Selective binding of amino acid residues to trna molecules detected by anticodon-anticodon interactions

Anticodon-anticodon pairing of complementary tRNA’s has been studied by fluorescence temperature jump measurements in the presence of different ligands as an approach for the evaluation of ligand binding to tRNA. This procedure is particularly useful for ligands which do not show spectroscopic changes upon binding, but affect the pairing potential of anticodons. Addition of phenylalanine-, tyrosine- and tryptophan-amide leads to a substantial decrease of the tRNA^Phe-tRNA^Glu pairing constant K_p, whereas K_p remains almost unaffected by addition of leucine amide and increases upon addition of glycine amide. The effects observed for the aromatic amino acid amides can be described quantitatively by a site binding model with preferential binding of the amides to tRNA^Phe. The binding constants evaluated according to this model (Pheamide 120 M^-1, Tyr-amide 160 M^-1 and Trp-amide 580 M^-1) are consistent with values obtained independently by fluorescence titrations with tRNA^Phe. Selective binding of these amino acid residues to tRNA^Phe is deduced from the observed concentration dependence, which is not compatible with a corresponding binding process to tRNA^Glu. Addition of glutamic acid diamide induces an increase of the tRNA^Phe • tRNA^Glu pairing constant, which is however equivalent to that observed for tRNA^Phe-tRNA^Lys pairing and thus does not demonstrate a selective binding to tRNA °1 u . The pairing of tRNA^Phe with tRNA^Glu is strongly enhanced by addition of Mg²⁺ or spermine. Evaluation of the Mg²⁺ data by a site model leads to constants of 360 M^-1 for the binding of Mg²⁺ to monomer tRNA and 3000 M^-1 for the binding of Mg²⁺ to the tRNA^PhetRNA^Glu dimer. A comparison of the enhanced pairing observed in the presence of Mg²⁺, which is known to induce a 3′-stack conformation of the anticodon loop, with the reduced pairing affinity observed in the presence of aromatic amino acid amides suggests induction of a 5′-stack conformation by the latter ligands