FIGURE 7.

Direct evidence of the transient quaternary complex. A, schematic illustrating the FRET assay used to detect quaternary complex. Cy3B-labeled Phe-tRNA^Phe (white) is mixed with EF-Tu/Ts·GTP harboring a Cy5 fluorophore linked to EF-Ts. B, simulation of the time-dependent formation of quaternary complex (red curve; QC) and ternary complex (green curve; TC) based on the schematic presented in A and outlined under “Experimental Procedures.” C, transient FRET observed upon mixing 2 μm EF-Tu/Ts(Cy5) and 250 nm Phe-tRNA^Phe (Cy3B) in the presence of 1 mm GTP (black). These data were fit to a double exponential function (red). This experiment was repeated with deacylated tRNA^Phe (Cy3B) (gray). Error bars represent the S.D. of three separate experiments.