MOLECULAR CHARACTERIZATION OF THE PROKARYOTIC EFP GENE-PRODUCT INVOLVED IN A PEPTIDYLTRANSFERASE REACTION

The translation factor EF-P is required for efficient prokaryotic pept ide bond synthesis on 70S ribosomes from fMet-tRNA(Met)(f). This prote in has been purified from Escherichia coli cells and the gene, efp, en coding it has been cloned and sequenced, We have isolated recombinant clones which overexpress a protein that co-migrates with purified EF-P upon SDS-PAGE analysis. Using these-clones, we report the purificatio n, crystallization and initial characterization of the efp gene produc t. The mechanism by which EF-P stimulates peptide-bond synthesis was s tudied using several antibiotics that inhibit translocation, peptide-b ond synthesis and decoding. The stimulation of peptidyltransferase by EF-P was not inhibited by antibiotics that affect translocation and oc cupation of the A site (in the elongation state), ie thiostrepton, vio mycin, neomycin and fusidic acid but was inhibited by streptomycin as well as by inhibitors of peptidyltransferase, chloramphenicol and linc omycin. This observation and the requirement for L16 but not for the L 7/L12 nor L6 or L11 r-proteins suggest that the binding site for EF-P may overlap the peptidyltransferase center of the ribosome.