Structural equilibria in RNA as revealed by 19F NMR.
We have incorporated 5-fluorouridine into several sites within a 19-mer RNA modelled on the translational operator of the MS2 bacteriophage. The 19F NMR spectra demonstrate the different chemical shifts of helical and loop fluorouridines of the hairpin secondary structure. Addition of salt gives rise to a species in which the loop fluorouridine gains the chemical shift of its helical counterparts, due to the formation of the alternative bi-molecular duplex form. This is supported by UV thermal melting behaviour which becomes highly dependent on the RNA concentration. Distinct 19F NMR signals for duplex and hairpin forms allow the duplex-hairpin equilibrium constant to be determined under a range of conditions, enabling thermodynamic characterisation and its salt dependence to be determined. Mg2+ also promotes duplex formation, but more strongly than Na+, such that at 25 degrees C, 10 mM MgCl2 has a comparable duplex-promoting effect to 300 mM NaCl. A similar effect is observed with Sr2+, but not Ca2+ or Ba2+. Additional hairpin species are observed in the presence of Na+ as well as Mg2+, Ca2+, Sr2+ and Ba2+ ions. The overall, ensemble average, hairpin conformation is therefore salt-dependent. Electrostatic considerations are thus involved in the balance between different hairpin conformers as well as the duplex-hairpin equilibrium. The data presented here demonstrate that 19F NMR is a powerful tool for the study of conformational heterogeneity in RNA, which is particularly important for probing the effects of metal ions on RNA structure. The thermodynamic characterisation of duplex-hairpin equilibria will also be valuable in the development of theoretical models of nucleic acid structure.
Arnold
J R
JR
School of Biology, University of Leeds, UK. genjrpa@leeds.ac.uk
Fisher
J
J
D005461:Fluorine; D006863:Hydrogen-Ion Concentration; D007477:Ions; D007700:Kinetics; D015636:Magnesium Chloride / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D009690:Nucleic Acid Conformation; D010710:Phosphates / Q000378:metabolism; D012313:RNA / Q000737:chemistry*; D012965:Sodium Chloride / Q000378:metabolism; D013696:Temperature; D013816:Thermodynamics; D014466:Ultraviolet Rays; D014529:Uridine / Q000031:analogs & derivatives / Q000378:metabolism
D007477:Ions; D010710:Phosphates; D015636:Magnesium Chloride; D005461:Fluorine; D012965:Sodium Chloride; C001943:5-fluorouridine; D012313:RNA; C018279:sodium phosphate; D014529:Uridine