Interaction of elongation factor‐Tu from Escherichia coli with aminoacyl‐tRNA carrying a fluorescent reporter group on the 3′ terminus

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Abstract
Transfer ribonucleic acids containing 2‐thiocytidine in position 75 ([s2C]tRNAs) were prepared by incorporation of the corresponding cytidine analogue into 3′‐shortened tRNA using ATP(CTP):tRNA nucleotidyl‐transferase. [s2C]tRNA was selectively alkylated with fluorescent N‐iodoacetyl‐N′‐(5‐sulfo‐1‐naphthyl)ethyl‐enediamine (1,5‐I‐AEDANS) on the 2‐thiocytidine residue. The product [AEDANS‐s2C]aminoacyl‐tRNA, forms a ternary complex with Escherichia coli elongation factor Tu and GTP, leading to up to 130% fluorescence enhancement of the AEDANS chromophore. From fluorescence titration experiments, equilibrium dissociation constants of 0.24 nM, 0.22 nM and 0.60 nM were determined for yeast [AEDANS‐s2C]Tyr‐tRNATyr, yeast Tyr‐tRNATyr, and the homologous E. coli Phe‐tRNAPhe, respectively, interacting with E. coli elongation factor Tu · GTP. The measurement of the association and dissociation rates of the interaction of [AEDANS‐s2C]Tyr‐tRNATyr with EF‐Tu · GTP and the temperature dependence of the resulting dissociation constants gave values of 55 J mol−1 K−1 for ΔS°′ and –34.7 kJ mol−1 for ΔH°′ of this reaction.