Structural features that give rise to the unusual stability of RNA hairpins containing GNRA loops.

The most frequently occurring RNA hairpins in 16S and 23S ribosomal RNA contain a tetranucleotide loop that has a GNRA consensus sequence. The solution structures of the GCAA and GAAA hairpins have been determined by nuclear magnetic resonance spectroscopy. Both loops contain an unusual G-A base pair between the first and last residue in the loop, a hydrogen bond between a G base and a phosphate, extensive base stacking, and a hydrogen bond between a sugar 2'-end OH and a base. These interactions explain the high stability of these hairpins and the sequence requirements for the variant and invariant nucleotides in the GNRA tetranucleotide loop family.

DNA conformation, dynamics, and interactions in solution.

The conformation and dynamics of the d(CGCGAATTCGCG) duplex, its analogs containing mismatched base pairs and helix interruptions, and its complexes with actinomycin and Netropsin, bound separately and simultaneously, have been investigated by nuclear magnetic resonance spectroscopy in aqueous solution. Structural information has been deduced from chemical shift and nuclear Overhauser effect parameters, while the kinetics have been probed from line width and saturation recovery experiments on proton and phosphorus markers at the individual base pair level. These studies lead to an improved understanding of the role of nucleic acid sequence on the structure, flexibility, and conformational interconversions in the duplex state. The nuclear magnetic resonance measurements readily identify helix modification and antibiotic binding sites on the nucleic acid and estimate the extent to which the observed conformational and dynamic perturbations are transmitted to adjacent base pair regions.

High-resolution molecular discrimination by RNA.

Species of RNA that bind with high affinity and specificity to the bronchodilator theophylline were identified by selection from an oligonucleotide library. One RNA molecule binds to theophylline with a dissociation constant Kd of 0.1 microM. This binding affinity is 10,000-fold greater than the RNA molecule's affinity for caffeine, which differs from theophylline only by a methyl group at nitrogen atom N-7. Analysis by nuclear magnetic resonance indicates that this RNA molecule undergoes a significant change in its conformation or dynamics upon theophylline binding. Binding studies of compounds chemically related to theophylline have revealed structural features required for the observed binding specificity. These results demonstrate the ability of RNA molecules to exhibit an extremely high degree of ligand recognition and discrimination.

NMR solution structure determination of RNAs.

During the past several years, there have been significant advances in NMR solution structure determination of macromolecules. The ability to easily measure residual dipolar couplings, to directly detect NHellipsisN hydrogen bonding interactions and to study much larger macromolecules by the application of heteronuclear experiments that select narrow lines in 2D and 3D spectra of isotopically labeled molecules promises to dramatically improve solution structure determination of nucleic acids.

Nuclear magnetic resonance studies of the hammerhead ribozyme domain. Secondary structure formation and magnesium ion dependence.

Proton nuclear magnetic resonance (n.m.r.) experiments were used to probe base-pair formation in several hammerhead RNA enzyme (ribozyme) domains. The hammerhead domains consist of a 34 nucleotide ribozyme bound to a complementary 13 nucleotide non-cleavable DNA substrate. Three hammerhead domains were studied that differ in the sequence and stability of one of the helices involved in recognition of the substrate by the ribozyme. The n.m.r. data show a 1:1 stoichiometry for the ribozyme-substrate complexes. The imino proton resonances in the hammerhead complexes were assigned by two-dimensional nuclear Overhauser effect experiments. These data confirm the presence of two of the three helical regions in the hammerhead domain, predicted from phylogenetic data; and are also consistent with the formation of the third helix. Since a divalent cation is required for efficient catalytic activity of the hammerhead domain, the magnesium ion dependence of the n.m.r. spectra was studied for two of the hammerhead complexes. One of the complexes showed very large spectral changes upon addition of magnesium ions. However, the complex that has the most C.G base-pairs in one of the recognition helices shows essentially no spectral (and therefore presumably structural) changes upon addition of magnesium. These data are consistent with a model where the magnesium binding site already exists in the magnesium-free complex, suggesting that the magnesium ion serves primarily a catalytic, and not a structural, role under the conditions used here.

Calibration of the angular dependence of the amide proton-C alpha proton coupling constants, 3JHN alpha, in a globular protein. Use of 3JHN alpha for identification of helical secondary structure.

