Conformational changes in yeast tRNA Tyr revealed by EPR spectra of spin-labelled [formula omitted] residue

Abstract

Temperature-induced conformational changes in the anticodon region of yeast tRNA Tyr were studied by EPR spectroscopy. The spin label 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl was attached to the N 6-(Δ 2- isopentenyl)-adenosine residue in tRNA Tyr, previously made reactive by iodination. The labelled tRNA Tyr gave an asymmetrical triplet spectrum typical of rapidly tumbling nitroxide, with a rotational correlation time ( τ c ) of 0.65 ns. Spin-labelled tRNA Tyr was exposed to heating and cooling in three different buffers each with or without MgCl 2. In each case the Arrhenius plot of − log τ c vs. inverse absolute temperature gave two straight lines, intersecting at a critical temperature ( t cr ). Above t cr , 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 · HCl (pH 7.0) were observed at approx. 37°C. When 0.01 M MgCl 2 was present in these buffers, transitions were shifted to 46° and 53°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°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.