Study of transfer ribonucleic acid unfolding by dynamic nuclear magnetic resonance.
Nuclear magnetic resonance (NMR) measurements of proton exchange were performed on yeast tRNAPhe, and in much less detail on Escherichia coli tRNAfMet, over a range of Mg2+ concentrations and temperatures, at neutral pH and 0.1 M NaCl. The resonances studied were those of ring nitrogen protons, resonating between 10 and 15 ppm downfield from sodium 3-(trimethylsilyl)-1-propanesulfonate, which partake in hydrogen bonding between bases of secondary and tertiary pairs. Methods include saturation--recovery, line width, and real-time observation after a change to deuterated solvent. The relevant theory is briefly reviewed. We believe that most of the higher temperature rates reflect major unfolding of the molecule. For E. coli tRNAfMet, the temperature dependence of the rate for the U8--A14 resonance maps well onto previous optical T-jump studies for a transition assigned to tertiary melting. For yeast tRNAPhe, exchange rates of several resolved protons could be studied from 30 to 45 degrees C in zero Mg2+ concentration and had activation energies on the order of 40 kcal/mol. Initially, the tertiary structure melts, followed shortly by the acceptor stem. At high Mg2+ concentration, relatively few exchange rates are measurable below the general cooperative melt at about 60 degrees C; these are attributed to tertiary changes. Real-time observations suggest a change in the exchange mechanism at room temperature with a lower activation energy. The results are compared with those obtained by other methods directed toward assaying ribonucleic acid dynamics.
D004926:Escherichia coli; D008274:Magnesium; D009682:Magnetic Resonance Spectroscopy; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D012343:RNA, Transfer; D012346:RNA, Transfer, Amino Acyl; D012358:RNA, Transfer, Met; D012441:Saccharomyces cerevisiae
D012346:RNA, Transfer, Amino Acyl; D012358:RNA, Transfer, Met; C024988:tRNA, formylmethionine-; D012343:RNA, Transfer; D008274:Magnesium