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Fluorescence labeling of an aminoacyl‐tRNA at the 3'‐end and its interaction with elongation factor Tu·GTP
Author(s) -
Joshi R.L.,
Faulhammer H.G.,
Haenni A.L.,
Sprinzl M.
Publication year - 1986
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(86)81015-8
Subject(s) - aminoacyl trna , ef tu , elongation factor , elongation , fluorescence , gtp' , chemistry , gtpase , biophysics , transfer rna , biochemistry , biology , rna , physics , materials science , enzyme , ribosome , quantum mechanics , gene , ultimate tensile strength , metallurgy
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 tRNA Tyr ‐C‐C‐A was replaced by 2‐thiocytidine (s 2 C). The resulting tRNA Tyr ‐C‐s 2 C‐A was aminoacylated and then alkylated at the s 2 C 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‐tRNA Tyr ‐C‐s 2 C(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‐tRNA Tyr ‐C‐C‐A is identical to that of the modified Tyr‐tRNA Tyr . 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.