No abstract

The 3'-5' circular trinucleotide cr(GpGpGp) was studied by means of 1D and 2D high resolution NMR techniques and molecular mechanics calculations. Analysis of the J-couplings, obtained from the 1H and 13C-NMR spectra, allowed the determination of the conformation of the sugar rings and of the 'circular' phosphate backbone. In the course of the investigations it was found that the Karplus-equation most recently parametrized for the CCOP J-coupling constants could not account for the measured J(C4'P) of 11.1 Hz and a new parametrization for both HCOP and CCOP coupling constants is therefore presented. Subsequent analysis of the coupling constants yielded 'fixed' values for the torsion angles beta and delta (with beta = 178 degrees and delta = 139 degrees). The value of the latter angle corresponds to an S-type sugar conformation. The torsion angles gamma and epsilon are involved in a rapid equilibrium in which they are converted between the gauche(+) and trans and between the trans and gauche(-) domain respectively. We show that the occurrence of epsilon in the gauche(-) domain necessitates S-type sugar conformations. Given the aforementioned values for beta, gamma, delta and epsilon the ring closure constraints for the ring, formed by the phosphate backbone can only be fulfilled if alpha and zeta adopt some special values. After energy minimization with the CHARMm force field only two combinations of alpha and zeta result in energetically favourable structures, i.e. the combination alpha (t)/zeta(g-) in case gamma is in a gauche(+) and epsilon is in a trans conformation, and the combination alpha (t)/zeta (g+) for the combination gamma (t)/epsilon (g-). The results are discussed in relation to earlier findings obtained for cd(ApAp) and cr(GpGp), the latter molecule being a regulator of the synthesis of cellulose in Acetobacter xylinum.

The three-dimensional structure of the hairpin formed by d(ATCCTA-GTTA-TAGGAT) has been determined by means of two-dimensional NMR studies, distance geometry and molecular dynamics calculations. The first and the last residues of the tetraloop of this hairpin form a sheared G-A base pair on top of the six Watson-Crick base pairs in the stem. The glycosidic torsion angles of the guanine and adenine residues in the G-A base pair reside in the anti and high- anti domain ( approximately -60 degrees ) respectively. Several dihedral angles in the loop adopt non-standard values to accommodate this base pair. The first and second residue in the loop are stacked in a more or less normal helical fashion; the fourth loop residue also stacks upon the stem, while the third residue is directed away from the loop region. The loop structure can be classified as a so-called type-I loop, in which the bases at the 5'-end of the loop stack in a continuous fashion. In this situation, loop stability is unlikely to depend heavily on the nature of the unpaired bases in the loop. Moreover, the present study indicates that the influence of the polarity of a closing A.T pair is much less significant than that of a closing C.G base pair.

1D and 2D NMR investigations of the 15 residue deoxynucleotide sequence d(TCTCTC-TTT-GAGAGA) show that above pH = 6.5 the molecule adopts a B-form hairpin conformation. As the pH is lowered below 6.5 molecules progressively associate in pairs to form a partially triple helical, partially single stranded structure in which the bases of the oligopyrimidine d(TC)3 tract from one molecule form Hoogsteen pairs with the d(G-A)3 tract of the other. Imino protons of protonated cytosines can be observed at very low field (approximately 15 ppm). The enthalpy of triplex formation was estimated by NMR techniques to be -16 kcal mol-1. Intense H6 to H3' cross peaks from residues in all three strands suggest the presence of N-type sugars at some but not at all possible sites. Surprisingly strong cross peaks between H5' or H5" and non-exchangeable base protons are also observed. These suggest that certain of the O5'-C5'-C4'-C3' phosphate backbone torsion angles (gamma) are unusual.

The 15-residue oligonucleotide d(TCTCTC-TTT-GAGAGA) forms a hairpin structure with a loop of three thymidine residues at neutral pH or above. The three-dimensional solution structure of this oligonucleotide has been determined by means of two-dimensional nuclear magnetic resonance methods. Interproton distance constraints derived from NOEs, in combination with torsion angle constraints obtained from J-coupling constants were used in the variable target function program DIANA to derive the hairpin structure. It was found that hairpins with two different loop conformations fit the NMR data, i.e. an equilibrium between these two conformational states can only fully explain the NOE data available. In one state, loop residue T7 is turned into the minor groove, while in the second state residue T8 is in the minor groove. In both conformations the phosphate backbone changes its direction by 180 degrees between residues T9 and G10. Concomitantly, torsion angles zeta of T9 and alpha of G10 both adopt a gauche(+) conformation and gamma of residue G10 adopts a trans conformation to induce this complete change in the direction of the backbone.