Structure of Poly (U).poly (A).poly (U)

The molecular structure of poly (U).poly (A).poly (U) has been determined and refined using the continuous x-ray intensity data on layer lines in the diffraction pattern obtained from an oriented fiber of the RNA. The final R-value for the preferred structure is 0.24, far lower than that for the plausible alternatives. The polymer forms an 11-fold right-handed triple-helix of pitch 33.5A and each base triplet is stabilized by Crick-Watson-Hoogsteen hydrogen bonds. The ribose rings in the three strands have C3'-endo, C2'-endo and C2'-endo conformations, respectively. The helix derives additional stability through systematic interchain hydrogen bonds involving ribose hydroxyls and uracil bases. The relatively grooveless cylindrical shape of the triple-helix is consistent with the lack of lateral organization.     

The structure of triple helical poly(U).poly(A).poly(U) studied by Raman spectroscopy

Using Raman spectroscopy, we examined the ribose-phosphate backbone conformation, the hydrogen bonding interactions, and the stacking of the bases of the poly(U).poly(A).poly(U) triple helix. We compared the Raman spectra of poly(U).poly(A).poly(U) in H2O and D2O with those obtained for single-stranded poly(A) and poly(U) and for double-stranded poly(A).poly(U). The presence of a Raman band at 863 cm-1 indicated that the backbone conformations of the two poly(U) chains are different in the triple helix. The sugar conformation of the poly(U) chain held to the poly(A) by Watson-Crick base pairing is C3' endo; that of the second poly(U) chain may be C2' endo. Raman hypochromism of the bands associated with base vibrations demonstrated that uracil residues stack to the same extent in double helical poly(A).poly(U) and in the triple-stranded structure. An increase in the Raman hypochromism of the bands associated with adenine bases indicated that the stacking of adenine residues is greater in the triple helix than in the double helical form. Our data further suggest that the environment of the carbonyls of the uracil residues is different for the different strands.     

A family of poly(U) polymerases

The GLD-2 family of poly(A) polymerases add successive AMP monomers to the 3' end of specific RNAs, forming a poly(A) tail. Here, we identify a new group of GLD-2-related nucleotidyl transferases from Arabidopsis, Schizosaccharomyces pombe, Caenorhabditis elegans, and humans. Like GLD-2, these enzymes are template independent and add nucleotides to the 3' end of an RNA substrate. However, these new enzymes, which we refer to as poly(U) polymerases, add poly(U) rather than poly(A) to their RNA substrates.     

Conformation of poly(L-lysine) homologs in solution

The effect of the number of methylene groups in the side chains on the conformation of polypeptides is assessed for three poly(L -lysine) homologs with R = –(CH₂)_nNH₂. Circular dichroism studies show a pH-induced helix–coil transition in 0.05 M KCl with midpoints at 9.6, 9.0, and 8.7 for n = 5, 6, and 7, respectively, as compared with 10.1 for (Lys)_x (n = 4). Homologs with n = 6 and 7 could be partially helical even when the side groups are fully charged (with n = 7, the compound is highly aggregated above pH 9.1). Thus, the longer the number of methylene groups the more stable is the helical conformation of these homologs. Potentiometric titration of the n = 5 homolog gives a ΔG° of ?310 cal/mol (residue) for the uncharged coil-to-helix transition at 25°C. The corresponding ΔH° and ΔS° are ?1740 cal/mol (residue) and ?4.8 e.u./mol (residue). Unlike (Lys)_x, the uncharged helix-to-β transition is slow and incomplete even after heating at 80°C for 1 hr. Addition of methanol enhances the helical formation in neutral solution with midpoints at 72, 52, and 27% methanol (v/v) for n = 5, 6, and 7, respectively [cf. 88% for (Lys)_x]. Addition of sodium dodecyl sulfate induces a coil-to-helix transition for all three homologs in contrast with the β form of (Lys)_x under similar conditions.     

Study of specific interactions of amino acid esters with the synthetic polynucleotides poly(A) x 2 poly(U), poly(A) x poly(U) and poly(A) by thermal denaturation

The interactions of amino acid esters with poly(A)x2poly(U) and poly(A)xpoly(U) have been investigated by means of thermal denaturation of these polynucleotides. The esters under consideration raised the melting point, revealing the preferable binding to helical polynucleotide structures. The melting point shifts demonstrate the following sequence of the stabilities of these complexes: Arg greater than Lys much greater than His greater than Met greater than Ser greater than Gly. The same stability order is observed when studying the polynucleotide renaturation in the presence of esters. This order coincides with that previously obtained for the nucleotide base--amino acid ester complexes excepting basic amino acid esters. The ester interactions with poly(A) and poly(U) also reveal the specificity of monomer--monomer interactions. Some dynamic contributions into the studied specificity are also discussed.     

