Dependence of tRNA Structure in Solution upon Ionic Condition of the Solvent

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Abstract
Barley phenylalanine tRNA (tRNAphe) exhibits a 4.7‐fold enhancement of fluorescence intensity in response to the addition of MgCl2 in 0.01 M Tris‐HCl, pH 7.5, 0.001 M Na2 EDTA. In the case of 2.0 M NaCl about 60% of this effect was observed. The change of fluorescence intensity was used as a measure of bound ligand concentration. From fluorescence Scatchard plots a different character of Mg2+ binding to tRNA was revealed, depending upon the initial conformation of tRNA in solution submitted to titration. In NaCl‐free tRNAphe solution strong interacting (Ks= 1.5 × 105 M−1n= 2.4) and weak independent (Kw= 9 x 103 M−1) Mg2+ binding were observed. In solution containing 0.1 M NaCl both the strong and the weak Mg2+ binding demonstrated independent behaviour. Ks and Kw, in the presence of NaCl, had lower values than in NaCl‐free solution, since Na+ was found to be a competitive inhibitor of Mg2+ binding. Fluorescence titrations of tRNAphe solutions containing Mg2+ evidenced 2 Na+ binding sites/Mg2+ binding site (at least in the region of weak sites of Mg2+) characterized by inhibitory binding constants K̄i1 Na= 45 M−1 and K̄i 1 Na= 8.5 M−1 (mean values) for first and second Na+ bindings respectively. Na+ exerts a destabilizing effect on tRNA conformation induced by Mg2+. A model of Mg2+ binding and concomitant conformational changes of the tRNA molecule is discussed: one class of Mg2+ binding sites in unfolded tRNA and two classes of sites, strong and weak, in folded conformation are postulated.