Characterization of a high-throughput screening assay for inhibitors of elongation factor p and ribosomal peptidyl transferase activity.
Elongation Factor P (EF-P) is an essential component of bacterial protein synthesis, enhancing the rate of translation by facilitating the addition of amino acids to the growing peptide chain. Using purified Staphylococcus aureus EF-P and a reconstituted Escherichia coli ribosomal system, an assay monitoring the addition of radiolabeled N-formyl methionine to biotinylated puromycin was developed. Reaction products were captured with streptavidin-coated scintillation proximity assay (SPA) beads and quantified by scintillation counting. Data from the assay were used to create a kinetic model of the reaction scheme. In this model, EF-P binding to the ribosome essentially doubled the rate of the ribosomal peptidyl transferase reaction. As described here, EF-P bound to the ribosomes with an apparent K(a) of 0.75 microM, and the substrates N-fMet-tRNA and biotinylated puromycin had apparent K(m)s of 19 microM and 0.5 microM, respectively. The assay was shown to be sensitive to a number of antibiotics known to target ribosomal peptide bond synthesis, such as chloramphenicol and puromycin, but not inhibitors that target other stages of protein synthesis, such as fusidic acid or thiostrepton.
Swaney
Steven
S
Biology I, Pfizer, Kalamazoo, MI, USA.
McCroskey
Mark
M
Shinabarger
Dean
D
Wang
Zhigang
Z
Turner
Benjamin A
BA
Parker
Christian N
CN
D000900:Anti-Bacterial Agents / Q000032:analysis* / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D008826:Microbial Sensitivity Tests; D010445:Peptide Elongation Factors / Q000037:antagonists & inhibitors*; D010458:Peptidyl Transferases / Q000037:antagonists & inhibitors*; D015203:Reproducibility of Results; D012269:Ribosomal Proteins / Q000037:antagonists & inhibitors*; D012270:Ribosomes / Q000378:metabolism; D013211:Staphylococcus aureus / Q000378:metabolism; D013997:Time Factors
D000900:Anti-Bacterial Agents; D010445:Peptide Elongation Factors; D012269:Ribosomal Proteins; C020746:factor EF-P; D010458:Peptidyl Transferases