Topographic modeling of free and methionyl-tRNA synthetase bound tRNAfMet by singlet-singlet energy transfer: bending of the 3'-terminal arm in tRNAfMet.
Conformations of tRNAfMet, free and methionyl-tRNA synthetase bound forms, are analyzed by using singlet-singlet energy transfer as a spectroscopic ruler. tRNAfMet(8-13,3'-Flc), tRNAfMet(8-13,D-Etd), and tRNAfMet(3'-Flc,D-Etd) are prepared by sequential chemical modifications. The methionyl-tRNA synthetase binding affinity of these double-labeled tRNAfMets is similar to those of unmodified tRNAfMet. The fluorescence properties of the individual fluorophore in these tRNAs, including emission spectra, anisotropy, and quenching by methionyl-tRNA synthetase, are similar to those of single-labeled tRNAfMet. The transfer efficiencies of double-labeled tRNAfMets, as determined by both donor quenching and sensitized emission, showed efficient energy transfer in all cases. Random orientation being assumed, the apparent distances are 25 A between 8-13 and D20, 44 A between 8-13 and the 3'-terminus, and 49 A between the 3'-terminus and D20, respectively, in free tRNAfMet. Upon binding of methionyl-tRNA synthetase, the apparent distances are 25 A between 8-13 and D20, 45 A between 8-13 and the 3'-terminus, and 54 A between the 3'-terminus and D20, respectively. These results provide topographic models of these specific locations in free and methionyl-tRNA synthetase bound tRNAfMet and suggest that the immobilized 3'-terminal arm in the amino acid acceptor stem bends toward the inner loop of the L-shaped tRNA upon binding of methionyl-tRNA synthetase.
D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D004735:Energy Transfer; D007700:Kinetics; D008718:Methionine-tRNA Ligase / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D012358:RNA, Transfer, Met; D013050:Spectrometry, Fluorescence
D012346:RNA, Transfer, Amino Acyl; D012358:RNA, Transfer, Met; C024988:tRNA, formylmethionine-; D000604:Amino Acyl-tRNA Synthetases; D008718:Methionine-tRNA Ligase