Properties of Escherichia coli EF-Tu mutants designed for fluorescence resonance energy transfer from tRNA molecules

Abstract

Here we describe the design, preparation and characterization of 10 EF-Tu mutants of potential utility for the study of Escherichia coli elongation factor Tu (EF-Tu) interaction with tRNA by a fluorescence resonance energy transfer assay. Each mutant contains a single cysteine residue at positions in EF-Tu that are proximal to tRNA sites within the aminoacyl-tRNA.EF-Tu.GTP ternary complex that have previously been labeled with fluorophores. These positions fall in the 323-326 and 344-348 regions of EF-Tu, and at the C terminus. The EF-Tus were isolated as N-terminal fusions to glutathione S-transferase (GST), which was cleaved to yield intact EF-Tus. The mutant EF-Tus were tested for binding to GDP, binding to tRNA in gel retardation and protection assays, and activity in poly-U translation in vitro. The results indicate that at least three EF-Tu mutants, K324C, G325C and E348C, are suitable for further studies. Remarkably, GST fusions that were not cleaved were also active in the various assays, despite the N-terminal fusion.

Fig. 1

Structure of the EF-Tu·GTP·aa-tRNA complex. (A) Highlighted are positions of three key EF-Tu mutations and three nucleotide residues in tRNA commonly used for labeling with fluorescent dyes. The C-terminal residue is marked with a star. Symbols D1, D2 and D3 denote Domains 1, 2 and 3, respectively. Only fragments of Domain 2 can be seen in this drawing. The drawing is based on the coordinates from 1ob2.pdb (R.C. Nielsen, O. Kristensen, M. Kjeldgaard, S. Thirup and P. Nissen, to be published). Phe-tRNA^Phe is from baker's yeast, and EF-Tu is from E.coli. The figure was created in PyMol. (B) Close-up view of the mutated regions 323–326, 344–348 and residue 393 showing positions of the amino acid side-chains.

Fig. 2

Native PAGE of the ternary complex formed by wt EF-Tu with a His₆ tag, EF-Tu_SAVK324C and EF-Tu_SAVE348C. The molar ratio of Phe-tRNA^Phe to EF-Tu is given above each lane. The upper band is EF-Tu·GTP, while the lower band is the ternary complex, EF-Tu·GTP·Phe-tRNA^Phe.

Fig. 3

Sequence alignment of the domain 3 fragment from E.coli EF-Tu subjected to labeling with the corresponding fragments from selected prokaryotes and eukaryotes (mitochondrial EF-Tus). The computational analysis was performed using BLAST network service (Altschul et al., 1997). The amino acid residues are numbered as in E.coli EF-Tu. The residues mutated in this study are in the 323–326 and 344–348 regions, as indicated in the figure.