The vicinal amide proton-C alpha proton spin-spin coupling constants, JHN alpha, in the globular protein basic pancreatic trypsin inhibitor (BPTI) have been measured using phase-sensitive correlated spectroscopy at high digital resolution. In conjunction with the crystal structure of BPTI, these data were used to calibrate the correlation between 3JHN alpha and the dihedral angle phi. The resulting "BPTI curve" is 3JHN alpha = 6.4 cos2 theta - 1.4 cos theta + 1.9 (theta = [phi - 60 degrees]). It is further shown that measurement of the spin-spin couplings 3JHN alpha presents an independent, reliable method for identification of the location of helical structure in the amino acid sequence of proteins.

An efficient procedure for assignment of the proton, carbon and nitrogen resonances in 13C/15N labeled nucleic acids.

An efficient method is presented for the assignment of the proton, carbon, and nitrogen resonances in the NMR spectra of isotopically labeled nucleic acids. The assignment strategy starts by identifying all protons and carbons belonging to the same sugar ring through application of a set of 2D or 3D heteronuclear HCCH NMR experiments. Next the individual sugar rings are connected to their corresponding bases through intra-residue 1H-1H nuclear Overhauser effects (NOEs) observed in a 3D (1H, 13C, 1H) NOESY-HMQC experiment. Sequential NOE connectivities observed in this experiment are then used to assign each residue in the nucleotide sequence. The imino protons and nitrogens, and the cytidine amino protons and nitrogens, are assigned by 2D (15N, 1H) HMQC and 3D (1H, 15N, 1H) NOESY-HMQC experiments in H2O. This assignment procedure is illustrated on the 99% 13C/15N labeled RNA duplex r(GGCGCUUGCGUC)2. The application of these multi-dimensional heteronuclear magnetic resonance experiments enormously simplifies the resonance assignment of nucleic acids and allows assignment of many more protons, carbons and nitrogens than was possible using standard techniques on unlabeled molecules. Since a larger percentage of the protons can now be assigned by these experiments, much more NMR structural information can be obtained which will significantly extend the size limit for solution structure determinations of RNAs.

NMR solution structure of the lead-dependent ribozyme: evidence for dynamics in RNA catalysis.

The NMR solution structure of a lead-dependent ribozyme, known as the leadzyme, is presented. This ribozyme is among the smallest of the known catalytic RNAs, with an active site consisting of a six-nucleotide asymmetric internal loop. This loop has a roughly double-helical structure, including a protonated adenine-cytosine wobble base-pair, that positions the cytosine base 5' to the cleavage site in a double-helical conformation. The deviations from helical structure consist of two bulged guanosine residues, G7 and G9, where G7 is the residue 3' to the cleavage site. The scissile phosphate group of the leadzyme is not positioned for in-line nucleophilic attack. Therefore, a conformational rearrangement in the active site is required to reach the proposed transition state for this ribozyme. This is similar to previous observations in X-ray studies of the hammerhead ribozyme, and emphasizes the necessity for dynamic structural fluctuations in the catalytic mechanism of small ribozymes. A model for metal-binding in the leadzyme is proposed in which a lead ion binds to a bulged guanine base that is critical for leadzyme function.

Order, dynamics and metal-binding in the lead-dependent ribozyme.

The in vitro selected lead-dependent ribozyme is among the smallest and simplest of the known catalytic RNA motifs and has a unique metal ion specificity for divalent lead. The conformation and dynamics of this ribozyme are analyzed here by NMR and chemical probing experiments. Complete assignments of the 1H, 13C, and 15N resonances have been made, and the NMR chemical shift changes in the presence of Pb2+, Mg2+ or high concentrations of Na+ show that there is no significant structural change upon addition of either activating (Pb2+) or inhibitory (Mg2+) divalent ions. The 13C NMR relaxation data indicate substantial dynamic fluctuations on various time-scales for active-site residues in this ribozyme. The combination of chemical probing and NMR experiments reveals a picture of the active site for the lead-dependent ribozyme that has both ordered and dynamic features.

Structure and dynamics of the iron responsive element RNA: implications for binding of the RNA by iron regulatory binding proteins.

The iron responsive element (IRE) is a approximately 30 nucleotide RNA hairpin that is located in the 5' untranslated region of all ferritin mRNAs and in the 3' untranslated region of all transferrin receptor mRNAs. The IREs are bound by two related IRE-binding proteins (IRPs) which help control intracellular levels of iron by regulating the expression of both ferritin and transferrin receptor genes. Multi-dimensional NMR and computational approaches were used to study the structure and dynamics of the IRE RNA in solution. The NMR data are consistent with formation of A-form helical stem regions, a one-base internal bulge and a Watson-Crick C.G base-pair between the first and fifth nucleotides in the loop. A superposition of refined structures indicates that the conserved C in the internal bulge, and three residues in the six-nucleotide hairpin loop are quite dynamic in this RNA. The structural roles of the stems, the loop and the bulge in the function of the IRE RNA and in possible interactions with the iron regulatory protein are discussed.

