Fluorescence labeling of an aminoacyl-tRNA at the 3'-end and its interaction with elongation factor Tu.GTP.
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 greater than 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 X 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.
D003562:Cytidine / Q000031:analogs & derivatives; D004926:Escherichia coli; D006160:Guanosine Triphosphate / Q000378:metabolism; D009282:Naphthalenesulfonates; D010444:Peptide Elongation Factor Tu / Q000378:metabolism*; D011485:Protein Binding; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D009282:Naphthalenesulfonates; D012346:RNA, Transfer, Amino Acyl; C002908:2-thiocytidine; D003562:Cytidine; C006421:1,5-I-AEDANS; D006160:Guanosine Triphosphate; D010444:Peptide Elongation Factor Tu