Formation of triple-helical nucleic acids studied by using antibodies specific for poly(A).poly(U).poly(U)

The formation of the triple helix of poly(A).poly(U).poly(U) was studied by using antibodies specific to poly(A).poly(U).poly(U). the 10-11 base chain length for oligo(A) and the 20-30 base chain length for oligo(U) may be the minimum sizes required to maintain a stable triple helix. Double-stranded poly(A).poly(U) which was the core of triple-stranded poly(A).poly(U).poly(U) could bind poly(U) and produce an analogue of poly(A).poly(U).poly(U) reactive with the antibodies even if the poly(A) or poly(U) was brominated or acetylated to the extent of 35-55%. However, brominated or acetylated poly(U) did not produce a stable triple helix with double-stranded poly(A).poly(U).     

The Cid1 poly(U) polymerase

The Schizosaccharomyces pombe cytoplasmic protein Cid1 acts as a poly(U) polymerase (PUP). Polyadenylated actin mRNA, a target of this activity, is uridylated upon arrest in S phase and is likely to be one of many such Cid1 targets. This RNA uridylation pathway appears to be conserved, as Cid1 orthologs in Arabidopsis thaliana, Caenorhabditis elegans and humans display PUP activity either in vitro or in Xenopus laevis oocytes. Here, we review the literature on Cid1, other PUPs and uridylation, a conserved and previously under-appreciated mechanism of RNA regulation.     

Radiolysis of human gastric glycopolypeptides in aqueous solution

The degradation of human gastric glycopolypeptides by hydroxyl radicals formed in irradiated N2O-saturated aqueous solution has been investigated. Gel exclusion chromatography shows the formation of lower molecular weight degradation products after irradiation and the appearance of unsaturated carbonyl-containing products which absorb in the ultra-violet. The radiation-induced destruction of individual monosaccharides in three human glycopolypeptides having different oligosaccharide chains has been measured. The results indicate that the structure of the oligosaccharide chain determines the extent of destruction of each type of monosaccharide present.     

Titration of poly(dA-dT) . poly(dA-dT) in solution at variable NaCl concentration

CD and uv absorption data showed that high molecular weight poly(dA-dT) . poly(dA-dT), at 298 K, undergoes an acid-induced transition from B-double helix to random coil in NaCl solutions of different concentrations, ranging from 0.005 to 0.600M. Similarly, titration of the polynucleotide with a strong base causes duplex-to-single strands transition. The base- and acid-induced transitions were both reversible by back-titration (with an acid or, respectively, with a base): the apparent pKa were the same in both directions. However, the number of protons per titratable site (adenine N1) required to reach half-denaturation was in great excess over the stoichiometric value; to a much larger extent, the same effect was observed also for the deprotonation of the N3H sites of thymine. Moreover, in the basic denaturation experiments, at low salt concentrations ([NaCl]< or =0.300M) less acid than calculated was needed to back-titrate the base excess to half-denaturation. Both effects could be qualitatively justified on the basis of the counterion condensation theory of polyelectrolytes and considering the energy barrier created by the negatively charged phosphodiester groups to the penetration of the OH- ions inside the double helix and the screening effect of the Na+ ions on such charges, in the deprotonation experiments.     

A Z-like form of poly(dA-dC).poly(dG-dT) in solution?

Circular dichroism was used to study changes in conformation of poly(dA-dC).poly(dG-dT) caused by a high concentration of various monovalent salts. It was found that CsF induced the gradual appearance of a negative band in the long wavelength part of the CD spectrum of poly(dA-dC).poly(dG-dT), which might reflect a transition of this DNA toward a Z-like structure.     

Hydroxyl radical-induced strand break formation of poly(U) in anoxic solution. Effect of dithiothreitol and tetranitromethane

The role of dithiothreitol (DTT) and tetranitromethane (TNM) on the yields of radiation-induced strand break formation in polyuridylic acid (poly(U] was studied in anoxic aqueous solutions at neutral pH by low-angle laser light-scattering. From G (single-strand breaks) as a function of DTT concentration it follows that two different processes lead to OH radical-induced single-strand break (ssb) formation. Only one of the two processes, which accounts for 80 per cent of the ssb formation, is inhibited by DTT, the other one, 20 per cent, is not inhibited. The 'repair' process is attributed to H-donation to the C-6-yl radical of the uracil moiety. The C-6-yl radical is produced by OH addition to the C-5 position of the uracil moiety. It follows that the sugar radicals, in contrast to earlier suggestions, do not seem to be repaired by DTT at the low concentrations used. The strand break formation not inhibited by DTT is induced by radicals other than the uracil-6-yl radical, e.g. the uracil-5-yl or the OH radicals reacting with the sugar moiety. The strong reduction of G(ssb) from 2.3 to 0.2 on addition of TNM is also discussed.     

