Design and properties of efficient tRNA:EF-Tu FRET system for studies of ribosomal translation

Abstract

Formation of the ternary complex between GTP-bound form of elongation factor Tu (EF-Tu) and aminoacylated transfer RNA (aa-tRNA) is a key event in protein biosynthesis. Here we show that fluorescently modified Escherichia coli EF-Tu carrying three mutations, C137A, C255V and E348C, and fluorescently modified Phe-tRNA(Phe) form functionally active ternary complex that has properties similar to those of the naturally occurring (unmodified) complex. Similarities include the binding and binding rate constants, behavior in gel retardation assay, as well as activities in tRNA protection and in vitro translation assays. Proper labeling of EF-Tu was demonstrated in MALDI mass spectroscopy experiments. To generate the mutant EF-Tu, a series of genetic constructions were performed. Two native cysteine residues in the wild-type EF-Tu at positions 137 and 255 were replaced by Ala and Val, respectively, and an additional cysteine was introduced either in position 324 or 348. The assembly FRET assay showed a 5- to 7-fold increase of Cy5-labeled EF-Tu E348C mutant fluorescence upon formation of ternary complex with charged tRNA(Phe)(Cy3-labeled) when the complex was excited at 532 nm and monitored at 665 nm. In a control experiment, we did not observe FRET using uncharged tRNA(Phe)(Cy3), nor with wild-type EF-Tu preparation that was allowed to react with Cy5 maleimide, nor in the absence of GTP. The results obtained demonstrate that the EF-Tu:tRNA FRET system described can be used for investigations of ribosomal translation in many types of experiments.

Hierarchical mechanism of amino acid sensing by the T-box riboswitch

“…7a, b and Supplementary Fig. 5e ), comparable to previously measured affinities based on a similar assay 31 . As expected, the ternary complex was not observed for non-aminoacylated tRNA Gly or Cy5-tRNA Gly (Fig.…”

“…Unlabeled tRNA Gly was produced by in vitro transcription and gel purified before charging with glycine. Charging of EF-Tu with GTP was performed based on published protocols 31 and was prepared freshly for each experiment, under the following conditions: 70 mM NH 4 Cl, 7 mM MgCl 2 , 30 mM KCl; and 50 mM Tris-acetate, pH 7, 5 mM β‐mercaptoethanol (BME), 12 μM EF-Tu, 8 mM GTP, 5 U/ml pyruvate kinase from rabbit muscle (Sigma Aldrich), and 7 mM phosphoenolpyruvate. The mixture was incubated for 15 min at 37 °C, and kept on ice before use.…”

“…Electrophoretic mobility shift assays were performed as previously described 31 . Briefly, tRNAs were incubated in 8 mM GTP at concentrations ranging between 0.4 to 1 µM in 50 mM Tris-acetate, pH 6.0, 5 mM BME, 70 mM NH 4 Cl, 7 mM MgCl 2 , and 30 mM KCl.…”

Labeled EF-Tus for Rapid Kinetic Studies of Pretranslocation Complex Formation

“…The latter value (76%) is essentially identical to the FRET efficiency (74%) obtained for EF-Tu AV-Cy5 interaction with a Cy3 derivative of Phe-tRNA Phe labeled at position 47. 22 The similarity in the FRET efficiency values for TC QSY9/Cy3 and TC AV-Cy5/Cy3 is expected based on the similar Förster R o values for the pairs Cy3/QSY9 (55 Å) 26 and Cy3/Cy5 (55–60 Å). 27 Fluorescence quenching within TC QSY9/Cy3 is due solely to the QSY9 bound at position 348, since added wt-EF-Tu QSY9 , which lacks QSY9 bound at EF-Tu position 348 ( Supporting Information Table 1 ), has no effect on Phe-tRNA Phe (Cy3) fluorescence ( Supporting Information Figure S1c ).…”

“…The second variant, C137A/C255V/E348C-EF-Tu, in which the two nonconserved Cys residues are replaced, is labeled with Cy5-maleimide, yielding the labeled product denoted EF-Tu AV-Cy5 , which has 0.9 Cy5/EF-Tu, as compared with 0.2 Cy5/EF-Tu for wt-EF-Tu ( Supporting Information Table 1 ), consistent with earlier results. 22 …”