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

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.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.EF-Tu.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.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.(ABSTRACT TRUNCATED AT 250 WORDS)

Many of the conserved nucleotides of tRNA(Phe) are not essential for ternary complex formation and peptide elongation.

“…As noted above, several of the GTP analogues also reduce the association rate. These facts are consistent with the suggestion that binding may be partially rate-limited by a conformational rearrangement (Weygand-Durasevic et al, 1981;Adkins et al, 1983;Hazlett et al, 1989). 2468 1-.;.…”

Restrained Torsional Dynamics of Nuclear DNA in Living Proliferative Mammalian Cells

“…tional intermediate lifetime of about 10 ns, as had been observed before with hairpin structures of DNA (Hernandez et al, 1994) and with tRNA in solution (Hazlett et al, 1989). The contribution of this component, however, never exceeded 10% in living cells ( Table 3).…”

Bacterial elongation factors EF-Tu, their mutants, chimeric forms, and domains: Isolation and purification

“…At present, apparently the most precise EFTu protein quantification can be carried out by quantitative amino acid analysis; according to our experience the values obtained are smaller than those obtained by the Bradford or Lowry procedures (H.Šanderová and J. Jonák, unpublished results). Another way to determine the EF-Tu concentration is by a spectroscopic measurement at 280 m. The extinction coefficient ε 280 of EF-Tu.GDP from E. coli was found to be 29200 M −1 cm −1 by quantitative amino acid analysis [18]. This extinction coefficient is, in contrast to some previous estimations of e.g.…”