.     

Theoretical studies of DNA-RNA hybrid conformations.

Molecular modelling has been used to probe the conformational preferences of double stranded DNA-RNA hybrids. As might be expected, the sugars of the DNA strand have higher conformational flexibility, but, for the majority of the repetitive sequences studied, these sugars prefer a C2-endo pucker, while ribose sugars uniformly adopt a C3-endo pucker. This gives rise to a strongly heteronomous duplex conformation. One exception to this rule involves the thymidine strand of poly(dT).poly(rA), which marginally prefers a C3-endo pucker. Our study further indicates that the DNA strands of the hybrids favour backbone torsions in the canonical B domain, rather than the modified values proposed on the basis of fibre diffraction studies. Backbone conformational transitions can nevertheless be induced leading to an alpha gamma-flip (alpha:gamma, g-/g(+)-->t/t) or to the alpha beta gamma-flip form proposed from fibre studies (alpha:beta:gamma, g-/t/g(+)-->t/g+/t). The latter transition is also found to be linked to BI-->BII transitions (epsilon:zeta, t/g(-)-->g-/t).     

Renaturation of bacteriophage phiX174 DNA-RNA hybrid: RNA length effect and nucleation rate constant

A one to one RNA-DNA hybrid was synthesized using bacteriophage φX174 DNA as a template for the Escherichia coli RNA polymerase system. The renaturation rate for the hybrid was found to vary as the square root of the molecular weight of the RNA. The RNA molecular weights were determined using a standardized dimethylsulfoxide, cesium sulfate equilibrium density gradient. The optimum nucleation rate constant for the renaturation of the hybrid in 0.4 m-sodium chloride was found to be 1.3 × 10⁵, as compared with to 1.7 × 10⁵ for DNA renaturation.     

DNA-RNA hybridization.

Interest in nucleic acid hybridization stems mainly from its great power as a tool in biological research. It is used in several quite distinct ways. Because of the high degree of specificity that they show, hybridization techniques can be used to measure the amount of one specific sequence within a very heterogeneous mixture of sequences. Measurements of 1/10(6)-10(7) have been recorded. In extension of this, various properties of a specific sequence can often be studied. Secondly, because the kinetics of nucleic acid hybridization are quite well understood, it can be used to characterize both a pure sequence and a very complex mixture of sequences, like the genome of a vertebrate. Thirdly, again because of its specificity, it can be used to measure homologies between different populations of nucleic acids. Lastly, in conjunction with other techniques, it can be used as a basis for the fractionation of nucleic acid populations and the purification of specific sequences. Specific examples of these applications are given, with special reference to the organization of the genome in higher eukaryotes.     

Infrared absorption spectrum of keratin. II. Deuteration studies

A number of new bands have been found in the spectra of deuterated α- and β-keratin. In particular, the deuteration difference spectrum has been useful for the determination of frequencies of previously unsuspected bands. Thus it is found that the amide A and II frequencies of the nonhelical component in α-keratin occur at 3310–3320 and 1520 cm.^?1, respectively, and that both bands exhibit dichroism consistent with polypeptide chains which have a measure of alignment parallel to the fiber axis. The parallel dichroism of the amide II′ band of this phase at about 1435 cm.^?l also indicates some alignment. A nondichroic residual band at 1513 cm.^?1 in highly deuterated α-keratin is assigned to the tyrosine residue, as a sharp band near this frequency is found in the spectrum of polytyrosine. The ν‖(o) component of the α-helix is weak or absent in α-keratin, and the relatively sharp band observed near this frequency is thought to be due to the tyrosine residue, while its dichroism is caused by the presence of dichroic nonhelical material. A band near 1575 cm.^?1 in deuterated α- and β-keratin is tentatively assigned to the deuterated guanidinium group of arginine. This band becomes progressively more prominent during deuteration, which indicates that some arginine side chains arc slow to exchange, possibly because their environment prevents interaction with D₂O. The deuteration difference spectrum also shows that, contrary to earlier views, helical material in α-keratin exchanges significantly during the early stages of deuteration, although at a slower rate than the nonhelical material, while part of the nonhelical phase does not exchange as rapidly as had been thought and makes a contribution even after many hours or days.     

High-resolution NMR study of a synthetic DNA-RNA hybrid dodecamer containing the consensus pribnow promoter sequence: d(CGTTATAATGCG).r(CGCAUUAUAACG)

The nonsymmetrical double-helical hybrid dodecamer d(CGTTATAATGCG).r(CGCAUUAUAACG) was synthesized with solid-phase phosphoramidite methods and studied by high-resolution 2D NMR. The imino protons were assigned by one-dimensional nuclear Overhauser methods. All the base protons and H1', H2', H2", H3', and H4' sugar protons of the DNA strand and the base protons, H1', H2', and most of the H3'-H4' protons of the RNA strand were assigned by 2D NMR techniques. The well-resolved spectra allowed a qualitative analysis of relative proton-proton distances in both strands of the dodecamer. The chemical shifts of the hybrid duplex were compared to those of the pure DNA double helix with the same sequence (Wemmer et al., 1984). The intrastrand and cross-strand NOEs from adenine H2 to H1' resonances of neighboring base pairs exhibited characteristic patterns that were very useful for checking the spectral assignments, and their highly nonsymmetric nature reveals that the conformations of the two strands are quite different. Detailed analysis of the NOESY and COSY spectra, as well as the chemical shift data, indicate that the RNA strand assumes a normal A-type conformation (C3'-endo) whereas the DNA strand is in the general S domain but not exactly in the normal C2'-endo conformation. The overall structure of this RNA-DNA duplex is different from that reported for hybrid duplexes in solution by other groups (Reid et al., 1983a; Gupta et al., 1985) and is closer to the C3'-endo-C2'-endo hybrid found in poly(dA).poly(dT) and poly(rU).poly(dA) in the fiber state (Arnott et al., 1983, 1986).