Characterization and site of action of a soluble protein that stimulates peptide-bond synthesis

A recently identified soluble protein, named EF-P, stimulates peptide bond synthesis from ribosomal-bound N-formylmethionyl-tRNA and the aminoacyl-tRNA analog, puromycin. Using this model of peptide bond formation we have purified this activity approximately 100-fold from ribosome-free extracts of Escherichia coli. In order to study the mechanism by which the EF-P factor stimulates peptide bond formation, we examined and compared the requirements and site of action of the spontaneous and the EF-P-mediated synthesis of peptide bonds. We find that "enzymic" peptide bond synthesis (+EF-P) is characterized by relatively broad temperature and NH4Cl optima, a sharp Mg2+ optimum at 12 mM, and an apparent pKa of approximately 8.5. The characteristics of enzymic peptide bond synthesis closely resemble those reported for native peptidyl-puromycin formation rather than other models of peptide synthesis. Factor EF-P requires both 30-S and 50-S subunits for activity. The 30-S particle is inactive by itself and may function in the reaction merely to bind the fMet-tRNA substrate. Both the peptidyl transferase and the EF-P binding site may be part of the 50-S subunit. Unlike all other propagation factors, EF-P does not require the 50-S ribosomal proteins L7 and L12 and may therefore occupy a different ribosomal site.