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A Pardi

Publications and source records attributed to A Pardi.

At least 19 recordsLinked to original sources
PubMed1997

A conformational change in the catalytic core of the hammerhead ribozyme upon cleavage of an RNA substrate.

Heteronuclear multidimensional NMR structural studies have been performed on a hammerhead ribozyme complexed with a cleaved and an uncleaved substrate. The NMR data demonstrate that the three helices surrounding the conserved catalytic core hammerhead are stably formed in both complexes. Evidence is also presented that indicates that the sheared G-A base pairs in the conserved core are formed in the absence of Mg2+. The NMR structural data demonstrate that there is a significant structural change of the conserved core of the hammerhead ribozyme-substrate complex upon cleavage of the substrate. Molecular dynamics calculations were performed to generate models of the ribozyme-cleaved substrate complex, and these results are used to help understand the mechanism of the hammerhead cleavage reaction.

Base Sequence↗
PubMed1996

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.

Base Composition↗
PubMed1996

A curved RNA helix incorporating an internal loop with G.A and A.A non-Watson-Crick base pairing.

The crystal structure of the RNA dodecamer 5'-GGCC(GAAA)GGCC-3' has been determined from x-ray diffraction data to 2.3-A resolution. In the crystal, these oligomers form double helices around twofold symmetry axes. Four consecutive non-Watson-Crick base pairs make up an internal loop in the middle of the duplex, including sheared G.A pairs and novel asymmetric A.A pairs. This internal loop sequence produces a significant curvature and narrowing of the double helix. The helix is curved by 34 degrees from end to end and the diameter is narrowed by 24% in the internal loop. A Mn2+ ion is bound directly to the N7 of the first guanine in the Watson-Crick region following the internal loop and the phosphate of the preceding residue. This Mn2+ location corresponds to a metal binding site observed in the hammerhead catalytic RNA.

Adenine↗
PubMed1996

Correlation of the guanosine exchangeable and nonexchangeable base protons in 13C-/15N-labeled RNA with an HNC-TOCSY-CH experiment.

A triple resonance HNC-TOCSY-CH experiment is described for correlating the guanosine imino proton and H8 resonances in 13C-/15N-labeled RNAs. Sequential assignment of the exchangeable imino protons in Watson-Crick base pairs is generally made independently of the assignment of the nonexchangeable base protons. This H(NC)-TOCSY-(C)H experiment makes it possible to unambiguously link the assignment of the guanosine H8 resonances with sequential assignment of the guanosine imino proton resonances. 2D H(NC)-TOCSY-(C)H spectra are presented for two isotopically labeled RNAs, a 30-nucleotide lead-dependent ribozyme known as the leadzyme, and a 48-nucleotide hammerhead ribozyme-RNA substrate complex. The results obtained on these two RNAs demonstrate that this HNC-TOCSY-CH experiment is an important tool for resonance assignment of isotopically labeled RNAs.

Base Sequence↗
PubMed1995

Solution structure of the CUUG hairpin loop: a novel RNA tetraloop motif.

The solution structure of a uniformly 13C/15N-labeled CUUG RNA hairpin loop has been determined by multidimensional heteronuclear magnetic resonance spectroscopy in combination with distance geometry and restrained molecular dynamics calculations. The structure of this CUUG tetraloop represents a novel RNA loop motif where the first and last loop nucleotides form a standard Watson-Crick C-G base pair and the second loop nucleotide interacts directly with the closing base pair of the stem by folding into the minor groove. This structure helps explain why the closing base pair is phylogenetically conserved and indicates a six-nucleotide G(CUNG)C motif for the CUUG RNA tetraloop. Implications for the function of this CUUG tetraloop in ribosomal RNA and in RNA tertiary interactions are discussed.

Base Sequence↗
PubMed1995

Solution structure of bovine neutrophil beta-defensin-12: the peptide fold of the beta-defensins is identical to that of the classical defensins.

