Fluorescence labeling of an aminoacyl‐tRNA at the 3'‐end and its interaction with elongation factor Tu·GTP

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Abstract
A new approach for the fluorescence labeling of an aminoacyl‐tRNA at the 3'‐end is applied to study its interaction with bacterial elongation factor Tu (EF‐Tu) and GTP at equilibrium. The penultimate cytidine residue in yeast tRNATyr‐C‐C‐A was replaced by 2‐thiocytidine (s2C). The resulting tRNATyr‐C‐s2C‐A was aminoacylated and then alkylated at the s2C residue with N‐(iodoacetylaminoethyl)‐5‐naphthylamine‐1‐sulfonic acid (1,5‐I‐AEDANS). A > 100% increase in the intensity of fluorescence emission of the modified Tyr‐tRNATyr‐C‐s2C(AEDANS)‐A was observed upon interaction with EF‐Tu · GTP. A ternary complex dissociation constant of 1.27 × 10−8 M was calculated from this direct interaction. Using such fluorescent aminoacyl‐tRNA the affinity of any unmodified aminoacyl‐tRNA can be determined by competition experiments. By this approach we show here that the affinity of unmodified Tyr‐tRNATyr‐C‐C‐A is identical to that of the modified Tyr‐tRNATyr. This indicates that the fluorescence labeling procedure applied does not alter the affinity of the aminoacyl‐tRNA for EF‐Tu·GTP. The introduction of 2‐thiocytidine into nucleic acids and their labeling with spectroscopic reporter groups may provide a unique means of investigating various types of nucleic acid‐protein interactions.