Anticodon Loop of tRNA^phe: Structure, Dynamics, and Mg²⁺ Binding

The structure, dynamics, and Mg²⁺ binding reactions of the isolated anticodon hairpin loop from tRNA^Phe (yeast) have been analyzed by fluorescence-detected temperature-jump relaxation, melting experiments, and equilibrium sedimentation. Most of the measurements were performed at an ionic strength of 0.15 M and at temperatures below 25 °C, where the hairpin loop proved to be stable. A relaxation effect with a time constant of ~100 μs, indicated by the Wye base fluorescence, is attributed to a conformational change of the anticodon loop and is very similar to a corresponding transition observed previously for the whole tRNA^phe molecule. A Mg²⁺ binding site reflected by an inner-sphere relaxation process and associated with a strong increase of the Wye base fluorescence closely resembles a corresponding site observed in the complete tRNA^phe and is attributed to a site in the anticodon loop identified by X-ray analysis. In addition to the Mg²⁺ site in the loop, which is associated with a binding constant of 2 X 10³M^-1 the existence of sites with a higher affinity is demonstrated by an unusual relaxation effect, showing a minimum in the reciprocal time constant with increasing Mg²⁺ concentration. The experimental data can be described by a transition between two states and Mg²⁺ binding to both states resulting in a reaction cycle, which is extended by an additional Mg²⁺ binding reaction to one of the states. The unusual effect has not been observed for the complete tRNA^phe and is also not observed when Ca²⁺ is added instead of Mg²⁺. This result indicates the existence of a conformational change involving Mg²⁺ inner-sphere complexation. None of the relaxation effects is observed for a hexamer, which is excised from the anticodon loop and contains the Wye base but does not form the loop structure. Thus, the presence of the hairpin loop is necessary for the anticodon loop transition, the Mg²⁺ inner-sphere complexation in the anticodon loop, and the special transition coupled to the Mg²⁺ sites with high affinity; apparently, the hairpin loop structure is required for a specific arrangement of molecular contacts.