Use of ultra stable UNCG tetraloop hairpins to fold RNA structures: thermodynamic and spectroscopic applications.

RNA molecules of > 20 nucleotides have been the focus of numerous recent NMR structural studies. Several investigators have used the UNCG family of hairpins to ensure proper folding. We show that th UUCG hairpin has a minimum requirement of a two base-pair stem. Hairpins with a CG loop closing base pair and an initial 5'CG or 5'GC base pair have a melting temperature approximately 55 degrees C in 10 mM sodium phosphate. The high stability of even such small hairpins suggests that the hairpin can serve as a nucleation site for folding. For high resolution NMR work, the UNCG loop family (UACG in particular) provides excellent spectroscopic markers in one-dimensional exchangeable spectra, in two-dimensional COSY spectra and in NOESY spectra that clearly define it as forming a hairpin. This allows straightforward initiation of chemical shift assignments.     

A mutant RNA pseudoknot that promotes ribosomal frameshifting in mouse mammary tumor virus.

A single A-->G mutation that changes a potential A.U base pair to a G.U pair at the junction of the stems and loops of a non-frameshifting pseudoknot dramatically increases its frameshifting efficiency in mouse mammary tumor virus. The structure of the non-frameshifting pseudoknot APK has been found to be very different from that of pseudoknots that cause efficient frameshifting [Kang,H., Hines,J.V. and Tinoco,I. (1995) J. Mol. Biol. , 259, 135-147]. The 3-dimensional structure of the mutant pseudoknot was determined by restrained molecular dynamics based on NMR-derived interproton distance and torsion angle constraints. One striking feature of the mutant pseudoknot compared with the parent pseudoknot is that a G.U base pair forms at the top of stem 2, thus leaving only 1 nt at the junction of the two stems. The conformation is very different from that of the previously determined non-frameshifting parent pseudoknot, which lacks the A.U base pair at the top of the stem and has 2 nt between the stems. However, the conformation is quite similar to that of efficient frameshifting pseudoknots whose structures were previously determined by NMR. A single adenylate residue intervenes between the two stems and interrupts their coaxial stacking. This unpaired nucleotide produces a bent structure. The structural similarity among the efficient frameshifting pseudoknots indicates that a specific conformation is required for ribosomal frameshifting, further implying a specific interaction of the pseudoknot with the ribosome.     

Circular dichroism of polynucleotides: A general method applied to dimers

A method is described which relates the circular dichroism of a polymer to its conformation. The method takes into account near and accidental degeneracies and eliminates the artificial distinction between degenerate and nondegenerate systems. Comparison of this method with perturbation theory indicates that the errors inherent in nondegenerate perturbation theory tend to cancel when a circular dichroism spectrum is calculated. The method is applied to dinucleoside phosphates.     

Fluorescence-detected circular dichroism of dinucleoside phosphates. A study of solution conformations and the two-state model

Three fluorescent dinucleoside phosphates containing 1,N⁶-ethenoadenosine (εA), εA_pεA, εA_pεC, and εA_pU were studied using fluorescence-detected circular dichroism (FDCD), circular dichroism (CD), and absorption measurements. The FDCD data indicate that εA_pεC and εA_pU can be described as two-state systems consisting of a fluorescent species and a stacked, nonfluorescent species. Thermodynamic stacking parameters are calculated for these molecules using the van't Hoff equation. εA_pεA is found to be a more complicated system with a fluorescent CD which is different in shape, but comparable in magnitude, to the conventional CD of the dimer. This molecule, unlike the other two dinucleoside phosphates, cannot be characterized as a two-state system; it is described as consisting of at least three states at temperatures above 35°C. The CD data were subjected to a linear analysis in order to determine the minimum number of states present. In agreement with the FDCD data, εA_pεC and εA_pU are found to consist of a minimum of two states, while εA_pεA is indicated to have at least three. The more complicated behavior of the latter dimer is also indicated by the values of the unstacked CD obtained in the van't Hoff analysis.     

Fatty acid changes in liver and plasma lipid fractions after safflower oil was fed to rats deficient in essential fatty acids

1. Fatty acid patterns of liver and plasma triglycerides, phospholipids and cholesteryl esters were determined at intervals during 24hr. after essential fatty acid-deficient rats were given one feeding of linoleate (as safflower oil). 2. Liver triglyceride, phospholipid and cholesteryl ester fatty acid compositions did not change up to 7hr. after feeding. Between 7 and 10hr., linoleic acid began to increase in all fractions, but arachidonic acid did not begin to rise in the phospholipid until 14-19hr. after feeding. 3. Oleic acid and eicosatrienoic acid in liver phospholipid began to decline at about the time that linoleic acid increased, i.e. about 9hr. before arachidonic acid began to increase. 4. Changes in linoleic acid, arachidonic acid and eicosatrienoic acid in phosphatidylcholine resembled those of the total phospholipid. Phosphatidylethanolamine had a higher percentage content of arachidonic acid before the linoleate was given than did phosphatidylcholine, and after the linoleate was given the fatty acid composition of this fraction was little changed. 5. The behaviour of the plasma lipid fatty acids was similar to that of the liver lipids, with changes in linoleic acid, eicosatrienoic acid and arachidonic acid appearing at the same times as they occurred in the liver. 6. The results indicated that linoleic acid was preferentially incorporated into the liver phospholipid at the expense of eicosatrienoic acid and oleic acid. The decline in these fatty acids apparently resulted from their competition with linoleic acid for available sites in the phospholipids rather than from any direct replacement by arachidonic acid.     