Determining local conformational variations in DNA. Nuclear magnetic resonance structures of the DNA duplexes d(CGCCTAATCG) and d(CGTCACGCGC) generated using back-calculation of the nuclear Overhauser effect spectra, a distance geometry algorithm and constrained molecular dynamics.

Two-dimensional nuclear magnetic resonance (n.m.r.) spectroscopy and a variety of computational techniques have been used to generate three-dimensional structures of the two DNA duplexes d(CGCCTAATCG) and d(CGTCACGCGC). The central six base-pairs in these two decamers contain all ten dinucleotide pairs in DNA and thus, represent a model system for investigating how the local structure of DNA varies with base sequence. Resonance assignments were made for the non-exchangeable base protons and most of the C-1'-C-4' sugar protons in both decamers. Three-dimensional structures were generated using a distance geometry algorithm and these initial structures were refined by optimizing the fit of back-calculated spectra against the experimental two-dimensional nuclear Overhauser effect (NOE) spectra. This back-calculation procedure consists of calculating NOE cross relaxation rates for a given structure by solution of the Bloch equations, and directly accounts for spin diffusion effects. Use of this refinement procedure eliminates some assumptions that have been invoked when generating structures of DNA oligomers from n.m.r. data. Constrained energy minimization and constrained quenched molecular dynamics calculation were also performed on both decamers to help generate energetically favorable structures consistent with the experimental data. Analysis of the local conformational parameters of helical twist, helical rise, propeller twist, displacement and the alpha, beta, gamma, epison and zeta backbone torsion angles in these structures shows that these parameters span a large range of values relative to the X-ray data of nucleic acids. However, the glycosidic and pseudorotation angles are quite well defined in these structures. The implications that these results have for determination of local structural variations of DNA in solution, such as those predicted by Callidine's rules, are discussed. Our results differ significantly from some previous studies on determining local conformations of nucleic acids and comparisons with these studies are made.

Solution structures of the rabbit neutrophil defensin NP-5.

Solution structures of the rabbit neutrophil defensin NP-5 have been determined by 1H nuclear magnetic resonance (n.m.r.) spectroscopy and distance geometry techniques. This 33 amino acid peptide is part of the oxygen-independent mammalian defense system against microbial infection. The structures were generated from 107 n.m.r. derived inter-residue proton-proton distance constraints. A distance geometry algorithm was then used to determine the range of structures consistent with these distance constraints. These distance geometry calculations employed an improved algorithm that allowed the chirality constraints to be relaxed on prochiral centers when it was not possible to make stereo-specific assignments of protons on these centers. This procedure gave superior results compared with standard distance geometry methods and also produced structures that were more consistent with the original n.m.r. data. Analysis of the NP-5 structures shows that the overall folding of the peptide backbone is well defined by the n.m.r. distance information but that the side-chain group conformations are generally less well defined.

A network of heterogeneous hydrogen bonds in GNRA tetraloops.

RNA hairpin loops containing a GNRA consensus sequence are the most frequently occurring hairpins in a variety of prokaryotic and eukaryotic RNAs. These tetraloops play important functional roles in RNA folding, in RNA-RNA tertiary interactions and as protein binding sites. Homo and heteronuclear NMR spectroscopy have been used to determine the structures of the most abundant members of the GNRA tetraloop family: the GAGA, GCAA and GAAA loops closed by a C-G base pair. Analysis of the structures of these three hairpin loops reveals a network of heterogeneous hydrogen bonds. The loops contain a G-A base pair, a G base-phosphate hydrogen bond and several 2' OH-base hydrogen bonds. These intramolecular interactions and the extensive base stacking in the loop help explain the high thermodynamic stability and give insight into the diverse biological roles of the GNRA RNA hairpins.

RNA aptamers to the peptidyl transferase inhibitor chloramphenicol.

