FIG. 3.
ADP-ribosylation of EF-Tu prevents the formation of the stable ternary EF-TuGTPaminoacyl-tRNA complex. [14C]Phe-tRNAPhe (0.5 μM) was incubated with 1 μM EF-Tu-GTP ADP-ribosylated by MTX30-264 (⧫) or 1 μM native EF-Tu-GTP (▴) in a total volume of 60 μl. As a control, the assays were performed in the absence of EF-Tu (▪) and in the presence of EF-Tu treated with catalytically inactive MTX30-264E197Q (○). At the indicated times, 10-μl aliquots of the reaction mixtures were transferred to trichloroacetic acid-soaked filter paper, and the amount of precipitated [14C]Phe-tRNAPhe was quantified by scintillation counting. Time zero was set to 0% hydrolysis.