The solution structure is reported for bovine neutrophil beta-defensin-12 (BNBD-12), a member of the beta-defensin family of antimicrobial peptides. Structural constraints in the form of proton-proton distances, dihedral angles, and hydrogen bond constraints were derived from two-dimensional, homonuclear magnetic resonance spectroscopy experiments. The three-dimensional structure of BNBD-12 was calculated using distance geometry and restrained molecular dynamics. An ensemble of structures with low NOE constraint violation energies revealed a precisely defined triple-stranded, antiparallel beta-sheet as the structural core of the peptide. The N-terminal beta-strand and three locally well-defined tight turns form a hydrophobic face. Conserved isoleucine and glycine residues form a beta-bulge structure which initiates a beta-hairpin secondary structure motif composed of the second and C-terminal beta-strands. The beta-hairpin contains numerous charged residues and forms the cationic face of BNBD-12. The N-terminal residues were found to be disordered, due to an absence of tertiary NOEs. The triple-stranded beta-sheet, the beta-bulge preceding the hairpin, and the cationic/hydrophobic amphiphilic character are definitive features of all defensin structures determined to date. Further, we predict that the tracheal antimicrobial peptide (TAP) and the recently described gallinacins will have tertiary structures similar to that of BNBD-12.

Amino Acid Sequence↗
PubMed1995

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.

Base Sequence↗
PubMed1995

Triple resonance HNCCCH experiments for correlating exchangeable and nonexchangeable cytidine and uridine base protons in RNA.

A set of triple resonance experiments is presented, providing through-bond H2N/HN to H6 connectivities in uridines and cytidines in 13C-/15N-labeled RNAs. These connectivities provide an important link between the sequential assignment pathways for the exchangeable and nonexchangeable proton resonances in nucleic acids. Both 2D and pseudo-3D HNCCCH experiments were applied to a 30-nucleotide lead-dependent ribozyme, known as the leadzyme. The HN to H6 connectivities for three uridines in the leadzyme were identified from one 2D H(NCCC)H experiment, and the H2N to H6 connectivities were identified for seven of the eight cytidines from the combination of a 2D H(NCCC)H and a pseudo-3D H(NCC)CH experiment.

Animals↗
PubMed1995

GNRA tetraloops make a U-turn.

The U-turn (uridine turn) is an RNA structural motif that contains a change in backbone direction stabilized by specific interactions across the bend. It was first identified in the anticodon loop and the T-loop of yeast tRNA(Phe) (Quigley & Rich, 1976, Science 194:796-806) and has recently also been found in the crystal structure of the hammerhead ribozyme (Pley HW, Flaherty KM, McKay DB, 1994a, Nature 372:68-74). These U-turn motifs follow a UNR consensus sequence (where N is any nucleotide and R is G or A). Here we report that the frequently occurring GNRA tetraloops also contain a U-turn motif, and we discuss the role of U-turns as abundant tertiary structural motifs in RNA.

Nucleic Acid Conformation↗
PubMed1995

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.

Adult↗
PubMed1994

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.

Base Composition↗
PubMed1994

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.

Base Sequence↗
PubMed1994

Structure and dynamics of the neutrophil defensins NP-2, NP-5, and HNP-1: NMR studies of amide hydrogen exchange kinetics.

The exchange kinetics for the slowly exchanging amide hydrogens in three defensins, rabbit NP-2, rabbit NP-5, and human HNP-1, have been measured over a range of pH at 25 degrees C using 1D and 2D NMR methods. These NHs have exchange rates 10(2) to 10(5) times slower than rates from unstructured model peptides. The observed distribution of exchange rates under these conditions can be rationalized by intramolecular hydrogen bonding of the individual NHs, solvent accessibility of the NHs, and local fluctuations in structure. The temperature dependencies of NH chemical shifts (NH temperature coefficients) were measured for the defensins and these values are consistent with the defensin structure. A comparison is made between NH exchange kinetics, NH solvent accessibility, and NH temperature coefficients of the defensins and other globular proteins. Titration of the histidine side chain in NP-2 was examined and the results are mapped to the three-dimensional structure.

Amides↗
PubMed1994

Unambiguous through-bond sugar-to-base correlations for purines in 13C,15N-labeled nucleic acids: the HsCsNb,HsCs(N)bCb, and HbNbCb experiments.

A set of three 3D (1H,13C,15N) triple-resonance correlation experiments has been designed to provide H1'-H8 intraresidue sugar-to-base correlations in purines in an unambiguous and efficient manner. Together, the HsCsNb, HsCs(N)bCb, and HbNbCb experiments correlate the H1' sugar proton to the H8 proton of the attached base by means of the (H1', C1', N9, C8, H8) heteronuclear scalar coupling network. The assignment strategy presented here allows for unambiguous H1'-H8 intraresidue correlations, provided that no two purines have both the same H1' and C1' chemical shifts and the same C8 and N9 chemical shifts. These experiments have yielded H1'-H8 intraresidue sugar-to-base correlations for all five guanosines in the [13C,15N] isotopically labeled RNA duplex r(GGCGCUUGCGUC)2.

Base Sequence↗