31P Magnetic relaxation studies of yeast transfer RNAPhe

The molecular mechanism of thermal unfolding of yeast tRNAPhe in 20 mM NaCl, 1 mM EDTA, and 10 mM MgSO4, pH 7.1 ± 0.1, has been examined by 31P magnetic relaxation and the nuclear Overhauser effect methods at 40.48 MHz in the temperature range of 22.5–80°C. Two partially resolved 31P resonance peaks of yeast tRNAPhe have been found to behave distinctively different in their longitudinal relaxation times. Individual intensities of the two partially resolved peaks have been quantitatively estimated by the use of relaxation data and the nuclear Overhauser effect as a function of temperature. The results of these observations largely support the earlier suggestion by Gueron and Shulman that the high- and low-field parts of the main 31P resonance cluster originate from phosphorus nuclei belonging to the double-helical and nonhelical regions of the tRNA, respectively. The spin-lattice relaxation of the phosphorus nucleus has been found to be determined dominantly by the dipolar interaction with the surrounding ribose protons at this observing frequency. Rotational correlation times for the two portions of the ribose-phosphate backbone of the tRNA have been separately deduced from the quantitative treatment of the 31P nuclear spin-lattice relaxation times (T1) and the nuclear Overhauser effect. The result indicates that the two portions undergo internal motions at distinctively different rates of 108–1010 sec−1 order in the temperature range of 22.5–80°C, and that the thermal activation of these motions occurs at least in three distinctive steps, i.e., 22.5–31, 31–40, and 40–80°C. The rates of the internal motions and the associated activation energies in respective steps give some insight into the thermo-induced change of the yeast tRNAPhe structure.