Thermodynamic studies of base pairing involving 2,6-diaminopurine.

The thermal stabilities of oligodeoxyribonucleotide duplexes containing 2,6-diaminopurine (D) matched with each of the four normal DNA bases were determined by optical melting techniques. Comparison of optical melting curves yielded relative stabilities for the D-containing standard base pairs in an otherwise identical base-pair sequence. The D:T pair was found to be more stable than the A:T pair in dC3DG3:dC3TG3, as stable as the A:T in dCT3DT3G:dCA3TA3G, and less stable than the A:T in dCA3DA3G:dCT7G. The order of stabilities for X:Y in the DNA duplex dCA3XA3G:dCT3YT3G is: (A:T) greater than (T:D) congruent to (D:T) greater than or equal to (T:A) greater than (C:D) congruent to (D:A) congruent to (D:G) greater than or equal to (D:C) congruent to (G:D) congruent to (D:D) greater than or equal to (A:D). Implications of these results for design of DNA oligonucleotide probes are discussed.     

Raman spectroscopic study of left-handed Z-RNA

The solvent conditions that induce the formation of a left-handed Z form of poly[r(G-C)] have been extended to include 6.5 M NaBr at 35 degrees C and 3.8 M MgCl2 at room temperature. The analysis of the A----Z transition in RNA by circular dichroism (CD), 1H and 31P NMR, and Raman spectroscopy shows that two distinct forms of left-handed RNA exist. The ZR-RNA structure forms in high concentrations of NaBr and NaClO4 and exhibits a unique CD signature. ZD-RNA is found in concentrated MgCl2 and has a CD signature similar to the Z form of poly[d(G-C)]. The loss of Raman intensity of the 813-cm-1 A-form marker band in both the A----ZR-RNA and A----ZD-RNA transitions parallels the loss of intensity at 835 cm-1 in the B----Z transition of DNA. A guanine vibration that is sensitive to the glycosyl torsion angle shifts from 671 cm-1 in A-RNA to 641 cm-1 in both ZD- and ZR-RNA, similar to the B----Z transition in DNA in which this band shifts from 682 to 625 cm-1. Significant differences in the glycosyl angle and sugar pucker between Z-DNA and Z-RNA are suggested by the 16-cm-1 difference in the position of this band. The Raman evidence for structural difference between ZD- and ZR-RNA comes from two groups of bands: First, Raman intensities between 1180 and 1600 cm-1 of ZD-RNA differ from those for ZR-RNA, corroborating the CD evidence for differences in base-stacking geometry. Second, the phosphodiester stretching bands near 815 cm-1 provide evidence of differences in backbone geometry between ZD- and ZR-RNA.     

Binding of ethidium ion to left-handed Z-RNA induces a cooperative transition to right-handed RNA at the intercalation site

The equilibrium binding of the ethidium cation (Etd+) to the right-handed A-form of poly-[r(C-G)], the B-form of poly[d(C-G)], and the left-handed Z-forms of Br-poly[r(C-G)] and Br-poly[d(C-G)] was investigated in 0.22 M NaCl by optical methods. Scatchard analysis indicates that Etd+ intercalates into right-handed forms of poly[r(C-G)] and poly[d(C-G)] in a noncooperative manner. Correlation of Etd+ absorbance binding isotherms and polynucleotide circular dichroism data indicates that drug binding to Br-poly[r(C-G) and Br-poly[d(C-G)] results in cooperative conversion from left-handed Z-forms to right-handed intercalated conformations. Approximate stoichiometries necessary to induce the left- to right-handed transitions are 1 Etd+/9 base pairs (bp) for Z-RNA and 1 Etd+/6 bp for Z-DNA. The apparent limiting binding stoichiometries are approximately 1 Etd+/3 bp for RNA and 1 Etd+/2 bp for DNA. The equilibrium binding constants for binding to the right-handed forms decrease in the order Br-poly[d(C-G)], Br-poly[r(C-G)], poly[d(C-G)], and poly[r(C-G)]. Thermodynamic parameters are obtained by van't Hoff analysis of Etd+ absorbance thermal dissociation data. Enthalpy values for all four polynucleotides are negative and of similar magnitude. Negative entropy values indicate that the binding processes are primarily enthalpically driven.(ABSTRACT TRUNCATED AT 250 WORDS)