The structure of the DNA.RNA hybrid (GTCACATG).(caugugac), where lowercase letters designate RNA residues, has been determined on the basis of J-coupling analysis and 2D-NOE studies. The central hexamer in this sequence has been previously studied [Reid, D. G., Salisbury, S. A., Brown, T., Williams, D. H., Vasseur, J.-J., Rayner, B., & Imabach, J.-L. (1983) Eur. J. Biochem. 135, 307-314] via one-dimensional NOE methods and circular dichroism studies. Contrary to their results, we find that this duplex does not assume a B-form conformation in solution. Instead, the RNA residues retain their C3'-endo (A-form) conformation, as indicated by the absence of H1'-H2' couplings and by strong H6/H8 to (n-1) H2'NOEs. The sugars of the DNA residues, on the other hand, do not assume an A-form (or a B-form) conformation but an intermediate conformation in the O4'-endo range (P approximately 72-110 degrees), as indicated by the presence of strong H1'-H4' NOEs, medium-strength H2"-H3' COSY cross peaks, strong H3'-H4' DQF-COSY cross peaks, and H1'-H2' coupling constants that are of approximately the same magnitude as the H1'-H2" coupling constants. These results suggest that the RNA strand not only retains its N-type structure but also exerts an influence on the conformation of the DNA strand. Our results provide strong evidence that DNA.RNA hybrid duplexes do not assume an all-C2'-endo B-type conformation; neither do they assume an all-C3'-endo A-type conformation in solution. Furthermore, although not the main focus of this study, a comparison of the longitudinal relaxation times of the DNA and RNA residues indicates the need for extended relaxation delays in two-dimensional NMR spectra of hybrid duplexes, as has been previously observed for DNA.RNA chimeric duplexes (Wang, A. C., Kim, S.-G., Chou, S.-H., Orban, J., Flynn, P., & Reid, B. R. (1992) Biochemistry 31, 3940-3946).

Nuclear magnetic resonance and optical experiments are combined to determine the rms amplitude of local angular motion of purines in DNA in solution. A 12 base-pair duplex DNA with the sequence d(CGCGAATTCGCG)2 is deuterated at the H8 positions of adenine and guanine by exchange with solvent at 55 degrees C. The deuterium nmr spectrum of this DNA is measured at 30 mg/mL at 30 degrees C in an 11.76 Tesla magnet (76.75 MHz). The time-resolved fluorescence polarization anisotropies (FPA) of this same sample and also a greatly diluted sample (0.215 mg/mL) were measured after addition of ethidium. FPA measurements of the dilute sample yield the hydrodynamic radius, RH = 9.94 +/- 0.2 A, while those at the nmr concentration are employed to characterize the collective motions in terms of either an enhanced viscosity or dimer formation. The rms amplitude of local angular motion was determined by analyzing the 2H-nmr spectrum, in particular the line width, using recently developed theory for the transverse relaxation rate (RQ2) together with essential information about the collective motions from these and other optical studies. When the principal-axis frame of the electric field gradient tensor is assumed to undergo overdamped libration around each of its three body-fixed axes in an isotropic deflection potential, then the rms amplitude of local angular motion around any single axis is found to lie in the range 10 degrees-11 degrees, provided the high DNA concentration acts to enhance the viscosity, and is about 9 degrees-11 degrees, if it acts to produce end-to-end dimers. The proton nmr relaxation data of Eimer et al. are reanalyzed and shown to yield an rms amplitude of angular motion of the cytosine H5-H6 internuclear vector of 9 degrees-10 degrees, depending upon its orientation with respect to the helix axis. In all of these analyses, full account is taken of the collective twisting and bending deformations, which have a small but significant effect on the results. It is shown that the rms amplitudes of local angular motion do not depend strongly on the model (potential), provided that isotropic rotation around the same number of axes is allowed and that one compares rms angles of the same dimensionality. The rms amplitudes of local angular motion in solution are comparable to those observed for the same sequence at low levels of hydration in the solid state.

The imino proton spectrum of Escherichia coli valine tRNA has been studied by two-dimensional nuclear Overhauser effect spectroscopy (NOESY) in H2O solution. The small nuclear Overhauser effects from the imino proton of an internal base pair to the imino protons of each nearest neighbor can be observed as off-diagonal cross-peaks. In this way most of the sequential NOE connectivity trains for all the helices in this molecule can be determined in a single experiment. AU resonances can be distinguished from GC resonances by the AU imino NOE to the aromatic adenine C2-H, thus leading to specific base-pair assignments. In general, the NOESY spectrum alone is not capable of assigning every imino proton resonance even in well-resolved tRNA spectra. Multiple proton peaks exhibit more than two cross-peaks, resulting in ambiguous connectivities, and coupling between protons with similar chemical shifts produces cross-peaks that are incompletely resolved from the diagonal. The sequence of the particular tRNA determines the occurrence of the latter problem, which can often be solved by careful one-dimensional experiments. The complete imino proton assignments of E. coli valine tRNA are presented.

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The low-field hydrogen-bond ring NH proton nuclear magnetic resonance (NMR) spectra of several transfer ribonucleic acids (tRNAs) related to yeast tRNAPhe have been examined in detail. Several resonances are sensitive to magnesium ion and temperature, suggesting that they are derived from tertiary base pairs. These same resonances cannot be attributed to cloverleaf base pairs as shown by experimental assignment and ring current shift calculation of the secondary base pair resonances. The crystal structure of yeast tRNAPhe reveals at least six tertiary base pairs involving ring NH hydrogen bonds, which we conclude are responsible for the extra resonances observed in the low-field NMR spectrum. In several tRNAs with the same tertiary folding potential and dihydrouridine helix sequence as yeast tRNAPhe, the extra resonances from tertiary base pairs are observed at the same position in the spectrum.