Localization of noncovalently bound ethidium in free and methionyl-tRNA synthetase bound tRNAfMet by singlet-singlet energy transfer.
Ethidium binds tRNAfMet with 17-fold enhancement in the emission intensity at 600 nm. Fluorescence titration of tRNAfMet with ethidium indicates a single high-affinity site in tRNAfMet with a dissociation constant of 5 microM. Ethidium is apparently rigidly bound to tRNAfMet and effectively shielded from solvent. tRNAfMet(8-13), tRNAfMet(3'-Flc), and tRNAfMet(D-PF) with fluorophores at thiouridine, the 3'-terminus, and dihydrouridine, respectively, are prepared, and the singlet-singlet energy-transfer efficiencies between these fluorophores and noncovalently bound ethidium are determined. The transfer efficiency between bound ethidium and the fluorophore in tRNAfMet(8-13) determined by donor quenching and sensitized emission is the same, strongly suggesting that there is only one bound ethidium per tRNAfMet molecule. The apparent distances between ethidium and various fluorophores including 3'-fluorescein, the 8-13 photo-cross-link, and D-proflavin are 41, 19, and 30 A, respectively, assuming random orientation between the donor and the acceptor. The results suggest that noncovalently bound ethidium is intercalated in the amino acid acceptor stem. In the complex of tRNAfMet and methionyl-tRNA synthetase, the transfer efficiencies for the tRNAfMet(8-13), tRNAfMet(3'-Flc), and tRNAfMet(D-PF) are reduced, enhanced, and little changed, respectively. These methionyl-tRNA synthetase induced changes suggest changes in the conformation of the 3'-terminal unpaired bases and the relative orientation or location between tRNAfMet and ethidium upon binding of methionyl-tRNA synthetase.
D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D004735:Energy Transfer; D004996:Ethidium / Q000378:metabolism*; D007700:Kinetics; D008433:Mathematics; D008718:Methionine-tRNA Ligase / Q000378:metabolism*; D011485:Protein Binding; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D012358:RNA, Transfer, Met
D012346:RNA, Transfer, Amino Acyl; D012358:RNA, Transfer, Met; C024988:tRNA, formylmethionine-; D000604:Amino Acyl-tRNA Synthetases; D008718:Methionine-tRNA Ligase; D004996:Ethidium