Spin-labeling experiments have been carried out in which an organic free radical is linked to Escherichia coli Val- or Phe-tRNA by acylation of the α-amino group. The chemistry of the labeling reaction is studied in detail, and special attention is paid to the specificity of the reaction. Nonspecific labeling of tRNA can be decreased considerably by fractionating the tRNA after the spin-labeling reaction. During this process, a single peak of spin-labeled Val-tRNA and two peaks of Phe-tRNA were recovered. The two species of Phe-tRNA differ by their stability to heat denaturation. A change in mobility of the spin label occurs after ribonuclease treatment and after thermal denaturation. The latter process is reversible, and the spin-melting temperature measured in this process is sensitive to ionic strength. The spin-melting phenomenon is interpreted as indicating two molecular states of tRNA with different activation energies for motion of the spin label at temperatures, respectively, above and below the sharp transition which occuss at the spin-melting temperature.
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