A poly(U) polymerase in tobacco leaves.

A poly(U) polymerizing enzyme has been found in healthy and tobacco mosaic virus-infected tobacco leaves and has been partially purified by affinity chromatography on a gel prepared from agarose with chemically coupled RNA. The enzyme is stimulated by Mn-2+ and dependent on a polynucleotide, preferentially poly(A). The synthesis proceeds optimally at pH 7.6 and 25 degrees C. The enzyme is highly specific for UTP and is inhibited by other ribonucleoside triphosphates. The product was partly sensitive to pancreatic ribonuclease. The synthetic reaction is inhibited in the presence of pyrophosphate but insensitive to 10 mM orthophosphate and high levels of cordycepin, rifampicin and actinomycin D. A molecular weight of about 40,000 has been estimated by sucrose gradient analysis and partition cell ultracentrifugation.     

Interaction of sodium decyl sulfate with poly(L-ornithine) and poly(L-lysine) in aqueous solution

The binding isotherms of sodium decyl sulfate to poly(L -ornithine), poly(D ,L -ornithine), and poly(L -lysine) at neutral pH were determined potentiometrically. The nature of a highly cooperative binding in all three cases suggests a micelle-like clustering of the surfactant ions onto the polypeptide side groups. The hydrophobic interaction between the nonpolar groups overshadows the coulombic interaction between the charged groups. The titration curves can be interpreted well by the Zimm–Bragg theory. The average cluster size of bound surfactant ions is sufficiently large to promote the β-structure of (L -Lys)_n even at a very low binding ratio of surfactant to polypeptide residue, whereas the onset of the helical structure for (L -Orn)_n begins after about 7 surfactant ions are bound to two turns of the helix. The CD results are consistent with this explanation.     

Binding of novel 9-O–N-aryl/arylalkyl amino carbonyl methyl berberine analogs to poly(U)-poly(A)·poly(U) triplex and comparison to the duplex poly(A)-poly(U)

Interaction of the 9-O–N-aryl/arylalkyl amino carbonyl methyl substituted analogs of the anticancer isoquinoline alkaloid berberine with RNA triplex, poly(U)-poly(A)·poly(U) has been studied in comparison to the duplex poly(A)-poly(U), using multiple biophysical techniques. Spectrophotometric and spectrofluorimetric studies established the non-cooperative binding mode of all the analogs with both the duplex and the triplex. However, berberine exhibited cooperative binding with poly(A)-poly(U) and non-cooperative binding with poly(U)-poly(A)·poly(U). Analog BER1 showed the highest affinity to both the duplex and the triplex followed by BER2 and BER3. The overall binding affinity varied as BER1 > BER2 > BER3 > BER. The magnitude of the quantum efficiency values (Q > 1) revealed that energy was transferred from the bases of the triplex and the duplex to the analogs. Comparative ferrocyanide quenching and viscosity studies unambiguously established a stronger intercalative geometry of the analogs to both the triplex and the duplex in comparison to berberine. Circular dichroism studies revealed that the alkaloids perturbed the conformation of both RNA helices. The binding of all the alkaloids was found to be exothermic from isothermal titration studies. Binding of the analogs was highly entropy driven while that of berberine was enthalpy dominated. The results presented here reveal strong and specific binding of these new berberine analogs to the RNA triplex and duplex and highlight the remarkable influence of the 9-substitution on the interaction profile.     

Structure of the beta-form of poly d(A).poly d(U)

The crystalline beta-form of the sodium salt of poly d(A).poly d(U) trapped in oriented fibers forms a Watson-Crick base-paired, 10(1) double-helix of pitch 3.2 nm. Two molecules are present in a monoclinic unit cell apparently isomorphous with beta-poly d(A).poly d(T). The two chains in each molecule both carry C2'-endo puckered furanose rings but are conformationally not identical. The orientations of the A:U base-pairs relative to the helix-axis are distinctly different from those in classical B-DNA and the overall morphology of the duplex in which they reside resembles that of the alpha-forms of poly (purine).poly (pyrimidine) DNA duplexes previously reported.