FIGURE 1.
Ternary complex structure and steady state measurements of ternary complex formation. A, structure of ternary complex stabilized by the antibiotic kirromycin (Protein Data Bank code 1OB2). E. coli EF-Tu (blue) bound to GDPNP (carbon atoms in gray, nitrogen in blue, oxygen in red, and phosphorous atoms in orange) is complexed with Saccharomyces cerevisiae Phe-tRNAPhe (wheat). Domains of EF-Tu are represented as D1, D2, and D3. For simplicity, kirromycin is not shown. B, specific functional elements are highlighted: switch 1 (S1) is in red, switch 2 (S2) is in orange, His-66 is in green, and the Phe amino acid is in purple. C, the affinity of EF-Tu for aminoacyl-tRNA was determined by titrating EF-Tu (open diamonds) or EF-Tu·EF-Ts (closed squares) into a solution of Cy3-labeled Phe-tRNAPhe and 10 μm GTP. An identical titration of EF-Tu was performed either with deacylated tRNAPhe (circles) or in the absence of GTP (blue squares). D, the apparent nucleotide affinity was measured by titrating GTP into a cuvette containing Phe-tRNAPhe (Cy3-acp3U47) and EF-Tu (open diamonds) or EF-Tu·EF-Ts (closed diamonds). Identical titration experiments of GDPNP with EF-Tu (open triangles) and EF-Tu·EF-Ts (closed triangles) and GTPγS with EF-Tu (open squares) or EF-Tu·EF-Ts (closed squares) are shown. Error bars represent the S.E. of three separate experiments. Estimates of the apparent KD were obtained by fitting the titration data as described under “Experimental Procedures.” Data points were splined for clarity.