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

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 nucleotidyltransferase. [s2C]tRNA was selectively alkylated with fluorescent N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (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 delta S degrees' and -34.7 kJ mol-1 for delta H degrees' of this reaction.