Structure of the 3''-hairpin of the TYMV pseudoknot: preformation in RNA folding.

The solution structure of an RNA-hairpin present in the pseudoknot, which is found at the 3'-terminus of turnip yellow mosaic virus genomic RNA, has been solved by nuclear magnetic resonance spectroscopy. The loop, which contains the sequence 5'-GGGUCA-3', was found to be highly structured and, contrary to expectations, does not attain its stability through GA or GC base pair formation but by triple interactions between the tilted adenosine and the minor groove sides of the first two guanosines. Interestingly, a very similar conformation was found for the cognate pseudoknot, implying that the 3'-hairpin is preformed for folding into a pseudoknotted structure. These findings suggest a mechanism of 'predetermined-fit' as a principle in RNA folding.     

1H-NMR studies on the gene-5-encoded single-stranded DNA binding protein of the filamentous bacteriophage IKe. General spectral and structural features

Under physiological conditions and at concentrations needed for NMR studies, severe aggregation of the gene-5 protein of the filamentous phage IKe occurs. Conditions are described for which well-resolved 1H-NMR spectra of the protein can be obtained. The aromatic part of the spectrum is analyzed by means of two-dimensional NMR techniques; a complete interpretation is presented. Oligonucleotide binding studies reveal that just one phenylalanyl residue and one tyrosyl residue are influenced by the binding of rAMP, (dA)2, (dA)3, (dA)4, (dA)6, d(pT)3 or (dT)4. Upon binding, the aromatic resonances of these amino acid residues are shifted upfield by about 0.4-0.5 ppm. NMR measurements at different pH values demonstrate that only one of the two histidyl residues is freely titratable. From CIDNP experiments it is concluded that three out of five tyrosyl residues are located at the surface of the protein. Measurements carried out as a function of protein concentration indicate the occurrence of specific protein-protein interactions between dimeric gene-5-protein molecules. The data obtained are compared with those available for the gene-5 protein of M13. It follows from the comparison that these proteins mimic each other in almost every respect.     

Residue-specific assignments of resonances in the 1H nuclear magnetic resonance spectrum of ribosomal protein E-L30 by systematic application of two-dimensional Fourier transform nuclear magnetic resonance methods

A two-dimensional Fourier transform nuclear magnetic resonance study of the ribosomal protein E-L30 is reported. Five two-dimensional techniques, namely: nuclear magnetic resonance J-resolved spectroscopy, correlated spectroscopy, double quantum spectroscopy, relayed coherence transfer and nuclear Overhauser enhancement spectroscopy were used. Qualitative inspection of the spectra obtained by these techniques provided evidence that the E-L30 molecule has a well-defined structure in solution. This analysis indicated that, despite the fact that the protein is stable only at moderate temperatures and neutral pH, a structural analysis of the molecule would be feasible. A detailed analysis of the spectra permitted unambiguous discrimination between the spin systems of different amino acids, resulting in residue-specific resonance assignments. We were able to assign all resonances of all six threonine, four valine, five alanine, two histidine, two serine, one phenylalanine, one asparagine and one aspartic acid residue of E-L30. Complete resonance assignment was obtained for two glycine residues. Partial assignments became available for all six isoleucine, three glycine and one glutamine residue. These results form a sound basis for the structure determination of the protein described in the accompanying paper.     

On loop folding in nucleic acid hairpin-type structures

In a series of studies, combining NMR, optical melting and T-jump experiments, it was found that DNA hairpins display a maximum stability when the loop part of the molecule comprises four or five nucleotide residues. This is in contrast with the current notion based on RNA hairpin studies, from which it had been established that a maximum hairpin stability is obtained for six or seven residues in the loop. Here we present a structural model to rationalize these observations. This model is based on the notion that to a major extent base stacking interactions determine the stability of nucleic acid conformations. The model predicts that loop folding in RNA is characterized by an extension of the base stacking at the 5'-side of the double helix by five or six bases; the remaining gap can then easily be closed by two nucleotides. Conversely, loop folding in DNA is characterized by extending base stacking at the 3'-side of the double helical stem by two or three residues; again bridging of the remaining gap can then be achieved by one or two nucleotides. As an example of loop folding in RNA the anticodon loop of yeast tRNAPhe is discussed. For the DNA hairpin formed by d(ATCCTAT4TAGGAT) it is shown that the loop structure obtained from molecular mechanics calculations obeys the above worded loop folding principles.     

Polypurine/polypyrimidine hairpins form a triple helix structure at low pH.

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.     

Resonance assignments of non-exchangeable protons in B type DNA oligomers, an overview.

The chemical shifts of 1H resonances of non exchangeable protons (except H5', H5" and adenine H2) of over six hundred nucleotides have been collected. The influence which the base of the nucleotide itself as well as the bases on its 5' and 3' side exert on the chemical shifts of the various resonances has been investigated. Most of the resonances appear to be predominantly influenced by only one base. For H2', H2", H3', H4' and H6/H8 this is the base of the central nucleotide, for H5(C) and CH3(T) it is the one on the 5' side and for H1' it is the one on the 3' side. Chemical shift distribution profiles are presented which allow an estimation of the probability of finding a particular resonance at a particular position in the spectrum.     

Pentosan polysulfate promotes proliferation and chondrogenic differentiation of adult human bone marrow-derived mesenchymal precursor cells

Introduction  

This study was undertaken to determine whether the anti-osteoarthritis drug pentosan polysulfate (PPS) influenced mesenchymal precursor cell (MPC) proliferation and differentiation.     

Experimental stress in inflammatory rheumatic diseases: a review of psychophysiological stress responses

Introduction  

Stressful events are thought to contribute to the aetiology, maintenance and exacerbation of rheumatic diseases. Given the growing interest in acute stress responses and disease, this review investigates the impact of real-life experimental psychosocial, cognitive, exercise and sensory stressors on autonomic, neuroendocrine and immune function in patients with inflammatory rheumatic diseases.