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A newly implemented G-matrix Fourier transform (GFT) (4,3)D HC(C)CH experiment is presented in conjunction with (4,3)D HCCH to efficiently identify (1)H/(13)C sugar spin systems in (13)C labeled nucleic acids. This experiment enables rapid collection of highly resolved relay 4D HC(C)CH spectral information, that is, shift correlations of (13)C-(1)H groups separated by two carbon bonds. For RNA, (4,3)D HC(C)CH takes advantage of the comparably favorable 1'- and 3'-CH signal dispersion for complete spin system identification including 5'-CH. The (4,3)D HC(C)CH/HCCH based strategy is exemplified for the 30-nucleotide 3'-untranslated region of the pre-mRNA of human U1A protein.

Molecular motion and dynamics play an essential role in the biological function of many RNAs. An important source of information on biomolecular motion can be found in residual dipolar couplings which contain dynamics information over the entire ms-ps timescale. However, these methods are not fully applicable to RNA because nucleic acid molecules tend to align in a highly collinear manner in different alignment media. As a consequence, information on dynamics that can be obtained with this method is limited. In order to overcome this limitation, we have generated a chimeric RNA containing both the wild type TAR RNA, the target of our investigation of dynamics, as well as the binding site for U1A protein. When U1A protein was bound to the portion of the chimeric RNA containing its binding site, we obtained independent alignment of TAR by exploiting the physical chemical characteristics of this protein. This technique can allow the extraction of new information on RNA dynamics, which is particularly important for time scales not covered by relaxation methods where important RNA motions occur.

Formation of the complex between human immunodeficiency virus type-1 Tat protein and the transactivation response region (TAR) RNA is vital for transcriptional elongation, yet the structure of the Tat-TAR complex remains to be established. The NMR structures of free TAR, and TAR bound to Tat-derived peptides have been obtained by solution NMR, but only a small number of intermolecular NOEs could be identified unambiguously, preventing the determination of a complete structure. Here we show that a combination of multiple solid state NMR REDOR experiments can be used to obtain multiple distance constraints from (15)N to (13)C spins within the backbone and side chain guanidinium groups of arginine in a Tat-derived peptide, using (19)F spins incorporated into the base of U23 in TAR and (31)P spins in the P22 and P23 phosphate groups. Distances between the side chain of Arg52 and the base and phosphodiester backbone near U23 measured by REDOR NMR are comparable to distances observed in solution NMR-derived structural models, indicating that interactions of TAR RNA with key amino acid side chains in Tat are the same in the amorphous solid state as in solution. This method is generally applicable to other protein-RNA complexes where crystallization or solution NMR has failed to provide high resolution structural information., (© Springer Science+Business Media B.V. 2011)

The essential role played by local and collective motions in RNA function has led to a growing interest in the characterization of RNA dynamics. Recent investigations have revealed that even relatively simple RNAs experience complex motions over multiple time scales covering the entire ms-ps motional range. In this work, we use deuterium solid-state NMR to systematically investigate motions in HIV-1 TAR RNA as a function of hydration. We probe dynamics at three uridine residues in different structural environments ranging from helical to completely unrestrained. We observe distinct and substantial changes in (2)H solid-state relaxation times and lineshapes at each site as hydration levels increase. By comparing solid-state and solution state (13)C relaxation measurements, we establish that ns-micros motions that may be indicative of collective dynamics suddenly arise in the RNA as hydration reaches a critical point coincident with the onset of bulk hydration. Beyond that point, we observe smaller changes in relaxation rates and lineshapes in these highly hydrated solid samples, compared to the dramatic activation of motion occurring at moderate hydration.

Photoexcitation under mild temperature and pressure conditions of FeCl4- heterogenized with Amberlite causes the conversion of several cycloalkanes to the corresponding monochlorinated products with selectivity higher than 95%.

The structures of 113 eukaryotic cytochrome c proteins of known sequence have been modeled in the oxidized state based on the existing crystallographic and NMR structures. The secondary structural elements and the overall three-dimensional structure were found to be maintained throughout the super-family, despite variability in the sequence of individual proteins. The iron axial ligands and their reciprocal orientation were found to be nearly universally conserved. Residues constituting the hydrophobic core of the protein are also very highly conserved or conservatively substituted. Certain surface-exposed charged as well as hydrophobic groups have also been found to be conserved to the same degree as core residues. Patterns of conservation of exposed residues identify regions of the protein that are likely to be critical for its function in electron transfer.

A new experiment, the forward directed quantitative gamma-HCCH-TOCSY for the measurement of the conformation of the five-membered ribosyl unit in RNA oligonucleotides, is presented. The experiment relies on quantification of cross peak intensities caused by evolution of CH, CH-dipole-dipole cross correlated relaxation in non-evolution periods and the resolution enhancement obtainable in forward directed HCC-TOCSY transfer. Cross correlated relaxation rates are interpreted to reveal the sugar conformation of 22 out of 25 nucleotides in an isotopically labelled 25-mer RNA. The results obtained with this new method are in agreement with the conformational analysis derived from 3J(H,H) coupling constants.

The main limitation in NMR-determined structures of nucleic acids and their complexes with proteins derives from the elongated, non-globular nature of physiologically important DNA and RNA molecules. Since it is generally not possible to obtain long-range distance constraints between distinct regions of the structure, long-range properties such as bending or kinking at sites of protein recognition cannot be determined accurately nor precisely. Here we show that use of residual dipolar couplings in the refinement of the structure of a protein-RNA complex improves the definition of the long-range properties of the RNA. These features are often an important aspect of molecular recognition and biological function; therefore, their improved definition is of significant value in RNA structural biology.

RNA-protein recognition is critical to post-transcriptional regulation of gene expression, yet poorly understood at the molecular level. The relatively slow progress in understanding this important area of molecular biology is due to difficulties in obtaining good-quality crystals and derivatives, and in preparing samples suitable for NMR investigation. The determination of the structure of the complex between the human U1A protein and its polyadenylation inhibition element is described here. In this paper, we describe the sample preparation, spectral assignments, construction of the NOE-based distance constraints and methodology for calculating the structure of the complex. The structure was determined to an overall precision of 2.03 A (for all ordered regions), and 1.08 A for the protein-RNA interface. The patterns of hydrogen bonding and hydrophobic interactions at the interface were analysed statistically using the final ensemble of 31 structures.

Backbone-driven assignment methods that utilize covalent connectivities have greatly facilitated spectral assignments of proteins. In nucleic acids, 1H-13C-31P correlations could play a similar role, and several related experiments (HCP) have recently been presented for backbone-driven sequential assignments in RNA. The three-dimensional extension of 1H-31P Het-Cor (P,H-COSY-H,C-HMQC) and Het-TOCSY (P,H-TOCSY-H,C-HMQC) experiments presented here complements HCP experiments as tools for spectral assignments and extraction of dihedral angle constraints. By relying on 1H-31P rather than 13C-31P couplings to generate cross peaks, the strongest connectivities are observed in different spectral regions, increasing the likelihood of resolving spectral overlap. In addition, semiquantitative estimates of 1H-31P and 13C-31P couplings provide dihedral angle constraints for RNA structure determination.