PubMed · 13676213
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J MATHIEU. 1959. [The magnesium].. https://pubmed.ncbi.nlm.nih.gov/13676213/
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Given the essential cellular roles for ribonucleic acids (RNAs) it is important to understand the stability of three-dimensional structures formed by these molecules. This study aims to investigate the dissociation energy landscape for simple RNA structures via atomic-force-microscopy-based single-molecule force-spectroscopy measurements. This approach provides details on the locations and relative heights of the energy barriers to dissociation, and thus information upon the relative kinetic stabilities of the formed complexes. Our results indicate that a simple dodecamer RNA helix undergoes a forced dissociation process similar to that previously observed for DNA oligonucleotides. Incorporating a UCU bulge motif is found to introduce an additional energy barrier closer to the bound state, and also to destabilize the duplex. In the absence of magnesium ions a duplex containing this UCU bulge is destabilized and a single, shorter duplex is formed. These results reveal that a bulge motif impacts upon the forced dissociation of RNA and produces an energy landscape sensitive to the presence of magnesium ions. Interestingly, the obtained data compare well with previously reported ensemble measurements, illustrating the potential of this approach to improve our understanding of RNA stability and dissociation kinetics.
Tertiary folding of the hammerhead ribozyme has been analyzed by hydroxyl radical footprinting. Three hammerhead constructs with distinct noncore sequences, connectivities, and catalytic properties show identical protection patterns, in which conserved core residues (G5, A6, U7, G8, and A9) and the cleavage site (C17, G1.1, and U1.2) become reproducibly protected from nucleolytic attack by radicals. Metal ion titrations show that all protections appear together, suggesting a single folding event to a common tertiary structure, rather than an ensemble of different folds. The apparent binding constants for folding and catalysis by Mg(2+) are lower than those for Li(+) by 3 orders of magnitude, but in each case the protected sites are identical. For both Mg(2+) and Li(+), the ribozyme folds into the protected tertiary structure at significantly lower cation concentrations than those required for cleavage. The sites of protection include all of the sites of reduced solvent accessibility calculated from two different crystal structures, including both core and noncore nucleotides. In addition, experimentally observed protected sites include additional sequences adjacent to those predicted by the crystal structures, suggesting that the solution structure may be folded into a more compact shape. A 2'-deoxy substitution at G5 abolishes all protection, indicating that the 2'-OH is essential for folding. Together, these results support a model in which low concentrations of metal ions fold the ribozyme into a stable ground state tertiary structure that is similar to the crystallographic structures, and higher concentrations of metal ions support a transient conformational change into the transition state for catalysis. These data do not themselves address the issue as to whether a large- or small-scale conformational change is required for catalysis.
Aqueous extracts of birch leaves ( folium Betulae), peppermint leaves ( folium Menthae), sage leaves ( folium Salviae), valerian roots ( radix Valerianae), and dandelion roots ( radix Taraxaci) are analysed for the three essential elements magnesium, manganese and zinc. Ultrafiltration reveals that 60-100% of these metals are present as low molecular weight species (<5000 Da). Further characterisation of the low molecular weight fraction is done by using size exclusion chromatography and different detectors, namely element specific AAS detection, diode array UV-VIS detection and electrochemical detection. The similarities and differences of the plant extracts are well reflected by the respective chromatograms, and typical plant constituents can be identified by their spectral and electrochemical properties (e.g. flavonoids in Betula). Mg and Mn species are selectively detected by AAS in closely neighbouring chromatographic regions for all five samples. However, there are significant differences between the samples investigated. In all cases a good correlation exists between detection of these metals (Mg, Mn) and pulsed amperometric detection (PAD), which is selective for carbohydrates. The respective molecular weight of carbohydrate species is in the range of approximately 300-600 Da. The distribution of zinc species (detectable only in the Betula extract) is totally different from that of Mg and Mn species. For zinc, many more species are detected, and there is no correlation to carbohydrates; instead (poly)phenols are involved in zinc complexation.