BACKGROUND: The problem of how macromolecules adopt specific shapes to recognize small molecules in their environment is readily addressed through in vitro selections (the SELEX protocol). RNA-antibiotic interactions are particularly attractive systems for study because they provide an opportunity to expand our understanding of molecular recognition by RNA and to facilitate ribosomal modeling. Specifically, the antibiotic chloramphenicol (Cam) naturally binds bacterial ribosomes in the 'peptidyl transferase loop' of 23S ribosomal RNA to inhibit peptide bond formation. RESULTS: We identified Cam-binding RNA molecules ('aptamers') from two independent initial random RNA populations. Boundary determinations, ribonuclease S1 sensitivity analyses and the activity of truncated minimal RNAs identified a structural motif that is shared by sequences from both selections. The pseudosymmetric motif consists of a highly conserved central helix of five to six base pairs flanked by A-rich bulges and additional helices. Addition of Cam prior to ribonuclease S1 protected nucleotides in the conserved cores from cleavage. Reselection from a pool of mutated variants of the minimal aptamer further refined the sequence requirements for binding. Finally, we used proton nuclear magnetic resonance (NMR) to establish a 1:1 RNA: Cam stoichiometry of the complex. Both the protection and NMR data both show that Cam stabilizes the active fold of this aptamer. CONCLUSIONS: There are many different RNA sequences that can bind Cam. The Cam aptamers that we examined have a well-defined secondary structure with a binding pocket that appears to be stabilized by Cam. This RNA motif superficially resembles the Cam-binding site in 23S rRNA, although further work is needed to establish the significance of these similarities.

Solution structure of the calcium channel antagonist omega-conotoxin GVIA.

The three-dimensional solution structure is reported for omega-conotoxin GVIA, which is a potent inhibitor of presynaptic calcium channels in vertebrate neuromuscular junctions. Structures were generated by a hybrid distance geometry and restrained molecular dynamics approach using interproton distance, torsion angle, and hydrogen-bonding constraints derived from 1H NMR data. Conformations of GVIA with low constraint violations converged to a common peptide fold. The secondary structure in the peptide is an antiparallel triple-stranded beta-sheet containing a beta-hairpin and three tight turns. The NMR data are consistent with the region of the peptide from residues S9 to C16 being more dynamic than the rest of the peptide. The peptide has an amphiphilic structure with a positively charged hydrophilic side and an opposite side that contains a small hydrophobic region. Residues that are thought to be important in binding and function are located on the hydrophilic face of the peptide.

Improved measurement of 13C, 31P J coupling constants in isotopically labeled RNA.

3JCP coupling constants have been measured in a 99% 13C,15N labeled lead-dependent ribozyme, known as the leadzyme. These coupling constants were determined by analysis of the intensity of individual crosspeaks in a spin-echo difference constant time HSQC experiment. This procedure permits improved measurement of the 3JC2'P and 3JC4'P coupling constants in isotopically labeled RNA and yielded valuable information on the beta and epsilon backbone torsion angles in the leadzyme.

Distinguishing between duplex and hairpin forms of RNA by 15N-1H heteronuclear NMR.

A general method is described for distinguishing RNA hairpins from RNA duplexes by application of two-dimensional filtered nuclear Overhauser enhancement spectra on a 1:1 mixture of unlabeled and 99% 15N-labeled molecules. The method is applied to the RNA dodecamer rGGCGCUUGCGUC which can form an intramolecular hairpin under low salt conditions and a duplex in high salt. This procedure allows unambiguous identification of RNA hairpins or duplexes under the same conditions that are used in the NMR solution structure determination.

Measurement of carbon-phosphorus J coupling constants in RNA using spin-echo difference constant-time HCCH-COSY.

We report a novel NMR technique for the measurement of carbon-phosphorus coupling constants in RNA oligomers. This method, spin-echo difference constant-time HCCH-COSY, takes advantage of the well-dispersed H1' and C1' resonances to analyze couplings involving the more poorly dispersed ribose carbon and phosphorus resonances. The technique was applied to analysis of the 3JC2'P coupling constants related to backbone epsilon torsion angles in a 30-nucleotide lead-dependent ribozyme. 3JC2'P coupling constants were obtained for approximately 90% of the residues in this RNA, which is over twice as many as could be obtained with previous methods.

Persistent median artery in carpal tunnel syndrome.

Persistent median artery of the forearm and wrist is not very frequently observed. Only a few cases of persistent median artery thrombosis associated with compression of the median nerve in the carpal tunnel have been reported: in these cases symptoms arise acutely and surgery consists in the excision of the thrombosed arterial branch. In a patient with recurrent carpal tunnel syndrome, with a patent median artery and duplication of the median nerve, we performed neurolysis of the nerve and repositioning of the artery to the ulnar side. Electromyography, arteriography and clinical examinations performed six months later showed that irritative phenomena of the median nerve had regressed and the artery was still patent.