PubMed1979
The solution structure of yeast tRNAPhe was investigated by using ethidium as a fluorescent probe in the D loop and the anticodon loop. For this purpose the dihydrouracils in position 16/17 and wybutine in position 37 were substituted by ethidium. The lifetimes and the time-dependent anisotropy of ethidium fluorescence were measured by pulsed nanosecond fluorometry. The kinetics of the transitions between different states of the tRNAPheEtd derivatives were determined by chemical relaxation measurements. It was found that the ethidium label irrespective of its position exhibits three different states called T1, T2 and T3 characterized by lifetimes tau 1 = 30 ns, tau 2 = 12 ns, and tau 3 = 3 ns. The lifetime differences are due to different accessibilities of ethidium for solvent quenching in the three states. Thus, there are three different defined structural environments of the ethidium in both the anticodon and the D loop. The distribution of the three states was measured as a function of Mg2+ concentration and temperature; it was found that state T3 is favored over states T2 and T1 by both increasing Mg2+ concentration and increasing temperature. The chemical relaxation kinetics exhibit a fast transition between T1 and T2 (10--100 ms) and a slow transition between T2 and T3 (100--1000 ms). The rates of both transitions depend likewise on Mg2+ concentration and temperature. The equilibrium and kinetic data clearly show the presence of strong and weak interactions between Mg2+ and tRNA. A cooperative model accounting for this behavior is developed. The ethidium probe behaves identically when located in different regions of the tRNA regarding both its distribution of states and its transition kinetics. This suggests that the different spectroscopic states report different conformations of the tRNA structure. The dependence of the three states on Mg2+ and spermine indicates that conformation T3 is closely related to or identical with the crystal structure. The rotational diffusion constants indicate that of all three states T3 is most extended while T2 is most compact. The thermodynamic analysis reveals that the strongly bound Mg2+ ions reduce both the activation entropy and enthalpy of all transitions. The weakly bound Mg2+ ions increase both the activation enthalpy and entropy of the slow transition between T2 and T3. It is suggested that the breaking of several intramolecular bonds, e.g., hydrogen bonds, is involved in this transition.