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2-Thiocytidine incorporated into tRNAPhe from yeast by tRNA nucleotidyl transferase can be alkylated by iodoacetamide and its derivatives. Using this method a spin label was introduced specifically into tRNAPhe and the product, tRNAPhe-C-(s.l.)s²C-A, can still be aminoacylated by phenylalanyl-tRNA synthetase. Electron paramagnetic resonance spectra obtained between 10° and 70°C for tRNAPhe-C-(s.l.)s²C-A and the pentanucleotide C-A-C-(s.1.)s²C-A (a model compound) were evaluated for the correlation time, τc, of the reorientation of the spin label in its environment and for the motional anisotropy parameter, ε. Spin-labeled pentanucleotide is characterized by a spin label motion with an activation energy, EA, of 1-67 × 10-3 J × mol-1 × K-1 (7.0 kcal × mol-1 × °C-1) ) and a strongly anisotropic motion with a long axis of the rotational ellipsoid along the N-O bond of the radical. This motion is temperature independent. In comparison spin label bound to the tRNA molecule is more immobilised and its motion is more isotropic. In addition, the slope of log τc(T) and the nature of the motion changes at a critical temperature Tcr which, in the absence of magnesium, varies with ionic strength. In the presence of magnesium Tcr is 47.5 °C, whereas the midpoint temperature of the optical melting curve, Tm, is 75 °C. The melting transition monitored by the spin label is a cooperative process associated with the melting of a higher-ordered structure of the tRNAPhe molecule. [ABSTRACT FROM AUTHOR]