| Popis: |
Temperature-induced conformational changes in the anticodon region of yeast tRNATyr were studied by EPR spectroscopy. The spin label 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl was attached to the N6-(delta2-isopentenyl)-adenosine residue in tRNATyr, previously made reactive by iodination. The labelled tRNATyr gave an asymmetrical triplet spectrum typical of rapidly tumbling nitroxide, with a rotational correlation time (tauc) of 0.65 ns. Spin-labelled tRNATyr was exposed to heating and cooling in three different buffers each with or without MgCl2. In each case the Arrhenius plot of --log tauc vs. inverse absolute temperature gave two straight lines, intersecting at a critical temperature (tcr). Above tcr, the anisotropy of the spectrum was not reduced and the activation energy of motion increased, indicating that the transition is associated with a conformational change of the macromolecule. Transitions in 0.05 M potassium phosphate (pH 8.0) and 0.02 M Tris - HC1 (pH 7.0) were observed at potassium phosphate (pH 8.0) and 0.02 M Tris - Hc1 (pH 7.0) were observed at approx. 37 degrees C. When 0.01 M mgCl2 was present in these buffers, transitions were shifted to 46 degrees and 53 degrees C, respectively. Transitions in 0.01 M sodium cacodylate were observed at temperatures which are significantly lower. Since all these transitions occur at temperatures considerably below those required to melt the helical regions of tRNA, and at least approximately 10 degrees C below those reported to break tertiary interactions, it is supposed that they reflect some reorientation of the anticodon region, e.g. a change in tilt of the bases. |
