Time-resolved fluorescence studies on the ternary complex formed between bacterial elongation factor Tu, guanosine 5'-triphosphate, and phenylalanyl-tRNAPhe

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Abstract
Time-resolved fluorescence spectroscopy was used to investigate the solution dynamics of Escherichia coli tRNAPhe, Phe-tRNAPhe, and Phe-tRNAPHe associated with GTP and elongation factor Tu (EF-Tu) in a ternary complex. Two fluorescence probes were employed: fluorescein, covalently bound to Phe-tRNAPhe at the s4U8 base (Phe-tRNAPhe-Fl8), and ethidium bromide, noncovalently associated with the tRNA (EB.cntdot.Phe-tRNAPhe). The lifetimes observed for ethidium bromide were 1.89 ns, free in solution, and 26.3 ns, bound to its tight binding site on tRNA. Fluorescein-labeled tRNA had a lifetime of 4.3 ns, with no significant difference among the values for aminoacylated, unacylated, and EF-Tu-bound Phe-tRNAPhe-Fl8. Differential phase and modulation data for each fluorophore-tRNA system were fit with local and global Debye rotational relaxation times. Local motion of the labeled fluorescein in Phe-tRNAPhe-Fl8, tRNAPhe-Fl8, and Phe-tRNAPhe-Fl8.cntdot.EF-Tu.cntdot.GTP was characterized by rotational relaxation times of 2.7 .+-. 0.5, 2.4 .+-. 0.4, and 2.4 .+-. 0.1 ns, respectively. These values are equal, within experimental error, and suggest that the rotational mobility of the s4U8-conjugated dye is unaffected by either tRNAPhe aminoacylation or ternary complex formation. Global rotational relaxation times for Phe-tRNAPhe-Fl8, 97 ns, and EB .cntdot. Phe-tRNAPhe, 140 ns, were equivalent to those determined for the unacylated species, denoting little change in the overall size or shape of the tRNA molecule upon aminoacylation. These values for (Phe-)tRNA were larger than expected for a hydrated sphere of equivalent volume, 83 ns, and therefore confirm the asymmetric nature of the tRNA structure in solution. In the presence of EF-Tu the global rotational relaxation times increased to 175 ns (fluorescein) and to 223 ns (ethidium bromide). The ratio of the observed rotational relaxation time to that predicted for a hydrated sphere of equivalent volume (163 ns for the ternary complex) is less for the ternary complex than for either Phe-tRNAPhe-Fl8 or EF-Tu by itself. This result indicates that the ternary complex is more symmetrical than either macromolecular subunit, which suggests that the molecular arrangement of the asymmetric components in this complex is more side by side than end to end.