Methionyl-tRNA synthetase induced 3'-terminal and delocalized conformational transition in tRNAfMet: steady-state fluorescence of tRNA with a single fluorophore.
Five species of tRNAfMet labeled with a single fluorophore are prepared to analyze the conformational changes at the 3'-end, at dihydrouridine, and at thiouridine in tRNAfMet upon binding of methionyl-tRNA synthetase. The emission and excitation spectra, anisotropy, and solvent accessibility of the fluorophore in each of the modified tRNAfMet's are determined in the absence and presence of methionyl-tRNA synthetase. The results are consistent with the following. The probes at the 3'-end are in a nonpolar environment, mobile relative to the tRNA molecule, and fully exposed to the solvent. The probes at dihydrouridine are partially stacked over the neighboring bases, nearly immobile, and relatively inaccessible. The S8-C13 cross-linked product is rigid. Upon binding of methionyl-tRNA synthetase, the probes at the 3'-terminus become localized in a less polar environment, highly immobilized, and effectively shielded against solvent access, while the probes at dihydrouridine appear to be partially unstacked from the neighboring base and become slightly more accessible for solvent. Singlet-singlet energy transfer between the intrinsic protein fluorescence and the fluorophores in modified tRNA's was observed by sensitized emission for tRNAfMet modified at the 3'-end and for S8-C13 but not for tRNAfMet's modified at dihydrouridine. These results suggest that dihydrouridine in tRNAfMet is oriented away from methionyl-tRNA synthetase in the tRNA-enzyme complex.
D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D005456:Fluorescent Dyes; D007700:Kinetics; D008433:Mathematics; D008718:Methionine-tRNA Ligase / Q000378:metabolism*; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D012358:RNA, Transfer, Met; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D012346:RNA, Transfer, Amino Acyl; D012358:RNA, Transfer, Met; C024988:tRNA, formylmethionine-; D000604:Amino Acyl-tRNA Synthetases; D008718:Methionine-tRNA Ligase