FIGURE 5.

Comparison of the activities of unmodified and modified recombinant EF-P, mutant EF-P (K34A) and native EF-P in N-formyl-methionyl-puromycin synthesis. The assays were carried out as described under “Experimental Procedures.” using 0.5 μg of each purified EF-P protein shown in Fig. 1A. At the indicated time points (1.5, 3, 5, and 10 min), the reaction was stopped, and the N-formyl-methionyl-puromycin formed was measured (A) and the values at 3 min are compared as a bar graph (B). The assay was repeated three times independently, and data are shown with error bars representing S.D. From experiments illustrated in Figs. 1 and 4, His-EF-P1 is mostly unmodified EF-P, His-EF-P2 is estimated to contain α-lysyl-EF-P (>85%) and unmodified EF-P, and His-EF-P3 is estimated to contain β-lysyl-EF-P (>70%) and α-lysyl-EF-P.